HK1210425B - Compositions containing hc-ha/ptx3 complexes and methods of use thereof - Google Patents

Description

Compositions containing HC-HA/PTX3 complexes and methods of use thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application Serial No. 61/670,571, filed July 11, 2012, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The work described herein was funded in part by federal research grants RO1 EY06819, R44EY017497, and R43 EY021045 from the National Eye Institute, National Institutes of Health (Bethesda, MD, USA).

Incorporation by Reference of Sequence Listings Submitted as Text Files via EFS-WEB

This application contains a sequence listing, which has been submitted as an ASCII-formatted computer-readable text file via EFS-Web and is incorporated herein by reference in its entirety. The text file was created on July 8, 2013, is named 34157-732-601SEQ.txt, and is 462KB in size.

Background Art

The amniotic membrane (AM) is a nonvascular membrane sac filled with amniotic fluid that surrounds the fetus. Like the placenta, the AM is derived from the epiblast that forms during the development of the fertilized egg. The AM forms the innermost membrane surrounding the fetus in the amniotic cavity. In placental mammals, the umbilical cord (i.e., funiculus umbilicalis) connects the developing fetus to the placenta. The umbilical cord is composed of the amniotic membrane (UCAM) and Wharton's jelly. The amniotic membrane forms the outer layer of the umbilical cord. The UCAM serves to regulate fluid pressure within the UC. Wharton's jelly is a gel-like substance within the umbilical cord, primarily composed of mucopolysaccharides (hyaluronic acid and chondroitin sulfate). It also contains some fibroblasts and macrophages. The umbilical cord further contains two arteries (umbilical arteries) and one vein (umbilical vein) embedded in the Wharton's jelly.

Summary of the Invention

Described herein are methods for identifying HC-HA/PTX3 complexes in fetal tissues, such as amnion and umbilical cord. Also described herein are methods for isolating native HC-HA/PTX3 complexes from fetal tissues, such as amnion and umbilical cord. Also described herein are methods for producing reconstituted HC-HA/PTX3 complexes. Also described herein are methods for using the native and reconstituted HC-HA/PTX3 complexes provided herein.

In certain embodiments, methods of producing HC-HA/PTX3 complexes are described herein. In some embodiments, the methods include isolating native HC-HA/PTX3 (nHC-HA/PTX3) complexes from an extract prepared from tissue or cells. In some embodiments, the methods include generating reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complexes.

In certain embodiments, described herein are methods for isolating a native HC-HA/PTX3 (nHC-HA/PTX3) complex from amniotic tissue, such as the umbilical cord or amniotic membrane. In some embodiments, the nHC-HA/PTX3 complex is isolated from isolated cells. In some embodiments, the nHC-HA/PTX3 complex is isolated from cultured cells. In some embodiments, the nHC-HA/PTX3 complex is isolated from stem cells. In some embodiments, the nHC-HA/PTX3 complex is isolated from the water-soluble fraction of an extract prepared from tissue, such as the umbilical cord or amniotic membrane. In some embodiments, the water-soluble fraction is extracted using an isotonic saline solution. In some embodiments, the nHC-HA/PTX3 complex is isolated from the water-insoluble fraction of an extract prepared from tissue, such as the umbilical cord or amniotic membrane. In some embodiments, the insoluble fraction is extracted using GnHCl.

In certain embodiments, described herein are methods for producing reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complexes in vitro: (a) contacting (i) high molecular weight hyaluronan (HMW HA) immobilized on a solid support, and (ii) pentraxin 3 (PTX3) protein to form a complex of immobilized PTX3 and HMW HA (immobilized PTX3/HA); and (b) contacting the immobilized PTX3/HA with inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HCl) and tumor necrosis factor α-stimulated gene 6 (TSG-6) to form an immobilized rcHC-HA/PTX3 complex. In some embodiments, steps (a) and (b) of the method are performed sequentially. In some embodiments of the method, the method comprises simultaneously contacting high molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) protein, inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HCl), and tumor necrosis factor α-stimulated gene 6 (TSG-6). In some embodiments of the method, TSG-6 catalyzes the covalent attachment of IαI HCl to HA. In some embodiments, the method further comprises removing unbound PTX3 protein after step (a) and before performing step (b). In some embodiments, the method further comprises removing unbound TSG-6 after step (b). In some embodiments, the PTX3 protein used in the method is a naturally occurring PTX3 protein isolated from a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an amniotic cell. In some embodiments, the cell is an umbilical cord cell. In some embodiments, the cell is an amniotic cell from the umbilical cord. In some embodiments, the amniotic cell is an amniotic epithelial cell. In some embodiments, the amniotic cell is an umbilical cord epithelial cell. In some embodiments, the amniotic cell is an amniotic stromal cell. In some embodiments, the amniotic cell is an umbilical cord stromal cell. In some embodiments, the PTX3 protein is a recombinant protein. In some embodiments, the PTX3 protein used in the method comprises a polypeptide having the sequence shown in SEQ ID NO: 33 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO: 33. In some embodiments, the PTX3 protein used in the method comprises a polypeptide having the sequence shown in any one of SEQ ID NOs: 32-45 or a species variant or allelic variant thereof. In some embodiments, the PTX3 protein used in the method is a multimeric protein. In some embodiments, the PTX3 protein used in the method is a homomultimer (i.e., a multimeric protein composed of two or more identical components). In some embodiments, the PTX3 homomultimer is a dimer, trimer, tetramer, pentamer, hexamer, or octamer. In some embodiments, the PTX3 homomultimer is an octamer. In some embodiments, the PTX3 protein comprises a modified multimerization domain or a heterologous multimerization domain. In some embodiments, the TSG-6 protein used in the method is a native TSG-6 protein isolated from a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an amniotic cell. In some embodiments, the cell is an umbilical cord cell. In some embodiments, the cell is an amniotic cell from the umbilical cord. In some embodiments, the amniotic cell is an amniotic epithelial cell. In some embodiments, the amniotic cell is an umbilical cord epithelial cell. In some embodiments, the amniotic cell is an amniotic stromal cell. In some embodiments, the amniotic cell is an umbilical cord stromal cell. In some embodiments, the TSG-6 protein is a recombinant protein. In some embodiments, the TSG-6 protein comprises a polypeptide having a sequence as set forth in SEQ ID NO: 2, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with a polypeptide having a sequence as set forth in SEQ ID NO: 2. In some embodiments, the TSG-6 protein used in the method comprises a polypeptide having a sequence as set forth in any one of SEQ ID NOs: 1-31, or a species variant or allelic variant thereof. In some embodiments, the HCl used in the method comprises a polypeptide having a sequence as set forth in SEQ ID NO: 47, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with a polypeptide having a sequence as set forth in SEQ ID NO: 47. In some embodiments, the inter-α-inhibitor (IαI) protein used as a source of HCl in the method further comprises HC2 and urotryptamine linked by a chondroitin sulfate chain. In some embodiments, the HCl comprises a polypeptide having a sequence as set forth in any one of SEQ ID NOs: 46-47, or a species variant or allelic variant thereof. In some embodiments, the HC2 comprises a polypeptide having a sequence as set forth in SEQ ID NO: 49, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with a polypeptide having a sequence as set forth in SEQ ID NO: 49. In some embodiments, the HC2 used in the method comprises a polypeptide having a sequence as set forth in any one of SEQ ID NOs: 48-49, or a species variant or allelic variant thereof. In some embodiments, the uropancreatin comprises a polypeptide having a sequence as set forth in SEQ ID NO: 53, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with a polypeptide having a sequence as set forth in SEQ ID NO: 53. In some embodiments, the uropancreatin used in the method comprises a polypeptide having a sequence as set forth in any one of SEQ ID NOs: 52-53, or a species variant or allelic variant thereof. In some embodiments, the IαI protein used in the method is isolated from blood, serum, plasma, amnion, chorion, amniotic fluid, or a combination thereof. In some embodiments, the IαI protein used in the method is isolated from serum. In some embodiments, the IαI protein used in the method is isolated from human serum. In some embodiments, the IαI protein used in the method is produced by mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are amniotic cells. In some embodiments, the cells are umbilical cord cells. In some embodiments, the cells are amniotic cells from the umbilical cord. In some embodiments, the amniotic cells are amniotic epithelial cells. In some embodiments, the amniotic cells are umbilical cord epithelial cells. In some embodiments, the amniotic cells are amniotic stromal cells. In some embodiments, the amniotic cells are umbilical cord stromal cells. In some embodiments, the IαI and TSG-6 protein are contacted at a molar ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1. In some embodiments, the IαI and TSG-6 protein are contacted at a molar ratio of 3:1. In some embodiments of the method, the weight average molecular weight of the HMW HA is about 500 kDa to about 10,000 kDa, about 800 kDa to about 8,500 kDa, about 1100 kDa to about 5,000 kDa, or about 1400 kDa to about 3,500 kDa. In some embodiments of the method, the weight average molecular weight of the HMW HA is 3,000 kDa. In some embodiments, the HMW HA is immobilized to the solid support by direct attachment. In some embodiments of the method, the HMW HA is immobilized to the solid support by indirect attachment. In some embodiments of the method, HMW HA is fixed to the solid support by covalent attachment. In some embodiments of the method, HMW HA is fixed to the solid support by non-covalent attachment. In some embodiments of the method, HMW HA is fixed to the solid support by connection via a cleavable linker. In some embodiments, the linker is a chemically or enzymatically cleavable linker. In some embodiments, the method further comprises dissociating the rcHC-HA/PTX3 complex from the solid support after step (b). In some embodiments, the dissociation comprises cleavage of the cleavable linker. In some embodiments, the method further comprises purification of the dissociated rcHC-HA/PTX3 complex. In some embodiments, purification comprises affinity purification, centrifugation, filtration, chromatography, or a combination thereof. In some embodiments of the method, the PTX3, IαI HCl, or TSG-6 polypeptide comprises an affinity tag. In some embodiments, the affinity tag is selected from a hemagglutinin tag, a polyhistidine tag, a myc tag, a FLAG tag, or a glutathione-S-transferase (GST) tag. In some embodiments, HMW HA is immobilized by binding HMW HA to an intermediate polypeptide. In some embodiments, the intermediate polypeptide is covalently attached to a solid support. In some embodiments, the binding of HMW HA to the intermediate polypeptide is non-covalent. In some embodiments, the intermediate polypeptide is an HA binding protein (HABP). In some embodiments, the intermediate polypeptide is selected from HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, CD44, stabilin-1, stabilin-2, or a HABP sufficient to bind a portion thereof to HA. In some embodiments, the intermediate polypeptide is versican. In some embodiments, the intermediate polypeptide comprises a linking module. In some embodiments, the intermediary polypeptide comprises a linking module of HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphocan, TSG-6, CD44, stabilin-1, or stabilin-2. In some embodiments, the intermediary polypeptide comprises a linking module of versican. In some embodiments, the intermediary polypeptide comprises a polypeptide set forth in any one of SEQ ID NOs: 54-99. In some embodiments, the intermediary polypeptide comprises a polypeptide linker. In some embodiments, the linker is attached to a solid support. In some embodiments, the method further comprises dissociating the rcHC-HA/PTX3 complex from the intermediary polypeptide after step (b). In some embodiments, dissociating the rcHC-HA/PTX3 complex from the intermediary polypeptide comprises contacting the complex with a dissociating agent. In some embodiments, the dissociating agent is guanidine hydrochloride or urea. In some embodiments, the dissociating agent is about 4M to about 8M guanidine hydrochloride. In some embodiments, the intermediary polypeptide or linker comprises a proteolytic cleavage sequence. In some embodiments, dissociation of the rcHC-HA/PTX3 complex comprises cleaving the intermediary polypeptide or linker at the proteolytic cleavage sequence. In some embodiments, cleavage comprises contacting the proteolytic cleavage sequence with a protease. In some embodiments, the protease is selected from furin, 3C protease, caspase, matrix metalloprotease, and TEV protease.

In certain embodiments, described herein are methods for producing a reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex in vitro, comprising contacting a PTX3/HA complex immobilized on a solid support with an inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1) and TSG-6. In some embodiments, the PTX3/HA complex is produced by contacting high molecular weight hyaluronan (HMW HA) with a pentraxin 3 (PTX3) protein under conditions effective to form a complex of PTX3 and HMW HA (PTX3/HA), wherein the HMW HA is immobilized on a solid support. In some embodiments, the method further comprises removing unbound PTX3 protein prior to contacting the PTX3/HA complex with IαI and TSG-6. In some embodiments, the method further comprises removing unbound TSG-6. In some embodiments, the PTX3 protein is a recombinant protein. In some embodiments, the PTX3 protein comprises a polypeptide having the sequence set forth in SEQ ID NO:33, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to the polypeptide having the sequence set forth in SEQ ID NO:33. In some embodiments, the PTX3 protein used in the method is a multimeric protein. In some embodiments, the PTX3 protein used in the method is a homomultimer. In some embodiments, the PTX3 homomultimer is a dimer, trimer, tetramer, pentamer, hexamer, or octamer. In some embodiments, the PTX3 homomultimer is an octamer. In some embodiments, PTX3 comprises a modified multimerization domain or a heteromultimerization domain. In some embodiments, TSG-6 is a recombinant protein. In some embodiments, TSG-6 comprises a polypeptide having the sequence set forth in SEQ ID NO:2, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to a polypeptide having the sequence set forth in SEQ ID NO:2. In some embodiments, HCl comprises a polypeptide having the sequence set forth in SEQ ID NO:47, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to a polypeptide having the sequence set forth in SEQ ID NO:47. In some embodiments, the IαI protein further comprises HC2. In some embodiments, HC2 comprises a polypeptide having the sequence set forth in SEQ ID NO:49, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to a polypeptide having the sequence set forth in SEQ ID NO:49. In some embodiments, the IαI protein further comprises uropancreatin. In some embodiments, uropancreatin comprises a polypeptide having the sequence set forth in SEQ ID NO:53, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to the polypeptide having the sequence set forth in SEQ ID NO:53. In some embodiments, IαI further comprises a chondroitin sulfate chain. In some embodiments, the IαI protein is a recombinant protein. In some embodiments, the IαI protein is isolated from blood, plasma, serum, amnion, chorion, amniotic fluid, or a combination thereof. In some embodiments, the IαI protein is isolated from serum. In some embodiments, the IαI protein is isolated from human serum. In some embodiments, the IαI and TSG-6 protein are contacted at a molar ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1. In some embodiments, the IαI and TSG-6 protein are contacted at a molar ratio of 3:1. In some embodiments, the HMW HA has a weight average molecular weight of about 500 kDa to about 10,000 kDa, about 800 kDa to about 8,500 kDa, about 1100 kDa to about 5,000 kDa, or about 1400 kDa to about 3,500 kDa. In some embodiments, the HMW HA has a weight average molecular weight of 3,000 kDa. In some embodiments, the HMW HA is immobilized to the solid support by direct attachment. In some embodiments, the HMW HA is immobilized to the solid support by indirect attachment. In some embodiments, the HMW HA is immobilized on a solid support by covalent attachment. In some embodiments, the HMW HA is immobilized on a solid support by non-covalent attachment. In some embodiments, the method further comprises dissociating the rcHC-HA/PTX3 complex from the solid support. In some embodiments, the method further comprises purifying the dissociated rcHC-HA/PTX3 complex. In some embodiments, purification comprises affinity purification, centrifugation, filtration, chromatography, or a combination thereof. In some embodiments, the PTX3, IαI HCl, or TSG-6 polypeptide comprises an affinity tag. In some embodiments, the affinity tag is selected from a hemagglutinin tag, a polyhistidine tag, a myc tag, a FLAG tag, or a glutathione-S-transferase (GST) tag. In some embodiments, the HMW HA is immobilized by binding the HMW HA to an intermediary polypeptide. In some embodiments, the intermediary polypeptide is covalently attached to a solid support. In some embodiments, the binding of the HMW HA to the intermediary polypeptide is non-covalent. In some embodiments, the intermediary polypeptide is an HA binding protein (HABP). In some embodiments, the intermediary polypeptide is selected from HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, CD44, stabilin-1, stabilin-2, or a portion thereof sufficient to bind to HA. In some embodiments, the intermediary polypeptide is versican. In some embodiments, the intermediary polypeptide comprises a linking module. In some embodiments, the intermediary polypeptide comprises a linking module of HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, CD44, stabilin-1, or stabilin-2. In some embodiments, the intermediary polypeptide comprises a linking module of versican. In some embodiments, the intermediary polypeptide comprises a polypeptide set forth in any one of SEQ ID NOs: 54-99. In some embodiments, the intermediary polypeptide comprises a polypeptide linker. In some embodiments, the linker is attached to a solid support. In some embodiments, the method further comprises dissociating the rcHC-HA/PTX3 complex from the intermediary polypeptide. In some embodiments, dissociating the rcHC-HA/PTX3 complex from the intermediary polypeptide comprises contacting the complex with a dissociating agent. In some embodiments, the dissociating agent is guanidine hydrochloride or urea. In some embodiments, the dissociating agent is about 4M to about 8M guanidine hydrochloride. In some embodiments, the intermediary polypeptide or linker comprises a proteolytic cleavage sequence. In some embodiments, dissociation comprises cleaving the intermediary polypeptide or linker at the proteolytic cleavage sequence. In some embodiments, cleavage comprises contacting the proteolytic cleavage sequence with a protease. In some embodiments, the protease is selected from furin, 3C protease, caspase, matrix metalloprotease, and TEV protease.

Described herein, in certain embodiments, are reconstituted HC-HA (rcHC-HA/PTX3) complexes produced by any of the methods provided herein for generating rcHC-HA/PTX3 complexes.

In certain embodiments, described herein are reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA/PTX3 complex promotes M2 polarization of macrophages. In certain embodiments, described herein are native HC-HA/PTX3 (nHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαI HC1, wherein the nHC-HA/PTX3 complex promotes M2 polarization of macrophages. In certain embodiments, described herein are methods of inducing M2 polarization of macrophages comprising contacting a macrophage with an rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex.

In certain embodiments, described herein are reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαIHC1, wherein the rcHC-HA/PTX3 complex reduces IL-12p40 expression in LPS-stimulated macrophages, wherein the level of IL-12p40 expressed by the LPS-stimulated macrophages is lower when the LPS-stimulated macrophages are contacted with the rcHC-HA/PTX3 complexes compared to the level of IL-12p40 expressed by the LPS-stimulated macrophages in the absence of the rcHC-HA/PTX3 complexes. In some embodiments, the level of IL-12p40 mRNA is reduced. In some embodiments, the level of IL-12p40 protein is reduced.

In certain embodiments, described herein are native HC-HA/PTX3 (nHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαIHC1, wherein the nHC-HA/PTX3 complex reduces IL-12p40 expression in LPS-stimulated macrophages, wherein the level of IL-12p40 expressed by the LPS-stimulated macrophages when the LPS-stimulated macrophages are contacted with the nHC-HA/PTX3 complex is lower compared to the level of IL-12p40 expressed by the LPS-stimulated macrophages in the absence of the nHC-HA/PTX3 complex. In some embodiments, the level of IL-12p40 mRNA is reduced. In some embodiments, the level of IL-12p40 protein is reduced.

In certain embodiments, described herein are methods for reducing the level of IL-12p40 expressed by LPS-stimulated macrophages, comprising contacting the LPS-stimulated macrophages with rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complexes. In some embodiments, the level of IL-12p40 mRNA is reduced. In some embodiments, the level of IL-12p40 protein is reduced.

Described herein, in certain embodiments, are reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαIHC1, wherein the rcHC-HA/PTX3 complexes reduce expression of IL-12p70 protein in LPS-stimulated macrophages, wherein the amount of IL-12p70 protein expressed by the LPS-stimulated macrophages is lower when the LPS-stimulated macrophages are contacted with the rcHC-HA/PTX3 complexes as compared to the amount of IL-12p70 expressed by the LPS-stimulated macrophages in the absence of the rcHC-HA/PTX3 complexes.

Described herein, in certain embodiments, are native HC-HA/PTX3 (nHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαIHC1, wherein the nHC-HA/PTX3 complex reduces expression of IL-12p70 protein in LPS-stimulated macrophages, wherein the amount of IL-12p70 protein expressed by the LPS-stimulated macrophages is lower when the LPS-stimulated macrophages are contacted with the nHC-HA/PTX3 complexes as compared to the amount of IL-12p70 protein expressed by the LPS-stimulated macrophages in the absence of the nHC-HA/PTX3 complex.

Described herein, in certain embodiments, are methods of reducing the level of IL-12p70 protein expressed by LPS-stimulated macrophages, comprising contacting the LPS-stimulated macrophages with rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complexes.

In certain embodiments, described herein are reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαIHC1, wherein the rcHC-HA/PTX3 complex reduces IL-23 expression in LPS-stimulated macrophages, wherein the level of IL-23 expressed by the LPS-stimulated macrophages is lower when the LPS-stimulated macrophages are contacted with the rcHC-HA/PTX3 complex as compared to the level of IL-23 expressed by the LPS-stimulated macrophages in the absence of the rcHC-HA/PTX3 complex. In some embodiments, the level of IL-23 mRNA is reduced. In some embodiments, the level of IL-23 protein is reduced.

In certain embodiments, described herein are native HC-HA/PTX3 (rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαIHC1, wherein the complex reduces IL-23 expression in LPS-stimulated macrophages, wherein the level of IL-23 expressed by the LPS-stimulated macrophages is lower when the LPS-stimulated macrophages are contacted with the nHC-HA/PTX3 complex compared to the level of IL-23 expressed by the LPS-stimulated macrophages in the absence of the nHC-HA/PTX3 complex. In some embodiments, the level of IL-23 mRNA is reduced. In some embodiments, the level of IL-23 protein is reduced.

In certain embodiments, described herein are methods of reducing the level of IL-23 expressed by LPS-stimulated macrophages, comprising contacting the LPS-stimulated macrophages with rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complexes. In some embodiments, the level of IL-23 mRNA is reduced. In some embodiments, the level of IL-23 protein is reduced.

In certain embodiments, described herein are reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαIHC1, wherein the rcHC-HA/PTX3 complex increases IL-10 expression in LPS-stimulated macrophages, wherein the level of IL-10 expressed by the LPS-stimulated macrophages is higher when the LPS-stimulated macrophages are contacted with the rcHC-HA/PTX3 complex as compared to the level of IL-10 expressed by the LPS-stimulated macrophages in the absence of the rcHC-HA/PTX3 complex. In some embodiments, the level of IL-10 mRNA is increased. In some embodiments, the level of IL-10 protein is increased.

In certain embodiments, described herein are native HC-HA/PTX3 (nHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαIHC1, wherein the complex increases IL-10 expression in LPS/IFNγ-stimulated macrophages, wherein the amount of IL-10 expressed by the LPS/IFNγ-stimulated macrophages is higher when the LPS-stimulated macrophages are contacted with the nHC-HA/PTX3 complex compared to the amount of IL-10 expressed by the LPS/IFNγ-stimulated macrophages in the absence of the nHC-HA/PTX3 complex. In some embodiments, the level of IL-10 mRNA is increased. In some embodiments, the level of IL-10 protein is increased.

In certain embodiments, described herein are methods for increasing the level of IL-10 expressed by LPS-stimulated macrophages, comprising contacting the LPS-stimulated macrophages with rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complexes. In some embodiments, the level of IL-10 mRNA is increased. In some embodiments, the level of IL-10 protein is increased.

In certain embodiments, described herein are reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA/PTX3 complex promotes apoptosis in LPS-stimulated neutrophils, but not in resting neutrophils. In some embodiments, a reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαI HC1 promotes apoptosis in LPS-stimulated neutrophils, wherein the number of LPS-stimulated neutrophils that become apoptotic in a sample of LPS-stimulated neutrophils is higher when the sample is contacted with the rcHC-HA/PTX3 complex as compared to the number of LPS-stimulated neutrophils that become apoptotic in the sample in the absence of the rcHC-HA/PTX3 complex.

In certain embodiments, described herein are native HC-HA/PTX3 (nHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαI HC1, wherein the nHC-HA/PTX3 complex promotes apoptosis in LPS-stimulated neutrophils, but not in resting neutrophils. In some embodiments, the nHC-HA/PTX3 complex comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαI HC1 promotes apoptosis in LPS-stimulated neutrophils, wherein the number of apoptotic LPS-stimulated neutrophils in a sample of LPS-stimulated neutrophils is higher when the sample is contacted with the nHC-HA/PTX3 complex as compared to the number of apoptotic LPS-stimulated neutrophils in the sample in the absence of the nHC-HA/PTX3 complex.

Described herein, in certain embodiments, are methods of inducing apoptosis in LPS-stimulated neutrophils, comprising contacting the LPS-stimulated neutrophils with rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complexes.

Described herein, in certain embodiments, are reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαIHC1, wherein the rcHC-HA/PTX3 complex promotes phagocytosis of apoptotic neutrophils, wherein when a sample of apoptotic neutrophils and LPS-stimulated macrophages is contacted with the rcHC-HA/PTX3 complex, the number of apoptotic neutrophils that are phagocytosed by LPS-stimulated macrophages in the sample is higher compared to the number of neutrophils that are phagocytosed by LPS-stimulated macrophages in the sample in the absence of the rcHC-HA/PTX3 complex. Described herein, in certain embodiments, are reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαI HCl, wherein the complex promotes phagocytosis of apoptotic neutrophils, wherein when a sample of apoptotic neutrophils and resting macrophages is contacted with the rcHC-HA/PTX3 complex, the number of apoptotic neutrophils that are phagocytosed by resting macrophages in the sample is higher compared to the number of neutrophils that are phagocytosed by resting macrophages in the sample in the absence of the rcHC-HA/PTX3 complex.

Described herein, in certain embodiments, are native HC-HA/PTX3 (nHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαIHC1, wherein the nHC-HA/PTX3 complex promotes phagocytosis of apoptotic neutrophils, wherein when a sample of apoptotic neutrophils and LPS-stimulated macrophages is contacted with the nHC-HA/PTX3 complex, the number of apoptotic neutrophils that are phagocytosed by LPS-stimulated macrophages in the sample is higher compared to the number of neutrophils that are phagocytosed by LPS-stimulated macrophages in the sample in the absence of the nHC-HA/PTX3 complex. Described herein, in certain embodiments, are native HC-HA/PTX3 (nHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαI HCl, wherein the nHC-HA/PTX3 complex promotes phagocytosis of apoptotic neutrophils, wherein when a sample of apoptotic neutrophils and LPS-stimulated macrophages is contacted with the nHC-HA/PTX3 complex, the number of apoptotic neutrophils that are phagocytosed by resting macrophages in the sample is higher compared to the number of neutrophils that are phagocytosed by resting macrophages in the sample in the absence of the nHC-HA/PTX3 complex.

Described herein, in certain embodiments, are methods of inducing phagocytosis of apoptotic neutrophils, comprising contacting a sample comprising apoptotic neutrophils and LPS-stimulated or resting macrophages with an rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex.

In certain embodiments, described herein are reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαIHC1, wherein the rcHC-HA/PTX3 complex promotes LPS-stimulated macrophage attachment to at least the same level as native HC-HA/PTX3 (nHC-HA/PTX3) complexes isolated from human umbilical cord, human amniotic membrane, or a combination of nHC-HA/PTX3 complexes from both human umbilical cord and human amniotic membrane, wherein attachment comprises contacting LPS-stimulated macrophages with rcHC-HA/PTX3 or nHC-HA/PTX3 complexes immobilized on a solid support. In some embodiments, the nHC-HA/PTX3 is isolated from human umbilical cord. In some embodiments, the nHC-HA/PTX3 is isolated from human amniotic membrane. In some embodiments, the nHC-HA/PTX3 is isolated from a combination of nHC-HA/PTX3 complexes from both human umbilical cord and human amniotic membrane.

In some embodiments, the weight average molecular weight of the HMW HA of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is about 500 kDa to about 10,000 kDa, about 800 kDa to about 8,500 kDa, about 1100 kDa to about 5,000 kDa, or about 1400 kDa to about 3,500 kDa. In some embodiments, the weight average molecular weight of the HMW HA of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is about 3,000 kDa.

In some embodiments, the HCl of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is covalently linked to HA.

In some embodiments, the PTX3 protein of the rcHC-HA/PTX3 complex is a recombinant protein. In some embodiments, the PTX3 of the rcHC-HA/PTX3 complex comprises a polypeptide having the sequence set forth in SEQ ID NO: 33, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to the polypeptide having the sequence set forth in SEQ ID NO: 33. In some embodiments, the PTX3 protein used in the method is a multimeric protein. In some embodiments, the PTX3 protein used in the method is a homomultimer. In some embodiments, the PTX3 homomultimer is a dimer, trimer, tetramer, pentamer, hexamer, or octamer. In some embodiments, the PTX3 homomultimer is a trimer, tetramer, or octamer. In some embodiments, the PTX3 homomultimer is an octamer.

In some embodiments, the IαI HCl of the rcHC-HA/PTX3 complex comprises a polypeptide having the sequence set forth in SEQ ID NO: 47, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to the polypeptide having the sequence set forth in SEQ ID NO: 47. In some embodiments, the IαI HCl of the rcHC-HA/PTX3 complex is a recombinant protein.

In some embodiments, the rcHC-HA/PTX3 complex comprises TSG-6. In some embodiments, the TSG-6 protein is a recombinant protein. In some embodiments, the TSG-6 protein comprises a polypeptide having the sequence set forth in SEQ ID NO: 2, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to a polypeptide having the sequence set forth in SEQ ID NO: 2.

In some embodiments, the PTX3, IαI HCl, or TSG-6 polypeptide of the rcHC-HA/PTX3 complex comprises an affinity tag. In some embodiments, the affinity tag is selected from a hemagglutinin tag, a polyhistidine tag, a myc tag, a FLAG tag, or a glutathione-S-transferase (GST) tag.

In certain embodiments, described herein is a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is in the form of a solution, suspension, powder, ointment, tablet, capsule, or aerosol. In some embodiments, the pharmaceutical composition is in the form of a solid, a cross-linked gel, or a liposome. In some embodiments, the pharmaceutical composition is in the form of a cross-linked hyaluronan hydrogel. In some embodiments, the pharmaceutical composition comprises a natural polymer. In some embodiments, the natural polymer comprises fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate, or a combination thereof. In some embodiments, the pharmaceutical composition further comprises an anti-inflammatory agent, an anti-scarring agent, an anti-tumor agent, a chemotherapeutic agent, an immunosuppressant, a cytotoxic agent, an antimicrobial agent, or a combination thereof.

Described herein, in certain embodiments, are uses of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein for the production of a medicament.

Described herein, in certain embodiments, is a combination comprising: (a) an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein; and (b) an anti-inflammatory agent, an anti-scarring agent, an anti-tumor agent, a chemotherapeutic agent, an immunosuppressive agent, a cytotoxic agent, an antimicrobial agent, or a combination thereof.

In certain embodiments, described herein are methods of treatment comprising administering a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein.

In certain embodiments, described herein are methods for preventing or reversing scarring or fibrosis in a tissue, comprising administering to a subject in need thereof an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting the tissue with an effective amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, the scar is a dermatitis scar, a keloid scar, a contracture scar, a hypertrophic scar, or a scar caused by acne. In certain embodiments, described herein are uses of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein for reducing or preventing scarring. In some embodiments, administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein reduces or prevents scarring or fibrosis by reducing or inhibiting TGF-β signaling in the tissue.

In certain embodiments, described herein are methods for preventing or reducing inflammation in a subject in need thereof, comprising administering to the subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting inflamed tissue with the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, the inflammation is acute inflammation or chronic inflammation. In some embodiments, the subject suffers from an inflammatory disorder. In some embodiments, the inflammatory disorder is a macrophage-mediated inflammatory disorder, a Th-17-mediated immune disorder, or a T-cell-mediated inflammatory disorder. In some embodiments, the subject suffers from an autoimmune disease, an allergy, a leukocyte deficiency, an infection, graft-versus-host disease, tissue transplant rejection, or a combination thereof. In some embodiments, the inflammatory disorder is rheumatoid arthritis. In some embodiments, the inflammatory disorder is an inflammatory disorder of the eye. In some embodiments, the inflammatory condition is conjunctivitis, keratitis, blepharitis, blepharoconjunctivitis, scleritis, episcleritis, uveitis, retinitis, or choroiditis. In some embodiments, the acute inflammation is caused by myocardial infarction, stroke, endotoxic shock, or sepsis. In some embodiments, the subject has atherosclerosis. In some embodiments, the subject has cancer. In some embodiments, the subject has inflammation of a solid tumor. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered to the subject in combination with an additional anti-inflammatory agent. In some embodiments, the additional anti-inflammatory agent is selected from an anti-TGF-β antibody, an anti-TGF-β receptor blocking antibody, an anti-TNF antibody, an anti-TNF receptor blocking antibody, an anti-IL1β antibody, an anti-IL1β receptor blocking antibody, an anti-IL-2 antibody, an anti-IL-2 receptor blocking antibody, an anti-IL-6 antibody, an anti-IL-6 receptor blocking antibody, an anti-IL-12 antibody, an anti-IL-12 receptor blocking antibody, an anti-IL-17 antibody, an anti-IL-17 receptor blocking antibody, an anti-IL-23 antibody, or an anti-IL-23 receptor blocking antibody. In some embodiments, the type 1 interferon is IFN-α or IFN-β. In certain embodiments, described herein are uses of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein or produced by the methods provided herein for reducing or preventing inflammation.

In certain embodiments, described herein are methods of treating a skin wound or ulcer in a subject in need thereof, comprising administering to the subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting the skin wound or ulcer with an effective amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In certain embodiments, described herein are uses of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein for treating a skin wound or ulcer. In some embodiments, the skin wound or ulcer is a non-healing ulcer.

In certain embodiments, described herein are methods of promoting or inducing bone formation in a subject in need thereof, comprising administering to the subject an effective amount of an rcHC-HA/PTX3 complex or nHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the subject has arthritis, osteoporosis, alveolar bone degradation, Paget's disease, or a bone tumor. In certain embodiments, described herein are uses of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein for promoting or inducing bone formation in a subject.

In certain embodiments, described herein are methods for preventing or reducing abnormal angiogenesis in a subject in need thereof, comprising administering to the subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the subject suffers from wet age-related macular degeneration (wARMD) or diabetic proliferative retinopathy. In some embodiments, the subject suffers from cancer. In some embodiments, the subject suffers from a solid tumor. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered to the subject in combination with an anti-cancer therapy. In some embodiments, the anti-cancer therapy comprises administration of an anti-tumor agent, a cytotoxic agent, an anti-angiogenic agent, a chemotherapeutic agent, or radiation therapy. In some embodiments, the anti-cancer therapy is administered sequentially, simultaneously, or intermittently with the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In certain embodiments, described herein are uses of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein for reducing or preventing angiogenesis.

In certain embodiments, described herein are methods for preventing transplant rejection in a transplant recipient, comprising administering to the transplant recipient an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting a transplant with the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered before, after, or during the transplant procedure. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered in combination with an immunosuppressant. In certain embodiments, described herein are uses of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein or produced by the methods provided herein for preventing transplant rejection in a transplant recipient. In some embodiments, the transplant is a corneal transplant.

In certain embodiments, described herein are methods for inducing stem cell expansion in a subject in need thereof, comprising administering to the subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting a stem cell with the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In certain embodiments, described herein are uses of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein or produced by the methods provided herein for inducing stem cell expansion. In some embodiments, administration of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein or produced by the methods provided herein induces stem cell expansion by inhibiting TGF-β signaling and/or upregulating the BMP signaling pathway. In some embodiments, administration of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein or produced by the methods provided herein induces stem cell expansion by reprogramming differentiated cells into stem cells (or induced progenitor cells, iPSCs).

Described herein, in certain embodiments, are uses of the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein or produced by the methods provided herein for cell therapy.

In certain embodiments, described herein are methods for performing cell therapy in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein, in combination with therapeutic cells. In some embodiments, the therapeutic cells and the nHC-HA/PTX3 or rcHC-HA/PTX3 complex are administered locally to damaged tissue. In some embodiments, the therapeutic cells and the nHC-HA/PTX3 or rcHC-HA/PTX3 complex are administered systemically. In some embodiments, the therapeutic cells are stem cells. In some embodiments, the therapeutic agent is a stem cell. In some embodiments, the cell therapy comprises administering stem cells. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the stem cells are induced progenitor stem cells. In some embodiments, the cell therapy comprises administering differentiated cells. In some embodiments, the therapeutic cells are insulin-producing cells. In some embodiments, the insulin-producing cells are pancreatic islet cells. In some embodiments, the subject suffers from type 1 diabetes.

In certain embodiments, described herein are methods for performing cell therapy in a subject in need thereof, comprising administering to the subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein, in combination with cells contained in a cell delivery device. In some embodiments, the cells are contained in a microcapsule. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached to a microcapsule. In some embodiments, the microcapsule is administered topically to damaged tissue. In some embodiments, the therapeutic cells and the nHC-HA/PTX3 or rcHC-HA/PTX3 complex are administered systemically. In some embodiments, the therapeutic cells are stem cells. In some embodiments, the therapeutic agent is a stem cell. In some embodiments, the cell therapy comprises administering stem cells. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the stem cells are induced progenitor stem cells. In some embodiments, the cell therapy comprises administering differentiated cells. In some embodiments, the therapeutic cells are insulin-producing cells. In some embodiments, the insulin-producing cells are pancreatic islet cells. In some embodiments, the subject has type 1 diabetes.

In certain embodiments, described herein are methods of preventing or reducing pain in a subject in need thereof, comprising administering to the subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein, wherein the pain is caused by chemical burns, severe bacterial keratitis, Stevens-Johnson syndrome, toxic epidermal necrolysis, or radiation to an ocular tumor. In certain embodiments, described herein are uses of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein for alleviating pain in a subject, wherein the pain is caused by chemical burns, severe bacterial keratitis, Stevens-Johnson syndrome, toxic epidermal necrolysis, or radiation to an ocular tumor.

In certain embodiments, described herein are methods for inducing or promoting tissue regeneration in a subject in need thereof, comprising administering to the subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting the subject's damaged tissue with an effective amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, the tissue is bone or gingival, corneal tissue, or conjunctival tissue. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered in combination with therapeutic cells, multiple therapeutic cells, or a tissue graft. In some embodiments, the tissue graft is an allogeneic graft or an autologous graft. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered in combination with a tissue-based therapy. In certain embodiments, described herein are uses of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein for inducing or promoting tissue regeneration in a subject.

In certain embodiments, described herein are methods of treating fibrosis in a subject in need thereof, comprising administering to the subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the treatment inhibits or prevents scarring. In certain embodiments, described herein are uses of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein for treating fibrosis in a subject.

In certain embodiments, described herein are methods for treating obesity or insulin resistance in a subject in need thereof, comprising administering to the subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the treatment inhibits or reduces the amount of M1 adipose tissue macrophages in the subject. In certain embodiments, described herein are methods for inhibiting or reducing the amount of M1 adipose tissue macrophages in a subject in need thereof, comprising administering to the subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the subject has been diagnosed with obesity or insulin resistance. In certain embodiments, described herein are uses of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein or produced by the methods provided herein for treating obesity or insulin resistance in a subject.

In certain embodiments, described herein are methods of treating conjunctivochalasis in a subject in need thereof, comprising administering to the subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting the conjunctiva of the subject with an effective amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In certain embodiments, described herein are uses of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein or produced by the methods provided herein for treating conjunctivochalasis in a subject.

In certain embodiments, described herein are cell cultures comprising a substrate suitable for culturing cells and an rcHA/PTX3 complex or nHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the rcHA/PTX3 complex or nHC-HA/PTX3 complex is immobilized to a substrate.

In certain embodiments, methods of treatment are described herein, wherein an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein is administered to a subject in combination with an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from an interferon, an anti-tumor necrosis agent, an interleukin-1 (IL-1) receptor antagonist, an interleukin-2 (IL-2) receptor antagonist, an interleukin-6 (IL-6) receptor antagonist, an interleukin-12 (IL-12) receptor antagonist, an interleukin-17 (IL-17) receptor antagonist, an interleukin-23 (IL-23) receptor antagonist, a cytotoxic agent, an antimicrobial agent, an interleukin, an immunomodulator, an antibiotic, a T-cell co-stimulatory blocker, a disease-modifying antirheumatic agent, an immunomodulator, an anti-inflammatory drug ... Inhibitors, anti-lymphocyte antibodies, anti-angiogenic agents, chemotherapeutic agents, anti-tumor agents, antimetabolites, Akt inhibitors, IGF-1 inhibitors, angiotensin II antagonists, cyclooxygenase inhibitors, heparanase inhibitors, lymphokine inhibitors, cytokine inhibitors, IKK inhibitors, P38MAPK inhibitors, anti-apoptotic pathway inhibitors, apoptotic pathway agonists, PPAR agonists, inhibitors of Ras, telomerase inhibitors, protease inhibitors, metalloproteinase inhibitors, aminopeptidase inhibitors, SHIP activators and combinations thereof. In some embodiments, the antimicrobial agent is an antiviral, antibacterial or antifungal agent.

In certain embodiments, described herein are methods of treatment wherein an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein is administered to a subject and the subject is a mammal. In some embodiments, the mammal is a human.

In certain embodiments, described herein are treatment methods wherein the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached to a solid surface. In some embodiments, the solid surface is the surface or a portion thereof of a nanoparticle, bead, microcapsule, or implantable medical device.

In certain embodiments, described herein are medical devices comprising a substrate coated with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the substrate comprises at least one of a stent, a joint, a screw, a rod, a pin, a plate, a staple, a shunt, a clamp, a clip, a suture, a suture anchor, an electrode, a catheter, a lead, a graft, a dressing, a pacemaker, a pacemaker housing, a cardioverter, a cardioverter housing, a defibrillator, a defibrillator housing, a prosthesis, an ear drain, an ophthalmic implant, an orthopedic device, a spinal disc, a bone substitute, an anastomotic device, a perivascular wrap, a colostomy bag attachment device, a hemostatic barrier, a vascular implant, a vascular support, a tissue adhesive, a tissue sealant, a tissue scaffold, and an intraluminal device.

In certain embodiments, described herein are devices comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein, immobilized on a surface. In some embodiments, the surface is a polystyrene, polyethylene, silica, metal, or polymer surface. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached to a microcapsule. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached to a nanoparticle. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached to a bead, a chip, a slide, or a filter. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached to a contact lens. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached to a surgical implant or prosthesis. In some embodiments, the implant or prosthesis is an artificial joint, a bone implant, a suture, or a stent. In some embodiments, the artificial joint is an artificial hip joint, an artificial knee joint, an artificial shoulder joint, or an artificial knee. In some embodiments, the stent is a coronary artery stent, a ureteral stent, a urethral stent, a prostate stent, an esophageal stent, or a bone stent.

In certain embodiments, described herein are medical devices comprising: a structure suitable for implantation into a patient, wherein a surface or portion of the surface of the structure is attached to a composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the attachment comprises covalent or non-covalent attachment of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex to the surface or portion of the surface of the structure. In some embodiments, the attachment comprises coating the surface or portion of the surface of the structure with a composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, the structure is a vascular stent, an artificial joint, a suture, or a microcapsule. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex inhibits bacterial biofilm formation. In some embodiments, the microcapsule contains therapeutic cells. In some embodiments, the therapeutic cells are stem cells.

In certain embodiments, methods for modulating macrophage activity are described herein, comprising contacting a macrophage with an amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein, the amount being sufficient to reduce or inhibit the expression of IL-12 or IL-23, but also promoting the expression of IL-10 in polarized macrophages from an M1 to an M2 phenotype. In some embodiments, the macrophage has been stimulated with a proinflammatory mediator. In some embodiments, the proinflammatory mediator is lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-α), interferon-γ (IFNγ), or a combination thereof. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex contacts the macrophage in a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the method is performed in vitro. In certain embodiments, treatment methods are described herein, comprising administering macrophages that have been modulated by the methods provided herein for modulating macrophage activity.

In certain embodiments, described herein is a kit comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by the methods provided herein, a device for administering the composition, and optionally instructions for administration.

In certain embodiments, described herein is a combination or mixture comprising: (a) a complex of PTX3 pre-bound to HMW HA (PTX3/HA); (b) inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1); and (c) TSG-6.

In certain embodiments, described herein is a combination or mixture comprising: (a) a complex of TSG-6 pre-bound to HC-HA; and (b) PTX3.

In certain embodiments, described herein are methods of producing a reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex in vitro, comprising: (a) contacting (i) high molecular weight hyaluronan (HMW HA) immobilized on a solid support, (ii) an inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1), and (iii) TSG-6 to form a rcHC-HA complex pre-bound to TSG-6; and (b) contacting the rcHC-HA complex pre-bound to TSG-6 with a pentraxin 3 (PTX3) protein to form the rcHC-HA/PTX3 complex.

Described herein, in certain embodiments, are methods for producing a reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex in vitro, comprising contacting (i) high molecular weight hyaluronan (HMW HA) immobilized on a solid support, (ii) pentraxin 3 (PTX3) protein, (iii) inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1), and (iv) tumor necrosis factor α-stimulated gene 6 (TSG-6) to form an immobilized rcHC-HA/PTX3 complex.

In certain embodiments, described herein is a complex comprising immobilized HA bound to PTX3.

In certain embodiments, described herein are methods for producing a complex comprising immobilized HA bound to PTX3 in vitro, comprising contacting high molecular weight hyaluronan (HMW HA) with a PTX3 protein under conditions effective to form a complex of PTX3 and HMW HA (PTX3/HA), wherein the HMW HA is immobilized on a solid support. In some embodiments, the PTX3 protein is a native PTX3 protein isolated from a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an amniotic cell. In some embodiments, the cell is an umbilical cord cell. In some embodiments, the cell is an amniotic cell from the umbilical cord. In some embodiments, the amniotic cell is an amniotic epithelial cell. In some embodiments, the amniotic cell is an umbilical cord epithelial cell. In some embodiments, the amniotic cell is an amniotic stromal cell. In some embodiments, the amniotic cell is an umbilical cord stromal cell. In some embodiments, the PTX3 protein is a recombinant protein. In some embodiments, the PTX3 protein comprises a polypeptide having the sequence set forth in SEQ ID NO:2, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to the polypeptide having the sequence set forth in SEQ ID NO:2. In some embodiments, the PTX3 protein used in the method is a multimeric protein. In some embodiments, the PTX3 protein used in the method is a homomultimer. In some embodiments, the PTX3 homomultimer is a dimer, trimer, tetramer, pentamer, hexamer, or octamer. In some embodiments, the PTX3 homomultimer is a trimer, tetramer, or octamer. In some embodiments, the PTX3 homomultimer is an octamer. In some embodiments, the PTX3 comprises a modified multimerization domain or a heteromultimerization domain. In some embodiments, immobilizing the HMW HA comprises non-covalent attachment to a solid support. In some embodiments, immobilizing HMW HA comprises binding HMW HA to an intermediary polypeptide. In some embodiments, the intermediary polypeptide is covalently attached to a solid support. In some embodiments, the binding of HMW HA to the intermediary polypeptide is non-covalent. In some embodiments, the intermediary polypeptide is an HA binding protein (HABP). In some embodiments, the intermediary polypeptide is an HABP selected from HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphocan, TSG-6, CD44, stabilin-1, stabilin-2, or a portion thereof sufficient to bind to HA. In some embodiments, the intermediary polypeptide is versican. In some embodiments, the intermediary polypeptide comprises a linking module. In some embodiments, the intermediary polypeptide comprises a linking module of HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphocan, TSG-6, CD44, stabilin-1, or stabilin-2. In some embodiments, the intermediary polypeptide comprises a linking module of versican. In some embodiments, the intermediary polypeptide comprises a polypeptide set forth in any one of SEQ ID NOs: 54-99. In certain embodiments, described herein are PTX3/HA complexes produced by the aforementioned methods. In certain embodiments, described herein are pharmaceutical compositions comprising a PTX3/HA complex produced by the aforementioned methods. In certain embodiments, described herein are uses of the PTX3/HA complex for the production of a medicament. In certain embodiments, described herein are methods of treatment comprising administering the PTX3/HA complex for preventing or inhibiting scarring, inflammation, angiogenesis, cancer, diabetes, obesity, or fibrosis.

In certain embodiments, described herein are methods of inducing or maintaining pluripotency in a cell, comprising culturing the cell with an nHC-HA/PTX3 complex or rcHC-HA/PTX3 complex, thereby inducing or maintaining pluripotency in the cell. In some embodiments, the cell heterologously expresses a protein selected from Sox2, myc, Oct4, and KLF4. In some embodiments, the cell heterologously expresses one, two, or three proteins selected from Sox2, myc, Oct4, and KLF4. In some embodiments, the nHC-HA/PTX3 complex or rcHC-HA/PTX3 complex is immobilized. In some embodiments, the cell is an adult differentiated cell. In some embodiments, the cell is a fibroblast. In some embodiments, the cell is a human corneal fibroblast. In some embodiments, the cell is an embryonic stem cell, an adult stem cell, a fetal stem cell, or an induced pluripotent stem cell. In some embodiments, the cell is a limbal epithelial progenitor cell, a limbal stromal septum cell, an umbilical cord stem cell, an amniotic membrane stem cell, or an adipose stem cell. In some embodiments, the nHC-HA/PTX3 complex is an amniotic membrane nHC-HA/PTX3 complex. In some embodiments, the nHC-HA/PTX3 is an umbilical cord nHC-HA/PTX3 complex. In some embodiments, the method further comprises purifying the nHC-HA/PTX3 complex by ultracentrifuging an amniotic membrane extract. In some embodiments, the method further comprises purifying the nHC-HA/PTX3 complex by ultracentrifuging an amniotic membrane extract prepared in PBS buffer to produce an nHC-HA/PTX3 extract (i.e., soluble nHC-HA/PTX3). In some embodiments, the method further comprises purifying the nHC-HA/PTX3 complex by ultracentrifuging an amniotic membrane extract prepared in GnHCl buffer to produce an nHC-HA/PTX3 extract (i.e., soluble nHC-HA/PTX3). In some embodiments, the method further comprises purifying the nHC-HA/PTX3 complex by ultracentrifuging an umbilical cord extract. In some embodiments, the umbilical cord extract comprises umbilical cord amniotic membrane, umbilical cord stroma, Wharton's jelly, or any combination thereof. In some embodiments, the method further comprises purifying the nHC-HA/PTX3 complex by ultracentrifuging the umbilical cord extract prepared in PBS buffer to produce an nHC-HA/PTX3 extract (i.e., soluble nHC-HA/PTX3). In some embodiments, the method further comprises purifying the nHC-HA/PTX3 complex by ultracentrifuging the umbilical cord extract prepared in GnHCl buffer to produce an nHC-HA/PTX3 extract (i.e., soluble nHC-HA/PTX3). In some embodiments, the method further comprises performing two, three, or four rounds of ultracentrifugation. In some embodiments, the method further comprises performing four rounds of ultracentrifugation. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex comprises PTX3. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex comprises small leucine-rich proteoglycan (SLRP). In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex comprises PTX3 and a small leucine-rich proteoglycan (SLRP). In some embodiments, the small leucine-rich proteoglycan is selected from decorin, biglycan, fibromodulin, lumican, PRELP (proline arginine-rich terminal leucine-rich protein), keratocan, osteoadherin, epipycan, and osteoglycin. In some embodiments, the small leucine-rich proteoglycan is covalently attached to a glycosaminoglycan. In some embodiments, the glycosaminoglycan is keratan sulfate.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the purification of native HC-HA/PTX3 (nHC-HA/PTX3) complexes from amniotic membrane extract (AME) and analysis of protein composition and HA size. (A-B) Total protein and HA concentrations in fractions obtained by CsCl/4 M guanidine hydrochloride ultracentrifugation. (C) HA stained with stains-all dye on a 0.5% agarose gel. (D-J) Analysis of proteins present in nHC-HA/PTX3 by Western blotting using antibodies against HCl (D and F), PTX3 (E and G), HC2 (H), HC3 (I), urotrypsin (J), TSG-6 (K), or TSP-1 (L). NaOH (N) = treatment with 0.05 N NaOH at 25°C for 1 hour. HAase (H) = treatment with 20 units/ml hyaluronidase at 60°C for 2 hours (F-K). Bar graph of relative protein amounts (M) determined by dot blot analysis using antibodies against various antigens including IGFBP1-3, PF4, or TIMP-1.

Figure 2 illustrates CD44 and TLR4 receptor-mediated LPS-stimulated macrophage attachment to immobilized nHC-HA/PTX3. (A) Cell attachment. RAW264.7 cells (2.5x10 ⁵ cells/ml in 100 μl) were seeded onto immobilized HA (2 μg/well) or nHC-HA/PTX3 (2 μg/well) (n=3) and stimulated with LPS (1 μg/ml). After 90 minutes of incubation, unattached cells were removed, and attached cells were counted by CyQuant analysis. The scale bar represents 100 μm. Asterisks (*) indicate p values <0.05 (HA or nHC-HA/PTX3 relative to PBS control, or nHC-HA/PTX3 relative to HA). (B) Cell viability. LPS-stimulated RAW264.7 cells were incubated on immobilized PBS control, HA, or nHC-HA/PTX3 for 24 hours (n=3). Cell viability was measured by MTT analysis. No significant differences in cell viability were observed between cells on these immobilized substrates (all p values>0.05). (C) CD44 and TLR4 receptors are responsible for the attachment of LPS-stimulated macrophages to immobilized nHC-HA/PTX3. RAW264.7 cells (2.5x10 ⁵ / ml) were pre-incubated on ice for 30 minutes (n=3) with blocking antibodies for CD44, TLR2, TLR4, integrin αv, β1, β2 or β3 or RGD peptide, as well as isotype control antibodies or RGD control peptides. After adding LPS (1 μg/ml), the cells were incubated for 90 minutes and subjected to the same cell attachment analysis as described in A. Asterisks (*) indicate p values <0.05.

FIG3 illustrates that immobilized nHC-HA/PTX3 polarizes LPS-stimulated macrophages toward an M2 phenotype. (A) Relative mRNA expression of M1 (TNF-α, IL-12p40) or M2 (IL-10, Arg-1, LIGHT, and SPHK1) markers in macrophages treated with PBS control or immobilized HA or nHC-HA/PTX3, as measured by quantitative PCR. (B) Relative TNF-α protein levels as measured by ELISA. (C) Western blot (left) and cellular localization (right) of IRF5 (an M1 marker) by immunofluorescence staining in macrophages treated with PBS control or immobilized nHC-HA/PTX3. (D) Apoptosis of resting, fMLP-stimulated, or LPS-stimulated neutrophils after incubation with immobilized nHC-HA/PTX3. Asterisks (*) indicate p < 0.05. (E) Phagocytosis of apoptotic neutrophils by resting or LPS-stimulated macrophages. Asterisks (*) indicate p < 0.05.

Figure 4 illustrates the role of CD44 in maintaining M2 macrophage polarization on immobilized nHC-HA/PTX3. (A) Relative mRNA expression of M1 (IL-12p40) and M2 (IL-10, LIGHT, and SPHK1) macrophage markers after binding to nHC-HA/PTX3 in macrophages pre-incubated with PBS or blocking antibodies against CD44, TLR4, or CD44/TLR4, as measured by qPCR. Asterisks (*) indicate p < 0.05 compared to no antibody treatment (None) in the same group. (B) IL-12 and IL-10 protein levels measured by ELISA. Asterisks (*) indicate p < 0.05 compared to no antibody treatment (None) in the same group.

Figure 5: nHC-HA/PTX3 (4th) promotes cell aggregation, but both nHC-HA/PTX3 (2nd) and nHC-HA/PTX3 (4th) inhibit IL-12p40 and IL-23 protein production in IFN-γ/LPS-stimulated macrophages. RAW264.7 cells (2.5x10 ⁵ /ml) were cultured on an immobilized substrate (PBS as a control) and stimulated with IFN-γ/LPS for 4 hours (A) or 24 hours (B and C). (A) Cell morphology 4 hours after inoculation. Alternatively, cells were stimulated with LPS for 24 hours, and IL-10 and IL-12p70 protein levels were measured in the cell culture supernatants by respective ELISAs (B and C). p values are shown in B and C.

Figure 6 illustrates the dose-dependent and covalent coupling of HMW HA and nHC-HA/PTX3 to the surface of a 96-well CovaLink ^™ plate. (A) HA ELISA of bound HMW HA and nHC-HA/PTX3 after removal of unbound HMW HA and nHC-HA/PTX3. (B) HA ELISA of bound and unbound HA from HMW HA and nHC-HA/PTX3.

Figure 7 illustrates the dose-dependent binding of TSG-6 to immobilized HA (iHA) and resistance to various dissociation and reducing agents. (A) TSG-6 binding to iHA measured by TSG-6 ELISA. (B) TSG-6 binding to iHA measured by TSG-6 ELISA after treatment with 6M guanidine hydrochloride, 8M guanidine hydrochloride, 2% SDS, 100mM DTT, or 25mM NaOH.

Figure 8 illustrates the dose-dependent binding of PTX3 to immobilized HA (iHA) and resistance to various dissociation and reducing agents. (A) PTX3 binding to iHA measured by PTX3 ELISA. (B) PTX3 binding to iHA measured by PTX3 ELISA after treatment with 6 M guanidine hydrochloride, 8 M guanidine hydrochloride, 2% SDS, 100 mM DTT, or 25 mM NaOH.

Figure 9 illustrates the lack of competition or synergy between TSG-6 and PTX3 for binding to iHA. Relative absorbance measured by ELISA is shown for bound TSG-6 or PTX3, either alone or in combination with iHA. No statistical significance was found for the binding of TSG-6 or PTX3 to iHA (p>0.05).

Figure 10 illustrates partial inhibition of PTX3 binding to iHA pre-bound with TSG-6, and lack of inhibition of TSG-6 binding to iHA pre-bound with PTX3. The figure shows TSG-6 and PTX3 ELISA results for pre-bound TSG-6/iHA (A) or pre-bound PTX3/iHA (B), followed by binding of TSG-6 or PTX3. P values are shown in (A), and no statistical significance was seen between the groups in (B).

Figure 11 illustrates the attachment of LPS-stimulated RAW264.7 macrophages to PBS (control), HA (iHA), nHC-HA/PTX3, TSG-6/iHA, or PTX3/iHA. Cells were photographed 24 hours after incubation.

Figure 12 illustrates relative gene expression in RAW264.7 macrophages after incubation on PBS (control), HA (iHA), nHC-HA/PTX3, TSG-6/iHA or PTX3/iHA. Total RNA was isolated, and mRNA expression of IL-12p40 (A) and IL-10 (D) was measured by quantitative PCR. Alternatively, cells were stimulated with LPS (B and E) or IFN-γ/LPS (C) for 24 hours, and protein expression of IL-12p70 (B), IL-23 (C) and IL-10 (E) was measured in cell culture medium using respective ELISAs. Asterisks (*) indicate p<0.05 compared to controls.

Figure 13 illustrates the efficiency of free TSG-6 in solution compared to bound TSG-6 for transferring HCl and HC2 from IαI to iHA. (A, B) Relative bound HCl (A) or IαI (B) measured by respective ELISAs after simultaneous or sequential addition of TSG-6 and IαI to iHA. Asterisks (*) indicate p < 0.05 at the same TSG-6 concentration when added simultaneously and sequentially. (C) Western blot of samples from A digested with hyaluronidase (HAase) and analyzed with anti-TSG-6 antibodies. (D) Relative HCl and PTX3 bound to iHA measured by ELISA after simultaneous incubation of PTX3 and IαI with iHA.

FIG14 illustrates complexes formed in solution after simultaneous incubation of IαI and TSG-6 with or without PTX3. (A-D) Western blots obtained with antibodies against HCl (A), HC2 (B), TSG-6 (C), or urotrypsin (D). HAase = hyaluronidase treatment. (E) Schematic representation of the interaction of TSG-6 with IαI. (F) Schematic representation of PTX3 inhibition of HC2·TSG-6 formation. (G) Western blot obtained with anti-IαI antibody.

Figure 15 illustrates the complex formed on iHA after IαI and TSG-6 were incubated with or without PTX3. After washing with 8M GnHCl and PBS, the bound HCl, TSG-6, and PTX3 (A, D, F) were determined by their respective ELISAs. Asterisks (*) indicate p<0.05 compared to 1 μg/ml of PTX3. The complex was washed again with 8M GnHCl and PBS, and the bound components were digested with 1 unit/ml of hyaluronidase for 2 hours. The digested samples were analyzed by Western blotting using antibodies against HCl (B), HC2 (C), TSG-6 (E), and PTX3 (G).

Figure 16 illustrates the complex formed on iHA after the sequential addition of IαI and TSG-6 and subsequent addition of PTX3. Bound HCl, TSG-6, and PTX3 were measured by respective ELISAs (A, D, F). The complex was washed again with 8M GnHCl and PBS, and the bound components were digested with 1 unit/ml of hyaluronidase for 2 hours. The digested samples were analyzed by Western blotting using antibodies against HCl (B), HC2 (C), TSG-6 (E), and PTX3 (G).

Figure 17 illustrates the complex formed on iHA after the sequential addition of PTX3 and then IαI and TSG-6. Bound HCl, TSG-6, and PTX3 were measured by respective ELISAs (A, C, E). The complex was washed again with 8M GnHCl and PBS, and the bound components were digested with 1 unit/ml of hyaluronidase for 2 hours. The digested samples were analyzed by Western blotting using antibodies against PTX3 (B), TSG-6 (D), HCl (F), and HC2 (G).

Figure 18 exemplifies the attachment of LPS-stimulated RAW264.7 macrophages to PBS (control), HA (iHA), nHC-HA/PTX3, (IαI/TSG-6/PTX3)/iHA (IαI, TSG-6, or PTX3 simultaneously bound to iHA), (IαI/TSG-6)/PTX3/iHA (PTX3 was sequentially added to iHA pre-incubated with IαI and TSG-6), or (PTX3)/IαI/TSG-6/iHA (IαI and TSG-6 were sequentially added to iHA pre-incubated with PTX3). Cells were photographed 24 hours after incubation.

Figure 19 illustrates gene expression in RAW264.7 macrophages cultured on an immobilized substrate and stimulated with LPS. Total RNA was isolated and the expression of IL-10 and IL-12p40 mRNA was determined by quantitative PCR (A and C). IL-10 and IL-12p70 proteins in cell culture supernatants were determined by respective ELISAs (B and D). (E) IL-23 protein was measured by IL-23 ELISA in the cell culture supernatants of RAW264.7 cells that were resting (none) or stimulated for 24 hours with IFN-γ (200 units/ml), LPS (1 μg/ml), IFN-γ/LPS, LPS containing immune complexes (LPS/IC), or IL-4 (10 ng/ml). (F) IL-23 was measured by IL-23 ELISA in the cell culture supernatants of RAW264.7 cells that were cultured on an immobilized substrate and stimulated for 24 hours with IFN-γ/LPS. Asterisk (*) indicates p < 0.05.

Figure 20 illustrates immunostaining of HA, PTX3, TSG-6, HC, and urotrypsin in human umbilical cord (A) or amniotic membrane (B). Cryosections of human umbilical cord were probed with biotinylated HABP using antibodies against PTX3 and TSG-6 and chain-specific antibodies against the indicated IαI and PαI components, with or without hyaluronidase digestion. Nuclei were counterstained with Hoechst 33342 (blue). Epi, epithelium. Bars represent 100 μm.

FIG21 illustrates a comparison of PTX3 levels in sequential PBS and GnHCl extracts from AM, CH, and UC. A, Lanes 2 and 3 contain 2 μg of HA each, while lanes 4-11 contain 20 μg of total protein each. B, Lanes 3-10 contain 40 μg of total protein each.

Figure 22 illustrates a comparison of HCl, urotrypsin, and IαI in sequential PBS and GnHCl extracts from AM, CH, and UC. In A and C, each lane contains 20 μg of total protein, while in B, each lane contains 40 μg of total protein, except for the positive control.

Figure 23 illustrates a comparison of TSG-6 in AM and UC GnHCl extracts. Each lane contained 40 μg of total protein, except for the positive TSG-6 control.

Figure 24 illustrates Western blot analysis of PTX3 (A), HC1 (B), HC2 (C), and TSG-6 (D) in the 1st to 4th AM HC·HA complexes. Each lane contained 4 μg of HA, except for the positive control.

Figure 25 illustrates Western blot analysis of TSP-1 in AME, AM GnHCl, and HC-HA complexes 1 to 4. Lanes 3, 4, 10, and 11 contain 30 μg of total protein each. Lanes 5-8 and 12-15 contain 4 μg of HA each.

Figure 26 illustrates Western blot analysis of PTX3 (A), HC1 (B), HC2 (C), HC3 (D), and TSG-6 (E) in the 4UC HC-HA complex. Except for the positive control, each lane contained 4 μg of HA.

Figure 27 illustrates a comparison of HCl (A) and PTX3 (B) in the 4th HC·HA complex from PBS and GnHCl extracts. Except for the positive control, each lane contained 4 μg of HA.

Figure 28 illustrates a comparison of the 4th HC-HA complex from PBS and GnHCl in agarose gel. Each lane contains 15 μg HA, except for the positive HA control.

Figure 29 illustrates Coomassie blue staining of SDS-PAGE gels of GnHCl HC-HA and PBS HC-HA. A, AMPBS and GnHCl HC-HA. B, UCPBS and GnHCl HC-HA. Each lane contains 30 μg of HA.

Figure 30 illustrates the presence of keratan sulfate and osteoadherin in AM GnHCl HC-HA, but not in PBS HC-HA. A, Coomassie blue staining. Each lane contains 30 μg HA. B and C, Western blots of keratan sulfate (B) and osteoadherin (C). Each lane contains 4 μg HA. D, Immunostaining of keratan sulfate in AM.

FIG31 illustrates deglycosylation of AM GnHCl HC-HA and analysis by SDS-PAGE or Western blotting stained with CB (Coomassie Blue). A, Coomassie Blue staining. Each lane contains 30 μg of HA, except lane 6, which contains 5 μg of HA. B-H, Western blots of osteoadherin (B), decorin (C, D), biglycan (E, F), keratan sulfate (G), and PTX3 (H). H: hyaluronidase; C: chondroitinase (Cabc); K: keratinase (keratan sulfate endo-β-galactosidase); T: TFMSA (trifluoromethanesulfonic acid). Each lane contains 4 μg of HA.

