WO2012159216A1 - Bone tissue regenerating peptides - Google Patents

Abstract

The present invention provides a recombinant or synthetic fusion polypeptide comprising a peptide component with bone tissue renerating activity, a linker peptide and an antimicrobial competence stimulating peptide with bone tissue renerating activity. The fusion polypeptide or antimicrobial competence stimulating peptide, which enhances osteogenic cell binding, is useful for repairing dental bony defects such as those caused by bone loss resulting from moderate to severe periodontitis, augmenting of bony defects of the alveolar ridge, filling tooth extraction sites, or sinus elevation grafting. The repair material includes a porous, resorbable particulate that is bone derived or derived from bone-like hydroxyapatite or synthetic hydroxylapatite and/or a resorbable carrier such as high molecular weight polysaccharides. Furthermore, the fusion polypeptide or antimicrobial competence stimulating peptide alone of this invention is useful for treatment of fractures, as filler in deficient sites of bone, for inhibition of decrease in bone substance related to osteoporosis, and also for prevention of fractures associated with osteoporosis and rheumatoid arthritis. The fusion polypeptide or antimicrobial competence stimulating peptide can be combined with a bone-compatible matrix to facilitate slow release of the peptide to a treatment site and/or provide a structure for developing bone.

Description

BONE TISSUE REGENERATING PEPTIDES

FIELD OF THE INVENTION This application claims priority to and the benefit of United States Patent Application

Serial Number 61/489,947 filed May 25, 2011, the subject matter of which is incorporated herein by reference.

The present invention relates to biologically active peptides, and more particularly relates to a composition comprising an antimicrobial competence stimulating peptide having bone tissue regenerating activity and a bone regenerating peptide. The present invention also includes methods of stimulating bone growth comprising administering a composition comprising an antimicrobial competence stimulating peptide peptide having bone tissue regenerating activity and a bone regenerating peptide.

BACKGROUND OF THE INVENTION

In the repair of a dental bone defect such as periodontal bone loss, a treatment may include application of a composition or formulation to the defect site to enhance repair and bone healing. The composition typically includes: (i) a particulate material to provide structural support and filling of the defect; (ii) compounds or medicaments to enhance repair of bone; and (iii) a carrier system to facilitate delivery to and retention of the composition at the defect site for the duration of the treatment.

Where it is desired to generate new bone to repair a defect and where immediate and continued structural support is not a limiting factor, regeneration of bone by natural body mechanisms is most desirable. The natural repair and regeneration process has long been thought to be enhanced by filling the defect with various bone derived or bone-related synthetic particulates. Of the useful bone particulates, autologous derived material, while effective and safe, is generally of impractical availability. Allogenic material is readily available and, alternatively, xenogenic bone sources are utilized as well. Synthetic materials, principally hydroxyapatite are also available. The various particulate bone derived materials may include naturally occurring organic components that function to induce and mediate replacement bone growth. However, there are concerns for biocompatibility and safety in allowing organic components to remain in the bone particulate material. Hence, the bone particulate may be treated by sintering process to reduce such risks. Alternatively, the bone particulate source material may be replaced by a completely synthetic hydroxyapatite material that includes no organic residue. The difficulty arising for synthetics is that the resulting material may not resorb or otherwise lacks activity in the remodeling process.

Some researchers have focused upon providing bone or substitute particulates that have porous structures that enhance bone growth or integration. U.S. Patent No. 4,770,860 describes a resorbable porous hydroxyapatite material derived from lime-containing algae by means of a hydrothermal process in the presence of phosphate. Hydroxyapatite material can be provided in the form of a gel obtained by a unique sol-gel process.

Formulations thought to enhance repair of bone tissue may include bone growth agents. Bhatnagar in U. S. Patent No. 5,635,482 described a synthetic collagen-like agent that mimics autogenous cell attachment factors that promote bone growth. Bhatnagar identified and synthesized a fifteen amino acid sequence of Type I collagen that promotes migration of reparative cells from surrounding tissues; directs cell attachment, orients migration, and facilitates a biomimetic environment for bone generation. These and related polypeptide materials, called P-15, are bound to a particulate hydroxyapatite which may be natural, microporous xenogenic bone mineral, such as OsteoGraf^® N-300 manufactured by Dentsply Friadent CeraMed of Lakewood, Colo. In order for the P-15 cell binding irreversible to be active, it must be bound irreversibly to the particulate. Bhatnagar teaches that the resulting dry particulate matrix including P-15, trade marked PepGen P-15^®.

In a typical periodontal surgical bone repair procedure or method, an incision is made in the gum tissue to expose a bone defect adjacent to a tooth root. Once the root and defect is debrided, a bone repair material, such as the aforementioned PepGen P-15^® bone graft material, suspended in a suitable carrier is placed. The gum tissue is closed, maintaining the repair material in place. Optionally, a carrier material may be utilized to retain the repair formulation in contact with the defect. In addition, a bone repair material may contain antimicrobial agents or antimicrobial peptides fused with PepGen P-15 ^E or other osteogenic compounds to prevent infection at the surgical site.

SUMMARY OF THE INVENTION

The present invention includes a bone tissue regenerating composition comprising (a) an antimicrobial competence stimulating peptide with bone tissue regenerating activity and (b) a bone tissue regenerating peptide. An embodiment of the present invention provides a bone tissue regenerating composition comprising (a) an antimicrobial competence stimulating peptide with bone tissue regenerating activity having an amino acid sequence of at least one of SEQ ID NOs: 1 to 15, and (b) a bone tissue regenerating peptide having an amino acid sequence of at least one of SEQ ID NOs: 16 to 23.

The present invention includes a fusion polypeptide where an antimicrobial competence stimulating peptide with bone tissue regenerating activity is fused to a bone tissue regenerating peptide. In an embodiment, a fusion polypeptide comprises (a) an antimicrobial competence stimulating peptide with bone tissue regenerating activity having an amino acid sequence of at least one of SEQ ID NOs: 1 to 15, and (b) a bone tissue regenerating peptide having an amino acid sequence of at least one of SEQ ID NOs: 16 to 23. In an embodiment, an antimicrobial competence stimulating peptide with bone tissue regenerating activity is linked to one terminus of a linker peptide, wherein the other terminus of a linker peptide is linked to a bone tissue regenerating peptide to form a fusion polypeptide. In an embodiment, a peptide linker includes one of SEQ ID NOs: 16 to 23. In another embodiment, a fusion polypeptide with both the bone tissue regenerating and antimicrobial activity is recombinant or synthetic. In another embodiment, the antimicrobial competence stimulating peptide alone has both the antimicrobial and bone tissue regenerating activity.

An embodiment includes an isolated nucleic acid encoding a fusion polypeptide described herein. The nucleic acid can be DNA or RNA. An embodiment of the invention includes a vector comprising DNA encoding a fusion polypeptide of the invention. Another embodiment of the invention includes a host cell comprising the vector comprising DNA encoding a fusion polypeptide of the invention. A further embodiment of the invention provides a method of expressing a recombinant fusion polypeptide of the invention comprising culturing a host cell comprising a nucleic acid encoding a fusion polypeptide of the invention. Yet another embodiment includes a method of expressing a fusion polypeptide of the invention comprising culturing a host cell comprising a vector comprising DNA encoding a fusion polypeptide of the invention.

A further embodiment of the invention provides the method of synthesizing the antimicrobial competence stimulating peptide and fusion polypeptide.

Another embodiment of the invention provides a treatment method for promoting adhesion and proliferation of osteoblasts comprising administering a bone tissue regenerating composition to a patient in need thereof comprising a recombinant or synthetic fusion polypeptide or synthetic antimicrobial competence stimulating peptide alone.

Another embodiment includes a delivery vehicle for the peptide component, which is either fusion polypeptide or antimicrobial competence stimulating peptide alone. A delivery vehicle is preferably a bone-compatible matrix which provides for slow release of peptide component to patient in need of said composition. A bone-compatible matrix can be biodegradable polymer, demineralized bone matrix, ceramic, β-tricalcium phosphates, calcined or sintered bovine bone (hydroxyapatite), an inorganic component of bovine bone, algae-derived hydroxyapatite, synthetic hydroxyapatite, nanocrystalline precipitated hydroxyapatite, or combinations thereof.

