Methods and compositions for regenerating connective tissue

Abstract

Connective tissue regenerative compositions and methods of repairing and regenerating connective tissue using such compositions are provided. The compositions generally comprise a bioactive hydrogel matrix comprising a polypeptide, such as gelatin, and a long chain carbohydrate, such as dextran. The hydrogel matrix may further include polar amino acids, as well as additional beneficial additives. Advantageously, the compositions include further components, such as osteoinductive or osteoconductive materials, medicaments, stem or progenitor cells, and three-dimensional structural frameworks. The compositions are useful for regenerating connective tissue, and can be administered to an area having injury to, or a loss of, connective tissue, such as bone, cartilage, tendon, and ligament.

1 . A bioactive hydrogel matrix comprising a polypeptide, a long chain carbohydrate, and at least one osteoinductive or osteoconductive material selected from the group consisting of demineralized bone matrix (DBM), bone morphogenetic proteins (BMPs), transforming growth factors (TGFs), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), platelet-derived growth factors (PDGFs), epidermal growth factors (EGFs), vascular endothelial growth factors (VEGFs), and vascular permeability factors (VPFs). 
     
     
         2 . The hydrogel matrix of  claim 1 , wherein the polypeptide is a tissue-derived or synthetic polypeptide. 
     
     
         3 . The hydrogel matrix of  claim 2 , wherein the polypeptide is a tissue-derived polypeptide derived from tissue selected from the group consisting of collagens, gelatins, agarose, alginate, keratin, decorin, aggrecan, and glycoproteins. 
     
     
         4 . The hydrogel matrix of  claim 2 , wherein the polypeptide is a tissue-derived polypeptide derived from extracts of tissue selected from the group consisting of submucosal tissues, arteries, vocal chords, pleura, trachea, bronchi, pulmonary alveolar septa, ligaments, auricular cartilage, abdominal fascia, liver, kidney, neurilemma, arachnoid, dura mater, and pia mater. 
     
     
         5 . The hydrogel matrix of  claim 1 , wherein the polypeptide has a molecular mass of about 3,000 to about 3,000,000 Da. 
     
     
         6 . The hydrogel matrix of  claim 5 , wherein the polypeptide has a molecular mass of about 30,000 to about 300,000 Da. 
     
     
         7 . The hydrogel matrix of  claim 1 , wherein the long chain carbohydrate is a polysaccharide or a sulfated polysaccharide. 
     
     
         8 . The hydrogel matrix of  claim 7 , wherein the long chain carbohydrate is a polysaccharide comprising more than about 10 monosaccharide residues joined to each other by glycosidic linkages. 
     
     
         9 . The hydrogel matrix of  claim 7 , wherein the polysaccharide is selected from the group consisting of glycosaminoglycans and glucosaminoglycans. 
     
     
         10 . The hydrogel matrix of  claim 7 , wherein the polysaccharide is selected from the group consisting of dextran, dextrin, heparan, heparin, hyaluronic acid, chondroitin, alginate, agarose, carageenan, amylopectin, amylose, glycogen, starch, cellulose, chitin, and chitosan. 
     
     
         11 . The hydrogel matrix of  claim 7 , wherein the sulfated polysaccharide is selected from the group consisting of heparan sulfate, heparin sulfate chondroitin sulfate, dextran sulfate, dermatan sulfate, and keratan sulfate. 
     
     
         12 . The hydrogel matrix of  claim 1 , wherein the long chain carbohydrate has a molecular mass of about 2,000 to about 8,000,000 Da. 
     
     
         13 . The hydrogel matrix of  claim 12 , wherein the long chain carbohydrate has a molecular mass of about 20,000 to about 1,000,000 Da. 
     
     
         14 . The hydrogel matrix of  claim 1 , wherein the hydrogel matrix further comprises one or more components selected from the group consisting of polar amino acids, polar amino acid analogs or derivatives, divalent cation chelators, and combination thereof. 
     
     
         15 . The hydrogel matrix of  claim 14 , wherein the hydrogel matrix comprises one or more polar amino acids selected from the group consisting of tyrosine, cysteine, serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, lysine, histidine, and mixtures thereof. 
     
     
         16 . The hydrogel matrix of  claim 15 , wherein the one or more polar amino acids are present at a concentration of about 3 to about 150 mM. 
     
     
         17 . The hydrogel matrix of  claim 16 , wherein the one or more polar amino acids are present at a concentration of about 10 to about 65 mM. 
     
     
         18 . The hydrogel matrix of  claim 15 , wherein the one or more polar amino acids are selected from the group consisting of L-cysteine, L-glutamic acid, L-lysine, L-arginine, and mixtures thereof. 
     
     
         19 . The hydrogel matrix of  claim 15 , wherein the hydrogel matrix comprises one or more of the following:
 L-glutamic acid at a concentration of about 2 to about 60 mM;   L-lysine at a concentration of about 0.5 to about 30 mM;   L-arginine at a concentration of about 1 to about 40 mM; and   L-cysteine at a concentration of about 0.005 to about 5 mM.   
     
     
         20 . The hydrogel matrix of  claim 14 , wherein the hydrogel matrix comprises ethylenediaminetetraacetic acid or a salt thereof. 
     
     
         21 . The hydrogel matrix of  claim 20 , wherein the ethylenediaminetetraacetic acid or salt thereof is present at a concentration of about 0.01 to about 10 mM. 
     