FIG32 illustrates that decorin and biglycan are abundantly present in UC GnHCl HC-HA, but not in PBS HC-HA. Keratan sulfate, osteoadherin, and uropancreatin are also present in UC GnHCl HC-HA, but not in PBS HC-HA (except keratan sulfate). A, Coomassie blue staining. Each lane contains 30 μg of HA, except lane 6, which contains 5 μg of HA. B-H, Western blots of decorin (B), biglycan (C), uropancreatin (D), PTX3 (E), keratan sulfate (F), and osteoadherin (G). H: hyaluronidase; C: chondroitinase (Cabc); K: keratinase (keratan sulfate endo-β-galactosidase). Each lane contains 4 μg of HA.

Figure 33 illustrates immunolocalization of PTX3 in human AM. Cryosections of human fetal membranes were probed with anti-PTX3, biotinylated HABP, with or without hyaluronidase digestion and using chain-specific antibodies against the IαI component. Nuclei were counterstained with Hoechst 33342 (blue). AM, amnion; Epi, epithelium; CH, chorion. Bars represent 100 μm.

FIG34 illustrates the presence of PTX3 in AM soluble extracts and purified HC-HA complexes. Purified PTX3, AM extracts (AME), and AM HC-HA complexes were treated with or without 50 mM NaOH at 25°C for 1 hour, or with hyaluronidase (HAase) at 37°C for 1 hour, followed by Western blot analysis using anti-PTX3 (A) and anti-HCl (B) antibodies and analysis on 0.5% agarose gel electrophoresis followed by staining with panstain (C). PTX3 species and their multimeric forms were found in AM soluble extracts and purified HC-HA complexes. M, protein ladder molecular weight standard.

Figure 35 illustrates PTX3 mRNA and protein expression in AMECs and AMSCs. RNA and protein were extracted from human skin fibroblasts (SkinFib.) as well as AMECs and AMSCs. PTX3 mRNA (A) and protein expression in supernatants and cell lysates (B) were compared. PTX3 siRNA transfection was performed to confirm PTX3 expression in AMECs and AMSCs (C).

Figure 36 illustrates morphological changes in human dermal fibroblasts (HSF, A), AMSCs (B), and AMECs (C) after overlaying with agarose. HSFs, AMSCs, and AMECs were cultured in serum-free and serum-supplemented conditions for five days with or without overlaying with 3% agarose, and cell morphology was photographed. Scale bar, 50 μm.

Figure 37 illustrates that agarose overlay reduces the release of HA into the culture medium from HSF, AMSC and AMEC cultures. HA concentrations were determined by ELISA assay in culture medium from HSF, AMSC and AMEC under serum-free and serum-containing conditions with or without agarose overlay.

Figure 38 illustrates immunolocalization of PTX3, HA, and HCl in agarose-coated cell cultures. HSF, AMSC, and AMEC were cultured in agarose-coated cultures with or without TNF treatment and probed for hyaluronan (red), PTX3 (green, A-F; red, J-L), and HCl (green) (nuclei are blue). Colocalization of HCl with HA was observed in all cultures, but colocalization of PTX3 with either HA or HCl was only observed in AMSC and AMEC. Scale bar, 50 μm.

FIG39 illustrates the HC-HA/PTX3 complex in AMSCs but not HSFs under agarose overlay. GnHCl extracts from the cell layers of HSF and AMSC cultures overlaid with agarose were Western blotted for PTX3 with or without NaOH treatment. M, protein ladder molecular weight standard.

Figure 40 illustrates the reconstitution of HC-HA/PTX3 complexes on immobilized HA in vitro. iHA (~14 μg/ml), IαI (5 μg/ml), and TSG-6 (12 μg/ml) were incubated simultaneously with or without PTX3 (1, 5, or 20 μg/ml) at 37°C for 2 hours. Sequentially, iHA (~14 μg/ml), IαI (12 μg/ml), and TSG-6 (12 μg/ml) were incubated in reaction buffer at 37°C for 2 hours, followed by the addition of PTX3 (1, 5, or 20 μg/ml) and an additional 2 hours at 37°C. After washing with 8M GnHCl and PBS, iHA with bound components was digested with 1 unit/ml of hyaluronidase at 60°C for 2 hours. Samples were analyzed by Western blotting using antibodies against IαI (A), TSG-6 (B), and PTX3 (C).

FIG41 exemplifies the cell morphology of human corneal fibroblasts cultured in DMEM/10% FBS for 2 days (A) or in DMEM/10% FBS for 2 days ± TGF-β1 for 3 days (B) up to day 3.

FIG42 exemplifies that soluble HC-HA (PBS) inhibits TGFβ1 but activates TGFβ3 signaling, whereas insoluble HC-HA (GnHCl) activates both TGFβ1 and TGFβ3 signaling under serum-free conditions and is further enhanced upon TGFβ1 stimulation.

Figure 43 illustrates that both soluble HC-HA (PBS) and insoluble HC-HA (GnHCl) inhibit TGFβR2 and TGFβR3 expression in response to TGFβ1 stimulation. A, TGFβR mRNA expression. B, TGFβR protein expression.

FIG. 44 exemplifies inhibition of nuclear translocation of pSMAD2/3 signaling by inhibition of TGFβ1 signaling by HC-HA.

FIG45 exemplifies HC-HA inhibition of α-smooth muscle actin formation.

Figure 46 illustrates that HA and soluble/insoluble HC-HA activate BMP6 transcripts. Addition of TGFβ1 activated BMP6 transcript expression on plastic, but significantly activated BMP4/6 mRNA expression on HA and soluble and insoluble HC-HA in HCFs.

FIG47 exemplifies that HC-HA, but not HA, activates transcript expression of BMPR1A in HCFs challenged with TGFβ1, whereas additional TGFβ1 non-specifically activates mRNA expression of BMPR1B and BMPR2 in HCFs.

FIG. 48 exemplifies that soluble HC-HA (PBS) and insoluble HC-HA (GnHCl) activate BMP4/6 signaling through pSMAD1/5/8.

FIG49 illustrates that activation of SMAD/1/5/8 leads to upregulation of its downstream genes, inhibitors of DNA binding 1, 3, and 4 (ID1, ID3, and ID4), downstream targets of BMP signaling.

FIG50 illustrates that HC-HA(PBS) and HC-HA(GnHCl) promoted keratan mRNA expression by 14-fold and 16-fold, respectively, which was significantly downregulated by the addition of TGFβ1.

FIG. 51 exemplifies that HC-HA(PBS) and HC-HA(GnHCl) promote protein expression of keratin.

Figure 52 illustrates that HCF express more ESC markers on 4X HC-HA (PBS) and insoluble HC-HA (GnHCl) than on plastic and when cells are stimulated by the addition of TGFβ1.

FIG. 53 exemplifies cell viability of MC3T3-E1 cells measured by MTT.

FIG54 exemplifies mineralization of MC3T3-E1 cells measured by Alizarin Red staining.

FIG55 exemplifies the morphology of MC3T3-E1 cells treated with HC-HA (A) or AMP (B) on day 13 of induction.

FIG. 56 exemplifies Alizarin Red staining of MC3T3-E1 cells treated with HC-HA (A) or AMP (B) on day 13 of induction.

FIG57 exemplifies quantitative analysis of ARS staining of MC3T3-E1 cells treated with HC-HA (A) or AMP (B) on day 13 of induction.

FIG. 58 exemplifies ALP activity (IU/L) of cells treated with HC-HA (A) and AMP (B) on day 13 of induction.

Figure 59 illustrates phase contrast microscopy of morphological changes in MC3T3-E1 cells after induction from day 3. (A) Uninduced cells were cultured in αMEM containing 10% FBS for 7 days. (B) MC3T3-E1 cells were cultured to confluence in flat-bottom 96-well plates one day after inoculation (day 0). Cells were then induced with ascorbic acid and β-glycerophosphate.

FIG60 exemplifies phase contrast microscopy examination of morphological changes of MC3T3-E1 cells induced by treatment with HC-HA (A) or AMP (B) from day 1 to day 7. ...

Figure 61 exemplifies phase contrast microscopy examination of spindle ring formation in induced MC3T3-E1 cells from day 3 of induction.

FIG62 exemplifies phase contrast microscopy of spindle ring formation in induced MC3T3-E1 cells treated with HC-HA (A) and AMP (B) (day 0 to day 6).

FIG. 63 exemplifies ARS staining of induced MC3T3-E1 cells treated with HC-HA and AMP and extraction using GnHCl.

Figure 64 illustrates the extraction and quantification of ARS in MC3T3-E1 cells using acetic acid and 10% ammonium hydroxide. The ARS extract obtained by acetic acid treatment was neutralized with 10% ammonium hydroxide and then added to a 96-well clear bottom assay plate (A) for reading on a spectrophotometer (B). * indicates statistical significance.

Figure 65 illustrates the extraction and quantification of ARS in MC3T3-E1 cells using GnHCl. ARS extracts obtained by GnHCl treatment were added to 96-well clear bottom assay plates (A) for reading on a spectrophotometer (B). * indicates statistical significance.

Figure 66 illustrates ARS staining (A) and quantification (B) of MC3T3-E1 cells treated with HC-HA on day 19 (day 18 of induction). ARS staining was performed on day 19 of culture (day 18 of induction).

Figure 67 illustrates ARS staining (A) and quantification (B) of MC3T3-E1 cells treated with AMP.ARS staining was performed on day 19 of culture (day 18 of induction).

FIG. 68 exemplifies spindle-like rings observed in induced MC3T3-E1 cells on day 14 of induction.

FIG69 exemplifies phase contrast microscopy of cell morphology of induced MC3T3-E1 cells cultured (A) without or (B) with Transwells and treated with AMP 14 days after induction.

FIG. 70 exemplifies ARS staining of induced MC3T3-E1 cells on day 14 of induction.

FIG. 71 exemplifies quantification of ARS staining of induced MC3T3-E1 cells at day 14 of induction.

Figure 72 illustrates ARS staining and quantification of MC3T3-E1 cells treated with AMP. (A) Phase contrast and ARS staining images of MC3T3-E1 cells taken on day 21 of culture (day 20 of induction). (B) ARS staining was quantified on day 21 of culture (day 20 of induction). * indicates statistical significance at p < 0.05.

Figure 73 shows images of the differential differentiation of progenitor cells into osteoblasts, showing common factors added to the culture medium.

Figure 74 illustrates ARS staining and quantification in MC3T3-E1 cells. (A) Phase contrast micrographs of HUVEC, hBMMSC, and hAM mesenchymal stem cells with and without ARS staining from day 4 to day 21. (B) Quantification of ARS staining. * indicates statistical significance at p < 0.05 compared to the negative control.

FIG75 illustrates the timeline of osteogenesis in MC3T3-E1 cells.

Figure 76 illustrates ARS staining and quantification in MC3T3-E1 cells. (A) Cell morphology and ARS staining of MC3T3-E1 cells treated with AMP (days 1, 2, 7, and 10). (B) ARS quantification of MC3T3-E1 cells treated with AMP (days 1, 2, 7, and 10). * Symbol indicates statistical significance at p < 0.05.

Figure 77 illustrates a timeline of cell viability and proliferation measured by MTT assay. (A) MTT assay of MC3T3-E1 cell viability and metabolic activity at day 1, day 2, and day 4. * indicates statistical significance relative to p < 0.05 at day 1. (B) BrdU assay of MC3T3-E1 cell proliferation at day 1, day 2, and day 16. * indicates statistical significance relative to p < 0.05 at day 1.

Figure 78 illustrates the mRNA expression of multiple genes measured by QPCR after differentiation induction with or without AMP treatment. (A) hMSC and (B) MC3T3-E1 cell expression.

Figure 79 illustrates phase contrast microscopy and quantification of ARS analysis. MC3T3-E1 cells deposited mineralization over the duration of the experiment (8 days). ARS was then used for qualitative analysis. (A) CovaLink-NH 96-well plates with PBS, HA, and HC-HA immobilized therein. (B) Conventional 96-well plates with negative controls and AGM (inducer) and AMP as positive controls. (C) Quantification of ARS analysis. * Symbols indicate statistical significance relative to day 1 p < 0.05.

Figure 80 illustrates the mRNA expression of multiple genes tested by QPCR after differentiation induction with or without HC-HA/PTX3 treatment. (A) Runx2 and Sox9, master transcription factors for osteogenesis and chondrogenesis, at days 1, 7, and 14. (B) Bone morphogenetic protein (BMP) expression at day 14. (C) Chondrogenesis marker collagen 2 (COL2) and osteogenic marker alkaline phosphatase (ALPL) expressed at days 7 and 14. (D) Hypertrophy markers collagen 10 (COL10) and MMP13 expressed at day 14. (E) Osteogenesis markers collagen 1 (COL1), osterix (OSX), and bone sialoprotein (BSP) at day 14.

FIG81 exemplifies phase microscopy (A) and quantification (B) of ARS analysis at day 14 following differentiation induction with or without HC-HA/PTX3 (soluble or insoluble) treatment.

Figure 82 illustrates CD4 ⁺ T cell activation and differentiation. Under different stimuli, naive CD4 ⁺ T helper cells (Th) differentiate into Th1, Th2, Th17, or Treg and secrete different cytokines. Th1-type cytokines (e.g., IFN-γ and IL-2) tend to produce pro-inflammatory responses.

Figure 83 illustrates the procedure for measuring cell proliferation and cytokine production. Splenocytes were isolated from OT-II mice expressing an ovalbumin (OVA)-specific transgenic TCR and stimulated with OVA for up to 4 days. Cell proliferation was measured by BrdU labeling, and cytokine (IFN-γ and IL-2) expression was measured by respective ELISAs.

Figure 84 illustrates the inhibition of CD4 ⁺ T cell proliferation by nHC-HA/PTX3. Splenocytes isolated from Ova T cell receptor transgenic mice were stimulated with OVA (0-10 μM) for 4 days. AM extract (AME, 25 μg/ml) and nHC-HA/PTX3 (25 μg/ml) inhibited the clonal growth of activated T cells induced by increased OVA concentrations (top). The proliferation of splenocytes treated with HA, AME or nHC-HA/PTX3 and labeled with CFSE for 4 days was determined by flow cytometry. Both AME and nHC-HA/PTX3 inhibited cell proliferation in a dose-dependent manner (middle). 25 μg/ml nHC-HA/PTX3 inhibited the proliferation of CD4 ⁺ T cells labeled with BrdU (bottom) (*, p<0.05 compared to the control).

Figure 85 illustrates the inhibition of Th1 cytokines IFN-γ and IL-2 by nHC-HA/PTX3. Splenocytes treated with PBS, 25 μg/ml HA, 25 μg/ml AME, or 25 μg/ml nHC-HA/PTX3 were stimulated with 10 μM OVA for 4 days. IFN-γ and IL-2 were measured in the culture supernatant by respective ELISAs. Both AME and nHC-HA/PTX3 inhibited the production of IFN-γ and IL-2 (*p < 0.05 compared to control).

Figure 86 illustrates that nHC-HA/PTX3 reduces the influx of macrophages (labeled by enhanced green fluorescent protein or EGFP). LPS (2 μl, 2 μg/ml) was injected into the corneal stroma of Mafia mice. 5 μl PBS or nHC-HA/PTX3 (1 mg/ml) was immediately injected through the subconjunctival tissue into each quadrant of a cornea from the same mouse. In vivo live microscopy was used to monitor the influx of EGFP ⁺ macrophages on the 1st, 2nd, 3rd, and 6th days after LPS treatment (top). Alternatively, PBS or nHC-HA/PTX3 were used to simultaneously treat mouse corneas with LPS (pretreatment (-) or three days before LPS treatment (pretreatment (+)). On the 4th day after LPS treatment, cells in the cornea were isolated by collagenase digestion and sorted by FACS into ^EGFP- or EGFP ⁺ (macrophages) (* and ** were p<0.05 and p<0.01, respectively, compared to the control).

Figure 87 illustrates that nHC-HA/PTX3 polarizes macrophages toward an M2 phenotype. The mRNA expression of M2 markers (Arg-1 and IL-10) and M1 markers (IL-12p40 and IL-12p35) in macrophages (EGFP ⁺ ) infiltrating LPS-primed mouse corneas was quantified by qPCR (* and **p < 0.05 and p < 0.01, respectively, compared to control).

Figure 88 illustrates the improvement of corneal allograft survival by nHC-HA/PTX3. The survival of mouse corneal allografts was significantly improved by injection of nHC-HA/PTX3 (10 μg/time, 2 times/week, upper left panel) at one quadrant of the subconjunctival site, but was more significantly improved by injection of nHC-HA/PTX3 (20 μg/time, 2 times/week, upper right panel) at four quadrants of the subconjunctival site. Below, photos of corneal allografts treated with PBS (postoperative day 21, left) or nHC-HA/PTX3 (postoperative day 40, right).

FIG89 exemplifies classical M1 activation of macrophages (e.g., induced by IFN-γ and/or TLR ligands such as LPS) to express high levels of proinflammatory cytokines (such as TNF-α, IL-12, and IL23), which activate Th1 and Th17 lymphocytes, leading to many chronic inflammatory diseases.

Figure 90 illustrates the infiltration of LPS-induced macrophages into the mouse cornea using PBS (control), HC-HA/PTX3 or AMP. Both eyes of Mafia mice (macrophages are EGFP+) were injected intrastromally with LPS (5 μg). OS was treated with PBS, and OD was treated with HC-HA (2 injection sites) (A), HC-HA (4 injection sites) (B), AMP (2 injection sites) (C), and AMP (4 injection sites) (D), with each injection being 5 μl. Treatment was performed immediately after LPS injection. Pictures were taken on day 1, day 2, day 3, and day 6. Cells were counted based on the intensity of green fluorescence.

Figure 91 illustrates the infiltration of LPS-induced macrophages into the mouse cornea and their subtypes (M1 and M2) treated or pretreated with PBS (control), nHC-HA/PTX3 or AMP. The pretreatment and treatment of Mafia mice are the same as those described in Figure 86. On day 4, the corneal button was cut and digested with collagenase at 37°C for 2 hours. EGFP positive/negative cells were sorted by FACS. The ratio of EGFP-positive macrophages to EGFP-negative cells was calculated and used as an arbitrary unit to determine the degree of macrophage infiltration (A). Total RNA was extracted from the sorted EGFP-positive macrophages and converted into cDNA. The expression of Arg-1, IL-10, IL-12p40 and IL-12p35 was determined by quantitative PCR (B).

Figure 92 is a diagram of the injection site. The injection site will be in the subconjunctival region close to the fornix. nHC-HA/PTX3 or AMP can reduce DS-induced ALKC in a murine experimental dry eye model.

FIG93 illustrates HC-HA activation of IGF1-HIF1α-VEGF signaling to promote angiogenesis. When cells were in a resting state, HC-HA induced a 2- to 6-fold increase in IGF1 mRNA and a 2-fold increase in VEGF mRNA. When cells were stimulated with TGFβ (10 ng/ml), HC-HA induced a 5- to 12-fold increase in IGF1 mRNA and a 5- to 9-fold increase in VEGF mRNA. n=4, *p<0.05, **p<0.01. IGF1, insulin-like growth factor 1; HIF1α, hypoxia-inducible factor 1-α; VEGF, vascular endothelial growth factor.

DETAILED DESCRIPTION

Specific terms

Unless otherwise defined, the meaning of all technical and scientific terms used herein is the same as that generally understood by those skilled in the art as to the subject matter to which protection is sought. Unless otherwise indicated, all patents, patent applications, disclosed applications and publications, GENBANK sequences, websites and other published materials related to the present invention in full are incorporated herein by reference in their entirety. When there are multiple definitions for a term in this article, the definitions in this section shall prevail. When referring to a URL or other such identifiers or addresses, it should be understood that such identifiers may change, and specific information on the Internet may vary, but equivalent information is known and can be easily accessed, such as by searching the Internet and/or appropriate databases. References have been made to the availability and public dissemination of this information.

As used herein, ranges and amounts can be expressed as "about" a particular value or range. "About" also includes the exact amount. Thus, "about 5 μg" means "about 5 μg" and also refers to "5 μg." In general, the term "about" includes amounts that are expected to be within experimental error.

As used herein, a reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex is an HC-HA/PTX3 complex formed by assembling the component molecules of the complex in vitro. Methods for assembling rcHC-HA/PTX3 include: reconstitution using purified natural proteins or molecules from biological sources, producing recombinant proteins by recombinant methods, or synthesizing molecules by in vitro synthesis. In some cases, the purified natural proteins used to assemble rcHC-HA/PTX3 are proteins in complexes (i.e., multimers, multi-chain proteins, or other complexes) with other proteins. In some cases, PTX3 is purified from cells as a multimer (e.g., a homomultimer) and used to assemble the rcHC-HA/PTX3 complex.

As used herein, purified native HC-HA/PTX3 (nHC-HA/PTX3) complexes refer to HC-HA/PTX3 complexes purified from biological sources (e.g., cells, tissues, or biological fluids). Such complexes are typically assembled in vivo in a subject, or ex vivo in cells, tissues, or biological fluids from a subject (including humans or other animals).

As used herein, PTX3/HA complex refers to the intermediate complex formed by contacting PTX3 with immobilized HA. In the methods provided herein, the PTX3/HA complex is generated before hydrochloric acid is added to HA.

As used herein, "hyaluronan," "hyaluronic acid," or "hyaluronate" (HA) are used interchangeably and refer to a substantially non-sulfated linear glycosaminoglycan (GAG) having repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine (D-glucuronyl-N-acetylglucosamine).

As used herein, the term "high molecular weight" or "HMW," as in high molecular weight hyaluronan (HMW HA), means HA having a weight average molecular weight greater than about 500 kilodaltons (kDa), for example, HA having a weight average molecular weight of about 500 kDa to about 10,000 kDa, about 800 kDa to about 8,500 kDa, about 1100 kDa to about 5,000 kDa, or about 1400 kDa to about 3,500 kDa. In some embodiments, HMW HA has a weight average molecular weight of 3000 kDa or greater. In some embodiments, HMW HA has a weight average molecular weight of 3000 kDa. In some embodiments, HMW HA is a HA having a weight average molecular weight of about 3000 kDa. In some embodiments, HMW HA has a molecular weight of about 500 kDa to about 10,000 kDa. In some embodiments, HMW HA has a molecular weight of about 800 kDa to about 8,500 kDa. In some embodiments, the HMW HA has a molecular weight of about 3,000 kDa.

As used herein, the term "low molecular weight" or "LMW," as in low molecular weight hyaluronan (LMW HA), refers to HA having a weight average molecular weight of less than 500 kDa, e.g., less than about 400 kDa, less than about 300 kDa, less than about 200 kDa, about 200-300 kDa, or about 1-300 kDa.

As used herein, pentraxin 3 or PTX3 protein or polypeptide refers to any PTX3 protein, including but not limited to recombinantly produced proteins, synthetically produced proteins, native PTX3 proteins, and PTX3 proteins extracted from cells or tissues. PTX3 includes multimeric forms of PTX3 (e.g., homomultimers), including but not limited to dimers, trimers, tetramers, pentamers, hexamers, tetramers, octamers, and other naturally or artificially produced multimeric forms.

As used herein, tumor necrosis factor-stimulated gene-6 (TSG-6) refers to any TSG-6 protein or polypeptide, including but not limited to recombinantly produced proteins, synthetically produced proteins, naturally occurring TSG-6 proteins, and TSG-6 proteins extracted from cells or tissues.

As used herein, inter-α-inhibitor (IαI) refers to an IαI protein consisting of a light chain (i.e., urokinin) and one or two heavy chains of the HCl or HC2 type covalently linked by a chondroitin sulfate chain. In some embodiments, the source of IαI is from serum, or from cells producing IαI, e.g., hepatocytes or amniotic epithelial or stromal cells or umbilical cord epithelial or stromal cells, under constitutive stimulation with proinflammatory cytokines such as IL-1 or TNF-α.

As used herein, "hyaluronan binding protein," "HA binding protein," or "HABP" refers to any protein that specifically binds to HA.

As used herein, "linking module" refers to a hyaluronan binding domain.

As used herein, "biological activity" refers to the in vivo activity or physiological response of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex that occurs upon in vivo administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex, or a composition or mixture comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. Thus, biological activity includes the therapeutic effects and pharmaceutical activities of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes, and compositions and mixtures thereof.

As used herein, the terms "subject," "individual," and "patient" are used interchangeably. None of these terms should be construed as requiring supervision by a medical professional (e.g., a doctor, nurse, physician assistant, caregiver, hospice staff). As used herein, a subject is any animal, including mammals (e.g., humans or non-human animals) and non-mammals. In one embodiment of the methods and compositions provided herein, the mammal is a human.

As used herein, the terms "treat," "treating," or "treatment," and other grammatical equivalents, include alleviating, alleviating, or ameliorating one or more symptoms of a disease or condition, ameliorating, preventing, or reducing the appearance, severity, or frequency of one or more additional symptoms of a disease or condition, ameliorating or preventing the underlying metabolic cause of one or more symptoms of a disease or condition, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, alleviating a condition caused by the disease or condition, or prophylactically and/or therapeutically inhibiting the symptoms of a disease or condition. In one non-limiting example, to obtain a prophylactic benefit, an rcHC-HA/PTX3 complex or composition disclosed herein is administered to an individual at risk for, prone to, or reporting one or more physiological symptoms of a disorder.

As used herein, "placenta" refers to the organ that connects the developing fetus to the wall of the mother's uterus to allow for nutrient absorption, waste elimination, and gas exchange through the mother's blood supply. The placenta is composed of three layers. The innermost placental layer surrounding the fetus is called the amnion. The allantois is the middle layer of the placenta (derived from the embryonic hindgut); blood vessels originating from the umbilicus pass through this membrane. The outermost layer of the placenta, the chorion, contacts the endometrium. The chorion and allantois fuse to form the chorioallantoic membrane.

As used herein, "chorion" refers to a membrane formed by the extraembryonic mesoderm and two layers of trophoblast. The chorion consists of two layers: an outer layer formed by the trophoblast and an inner layer formed by the parietal mesoderm; the amnion is in contact with the latter. The trophoblast consists of an inner layer of cubic or prismatic cells, the cytotrophoblast or Langhans layer, and an outer layer of nuclear-rich protoplasm that lacks cell borders (i.e., the syncytiotrophoblast). The avascular amnion is attached to the inner layer of the chorion.

As used herein, "amnion-chorion" refers to a product comprising the amnion and the chorion. In some embodiments, the amnion and the chorion are not separated (i.e., the amnion is naturally attached to the inner layer of the chorion). In some embodiments, the amnion is initially separated from the chorion and is subsequently combined with the chorion during processing.

As used herein, "umbilical cord" refers to the organ that connects the developing fetus to the placenta. The umbilical cord is composed of Wharton's jelly, a jelly-like substance made primarily of mucopolysaccharides. It contains a vein, which carries oxygenated, nutrient-rich blood to the fetus, and two arteries, which carry deoxygenated, nutrient-depleted blood away.

As used herein, "placental amniotic membrane" (PAM) refers to amniotic membrane derived from the placenta. In some embodiments, the PAM is substantially isolated.

As used herein, "umbilical cord amniotic membrane" (UCAM) means the amniotic membrane derived from the umbilical cord. UCAM is a translucent membrane. UCAM has multiple layers: an epithelial layer (basement membrane); a dense layer; a fibroblast layer; and a spongy layer. It lacks blood vessels or a direct blood supply. In some embodiments, UCAM includes Wharton's jelly. In some embodiments, UCAM includes blood vessels and/or arteries. In some embodiments, UCAM includes Wharton's jelly and blood vessels and/or arteries.

As used herein, the terms "purified" and "isolated" mean a material (e.g., an nHC-HA/PTX3 complex) that is substantially or essentially free from components that normally accompany it in its native state. In some embodiments, "purified" or "isolated" means about 50% or more free from components that normally accompany it in its native state, for example, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% free from components that normally accompany it in its native state.

Overview: nHC-HA/PTX3 and rcHC-HA/PTX3 Complexes

Hyaluronan (HA) is a substantially non-sulfated linear glycosaminoglycan (GAG) composed of repeated disaccharide subunits of D-glucuronic acid and N-acetyl-D-glucosamine through GlcUA-β1,3-GlcNAc-β1,4-bonds. HA is synthesized by HA synthase (e.g., HAS1, HAS2, and HAS3) and deposited into the extracellular matrix, where it contributes to the structural integrity of the tissue and also regulates many cellular processes by interacting with proteins (including cell surface receptors). The molecular weight range of HA is typically from about 200 to about 10,000 kDa. The normal level of HA in the tissue is maintained by a balance between the biosynthesis performed by the HAS enzyme and the catabolism performed by hyaluronidase such as Hyal1.

High molecular weight HA (HMW HA), typically greater than 500 kDa, promotes cell quiescence and structural integrity in tissues such as cartilage and vitreous humor (body fluid) in the eye and is associated with scarless fetal wound healing. In certain instances, HMW HA inhibits gene expression of pro-inflammatory mediators and angiogenesis.

Under certain pathogenic conditions, HMW HA is degraded into smaller fragments and oligosaccharides (e.g., by hyaluronidase or free radical oxidation). LMW HA fragments stimulate vascular endothelial cell proliferation, migration, collagen synthesis, sprout formation, and angiogenesis in rat skin, myocardial infarction, and cryoinjured skin transplant models by promoting gene expression of pro-inflammatory and pro-angiogenic mediators.

The biological function of HA is mediated by the interaction of HA with HA binding protein (HABP) (also known as hyaluronan adhesin). Such proteins include, but are not limited to, tumor necrosis factor-α stimulated gene 6 (TSG-6), aggrecan, versican, neurocan, brevican, LYVE-1, CD44, and inter-α-inhibitor (IαI). In some cases, HABP comprises a connecting module domain that binds to HA. TSG-6, aggrecan, versican, neurocan, brevican, LYVE-1, and CD44 are exemplary HABPs that comprise a connecting module.

IαI comprises two heavy chains (HC1 and HC2) ester-linked to a chondroitin sulfate chain attached to a light chain (i.e., urokinin). In some cases, HA forms a covalent complex with one or both HCs of IαI by covalently linking to the IαI heavy chain (hereinafter referred to as "HC-HA"). In some cases, IαI is found in serum and/or is obtained from IαI-producing cells, e.g., hepatocytes or amniotic epithelial or stromal cells or umbilical cord epithelial or stromal cells, under constitutive stimulation with proinflammatory cytokines such as IL-1 or TNF-α.

In certain instances, TSG-6 promotes the transfer of HC1 and HC2 of IαI to HA, catalyzes this transfer, and/or transfers HC1 and HC2 of IαI to HA. TSG-6 forms a stable complex with immobilized HA (TSG-6·HA), resulting in the transfer of HC1 and HC2 to HA, thereby forming an HC-HA complex and releasing TSG-6 from the complex. TSG-6 expression is typically induced by inflammatory mediators such as TNF-α and interleukin-1 during inflammatory-like processes such as ovulation and cervical ripening.

Amniotic membrane (AM) regulates adult wound healing and promotes tissue regeneration. In some cases, AM promotes epithelialization while suppressing stromal inflammation, angiogenesis and scarring. AM has been successfully used as a surgical implant or temporary biological patch for the treatment of ophthalmic conditions requiring corneal and conjunctival surface reconstruction, including but not limited to persistent epithelial defects, deep corneal ulcers, infectious keratitis, symptomatic bullous keratopathy, acute Stevens Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), limbal stem cell deficiency, pterygium, conjunctival macula, conjunctival laxity, symblepharon, fornix (formix) reconstruction and conjunctival tumors.

The avascular stromal matrix of AM contains a high content of HA and constitutively expresses IαI (Zhang et al. (2012), J. Biol. Chem. 287(15):12433-44). HMW HA in AM forms an nHC-HA complex (He et al. (2009), J. Biol. Chem. 284(30):20136-20146). As shown herein, in the examples provided, the nHC-HA complex also contains pentraxin 3, PTX3 (Figure 1), and is therefore referred to herein as the "nHC-HA/PTX3 complex". Natural HC-HA/PTX3 complex extracted from AM has been shown to inhibit TGF-β promoter activity, promote macrophage cell death, and inhibit vascular development. Therefore, the nHC-HA/PTX3 complex of AM plays a positive role in the anti-inflammatory, anti-scarring, and anti-angiogenic effects of AM.

As described herein, nHC-HA/PTX3 complexes are also found in umbilical cord (UC). These UC HC-HA/PTX3 complexes differ in their biochemical composition with respect to HA content and the presence and/or relative abundance of various components of the complex, including proteoglycans, such as small leucine-rich proteoglycans (SLRPs). In some embodiments, the SLRPs are decorin, biglycan, and/or osteoadherin. As described herein, these complexes also differ in the presence and content of specific sulfated glycosaminoglycans, such as keratan sulfate. Furthermore, as described herein, complexes isolated from AM or UC using different extraction methods (e.g., PBS versus GnHCl extraction) result in complexes with different biochemical compositions and biological properties. In some cases, complexes isolated from the insoluble fraction obtained from umbilical cord tissue by GnHCl extraction were found to exhibit improved properties.

PTX3 is a multimeric protein that has been shown to directly interact with TSG-6 and IαI HC. PTX3 is upregulated in response to inflammatory mediators and has been shown to play an important role in the organization of HA in the extracellular matrix of the cumulus oophorus during oocyte maturation. As demonstrated herein, PTX3 is also found within nHC-HA complexes (i.e., nHC-HA/PTX3) in the amnion and umbilical cord and plays a key role in M2 macrophage polarization.

M1 macrophages, or classically activated proinflammatory macrophages, are induced by interferon (IFN) alone or in combination with lipopolysaccharide (LPS) or tumor necrosis factor (TNF) alpha. M1 macrophages are typically characterized by high expression of interleukin-12 (IL-12) and IL-23 and low levels of IL-10. In contrast, M2 macrophages, or "alternatively activated" macrophages, exhibit wound healing and tissue regeneration properties and are characterized by low IL-12/IL-23 and high IL-10 or roughly equal ratios of IL-12 to IL-10. In some cases, M2 macrophages also have high expression of TGF-β.

The examples provided herein demonstrate that PTX3 directly binds to immobilized HA, as demonstrated by resistance to dissociation agents. It is demonstrated herein that the in vitro reconstituted complex of HA bound to PTX3 exhibits different properties than the in vitro reconstituted complex of HA bound to TSG-6. For example, in some embodiments, the PTX3/HA complex promotes the attachment of LPS-stimulated macrophages without aggregation and induces the expression of IL-10 in LPS-stimulated macrophages. In some embodiments, the PTX3/HA complex disclosed herein increases the expression of IL-10 in LPS-stimulated macrophages by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the expression of IL-10 in the absence of the PTX3/HA complex. In contrast, in some embodiments, the TSG-6/HA complex reduces cell attachment and promotes aggregation of LPS-stimulated macrophages, and does not induce IL-10 expression in LPS-stimulated macrophages. In addition, in some embodiments, TSG-6 pre-bound to HA inhibits subsequent binding of PTX3 to the complex. In some embodiments, both the TSG-6/HA complex and the PTX3/HA complex reduce the expression of IL-12 in LPS-stimulated macrophages. In some embodiments, the PTX3/HA complex or TSG-6/HA complex disclosed herein reduces or inhibits the expression of IL-12 in LPS-stimulated macrophages by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, compared to the expression of IL-12 in the absence of the PTX3/HA complex or TSG-6/HA complex. In some embodiments, both the TSG-6/HA complex and the PTX3/HA complex increase the expression of IL-23 in LPS/IFNγ-stimulated macrophages. In some embodiments, the PTX3/HA complex or TSG-6/HA complex disclosed herein reduces or inhibits the expression of IL-23 in LPS/IFNγ-stimulated macrophages by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the expression of IL-23 in the absence of the PTX3/HA complex or TSG-6/HA complex.

Furthermore, it is demonstrated herein that rcHC-HA/PTX3 complexes reconstituted in vitro have different biological activities depending on whether the rcHC-HA/PTX3 complexes are formed using the following steps: pre-binding HA to TSG-6 in the presence of IαI, followed by the addition of PTX3 or HA pre-bound to PTX3, followed by the addition of TSG-6 with IαI. Provided herein are exemplary methods for reconstitution of rcHC-HA/PTX3 complexes formed using HA pre-bound to TSG-6 or HA pre-bound to PTX3. In some embodiments, rcHC-HA/PTX3 complexes formed using immobilized HA pre-bound to TSG-6 result in the aggregation of LPS-stimulated macrophages. In some embodiments, rcHC-HA/PTX3 complexes formed using immobilized HA pre-bound to PTX3 promote the attachment of LPS-stimulated macrophages without aggregation.

In some embodiments, the rcHC-HA/PTX3 complex formed using immobilized HA pre-bound to PTX3 reduces or inhibits the expression of M1 macrophage markers such as IL-12 and IL-23. In some embodiments, the rcHC-HA/PTX3 complex formed using immobilized HA pre-bound to PTX3 reduces the expression of IL-12 in LPS-stimulated macrophages compared to the expression of IL-12 in the absence of the rcHC-HA/PTX3 complex. In some embodiments, the rcHC-HA/PTX3 complex disclosed herein reduces or inhibits the expression of IL-12 in LPS-stimulated macrophages by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the expression of IL-12 in the absence of the rcHC-HA/PTX3 complex. In some embodiments, rcHC-HA/PTX3 complexes formed with immobilized HA pre-bound to PTX3 reduce or inhibit IL-23 expression in LPS/IFNγ-stimulated macrophages compared to IL-12 expression in the absence of rcHC-HA/PTX3 complexes. In some embodiments, rcHC-HA/PTX3 complexes formed with immobilized HA pre-bound to PTX3 reduce or inhibit IL-23 expression in LPS/IFNγ-stimulated macrophages by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to IL-23 expression in the absence of rcHC-HA/PTX3 complexes. In some embodiments, the activity of nHC-HA/PTX3 complexes isolated from amniotic membrane is replicated using immobilized HA pre-bound to PTX3 to form rcHC-HA/PTX3 complexes.

In some embodiments, rcHC-HA/PTX3 complexes formed with immobilized HA pre-bound to TSG-6 reduce or inhibit the expression of M1 macrophage markers such as IL-12, but increase the expression of IL-23. In some embodiments, rcHC-HA/PTX3 complexes formed with immobilized HA pre-bound to TSG-6 reduce or inhibit the expression of IL-12. In some embodiments, rcHC-HA/PTX3 complexes formed with immobilized HA pre-bound to TSG-6 increase the expression of IL-23.

Provided herein are methods for preparing reconstituted HC-HA/PTX3 complexes using immobilized HA pre-bound to PTX3 and their uses. Also provided herein are complexes of immobilized HA pre-bound to PTX3 and their uses. Also provided herein are methods for preparing reconstituted HC-HA/PTX3 complexes using immobilized HA pre-bound to TSG-6 and their uses. In some embodiments, the reconstituted HC-HA/PTX3 complexes provided herein are administered to treat a wide variety of diseases or conditions, including but not limited to treatments such as suppressing, reducing, preventing, or reducing inflammation, immune responses leading to autoimmunity or immune rejection, adhesions, scarring, angiogenesis, conditions requiring cell or tissue regeneration, tissue reperfusion injury due to ischemia (including myocardial infarction and stroke), and the risk of symptoms resulting therefrom. In some embodiments, the reconstituted HC-HA/PTX3 complexes provided herein are administered to treat inflammation. In some embodiments, the reconstituted HC-HA/PTX3 complexes provided herein are administered to treat scarring. In some embodiments, the reconstituted HC-HA/PTX3 complexes provided herein are administered to treat angiogenesis. In some embodiments, the reconstituted HC-HA/PTX3 complexes provided herein are administered to treat immune responses that lead to autoimmunity or immune rejection. In some embodiments, the reconstituted HC-HA/PTX3 complexes provided herein are administered to treat conditions requiring inhibition of cell adhesion. In some embodiments, the reconstituted HC-HA/PTX3 complexes provided herein are administered to treat conditions requiring cell or tissue regeneration.

Furthermore, the examples provided herein demonstrate the ability of HC-HA/PTX3 complexes to maintain stem cells in an undifferentiated state and to induce adult differentiated fibroblasts into younger progenitor cells in a human corneal fibroblast model. These human corneal fibroblasts are differentiated from keratocytes upon addition of exogenous TGF-β1, which further differentiate into scar-forming myofibroblasts. The data provided herein demonstrate that culturing cells in the presence of HC-HA prevents the cells from differentiating into myofibroblasts upon stimulation with TGF-β1. In the absence of TGF-β1, the HC-HA/PTX3 complex reverts human corneal fibroblasts to keratocytes expressing keratin and CD34. In the presence of TGF-β1, human corneal fibroblasts were further reprogrammed into younger progenitor cells that lacked keratin expression but expressed multiple neural crest cell markers (such as Osr2, FGF10, and Sox9) and embryonic stem cell markers (such as c-myc, KLF4, Nanog, nestin, Oct 4, Rex-1, Sox-2, and SSEA-4).

It is known that the transcription factors Sox2, Oct4, c-Myc, and KLF4 play an important role in the process of inducing adult differentiated cells into progenitor stem cells (iPSCs). Therefore, in some embodiments, the HC-HA/PTX3 complex provided herein is used to reprogram adult differentiated cells into iPSCs. In some embodiments, the induction of iPSCs using a combination of the HC-HA/PTX3 complex and one or more of Sox2, Oct4, c-Myc, and KLF4 is much more efficient than conventional methods using these four transcription factors without HC-HA/PTX3. In some embodiments, the addition of the HC-HA/PTX3 complex promotes stem cell induction by shutting down TGF-β signaling to prevent differentiation and by turning on BMP signaling to promote reprogramming into young progenitor cells such as iPSCs. In some embodiments, the addition of the HC-HA/PTX3 complex promotes stem cell induction by reprogramming cells into younger progenitor cells and inducing stem cell markers. In some embodiments, the addition of the HC-HA/PTX3 complex helps maintain the characteristics of stem cells during in vitro expansion, thereby eliminating the need to use a feeder layer composed of mouse embryonic fibroblasts. Therefore, in some embodiments, the HC-HA/PTX3 complex is used as a carrier or scaffold to facilitate the delivery of stem cells that have been expanded in vitro to human patients, thereby enhancing the efficacy of the stem cell therapy.

Methods for preparing isolated nHC-HA/PTX3 complexes

Disclosed herein are methods of producing isolated native HC-HA/PTX3 complexes (nHC-HA/PTX3).

In some embodiments, the isolated nHC-HA/PTX3 complex is isolated from amniotic tissue. In some embodiments, the isolated nHC-HA/PTX3 complex is isolated from amniotic membrane or umbilical cord. In some embodiments, the isolated nHC-HA/PTX3 complex is isolated from fresh, frozen, or previously frozen placental amniotic membrane (PAM), fresh, frozen, or previously frozen umbilical cord amniotic membrane (UCAM), fresh, frozen, or previously frozen placenta, fresh, frozen, or previously frozen umbilical cord, fresh, frozen, or previously frozen chorion, fresh, frozen, or previously frozen amniotic-chorionic membrane, or any combination thereof. Such tissues can be obtained from any mammal, for example, but not limited to, humans, non-human primates, cattle, or pigs.

In some embodiments, nHC-HA/PTX3 is purified by any suitable method. In some embodiments, the nHC-HA/PTX3 complex is purified by centrifugation (e.g., ultracentrifugation, gradient centrifugation), chromatography (e.g., ion exchange chromatography, affinity chromatography, size exclusion chromatography, and hydroxyapatite chromatography), gel filtration or differential solubility, ethanol precipitation, or by any other available technique for protein purification (see, e.g., Scopes, Protein Purification Principles and Practice, 2nd ed., Springer-Verlag, New York, 1987; Higgins, S.J. and Hames, B.D. (Eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, M.P., Simon, M.I., Abelson, J.N. (Eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol 182), Academic Press, 1997, all of which are incorporated herein by reference).

In some embodiments, nHC-HA/PTX3 is isolated from an extract. In some embodiments, the extract is prepared from an amniotic membrane extract. In some embodiments, the extract is prepared from an umbilical cord extract. In some embodiments, the umbilical cord extract comprises umbilical cord stroma and/or Wharton's jelly. In some embodiments, the nHC-HA/PTX3 complex is contained in an extract prepared by ultracentrifugation. In some embodiments, the nHC-HA/PTX3 complex is contained in an extract prepared by ultracentrifugation using a CsCl/4-6 M guanidine hydrochloride gradient. In some embodiments, the extract is prepared by at least two rounds of ultracentrifugation. In some embodiments, the extract is prepared by more than two rounds of ultracentrifugation (i.e., nHC-HA/PTX3, second round). In some embodiments, the extract is prepared by at least four rounds of ultracentrifugation (i.e., nHC-HA/PTX3, fourth round). In some embodiments, the nHC-HA/PTX3 complex comprises small leucine-rich proteoglycans. In some embodiments, the nHC-HA/PTX3 complex comprises HCl, HA, PTX3, and/or small leucine-rich proteoglycans.

In some embodiments, the extract prepared by extraction is subjected to ultracentrifugation in an isotonic solution. In some embodiments, the isotonic solution is PBS. For example, in some embodiments, tissue is homogenized in PBS to prepare a homogenate sample. The homogenate sample is then separated by centrifugation into a soluble portion and an insoluble portion. In some embodiments, the soluble portion of the PBS-extracted tissue is subjected to ultracentrifugation. In such embodiments, nHC-HA/PTX3 purified by ultracentrifugation of the PBS-extracted tissue is referred to as an nHC-HA/PTX3 soluble complex. In some embodiments, the nHC-HA soluble complex comprises small leucine-rich proteoglycans. In some embodiments, the nHC-HA/PTX3 soluble complex comprises HCl, HA, PTX3, and/or small leucine-rich proteoglycans.

In some embodiments, an extract prepared by direct guanidine hydrochloride extraction (e.g., 4-6 M GnHCl) of amniotic and/or umbilical cord tissue is subjected to ultracentrifugation. In some embodiments, the GnHCl extract tissue is subsequently centrifuged to produce GnHCl-soluble and GnHCl-insoluble fractions. In some embodiments, the GnHCl-soluble fraction is subjected to ultracentrifugation. In such embodiments, nHC-HA/PTX3 purified by ultracentrifugation of guanidine hydrochloride-extracted tissue is referred to as an nHC-HA/PTX3 insoluble complex. In some embodiments, the nHC-HA insoluble complex comprises small leucine-rich proteoglycans. In some embodiments, the nHC-HA/PTX3 insoluble complex contains HCl, HA, PTX3, and/or small leucine-rich proteoglycans.

In some embodiments, an extract prepared by further guanidine hydrochloride extraction of the insoluble portion of the PBS-extracted tissue is subjected to ultracentrifugation. For example, in some embodiments, the tissue is homogenized in PBS to prepare a homogenate sample. The homogenate sample is then separated by centrifugation into a soluble portion and an insoluble portion. The insoluble portion is then further extracted in guanidine hydrochloride (e.g., 4-6M GnHCl) and centrifuged to produce a guanidine hydrochloride-soluble portion and an insoluble portion. In some embodiments, the guanidine hydrochloride-soluble portion is subjected to ultracentrifugation. In such embodiments, nHC-HA/PTX3 purified by ultracentrifugation of guanidine hydrochloride-extracted tissue is referred to as an nHC-HA/PTX3 insoluble complex. In some embodiments, the nHC-HA insoluble complex comprises small leucine-rich proteoglycans. In some embodiments, the nHC-HA/PTX3 insoluble complex comprises HCl, HA, PTX3, and/or small leucine-rich proteoglycans.

In some embodiments, methods of purifying an isolated nHC-HA/PTX3 extract comprise: (a) dissolving the isolated extract (e.g., prepared by the soluble or insoluble methods described herein) in CsCl/4-6 M guanidine hydrochloride at an initial density of 1.35 g/ml to produce a CsCl mixture, (b) centrifuging the cesium chloride mixture at 125,000 x g for 48 hours at 15°C to produce a first purified extract, (c) extracting the first purified extract and dialyzing it against distilled water to remove cesium chloride and guanidine hydrochloride, thereby producing a dialysate. In some embodiments, the method of purifying the isolated extract further comprises: (d) mixing the dialysate with 3 volumes of 95% (v/v) ethanol containing 1.3% (w/v) potassium acetate at 0°C for 1 hour to produce a first dialysate/ethanol mixture, (e) centrifuging the first dialysate/ethanol mixture at 15,000 x g to produce a second purified extract, and (f) extracting the second purified extract. In some embodiments, the method of purifying the isolated extract further comprises: (g) washing the second purified extract with ethanol (e.g., 70% ethanol) to produce a second purified extract/ethanol mixture; (h) centrifuging the second purified extract/ethanol mixture to produce a third purified extract; and (i) extracting the third purified extract. In some embodiments, the method of purifying the isolated extract further comprises: (j) washing the third purified extract with ethanol (e.g., 70% ethanol) to produce a third purified extract/ethanol mixture; (k) centrifuging the third purified extract/ethanol mixture to produce a fourth purified extract; and (l) extracting the fourth purified extract. In some embodiments, the purified extract contains nHC-HA/PTX3 complex.

In some embodiments, the nHC-HA/PTX3 complex is purified by immunoaffinity chromatography. In some embodiments, anti-HCl antibodies, anti-HC2 antibodies, or both are generated and attached to a fixed support. In some embodiments, unpurified HC-HA complexes (i.e., mobile phase) are passed over the support. In some cases, the HC-HA complex binds to the antibody (e.g., through interaction of (a) anti-HCl antibody and HCl, (b) anti-HC2 antibody and HC2, (c) anti-PTX antibody and PTX3, (d) anti-SLRP antibody and SLRP, or (e) any combination thereof). In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is subsequently washed with a solution capable of eluting the nHC-HA/PTX3 complex from the support (e.g., 1% SDS, 6 M guanidine hydrochloride, or 8 M urea).

In some embodiments, the nHC-HA/PTX3 complex is purified by affinity chromatography. In some embodiments, HABP is produced and attached to a fixed support. In some embodiments, unpurified nHC-HA/PTX3 complex (i.e., mobile phase) is passed over the support. In some cases, the nHC-HA/PTX3 complex binds to the HABP. In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is subsequently washed with a solution capable of eluting the HC-HA complex from the support.

In some embodiments, the nHC-HA/PTX3 complex is purified by HABP affinity chromatography and immunoaffinity chromatography using an anti-HCl antibody, an anti-HC2 antibody, an anti-PTX3 antibody, an antibody to a SLRP or a combination of SLRPs, or any combination of antibodies thereto.

In some embodiments, one or more antibodies are used to purify the nHC-HA/PTX3 complex from the insoluble fraction as described herein. In some embodiments, an anti-SLRP antibody is used to purify the nHC-HA/PTX3 complex from the insoluble fraction as described herein.

In some embodiments, the nHC-HA/PTX3 complex is purified from a soluble fraction as described herein. In some embodiments, the nHC-HA/PTX3 complex is purified from a soluble fraction as described herein using an anti-PTX3 antibody.

In some embodiments, the nHC-HA/PTX3 complex comprises a small leucine-rich proteoglycan (SLRP). In some embodiments, the nHC-HA/PTX3 complex comprises a class I, class II, or class III SLRP. In some embodiments, the small leucine-rich proteoglycan is selected from a class I SLRP, such as decorin and biglycan. In some embodiments, the small leucine-rich proteoglycan is selected from a class II SLRP, such as fibromodulin, lumican, PRELP (proline arginine-rich terminal leucine-rich protein), keratocan, and osteoadherin. In some embodiments, the small leucine-rich proteoglycan is selected from a class III SLRP, such as epiphyseal and osteoglycan. In some embodiments, the small leucine-rich proteoglycan is selected from uropancreatin, decorin, biglycan, and osteoadherin. In some embodiments, the small leucine-rich protein comprises a glycosaminoglycan. In some embodiments, the small leucine-rich proteoglycan comprises keratan sulfate.

Methods for producing rcHC-HA/PTX3 complexes

Disclosed herein are methods for producing reconstituted HC-HA/PTX3 complexes (rcHC-HA/PTX3) with or without SLRPs. Also disclosed herein are rcHC-HA/PTX3 complexes produced by such methods and intermediate combinations of components.

In some embodiments, a method for producing a reconstituted HC-HA/PTX3 complex comprises: (a) contacting immobilized high molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) under appropriate conditions to form a PTX3/HA complex, and (b) contacting the PTX3/HA complex with IαI and tumor necrosis factor-stimulated gene 6 (TSG-6). Provided herein are rcHC-HA/PTX3 complexes produced by this method. In some embodiments, TSG-6 catalyzes the transfer of heavy chain 1 (HCl) of inter-α-inhibitor (IαI) to HA. In some embodiments, HCl of IαI forms a covalent bond with HA. In some embodiments, steps (a) and (b) of the method are performed sequentially.

In some embodiments, a method for producing a reconstituted HC-HA/PTX3 complex comprises contacting the PTX3/HA complex with IαI and TSG-6. In some embodiments, TSG-6 catalyzes the transfer of heavy chain 1 (HCl) of inter-α-inhibitor (IαI) to HA. Provided herein are rcHC-HA/PTX3 complexes produced by this method. In some embodiments, HCl of IαI forms a covalent bond with HA.

In some embodiments, a method for producing a complex of HA bound to PTX3 comprises contacting immobilized high molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) under appropriate conditions to form a PTX3/HA complex. Provided herein are PTX3/HA complexes produced by this method.

In some embodiments, a method for producing a reconstituted HC-HA/PTX3 complex comprises (a) contacting immobilized high molecular weight hyaluronan (HMW HA) with IαI and TSG-6 directed against HA to form an HC-HA complex pre-bound to TSG-6, and (b) contacting the HC-HA complex with pentraxin 3 (PTX3) under appropriate conditions to form a rcHC-HA/PTX3 complex. Provided herein are rcHC-HA/PTX3 complexes produced by this method. In some embodiments, HCl of IαI forms a covalent bond with HA. In some embodiments, steps (a) and (b) of the method are performed sequentially. In some embodiments, the method comprises contacting the HC-HA complex pre-bound to TSG-6 with PTX3.

In some embodiments, the method comprises first contacting high molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) under appropriate conditions to form a PTX3/HA complex, and then contacting the PTX3/HA complex with IαI and TSG-6.

In some embodiments, the IαI protein and the TSG-6 protein are contacted at a molar ratio of about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1 (IαI:TSG-6) to form a complex. In some embodiments, the IαI:TSG-6 ratio ranges from about 1:1 to about 20:1, such as about 1:1 to about 10:1, such as about 1:1 to about 5:1, such as about 1:1 to about 3:1. In some embodiments, the IαI:TSG-6 ratio ranges from 3:1 or more. In some embodiments, the IαI:TSG-6 ratio is 3:1.

In some embodiments, steps (a) and (b) of the method are performed sequentially. In some embodiments, the method comprises contacting the PTX3/HA complex with IαI and TSG-6.

In some cases, TSG-6 interacts with IαI and forms a covalent complex with the HCl and HC2 of IαI (i.e., HCl·TSG-6 and HC2·TSG-6). In some cases, in the presence of HA, HC is transferred to HA to form rcHC-HA. In some embodiments, the TSG-6·HCl complex is added to the pre-bound PTX3/HA complex to catalyze the transfer of HCl to HA. In some embodiments, the method comprises: first contacting immobilized high molecular weight hyaluronan (HMWHA) with pentraxin 3 (PTX3) under appropriate conditions to form a PTX3/HA complex, and then contacting the PTX3/HA complex with the HCl·TSG-6 complex. In some embodiments, a combination of the HCl·TSG-6 complex and the HC2·TSG-6 complex is added to the PTX3/HA complex.

In some embodiments, the step of contacting PTX3 with immobilized HMW HA is performed for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting PTX3 with immobilized HMW HA is performed for at least 2 hours or longer. In some embodiments, the step of contacting PTX3 with immobilized HMW HA is performed for at least 2 hours. In some embodiments, the step of contacting PTX3 with immobilized HMW HA is performed at 37° C. In some embodiments, the step of contacting PTX3 with immobilized HMW HA is performed in 5 mM MgCl ₂ in PBS.

In some embodiments, the step of contacting the PTX3/HA complex with IαI and TSG-6 for HA is performed for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting the PTX3/HA complex with the HCl·TSG-6 complex and/or the HC2·TSG-6 complex is performed for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting the PTX3/HA complex with the HCl·TSG-6 complex and/or the HC2·TSG-6 complex is performed for at least 2 hours or longer. In some embodiments, the step of contacting the PTX3/HA complex with the HCl·TSG-6 complex and/or the HC2·TSG-6 complex is performed for at least 2 hours. In some embodiments, the step of contacting the PTX3/HA complex with the HCl·TSG-6 complex and/or the HCl·TSG-6 complex is performed at 37° C. In some embodiments, the step of contacting the PTX3/HA complex with the HCl·TSG-6 complex and/or the HCl·TSG-6 complex is performed in 5 mM MgCl ₂ in PBS.

In some embodiments, the method comprises contacting high molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) protein, inter-α-inhibitor (IαI) protein containing heavy chain 1 (HCl), and tumor necrosis factor α-stimulated gene 6 (TSG-6) simultaneously under appropriate conditions to form a HC-HA/PTX3 complex. In some embodiments, contacting HMW HA with PTX3, IαI, and TSG-6 is performed for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting HMW HA, PTX3, IαI, and TSG-6 is performed at 37° C. In some embodiments, the step of contacting HMW HA, PTX3, IαI, and TSG-6 is performed in 5 mM MgCl ₂ in PBS.

In some embodiments, the method comprises contacting high molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) protein, inter-α-inhibitor (IαI) protein containing heavy chain 1 (HCl), and tumor necrosis factor α-stimulated gene 6 (TSG-6) sequentially in any order under appropriate conditions to form a HC-HA/PTX3 complex. In some embodiments, contacting HMW HA with PTX3, IαI, and TSG-6 is performed for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting HMW HA, PTX3, IαI, and TSG-6 is performed at 37° C. In some embodiments, the step of contacting HMW HA, PTX3, IαI, and TSG-6 is performed in 5 mM MgCl ₂ in PBS.

In some embodiments, the method for producing a rcHC-HA/PTX3 complex further comprises adding one or more small leucine-rich proteoglycans (SLRPs). In some embodiments, the method for producing a reconstituted HC-HA/PTX3 complex comprises: (a) contacting immobilized high molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) under appropriate conditions to form a PTX3/HA complex, (b) contacting the PTX3/HA complex with IαI and tumor necrosis factor-stimulated gene 6 (TSG-6), and (c) contacting the PTX3/HA complex with one or more SLRPs. Provided herein are rcHC-HA/PTX3 complexes produced by this method. In some embodiments, TSG-6 catalyzes the transfer of heavy chain 1 (HCl) of inter-α-inhibitor (IαI) to HA. In some embodiments, HCl of IαI forms a covalent bond with HA. In some embodiments, steps (a), (b), and (c) of the method are performed sequentially. In some embodiments, steps (a), (b), and (c) of the method are performed simultaneously. In some embodiments, step (a) of the method is performed, and then steps (b) and (c) of the method are performed sequentially. In some embodiments, step (a) of the method is performed, and then steps (b) and (c) of the method are performed simultaneously.

In some embodiments, a method for producing a reconstituted HC-HA/PTX3 complex comprises: (a) contacting immobilized high molecular weight hyaluronan (HMW HA) with IαI and TSG-6 directed against HA to form a HC-HA complex pre-bound to TSG-6, (b) contacting the HC-HA complex with pentraxin 3 (PTX3), and (c) contacting the HC-HA complex with one or more SLRPs under appropriate conditions to form a rcHC-HA/PTX3 complex. Provided herein are rcHC-HA/PTX3 complexes produced by this method. In some embodiments, the HCl of IαI forms a covalent bond with HA. In some embodiments, the method comprises contacting the HC-HA complex pre-bound to TSG-6 with PTX3. In some embodiments, steps (a), (b), and (c) of the method are performed sequentially. In some embodiments, steps (a), (b), and (c) of the method are performed simultaneously. In some embodiments, step (a) of the method is performed, and then steps (b) and (c) of the method are performed sequentially. In some embodiments, step (a) of the method is performed, and then steps (b) and (c) of the method are performed simultaneously.

In some embodiments, the SLRP is selected from Class I, Class II, or Class II SLRPs. In some embodiments, the SLRP is selected from Class I SLRPs, such as decorin and biglycan. In some embodiments, the small leucine-rich proteoglycan is selected from Class II SLRPs, such as fibromodulin, lumican, PRELP (proline arginine-rich terminal leucine-rich protein), keratocan, and osteoadherin. In some embodiments, the small leucine-rich proteoglycan is selected from Class III SLRPs, such as epiphyseal and osteoglycan. In some embodiments, the small leucine-rich proteoglycan is selected from urotrypsin, decorin, biglycan, and osteoadherin. In some embodiments, the small leucine-rich protein comprises a glycosaminoglycan. In some embodiments, the small leucine-rich proteoglycan comprises keratan sulfate.

PTX3

In some embodiments, the PTX3 used in the method is isolated from one or more cells (e.g., tissue extracts). Exemplary cells suitable for expressing PTX3 include, but are not limited to, animal cells (including, but not limited to, mammalian cells, primate cells, human cells, rodent cells, insect cells, bacteria, and yeast) and plant cells (including, but not limited to, algae, angiosperms, gymnosperms, ferns, and mosses). In some embodiments, the PTX3 used in the method is isolated from human cells. In some embodiments, the PTX3 used in the method is isolated from cells that have been stimulated with one or more proinflammatory cytokines to upregulate PTX3 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, the PTX3 used in the methods is isolated from amniotic cells. In some embodiments, the PTX3 used in the methods is isolated from amniotic cells from the umbilical cord. In some embodiments, the amniotic cells are stimulated with one or more proinflammatory cytokines to upregulate PTX3 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, the PTX3 used in the method is isolated from umbilical cord cells. In some embodiments, the umbilical cord cells are stimulated with one or more proinflammatory cytokines to upregulate PTX3 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, the PTX3 used in the methods is isolated from amniotic epithelial cells. In some embodiments, the PTX3 used in the methods is isolated from umbilical cord epithelial cells. In some embodiments, the amniotic epithelial cells or umbilical cord epithelial cells are stimulated with one or more proinflammatory cytokines to upregulate PTX3 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, the PTX3 used in the methods is isolated from amniotic stromal cells. In some embodiments, the PTX3 used in the methods is isolated from umbilical cord stromal cells. In some embodiments, the amniotic stromal cells or umbilical cord stromal cells are stimulated with one or more proinflammatory cytokines to upregulate PTX3 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, the PTX3 used in the method is a naturally occurring PTX3 protein isolated from cells. In some embodiments, the cells are stimulated with one or more proinflammatory cytokines to upregulate PTX3 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, PTX3 is produced by recombinant techniques. In some embodiments, PTX3 is expressed from a recombinant expression vector. In some embodiments, the nucleic acid encoding PTX3 is operably linked to a constitutive promoter. In some embodiments, the nucleic acid encoding PTX3 is operably linked to an inducible promoter. In some embodiments, PTX3 is expressed in transgenic animals. In some embodiments, PTX3 is a recombinant protein. In some embodiments, PTX3 is a recombinant protein isolated from cells. In some embodiments, PTX3 is a recombinant protein produced in a cell-free extract.

In some embodiments, PTX3 is purified from amniotic membrane, umbilical cord, umbilical cord amniotic membrane, chorion, amniotic fluid, or a combination thereof. In some embodiments, PTX3 is purified from amniotic cells. In some embodiments, the amniotic cells are amniotic epithelial cells. In some embodiments, the amniotic cells are umbilical cord epithelial cells. In some embodiments, the amniotic cells are amniotic stromal cells. In some embodiments, the amniotic cells are umbilical cord stromal cells. In some embodiments, the amniotic cells are stimulated with one or more proinflammatory cytokines to upregulate PTX3 expression. In some embodiments, the proinflammatory cytokines are IL-1 or TNF-α.

In some embodiments, PTX3 is not isolated from a cell or cells (eg, a tissue extract).

In some embodiments, PTX3 comprises a polypeptide having a sequence as set forth in SEQ ID NO: 33, or a variant thereof having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having a sequence as set forth in SEQ ID NO: 33. Exemplary variants include, for example, species variants, allelic variants, and variants comprising conservative and non-conservative amino acid mutations. In some embodiments, PTX3 comprises a PTX3 fragment sufficient to bind to HA and promote formation of the rcHC-HA/PTX3 complex. In some embodiments, PTX3 comprises Glu18 to Ser277 of human PTX3. Variants of PTX3 for use in the provided methods include variants having amino acid modifications that are amino acid substitutions (alternatives), deletions, or insertions. In some embodiments, the modification improves one or more properties of the PTX3 polypeptide, such as improving one or more therapeutic properties of the rcHC-HA/PTX3 complex (e.g., anti-inflammatory, anti-immune, anti-angiogenic, anti-scarring, anti-adhesion, regenerative, or other therapeutic activity as described herein).

In some embodiments, the PTX3 protein is obtained from a commercial source. An exemplary commercial source of PTX3 is, but is not limited to, PTX3 (Catalog No. 1826-TS; R&D Systems, Minneapolis, MN).

In some embodiments, the PTX3 protein used in the methods is a multimeric protein. In some embodiments, the PTX3 protein used in the methods is a homomultimer. In some embodiments, the homomultimer is a dimer, trimer, tetramer, hexamer, pentamer, or octamer. In some embodiments, the PTX3 homomultimer is a trimer, tetramer, or octamer. In specific embodiments, the PTX3 homomultimer is an octamer. In some embodiments, the multimerization domain is modified to improve the multimerization of the PTX3 protein. In some embodiments, a heterologous multimerization domain (e.g., an Fc multimerization domain or a leucine zipper) that improves the multimerization of PTX3 when fused to PTX3 is used in place of the multimerization domain.