In a further embodiment, the invention provides a method of preparing a bone tissue regenerating composition comprising combining a bone -compatible matrix with a fusion polypeptide of the invention or antimicrobial competence stimulating peptide (CSP); and immobilizing the fusion polypeptide to or CSP within the bone compatible matrix.

In a still further embodiment, the composition of the present invention is administered to stimulate bone growth. Such treatments can be administered to subjects in need of bone repair due to bone damage and for tooth implants in reconstructive surgeries.

In a still further embodiment, the present invention relates to the use of a composition comprising a peptide component with both the bone tissue regenerating and antimicrobial activity to control dental plaque-associated bacteria and thus the plaque-associated conditions such dental cavities and periodontal diseases BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is the plasmid construct expressing fusion polypeptide (P15-CSP) comprising an antimicrobial competence stimulating peptide (CSP) and an osteogenic peptide (P-15) with a helical linker peptide and His-tag.

FIG. 2 is the plasmid construct expressing fusion polypeptide (P15-CSP) comprising an antimicrobial competence stimulating peptide (CSP) and an osteogenic peptide (P-15) with a flexible linker peptide and His-tag.

FIG. 3 is the plasmid construct expressing fusion polypeptide (CSP-Osteogenic Peptide OP8) comprising an antimicrobial competence stimulating peptide (CSP) and an osteogenic peptide (OP8) with a helical linker peptide and His-tag.

FIG. 4 is the plasmid construct expressing fusion polypeptide (CSP-Osteogenic Peptide OP8) comprising an antimicrobial competence stimulating peptide (CSP) and an osteogenic peptide (OP8) with a flexible linker peptide and His-tag.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes a bone tissue regenerating composition comprising (a) an antimicrobial competence stimulating peptide with bone tissue regenerating activity and (b) a bone tissue regenerating peptide. An embodiment of the present invention provides a bone tissue regenerating composition comprising (a) an antimicrobial competence stimulating peptide with bone tissue regenerating activity having an amino acid sequence of at least one of SEQ ID NOs: 1 to 15, and (b) a bone tissue regenerating peptide having an amino acid sequence of at least one of SEQ ID NOs: 16 to 23.

An embodiment includes an antimicrobial competence stimulating peptide component of recombinant or synthetic fusion polypeptide selected from a family of peptides with 8 to 21 amino acid residues (SEQ ID NO: 1 to SEQ ID NO: 15), which is summarized in Table 1. Table 1: Antimicrobial Competence Stimulating Peptides (CSP)

With or Without Osteogenic Activity

* CSP analog (one amino acid substituted)

The present invention includes a fusion polypeptide where an antimicrobial competence stimulating peptide with bone tissue regenerating activity is fused to a bone tissue regenerating peptide. In an embodiment, a fusion polypeptide comprises (a) an antimicrobial competence stimulating peptide with bone tissue regenerating activity having an amino acid sequence of at least one of SEQ ID NOs: 1 to 15, and (b) a bone tissue regenerating peptide having an amino acid sequence of at least one of SEQ ID NOs: 16 to 23. In an embodiment, an antimicrobial competence stimulating peptide with bone tissue regenerating activity is linked to one terminus of a linker peptide, wherein the other terminus of a linker peptide is linked to a bone tissue regenerating peptide to form a fusion peptide. In an embodiment, a peptide linker includes one of SEQ ID NOs: 16 to 23.

Another embodiment includes the recombinant or synthetic bone tissue regenerating peptide component of fusion polypeptide selected from a family of peptides that mimic cell binding domain of collagen with 5 to 15 amino acid residues (SEQ ID NO: 16 to SEQ ID NO: 22) and a bone tissue regenerating peptide (SEQ ID NO: 23), which is summarized in Table 2. Table 2: Bone Tissue Regenerating Peptides

In another embodiment, a linker peptide is selected from the group consisting of SEQ ID NO: 24 to SEQ ID NO: 37 as summarized in Table 3 or a combination (a multimer) of any two (dimmer), three (trimer), four (tetramer), five (pentamer) or more than five thereof.

Table 3: Linker Peptides

Another aspect of this invention includes fusion polypeptides comprising (a) an antimicrobial with bone tissue regenerating activity selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 5, (b) a bone tissue regenerating peptide having SEQ ID NO: 16 or 23, and (c) a linker peptide selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 34, and SEQ ID NO: 35. These fusion polypeptides comprising various possible combinations of sequences of selected antimicrobial peptides, bone regenerating peptides and linkers plus polyhistidine peptides (MGHHHHHH (SEP ID 126), MHHHHHH (SEP ID 127), and HHHHHH (SEP ID 128)) are summarized in Tables 4a and 4b.

Table 4a: Fusion Polypeptides

Table 4b: Fusion Polypeptides

Table 4c: Fusion Polypeptides

Table 4d: Fusion Polypeptides

The term "fusion polypeptide" refers to a chimeric polypeptide that comprises an antimicrobial competence stimulating peptide and a bone tissue regenerating peptide. In an embodiment, a bone tissue regenerating peptide is covalently linked or conjugated (e.g., via a peptide bond) to an antimicrobial peptide either at the C-terminal or N-terminal of the targeting peptide. For example, a fusion polypeptide may comprise a bone tissue regenerating peptide with its C-terminal covalently linked to N-terminal of an antimicrobial peptide [Amino terminus- osteogenic peptide-peptide bond-antimicrobial peptide-peptide bond-carboxyl terminus], or an antimicrobial peptide with its C-terminal covalently linked to the N-terminal of a bone tissue regenerating peptide [Amino terminus-antimicrobial peptide-peptide bond-osteogenic peptide- carboxyl terminus].

In one embodiment of the present invention, a fusion polypeptide comprises a peptide linker by which an antimicrobial competence stimulating peptide is covalently linked or conjugated to a bone tissue regenerating peptide. For example, a fusion polypeptide may comprise an antimicrobial peptide with its C-terminal covalently linked to the N-terminal of a linker peptide and a bone tissue regenerating peptide with its N-terminal covalently linked to the C-terminal of the linker peptide (Amino terminus-osteogenic peptide-peptide bond-linker peptide-antimicrobial peptide-peptide bond-carboxyl terminus). In another embodiment, an antimicrobial peptide with its N-terminal covalently linked to the C-terminal of a linker peptide and an osteogenic peptide with its C-terminal covalently linked to the N-terminal of the linker peptide (Amino terminus-antimicrobial peptide-peptide bond-linker peptide-peptide bond- osteogenic peptide-carboxyl terminus).

A fusion polypeptide or antimicrobial competence stimulating peptide with bone tissue regenerating activity may be the sole active ingredient in a bone tissue regenerating composition. The composition may be used for preventing or treating bone fractures. A fusion polypeptide or antimicrobial competence stimulating peptide with bone tissue regenerating activity may also be used as a bone tissue regeneration accelerator obtained by fixing, mixing, dissolving or suspending the peptide in a pharmaceutically acceptable carrier or an aqueous solvent. For example, suitable examples of carriers or aqueous solvents include, but are not limited to, clinical grade sterile water, sterile saline, sterile buffered saline, dextrose in sterile water, sterile liquid media or other physiologically acceptable isotonic liquids. A bone tissue regenerating composition of the present invention can contain a variety of pharmacologically acceptable additives, such as a stabilizer, a preservative, a thickener, a solubilizer and the like, which can be combined with the carrier or aqueous solvent.