     
         22 . The hydrogel matrix of  claim 1 , wherein the bioactive hydrogel matrix further comprises at least one medicament selected from the group consisting of antivirals, antibacterials, anti-inflammatories, immunosuppressants, analgesics, anticoagulants, and healing promotion agents. 
     
     
         23 . The hydrogel matrix of  claim 1 , wherein the bioactive hydrogel matrix further comprises cells selected from the group consisting of stem cells, progenitor cells, and mixtures thereof. 
     
     
         24 . The hydrogel matrix of  claim 1 , wherein the bioactive hydrogel matrix is at least partially contained within a three-dimensional structural framework. 
     
     
         25 . The hydrogel matrix of  claim 24 , wherein the three-dimensional structural framework includes a bioreabsorbable material. 
     
     
         26 . The hydrogel matrix of  claim 24 , wherein the three-dimensional framework includes a material selected from the group consisting of coralline, metals, sponge, bioactive glass, ceramics, calcium salts, collagen, keratin, fibrinogen, alginate, chitosan, allogenic bone, autologous bone, hyaluronan, polyethylene, poly(vinylidene fluoride), poly(tetrafluoroethylene), poly(vinyl alcohol), poly(hydroxyalkanoate), poly(ethylene terephthalate), poly(butylene terephthalate), poly(methyl methacrylate), poly(hydroxyethyl methacrylate), poly(N-isopropylacrylamide), poly(dimethyl siloxane), polydioxanone, and polypyrrole, poly(glycolic acid), poly(lactic acids), poly(ethylene oxides), poly(lactide-co-glycolides), poly(s-caprolactone), polyanhydrides, polyphosphazenes, poly(ortha-esters), and polyimides, and combinations thereof. 
     
     
         27 . The hydrogel matrix of  claim 24 , wherein the three-dimensional structural framework comprises a metal cage or a collagen sponge. 
     
     
         28 . The hydrogel matrix of  claim 1 , wherein the bioactive hydrogel matrix is in dehydrated form. 
     
     
         29 . The hydrogel matrix of  claim 28 , wherein the dehydrated bioactive hydrogel matrix is in particulate form. 
     
     
         30 . The hydrogel matrix of  claim 1 , wherein at least a portion of the bioactive hydrogel matrix is in crosslinked form, the long chain carbohydrate being covalently crosslinked to the polypeptide. 
     
     
         31 . The hydrogel matrix of  claim 1 , wherein the osteoinductive or osteoconductive material is demineralized bone matrix (DBM) or a bone morphogenetic protein (BMP). 
     
     
         32 . The hydrogel matrix of  claim 1 , wherein the at least one osteoinductive or osteoconductive material is present at a concentration of about 0.01 volume percent to about 90 volume percent, based upon the total volume of the hydrogel matrix. 
     
     
         33 . The hydrogel matrix of  claim 32 , wherein the at least one osteoinductive or osteoconductive material is present at a concentration of about 50 volume percent to about 80 volume percent, based upon the total volume of the hydrogel matrix. 
     
     
         34 . A bioactive hydrogel matrix comprising gelatin, a polysaccharide, at least one polar amino acid or divalent cation chelator, and at least one osteoinductive or osteoconductive material selected from the group consisting of demineralized bone matrix (DBM), bone morphogenetic proteins (BMPs), transforming growth factors (TGFs), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), platelet-derived growth factors (PDGFs), epidermal growth factors (EGFs), vascular endothelial growth factors (VEGFs), and vascular permeability factors (VPFs). 
     
     
         35 . The hydrogel matrix of  claim 34 , wherein the gelatin is skin-derived gelatin or bone-derived gelatin. 
     
     
         36 . The hydrogel matrix of  claim 34 , wherein the gelatin has a molecular mass of about 80,000 to about 200,000 Da and the polydispersity of the molecular mass of the gelatin is 1 to about 3. 
     
     
         37 . The hydrogel matrix of  claim 34 , wherein the gelatin is present at a concentration of about 0.01 to about 40 mM. 
     
     
         38 . The hydrogel matrix of  claim 34 , wherein the polysaccharide is dextran. 
     
     
         39 . The hydrogel matrix of  claim 38 , wherein the dextran has a molecular mass of about 300,000 to about 600,000 Da and the polydispersity of the molecular mass of the dextran is about 1 to about 3. 
     
     
         40 . The hydrogel matrix of  claim 38 , wherein the dextran is present at a concentration of about 0.01 to about 10 mM. 
     
     
         41 . The hydrogel matrix of  claim 34 , wherein the hydrogel matrix comprises one or more components selected from the group consisting of tyrosine, cysteine, serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, lysine, histidine, ethylenediaminetetraacetic acid or a salt thereof, and mixtures thereof. 
     
     
         42 . The hydrogel matrix of  claim 34 , wherein the bioactive hydrogel matrix is at least partially contained within a three-dimensional structural framework. 
     
     
         43 . A method for regenerating connective tissue selected from bone, cartilage, ligament, and tendon, comprising administering a bioactive hydrogel matrix according to  claim 1  to a site in need of connective tissue regeneration. 
     
     
         44 . A method for regenerating connective tissue selected from bone, cartilage, ligament, and tendon, comprising administering a bioactive hydrogel matrix according to  claim 34  to a site in need of connective tissue regeneration.