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WO2016172004A1 - Système de cicatrisation osseuse, d'accélération de l'angiogenèse et de production de vasculogenèse - Google Patents

Système de cicatrisation osseuse, d'accélération de l'angiogenèse et de production de vasculogenèse Download PDF

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WO2016172004A1
WO2016172004A1 PCT/US2016/027729 US2016027729W WO2016172004A1 WO 2016172004 A1 WO2016172004 A1 WO 2016172004A1 US 2016027729 W US2016027729 W US 2016027729W WO 2016172004 A1 WO2016172004 A1 WO 2016172004A1
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Prior art keywords
bone
mdc
scaffold
subject
cells
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Anand Kumar
Denver LOUGH
Chris Madsen
Qiongyu Guo
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Johns Hopkins University
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Johns Hopkins University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • This invention relates generally to methods and compositions for treating and/or mending bone defects and/or injuries, or for treating damaged, diseased or otherwise aberrant tissue in need of angiogenesis and/or vasculogenesis.
  • Heterotopic ossification is a naturally occurring pathologic process of mature bone formation in muscle tissues that occurs via an endochondral ossification pathway, and is a robust, naturally occurring process of de novo bone formation.
  • Muscle-derived stem cells are the putative source of heterotopic ossification.
  • a lack of a translational bone regeneration system that employs endochondral ossification has to date prevented deployment of MDSc-involving regenerative cranial human trials.
  • the invention is based, at least in part, upon the identification of a system that includes muscle-derived cells (MDCs, e.g., muscle-derived stem cells (MDScs)), a bone induction agent (e.g., a bone morphogenetic protein, i.e., BMP2) and a scaffold (i.e., a collagen scaffold), as a system that effects surprisingly enhanced levels of bone defect healing and also imparts dramatic angiogenesis and/or vasculogenesis at the site of application.
  • MDCs muscle-derived cells
  • MDScs muscle-derived stem cells
  • BMP2 bone morphogenetic protein
  • a scaffold i.e., a collagen scaffold
  • MDScs to promote angiogenesis and/or vasculogenesis at the site of application of a scaffold
  • a scaffold e.g., a collagen disk
  • a scaffold e.g., a collagen disk
  • degeneration and/or loss e.g., joint treatments, muscle -rebuilding treatments and cardiac treatments, e.g., for effecting angiogenesis and/or vasculogenesis at the site of a coronary blockage, are among those specifically contemplated).
  • the invention provides a method for treating a bone defect in a subject, the method involving obtaining a muscle-derived cell (MDC) population; contacting the MDC population with a bone induction agent; applying the MDC population to a scaffold, thereby forming a MDC-scaffold composition; and contacting the MDC-scaffold composition to a bone of a subject, wherein the bone possesses a bone defect, thereby treating the bone defect in the subject.
  • MDC muscle-derived cell
  • the MDC population is enriched for muscle-derived stem cells (MDScs). In another embodiment, the MDC population is derived from the subject having the bone defect that is treated.
  • MDScs muscle-derived stem cells
  • the MDC population is isolated from a preplate derived by the method of Gharaibeh, B. et al. Nature Protocols 3: 1501-1509, and optionally is a preplate 1 (PP1) to preplate 6 (PP6) cellular population.
  • the MDC population is isolated from preplate 2 (PP2) or preplate 3 (PP3).
  • the MDC population is an ex vivo expanded MDC population.
  • the MDC population is fibroblast-depleted, as compared to an unexpanded control MDC population.
  • the bone induction agent is a bone morphogenetic protein, optionally BMP2, e.g., rhBMP2, or a fragment thereof.
  • the bone induction agent is present at a concentration sufficient to osteo-induce the MDC population. In one embodiment, 1-10 ⁇ g of the bone induction agent is present in the MDC- scaffold composition, optionally 5 ⁇ g of the bone induction agent is present in the MDC- scaffold composition, and/or optionally the bone induction agent is present in the MDC- scaffold composition at a concentration of at least 10 ng/ml, at least 100 ng/ml, and/or at least 1 mg/ml.
  • the scaffold is a Collagen I scaffold, optionally a Collagen I scaffold disk, optionally a Collagen I scaffold disk of 1 to 10 mm diameter, optionally of 5 mm diameter.
  • the bone defect is a defect of 1 to 10 mm diameter, optionally of 5 mm diameter.
  • the MDC population includes at least 1 x 10 4 cells, optionally at least 1 x 10 5 cells, optionally at least 1 x 10 6 cells, optionally about 2 x 10 6 cells.
  • the bone defect is significantly reduced in size and/or is healed within eight weeks of contacting the MDC-scaffold composition to the bone of the subject, optionally within four weeks of contacting the MDC-scaffold composition to the bone of the subject, optionally within three weeks of contacting the MDC-scaffold composition to the bone of the subject.
  • contacting the MDC-scaffold composition to a bone of the subject results in functionally polarized healing of the bone defect of the subject.
  • the subject is a mammalian subject, optionally a human.
  • Another aspect of the invention provides a method for promoting angiogenesis and/or producing vasculogenesis in a subject, the method involving obtaining a muscle-derived cell (MDC) population; contacting the MDC population with a bone induction agent; applying the MDC population to a scaffold, thereby forming a MDC-scaffold composition; and contacting the MDC-scaffold composition to a tissue of a subject, where the tissue possesses a region of injury, disease, disorder and/or lack of blood vessels in need of angiogenesis and/or vasculogenesis, thereby promoting angiogenesis and/or vasculogenesis in the subject.
  • MDC muscle-derived cell
  • the tissue is a cardiac tissue, a bone tissue, a muscle tissue, a wounded tissue and/or a joint tissue, optionally a knee joint tissue.
  • a further aspect of the invention provides a composition for treating a bone defect in a subject, promoting angiogenesis and/or producing vasculogenesis in a tissue of a subject that includes a muscle-derived cell (MDC) population; a bone induction agent; and a scaffold, where the composition is capable of treating a bone defect, promoting angiogenesis and/or producing vasculogenesis in a subject when applied to a bone or other tissue of the subject, as compared to an appropriate control composition.
  • MDC muscle-derived cell
  • kits for treating a subject having a bone defect includes a muscle-derived cell (MDC) population, a scaffold and instructions for its use.
  • MDC muscle-derived cell
  • the kit also includes a bone induction agent, optionally BMP2, optionally rhBMP2 or a fragment thereof.
  • the kit includes a muscle-derived cell population is enriched for muscle-derived stem cells (MDScs).
  • MDScs muscle-derived stem cells
  • the scaffold of the kit is a Collagen I scaffold, optionally a Collagen I scaffold disk, optionally a Collagen I scaffold disk of 1 to 10 mm diameter, optionally of 5 mm diameter.
  • An additional aspect of the invention provides a method for treating a bone defect in a subject, the method involving identifying a bone defect in the subject; and using a kit of the invention to treat the bone defect in the subject.
  • Figures 1A to ID demonstrate regeneration of polarized bone using MDScs in osteo-enriched collagen scaffold in vivo.
  • the addition of type-1 collagen and BMP2 in MDScs promoted formation of apparent cortical and cancellous bone elements in 2D co-culture ( Figures 1A and IB) and 3D culture ( Figures 1C and ID). Alizarin red staining was applied in Figure IB. Scale: 200 ⁇ .
  • Figures 2 A and 2B show that a three-principled regenerative healing strategy of the invention (here involving MDScs + BMP2 + collagen I scaffold) enhanced bone regeneration in critically-sized cranial defects in a murine model.
  • Figure 2A shows micro CT images of an osteo-induced MDSc construct implanted for 3 weeks (right) vs. empty control (left).
  • Figure 2B shows retention of eGFP-MDScs during bone healing confirmed by fluorescent imaging.
  • Figures 3 A and 3B show nascent bone formation within a murine cranial defect wound bed model at 8 weeks.
  • Figure 3A shows a result where 5 mm full-thickness cranial bone defects in murine skull were treated with a BMP2 bound collagen I scaffold implant, in the absence of MDScs.
  • Figure 3B shows a result where 5 mm full-thickness cranial bone defects in murine skull were treated with a BMP2 bound collagen 1 scaffold seeded with MDSc (2xl0 6 ).
  • Upper panels (transverse view) show a CT reconstruction of murine skull at 8 weeks following creation of 5 mm bone defect, while the white solid arrow marks ingrowth of petri- defect native bone elements.
  • Middle panels show CT reconstruction through a central defect (white dotted line).
  • the solid arrow in the middle panels indicates neo-diploic space formation following implant of MDSc seeded graft after 8 weeks.
  • the lower panel shows a 4x magnification of the middle panel region of interest ("ROI"; white dotted box).
  • Figures 4A to 4C show the MDSc enrichment process and images of cellular structures resulting upon treatment with various components of the system of the instant invention.
  • Figure 4A shows a drawing of the MDSc enrichment process, showing an expansion/passage process, while
  • Figures 4B and 4C show additional images of polarity establishment and structures formed upon combination of collagen 1 scaffold + BMP2 + MDSc population.
  • Figure 5 shows images of the testing process, including cranial drilling used to create consistently sized cranial defects for test treatments.
  • Figures 6A to 6C show cellular images and markers of cell cycle progression in a MDSc population in vitro, showing that increasing levels of BMP2 accelerated cell cycle progression. Specifically, as shown in Figures 6B and 6C, BMP2 + collagen I scaffold MDScs exhibited greater multiplication and proliferation.
  • Figures 7 A and 7B show that MDScs subjected to both BMP2 and collagen I contact in vitro were multipotent, with Figure 7A showing FACs analysis for cell surface markers of differentiation and Figure 7B showing cellular images of differentiated cells.
  • Figure 8 shows both scaffold and migratory kinetics of MDScs of the system of the invention, which established that MDScs of the invention could migrate into bone, performing repair of bone defects in a functionally polarized manner capable of complete repair of such defects.
  • Figures 9 A to 9E show that the combination of BMP2 and type 1 collagen acted as a mitogen in MDSc enriched populations, leading to polarized bone formation.