Peptides of the present invention can be useful in clinical applications in conjunction with a suitable matrix that acts as a delivery or support system. A successful matrix for a bone tissue regenerating peptide desirably performs several important functions. It desirably binds the bone tissue regenerating peptide and acts as a slow release delivery system, accommodates each step of the cellular response during bone development, and protects a bone tissue regenerating peptide from nonspecific proteolysis. In addition, selected materials should be biocompatible in vivo, porous and preferably biodegradable. In bones, dissolution rates can vary according to whether an implant is placed in cortical or trabecular bone. A matrix also desirably acts as a temporary scaffold until replaced by new bone formation. Therefore, in one embodiment, a bone- compatible matrix provides for slow release of a peptide component to a patient in need of a bone tissue regenerating composition and/or provides a structure for developing bone in the patient.

A matrix is preferably ceramic, biodegradable biopolymer, demineralized bone matrix, β- tricalcium phosphates, calcined and sintered bovine bone (hydro xyapatite), inorganic component of bovine bone, algae-derived hydroxyapatite, synthetic hydroxyapatite, nanocrystalline precipitated hydroxyapatite, or combinations thereof. In one embodiment, a bone-compatible matrix is a woven or non-woven porous structure. In another embodiment, a bone-compatible matrix is a powder, microparticles, microspheres, microfibers, microfibrils, a strip, a gel, a web, a sponge, or combinations thereof.

Suitable ceramics for use as a bone-compatible matrix include, but are not limited to, calcium sulfate, hydroxyapatite, tricalcium phosphate, and combinations thereof. Other ceramics used as artificial bone are also suitable. A ceramic can be in particulate form or can be a structurally stable, three-dimensional implant (e.g., a scaffold). An implant can be, for example, a cube, cylinder, block or an appropriate anatomical form.

A bone-compatible matrix may comprise natural, modified natural or synthetic biodegradable polymers, copolymers, block polymers, or combinations thereof. Examples of suitable biodegradable polymers or polymer classes include fibrin, collagen, elastin, celluloses, gelatin, vitronectin, fibronectin, laminin, reconstituted basement membrane matrices, starches, dextrans, alginates, hyaluron, chitin, chitosan, agarose, polysaccharides, hyaluronic acid, poly(lactic acid), poly(glycolic acid), polyethylene glycol, decellularized tissue, self-assembling peptides, polypeptides, glycosaminoglycans, their derivatives and mixtures thereof. For both glycolic acid and lactic acid, an intermediate cyclic dimer can be prepared and purified, prior to polymerization. Such intermediate dimers are called glycolide and lactide, respectively. Self- assembling peptides are described in U.S. Pat. Nos. 5,670,483 and 5,955,343. Other useful biodegradable polymers or polymer classes include polydioxanones, polycarbonates, polyoxalates, poly(alpha-esters), polyanhydrides, polyacetates, polycaprolactones, poly(orthoesters), polyamino acids, polyamides and mixtures and copolymers thereof. Additional useful biodegradable polymers include, stereopolymers of L- and D-lactic acid, copolymers of bis(p-carboxyphenoxy) propane acid and sebacic acid, sebacic acid copolymers, copolymers of caprolactone, poly(lactic acid)/poly(glycolic acid)/polyethyleneglycol copolymers, copolymers of polyurethane and (poly(lactic acid), copolymers of polyurethane and poly(lactic acid), copolymers of alpha-amino acids, copolymers of alpha-amino acids and caproic acid, copolymers of alpha-benzyl glutamate and polyethylene glycol, copolymers of succinate and poly(glycols), polyp ho sphazene, polyhydroxy-alkanoates, and mixtures thereof. Binary and ternary systems are contemplated.

In one aspect of the invention, a polymer used to form a bone-compatible matrix is a hydrogel. Preferably, a hydrogel is produced from a synthetic polymeric material. Such synthetic polymers can be tailored to a range of properties and predictable lot-to-lot uniformity, and represent a reliable source of material and one generally free from concerns of immunogenicity. In general, hydrogels are polymeric materials that can absorb more than 20% of their weight in water while maintaining a distinct three-dimensional structure. This definition includes dry polymers that will swell in aqueous environments, as well as to water-swollen materials. Many hydrophilic polymers can be cross-linked to produce hydrogels, whether the polymer is of biological origin, semi-synthetic, or wholly synthetic.

In general, a suitable biodegradable polymer for use as a bone-compatible matrix is desirably configured so that it has mechanical properties that match the application, remaining sufficiently intact until bone tissue has in-grown and healed, does not invoke an inflammatory or toxic response, is metabolized in the body after fulfilling its purpose, leaving no trace, is easily processible into the final product formed, demonstrates acceptable shelf-life, and is easily sterilized.

Properties that make hydrogels valuable in drug delivery applications include the equilibrium swelling degree, sorption kinetics, solute permeability, and their in vivo performance characteristics. Permeability to compounds, including the fusion polypeptide, depends in part upon the swelling degree or water content and the rate of biodegradation. Since the mechanical strength of a gel declines in direct proportion to the swelling degree, it is also well within the contemplation of the present invention that a hydrogel can be attached to a substrate so that a composite system enhances mechanical strength. In alternative embodiments, a hydrogel can be impregnated within a porous substrate, such as a ceramic scaffold, so as to gain the mechanical strength of the substrate, along with the useful delivery properties of the hydrogel for the poly peptide.

Factors affecting the mechanical performance of in vivo biodegradable polymers are well known and include monomer selection, initial process conditions, and the presence of additives. Biodegradation can be accomplished by synthesizing polymers that have unstable linkages in the backbone, or linkages that can be safely oxidized or hydrolyzed in the body. The most common chemical functional groups having this characteristic are ethers, esters, anhydrides, orthoesters and amides. Therefore, in one embodiment, a peptide component is controllably released from a biodegradable polymer to a site where it is needed by hydrolysis of chemical bonds in the biodegradable polymer. Biodegradable polymers are preferably in the form of a powder, microparticle, microsphere, strip, gel, web or sponge.

As described above, a bone-compatible matrix can be a demineralized bone matrix (DBM). This is produced by decalcifying cortical bone, and represents a form of allograft processing (Trumees, E. and Herkowitz, H. (1999) Univ. of Penn. Orthop. J. 12:77 88). The resulting matrix is more bone tissue regenerating than ordinary allograft. One commercially available preparation of a demineralized bone matrix gel is Grafton^® gel (Osteotech, Inc., Eatontown, J), which combines DBM with a glycerol carrier.

A matrix medium, vehicle excipient or carrier can be any of those known to be pharmaceutically acceptable for administration to a patient, particularly locally at the site at which new bone growth is to be induced. Examples include liquid media, for example, Dulbeccos Modified Eagles Medium (DMEM), sterile saline, dextrose in sterile water and any other physiologically acceptable isotonic liquid.

In one embodiment, one or more of the peptides of the present invention is immobilized to the bone-compatible matrix. In another embodiment, one or more of the inventive peptides is impregnated or encapsulated within the bone-compatible matrix so as to be immobilized there within. Furthermore, cells which have been genetically engineered to include a nucleic acid sequence encoding a peptide of the present invention can be impregnated or encapsulated within the bone-compatible matrix so as to produce the peptide at the treatment site.

A fusion polypeptide or antimicrobial competence stimulating peptide with bone tissue regenerating activity can be impregnated within a porous bone-compatible matrix. For example, it is contemplated that a fusion polypeptide or antimicrobial competence stimulating peptide may be blended with a fluid material such as an aqueous solvent or a hydrogel to form a mixture which is used to impregnate pores of a porous bone-compatible matrix, such as a ceramic scaffold. Alternatively, it is contemplated that pores of the bone-compatible matrix may first be filled with a fluid material and that air pressure or other suitable means may then be employed to disperse a dry peptide of the invention substantially evenly within the filled pores of the bone- compatible matrix.

In a further embodiment, a fusion polypeptide may be encapsulated in a polymer or a lipid-containing vesicle, such as a liposome, to allow for a controlled release of the peptide to a site where it is needed. For example, a polymeric matrix containing one or more peptides according to the invention may include, without limitation, microparticles, microspheres, microfibers or microfibrils. In one example, a microsphere could be contained within a mesh of a polymeric scaffold or other implant or device for peptide delivery. Microspheres containing a fusion polypeptide may be incorporated within a polymeric scaffold by adhesively positioning them onto a scaffold. Alternatively, microspheres may be mixed with a fluid or gel and allowed to flow into a polymeric matrix of the scaffold. Moreover, microfibers or microfibrils, which may be peptide loaded by extrusion, can be adhesively layered or woven into the polymeric material included in a surface of a scaffold for peptide delivery.