TSG-6

In some embodiments, the TSG-6 used in the method is isolated from one or more cells (e.g., tissue extracts). Exemplary cells suitable for expressing TSG-6 include, but are not limited to, animal cells (including, but not limited to, mammalian cells, primate cells, human cells, rodent cells, insect cells, bacteria, and yeast) and plant cells (including, but not limited to, algae, angiosperms, gymnosperms, ferns, and mosses). In some embodiments, the TSG-6 used in the method is isolated from human cells. In some embodiments, the TSG-6 used in the method is isolated from cells that have been stimulated with one or more proinflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, TSG-6 used in the methods is isolated from amniotic cells. In some embodiments, TSG-6 used in the methods is isolated from amniotic cells from the umbilical cord. In some embodiments, TSG-6 used in the methods is isolated from amniotic cells stimulated with one or more proinflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, the TSG-6 used in the methods is isolated from umbilical cord cells. In some embodiments, the TSG-6 used in the methods is isolated from umbilical cord cells that have been stimulated with one or more proinflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, TSG-6 used in the methods is isolated from amniotic epithelial cells. In some embodiments, TSG-6 used in the methods is isolated from umbilical cord epithelial cells. In some embodiments, TSG-6 used in the methods is isolated from amniotic epithelial cells or umbilical cord epithelial cells that have been stimulated with one or more proinflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, the TSG-6 used in the methods is isolated from amniotic stromal cells. In some embodiments, the TSG-6 used in the methods is isolated from umbilical cord stromal cells. In some embodiments, the TSG-6 used in the methods is isolated from amniotic stromal cells or umbilical cord stromal cells that have been stimulated with one or more proinflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, the TSG-6 used in the method is a naturally occurring TSG-6 protein isolated from cells. In some embodiments, the cells are stimulated with one or more proinflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, TSG-6 is produced by recombinant techniques. In some embodiments, TSG-6 is expressed from a recombinant expression vector. In some embodiments, the nucleic acid encoding TSG-6 is operably linked to a constitutive promoter. In some embodiments, the nucleic acid encoding TSG-6 is operably linked to an inducible promoter. In some embodiments, TSG-6 is expressed in transgenic animals. In some embodiments, TSG-6 is a recombinant protein. In some embodiments, TSG-6 is a recombinant protein isolated from cells. In some embodiments, TSG-6 is a recombinant protein produced in a cell-free extract.

In some embodiments, TSG-6 is purified from amniotic membrane, amniotic membrane, chorion, amniotic fluid, or a combination thereof. In some embodiments, PTX3 is purified from amniotic cells. In some embodiments, the amniotic cells are amniotic epithelial cells. In some embodiments, the amniotic epithelial cells are umbilical cord epithelial cells. In some embodiments, the amniotic cells are amniotic stromal cells. In some embodiments, the amniotic cells are umbilical cord stromal cells. In some embodiments, the amniotic cells are stimulated with one or more proinflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the proinflammatory cytokines are IL-1 or TNF-α.

In some embodiments, TSG-6 is not isolated from a cell or cells (eg, a tissue extract).

In some embodiments, TSG-6 includes a TSG-6 fragment sufficient to promote or catalyze the transfer of HCl of IαI to HA. In some embodiments, TSG-6 includes a TSG-6 linker module. In some embodiments, TSG-6 comprises amino acids Trp18 to Leu277 of TSG-6. In some embodiments, TSG-6 includes a polypeptide having a sequence as set forth in SEQ ID NO: 2 or a variant thereof, which has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence amino acid identity with the polypeptide having a sequence as set forth in SEQ ID NO: 2. Exemplary variants include, for example, species variants, allelic variants, and variants containing conservative and non-conservative amino acid mutations. Natural allelic variants of human TSG-6 include, for example, TSG-6 containing the amino acid substitution Q144R. Variants of TSG-6 or HA-binding fragments thereof for use in the provided methods include variants having amino acid modifications (the modifications being amino acid substitutions (alternatives), deletions, or insertions). In some embodiments, the modification improves one or more properties of the TSG-6 polypeptide, such as improved transfer of the HCl of IαI to HA or improved release of the TSG-6 polypeptide from the rcHC-HA/PTX3 complex following transfer of the HCl of IαI to HA.

In some embodiments, TSG-6 comprises an affinity tag. Exemplary affinity tags include, but are not limited to, hemagglutinin tags, polyhistidine tags, myc tags, FLAG tags, and glutathione-S-transferase (GST) tags. Such affinity tags are well known in the art for use in purification. In some embodiments, such affinity tags are introduced into the TSG-6 polypeptide as a fusion protein or via a chemical linker. In some embodiments, TSG-6 comprises an affinity tag, and unbound TSG-6 is removed from the rcHC-HA/PTX3 complex by affinity purification.

In some embodiments, the TSG-6 protein is obtained from a commercial source. An exemplary commercial source of TSG-6 is, but is not limited to, TSG-6 (Cat. No. 2104-TS R&D Systems, Minneapolis, MN).

IαI

In some embodiments, the IαI comprises an HCl chain. In some embodiments, the IαI comprises an HCl and an HC2 chain. In some embodiments, the IαI comprises an HCl and uropancreatin. In some embodiments, the IαI comprises an HCl and HC2 chain and uropancreatin. In some embodiments, the IαI comprises an HCl and HC2 chain connected by a chondroitin sulfate chain and uropancreatin.

In some embodiments, IαI is isolated from a biological sample. In some embodiments, the biological sample is a biological sample from a mammal. In some embodiments, the mammal is a human. In some embodiments, the biological sample is a blood, serum, plasma, liver, amnion, chorion, or amniotic fluid sample. In some embodiments, the biological sample is a blood, serum, or plasma sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, the IαI is purified from human blood, plasma, or serum. In some embodiments, IαI is isolated from human serum. In some embodiments, IαI is not isolated from serum. In some embodiments, the IαI used in the methods is produced in amniotic cells. In some embodiments, the IαI used in the methods is produced in umbilical cord cells. In some embodiments, the IαI used in the methods is produced in amniotic cells from the umbilical cord. In some embodiments, the IαI used in the methods is produced in amniotic epithelial cells. In some embodiments, the IαI used in the methods is produced in umbilical cord epithelial cells. In some embodiments, the IαI used in the methods is produced in amniotic stromal cells. In some embodiments, the IαI used in the methods is produced in umbilical cord stromal cells. In some embodiments, the IαI used in the methods is produced in hepatocytes. In some embodiments, the IαI used in the methods is produced by recombinant technology.

In some embodiments, the HCl of IαI is isolated from a biological sample. In some embodiments, the biological sample is a biological sample from a mammal. In some embodiments, the mammal is a human. In some embodiments, the biological sample is a blood, serum, plasma, liver, amnion, chorion, or amniotic fluid sample. In some embodiments, the biological sample is a blood, serum, or plasma sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, the HCl of IαI is purified from human blood, plasma, or serum. In some embodiments, IαI is isolated from human serum. In some embodiments, the HCl of IαI is not purified from serum. In some embodiments, the HCl of IαI is produced by recombinant technology. In some embodiments, the HCl of IαI is purified from hepatocytes. In some embodiments, the HCl of IαI is purified from amniotic cells. In some embodiments, the HCl of IαI is purified from amniotic epithelial cells or umbilical cord epithelial cells. In some embodiments, the HCl of IαI is purified from amniotic membrane stromal cells or umbilical cord stromal cells.

In some embodiments, HCl comprises a polypeptide having the sequence set forth in SEQ ID NO: 47, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to a polypeptide having the sequence set forth in SEQ ID NO: 47.

In some embodiments, the HC2 of IαI is isolated from a biological sample. In some embodiments, the biological sample is a biological sample from a mammal. In some embodiments, the mammal is a human. In some embodiments, the biological sample is a blood, serum, plasma, liver, amnion, chorion, or amniotic fluid sample. In some embodiments, the biological sample is a blood, serum, or plasma sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, the HC2 of IαI is purified from human blood, plasma, or serum. In some embodiments, the HC2 of IαI is isolated from human serum. In some embodiments, the HC2 of IαI is isolated from human serum. In some embodiments, the HC2 of IαI is not isolated from serum. In some embodiments, the HC2 of IαI is produced by recombinant technology. In some embodiments, the HC2 of IαI is purified from hepatocytes. In some embodiments, the HC2 of IαI is purified from amniotic cells. In some embodiments, the HC2 of IαI is purified from amniotic epithelial cells or umbilical cord epithelial cells. In some embodiments, the HC2 of IαI is purified from amniotic stromal cells or umbilical cord stromal cells.

In some embodiments, HC2 comprises a polypeptide having the sequence set forth in SEQ ID NO:49, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to a polypeptide having the sequence set forth in SEQ ID NO:49.

In some embodiments, IαI comprises uropancreatin. In some embodiments, uropancreatin comprises a polypeptide having the sequence set forth in SEQ ID NO: 53, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to the polypeptide having the sequence set forth in SEQ ID NO: 53. In some embodiments, IαI comprises a chondroitin sulfate chain.

HA

In some embodiments, HA is purified from cells, tissues or fluid samples. In some embodiments, HA is obtained from commercial suppliers (e.g., Sigma Aldrich or Advanced Medical Optics, Irvine, CA (e.g., Healon)). In some embodiments, HA is obtained from commercial suppliers as powder. In some embodiments, HA is expressed in cells. Exemplary cells suitable for expressing HA include but are not limited to animal cells (including but not limited to mammalian cells, primate cells, human cells, rodent cells, insect cells, bacteria and yeast) and plant cells (including but not limited to algae, angiosperms, gymnosperms, ferns and mosses). In some embodiments, HA is expressed in human cells. In some embodiments, HA is expressed in transgenic animals. In some embodiments, HA is obtained from cells expressing hyaluronan synthase (e.g., HAS1, HAS2 and HAS3). In some embodiments, the cells contain a recombinant expression vector expressing HA synthase. In certain instances, HA synthase elongates hyaluronan by repeatedly adding glucuronic acid and N-acetylglucosamine to the nascent polysaccharide as it is extruded across the cell membrane into the extracellular space.

The HA used in the methods is typically high molecular weight (HMW) HA. In some embodiments, the HMW HA has a weight average molecular weight greater than about 500 kilodaltons (kDa), for example, from about 500 kDa to about 10,000 kDa, from about 800 kDa to about 8,500 kDa, from about 1100 kDa to about 5,000 kDa, or from about 1400 kDa to about 3,500 kDa. In some embodiments, the HMW HA has a weight average molecular weight of about 3000 kDa.

Additional components

In some embodiments, one or more additional components are added to produce the rcHC-HA/PTX3 complex. In some embodiments, a small leucine-rich proteoglycan (SLRP) is added to produce the rcHC-HA/PTX3 complex. In some embodiments, the SLRP is a Class I, Class II, or Class III SLRP. In some embodiments, the SLRP is selected from Class I SLRPs, such as decorin and biglycan. In some embodiments, the SLRP is selected from Class II SLRPs, such as fibromodulin, lumican, PRELP (proline arginine-rich terminal leucine-rich protein), keratocan, and osteoadherin. In some embodiments, the SLRP is selected from Class III SLRPs, such as epiphyseal and osteoglycan. In some embodiments, the SLRP is selected from uropancreatin, decorin, biglycan, and osteoadherin. In some embodiments, the SLRP comprises a glycosaminoglycan. In some embodiments, the SLRP comprises keratan sulfate.

HA immobilization

In some embodiments, HMW HA is immobilized by any suitable method. In some embodiments, HMW HA is fixed to a solid support, such as a culture dish, a bead, a column or other suitable surface, for example, the surface of an implantable medical device or a portion thereof, or is fixed on a surface subsequently connected or merged with an implantable medical device as described herein. In some embodiments, HMW HA is directly fixed to a solid support, for example, by chemical connection. In some embodiments, HMW HA is indirectly attached to a solid support via a connector or an intermediate protein. Numerous heterobifunctional cross-linking reagents for forming a covalent bond between an amino group and a thiol group and introducing a thiol group into a protein are known to those skilled in the art. In some embodiments, HMW HA is directly fixed to a solid support by cross-linking with a solid support. In some embodiments, HMW HA is directly fixed to a solid support without cross-linking with a solid support. In some embodiments, HMW HA is directly fixed to a solid support as a coating. In some embodiments, HMW HA is fixed on a Covalink ^™ -NH surface. In some embodiments, HMW HA is directly fixed to a solid support as a coating. In some embodiments, HMW HA is immobilized on a Covalink ^™ -NH surface at 4°C for about 16 h.

In some embodiments, the method comprises immobilizing HMW HA on a solid surface by direct attachment to the solid support (i.e., without an intervening protein). In some embodiments, the solid support is washed to remove unbound HMW HA prior to contacting the immobilized HA with PTX3. In some embodiments, the solid support is washed with a wash solution of 8M GnHCl and PBS prior to contacting the immobilized HA with PTX3 to remove unbound HMW HA.

In some embodiments, the method includes fixing HA on a solid surface through an intermediate protein or a connector. In some embodiments, the connector is a peptide connector. In some embodiments, the intermediate protein is an HA-binding protein (HABP). In some embodiments, HABP is first attached to a solid support (e.g., by cross-linking, chemical connection, or by a chemical connector). In some embodiments, a solid support comprising HABP is subsequently contacted with HA (e.g., HMW HA) to fix HA on a solid support by binding HABP to HA. In some embodiments, the solid support is washed to remove unbound HMW HA before immobilized HMW HA is contacted with PTX3. In some embodiments, the solid support is washed with a washing solution of 8M GnHCl and PBS before immobilized HA is contacted with PTX3 to remove unbound HMW HA.

In some embodiments, the method comprises immobilizing HA on a solid surface by attaching a peptide linker to a solid support and attaching HA to the peptide linker. In some embodiments, the peptide linker comprises a protease cleavage site.

In some embodiments, the method comprises immobilizing HA on a solid surface by attaching a cleavable chemical linker, such as, but not limited to, a disulfide chemical linker.

In some embodiments, the HABP selected for use in the method is one that dissociates from HA after the rcHC-HA/PTX3 complex is formed. In some embodiments, the HABP is non-covalently bound to HA. In some embodiments, the method further comprises dissociating the rcHC-HA/PTX3 complex from the HABP using one or more dissociating agents. Dissociating agents for disrupting non-covalent interactions (e.g., guanidine hydrochloride, urea, and various detergents, e.g., SDS) are known in the art. In some embodiments, the dissociating agent is urea. In some embodiments, the dissociating agent is guanidine hydrochloride. In some embodiments, the dissociating agent is about 4M to about 8M guanidine hydrochloride. In some embodiments, the dissociating agent is about 4M, about 5M, about 6M, about 7M, or about 8M guanidine hydrochloride. In some embodiments, the dissociating agent is about 4M to about 8M guanidine hydrochloride in PBS at pH 7.5.

In some embodiments, such a dissociating agent is used to dissociate the rcHC-HA/PTX3 complex from the intermediate HABP. The HABP used in the method is selected so that the binding affinity for HA is strong enough to allow the assembly of the rcHC-HA/PTX3 complex, but can be dissociated from the rcHC-HA/PTX3 complex using a suitable dissociating agent. In some embodiments, the dissociating agent is guanidine hydrochloride.

Exemplary HABPs for use with the methods provided herein include, but are not limited to, HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphocan, TSG-6, CD44, stabilin-1, stabilin-2, or a portion thereof sufficient to bind to HA (e.g., a connecting module thereof). In some embodiments, the HABP comprises a polypeptide having a sequence as set forth in any one of SEQ ID NOs: 54-99, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with a polypeptide having a sequence as set forth in any one of SEQ ID NOs: 54-99. In some embodiments, the HABP is versican. In some embodiments, the HABP is a recombinant protein. In some embodiments, the HABP is a recombinant mammalian protein. In some embodiments, the HABP is a recombinant human protein. In some embodiments, the HABP is a recombinant versican protein or a portion thereof sufficient to bind to HA. In some embodiments, the HABP is a recombinant aggrecan protein or a portion thereof sufficient to bind to HA. In some embodiments, the HABP is a native HABP or a portion thereof sufficient to bind to HA. In some embodiments, the native HABP is isolated from mammalian tissue or cells. In some embodiments, the HABP is isolated from bovine nasal cartilage (e.g., a HABP from Seikagaku containing the HA binding domain of aggrecan and a linker protein).

In some embodiments, the HABP comprises a linking module of HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphocan, TSG-6, CD44, stabilin-1, or stabilin-2. In some embodiments, the HABP comprising the linking module comprises a polypeptide having a sequence as set forth in any of the linking domains of SEQ ID NOs: 54-99, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to a polypeptide having a sequence as set forth in any of the linking domains of SEQ ID NOs: 54-99. In some embodiments, the HABP comprises a linking module of versican. In some embodiments, the HABP comprising a linking module is a recombinant protein. In some embodiments, the HABP comprising a linking module of versican is a recombinant protein.

In some embodiments, the intermediate protein, such as HABP, comprises a protease cleavage sequence that is recognized and hydrolyzed by a site-specific protease (e.g., furin, 3C protease, caspase, matrix metalloproteinase, or TEV protease). In such embodiments, the assembled rcHC-HA/PTX3 complex is released from the solid support by contacting the immobilized complex with a protease that cleaves the specific cleavage sequence.

In some embodiments, the rcHC-HA/PTX3 complex is purified. In some embodiments, the rcHC-HA/PTX3 complex is purified by any suitable method or combination of methods. The embodiments described below are not exclusive and are merely exemplary.

In some embodiments, the rcHC-HA/PTX3 complex is purified by chromatography (e.g., ion exchange chromatography, affinity chromatography, size exclusion chromatography, and hydroxyapatite chromatography), gel filtration, centrifugation (e.g., gradient centrifugation) or differential solubility, ethanol precipitation, or by any other available technique for protein purification.

In some embodiments, the rcHC-HA/PTX3 complex is purified by immunoaffinity chromatography. In some embodiments, antibodies to components of the rcHC-HA/PTX3 complex (e.g., anti-HCl, anti-PTX, anti-one or more SLRPs of the rcHC-HA/PTX3 complex, e.g., anti-urotrypsin, anti-decorin, anti-biglycan, or anti-osteoadherin) are generated and attached to a solid support. In some embodiments, unpurified rcHC-HA/PTX3 complex (i.e., mobile phase) is passed over the support. In some cases, the rcHC-HA/PTX3 complex binds to the antibody. In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is then washed with a solution capable of eluting the rcHC-HA/PTX3 complex from the support (e.g., 1% SDS, 6 M guanidine hydrochloride, or 8 M urea). In some embodiments, the dissociating agent is removed from the dissociated rcHC-HA/PTX3 complex. In some embodiments, the dissociating agent is removed from the dissociated rcHC-HA/PTX3 complex by methods including, but not limited to, ion exchange chromatography, dialysis, gel filtration chromatography, ultrafiltration, or diafiltration.

In some embodiments, the rcHC-HA/PTX3 complex is purified by affinity chromatography. In some embodiments, HABP is used to bind to the rcHC-HA/PTX3 complex to purify the complex and is attached to a fixed support. In some embodiments, unpurified rcHC-HA/PTX3 complex (i.e., mobile phase) is passed through the support. In some cases, the rcHC-HA/PTX3 complex is bound to HABP. In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is then washed with a solution (e.g., a dissociating agent) capable of eluting the rcHC-HA/PTX3 complex from the support. In some embodiments, the dissociating agent is removed from the dissociated rcHC-HA/PTX3 complex by methods including, but not limited to, ion exchange chromatography, dialysis, gel filtration chromatography, ultrafiltration, or diafiltration.

In some embodiments, the rcHC-HA/PTX3 complex is purified by a combination of HABP affinity chromatography and immunoaffinity chromatography using antibodies to one or more components of the rcHC-HA/PTX3 complex.

In some embodiments, one or more components of the rcHC-HA/PTX3 complex disclosed herein comprise an affinity tag (e.g., a fusion protein of PTX3 or HCl with an affinity tag). In some embodiments, exemplary affinity tags introduced into one or more components of the rcHC-HA/PTX3 complex include, but are not limited to, a hemagglutinin tag, a polyhistidine, a myc tag, a FLAG tag, or a glutathione-S-transferase sequence. In some embodiments, the ligand of the affinity tag is attached to a solid support. In some embodiments, unpurified rcHC-HA/PTX3 complex is passed over the support. In some cases, the rcHC-HA/PTX3 complex binds to the ligand. In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is subsequently washed with a solution capable of eluting the rcHC-HA/PTX3 complex disclosed herein from the support. In some embodiments, the eluent is removed from the dissociated rcHC-HA/PTX3 complex by methods including, but not limited to, ion exchange chromatography, dialysis, gel filtration chromatography, ultrafiltration, or diafiltration.

In some embodiments, PTX3, TSG-6 and/or HCl are coupled to a label. A "label" refers to a detectable compound or composition that is directly or indirectly coupled to a polypeptide to produce a labeled polypeptide. In some embodiments, the label is self-detectable (e.g., radioisotope labeling or fluorescent labeling), or in the case of an enzyme label, catalyzes a chemical change in the composition of a detectable substrate compound. Non-limiting examples of labels include fluorescent moieties, dyes, fluorescent tags, green fluorescent protein, or luciferase.

Methods for Assessing the Activity of nHC-HA/PTX3 and rcHC-HA/PTX3 Complexes

The properties of the nHC-HA/PTX3 and rcHC-HA/PTX3 complexes provided herein are evaluated by any suitable method, including in vitro and in vivo methods. Exemplary in vitro methods are provided herein and include, but are not limited to, cell culture methods for evaluating the ability of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes to promote macrophage attachment to immobilized nHC-HA/PTX3 or rcHC-HA/PTX3 complexes, inhibit or reduce macrophage aggregation, promote neutrophil apoptosis, macrophage phagocytosis of apoptotic neutrophils, and M2 polarization of stimulated macrophages. In some embodiments, the macrophages used in the assay are stimulated, for example, by exposure to LPS or IFN-γ. In some embodiments, gene or protein expression in stimulated macrophages is evaluated after contact with the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In such methods for evaluating the activity of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes, appropriate controls are used for comparison. In some embodiments, the control lacks treatment with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex (i.e., a negative control).

In some embodiments, the activity of the rcHC-HA/PTX3 complex is compared to the activity of a native HC-HA/PTX3 complex. In some embodiments, the native HC-HA/PTX3 is isolated from amniotic membrane.

In some embodiments, gene expression in treated macrophages is assessed by PCR, RT-PCR, Northern blotting, Western blotting, dot blotting, immunohistochemistry, chromatography, or other suitable methods for detecting proteins or nucleic acids. In some embodiments, the expression levels of IL-10, IL-12, IL23, LIGHT, and SPHK1 are assessed.

Exemplary in vitro methods for evaluating the activity of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein include, but are not limited to, animal models of various diseases and conditions. A variety of animal models for diseases and conditions are available and well known in the art, including, but not limited to, animal models for various inflammatory and autoimmune diseases and disorders, including, but not limited to, ischemia-reperfusion injury, type 1 and type 2 diabetes, inflammatory diseases, collagen-induced arthritis, rheumatoid arthritis, antigen-induced autoimmune diseases, such as collagen-induced arthritis and myelin peptide-induced experimental allergic encephalomyelitis, inflammatory bowel disease (IBD)/ulcerative colitis, multiple sclerosis, surgery-induced osteoarthritis and nephritis, psoriasis, inflammatory skin diseases, LPS-induced endotoxic shock, LPS-induced lung injury, allergic rhinitis, liver injury, chronic stress, asthma (e.g., rodent and primate models), and xenograft and allograft models for various cancers.

In some embodiments, the animal model is inflammation, such as a rodent model of chronic graft-versus-host disease (cGVHD), HSV1-induced necrotizing stromal keratitis, or high-risk corneal transplantation. In some embodiments, the reduction in inflammation caused by nHC-HA/PTX3 or rcHC-HA/PTX3 treatment is assessed by measuring the proliferation and activation of T cells and the production of immune cytokines such as IL-1α, IL-2, IL-6, IFN-γ, and TNF-α. In some embodiments, the animal model is scarring, such as a rodent model of excimer laser-assisted photorefractive keratectomy (PRK). Exemplary methods for such animal models are provided in the Examples provided herein.

In some embodiments, the animal model is a genetic model of inflammatory and autoimmune diseases and conditions that comprises one or more genetic modifications that cause the disease or condition. In some embodiments, such models are obtained from commercial sources. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex provided herein is administered to an animal model of a particular disease or condition, and the ability of the rcHC-HA/PTX3 complex to inhibit or reduce one or more symptoms of the disease or condition is assessed.

Pharmaceutical composition

In certain embodiments, disclosed herein are pharmaceutical compositions comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein. In certain embodiments, disclosed herein are pharmaceutical compositions comprising nHC-HA/PTX3 or rcHC-HA/PTX3 complexes produced by the methods provided herein. In some embodiments, the pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers (including excipients and adjuvants that facilitate processing of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex into a formulation suitable for pharmaceutical use). Appropriate formulations depend on the chosen route of administration. Any known techniques, carriers, and excipients may be used as suitable techniques, carriers, and excipients, as understood in the art.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein. In some embodiments, the pharmaceutical compositions further comprise at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions further comprise adjuvants, excipients, preservatives, agents for delayed absorption, fillers, binders, adsorbents, buffers, and/or solubilizers. Exemplary pharmaceutical compositions formulated to contain the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein include, but are not limited to, solutions, suspensions, emulsions, syrups, granules, powders, ointments, tablets, capsules, pills, or aerosols.

Dosage form

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as an aqueous suspension. In some embodiments, the aqueous suspension comprises water, Ringer's solution, and/or isotonic sodium chloride solution. In some embodiments, the aqueous suspension comprises a sweetener or flavoring, a coloring matter or dye, and, if desired, an emulsifier or suspending agent, along with the diluents water, ethanol, propylene glycol, glycerol, or a combination thereof. In some embodiments, the aqueous suspension comprises a suspending agent. In some embodiments, the aqueous suspension comprises sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth, and/or gum arabic. In some embodiments, the aqueous suspension comprises a dispersant or wetting agent. In some embodiments, the aqueous suspension comprises a naturally occurring phospholipid (e.g., lecithin), or condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitan monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). In some embodiments, the aqueous suspension comprises a preservative. In some embodiments, the aqueous suspension comprises ethyl parahydroxybenzoate or n-propyl parahydroxybenzoate. In some embodiments, the aqueous suspension comprises a sweetener. In some embodiments, the aqueous suspension comprises sucrose, saccharin, or aspartame.

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as an oily suspension. In some embodiments, the oily suspension is prepared by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or a mineral oil (such as liquid paraffin). In some embodiments, the oily suspension contains a thickener (e.g., beeswax, hard paraffin, or cetyl alcohol). In some embodiments, the oily suspension contains a sweetener (e.g., those listed above). In some embodiments, the oily suspension contains an antioxidant (e.g., butylated hydroxyanisole or α-tocopherol).

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated for parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardial, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural, and/or subcutaneous). In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as a sterile solution, suspension, or emulsion.

In some embodiments, formulations for parenteral administration include aqueous and/or non-aqueous (oily) sterile injection solutions of the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein (which, in some embodiments, contain antioxidants, buffers, bacteriostats, and/or solutes that render the formulation isotonic with the blood of the intended recipient); and/or aqueous and/or non-aqueous sterile suspensions (which, in some embodiments, include suspending agents and/or thickening agents). In some embodiments, formulations for parenteral administration include suitable stabilizers or agents that increase the solubility of the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein to allow for the preparation of highly concentrated solutions.

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as an oil-in-water microemulsion (wherein the active ingredient is dissolved in the oil phase). In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is dissolved in a fatty oil (e.g., sesame oil or a synthetic fatty acid ester (e.g., ethyl oleate or triglycerides)) or liposomes. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is dissolved in a mixture of soybean oil and/or lecithin. In some embodiments, the oil solution is introduced into a mixture of water and glycerol and processed to form a microemulsion.

In some embodiments, a composition formulated for parenteral administration is administered as a single bolus injection.In some embodiments, a composition formulated for parenteral administration is administered via a continuous intravenous delivery device (eg, a Deltec CADD-PLUS ^™ model 5400 intravenous pump).

In some embodiments, the preparations for injection are presented in unit dose form, e.g., in ampoules or in multi-dose containers, with preservatives added. In some embodiments, the preparations for injection are stored in powder form or under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier (e.g., saline or sterile pyrogen-free water) immediately before use.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated for topical administration. Topical formulations include, but are not limited to, ointments, creams, lotions, solutions, pastes, gels, films, sticks, liposomes, and nanoparticles. In some embodiments, the topical formulation is administered using a patch, bandage, or wound dressing.

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are formulated as compositions in the form of solids, cross-linked gels, or liposomes. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are formulated as insoluble cross-linked hydrogels.

In some embodiments, the topical formulation comprises a gelling (or thickening) agent. Suitable gelling agents include, but are not limited to, cellulose, cellulose derivatives, cellulose ethers (e.g., carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, methyl cellulose), guar gum, xanthan gum, locust bean gum, alginates (e.g., alginic acid), silicates, starches, tragacanth gum, carboxyvinyl polymer, carrageenan, paraffin, petrolatum, gum arabic (gum arabic), agar, magnesium aluminum silicate, sodium alginate, sodium stearate, fucus vesiculosus, bentonite, carbomer, carrageenan, carbopol, xanthan gum, cellulose, microcrystalline cellulose (MCC), carob bean gum, chondrus, glucose, furcellaran, gelatin, ghatti gum (gum arabic), cellulose derivatives, cellulose ethers (e.g., carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, methyl cellulose), guar gum, xanthan gum, locust bean gum, locust bean gum, chondrus, glucose, furcellaran, gelatin, ghatti gum (gum arabic), cellulose derivatives, cellulose ethers (e.g., carboxymethyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, methyl cellulose), guar gum, xanthan gum, locust bean gum, chondrus, glucose, furcellaran, gelatin, ghatti gum (gum arabic), cellulose derivatives, cellulose ethers (e.g., carboxymethyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, methyl cellulose), gum), guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, corn starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, polyethylene glycol (e.g., PEG 200-4500), gum tragacanth, ethylcellulose, ethylhydroxyethylcellulose, ethylmethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose, poly(hydroxyethyl methacrylate), oxidized polygelatin, pectin, polygelin, povidone, propylene carbonate, methyl vinyl ether/maleic anhydride copolymer (PVM/MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropylcellulose, hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), or a combination thereof.

In some embodiments, the topical formulations disclosed herein comprise an emollient. Emollients include, but are not limited to, castor oil esters, cocoa butter esters, safflower oil esters, cottonseed oil esters, corn oil esters, olive oil esters, cod liver oil esters, almond oil esters, avocado oil esters, palm oil esters, sesame oil esters, squalene esters, kikui oil esters, soybean oil esters, acetylated monoglycerides, ethoxylated monostearate glyceryl, hexyl laurate, hexyl isolaurate, isohexyl palmitate, isopropyl palmitate, methyl palmitate, decyl oleate, isodecyl oleate, cetyl decyl stearate, isopropyl isostearate, methyl isostearate, diisopropyl adipate, diisohexyl adipate, dihexyldecyl adipate, diisohexyl sebacate Isopropyl betaine, lauryl lactate, myristyl lactate, and cetyl lactate, oleyl myristate, oleyl stearate, and oleyl oleate, pelargonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, hydroxystearic acid, oleic acid, linoleic acid, ricinoleic acid, arachidic acid, behenic acid, erucic acid, lauryl alcohol, myristyl alcohol, cetyl alcohol, cetyl alcohol, stearyl alcohol, isostearyl alcohol, hydroxystearyl alcohol, oleyl alcohol, ricinoleyl alcohol, behenyl alcohol, erucyl alcohol, 2-octyldodecyl alcohol, lanolin and lanolin derivatives, beeswax, spermaceti wax, myristyl myristate, stearyl stearate, carnauba wax, candelilla wax, lecithin, and cholesterol.

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are formulated with one or more natural polymers. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are formulated with natural polymers such as fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are formulated with a polymer gel formulated from natural polymers. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are formulated with a polymer gel formulated from natural polymers such as, but not limited to, fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate, and combinations thereof. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a cross-linked polymer. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a non-cross-linked polymer. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a non-cross-linked polymer and a cross-linked polymer. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a cross-linked hyaluronan gel. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with an insoluble cross-linked HA gel. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a non-cross-linked hyaluronan gel. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a collagen matrix. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a fibrin matrix. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a fibrin/collagen matrix.

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated for administration to the eye or tissue associated therewith. Formulations suitable for administration to the eye include, but are not limited to, solutions, suspensions (e.g., aqueous suspensions), ointments, gels, creams, liposomes, vesicles, pharmacoplasts, nanoparticles, or combinations thereof. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein for topical administration to the eye is administered by spraying, washing, or a combination thereof. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to the eye via an injectable depot formulation.

As used herein, a "depot formulation" is a controlled-release formulation that is implanted into the eye or tissue associated therewith (e.g., sclera), e.g., subcutaneously, intramuscularly, intravitreally, or subconjunctivally. In some embodiments, the depot formulation is formulated by forming a microencapsulation matrix (also referred to as a microcapsule matrix) of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein within a biodegradable polymer. In some embodiments, the depot formulation is formulated by encapsulating the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein in a liposome or microemulsion.

The preparation for application to the eye has an ophthalmologically acceptable osmotic pressure (tonicity). In some cases, tear fluid has an isotonic value equivalent to a 0.9% sodium chloride solution. In some embodiments, an isotonic value of about 0.6% to about 1.8% sodium chloride is suitable for topical application to the eye. In some embodiments, the preparation disclosed herein for application to the eye has an osmotic pressure concentration of about 200 to about 600mOsm/L. In some embodiments, the preparation disclosed herein for application to the eye is hypotonic, therefore it is necessary to add any suitable to reach an appropriate osmotic pressure range. Ophthalmologically acceptable substances for regulating osmotic pressure include but are not limited to sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

The formulation for administration to the eye has an ophthalmologically acceptable clarity. Examples of ophthalmologically acceptable clarifying agents include, but are not limited to, polysorbate 20, polysorbate 80, or a combination thereof.

In some embodiments, the formulation for administration to the eye comprises an ophthalmologically acceptable viscosity enhancer. In some embodiments, the viscosity enhancer increases the time that the formulation disclosed herein remains in the eye. In some embodiments, increasing the time that the formulation disclosed herein remains in the eye allows for better drug absorption and effect. Non-limiting examples of mucoadhesive polymers include carboxymethyl cellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate, and dextran.

In some embodiments, the formulation for administration to the eye is administered or delivered to the posterior segment of the eye (e.g., retina, choroid, vitreous body, and optic nerve). In some embodiments, the topical formulations disclosed herein for administration to the eye, for delivery to the posterior segment of the eye, comprise a solubilizing agent, e.g., dextran sulfate and/or cyclodextrin. Dextran sulfates used in some embodiments include, but are not limited to, dextran sulfate, cyclodextrin sulfate, and β-1,3-glucan sulfate (natural and their derivatives), or any compound that is temporarily bound and retained in the tissue, comprising fibroblast growth factor (FGF), which improves the stability and/or solubility of the drug, and/or improves the penetration and ocular absorption of the topical formulations disclosed herein for administration to the eye. Cyclodextrin derivatives for use as solubilizing agents in some embodiments include, but are not limited to, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyethyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, hydroxypropyl β-cyclodextrin, sulfated α-cyclodextrin, sulfated β-cyclodextrin, and sulfobutyl ether β-cyclodextrin.

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated for rectal or vaginal administration. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as a suppository. In some embodiments, a composition suitable for rectal administration is prepared by mixing the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein with a suitable non-irritating excipient (which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug). In some embodiments, a composition suitable for rectal administration is prepared by mixing the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein with cocoa butter, glycerinated gelatin, hydrogenated vegetable oil, a mixture of polyethylene glycols of various molecular weights, or fatty acid esters of polyethylene glycol.

In certain embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are optionally blended with controlled-release particles, lipoplexes, liposomes, nanoparticles, microspheres, microparticles, nanocapsules, or other agents that enhance or promote topical delivery to the skin. Examples of conventional microencapsulation processes for preparing pharmaceutical formulations are described in U.S. Patent No. 3,737,337, which is incorporated herein by reference.

dose

The amount of the pharmaceutical composition administered depends in part on the individual being treated. Where the pharmaceutical composition is administered to a human subject, the daily dosage is generally determined by the prescribing physician, with dosage generally varying according to age, sex, diet, weight, general health and individual response, severity of individual symptoms, precise disease or condition being treated, severity of the disease or condition being treated, time of administration, route of administration, handling of the composition, rate of excretion, drug combination, and the judgment of the prescribing physician.

In some embodiments, the dosage of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is about 0.001 to about 1000 mg/kg body weight/day. In some embodiments, the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is about 0.5 to about 50 mg/kg/day. In some embodiments, the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is about 0.001 to about 7 g/day. In some embodiments, the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is about 0.01 to about 7 g/day. In some embodiments, the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is about 0.02 to about 5 g/day. In some embodiments, the amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is about 0.05 to about 2.5 g/day. In some embodiments, the amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is about 0.1 to about 1 g/day.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered before, during, or after the onset of a disease or condition. In some embodiments, a combination therapy is administered before, during, or after the onset of a disease or condition. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a combination therapy are administered before, during, or after the onset of a disease or condition. In some embodiments, the timing of administration of a composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 disclosed herein varies. Thus, in some examples, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as a prophylactic and continuously to a subject predisposed to developing a condition or disease to prevent the onset of the disease or condition. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a subject during or as soon as possible after the onset of symptoms. In some embodiments, administration of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is initiated within 48 hours of the onset of symptoms, preferably within 48 hours of the onset of symptoms, more preferably within 6 hours of the onset of symptoms, and most preferably within 3 hours of the onset of symptoms. In some embodiments, the initial administration is by any practical route, such as, for example, intravenous injection, a single bolus injection, an infusion over about 5 minutes to about 5 hours, a pill, a capsule, a transdermal patch, oral delivery, or a combination thereof. The nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is preferably administered as soon as possible after the disease or condition is detected or suspected and for the desired duration of treatment, such as, for example, about 1 month to about 3 months. In some embodiments, the duration of treatment varies for each subject and is determined using known standards. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein or a formulation comprising the complex is administered for at least 2 weeks, preferably about 1 month to about 5 years, and more preferably about 1 month to about 3 years.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as a single dose once daily. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as multiple doses more than once daily. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered twice daily. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered three times daily. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered four times daily. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered more than four times daily.

In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are administered for prophylactic and/or therapeutic treatments. In therapeutic applications, in some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are administered to an individual already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. Amounts effective for this use depend on the severity and course of the disease or condition, previous treatments, the individual's health, weight, and response to medications, and the judgment of the treating physician.

In prophylactic applications, in some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to an individual at risk for a particular disease. Such an amount is defined as a "prophylactically effective amount or dose." In such uses, the precise amount also depends on the individual's health, weight, and other physical parameters of the individual.

In cases where the individual's condition does not improve, at the physician's discretion, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered chronically, i.e., for an extended period of time, including throughout the duration of the individual's life, to ameliorate or otherwise control or limit the symptoms of the individual's disease or condition.

In some embodiments, if the individual's condition does improve, based on the physician's discretion, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is continuously administered or the dose of the administered drug is temporarily reduced or suspended for a certain length of time (i.e., a "drug holiday"). In some embodiments, the length of the drug holiday ranges from 2 days to 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. In some embodiments, the dosage during the drug holiday is reduced by between 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the individual's condition occurs, a maintenance dose is administered as needed. In some embodiments, the dosage or frequency of administration, or both, is subsequently reduced as a function of symptoms to a level at which the improved disease, disorder, or condition is maintained. In some embodiments, upon any recurrence of symptoms, the individual requires intermittent treatment on a long-term basis.

In some embodiments, the pharmaceutical compositions disclosed herein are in unit doses suitable for single administration of precise dosages. In unit dosage forms, the formulation is divided into unit doses containing an appropriate amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein. In some embodiments, the unit dose is in the form of a package containing separate amounts of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. In some embodiments, aqueous suspension compositions are packaged in single-dose non-reclosable containers. In some embodiments, multi-dose reclosable containers are used, in which case it is typical to include a preservative in the composition. In some embodiments, formulations for parenteral injection are in unit dose form, where unit dose forms include but are not limited to ampoules, or in multi-dose containers, with the addition of a preservative.

Suitable daily dosages for the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are, for example, about 0.01 to 2.5 mg/kg per body weight. Indications for daily dosages in larger mammals, including but not limited to humans, range from about 0.5 mg to about 100 mg, conveniently administered in divided doses, including but not limited to up to four times a day or in an extended-release form. Suitable unit dosage forms for oral administration include about 1 to 50 mg of active ingredient. The above ranges are merely suggestive, as the number of variables associated with individual treatment regimens is vast, and considerable deviations from these recommended values are not uncommon. In some embodiments, dosages vary depending on a number of variables, including but not limited to the activity of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex used, the disease or condition being treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the physician's discretion.

In some embodiments, the toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determination of the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (the dose therapeutically effective in 50% of the population). In some embodiments, the dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio between LD ₅₀ and ED _50. nHC-HA/PTX3 or rcHC-HA/PTX3 complexes exhibiting high therapeutic indices are preferred. In some embodiments, data obtained from cell culture assays and animal studies are used to formulate a dosage range for use in humans. The dosage of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein preferably lies within a range of circulating concentrations that include the ED ₅₀ with minimal toxicity. In some embodiments, the dosage varies within this range depending on the dosage form employed and the route of administration utilized.

In some embodiments, a pharmaceutical composition of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is packaged as an article of manufacture comprising packaging material, a pharmaceutical composition effective for preventing and/or treating a disease or condition, and a label indicating that the pharmaceutical composition is used to treat the disease or condition. In some embodiments, the pharmaceutical composition is packaged in a unit dosage form containing an amount of the pharmaceutical composition for a single dose or multiple doses. In some embodiments, the packaged composition comprises a lyophilized powder of the pharmaceutical composition that is reconstituted (e.g., with water or saline) prior to administration.

Medical devices and biomaterial compositions

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is assembled directly on a surface or formulated as a coating for an implantable medical device. Methods for covalently attaching hyaluronan to surfaces (such as, but not limited to, metal, polymer, ceramic, silica, and composite surfaces) are well known in the art and, in some embodiments, are used in conjunction with the methods provided herein for assembling nHC-HA/PTX3 or rcHC-HA/PTX3 complexes on these surfaces (see, e.g., U.S. Patent Nos. 5,356,433, 5,336,518, 4,613,665, 4,810,784, 5,037,677, 8,093,365). In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is assembled directly on the surface of an implantable medical device or portion thereof. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex produced according to the methods provided herein is purified and then directly attached to the surface of an implantable medical device or portion thereof. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex produced according to the methods provided herein is purified and then formulated into a coating for attachment to a medical device or portion thereof. In some embodiments, the coating is applied directly to a surface or to a pretreated or coated surface, wherein the pretreatment or coating is designed to aid adhesion of the coating to the substrate. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex produced according to the methods provided herein is purified and then attached to a medical device or portion thereof that has been coated with a substance capable of promoting attachment of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. For example, in some embodiments, the medical device or portion thereof is coated with an adhesive polymer that provides functional groups of hyaluronan on its surface for covalent attachment of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, a coupling agent, such as, but not limited to, a carbodiimide, is used to attach an nHC-HA/PTX3 or rcHC-HA/PTX3 complex to a polymer coating. In some embodiments, photoimmobilization is used to covalently attach an nHC-HA/PTX3 or rcHC-HA/PTX3 complex produced according to the methods provided herein to a medical device or portion thereof. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex produced according to the methods provided herein is attached to a medical device or portion thereof using a spacer molecule comprising a photochemically or thermochemically reactive group.

In some embodiments, a coating formulation containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied to a substrate by, for example, dip coating. Other methods of application include, but are not limited to, spraying, washing, vapor deposition, brushing, roller coating, curtain coating, spin coating, and other methods known in the art.

Exemplary implantable medical devices include, but are not limited to, artificial joints, orthopedic devices, bone implants, contact lenses, sutures, surgical staples, surgical clips, catheters, angioplasty balloons, sensors, surgical devices, electrodes, needles, syringes, wound drains, shunts, urinary inserts, metal or plastic implants, heart valves, artificial organs, sutured bands, annuloplasty rings, guidewires, Kirschner wires or Denham pins, stents, stent grafts, vascular grafts, pacemakers, pills, wafers, medical catheters, infusion cannulas, implantable defibrillators, neurostimulators, glucose sensors, cerebrospinal fluid shunts, implantable drug pumps, spinal cages, artificial spinal discs, eye implants, cochlear implants, breast implants, nucleus pulposus replacement devices, ear tubes, intraocular lenses, drug delivery systems, microparticles, nanoparticles, and microcapsules.

In specific embodiments, the implantable medical device is an implant or prosthesis comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein. In specific embodiments, the prosthesis is an artificial joint. In some embodiments, the prosthesis is an artificial hip, artificial knee, artificial glenohumeral joint, or artificial ankle.

In a specific embodiment, the implant is a stent. In a specific embodiment, the implant is a coronary stent, a ureteral stent, a urethral stent, a prostate stent, a bone stent, or an esophageal stent. In a specific embodiment, the implant is a bone implant, such as, but not limited to, an osseointegrated implant or a craniofacial prosthesis (e.g., an artificial ear, an orbital prosthesis, a nasal prosthesis).

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is assembled directly on a microparticle or nanoparticle for delivery of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex to a subject (see, e.g., WO 03/015755 and US2004/0241248).

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex provided herein is attached to, assembled on, or provided as a coating on a surface or portion of any such implantable medical device provided herein or known in the art. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex elutes from the coating and enters the surrounding tissue after implantation.

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are directly assembled onto scaffolds, microparticles, microcapsules for delivering biomaterials, or microcarriers, such as stem cells or insulin-producing cells. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are directly attached to or assembled onto microcapsules. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are combined with materials for forming microcapsules, and microcapsules containing the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are produced. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are used to coat the interior surface of a microcapsule. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are used to coat the exterior surface of a microcapsule. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are used to coat both the interior and exterior surfaces of a microcapsule.

Exemplary materials for encapsulating cells include, but are not limited to, thermosetting hydrogels such as agarose, alginate, and artificial polymers such as poly(NiPAAm-co-AAC), poly(ethylene glycol) (PEG), and PEG derivatives such as PEG diacrylate and oligo(poly(ethylene glycol))fumaric acid. In some embodiments, methods for culturing and microencapsulating stem cells are known in the art and are used to produce microcapsules containing nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein.

In some embodiments, the microcapsule comprises cells, multiple cells, or other biomaterials. In some embodiments, the cells or multiple cells are stem cells, such as, but not limited to, mesenchymal stem cells. In some embodiments, the cells or multiple cells are differentiated cells, such as, but not limited to, insulin-producing cells. In some embodiments, the cells or multiple cells are autologous cells (i.e., cells from or derived from the recipient of the cells). In some embodiments, the cells or multiple cells are allogeneic cells (i.e., cells not from or derived from the recipient of the cells). In some embodiments, the microcapsule comprises cells, multiple cells, or other biomaterials and the inner surface of the microcapsule is coated with nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein. In some embodiments, the microcapsule comprises cells, multiple cells, or other biomaterials and the outer surface of the microcapsule is coated with nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein. In some embodiments, the microcapsule comprises cells, multiple cells, or other biomaterials and both the outer and inner surfaces of the microcapsule are coated with nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein. In some embodiments, the microcapsules are administered to treat a disease or condition.Exemplary diseases and conditions and treatments for which microcapsules can be administered are described elsewhere herein and include, but are not limited to, inflammatory and immune-related diseases.

Treatment

In certain embodiments, disclosed herein are methods of treating an individual in need thereof, comprising administering to the individual an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein. In certain embodiments, disclosed herein are methods of treating an individual in need thereof, comprising administering to the individual an nHC-HA/PTX3 or rcHC-HA/PTX3 complex produced by the methods described herein. The following are non-limiting examples of treatment methods comprising administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to inhibit at least one of the following: scarring, inflammation, an immune response leading to autoimmune disease or immune rejection, adhesion, angiogenesis, and a condition requiring cell or tissue regeneration. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to promote wound healing. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are used to promote stem cell expansion. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are used to promote tissue regeneration.

In some embodiments, a method of treating an individual in need thereof comprises administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein to the individual by any suitable method. In some embodiments, a method of treating an individual in need thereof comprises administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein to the individual by any suitable route. The appropriate method for administration will depend on the disease or condition to be treated. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered topically to the treatment site. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered systemically. Exemplary methods of administering the nHC-HA/PTX3 or rcHC-HA/PTX3 complex provided herein include, but are not limited to, parenteral, enteral, subcutaneous, transdermal, transdermal, intradermal, intravenous, topical, inhalation, or implantation.

scarring

Described herein, in certain embodiments, are methods of preventing, reducing, or reversing scarring in a subject in need thereof, comprising administering to the subject a composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein.

As used herein, "scarring" refers to the formation of a scar. In one aspect, the scar is a hypertrophic scar, a keloid scar, or a scar caused by acne. As used herein, a "scar" is an area of fibrous tissue caused by an overproduction of collagen. In certain instances, wound healing involves the migration of fibroblasts to the site of injury. In certain instances, fibroblasts deposit collagen. In certain instances, fibroblasts deposit excess collagen at the site of injury, resulting in a scar.

In some cases, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein prevent or inhibit TGF-β signaling. In some cases, TGF-β regulates the extracellular matrix by stimulating cellulose proliferation and collagen deposition and inhibiting extracellular matrix degradation (by upregulating the synthesis of protease inhibitors). In some cases, preventing or inhibiting the expression of TGF-β leads to preventing a decrease in scar intensity. In some embodiments, administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein prevents or reduces scar formation.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein inhibits or prevents the ability of fibroblasts to differentiate into myofibroblasts. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein reverses the differentiation of myofibroblasts into fibroblasts.

In some embodiments, the methods disclosed herein are used to prevent, reduce, or reverse the formation of scars. In some embodiments, the methods disclosed herein include administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein to an individual suffering from a condition that causes scarring (e.g., dermatitis scars, keloid scars, contracture scars, hypertrophic scars, or scars caused by acne). In some embodiments, the methods disclosed herein include administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein to an individual in need before or after trauma. In some embodiments, the methods disclosed herein include administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein to an individual in need before or after surgery.

In some embodiments, the methods disclosed herein are used to prevent or reduce scarring on the eye or its surrounding tissues. In some embodiments, the methods disclosed herein include administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein to an individual suffering from a condition caused by scarring of the eye or surrounding tissues (e.g., retinopathy of prematurity). In some embodiments, the methods disclosed herein include administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein to an individual in need thereof before or after trauma to the eye or surrounding tissues. In some embodiments, the methods disclosed herein include administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein to an individual in need thereof before or after surgery on the eye or surrounding tissues.

inflammation

In certain embodiments, described herein are methods of preventing or reducing inflammation in a subject in need thereof, the methods comprising administering to the subject a composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein. As used herein, "inflammation" refers to the physiological response resulting from the migration of plasma and/or leukocytes (e.g., lymphocytes, macrophages, granulocytes, and neutrophils) to the site of infection or trauma (e.g., blunt force trauma, penetrating trauma, or surgery).

In some cases, leukocytes secrete cytokines after contact with antigens. As used herein, "cytokine" is a signaling protein or glycoprotein. In some cases, cytokines bind to cell surface receptors. In some cases, cytokines induce chemotaxis of leukocytes to the site of infection. In some cases, cell surface receptors on leukocytes detect the chemical gradient of cytokines. In some cases, leukocytes follow the gradient to the site of infection. In some cases, the combination of cytokines and cell surface receptors causes the increase or decrease of certain genes and their transcription factors. In some cases, the change in gene expression causes the increase of the production of cytokines, the increase of cytokine production or the presence of cell surface receptors.

As non-limiting examples, cytokines include interleukin IL-1, IL-6, IL-8, MCP-1 (also referred to as CCL2) and TNF-α. Interleukin 1 exists in the body in two subtypes: IL-1α and IL-1β. In some cases, the presence of IL-1 increases the expression of adhesion factors on endothelial cells. In turn, this enables leukocytes to migrate to the site of infection. In some cases, IL-8 induces the chemotaxis of leukocytes. In some cases, TNF-α induces the chemotaxis of leukocytes. In some cases, MCP-1 recruits leukocytes to tissue damage and sites of infection.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein inhibits the production and/or activity of cytokines. In certain instances, the reduction in cytokine concentration reduces or prevents inflammation by reducing the number of leukocytes and/or the rate at which leukocytes migrate to the site of injury.

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein induce apoptosis in leukocytes (e.g., macrophages, neutrophils, or lymphocytes). In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein reduce the number of activated leukocytes or the rate at which the leukocytes are activated. In certain instances, the reduction in the concentration of leukocytes reduces or prevents inflammation by reducing the number of cells that migrate to the site of injury (e.g., promoting the death of such cells by apoptosis).

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein inhibits the polarization of macrophages toward an inflammatory phenotype (i.e., M1 type). In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein reduces or inhibits the expression of IL-12 or IL-23 in stimulated macrophages. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein promotes the polarization of stimulated macrophages toward a regulator or wound healing M2 phenotype. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein inhibits or reduces inflammation in a subject. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein inhibits or reduces tissue damage caused by a condition or disease that induces inflammation in a subject.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a subject with inflammation. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a subject with acute inflammation. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a subject with chronic inflammation. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a subject with an inflammatory condition. In some embodiments, the inflammatory condition is a macrophage-mediated inflammatory condition. In some embodiments, the inflammatory condition is a T cell-mediated inflammatory condition. In some embodiments, the inflammatory condition is a Th-17-mediated immune condition.

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a subject suffering from an acute inflammatory response. In some embodiments, the acute inflammatory response is caused by, for example, allergic reaction, sepsis, endotoxic shock, or ischemia, such as, but not limited to, myocardial infarction and stroke. In some embodiments, the acute inflammatory response is caused by bacterial infection, protozoal infection, protozoal infection, viral infection, fungal infection, or a combination thereof. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex inhibits or reduces acute inflammation. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex inhibits or reduces tissue damage caused by acute inflammation. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex inhibits or reduces tissue reperfusion injury caused by ischemia, including myocardial infarction and stroke.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a subject with chronic inflammation associated with activation of lymphocytes through adaptive immunity. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a subject with a Th1 response. In some embodiments, the Th1 response leads to immune rejection of a biological transplant. In some embodiments, the transplant is an allogeneic transplant. In some embodiments, the transplant is an autologous transplant. In some embodiments, the inflammatory condition is graft-versus-host disease or tissue transplant rejection. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex inhibits or reduces chronic inflammation in a subject.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a subject with chronic inflammation associated with a Th17 cell immune response associated with an inflammatory disorder. In some embodiments, the inflammatory disorder is an autoimmune disease or a leukocyte deficiency.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a subject having an inflammatory disorder selected from the group consisting of acute disseminated encephalomyelitis, Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, Chagas disease, chronic obstructive pulmonary disease, celiac disease, dermatomyositis, type 1 diabetes, type 2 diabetes, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, idiopathic thrombocytopenic purpura, interstitial cystitis, and inflammatory bowel disease. , systemic lupus erythematosus (SLE), metabolic syndrome, multiple sclerosis, myasthenia gravis, myocarditis, narcolepsy, obesity, pemphigus vulgaris, pernicious anemia, polymyositis, primary biliary cirrhosis, rheumatoid arthritis, schizophrenia, scleroderma, syndrome, vasculitis, vitiligo, Wegener's granulomatosis, allergic rhinitis, prostate cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, gastric cancer, colorectal cancer, brain cancer, metastatic bone disease, pancreatic cancer, lymphoma, nasal polyps, digestive tract tumors, ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome ( syndrome), infectious enteritis, indeterminate colitis, inflammatory liver disease, ischemia, myocardial infarction, stroke, endotoxic shock, septic shock, rheumatoid spondylitis, ankylosing spondylitis, gouty arthritis, polymyalgia rheumatica, Alzheimer's disease, Parkinson's disease, epilepsy, AIDS dementia, asthma, adult respiratory distress syndrome, bronchitis, cystic fibrosis, acute leukocyte-mediated lung injury, distal proctitis, Wegener's granulomatosis, fibromyalgia, uveitis, conjunctivitis, psoriasis, eczema, dermatitis, smooth muscle proliferative disorders, meningitis, herpes zoster, encephalitis, nephritis, tuberculosis, retinitis, atopic dermatitis, pancreatitis, periodontitis, coagulative necrosis, liquefactive necrosis, fibrinoid necrosis, neointimal hyperplasia, or a combination thereof.

In some embodiments, the inflammatory condition is an inflammatory condition of the eye or surrounding tissues. In some embodiments, the inflammatory condition is conjunctivitis. In some cases, conjunctivitis is caused by exposure to allergens. In some cases, conjunctivitis is caused by bacterial infection. In some embodiments, the inflammatory condition is keratitis. As used herein, "keratitis" is a condition characterized by inflammation of the cornea. In some embodiments, the inflammatory condition is keratoconjunctivitis (i.e., a combination of conjunctivitis and keratitis (i.e., corneal inflammation)). In some embodiments, the inflammatory condition is blepharitis. As used herein, "blepharitis" is an ophthalmic disease characterized by inflammation of the eyelid margin. In some embodiments, the inflammatory condition is blepharoconjunctivitis (i.e., a combination of conjunctivitis and blepharitis (i.e., blepharitis)). In some embodiments, the inflammatory condition is scleritis. As used herein, "scleritis" is a condition characterized by inflammation of the sclera. In some embodiments, the inflammatory condition is episcleritis. As used herein, "episcleritis" is an inflammatory condition of the outer sclera characterized by hyperemia and chemosis. In some embodiments, the inflammatory condition is uveitis. As used herein, "uveitis" is an inflammatory condition characterized by the uvea. In some embodiments, the condition is retinitis. As used herein, "retinitis" is an inflammatory condition of the retina. In some embodiments, the condition is choroiditis. As used herein, "choroiditis" is an inflammatory condition of the uvea, ciliary body, and choroid.

Abnormal angiogenesis

In certain embodiments, disclosed herein are methods for preventing or reducing angiogenesis in a subject in need thereof, the methods comprising administering to the subject a composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein. As used herein, "angiogenesis" refers to the formation of new blood vessels. In certain instances, angiogenesis promotes tumor growth and metastasis. Furthermore, in certain instances, abnormal angiogenesis underlies wet age-related macular degeneration (wARMD) and diabetic proliferative retinopathy. In certain instances, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein prevents or reduces angiogenesis.

In certain instances, ligand binding to VEGF receptor-2 (VEGFR-2) initiates a tyrosine kinase signaling cascade that stimulates the production of factors that variously stimulate vascular permeability (eNOS, producing NO), proliferation/survival (bFGF), migration (ICAM/VCAM/MMP), and ultimately differentiation into mature blood vessels. In certain instances, following binding of VEGFR-2 to its ligand, endothelial cells form tubular structures that resemble capillaries.

As used herein, "wet age-related macular degeneration," "wARMD," or "wet ARMD" refers to an eye disorder characterized by the proliferation of blood vessels originating from the choroid. In certain instances, wet ARMD causes vision loss due to leakage of blood and protein beneath the macula. In certain instances, if left untreated, bleeding, leakage, and scarring of these blood vessels lead to irreversible damage to photoreceptors and rapid vision loss.

As used herein, "diabetic proliferative retinopathy" refers to an eye disorder characterized by insufficiency of blood vessel walls. In certain instances, a lack of oxygen in the retina leads to the growth of blood vessels along the retina and in the vitreous humor. In certain instances, the new blood vessels bleed, obscuring vision and damaging the retina.

In some cases, the proliferation of capillaries provides nutrients to tumors, allowing them to expand. In some cases, the proliferation of capillaries can rapidly remove cellular waste products, allowing tumors to grow. In some cases, angiogenesis promotes metastasis. In some cases, the proliferation of capillaries increases the likelihood that cancer cells will be able to enter the blood vessels and thus establish new tumors at new sites.

In some embodiments, exemplary cancer types treated using the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein include, but are not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial adenoma, Burkitt's Lymphoma, carcinoid tumor, carcinoma, central nervous system lymphoma, cerebellar astrocytoma, Cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, extragonadal germ cell tumor, eye cancer, intraocular melanoma, eye cancer, retinoblastoma, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor (extracranial), germ cell tumor (extragonadal), germ cell tumor (ovarian), gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin’s lymphoma Lymphoma, Hypopharyngeal Cancer, Hypothalamic and Visual Pathway Glioma, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi's Sarcoma, Kidney (Renal Cell) Cancer, Laryngeal Cancer, Leukemia (Acute Lymphocytic), Leukemia (Acute Myeloid), Leukemia (Chronic Lymphocytic), Leukemia (Chronic Myeloid), Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell), Lung Cancer (Small Cell), Lymphoma (Cutaneous T-Cell), Lymphoma (Non-Hodgkin), Malignant Fibrous Histiocytoma/Osteosarcoma of Bone, Medulloblastoma, Melanoma, Markel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary Metastasis Primary), Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndrome, Myelodysplastic/Myeloproliferative Disorders, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low-Grade Potential Tumor, Pancreatic Cancer, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pineoblastoma, and Supratentorial Primary Neuroectodermal Tumor Tumors), pituitary tumors, plasma cell neoplasms/multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (Kaposi's), sarcoma (uterine), Sezary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), skin cancer (Merkel cell), small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, trophoblastic tumor, pregnancy, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic gliomas, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

Wound repair and tissue regeneration

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as a wound covering or to promote wound repair. In some embodiments, the tissue is damaged, impaired, or missing due to injury (e.g., burns; surgical incisions; necrotic areas caused by infection, trauma, or toxins; lacerations). In some embodiments, the tissue is damaged, impaired, or missing due to burns. In some embodiments, the tissue is damaged, impaired, or missing due to trauma (e.g., incisions, lacerations, abrasions). In some embodiments, the tissue is damaged, impaired, or missing due to necrosis. In some embodiments, the tissue is damaged, impaired, or missing due to ulcers.

burn

In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a burn. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a first-degree burn. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a second-degree burn. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a third-degree burn. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate prior to placement on the burn site.

trauma

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a wound (e.g., an incision, laceration, abrasion, ulcer, puncture, infiltration) of the skin. In some embodiments, the wound is an ischemic wound. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied to a substrate prior to placement on the wound. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein treats the wound.

In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to an incision in an organ (e.g., skin, brain, stomach, kidney, liver, intestine, lung, bladder, trachea, esophagus, vagina, ureter, and blood vessel wall). In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a surgical incision. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to the site of a colectomy. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to the site of a gastrectomy. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to the site of a breast surgery (e.g., breast reduction surgery, breast augmentation surgery, and mastectomy). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied to a substrate prior to placement on the wound.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as a covering over an incision in the skin (e.g., an incision in the epidermis, dermis, and/or subcutaneous tissue). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to repair or replenish the skin after hemorrhoid surgery. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate prior to placement on a wound. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein treats a wound.

Necrosis

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as a protective graft on an area of necrotic tissue (e.g., from an infection). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as a protective graft on an area of necrotic skin. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is placed on an area of necrotic tissue. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate prior to placement on the necrotic tissue. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein treats necrotic tissue.

ulcer

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as a protective covering on an ulcer. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein treats an ulcer. In some embodiments, the ulcer is a diabetic ulcer, such as a diabetic foot or leg ulcer. In some embodiments, the ulcer is an ischemic wound. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied to a substrate before being placed on the ulcer. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein treats an ulcer. In some embodiments, the ulcer is an unhealed ulcer. For example, in some embodiments, the unhealed ulcer is a wound or ulcer on the skin that has persisted for about 3-4 weeks without healing.

In some embodiments, the ulcer is a foot ulcer (e.g., a diabetic foot ulcer or an arterial insufficiency ulcer). In some embodiments, treating a foot ulcer comprises: (a) preparing the wound (e.g., cleaning the wound); and (b) placing a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein on the wound. In some embodiments, treating a foot ulcer comprises: (a) preparing the wound (e.g., cleaning the wound); (b) placing a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein on the wound; and (c) covering the pharmaceutical composition with a protective barrier (e.g., a silver cell dressing, metipel, gauze, or bandage). In some embodiments, the pharmaceutical composition containing the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied to a substrate prior to placement on the ulcer. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein treats ulcers.

In some embodiments, the ulcer is a venous stasis (VS) ulcer. In some embodiments, treating a VS ulcer comprises: (a) preparing the wound (e.g., cleaning the wound); and (b) placing a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein on the wound. In some embodiments, treating a VS ulcer comprises: (a) preparing the wound (e.g., cleaning the wound); (b) placing a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein on the wound; and (c) covering the pharmaceutical composition with a protective barrier (e.g., a wound tissue, an antimicrobial dressing, gauze, or a bandage). In some embodiments, the pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied to a substrate prior to placement on the wound. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein treats the ulcer.

In some embodiments, the ulcer is a corneal ulcer (i.e., ulcerative keratitis). In some embodiments, treating a corneal ulcer comprises: (a) preparing the wound (e.g., cleaning the wound); and (b) placing a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein on the wound. In some embodiments, treating a corneal ulcer comprises: (a) preparing the wound (e.g., cleaning the wound); (b) placing a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein on the wound; and (c) covering the pharmaceutical composition with a protective barrier (e.g., a contact lens or a bandage). In some embodiments, the pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied to a substrate prior to placement on the wound. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein treats an ulcer.

Therapeutic cell therapy

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used in combination with a cell therapy. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administered with therapeutic cells. Therapeutic cells include any cells that exhibit therapeutic properties for treating a disease or condition. In some embodiments, the therapeutic cells are recombinant cells that heterologously express one or more therapeutic gene products. In some embodiments, the therapeutic cells are transplanted cells. In some embodiments, the therapeutic cells are stem cells. In some embodiments, the therapeutic cells are cells that express one or more stem cell markers (e.g., Oct-3/4 (Pou5f1), Sox2, c-Myc, and Klf4).

In some embodiments, the cell therapy is stem cell transplantation. In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are administered to promote the expansion of stem cells for transplantation and tissue regeneration. In some examples, nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are used to reduce or inhibit inflammation, scarring, and abnormal angiogenesis caused by stem cell transplantation. In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are used to maintain the characteristics of stem cells during in vitro expansion by replacing feeder layers. In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are used to reprogram differentiated cells into stem cells. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are used to expand and culture stem cells in vitro for subsequent transplantation into a subject.