  • Figure 9A shows DIC and fluorescence channels monitoring the real-time proliferation index of MDSc enriched populations plated at 10 6 over 18 hours using live confocal imaging. Populations were treated with a spectrum of BMP2 as indicated on either plastic or type I collagen. Red fluorescence indicated presence of Cdtl or the Gl phase of the cell cycle. Green
  • FIG. 9B shows exemplary images used to determine the migration index and volume index of confocal acquired image files.
  • Figure 9C shows the correlative migration index and cell volume index for populations in the presence or absence of type 1 collagen and a spectrum of BMP2 concentrations.
  • Figure 9D shows relative quantification of cells within a defined cell cycle parameter (Gl, S and G2 phases) at 18 hours in the presence or absence of type I collagen and a spectrum of BMP2 concentrations.
  • Figure 9E shows MDSc population topography and linear tag tracking over time while in the presence of type 1 collagen and BMP2. Black arrow indicates region of mitosis. White arrow indicates limited radial migration. Dotted lines indicate repeated radial migratory vectors by MDSc population undergoing bone formation.
  • Figure 10 shows that following optimization of migratory kinetics and lineage induction, MDSc were applied to a spectrum of engineered scaffolds for traceable, real-time in vivo studies within defect models.
  • Figures 11A and 11B show that the process of in vitro and organotypic optimization permitted the application of such findings to real-time regenerative living studies, which showed efficacy in augmenting the healing of bone defects with functional polarized bone.
  • Figures 12A and 12B show that MDScs augmented polarized bone healing and diploic space formation in a treated subject, with in vivo cortical and cancellous bone formation following delivery of MDSc seeded scaffolds at week three in an explant specifically observed.
  • Figure 12A shows murine eGFP expressing MDSc (2xl0 6 ) seeded onto BMP2 bound Col-1 (collagen I) scaffolds produced a polarized bone construct (cortical and cancellous bone architecture) within 14-21 days following implantation into 5 mm diameter full-thickness skull defects.
  • White hollow arrow indicates cortical bone, while white solid arrow indicates cancellous bone.
  • Scale in ⁇ Figure 12B shows a Z-stack through the nascent diploic space with explanted MDSc collagen scaffold at eight weeks.
  • FIG 13 shows that MDSc compositions of the invention (implants comprising collagen I scaffold-MDSc and BMP2) were identified to augment both localized angiogenesis and localized vasculogenesis in vivo.
  • Figures 14A and 14B show that MDSc populations possess multi-lineage cellular potency.
  • Figure 14A shows that MDSc populations harvested from C57BL/6 skeletal muscle maintained the capacity to undergo myocyte differentiation following primary culture. Linear fused pre-myocytic cellular entities were distinguishable at 24 hours, and were able to beat/pulse within 48 hours. Full myocyte phenotype and motion was maintained even at confluence. Fewer linear fused pre-myocytes were detectable under non-myogenic (MSC basal media) conditions.
  • Figure 14B shows that MDSc populations harvested from skeletal muscle displayed a multi-lineage capacity of differentiation when induced in basic adipogenic, chondrogenic and osteogenic media elements. Characterization of tri-lineage cellular potency was conducted using Oil Red, Alcian Blue and Alizarin Red staining solutions to indicate adipogenesis, chondrogenesis and osteogenesis, respectively, and imaged at lOx on an inverted phase filtered epifluorescent Zeiss Axio X10 microscope.
  • Figures 15A and 15B show that MDSc populations maintained stem cell expansion foci which possessed the intrinsic capacity to form corticocancellous bone while within a closed, 2D in vitro culture system.
  • Figure 15A shows plating and growth/differentiation characteristics of adipose-derived stem cell (ADSC), bone marrow-derived mesenchymal stem cell (BM-MSC) and MDSc populations surgically harvested from C57BL/6 murine tissues and cultured separately on type- 1 collagen coated plates in MSC basal media conditions. Adherent stem cell expansion focal aggregates were quantified and measured at 24 and 48 hours. ADSC, BM-MSC and MDSc populations all displayed mitogenic activity within the aggregates and subsequent migration of cells away from the centralized foci.
  • ADSC adipose-derived stem cell
  • BM-MSC bone marrow-derived mesenchymal stem cell
  • MDSc populations surgically harvested from C57BL/6 murine tissues and cultured separately on type- 1 collagen coated plates in MSC basal
  • MDSc populations also displayed forms of multi-cellular structures which organized into non-random linear bridges (black arrows) which joined the stem cell proliferative focal aggregates and permitted other cells to adhere to and migrate vectorally along the cell-based scaffold.
  • Figure 15B shows plating and growth/differentiation characteristics of ADSC, BM-MSC and MDSc populations surgically harvested from C57BL/6 murine tissues and cultured separately on type-1 collagen-coated plates in osteoinductive media conditions. Alizarin Red staining solution was used to indicate osteogenic differentiation.
  • ADSC populations exhibited a typical dispersed form of micro bone aggregates
  • BM-MSC populations demonstrated satellite forms of micro bone aggregates surrounding larger centralized ossified foci.
  • the MDSc population was capable of forming non-random corticocancellous ultrastructures, which readily bound Alizarin Red staining solution.
  • Cortical/dense bone is indicted by a black pentagon.
  • Cancellous/trabecular bone elements are indicated by the black, dot-ended arrows. Images were collected at lOx on an inverted phase filtered epifluorescent Zeiss Axio X10 microscope.
  • Figures 16A to 16C show that MDSc populations were capable of generating 3D organized bone on deployable implant constructs, for delivery into in vivo systems.
  • Figure 16A shows results obtained when 2 x 10 6 MDScs were seeded onto 1 cm x 1 cm type-1 collagen constructs and cultured under osteoinductive media conditions.
  • stem cell focal aggregates were observed that were capable of multi-dimensional organization, with MDSc structures growing directly away from the collagen and into the media (white arrow), acquiring a form of intrinsic rigidity.
  • the MDSc-derived structures were capable of spanning the aqueous media environment to adhere to and join separate collagen constructs (black arrow) while also binding the plastic culture vessel (black ball tip arrow).
  • FIG. 16B shows results utilizing tissues harvested from C57BL/6-Tg(CAG-EGFP)10sb cells, which intrinsically express eGFP.
  • 2 x 10 6 ADSCs eGFP , BM-MSCs eGFP or MDScs eGFP were separately seeded onto 1 cm x 1 cm type-1 collagen constructs and cultured under osteoinductive media conditions for 7 days in order to compare population phenotype tendencies through induction while in a 3D culture arrangement, as well as deliverability into living systems.
  • FIG. 16C shows that full- thickness 5 mm diameter defects were created in the left parietal skulls of C57BL/6 mice using a powered hollow-bore drill bit, and 5 mm collagen constructs containing 2x 10 6 ADSCs eGFP , BM-MSCs eGFP or MDScs eGFP were implanted within the void. Cellular eGFP emission was monitored and compared with acellular control constructs. Bio-fluorescent image of mouse depicted an example of the left parietal defect containing a MDScs eGFP construct, while the right side of the dotted line contains a simple acellular construct at 1 week post implant.
  • Figures 17 A and 17B show that osseous defects that received MDSc biologic implants were capable of regenerating vascularized corticocancellous bone with structure comparable to native architecture, including the cranial diploic space found within living systems.
  • Figure 17A shows results for 5mm defects of the C57BL/6 mice which received either implant control or implants containing 2x 10 6 of ADSCs eGFP , BM-MSCs eGFP or MDScs eGFP and were assessed for relative healing, bone formation and vascular ingrowth at 8 weeks.
  • Implant controls contained non-rigid and sunken soft scar tissue over the defect (white pentagon).
  • ADSCs eGFP -containing implants developed fibrotic rims around the defect with particles of osseous aggregate intermixed with scar tissue.
  • BM-MSCs eGFP also developed fibrotic rims around the defect with larger particles of osseous aggregates intermixed within scar tissue; however, the population also notably developed a higher grade blood vessel ingrowth which resulted in more hemorrhagic tissues.
  • MDScs eGFP populations showed significantly less fibrotic material at the rim of the defect and significantly more bone regeneration.
  • the bone developed within the MDScs eGFP had more organized blood vessel formation and a less irregularity at the construct-native bone interface.
  • Figure 17B shows that laser scanning multi-photon confocal microscopy of in vivo constructs prior to explant revealed significant vascular ingrowth (white arrows) of vessels into construct still containing green fluorescent MDScs eGFP .
  • Corticocancellous ultrastructure was also appreciated in defects which received MDScs eGFP while no other implants provided similar cellular organization.
  • Cancellous bone is shown as the white open arrow, while cortical bone region is indicated by a white bracket.
  • Z scanning of the construct within the skull exhibited a diploic space (dual headed arrows) comparable to native bone tissues in only those defects receiving MDScs eGFP -enriched constructs.
  • the formation of diploic space and relative bone formation was further investigated and validated using a miniCT scan imaging system.
  • the white arrow indicates a lower cortical plate of diploic space on coronal cross-section and correlative location on the sagittal slice.
  • Figure 18 shows that a relative reduction of the fibroblast population led to an increase in CD34-expressing cellular aggregates. Specifically, the results of pre-plating of muscle- derived cellular suspension following 72 hours of contact is shown. Labels: vimentin/red, CD34/green and DNA/blue. PP1, PP2 and PP3 showed progressive reduction in fibroblasts expressing vimentin, while inversely increasing the relative number of CD34-expressing cells and CD34 cellular aggregates. PP3 (solid white arrows) showed radial outgrowth from CD34-expressing aggregates (white hollow arrows).
  • Figures 19A and 19B show that osteogenic induction of MDScs resulted in the formation of bone ultrastructure.
  • Figures 20A and 20B show results for treatment of MDScs with collagen + bmp2 only, where blue staining in the middle of the image is DAPI (sub-optimal).
  • the right side image shows angiogenesis into a bone construct, where actin is red, while also showing GFP staining (from stem cells), which indicated that true vasculogenesis was occurring.