One or more peptides according to the invention can be encapsulated within a liposome. Liposomes are spherical vesicles prepared from either natural or synthetic phospholipids or cholesterol. These vesicles can be composed of either one (unilamellar liposomes) or several (oligo- or multilamallar liposomes) lipid bilayes surrounding internal aqueous volumes. It is known to entrap drugs, proteins and nucleic acids within the internal aqueous space of a liposome. For example, U.S. Pat. No. 5,567,433 discloses a liposome preparation including encapsulated granulocyte-colony stimulating factor (G-CSF), a relatively unstable protein. In addition, U.S. Pat. No. 4,241,046 describes a method for encapsulating an enzyme within a synthetic liposome, the product liposomes being useful for enzyme replacement therapy. Liposomes allow the parenteral administration of the therapeutic agent. On the cellular level, liposomes interact with cell membranes by adsorption, endocytose, membrane fusion, and lipid exchange, or by a combination of these mechanisms as described by Pagano and Weinstein in Ann. Rev. Biophys. Bioeng. (1978) 7:435. Fast elimination of a therapeutic agent and its metabolism can be impeded by shielding the therapeutic agent in a liposome.

One or more of the peptides described herein can be combined with a variety of orthopedic devices, including, but not limited to, bone graft material, replacement knees, hips, joints, pins, rods, plates, crews, fasteners, darts, arrows and staples. There are many methods of immobilizing a peptide to a bone-compatible matrix. It is possible to adopt an immobilization method allowing formation of a covalent bond, ionic bond, hydrophobic bond, hydrogen bond, sulfur-sulfur bond or the like, for example, an immersion, impregnation, spray, application and dropping method with use of a solution containing the peptide. Among these immobilization methods, fixation by covalent bond is preferred due to its stability and continuity of effect. Such fixation can be done by a method usually used for fixing a physiologically active protein such as an enzyme.

For example, in one embodiment, free carboxyl groups on a biocompatible, biodegradable polymer forming the bone-compatible matrix may be chemically cross-linked to a free amino group on the peptide using carbodiimide as a cross-linker agent. Other standard immobilization chemistries are known by those of skill in the art and can be used to join the peptides of the present invention to the bone-compatible matrix. For example, see Protein Immobilization: Fundamentals and Applications Taylor, R. (Ed.) M. Dekker, NY, (1991).

An embodiment includes a therapeutic method comprising administering a composition comprising (a) an antimicrobial competence stimulating peptide with bone tissue regenerating activity and (b) a bone tissue regenerating peptide. An embodiment includes a therapeutic method comprising administering a fusion polypeptide described herein. A composition can be administered topically, systematically, or locally (e.g., as an implant or device). When administered, a therapeutic composition of the invention is in a pyrogen-free, physiologically acceptable form. Further, a composition of the invention may be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than the fusion polypeptide of the current invention, which may also optionally be included in the inventive fusion polypeptide composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the fusion polypeptide composition in the methods of the invention. Dosages of therapeutic compositions described herein can vary as required depending upon the weight of bone desired to be formed, the site of injured bone, the condition of bone, and the age, sex and weight of a patient and the like.

A fusion polypeptide of the invention can also be administered in combination with additional components, such as bone tissue regenerating factors. Bone tissue regenerating factors include, for example, dexamethasone, ascorbic acid-2-phosphate, beta-glycerophosphate and combinations thereof. A composition can also contain antibiotic, antimycotic, antiinflammatory, immunosuppressive and other types of therapeutic, preservative and excipient agents.

Furthermore, a fusion polypeptide of the invention can be administered in combination with a bone tissue regenerating substance such as growth factors, cytokines, hormones, enzymes, enzyme inhibitors, bone matrix components, growth differentiation factors, and combinations thereof.

It is expected that the peptides of the invention may act in concert with other related proteins and growth factors. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), members of the transforming growth factor superfamily of proteins (e.g., TGF-. alpha, and TGF-.beta.), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), bone morphogenic proteins (BMPs), and combinations thereof.

For example, the following molecules have a mitogenic effect and are polypeptides that exhibit heparin- binding affinity: acidic fibroblast growth factor, basic fibroblast growth factor, platelet-derived growth factor, and an insulin- like growth factor II, originally called skeletal growth factor. Moreover, it has been demonstrated that TGF~p₂ is effective in promoting bone mass in several animal models. Furthermore, BMPs are members of the transforming growth factor (TGF) P family. BMP has the function of acting on undifferentiated mesenchymal cells, inducing differentiation to chondroblasts and osteoblasts and effecting chondrogenesis and osteogenesis. Moreover, BMPs are characterized by the presence of several interchain disulfide bonds essential to bioactivity (they exist as a homodimer in their active form) and moderate affinity for heparin.

Bone tissue regenerating peptides disclosed herein will permit the physician to obtain optimal predictable bone formation to correct, for example, acquired and congenital craniofacial and other skeletal or dental anomalies (Glowacki et al, Lancet 317: 959-963, 1981). Devices may be used to induce local endochondral bone formation in non-union fractures as demonstrated in animal tests, and in other clinical applications including dental and periodontal applications where bone formation is required. Another potential clinical application is in cartilage repair, for example, in the treatment of osteoarthritis. Thus, the peptides of the present invention, either alone or in combination with a pharmaceutically acceptable carrier, implant or device can promote treatment of fractures by being administered to patients with fractures caused by rheumatoid arthritis and osteoporosis or by being filled or implanted in a defective site in bone. Also, they can inhibit a decrease in bone substance and prevent fractures by being administered to patients with rheumatoid arthritis, osteoporosis and periodontic diseases.

Mesenchymal stem cell (MSC) therapy can serve as a means to deliver high densities of repair-competent cells to a defect site when adequate numbers of MSC and MSC lineage- specific cells are not present in vivo, especially in older and/or diseased patients. In order to efficiently deliver high densities of MSC to a defect site, methods for rapidly producing large numbers of MSC are necessary. Methods that increase the ex vivo proliferation rate of MSC will greatly increase the utility of MSC therapy. Similarly, methods that increase in vivo proliferation rate of MSC will enhance the utility of MSC therapy by rapidly increasing local concentrations of MSC at the repair site. Furthermore, methods that enhance the proliferation rate of lineage- specific descendants of MSC, including, but not limited to, bone marrow stromal cells, osteoclasts, chondrocytes, and adipocytes, will enhance the therapeutic utility of MSC therapy by increasing the concentration of lineage- specific cell types at appropriate repair sites.

Bone tissue regeneration (i.e., the production of new bone) can occur directly from osteoblasts and osteoprognitor cells. For example, circulating mesenchymal stem cells and osteoinductive growth factors can migrate and adhere to a bone-compatible matrix, such as a ceramic scaffold, in the body. Within the scaffold, progenitor cells can differentiate into functioning osteoblasts. In one embodiment of the present invention, an orthopedic implant or device is provided which includes one or more of the peptides of the present invention, and which also includes osteogenic cells, such as osteoprogenitor stem cells and/or osteoblasts so as to increase the bone tissue regenerating potential associated with bone-graft substituents like ceramic scaffolds. Mesenchymal stem cells are described by Minguell, J., et al, Exp. Biol. Med. 226; 507-520, 2001 and Fibbe, W. Ann Rheum Dis 61 (Suppl II): ii29- ii31, 2002) These cells can be incorporated into an implant or device prior to, during, or following implantation. The implant or device may further incorporate other bone tissue regenerating substances, such as those described herein.