In some embodiments, the stem cell therapy is embryonic stem cell therapy. In some embodiments, the stem cell therapy is adult stem cell therapy. In some embodiments, the stem cell therapy is mesenchymal stem cell therapy. In some embodiments, the stem cell therapy is administered to treat a disease or condition such as, but not limited to, cardiovascular disease, cancer, diabetes, spinal cord injury, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, Duchenne muscular dystrophy, muscle injury or malnutrition, stroke, burns, lung disease, retinopathy, kidney disease, osteoarthritis, and rheumatoid arthritis.

In some embodiments, stem cells are used to treat diabetes. Type 1 diabetes is caused by autoimmune-mediated destruction of insulin-secreting β cells in the islets of Langerhans of the pancreas. Type 2 diabetes is caused by systemic insulin resistance and reduced insulin secretion of pancreatic β cells. It has been demonstrated in vitro that stem cells differentiate into insulin-producing cells (see, e.g., Schuldiner et al. (2000) Proc. Natl. Acad. Sci. USA. 97: 11307–11312; Guo et al. (2009) Endocr Rev 30: 214-227). Therefore, in some embodiments, stem cells, including ESCs and ASCs and derivatives thereof, such as partially differentiated stem cells, are used for regeneration of pancreatic β cells in stem cell therapy.

In some embodiments, stem cells or differentiated cells for treatment are encapsulated in a microcapsule device. In some embodiments, the microcapsules contain nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are covalently attached to the microcapsules. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are assembled on the surface of the microcapsules, for example, on the inner surface or the outer surface or both. In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 are formulated as coated microcapsules. In some embodiments, the microcapsules contain pores to allow nutrients to enter the microcapsules through the cells and/or to allow proteins and molecules (e.g., insulin) secreted by the encapsulated cells to flow out of the microcapsules. In some embodiments, the cells are first immobilized on a microcarrier (e.g., with coated beads) and then encapsulated within the microcapsules. Methods for encapsulating cells, such as stem cells, are known in the art and described in, for example, Serra et al., (2011) PLoS ONE 6(8):e23212. In some embodiments, any method of encapsulating cells is used in combination with the methods provided herein.

In some embodiments, allogeneic therapeutic stem cells (e.g., insulin-producing islet cells) are encapsulated in a microcapsule device for the production of insulin. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex promotes the expansion of stem cells. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex inhibits or reduces the inflammatory response to microcapsules containing stem cells for the treatment of diabetes. In some embodiments, the microcapsules contain nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex inhibits or reduces the inflammatory response to microcapsules containing stem cells.

Soft tissue applications

In certain embodiments, disclosed herein are nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein for use in repairing, reconstructing, replacing, or supplementing damaged, injured, or missing soft tissue (e.g., tendon) of a recipient. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered directly to a tissue. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered in combination with a cell- or tissue-based therapy. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is mixed with a single cell, multiple cells, or tissue and administered as part of a tissue-based therapy. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to coat a single cell, multiple cells, or tissue and administered as part of a tissue-based therapy.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as a covering over an incision in soft tissue (e.g., the eyelid forms a tissue plane between different soft tissue layers). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied to a substrate and then used as a covering over an incision in soft tissue (e.g., the eyelid forms a tissue plane between different soft tissue layers).

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as a structural (tectonic) support for soft tissue. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein prevents adhesions in joint or tendon repair.

In some embodiments, pharmaceutical compositions containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein are used to repair tendons or joints (e.g., rotator cuff repair, hand tendon repair). In some embodiments, pharmaceutical compositions containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein are used to strengthen tendons or joints. In some embodiments, pharmaceutical compositions containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein are used to prevent adhesion of a healing tendon to surrounding tissues, tendons, or joints. In some embodiments, pharmaceutical compositions containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein are used to prevent the formation of scar tissue on a tendon.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to augment the smaller tendons and ligaments of the foot and ankle, including the posterior tibial tendon, peroneal tendon, flexor tendon, and extensor tendon, as well as the ligaments of the lateral malleolus complex. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to strengthen the primary repair of the quadriceps and patellar tendons around the knee. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a periosteal patch for bone graft in joint replacement. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 is used to augment insufficient capsular tissue of the hip and knee joints following total joint revision surgery.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to repair a torn rotator cuff. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a patch on a rotator cuff muscle or tendon (e.g., supraspinatus tendon). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to reconstruct a rotator cuff muscle or tendon (e.g., supraspinatus tendon). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to augment a rotator cuff muscle or tendon (e.g., the supraspinatus tendon). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to strengthen a rotator cuff muscle or tendon (e.g., the supraspinatus tendon). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to prevent soft tissue adhesions to a rotator cuff muscle or tendon (e.g., the supraspinatus tendon).

In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to repair gums. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to repair gum recession. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate and used as a patch on the gums. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate and used as a patch on an exposed root surface. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to reconstruct gums. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to augment gums. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to strengthen the gums. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to prevent adhesion of soft tissue to the gums.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a protective graft for an incision or tear in the fascia. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural (tectonic) support for the fascia. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a replacement or supplement for the fascia. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to repair a hernia (e.g., for repairing fascia). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to repair an inguinal hernia. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to repair a femoral hernia. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to repair an umbilical hernia. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to repair an incisional hernia. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to repair a diaphragmatic hernia. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to repair a Cooper's hernia, epigastric hernia, hiatal hernia, Littre hernia, lumbar hernia, Maydl hernia, obturator hernia, pantaloon hernia, paraesophageal hernia, paraumbilical hernia, perineal hernia, propertoneal hernia, Richter's hernia, sliding hernia, ischial hernia, semilunar hernia, sports hernia, Velpeau hernia, or Amyand's hernia.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to repair a herniated intervertebral disc. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a protective graft over an incision or tear in an intervertebral disc. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a protective graft over an incision or tear in an annulus fibrosus. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural (tectonic) support for an intervertebral disc. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural (tectonic) support for the annulus fibrosus. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a replacement or supplement for an intervertebral disc. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural (tectonic) support for an intervertebral disc. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a replacement or supplement for the annulus fibrosus.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used over an incision in the brain or one (or all) of the meninges (i.e., dura mater, pia mater, and/or arachnoid mater). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural (tectonic) support for one (or all) of the meninges (i.e., dura mater, pia mater, and/or arachnoid mater). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a replacement for one (or all) of the meninges (i.e., dura mater, pia mater, and/or arachnoid mater).

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used on an incision in the lung or pleura. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural (or constructive) support for the pleura. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a replacement for the pleura.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used over an incision in the tympanic membrane. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural (tectonic) support for the tympanic membrane. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a replacement for the tympanic membrane.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a protective graft over an incision in the heart or pericardium. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural (constructive) support for the pericardium. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a replacement for the pericardium.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a protective graft over an incision in the peritoneum. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural (tectonic) support for the peritoneum. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a replacement for the peritoneum.

Ophthalmic applications

Disclosed herein, in certain embodiments, are uses of pharmaceutical compositions containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein for repairing, reconstructing, replacing, or supplementing damaged, injured, or missing ocular tissue of a recipient.

Treatment of glaucoma

As used herein, "glaucoma" refers to a condition characterized by loss of retinal ganglion cells in the optic nerve. In certain instances, glaucoma is caused in part or in whole by an increase in intraocular pressure in the anterior chamber (AC). Intraocular pressure varies depending on the production of aqueous humor, a liquid by the ciliary processes of the eye, and its drainage through the trabecular meshwork.

A glaucoma drainage device (GDD) is a medical device implanted in the eye to relieve intraocular pressure by providing a bypass pathway for the drainage of aqueous humor. GDD tubes, if left uncovered, will corrode and make the eye susceptible to intraocular infection. Therefore, GDD tubes need to be covered. Currently, the patches used to cover GDD tubes are made of pericardium, sclera, and cornea. These patches are approximately 400-550 microns thick. These thin patches result in 25% melting over a two-year period, potentially exposing the shunt tube to new light.

In some embodiments, a pharmaceutical composition disclosed herein containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to cover a GDD tube. In some embodiments, the substrate/nHC-HA/PTX3 or rcHC-HA/PTX3 complex is 300-600 microns thick. In some embodiments, the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex does not melt 25% in 2 years.

Treatment of eye ulcers

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to cover persistent epithelial defects and/or ulcers of the eye.

In some embodiments, a surgical sponge is used to debride the substrate of the ulcer and remove the poorly adherent epithelium adjacent to the edge of the ulcer (e.g., to obtain a portion of the eye where epithelial adhesion becomes significant). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to the substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is transferred to the recipient eye. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to the substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is then secured to the eye with a suture (e.g., an interrupted 10-0 nylon suture or a continuous 10-0 nylon suture) and the suture knot is buried. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is secured to the eye using fibrin glue. In some embodiments, a protective layer is applied over the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex or the entire eye (e.g., a contact lens). In some embodiments, the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex further comprises an antibiotic (e.g., neomycin, polymyxin B sulfate, and dexamethasone).

Reconstruction of the conjunctiva, sclera, eyelid, and orbital rim surfaces

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used to reconstruct the conjunctival, scleral, eyelid, and orbital rim surfaces. In some embodiments, the damage to the conjunctival surface is caused by symblepharon cell lysis; surgical removal of tumors, lesions, and/or scar tissue; excimer laser photorefractive keratectomy and therapeutic keratectomy, or a combination thereof.

Coronary artery applications

Disclosed herein, in certain embodiments, are uses of pharmaceutical compositions containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein for repairing, reconstructing, replacing, or supplementing damaged, injured, or missing coronary artery tissue of a recipient.

Prevention of ischemia-reperfusion injury

Disclosed herein are uses of pharmaceutical compositions containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein for inhibiting or reducing tissue damage caused by acute inflammation resulting from ischemia (e.g., myocardial infarction or stroke). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a subject suffering from an ischemic condition (e.g., but not limited to, myocardial infarction or stroke).

Coronary artery bypass surgery

Disclosed herein are uses of pharmaceutical compositions containing nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein for coronary artery bypass surgery. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is transplanted onto a coronary artery to bypass a portion of the artery characterized by atherosclerosis.

heart valves

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is applied to a heart valve. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural support for a heart valve. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a replacement for a heart valve.

Veins and arteries

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is applied to a vein or artery. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural (tectonic) support for a vein or artery.

Neural Applications

Disclosed herein, in certain embodiments, are uses of pharmaceutical compositions containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein for repairing, reconstructing, replacing, or supplementing damaged, injured, or missing neural tissue of a recipient.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a covering on a nerve (e.g., a peripheral nerve). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a covering on a nerve graft, nerve transfer, or repaired nerve. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a covering on an incision in a nerve (e.g., a peripheral nerve). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural (tectonic) support for a nerve (e.g., a peripheral nerve). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein prevents adhesions in nerve repair.

In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a non-constricting encasement for damaged nerves. In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein prevents or minimizes scar formation, encapsulation of nerves, chronic compression, constriction, and nerve entrapment. In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein prevents or minimizes the formation of neuromas. In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein prevents or minimizes the migration of endogenous growth factors (nerve growth factors) that occur during nerve repair.

Spinal applications

Disclosed herein, in certain embodiments, are uses of pharmaceutical compositions containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein in spinal surgery.

In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is used during laminectomy. In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is used to reduce or prevent epidural fibrosis and/or scar adhesions after spinal surgery (e.g., laminectomy). In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is implanted between the dura mater and the overlying tissue after spinal surgery (e.g., laminectomy). In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is implanted between the dura mater and the overlying tissue after spinal surgery (e.g., laminectomy) to reduce or prevent fibroblast migration to the dura mater and collagen deposition on the dura mater.

In some embodiments, pharmaceutical compositions containing nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein are used to reduce or prevent the growth of hypertrophic scars following spinal surgery (e.g., laminectomy). In some embodiments, pharmaceutical compositions containing nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein are used to reduce or prevent the growth of epidural/epidural/perineural scars following surgery (e.g., laminectomy). In some embodiments, pharmaceutical compositions containing nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein are used to reduce or prevent the growth of hypertrophic scars following spinal surgery (e.g., laminectomy). In some embodiments, pharmaceutical compositions containing nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are used to reduce or prevent the growth of membranes following laminectomy.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is used to reduce or prevent the development of epidural compression or dural tethering following spinal surgery (e.g., laminectomy). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is used to reduce or prevent the growth of tethered nerve roots following spinal surgery (e.g., laminectomy). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is used to reduce or prevent the development of arachnoiditis following spinal surgery (e.g., laminectomy).

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein further includes morcellated bone tissue. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and morcellated bone tissue is used in a spinal fusion procedure. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and morcellated bone tissue is implanted between adjacent vertebrae. In some embodiments, implanting a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and morcellated bone tissue between two adjacent vertebrae promotes fusion of the vertebrae.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as a protective graft over an incision in the dura mater. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a structural support for the dura mater. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate, and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as a replacement for the dura mater.

Multifaceted applications of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a patch or wound dressing.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as a dermal filler. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is injected into subcutaneous facial tissue. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is injected beneath facial wrinkles and aging lines (e.g., nasolabial furrows, nasolabial furrows, crow's feet, and forehead lines). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used for lip augmentation. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is injected into the lips.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to treat arthritis (e.g., osteoarthritis, rheumatoid arthritis, septic arthritis, ankylosing spondylitis, spondylosis). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is injected into a joint (e.g., knee) affected by arthritis.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to treat arthritis of the foot. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to treat arthritis of the first metatarsophalangeal (MTP) joint (e.g., hallux rigidus). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to the MTP joint following a dorsal lip resection procedure. In some embodiments, administration of a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein reduces one or more adverse symptoms associated with hallux rigidus or dorsal lip resection procedures (e.g., scarring, joint stiffness, swelling, inflammation, and pain).

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to treat one or more symptoms associated with bone spurs (e.g., scarring, joint stiffness, swelling, inflammation, and pain).

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to inhibit bone resorption in an individual in need thereof. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease, or a bone tumor. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is injected into a joint. In some embodiments, the pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is contacted with a bone (e.g., by using a wound dressing or bandage). In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex coats a bone scaffold, bone implant, or bone prosthesis (e.g., an osseointegrated implant). As used herein, an "osseointegrated implant" means a three-dimensional implant that contains pores into which osteoblasts and supporting connective tissue migrate. In some embodiments, the bone scaffold is inserted into the intramedullary canal of a bone. In some embodiments, the bone scaffold is placed in the sinus tarsi. In some embodiments, the bone scaffold is placed in the knee or joint. In some embodiments, the bone scaffold is placed at a fracture. In some embodiments, the bone scaffold is expandable or retractable.

In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to promote or induce bone formation in an individual in need thereof. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease, or a bone tumor. In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is injected into a joint. In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is brought into contact with the bone (e.g., by using a wound dressing or bandage). In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is covered with a bone scaffold, bone implant, or bone prosthesis (e.g., an osseointegrated implant). As used herein, an "osseointegrated implant" means a three-dimensional implant comprising holes for osteoblasts and supporting connective tissue to migrate into. In some embodiments, the bone scaffold is inserted into the intramedullary canal of the bone. In some embodiments, the bone scaffold is placed in the sinus tarsi. In some embodiments, the bone scaffold is placed in the knee or joint. In some embodiments, the bone scaffold is placed at a fracture. In some embodiments, the bone scaffold is expandable or retractable.

In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to inhibit osteoclast differentiation. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease, or a bone tumor. In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is injected into a joint. In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is contacted with the bone (e.g., by using a wound dressing or bandage). In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is covered with a bone scaffold, bone implant, or bone prosthesis (e.g., an osseointegrated implant). As used herein, an "osseointegrated implant" means a three-dimensional implant comprising pores into which osteoblasts and supporting connective tissue migrate. In some embodiments, the bone scaffold is inserted into the intramedullary canal of the bone. In some embodiments, the bone scaffold is placed in the tarsal sinus. In some embodiments, the bone scaffold is placed in the knee or joint. In some embodiments, the bone scaffold is placed at a fracture. In some embodiments, the bone scaffold is expandable or retractable.

In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to promote mineralization by osteoblasts in an individual in need thereof. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease, or a bone tumor. In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is injected into a joint. In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is brought into contact with the bone (e.g., by using a wound dressing or bandage). In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is covered with a bone scaffold, bone implant, or bone prosthesis (e.g., an osseointegrated implant). As used herein, an "osseointegrated implant" means a three-dimensional implant comprising holes into which osteoblasts and supporting connective tissue migrate. In some embodiments, the bone scaffold is inserted into the intramedullary canal of the bone. In some embodiments, the bone scaffold is placed in the sinus tarsi. In some embodiments, the bone scaffold is placed in the knee or joint. In some embodiments, the bone scaffold is placed at a fracture. In some embodiments, the bone scaffold is expandable or retractable.

In some embodiments, a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to balance bone resorption and bone formation in an individual in need thereof. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease, or a bone tumor. In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is injected into a joint. In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is contacted with the bone (e.g., by using a wound dressing or bandage). In some embodiments, the pharmaceutical composition comprising the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is covered with a bone scaffold, bone implant, or bone prosthesis (e.g., an osseointegrated implant). As used herein, an "osseointegrated implant" means a three-dimensional implant comprising pores into which osteoblasts and supporting connective tissue migrate. In some embodiments, the bone scaffold is inserted into the intramedullary canal of the bone. In some embodiments, the bone scaffold is placed in the sinus tarsi. In some embodiments, the bone scaffold is placed in the knee or joint. In some embodiments, the bone scaffold is placed at a fracture. In some embodiments, the bone scaffold is expandable or retractable.

In some embodiments, pharmaceutical compositions containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein are used to treat orthodontic or periodontal conditions. In some embodiments, the periodontal condition is selected from gingivitis, gum recession, or periodontitis. In some embodiments, pharmaceutical compositions containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein are used as anti-inflammatory agents or for promoting osseointegration or healing. In some embodiments, pharmaceutical compositions containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein are used in conjunction with dental implants to promote osseointegration, anti-inflammation, and healing of the implant.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to treat hoarseness or voice disorders. In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used for injection laryngoplasty to repair the vocal cords.

In some embodiments, a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to a medical implant (e.g., a stent). In some embodiments, a medical implant/nHC-HA/PTX3 or implant/rcHC-HA/PTX3 complex disclosed herein is implanted into an individual in need thereof, wherein the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is partially or completely released to the individual. In some embodiments, the medical implant is a stent (e.g., a bone stent or a coronary stent). In some embodiments, the medical implant is a bone stent. In some embodiments, the medical implant is a coronary stent.

combination

In some embodiments, the compositions and methods described herein are used in combination with a second therapeutic agent. In some embodiments, the compositions and methods described herein are used in combination with two or more therapeutic agents. In some embodiments, the compositions and methods described herein are used in combination with one or more therapeutic agents. In some embodiments, the compositions and methods described herein are used in combination with 2, 3, 4, 5, 6, 7, 8, 9, 10 or more therapeutic agents.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second therapeutic agent are administered in the same dosage form. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second therapeutic agent are administered in separate dosage forms.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered concurrently (e.g., simultaneously, substantially simultaneously, or within the same treatment regimen) with a second therapeutic agent.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second therapeutic agent are administered sequentially. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered before or after the second therapeutic agent. In some embodiments, the time period between administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second active agent ranges from a few minutes to several hours, depending on the characteristics of each agent, such as potency, solubility, bioavailability, plasma half-life, and pharmacokinetic profile. In some embodiments, diurnal variations in target molecule concentrations determine the optimal dosing interval. In some embodiments, the time between administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second active agent is about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, or more.

In some embodiments, co-administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein results in a lower required dose of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex than when the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered alone. In some embodiments, co-administration of a second therapeutic agent results in a lower required dose of the second dose than when the second dose is administered alone. Methods for experimentally determining therapeutically effective doses of drugs and other agents used in combination therapy regimens are known and described in the art. For example, the use of metronomic dosing, i.e., providing more frequent, lower doses to minimize toxic side effects, has been widely described in the art. Combination therapy further includes periodic treatments that are started and stopped at different times to assist in the clinical management of the individual.

In some embodiments, combination therapies of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex and one or more additional therapeutic agents are improved. In some embodiments, the combination therapies are improved such that the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is increased relative to the amount of the second therapeutic agent. In some embodiments, the combination therapies are improved such that the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is decreased relative to the amount of the second therapeutic agent. In some embodiments, the combination therapies are improved such that the amount of the second therapeutic agent is increased relative to the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, the combination therapies are improved such that the amount of the second therapeutic agent is decreased relative to the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex.

In some embodiments, the second therapeutic agent is selected from cytotoxic agents, antimicrobial agents, anti-angiogenic agents, chemotherapeutic agents, anticancer agents or radiation therapy. In some embodiments, the second therapeutic agent is selected from alkylating agents, antimetabolites, teniposide; anti-tumor enzymes, topoisomerase inhibitors, procarbazine, mitoxantrone, platinum coordination complexes, biological response modifiers and growth inhibitors, hormone/anti-hormonal therapeutic agents, hematopoietic growth factors, aromatase inhibitors, antiestrogens, antiandrogens, corticosteroids, gonadorelin agonists, microtubule active agents, nitrosoureas, lipid or protein kinase targeting agents, immunomodulatory drugs (IMiD), protein or lipid phosphatase targeting agents, anti-angiogenic agents, Akt inhibitors, IGF-I inhibitors, FGF3 regulators, mTOR inhibitors, Smac mimetics, HDAC inhibitors, agents that induce cell differentiation , bradykinin 1 receptor antagonists, angiotensin II antagonists, cyclooxygenase inhibitors, heparanase inhibitors, lymphokine inhibitors, cytokine inhibitors, IKK inhibitors, P38MAPK inhibitors, HSP90 inhibitors, multikinase inhibitors, bisphosphonates, rapamycin derivatives, anti-apoptotic pathway inhibitors, apoptotic pathway agonists, PPAR agonists, RAR agonists, Ras subtype inhibitors, telomerase inhibitors, protease inhibitors, metalloproteinase inhibitors, aminopeptidase inhibitors, SHIP activator-AQX-MN100, Humax-CD20 (ofatumumab), CD20 antagonists, IL2-diphtheria toxin fusions, or combinations thereof. In some embodiments, the antibacterial agent is an antiviral, antibacterial, or antifungal agent. Non-limiting exemplary antibacterial agents include those classified as aminoglycosides, β-lactams, quinolones or fluoroquinolones, macrolides, sulfonamides, sulfamethoxazole, tetracyclines, streptomycins, oxazolidinones (such as linezolid), clindamycin, lincomycin, rifamycins, glycopeptides, polymyxins, lipopeptide antibiotics, and pharmaceutically acceptable sodium salts, pharmaceutically acceptable calcium salts, pharmacologically acceptable potassium salts, lipid formulations, derivatives and/or analogs of the foregoing. Non-limiting exemplary classes of antifungal agents include: imidazoles or triazoles, such as clotrimazole, miconazole, ketoconazole, econazole, butoconazole, omoconazole, oxiconazole, terconazole, itraconazole, fluconazole, voriconazole (UK 109,496), posaconazole, ravuconazole or flutrimazole; polyene antifungals, such as amphotericin B, liposomal amphotericin B, natamycin, nystatin and lipid formulations of nystatin; cell wall-active cyclic lipopeptide antifungals, which include the echinocandins, such as caspofungin, micafungin, androfungin, cilofungin; LY121019; LY303366; the allylamine group of antifungals, such as terbinafine. Other non-limiting examples of antifungal agents include naftifine, tolnaftate, midibacterium, candidiasis, anti-trichomoniasis, hamycin, aureomycin, ascomycin, anti-zymosan, anti-mycotic, anti-trichomoniasis, zymosan, heptamycin, anidomycin, griseofulvin, BF-796, MTCH 24, BTG-137586, pramixin (MNS 18184), benamicin; amphotericin B liposomal injection; nikkomycin Z; flucytosine or fungal mycin. Non-limiting examples of antiviral agents include cidofovir, amantadine, rimantadine, acyclovir, ganciclovir, penciclovir, famciclovir, foscarnet, ribavirin or valacyclovir. In some embodiments, the antibacterial agent is an innate immune peptide or protein. Some exemplary classes of innate peptides or proteins are transferrin, lactoferrin, defensins, phospholipases, lysozymes, endogenous antimicrobial peptides, serprocidins, bactericidal permeability increasing proteins, amphipathic alpha helical peptides, and other synthetic antimicrobial proteins. In some embodiments, the antimicrobial agent is a preservative.

In some embodiments, the second therapeutic agent is selected from ARRY-797, dacarbazine (DTIC), actinomycin C2, C3, D, and F1, cyclophosphamide, melphalan, estramustine, maytansinol, rifamycin, streptothricin, doxorubicin, daunorubicin, epirubicin, idarubicin, detoximum rubicin, carminomycin, esorubicin, mitoxantrone, bleomycin A, A2, and B, camptothecin, irinotecan, topotecan, 9-aminocamptothecin, 10,11-methylenedioxycamptothecin, 9-nitrocamptothecin, bortezomib, temozolomide, TAS103, NPI0052, combretastatin, combretastatin A-2, combretastatin A-4, calicheamicin, neocarzinostatin, epothilone A B, C and semisynthetic variants, Herceptin, Rituximab, CD40 antibody, asparaginase, interleukins, interferons, leuprorelin, and pegaspargase, 5-fluorouracil, fluorodeoxyuridine, furofuridine, 5'-deoxyfluorouridine, UFT, MITC, S-1 capecitabine, diethylstilbestrol, tamoxifen, toremefine, tolmudex, nolatrexed hydrochloride, flutamide, fluoxymesterone, Bicalutamide, finasteride, estradiol, troloxifene, dexamethasone, leuprolide acetate, estramustine, droloxifene, medroxyprogesterone acetate, megestrol acetate, aminoglutethimide, testolactone, testosterone, diethylstilbestrol, hydroxyprogesterone, mitomycins A, B, and C, methyl mitomycin, cisplatin, carboplatin, oxaliplatin, tetraplatin, platinum-DACH, ormaplatin, thalidomide, lenalidomide, CI-973, telomerase, CHIR258, Rad 001, SAHA, Tubacin, 17-AAG, sorafenib, JM-216, podophyllotoxin, epipodophyllotoxin, etoposide, teniposide, Tarceva, Iressa, imatinib, miltefosine, perifosine, aminopterin, methotrexate, methotrexate, dichloro-methotrexate, 6-mercaptopurine, thioguanine, azattuoprine, allopurinol, cladribine, fludarabine, pentostatin, 2-chloroadenosine, deoxycytidine, cytosine arabinoside, cytarabine, azacitidine, 5-azacytosine, gemcitabine, 5-azacytosine-arabinoside, vincristine, vinblastine, vinorelbine, vinblastine, isovinblastine, and vindesine, paclitaxel, taxotere, and/or docetaxel.

In some embodiments, the second active agent is niacin, a fibrate, a statin, an Apo-A1 mimetic polypeptide (e.g., DF-4, Novartis), an apoA-I transcriptional upregulator, an ACAT inhibitor, a CETP modulator, a glycoprotein (GP) IIb/IIIa receptor antagonist, a P2Y12 receptor antagonist, an Lp-PLA2-inhibitor, an anti-tumor necrosis factor (TNF) agent, an interleukin-1 (IL-1) receptor antagonist, an interleukin-2 (IL-2) receptor antagonist, an interleukin-6 ( IL-6) receptor antagonist, interleukin-12 (IL-12) receptor antagonist, interleukin-17 (IL-17) receptor antagonist, interleukin-23 (IL-23) receptor antagonist, cytotoxic agent, antibacterial agent, immunomodulator, antibiotic, T cell co-stimulation blocker, disease-modifying antirheumatic agent, B cell depleting agent, immunosuppressant, antilymphocyte antibody, alkylating agent, antimetabolite, plant alkaloid, terpenoid, topoisomerase inhibitor, antitumor antibiotic, monoclonal antibody, hormonal therapy (e.g., aromatase inhibitor), or a combination thereof.

In some embodiments, the second active agent is an anti-TGF-β antibody, an anti-TGF-β receptor blocking antibody, an anti-TNF antibody, an anti-TNF receptor blocking antibody, an anti-IL1β antibody, an anti-IL1β receptor blocking antibody, an anti-IL-2 antibody, an anti-IL-2 receptor blocking antibody, an anti-IL-6 antibody, an anti-IL-6 receptor blocking antibody, an anti-IL-12 antibody, an anti-IL-12 receptor blocking antibody, an anti-IL-17 antibody, an anti-IL-17 receptor blocking antibody, an anti-IL-23 antibody, or an anti-IL-23 receptor blocking antibody.

In some embodiments, the second active agent is niacin, bezafibrate, ciprofibrate, clofibrate, gemfibrozil, fenofibrate, DF4 (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2), DF5, RVX-208 (Resverlogix), avasimib, pactimib sulfate (CS-505), CI-1011 (2,6-diisopropyl[(2,4,6-triisopropyl)acetyl]sulfamate), CI-976 (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecaneamide), VULM1457 (1-(2,6-diisopropylphenyl)-3-[4-(4'-nitrophenyl)phenyl]sulfamate), ]urea), CI-976 (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl) dodecaneamide), E-5324 (n-butyl-N'-(2-(3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy)-6-methylphenyl)urea), HL-004 (N-(2,6-diisopropylphenyl)tetradecylthioacetamide), KY-455 (N-(4,6-dimethyl-1-pentindolyl-7-yl)-2,2-dimethylpropionamide), FY-087 (N-[2-[N'-pentyl-(6,6-dimethyl-2,4-heptadiynyl)amino]ethyl]-(2-methyl-1-naphthyl-thio)acetamide), MCC-147 (Mitsubishi Pharma), F12511 ((S)-2',3',5'-trimethyl-4'-hydroxy-α-dodecylthioacetanilide), SMP-500 (Sumitomo Pharmaceuticals), CL 277082 (2,4-difluoro-phenyl-N[[4-(2,2-dimethylpropyl)phenyl]methyl]-N-(heptyl)urea), F-1394 ((1s,2s)-2-[3-(2,2-dimethylpropyl)-3-nonylurea]aminocyclohexan-1-yl 3-[N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propanoate), CP-113818 (N-(2,4-bis(methylthio)-6-methylpyridin-3-yl)-2-(hexylthio)decanoic acid amide), YM-750, torcetrap, anacetrapid, JTT-705 (Japan Tobacco/Roche), abciximab, eptifibatide, tirofiban, loxifiban, variabilin, XV 459 (N(3)-(2-(3-(4-carbamimidophenyl)isoxazolin-5-yl)acetyl)-N(2)-(1-butoxycarbonyl)-2,3-diaminopropanoate), SR 121566A (3-[N-{4-[4-(aminoiminomethyl)phenyl]-1,3-thiazol-2-yl}-N-(1-carbonylmethylpyridin-4-yl)amino]propionic acid, trihydrochloride), FK419 ((S)-2-acetylamino-3-[(R)-[1-[3-(piperidin-4-yl)propanoyl]piperidin-3-ylcarbonyl]amino]propionic acid trihydrate), clopidogrel, prasugrel, cangrelor, AZD6140 (AstraZeneca), MRS 2395 (2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxy)-propyl ester), BX 667 (Berlex Biosciences), BX 048 (Berlex Biosciences), Dalarifene (SB 480848), SB-435495 (GlaxoSmithKline), SB-222657 (GlaxoSmithKline), SB-253514 (GlaxoSmithKline), alefacept, efalizumab, methotrexate, acitretin, isotretinoin, hydroxyurea, mycophenolate mofetil, sulfasalazine, 6-thioguanine, calcipotriol, Taclonex, betamethasone, tazarotene, hydroxychloroquine, sulfasalazine, etanercept, adalimumab, infliximab, abatacept, rituximab, trastuzumab, anti-CD45 monoclonal antibody AHN-12 (NCI), iodine-131 anti-B1 antibody (Corixa), anti-CD66 monoclonal antibody BW 250/183 (NCI, Southampton General Hospital), anti-CD45 monoclonal antibody (NCI, Baylor College of Medicine), antibody anti-anb3 integrin (NCI), BIW-8962 (BioWa Inc.), antibody BC8 (NCI), antibody muJ591 (NCI), indium In 111 monoclonal antibody MN-14 (NCI), yttrium Y 90 monoclonal antibody MN-14 (NCI), F105 monoclonal antibody (NIAID), monoclonal antibody RAV12 (Raven Biotechnologies), CAT-192 (human anti-TGF-β1 monoclonal antibody, Genzyme), antibody 3F8 (NCI), 177Lu-J591 (Weill Medical College of Cornell University), TB-403 (BioInvent International AB), anakinra, azathioprine, cyclophosphamide, cyclosporine A, leflunomide, d-penicillamine, amitriptyline, or nortriptyline, chlorambucil, mechlorethamine, prasterone, LJP 394 (abetimus sodium), LJP 1082 (LaJolla Pharmaceutical), eculizumab, belimumab, rhuCD40L (NIAID), epratuzumab, sirolimus, tacrolimus, pimecrolimus, thalidomide, antithymocyte globulin - horse (Atgam, Pharmacia Upjohn), antithymocyte globulin - rabbit (Thymoglobulin, Genzyme), muromonab CD3 (FDA Office of Orphan Products Development), basiliximab, zenapax, riluzole, cladribine, natalizumab, interferon beta-1b, interferon beta-1a, tizanidine, baclofen, mesalazine, Ansacco, podesan, aminosalicylic acid, balsalazide, olsalazine, 6-mercaptopurine, AIN457 (anti-IL-17 monoclonal antibody, Novartis), theophylline, D2E7 (human anti-TNF mAb, Knoll Pharmaceuticals), mepolizumab (anti-IL-5 antibody, SB 240563), canakinumab (anti-IL-1β antibody, NIAMS), anti-IL-2 receptor antibody (daclizumab, NHLBI), CNTO 328 (anti-IL-6 monoclonal antibody, Centocor), ACZ885 (fully human anti-interleukin-1β monoclonal antibody, Novartis), CNTO 1275 (fully human anti-IL-12 monoclonal antibody, Centocor), (3S)-N-hydroxy-4-({4-[(4-hydroxy-2-butynyl)oxy]phenyl}sulfonyl)-2,2-dimethyl-3-thiomorpholinecarboxamide (apratastat), golimumab (CNTO 148), onarcept, BG9924 (Biogen Idec), Certolizumab Pegol (CDP870, UCB Pharma), AZD9056 (AstraZeneca), AZD5069 (AstraZeneca), AZD9668 (AstraZeneca), AZD7928 (AstraZeneca), AZD2914 (AstraZeneca), AZD6067 (AstraZeneca), AZD3342 (AstraZeneca), AZD8309 (AstraZeneca), [(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propionyl}amino)butyl]boronic acid (bortezomib), AMG-714 (anti-IL 15 human monoclonal antibody, Amgen), ABT-874 (anti-IL-12 monoclonal antibody, Abbott Labs), MRA (tocilizumab, anti-IL-6 receptor monoclonal antibody, Chugai Pharmaceutical), CAT-354 (human anti-interleukin-13 monoclonal antibody, Cambridge Antibody Technology, MedImmune), aspirin, salicylic acid, gentisic acid, choline magnesium salicylate, choline salicylate, choline magnesium salicylate, choline salicylate, magnesium salicylate, sodium salicylate, diflunisal, carprofen, fenoprofen, fenoprofen calcium, flurbiprofen, ibuprofen, ketoprofen, nabutone, norketorolac, ketorolac tromethamine, naproxen, oxaprozin, diclofenac, etodolac, indomethacin, sulindac, tolmetin, meclofenamate, meclofenamate sodium, mefenamic acid, piroxicam, meloxicam, celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib, lumiracoxib (Sankyo), JTE-522 (Japan Tobacco Inc.), L-745,337 (Almirall), NS398 (Sigma), betamethasone (Celestone), prednisone (Deltasone), alclomethasone, aldosterone, amcinonide, beclomethasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednisolone, cortisone, cortivazole, deflazacort, deoxycorticosterone, desonide, desoximetasone, deoxycorticosterone, dexamethasone, diflorasone, diflucortolone, difluprednisolone Fluprednisolone, flucloronide, fludrocortisone, fludrocortisone acetonide, flumethasone, flunisolide, fluocinolone acetonide, fluocinolone acetonide, fluocinolone, flucortolone, fluorometholone, fluperonolone, fluprednidine, fluticasone, formocortal, formoterol, halcinonide, halometasone, hydrocortisone, hydrocortisone acetonide, hydrocortisone, butyrate, hydrocortisone butyrate, loteprednol, medrysone, methylprednisolone, methylprednisolone, methylprednisolone acetonide, mometasone furoate, paramethasone, prednicasone, prednisone, rimexolone, Cortisone, triamcinolone, ubetasol, cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, vincristine, vinblastine, vinorelbine, vindesine, azathioprine, mercaptopurine, fludarabine, pentostatin, cladribine, 5-fluorouracil (5FU), floxuridine (FUDR), cytarabine, methotrexate, trimethoprim, pyrimethamine, pemetrexed, paclitaxel, docetaxel, etoposide, teniposide, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate Toposide, teniposide, dactinomycin, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin, mitomycin, trastuzumab, cetuximab, rituximab, bevacizumab, finasteride, goserelin, aminoglutethimide, anastrozole, letrozole, vorozole, exemestane, 4-androstene-3,6,17-trione ("6-OXO"; 1,4,6-androstriatriene-3,17-dione (ATD), formestane, testolactone, fadrozole, or a combination thereof.

In some embodiments, the second therapeutic agent is an antibiotic. In some embodiments, the second therapeutic agent is an antibacterial agent. In some embodiments, the second therapeutic agent is amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin, geldanamycin, herbimycin, loracarbef, ertapenem, doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cephalothin, cephalexin, cefaclor, cefamandole, cefoxitin, defprozil, Cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftobiprole, teicoplanin, vancomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin, aztreonam, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dimethoate Cloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin, piperacillin, ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanimilimde, sulfasalazine, sulfone Amindimethoprim, trimethoprim, demeclocycline, doxycycline, minocycline, oxtetracycline, tetracycline, arsphenamine, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platamicin, pyrazinamide, quinupristin/dalfopristin, rifampicin, tinidazole, and combinations thereof.

In some embodiments, the second therapeutic agent is an antiviral agent. In some embodiments, the second therapeutic agent is acyclovir, famciclovir, valacyclovir, abacavir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, artipla, brivudine, cidofovir, dipyridamole, edoxuridine, efavirenz, emtricitabine, enfuvir, entecavir, fomvirsen, fosamprenavir, foscarnet, fosfoacetic acid, ganciclovir, gardasi, ibacitabine, creatinine, pranobib, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferons, including interferon type I (such as IFNα and IFNβ), interferon type II, interferon Type III, lamivudine, lopinavir, loviride, MK-0518, maraviroc, morphine, nelfinavir, nevirapine, sorafenib, nucleoside analogs, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, acetamide, Truvada, valganciclovir, virimide, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine, and combinations thereof.

In some embodiments, the second therapeutic agent is an antifungal agent. In some embodiments, the second therapeutic agent is amrolfine, butenafine, naftifine, terbinafine, flucytosine, fluconazole, itraconazole, ketoconazole, posaconazole, ravuconazole, voriconazole, clotrimazole, econazole, miconazole, oxiconazole, sulconazole, terconazole, tioconazole, nikkomycin Z, caspofungin, micafungin, anidulafungin, amphotericin B, liposomalnystastin, pimaricin, griseofulvin, ciclopirox olamine, haloproquin, tolnaftate, undecylenic acid, clioquinol, and combinations thereof.

In some embodiments, the second therapeutic agent is an antiparasitic agent. In some embodiments, the second therapeutic agent is amitraz, nitrothiocyanate, avermectin, carbadox, diethylcarbamazine, dimetridazole, diamidinazine, ivermectin, macrofilaricide, malathion, mitaban, oxamectin, permethrin, praziquantel, pyrantel pamoate, selamectin, sodium stibogluconate, thiabendazole, and combinations thereof.

Combination with cells and tissues

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administered with a cell, a plurality of cells, or a tissue.

In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are co-administered with therapeutic cells. In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are co-administered with tissue metastasis. In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are co-administered with stem cell metastasis. In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are co-administered with organ metastasis.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered concurrently with a tissue metastasis (e.g., simultaneously, substantially simultaneously, or within the same treatment regimen). In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered before or after a tissue metastasis. In some embodiments, the time period between administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a tissue metastasis varies from a few minutes to several hours, depending on the characteristics of each agent, such as potency, solubility, bioavailability, plasma half-life, and pharmacokinetic profile. In some embodiments, diurnal variations in target molecule concentrations determine the optimal dosage interval. In some embodiments, the time between administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second active agent is approximately less than 1 hour, less than 1 day, less than 1 week, or less than 1 month.

In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are co-administered with tissue metastasis and immunosuppressive agents. In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 complexes disclosed herein are co-administered with tissue metastasis and calcineurin inhibitors (e.g., cytochrome P60 or tacrolimus), mTOR inhibitors (rapamycin; everolimus); antiproliferative agents (azathioprine or mycophenolic acid); corticosteroids (e.g., prednisone or hydrocortisone); monoclonal anti-IL-2Rα receptor antibodies (e.g., basiliximab or daclizumab); polyclonal anti-T-cell antibodies (e.g., anti-thymocyte globulin (ATG) or anti-lymphocyte globulin (ALG)), or a combination thereof.

In some embodiments, a tissue is coated with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein. In some embodiments, a plurality of stem cells are coated with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein. In some embodiments, an organ is coated with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein. In some embodiments, coating a tissue with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein prevents the host immune system from acting on the tissue.

In some embodiments, an organ, tissue, or multiple stem cells are contacted with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein. In some embodiments, an organ, tissue, or multiple stem cells are contacted with a composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein. In some embodiments, the composition has a pH of about 7.0 to about 7.5. In some embodiments, the composition has a pH of 7.4. In some embodiments, the composition further comprises potassium, magnesium, and raffinose. In some embodiments, the composition further comprises at least one of adenosine, glutathione, allopurinol, and hydroxyethyl starch. In some embodiments, the composition is a UW solution supplemented with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein.

In some embodiments, an organ, tissue, or a plurality of stem cells is contacted with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein for about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, or about 48 hours. In some embodiments, contact occurs at a temperature that protects tissue and vascular conditioning (e.g., less than ambient temperature). In some embodiments, contact occurs at 4°C.

Combination of medical devices

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administered with a medical device. In some embodiments, a medical device or portion thereof is contacted with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to coat a medical device or portion thereof, as described elsewhere herein. In some embodiments, the co-administration of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein with a medical device reduces, inhibits, or prevents an inflammatory response to an implanted device. In some embodiments, the co-administration of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein with a medical device reduces, inhibits, or prevents the formation of infectious biofilms (i.e., chronic biofilm infections) produced by microbial growth on an implanted medical device. Examples of such biofilms are those produced by bacteria, such as, but not limited to, Staphylococcus aureus.

Products and kits

Preparation provided herein includes packaging material.Packaging material for packaging pharmaceutical products is well known to those skilled in the art.The example of pharmaceutical packaging material includes but is not limited to, blister pack, bottle, tube, inhaler, inhaler (for example, pressurized metered dose inhaler (MDI), dry powder inhaler (DPI), nebulizer (for example, jet or ultrasonic nebulizer) and other single-breath liquid systems), pump, bag, vial, container, syringe, bottle and any packaging material applicable to selected preparation and expected administration and treatment mode.In some embodiments, pharmaceutical composition is incorporated into, applied to or coated to medical device, as implant, conduit, artificial joint, stent, valve, nanoparticle or microcapsule.

In some embodiments, the pharmaceutical compositions or combinations provided herein are provided as kits. The kits optionally include one or more components, such as instructions for use, devices, and additional reagents (e.g., sterile water or saline solution for diluting the composition and/or reconstituted lyophilized protein), as well as components such as tubes, containers, and syringes for implementing the method. Exemplary kits include a pharmaceutical composition or combination provided herein, and optionally include instructions for use, a device for administering the pharmaceutical composition or combination to a subject, a device for detecting an nHC-HA/PTX3 or rcHC-HA/PTX3 complex in a subject, a device for detecting an nHC-HA/PTX3 or rcHC-HA/PTX3 complex in a sample obtained from a subject, and a device for administering an additional therapeutic agent to a subject.

The kit may optionally include instructions. The instructions typically include a tangible representation describing the nHC-HA/PTX3 or rcHC-HA/PTX3 complex, and optionally other components contained in the kit, and methods of administration, including methods for determining a subject's normal state, appropriate dosage, dosing regimen, and appropriate dosing methods for administering the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, the instructions include guidance for monitoring the subject over the duration of the treatment period.

In some embodiments, a kit includes a pharmaceutical composition described herein and a diagnostic item. For example, such a kit includes items for measuring the concentration, amount, or activity of a selected nHC-HA/PTX3 or rcHC-HA/PTX3 complex in a subject.

In some embodiments, the kits provided herein include a device for administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex to a subject. In some embodiments, any of a variety of devices known in the art for administering a drug to a subject are included in the kits provided herein. Exemplary devices include, but are not limited to, inhalers (e.g., pressurized metered dose inhalers (MDIs), dry powder inhalers (DPIs), nebulizers (e.g., jet or ultrasonic nebulizers), and other single-breath liquid systems), hypodermic needles, intravenous needles, catheters, and liquid dispensers, such as eyedroppers. Typically, the device used to administer the nHC-HA/PTX3 complex of the kit will be compatible with the desired method of administration of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex.

Expansion of stem cell cultures

Disclosed herein, in certain embodiments, are methods for expanding isolated stem cells on a substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex provided herein. As described herein, the HC-HA/PTX3 complex promotes stem cell aggregation, prevents cell differentiation, and protects expression of stem cell markers.

In some embodiments, expansion on nHC-HA/PTX3 complexes or rcHC-HA/PTX3 complexes preserves one or more embryonic stem cell (ESC) markers (e.g., Oct4, Nanog, Sox2 (SRY (sex determining region Y)-box 2), Rex1 (Zfp42), SSEA4 (stage-specific embryonic antigen-4), MYC/c-Myc and KLF4, pericyte markers (e.g., NG2 (neuronal glial antigen 2/chondroitin sulfate proteoglycan 4 (CSPG4)), PDGFR-β (platelet-derived growth factor receptor B) and α-SMA (α-smooth muscle actin)), and angiogenesis markers (e.g., CD133/2, FLK-1 (VEGF-R2, Ly-73), vWF (von Willebrand factor), CD34, CD31 (PECAM-1), and CD146). In some embodiments, the expression of stem cell markers is determined by conventional methods such as protein expression analysis (e.g., Western blot analysis, immunofluorescence, immunohistochemistry, fluorescence-activated cell sorting) or mRNA analysis (e.g., polymerase chain reaction (PCR) or Northern).

In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 inhibits TGF-β signaling in cultured cells. In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 inhibits TGF-β signaling in cultured stem cells. In some embodiments, inhibition of TGF-β signaling refers to a decrease in the activity or expression of one or more proteins or markers in the TGF-β cell signaling pathway, such as pSMAD 2/3 signaling, alpha smooth muscle formation, in a cell in the presence of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex compared to the absence of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex.

In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 induces BMP signaling in cultured cells. In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 induces BMP signaling in cultured stem cells. In some embodiments, inhibition of TGF-β signaling refers to an increase in the activity or expression of one or more proteins in the BMP signaling pathway, such as BMP-2, BMP-4, BMP-6, and pSMAD1/5/8, in a cell in the presence of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex compared to the absence of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex.

In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are embryonic stem cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are adult stem cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are fetal stem cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are induced pluripotent stem/progenitor stem cells (IPSCs).

In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are mesenchymal stem cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are adipose-derived stem cells (ASCs). In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are umbilical cord stem cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are amniotic membrane stem cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are limbal cells, such as limbal niche cells or limbal epithelial cell progenitors. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are endothelial stem cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are hematopoietic stem cells. In some embodiments, the isolated stem cells are bone marrow stem cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are neural stem cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are endothelial progenitor cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are skeletal muscle stem cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are mammary stem cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are intestinal stem cells.

In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are induced pluripotent stem cells/progenitor stem cells (iPSCs). In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are induced pluripotent stem cells derived from adult differentiated or partially differentiated cells. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are induced pluripotent stem cells derived from fibroblasts. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are induced pluripotent stem cells derived from human corneal fibroblasts.

In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from fetal tissue, such as placental tissue or umbilical cord tissue. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from amniotic membrane. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from adipose tissue. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from limbal tissue. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from bone marrow. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from endothelial tissue. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from limbal tissue. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from neural tissue. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from limbal tissue. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from skeletal muscle. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from the skin. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from the digestive system. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from the pancreas. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from the liver. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from olfactory mucosa. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from a germ cell population. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from blood. In some embodiments, the isolated stem cells cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 are derived from umbilical cord blood.

In some embodiments, the HC-HA/PTX3 complex is nHC-HA/PTX3 isolated from amniotic membrane or umbilical cord. In some embodiments, the HC-HA/PTX3 complex is a reconstituted HC-HA complex. In some embodiments, the HA is covalently linked to the HC. In some embodiments, the HC of IαI is heavy chain 1 (HCl). In some embodiments, the HC-HA complex includes pentraxin-3 (PTX3).

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex comprises a small leucine-rich proteoglycan (SLRP). In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex comprises a class I, class II, or class III SLRP. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex comprises PTX3 and a small leucine-rich proteoglycan (SLRP). In some embodiments, the small leucine-rich proteoglycan is selected from class I SLRPs, such as decorin and biglycan. In some embodiments, the small leucine-rich proteoglycan is selected from class II SLRPs, such as biglycan, lumican, PRELP (proline arginine-rich terminal leucine-rich protein), keratocan, and osteoadherin. In some embodiments, the small leucine-rich proteoglycan is selected from class III SLRPs, such as epiphyseal and osteoglycan.

In some embodiments, the isolated stem cells are expanded on a substrate comprising immobilized nHC-HA/PTX3 or rcHC-HA/PTX3. In some embodiments, the immobilized nHC-HA/PTX3 or rcHC-HA/PTX3 comprises one or more small leucine-rich proteoglycans (SLRPs). In some embodiments, the SLRP is selected from urotrypsin, decorin, biglycan, and osteoadherin. In some embodiments, the small leucine-rich protein comprises a glycosaminoglycan. In some embodiments, the small leucine-rich proteoglycan comprises keratan sulfate.

In some embodiments, the isolated stem cells are expanded in a culture medium containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 comprises one or more small leucine-rich proteoglycans (SLRPs). In some embodiments, the SLRP is selected from urokinin, decorin, biglycan, and osteoadherin. In some embodiments, the small leucine-rich protein comprises a glycosaminoglycan. In some embodiments, the small leucine-rich proteoglycan comprises keratan sulfate. In some embodiments, the culture medium is embryonic stem cell culture medium, modified embryonic stem cell culture medium, hormone-supplemented epithelial culture medium, and/or a combination thereof. In some embodiments, the culture medium is supplemented with one or more growth factors. In some embodiments, the culture medium is supplemented with EGF, bFGF, and/or LIF. In some embodiments, the culture medium is supplemented with an inhibitor of Rho-associated kinase (ROCK inhibitor).

Induction and maintenance of pluripotency

Disclosed herein, in certain embodiments, are methods for inducing pluripotency in cells or maintaining pluripotency in stem cells on a substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. As described herein, the HC-HA/PTX3 complex helps maintain the expression of stem cell markers and prevents cell differentiation during serial passage of a stem cell population. Furthermore, as described herein, the HC-HA/PTX3 complex promotes the induction of stem cell properties in differentiated or partially differentiated populations of cells.

In certain embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex promotes or induces pluripotency in differentiated or partially differentiated cells. In certain embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex promotes or induces pluripotency in differentiated or partially differentiated cells compared to differentiated or partially differentiated cells cultured in the absence of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In an exemplary method, differentiated cells or partially differentiated cells are cultured on a substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex, thereby inducing pluripotency in the cells.

In certain embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex further promotes or induces pluripotency in stem cells. In certain embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex further promotes or induces pluripotency in stem cells compared to stem cells cultured in the absence of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In exemplary methods, stem cells are cultured on a substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex, thereby maintaining pluripotency in the stem cells. In exemplary methods, stem cells are cultured on a substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex, thereby further inducing pluripotency in the stem cells.

Using genetic reprogramming under protein factors, pluripotent stem cells equivalent to embryonic stem cells have been derived from human adult skin tissue. iPSC is usually derived from non-pluripotent cells, such as adult fibroblasts, by transfection of certain stem cell-related genes. Transfection is usually completed by viral vectors such as retroviruses, in which the pluripotency gene is operably linked to a promoter for gene expression. The four key pluripotency genes necessary for producing pluripotent stem cells are Oct-3/4 (Pou5f1), Sox2, c-Myc and Klf4. Other genes can enhance induction efficiency. In some studies, Oct4, Sox2, Nanog and Lin28 have been used to induce pluripotency. In some cases, after 3-4 weeks, a small amount of transfected cells begin to become similar to pluripotent stem cells morphologically and biochemically, and are usually separated by morphological selection, doubling time or by reporter gene and antibiotic selection.

In some embodiments, methods for inducing pluripotency in differentiated or partially differentiated cells using heterologous expression of less than four essential transcription factors, Oct-3/4 (Pou5f1), Sox2, c-Myc, and Klf4, are provided. In some embodiments, methods for inducing pluripotency are provided, wherein the use of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes enhances the pluripotency of differentiated or partially differentiated cells that express at least one of Oct-3/4 (Pou5f1), Sox2, c-Myc, and/or Klf4 via heterologous gene transfer. In some embodiments, methods for inducing pluripotency are provided, wherein the use of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes enhances the pluripotency of differentiated or partially differentiated cells that express one, two, or three factors selected from Oct-3/4 (Pou5f1), Sox2, c-Myc, and/or Klf4 via heterologous gene transfer. In some embodiments, methods for inducing pluripotency are provided, wherein the use of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex enhances the pluripotency of induced differentiated or partially differentiated cells that express Oct-3/4 (Pou5f1) by heterologous gene transfer. In some embodiments, methods for inducing pluripotency are provided, wherein the use of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex enhances the pluripotency of induced differentiated or partially differentiated cells that express Sox2 by heterologous gene transfer. In some embodiments, methods for inducing pluripotency are provided, wherein the use of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex enhances the pluripotency of induced differentiated or partially differentiated cells that express c-Myc by heterologous gene transfer. In some embodiments, methods for inducing pluripotency are provided, wherein use of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex enhances the induction of pluripotency in differentiated or partially differentiated cells that express Klf4 by heterologous gene transfer.

In some embodiments, differentiated or partially differentiated cells are transduced to express one or more of Oct-3/4 (Pou5f1), SOX2, c-Myc, and Klf4; and the transduced cells are cultured on a substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, differentiated or partially differentiated cells are transduced to express at least one of Oct-3/4 (Pou5f1), SOX2, c-Myc, and Klf4; and the transduced cells are cultured on a substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, differentiated or partially differentiated cells are transduced to express one, two, or three of Oct-3/4 (Pou5f1), SOX2, c-Myc, and Klf4; and the transduced cells are cultured on a substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, differentiated or partially differentiated cells are transduced for expression of Oct-3/4 (Pou5f1), SOX2, c-Myc, and Klf4; and the transduced cells are cultured on a substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, differentiated or partially differentiated cells are transduced for expression of Oct-3/4 (Pou5f1); and the transduced cells are cultured on a substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, differentiated or partially differentiated cells are transduced for expression of SOX2; and the transduced cells are cultured on a substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, differentiated or partially differentiated cells are transduced for expression of c-Myc; and the transduced cells are cultured on a substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, differentiated or partially differentiated cells are transduced to express Klf4, and the transduced cells are cultured on a substrate containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, cells are transduced to express one or more additional genes such as, for example, Nanog, Fbx15, Eras, ECAT15-2, Tcl1, and β-catenin.

In some embodiments, differentiated or partially differentiated cells are transduced with a viral vector comprising one or more genes encoding one or more Oct-3/4 (Pou5f1), SOX2, c-Myc, and Klf4. In some embodiments, differentiated or partially differentiated cells are transduced with two or more viral vectors comprising one or more genes encoding one or more Oct-3/4 (Pou5f1), SOX2, c-Myc, and Klf4.

Various methods for inducing, culturing, and maintaining induced pluripotent stem cells and assessing the pluripotency of induced stem cells (including assessing stem cell markers and inducing different cell lineages) are known in the art and include methods described in U.S. Patent Nos. 7,682,828, 8,048,999, 8,211,697, 7,951,592, and U.S. Patent Publication Nos. 2009/0191159 and 2010/000375.

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex reduces the time to induction of pluripotency in transduced cells compared to transduced cells cultured in the absence of nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex increases the percentage of transduced cells in which pluripotency is induced compared to transduced cells cultured in the absence of nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex increases the level of pluripotency in transduced cells. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex reduces the number of heterologous transcription factors required to induce pluripotency in transduced cells.

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein inhibit TGFβ1 signaling in differentiated cells, stem cells, or iPSCs. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein inhibit nuclear translocation of SMAD2 or SMAD3 in differentiated cells, stem cells, or iPSCs. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein inhibit the formation of α-smooth muscle actin in differentiated cells, stem cells, or iPSCs. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein activate BMP4 signaling in differentiated cells, stem cells, or iPSCs. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein activate BMP6 signaling in differentiated cells, stem cells, or iPSCs. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein induce expression of embryonic cell markers in differentiated cells, stem cells, or iPSCs. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein induce expression of c-myc, KLF-4, Nanog, nestin, Oct4, Rex-1, Sox-2, and SSEA-4 in differentiated cells, stem cells, or iPSCs.

Example

The following examples are included for illustrative purposes only and are not intended to limit the scope of the claimed subject matter.

Example 1: Purification of native HC-HA/PTX3 (nHC-HA/PTX3) complex from human amniotic membrane extract (AME) change

Preparation of amniotic membrane extract (AME) and AM powder (AMP)

Frozen human AM obtained from Bio-tissue (Miami, FL) was washed 2-3 times with PBS to remove the storage medium. To prepare AME, AM was transferred to a sterile 50 ml centrifuge tube and centrifuged at 5000 × g for 5 minutes at 4 ° C to remove excess liquid. AM was weighed (~10 mg/cm ² ), transferred to a 100 mm or 150 mm sterile culture dish, and frozen in the vapor phase of a liquid nitrogen container for 20 minutes, after which it was cut into small pieces with a disposable scalpel and homogenized in PBS with a Tissue-Tearor (Biospec Products, Inc., Bartlesville, OK). The homogenate was mixed at 4 ° C for 30 minutes and centrifuged at 48,000 × g for 30 minutes. The supernatant, called AME, was collected and used for nHC-HA/PTX3 purification or stored at -80 ° C.

To prepare lyophilized AM powder (AMP), AM frozen in a -80°C freezer was transferred to a benchtop freeze dryer (Freezone 4.5, Labconco, Kansas City, MO) and lyophilized for 16 hours. The lyophilized AM was then micronized to its matrix form (AMP) using a mixer mill (Retsch, Newtown, PA). AMP was stored below -20°C for further analysis.

Purification of native HC-HA/PTX3 (nHC-HA/PTX3) complex

AME was dissolved in a CsCl/4M guanidine hydrochloride mixture with an initial density of 1.35 g/ml and centrifuged at 125,000 × g for 48 hours at 15°C. A total of 15 fractions (0.8 ml/fraction) were collected from top to bottom in each tube. The total protein concentration of each fraction was determined by a BCA protein assay kit. The hyaluronan (HA) concentration of each fraction was determined by an ELISA-based HA quantitative test kit from Corgenix (Westminster, CO) (Figure 1A). Fractions 8-15 containing HA but not containing detectable protein were merged and used for a second ultracentrifugation. Samples of the merged fractions (referred to as the first AM) were preserved for analysis. The merged fractions were adjusted with CsCl/4M guanidine hydrochloride having an initial density of 1.40 g/ml, centrifuged, and fractionated (Figure 1B) in the same manner as described above. Fractions 3-15, containing HA but no detectable protein, were combined (referred to as the second AM) and dialyzed against distilled water to remove CsCl and guanidine hydrochloride. The dialysate was lyophilized in the same manner as described above for AMP. Alternatively, the dialysate was mixed with 3 volumes of 95% (v/v) ethanol containing 1.3% (w/v) potassium acetate at 0°C for 1 hour. After centrifugation at 15,000 × g, the precipitate was washed with 70% (v/v) ethanol and centrifuged again. The precipitate was briefly air-dried and stored at -80°C. The powder and precipitate are referred to as nHC-HA/PTX3 complexes.

In some cases, the pooled samples were subjected to three or four ultracentrifugations. In these ultracentrifugations, only fractions 7-12 were pooled, and the initial density of CsCl/4M guanidine hydrochloride was 1.42 g/ml. After three or four ultracentrifugations, the pooled fractions 7-12 were referred to as nHC-HA/PTX3 (3rd) or nHC-HA/PTX3 (4th).

After the first, second, third, or fourth ultracentrifugation, the pooled fractions of AME were treated with or without 0.05 N NaOH at 25° C. for 1 hour. The pooled fractions from the first, second, third, or fourth ultracentrifugation were also digested with 20 units/ml of hyaluronidase (HAase) (Seikagaku Biobusiness Corporation, Tokyo, Japan) at 60° C. for 2 hours.

Samples from the pooled fractions and the NaOH- and HAase-treated samples were then run on 0.5% agarose gels and analyzed by staining with whole-stain dye ( FIG1C ) or by Western blotting using antibodies against IαI heavy chain 1 (HCl) ( FIG1D and 1F ), pentraxin 3 (PTX3) ( FIG1E and 1G ), IαI heavy chain 2 (HC2) ( FIG1H ), IαI heavy chain 3 (HC3) ( FIG1I ), urokinin ( FIG1J ), TNF-stimulated gene 6 (TSG-6) ( FIG1K ), thrombospondin-1 (TSP-1) ( FIG1L ), or IGFBP1-3 and PF4 ( FIG1M ), the latter two of which were analyzed by protein spotting using human angiogenesis arrays (each array containing 56 different angiogenic proteins, R&D SYSTEMS, Minneapolis, MN)). Briefly, 1.5 ml of human AM extract (25 μg / ml HA) and purified nHC-HA / PTX3 (second) (25 μg / ml HA) were incubated with detection antibodies pre-coated on the membrane overnight at 4 ° C, followed by incubation with secondary antibodies overnight. The signal was detected by chemiluminescence exposed to X-ray film. The array data on the developed X-ray film was quantified by scanning the film on a transmission scanner, and the array image file was analyzed by ImageJ1.46 software (National Institutes of Health, Bethesda, MD).

Biochemical characterization revealed that nHC-HA/PTX3 consisted of high-molecular-weight HA (HMW HA) covalently linked to the heavy chain (HCl) of IαI and PTX3 (Figure 1C). HCl and PTX3 in nHC-HA/PTX3 were released only after treatment with hyaluronidase (HAase) or NaOH (Figures 1D-G), indicating that HCl is linked to HA via an ester bond, as reported.

In contrast, nHC-HA/PTX3 lacked HC2 (Figure 1H), HC3 (Figure 1I, the band at ~12 kDa detected only after NaOH treatment was likely nonspecific), urotrypsin (Figure 1J), TSG-6 (Figure 1K), and TSP-1 (Figure 1L). Insulin-like growth factor binding proteins 1-3 (IGFBP1-3) and platelet factor 4 (PF4) were detected in nHC-HA/PTX3 (2) by a protein spot assay (similar to ELISA) (Figure 1J); it is unclear whether they are still present in nHC-HA/PTX3 (4).

Example 2: Preparation of immobilized HA (iHA) by covalent linkage

A range of hyaluronan (HA) amounts (0, 0.25, 0.5, 1.0, 2.5, 5, 10, and 25 μg/well) from HMW HA (Healon, Advanced Medical Optics, Santa Ana, CA) or nHC-HA/PTX3 (No. 2) was added to a conjugation solution containing sulfo-NHS (0.184 mg/ml) and EDAC (0.123 mg/ml) (both from Thermo Fisher Scientific, Rockford, IL) and incubated in Covalink ^™ -NH 96-well plates (Thermo Fisher Scientific Inc.) at 4°C for 16 hours. After washing three times with 8M guanidine hydrochloride (GnHCl) and then with PBS, the conjugated HA from HMW HA or nHC-HA/PTX3 was quantitatively measured by HA ELISA from Corgenix (Westminster, CO) according to the manufacturer's protocol ( FIG. 6A ). Purified HMW HA and nHC-HA/PTX3 from AM were dose-dependently and covalently coupled to the surface of Covalink-NH 96-well plates. The resulting iHA or immobilized nHC-HA/PTX3 were resistant to washing with 8 M guanidine hydrochloride. HMW HA or nHC-HA/PTX3 HA was maximally coupled at an HA equivalent input of 2 μg/well (Figure 6A).

To determine the coupling efficiency, HA from HMW HA or nHC-HA/PTX3 was coupled to Covalink ^™ -NH in each well of a 96-well plate, and unbound and bound HA from HMW HA or nHC-HA/PTX3 was measured by HA ELISA ( FIG6B ). 2 μg of HA from HMW HA or nHC-HA/PTX3 was added to a _coupling solution containing sulfo-NHS (0.184 mg/ml) and EDAC (0.123 mg/ml) [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] in H 2 O and incubated in Covalink ^™ -NH 96-well plates (Thermo Fisher Scientific, Rockford, IL) for 16 hours at 4°C. HA coupled to the wells or unbound in the wash solution (pooled) was measured using an HA ELISA from Corgenix (Westminster, CO) according to the manufacturer's protocol. The total amount of coupled or unbound HA in each well was divided by the input HA amount (2 μg/well) to calculate the coupling efficiency or the percentage of unbound. The average coupling efficiency of HMW HA was determined to be 70.5 ± 13.4%, while the average coupling efficiency of nHC-HA/PTX3 was determined to be 69.0 ± 5.7% (Figure 6B). Therefore, 2 μg/well input HA resulted in approximately 1.4 μg iHA.