  • These data showed the muscle-forming properties of these compositions, for generating vascularized muscle and/or cardiac cells with myotubes, projected as therapeutically useful for conditions of muscle degeneration, whether peripheral or cardiac.
  • Figure 21 shows explant confocal imaging of the cranium, which demonstrated localized angiogenesis and vasculogenesis following administration of implants containing MDScs and rhBMP2.
  • Figures 22A and 22B show that increased concentrations of VEGF enhanced MDSc migration.
  • Figure 22A shows a wound healing assay of GFP-expressing MDScs treated with various concentrations of VEGF (0 - 250ng/ml). Wounds were created via removal of ibidi culture inserts when cells reached confluence. Migration was assessed every 6 hours, and yellow outline indicated the wound edge.
  • Figure 22B shows results for quantification of wound closure. Percent reduction from initial wound area was calculated at each time point using ImageJ. 50 and 250 ng/ml of VEGF led to significantly higher migration rates at 6 and 12 hours, as compared to the control group. Each point refers to mean + SEM. Two-way ANOVA was used to evaluate statistical differences among groups (* indicates p ⁇ 0.05).
  • Figures 23A and 23B show the upregulation of endothelial marker expression in MDScs following VEGF stimulation.
  • Figure 23B shows quantification of the percent of cells that exhibited positive CD31 expression. VEGF stimulation of MDScs led to a significantly higher percent of CD31 expressing cells.
  • Figures 24A and 24B show that VEGF stimulation of MDSc promoted capillary tube formation.
  • Figure 24A shows fluorescent images of capillary tube formation.
  • HUVECs were used as a positive control.
  • Figure 24B shows quantification of capillary tube formation.
  • VEGF-stimulated MDScs exhibited a significantly greater number of branches/mm 2 , junctions/mm 2 , and total length, as compared to control MDScs.
  • a student's t-test was performed for statistical analysis (* indicated p ⁇ 0.05).
  • Figures 25A and 25B show that MDSc myogenesis was unimpaired following VEGF stimulation.
  • Figure 25B shows quantification of myotube formation. Fusion index was calculated as [(# myotube nuclei/# of total nuclei) x 100]. No significant differences were noted between control MDSc and MDSc stimulated with various concentrations of VEGF. Two-way ANOVA was performed for statistical analysis.
  • Figure 26 shows compression moduli of regenerated bones as compared with normal bone and collagen showing that both Bone2M5B (MDSC with BMP Group) and Bone-Collagen (Collagen-only group) were stronger than normal bones. Bone 346.9+-33.6 kPa, Bone2M5B 606.4 kPa, Bone-Collagen 512.9 kPa, and Collagen 1 kPa.
  • the present invention relates, at least in part, to the observation that a combined system comprising muscle derived cells (MDCs, e.g., muscle derived stem cells (MDScs)), bone induction agent(s) and a scaffold(s) produced both remarkable localized bone healing and angiogenic/vasculogenic effects when the combined system was employed to contact a region of bone defect in a mammalian subject.
  • MDCs muscle derived cells
  • MDScs muscle derived stem cells
  • the invention relates to harnessing heterotropic ossification by employing the newly-identified bone healing system described herein.
  • the capacity of an MDSc population to form operatively useful and deployable bone substrate in vitro for translational regenerative medicine applications and the utility of such an implant to perform within an in vivo model is disclosed herein.
  • VCA vascularized composite allo-transplantation
  • BM-MSC bone marrow-derived mesenchymal stem cell
  • ADSC adipose-derived stem cell
  • the muscle-derived stem cell (MDSc) employed herein which is a precursor to the well-defined satellite cell (committed to the regeneration of skeletal muscle), is distinct in that it may not be restricted to the development of myogenic or even mesenchymal tissue lineages (Jankowski RJ, et al., Gene Ther. 2002 May;9(10):642-7, Cooper RN, et al., Curr Opin Pharmacol. 2006 Jun;6(3):295- 300, Wada MR, et al., Development. 2002 Jun;129(12):2987-95, Usas A, et al., Biomaterials. 2007 Dec;28(36):5401-6, Deasy BM, et al., Blood Cells Mol Dis.
  • the system of the instant invention includes muscle-derived cells (MDCs).
  • the MDCs of the invention include early progenitor cells (also termed muscle- derived progenitor cells or muscle-derived stem cells (MDScs) herein) that show long-term survival rates following transplantation into body tissues, e.g., soft tissues and/or bone, either alone or as a component of the system described herein.
  • MDCs of this invention a muscle explant, preferably skeletal muscle, is obtained from an animal donor, preferably from a mammal, including rats, dogs and humans. This explant serves as a structural and functional syncytium including "rests" of muscle precursor cells (T. A.
  • Cells isolated from primary muscle tissue contain a mixture of fibroblasts, myoblasts, adipocytes, hematopoietic, and muscle-derived stem cells (MDScs).
  • the stem cells of a muscle-derived population can be enriched using differential adherence characteristics of primary muscle cells on collagen coated tissue flasks, such as described in U.S. Pat. No. 6,866,842 of Chancellor et al. Cells that are slow to adhere tend to be morphologically round, express high levels of desmin, and have the ability to fuse and differentiate into multinucleated myotubes (U.S. Pat. No. 6,866,842 of Chancellor et al.).
  • a preplating procedure can be used to differentiate rapidly adhering cells from slowly adhering cells (MDCs).
  • MDCs slowly adhering cells
  • the preplating technique can be used to differentiate populations of isolated muscle-derived cells based on their adhesion
  • populations of rapidly adhering cells PP1-4, or, in certain instances, only PP1, optionally including in certain embodiments PP2
  • intermediate adhering populations e.g., PP2 and/or PP3
  • slowly adhering, round MDCs PP6
  • the PP6 cells expressed myogenic markers, including desmin, MyoD, and Myogenin.
  • the PP6 cells also expressed c-met and MNF, two genes which are expressed at an early stage of myogenesis (J. B. Miller et al., 1999, Curr. Top. Dev. Biol. 43:191-219).
  • the PP6 showed a lower percentage of cells expressing M-cadherin, a satellite cell-specific marker (A. Irintchev et al., 1994, Development Dynamics 199:326-337), but a higher percentage of cells expressing Bcl-2, a marker limited to cells in the early stages of myogenesis (J. A. Dominov et al., 1998, J. Cell Biol.
  • the PP6 cells also expressed CD34, a marker identified with human hematopoietic stem cells, as well as stromal cell precursors in bone marrow (R. G. Andrews et al., 1986, Blood 67:842-845; C. I. Civin et al., 1984, J. Immunol. 133: 157-165; L. Fina et al, 1990, Blood 75:2417-2426; P. J. Simmons et al, 1991, Blood 78:2848-2853).
  • the PP6 cells also expressed Flk-1, a mouse homologue of human KDR gene which was recently identified as a marker of hematopoietic cells with stem cell-like characteristics (B. L.
  • the PP6 cells expressed Sca-1, a marker present in hematopoietic cells with stem cell-like characteristics (M. van de Rijn et al., 1989, Proc. Natl Acad. Sci. USA 86:4634-8; M. Osawa et al., 1996, J. Immunol. 156:3207-14).
  • the PP6 cells did not express the CD45 or c-Kit hematopoietic stem cell markers (reviewed in L K. Ashman, 1999, Int. J. Biochem. Cell. Biol. 31 : 1037-51; G. A. Koretzky, 1993, FASEB J. 7:420-426).
  • PP6 cells can be isolated by previously described techniques (see, e.g., US Patent No. 8,105,834) to obtain a population of muscle-derived stem cells that have long-term survivability following transplantation.
  • the PP6 muscle-derived stem cell population comprises a significant percentage of cells that express stem cell markers such as desmin, CD34, and Bcl-2,
  • stem cell markers such as desmin, CD34, and Bcl-2
  • PP6 cells express the Flk- 1 and Sca- 1 cell markers, but do not express the CD45 or c-Kit markers.
  • greater than 95% of the PP6 cells express the desmin, Sca-1, and Flk-1 markers, but do not express the CD45 or c-Kit markers.
  • the PP6 cells are utilized within about 1 day or about 24 hours after the last plating.
  • adhering populations of preplate 2 and preplate 3 (PP2 and PP3) (alterna ively, such fractions are also termed herein the "intermediate adherence population” or “intermediate adherence cells”) were isolated and enriched from muscle explants, and tested for the expression of various markers.
  • the "muscle -derived aggregate colonies” (MDAC) resulting from the preplating technique at PP2 and PP3, for example, can exhibit characteristic profiles presenting different morphology, marker profiles and possessing superior regenerative capabilities, as compared with rapidly adhering cells (or fast adhering cell populations), or also as compared with slow-adhering cells.
  • the plates can be further replated (and, in some embodiments, the replating can optionally be repeated several times).
  • Such intermediate adherence cells were also identified as characterized by CD markers (e.g., Sca-1, CD29, CD105, CD73 and CD34 expression and low CD3 I, CD56, Cdl44, and CD146 expression).
  • Intermediate adherence cells can be isolated by previously described techniques ⁇ see, e.g., US Patent No. 8, 105,834) to obtain a population of muscle-derived stem cells that possess long-term survivability following transplantation.
  • the intermediate adherence muscle-derived stem cell population comprises a significant percentage of cells that express stem cell markers such as desmin, CD34, and Bcl-2.
  • Intermediate adherence cells can be utilized within about 1 day - i.e., about 24 hours after the last plating.
  • the rapidly adhering cells, the intermediate adherence cells and slowly adhering cells are separated from each other using a single plating technique.
  • cells are provided from a skeletal muscle biopsy. The biopsy need only contain about 100 mg of cells.
  • Biopsies ranging in size from about 50 mg to about 500 mg can be used according to both pre-plating and single plating methods of obtaining and expanding MDCs (e.g., MDScs). Further biopsies of 50, 100, 110, 120, 130, 140, 150, 200, 250, 300, 400 and 500 mg can be used according to both pre- plating and single plating methods.