Therapeutic compositions of the invention may also be used for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with peptides of the present invention. Peptides described herein may be prepared by methods known in the art. Such methods include synthesizing a fusion polypeptide or a single peptide chemically from individual amino acids or synthesizing a nucleic acid encoding the fusion polypeptide and using the nucleic acid to produce recombinant fusion polypeptide ex vivo or in vivo.

Fusion polypeptides of the invention and nucleic acids encoding the fusion polypeptides may be chemically synthesized by methods known in the art. Suitable methods for synthesizing the peptide are described by Stuart and Young (1984), "Solid Phase Peptide Synthesis," Solid Phase Peptide Synthesis, Methods Enzymol, Second Edition, Pierce Chemical Company, 289, Academic Press, Inc., NY (1997). For example, a solid phase synthesis method or a liquid phase synthesis method may be used. The solid phase synthesis is usually carried out by protecting amino groups with appropriate protecting groups. For example, either Boc (tert-butoxycarbonyl) or Fmoc (9-fluorenylmethyloxycarbonyl), or a combination thereof may be used.

Reverse phase liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, partition chromatography, counter current distribution or other similar techniques can be utilized to purify an obtained peptide. Either or both of the N- and C- terminals of peptides of the invention may optionally be modified chemically. For example, an N-terminal may be acetylated and a C-terminal may be amidated.

Nucleic acids encoding peptides of the invention may be replicated. For instance, DNA encoding peptides of the invention can be used to express a recombinant peptide following insertion into a wide variety of host cells in a wide variety of cloning and expression vectors. A host cell may be prokaryotic or eukaryotic. Additionally, nucleic acids may be chemically synthesized. Suitable methods for synthesizing DNA are described by Caruthers in Science (1985) 230:281-285 and DNA Structure, Part A: Synthesis and Physical Analysis of DNA, Lilley. D. and Dahlberg, J. (Eds.). Methods Enzymol, 211, Academic Press, Inc., NY (1992).

Cloning vectors may comprise segments of chromosomal, non-chromosomal and synthetic DNA sequences. Some suitable prokaryotic cloning vectors include plasmids from E. coli, such as colEl, pCRl, pBR322B9, pUC, pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA such as M13 fd, and other filamentous single- stranded DNA phages.

Vectors for expressing proteins in bacteria, especially E. coli, are also known. Such vectors include the pK233 (or any of the tac family of plasmids), T7, pBluescript II, bacteriophage lamba ZAP, and lambda P.sub.L. For example, see Recombinant DNA Methodology II, Methods Enzymol, Wu, R. (Ed.), Academic Press, Inc., NY, (1995). Examples of vectors that express fusion proteins are PATH vectors described by Dieckmann and Tzagoloff (J. Biol. Chem. 260: 1513 1520, 1985). These vectors contain DNA sequences that encode anthranilate synthetase (TrpE) followed by a polylinker at the carboxy terminus. Other expression vector systems are based on .beta.-galactosidase (pEX); maltose binding protein (pMAL); glutathione S-transferase (pGST or PGEX) (Smith, D., Methods Mol. Cell Biol. 4:220- 229, 1993; Smith, D. and Johnson, K. Gene 67:31-40, 1988; and Peptide Res. 3: 167, 1990; and TRX (thioredoxin) fusion protein (LaVallie, R., et al, Bio/Technology 11: 187-193, 1993).

Suitable cloning/expression vectors for use in mammalian cells are also known. Such vectors include well-known derivatives of SV-40, adenovirus, cytomegalovirus (CMV) retrovirus-derived DNA sequences. Any such vectors, when coupled with vectors derived from a combination of plasmids and phage DNA, i.e. shuttle vectors, allow for the isolation and identification of amino acid coding sequences in prokaryotes.

Expression vectors can contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed. A control sequence is inserted in the vector in order to control and regulate expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the tet system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the glycolytic promoters of yeast, e.g., the promoter for 3- phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters or SV40, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.

Expression hosts include well-known prokaryotic and eukaryotic cells. Suitable prokaryotic hosts include, for example, E. coli, such as E. Coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coli DH1, E. coli DH5aF, and E. coli MRC1, Bacilus, such as Bacillus subtilis, and Streptomyces. Suitable eukaryotic cells include yeasts and other fungi, insect, animal cells, such as COS cells and CHO cells, human cells and plant cells in tissue culture. A recombinant fusion polypeptide can be purified by methods known in the art (e.g., utilizing a his-tag). Such methods include affinity chromatography using specific antibodies. Alternatively, a recombinant fusion polypeptide of the invention may be purified using a combination of ion-exchange, size-exclusion, hydrophobic interaction chromatography and reverse phase liquid chromatography using methods known in the art. These and other suitable methods are described by Marston, "The Purification of Eukaryotic Proteins Expressed in E. coli" DNA Cloning, D. Glover (Ed.), Volume III, IRL Press Ltd., England (1987); "Guide to Protein Purification", M. Deutscher (Ed.), Methods Enzymol, Academic Press, Inc., (Ed.), NY (1990); and Protein Purification, Scopes, R. and Cantor, C. (Eds.), (3d), Springer- Verlag, NY (1994).

A composition of the present invention also provides antimicrobial effect and can be used to prevent and treat infection associated with oral pathogens. An antimicrobial effect refers to interfering with any biological function of a target microorganism. An antimicrobial effect includes killing or inhibiting the growth of target microorganisms. In one embodiment, compositions of the present invention are administered to treat a disease or infection on an implant site containing a biofilm. Target microorganisms for the antimicrobial peptide in the fusion polypeptide include, without limitation, Streptococcus mutans, Streptococcus sobrinus, Streptococcus gordonii, Streptococcus mitis, Streptococcus sanguis, Streptococcus sanguinis, Streptococcus parasanguis, Streptococcus crista, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus milleri and Streptococcus oralis, Fusobacterium nucleatum, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Treponema denticola, and Bacteroides forsythus.

EXAMPLES

Example 1: Preparation of plasmid constructs to express P15-CSP fusion polypeptide or OP8 (an osteogenic peptide)-CSP fusion polypeptide with helical or flexible linker in

Escherichia coli Nucleotide sequence of the coding region

ATGGGCCATCATCATCATCATCATGGCACCCCGGGCCCGCAGGGCATTGCGGGCCA GCGCGGCGTGGTGGCGGAAGCGGCGGCGAAAGAAGCGGCGGCGAAAGAAGCGGCG GCGAAAGCGAGCGGCAGCCTGAGCACCTTTTTTCGCCTGTTTAACCGCAGCTTTACC CAGGCGCTGGGCAAATAA (SEQ ID NO: 106)

Amino acid sequence

MGHHHHHHGTPGPQGI AGQRG V V AE A A AKE A A AKE A A AKAS G S LS TFFRLFNRS FTQ A LGK (SEQ ID NO: 38)

Construction of plasmid pQEFUP2

The nucleotide sequence encoding the fusion peptide (N-terminal Histidine tag, pl5, helical linker, CSP) was constructed by two step PCR using two sets of long oligonucleotide primers. The first PCR was conducted using forward primer 5-GGCATTGCGGGCCAGCGCGGCGT GGTGGCGGAAGCGGCGGCGAAAGAAGCGGCGGCGAAAG-3' (SEQ ID NO: 107) and reverse primer 5 ' - AGGTGCTC AGGCTGCCGCTCGCTTTCGCCGCCGCTTCTTTCGCCG CCGCTTCTTTCGCCGCCGCTTCTTTCGC-3' (SEQ ID NO: 108), which are complimentary at their 3' ends. The second PCR reaction used 1/10^th of the 1^st PCR reaction as the template and forward primer 5'-ATAATTCCATGGGCCATCATCATCATCATCATGGCACCCCGGGCCC GCAGGGCATTGCGGGCCAGCGCGG-3' (SEQ ID NO: 109) and reverse primer 5'- ATAATTGATCCTTATTTGCCCAGCGCCTGGGTAAAGCTGCGGTTAAACAGGCGAAAA AAGGTGCTCAGGCTGCCGCTCG-3' (SEQ ID NO: 110). The PCR product were digested with Ncol and Bamlil and ligated to Ncol and BamHl digested pQE60 vector(Qiagen) to yield PQEFUP2 (Fig. 1).