Example 3: Activity of purified native HC-HA/PTX3 (nHC-HA/PTX3) complex

Attachment of LPS-stimulated macrophages to immobilized nHC-HA/PTX3

RAW264.7 cells (100 μl, 2.5×10 ⁵ cells/ml) [American Type Culture Collection (ATCC), Manassas, VA] in DMEM/10% FBS (Life Technologies, Grand Island, NY) were seeded in 96-well plates containing immobilized HA (Advanced Medical Optics, Santa Ana, CA, 2 μg/well), nHC-HA/PTX3 (2 μg/well), or PBS control and stimulated with lipopolysaccharide (LPS) (1 μg/ml) (n=3) [LPS-EB Ultrapure, InvivoGen, San Diego, CA]. Similarly, immobilization of HA and nHC-HA/PTX3 on the surface of Covalink-NH 96-well plates was performed as described above. Briefly, Covalink-NH 96-well plates were sterilized in 70% ethanol for 2 hours, washed three times with distilled water, and 100 μl of 0.184 mg/ml sulfo-NHS (Thermo Fisher Scientific, Rockford, IL) and 0.123 mg/ml EDAC (Thermo Fisher Scientific, Rockford, IL) in distilled water containing 20 μg/ml HA or nHC-HA/PTX3 was added to each 96-well plate (PBS control wells contained all reagents except HA and nHC-HA/PTX3). Before removing the coupling solution, the plate was incubated at 4°C overnight or at 25°C for 2 hours, washed three times with PBS containing 2M NaCl and 50mM _MgSO4 , and then washed three times with PBS. After incubation for 90 minutes, unattached cells were removed, and attached cells were photographed and counted by CyQuant assay (Figure 2A). Compared to control wells, wells containing immobilized nHC-HA/PTX3 showed a >3-fold increase in LPS-stimulated macrophage attachment. Wells containing immobilized HA inhibited LPS-stimulated macrophage attachment.

The cell viability of the attached LPS-stimulated macrophages was then examined. LPS-stimulated RAW264.7 cells (100 μl, 2.5 × ¹⁰ cells/ml) were incubated in DMEM/10% FBS for 24 hours on immobilized PBS controls, HA, or nHC-HA/PTX3 as described above (n=3). After incubation, the cell viability of the attached macrophages was measured by MTT analysis. No significant differences in cell viability were observed between cells on these immobilized substrates (all p values>0.05) (Figure 2B).

The ability of blocking antibodies and peptides to inhibit LPS-stimulated macrophage attachment to immobilized nHC-HA/PTX3 was then examined. RAW264.7 cells (at a concentration of 2.5 x 10 ⁵ cells/ml) were pre-incubated with blocking antibodies against CD44 (10 μg/ml), TLR2 (10 μg/ml), TLR4 (10 μg/ml), integrin αv (20 μg/ml), β1 (20 μg/ml), β2 (20 μg/ml), or β3 (20 μg/ml), or RGD peptide (SDGRG, RGDS, GRGDS, all 1 mg/ml), as well as isotype control antibodies [rat IgG (10 μg/ml), mouse IgG (10 μg/ml), or Armenian hamster IgG (20 μg/ml)] or RGD control peptide (1 mg/ml) in DMEM/10% FBS for 30 minutes on ice (n=3). (Anti-CD44 antibodies and rat IgG were from BD Pharmingen, San Diego, CA; anti-TLR2, TLR4 antibodies and mouse IgG were from InvivoGen, San Diego, CA; anti-integrin αv, β1, β2, β3 antibodies and Armenian hamster IgG were from Biolegend, San Diego, CA; RGD peptide was from Sigma-Aldrich, St Louis, MO). After the addition of LPS (1 μg/ml), cells were seeded into plates containing immobilized HA (2 μg/well), nHC-HA/PTX3 (2 μg/well) or PBS control and incubated for 90 minutes (n=3). After incubation, non-attached cells were removed, and attached cells were photographed and counted by CyQuant assay (Figure 2C). The results showed that antibodies against CD44 and TLR4 significantly inhibited the attachment of LPS-stimulated macrophages, indicating that these antibodies are involved in the attachment of LPS-stimulated macrophages to immobilized nHC-HA/PTX3.

Polarization of LPS-stimulated macrophages

The polarization of LPS-stimulated macrophages toward an M1 or M2 phenotype by immobilized nHC-HA/PTX3 was examined by determining the expression of genes encoding M1 and M2 markers by RNA and protein analysis.

RAW264.7 cells (100 μl, 2.5×10 ⁵ cells/ml) in DMEM/10% FBS were seeded into 96-well plates containing immobilized HA (2 μg/well), nHC-HA/PTX3 (2 μg/well), or PBS control and stimulated with LPS (1 μg/ml) for 4 hours (n=3). After incubation, unattached cells were removed, and total RNA was extracted from attached cells. The mRNA expression of M1 markers (tumor necrosis factor α (TNF-α) (Mm00443258_m1) and interleukin 12 subunit p40 (IL-12p40) (Mm00434165_m1)) and M2 markers (interleukin-10 (IL-10) (Mm00439614_m1), arginase-1 (Arg-1) (Mm00475988_m1), LIGHT/TNSF14 (Mm00444567_m1), and sphingosine kinase-1 (SPHK1) (Mm0044884_g1)) was determined by quantitative real-time PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Mm99999915_g1) as an endogenous control. The mRNA expression of the M1 markers was determined by quantitative real-time PCR on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, MD). Real-time PCR was performed on a 40-well plate in San Diego, CA. The amplification protocol consisted of an initial activation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing and extension at 60°C for 1 minute. Relative gene expression data were analyzed by the comparative CT method (ΔΔCT). All experiments were performed in triplicate; the results were normalized using GAPDH as an internal control. All primers were from Applied Biosystems. Compared to the control, significant induction of expression of M2 markers IL-10, Arg-1, LIGHT, and SPHK1 was observed in cells attached to immobilized nHC-HA/PTX3 rather than HA (Figure 3A). In addition, expression of M1 markers, TNF-α, and IL-12p40 was reduced.

As described above, the amount of secreted TNF-α protein was determined in the culture supernatant of cells treated with LPS (1 μg/ml) in DMEM/10% FBS for 4 hours on plates containing immobilized HA (2 μg/well), nHC-HA/PTX3 (2 μg/well), or PBS control (n=3). The amount of TNF-α was determined by ELISA according to the manufacturer's protocol (R&D Systems, Minneapolis, MN).

A decrease in TNF-α levels was observed in the cell culture supernatants of cells incubated on plates containing immobilized nHC-HA/PTX3 compared to PBS controls (Figure 3B). No changes in TNF-α levels were observed on immobilized HA plates.

High expression of IRF-5 is a characteristic of M1 macrophages. IRF-5 directly activates the transcription of genes encoding IL-12p40, IL-12p35, and IL-23p19 and inhibits the gene encoding IL-10. The expression of IRF-5 protein and its cellular localization on immobilized nHC-HA/PTX3 were examined. Cells seeded onto immobilized control or nHC-HA/PTX3 were stimulated with LPS (1 μg/ml) in DMEM/10% FBS for 4 hours or 24 hours. The expression of IRF-5 protein in cell lysates (LPS stimulation for 24 hours) was detected by Western blotting (Figure 3C, left) (primary antibody: abcam, Cambridge, MA; secondary antibody: DAKO, Carpinteria, CA). In a parallel experiment, cells (LPS stimulation for 4 hours) were fixed and immunostained with anti-IRF-5 antibody. The cellular localization of IRF-5 was examined by confocal immunofluorescence microscopy (LSM 700 confocal microscope, Zeiss, Oberkochen, Germany) (Figure 3C, right). Immobilized nHC-HA/PTX3 reduced the expression of IRF-5 and prevented its nuclear localization. These results are consistent with the inhibition of the M1 phenotype by immobilized nHC-HA/PTX3.

Apoptosis of activated neutrophils and phagocytosis of apoptotic neutrophils by macrophages

Neutrophils were isolated from normal human peripheral blood using dextran density [Lymphocyte Poly (R), Cedarlane USA, Burlington, NC] centrifugation according to the manufacturer's protocol. The isolated neutrophils were seeded into IMDM (Iscove modified Dulbecco's medium, Life Technologies, Grand Island, NY) on immobilized HA ( ² μg/well), nHC-HA/PTX3 (2 μg/well) or PBS controls with 2 × 106 cells/ml, and treated with PBS (resting), N-formyl-methionyl-leucyl-phenylalanine (fMLP) (1 μM) (Sigma-Aldrich, St Louis, MO) or LPS (1 μg/ml) for 24 hours (n=3). The apoptosis of neutrophils in cell lysates was determined by cell death detection ELISA (Roche Applied Science, Indianapolis, IN) according to the manufacturer's protocol. Immobilized nHC-HA/PTX3, but not HA, promoted apoptosis of fMLP- or LPS-activated neutrophils, but not resting neutrophils ( FIG3D ).

The phagocytosis of apoptotic neutrophils by resting or LPS-stimulated macrophages was then examined. RAW264.7 cells (1×10 ⁵ cells/ml) were cultured for 6 days (n=3) in DMEM/10% FBS with or without LPS (1 μg/ml) stimulation on immobilized HA (2 μg/well), nHC-HA/PTX3 (2 μg/well) or PBS controls. The cell culture medium was then removed, and 100 μl of IMDM containing 2×10 ⁶ cells/ml of apoptotic neutrophils (prepared by treating isolated resting human neutrophils with roscovitine (20 μM) (Sigma-Aldrich, St Louis, MO) for 8 hours) was added to each well containing resting or LPS-stimulated macrophages. After incubation at 37°C for 30 minutes, each well was washed three times with cold PBS, and cell lysates (containing macrophages and engulfed neutrophils) were collected to determine the activity of human myeloperoxidase (MPO) by ELISA assay measuring neutrophils engulfed by macrophages. Cell lysates were collected and subjected to human myeloperoxidase (MPO) ELISA assay (n=4) (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. MPO was then normalized using total protein measured in each cell lysate by BCA protein assay (Thermo Fisher Scientific, Rockford, IL) and expressed as the phagocytic index. The phagocytic index of resting cells without LPS (-LPS) stimulation was defined as 100% in this experiment. Immobilized nHC-HA/PTX3, but not HA, promoted phagocytosis of apoptotic neutrophils by resting or LPS-treated macrophages (Figure 3E).

These results demonstrate that immobilized nHC-HA/PTX3 (second) enhances the apoptosis of activated neutrophils and the phagocytosis of apoptotic neutrophils by macrophages.

Analysis of receptors involved in the polarization of M2 macrophages by immobilized nHC-HA/PTX3

To determine the involvement of specific receptors in M2 macrophage polarization, quantitative mRNA expression of M1 and M2 macrophage markers was performed in the presence or absence of receptor blocking antibodies. RAW264.7 cells (2.5×10 ⁵ cells/ml) in DMEM/10% FBS were pre-incubated on ice for 30 minutes with PBS (control) or blocking antibodies for CD44 (10 μg/ml), TLR4 (10 μg/ml) or CD44/TLR4 (10 μg/ml each) (n=3). The cells were then stimulated with LPS (1 μg/ml) and incubated for 4 hours at 37°C on immobilized HA (2 μg/well), nHC-HA/PTX3 (2 μg/well) or PBS control. Total RNA was extracted from total cells. As described above, relative mRNA expression of M1 markers (IL-12p40) and M2 markers (IL-10, LIGHT, and SPHK1) was determined by quantitative PCR using GAPDH as an endogenous control (Figure 4A). Immobilized nHC-HA/PTX3, but not HA, abolished IL-12p40 expression and promoted IL-10, LIGHT, and SPHK1 expression. CD44-blocking antibodies inhibited this expression pattern more than TLR4-blocking antibodies. Conversely, anti-TLR4 blocking antibodies affected the expression of IL-12p40 and IL-10 transcripts induced by immobilized HA more than anti-CD44 blocking antibodies.

Protein expression of IL-12 and IL-10 was also determined. Cell culture supernatants were collected from cells cultured on immobilized HA (2 μg/well), nHC-HA/PTX3 (2 μg/well) or PBS controls (n=3) except for 24 hours (instead of 4 hours). The amount of IL-12 or IL-10 protein in the cell culture supernatant was determined by ELISA (Biolegend, San Diego, CA) according to the manufacturer's protocol (Figure 4B). Immobilized nHC-HA/PTX3 eliminated the expression of IL-12 protein and significantly promoted the expression of IL-10 protein. Anti-CD44 blocking antibodies inhibited this expression pattern. In contrast, immobilized HA promoted the expression of IL-12 protein and inhibited the expression of IL-10, and anti-TLR4 blocking antibodies were more able to affect this expression pattern.

Comparison of nHC-HA/PTX3 (2nd) and nHC-HA/PTX3 (4th) complexes

The nHC-HA/PTX3 (2nd) and nHC-HA/PTX3 (4th) complexes were compared by examining the ability of each complex to induce macrophage cell aggregation (indicating poor cell attachment) and/or promote M2 macrophage polarization. RAW264.7 cells (2.5×10 ⁵ cells/ml) were cultured in DMEM/10% FBS on immobilized HA (2 μg/well), nHC-HA/PTX3 (2 μg/well), or PBS control and stimulated with 200 units/ml IFN-γ/1 μg/ml LPS (both from InvivoGen, San Diego, CA) for 4 hours or 24 hours (n=3). After 4 hours, cell aggregation was examined by light microscopy and photographed ( FIG5A ). Immobilized nHC-HA/PTX3(4th), but not nHC-HA/PTX3(2nd), promoted cell aggregation of macrophages, indicating that nHC-HA/PTX3(4th) did not promote cell attachment to the plate, whereas nHC-HA/PTX3(2nd) did.

After 24 hours, samples were obtained from the cell culture supernatant and the IL-12p40 protein and IL-23 protein concentrations were determined by respective ELISAs (Biolegend, San Diego, CA) according to the manufacturer's protocol (Figures 5B and 5C). Both nHC-HA/PTX3 (No. 2) and nHC-HA/PTX3 (No. 4) inhibited the production of IL-12p40 and IL-23 proteins in IFN-γ/LPS-stimulated macrophages.

Example 4: In vitro binding of TSG-6 and PTX3 to immobilized HA (iHA) in the absence of IαI

Binding of TSG-6 to iHA

Immobilized HA (2 μg/well input) was prepared as described above. A range of human TSG-6 (overexpressed in the mouse myeloma cell line NS0, with Trp18 to Leu277 of human TSG-6, with a C-terminal 10 His tag, accession number: P98066; R&D SYSTEMS, Minneapolis, MN, catalog number: 2104-TS) concentrations (0, 0.24, 1.2, 6, 12 and 24 μg/ml, 100 μl volume/well) were incubated with iHA in reaction buffer (PBS containing 5 mM MgCl ₂ , pH 7.5) at 37° C. for 2 hours. Unbound TSG-6 was removed by washing with 8 M guanidine hydrochloride and PBS. Bound TSG-6 was measured by a modified TSG-6 ELISA (R&D SYSTEMS, Minneapolis, MN). Since TSG-6 was already bound to the iHA coupled in the wells, the step of incubating the sample with the pre-coated TSG-6 antibody was omitted. Subsequent steps were performed according to the manufacturer's protocol (Figure 7A). When iHA was approximately 1.4 μg (2 μg HA/well, based on a coupling efficiency of 70%), TSG-6 dose-dependently bound to iHA and reached its maximum binding capacity at approximately 6 μg/ml (or 0.6 μg in 0.1 ml of reaction solution). The molar ratio of TSG-6 to HA was approximately 37:1, based on TSG-6 being 35 kDa and HA being 3,000 kDa.

The ability of the TSG-6/iHA complex to resist dissociation was then examined. iHA (2 μg/well input) was prepared as described above. TSG-6 (6 μg/ml, 100 μl) was incubated with iHA at 37°C for 2 hours. Unbound TSG-6 was removed by washing with PBS (as a control) or with different dissociation agents or reducing agents: 6M guanidine hydrochloride/PBS, 8M guanidine hydrochloride/PBS, 2% SDS/PBS, 100mM DTT/PBS and 25mM NaOH/H ₂ O. As described above, bound TSG-6 was measured by a modified TSG-6 ELISA ( FIG. 7B ). The TSG-6/iHA complex formed was stable and resistant to treatment with various dissociation agents and/or reducing agents. No statistical significance was seen between all groups.

Binding of PTX3 to iHA

Immobilized HA (2 μg/well input) was prepared as described above. A series of PTX3 (overexpressed in the mouse myeloma cell line NS0, with Glu18 to Ser277 of human PTX3, with a C-terminal 6 His tag, accession number: P26022; R&D SYSTEMS, Minneapolis, MN) concentrations (0, 0.04, 0.2, 1, 5 and 25 μg/ml, 100 μl volume/well) were incubated with iHA in reaction buffer (PBS containing 5 mM MgCl ₂ , pH 7.5) at 37 ° C for 2 hours. Unbound PTX3 was removed by washing with 8M GnHCl and PBS. The bound PTX3 was determined by a modified PTX3 ELISA (R&D SYSTEMS, Minneapolis, MN). Because PTX3 was already bound to the iHA coupled in the well, the step of incubating the sample with the pre-coated PTX3 antibody was omitted. Subsequent steps were performed according to the manufacturer's protocol (Figure 8A). PTX3 bound to iHA in a dose-dependent manner at approximately 1.4 μg of iHA (2 μg HA/well, based on a coupling efficiency of ∼70%) and reached its maximum binding capacity at approximately 5 μg/ml (or 0.5 μg in 0.1 ml of reaction solution). The molar ratio of PTX3 to HA was approximately 24:1, based on a PTX3 concentration of 45 kDa and a HA concentration of ∼3,000 kDa.

The ability of the PTX3/iHA complex to resist dissociation was then examined. iHA (2 μg/well input) was prepared as described above. PTX3 (5 μg/ml, 100 μl) was incubated with iHA at 37°C for 2 hours. Unbound PTX3 was removed by washing with PBS (as a control) or with different dissociation agents or reducing agents: 6M guanidine hydrochloride/PBS, 8M guanidine hydrochloride/PBS, 2% SDS/PBS, 100mM DTT/PBS, and 25mM NaOH/H ₂ O. As described above, bound PTX3 was measured by a modified PTX3 ELISA ( FIG. 8B ). The formed PTX3/iHA complex was stable and resistant to treatment with various dissociation agents and/or reducing agents. No statistical significance was seen between all groups.

Simultaneous binding of TSG-6 and PTX3 to iHA

iHA (2 μg/well input) was prepared as described above. 6 μg/ml of TSG-6 and 5 μg/ml of PTX3 (maximum binding concentration as described above) were incubated with iHA alone or in combination in a reaction buffer (PBS containing 5 mM MgCl ₂ , pH 7.5). The bound TSG-6 or PTX3 was measured by respective improved ELISAs as described above. Compared to when TSG-6 or PTX3 was added alone, when the two proteins were incubated with iHA simultaneously, there was no competition or synergy between TSG-6 or PTX3 and the binding of iHA (p>0.05). These data indicate that the TSG-6 and PTX3 binding sites on iHA are different and may not overlap ( FIG. 9 ).

Sequential binding of TSG-6 and PTX3 to iHA

TSG-6 and PTX3 were added to iHA in an orderly manner to determine whether pre-bound TSG-6 or PTX3 would inhibit the binding of other proteins to iHA. 6 μg/ml of TSG-6 or 5 μg/ml of PTX3 were pre-bound to iHA prepared as described above. After washing with 8M GnHCl and PBS, a series of concentrations of PTX3 (0, 5, or 5 μg/ml) or TSG-6 (0, 1.2, or 6 μg/ml) were then incubated with pre-bound TSG-6/iHA or pre-bound PTX3/iHA in reaction buffer (PBS, pH 7.5 containing 5mM MgCl ₎ . TSG-6 and PTX3 subsequently bound were measured by respective improved ELISAs.

Pre-bound TSG-6 (6 μg/ml) partially blocked subsequent PTX3 binding to iHA ( FIG. 10A ) (p = 0.05 and 0.01 when subsequent PTX3 was added at 1 μg/ml and 5 μg/ml, respectively) ( FIG. 5A ). Pre-bound PTX3 (5 μg/ml) did not interfere with subsequent TSG-6 binding to iHA (p = 0.56 and 0.74 when subsequent TSG-6 was added at 1.2 μg/ml and 6 μg/ml, respectively) ( FIG. 10B ). These data suggest that iHA undergoes structural changes upon TSG-6 binding, thereby interfering with subsequent PTX3 binding.

Example 5: Adhesion of LPS-stimulated macrophages to immobilized TSG-6/iHA and PTX3/iHA complexes

As described above, 96 wells of Covalink-NH were covalently coupled with PBS (control), HA (iHA), or native HC-HA/PTX3 (nHC-HA/PTX3). TSG-6 (6 μg/ml) or PTX3 (5 μg/ml) were then added and allowed to bind to iHA. RAW264.7 macrophages (100 μl, 1×10 ⁵ cells/ml) in DMEM/10% FBS were seeded into each coupled well and treated with 1 μg/ml LPS. After incubation for 24 hours, cell morphology was photographed.

When compared to plastic controls, macrophages attached poorly to iHA (i.e., resulting in more aggregated cells). TSG-6/iHA further impeded this attachment, resulting in numerous and larger cell aggregates. In contrast, PTX3/iHA promoted cell attachment, resulting in a pattern similar to that shown on immobilized nHC-HA/PTX3 ( FIG. 11 ).

Example 6: Regulation of M1 and M2 markers by TSG-6/iHA and PTX3/iHA complexes

RAW264.7 cells were cultured as described in Example 5 and stimulated for 4 hours with 1 μg/ml LPS in PBS (control), immobilized HA (iHA), TSG-6/iHA, PTX3/iHA, or nHC-HA/PTX3. Total RNA was isolated from the cells, and mRNA expression of the M2 marker IL-10 and the M1 marker IL-12p40 was measured by quantitative PCR as described above (Figures 12A and 12D). Alternatively, cells were stimulated for 24 hours with 1 μg/ml LPS (Figures 12B and 12E) or IFN-γ/LPS (Figure 12C), and protein expression of IL-10, IL-12p70, and IL-23 was measured in the cell culture medium using respective ELISAs.

Compared to the expression of the control (Ctrl), the expression of IL-12p40 mRNA (one of the two subunits of IL-12p70) on iHA (none) did not change significantly (p>0.05) (Figure 12A). In contrast, IL-12p40 mRNA was significantly upregulated on TSG-6/iHA (p<0.01), but significantly downregulated on PTX3/iHA and nHC-HA/PTX3 (p<0.05) (Figure 12A). However, the expression of IL-12p70 protein was only detectable on the control or iHA alone without any significant difference (p>0.05), but was not detected on TSG-6/iHA, PTX3/iHA and nHC-HA/PTX3 (Figure 12B). IL-12p40 also acts as a subunit of IL-23. It was observed that IL-23 protein expression was significantly upregulated on TSG-6/iHA and PTX3/iHA (p < 0.01), but was not detected on nHC-HA/PTX3 (p < 0.05) (Figure 12C). These data indicate that both TSG-6/IHA and PTX3/iHA effectively inhibit IL-12 but not IL-23.

Compared to the control, IL-10 mRNA expression in RAW264.7 cells was not significantly altered on iHA alone (p>0.05), but was significantly upregulated on TSG-6/iHA, PTX3/iHA, and nHC-HA/PTX3 (p<0.05) (Figure 12D). However, similar to the positive control of immobilized nHC-HA/PTX3, IL-10 protein expression was only significantly upregulated on PTX3/iHA (p<0.05) (Figure 12E). These data indicate that following different modes of cell attachment (Example 5), the resulting cells exhibit different functions, and that PTX3/iHA is more effective than TSG-6/iHA in upregulating IL-10.

Example 7: In vitro transfer of HCl and HC2 from IαI to immobilized HA

As described above, Covalink-NH 96 wells were covalently coupled to PBS (control), HA (iHA), or native HC-HA/PTX3 (nHC-HA/PTX3). A series of TSG-6 concentrations (0, 0.24, 1.2, 6, 12 μg/ml in 100 μl) were incubated separately with iHA in reaction buffer (PBS containing ₅ mM MgCl2, pH 7.5). Human IαI (5 μg/ml) (prepared from human plasma according to Blom et al. 1999) J. Biol. Chem. 274, 298–304) was added simultaneously with TSG-6 or sequentially (after 2 hours). Bound HCl, HC2 (antibodies to HCl and HC2 were from abcam, Cambridge, MA), or IαI (DAKO, Carpinteria, CA) was measured by modified ELISAs similar to those described above for TSG-6 and PTX3.

The data show that when TSG-6 was pre-bound to iHA and then IαI was added, the amount of HCl (Figure 13A) or IαI (Figure 13B) bound to iHA was lower at higher TSG-6 concentrations (6 and 12 μg/ml). HC2 was not detected in the samples (data not shown). The wells were incubated with hyaluronidase to digest the bound HA and release the bound protein from HA, and the samples were analyzed by Western blotting with anti-TSG-6 antibodies (R&D Systems, Minneapolis, MN); the amount of TSG-6 bound to iHA was lower when IαI was added simultaneously with TSG-6 compared to when IαI was added subsequently (ie, after TSG-6 was bound to iHA, Figure 13C). When 5 μg/ml of PTX3 and 5 μg/ml of IαI were incubated simultaneously with iHA, PTX3, but not IαI, bound to iHA (Figure 13D).

These data indicate that free TSG-6 in solution transfers HCl from IαI to iHA more efficiently than TSG-6 bound to iHA (Figures 13A and 13B). When TSG-6 was pre-bound to iHA alone, more TSG-6 bound to iHA than when TSG-6 and IαI were incubated with iHA simultaneously (Figure 13C), suggesting that IαI prevents TSG-6 from binding to iHA if added simultaneously with TSG-6, and that TSG-6 may have a higher affinity for binding to IαI than to iHA. In addition, PTX3, free or bound to iHA in solution, did not transfer HC from IαI to iHA (Figure 13D).

Example 8: Effect of PTX3 on the formation of HC1·TSG-6 and HC2·TSG-6 complexes

IαI (40 μg/ml) and TSG-6 (6 μg/ml) were incubated with or without PTX3 (20 μg/ml or 120 μg/ml) in reaction buffer (PBS containing 5 mM MgCl ₂ , pH 7.5) at 37° C. for 2 hours. The reaction samples were analyzed by Western blotting using antibodies against HCl ( FIG. 14A ), HC2 ( FIG. 14B ), TSG-6 ( FIG. 14C ), urotrypsin (abcam, Cambridge, MA) ( FIG. 14D ), and PTX3 (data not shown). In a solution without HMW HA, TSG-6 formed HCl·TSG-6 and HC2·TSG-6 complexes and produced HMW IαI ( FIG. 14A and FIG. 14B ). The formation of HMW IαI is shown in FIG. 14E . This data indicates that both HCl and HC2 are transferred to HMW HA by TSG-6 in solution.

The simultaneous addition of PTX3 dose-dependently inhibited the formation of HC2·TSG-6, but not HC1·TSG-6 (Figures 14A and 14B). In contrast, HMW IαI containing HC2 increased while HMW IαI containing HC1 decreased (Figures 14A and 14B). When TSG-6 was added to a solution without HMW HA and with or without PTX3, TSG-6 formed a dimer (Figure 14C). These results indicate that: 1) HC1 and HC2 in IαI either form a complex with TSG-6 or form HMW IαI through the action of TSG-6; 2) PTX3 interferes differently with the transfer of HC1 and HC2 by TSG-6. There are no truncated forms of HC1 or HC2. Figure 14F shows the inhibition of HC2·TSG-6 by PTX3.

TSG-6 and PTX3 release glycosylated and glycosylated forms of uropancreatin of approximately 40 kDa and 45 kDa, respectively (Figure 14D). This data is consistent with published data, indicating that both TSG-6 and PTX3 interact with IαI, and also indicates that TSG-6 and PTX3 interact differently with IαI, resulting in different consequences for uropancreatin.

In a separate experiment, HMW HA (250 μg/ml), IαI (40 μg/ml), and TSG-6 (6 μg/ml) were incubated with or without PTX3 (1, 2.5, and 5 μg/ml) in a reaction buffer (PBS, pH 7.5 containing 5 mM MgCl ₂ ) at 37° C. for 24 hours. Reaction samples were analyzed by Western blotting using an anti-IαI antibody ( FIG14G ). In solutions containing HMW HA but not PTX3, HC from IαI was completely transferred to HMW HA by TSG-6. In the presence of PTX3, TSG-6-mediated HC transfer was dose-dependently inhibited, resulting in the accumulation of LMW intermediates (~130 kDa and likely composed of HC1-TSG-6) or unprocessed pre-IαI (~130 kDa, unprocessed IαI (220 kDa), and HMW IαI (remaining in the well) ( Figure 14G ). These data are consistent with the results that PTX3 specifically prevents the formation of HC2-TSG-6, leading to the inhibition of HC2 transfer and possibly HC1 transfer.

Example 9: Formation of reconstituted HC-1 from immobilized HA in vitro with simultaneous addition of TSG-6, PTX3, and IαI HA/PTX3 (rcHC-HA/PTX3) complex

Immobilized HA (~14 μg/ml in each well or 1.4 μg in 100 μl) was prepared as described in Example 3. IαI (5 μg/ml) and TSG-6 (12 μg/ml) were simultaneously incubated on iHA with or without PTX3 (1, 5, or 20 μg/ml) in reaction buffer (PBS containing ₅ mM MgCl2, pH 7.5) at 37°C for 2 hours. After washing with 8M GnHCl and PBS, bound HCl, TSG-6, and PTX3 were measured by respective modified ELISAs (Figures 15A, 15D, and 15F, respectively). Each well was washed again with 8M GnHCl and PBS, and iHA with bound components was digested with 1 unit/ml of hyaluronidase at 60°C for 2 hours in 10 mM acetate buffer (containing 75 mM NaCl, pH 6.0). Samples were analyzed by Western blotting using antibodies against HCl (FIG. 15B), HC2 (FIG. 15C), TSG-6 (FIG. 15E), and PTX3 (FIG. 15G).

Simultaneous addition of TSG-6, PTX3, and IαI to iHA resulted in rcHC-HA/PTX3 complexes containing HMW HCl without HC2 and truncated HCl and HC2 (Figures 15A-C). PTX3 dose-dependently reduced the amount of HMW HCl in the complex. These data show that PTX3 dose-dependently interfered with TSG-6 transfer of HCl and HC2 to iHA (Figures 15A-C), resulting in less HCl/truncated HCl and truncated HC2 (Figures 15B and 15C). TSG-6 monomers decreased while HMW TSG-6 (either multimeric or complexed with PTX3 and/or HC) remained unchanged (Figures 15D and E).

The data also demonstrate that PTX3 does not interfere with TSG-6 binding to iHA in the presence or absence of IαI. Published data demonstrate that TSG-6 forms dimers with iHA when smaller MW HA is tested (Baranova et al. (2011) J Biol Chem. 286(29):25675-86). The data presented herein demonstrate that TSG-6 complexes with the HMW HC-HA/PTX3 complex in the presence of IαI. Since PTX3 reduces free TSG-6 in a dose-dependent manner, this further demonstrates that PTX3 promotes TSG-6 binding to the HC-HA/PTX3 complex in the presence of IαI. In this case, the majority of PTX3 is present in the HC-HA/PTX3 complex as multimers, similar to what has been observed with NHC-HA/PTX3, with decreasing amounts of monomers, dimers, or trimers (Figures 15F and G).

Example 10: Sequential addition of PTX3 affects the reconstitution of TSG-6 and IαI on immobilized HA in vitro Effects of HC-HA/PTX3 (rcHC-HA/PTX3) complexes

Immobilized HA (~14 μg/ml) was prepared as described in Example 3. IαI (5 μg/ml) and TSG-6 (12 μg/ml) were incubated on iHA in reaction buffer (PBS containing 5 mM _MgCl2 , pH 7.5) at 37°C for 2 hours. After removing unbound IαI and TSG-6, reaction buffer containing or not containing PTX3 (1, 5 or 20 μg/ml) was incubated with pre-bound HC and TSG-6 at 37°C for 2 hours. After washing with 8M GnHCl and PBS, the bound HCl, TSG-6 and PTX3 were determined by ELISA (Figures 16A, 16D and 16F, respectively). The wells were then washed again with 8M GnHCl. The PBS control and iHA with bound components were then digested with 1 unit/ml of hyaluronidase at 60°C for 2 hours. The samples were analyzed by Western blotting using antibodies against HCl ( FIG. 16B ), HC2 ( FIG. 16C ), TSG-6 ( FIG. 16E ), and PTX3 ( FIG. 16G ).

When PTX3 was added after TSG-6 and HC were pre-bound to iHA, PTX3 dose-dependently reduced the transfer of HCl to the HMW complex (both intact and truncated HCl were reduced), but increased the amount of truncated HC2 in the complex. Consistent with the data shown in Example 7, bound TSG-6 was less efficient than free TSG-6 in transferring HC to iHA.

Similar to the data shown in Example 8, PTX3 also dose-dependently reduced HMW TSG-6 and monomeric TSG-6 (Figures 16D and 16E), indicating that subsequent addition of PTX3 continuously depleted pre-bound TSG-6. However, PTX3 was no longer able to be incorporated into the TSG-6/HC-HA complex (Figures 16F and 16G). Because pre-bound TSG-6 in iHA also partially prevented PTX3 from binding to iHA (see Example 4), this finding suggests that the rcHC-HA/PTX3 complex formed by TSG-6 and IαI is structurally different from TSG-6/iHA to the extent that it completely excludes PTX3 binding to iHA.

Example 11: In vitro formation of reconstituted PTX3 by pre-bound PTX3 on immobilized HA and subsequent addition of TSG-6 and IαI HC-HA/PTX3 (rcHC-HA/PTX3) complex

Immobilized HA (~14 μg/ml) was prepared as described in Example 3. PTX3 (5 μg/ml) and iHA were incubated in reaction buffer (PBS containing ₅ mM MgCl2, pH 7.5) at 37 ° C for 2 hours. After removing unbound PTX3, reaction buffer containing TSG-6 (6 μg/ml) and IαI (5, 25 and 125 μg/ml) was incubated at 37 ° C for 2 hours. After washing with 8M GnHCl and PBS, the bound HCl, TSG-6 and PTX3 (17A, 17C and 17E, respectively) were determined by ELISA. The wells were then washed again with 8M GnHCl. The PBS or iHA with the bound components were then digested with 1 unit/ml of hyaluronidase in 10 mM acetate buffer (pH 6.0) containing 75 mM NaCl at 60 ° C for 2 hours. Samples were analyzed by Western blotting using antibodies against PTX3 ( FIG. 17B ), TSG-6 ( FIG. 17D ), HCl ( FIG. 17F ), and HC2 ( FIG. 17G ).

In the presence of IαI and TSG-6, pre-bound PTX3 dose-dependently increased ELISA immunoreactivity to PTX3 and the amount of multimeric PTX3, but reduced the amount of monomeric PTX3 in the HC-HA/PTX3 complex ( Figures 17A and 17B ). This data suggests that multimeric PTX3 promotes immune reactivity against this antibody.

Pre-bound PTX3 dose-dependently excluded monomeric TSG-6 and reduced TSG-6 in the rcHC-HA/PTX3 complex (Figures 17C and 17D). When the molar ratio of IαI to TSG-6 was 3:1, a significant reduction in bound TSG-6 (both monomeric and HMW forms) was detected, among which the bound multimeric PTX3 was also maximized.

Based on HCl ELISA data, there was no significant change in bound HCl ( FIG17E ). HC2 transfer was dose-dependently increased by increasing IαI concentrations.

Example 12: Reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) Complexes Formed in Vitro by Pre-bound TSG-6 Comparison of macrophage attachment activity between the complex and pre-bound PTX3 on immobilized HA

Covalink-NH 96 wells were covalently coupled to PBS (control), HA (iHA), or nHC-HA/PTX3 as described in Example 3. IαI (5 μg/ml), TSG-6 (6 μg/ml), or PTX3 (5 μg/ml) were conjugated to iHA simultaneously or sequentially as follows: (1) (IαI/TSG-6/PTX3)/iHA: IαI, TSG-6, and PTX3 were incubated simultaneously with iHA in reaction buffer at 37°C for 2 hours; (2) (IαI/TSG-6)/PTX3/iHA: IαI and TSG-6 were first incubated with iHA in reaction buffer at 37°C for 2 hours. After removing unbound IαI/TSG-6 and washing with 8M GnHCl and PBS, PTX3 was added and incubated in reaction buffer at 37°C for 2 hours; (3) (PTX3)/IαI/TSG-6/iHA: PTX3 was first incubated with iHA in reaction buffer at 37°C for 2 hours. After removing unbound PTX3 and washing with 8M GnHCl and PBS, IαI/TSG-6 was added and incubated in reaction buffer at 37°C for 2 hours. After complex formation, 100 μl of RAW264.7 cells (1×10 ⁵ cells/ml) were seeded into each coupled well and treated with 1 μg/ml LPS. After incubation for 24 hours, cell morphology was photographed.

As with the control, macrophages attached poorly to iHA. In the presence of IαI, TSG-6 and iHA simultaneously or TSG-6 pre-bound to iHA ((IαI/TSG-6/PTX3)/iHA or (IαI/TSG-6)/PTX3/iHA) inhibited cell attachment and promoted cell aggregation ( FIG. 18 ), similar to the absence of IαI (see Example 5). Conversely, PTX3 pre-bound to iHA [(PTX3)/IαI/TSG-6/iHA] promoted cell attachment, similar to pre-bound PTX3 without IαI as shown in Example 5. The latter was similar to the positive control of nHC-HA/PTX3 ( FIG. 18 ).

Example 13: Reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) Complexes Formed in Vitro by Pre-bound TSG-6 Comparison of the modulation of M1 and M2 marker expression between the complex and pre-bound PTX3 on immobilized HA

Expression of IL-10 and IL-12p40 in macrophages cultured on rcHC-HA/PTX3 complexes

As described in Example 12, RAW264.7 cells were cultured on immobilized substrates in DMEM/10% FBS and stimulated with 1 μg/ml LPS for 4 hours. Total RNA was isolated, and IL-10 and IL-12p40 mRNA expression was measured by quantitative PCR as described above ( Figures 19A and 19C ). Alternatively, cells were stimulated with 1 μg/ml LPS for 24 hours, and IL-10 and IL-12p70 protein were measured in the cell culture supernatants by respective ELISAs ( Figures 19B and 19D ).

Compared to the PBS control, iHA did not significantly alter IL-10 mRNA expression (p = 0.56), but was significantly upregulated in the complex formed by the simultaneous addition of TSG-6, IαI, and PTX3 to iHA (IαI/TSG-6/PTX3 (a) in Figure 19) (p = 0.0008). Similarly, IL-10 mRNA expression was significantly upregulated in the complex formed by pre-binding TSG-6 to iHA and then adding IαI and PTX3 (IαI/TSG-6/PTX3 (b) in Figure 19) (p = 0.04) and in the positive control nHC-HA/PTX3 (p = 0.008). IL-10 mRNA expression was significantly higher on nHC-HA/PTX3 than on IαI/TSG-6/PTX3 (a) (p=0.04), but not significantly higher on IαI/TSG-6/PTX3 (b) (p=0.55). In contrast, IL-10 mRNA expression was not significantly upregulated on the complex formed by pre-bound PTX3 to iHA (IαI/TSG-6/PTX3 (c) in Figure 19) (p=0.74) (Figure 19A). As measured by ELISA, IL-10 protein expression was significantly upregulated only by nHC-HA/PTX3 (p=0.03) (Figure 19B).

Compared to the control, iHA did not significantly change the expression of IL-12p40 (IL-12p40 is one of the two subunits of IL-12p70, and the other subunit is IL-12p35) mRNA (p = 0.1). In contrast, the expression of IL-12p40 mRNA was significantly upregulated in the complex formed by the simultaneous addition of TSG-6, IαI and PTX3 to iHA (IαI/TSG-6/PTX3 (a) in Figure 19) (p = 0.05) and the complex formed by TSG-6 pre-bound to iHA (IαI/TSG-6/PTX3 (b) in Figure 19) (p = 0.04). In contrast, the expression of IL-12p40 mRNA was completely abolished in the complex formed by pre-bound PTX3 (IαI/TSG-6/PTX3 (c) in Figure 19) and was significantly downregulated by nHC-HA/PTX3 (p = 0.01). There was a statistically significant difference between the latter two conditions (p = 0.04) (Figure 19C). Compared to the control, the expression of IL-12p70 protein was not significantly changed by iHA (p = 0.32), but was significantly downregulated on the complex formed by pre-bound PTX3 (IαI/TSG-6/PTX3 (c)) (p = 0.03) (Figure 19D). In contrast, the expression of IL-12p70 protein was eliminated on the complex formed by adding TSG-6, IαI and PTX3 to iHA at the same time (IαI/TSG-6/PTX3 (a)), on the complex formed by pre-bound PTX3 (IαI/TSG-6/PTX3 (c)) and on nHC-HA/PTX3 (p = 0.05, 0.02 and 0.01 respectively).

Expression of 1L-23 in macrophages cultured in the presence of various stimuli

In separate experiments, IL-23 protein was measured in the cell supernatants of resting RAW264.7 cells (none) or cells stimulated for 24 hours in DMEM/10% FBS with IFN-γ (200 units/ml), LPS (1 μg/ml), IFN-γ/LPS, LPS (1 μg/ml) containing immune complexes or IC (LPS/IC) [the IC contained 150 μg/ml IgG-opsonized OVA (IgG-OVA) and was prepared by mixing a ten-fold molar excess of rabbit anti-OVA IgG (Cappel, Durham, NC) with OVA (Worthington Biochemical Corp., Lakewood, NJ) at 25°C for 30 minutes] or IL-4 (10 ng/ml) (R&D Systems, Minneapolis, MN). IL-23 protein was measured in cell culture supernatants by IL-23 ELISA (Biolegend, San Diego, CA) according to the manufacturer's protocol (Figure 19E). IL-23 protein was not detected in the cell culture supernatants of resting RAW264.7 cells and in the cell culture supernatants of cells stimulated for 24 hours with LPS (1 μg/ml), LPS containing immune complexes (LPS/IC), or IL-4 (10 ng/ml), but became detectable after 24 hours of stimulation with IFN-γ (200 units/ml) and IFN-γ/LPS (Figure 19E).

IL-23 expression in macrophages cultured on rcHC-HA/PTX3 complexes

In a separate experiment, RAW264.7 cells were cultured on immobilized substrates as described above and stimulated with IFN-γ/LPS for 24 hours. IL-23 was measured in cell culture supernatants by IL-23 ELISA as described above ( FIG. 19F ).

Compared to the control, IL-23 protein in the cell culture supernatant of RAW264.7 cells stimulated with 200 units/ml IFN-γ/1 μg/ml LPS for 24 hours was not significantly affected by iHA (p=0.02), but was significantly upregulated in the complex formed by the simultaneous addition of TSG-6, IαI, and PTX3 to iHA (IαI/TSG-6/PTX3(a)) (p=0.002) and in the complex formed by TSG-6 pre-bound to iHA (IαI/TSG-6/PTX3(b)) (p=0.0005). In contrast, similar to nHC-HA/PTX3 (p=0.05), IL-23 protein was completely eliminated in the complex formed by pre-bound PTX3 (IαI/TSG-6/PTX3(c)) (p=0.05) (Figure 19F).

Example 14: Use of HC-HA/PTX3 for the treatment of chronic graft-versus-host disease

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for hematological malignancies. However, chronic graft-versus-host disease (cGVHD) remains a major complication. GVHD causes ocular manifestations in 45-60%, with dry eye being the most common complication, occurring in nearly 50% of allogeneic HSCT recipients. In fact, dry eye is a distinguishing sign and symptom for the diagnosis of cGVHD. Patients with cGVHD either present with early, mild dry eye associated with cGVHD or with so-called "distinguishing features of cGVHD" according to the NIH consensus conference classification. Two types of dry eye have been noted after HSCT; one with severe damage to the ocular surface and tear function, in which reflex tearing occurs shortly after the onset of dry eye, while the other is mild with normal reflex tearing. Dry eye usually develops 6 months after transplantation, and its severity has been reported to be associated with the presence of cGVHD and meibomian gland disease. The onset of severe dry eye associated with cGVHD is earlier than that of mild dry eye. For example, severe dry eye developed 6.8 ± 2.5 months after HSCT, whereas mild dry eye developed 13.2 ± 9.1 months after HSCT. A comparative study of 50 eyes of 25 post-HSCT patients and 28 eyes of 14 age-matched healthy controls showed that MG obstruction, decreased corneal sensitivity, enhanced tear evaporation rate, decreased conjunctival GCD, increased conjunctival squamous metaplasia, and inflammatory cells were more frequently noted in cGVHD-associated dry eye than in normal controls and subjects without dry eye after HSCT. In addition, conjunctival inflammatory cells were significantly higher in severe dry eye than in mild dry eye (P < 0.03). Moreover, the majority of patients with severe dry eye had systemic cGVHD, whereas only a minority of patients in the mild dry eye group had systemic cGVHD. These findings suggest different pathological processes in severe and mild dry eye associated with cGVHD. Due to the comprehensive ocular surface changes noted in patients (regardless of whether they suffer from the dry eye associated with cGVHD) after HSCT, their results show that the degree of inflammatory process seems to play a key role in the result of the dry eye associated with cGVHD. Compared with the subject without dry eye after normal control and HSCT, in the severe dry eye associated with cGVHD and mild dry eye patients, conjunctival brush cytology specimens show the inflammatory cell number significantly increased. Moreover, the number of inflammatory cells in severe dry eye specimens is significantly higher than the inflammatory cell number in mild dry eye specimens. In addition, many inflammatory markers are expressed in the biopsy samples of the conjunctiva and lacrimal gland of the dry eye patient associated with cGVHD, it is confirmed that inflammation is relevant to the pathogenesis of the dry eye associated with cGVHD.

A possible reason for producing multiple scarring complications in cGVHD is the EMT of the conjunctival basal epithelium and the lacrimal gland myoepithelial cell due to infiltration of donor lymphocytes by the cytokines released via chronic inflammation. Previously, it has been recognized that inflammation and excessive fibrosis are the significant histological features of chronic graft-versus-host disease (cGVHD), but the basic mechanism of these changes is still unknown. cGVHD shows a feature similar to scleroderma, showing significant fibrosis, pulmonary fibrosis and chronic immunodeficiency in skin damage. The clinical symptoms of eye cGVHD include the outbreak of dry eye, trachoma or eye pain, cicatricial conjunctivitis including subconjunctival fibrovascular tissue formation, and sclera shortening (it is the unique feature of conjunctival fibrosis). Except the sclerotic feature in skin damage, the mucosal atrophy in mouth, the narrowing or stenosis of the upper third to middle third of the esophagus, the joint stiffness or contracture caused by sclerosis and the obliterative bronchitis of lung also show the fibrotic feature of systemic GVHD-mediation together. The main histological findings in the affected exocrine glands and mucosa are the significant increase in the number of obvious fibrosis and fibroblasts in the interstitium, with mild lymphocyte infiltration. Clinically, the severity of dry eye is related to the degree of fibrosis change, but not to the degree of lymphocyte infiltration, which shows that excessive extracellular matrix accumulation mainly causes exocrine dysfunction. Fibroblasts in the interstitium also play a role in inflammation by attaching to lymphocytes and expressing class II human leukocyte antigens and co-stimulatory molecules. These findings together show that fibroblasts play an important role in the pathogenesis of cGVHD. Moreover, we have found that the fibroblasts accumulated in the lacrimal glands of cGVHD patients have a mosaic state. Therefore, fibroblasts originating from circulating donor-derived precursors and fibroblasts derived from receptors can participate in the excessive fibrosis in cGVHD patients by interacting T cells. It is not clear whether controlling inflammation by inhibiting T cell infiltration will lead to less cicatricial complications in cGVHD.

Previously, by combining immunohistochemistry with Y-chromosome fluorescence in situ hybridization (FISH) method to detect donor-derived fibroblasts in human cGVHD tissue samples. Using the mouse model established by Zhang et al. ((2002) J Immunol.168:3088-3098), the above results can be reproduced. In this model, the tear volume begins to decrease 3 weeks after transplantation. Early fibrosis and progressive fibrosis around the lacrimal duct were detected as early as 3 weeks after transplantation and gradually progressed in a manner similar to that of human samples, up to 8 weeks. In order to successfully create GVHD groups and control groups, we have performed this experiment more than 20 times, and based on the analysis of lacrimal tissue samples and tear volume, the total reproducibility was 70-80%.

In a typical transplantation experiment, 7 to 8 week old male and female B10.D2 (H-2d) and BALB/c (H-2d, Sankyo Laboratory, Ltd) mice were used as donors and recipients, respectively, using added splenocytes as a source of mature T cells. In brief, female recipient mice received lethal radiation of 700 cGy from a Gammacel 137Cs source (JL Shepherd & Associates, San Fernando, CA). About 6 hours later, male recipient bone marrow (1 × 10 ⁶ / mouse) and spleen (2 × 10 ⁶ / mouse) cells suspended in RPMI1640 (BioWhittaker, Walkersville, MD) were injected into the tail vein. The control group (syngeneic BMT) consisted of female BALB/c recipient mice that received the same amount of female BALB/c spleen cells and bone marrow cells. (Zhang et al. (2002) J Immunol. 168: 3088-3098). For HC-HA/PTX3 treatment, the HC-HA/PTX3 complex is administered by subconjunctival injection at predetermined times after bone marrow transplantation, such as 7, 14, 21, and 28 days after bone marrow transplantation.

The efficacy of treatment was assessed using tests including, but not limited to, Mallory staining to measure lacrimal gland fibrosis, determination of the number of activated fibroblasts per field using HSP47 (a collagen-specific molecular chaperone) as a marker of activated fibroblasts, measurement of lacrimal gland tear production under pilocarpine stimulation using the cotton thread test, and determination of the levels of fibrogenic cytokines such as HSP47, IL-4, IL-6, and TGF-β using RT-PCR.

It is expected that treatment with the HC-HA/PTX3 complex will lead to a reduction in lacrimal gland fibrosis in the mouse model. The HC-HA/PTX3 complex can then be administered to human subjects via subconjunctival injection in clinical settings for the treatment of dry eye caused by cGVHD.

In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3). In some examples, the nHC-HA/PTX3 is isolated from umbilical cord tissue. In some examples, the nHC-HA/PTX3 is isolated from amniotic membrane. In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is a reconstituted HC-HA/PTX3 complex (rcHC-HA/PTX3).

Example 15: Use of HC-HA/PTX3 in treating inflammation in a mouse model

In this example, anti-inflammatory efficacy was tested in a murine model of HSV-1 necrotizing stromal keratitis. A total of 240 female BALB/c mice (6-8 weeks old) obtained from Charles River Wiga (Sulzfeld, Germany) were anesthetized by intraperitoneal injection of 2 mg of ketamine hydrochloride and 400 ng of mepivacaine hydrochloride. For each mouse, the central cornea of one eye was scratched using a 27-gauge needle under a surgical microscope with a staggered pattern of 8 horizontal and 8 vertical scratches. 5 μl of a suspension containing 1×10 ⁵ plaque-forming units of HSV-1 virus (KOS strain), which had been routinely propagated in Vero cells and stored at -80°C, was applied to each injured cornea and quantified by a standard plaque-forming assay. On day 14 after HSV-1 inoculation, mouse corneas that had developed severe ulcerative stromal keratitis were included in the study (approximately 50% yield) and subdivided into 3 groups, each including 40 corneas (n=6 for clinical examination, n=5 for histology, n=5 for immunostaining, n=6 for cytokine ELISA, n=5 for TUNEL, and n=10 for flow cytometry, and n=3 for depletion/backup). Uninfected contralateral eyes served as negative controls. The positive control group received tarsal sutures to close the eyelids using 210-O nylon sutures. The experimental HC-HA/PTX3 group received the same tarsal sutures as the positive control and received topical application of a composition comprising a purified HC-HA/PTX3 complex 4 times daily. The experimental HA group received the same tarsal sutures, but received topical application of a composition containing only HA 4 times daily. After two days, tarsal sutures were canceled in all three groups. The severity of stromal inflammation of each cornea was assessed using an operating microscope (Zeiss, Germany) on a scale of 0-4+, with 1+ being less than 25% corneal opacity with corneal neovascularization, edema, and thinning, 2+ being less than 50%, 3+ being less than 75%, and 4+ being 75-100%. After euthanasia by _CO2 chamber followed by cervical dislocation, 5 corneas from each group were immunostained for frozen sections using primary antibodies against CD11b (neutrophils and macrophages), F4/80 (macrophages), GR-1 (PMN), and CD3 (T cells) (see Methods), and another 5 corneas from each group were stained with hematoxylin-eosin and TUNEL. In addition, corneal homogenates prepared from 6 corneas from each group were subjected to ELISA assays for IL-1α, IL-2, IL-6, IFN-γ, and TNFα levels. Cells released with collagenase from 10 corneas per group were prepared for flow cytometry to quantify viable cells by MTT assay and apoptotic cells by Annexin V-PE apoptosis detection kit (BD-Pharmingen, Heidelberg, Germany).

It is expected that within two weeks after inoculation, 50% of the HSV-1 infected corneas of the mice included in the study will develop severe stromal keratitis (inflammation), edema, and ulcers. After two days, the uninfected corneas will remain normal, while the infected corneas in the control group will maintain similar severe inflammation when the tarsorrhaphy is removed. Similar to the control group, the corneas in the experimental HA group will show similar severe inflammation. In contrast, the corneas of the experimental HC-HA/PTX3 group showed reduced inflammation, which was correlated with and confirmed by the following results: compared with the positive control and experimental HA groups, based on histology and CD11b, F4/80, Gr-1 and CD3 immunostaining, there was a significant decrease in inflammatory (PMN/macrophage) and immune (T-cell) infiltration; based on ELISA, there was a significant decrease in inflammatory and immune cytokines such as IL-1α, IL-2, IL-6, IFN-γ and TNF-α; and a significant increase in TUNEL-positive cells in corneal tissue and in the release of dead cells (MTT) and apoptotic cells from the cornea using collagenase (flow cytometry using Annexin-V/7-AAD). Overall, these data support the concept that the HC-HA/PTX3 complex exerts clinical anti-inflammatory efficacy in this murine HSV-1 model.

In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3). In some examples, the nHC-HA/PTX3 is isolated from umbilical cord tissue. In some examples, the nHC-HA/PTX3 is isolated from amniotic membrane. In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is a reconstituted HC-HA/PTX3 complex (rcHC-HA/PTX3).

Example 16: Use of HC-HA/PTX3 to inhibit scar formation in a rabbit model

In this embodiment, the anti-scarring efficacy was tested in a rabbit model of excimer laser-assisted photorefractive keratectomy (PRK). A total of 30 New Zealand white rabbits (either sex) weighing 2.5-3.0 kg (BW) were used and subdivided into 3 groups (n=10 per group): a non-PRK control group, a PRK HA group, and a PRK HC-HA/PTX3 group. Before PRK and for all CMTF examinations, rabbits were anesthetized by intramuscular injection of 5 mg/kg BW xylazine and 30 mg/kg BW ketamine and topical application of 0.5% tetracaine hydrochloride ophthalmic solution (Ortopics Laboratories Corp., Fairton, NJ). For both PRK groups, the corneal epithelium of one eye of each animal was manually removed by gently scraping with a blunt spatula in an area just larger than the ablation zone, the exposed stroma was rinsed with saline, and excess fluid was gently removed with a cellulose sponge. Standard 6 mm diameter, 9.0 D PRK myopia correction PRK was performed using a LaddarVision excimer laser (Alcon, Ft. Worth, TX) to achieve a predicted theoretical stromal ablation depth of 118 μm. Immediately after and following PRK, the PRK HC-HA/PTX3 group was administered a composition containing the HC-HA/PTX3 complex, while the PRK HA group was administered a composition containing only HA, both groups being administered four times daily for three weeks. In addition, all PRK-treated eyes received topical 0.1% diclofenac sodium (one drop immediately after PRK) and 0.3% gentamicin sulfate (three times daily for three days).

In vivo CMTF was performed on all postoperative eyes (n=6 from each group) before PRK and one, two, three and four weeks, two and four months after PRK using a modified Tandem scanning confocal microscope (Tandem Scanning Corporation, Reston, VA) equipped with a 24X surface contact objective. After standard confocal examination of corneal morphology, the camera settings (gain, kilovolts and black plane level) were switched to manual and kept unchanged during the study to allow direct comparison of all scans. CMTF was performed as a continuous z-axis scan through the entire cornea. Corneal, epithelial and stromal thicknesses were mapped to the central 3 mm area by performing 10 continuous CMTF scans in an area covering all regions. Subsequent calculations used only data obtained from the thinnest stromal area corresponding to the center of the photoablation profile. A CMTF atlas based on image intensity depth was generated from the CMTF video recordings. Corneal reflectivity was measured by the CMTF atlas and expressed in arbitrary units (U), which are defined as μm*pixel intensity, as an estimate of corneal opacity.

To identify and measure the presence of stromal fibrotic tissue, three PRK-treated animals from each group (control and treatment) were vitally stained with 0.5% 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) dissolved in 0.2 M sodium bicarbonate as previously reported. After 2 minutes of staining, the eyes were thoroughly rinsed to remove excess dye before topical antibiotic administration. Four months after PRK, animals were euthanized by intravenous injection of sodium pentobarbital (120 mg/kg BW). After euthanasia, all corneas were fixed in situ by anterior chamber perfusion with PBS (pH 7.2) containing 2% paraformaldehyde for 3 minutes, excised, placed in fresh fixative, and stored at 4°C. The tissue was then embedded in OCT, snap-frozen in liquid nitrogen, and sectioned using a freezing microtome. The tissue was serially sectioned to identify the central and deepest portions of the photoablation and immunostained with antibodies against keratin, CD3434, FITC-conjugated phalloidin, ED-A fibronectin, S-100A4, and α-smooth muscle actin (α-SMA) to correlate changes in corneal opacity (based on CMTF light reflectance) with phenotypic changes from keratocytes to fibroblasts and myofibroblasts. In addition, in eyes stained with DTAF, the thickness of the deposited fibrotic tissue was measured by determining the distance between the basal epithelial cells and the DTAF-stained corneal tissue (representing the original, undamaged corneal stroma).

It is expected that in vivo confocal microscopy will reveal characteristic epithelial, basal, stroma and endothelial features that will be well correlated with well-defined peaks whose intensity and position change over time when analyzing the in vivo CMTF-atlas for non-PRK controlled corneas and corneas that receive PRK. According to published data, 1 week after PRK, the PRK-treated cornea will show four peaks from the surface epithelium, the photoablated stroma surface, the spindle-shaped fibroblast layer and the endothelium, while the non-PRK controlled cornea will show three peaks from the surface epithelium, the basal layer and the endothelium. It is expected that there will not be much difference between the two experimental groups 1 week after PRK. 2 weeks after PRK, the experimental PRK HA group will show an enhanced peak intensity close to the photoablated stroma surface due to the continued cell migration of spindle-shaped fibroblasts. However, it is expected that in the experimental PRK HC-HA/PTX3 group, the intensity of the repopulated fibroblasts (based on the height of the peak) will be greatly reduced. Between three weeks and four months after PRK, in the experimental PRK HA group, due to the completion of the repopulation of the acellular pre-stroma, the peak corresponding to the layer of spindle-shaped fibroblasts will merge with the peak derived from the photoablated stromal surface, and will result in a sharp increase in the reflectivity of the peak corresponding to the photoablated stromal surface. In contrast, there will be no such sharp increase in reflectivity in the experimental PRK HC-HA/PTX3 group. This difference in light reflectivity can also be quantified by calculating the area of the CMTF peak derived from specific intracorneal structures. It is expected that the experimental PRK HA group will show a substantial linear increase in reflectivity intensity within the first 2-3 weeks after PRK, and a slow linear decrease in reflectivity thereafter. In contrast, it is expected that there will be a significant decrease in reflectivity in the experimental PRK HC-HA/PTX3 group during these two periods. Collectively, these CMTF data support that the HC-HA/PTX3 complex exerts an inhibitory effect on keratocyte activation, migration, and cell recruitment during repopulation of the acellular anterior stroma, explaining why corneal light scatter (turbidity) is reduced similarly to that previously reported with anti-TGF-β antibodies. Thus, there is lower cellularity and reflectivity of activated, migrating, intrastromal wound-healing keratocytes within the anterior stroma, less deposition of new stromal extracellular matrix, and more rapid establishment of a normal, quiescent keratocyte population. This conclusion is supported by the following results: the experimental PRK HC-HA/PTX3 group showed a significant decrease in activated keratocytes (F-actin in cells expressing keratin), fibroblasts (cytoplasmic staining of S-100A4, membrane expression of ED-A fibronectin), and myofibroblasts (nuclear expression of S100A4 and cytoplasmic expression of α-SMA) compared to the experimental PRK HA group 2-3 weeks after PRK. It was also expected that the distance between the basal epithelial cells and the DTAF-stained corneal tissue would be significantly reduced in the PRK HC-HA/PTX3 group compared to the PRK HA group, indicating significantly reduced fibrotic tissue.

In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3). In some examples, the nHC-HA/PTX3 is isolated from umbilical cord tissue. In some examples, the nHC-HA/PTX3 is isolated from amniotic membrane. In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is a reconstituted HC-HA/PTX3 complex (rcHC-HA/PTX3).

Example 17: Use of HC-HA/PTX3 for the treatment of atherosclerosis

In this example, HC-HA/PTX3 complexes produced by the methods described herein are administered for the treatment of atherosclerosis.

Atherosclerosis includes the participation of inflammatory cell colonies, especially macrophages. Phenotypic conversion of macrophages is observed in disease progression. In atherosclerosis, circulating monocytes are recruited to the fatty deposit accumulation sites in the vascular intima and subintima through a mechanism mediated by CCR2 and endothelial cell adhesion. Once arrived, these cells become activated and differentiate into macrophages. Subsequently, fatty deposits begin to mature into plaques, accompanied by the continued recruitment of inflammatory cells and smooth muscle cells and the production of extracellular matrix. The initial infiltrating macrophage population in early atherosclerosis is heterogeneous, but has a phenotype that is mainly M2-like. While the lesion progresses and expands, a conversion to a phenotype that is mainly M1 is observed. This phenotypic conversion may be due to the production of IFN-γ by macrophages that engulf excessive oxidized low-density lipoprotein (LDL) and local Th1 cells in the plaque, thereby leading to the development of foam cell macrophages. Foam cell macrophages show a highly activated phenotype, resulting in the production of proinflammatory mediators and MMPs that make plaques unstable, potentially leading to thromboembolism. Therapies that prevent the conversion of M2 to M1 macrophages or selectively deplete M1 macrophages are clinically used to stabilize atherosclerotic plaques.

The HC-HA/PTX3 complex produced by the methods described herein is administered to a subject suffering from atherosclerosis. For example, the HC-HA/PTX3 complex can be used to coat an implantable medical device, such as a stent, for implantation at or near a site of inflammation. Treatment of atherosclerosis with the HC-HA/PTX3 complex is expected to reduce inflammation and prevent thromboembolism.

In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3). In some examples, the nHC-HA/PTX3 is isolated from umbilical cord tissue. In some examples, the nHC-HA/PTX3 is isolated from amniotic membrane. In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is a reconstituted HC-HA/PTX3 complex (rcHC-HA/PTX3).

Example 18: Use of HC-HA/PTX3 for the treatment of obesity and insulin resistance

In this example, HC-HA/PTX3 complexes produced by the methods described herein are administered for the treatment of obesity and insulin resistance.

In both lean and obese states, adipose tissue macrophages (ATMs) comprise a significant proportion of the cellular components of adipose tissue. In normal individuals, ATMs constitute up to 10% of the cellular components of this tissue. In comparison, this figure rises to up to 40% in obese subjects. In normal, non-obese subjects, ATMs have a polarized M2 phenotype, characterized by elevated baseline STAT6 and PPAR-γ expression. These cells play an important and beneficial role in nutrient metabolism. The lack of PPAR-γ leads to impaired M2 macrophage function and susceptibility to diet-induced inflammation and insulin resistance. In contrast, ATMs accumulate in adipose tissue during obesity and have a strongly polarized pro-inflammatory M1 phenotype. These cells produce high levels of TNFα, IL-6, and IL-1β, all of which are observed in adipose tissue from elevated levels of insulin-resistant individuals. High levels of pro-inflammatory mediators locally impair the function of intrinsic insulin-processing cells.

The HC-HA/PTX3 complex produced by the methods described herein is administered to a subject suffering from obesity or insulin resistance. For example, the HC-HA/PTX3 complex is administered as a gel solution for treatment. It is expected that treatment with the HC-HA/PTX3 complex will promote phenotypic switching of adipose tissue macrophages (ATMs) from a pro-inflammatory M1 phenotype to an M2 phenotype and restore normal insulin processing.

In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3). In some examples, the nHC-HA/PTX3 is isolated from umbilical cord tissue. In some examples, the nHC-HA/PTX3 is isolated from amniotic membrane. In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is a reconstituted HC-HA/PTX3 complex (rcHC-HA/PTX3).

Example 19: Use of HC-HA/PTX3 for the treatment of type 1 diabetes

In this example, HC-HA/PTX3 complexes produced by the methods described herein are administered for the treatment of type 1 diabetes.

Type 1 diabetes mellitus (T1DM, IDDM, or formerly known as juvenile diabetes) is a form of diabetes caused by autoimmune destruction of the insulin-producing beta cells in the pancreas. The resulting insulin deficiency leads to increased blood and urine glucose. Typical symptoms are polyuria (frequent urination), polydipsia (increased thirst), polyphagia (increased hunger), and weight loss.

The HC-HA/PTX3 complexes produced by the methods described herein are administered to a subject suffering from type 1 diabetes in the form of microcapsules containing autologous or allogeneic insulin-producing cells coated with the HC-HA/PTX3 complexes. For example, the microcapsules are administered to the subject by injection. It is expected that treatment with the HC-HA/PTX3-coated microcapsules will allow for the production of insulin released in the subject and prevent or reduce inflammatory responses to the cell therapy or microcapsules, thereby alleviating type 1 diabetes and its symptoms.