  • the tissue from the biopsy is then stored for 1 to 7 days.
  • This storage is at a temperature from about room temperature to about 4°C.
  • This waiting period causes the biopsied skeletal muscle tissue to undergo stress. While this stress is not necessary for the isolation of MDCs using a single plate technique, using a wait period can result in a greater yield of MDCs.
  • Tissue from the biopsies is minced and centrifuged.
  • the pellet is resuspended and digested using a digestion enzyme.
  • Enzymes that may be used include collagenase, dispase or combinations of these enzymes. After digestion, the enzyme is washed off of the cells.
  • the cells are transferred to a flask in culture media for the isolation of the rapidly adhering cells.
  • Many culture media may be used.
  • Exemplary culture-media include those that are designed for culture of endothelial cells including Cambrex Endothelial Growth Medium. This medium may be supplemented with other components including fetal bovine serum, IGF- 1 , bFGF, VEGF, EGF, hydrocortisone, heparin, and/or ascorbic acid.
  • Other media that may be used in a single plating technique include InCell M310F medium. This medium may be
  • the step for isolation of the rapidly adhering cells may require culture in flask for a period of time from about 30 to about 120 minutes.
  • the rapidly adhering cells adhere to the flask in 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 minutes. After they adhere, the slowly adhering cells are separated from the rapidly adhering cells by removing the culture media from the flask to which the rapidly adhering cells are attached.
  • the culture medium removed from this flask is then transferred to a second flask.
  • the cells may be centrifuged and resuspended in culture medium before being transferred to the second flask.
  • the cells are cultured in this second flask for between 1 and 3 days.
  • the cells are cultured for two days.
  • the slowly adhering cells (MDCs) adhere to the flask.
  • the culture media is removed and new culture media is added so that the MDCs can be expanded in number.
  • the MDCs may be expanded in number by culturing them for from about 10 to about 20 days.
  • the MDCs may be expanded in number by culturing them for 1.0, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.
  • the MDCs are subject to expansion culture for 17 days.
  • the MDCs of the present invention can be isolated by fluorescence- activated cell sorting (FACS) analysis using labeled antibodies against one or more of the cell surface markers expressed by the MDCs (C. Webster et al., 1988, Exp. Cell. Res. 174:252-65; J. R. Blanton et al., 1999, Muscle Nerve 22:43-50).
  • FACS fluorescence- activated cell sorting
  • FACS analysis can be performed using labeled antibodies directed to CD34, Flk- 1, Sca- 1, and/or other cell-surface markers to select a population of PI T>- like cells that exhibit long-term survivability when introduced into a host tissue.
  • fluorescence-detection labels for example, fluorescein or rhodamine, can also be used for antibody detection of different cell marker proteins.
  • MDCs that are to be transported, or are not going to be used for a period of time may be preserved using methods known in the art. More specifically, the isolated MDCs may be frozen to a temperature ranging from about -25 to about -90°C. Optionally, the MDCs are frozen at about -80°C, on dry ice for delayed use or transport. The freezing may be done with any cryopreservation medium known in the art.
  • the MDCs are isolated from a skeletal muscle source and, using the MDC-bone induction agent-scaffold system of the invention, introduced or transplanted into bone structures or into a muscle or non-muscle soft tissue site of interest.
  • the MDCs of the present invention are isolated and enriched to contain a large number of stem cells showing long-term survival following transplantation.
  • the muscle-derived stem cells used in the invention can express a number of characteristic cell markers, such desmin, CD34, and Bcl-2.
  • the muscle-derived stem cells of the invention express the Sca-1, and Flk-1 cell markers, but do not express the CD45 or c-Kit cell markers.
  • MDCs and compositions comprising MDCs of the present invention can be used to repair, treat, or ameliorate various aesthetic or functional conditions (e.g. defects) through the augmentation of muscle, non- muscle soft tissues and/or bone.
  • various aesthetic or functional conditions e.g. defects
  • compositions can be used for the treatment of bone defects and/or injury and/or muscle or soft tissue weakness, disease, injury, or dysfunction.
  • MDCs and compositions thereof can be used for promoting angiogenesis and/or producing vasculogenesis in the location at which a system of the invention is contacted to a subject, tissue, bone and/or organ.
  • angiogenesis-promoting and/or vasculogenesis-producing applications of the instant invention include joint repair (e.g., use in joint repair surgeries (e.g., ACL repair) and/or treatment of osteoarthritic joints), rebuilding of muscle cells and/or use in cardiac surgery, e.g., via placement at locations of coronary artery blockage and/or regions of damage.
  • the systems of the invention can also be used to augment soft tissue not associated with injury by adding bulk to a soft tissue area, opening, depression, or void in the absence of disease or trauma, such as for "smoothing".
  • Multiple and successive applications of MDCs/MDScs within the scaffold -bone induction agent system of the invention are also contemplated, as are use of the system in combination with other therapeutic agents, and/or use of the MDCs MDScs of the invention not only as an agent for angiogenesis promotion, vasculogenesis production and/or repair of local damage via promotion of growth of functionally polarized bone and/or cartilage within areas of critical size bone defects (defects of such size that they would not heal spontaneously within the lifetime of a subject), but also as a possible vector for gene therapy via, e.g., lentiviral transformation of MDCs/MDScs with constructs comprising therapeutic genetic agents, as has been described previously in the art.
  • a skeletal muscle explant is optionally obtained from an autologous or heterologous human or animal source.
  • An autologous animal or human source is advantageous as the least likely to produce a deleterious immune reaction upon
  • MDC compositions are then prepared and isolated as described herein or as known in the art.
  • a suspension of mononucleated muscle cells can optionally first be prepared.
  • Such suspensions contain concentrations of the muscle -derived stem cells of the invention in a physiologically - acceptable carrier, excipient, or diluent.
  • suspensions of MDCs for administering to a subject can comprise 10 8 to 10 9 cells/ml in a sterile solution of complete medium modified to contain the subject's serum, as an alternative to fetal bovine seram.
  • MDC suspensions can be in serum-free, sterile solutions, such as cryopreservation solutions (Celox Laboratories, St. Paul, Minn.).
  • the MDC suspensions can then be introduced into the scaffold that is introduced into a subject, with additional scaffold details described below.
  • the described cells can be administered as a pharmaceutically or physiologically acceptable preparation or composition containing a physiologically acceptable carrier, excipient, or diluent, used to contact and/or impregnate the scaffold of the system of the invention, and then used to contact the tissues (e.g., bone and/or soft tissues or muscles) of the recipient organism of interest, including humans and non-human animals.
  • the MDC- containing component of the system composition can be prepared by resuspending the cells in a suitable liquid or solution such as sterile physiological saline or other physiologically acceptable aqueous liquids, suitable for use in a scaffold system that is introduced into a subject for therapeutic purpose.
  • a suitable liquid or solution such as sterile physiological saline or other physiologically acceptable aqueous liquids, suitable for use in a scaffold system that is introduced into a subject for therapeutic purpose.
  • suitable liquid or solution such as sterile physiological saline or other physiologically acceptable aqueous liquids
  • the MDCs or compositions thereof can be administered by placement of the MDC suspensions onto absorbent or adherent material, i.e., a collagen sponge matrix, and insertion of the MDC-containing material into or onto the site of interest.
  • the MDC/MDSc systems of the instant invention also include a bone inducing agent, and it is the combination of all three components of the system, i.e., (1) MDC/MDSc, (2) bone inducing agent (e.g., BMP2) and (3) scaffold (e.g., collagen I scaffold/disk) that have herein been identified to produce surprising bone healing and angiogenesis/vasculogenesis results, thereby distinguishing the MDC/MDSc-collagen matrix compositions of the instant invention from previously contemplated MDC-collagen matrix compositions.
  • administration of the MDCs 4- bone inducing agent within a scaffold can be mediated by endoscopic surgery.
  • MDC solutions or suspensions of MDCs can be prepared during preparation of the system compositions of the invention.
  • Such MDC solutions or suspensions can include pharmaceutically- and physiologically-acceptable aqueous or oleaginous vehicles, which may contain preservatives, stabilizers, and material for rendering the solution or suspension isotonic with body fluids (i.e. blood) of the recipient.
  • excipients suitable for use include water, phosphate buffered saline, pH 7.4, 0.15 M aqueous sodium chloride solution, dextrose, glycerol, dilute ethanol, and the like, and mixtures thereof.
  • Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids, which may be sed either on their own or as admixtures.
  • the amounts or quantities, as well as the sites targeted, are determined on an individual basis, and correspond to the amounts used in similar types of applications or indications known to those of skill in the art.
  • immunosuppressive or immunomodulatory therapy can be started before, during, and/or after the transplant procedure.
  • cyclosporin A or other immunosuppressive drugs can be administered to the transplant recipient.
  • Immunological tolerance may also be induced prior to transplantation by alternative methods known in the ait (D. J. Watt et al., 1984, Clin. Exp. Immunol. 55:419; D. Faustman eL al., 1991 , Science 252: 1701).
  • the MDC-bone induction agent-scaffold system of the invention can be administered to body tissues, including not only bone tissue but also epithelial tissue (i.e., skin, lumen, etc.) muscle tissue (i.e. smooth muscle), and various organ or joint tissues.
  • body tissues including not only bone tissue but also epithelial tissue (i.e., skin, lumen, etc.) muscle tissue (i.e. smooth muscle), and various organ or joint tissues.
  • the number of cells in an MDC/MDSc suspension/composition and the mode of administration may vary depending on the site and condition being treated.
  • about 2xi0 6 MDCs are used within a collagen disk/collagen scaffold of an exemplary system of the invention for the treatment of a critical size bone defect of a subject.
  • a skilled practitioner can modulate the amounts and methods of MDC-bone inducing agent-scaffold system treatments according to requirements, limitations, and/or optimizations determined for each case.
  • the MDCs and compositions thereof according to the present invention have utility as treatments for conditions of the lumen and/or of voids or defects in an animal or mammal subject, including humans.