Nucleotide Sequence of the coding region

ATGGGCCATCATCATCATCATCATAGCGGTGGTGGCAGCGGTACTCCAGGTCCTCAA GGTATTGCAGGTCAACGTGGTGTTGTGTCTGGTGGCGGTGGATCGGGCGGTGGTGGT TCGGGTGGTGGCGGATCTGGCAGCCTGAGTACCTTCTTTCGCCTCTTCAACCGTTCGT TCACGCAGGCGCTTGGTAAATAA (SEQ ID NO: 111)

Amino acid sequence

MGHHHHHHSGGGSGTPGPQGIAGQRGVVSGGGGSGGGGSGGGGSGSLSTFFRLFNRSF TQALGK (SEQ ID NO: 46) Construction of plasmid pQEP15CSP-l

The nucleotide sequence encoding fusion polypeptide (N-terminal Histidine tag, Flexible linker, pl5, Flexible linker, CSP) was constructed by two step PCR using two sets of long oligonucleotide primers. The first PCR was conducted using forward primer 5'- GCGGTACTCCAGGTCCTCAAGGTATTGCAGGTCAACGTGGTGTTGTGTCTGGTGGCG GTGGATC-3' (SEQ ID NO: 112) and reverse primer 5'- AGGTACTCAGGCTGCCAGATCCGCCACCACCCGAACCACCACCGCCCGATCCACCG CCACCAG-3' (SEQ ID NO: 113), which are complimentary at their 3' ends. The second PCR reaction used l/10^th of the 1^st PCR reaction as the template and forward primer 5'- ATAATACCATGGGCCATCATCATCATCATCATAGCGGTGGTGGCAGCGGTACTCCAG GTCCTC-3' (SEQ ID NO: 114) and reverse primer 5 '-TATTATGGATCCT TATTTACCAAGCGCCTGCGTGAACGAACGGTTGAAGAGGCGAAAGAAGGTACTCAG GCTGCCAG-3' (SEQ ID NO: 115). The PCR product were digested with Ncol and Bamlil and ligated to Ncol and Bamlil digested pQE60 vector (Qiagen) to yield pQEP15CSP-l (Fig. 2).

Nucleotide sequence of the coding region

ATGGGCCATCATCATCATCATCATTGCGGCGGTGGTCGCTGGTGCGGTGCGGAAGCG GCGGCGAAAGAAGCGGCGGCGAAAGAAGCGGCGGCGAAAGCGAGCGGCAGCCTGA GCACCTTTTTTCGCCTGTTTAACCGCAGCTTTACCCAGGCGCTGGGCAAATAA (SEQ ID NO: 116)

Amino acid sequence

MGHHHHHHCGGGRWCGAEAAAKEAAAKEAAAKASGSLSTFFRLFNRSFTQALGK

(SEQ ID NO: 72)

Construction of plasmid pQEOPHeCSP

The nucleotide sequence encoding a fusion polypeptide (N-terminal Histidine tag, osteogenic peptide, helical linker, CSP) was constructed by two step PCR using two sets of long oligonucleotide primers. The first PCR was conducted using forward primer 5'-GGTCGCTG GTGCGGTGCGGAAGCGGCGGCGAAAGAAGCGGCGGCGAAAGAAGCGGCGGCG-3' (SEQ ID NO: 117). and reverse primer 5 ' -TAAAC AGGCG AAAAAAGGTGCTC AGGCT GCCGCTCGCTTTCGCCGCCGCTTCTTTCGCCG-3' (SEQ ID NO: 118), which are complimentary at their 3' ends. The second PCR reaction used l/10^th of the 1^st PCR reaction as the template and forward primer 5'-TATAATCCATGGGCCATCATCATCATCATCATTGC GGCGGTGGTCGCTGGTGCGGTGCGG AAG-3 ' (SEQ ID NO: 119) and reverse primer 5'- ATTATAGGATCCTTATTTGCCCAGCGCCTGGGTAAAGCTGCGGTTAAACAGGCGAAA AAAGGTG-3' (SEQ ID NO: 120). The PCR product were digested with Ncol and BamUl and ligated to Ncol and BamHl digested pQE60 vector(Qiagen) to yield pQEOPHeCSP (Fig. 3). Nucleotide Sequence of the coding region

ATGGGCCATCATCATCATCATCATTGCGGCGGTGGTCGCTGGTGCGGTTCTGGTGGC GGTGGATCGGGCGGTGGTGGTTCGGGTGGTGGCGGATCTGGCAGCCTGAGTACCTTC TTTCGCCTCTTCAACCGTTCGTTCACGCAGGCGCTTGGTAAATAA (SEQ ID NO: 121) Amino acid sequence

MGHHHHHHCGGGRWCGSGGGGSGGGGSGGGGSGSLSTFFRLFNRSFTQALGK (SEQ ID NO: 81)

Construction of plasmid pQEOPFxCSP

The nucleotide sequence encoding fusion polypeptide (N-terminal Histidine tag, Flexible linker, Osteogenic peptide, Flexible linker, CSP) was constructed by two step PCR using two sets of long oligonucleotide primers. The first PCR was conducted using forward primer 5'-GCTGG TGCGGTTCTGGTGGCGGTGGATCGGGCGGTGGTGGTTCGGGTGGTGGCGGATCTG-3' (SEQ ID NO: 122) and reverse primer 5'-GGCGAAAGAAGGTACTCAGGCTGCCAGATCC GCCACCACCCGAACCACCACCGCCCGATC-3' (SEQ ID NO: 123), which are complimentary at their 3' ends. The second PCR reaction used l/10^th of the 1^st PCR reaction as the template and forward primer 5'-TATAATCCATGGGCCATCATCATCATCATCATTGC GGCGGTGGTCGCTGGTGCGGTTCTGGTGGC-3' (SEQ ID NO: 124) and reverse primer 5'- ATTATAGGATCCTTATTTACCAAGCGCCTGCGTGAACGAACGGTTGAAGAGGCGAA AG AAGGTACTC AGGCTGC-3 ' (SEQ ID NO: 125). The PCR product were digested with Ncol and BamHl and ligated to Ncol and BamHl digested pQE60 vector (Qiagen) to yield pQEOPFxCSP (Fig. 4).

Example 2: Isolation and purification of Recombinant P15-CSP and OP8-CSP fusion polypeptides

E. coli Tuner (DE3)pLacI strain bearing the plasmid pQEFUP2, pQEP15CSP-l, pQEOPHeCSP, or pQEOPFxCSP was grown in Luria-Bertani (LB) medium at 37°C. The expression of the P15-CSP fusion polypeptide was induced with ImM IPTG at exponential growth phase. The cells were harvested by centrifugation 4 hrs post-induction, resuspended in extraction buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl) containing ImM PMSF, 2 mg/mL lysozyme and 0.1% Igepal^®, ruptured by sonication, and treated with DNasel and RNaseA. P15-CSP was captured by passing the cleared lysate through a column of His-Select™ Nickel Affinity Gel equilibrated with extraction buffer. The column was washed twice with extraction buffer containing 5 mM imidazole and then 20 mM imidazole. P15-CSP was eluted with extraction buffer containing lOOmM imidazole, dialyzed against deionized water, and then lyophilized.