In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3). In some examples, the nHC-HA/PTX3 is isolated from umbilical cord tissue. In some examples, the nHC-HA/PTX3 is isolated from amniotic membrane. In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is a reconstituted HC-HA/PTX3 complex (rcHC-HA/PTX3).

Example 20: Use of HC-HA/PTX3 for the treatment of fibrosis

In this example, HC-HA/PTX3 complexes produced by the methods described herein are administered for the treatment of fibrosis or a fibrotic disorder.

Progressive fibrotic conditions, such as idiopathic pulmonary fibrosis (IPF), liver fibrosis, and systemic sclerosis, are tightly regulated by macrophages. "Profibrotic" macrophages exhibit M1 properties and produce a variety of mediators, including TGFβ1, PDGF, and insulin-like growth factor 1, which directly activate fibroblasts and myofibroblasts, which control ECM deposition. Profibrotic macrophages also produce MMPs, TIMPs, and IL-1β. IL-1β stimulates TH17 cells to produce IL-17, which is an important inducer of bleomycin-induced pulmonary fibrosis, a fibrotic condition with similar features to IPF. M2-like macrophages produce IL-10, RELMα, and ARG1 to inhibit fibrosis.

The HC-HA/PTX3 complex produced by the methods described herein is administered to a subject suffering from fibrosis or a fibrotic condition. For example, the HC-HA/PTX3 complex is administered as a solution, a gel, or as a coating on an implantable medical device. Treatment with the HC-HA/PTX3 complex is expected to reduce the activation of M1 macrophages, as well as fibroblasts and myofibroblasts, and increase the amount of M2 macrophages present at the affected site of the subject, thereby inhibiting fibrosis and its symptoms, such as scarring.

In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3). In some examples, the nHC-HA/PTX3 is isolated from umbilical cord tissue. In some examples, the nHC-HA/PTX3 is isolated from amniotic membrane. In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is a reconstituted HC-HA/PTX3 complex (rcHC-HA/PTX3).

Example 21: Use of HC-HA/PTX3 for the treatment of chronic inflammation

In this example, HC-HA/PTX3 complexes produced by the methods described herein are administered for the treatment of chronic inflammatory disorders, such as rheumatoid arthritis.

Many autoimmune diseases, including rheumatoid arthritis, involve an inflammatory response to autoantibodies, which activate Fc receptors, triggering the activation of mast cells and macrophages, as well as the invasion of neutrophils. This results in an intense local inflammatory response that, if not resolved, leads to tissue damage over time through a cycle of repair and destruction. In rheumatoid arthritis, CSF1 is constitutively produced by synovial fibroblasts and recruits tissue-infiltrating monocytes and macrophages. Furthermore, locally produced CSF1, together with RANKL, induces monocyte differentiation into osteoclasts, which triggers bone loss.

The HC-HA/PTX3 complex produced by the methods described herein is administered to patients suffering from chronic inflammatory conditions, such as rheumatoid arthritis. For example, the HC-HA/PTX3 complex is administered as a solution, a gel, or as a coating on an implantable medical device. Treatment with HC-HA/PTX3 is expected to suppress M1 pro-inflammatory macrophages, induce neutrophil apoptosis, and inhibit osteoclast differentiation, thereby treating inflammatory conditions and their symptoms.

In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3). In some examples, the nHC-HA/PTX3 is isolated from umbilical cord tissue. In some examples, the nHC-HA/PTX3 is isolated from amniotic membrane. In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is a reconstituted HC-HA/PTX3 complex (rcHC-HA/PTX3).

Example 22: Use of HC-HA/PTX3 for the treatment of acute inflammatory responses

In this example, the HC-HA/PTX3 complex produced by the methods described herein is administered to treat an acute inflammatory response caused by a condition such as myocardial infarction, stroke, or sepsis. The HC-HA/PTX3 complex produced by the methods described herein is administered to a subject suffering from an acute inflammatory response caused by a condition such as myocardial infarction, stroke, or sepsis. For example, the HC-HA/PTX3 complex is administered as a solution by intravenous infusion. It is expected that the HC-HA/PTX3 complex will reduce or prevent damage caused by acute inflammation by inhibiting M1 inflammatory macrophages.

In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3). In some examples, the nHC-HA/PTX3 is isolated from umbilical cord tissue. In some examples, the nHC-HA/PTX3 is isolated from amniotic membrane. In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is a reconstituted HC-HA/PTX3 complex (rcHC-HA/PTX3).

Example 23: Use of HC-HA/PTX3 for the treatment of cancer

In this example, HC-HA/PTX3 complexes produced by the methods described herein are administered for the treatment of cancer.

It has been observed that a large number of inflammatory cells are involved in tumor development and progression, and are often described as "smoldering inflammation". These findings have led to a well-established connection between inflammatory cells (particularly macrophages) and cancer. Initially, it was believed that the development of oncogenes led to a hallmark microenvironment of cancer, in which transformed cells secrete cytokines and chemokines that promote tissue development and prevent apoptosis and suppress cytotoxic immune responses (called the "intrinsic pathway"). It is now recognized that another pathway leads to tumorigenesis. This "extrinsic pathway" was initially characterized by a chronic proinflammatory environment caused by persistent microbial infection, autoimmune disease, or other causes of unknown origin. In these cases, the long-term production of large amounts of inflammatory mediators can lead to tumor cell proliferation and survival, or lead to the induction of genetic instability in normal cells, as well as the expression of oncogenes and the production of immunosuppressive cytokines. Therefore, in many cases, early tumor development is characterized by a polarized inflammatory, M1-like macrophage environment.

The HC-HA/PTX3 complex produced by the methods described herein is administered to a subject suffering from cancer, such as a solid tumor cancer. For example, the HC-HA/PTX3 complex is administered as a solution, a gel, or as a coating on an implantable medical device for topical, injection, or implantable applications. Because HC-HA/PTX3 can inhibit the polarization of M1 macrophages, treatment with HC-HA/PTX3 is expected to inhibit or prevent cancer or its progression to an advanced phenotype.

In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3). In some examples, the nHC-HA/PTX3 is isolated from umbilical cord tissue. In some examples, the nHC-HA/PTX3 is isolated from amniotic membrane. In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is a reconstituted HC-HA/PTX3 complex (rcHC-HA/PTX3).

Example 24: Use of HC-HA/PTX3 for treating unhealed skin wounds or ulcers

In this example, HC-HA/PTX3 complexes produced by the methods described herein are administered to treat non-healing wounds or ulcers on the skin.

A non-healing wound or sore on the skin that has been there for about 3-4 weeks without healing is called a non-healing ulcer. Diseases that commonly cause non-healing ulcers are vascular disease, diabetes, skin cancer, and some infections.

The HC-HA/PTX3 complex produced by the methods described herein is administered to a subject with an unhealed wound or ulcer on the skin. For example, the HC-HA/PTX3 complex is administered topically or subcutaneously as a solution, gel, or for treatment at the site of the wound or ulcer. It is expected that treatment with HC-HA/PTX3 will promote wound or ulcer healing by promoting the M2 phenotype of wound-healing and tissue-regenerating macrophages.

In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3). In some examples, the nHC-HA/PTX3 is isolated from umbilical cord tissue. In some examples, the nHC-HA/PTX3 is isolated from amniotic membrane. In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is a reconstituted HC-HA/PTX3 complex (rcHC-HA/PTX3).

Example 25: Use of HC-HA/PTX3 for the treatment of high-risk corneal transplantation

In this example, HC-HA/PTX3 was administered to treat high-risk corneal transplants. Mafia mice were injected intrastromally with EGFP+ macrophages and LPS (5 μg per eye). In each eye, OS (left eye; left eye) was treated with PBS (2 or 4 injection sites), while OD (right eye, right eye) was treated once with HC-HA/PTX3 immediately after LPS injection (2 or 4 injection sites; 5 μl of 1 mg/ml HA composition containing HC-HA/PTX3 per injection site). The entire cornea was photographed using in vivo intravital microscopy on days 1, 2, 3, 4, 5, 6, and 7. EGFP-positive cells were counted based on the intensity of green fluorescence to determine the level of EGFP infiltration. In the corneal transplant mouse model, injection of HC-HA/PTX3 into the subconjunctival area is expected to reduce inflammation (i.e., infiltration of macrophages) and improve the survival rate of the transplanted cornea compared to the PBS vehicle control.

In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3). In some examples, the nHC-HA/PTX3 is isolated from umbilical cord tissue. In some examples, the nHC-HA/PTX3 is isolated from amniotic membrane. In some examples, the HC-HA/PTX3 complex used in the treatment methods described herein is a reconstituted HC-HA/PTX3 complex (rcHC-HA/PTX3).

Example 26: Distribution of HA, PTX3, TSG-6, HCl, HC2, HC3 and Urotrypsin in Umbilical Cord (UC)

In this example, the in vivo distribution of HA, PTX3, TSG-6, HCl, HC2, HC3, and uropancreatin was detected in the umbilical cord (UC) by immunostaining. UC tissue cryosections were immunostained for HA, PTX3, TSG-6, and various components of IαI, including HC and uropancreatin. UC consists of an epithelium and a matrix comprising the subamnion and Wharton's jelly, which contains three blood vessels, i.e., one vein and two arteries ( FIG. 20 a, with one arterial vessel, stage). Strong positive HA staining was observed in the UC epithelium, subamnion, and Wharton's jelly, and weak HA staining was observed in the vascular wall ( FIG. 20 a, HA). The HA staining disappeared upon digestion with HAase ( FIG. 20 a, HA (+HAase)), which is consistent with specific staining for HA.

There was strong positive immunostaining for PTX3 in Wharton's jelly, and weak PTX3 staining in the subamnion and epithelium (Figure 20a, PTX3). HAase digestion did not enhance PTX3 staining in the subamnion and epithelium (not shown), suggesting that the weak PTX3 staining was not due to a masking effect of HA. Positive PTX3 staining was also observed in the vascular endothelium (not shown), but not in the vessel walls of arteries and veins.

Both TSG-6 and uropancreatin were present throughout UC, with TSG-6 primarily present in and around cells and more in the epithelium compared to Wharton's jelly. HC1 also had a similar localization to HA, with the exception of weak HC1 staining in the epithelium and vessel walls. Weak to absent staining of HC2 and HC3 was present in the epithelium but absent in the stroma of UC. These results indicate that UC produces abundant HA, PTX3, TSG-6, HC1, and uropancreatin, and disproportionately less HC2 and HC3, compared to AM. This confirms that UC constitutively expresses the aforementioned proteins and the HC-HA/PTX3 complex.

Furthermore, PTX3 was present in UC with a distribution pattern that differed from that reported in AM. There was more PTX3 in UC Wharton's jelly, and less in the epithelium and subamnion. In contrast, more PTX3 was present in the epithelium and stratum compactum of AM. For the following markers, UC had a similar pattern to AM: more HA was present throughout the stroma of UC, and less HA was present in the epithelium of UC. This is similar to the distribution pattern of HA in AM. TSG-6 was primarily localized in the epithelium and subamnion of UC, and urotrypsin was found throughout UC.

Example 27: Comparison of PBS and GnE extracts obtained sequentially from AM and UC

This example determined whether the insoluble fraction from AM after PBS extraction still contained any PTX3, TSG-6, IαI, and HC-HA/PTX3 complexes. Proteins were extracted from the insoluble fraction of AM using 4M GnHCl after PBS extraction to examine the presence of PTX3, TSG-6, and IαI. In addition, UC with PBS was sequentially extracted with 4M GnHCl to detect PTX3, TSG-6, HC, and urotrypsin in these two different extracts.

According to the method described in He et al. (2009) J. Biol. Chem. 284:20136-20146, AM, CH, and UC were homogenized in cold PBS using a blender at a 1:1 (g/ml) ratio for AM or a 1:1.5 (g/ml) ratio for UC and mixed at 4°C for 1 hour. The mixture was centrifuged at 48,000 g for 30 minutes at 4°C. The supernatants of the PBS extracts were designated AME, CHE, and UCE, respectively. Separately, a Wharton's gel mixture from UC was extracted with PBS, and this extract was designated UJE. The insoluble precipitates from AM, CH, UC, and the UC gel mixture after PBS extraction were further extracted with 4M GnHCl buffer (100 mM sodium acetate, pH 5.8, 4M GnHCl, 10 mM EDTA, 1% Triton X-100) at 4°C for 24 hours. After centrifugation at 48,000 g for 30 min at 4°C, the supernatants were collected and named AMGnE, CHGnE, UCGnE, and UJGnE, respectively. HA and protein concentrations in each extract were determined by HA ELISA and BCA assay, respectively.

GnHCl further extracted the abundant HA and proteins from the insoluble precipitate after PBS extraction

The HA and protein concentrations in the sequential PBS and GnHCl extracts are summarized in Table 1, which also compares the HA/protein ratio between the two extracts. In general, 4 M GnHCl further extracted abundant protein and HA from the insoluble precipitates of AM, CH, UC, and the UC gel mixture after PBS extraction. GnHCl buffer extracted more protein but less HA from the insoluble precipitate than PBS. However, UC CGnE still contained similar amounts of protein and HA as AME and CHE. That is, UC contained more HA than AM and CH in both PBS and GnHCl extracts.

Table 1. Quantification of proteins and HA in 4 M GnHCl extracts (GnE) from the insoluble pellets of AM, CH, UC, and UC gel mixtures after PBS extraction.

Compared with AM cells, higher amounts of monomer, dimer, and HMW PTX3 were present in PBS extracts of UC cells, whereas HMW PTX3 was present in higher amounts in GnHCl extracts of AM cells.

AME analysis using an anti-PTX3 antibody revealed a ~45 kDa band corresponding to the size of a native PTX3 monomer and a HMW band at the bottom of the well (Figure 21A, lane 4). NaOH treatment did not affect the 45 kDa band, but completely eliminated the HMW band, resulting in a HMW smear of PTX3 (Figure 21A, lane 5), a notable characteristic of PTX3 in NaOH-treated HC-HA complexes. No monomeric PTX3 was detected with or without NaOH treatment, but a 90 kD dimer was detected (Figure 21A, lanes 2 and 3). The HMW smear of PTX3 represents the complex formed between PTX3 and HC-HA. CHE showed the same PTX3 banding pattern, but with less PTX3 smearing after NaOH treatment, consistent with the immunostaining results. Placental extracts showed similar results to CHE. Notably, in addition to the monomer in UCE, PTX3 was present more as dimers and HMW smears, and their intensities further increased after NaOH treatment. Similar to AME, UCE also produced more HMW smear patterns than AME. These results indicate that UCE contains more PTX3 than AME, while CHE and placental extracts contain almost no PTX3.

Compared with AME (Figure 21A), AMGnE showed strong HMW PTX3 slabs, weak dimer and monomer levels of PTX3, and the intensity of the HMW PTX3 slabs further increased after NaOH treatment (Figure 21B, lanes 3 and 4), indicating that AMGnE contains more HMW PTX3 than AME. More PTX3 is present in the water-insoluble portion of AM. Regardless of whether it was treated with NaOH, CHGnE only had HMW bands in the sample wells, but no HMW PTX3 slabs. With or without NaOH treatment, UCGnE and UJGnE had the same PTX3 pattern as AMGnE, except that the intensity of the PTX3 slabs was slightly weaker than that of AMGnE (Figure 21B, lanes 3 and 4). The intensity of the PTX3 smear band in UCGnE was also weaker than that in UCE, indicating that UCGnE contains less HMW PTX3 than UCE; that is, more PTX3 is present in the water-soluble fraction of UC.

The above results showed that both AM and UC contained HMW PTX3. In AM, more HMW PTX3 was insoluble in water and could be extracted by GnHCl after PBS extraction, while more PTX3 in UC was water-soluble, most of which could be extracted by PBS.

IαI and HCl were mainly in the AM PBS extract, but in UC, most of IαI was in the PBS extract and HCl was in the GnHCl extract, while more urotrypsin was present in CGnE than in UCE and was not different in the other two extracts.

Figure 22 shows that an 80 kDa HCl band was present in all PBS extracts except UCE and all GnE extracts except AMGnE. NaOH increased this band in all PBS extracts, but not in GnE, suggesting that AM contains both free and bound water-soluble HCl (i.e., esters bound to HA in HC-HA and urokinin in IαI) that are released by NaOH, consistent with Zhang et al. (2012) J. Biol. Chem. 287:12433-12444. UC contains water-soluble bound HCl released by NaOH and also contains water-insoluble free HCl bound to water-insoluble extracellular components, which are dissociated by SDS and 2-ME but unaffected by NaOH. This is consistent with the strong positive HCl staining in UC. The HMW HCl band was present in all PBS and GnHCl extracts in the sample wells and was reduced by NaOH, indicating that it is a HC-HA complex. The HMW HCl band was weaker in UCGnE than in UCJGnE, indicating that Wharton's jelly contains more water-insoluble HC-HA complexes. Free IαI was found in all PBS extracts but absent in all GnE extracts, indicating that it is water-soluble. However, free PαI was found in all PBS and GnHCl extracts, suggesting that PαI has distinct interactions from IαI. Uropancreatin was found in greater amounts in UCGnE than in UCE, with no difference in the other two extracts, highlighting that the majority of uropancreatin is bound to other water-insoluble molecules in UC and indicating distinct functions.

TSG-6 is present in the HMW complex of AM but not in UC GnHCl extract

Figure 23 shows that the 35 kDa TSG-6 band reported in AME (Zhang et al. (2012) J. Biol. Chem. 287: 12433-12444) was present in AMGnE but not in all other extracts, indicating that TSG-6 was not present in GnE from UC. This band was not affected by NaOH, confirming that TSG-6 was not bound to HMW species that could be cleaved by NaOH. However, the HMW TSG-6 band was found in AMGnE and CHGnE, but not in UCGnE and UJGnE. In addition, this band was not altered by NaOH, indicating that TSG-6 strongly binds to HMW species. TSG-6 was not detected in HC-HA purified from GnE by 4X ultracentrifugation, indicating that although TSG-6 was still bound in the insoluble matrix, it could be separated by GnHCl during ultracentrifugation.

In summary, GnHCl further extracted abundant HA and proteins from the insoluble fractions of AM and UC after PBS extraction. In both PBS and GnHCl extracts, UC contained more HA than AM and CH. HMW PTX3 was present at higher levels in the AM GnHCl extracts and at higher levels in the UC PBS extracts. More HMW PTX3 was retained in the insoluble fraction after PBS extraction. HCl was mainly found in the AM PBS extract but not in the UC GnHCl extract. HMW TSG-6 was found in the AMGnE but not in the UCGnE and UJGnE, indicating that TSG-6 remained bound in the insoluble matrix but could be separated by GnHCl during ultracentrifugation.

Example 28: Purification of HC-HA complex from AM and UC PBS extracts by four consecutive ultracentrifugations, and Detection of the presence of PTX3, HC, urotrypsin, and TSG-6 in HC-HA complexes

In this example, HC-HA complexes were purified from AME and UCE by four consecutive ultracentrifugations, and the presence of PTX3 and HC, urotrypsin, and TSG-6 in AM and UC HC-HA complexes were detected by Western blotting.

The 4th AM HC-HA complex contained more HMW PTX3 and HC1 and was purer than the 2nd and 3rd HC-HA complexes.

Using an anti-PTX3 antibody, Western blot analysis of fractions 1 to 4 of HC-HA revealed a 90 kDa band (dimer), compared to the monomer found in the PBS extract. This suggests that further extraction in 4M GnHCl by ultracentrifugation deconstructs the dimeric state, revealing HMW bands at the top of the gels for the 1st, 2nd, 3rd, and 4th HC-HA complexes ( FIG. 24A ). Compared to the purified PTX3 control, the 90 kDa band represents the PTX3 dimer, and the high molecular weight band represents the HC-HA complex containing PTX3. After HAase treatment, the 90 kDa band remained unchanged in all HC-HA complexes, but HMW smears were faintly detected in the 3rd and 4th fractions. That is, from fractions 1 to 4, the HMW band gradually disappeared, while the smears gradually emerged, which were more pronounced in the 4th HC-HA complex. The 45 kDa PTX3 monomer band was absent in all HC-HA complexes. The results showed that the HC-HA complex contained multimeric forms of PTX3 capable of binding to HC-HA, and that the HC-HA complex containing PTX3 became purer with increasing ultracentrifugation cycles. The presence of a 90 kDa PTX3 dimer in the HC-HA complex with or without HAase indicated that: 1) the PTX3 dimer present in the HC-HA complex was dissociated by SDS and 2-ME, and 2) HMW PTX3 was resistant to SDS and 2-ME. The PTX3 dimer was further confirmed as a product obtained with 2-ME below ( FIG24A ).

Using an anti-HCl antibody, the 80 kDa HCl band was detected only in the early first and second fractions from all four HC-HA complexes ( FIG. 24B ). After HAase treatment, the HCl band was enhanced, and multiple smaller bands also appeared in the first to fourth HC-HA complexes. The results indicate that the purified HC-HA complex does not contain free HC and that the HC-HA is made of HCl. Consistent with the PTX3 Western blot results described above, the HC-HA complex becomes purer with increasing ultracentrifugation times. HC2 ( FIG. 24Cc ), HC3, and TSG-6 ( FIG. 24D ) were not found in any of the HC-HA complexes.

TSP-1 was only present in AM GnHCl extracts but not in PBS extracts and the 1st to 4th HC-HA complexes.

Thrombospondin-1 (TSP-1) was detected only as a trimer in AM GnHCl extracts and not in PBS extracts or HC-HA complexes 1 to 4 (Figure 25). After HAase treatment, it appeared as a smear, indicating that TSP-1 is water-insoluble and strongly binds to HC-HA. However, this binding to HC-HA in an insoluble matrix can be dissociated by GnHCl and CsCl.

Similar to the 4AM HC-HA complex, the 4UC HC-HA complex contains PTX3 and HC1 but lacks HC2, HC3, urokinin, and TSG-6, and the absence of 2ME does not produce PTX3 dimers but produces HC1.

Western blot analysis of 4UC HC-HA with or without HAase treatment using an anti-PTX3 antibody revealed that the PTX3 banding pattern in the 4UC HC-HA complex (Figure 26a, lanes 5 and 6) was similar to that in the 4AM HC-HA complex (Figure 26a, lanes 3 and 4). When the sample buffer did not contain 2-ME, the 90 kDa PTX3 dimer disappeared from the UC HC-HA complex with or without HAase (Figure 26a, lanes 7 and 8), indicating that the appearance of the 90 kDa PTX3 dimer in the HC-HA complex was due to the reduction of PTX3 bound to HC-HA by 2-ME in the sample buffer. Using an anti-HCl antibody, only a high molecular weight HC-HA band was detected in the 4UC HC-HA complex (Figure 26b, lane 6), similar to the band in the 4AM HC-HA complex (Figure 26b, lane 4). After HAase treatment, the HC-HA band disappeared and the HCl band increased (lane 7), similar to the 4 AM HC-HA complex (lane 5), but its intensity was slightly weaker than that in the 4 AM HC-HA. This indicates that HCl forms a complex with PTX3 through S-S. When the sample buffer does not contain 2-ME, a stronger band with a slightly higher MW than that of ordinary HCl appears, indicating that HCl is linked to PTX3 through S-S. HC2, HC3 (Figure 26c), urotrypsin (Figure 26d), and TSG-6 (Figure 26e) were not detected in the 4 AM and UC HC-HA complexes.

Example 29: Purification of HC-HA complex from total AM and UC GnHCl extracts by four consecutive ultracentrifugation steps and Comparison with PBS extract

This example demonstrates that more HC-HA complexes can be obtained from AM and UC, with more rational PTX3 and HC-HA compositions, and clinically more effective therapeutic effects. AM and UC were extracted using 6M GnHCl buffer (200 mM Tris-HCl, pH 8.0, 6 M GnHCl, 10 mM EDTA, 10 mM aminocaproic acid, 10 mM N-ethylmaleimide, 2 mM PMSF): GnHCl was extracted from AM and UC by adding 6M GnHCl buffer to AM and UC powder at a 1:4 ratio (g/ml). The samples were mixed overnight at 4°C and centrifuged at 48,000 g for 30 minutes at 4°C. The supernatant was the GnHCl extract. The fourth HC-HA complex was purified from AM and UC GnHCl extracts using the same procedure as for the purification of the fourth HC-HA from PBS extracts. Characterization of the HC-HA complex was performed by Western blot analysis to examine PTX3, HC, urotrypsin, TSG-6, and other possible proteins. Agarose gels of HC-HA were run to observe the content and molecular weight of HA.

GnHCl extracted more HC-HA complexes from AM and UC than PBS

GnHCl extracts from AM and UC were designated AMEG and UCEG, and their HA and protein contents were assayed by BCA assay and HA ELISA, respectively. The 4th HC-HA complex was purified from the GnHCl extracts and similarly assayed for HA and protein content. Table 2 summarizes the protein and HA contents of the PBS and GnHCl extracts and their 4th HC-HA complexes. The results showed that the AM and UC GnHCl extracts contained more HA and had a higher HA/protein ratio than the corresponding PBS extracts. Furthermore, more HC-HA complexes were purified from the GnHCl extracts.

Table 2. Quantification of proteins and HA in extracts from AM and UC and in 4th HC-HA.

AM4 GnHCl HC-HA complexes contained more HCl and HMW PTX3, but less than PBS HC-HA with or without HAase or NaOH treatment, and neither PBS nor GnHCl HC-HA contained TSP-1.

Using anti-HCl antibodies, GnHCl HC-HA showed the same HMW bands as PBS HC-HA in the sample wells, but HAase digestion only released weaker HCl (Figure 27a, lanes 6 and 7). NaOH treatment also released a weaker HCl band, which had a slightly higher MW than the band released by HAase (Figure 27a, lane 8), which was not seen in PBS HC-HA after NaOH treatment. These results indicate that GnHCl HC-HA contains HCl but in less amount than PBS HC-HA. Similarly, unlike PBS HC-HA, using anti-PTX3 antibodies, GnHCl HC-HA showed only dimeric PTX3 without obvious HMW PTX3 smears, with or without HAase digestion (Figure 27b, lanes 6 and 7). NaOH also caused the appearance of HMW and dimeric PTX3. These results indicate that GnHCl HC-HA contains less HMW PTX3 than PBS HC-HA.

Similar to the HCl blot, higher MW PTX3 dimers were present in GnHCl HC-HA after NaOH treatment compared to HAase treatment. Together, these results suggest that NaOH releases the ester bond that links HCl to HA and allows for association with PTX3. Because GnHCl HC-HA has a higher HA content than PBS HC-HA, the GnHCl HC-HA complex contains HA that is not bound by PTX3 or HCl, resulting in a decrease in the actual HC-HA/PTX3 complex content and less HCl and HMW PTX3 in the purified product. TSP-1 was not detected in PBS HC-HA using anti-TSP-1. It should be noted that TSP-1 was also not detected in GnHCl HC-HA. Since the GnHCl extract contains TSP-1, these results suggest that TSP-1 was dissociated by ultracentrifugation and therefore not present in GnHCl HC-HA.

Agarose gel showed that HA was abundant in GnHCl HC-HA

PBS HC-HA showed a "continuous HA smear band" from the top loading well to the bottom of the agarose gel, and HAase completely eliminated the HA smear band (Figure 28, lanes 3 and 4). GnHCl HC-HA showed a band and a HA smear band in the loading well, which started at the 4,570 kDa position to the bottom of the agarose gel (Figure 28, lane 5). There was a break in the HA in the GnHCl HC-HA between the loading well and the beginning of the HA smear band, although its intensity was stronger than that in PBS HC-HA. In addition, HAase did not completely eliminate the HA smear band and HMW HA band in GnHCl HC-HA (Figure 28, lane 6). The top fractions (fractions 1-6) from the GnHCl extract after the 4th ultracentrifugation also showed the same HA smear band pattern as the "bottom fraction" of GnHCl HC-HA (Figure 28, lanes 7 and 8). These results indicate that GnHCl HC-HA contains more HMW HA (MW is smaller than PBS HC-HA), but lacks a portion of the HMW HA smear band, which corresponds to the lack of HMWPTX3 smear bands in the Western blot of GnHCl HC-HA. This suggests that the disappeared HMW HA smear band (which is present in PBS HC-HA) is at least partially formed by cross-linking of PTX3 and HC-HA, and that the HMW HA in the GnHCl HC-HA sample wells is complexed with components other than PTX3.

GnHCl HC-HA contained some proteins that were not found in PBS HC-HA by Coomassie blue staining.

Figure 29A shows that the bands in the top loading wells, a major 140 kDa band and some minor 70 kDa, double 55 kDa, and 20 kDa bands, are present in the top fractions of AM GnHCl HC-HA and GnHCl HC-HA, but absent from all other PBS HC-HA. This suggests that AM GnHCl HC-HA contains some proteins not present in PBS HC-HA. In addition, 90 kDa and 25 kDa bands are also visible in the top fractions of GnHCl HC-HA. Because Western blotting detected HCl in PBS HC-HA, HCl should also be present in PBS HC-HA by Coomassie blue staining. Its absence in PBS HC-HA is due to the fact that the loaded HC-HA did not enter the gel due to overloading. The 140 kDa band appears as a broad band, indicating that it contains a sugar moiety. Furthermore, HAase did not affect these bands, indicating that these substances were dissociated by SDS and 2-ME.

Compared to AM GnHCl HC-HA, UC GnHCl HC-HA showed bands in the top loading wells, 90 kDa, 70 kDa, two 55 kDa, 35 kDa, and 20 kDa bands ( FIG. 29B , lanes 4 and 5). A faint 140 kDa band was also present. These bands were not affected by HAase. In addition, the top fraction of GnHCl HC-HA also showed a patch of bands from the top well to the 200 kDa position, which decreased after HAase treatment. All bands were absent in UC PBS HC-HA. These results indicate that UC GnHCl HC-HA also contains some proteins that are not present in PBS HC-HA, and that UC GnHCl HC-HA differs from AM GnHCl HC-HA in the protein bands it contains.

The above results indicate that GnHCl HC-HA differs from PBS HC-HA from both AM and UC in several respects: (1) GnHCl HC-HA contains less HCl and HMW PTX3 than PBS HC-HA (from Western blot), while TSG-6, HC2, and HC3 are absent in GnHCl HC-HA, similar to PBS HC-HA. (2) GnHCl HC-HA contains more HMW HA but lacks a patch of HMW PTX3, which corresponds to the smear of HMW PTX3 in the Western blot revealed by PBS HC-HA (from agarose gel). (3) GnHCl HC-HA contains some proteins, mainly MW 140 kDa, which are not found in PBS HC-HA (from Coomassie blue stained gel).

In summary, GnHCl extracted more HA and protein from AM and UC tissues compared to PBS extracts, resulting in a higher HA/protein ratio. For both AM and UC, more HC-HA complexes were purified from GnHCl extracts (based on HA content). GnHCl HC-HA contained HCl and HMW PTX3, but much less for both AM and UC than in PBS HC-HA. GnHCl HC-HA lacked HMW HA species in agarose gels, which corresponded to the HMW PTX3 smear in Western blot analysis shown by PBS HC-HA. GnHCl HC-HA contained some proteins that were not found in PBS HC-HA.

Example 30: GnHCl purified from AM and UC Determination of the identity of unknown protein bands in the HC-HA complex

This embodiment is by running SDS-PAGE gel, then carries out CB staining or Western blot analysis to GnHCl HC-HA from AM and UC under the condition of having or not deglycosylation, thereby determines the identity of unknown band in GnHCl HC-HA.This sample is freeze-dried AM and UC 4x HC-HA (comprising 30 μ g HA) from both PBS and GnHCl extract.Freeze-dried HC-HA is incubated on ice for 3h with 50μl TFMS and 20μl anisole and neutralized using TFMS containing 125μl N-ethylmorpholine.Use 5-10 times volume of acetone to precipitate the sample at -20 ℃ overnight or at -80 ℃ for 1h.The sample is centrifuged and the dried precipitate is dissolved in SDS sample loading buffer for electrophoresis.Use keratinase (endo-β-galactosidase) to carry out enzymatic deglycosylation to remove keratan sulfate chains and N-connected oligosaccharides, or use chondroitinase (Cabc) to remove chondroitin sulfate chains. HC-HA (containing 30 μg HA) was incubated with 0.1 U/ml keratinase in 50 mM sodium acetate (pH 5.8) for 2 h at 37° C. or with 5 U/ml Cabc in PBS for 2 h at 37° C. SDS-PAGE gels were run to test CB staining, followed by Western blot analysis.

Keratan sulfate and osteoadhesin were present in AM GnHCl HC-HA but not in PBS HC-HA.

Western blot analysis was performed. The results are shown in Figures 30B and C. Western blots using anti-keratan sulfate antibodies in AM GnHCl HC-HA with or without HAase digestion showed a broad 70 kDa (60-80 kDa) band (Figure 30B, lanes 6-8), but not in PBS HC-HA, indicating that this ~70 kD keratan sulfate proteoglycan is responsible for the positive immunostaining shown in Figure 30D. This 70 kD band corresponds to the same band noted in GnHCl HC-HA with or without HAase treatment as shown in the Coomassie blue-stained gel (Figure 30A).

To further determine whether this 70kD keratan sulfate proteoglycan is an SLRP, anti-lumencan, anti-fibromodulin, and anti-osteoadherin antibodies were used in Western blotting. The anti-osteoadherin antibody recognized the 60kD band, but not the 70kD band in GnHCl HC-HA with or without HAase digestion ( FIG. 30C , lanes 6-8), but not in PBS HC-HA. Osteoadherin with keratan sulfate chains has a molecular weight of ˜80kD, while its non-keratan sulfate protein is ˜60kD. A keratan sulfate band of 60kD was detected, but only as a broad band of 70kD, indicating that the 60kD band detected by anti-osteoadherin is a non-keratan sulfate osteoadherin. AM GnHCl HC-HA contains non-keratan sulfate osteoadhesin proteoglycans closely related to HC-HA and can withstand four ultracentrifugations in the presence of 6MGnHCl and cesium chloride. However, it is released by SDS and 2-ME in the sample buffer. The results also indicate that the 70kD keratan sulfate proteoglycan is not an osteoadhesin proteoglycan. Neither luminin nor fibromodulin was detected in GnHCl HC-HA, indicating that the 80kD proteoglycan is neither luminin nor fibromodulin.

Deglycosylation and analysis of AM GnHCl HC-HA

HC-HA was deglycosylated by TFMSA to remove all glycans using keratinase and chondroitinase to remove specific glycans to observe whether there were any changes in the 140 kD and ∼80 kD bands in AM GnHCl HC-HA and to further determine whether the ∼80 kD keratan sulfate proteoglycan was keratan, PRELP, or osteoproteoglycan. As a first step to confirm the effects of the above deglycosylation, Coomassie blue staining was performed.

In Figure 31 A, the gel of Coomassie blue (CB) staining, AM GnHCl HC-HA (Figure 31 A, swimming lane 2) demonstrates the identical band of main 140kDa, 70kDa, double 50kDa, 20kDa and weak 35kDa band and the HMW band at the gel top.K/C/H does not produce a great impact on these bands, except producing main 80kDa, weak 100kDa and 30kDa band outward appearance (Figure 31 A, swimming lane 3).This pattern is similar to the pattern (Figure 31 A, swimming lane 6) obtained from the top fraction of AM GnHCl HC-HA under C/K/H.These results show that 140kDa, 70kDa and 55kDa bands are not keratan sulfate and/or chondroitin sulfate, and in GnHCl HC-HA, there are other keratan sulfate and/or chondroitin sulfate proteins that are released as main 80kDa material after C/K/H. TFMSA treatment resulted in the disappearance of all bands except the 20 kDa band, and the generation of a clear new 50 kDa band and HMW smear bands ( FIG. 31A , lane 4). TFMSA/H caused the smear band to disappear, thereby generating a new 25 kDa band but did not alter the 50 kDa band ( FIG. 31A , lane 5). This result indicates that the 140 kDa, 70 kDa, 55 kDa, and 80 kDa bands are the same 50 kDa species with different amounts of glycan chains.

To determine whether the 50 kDa band is derived from the 60 kDa osteoadhesin, Western blot analysis was performed using an anti-osteoadhesin antibody. The results showed a 60 kDa substance in AM GnHCl HC-HA (Figure 31B, lane 4), which is consistent with the findings shown in Figure 31C. C/K/H did not change its molecular weight (Figure 31B, lanes 5 and 6), but TFMSA with or without HAase (T/H) treatment completely changed it to a 55 kDa substance with lower intensity (Figure 31, lanes 7 and 8). Compared with the absence of T/H, the top fraction of AM GnHCl HC-HA under T/H conditions showed a stronger band but a smaller MW, and no intensity change (Figure 31B, lanes 9 and 10). These results further confirm that AM GnHCl HC-HA contains osteoadhesin, which does not contain keratan sulfate and chondroitin sulfate. The decrease in the intensity of the osteoadherin band after TFMSA treatment is due to (1) degradation of the protein by TFMSA and (2) its blocking by a large amount of other proteins with the same MW that are also released after TFMSA treatment. Osteoderm proteoglycan was undetectable in PBS HC-HA without any treatment, but a double band of 60 kDa appeared after TFMSA/HAase treatment, indicating that PBS HC-HA contains trace amounts of osteoadherin that are tightly bound to HA.

Western blotting using an anti-decorin antibody revealed a very strong, broad 140 kDa species (80-160 kDa) and a weak double 50 kDa species in AM GnHCl HC-HA (Figure 31C, lane 4; 31D, lanes 4 and 5), but not in PBS HC-HA (Figure 31C, lane 2; 31D, lanes 2 and 3). The broad 140 kDa species corresponds to decorin with a single chondroitin sulfate or dermatan sulfate chain and varying numbers of glycans, while the double 50 kDa species likely corresponds to less glycosylated decorin. Since HAase did not affect the decorin species, this suggests that they can be released by SDS and 2-ME. This was confirmed by C/H, which increased the 70 kDa species (Figure 31C, lane 5), and by C/H/K, which gave similar results (Figure 31B, lane 6). Therefore, the 70 kDa species is decorin without chondroitin. This 70 kDa species is a minor component, as neither C/H nor A/H/K significantly altered the major broad 140 kDa species. TFMSA treatment completely eliminated the broad 140 kDa species, while generating a major 43 kDa species corresponding to the deglycosylated decorin core protein, as well as minor 95 kDa, 80 kDa, and weak 30 kDa species (Figure 31C, lane 7). TFMSA/H treatment showed the same species pattern as TFMSA alone, except that the intensity of all these species was enhanced, indicating that decorin was tightly bound to HA. TFMSA/H also resulted in the release of a faint 43 kDa species from PBS HC-HA, indicating that AM PBS HC-HA also contains a small amount of decorin tightly bound to HA. The top fraction of AM GnHCl HC-HA with or without TFMSA/H showed the same pattern as the bottom fraction, with a stronger intensity than the latter (Figure 31C, lanes 9 and 10), indicating that the top fraction also contained abundant decorin. The above results indicate that the major 140 kDa, 70 kDa, and double 50 kDa species in the CB-stained gel are formed by decorin through CS and mainly by non-CS and non-KS.

Unlike decorin, Western blotting using an anti-biglycan antibody revealed a strong HMW smear at 400 kDa and a weak 45 kDa species in AM GnHCl HC-HA with or without HAase treatment (Figure 31E, lane 4; 2F, lanes 4 and 5), but not in PBS HC-HA (Figure 31D, lane 2; F, lanes 2 and 3). The 45 kDa species corresponds to the biglycan core protein, while the HMW smear is glycosylated biglycan with chondroitin sulfate or dermatan sulfate chains. HAase enhanced the 400 kDa region above 400 kDa with less HMW smear, indicating that some biglycan also binds to HC-HA. C/H or C/K/H did not significantly alter the HMW smear and 45 kDa species, but increased a 70 kDa species that may be chondroitin-free biglycan (Figure 31E, lanes 5 and 6). Since the amount of 70 kDa species was very small and the above treatments did not significantly change the major HMW smear, most of the biglycan in AM GnHCl HC-HA was not associated with CS or KS. TFMSA treatment completely eliminated the HMW smear and produced a major 45 kDa species corresponding to the deglycosylated decorin core protein, as well as weak 95 kDa, 80 kDa, and 30 kDa species (Figure 31E, lane 7), indicating the presence of biglycan in AM GnHCl HC-HA. The 95 kDa and 80 kDa species are partially deglycosylated biglycan, while the 30 kDa species is degraded biglycan. TFMSA treatment with HAase showed the same pattern as TFMSA alone, except that the intensity of all of these substances was enhanced, indicating that biglycan was also tightly bound to HA. The top fraction of AM GnHCl HC-HA with or without TFMSA/HAase treatment showed the same pattern as the bottom fraction, with its intensity being stronger than the latter (Figure 31E, lanes 9 and 10), indicating that the top fraction also contained abundant biglycan. Western blot analysis using an anti-keratan sulfate antibody showed the presence of a 70 kDa keratan sulfate protein in AM GnHCl HC-HA with or without keratinase or chondroitinase treatment, with no change in molecular size (Figure 31G), indicating that keratinase did not completely eliminate keratan sulfate or that the amount of KS in this material was minimal. Western blot analysis using an anti-PTX3 antibody showed an increase in HMW PTX3 smears, as compared to the presence or absence of HAase digestion alone ( FIG. 31G , lanes 4 and 5), which was shown in the AMGnHCl HC-HA with K, and even more so when K/H was used ( FIG. 31H , lanes 6 and 8). Chondroitinase had no such effect, suggesting that some KS-containing substances bind to PTX3 in the GnHCl HC-HA. Western blot analysis of UC GnHCl HC-HA with or without keratinase digestion yielded the same results (see below, FIG. 32G ). Western blot analysis demonstrated the absence of fibromodulin, lumican, keratocan, PRELP, osteoglycan, epiphyseal proteoglycan, periostin, and TSG-6, as well as urotrypsin, in the AM GnHCl HC-HA.

In summary, AM GnHCl HC-HA contains large amounts of decorin and biglycan bound to the HC-HA, whereas PBS HC-HA contains only trace amounts of decorin and no biglycan. AM GnHCl HC-HA contains osteoadhesin and keratan sulfate-containing substances, whereas PBS HC-HA does not. The very small amounts of decorin and biglycan in AM GnHCl HC-HA contain chondroitin sulfate chains.

Deglycosylation and analysis of UC GnHCl HC-HA revealed the presence of abundant decorin and biglycan in UC GnHCl HC-HA, which were absent in PBS HC-HA. Keratan sulfate, osteoadherin, and urotrypsin were also present in UC GnHCl HC-HA.

CB staining (Figure 32 A) shows the same bands of 160kDa, 90kDa, 70kDa, double 50kDa, 35kDa and 20kDa bands in the top sample wells in UC GnHCl HC-HA (Figure 32 A, lane 4). C/H or C/H/K do not significantly affect these bands, except causing the appearance of main 80kDa and weak 30kDa bands (Figure 32 A, lanes 5 and 6). TFMSA treatment reduces all of the above-mentioned bands except the 20kDa band, but increases main 50kDa band, less important 80kDa band and HMW smear bands (Figure 32 A, lane 7). TFMSA/H makes smear bands and 80kDa bands disappear but causes the appearance of weak 25kDa and reduces the intensity of the 50kDa band (Figure 32 A, lane 8). These results are similar to those obtained from AM GnHCl HC-HA ( FIG. 31A ), indicating that UC GnHCl HC-HA has a similar composition to AM GnHCl HC-HA. The top fractions of UC GnHCl HC-HA with or without TFMSA/H treatment showed the same pattern as the bottom fractions ( FIG. 32A , lanes 9 and 10), indicating that they have the same components as the bottom fractions. Undeglycosylated UC PBS HC-HA showed only HMW bands in or below the wells, as well as a 20 kDa band. TFMSA/H deleted the HMW bands but predominantly increased the 50 kDa band in addition to weak 80 kDa and 25 kDa bands, indicating that UC PBS HC-HA contains some glycosylated proteins that are only released by complete deglycosylation and HA degradation.

Western blot analysis using an anti-decorin antibody revealed a broad 160 kDa species in UC GnHCl HC-HA with or without HAase ( FIG32B , lanes 4 and 5), but not in PBS HC-HA ( FIG32B , lanes 2 and 3), indicating that UC GnHCl HC-HA, like AM GnHCl HC-HA, also contains decorin. Due to the different levels of glycosylation, its molecular weight differs from that in AM GnHCl HC-HA. HAase significantly reduced the 160 kDa species, indicating that it is bound to HC-HA. Keratinase also reduced the intensity of the 160 kDa species, with or without HAase, indicating that it also contains some KS. Notably, C with or without HAase digestion resulted in the disappearance of the 160 kDa species and the material in the top well, but produced strong 50 kDa and 90 kDa species as well as HMW smears, indicating that chondroitin sulfate was predominant in UC GnHCl HC-HA compared to AM GnHCl HC-HA, with a minor fraction containing chondroitin sulfate chains. These results further confirm that UC GnHCl HC-HA contains decorin and that this decorin in UC differs from AM in (1) glycosylation, (2) type of attached glycosaminoglycans, and (3) total amount.

In UC GnHCl HC-HA with or without HAase, Western blots using anti-biglycan antibodies showed strong HMW species in the top wells, HMW smears in the 400 kDa region and the 140 kDa region, and 45 kDa species ( FIG. 31C , lanes 4 and 5), while there was no HMW species in PBS HC-HA ( FIG. 31C , lanes 2 and 3). HAase enhanced the 400 kDa region without affecting the HMW species in the wells, indicating that some species were tightly bound to HA. Keratinase did not significantly alter these species with or without HAase, except for reducing the 45 kDa species ( FIG. 32C , lanes 6 and 8), suggesting that the 45 kDa species contained KS while the others did not. Chondroitinase alone eliminated the HMW species in the top well and reduced the intensity of the 400 kDa region, but increased strong and broad 50 kDa, 100 kDa, and 28 kDa species, with smears between them ( FIG32C , lane 7). Chondroitinase plus HAase had the same results, with all smears intensified and the 28 kDa species eliminated ( FIG32C , lane 9). These results indicate that, similar to decorin, biglycan in UCGnHCl HC-HA primarily carries chondroitin sulfate chains. This finding differs from the results in AM HC-HA, where a small fraction was chondroitin sulfate. Similarly, in HC-HA, most biglycan formed HMW complexes, with some bound to HC-HA.

Western blots using anti-uropancreatin antibodies in UC GnHCl HC-HA with or without HAase digestion showed a broad 35 kDa band, whereas in PBS HC-HA, there was none ( FIG. 32D , lanes 4 and 5). The 35 kDa band corresponds to the MW of native uropancreatin. Keratinase with or without HAase digestion sharpened the 35 kDa band but did not change its MW ( FIG. 32D , lanes 6 and 8), while chondroitinase with or without HAase digestion converted the 35 kDa uropancreatin into a 25 kDa core uropancreatin ( FIG. 32D , lanes 7 and 9), further confirming the presence of uropancreatin in UC GnHCl HC-HA and the inclusion of CS as reported. Because HMW bands were also formed after chondroitinase treatment, this suggests that uropancreatin is tightly bound to HC-HA via CS. These results are different from AMGnHCl HC-HA, which does not contain uropancreatin.

Western blotting using an anti-PTX3 antibody showed an increase in HMW PTX3 smears in UC GnHCl HC-HA after H, K, C, and especially K/H treatment ( FIG. 32E , lanes 6 and 8), compared to treatment with or without HAase digestion ( FIG. 32E , lanes 4 and 5). These results confirm the presence of HMW PTX3 in GnHCl HC-HA and its strong binding to GnHCl HC-HA. In addition, the presence of a substance containing KS further aids such strong binding, the identity of which remains unclear. This also explains that our previous data (without enzyme digestion) may have underestimated the amount of HMW PTX3 in UC GnHCl HC-HA.

Western blots using anti-keratan sulfate antibodies revealed HMW species in the wells and a faint 55 kDa band in UC PBS HC-HA. HAase did not alter this band but made the 60 kDa band more prominent ( FIG. 32F , lanes 2 and 3). However, a 140 kDa band was recognized by anti-keratan sulfate antibodies in UC GnHCl HC-HA with or without HAase ( FIG. 32F , lanes 4 and 5). Keratinase, with or without HAase treatment, did not delete the 140 kDa band but added a major 35 kDa band and several other bands between them, including the 60 kDa and 55 kDa bands seen in PBS HC-HA ( FIG. 32F , lanes 6 and 8).

Chondroitinase, with or without HAase treatment, also did not delete the 140 kDa band, but increased the 90 kDa band and the 60 kDa and 55 kDa bands seen in PBS HC-HA (Figure 32F, lanes 7 and 9). Chondroitinase treatment also resulted in the appearance of a HMW smear, which decreased after HAase treatment, indicating that UC GnHCl HC-HA contains abundant chondroitin sulfate proteins in addition to keratan sulfate proteins. Clearly, the keratan sulfate proteins in UC GnHCl HC-HA (140 kDa) have different MWs than those in AM GnHCl HC-HA (~80 kDa), and this may be due to the different amounts of glycans in the chains.

Using an anti-osteoadherin antibody, a major 60 kD band and a weak 80 kD band were detected in UC GnHCl HC-HA (Figure 32G, lane 4), but not in PBS HC-HA. These two bands were not significantly affected by keratinase, chondroitinase, or HAase. The 80 kD band should be keratan sulfate osteoadherin, while the ~60 kD band should be non-keratan sulfate osteoadherin. These results indicate that UC GnHCl HC-HA contains both keratan sulfate and non-keratan sulfate osteoadherin.

In summary, fibromodulin, lumican, keratocan, PRELP, osteoglycan, epiphyseal cane, periostin, and TSG-6 were not detected in UC GnHCl HC-HA. UC GnHCl HC-HA contained decorin, biglycan, osteoadherin, keratan sulfate, and uropancreatin. Among them, biglycan and decorin were abundant, while PBS HC-HA did not contain these substances in abundance.

Summarize

We purified the HC-HA complex from PBS and GnHCl extracts of AM and UC by 4x ultracentrifugation. HC-HA purified from GnE differed significantly from HC-HA purified from PBS extracts in terms of yield and chemical composition (see Table 1). Quantitatively, HC-HA purified from GnE contained more HA than that purified from PBS. Chemically, GnHCl HC-HA contained more HMW HA (whose MW was slightly smaller than that of PBS HC-HA) but lacked a patch of HMW HA corresponding to the HMW PTX3 smear in Western blots revealed by PBS HC-HA (from agarose gel). GnHCl HC-HA contained less HCl and HMW PTX3 than PBS HC-HA, with or without HAase digestion, but more PTX3 was detected after keratinase plus HAase treatment (from Western blots), indicating that HMW PTX3 is tightly bound to keratan sulfate proteins in GnHCl HC-HA. Neither PBS HC-HA nor GnHCl HC-HA contained TSG-6, HC2, or HC3. Urotrypsin was present in UC GnHCl HC-HA but not in UC PBS HC-HA or AM PBS and GnHCl HC-HA. AM GnHCl HC-HA contained abundant decorin, relatively more than UC GnHCl HC-HA. Both AM and UC PBS HC-HA contained trace amounts of decorin. AM and UC GnHCl HC-HA contained abundant biglycan, particularly in UC GnHCl HC-HA. Biglycan was absent in PBS HC-HA. AM and UC GnHCl HC-HA contained osteoadhesin. AM and UC PBS HC-HA do not contain biglycan, keratan sulfate containing material, fibromodulin, lumican, keratocan, PRELP, osteoglycan, epiphyseal, periostin and osteopontin, TSP-1. Fibromodulin, lumican, keratocan, PRELP, osteoglycan, epiphyseal, periostin and osteopontin, TSP-1, asporidin are absent in AM and UC GnHCl HC-HA. GnHCl HC-HA contains visible protein bands, primarily MW 200 kDa, 80 kDa and 60 kDa, which are not found in PBS HC-HA (from Coomassie blue stained gel).

Table 3. Summary comparison of 4x HC-HA complexes purified from PBS and GnHCl extracts

*Note: This information differs from extracts, indicating the presence of TSP-1 that can be isolated by ultracentrifugation.

Table 4. Summary comparison of PBS and GnE extracts obtained sequentially from AM and UC

Example 31: Constitutive expression of PTX3 by human amniotic stromal cells leads to the formation of HC-HA/PTX3 complex

In this example, PTX3 expression in HC-HA purified from AM and its effect on HC-HA/PTX3 complex formation in AM were examined.

Experimental procedures

1. Materials

Guanidine hydrochloride, cesium chloride, EDTA, anhydrous ethanol, potassium acetate, sodium acetate, sodium chloride, sodium hydroxide, Tris base, Triton X-100, 3-(N,N-dimethylpalmitylammonio)propanesulfonate (Zwittergent ^3-16 ), protease inhibitor cocktail (including 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, aprotinin, bestatin hydrochloride, E-64, leupeptin, and pepstatin A), and phenylmethylsulfonyl fluoride were obtained from Sigma-Aldrich (St. Louis, MO). Streptomyces hyaluronidase (HAase) and biotinylated HA binding protein (HABP) were from Seikagaku Biobusiness Corporation (Tokyo, Japan). Dulbecco's modified Eagle's medium, Ham's F12 nutrient medium, fetal bovine serum, Hank's balanced salt solution, gentamicin, amphotericin B, and RIPA buffer were purchased from Invitrogen (Grand Island, NY). Slide-A-Lyzer dialysis cassettes (3.5K MWCO) were from Fisher Scientific (Pittsburgh, PA). The BCA protein assay kit was from Pierce (Rockford, IL). The HA quantification kit was from Corgenix (Westminster, CO). 4–15% gradient acrylamide precast gels and nitrocellulose membranes were from Bio-Rad (Hercules, CA). IαI was prepared from human plasma in our laboratory according to published methods (1, 38). PTX3 mAb (MNB4) and pAb were from Enzo Life Sciences, Inc. (Plymouth, PA). Recombinant human TNF, recombinant human pentraxin 3/TSG-14, and human/mouse TSG-6 mAbs (MAB2104) were from R&D Systems (Minneapolis, MN). Mouse anti-human ITIH1 polyclonal antibody against full-length ITIH1 and rabbit anti-human ITIH2 polyclonal antibody against amino acids 124–321 were obtained from Abcam Inc. (Cambridge, MA). HiPerFect transfection reagent and RNeasy Mini RNA isolation kit were obtained from QIAGEN (Valencia, CA). Small interfering RNA (siRNA) oligonucleotides targeting endogenous human PTX3 (ACACUUGAGACUAAUGAAAGAGAGA) and non-targeting siRNA control oligonucleotides (scrambled RNA) were obtained from OriGene Technologies, Inc. (Rockville, MD). Western Lighting ^™ chemiluminescent reagent was obtained from PerkinElmer, Inc. (Waltham, MA). An ultracentrifuge (LM8, SW41 rotor) was obtained from Beckman Coulter, Inc. (Fullerton, CA).

2. Cell Culture and Agarose Overlay

Human tissues were handled in accordance with the Declaration of Helsinki. Fresh human placentas were obtained from healthy mothers following elective cesarean sections at Baptist Hospital (Miami, FL) with approval from the Baptist Healthcare South Florida Institutional Review Board (protocol number 03-028). Primary human AM epithelial and stromal cells (referred to as AMEC and AMSC, respectively) were isolated from fresh placenta as described previously (Chen et al. (2007) Stem Cells. 25: 1995-2005; Li et al. (2008) J. Cell. Physiol. 215: 657-664) and cultured in hormone-supplemented epithelial medium (SHEM, which consists of DMEM/ _F12 (1:1, v/v), 5% (v/v) FBS, 0.5% (v/v) dimethyl sulfoxide, 2 ng/ml EGF, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, 0.5 μg/ml hydrocortisone, 0.1 nM cholera toxin, 50 μg/ml gentamicin, and 1.25 μg/ml amphotericin B) at 37°C in a humidified atmosphere with 5% CO2 (Chen et al. (2007) Stem Cells. 25: 1995-2005; Li et al. (2008) J. Cell. Physiol. 215: 657-664). Cells 25:1995-2005; Chen et al. (2011) Tissue Eng Part C Methods 17:537-548). The culture medium was changed every 2 days. 80% confluent cells were switched to DMEM/F1 containing 0.5% FBS for 48 h to allow the cells to rest and then treated with 20 ng/ml TNF or 20 ng/ml IL-1β for 4 h or 24 h, followed by RT-PCR and Western blot analysis. For agarose overlay culture, AMEC, AMSC and HSF were seeded in SHEM at a density of 2×10 ⁴ /cm ² in 12-well (1×10 ⁵ cells/well) and 6-well (2x10 ⁵ cells/well) plates. On day 1, the culture medium was changed to serum-free SHEM containing 5% KnockOut serum replacement and 2-phospho-L-ascorbic acid and cultured for another 2 days. After removing the culture medium, 3% agarose (low melting point type, type VII, Sigma, A9045) in DMEM/F12 containing 1 mM 2-phospho-L-ascorbic acid was layered at 1 ml or 0.5 ml to achieve a 1 mm thick gel layer at room temperature for 5-10 minutes, followed by addition of 3 ml or 1.5 ml of serum-free SHEM medium containing or not containing 5 ng/ml TNF to each 6 or 12 well plate, respectively. Cells were harvested on day 5 without changing the intermediate culture medium.

3. siRNA Transfection

AMECs and AMSCs were cultured in SHEM in 6-well plates until 80% confluence. Cells were switched to DMEM/F12 containing 0.5% FBS for 48 h and transfected with PepMute ^™ siRNA containing 100 nM PTX3 siRNA or scrambled (sc) RNA. After 48 h, cells were harvested and subjected to RT-PCR and Western blot analysis.

4. Purification of HC-HA Complexes from AM and Serum-free Medium by Ultracentrifugation

HC-HA complexes were purified from AM and cell culture medium as previously described (He et al. (2009) J. Biol. Chem. 284:20136-20146; Yoneda et al. (1990) J. Biol. Chem. 265:5247-5257; He et al. (2008) Invest. Ophthalmol. Vis. Sci. 49:4468-447532). Briefly, cryopreserved human AM obtained from Bio-tissue, Inc. (Miami, FL) was cut into small pieces, frozen in liquid nitrogen, and ground into a fine powder using a BioPulverizer. The powder was mixed with cold phosphate-buffered saline (PBS) at a 1:1 (g/ml) ratio. The mixture was maintained at 4°C for 1 hour with gentle stirring and then centrifuged at 48,000 g for 30 minutes at 4°C. The supernatant (referred to as AM extract) was then mixed with an 8M guanidine HCl/PBS solution containing 10mM EDTA, 10mM aminocaproic acid, 10mM N-ethylmaleimide, and 2mM PMSF (volume ratio of 1:1 v/v) and adjusted to a density of 1.35g/ml (AM extract) or 1.40g/ml (cell extract) using cesium chloride, and subjected to isopycnic centrifugation at 35,000rpm at 15°C for 48h. The resulting density gradient was divided into 12 tubes (1ml/tube), where the HA and protein contents were determined using a HA quantification test kit and a BCA protein test kit, respectively. The fractions from the first ultracentrifugation (which contained most of the HA) were pooled, brought to a concentration of 1.40g/ml by adding CsCl, ultracentrifuged, and fractionated in the same manner as described above. The fractions from the second ultracentrifugation (which contained HA but no detectable protein) were pooled and subjected to a third and fourth ultracentrifugation at a concentration of 1.42 g/ml by adding CsCl. The fractions from the second and fourth ultracentrifugations were dialyzed in distilled water and subsequently precipitated for 1 h at 0°C using 3 volumes of 95% (v/v) ethanol containing 1.3% (w/v) potassium acetate. After centrifugation at 15,000 g, the precipitates were briefly air-dried and stored at 80°C. They were designated AM 2nd HC-HA and 4th HC-HA, respectively.

5. Immunostaining

Human fetal membranes, including AM and chorionic villus sections and cell culture media, were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.2% (v/v) Triton X-100 in PBS for 20 minutes, with or without agarose overlay. After blocking with 0.2% (w/v) bovine serum albumin in PBS for 1 hour, sections were incubated with biotinylated HABP (5 μg/ml for HA), anti-PTX3, anti-HC1, or anti-HC2 antibodies (all diluted 1:200 in blocking solution) in a 4°C humidity chamber overnight. After washing with PBS, they were incubated with Alexa Fluor 488 streptavidin (1:200 for HA) or the respective secondary antibodies (i.e., Alexa Fluor 488 anti-mouse IgG or Alexa Fluor 555-conjugated anti-rat IgG) for 1 hour at room temperature. Isotype-matched nonspecific IgG antibodies were used as controls. Alternatively, sections were treated with 50 U/mL Streptomyces HAase for 4 h at 37° C. before fixation. Nuclei were stained with Hoechst 33342, and images were acquired using a Zeiss LSM700 confocal laser scanning microscope (Zeiss, Germany).

6. Real-time PCR

Total RNA was extracted from cell culture medium using the RNeasy Mini RNA Isolation Kit. cDNA was reverse-transcribed from 1 μg of total RNA using the Cloned AMW First-Strand cDNA Synthesis Kit with oligo(dT) primers. First-strand cDNA was amplified by qPCR using AmpliTaq Gold Fast PCR Master Mix and specific PTX3 primers (46-48). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was used to normalize the amount of amplified product.

7. Western blotting

The culture supernatant was collected and the cells were washed six times with cold PBS, followed by incubation at 4°C for 1 hour in RIPA buffer under gentle agitation and centrifugation at 14,000g for 30 minutes at 4°C to obtain cell lysates. The protein concentrations in the culture supernatant and cell lysates were quantified using a BCA protein assay kit. The samples were incubated at 25°C for 1 hour in 50mM NaOH or dissolved in 0.1M sodium acetate buffer (pH 6.0) and incubated at 60°C for 1 hour in the presence or absence of 20 units/ml Streptomyces HAase. The samples were then resolved by SDS-PAGE on a 4-15% (w/v) gradient acrylamide precast gel under denaturing and reducing conditions and transferred to a nitrocellulose membrane. The membrane was then blocked with 5% (w/v) skim milk in 50 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl and 0.05% (v/v) Tween-20, and then incubated with different primary antibodies in succession, followed by incubation with their respective HRP-conjugated secondary antibodies. Immunoreactive proteins were visualized by Western Lighting ^™ chemiluminescent reagent.

result

Positive PTX3 staining in AM epithelium and dense stroma

Anti-human PTX3 antibodies were used to carry out immunofluorescence staining on the cross section of fresh human fetal membranes. The human fetal membranes are composed of an epithelial layer and an avascular matrix, which can be further divided into a dense layer and a spongy layer, and the chorion (Figure 33, stage) rich in cells below. Positive PTX3 staining was found in the top surface of the epithelium and the dense matrix. On the contrary, PTX3 staining was significantly weakened in the spongy matrix and the chorion (Figure 33, PTX3). HAase digestion did not enhance the PTX3 staining in the latter, indicating that the weak PTX3 staining in these two areas was not due to a masking effect. Biotinylated HABP was used to find strong positive HA immunostaining in the AM matrix, and relatively weak staining was found in the AM epithelium (Figure 33, HA), and in the upper layer of the chorion under the AM matrix, weak staining of HA was noted, but strong staining was noted in the lower layer of the chorion. This staining disappeared when pre-digesting tissue sections with HAase (Figure 33, HA (+HAase)), indicating that HA staining was specific. Immunostaining of each HC also showed positive staining in AM epithelium, stromal cells and stroma and chorion (Figure 33, HC1 and HC2).These results indicate the presence of PTX3 in AM, mainly in the dense stroma and epithelium.

Presence of PTX3 in AM soluble extracts and purified HC-HA complexes

To further investigate the presence of PTX3 in AM, we first performed Western blot analysis of AM extracts using isotonic saline buffer before and after treatment with 50 mM NaOH to cleave the ester bond. Recombinant PTX3 appeared as 45 kDa, 90 kDa, 180 kDa, and HMW species (Figure 34, lane 2). Soluble AM extracts showed 45 kDa and HMW species (most of which did not enter the gel) at the bottom of the sample well (Figure 34, lane 3). NaOH treatment did not affect the 45 kDa species, but completely eliminated the HMW species, resulting in HMW bands of PTX3 (Figure 34, lane 4). These results indicate that PTX3 exists in AME as a monomer and HMW complex. Since the latter can be dissociated into HMW bands by NaOH, which can cleave the ester covalently linked between HC-HA, the substance containing HMW PTX3 in the sample well may be associated with HC-HA.

To further confirm whether PTX3 is associated with AM HC-HA complexes, as previously reported (He et al. (2009) J. Biol. Chem. 284:20136-20146; Zhang et al. (2012) J. Biol. Chem. 287:12433–12444), we purified HC-HA complexes from AM soluble extracts by two and four consecutive ultracentrifugations and performed Western blot analysis with or without HAase digestion. In contrast to the monomers found in soluble AM, a 90 kDa species, corresponding to the size of the native PTX3 dimer, was observed in the second and fourth AM HC-HA complexes, in addition to the HMW bands at the bottom of the wells (Figure 34, lanes 5 and 7). In addition, a weak HMW smear was observed in the second HC-HA complex, and a strong HMW smear was observed in the fourth HC-HA complex. After HAase treatment, the 90 kDa dimer remained in both HC-HA complexes, but the HMW smear was enhanced in the 4th HC-HA and increased in the 2nd HC-HA, and the HMW band at the top of the gel disappeared (Figure 34, lanes 6 and 8), similar to the results seen in AME. The presence of the 90 kDa PTX3 dimer in the HC-HA complex with or without HAase was found to be caused by dissociation from HC-HA by 2-ME, as the elimination of 2-ME resulted in the disappearance of this 90 kDa species (not shown). These results indicate that despite four ultracentrifugations, the HC-HA complex still contains PTX3 bound to HC-HA to form the HC-HA/PTX3 complex.