  • the muscle-derived stem cells are used for completely or partially blocking, enhancing, enlarging, sealing, repairing, bulking, or filling various biological voids within the body.
  • Voids may include, without limitation, various tissue wounds (i.e., loss of muscle and soft tissue bulk due to trauma; destruction of soft tissue due to penetrating projectiles such as a stab wound or bullet wound; loss of soft tissue from disease or tissue death due to surgical removal of the tissue including loss of breast tissue following a mastectomy for breast cancer or loss of muscle tissue following surgery to treat sarcoma, etc.), lesions, fissures, diverticulae, cysts, fistulae, aneurysms, and other undesirable or unwanted depressions or openings that may exist within the body of an animal or mammal, including humans.
  • tissue wounds i.e., loss of muscle and soft tissue bulk due to trauma; destruction of soft tissue due to penetrating projectiles such as a stab wound or bullet wound; loss of soft tissue from disease or tissue death due to surgical removal of the tissue including loss of breast tissue following a mastectomy for breast cancer or loss of muscle tissue following surgery to treat sarcoma, etc.
  • lesions
  • the MDCs are prepared in association with a bone induction agent and a scaffold as disclosed herein and then localized via transplant to the lumenal tissue to fill or repair the void.
  • the number of MDCs introduced is modulated to repair large or small voids in a soft tissue environment or in bone, cartilage or other tissue, as required.
  • Remarkable to the instant invention is the surprising efficacy of the currently described system to repair critical size tissue defects, e.g., critical size bone defects (those too large to heal
  • the MDCs and compositions thereof can be used to affect contractility and/or repair muscle tissues, including, e.g., smooth muscle tissue.
  • the present invention also embraces the use of the MDC system of the invention in restoring muscle contraction, and/or ameliorating or overcoming smooth muscle contractility problems.
  • the MDCs of the invention may be genetically engineered by a variety of molecular techniques and methods known to those having skill in the art, for example, transfection, infection, or transduction.
  • Transduction as used herein commonly refers to cells that have been genetically engineered to contain a foreign or heterologous gene via the introduction of a viral or non- viral vector into the cells.
  • Transfection more commonly refers to cells that have been genetically engineered to contain a foreign gene harbored in a piasmid, or non- viral vector.
  • MDCs can be transfected or transduced by different vectors and thus can serve as gene delivery vehicles to transfer the expressed products into muscle, bone or other treated tissues (further augmenting the therapeutic role of MDCs/MDScs of the instant system of the invention.
  • Viral vectors can be sed; however, those having skill in the art will appreciate that the genetic engineering of cells to contain nucleic acid sequences encoding desired proteins or polypeptides, cytokines, and the like, may be carried out by methods known in the art, for example, as described in U.S. Pat. No.
  • MDCs/MDScs include fusion, transfection, lipofection mediated by the use of liposomes, electroporation, precipitation with DEAE-Dextran or calcium phosphate, particle bombardment (biolistics) with nucleic acid- coated particles (e.g., gold particles), microinjection, and the like.
  • Exemplary dosing of MDCs/MDScs within the system of the invention includes, e.g., use of about 10 3 to about 10 8 cells per enr 1 of tissue to be treated, optionally about 10 J to 10 7 cells per cm 3 of tissue to be treated, within the scaffold/bone inducing agent composition of the system of the invention.
  • Cell count and viability for MDCs and/or enriched MDScs can be measured using a Guava flow cytometer and Viacount assay kit (Guava).
  • Cellular markers can be measured by flow cytometry (Guava) and conjugated anti-marker antibodies. Fluorescent labeling can be performed using conjugated anti-mouse IgG antibodies and other methods.
  • Myoblasts the precursors of muscle fibers, are mononucleated muscle cells which differ in many ways from other types of cells. Myoblasts naturally fuse to form post-mitotic multinucleated myotubes which result in the long-term expression and delivery of bioactive proteins (T. A. Partridge and K. E. Davies, 1995, Brit. Med. Bulletin, 51: 123- 137; J. Dhawan et al, 1992, Science, 254: 1509-1512; A. D. Grmnefi, 1994, In: Myology. Ed 2, Ed. Engel A G and Armstrong C F, McGraw-Hill, Inc, 303-304; S. Jiao and J. A. Wolff, 1992, Brain Research, 575: 143-147; H. Vandenburgh, 1996, Human Gene Therapy, 7:2195-2200).
  • Myoblasts have been used for gene delivery to muscle for muscle -related diseases, such as Duchenne muscular dystrophy (E. Gussoni et al, 1992, Nature, 356:435-438; J. Huard et al, 1992, Muscle & Nerve, 15:550-560; G. Karpati et al., 1993, Ann. Neurol., 34:8- 17; J. P. Tremblay et al., 1993, Cell Transplantation, 2:99-112), as well as for non -muscle-related diseases, e.g., gene delivery of human adenosine deaminase for the adenosine deaminase deficiency syndrome (C. M.
  • myoblasts to treat muscle degeneration, to repair tissue damage or treat disease is disclosed in U.S. Pat. Nos. 5,130,141 and 5,538,722. Also, myoblasts
  • muscle-derived cells may be primary cells, cultured cells, or cloned. They may be histocompatible (autologous) or nonhistocornpatible (allogeneic) to the recipient, including humans.
  • the MDCs are myoblasts and muscle-derived stem cells, optionally autologous myoblasts and muscle-derived stem cells which will not be recognized as foreign to the recipient, in this regard, the MDCs/myoblasts/MDScs used for the compositions of the invention will desirably be matched vis-a-vis the major histocompatibility locus (MHC or HLA in humans).
  • MHC or HLA-matched cells may be autologous.
  • the cells may be from a person having the same or a similar MHC or HLA antigen profile.
  • the patient may also be tolerized to the allogeneic MHC antigens.
  • the present invention also encompasses the use of cells lacking MHC Class I and/or II antigens, such as described in U.S. Pat. No. 5,538,722.
  • muscle-derived cells including MDScs
  • MDScs muscle-derived cells
  • Exemplary bone induction agents of the invention include bone morphogenetic proteins, particularly BMP2, BMP4 and BMP9, though also including BMPl , BMP3, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10 and/or BMP15.
  • BMP2 bone morphogenetic proteins
  • BMP4 bone morphogenetic proteins
  • BMP9 bone morphogenetic proteins
  • cytokines/growth factors are also contemplated for inclusion within the systems of the invention.
  • a human BMP2 preprotein sequence of accession number NP 001191.1 is:
  • BMP2 and/or rhBMP2 are used in an amount sufficient to osteo-induce MDC and/or MDSc cells.
  • use of active regions and/or fragments of the BMP2 polypeptide is also contemplated, as is use of variant and/or mutated forms of the BMP2 or rhBMP2 polypeptide, provided that osteo-induction of contacted MDC and/or MDSc cells is maintained.
  • contemplated bone induction agents of the present invention include compounds such as cholesterol derivatives (e.g., oxysterol), and certain polypeptide growth factors, such as, osteogenin, Insulin-like Growth Factor (IGF)-l, IGF-II, TGF- ⁇ , ⁇ - ⁇ 2, TGF-P3, ⁇ - ⁇ 4, TGF- 5, osteoinductive factor (OIF), basic Fibroblast Growth Factor (bFGF), acidic Fibroblast Growth Factor (aFGF), Platelet-Derived Growth Factor (PDGF), vascular endothelial growth factor (VEGF), Growth Hormone (GH), growth and
  • IGF Insulin-like Growth Factor
  • IGF-l Insulin-like Growth Factor
  • IGF-II Insulin-like Growth Factor
  • TGF- ⁇ fibroblast growth Factor
  • aFGF acidic Fibroblast Growth Factor
  • PDGF Platelet-Derived Growth Factor
  • VEGF vascular endothelial growth factor
  • GDF differentiation factors-5 through 9, as well as proteins including, osteopontin, osteonectin, osterix, and Runx-2.
  • Exemplary scaffolds of the invention include collagen scaffolds, e.g., a collagen I scaffold, such as a collagen I disk.
  • Collagen is commercially available, and exemplary methods for synthesis of a collagen scaffold (e.g., a collagen I sponge) are both known in the art and/or described herein.
  • a collagen scaffold is advantageous for use in the systems of the invention in supporting/promoting functionally polarized bone cell growth.
  • other forms of scaffold are alternatively or additionally contemplated for use in the instant invention, including, e.g., matrigel, hydrogel, gelatin sponge, calcium phosphate and/or calcium hydroxyapatite scaffolds, among other art-recognized scaffold materials. Combinations of different scaffold materials within a single scaffold are also contemplated.
  • exemplary scaffolds contemplated for the invention include, for example, ceramic scaffolds (e.g., hydroxyapatite and tri-calcium phosphate), synthetic polymers (e.g., poly-l-lactic acid (PLLA), polyglycolic acid (PGA), and poly-dl-lactic-co-glycolic acid (PLGA)), decellularized tissue and/or decalcified bone, allograft bone, biological materials (e.g., proteoglycans, alginate -based substrates, and chitosan), and a hybrid composition using any or all of the prior listed scaffold materials, among other art-recognized scaffold and/or biocompatible support compositions.
  • ceramic scaffolds e.g., hydroxyapatite and tri-calcium phosphate
  • synthetic polymers e.g., poly-l-lactic acid (PLLA), polyglycolic acid (PGA), and poly-dl-lactic-co-glycolic acid (PLGA)
  • a scaffold refers to a solid support capable of containing and/or otherwise supporting cells and compounds of the invention.
  • a scaffold of the invention can comprise, e.g, collagen, gelatin, matrigel, hydrogel, calcium phosphate, calcium hydroxyapatite, etc., as well as combinations thereof.
  • a "critical size bone defect” is a bone defect (e.g., void, fracture, etc.) that will not heal spontaneously within the lifetime of a subject).