Example 3: Synthesis of antimicrobial competence stimulating peptide (CSP) and P15-CSP fusion polypeptide

CSP ( S G S LS TFFRLFNRS FTQ ALG K) (SEQ ID NO: 1 } and P15-CSP (GTPGPQGI AGQRG V V AE A A AKE A A AKE A A AKAS G S LS TFFRLFNRS FTQ ALG K) ( S EQ ID NO: 41) were synthesized by Mimotopes (Roseville, MN, USA). The qualitative analysis of these two peptides were done by electrospray mass spectrometry (ESMS) and purified to 90% by reverse phase-high performance liquid chromatography (RP-HPLC) on a 150 x 4.6 mm Monitor C₁₈ column with a gradient of acetonitrile (10-66.6%) in 0.1% trifluoroacetic acid for 25 min. The peptides were stored at -20°C. Example 4: Cell adhesion activity of CSP, P15 peptide and recombinant P15-CSP fusion polypeptides The cell adhesion activity of CSP, P15 peptide and recombinant P15-CSP fusion polypeptide were tested as follows: A hydroxyapatite-based carrier was added to solutions containing CSP, P15 peptide and recombinant P15-CSP fusion polypeptide separately and was shaken for a desired period of time. The solution was decanted and the particles were washed 4-5 times with phosphate buffered saline (PBS) followed by water for injection 4-5 times. After the liquid was decanted, the samples were placed in a vacuum oven at 25 °C until dry. The samples were sterilized and tested for cell adhesion using short -term cell attachment assay as described by Vogler and Bussien (J. Biomed. Mater Res. 21: 1197, 1987). Samples of both the test material and parent particulate were placed in 96- well plate separately and the wells were seeded with fibroblast cells. Plates were placed in an incubator for 3 h to allow cellular attachment followed by washing with modified eagle medium to remove the unattached cells. The plates were further incubated for 3 days, followed by tissue culture medium-tetrazolium salt-phenazine metho sulphate reaction to demonstrate cell viability and to determine the number of adhered cells. Both CSP and P15-CSP fusion peptide showed unexpected level of increase in the cell (osteoblast) adhesion to hydroxyapatite carrier as compared to a modest increase in cell adhesion by P15 peptide alone. Particularly, recombinant P15-CSP fusion peptide showed two-fold increase in osteoblast adhesion compared to that by P15 peptide alone. Example 5: Osteogenic activity of CSP, P15 peptide and P15-CSP (synthetic and

recombinant) fusion polypeptides

The main objective of this study was to determine if CSP, P15 peptide, synthetic P15-CSP and recombinant P15-CSP polypeptides promote osteogenesis of osteoblast precursors such as human bone marrow stromal cells (hBMSCs) in mineralizing media with and without Dexamethasone as an osteogenic inducer. P15 peptide-only served as a control. Osteogenic activity was determined by the in situ staining intensity of alkaline phosphatase enzyme, calcium mineral deposition, and phosphate mineral accumulation after 3 weeks in confluent hBMSCs culture stimulated with the peptides in order to determine their level of differentiation.

Primary human bone marrow stromal cells (donor 800R1) were seeded at passage 3 in

24-well plates, at 4000 cells per well. Cells were allowed to proliferate for 1 week until confluent. The media was then changed to the test media (control media: 16% fetal bovine serum, alpha-Minimal Essential Medium, Pen-Strep, 100 μΜ L-ascorbate-2-phosphate, 5 mM disodium beta-glycerol phosphate; and osteogenic media (OSM): control media containing 10 nM dexamethasone) with or without peptides at different concentrations (1, 10, 100 μg/mL). The media was refreshed twice a week for a period of 3 weeks. The media volume was 0.5 mL per well. 2 petri dishes per peptide were treated with 0, 1, 10, and 100 μg/mL peptide in control media, or peptide in OSM.

Methods

The methods used for determining the osteoblast differentiation and calcium mineralization are described briefly as follows:

(i) Alkaline phosphatase staining for early osteoblast differentiation

After 3 weeks of culture, the 24 well plates were aspirated of media, rinsed in isotonic saline, and the cell monolayers or nodules fixed for 1 hour in 10% normal buffered formalin, then exposed to alkaline phosphatase reagent prepared according to the manufacturer (SigmaFast alkaline phosphatase enzymatic staining kit, Sigma-Aldrich, Oakville, ON, Canada, Product N° B5655) for 20 minutes at room temperature. After 20 minutes the substrate was aspirated and the plates rinsed in PBS with 20 mM EDTA to stop the reaction.

(ii) Alizarin red staining for calcium mineralization

After 3 weeks of culture, the plates were aspirated of media, rinsed in isotonic saline, fixed for 1 hour in 10% normal buffered formalin, and stained for 1 hour in alizarin red staining solution (1.37 g/100 mL adjusted to pH 4 with NH₄OH). The plates were washed at least 3 times with ddH₂0 to remove unbound alizarin red dye, and stored at 4°C covered in ddH₂0. For quantitative alizarin red spectroscopic analyses, 800 xL acetic acid was added to each of the alizarin red wells, the plates rocked for 30 minutes at room temperature, and all the well contents transferred to a 2-ml screw-cap tube. Samples were vortexed 30 seconds, 500 xL mineral oil added, heated for 10 minutes at 85°C in the oven, and iced. The samples were all yellow. Samples were centrifuged for 3000xg, and 450 xL transferred to a new vial containing 9 μΐ. NH₄OH. Sample absorbance was read using a plate reader at OD₄₂₅. Results (Table 5) and Conclusions

(i) CSP qualitatively led to more phosphate deposition (but not calcium) in osteogenic media, and may have stimulated cell proliferation (thicker monolayer). Furthermore, it inhibited phosphate deposition at the highest concentration in control media without Dex.

(ii) P15 peptide is not osteogenic in this assay based on macro scopically visible mineral deposition. P15 seems to aggravate cell detachment and to promote ball formation in absence of Dex (on Costar plates) and P15 led to a more fragmented monolayer sheet and did not enhance deposition of calcium or phosphate mineral. P15 did not enhance ALP activity by this BMSC culture in osteogenic media (with Dex).

(iii) Recombinant P15-CSP suppressed ALP in a dose-dependent manner in control cultures and promoted calcification in osteogenic cultures with Dex (higher calcium but not phosphate). The greater adhesion of the monolayer is consistent with the profile of lower MMP-2 activity. P15- CSP may have anti-inflammatory activity towards hBMSC. This peptide showed evidence of enhancing osteogenesis in Dex+ media.

(iv) Synthetic P15-CSP did not intensify monolayer adhesion (to Falcon plate) without Dex. Monolayers that stuck to Falcon dish without Dex had high calcium accumulation (with or without peptide). It seems to promote phosphate accumulation without Dex only at 100 μg/mL. Furthermore, it intensified calcium deposition and phosphate with Dex. No further increase was seen for ALP in the presence of peptide. This peptide showed evidence of enhancing osteogenesis in Dex+ media.

Table 5: Summary of the Effect of Peptides in Different Media Conditions

Control Osteogenic Activity adhesion ALP AR Pi adhesion ALP AR Pi

CSP low NR NR High* same* same* †*

NONE

P15 Peptide low NR NR NR high same same

same

P15-CSP High* II * †* †t* high * †* †*

(recombinant) same

P15-CSP (synthetic) low * NR * †* †* high * same *

NR: Not Relevant (data could not be collected on balls);†: Intensification of Signal; j: Less Signal; Same: No Difference from Control Monolayer; ALP: Alkaline Phosphatase; AR: Alizarin Red; Pi: Inorganic Phosphate; and ^ Evidence of Osteogenic Activity.

Claims

We claim:

1. A bone tissue regenerating composition comprising (a) an antimicrobial competence stimulating peptide with bone tissue regenerating activity, and (b) a bone tissue regenerating peptide.

2. A bone tissue regenerating composition comprising (a) an antimicrobial competence stimulating peptide with bone tissue regenerating activity having an amino acid sequence of at least one of SEQ ID NO: 1 to SEQ ID NO: 15, and (b) a bone tissue regenerating peptide having an amino acid sequence of at least one of SEQ ID NO: 16 to SEQ ID NO: 23.

3. The bone tissue regenerating composition of claim 1 or 2 comprising a fusion polypeptide comprising the antimicrobial stimulating peptide and the bone tissue regenerating peptide.

4. The bone tissue regenerating composition according to any one of claims 1-3 further comprising a linker peptide.

5. The bone tissue regenerating composition of claim 4, wherein the linker peptide has an amino acid sequence of one of SEQ ID NO: 24 to SEQ ID NO: 37.