Western blot analysis using an anti-HCl antibody revealed the presence of the HC-HA complex as a HMW species at the bottom of the well, which disappeared upon HAase digestion ( FIG. 34 , lanes 10-13), and also revealed the presence of HCl in the HC-HA complex, which was released from the HC-HA complex upon HAase digestion ( FIG. 34 , lanes 11 and 13). HC2 and TSG-6 were not detected (not shown). Together, these results confirm that the HC-HA purified from AM contained only HCl. We also noted a major difference between the second and fourth HC-HA complexes: a free 80 kDa HCl band was detected only in the second HC-HA complex ( FIG. 34 , lane 11), but not in the fourth HC-HA complex, indicating that the latter does not contain free HCl. The presence of HA in the fourth HC-HA was confirmed by agarose gel electrophoresis, which showed a continuous HA smear from the top well to the bottom of the gel, and this smear was resolved by HAase digestion.

AMECs and AMSCs express PTX3 mRNA and protein

We then determined that AM epithelial and stromal cell cultures synthesized PTX3. We cultured these cells as reported (Chen et al. (2007) Stem Cells 25(8):1995-2005; Li et al. (2008) J Cell Physiol. 215(3):657-64; Zhang et al. (2012) J. Biol. Chem. 287:12433–12444), extracted total RNA for RT-PCR, extracted protein for Western blot analysis, and compared them with human dermal fibroblasts (HCFs), which were reported to express PTX3 mRNA and protein only upon stimulation with proinflammatory cytokines such as TNF and IL-1. As expected, qRT-PCR results showed that expression of PTX3 transcripts was low in resting HSFs but was upregulated by TNF and IL-1β (Figure 35A). Although the expression of PTX3 transcripts in resting AMEC and AMSC is also very low, it is significantly increased by TNF or IL-1 β (Figure 35 A). Western blot analysis confirmed that PTX3 protein is very low (45kDa) in lysate and is not detected in the culture medium of resting HSF (Figure 35 B, lane 2), but is detected in lysate after adding TNF or IL-1 β 24h and is not detected in culture medium. On the contrary, PTX3 protein is detectable in resting AMEC, and it is significantly increased (Figure 35 B, lanes 5, 6 and 7) by TNF or IL-1b in lysate (45kDa) and culture medium (45kDa and 90kDa), wherein TNF is more effective than IL-1b. The same findings (Figure 34 B, lanes 8, 9 and 10) were noted in AMSC. To demonstrate that the 75kDa and 135kDa bands in lysates from all cells and the 50kDa band in the culture medium were nonspecific, as these bands did not change under TNF or IL-1b, PTX3 siRNA transfection was performed and indeed downregulated the 45kDa species in the lysates as well as the 45kDa and 90kDa species, but did not downregulate these nonspecific bands (Figure 35C). Together, these results indicate that resting AM cells do synthesize and secrete PTX3, and that it is further upregulated by proinflammatory stimuli.

AM stromal cells produce HC-HA/PTX3 complexes

Previous studies have shown that HC-HA (i.e., SHAP-HA) complexes can be isolated from mouse skin fibroblasts cultured in PBS-supplemented medium, and that the isolated HC-HA contains both HCl and HC2 derived from FBS-derived IαI (Yoneda et al. (1990) J. Biol. Chem. 265:5247-5257; Huang et al. (1993) J. Biol. Chem. 268:26725-26730). However, we reported that AM cells produce HC-HA using their endogenously produced IαI (Zhang et al. (2012) J. Biol. Chem. 287:12433–12444). Since AM cells synthesize the PTX3 protein, which can be further increased by TNF and IL-1, we aimed to determine whether they produce HC-HA that also contains PTX3. We used HSF as a control, which express PTX3 under proinflammatory stimuli (e.g., TNF and IL-1) only under serum-containing conditions (Yoneda et al. (1990) J. Biol. Chem. 265:5247-5257; Huang et al. (1993) J. Biol. Chem. 268:26725-26730) ( FIG35 ), and compared it with AMECs and AMSCs cultured under serum-free and serum-containing conditions with or without TNF. We also overlaid 3% agarose on the cell monolayer because this approach has been found to trap secreted procollagen at or near the cell surface rather than into the culture medium on corneal cell cultures (Hassell et al. (2008) Experimental Eye Resarch. 87:604-611; Etheredge et al. (2010) Matrix Boil. 29:519-524). After 5 days of coverage, HSF, AMSC and AMEC became more compact, especially under serum-containing conditions (Figure 36). Epithelial morphology became more distinct in AMEC.

We then determined whether 3% agarose overlays were equally effective in capturing secreted HA by measuring HA concentrations in the culture medium using an HA ELISA assay. Without agarose overlays, HA levels were readily detectable in serum-free culture medium for AMSC and AMEC, but not in serum-free culture medium for HSF. TNF significantly increased HA levels in all three cell culture media (Figure 37). This pattern was further enhanced in serum-containing culture medium. In all three cultures, agarose overlays reduced HA levels by more than 50% under both serum-free and serum-containing conditions. These results indicate that agarose overlays do reduce HA levels entering the culture medium.

To determine when the secreted HA after agarose coating is truly captured in the extracellular matrix, we used biotinylated HABP, specific antibodies for HCl and HC2, and two different anti-PTX3 antibodies, i.e., MNB4 and biotinylated pAb, to perform double immunostaining of PTX3/HA, HCl/HA, and HCl/PTX3. Under serum-free conditions, positive HA staining was noted in the pericellular region of HSF, while PTX3 staining was negative (Figure 38). Upon stimulation with TNF, positive PTX3 staining was observed in the cytoplasm (Figure 38B), confirming the inducible expression of PTX3 by TNF in HSF. TNF did not significantly increase HA staining intensity, but induced cable-like structures (Figure 38G), which is similar to what has been reported in cultured renal proximal tubular epithelial cells (Selbi et al. (2006) Kidney International.70:1287-1295). No colocalization of PTX3 and HA was observed, indicating that PTX3 is not associated with HA in HSF after TNF stimulation. On the contrary, positive HA staining was detected in resting AMSCs as a fiber network on the cell surface and extracellular matrix, colocalizing with HA in the extracellular matrix (Figures 38C and 6K). TNF further increased the intensity of PTX3 staining and the amount of HA fibrils (Figures 38D and 38K).

In resting AMEC, positive HA staining is also found in the extracellular space with some fibrillar appearance, but only in the less dense interspersed areas of the cells (Figures 38E and 38L). PTX3 and HA colocalization is also observed in AMEC. TNF treatment further increases the PTX3 staining intensity and the amount of HA fibrils (Figure 38F). These results further confirm that PTX3 is constitutively expressed by AMSC and AMEC, and its expression can be further increased by TNF. Under conditions of (Figures 38G and 38J) or without (not shown) TNF treatment, weak positive HCl staining is observed only in some HSFs, and especially colocalizes with HA in HA cable-like structures (Figure 38G). However, we did not notice colocalization between HCl and PTX3 (Figure 38J). On the contrary, strong positive HCl staining is noted in AMSC, and colocalizes with HA on the cell membrane (Figure 38H) and colocalizes with PTX3 in the cytoplasm (Figure 38K). AMECs also showed strong positive HCl staining and colocalized with HA in the cell membrane (Figure 38I) and with PTX3 in the cytoplasm and cell membrane (Figure 38L). These results together indicate that HA-rich matrix is effectively captured by agarose overlay in AMSCs, AMECs, and HSFs, and contains both HCl and PTX3 only in AMSCs and AMECs under serum-free conditions, further confirming that such HC-HA/PTX3 complexes are synthesized by endogenous IαI.

To further confirm the formation of HC-HA/PTX3 complexes under agarose overlay conditions, we extracted the cell layer with 6M GnHCl and performed Western blot analysis in serum-free and serum-containing AMSC and HSF cultures with or without NaOH treatment. Serum-free HSF showed 170 kDa and 140 kDa species, but no 45 kDa species corresponding to control PTX3, indicating that both bands were nonspecific (Figure 38D, lanes 2-5). Serum-containing HSF with TNF showed a faint 140 kDa species and some small MW species, similar to those seen in serum-free HSF (Figure 39, lanes 11 and 12). In contrast, serum-free AMSC showed a weak HMW PTX3 smear, 90 kDa and 45 kDa species (Figure 39, lane 6), the latter two corresponding to PTX3 dimers and monomers, respectively (Figure 39, lane 2). These results are similar to those seen in AME (Figure 34). The same nonspecific 170 kDa species and some small molecules as those seen in serum-free HSF were also observed. NaOH treatment increased the HMW PTX3 smear band but did not affect PTX3 monomer and dimer (Figure 39, lane 7) and other substances. TNF increased the HMW PTX3 smear band, PTX3 dimer and monomer, and other substances (Figure 39, lanes 8 and 9). Serum-containing AMSCs with TNF showed a strong HMW PTX3 smear band, 90 kDa PTX3 dimer, and 45 kDa PTX3 monomer (Figure 39, lane 13). NaOH treatment significantly increased the HMW PTX3 smear band and two substances at 60 kDa and 50 kDa (Figure 39, lane 14). Since NaOH breaks the ester bond between HC and HA, resulting in the dissociation of HC-HA, these results indicate that HMW PTX3 is released from the HC-HA complex. These results collectively indicate that AMSCs produce HC-HA complexes containing PTX3, while HSFs do not.

In vitro reconstitution of HC-HA/PTX3 complex (rcHC-HA/PTX3)

To further demonstrate how the HC-HA/PTX3 complex might be generated, we reconstituted the HC-HA/PTX3 complex in vitro using HA, TSG-6, IαI, and PTX3. We first immobilized HA on plastic and successfully added recombinant TSG-6, purified IαI, or serum as a source of IαI. It has been reported that TSG-6 can form a stable TSG-6/HA complex by binding to HA on an immobilized surface (Wisniewski et al. (2005) J Biol Chem. 280:14476-84), and that both free and HA-bound TSG-6 can transfer HC from IαI to immobilized HA to form HC-HA (Colon (2009) J Biol Chem. 284:2320-31). As expected, Western blotting using an anti-IαI antibody did not detect any material in the control iHA alone (Figure 40A, lane 2). When IαI and TSG-6 were added to iHA simultaneously, weak 220kDa IαI, 85kDa HC2, strong ~80kDa HCl, and 50kDa species were detected (Figure 40A, lane 3), indicating that in the presence of TSG-6, both HCl and HC2 were transferred to iHA to form HC-HA. Comparison of the band intensities of HCl and HC2 showed that more HCl was transferred to iHA by TSG-6 than HC2, resulting in a truncated 50kDa species. When PTX3 was added to iHA simultaneously with IαI and TSG-6, the intensity of the IαI species decreased, but the HCl intensity increased in a PTX3 dose-dependent manner (Figure 40A, lanes 4-6), while HC2 was undetectable, indicating that PTX3 preferentially promoted the transfer of HCl to immobilized HA without promoting HC2 transfer, catalyzed by TSG-6. When PTX3 was added after the simultaneous addition of IαI and TSG-6, HCl was detected but IαI or HC2 was not, and the HCl intensity also increased in a PTX3 dose-dependent manner (Figure 40A, lanes 7-9), confirming that PTX3 promoted the transfer of HCl to immobilized HA without promoting HC2 transfer. These results indicate that PTX3 uniquely promotes the transfer of primary HCl to immobilized HA to form an HCl-HA complex, whether simultaneously or sequentially. Western blotting using an anti-TSG-6 antibody showed that a 35 kDa TSG-6 monomer and a 75 kDa dimer were detected in rcHC-HA formed between IαI and TSG-6 on iHA by the simultaneous or sequential addition of PTX3. Since the intensity of the TSG-6 band decreased in a dose-dependent manner, it was shown that TSG-6 bound to immobilized HA could be competed out by PTX3 (Figure 40B). When PTX3 was added simultaneously with IαI and TSG-6, Western blotting using an anti-PTX3 antibody revealed prominent HMW PTX3 bands in the wells, with faint tetramer and dimer bands, and their intensities increased in a PTX3 dose-dependent manner (Figure 40C, lanes 4-6). This finding suggests that PTX3 preferentially and strongly binds to the rcHC-HA complex, with the binding resistant to 8M GnHCl washing. In contrast, when PTX3 was added sequentially after IαI and TSG-6, we did not detect HMW PTX3 and tetramer and dimer species (Figure 40C, lanes 7-9), indicating that the binding between PTX3 and pre-formed rcHC-HA PTX3 was not strong enough to withstand the 8M GnHCl wash. Therefore, these in vitro reconstitution experiments indicate that PTX3 must be produced in vivo simultaneously with HA, IαI, and TSG-6 to allow the formation of HC-HA/PTX3. This interpretation is supported by the immunological colocalization of HA, HC, and PTX3 in in vivo tissue sections and in the extracellular matrix formed by AMSCs.

Example 33: Effect of HC-HA Complex on TGFβ1 Signaling

Immobilized HC-HA inhibits TGFβ1 signaling by downregulating TGF-β1 expression but upregulating TGF-β3 signaling. This inhibition of TGFβ1 signaling, due to further inhibition by TGFβRII and TGFβRIII, resists stimulation by the addition of serum or exogenous TGF-β1. As a result, immobilized HC-HA prevents myofibroblast differentiation of corneal fibroblasts by inhibiting SMAD2/3 signaling and the expression of α-smooth muscle formation. This effect is also effective enough to revert corneal fibroblasts to keratocytes. HC-HA (insoluble (I)) differs from HC-HA (soluble (S)) in that it contains additional small leucine-rich proteins (SLRPs) and activates the expression of TGFβ1 and BMP signaling by upregulating the expression of BMPs and their receptors, thereby activating pSMAD1/5/8, which together further promote aggregate formation. These effects can further dedifferentiate corneal fibroblasts into neural crest progenitor cells.

In this example, the effects of immobilized soluble and insoluble HC-HA on TGF-β signaling in human corneal fibroblasts were examined with or without exogenous TGFβ1 stimulation. In addition, the effects of HC-HA complexes on the inhibition of SMAD2/3 signaling and αSMA expression were examined.

Experimental and clinical studies support an anti-scarring therapeutic effect of cryopreserved amniotic membrane (AM). Our studies demonstrate that a heavy-chain hyaluronan complex (HC-HA) is uniquely produced and purified from AM and inhibits TGF-β1 promoter activity in human corneal fibroblasts. It is unknown whether HC-HA inhibits TGF-β1 mRNA and protein expression and promotes TGF-β3 mRNA and protein expression, which are known to counteract TGF-β1 signaling, and, if so, whether this inhibition of TGF-β signaling by HC-HA translates into inhibition of nuclear translocation of pSMAD2/3.

If cultured on AM, mouse corneal cells can maintain an undifferentiated state (expressing corneal proteoglycans) in serum-free DMEM/ITS or DMEM/10% FBS on plastic. Treatment of corneal cells with 10% serum or 10 ng/ml TGF-β1 in serum-free DMEM/ITS induces Smad2/3 phosphorylation and nuclear localization (3 h) and α-SMA expression (5 days). The activation of Smad2/3 and α-SMA in corneal cells on AM treated with serum or TGF-β1 was inhibited (Kawakita et al. (2005) J Biol Chem. 280(29):27085-92). Our studies have shown that HC-HA inhibits TGF-β1 promoter activity in human corneal fibroblasts. It is expected that pSmad2/3 signaling and α-SMA formation will be inhibited by HC-HA.

As described above, human corneal fibroblasts (or Limbal alveolar cells, p3) were seeded in plastic dishes for 48 h with or without immobilized HA, soluble HC-HA (PBS) (2X or 4X) or insoluble HC-HA (GnHCl) (2X or 4X). The cells were then treated with or without TGFβ1 for 24 h and then harvested for mRNA quantification and signal transduction assays. For the determination of TGFβ receptor protein, cells were treated with or without TGFβ1 for 24 h and then protein samples were collected to allow time for protein expression from expressed mRNA. For TGF-β1 ELISA, cells were treated with or without TGF-β1 for 24 h and then cultured in fresh medium for 24 (and 48) h. Supernatants were collected for TGF-β1 ELISA. For TGF-β2 and TGF-β3 ELISA, cells were treated with or without TGF-β1 for 48 h. Supernatants were collected for TGF-β2 and TGF-β3 ELISA. Phase contrast images of different cultures were taken up to 72 h.

The following experiments were conducted:

1. Semi-quantification of mRNA for TGF-β1, TGF-β2, TGF-β3, and their receptors by real-time PCR: This assay is used to assess the expression of mRNA transcripts for the TGF-β family and its receptors. The real-time RT-PCR profile consists of an initial activation step at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 minutes, and annealing and extension at 60°C for 1 minute.

2. Determination of α-smooth muscle formation and SMAD2/3 signaling by immunostaining: Standard immunostaining procedures were used to monitor α-smooth muscle formation and SMAD2/3 signaling.

The experimental groups for Experiments 1 and 2 were:

PBS PBS+TGF-β1 HA HA+TGF-β1 2X HC-HA PBS 2X HC-HA PBS + TGF-β1 4X HC-HA PBS 4X HC-HA PBS + TGF-β1 2X HC-HA Gn 2X HC-HA Gn+TGF-β1 4X HC-HA Gn 4X HC-HA Gn+TGF-β1

3. Quantification of TGFβR by Western blotting: Used to quantify the protein concentrations of TGFβRI, TGFβRII, and TGFβRIII using their corresponding antibodies (R&D Systems). The order of loading is as follows:

4. ELISA for quantification of TGFβ in culture medium: The Quantikine Human TGF-β1 and TGF-β2 ELISA kits from R&D Systems and the TGF-β3 ELISA kit from Norvus Biologicals are solid phase ELISAs designed to measure TGF-β1, TGF-β2, and TGF-β13 in acid-activated cell culture supernatants, serum, plasma, and urine. They contain recombinant human TGF-β1, TGF-β2, and TGF-β3 and have been shown to accurately quantify the recombinant factors. The results obtained using native TGF-β1, TGF-β2, and TGF-β3 showed linear curves parallel to the standard curves obtained using the recombinant kit standards. These kits were used to determine TGF-β1, TGF-β2, and TGF-β3 in culture medium. The experimental groups for Experiment 4 were:

PBS PBS+TGF-β1 HA HA+TGF-β1 4X HC-HA PBS 4X HC-HA PBS + TGF-β1 4X HC-HA Gn 4X HC-HA Gn+TGF-β1

result

HCF seeded in DMEM/10% FBS formed aggregates only on insoluble HC-HA by 72 hours (Figure 41). These aggregates persisted even after 24 hours of serum starvation in DMEM/ITS (insulin/transferrin/selenium) medium. Cells were subsequently cultured in three different media: A - DMEM/ITS for 48 hours; B - 24 hours DMEM/ITS, 24 hours DMEM/10% FBS; and C - 24 hours DMEM/ITS, 24 hours in DMEM/ITS supplemented with 10 ng/ml TGF-β1. Cells cultured in HC-HA formed small aggregates on 4X HC-HA (Gn), but not in the other culture conditions (Figure 41). However, after 2 hours of seeding in DMEM/10% FBS on an immobilized substrate, only the HCF on the control adhered well. Cells on HA and HC-HA [HC-HA (4X, PBS) and 4X, GnHCl] were all rounded, indicating that they were not well attached. After 72 hours of incubation, all cells were well attached, however, fewer cells were present on immobilized HC-HA. The number of cells on these substrates was greater on HC-HA (4X, PBS) than on HC-HA (4X, GnHCl). HCF began to form aggregates on HC-HA (Gn) after 24 hours of culture and concentrated into larger aggregates after 72 hours of culture. Following TGFβ1 stimulation, aggregates were observed on HCF cultured on HC-HA (4X, PBS; 4X, Gn).

In conclusion, HC-HA(Gn) promoted HCF aggregation in the presence or absence of TGFβ1 stimulation, whereas HC-HA(PBS) promoted HCF aggregation under TGFβ1 stimulation. The significance of the aggregation is unknown.

In DMEM/ITS, expression of TGFβ1 and TGFβ3 transcripts was increased by HC-HA (Gn), but TGFβ3 transcripts were increased by HC-HA (PBS) (Figure 42). As expected from autoinduction, TGFβ1 and TGFβ3 mRNA were stimulated by TGFβ1 to increase 4-fold and 2-fold, respectively, in HCF cultured on plastic, and TGFβ1 protein increased accordingly from 60 pg/ml to 105 pg/ml (Due to experimental error, TGFβ3 protein was not detected in the experiment). Under serum-free conditions, soluble 4X HC-HA reduced TGFβ1 protein expression. Although it promoted TGFβ1 mRNA expression, insoluble HC-HA also reduced secreted TGFβ1, which remained higher than the control cultured in DMEM/ITS. In addition, significant inhibition of TGFβRII and TGFβRIII was observed by both soluble and insoluble HC-HA. Results showed that autocrine TGFβ signaling was inhibited in HCF cultured on either soluble or insoluble HC-HA, but paracrine TGFβ signaling was maintained in HCF cultured on insoluble HC-HA. Furthermore, under serum-free conditions without TGFβ1 stimulation, both soluble and insoluble 4X HC-HA upregulated TGFβ3 mRNA expression by 3-fold, a molecule known to counteract TGFβ1 signaling. Upon TGFβ1 stimulation, TGFβ3 mRNA expression increased 5- and 8-fold when HCF were cultured on soluble and insoluble HC-HA, respectively, indicating that soluble and insoluble HC-HA strongly promoted TGFβ3 transcript expression. These results also suggest that HC-HA purified from AM contributes to the anti-scarring effects of AM not only by inhibiting TGFβ1 signaling but also by significantly upregulating TGFβ3 expression. Our results suggest that HC-HA (PBS and Gn) do not appear to affect TGFβ2 expression at the mRNA or protein levels. In conclusion, HC-HA(PBS) inhibited TGFβ1 but activated TGFβ3 signaling in HCFs challenged with TGFβ1, whereas HC-HA(Gn) activated both TGFβ1 and TGFβ3 signaling.

In plastic controls, TGFβRII mRNA was upregulated 8-fold under TGFβ stimulation (Figure 43). TGFβII and TGFβIII receptor mRNA were upregulated 2-fold to 8-fold by HC-HA (PBS and Gn), respectively, under serum-free conditions, but were completely inhibited under TGFβ1 stimulation. The same results were noted for HA. When HCFs cultured on HC-HA (PBS and Gn, respectively) were stimulated with TGFβ, the corresponding protein expression of TGFβRII and TGFβRIII was downregulated 3-fold and 3-fold, and 2-fold and 3-fold, respectively. In this case, HA did not change the expression of these proteins. Such downregulation may partially explain the mechanism of the anti-inflammatory and anti-scarring effects of AM. In summary, when HCFs were cultured on HA and HC-HA (PBS and Gn), the mRNA expression of TGFβR2 and TGFβR3 was increased.

Immunostaining showed that HC-HA (PBS and Gn) inhibited pSMAD2/3 nuclear translocation in DMEM/ITS with or without TGFβ1 stimulation (Figure 44). This effect was more pronounced when TGFβ was used. This finding further confirms that inhibition of TGFβ1, TGFβRII, and TGFβRIII is translated into inhibition of SMAD-mediated signaling.

In addition, immunostaining results showed that both soluble and insoluble HC-HA inhibited the formation of α-SMA after TGFβ1 stimulation, further supporting that soluble and insoluble HC-HA inhibited TGF-β1 signaling in the presence or absence of TGFβ1, thereby inhibiting the differentiation of HCF into myofibroblasts (Figure 45).

In summary, under serum-free and TGFβ1-stimulated conditions, soluble HC-HA downregulated TGF-β1 but upregulated TGF-β3 expression, whereas insoluble HC-HA upregulated both TGF-β1 and TGF-β3 expression in HCFs. Because both soluble and insoluble HC-HA downregulated the expression of TGFβRII and TGFβRIII, these changes led to inhibition of TGFβ signaling, as evidenced by the lack of nuclear translocation of pSMAD2/3 and the inhibition of α smooth muscle formation.

Example 34: Effect of HC-HA Complex on BMP Signaling

In this example, the effects of immobilized HC-HA and additional TGF-β1 on BMP signaling were examined. Activation of BMP signaling through activation of pSMAD1/5/8 was also determined.

BMPs constitute a subgroup of the TGFβ superfamily, which includes BMP1-3, BMP3b, BMP4-7, BMP8a, BMP8b, and BMP9-15. BMPs bind to type II receptors (ALK2, ALK3, or ALK6), which activate type I receptors to phosphorylate Smad1, Smad5, and Smad8, leading to nuclear translocation of pSmad1/5/8 (reviewed in Massague 2000; Herpin, 2007). It is unclear which specific BMPs and BMP receptors are present in HCFs, and if so, whether BMP signaling can be activated by HC-HA (PBS and Gn) and additional TGFβ1 when TGFβ signaling is inhibited, and if so, which forms of BMPs and BMP receptors play a major role in controlling BMP signaling, and whether this activation of BMP signaling is through pSMAD1/5/8.

Human corneal fibroblasts were seeded on plastic containing or without immobilized HA or HC-HA PBS (4X) or HC-HAGn (4X) for 48 hours, followed by treatment with or without TGFβ1 for 24 hours as described above for mRNA quantification and determination of pSMAD1/5/8. To determine BMP receptor protein, cells were treated with or without TGF-β1 for 48 hours before collecting protein samples to allow protein expression. For BMP ELISA, cells were treated with or without TGF-β1 for 48 hours. Supernatants were collected for BMP ELISA. Phase contrast images of each culture were collected until 72 hours.

The cultures were subjected to the following experiments:

1. Semi-quantification of BMP and its receptor mRNA by real-time PCR: used to estimate the expression of mRNA transcripts of the BMP family and their receptors.

2. Determination of α-smooth muscle myogenesis and SMAD1/5/8 signaling by immunostaining: Immunostaining was performed to monitor α-smooth muscle myogenesis and SMAD2/3 signaling.

The experimental groups for Experiments 1 and 2 were:

PBS PBS+TGF-β1 HA HA+TGF-β1 4X HC-HA PBS 4X HC-HA PBS + TGF-β1 4X HC-HA Gn 4X HC-HA Gn+TGF-β1

3. Quantification of BMPR by Western blot analysis using BMPR1A, BMPR1B, and BMPR2 antibodies: Used to quantify the protein concentrations of BMPR1A, BMPR1B, and BMPR2, respectively. The order of sample addition is as follows:

4. ELISA for quantification of BMP in culture medium: We used a BMP ELISA kit (R&D Systems) to measure BMP in culture medium. The experimental groups for Experiment 4 were:

PBS PBS+TGF-β1 HA HA+TGF-β1 4X HC-HA PBS 4X HC-HA PBS + TGF-β1 4X HC-HA Gn 4X HC-HAGn+TGF-β1

result

Under resting conditions, HA, HC-HA (PBS), and HC-HA (Gn) activated BMP6 transcript expression by 7-fold and 4-fold, respectively (Figure 46). In the presence of TGFβ1, HA, HC-HA (PBS), and HC-HA (Gn) activated BMP4 transcript expression by 6-fold, 11-fold, and 6-fold, respectively, and BMP6 mRNA expression in HCFs was 30-fold, 37-fold, and 46-fold, respectively, indicating that HA and soluble and insoluble HC-HA can upregulate BMP4/6 expression, and additional TGFβ1 further significantly upregulates BMP4 and BMP6 signaling. BMP7 and BMP9 were not detected.

Although TGFβ1 itself did not activate BMPR1A transcript expression, HC-HA(PBS) and HC-HA(Gn), but not HA, activated BMPR1A transcript expression by 7-fold and 3-fold, respectively, in the presence of TGFβ1, suggesting that BMPR1A may play a major role in BMP signaling activated by HC-HA+TGFβ1 (Figure 47). Furthermore, TGFβ1 activated BMPR1B by 3-fold and BMPR2 by 3-fold, but activated 4-fold on plastic containing or without HA, HC-HA(PBS), and HC-HA(Gn), suggesting that TGFβ1 itself nonspecifically activates BMPR1B and BMPR2 mRNA expression. HC-HA(PBS) or HC-HA(Gn) enhanced BMPR2 transcript expression by 4-fold. BMP-BMPR1A is expected to activate SMAD1/5/8 signaling, while BMP-BMPRII activates non-SMAD signaling.

Immunofluorescence results showed that TGFβ1 itself moderately activated the nuclear translocation of pSMAD1/5/8 in HCFs, regardless of the substrate used (Figure 48). HC-HA (PBS and Gn) strongly promoted the activation of BMP4/6 signaling through the nuclear translocation of pSMAD1/5/8, as evidenced by more nuclei with pSMAD1/5/8 and stronger nuclear staining of pSMAD1/5/8.

ID1 is a helix-loop-helix (HLH) protein that forms heterodimers with members of the basic HLH family of transcription factors, downstream genes known to be regulated by SMAD1/5/8 signaling. Our results showed that when HCFs were cultured in HC-HA (PBS) and HC-HA (Gn), activation of SMAD1/5/8 led to a 4-fold and 8-fold upregulation of ID1 mRNA, respectively, indicating that SMAD1/5/8 signaling in HCFs is indeed activated by HC-HA + TGFβ (Figure 49). Since ID1 lacks DNA binding activity and thus inhibits the DNA binding and transcriptional activation abilities of the basic HLH proteins with which it interacts, we anticipate that ID1 plays an important role in cell growth, aging, and differentiation.

Example 35: Effect of HC-HA complex on myofibroblast differentiation and reversion of human corneal fibroblasts to corneal Effects of cells or younger progenitor cells

Keraocytes, a unique population of neural crest-derived cells embedded in the corneal stroma, express keratan sulfate-containing proteoglycans, including cornea-specific keratan. Kera is a cornea-specific keratan sulfate proteoglycan (KSPG) in the adult vertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP) gene family and is one of the major components of extracellular KSPG within the vertebrate corneal stroma. Corneal KSPG plays a key role in matrix assembly, which is responsible for corneal transparency. Luminal proteoglycan constitutes approximately half of corneal KSPG. The majority of the remaining corneal keratan sulfate-modified keratan. In adult tissue, keratin is confined to the corneal stroma, and keratin expression is considered a phenotypic marker of keratocytes (Liu et al. (2003) J. Biol Chem. 278(24):21672-7; Carlson et al. (2005) J Biol Chem. 280(27):25541-7). On plastic dishes, human, bovine, and rabbit keratocytes lose their characteristic dendritic morphology and keratin expression when cultured in serum-containing medium (Espana et al. (2003) Invest Ophthalmol Vis Sci. 44(12):5136-41; Espana et al. (2004) Invest Ophthalmol Vis Sci. 45(9):2985-91). These exposed cells downregulated the expression of keratan sulfate-containing proteoglycans, keratan, and CD34, and upregulated the expression of chondroitin-dermatan sulfate-containing proteoglycans and α-SMA, indicating that these cells became more differentiated.

Previous studies have shown that due to downregulation of the Smad signaling pathway, human (Espana et al. (2003) Invest Ophthalmol Vis Sci. 44(12):5136-41; Espana et al. (2004) Invest Ophthalmol Vis Sci. 45(9):2985-91) and murine (Kawakita et al. (2005) J Biol Chem. 280(29):27085-92) keratocytes can maintain their phenotype and not differentiate into myofibroblasts expressing α-SMA when cultured on the surface of AM stroma, even when TGF-β is added to serum-containing culture medium. Amniotic membrane stroma can maintain keratin expression in culture and prevent keratocytes from differentiating into myofibroblasts (Kawakita et al. (2005) J Biol Chem. 280(29):27085-92). Keratocytes maintain a dendritic morphology, continue to express corneal stroma-specific keratin for at least five passages (split 1:2), and do not express α-SMA in serum-containing conditions or with the addition of TGF-β1 (Espana et al. (2004) Invest Ophthalmol Vis Sci. 45(9):2985-91). Murine keratocytes can also be expanded on AM for at least eight passages without losing their normal phenotype, and inhibition of the Smad-mediated TGF-β signaling pathway is crucial for maintaining the keratin-expressing phenotype (Kawakita et al. (2005) J Biol Chem. 280(29):27085-92). In this example, we examined whether this was true for immobilized HC-HA and, if so, whether additional TGFβ1 could affect this outcome.

result

HA upregulates corneal proteoglycan mRNA expression by 4 times (Figure 50). Human corneal fibroblasts are seeded on plastic containing or not containing immobilized HA for 48 hours, serum-free for 24 hours, and then treated with or without TGFβ1 for 24 hours before being harvested for mRNA quantification and SMAD2/3 signaling assay. For the determination of TGF-β receptor protein, because the expression of protein lags behind mRNA expression, TGFβ1 is used or not to treat cells for 48 hours before collecting protein samples. For TGF-β1 ELISA, cells are treated with or without TGFβ1 for 24 hours and then cultured in fresh culture medium for 24 (and 48) hours. Supernatant is collected for TGF-β1 ELISA. For the ELISA of TGF-β2 and TGF-β3, cells are treated with or without TGFβ1 for 48 hours. Supernatant is collected for ELISA of TGF-β2 and TGF-β3. As expected, immobilized HC-HA increased keratin mRNA expression by 14- and 16-fold, respectively, indicating that these HCFs were younger when cultured on HC-HA without TGFβ1. Following TGFβ1 stimulation, keratin mRNA expression levels were significantly downregulated on both plastic and HA. However, keratin expression was maintained 3-fold on HC-HA (soluble, PBS). No keratin expression was observed on HC-HA (insoluble, Gn). We expected that the phenotype produced on HC-HA (I) would be more youthful than that of corneal cells.

Correspondingly, immobilized HC-HA(I/S) elevated keratin protein levels 8-fold and 10-fold, indicating that these HCFs actually reverted to keratocytes when cultured on HC-HA(S/I) (Figure 51). We did not see any corresponding keratin protein expression with the other treatments tested, including HA (4-fold increase in keratin mRNA) and 4X HC-HA(PBS) (3-fold increase in keratin mRNA), indicating that this moderate increase in keratin mRNA expression was insufficient to promote corresponding keratin protein expression.

Example 36: Effect of HC-HA Complex on ESC Marker Expression in HCF

Example 35 shows strong evidence that HCF not only prevents myofibroblast differentiation under stimulation by exogenous TGF-β1, but also restores corneal cells with keratin expression in the presence or absence of exogenous TGF-β1. Therefore, we investigated whether HCF could be further reprogrammed into younger progenitor cells, especially when seeded on immobilized HC-HA (insoluble, GnHCl) containing exogenous TGF-β1, which has been shown to inhibit TGF-β signaling, promote BMP signaling, but shut down keratin expression. We investigated the expression of some markers found in ESCs, endothelial progenitor cells, and pericytes, as we recently reported in angiogenic progenitor cells. To further investigate the potential reprogramming of HCF under these conditions regulated by HC-HA, we also investigated the expression of four key transcription factors, Sox2, Oct4, c-Myc, and KLF4, which have been reported to play a key role in reprogramming adult differentiated fibroblasts into iPSCs.

result

Gene expression was examined on HCF cultures as described in Example 35 above. The results showed that HCF expressed more (2 to 6 times) ESC markers such as cMYC, KLF-4, Nanog, Nestin, Oct4, Rex-1, SOX-2, and SSEA-4 on 4XHC-HA, and 2 to 4 times more ESC markers when HCF were stimulated with exogenous TGF-β1 compared to plastic controls (p < 0.05, n = 3) (Figure 52). These results indicate that HC-HA, especially HC-HA (insoluble), can reprogram HCF into younger progenitor cells.

Example 37: HC-HA in solution inhibits osteogenesis by affecting MC3T3-E1 cell viability and differentiation

In this example, we evaluated the effects of HC-HA (soluble fraction) and HA in solution on the viability and differentiation of undifferentiated MC3T3-E1 cells. MC3T3-E1 is an osteoblast cell line established from C57BL/6 mice. MC3T3-E1 cells have the ability to differentiate into osteoblasts and osteocytes and have been shown to form calcified bone tissue in vitro.

MC3T3-E1 cells were cultured in complete medium (α-MEM, 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin) containing various concentrations of HA (1, 5, 25, 100 μg/ml) or HC-HA (1, 5, 25 μg/ml), with PBS used as a vehicle control, and seeded at 1.6×10 ⁴ cells/ml in 96-well plates treated with plastic cell culture. Cell viability was measured by MTT assay. The results showed that the absorbance at 550 nm increased from 24 hours to 48 hours under all conditions except 25 μg/ml HC-HA, indicating that cell proliferation proceeded normally in the control group, 1 to 100 μg/ml HA, and 1 to 5 μg/ml HC-HA ( FIG. 53 ).

Next, the effect of HC-HA or HA on the differentiation of MC3T3-E1 into osteoblasts was studied. MC3T3-E1 cells were resuspended in non-induction medium (1.6×10 ⁵ /ml) and seeded in 96-well plates and incubated until confluent. The non-induction medium was removed and induction medium 1 (complete medium containing 0.2mM ascorbic acid-2-phosphate and 10mM glycerol-2-phosphate, manufacturer's instructions for the in vitro osteogenesis assay kit, catalog number ECM810, Millipore) was added. After 12 days of differentiation, osteoblast mineralization was measured and quantified using Alizarin red staining. The results showed that AR was actually promoted by the induction medium in the control. Consistent with previous reports (Kawano (2011) Biochemical and Biophysical Research Communications. 405: 575–580), 100μg/ml of HA, but not 25μg/ml of HA, further promoted AR staining compared to the control. In contrast, AR staining was not reduced by 5 μg/ml HC-HA (p=0.11), but was significantly reduced by 25 μg/ml HC-HA (p=0.00002) (Figure 54). These results indicate that increasing the concentration of HC-HA during induction also reduces bone formation.

Example 38: Dose-dependent effects of HC-HA and AMP on osteoblast differentiation using the MC3T3-E1 model system answer

Previous research results have shown that HC-HA and AMP dose-dependently inhibit osteoclast differentiation from RANKL-induced RAW264.7 cells (see International PCT Publication No. WO2012/149486). AMP (amniotic membrane powder) is a freeze-dried, then pulverized form of amniotic membrane. In this example, the IC50 values of HC-HA and AMP for osteogenesis were determined and compared with the IC50 values of HC-HA and AMP for osteoclastogenesis.

Alkaline phosphatase (ALP) assay and alizarin red staining (AR-S) are two assays for measuring MC3T3-E1 cell differentiation. ALP is secreted by osteoblasts and has long been a widely recognized biochemical marker of osteoblast activity (Sabokbar (1994) Bone Miner. 27 (1): 57-67), and is therefore used as an early bone formation marker. Alizarin red (AR) dye forms a chelate with calcium, and therefore AR-S is used to visualize mineralization. Because ARS dye can be easily extracted, it can also be used for the quantification of mineralization (Gregory et al. (2005) Analytical Biochemistry 329: 77–84).

Section A

Experimental design:

MC3T3-E1 culture

Adopt the MC3T3-E1 cell model system from Millipore external osteogenesis test kit, wherein this test kit is made up of α-MEM (Invitrogen, catalog number (Cat.No.) ICM810) basal culture medium comprising 10%FBS, 100 units/ml penicillin and 100 μ g/ml streptomycin.Cells are inoculated with 50,000 cells/ ^cm2 and cultured in 100mL cell culture dishes with 95% air and 5% _CO2 37.0 DEG C, and are passaged before converging.Once enough cell numbers are obtained, cells are subsequently inoculated in 96-well culture dishes with the basal culture medium of 100 μ L volumes in every hole (52 holes) with ^1.6x105 cells/ml.Each concentration is seeded in 4 holes, and wherein 2 holes are used for ALP determination, 2 holes are used for AR-S dyeing.In 5% _CO2 humidified air in 37 DEG C of culture cells and every 48-72 hours, change culture medium, until converging.

The dose range to be investigated was derived from preliminary data obtained in osteoblast differentiation (see above) and dose-response curves of HC-HA and AMP relative to osteoclast differentiation. Because 25 μg/ml of HC-HA significantly inhibited MC3T3-E1 cell proliferation and differentiation into osteoblasts, and completely inhibited osteoclast differentiation from RAW264.7 cells (see International PCT Publication No. WO2012/149486), it was selected as the highest concentration. Since 5 μg/ml of HC-HA showed less than 50% inhibition, suggesting that the IC50 of HC-HA for osteoblast differentiation may be higher than that of P-214, HC-HA concentrations ranging from 0.1, 0.5, 1, 5, 10, and 25 μg/ml were selected. Based on the preliminary data for HC-HA, the following concentrations of AMP were selected: 1, 5, 25, 125, and 250 μg/ml. Since ALP activity in MC3T3-E1 cells peaked on day 12 of differentiation (Maeda (2004) Journal of Cellular Biochemistry 92:458–471; Wang, (2008) J Dent Res. 87(7):650-654), we chose to study osteogenesis on day 12 after induction.

Once confluent, the medium from each well was replaced with 100 μL of osteogenic induction medium #1. Osteogenic induction medium contains 0.2 mM ascorbic acid-2-phosphate and 10 mM β-glycerophosphate (In Vitro Osteogenesis Assay Kit, catalog number ECM810, Millipore). 10 μL of AMP and HC-HA working solution was added to the induction medium #1. (Stock solutions of AMP (AMP-4; batch number CB102971, see International PCT Publication No. WO 2012/149486) (5 mg/ml) and HC-HA (He et al., (2009) J. Biol. Chem. 284(30):20136–20146) (250 μg/ml) in PBS were prepared and diluted accordingly with appropriate culture medium (osteogenesis induction medium #1) to various experimental concentrations (0.1, 0.5, 1, 5, 10, and 25 μg/ml for HC-HA and 1, 5, 25, 125, 250 μg/ml for AMP). The culture medium was changed every 3 days.

On the 6th day of differentiation, the culture medium was replaced with 100 μL fresh osteogenic induction medium #2 containing ascorbic acid, β-glycerophosphate and melatonin. 10 μL of AMP and HC-HA working solution was added to 100 μL of osteogenic induction medium #2 (0.2 mM ascorbic acid-2-phosphate, 10 mM glycerol-2-phosphate and 50 nM melatonin solution, manufacturer's instructions for in vitro osteogenesis assay kit, catalog number (Cat. No.) ECM810, Millipore) to reach the final test concentration in the culture wells. The culture medium was replaced every 2-3 days. Samples were subsequently assayed using ALP assay (H-156) and ARS staining assay according to manufacturer's instructions (in vitro osteogenesis assay kit (Cat. No.) ECM810).

Alizarin Red S staining

In the case of not disturbing the cells, the culture medium from each hole is sucked out. Wash the cells with 200 μ L PBS 1 time. Fix the cells by adding 100 μ L 70% ethanol and incubating at room temperature for 15 minutes. Remove the fixative then and rinse the cells 3 times (5 minutes each time) with excessive distilled water without disturbing the cell monolayer. Remove the water and add the alizarin red staining solution of 100 μ L/ hole. Incubate the holes at room temperature for 1 hour. Remove excessive dye and wash the cells with deionized water 4 times (shaking gently for 5 minutes each time). 0.1-0.15 mL water is added to each hole to prevent the cells from drying out. Under a microscope, the cells stained are taken pictures.

Excess water was then removed from each well. 100 μL of 10% acetic acid solution was added to each well and incubated with shaking for 30 minutes. Loosely attached monolayers were carefully removed with a cell scraper, and the cells and acetic acid were transferred to a 1.5 mL microcentrifuge tube and vortexed vigorously for 30 minutes. The samples were heated to 85°C for 10 minutes and then transferred to ice for 5 minutes to cool the tube. The slurry in the tube was centrifuged at 20,000xg for 15 minutes. 400 μL of the supernatant was removed and transferred to a new 1.5 mL centrifuge tube. The pH was neutralized to a range of 4.1-4.5 with 150 μL of 10% ammonium hydroxide. 150 μL of each sample was added to a clear bottom 96-well plate and read at OD405. A curve of Alizarin Red concentration versus OD405 was plotted.

ALP assay (BioAssay Systems: QuantiChrom ALP Assay Kit, catalog number: DALP-250 )

The cells in each well (96-well plate) are washed with PBS and shaken at room temperature for 20 minutes in 100 μL 0.2% Triton X-100 dissolved in distilled water to perform lysis. 200 μL distilled water and 200 μL calibration solution (provided by test kit) are transferred to the control independent well. 50 μL samples are transferred to independent wells. 150 μL working solution (200 μL assay buffer, 5 μL magnesium acetate (5mM), 2 μL pNPP liquid substrates (10mM)) are added to the sample wells (final reaction volume is 200 μL). Briefly, the plate is gently tapped to mix. At the 0th minute and the 4th minute, OD ₄₀₅ values are read on a plate reader.

result

Phase contrast microscopy

By 13 days of induction, the negative control remained a hexagon (Figure 55). The monolayer was smoother than the positive control, indicating that there were more cells or more cell accumulation in the latter. The cells in the positive control became spindle-shaped after the induction began. After a longer period of time, spindle-like rings were generated along the edge of the plastic culture dish (approximately 2-3 mm outside) on about the 4th day of induction.

At 0.1 μg/ml to 10 μg/ml of HC-HA, the cell monolayer did not differ from the positive control, indicating that these concentrations of HC-HA did not negatively affect induction ( FIG. 55A ). As with the positive control, cells still developed a spindle-shaped shape and the monolayer produced spindle-like rings. However, at 25 μg/ml, a significant decrease in cell density and changes in cell morphology were observed on day 13 of induction.

At concentrations greater than 25 μg/ml, AMP-deposited particles settle on the monolayer (Figure 55B). Since AMP concentrations are replenished after each culture medium change, AMP deposits on top of the monolayer increase during the induction period. Treatment with AMP below 125 μg/ml does not affect cell morphology, as cells also develop a spindle-shaped shape with spindle rings, indicating that AMP does not negatively affect induction. At concentrations above 125 μg/ml, AMP particle density increases to the extent that spindle ring formation is vaguely observed visually. However, cell density and morphology remain unchanged from the positive control, which also confirms that AMP does not negatively affect induction.

Alizarin red staining

The negative control produced a bluish-grey background with portions of the monolayer appearing light pink. In contrast, the positive control produced a rose-red background (Figure 56).

0.1 μg/ml HC-HA produced a rust-red color, with visible spindle rings stained reddish-brown, which was significantly different from the positive control ( FIG. 56A ). This trend persisted between 0.5 μg/ml and 1 μg/ml, with a slight decrease in color at 10 μg/ml, suggesting that mineralization was maintained at 0.1 to 5 μg/ml and that there was likely a dose-dependent relationship between 0 and 0.1 μg/ml. At 25 μg/ml HC-HA, the rust-red background disappeared and returned to a light purple-red color with distinct white gaps between cell junctions.

AMP at 1 μg/ml to 125 μg/ml produced a dose-dependent color change from a lighter rust-brown (1 μg/ml) (which appeared lighter than 0.1 μg/ml HC-HA, suggesting that the dose response was more gradual) to a rust-red (5 μg/ml and 25 μg/ml), reddish-brown (125 μg/ml) background, and significantly increased to a dark reddish-brown at 250 μg/ml ( FIG56B ). It should be noted that AMP particles appeared on top of the cell monolayer treated with AMP at greater than 5 μg/ml, and the particle size was smaller than the cells themselves and appeared to match the color of the stained background.

By quantitative analysis of OD values, ARS staining was increased 3-fold with 0.1 μg/ml HC-HA treatment relative to the positive control, which was statistically significant (p<0.05) (Figure 57A). Some variation was observed between concentrations of 0.1 μg/ml and 10 μg/ml, which may be due to the small sample size (N=2). A significant decrease in OD values was observed with 25 μg/ml HC-HA treatment. For AMP, treatment with 125 μg/ml AMP doubled the amount of mineralization compared to the positive control and was statistically significant (p<0.05) (Figure 57B). A slight decrease in OD values relative to the positive control was observed at 1 μg/ml AMP, and there was a slight dose-dependent increase in OD values from 1 μg/ml to 25 μg/ml. At 250 μg/ml AMP, OD ₄₀₅ was reduced compared to 125 μg/ml. Some variation can be attributed to the small sample size (N=2).

ALP staining

In the two AM derivative groups, the negative control showed 5-fold higher ALP activity than the positive control, which may have occurred due to sample loss in the negative control, which reduced the sample size (Figure 58). The smaller sample size resulted in an 8-fold higher standard deviation value for the negative control than for the positive control, and this much greater variation led to the increase.

Treatment of MC3T3-E1 with any amount of HC-HA reduced ALP activity compared to both positive and negative controls (Figure 58A). ALP activity varied between concentrations until 25 μg/ml, when it was significantly reduced. Notably, there was very little variation relative to the mean within each experimental group compared to the positive control.

At 1 μg/ml, ALP activity was almost 4-fold higher than the positive control ( FIG58B ). This phenomenon was blocked at 5 μg/ml and 25 μg/ml, with ALP activity reduced by almost 3-fold compared to the positive control. Increasing the concentration to 125 μg/ml to 250 μg/ml restored ALP activity to levels close to 1 μg/ml. Thus, ALP activity does not correspond to the amount of mineralization.

Summary of results

Compared to the negative control, the positive control showed more spindle-shaped cells and formed a "ring" around the edge of the plastic well (Figure 55A), with a color change observed by alizarin staining (Figure 56A), as well as a detectable but insignificant OD ₄₀₅ change (Figure 57A). Previously, MC3T3-E1 cells seeded at 5×10 ⁴ cells/35 mm in plastic culture dishes were also shown to form stratified collagen fibrils after day 4, layered fibrils by day 18, and nodular areas as early as day 21 of induction (Sudo (1983) J. Cell. Biol. 96:191-198). This previous study did not record the same ring formation as we observed. [Alternatively, they may have interpreted the ring as stratified collagen fibrils. If so, the ring area should be prone to mineralization.]

In the literature, alizarin red staining is described as representing the deep red color of mineralization. Mineralization and osteoblast nodules are described as being stained into deep red and the intensity of the color increases with mineralization (Wang, (2006) Biotechnol. Prog. 22 (6): 1697-701; Zhao, 2007). ARS staining is read at 405nm, which corresponds to purple in the visible spectrum. Unlike our results, the color photographs of ARS staining of MC3T3-E1 mineralization from the published data do not show rust red or reddish brown in the monolayer. The darker colors in our results represent more ARS staining, thereby having more osteogenesis compared with the published results. The OD change amount in ARS staining and quantification varies with culture conditions and cell types. Human mesenchymal stem cells (hMSCs) were cultured in 6-well plates (10 cm ² / well) for 30 days and an increase of OD ₄₀₅ from 0.5 to 4 was obtained (Gregory et al. (2005) Analytical Biochemistry 329: 77-84). Culturing MC3T3-E1 cells in 24-well plates for 28 days (α-MEM, ascorbic acid, β-glycerophosphate) yielded an OD value of 0.6. However, the OD values on days 16 and 26 were much lower than on day 28, falling below 0.05 and 0.2, respectively (Burkhardt (2006) University of Basel, Master's thesis). The lack of a clear color change in the positive control may be due to the 13th day, which, although optimal for ALP, is too short for ARS.

0.1 μg/ml HC-HA also induced a "ring change" ( FIG. 55A ), a significant increase in color ( FIG. 56A ), and an OD value 3-fold higher than the positive control group (p<0.05) ( FIG. 57A ), indicating that lower doses of HC-HA promote mineralization and that a dose-response curve exists between 0 and 0.1 μg/ml.

HC-HA at 0.5 to 10 μg/ml also showed a "ring" (Figure 55A), maintained the same color as 0.1 μg/ml (Figure 56A), and produced a non-statistically significant _OD405 . HC-HA at 25 μg/ml decreased cell density, changed cell morphology, lost the "ring" (Figure 55A), did not produce any color change (similar to the negative control) (Figure 56A), and produced a negligible _OD405 (like the negative control).

AMP from 25 μg / ml residual particles (Figure 55B), from 1 μg / ml AMP increased color in a positive dose-response relationship (Figure 56B), but _OD405 did not show a statistically significant increase (p < 0.05) until 125 μg / ml AMP, and then decreased at 250 μg / ml, which was not consistent with the color change. Unlike HC-HA, higher doses of AMP did not cause any adverse effects on cell morphology.

Section B

Subsequently, the alizarin red staining method was improved by increasing the sample size and merging the method ((2005) Analytical Biochemistry 329:77-84) and other methods known in the art for the alizarin staining of human MSCs by Gregory et al. The comparison between the method of Gregory et al., the above-mentioned previous method and the novel method outlined in this embodiment is provided in the table below. Previous studies have shown that MC3T3-E1 differentiation under induction can be subdivided into three stages, i.e., proliferation (day 1 to 9), ECM formation (day 9 to day 16) and mineralization (deposition of minerals in the ECM formed) (after day 16) (Quarles et al. (1992) Bone Miner Res. 7 (6): 683-92; Hong et al. (2010) Exp Cell Res. 316 (14): 2291-300). In order to compare studies from different groups, it is crucial to start timing events with confluence as day 0.

Table 5

*There are different steps among the 3 methods used for ARS staining and quantification.

Experimental design:

Cells were cultured and stimulated in differentiation medium as described in Section A and Table 5. Differentiation medium was changed every 3 days for 18 days. Assays were performed using 0.1 μg/ml HC-HA and 125 μg/ml AMP. ARS assays were performed as described in Section A, and changes are recorded in Table 5.

result

Cell morphology and ring formation

Uninduced cells acquired a flattened cuboid shape after seeding ( FIG. 59A ). Cell boundaries became more distinct with raised edges on day 4, and some cells formed a spindle shape on day 6. No spindle cells or multiple layers were generated.

From the 1st day to the 3rd day, the cells maintained a rectangular parallelepiped shape, and the monolayer remained flat (Figure 59B). By the 3rd day, the cell boundaries became clearer and the cell edges became raised. In addition, small round cell-like structures were visible on the monolayer (represented by black circles). The cell morphology became accompanied by the appearance of spindle cells and cells organized in multiple layers until the 4th day. The appearance of small round cell-like structures continued to increase to the 6th and 7th days.

The changes in cell morphology of cells induced by HC-HA treatment mirrored those of the positive control (Figure 60A). HC-HA did not leave particles on the monolayer like AMP. Like the positive control, small round cell-like structures (indicated by black circles) appeared on day 3 and continued to increase until day 7.

AMP particles (marked with black arrows) rest on top of the monolayer and obstruct observation of the underlying monolayer (Figure 60B). Areas not covered with AMP particles showed round and cuboidal shapes on days 0 and 1. On day 2, some spindle-shaped cells appeared on the monolayer. It was difficult to discern and distinguish small round cell-like structures from smaller AMP particles, and the generation of these structures remains unclear. On day 5, the spindle shapes elongated to form spindle-shaped cells. On day 6, the long spindle cells formed a network of interactions with the AMP particles (shown within the black circles).

On day 3, spindle-shaped cells were generated near the edges of the wells (1–2 mm from the edges) ( FIG. 61A ). Spindle cells and rings did not form from day 4 onwards. The cells appeared to overlap each other at the edges and grew into a spindle shape. From day 0 to day 2, there were no spindle-shaped cells along the edges of the wells ( FIG. 61B ). On day 3, similar spindle-shaped cells were noted stacked in a ring structure at the edges. From day 5 onwards, these cells were concentrated into a protruding ring approximately 2 mm from the edge. On day 6, the monolayer showed detachment from the plastic surface in certain areas near the edges (indicated by white →).

The cells remained smooth and cuboidal along the edges until day 2 ( FIG. 62A ). Spindle-shaped cells developed near the edges on day 2. Some small, round cell-like structures were also visible near the edges at this time. Spindle-shaped cells developed on day 3 and continued to thin out in a ring around the hole until day 5. On day 6, the monolayer was observed detaching from the plastic hole in an area near the edge (indicated by white arrows). Spindle-shaped cells appeared near the edge of the hole on day 2 ( FIG. 62B ). Spindle-shaped cells developed away from the edge (approximately 1 to 2 mm away) on day 3 and continued to form until day 5. On day 5, the monolayer was observed detaching from the plastic hole in some areas near the edge. Detachment continued on day 6, but the monolayer did not detach as much as the HC-HA-treated cells and the positive control cells.

ARS staining and quantification

The negative control monolayer is stained light pink in some areas (Figure 63). The positive control is stained light pink in the center, but presents a bright crimson color in the spindle ring area, indicating that MC3T3-E1 cells are heavily deposited and mineralized in the ring rather than in the rest of the monolayer. The intensity and color of the staining of the central monolayer and spindle ring of the 0.1 μg/ml HC-HA group and the positive control are all the same. The AMP particles on the top of the cells are stained reddish brown. The dyed particles hinder the visual observation of the cells below, but the opening shows a monolayer cell lacking a protrusion, which is stained with some sparse cells similar to the negative control and is stained light pink. Because the cells processed by AMP do not show visible spindle rings, ARS is not stained crimson around the edge as in the positive control.

In both the positive control and HC-HA-treated cells, GnHCl treatment resulted in the dissolution of the cell matrix and the removal of the deep red ARS dye, while leaving the monolayer intact. GnHCl digested and denatured cellular proteins, leaving the extracellular matrix behind. In the AMP experimental group, the density of AMP particles decreased, but most particles remained at the bottom of the wells. Over long culture times, AMP particles can form tight interactions with the ECM matrix that is not dissolved by GnHCl. The particles, which once stained a bright reddish-brown, now appear a light brown color, similar to the natural color of AMP. However, no clear monolayer structure was observed; this supports the observation of a cell monolayer in the gaps between the AMP particles. These cells may have migrated from the monolayer into the AMP particles and used them as a scaffold for differentiation and mineralization.

Through serial dilution from 2mM to 31.3μM, the ARS standard showed a gradient from deep red to creamy pink (Figure 64A). There was a clear color change between the HC-HA and AMP treated samples. The cell extracts treated with HC-HA showed a clear cream color, while the cell extracts treated with AMP showed a light creamy pink. The positive control also showed the same color and color intensity level as the HC-HA treated cells, while the negative control showed a lighter color and was similar to the blank (not shown). The OD ₄₀₅ value remained in the same range as the value in the previous example (Figure 64B). Compared to the negative control, the positive control showed a statistically significant 2-fold decrease in OD value. The HC-HA treated (0.1μg/ml) cells showed a slight increase in OD value compared to the positive control, but the difference was not statistically significant. When compared to the positive control, the AMP (125μg/ml) treatment OD value decreased slightly, but this decrease was not statistically significant.

By serial dilution from 2mM to 31.3μM, the ARS standard showed a gradient from deep red to milky pink (Figure 65A). The negative control extract showed a light cream color slightly darker than the blank (not shown). The positive control showed a light golden color, which was significantly darker than the negative control. HC-HA also showed a light golden color with the same intensity as the positive control. The AMP-treated extract showed a darker orange-gold color than the positive group and the HC-HA extract group. The OD ₄₀₅ value remained in the same range of approximately 0.25 (Figure 65B). The negative control showed a negligible OD value close to 0. The positive control and HC-HA (0.1μg/ml) treated extracts showed an average OD value close to 0.05. The AMP-treated (125μg/ml) extract showed a statistically significant 5-fold increase in OD compared to the positive group and the HC-HA treated group (p=0.039).

Summarize

Cell morphology

MC3T3-E1 cells cultured in αMEM w/10% FBS grew to confluence and became a rectangular parallelepiped shape. Like the results in targets #1 and #2, the cells did not differentiate when ascorbic acid, β-glycerophosphate and melatonin were not added. In the absence of induction medium, spindle cells and spindle rings (Figure 59) were not formed. MC3T3-E1 cells were induced to differentiate when ascorbic acid, β-glycerophosphate and melatonin were present. After inoculation, a smooth monolayer was formed by rectangular parallelepiped cells. After induction for 3 days, the cells acquired a spindle shape. By the 5th day, the cells were elongated and became spindle-shaped.

In this embodiment, spindle rings were formed on the 3rd day of induction, with spindle-shaped cells forming at a distance of 1 to 2 mm from the hole wall. On the 6th day, the monolayer was observed to detach from the hole edge and the plastic bottom (Figure 60). Small round cell-like structures began to appear on the monolayer on the 2nd day and increased in number to the 6th day (Figure 59). They did not float in the culture medium and were firmly attached to the monolayer, with the rest mostly between the cell boundaries. These structures may represent matrix vesicles (MVs), which are extracellular, membrane-embedded particles located at the initial calcification sites of cartilage and bone. Matrix vesicle synthesis occurs by budding and extrusion of membranes from specific regions of the extracellular plasma membrane of differentiated growth plate chondrocytes and osteoblasts (Anderson et al. (2003) Curr Rheumatol Rep. 5 (3): 222-6).

The MC3T3-E1 cell morphology changes (Figure 60) caused by differentiation were not changed by treating with HC-HA. After the reporting period of the positive control, fusiform cells, spindle cells and spindle rings (Figure 61) were formed. AMP particles stayed on the top of the cell monolayer similar to the previous results (Figure 60). This hindered the comprehensive observation of the cell monolayer and cell morphology changes of induction. However, some observations were recorded by openings where the AMP microparticles did not stay. Unlike HC-HA, AMP treatment accelerated cell morphology changes and some spindle cells were visible one day before induction on the 2nd day. The cells treated with AMP formed spindle rings similar to the cells treated with HC-HA and the positive control (Figure 62). However, the monolayer broke away from (Figure 62) earlier than the other two experimental groups (on the 5th day, rather than the 6th day).

ARS staining and quantification

ARS staining showed a significantly different color from that previously reported (Figure 66). In the negative control cell monolayer, ARS staining was light pink rather than bluish-gray. ARS staining also showed a bright crimson concentrated in the spindle ring, rather than the rust brown of the positive control and HC-HA treated cells in the previous results. AMP particles were stained reddish brown rather than dark brown, and the cells below were stained light pink rather than rust brown. Degradation of the ARS solution may have contributed to the color change.

After acetic acid extraction, the monolayer still showed a significant amount of staining remaining. Acetic acid was not effective in completely removing ARS staining from the monolayer. With acetic acid extraction, there was a statistically significant difference between the OD values of the positive and negative controls (Figure 68), which matched the visual observation that there was more ARS staining in the positive control (Figure 66). However, acetic acid extraction was not effective in showing a statistically significant increase in the OD value of the AMP group compared to the positive and HC-HA groups (Figure 66), although the visual observation of the AMP group had more color in the assay extract than the other two groups (Figure 67). AMP particle deposits may make it more difficult to remove ARS from the AMP group. Mineralized matrix and cells may also interact with the AMP particles that hinder the acetic acid extraction of ARS.

GnHCl removes ARS from the cell monolayer more thoroughly than acetic acid treatment (Figure 69). In addition, the color dissolves in the GnHCl solution without using a cell scraper to remove the monolayer. Both methods seem to leave particles that are invisible to the naked eye and are not affected by centrifugation. For GnHCl, this may be calcium and the dissolved matrix that forms microparticles. This causes variation between the repetitions of each sample. Reading at 670nm to remove particles solves the problem of this GnHCl extraction method.

GnHCl extraction failed to establish quantifiable statistical significance between the negative and positive controls (Figure 71) to match the visual observation of ARS staining. GnHCl extraction also showed that 0.1 μg / ml of HC-HA did not promote more mineralization in differentiating MC3T3-E1 cells (Figure 71), and this matched the results of acetic acid extraction. By observing the color of the assay extracts in a 96-well plate, the positive control and HC-HA wells showed no difference in color or intensity (Figure 70).

GnHCl extraction showed that 125 μg/ml of AMP promoted more mineralization in differentiating MC3T3-E1 cells compared to the positive control (Figure 71). This matched ARS staining of the monolayer (Figure 66) and visual observation of the extracts in the 96-well assay plate (Figure 70), where AMP showed a darker golden color than HC-HA or the positive control.

Based on these results, GnHCl is a better extraction method because it removes the ARS stain more thoroughly from the monolayer and leaves it intact; there is less technical error because the monolayer does not need to be scraped from the well. The color of the extract can be quantified by spectrophotometric reading.

Section C

The above preliminary study did not show statistically significant results for the dose-response curve of HC-HA and AMP on osteoblast differentiation due to the small sample size and incomplete performance of the ARS assay. This suggests that 0.1 μg/ml of HC-HA can enhance mineralization. Moreover, 10 μg/ml to 25 μg/ml of HC-HA may affect cell viability and reduce mineralization. The dose curve of HC-HA should include lower concentrations below 0.1 μg/ml and concentrations above 10 μg/ml. Unlike HC-HA, 5 μg/ml to 125 μg/ml of AMP can promote mineralization. In this experiment, the dose response of HC-HA and AMP was retested using the method of Part B. In this experiment, ARS staining was used for determination on day 15, and a modified protocol of 10% acetic acid staining and quantification was adopted.

Experimental design

The same model system based on 3T3-E1 cells as described in Part B was used by seeding 3×10 ⁴ cells/cm ² /well in 96-well plates in αMEM medium weight/10% FBS. Once confluent, cells were induced to differentiate by adding ascorbic acid, β-glycerophosphate, and melatonin induction medium. For each condition, N=3 were tested. After confluence (day 0 = seeding), HC-HA solutions were added at 0.02 μg/ml, 0.1 μg/ml, 1 μg/ml, 5 μg/ml, and 25 μg/ml, while AMP solutions were added at 1 μg/ml, 5 μg/ml, 25 μg/ml, and 125 μg/ml. For ARS staining, the optimized method of Part B was used.

result

Negative control MC3T3-E1 cells did not produce spindle-shaped shapes or spindle rings in culture (Figure 66A). The center and periphery of the monolayer were stained beige. Positive control cells produced spindle-shaped and spindle-shaped cells, with the appearance of spindle rings around the 5th day of culture (the 4th day of induction). ARS staining appeared light maroon in the center and was mostly concentrated in the spindle ring with a deep red color. Increasing the HC-HA concentration to 1.25 μg/ml had no effect on the morphology of the cells or the intensity and form of ARS staining. At 2.5 μg/ml, the spindle rings of MC3T3-E1 cells began to degrade, and the ARS color changed from deep red to reddish brown. At 20 μg/ml, the cells lost their spindle and spindle shapes; the cell edges were also less clear and raised. The cell density decreased, and the monolayer did not appear raised as before. GnHCl successfully extracted the ARS dye and the coefficient of variation of the OD ₄₅₀ value was 5% to 19%. The positive control showed a statistically significant increase in OD values (Figure 66B).