  • Sizes of bone and/or tissue defects, voids, fractures, etc. contemplated as treatable with the compositions of the invention include defects, voids, fractures, etc. of less than 2 mm in any relevant dimension, at least 1 mm in a relevant dimension (optionally, diameter), at least 2 mm in a relevant dimension, at least 3 mm in a relevant dimension, at least 4 mm in a relevant dimension, at least 5 mm in a relevant dimension, at least 6 mm in a relevant dimension, at least 7 mm in a relevant dimension, at least 8 mm in a relevant dimension, at least 9 mm in a relevant dimension, at least 10 mm in a relevant dimension, at least 20 mm in a relevant dimension, at least 30 mm in a relevant dimension, at least 40 mm in a relevant dimension, at least 50 mm or more in a relevant dimension; 10 cm or less in a relevant dimension, 9 cm or less in a relevant dimension, 8 cm or less in a relevant dimension, 7 cm or less in a relevant
  • BMP bone morphogenic protein
  • TGF- ⁇ superfamily polypeptide growth factors belonging to the TGF- ⁇ superfamily.
  • BMPs are widely expressed in many tissues, though many function, at least in part, by influencing the formation, maintenance, structure or remodeling of bone or other calcified tissues.
  • Members of the BMP family are potentially useful as therapeutics.
  • BMP-2 has been shown in clinical studies to be of use in the treatment of a variety of bone-related conditions.
  • bone induction agent and "osteogenic” as used herein refers to a material that stimulates growth of new bone tissue.
  • preplate refers to a technique used to isolate cells (e.g., stem cells or progenitor cells) from skeletal muscle, based on the ability of such cells to adhere to collagen-coated tissue flasks.
  • the preplating technique can be used to differentiate rapidly adhering cells from slowly adhering cells, or to identify an "intermediate adherence population" (alternatively referred to as an “intermediate adhering cell population”), based on differentiable surface markers and/or stem-like properties.
  • the preplate technique involves culturing digested muscle tissue for a set period of time to allow the fibroblastic cell fraction to attach, while transferring the supernatant containing the myogenic fraction into a new plate, thereby enriching for the desired cells (Gharaibeh, B. et al. Nature Protocols 3: 1501- 1509, incorporated herein by reference).
  • intermediate adherence population or “muscle-derived aggregate colonies (MDACs)” as used herein refers to a population of cells, for example, of preplate 2 or preplate 3 (PP2 or PP3), which are characterized by their "intermediate” adhesion characteristics (thus adhering at the PP2 or PP3 stage, rather than at, e.g., PP1 ("rapidly” or “early” adhering cells) or PP6 ("slowly” or “late” adhering cells) and expression of CD markers, including, for example, Sca-1, CD29, Cdl05, CD73, CD31, and CD34 expression, and low CD45, CD56, CD144, and CD146 expression.
  • the MDAC populations resulting from the preplating technique can have characteristic profiles that exhibit different morphology, marker profiles and possess superior regenerative capabilities, as compared with rapidly adhering cells or fast adhering cell populations, or as compared with slowly adhering cells or late adhering populations. If the MDAC population contains a high amount of fibroblast- like cells (i.e., large, flat cells versus small, refractive cells), the plates can be replated, effectively allowing for propagation of this select cell/aggregate population.
  • rapidly adhering cells or “fast adhering cell populations,” as used herein refers to the first cells to adhere during the early stages of the preplating technique (i.e., within minutes to hours of seeding, preplate 1 or, in certain embodiments, preplate 2 (PP1 or in certain embodiments PP2, respectively)).
  • these rapidly adhering cells may be comprised of mostly fibroblastic-like and myoblast cells.
  • slow adhering population refers to a population of cells, for example, of preplate 4, preplate 5, or preplate 6 (PP4, PP5, or PP6), that are characterized by their delayed adhesion characteristics during the preplating process, and expression of CD markers.
  • an effective amount includes an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., sufficient to produce significant reduction and/or healing of a bone defect and/or promote angiogenesis/produce
  • An effective amount of a system composition of the invention may vary according to factors such as the disease/injury state, age, and weight of the subject, and the ability of the MDC-bone inducing agent-scaffold composition to elicit a desired response in the subject. Administration regimens may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (e.g., side effects) of a system composition of the invention are outweighed by the therapeutically beneficial effects.
  • an effective amount of BMP2 within a composition of the invention can also be used in reference to, e.g., a bone inducing agent of the invention, e.g., an effective amount of BMP2 within a composition of the invention might be determined either in advance or empirically and might be confirmed via assessment of a phenotypic output/endpoint of administration of a system composition of the invention (e.g., the extent of bone healing and/or angiogenesis and/or vascularization observed in the subject administered the system of the invention).
  • a phenotypic output/endpoint of administration of a system composition of the invention e.g., the extent of bone healing and/or angiogenesis and/or vascularization observed in the subject administered the system of the invention.
  • “Ameliorate,” “amelioration,” “improvement” or the like refers to, for example, a detectable improvement or a detectable change consistent with improvement that occurs in a subject or in at least a minority of subjects, e.g., in at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% or in a range between any two of these values.
  • Such improvement or change may be observed in treated subjects as compared to subjects not treated with a scaffold-bone induction agent-MDC composition of the invention, where the untreated subjects have, or are subject to developing, the same or similar injury/condition, disease, symptom or the like.
  • Amelioration of an injury/condition, disease, symptom or assay parameter may be determined subjectively or objectively, e.g., via self-assessment by a subject(s), by a clinician's assessment or by conducting an appropriate assay or measurement, including, e.g., a quality of life assessment, a slowed progression of a disease(s) or condition(s), a reduced severity of a disease(s) or condition(s), or a suitable assay(s) for the level or activity(ies) of a biomolecule(s), cell(s), by detection of respiratory or inflammatory disorders in a subject, and/or by modalities such as, but not limited to photographs, video, digital imaging and pulmonary function tests.
  • Amelioration may be transient, prolonged or permanent, or it may be variable at relevant times during or after a scaffold-bone induction agent-MDC composition is applied to a subject or is used in an assay or other method described herein or a cited reference, e.g., within timeframes described infra, or about 12 hours to 24 or 48 hours after the contacting or use of a scaffold-bone induction agent-MDC composition to about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3, 6, 9 months or more after a subject(s) has received such treatment.
  • the "modulation" of, e.g., a symptom, level or biological activity of a molecule, or the like refers, for example, to the symptom or activity, or the like that is detectably increased or decreased. Such increase or decrease may be observed in treated subjects as compared to subjects not treated with a MDC-bone inducing agent-scaffold composition, where the untreated subjects have, or are subject to developing, the same or similar disease, condition, symptom or the like.
  • Such increases or decreases may be at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000% or more or within any range between any two of these values.
  • Modulation may be determined subjectively or objectively, e.g., by the subject's self-assessment, by a clinician's assessment or by conducting an appropriate assay or measurement, including, e.g., quality of life assessments, suitable assays for the level or activity of molecules, cells or cell migration within a subject and/or by modalities such as, but not limited to photographs, video, digital imaging and pulmonary function tests.
  • Modulation may be transient, prolonged or permanent or it may be variable at relevant times during or after a MDC-bone inducing agent-scaffold composition is administered to a subject or is used in an assay or other method described herein or a cited reference, e.g., within times described infra, or about 12 hours to 24 or 48 hours after the contacting or use of a scaffold- bone induction agent-MDC composition to about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3, 6, 9 months or more after a subject(s) has received such treatment.
  • fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
  • polypeptide and the terms “protein” and “peptide” which are used interchangeably herein, refers to a polymer of amino acids.
  • exemplary polypeptides include gene products, naturally-occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of the foregoing.
  • subject includes organisms which are capable of suffering from a defect, injury, disease and/or disorder treatable by a MDC-bone inducing agent-scaffold composition or who could otherwise benefit from the administration of a MDC-bone inducing agent-scaffold composition as described herein, such as human and non-human animals.
  • Preferred human animals include human subjects.
  • non-human animals includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non- mammals, such as non-human primates, e.g., sheep, dog, cow, chickens, amphibians, reptiles, etc.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • Various methodologies of the instant invention include a step that involves comparing a value, level, feature, characteristic, property, etc. to a "suitable control", referred to interchangeably herein as an "appropriate control".
  • a "suitable control” or “appropriate control” is a control or standard familiar to one of ordinary skill in the art useful for comparison purposes.
  • a "suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined prior to performing a methodology of the instant invention, as described herein. For example, a level or manner of healing of a critical size bone defect typical to an untreated or control-treated subject can be determined prior to or concurrent with contacting a composition of the invention to a tissue or organism/subject.
  • a "suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits.
  • a "suitable control” or “appropriate control” is a predefined value, level, feature, characteristic, property, etc.
  • sequences or subsequences refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • percent identity may be measured using sequence comparison software or algorithms or by visual inspection.
  • sequence comparison software or algorithms or by visual inspection.
  • Various algorithms and software are known in the art that may be used to obtain alignments of amino acid or nucleotide sequences.
  • One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al, Proc. Natl. Acad.
  • Gapped BLAST may be used as described in Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997).
  • BLAST-2 Altschul et al, Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences.
  • the percent identity between two nucleotide sequences is determined using the GAP program in GCG software (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6).
  • the GAP program in the GCG software package which incorporates the algorithm of Needleman and Wunsch (/.
  • Mol. Biol. (48):444-453 (1970)) may be used to determine the percent identity between two amino acid sequences (e.g., using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5).
  • the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)).
  • the percent identity may be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4.
  • Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art.
  • the default parameters of the alignment software are used.
  • the percentage identity "X" of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y/Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence.
  • whether any particular polynucleotide has a certain percentage sequence identity can, in certain embodiments, be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482 489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. It will be recognized in the art that some amino acid sequences of the invention can be varied without significant effect of the structure or function of the protein. Such mutants include deletions, insertions, inversions, repeats, and type substitutions.
  • polypeptides and analogs can be further modified to contain additional chemical moieties not normally part of the protein.
  • Those derivatized moieties can improve the solubility, the biological half-life or absorption of the protein.
  • the moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).
  • the isolated polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthetic methods to constructing a DNA sequence encoding isolated polypeptide sequences and expressing those sequences in a suitable transformed host.