6. The composition according to any one of claims 3-5, wherein the antimicrobial competence stimulating peptide with bone tissue regenerating activity is linked to one terminus of a linker peptide, wherein the other terminus of a linker peptide is linked to the bone tissue regenerating peptide.

7. The bone tissue regenerating composition of any one of claims 3 to 6 selected from one or more of SEQ. ID NO: 38 to SEQ. ID NO: 105.

8. The composition according to any one of claims 1-7, further comprising an osteoinductive substance selected from the group consisting of dexamethazone, ascorbic acid-2-phosphate, beta-glycophosphate, and combinations thereof.

9. The composition according to any one of claims 1-8, further comprising an osteoinductive substance selected from the group consisting of growth factors, cytokines, hormones, enzymes, enzyme inhibitors, bone matrix components, growth differentiation factors, and combinations thereof.

10. The composition according to any one of claims 1-9, further comprising a delivery vehicle, wherein the delivery vehicle is a bone-compatible matrix.

11. The composition of claim 10, wherein said bone-compatible matrix provides for slow release of said peptide component to a patient in need of said composition and/or provides a structure for developing bone in the patient.

12. The composition according to claims 10 or 11, wherein the CSP, fusion polypeptide, or both the CSP and fusion polypeptide is immobilized, encapsulated, or impregnated within said bone-compatible matrix.

13. The composition according to any one of claims 10-12, wherein said bone-compatible matrix is a porous structure.

14. The composition according to any one of claims 10-13, wherein said bone-compatible matrix is in a form selected from the group consisting of powder, microparticles, microspheres, granules, microfibers, strip, gel, sponge, and combinations thereof.

15. The composition according to any one of claims 10-14, wherein said bone-compatible matrix is selected from the group consisting of calcined or sintered bovine bone, synthetic β-tricalcium phosphates, algae-derived hydroxyapatite, synthetic hydroxyapatite, nanocrystalline precipitated hydroxyapatite, ceramics, and combinations thereof.

16. The composition of claim 15, wherein said calcined or sintered bovine bone is high temperature (1100 or >1200 °C) hydroxyapatite.

17. The composition of claim 14, wherein said ceramic is selected from the group consisting of calcium sulfate, hydroxyapatite, tricalcium phosphate, and combinations thereof.

18. The composition according to any one of claims 1-17, wherein the antimicrobial competence stimulating peptide is effective against dental plaque associated bacteria Streptococcus mutans, Streptococcus sobrinus, Streptococcus gordonii, Streptococcus mitis, Streptococcus sanguis, Streptococcus sanguinis, Streptococcus parasanguis, Streptococcus crista, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus milleri and Streptococcus oralis.

19. A bone tissue regenerating composition consisting essentially of an antimicrobial competence stimulating peptide (CSP) with bone tissue regenerating activity having an amino acid sequence of SEQ ID NO: 1.

20. The composition of claim 19, further comprising a delivery vehicle for said CSP, said delivery vehicle being a bone-compatible matrix.

21. The composition of claim 19, wherein said bone-compatible matrix provides for slow release of said peptide component to a patient in need of said composition and/or provides a structure for developing bone in the patient.

22. The composition of claim 20 or 21, wherein said peptide component is immobilized, encapsulated, or impregnated within said bone-compatible matrix.

23. The composition according to any one of claims 20-22, wherein said bone-compatible matrix is a porous structure.

24. The composition according to any one of claims 20-22, wherein said bone-compatible matrix is in a form selected from the group consisting of powder, microparticles, microspheres, granules, microfibers, strip, gel, sponge and combinations thereof.

25. The composition according to any one of claims 20-24, wherein said bone-compatible matrix is selected from the group consisting of calcined or sintered bovine bone, synthetic β-tricalcium phosphates, algae-derived hydroxyapatite, synthetic hydroxyapatite, nanocrystalline precipitated hydroxyapatite, ceramics and combinations thereof.

26. The composition of claim 25, wherein said calcined or sintered bovine bone is high temperature (1100 or >1200 °C) hydroxyapatite.

27. The composition of claim 25, wherein said ceramic is selected from the group consisting of calcium sulfate, hydroxyapatite, tricalcium phosphate and combinations thereof.

28. The composition according to any one of claims 19-27, wherein the antimicrobial CSP is effective against dental plaque associated bacteria Streptococcus mutans, Streptococcus sobrinus, Streptococcus gordonii, Streptococcus mitis, Streptococcus sanguis, Streptococcus sanguinis, Streptococcus parasanguis, Streptococcus crista, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus milleri and Streptococcus oralis.

29. The composition according to any one of claims 1-28 further comprising a pharmaceutically acceptable carrier or aqueous solvent.

30. A method for promoting osteoblast adhesion and proliferation comprising administering the composition according to any one of claims 1-29 to a patient in need thereof.

31. The method of claim 30, wherein the composition comprises the fusion polypeptide having an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 72

32. The method of claim 31, wherein the composition comprises the antimicrobial competence stimulating peptide having the amino acid sequence of SEQ ID NO: 1.

33. The method according to any one of claims 30-32, wherein the composition is administered locally, topically or systemically.

34. The method of claim 33, wherein local administration is provided via an implant or device.

35. The method according to any one of claims 30-33, wherein the treatment is useful for treating bone fractures.

36. The method according to any one of claims 30-33, wherein the treatment is useful for treating diseases or anomalies associated with deficient sites of bone.

37. The method of claim 36, wherein said anomalies are selected from the group consisting of craniofacial anomalies, dental anomalies and periodontal anomalies.

38. The method of claim 37, wherein said diseases are selected from the group consisting of osteoporosis and rheumatoid arthritis.

39. A method preparing a bone regenerative composition comprises of combining a bone- compatible matrix with a peptide component having an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 72 or SEQ ID NO: 1; and immobilizing said peptide component to or within said bone-compatible matrix.

40. A nucleic acid encoding a fusion polypeptide comprising (a) a nucleic acid encoding one of SEQ ID NO: 1 to SEQ ID NO: 15, and (b) a nucleic acid encoding one of SEQ ID NO: 16 to SEQ ID NO:23.

41. The nucleic acid encoding a fusion polypeptide of claim 40 further comprising a nucleic acid encoding a linker peptide.

42. The nucleic acid encoding a fusion polypetide of claim 41, wherein the nucleic acid encoding a linker peptide comprises a nucleic acid encoding one of SEQ ID NO: 24 to SEQ ID NO: 37.

43. A vector comprising the nucleic acid according to any one of claims 40-42.

44. The vector of claim 43, wherein the vector is an expression vector.

45. A host cell comprising the vector of claim 43 or 44.

46. The host cell of claim 45, wherein the host cell is Escherichia coli.

47. A method of producing a fusion polypeptide comprising culturing the host cell of claim 45 or 46.

48. A use of a composition according to any one of claims 1-29 for promoting osteoblast adhesion and proliferation.

49. The us of claim 48, wherein the composition comprises the fusion polypeptide having an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 72

50. The use of claim 48, wherein the composition comprises the antimicrobial competence stimulating peptide having the amino acid sequence of SEQ ID NO: 1.

51. The use according to any one of claims 48-50, wherein the composition is administered locally, topically or systemically.

52. The use of claim 51, wherein local administration is provided via an implant or device.

53. The use according to any one of claims 48-52, wherein the treatment is useful for treating bone fractures.

54. The use according to any one of claims 48-53, wherein the treatment is useful for treating diseases or anomalies associated with deficient sites of bone.

55. The use of claim 54, wherein said anomalies are selected from the group consisting of craniofacial anomalies, dental anomalies and periodontal anomalies.

56. The use of claim 54, wherein said diseases are selected from the group consisting of osteoporosis and rheumatoid arthritis.

57. A use of an an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 72 or SEQ ID NO: 1 for preparing a bone regenerative composition comprising a bone-compatible matrix with a peptide component, said peptide component immobilized to or within said bone-compatible matrix.