Negative control MC3T3-E1 cells did not produce spindle-shaped shapes or spindle rings in culture (Figure 67A). The center and periphery of the monolayer were stained beige. The positive control cells produced spindle-shaped and spindle-shaped cells, with the appearance of spindle rings on the 5th day of culture (the 4th day of induction). ARS staining showed light maroon in the center of the monolayer. AMP treatment produced AMP particles that stayed on the top of the cell monolayer, and blocked the observation of the monolayer from a concentration of more than 62.5 μg/ml. Only AMP-treated and uninduced MC3T3-E1 cells did not show spindle rings along the edge, and ARS staining showed a deep red with a light pink background. From 7.8 μg/ml to 31.25 μg/ml, AMP particles did not completely cover the monolayer and the cells showed spindle and spindle shapes. Along the edge, spindle rings can be seen. ARS staining shows that the central monolayer is stained light maroon and the dye is concentrated into a deep red color along the spindle ring like the positive control. GnHCl successfully extracted the ARS dye and the coefficient of variation of _OD450 values ranged from 3% to 10% (Figure 67B).

Summary of results

Cell morphology/ARS staining

Similar to previous results, MC3T3-E1 differentiation is carried out as cells are changed from cubic shape to spindle and spindle-shaped. Along with the increase of induction time, spindle rings are formed along the edge of the hole (about 2mm away), and monolayer is shrunk over time. Different from the positive control, the negative control never forms spindle-shaped cells or spindle rings. When the negative control monolayer is dyed into uniform beige, the positive control presents light maroon/pink in the center and presents concentrated crimson in the spindle ring.

At 10 μg/ml HC-HA, cell morphology changed from lower concentrations, with fewer cells displaying spindle and fusiform shapes. Cell density decreased, and the monolayer appeared less bulging. At 20 μg/ml HC-HA, cell density dropped dramatically, and fewer cells were spindle-shaped. The monolayer appeared smooth, similar to the negative control. For both concentrations, these changes began to be observed by day 5 of culture and induction. At both concentrations, the spindle ring was either poorly formed or absent. At concentrations of 2.5 μg/ml and above, increasing HC-HA concentrations caused the spindle ring to disintegrate.

In all AMP-treated cells, staining was deep red around the area where the AMP particles were retained. No monolayer was visible around the edges of the pores through the openings of the AMP particles. Uninduced MC3T3-E1 treated with 125 μg/ml AMP showed staining similar to MC3T3-E1 induced with 125 μg/ml AMP. Cells with retained AMP were stained as a blocky deep red, and no stained monolayer underneath was observed. From 7.8 μg/ml to 15.6 μg/ml AMP, cell morphology was visible. The cells exhibited spindle and spindle shapes, with spindle rings formed along the edges of the pores. ARS staining resembled the positive control with a light maroon center and deep red spindle ring area.

GnHCl treatment was successful for extracting ARS dye from the stained monolayer. Extraction showed a statistically significant (p < 0.01) 2-fold increase in OD ₄₅₀ from the negative control to the positive control. HC-HA treated cells showed a trend toward decreased mineralization with increasing HC-HA concentrations. At 10 μg/ml and 20 μg/ml HC-HA, mineralization was statistically significantly (p < 0.05) lower than the positive control. AMP dose-dependently increased the mineralization of differentiating MC3T3-E1 cells.

Uninduced cells treated with AMP also showed mineralization, and AMP-induced and promoted mineralization was more pronounced than the positive control (p<0.01). Furthermore, at 125 μg/ml AMP, uninduced cells showed greater mineralization than cells cultured in induction medium (p<0.05).

Example 39: Effect of AMP on osteoblast differentiation

Although ARS staining showed a clear dose-dependent increase in AMP staining and a statistically significant increase in mineralization at 125 μg/ml, it was unclear whether this change was caused by nonspecific binding of ARS to AMP. Because AMP acts differently from HC-HA, especially at high doses, i.e., promoting mineralization but not inhibiting it, it was important to rule out whether the effects of AMP depend on direct contact of cells with AMP. This issue was addressed by using Transwell plates with a pore size of 3 μm, which is sufficient for HC-HA to pass but small enough to block AMP particles. Because available Transwell plates with this pore size are suitable for 24-well plates, the culture conditions of the assay were changed accordingly. A concentration of 125 μg/ml AMP was used for the assay.

Experimental design:

MC3T3-E1 cells (cells at P2; ATCC, catalog number: CRL-2593 ^™ ) were seeded into 12-well flat, clear-bottomed wells at a density of 1×10 ⁵ cells/ml. The same AMP stock solution (AMP-4; lot number CB102971) as used above was prepared in PBS to 5 mg/ml. For wells not containing Transwell, 17.5 μL of AMP stock solution was added to 0.7 ml of culture medium (basal or induction medium) to achieve an AMP concentration of 125 μg/ml in α-MEM weight/10% FBS (without induction) or induction medium #1 followed by #2 (with induction). For wells containing Transwell, 17.5 μL of AMP stock solution was added directly to the center of the Transwell membrane in the same manner as described above. After day 0 of induction, the culture medium (α-MEM containing 10% FBS; induction medium #1 and #2 as described above) was replaced every 3 days. ARS staining and quantification procedures were performed as described above, except that 4% paraformaldehyde was used instead of 70% ethanol to fix the cell monolayers, and staining was performed for 2 hours instead of 1 hour.

result

Ring formation occurs on the 4th day of induction (Figure 68). The ring consists of a monolayer of cells around the edge of the plastic hole about 2-3 mm away. They grow in layers, curl up, and then the monolayer detaches from the plastic in many holes. Without induction, the cells maintain a roughly hexagonal shape, with some spindle-shaped shapes over time, and the monolayer remains smoother than the induced cells (Figure 69). Induction causes the spindle-shaped cells and monolayer to become raised and the boundaries between cells become clearer. Spindle-like rings were observed along the edge of the culture plate until the 4th day after induction. Treatment with AMP did not affect cell viability; nor did it affect the formation of rings, which indicates that AMP did not negatively affect induction. Addition of Transwell did not affect cell growth or morphology.

Without transwells, 125 μg/ml of AMP caused the particles to remain on the cell monolayer (Figure 69A). Without induction, AMP itself did not cause the cells to form a spindle shape and no rings were generated, indicating that AMP alone was not sufficient to cause induction, similar to the negative control. Under induction, 125 μg/ml AMP caused changes in cell morphology as the positive control, indicating that AMP itself did not negatively affect induction. Through Transwell, 125 μg/ml of AMP left negligible particles on the cell monolayer (Figure 69B). Without induction, AMP was not enough to induce cell differentiation and the cells were similar to the negative control. Under induction and 125 μg/ml AMP treatment, the cells produced spindle and spindle-shaped shapes and were similar to the positive control by day 14, again indicating that AMP did not negatively affect induction.

The negative control produced a gray-brown background color with patches of light pink within the monolayer, which is different from the blue-gray color of the negative control in the previous experiment (Figure 70). When not induced but treated with Transwell, i.e., with AMP, the monolayer only stained with a gray-brown background similar to the negative control, indicating that AMP itself did not cause induction. In the absence of Transwell, there were areas showing a significant decrease in background color compared to the negative control, indicating that AMP particles retained on the monolayer may block this color (marked by **), and AMP itself did not generate a positive color indicating the presence of mineralization.

The positive control produced a rose-pink color with a ring staining to rust-red, which was darker than the rose-red color of the positive control in the previous experiment. The addition of Transwell did not affect the color of the positive control, confirming that Transwell itself does not affect induction. When AMP was used but not Transwell, the monolayer produced a stronger rust-red color than the positive control, with the ring staining to a darker color, indicating that AMP exerted additional positive induction. In contrast, with Transwell, the intensity of the background color decreased to the level of the positive control, while the ring remained the same color, indicating that Transwell exerted a negative influence on the effect of AMP induction.

The quantitative results were not sufficient to provide statistical significance and did not match the visual analysis of ARS staining (Figure 71). However, the overall trend showed that the positive control showed more mineralization than the negative control. In addition, when Transwell was included in the presence of AMP and induction, there was a trend toward fewer OD ₄₀₅ .

Summary of results

It is speculated that due to the larger size of the dish, it may be easy to observe the formation of the ring (Figure 69A). However, the background color of the negative control is different from that of the previous experiment, probably due to the change of the fixative. In addition, there is a more dramatic color change between the negative and positive controls, especially in the ring area (Figure 70). Introducing Transwell in the positive control does not affect the cell morphology (Figure 69B) or the color of ARS staining (Figure 70).

Under the induction treatment, AMP without Transwell treatment clearly prevented positive ARS staining (Figure 70), and AMP after Transwell treatment showed the same color as the negative control. AMP itself does not cause nonspecific ARS and does not cause any induction. Under the induction treatment, AMP without Transwell treatment caused more color than the positive control. In contrast, AMP after Transwell treatment seemed to produce the same color as the positive control.

Previous ARS quantification results showed that 125 μg/ml AMP promoted mineralization more than 3-fold compared with the positive control group (p<0.05) ( FIG. 67 ), but this was not the case in FIG. 71 .

There was no difference in cell morphology between cells treated directly with AMP and cells treated with AMP and passed through Transwell (Figures 69A, 69B). Both cell groups produced ring formation and visual observation did not show a clear difference between the ring structures. ARS staining showed negligible differences in background color between the two experimental groups. However, cells treated directly with AMP showed more diffuse ring formation, which may be a scattering effect from the AMP particles (Figure 70).

AMP does not need to be in direct contact with MC3T3-E1 cells to affect mineralization. Although it is not clear from this experiment whether AMP promotes mineralization, AMP has been shown not to affect cell morphology or cell viability (Figure 69).

Section B

Our results showed that 125 μg/ml AMP significantly increased mineralization of MC3T3-E1 cells compared to the positive control grown and differentiated at day 15 ( Figure 65 ). AMP was delivered directly into the culture medium and AMP particles resided on the apical side of the cell monolayer; therefore, direct contact is required for AMP's effects in target 1B.

In Part A, we sought to investigate whether the effects of AMP acted as a scaffold in the differentiation of MC3T3-E1 cells or whether factors were released from the granules to promote mineralization. However, small sample size and technical errors in the extraction of ARS staining with 10% acetic acid affected the data, and no statistical significance was found. The experiment was repeated using a modified technical method of ARS staining and extraction using 4 M guanidine hydrochloride as in Example 38.

Experimental design:

As described above, MC3T3-E1 cells were seeded at 3×10 ⁴ cells/cm ² /well in αMEM medium supplemented with 10% FBS in a 24-well plate. Once confluent, the cells were induced to differentiate by adding ascorbic acid, β-glycerophosphate, and melatonin. For each condition, N=3 were tested. The day the cells were seeded was counted as day 0, and induction was performed after the cells were confluent. Total induction time = 15 days. There were two experimental groups: AMP was added directly to the induction medium and AMP was delivered to the Transwell. The AMP concentration was maintained at 125 μg/ml as before. The negative control (no induction) had or did not have AMP added but no insertion. ARS staining and quantification were performed on the 15th day of induction as described above.

result

In the absence of induction medium, negative control cells maintained a hexagonal shape with some spindle-shaped structures (Figure 72A). No spindle-like shapes were observed, and no spindle rings formed along the periphery. ARS stained the monolayer light pink. Under induction, positive control cells maintained a spindle-like shape. Cell borders became more prominent and raised; spindle rings formed along the periphery near the well edge. The center of the monolayer stained maroon, and ARS staining was concentrated in the spindle ring with an intense deep red color. Direct treatment with AMP caused AMP particles to remain on the monolayer and obscured the morphology of the cells. However, near the edge of the culture well, the spaces between AMP particles in both groups showed a lack of a prominent monolayer underneath. There was no significant difference in cell morphology between AMP treatment alone and AMP treatment plus induction. Visible cells had a spindle-like shape. No spindle rings were observed as in the positive control. ARS staining showed deep red staining in the center with reddish-brown staining along the periphery. The color and pattern of staining were difficult to distinguish between the induced and non-induced groups. Treatment with AMP through the Transwell did not cause AMP particles to remain on the monolayer. The cells were elongated and spindle-shaped, with spindle rings forming along the pore edges. Like the positive control, the center of the monolayer stained maroon, and the ARS dye was concentrated within the spindle rings, with an intense deep red color. GnHCl successfully extracted the ARS dye and the coefficient of variation of the OD ₄₅₀ values was 2% to 15% ( FIG. 72B ).

Summary of results

Morphology/ARS staining

The negative control cultured in α-MEM weight/10% FBS for 21 days did not produce spindle cells or spindle rings along the edge. In the positive control, after 20 days of induction with AA, β-glycerophosphate and melatonin, MC3T3-E1 cells produced spindle cells and spindle rings around the edge of the well (Figure 72). The ARS color and staining pattern of the cell monolayer were different from those of the negative and positive controls. The negative control monolayer failed to collect as much dye as the positive control and showed a uniform light pink color. The center of the positive control monolayer was stained maroon and the ARS concentrated in the spindle ring was stained intensely crimson (Figure 72).

Direct AMP treatment in the culture medium obstructed observation of the cell monolayer in the AMP-treated and AMP-treated plus induction groups due to the retention of AMP particles. However, no spindle rings were observed along the well edges, and some cells along the edges exhibited a spindle-like morphology. Openings through the AMP particles indicated the absence of the underlying monolayer. ARS staining showed no differences in color or pattern between the AMP-treated group and the AMP-treated plus induction group.

AMP treatment and Transwell culture did not produce AMP granules on the top of the monolayer. Cell morphology was similar to the positive control, with spindle-shaped cells and spindle ring formation. The color and pattern of ARS staining were also similar to the positive control, with the background monolayer staining maroon and ARS staining concentrated in the spindle ring appearing dark red.

ARS quantification

GnHCl is necessary and sufficient for extracting ARS staining from the monolayer. Compared to the control, there was a statistically significant 2-fold increase in OD ₄₅₀ from the negative control to the positive control (p < 0.01). In addition, there was an approximately 6-fold increase in OD values between the negative/positive control and AMP+ induced groups. There was a 6.5-fold increase in surface area from 96-well plates to 24-well plates, which may explain the increase in OD. We also performed ARS staining and quantification on day 20 of culture, which increased the culture time by 2 days and may have also increased mineralization.

AMP alone caused a statistically significant 10-fold (p < 0.01) and 3-fold (p < 0.01) increase in OD ₄₅₀ values compared to the negative and positive controls, respectively. Thus, AMP alone is sufficient to induce and promote differentiation. AMP-assisted induction resulted in a slight decrease in OD ₄₅₀ compared to AMP alone (p < 0.05), but showed a 3-fold increase in OD compared to the positive control (p < 0.01). Induction medium hindered differentiation and mineralization.

Transwell-delivered AMP-induced differentiation showed a 3-fold lower OD value than direct AMP delivery (p<0.01) and a slightly lower OD value than the positive control (p<0.05). AMP requires direct contact to promote differentiation. Without direct contact, AMP inhibits MC3T3-E1 mineralization.

Example 40: Effect of AMP on the induction of osteogenesis in MSCs

Our results indicate that AMP promotes MC3T3-E1 differentiation when in contact with pre-osteoblasts. However, it is unclear how AMP affects the growth and differentiation of less differentiated and committed cell lines, such as mesenchymal stem cells (MSCs), toward the osteoblast lineage.

MC3T3-E1 cells are pre-osteoblasts, unipotent, and therefore only need to be supplemented to promote their differentiation into osteoblasts. Other progenitor cell lines such as embryonic stem cells (ESC) or mesenchymal stem cells (MSC) are less differentiated and are oligopotent and multipotent, respectively. Therefore, by studying the effects of AMP on MSCs from different regions of the human body, we can better understand the role of AMP in osteoblast differentiation programming and the factors involved. This study will enable us to narrow down the cell types that AMP can affect and its effects on inducing osteogenesis in different progenitor cells.

Experimental design

The following cell lines were used: MC3T3-E1 (ATCC, Manassas, VA), mesenchymal stem cells from human bone marrow cells (Lonza, Wlakerfield, MD), marginal septal cells (Tissue Tech, Miami, FL), human amniotic membrane (hAM) stromal cells (Tissue Tech, Miami, FL), and human umbilical vein endothelial cells (HUVEC) (ATCC, Manassas, VA).

Cells were seeded at 3×10 ⁴ cells/cm ² /well in αMEM medium supplemented with 10% FBS in 96-well plastic culture plates. Once confluent, cells were induced to osteoblast differentiation by adding ascorbic acid, β-glycerophosphate and melatonin (AGM). For each condition, N=5 were tested. Total induction time = 20 days. Day 0 was counted as the day the cells were seeded, and induction was performed after the cells were confluent. There were 3 experimental groups for each cell type: negative control, positive control, and AMP treatment only. For the AMP treatment group, an AMP concentration of 125 μg/ml was used. The culture medium was changed every 3 days (100 μL) during the 20-day culture period. A negative control with AMP (no induction or cells) was added. ARS staining and quantification were performed as described above on day 20. The extracts were then read at 450 nm.

result

HUVEC cells formed a network of cell growth by day 4 (Figure 74A). However, significant cell death occurred by day 21, with dead cells remaining on top of the cell network. Most HUVEC cells could not be fixed with 10% paraformaldehyde, and the few cells stained with ARS were dark brown. Although AMP remained on top of the HUVEC cells and covered the cell network, AMP particles detached from the cells and from the plastic wells when stained with ARS; the few remaining AMP particles also stained dark brown.

Uninduced hBM MSCs maintain a long fibroblast shape (Figure 74A). With induction, MSCs become elongated with more raised cell edges by the 4th day. By the 10th day, induced MSCs produce spindle-shaped cells, and the cells grow overlapping each other on a monolayer. At the 17th day, overlapping spindle cells form a dense ring about 5 mm from the center of the hole. ARS staining shows that the uninduced MSC monolayer is stained cream-colored, and the spindle ring is stained reddish-orange. AMP-treated MSCs contain AMP particles covering the monolayer. Over time, the monolayer shrinks around the area where the AMP particles are concentrated. ARS staining is dark reddish-brown.

Uninduced hAM mesenchymal stem cells maintain a rectangular shape (Figure 74A). By the 4th day, under induction, cell morphology changes, and the cells elongate, with the formation of some spindle shapes. AMP particles stay and cover some monolayers in the stromal cells processed by AMP by the 4th day. The cells not covered by AMP particles are rectangular in shape on the 4th day. By the 17th day, the cells not covered by AMP particles elongate, similar to the induced cells in the positive control. By the 21st day, AMP particles cover the holes and cell morphology can not be observed. ARS staining shows that uninduced cells are stained cream, while induced cells are stained light pink. The cells processed by AMP are stained dark reddish brown, similar to the hBM MSC processed by AMP.

Extraction with 4M GnHCl resulted in a coefficient of variation in _OD450 values ranging from 2% to 15% (Figure 74B).

Summary of results

The results showed that no mineralization was observed in the negative control of HUVECs, regardless of whether the inducer or AMP was treated. For hBM MSCs and hAM mesenchymal stem cells, the inducer promoted mineralization, but the mineralization was significantly less than that promoted by AMP.

Example 41: Effects of AMP on Mineralization and Cell Proliferation during MC3T3-E1 Differentiation

MC3T3-E1 cells go through three main stages before becoming mature osteoblasts: proliferation, matrix deposition/maturation, and mineralization (Figure 75). Our results show that AMP promotes the mineralization in MC3T3-E1 cells. When viewing ARS staining, it shows that the cells treated with AMP are darker and denser (Figure 72) compared with the positive and negative controls. In addition, when AMP particles cover the monolayer, the monolayer is missing under the AMP particles (Figure 77). One possibility is that AMP serves as the scaffold of MC3T3-E1 cells and this interaction in the 3D matrix allows cell growth and mineralization. Therefore, AMP can promote mineralization by increasing the proliferation of MC3T3-E1 cells.

Experimental design

As described above, MC3T3-E1 cells were seeded at 3×10 ⁴ cells/cm ² /well in 24-well plates containing αMEM medium supplemented with 10% FBS. Once confluent, cell differentiation was induced by adding ascorbic acid, β-glycerophosphate, and melatonin. For each condition, N=3 was tested. The day the cells were inoculated was counted as day 1, and induction was performed after the cells converged. Total induction time = 20 days. Sampling was performed at four time points: day 1, day 2, day 7, day 10, day 13, and day 20. There were four groups at each time point: negative control, positive control, AMP treatment only, and AMP+ (with induction). The AMP concentration used was 125 μg/ml.

For experiments measuring proliferation using the MTT assay, the culture period was 9 days. Samples were collected at four time points: day 1, day 2, day 4, and day 9. Three groups were included at each time point: cells alone, AMP alone, and AMP + cells. The AMP concentration used was 125 μg/ml.

result

mineralization

On day 1, cells were inoculated for 24 hours and not treated with induction medium or AMP (Figure 76). The cells were round and appeared more prominent on the monolayer. ARS staining was light brown with no or almost no mineralization. On day 2, without induction, the negative control cells became hexagonal. ARS stained the monolayer into a light peach color. Under induction, the positive control cells looked identical to the negative control and had no mineralization, and ARS staining was light peach color. Treatment with AMP caused MC3T3-E1 cell morphology to change, with spindle cells and spindle cells observed. ARS staining showed increased mineralization from day 1 and the monolayer stained light pink. However, the area where AMP particles stayed stained reddish-brown. AMP treatment combined with induction also changed the morphology of some cells. Spindle cells appeared and the monolayer also stained light pink, and the area around the AMP particles stained reddish-brown. On day 7, the negative control cells appeared more hexagonal and the cell boundaries were more clearly defined. There was increased mineralization and the monolayer stained light brown. The positive control, under induction, produced spindle cells and spindle rings. ARS also stained dark pink and was associated with increased mineralization. AMP treatment and AMP treatment combined with induction resulted in the retention of AMP granules and the inability to observe cellular morphology. No cell monolayers containing single cells could be observed. ARS showed reddish-brown staining of the area surrounding the AMP granules and a light pink staining of the monolayer. GnHCl successfully extracted the ARS dye, and the coefficient of variation of _OD450 values ranged from 6% to 16%.

Summary of results

After the 2nd day, the negative control MC3T3-E1 cells showed that ARS staining increased as cell culture and therefore showed that mineralization increased (Figure 76). Similar to previous results, there was morphological change in the positive control MC3T3-E1 cells cultivated by induction medium and produced spindle cells and spindle rings (Figure 76) at the 7th day. AMP was processed one day later (the 2nd day), with or without induction, and the change of cell morphology could be seen. The cell became spindle-shaped and even the form of fibroblasts (Figure 76 A). This change was not observed in the negative or positive control. In addition, this change could be observed (Figure 76 A) in the MC3T3-E1 cells of induction at least after 4 days of cultivation.

As shown by ARS quantification (Figure 76B), cells treated with AMP and AMP binding induced by AMP showed a statistically significant increase in mineralization relative to the positive control by day 2. The monolayer stained light pink, but small areas where AMP particles resided showed increased mineralization and stained dark reddish brown (Figure 76A). This increase in mineralization persisted throughout the culture period.

AMP treatment alone did not promote mineralization compared with AMP combined induced treatment.

proliferation

To determine whether AMP promotes mineralization by promoting cell proliferation, MC3T3-E1 cells were seeded at 3 × 10 ⁴ cells/cm ² /well in 96-well plates in αMEM medium supplemented with 10% FBS. Once confluent, the AMP group was treated with fresh 125 μg/ml AMP, added to the culture medium every 3 days. MTT assays were performed on days 1, 2, and 4, while BrdU assays were performed on days 1, 2, and 16.

In untreated MC3T3-E1 cells, cell viability increased from day 1 to day 4 (Figure 77A). In cells treated with AMP, cell viability decreased on day 2, and then more than doubled on day 4, following the trend of the cell group alone. BrdU assay showed that cell proliferation in the cell-treated group and the AMP-treated cell group (Figure 77B) decreased after day 1. The proliferation of the cell group alone decreased by more than half on day 2 and continued to decrease on day 16. In cells treated with AMP, cell proliferation only showed statistically significant reduction on day 16. These results indicate that AMP did not promote proliferation during the 16-day culture period.

Summary of results

As shown by the MTT assay, cell viability increased from day 1 to day 4 in MC3T3-E1 cells. AMP-treated MC3T3-E1 cells showed a decrease in cell viability from day 1 to day 2, but then followed an upward trend similar to that of untreated cells. However, on day 4, the cell viability of AMP-treated cells was approximately half that of untreated MC3T3-E1 cells. Cell proliferation, as measured by BrdU assay, decreased from day 1 to day 16. Unlike AMP-treated cells, cell proliferation of untreated MC3T3-E1 cells decreased by more than half on day 2. On day 16, AMP-treated and untreated MC3T3-E1 cells showed the same level of cell proliferation.

Example 42: AMP-treated MC3T3-E1 and hBM Identification of genes expressed in MSCs during the early stages of osteogenesis Certainly.

Human bone marrow mesenchymal stem cells (hBMMSCs) are multipotent and can differentiate into a variety of tissue types such as osteoblasts, chondrocytes, and adipocytes (Born, 2012 J Cell Biochem. 113(1):313-21.). When transplanted in vivo, they are able to form new bone, and in vitro, hBMMSCs can be guided to osteogenesis by culturing in β-glycerophosphate, ascorbic acid, vitamin D ₃ , and low-dose dexamethasone. The osteogenesis of hBMMSCs is regulated by the expression of osteoblast-related genes, including specific transcription factors, adhesion molecules, and ECM proteins (Born (2012) J Cell Biochem. 113(1):313-21; Vater (2011) Acta Biomater. 7(2):463-77.). The progression to mature osteoblasts mirrors that of MC3T3-E1 and is accompanied by a loss of cell proliferation, an increase in expression of osteogenic markers, and mineralization of the ECM (Born (2012) J Cell Biochem. 113(1):313-21). First, cells initiate ECM synthesis through the expression of collagen I (Col I). Simultaneously, the expression of bone-specific alkaline phosphatase (bALP) increases, and on day 4, a significant increase in ALP levels was observed in induced cells (6x10 ⁴ cells/60 mm dish) compared to controls (Born (2012) J Cell Biochem. 113(1):313-21; Jaiswal (1997) J Cell Biochem. 64:295–312). As differentiation continues, cells produce proteins such as bone sialoprotein (BSP), osteopontin, osteonectin, and osteocalcin. Finally, much like osteogenesis in MC3T3-E1 cells, mineralization of the ECM indicates mature osteoblasts.

In this example, genes and transcription factors expressed in the early stages of osteogenesis were determined in three cell lines: MC3T3-E1 cells and hBM MSC cells. Our results showed that mineralization was statistically significantly increased in AMP-treated MC3T3-E1 cells compared to the positive control on day 2 (day 1 of treatment). Therefore, this experiment focused on the early stages of osteogenesis to identify the effects of AMP on specific genes after treatment.

Experimental design

MC3T3-E1 cells or human bone marrow MSCs were seeded in 24-well plates containing αMEM medium supplemented with 10% FBS at 3×10 ⁴ cells/cm ² /well as described above. Once confluent, cell differentiation was induced by adding ascorbic acid and β-glycerophosphate. N=2 cells were tested at each time point for each experiment. Samples were collected at four time points: day 0 (after confluence but before induction/treatment), day 1, day 2, day 4, and day 6. Three groups were included at each time point: negative control, positive control, and AMP treatment alone. The AMP concentration used was 125 μg/ml.

result

hMSC expression

AMP induced the strong expression of endogenous BMP2 and BMP6 transcripts (60 times and 5 times, respectively) (Figure 78 A) within 24 hours of cultivation. BMP2 reached its peak value (120 times) on the 1st day and remained at a higher level (between 60 and 10 times) until the 4th day before showing a decline. In contrast, BMP6 reached its peak value at 4 hours and then showed a gradual decline from the 1st day. As a comparison, AMP did not change the expression level of BMP4, BMP7 and BMP9. Nevertheless, AG only induced the mild rise of BMP2 and BMP6 (1-2 times) and the significant rise of BMP4 after the 1st day (4-10 times) on the 4th day. As a comparison, AGM did not change the expression of BMP7 and BMP9.

Runx2 peaked on day 1 for AMP and on day 2 for AGM ( Figure 78A ). Both groups then gradually declined before reaching another peak on day 6. Runx2 induces the differentiation of multipotent mesenchymal cells into immature osteoblasts, guiding immature bone formation. Furthermore, Runx2 triggers the expression of key bone matrix genes in the early stages of osteoblast differentiation, but Runx2 is not essential for maintaining the expression of these genes in mature osteoblasts ( Komori (2010) Adv Exp Med Biol. 658: 43-9 ).

ALP and Sox9 peaked on day 4, with levels upregulated by AMP being less than those in AGM (Figure 78A). This trend is consistent with the idea that ALP and Sox9 are downstream of Runx2. ALP is expressed in the early stages of osteogenesis and creates an alkaline environment, which causes calcium to precipitate from the solution and crystallize.

AMP upregulated VEGF, reaching a peak at 4 hours and on the 4th day, but AGM only upregulated VEGF on the 4th day (Figure 78A). On the 1st day, AMP upregulated CXCR4 while AGM upregulated SDF1, with the former declining rapidly and the latter more slowly. Kortesidis et al. ((2005) Blood. 105(10):3793-801) proposed that SDF-1 can position the original uncommitted BMSSC cell population in their perivascular microenvironment until it is needed to proliferate and differentiate in response to environmental factors, which may disrupt the SDF-1/CXCR4 interaction and allow proliferation and differentiation. In 7 pre-conditioning experiments of MSCs, the expression of CXCR4 is generally increased by about 2 to 4 times (Cencioni et al. ((2012) Cardiovasc Res. 94(3):400-7), but our AMP results show a 60-fold increase after the 1st day.

SDF-1 expression was very high in all MSC groups, although this was not shown in Figure 78A. SDF-1 was visible around cycle 20, and GAPDH was visible around cycle 17. Stromal cell-derived factor-1 is known to activate adhesion molecules on progenitor cells, and anti-SDF-1 mAbs inhibit transendothelial migration of hematopoietic progenitor cells (Imai et al. (1999) Blood 93(1):149-56). SDF-1 activates CXCR4+/CD34+ cells and leads to their adhesion and transendothelial migration (Bhakta et al. (2006) Cardiovasc Revasc Med. 7:19-24). GAPDH expression did increase in the AMP group from day 2 to day 6, so one would assume proliferation. This trend was not seen in the other samples. In addition, BMP9 was undetectable (after cycle 40), CXCR4 was detected around cycle 33, VEGF was detected around cycle 19, and SOX-9 was detected around cycle 27. BMP4 was detected around cycle 28, BMP7 was undetectable (after cycle 40), BMP2 was detected around cycle 20, Runx2 around cycle 25, BMP6 around cycle 27, and ALPL around cycle 24.

Expression of MC3T3-E1

It is understood that MSC is multipotent, wherein osteoprogenitor cells have been pushed down further along the lineage pathway and can only produce bone formation. Contrary to expectations, BMP2 is almost not expressed in mouse cells compared with human MSC (Figure 79). It has been shown that BMP is inefficient in promoting osteogenesis in humans, but is highly capable in mouse cells (Osyzka et al. (2004) Cells Tissues Organs.176 (1-3): 109-19., Skarzynska et al. (2011) Connect Tissue Res.52 (5): 408-14). Gene chip shows that the expression of BMP-2 is not prominent in MC3T3 cells (Beck et al. (2001) Cell Growth & Differentiation 12: 61-83). The difference between the observed human and rodent cells may indicate that, unlike the Smad1 in human cells, the Smad1 of rodents does not undergo ERK linker phosphorylation during osteogenesis. Alternatively, Smad1 activity in rodent cells may not be inhibited by ERK-mediated linker phosphorylation (Skarzynska et al. (2011) Connect Tissue Res. 52(5):408-14). BMP-induced osteogenesis in unresponsive human MSCs requires regulation (inhibition) of the ERK and phosphatidylinositol 3-kinase (PI3-K) pathways; inhibition of the insulin/IGF-I-activated PI3-K/Akt pathway in serum-free cultured human MSCs reduced BMP-induced alkaline phosphatase and osteopontin expression, but increased BMP-activated Smad (Osyzka et al. (2005) Endocrinology. 2005(8):3428-37).

Runx2 expression was also shown to be differentially expressed in MC3T3 cells compared to hMSCs. On day 2, an event leading to significant gene upregulation was observed in MC3T3 cells. The AGM group appeared to exhibit the opposite effect of the AMP group, potentially related to the Pi3K and MAPK pathways. Ibsp, also known as bone sialoprotein (BSP), was upregulated in the AMP group on day 2 but not at other times. In the positive group, BSP expression was high after day 2.

Bglap-rs1, also known as osteocalcin (OCN), was also upregulated in the AMP group on day 2 but not elsewhere. Consistent with BSP, OC was highly expressed in the positive group after day 2. Analysis of these results suggests that BSP and OCN need to be upregulated, as they are known to be expressed by mature osteoblasts. Consistent with our results, Runx2 regulates the expression of Col1, BSP, and OCN in MC3T3 cells. Like BSP and OCN, Runx2 expression increased on day 2 but not elsewhere. In summary, AMP downregulates the expression of osteogenic genes but promotes mineralization.

Example 43: Effect of nHC-HA/PTX3 purified from AM on mineralization using the MC3T3-E1 model system.

Previous experiments have clearly demonstrated the unique ability of AMP to promote mineralization independently of inducers such as ascorbic acid, β-glycerophosphate, and melatonin (AGM). In these experiments, AMP was added to a mouse osteoblast precursor cell line (MC3T3) in the presence or absence of AGM to determine the efficacy of AMP. Alizarin red staining using GnHCl extraction was also performed at multiple time points for further quantification. It was concluded that AMP not only enhances mineralization independently of AGM, but also enhances mineralization at a faster rate. Further studies of AMP have shown that the osteoinductive effect exhibited by AMP requires direct cell contact. In other words, when in direct contact with AMP, cells accelerate and enhance mineralization without the need for the introduction of AGM.

Previous studies have shown that MC3T3-E1 differentiation under induction can be divided into three stages, namely, proliferation (day 1 to day 9), ECM formation (day 9 to day 16), and mineralization (deposition of minerals in the formed ECM) (day 16+) (Quarles et al. (1992) J Bone Miner Res. 7(6): 683-92.; Hong et al. (2010) Exp Cell Res. 316(14): 2291-300). However, our previous goal 4 has shown that mineralization can be easily detected within 7 to 10 days using AMP. Therefore, we will perform our ARS experiment on day 8 to determine whether NHC-HA/PTXS purified from AM is responsible for promoting mineralization. Day 8 was chosen because the observed results should be obvious as in goal 4 and medium changes will only be required on days 0, 3, and 6.

nHC-HA/PTX3 is known to be responsible for the known anti-inflammatory, anti-angiogenic, and anti-scarring therapeutic responses in the amniotic membrane. Our hypothesis is that immobilized HC-HC/PTX3 is responsible for the mineralization-promoting effects of AMP. Because it also contains HA, we will also compare nHC-HA/PTX3 and HA to observe whether the hypothesized effects are unique to nHC-HA/PTX3 and not to HA. Hyaluronic acid (HA) is an unsulfated glycosaminoglycan composed of a single repeating disaccharide unit. It is an essential component of connective tissue, promoting matrix assembly and tissue hydration. Luben et al. hypothesize that HA, as a calcium binder, acts as a barrier to enzyme diffusion from the resorption site or regulates osteoclast mobility. Stern and Raisz note that HA appears to be the most suitable for study because it has been clearly linked to bone resorption. Due to its hygroscopic properties, HA can occupy 10,000 times its own volume. Therefore, HA allows proliferating cells to avoid inhibitory contacts. Hyaluronan is synthesized prior to mitosis and detaches the dividing cell from its substrate, allowing cell motility (Balazs (2001) Am J Physiol Regulatory Integrative Comp Physiol 280:R466-R472).

Experimental design:

Mouse MC3T3-E1 cells (C-136) were taken from a liquid nitrogen freezer and grown in ^{100 mm culture dishes (5 dishes) with αMEM medium plus 10% FBS, which was changed every three days until 80% confluence reached ~1.5 x 10 6} cells [4 * (3.1 x 10 ⁴ ) * 9 = 1,116,000 cells]. Cells were then seeded at 3.1 x 10 ⁴ cells/cm ² /96 plastic wells. Medium was changed every 2-3 days (100 μl per 96 well), i.e., on day 0 (Wednesday), day 2 (Friday), and day 5 (Monday), and culture was stopped on day 8. N = 4 cells were tested for each condition.

The summaries of each group are as follows:

Control group:

Negative control: no treatment on a regular 96-well plate

Positive control 1: AGM added to a regular 96-well plate

Positive control 2: Add 125 μg/ml AMP to a regular 96-well plate every 3 days

Experimental group:

Negative control: Covalink-NH 96-well plate

Experimental group 1: AGM was added to the Covalink-NH 96-well plate every 3 days

Experimental group 2: 20 μg/ml HA was immobilized on a Covalink-NH 96-well plate

Experimental group 3: 20 μg/ml HA was immobilized on Covalink-NH-96 well plates and AGM (H-124) was added every 3 days.

Experimental group 4: 20 μg/ml nHC-HC/PTX3 was immobilized on a Covalink-NH 96-well plate

Experimental group 5: 20 μg/ml nHC-HC/PTX3 was immobilized on a Covalink-NH 96-well plate and AGM was added every 3 days

For the AGM group: On days 0 and 3, osteogenesis induction medium #1 (ascorbic acid, β-glycerophosphate) was replaced. On day 6, osteogenesis induction medium #2 (ascorbic acid, β-glycerophosphate, melatonin) was replaced. On day 0, only 0.2 ml of 10X induction medium was prepared. On days 3 and 6, 10 ml of osteogenesis induction medium was freshly prepared. The instructions for the induction medium were obtained from the In Vitro Osteogenesis Assay Kit (Millipore).

Induction medium #1: 9.88 ml αMEM medium plus 10% FBS, 20 μl ascorbic acid 2-phosphate 500X (Millipore, Part. 2004011), 100 μl glycerol-2-phosphate 100X (Millipore, Part. 2004011).

Induction medium #2: 9.87 ml αMEM medium plus 10% FBS, 20 μl ascorbic acid 2-phosphate 500X (Millipore, Part. 2004011), 100 μl glycerol-2-phosphate 100X (Millipore, Part. 2004011), 10 μl 50 uM melatonin (Millipore, Part. 2004011). 500 μl dH ₂ O was added to 6 ug of the provided melatonin.

ARS staining and quantification were performed as described above. Photos were taken at 10X using a Nikon Eclipse CFI60.

result

MC3T3-E1 cells were cultured for 8 days in different well plates. On the 8th day, phase contrast photographs were taken in the wells and (Figure 79 A, 79B) can be seen below. The negative control wells showed round cells and ARS dyeing was very light pink. The wells using induction culture medium showed a more vivid red and spindle-shaped cells were seen in the periphery of the wells. More abundant ARS dyeing was seen in the peripheral rings. AMP treatment showed that ARS treatment was dark red and difficult to see cells because AMP precipitated thereon. Aggregation was not observed during the experiment (even if it had been observed that other cell types aggregated on immobilized HC-HA). Microphotographs were taken on the 6th, 7th and 8th days.

On day 8, guanidine hydrochloride extraction was performed and the plates were incubated overnight. Although difficult to detect with the naked eye, GnHCl was able to extract the ARS dye; all wells appeared to have the same light pink/red color. Because the plate reader was not capable of reading at 490 nm or less, the ARS extract was quantified at 450 nm. The results can be seen in Figure 79C. * indicates statistical significance at p < 0.05. (+ indicates the presence of AGM, - indicates the absence of AGM)

Consistent with previous results, AMP successfully promoted mineralization without the need for an inducer. This experiment lasted only 8 days, and the results were not as pronounced as those in the 20-day experiment in Objective 4. All conditions treated with AGM showed increased mineralization compared to the corresponding negative control. Our results also indicate that immobilized nHC-HA/PTXS is not responsible for promoting mineralization in AMP. Therefore, another active component of AMP must be involved in promoting mineralization.

Example 44: Effects of HC-HA/PTX3(PBS) and HC-HA/PTX3(Gn) on Endochondral Ossification

The master transcription factors for osteogenesis and chondrogenesis (Runx2 and Sox9, respectively) were expressed by cells in HC-HA-conditioned conditions over a 14-day culture period. HC-HA/PTX3, both soluble and insoluble, promoted the expression of BMP2 and, to a lesser extent, BMP6 without the need for the commercially available osteoinductive agent AGM (i.e., ascorbic acid, glycerophosphate, melatonin) (see below).

The chondrogenic marker collagen 2 was highly expressed by HC-HA/PTX3(PBS) without the need for AGM. HC-HA/PTX3(Gn) and the addition of AGM also upregulated collagen 2. Osteogenic markers (BSP, ALPL, OSX) were upregulated by HC-HA conditions on day 14, confirming the transition from a cartilage to a bone genotype.

Experimental design:

Culture conditions: Human bone marrow-derived mesenchymal stem cells purchased from Lonza (Basel, Switzerland) were taken out of a liquid nitrogen freezer and grown in 100 mm culture dishes containing the same culture medium that was changed every 3 days until 80% confluence. The cell culture medium was αMEM containing 10% fetal bovine serum and antibiotics. The culture medium was changed every 3 days (10 ml per 100 mm culture dish), and the cells were passaged to 80% confluence until they reached the desired cell number described above. The cells used for the experiment were seeded at 3.1 x 10 ⁴ cells/96 plastic wells on immobilized HA, HC-HA (PBS) or HC-HA (Gn) with or without the osteoinductive agents ascorbic acid 2-phosphate, glycerol 2-phosphate and melatonin (AGM). The final concentrations of AGM added were 0.2 mM, 10 mM and 50 nM, respectively. AGM was added while the cells were inoculated (day 0) and mRNA was extracted on days 1, 7 and 14. To quantify gene expression, qPCR was performed and the culture medium (100 μl per 96-well) was changed every 3 days.

The experimental group is summarized as follows:

Negative control: Covalink-NH 96-well plate

Experimental group 1: AGM was added to Covalink-NH 96-well plates every 3 days

Experimental group 2: 20 μg/ml HA was immobilized on a Covalink-NH 96-well plate

Experimental group 3: 20 μg/ml HA was immobilized on Covalink-NH 96-well plates and AGM was added every 3 days

Experimental Group 4: 20 μg/ml 4X nHC-HC/PTX3 was immobilized on a Covalink-NH-96-well plate

Experimental Group 5: 20 μg/ml 4X nHC-HC/PTX3 was immobilized on Covalink-NH-96-well plates and AGM was added every 3 days

Experimental Group 6: 20 μg/ml 4X nHC-HC/PTX3 (GuHCl extract) was immobilized on Covalink-NH-96 well plates

Experimental group 7: 20 μg/ml 4X nHC-HC/PTX3 (GuHCl extract) was immobilized on Covalink-NH-96 well plates and AGM was added every 3 days

For the AGM induction group: On days 0 and 3, the medium was replaced with Osteogenesis Induction Medium #1 (ascorbic acid, glycerophosphate). On day 6, the medium was replaced with Osteogenesis Induction Medium #2 (ascorbic acid, glycerophosphate, melatonin). On day 0, 10X induction medium was prepared. On days 3 and 6, 10 ml of fresh Osteogenesis Induction Medium was prepared. Instructions for preparing the induction medium were obtained from the In Vitro Osteogenesis Assay Kit (Millipore).

Induction medium #1: 9.88 ml of αMEM medium supplemented with 10% FBS, 20 μl of ascorbic acid 2-phosphate 500X (Millipore, Part. 2004011), and 100 μl of glycerol-2-phosphate 100X (Millipore, Part. 2004011).

Induction medium #2: 9.87 ml of αMEM medium supplemented with 10% FBS, 20 μl of ascorbic acid 2-phosphate 500X (Millipore, Part. 2004011), 100 μl of glycerol 2-phosphate 100X (Millipore, Part. 2004011), and 10 μl of 50 uM melatonin (Millipore, Part. 2004011). 500 μl of dH ₂ O was added to the 6 ug of melatonin provided.

mRNA was extracted from cells on days 1, 7, and 14 and gene expression was determined by qPCR (Figures 80A-E). The following genes were measured: osteogenesis markers Runx2, alkaline phosphatase (ALPL), markers collagen 1 (COL1), osterix (OSX), and bone sialoprotein (BSP), as well as chondrogenesis markers Sox9 and collagen 2 (COL2), hypertrophy marker collagen 10 (COL10), and MMP13. Cultures on day 14 were stained with ARS and quantified as described above (Figures 81A, 81B).

On plastic, AGM upregulated BMP4. HA upregulated BMP4 (early), but downregulated BMP6 (later) and had no effect on BMP2 ( FIG80B ). There is no literature data indicating that HA itself upregulated BMPs. However, addition of AGM upregulated BMP2 and BMP6.

4X soluble HC-HA initially upregulated BMP4 but later downregulated BMP4 (similar to HA) and significantly upregulated BMP2 (similar to AMP, but without transient BMP6) (Figure 80B). In contrast, the addition of AGM did not change the expression pattern of BMPs.

4X insoluble HC-HA initially upregulated BMP4 but downregulated BMP4 (later) (similar to HA) and significantly upregulated BMP2 (similar to soluble HCHA) (Figure 80B). [Consistent with soluble] Similarly, addition of AGM did not change the expression pattern.

Our results demonstrate that both soluble and insoluble HC-HA can induce bone differentiation and mineralization via endochondral ossification. Expression of bone markers (COL1, OSX, ALP, and BSP) was significantly similar to that of chondrocyte markers (Col2) and hypertrophy markers (Col10, MMP13) (Figures 80A-E). The difference between these HC-HA conditions is that insoluble HC-HA promoted greater gene expression and more pronounced bone nodules (even in the absence of AGM), while water-soluble HC-HA required AGM (data not shown). Therefore, both soluble and insoluble HC-HA/PTX3 were able to promote BMP2 expression in the absence of the osteoinductive agent AGM.

HA without AGM also showed chondrogenic markers (COL2) and also showed signs of bone formation as defined by ARS mineralization and slight increases in ALP and OC. However, HC-HA/PTX3(PBS) had higher expression of chondrogenic markers and higher expression of bone markers ALP, OSX, and BSP than HA. However, neither condition significantly expressed hypertrophic markers. HC-HA/PTX3(Gn) expressed significantly more ALP, OSX, and BSP than the above two conditions. The hypertrophic marker MMP13 was also expressed, and the chondrogenic marker COL2 was slightly expressed.

HA plus AGM promoted osteogenesis and increased the expression of BMP2, ALP, OSX, BSP, and OC, as well as the expression of hypertrophic markers COL10 and MMP13. However, HA produced less bone-specific mRNA expression and bone nodule formation than the HC-HA group. Another major difference was that HA downregulated SOX9 but later increased BMP6 expression.

All previous data indicate that insoluble HCHA is the strongest inducer of bone and, more importantly, induces endochondral ossification mechanisms.

Example 45: nHC-HA/PTX3 inhibits inflammation and immune responses and improves corneal transplant survival in mice

Experimental and clinical studies have shown that amniotic membrane (AM), AM extracts, and nHC-HA/PTX3 (a covalent complex of the heavy chain (HC) of inter-α-trypsin inhibitor (IαI) and hyaluronan (HA)) inhibit proinflammatory responses. This example demonstrates that nHC-HA/PTX3/PTX3 can modulate T cell responses and reduce corneal allograft rejection in mice.

T cell activation can be assessed by proliferation and the production of various cytokines (Figure 82). In this case, splenocytes were isolated from OT-II mice expressing transgenic TCRs specific for ovalbumin (OVA) and stimulated with OVA for 4 days (Figure 83). Cell proliferation was measured by BrdU labeling and the expression of cytokines (IFN-g and IL-2) was measured by respective ELISAs. 1 mg/ml of nHC-HA/PTX3, but not HA, significantly inhibited the proliferation of splenocytes treated with OVA peptides on days 2 and 4 (Figure 84) and the production of IFN-γ and IL-2 (Figure 85) (all p<0.05). In addition, corneal T cells were activated in vivo by injection of LPS.

The optimization of the injection site, volume, and frequency of nHC-HA/PTX3 was determined by the inward flux of green fluorescent protein-positive macrophages into the cornea of Mafia mice before or during intracorneal LPS injection. The injection protocol was further optimized by administering 5 μl to all four sectors per injection between the subconjunctiva and the fornix. After 4 days of nHC-HA/PTX3 treatment, the cornea was digested with 820 units/ml of collagenase at 37°C for 1 hour. EGFP- and EGFP+ cells were separated by FACS. Pretreatment with nHC-HA/PTX3 for 3 days before LPS injection significantly inhibited the inward flux of EGFP+ macrophages into the LPS-injected cornea (9.1±0.3 vs. 12.3±0.4, nHC-HA/PTX3 vs. PBS, p=0.02) (Figure 86). Importantly, even though EGFP+ macrophages do migrate into the cornea, some of them are converted into an M2 phenotype indicated by significantly upregulating Arg-1 and IL-10 but downregulating IL-12 (p < 0.05) (Figure 87). The mRNA expression of Arg-1, IL-10, and IL-12 was determined by qPCR. Finally, allogeneic corneal transplantation was performed using wild-type BALB/c mice as recipients and C57BL/6 mice as donors, and the transplant clarity was measured twice a week using a slit lamp biomicroscope to obtain the result score. Transplants with a score of ≥3 and no resolution for two consecutive times were considered rejections. Compared to the PBS control, allogeneic transplant rejection was significantly inhibited by injecting 10 μl of nHC-HA/PTX3 in one sector twice a week (p < 0.05), and further reduced by injecting 5 μl in 4 sectors twice a week (p < 0.002) (Figure 88).

These experiments demonstrated that nHC-HA/PTX3 significantly inhibited corneal transplant rejection in mice. This mechanism of action may be attributed to the ability of nHC-HA/PTX3 to downregulate proinflammatory macrophages and suppress T cell immune responses.

Example 46: Treatment of dry eye in mice induced by dry stress using nHC-HA/PTX3 and AMP

Dry eye, also known as tear dysfunction syndrome, is a common ocular surface disease with high prevalence and significant morbidity worldwide. It is an autoimmune inflammatory disease characterized by chronic autoreactive T cell-mediated inflammation and dysfunction of the lacrimal functional units (LFUs; cornea, conjunctiva, lacrimal glands, and meibomian glands). Dry eye syndrome (SS) is a pervasive, chronic autoimmune disease characterized by mononuclear cell infiltration of the salivary and lacrimal glands, leading to secondary damage to parenchymal tissues.

Keratoconjunctivitis sicca (KCS) in SS is a serious and potentially vision-threatening ocular epithelial disease characterized by infiltration of CD4+ T cells that produce IL-17 and interferon (IFN)-γ. Compounds that inhibit T cell activation (e.g., cyclosporin A) alleviate dry eye in animals and humans. Macrophages can undergo classic M1 activation (e.g., by IFN-γ and/or TLR ligands such as LPS) to express high levels of inflammatory cytokines (such as TNF-α, IL-12, and IL23), which activate Th1 and Th17 lymphocytes that lead to many chronic inflammatory diseases (Figure 89). This example demonstrates that the administration of nHC-HA/PTX3 and AMP is useful for the treatment of such conditions.

Four injection sites per eye inhibited macrophage infiltration into the cornea

MAFIA mice allow tracking of macrophage influx in vivo because they are labeled with green fluorescent protein. These mice were used to determine whether nHC-HA/PTX3 or AMP could block LPS-induced macrophage influx into the cornea, a model of keratitis. LPS was injected at an appropriate volume of 5 μl or less per injection site between the subconjunctiva and the fornix. Eyes of MAFIA mice (macrophages are EGFP+) were injected intrastromally with LPS (5 μg per eye). In each eye, the OS was treated with PBS (two or four injection sites) and the OD was treated once with nHC-HA/PTX3 (two or four injection sites; each injection site contained 5 μl of 1 mg/ml HA at nHC-HA) or AMP (two or four injection sites; each injection site contained 5 μl of 10 mg/ml protein at AMP). Treatments were performed immediately after LPS injection. The entire cornea was imaged using in vivo intravital microscopy on days 1, 2, 3, and 6. EGFP-positive cells were counted based on the intensity of green fluorescence.

EGFP-positive macrophages were detected in most of the corneal peripheral area after LPS injection and concomitant treatment with PBS on day 1. Treatment with nHC-HA/PTX3 or AMP did not significantly increase or decrease macrophages in the cornea.

On day 2, the number of macrophages in PBS-treated corneas was significantly increased compared to day 1 (p<0.05), as was the case with nHC-HA/PTX3 (two and four injection sites, p<0.05) and AMP (two and four injection sites, p<0.05). Specifically, two injections of nHC-HA/PTX3 resulted in more macrophage infiltration in the corneas than in those treated with PBS (p>0.05), but fewer in the corneas treated with four injections of nHC-HA/PTX3 (p>0.05). For AMP treatment, two injections had no significant effect, but four injections slightly reduced macrophage infiltration, suggesting that four injections of nHC-HA/PTX3 or AMP per eye are necessary to effectively reduce macrophage infiltration.

On day 3, the infiltration of macrophages continued under PBS, nHC-HA/PTX3, and AMP treatment.

Treatment with 2 injections, 4 injections of nHC-HA/PTX3, or 2 injections of AMP did not inhibit infiltration. The only treatment that showed inhibition was 4 injections of AMP (p < 0.05).

On day 6, macrophage infiltration was reduced. However, neither HC-HA/PTX3 nor AMP treatment had a significant inhibitory effect compared with the control.

These data indicate that EGFP-positive macrophages continue to infiltrate the LPS-injected cornea from day 1 to day 3, peak on day 4 or 5, and then decline on day 6, which is consistent with previously reported data (Figure 90). Treatment with four injections of nHC-HA/PTX3 per eye on day 2 or four injections of AMP on days 2 and 3 slightly inhibited macrophage infiltration. This suggests that AMP has better potency than nHC-HA/PTX3 in blocking LPS-induced macrophage infiltration.

Pretreatment with AMP significantly inhibited injury-induced macrophage infiltration due to additional injections and subsequent injections of nHC-HA/PTX3 or AMP.

The left eye (OS) of each MAFIA mouse was pretreated with PBS (5 μl) or AMP (5 μl of 10 mg/ml protein) at 4 sites under the conjunctiva/fornix as defined above. The right eye (OD) of each mouse was not treated. Three days later, each eye was injected with LPS (5 μg) at the cornea and then treated with PBS (5 μl), HC-HA/PTX3 (5 μl of 1 mg/ml HA) or AMP (5 μl of 10 mg/ml protein) at 4 sites. In vivo intravital microscopy was used to count the infiltration of EGFP+ macrophages, which did not show any significant reduction in macrophage infiltration of the mouse cornea (p>0.05) (data not shown). The accuracy of this quantitative method was then studied by in vivo fluorescence microscopy.

In order to more accurately measure the infiltration of macrophages and detect the phenotype of the macrophages produced (e.g., M1 relative to M2), we decided to quantify EGFP-positive macrophages by collagenase digestion and FACS of corneas removed on day 4. Then, the positive macrophages were normalized by the ratio of EGFP-negative macrophages to assess the extent of macrophage infiltration. In the group not pretreated, LPS injection in the PBS control group caused macrophage infiltration (Figure 91, A, blue bar). This infiltration was significantly inhibited by nHC-HA/PTX3 (9.1±0.3 relative to 12.3±0.4, p=0.02) or AMP (2.1±0.1 relative to 12.3±0.4, p=0.02). AMP treatment was better than nHC-HA/PTX3 treatment in inhibiting the infiltration of macrophages (p=0.02).

Compared to the untreated group, LPS injection in the PBS control group significantly induced macrophage infiltration in the pretreatment group (37.2±1.3 vs. 12.3±0.4, p=0.01) (Figure 91, A, red bar). This difference is expected, as the injury caused by the four subconjunctival injections during pretreatment caused inflammation, which enhanced macrophage infiltration into the cornea subsequently treated with LPS. However, this strongly increased infiltration was completely inhibited by AMP pretreatment and subsequent nHC-HA/PTX3 (8.2±0.3 vs. 37.2±1.3, p=0.02) or AMP treatment (2.3±0.1 vs. 37.2±1.3, p=0.02). Again, AMP pretreatment followed by AMP treatment was superior to AMP pretreatment followed by nHC-HA/PTX3 treatment in inhibiting macrophage infiltration (2.3±0.1 vs. 8.2±0.3, p=0.02). However, there was no significant difference in inhibition between the no pretreatment group and the AMP-pretreated nHC-HA/PTX3 or AMP groups (p>0.05). qPCR data showed that nHC-HA/PTX3 pretreatment reduced M1 markers IL-12p40 and IL-12p35 and increased M2 marker Arg-1 (Figure 91, B). AMP treatment and pretreatment significantly reduced IL-12p40 and IL-12p35 but greatly increased Arg-1 and IL-10. In summary, AMP pretreatment completely eliminated the infiltration of macrophages caused by additional damage during pretreatment. This effect was sustained by subsequent injection of nHC-HA/PTX3 or AMP accompanied by LPS injection. This benefit was noted after 4 days, and AMP was more effective than nHC-HA/PTX3.

nHC-HA/PTX3 or AMP reduced DS-induced ALKC in a mouse experimental dry eye model.

design

Species: C57BL/6 mice

Endpoint: Corneal epithelial barrier function (OGD staining)

Sample size: 15 per group

Groups: 2 control groups and 3 treated groups (PBS, nHC-HA/PTX3 and AMP): 1) non-dry eyes, untreated controls (UT) - kept in a separate animal housing room; 2) experimental dry eyes, untreated controls (EDE); 3) PBS; 4) nHA-HC/PTX; 5) AMP.

Desiccation stress (DS)

The dry stress model was created by blocking tear secretion with pharmacological choline and by being exposed to a ventilated and low-humidity controlled environment in a room for 5 days (Monday to Friday). Mice were placed in specially designed cages with holes, which contained standard mouse cages with wire mesh replaced on both sides to allow airflow through the cage. Each cage was placed in front of a constant airflow (electric fan). Lacrimal secretion was suppressed by subcutaneous injection of scopolamine (0.5 mg in 0.2 mL, Sigma-Aldrich) 4 times a day for a total of 5 days (8:30 am, 11:30 am, 1:30 pm, 4:30 pm). The humidity in the controlled environment room was maintained at a relative humidity of about 25-30%, which was achieved by 4 portable dehumidifiers and a dehumidifier unit on the ceiling.

Treatment course (5-day plan)

In each experiment, 3 controls were included:

Untreated controls consisted of a group of mice kept in an animal housing room at 40-70% relative humidity. These mice were never exposed to DS and did not receive any topical treatment.

Dry eye controls, consisting of a group of mice placed in an ambient dry eye chamber but not receiving treatment.

Vehicle control, consisting of a group of mice that underwent DS treatment but received PBS.

In addition, two experimental groups were included: nHA-HC/PTX3 and AMP.

For injections, we used nHC-HA/PTX3 (containing 1 mg/ml HMW HA) and AMP (containing 10 mg/ml total protein) with PBS as a solvent control. All solutions (PBS, nHC-HA/PTX3 and AMP) were drawn into a 30 G tuberculin syringe. The injection site was subconjunctival close to the fornix (Figure 92). Four (4) injections of 5 μl were injected at each injection site at 3, 6, 9 and 12 o'clock. The diffused solution completely covered the entire subconjunctival periphery, causing a small amount of conjunctival or overall congestion/swelling (if any, it should disappear within 15 minutes), which hindered eye closure and corneal surface rupture or inflammation. Injections were performed on days 1 and 3 (for all agents), i.e., 2 times in total. This injection schedule is summarized in Table 6.

Table 6. Experimental groups and reagents required for nHC-HA/PTX3 or AMP to reduce DS-induced ALKC in the mouse experimental dry eye model.

Measurement of corneal staining

On the morning of the 5th day, mice received 1 s.c. dose of scopolamine after measuring the tear volume. Two hours after this scopolamine dose, corneal staining was performed with Oregon Green Dextran (OGD-488), a conjugated fluorescent dye (molecular probe) with a molecular size of 70 kDa. The process included using a glass capillary pipette to instill 0.5 μl of OGD into the cornea 1 minute before euthanasia. Mice were then subjected to cervical dislocation after inhalation of isoflurane anesthesia gas. The eyes were then rinsed with 2 ml of BSS. Excess liquid was carefully blotted from the ocular surface with filter paper without touching the cornea. Digital images of the eyes were captured using a Nikon SMZ-1500 stereomicroscope with a CoolSnap HQ2 cooled CCD camera at an excitation wavelength of 470 nm and an emission wavelength of 488 nm, with an exposure time of 1 second. Both eyes of each animal were evaluated. Fluorescence intensity in a fixed area of interest (1-mm diameter circle) in the central cornea of three digital images was measured using Nikon Elements software, and the data were stored in a database (Excel, Microsoft). Results are expressed as mean ± standard deviation of grayscale. The results of three independent experiments were averaged for statistical comparison between groups.

The effects of nHC-HA/PTX3 and freeze-dried amniotic membrane powder (AMP) on the levels of T helper cell pathway regulators were compared in a 5-day experimental dry eye (EDE) model established in C57BL/6 mice. The expression of Th1 (IL-12, IFN-γ, and T-Bet), Th1-17 (IL-23, IL-17, ROR-γt, IL-6, TGF-β1, MMP-3, and MMP-9), and Th2 (IL-4, IL-13, and GATA3)-related factors in corneal epithelial cells and conjunctiva of the following groups was measured by real-time PCR.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Inc). One-way analysis of variance (ANOVA) was used to determine overall differences between groups, followed by post hoc tests (Tukey post hoc). Unpaired t-tests were used to assess statistical differences between the two experimental groups.

Example 47: HC-HA activates IGF1-HIF1α-VEGF signaling to promote angiogenesis by The effect of TGFβ1 on the membrane fibroblasts was further promoted.

In this example, the effect of the HC-HA complex on the induction of angiogenesis markers in human corneal fibroblasts was investigated.

Human corneal fibroblasts (3000 cells/well in a 96-well plate) were seeded in plastic dishes with or without immobilized HA, soluble HC-HA (PBS) (4X), or insoluble HC-HA (GnHCl) (4X) for 48 hours as described above. Cells were then treated with or without TGFβ1 for 24 hours before harvesting for quantification of IGF1, HIF1α, and VEGF mRNA. The experimental groups were:

PBS PBS+TGF-β1 HA HA+TGF-β1 4X HC-HA PBS 4X HC-HA PBS + TGF-β1 4X HC-HA Gn 4X HC-HA Gn+TGF-β1

Total RNA was extracted using the RNeasy Mini kit (Qiagen) and reverse transcribed using a high-capacity reverse transcription kit (Applied Biosystems). Real-time RT-PCR was performed to amplify the cDNA of each cellular component using a specific primer-probe mixture and DNA polymerase in a 7000 Real-time PCR System (Applied Biosystems). The real-time RT-PCR profile included an initial activation at 95°C for 10 minutes, followed by 40 cycles of annealing and extension at 95°C for 15 seconds and 60°C for 1 minute. The size of each PCR product (IGF1, HIF1α, and VEGF) was determined using a 2% agarose gel, subsequently stained with ethidium bromide along with PCR markers using an EC3 imaging system (BioImaging System).

When cells were in quiescent conditions, HC-HA induced a 2- to 6-fold increase in IGF1 mRNA and a 2-fold increase in VEGF mRNA (Figure 92). In contrast, when cells were treated with TGFβ (10 ng/ml), HC-HA induced a 5- to 12-fold increase in IGF1 mRNA and a 5- to 9-fold increase in VEGF mRNA. VEGF has been shown to play an important role in angiogenesis, increasing the number of capillaries in a given network. Activation of vascular endothelial growth factor is controlled by upstream regulators such as IGF1 and HIF1α. Our results suggest that HC-HA activates the IGF1-HIF1α-VEGF network to promote angiogenesis, which can be further enhanced by the addition of TGFβ1 to human corneal fibroblasts.

Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Various changes, modifications, and substitutions may be made. It should be understood that various alternatives to the embodiments of the present invention described herein may be used to practice the present invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims (15)

1. A reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex comprising high molecular weight acetylated hyaluronic acid (HMWHA), meso-α-inhibitor (IαI) protein heavy chain 1 (HC1) and heavy chain 2 (HC2), and pentacyclic protein 3 (PTX3). 2. A reconstituted HC-HA/PTX3/SLRP complex, comprising HMW HA, HC1, HC2, PTX3 and small leucine-rich proteoglycan (SLRP). 3. Use of an effective amount of the rcHC-HA/PTX3 complex as described in claim 1 in the preparation of a medicament for the prevention or reversal of scar formation or fibrosis in tissues. 4. Use of an effective amount of the rcHC-HA/PTX3 complex as described in claim 1 in the preparation of a medicament for the prevention or reduction of inflammation in subjects in need. 5. The use as claimed in claim 4, wherein the inflammation is associated with an autoimmune disease, allergic reaction, leukocyte deficiency, infection, graft-versus-host disease, or a combination thereof. 6. Use of an effective amount of the rcHC-HA/PTX3 complex as described in claim 1 in the preparation of a medicament for treating skin trauma or ulcers in subjects in need. 7. Use of an effective amount of the rcHC-HA/PTX3 complex of claim 1 in the preparation of a medicament for promoting or inducing bone formation in a subject in need, wherein the subject suffers from arthritis, osteoporosis, alveolar bone degradation, or Paget's disease. 8. Use of an effective amount of the rcHC-HA/PTX3 complex as described in claim 1 in the preparation of a medicament for the prevention or reduction of unwanted angiogenesis in a subject in need. 9. Use of an effective amount of the rcHC-HA/PTX3 complex as described in claim 1 in the preparation of a medicament for inhibiting or preventing transplant rejection in transplant recipients. 10. A method for inducing stem cell expansion in vitro, comprising contacting stem cells in vitro with an effective amount of the rcHC-HA/PTX3 complex as described in claim 1 or the reconstituted HC-HA/PTX3/SLRP complex as described in claim 2. 11. Use of an effective amount of the rcHC-HA/PTX3 complex as described in claim 1 in the preparation of a medicament for providing cell therapy to subjects in need. 12. The use as claimed in claim 11, wherein the cells are selected from stem cells and differentiated cells. 13. A medical device comprising a substrate coated with the rcHC-HA/PTX3 complex as described in claim 1. 14. A composition comprising the rcHC-HA/PTX3 complex of claim 1 and a pharmaceutically acceptable excipient or carrier. 15. A composition comprising the rcHC-HA/PTX3 complex of claim 1 and a pharmaceutically acceptable polymer selected from the following: fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate, and combinations thereof.