  • a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest (BMP2).
  • BMP2 wild-type protein of interest
  • the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81 :5662-5066 (1984) and U.S. Pat. No. 4,588,585.
  • the data obtained from cell culture assays and animal studies of the MDC-bone inducing agent-scaffold system of the invention can be used in formulating a range of dosage/relative concentrations of components of a scaffold-bone induction agent-MDC composition for use in humans.
  • the dosages of MDC/MDSc and/or BMP2 ranges to include within the scaffold system compositions of the invention can be determined based upon efficacy and/or toxicity profiling, as would be known within the art.
  • Therapeutically effective amounts of bone inducing agent are contemplated to include, e.g., in some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 ⁇ g, or 10, 30, 100, or 1000 mg, which may be included within a system composition of the invention.
  • a therapeutically effective amount of a compound of the compositions of the present invention can be determined by methods known in the art.
  • kits may include a muscle-derived cell (MDC) population of the invention, optionally also including a bone induction agent (e.g., BMP2, rhBMP2 or fragment thereof, or other induction agent as set forth elsewhere herein or known in the art), a scaffold (e.g., collagen I), and instructions for its use.
  • MDC muscle-derived cell
  • the muscle-derived cell population is optionally contacted with a bone induction agent and applied to a scaffold to form a MDC-scaffold composition.
  • the MDC-scaffold composition is then optionally contacted to a bone defect.
  • the MDC population optionally combined and/or packaged with a bone induction agent and/or scaffold, can be packaged in a suitable container.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a bone injury and/or a cardiac or other vascular injury, disease or disorder.
  • Treatment or “treating” as used herein, is defined as the application or
  • a system of the invention e.g., a MDSc + BMP2 + collagen disk composition as described herein
  • administration of a system of the invention to a patient, or application or administration of such a therapeutic agent to an isolated tissue or cell line from a patient who has the injury, disease and/or disorder and/or application or administration of such a therapeutic agent to an organ or tissue grown in vitro that is optionally derived from a patient who has the injury, disease and/or disorder (or, where an isolated tissue, cell line and/or organ or tissue grown in vitro is used, optionally from a subject not having the injury, e.g., for transfer to isolated tissue, cell or supernatant to a patient having the injury, disease or disorder) for a symptom of the injury, disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the injury, disease or disorder and/or the symptoms of the injury, disease or disorder.
  • a system of the invention e.g., a MDSc
  • compositions of the invention are provided as exemplary, specifically contemplated therapeutic uses of the compositions of the invention, and is not intended to be limiting in any manner.
  • Intramuscular bone formation is a poorly understood phenomenon. It can be present in the clinically pathologic states of heterotopic ossification, myositis ossificans,
  • BMPs bone morphogenetic proteins
  • TGF- ⁇ transforming growth factor ⁇
  • the MDC-bone induction agent (i.e., BMP2)-scaffold compositions of the invention were also identified as remarkably proficient in both promoting angiogenesis (inducing blood vessel growth in a treated tissue) and performing vasculogenesis (e.g., where the
  • MDCs/MDScs of a composition of the invention actually grow new blood vessels).
  • Use of such compositions to repair regions of cardiac damage e.g., application of the compositions of the invention to regions of the heart that have incurred damage related to, coronary artery disease, myocardial infarction, etc., is therefore contemplated as providing a new and effective therapeutic.
  • Degenerative and traumatic joint disorders are encountered frequently as the population becomes more active and lives longer. These disorders include arthritis of various etiologies, ligament disruptions, meniscal tears, and osteochondral injuries.
  • the clinician's tools consist primarily of surgical procedures aimed at biomechanically altering the joint, such as anterior cruciate ligament (ACL) reconstructions, total knee replacement, meniscal repair or excision, cartilage debridement, etc.
  • ACL anterior cruciate ligament
  • Intraarticular administration of the MDC-bone induction agent (i.e., BMP2)-scaffold compositions of the invention are contemplated for treatment of such conditions, noting both the remarkable efficacy of such compositions in promoting angiogenesis and performing vasculogenesis, as well as the potential for such compositions to repair cartilage
  • the ACL is the second most frequently injured knee ligament.
  • the ACL has a low healing capacity, in part because of the lack of blood flow to the knee joint and possibiy secondary to its encompassing synovial sheath or the surrounding synovial fluid.
  • complete tears of the ACL are incapable of spontaneous healing, current treatment options are limited to surgical reconstruction using autograft or allograft.
  • the replacement graft often either patella ligament or hamstring tendon in origin, undergoes ligamentizalion with eventual collagen remodeling (S. P. Arnosczky et al, 1982, Am. J. Sports Med., 10:90- 95). Therefore, augmentation of this ligamentization process using the compositions of the invention presents an additional and compelling therapeutic application of the compositions of the invention.
  • stem cell therapies While access to surgical applications utilizing stem cell therapies (where the stem cells are immediately available) increase the options for practicing real-time regenerative medicine, those employing such therapies will also need to remain aware of complexities surrounding "the unknown" that is harbored within deployable potent cell populations.
  • skeletal muscle-derived cells possessed a subgroup of MDScs which were capable of multi-lineage differentiation, including, but not limited to myogenic, adipogenic, chrondrogenic and osteogenic forms.
  • MDScs multi-lineage differentiation
  • BMP- 2 appears to have acted as a mitogen on MDSc populations, inducing the aggregation of proliferative focal expansion centers where cells divide and migrate radially, while a simple collagen scaffold provided the minimal necessary extracellular niche to eventually form a complex tissue that resembled functionally polarized bone.
  • MDSc populations containing progenitor/stem cell aggregates (optionally of a specific pre-plate fraction) in combination with other necessary entities (collagen scaffold and BMP-2) underwent early fate decisions that ultimately allowed for formation of a basic ex-vivo bone construct.
  • Such a construct was then delivered to a critical bone defect and remarkably underwent further propagation toward true organized bone formation and regenerative healing.
  • the simplicity of this autogenous MDSc-derived construct allowed for a streamlined harvest, processing and implantation into bone defects that required enhanced healing and/or neo-genesis of vascularized bone and provides a composition and means of overcoming the previously mentioned limitations in tissue regeneration.
  • the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed.
  • Enrichment and isolation of MDCs was performed by the following method, adapted from, e.g., U.S. Pat. No. 6,866,842 of Chancellor et al.
  • Muscle explants were obtained from the hind limbs of mice. Mice were sacrificed and sprayed with 70% ethanol to completely saturate the lower body. Skin/connective tissue was then removed from muscle, beginning at the ankle and moving up the leg. Muscle explants were placed in sterile PBS on ice while remaining mice were dissected (though explants were performed on no more than two mice at a time).
  • Tissue explants were washed with HBSS in a 50 mL conical tube to remove any fur that continued to be associated with the explants. The explant tissue was then transferred to a sterile tissue culture dish and a few drops of sterile PBS were added (to keep tissue moist). Tissue was minced with fine, sharp sterile scissors. Mincing proceeded for a few minutes, until tissue was a slurry. 1-2 mL of a collagenase/dispase/CaCl 2 solution (freshly mixed 50 ⁇ ⁇ CaCl 2 + 10 mL dispase + 10 mL collagenase, comprising 2.5 mM CaCl 2 ) were then added (2 mL per gram of tissue).
  • Mincing was continued for several minutes more, and minced tissue was then transferred to a sterile 15 mL tube. The tube was incubated on a shaker for 20 min at 37 °C. The suspension was then centrifuged for 5 min at 350 x g, pelleting cells. Supernatant was removed, and the cell pellet was re-suspended in 5 mL proliferative media (PM: DMEM, supplemented with 10% (vol/vol) FBS (Fetal Bovine Serum), 10% (vol/vol) HS (Horse Serum), 0.5% (vol/vol) CEE (Chick Embryo Extract) and 100 U ml_l penicillin/streptomycin, sterile-filtered by passing through 0.22 micron filter).
  • PM proliferative media
  • MDScs obtained by this process were then expanded in the following manner: the adherent cells were washed with 3 ml of PBS, 2 ml pre-warmed 0.1% (wt/vol) trypsin-EDTA solution was added, and the mixture was incubated at 37 °C for 2-3 min and subsequently examined under the microscope to ensure that all of the cells were detached. The reaction was stopped by adding 3 ml of PM, and the mixture was centrifuged at 930 x g at 4 °C for 5 min. Count the cells in the pellet were counted and replated at a density of 225-250 cells/cm 2 .
  • MDSc were harvested from murine skeletal muscle from the hind limb of C57BL/6 or C57BL/6-Tg(CAG-EGFP)10sb, depending on the need for fluorescent emission under aspetic conditions and were minced using sterile forceps and scalpel and further processed for culture on collagen coated plates as previously described by Lavasani M, et al., Methods Mol Biol. 976: 53-65. Identity of the MDSc population was characterized by flow cytometry directed at the following CD markers: Sca-1, CD29, CD105, CD73 and CD34 expression and low CD31, CD 56, CD 144 and CD 146. Additionally, immunofluorescent imaging was used to validate the finds in flow cytometry using the following CD markers and Alkaline
  • Cells undergoing in vitro tri-lineage differentiation were treated with the StemPro® Adipogenesis, Chondrogenesis or Osteogenesis Differentiation Kits (Life Technologies) per the manufacture's provided manual and protocols.
  • Cells utilized for collagen implant studies were seeded onto 1 cm x 1 cm x 0.2 cm type-1 collagen scaffolds under the same basal MSC proliferative media conditions as those plated on collagen coated culture dishes.
  • Experimental osteogenic induction studies performed on 2D and 3D cultured systems employed basal MSC proliferative media supplemented with 1.0 ⁇ g/ml of Bone Morphogenetic Protein-2 Human Recombinant ProSpecTM Bio Protein-Specialists.
  • ADSCs were harvested using previously published methods by Lough DM, et al., Plast Reconstr Surg. 2014 Mar;133(3):579-90. Briefly mice were shaved, cleansed with 70% ethanol and HBSS. Circumferential full thickness skin was harvested and underlying subcutaneous inguinal fat pads were collected. Fat was washed 3x in HBSS containing Penicillin/Streptomycin (1%) for 5 minutes each and then SVF isolation was carried out following the Pittsburg Protocol.
  • the bone marrow MSC were then harvested. Following dissection of skeletal muscle from disarticulated lower limb for MDSc isolation, a 30-gauge needle on a 10 cc syringe containing BMPM was used to flush the bone marrow cells from both ends of the bone shafts into a 50 ml conical top tube fitted with a 100 ⁇ filter (BD Falcon). Further cellular isolation utilized a previously published methods by Soleimani M and Nadri S. Nat Protoc. 2009;4(1): 102-6.
  • Example 3 Animal model and studies
  • CNT Carbon nanotube
  • Additional explant imaging studies utilized a Zeiss 510 Meta laser scanning microscope to determine vascular ingrowth of implants and relative quantity and location of ADSC eGFP , BM-MSC eGFP or MDSc eGFP populations within Z planar stack cross sections.
  • Example 4 Type-1 collagen and Bone Morphogenic Protein-2 (BMP2) augmented the rate of polarized bone formation from cellular expansion foci
  • Example 5 MDScs were viable throughout the healing process and mediated bone regeneration
  • Example 6 Defects treated with collagen scaffold only, and increasing BMP doses did not heal, whereas treatment with MDScs mediated more normal and robust bone formation
  • Example 7 MDSc-BMP2-Collagen I Scaffold Compositions Effected Functionally Polarized Bone Repair, Promoted Angiogenesis and Performed Vasculogenesis in Treated Mice
  • the BMP2 + collagen I scaffold MDScs were confirmed to have produced cell cycle progression, as well as enhanced multiplication and proliferation of treated cells, as compared to non-treated cells ( Figures. 6A to 6C). Notably, increasing doses of BMP2 in such- compositions showed progressively increased cell cycle progression response ( Figures. 6B and 6C).
  • MDScs were demonstrated as capable of the following forms of differentiation: reversion to muscle cells (if left untreated, data not shown), adipogenesis, chondrogenesis and osteogenesis ( Figure 7B).
  • MDScs employed in the invention were multipotent.
  • Polarized bone formation via use of the compositions of the invention was further documented. Specifically, the combination of BMP2 and type 1 collagen was further established to have acted as a mitogen in MDSc enriched populations, leading to polarized bone formation.
  • the real-time proliferation index and volume index, as well as mitosis and cell cycle progression of MDSc enriched populations plated at 10 6 over 18 hours was found to increase upon treatment with both BMP2 and Collagen I scaffold, with results obtained using live confocal imaging ( Figures 9A to 9E).
  • MDSc were applied to a spectrum of engineered scaffolds for traceable, real-time in vivo studies within defect models (Fig. 10).
  • MDSc-BMP2-collagen I scaffold composition treatments were specifically observed to have dramatically augmented polarized bone healing and diploic space formation in a treated subject, with in vivo cortical and cancellous bone formation observed following delivery of such MDSc seeded scaffolds at week three in an explant.
  • Murine eGFP expressing MDSc (2xl0 6 ) seeded onto BMP2 bound Col-1 (collagen I) scaffolds produced a polarized bone construct (cortical and cancellous bone architecture) within 14-21 days following implantation into 5 mm diameter full-thickness skull defects (Figure 12A).
  • a Z- stack through the nascent diploic space (which was remarkably re-established in cranial defects upon treatment with the exemplary compsitions of the invention) was observed for explanted MDSc collagen scaffold at eight weeks.
  • the collagen I scaffold-MDSc and BMP2 compositions of the invention achieve remarkable repair of bone defects, in a functionally polarized manner consistent with native bone morphology, the collagen I scaffold-MDSc and BMP2 compositions of the invention also both promoted angiogenesis and produced localized vasculogenesis in vivo (Figure 13).
  • compositions of the invention can be broadly and advantageously applied to a number of conditions, diseases and/or disorders, to achieve positive therapeutic outcomes.
  • Example 8 MDScs generated myogenic cellular entities while maintaining a multi- lineage potency comparable to other MSC populations
  • Skeletal muscle has remained a primary source of satellite and progenitor cells, which are primarily responsible for muscle regeneration following injury. Recently, however, muscle tissue has also been identified as a valuable source of adult MDScs, which appear to be distinct from satellite cells in that they intrinsically possess the ability to undergo multi- lineage differentiation (Usas A, et al., Biomaterials. 2007 Dec;28(36):5401-6, Deasy BM, et al., Blood Cells Mol Dis. 2001 Sep-Oct;27(5):924-33, and Cao B, et al., Nat Cell Biol. 2003 Jul;5(7):640-6).
  • MDScs possess a high myogenic capacity, which has been shown to effectively regenerate both skeletal and cardiac muscle, this unique population can also undergo adipogenic, chondrogenic and osteogenic differentiation (Asakura A, et al., Differentiation. 2001 Oct;68 (4-5):245-53, and Wada MR, et al., Development. 2002 Jun;129(12):2987-95).
  • multinucleated linear precursor cells were less evident in muscle-derived cell suspensions that were cultured in basal MSC proliferative media, confirming an apparent myogenic signal induction requirement for myocyte formation.
  • Example 9 MDScs generated self-organizing corticocancellous bone ultrastructure while expanding in culture
  • ADSC ADSC, BM-MSC and MDSc expansion foci in the presence of a biologic binding surface
  • ADSC, BM-MSC and MDSc groups were isolated and plated at 2 x 10 6 cells/mL on collagen-coated plates cultured in basal MSC proliferative media, and additionally supplemented with 1.0 ⁇ g/ml of BMP-2.
  • the cultures were prepared and stained with Alizarin Red solution to determine relative bone formation and generalized architecture of the cellular material generated (Figure 2).
  • ADSC replicates exhibited a typical dispersed form of micro-aggregate bone deposition
  • BM-MSC culture replicates demonstrated satellite forms of similar micro- aggregates surrounding larger centralized ossified foci.
  • the MDSc groups were capable of forming organized corticocancellous bone, which readily bound Alizarin Red staining solution throughout the resultant osseous ultrastructure.
  • Example 10 MDScs generated 3D organized bone on deploy able biologic implants for delivery into living systems
  • the MDSc population that readily formed corticocancellous bone structure in a 2D system was then evaluated for whether it could undergo similar self-assembling osteogenic differentiation in a 3D system, resulting in a bone construct capable of implantation into living systems.
  • ADSC, BM-MSC and MDSc groups were harvested and seeded at 2 x 10 6 cells/mL upon 3D collagen scaffolds and cultured in basal MSC proliferative media, supplemented with 1.0 ⁇ g/ml of BMP-2. Groups were monitored over 14 days to compare the above 2D culture findings to cellular activities in a 3D system.
  • an MDSc-enriched biologic scaffold was examined for possible function as a form of autologous bio-reactive implant similar to an autograft, within a well-established cranial defect model.
  • ADSC eGFP , BM-MSC eGFP and MDSc eGFP replicates all displayed similar structural arrangements as those seen in the above 2D and 3D systems for non-fluorescent cell types over the 14 days.
  • Intermediate day 7 was provided as an exemplary depiction of the cell-enriched scaffolds (Figure 16B).
  • ADSC eGFP , BM-MSC eGFP and MDSc eGFP were capable of viably seeding collagen-based scaffolds while maintaining the ability to undergo osteogenesis and surgical implantation into a living cranial defect system.
  • MDScs seeded onto a 3D collagen scaffold unlike ADSC and BM-MSC groups, appeared uniquely capable of self- assembly into a 3D tissue strongly resembling corticocancellous bone through apparent scaffold outgrowth, anchoring and multi-substrate spanning behaviors.
  • Example 11 Enriched biologic implants were capable of regenerating vascularized corticocancellous bone and even diploic space in a living system
  • an MDSc-enriched implant to regenerate 3D corticocancellous in vivo over an 8 week time period within the established cranial defect model was comparatively analyzed.
  • cranial defects that received either implant control(s) or cellular-enriched implants (2x 10 6 of ADSCs eGFP , BM-MSCs eGFP or MDScs eGFP ) were imaged and harvested, to assess relative healing indices, bone formation and vascular ingrowth ( Figures 17A and 17B).
  • ADSC eGFP -enriched implants developed fibrotic rims around the wound with particulate bone aggregates intermixed with scar tissue (white pentagons)( Figure 17A).
  • Defects administered implants containing BM-MSCs eGFP also developed fibrotic rims around the defect, with larger aggregates intermixed within thicker scar tissue.
  • BM- MSCs eGFP also notably developed a higher grade of blood vessel ingrowth, resulting in hemorrhagic tissues (white pentagon).

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Abstract

L'invention concerne un système de cicatrisation osseuse, d'accélération de l'angiogenèse et/ou de réalisation de la vasculogenèse et des procédés d'utilisation associés, qui emploient une stratégie de cicatrisation régénérative à trois principes unique de cellules dérivées du muscle (par exemple, cellules souches dérivées du muscle (MDSc) dépourvues de fibroblastes), un agent d'induction osseuse (par exemple, comprenant l'expansion ex vivode cellules progénitrices à ostéoinduction précoce), et un support d'échafaudage structuralement robuste (par exemple, un échafaudage de collagène) pour réaliser la régénération osseuse, l'accélération de l'angiogenèse et/ou les performances de vasculogenèse, ce qui permet de surmonter les limitations antérieures de la cicatrisation régénérative.
PCT/US2016/027729 2015-04-18 2016-04-15 Système de cicatrisation osseuse, d'accélération de l'angiogenèse et de production de vasculogenèse Ceased WO2016172004A1 (fr)

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CN111629765A (zh) * 2018-01-26 2020-09-04 两极组织工程公司 复合界面生物材料促进剂基质
EP3743080A4 (fr) * 2018-01-26 2021-10-27 Polarityte, Inc. Matériaux d'auto-assemblage coordinés à une interface vivante complexe (clicsam)

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