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 connective tissue regenerative composition comprising:
 a bioactive hydrogel matrix comprising a polypeptide and a long chain carbohydrate, the bioactive hydrogel matrix being in a dehydrated form; and   an osteoinductive or osteoconductive material comprising hydroxyapatite.   
     
     
         2 . The connective tissue regenerative composition of  claim 1 , wherein the osteoinductive or osteoconductive material is dispersed within the bioactive hydrogel matrix. 
     
     
         3 . The connective tissue regenerative composition of  claim 1 , wherein the bioactive hydrogel matrix is in particulate form. 
     
     
         4 . The connective tissue regenerative composition of  claim 3 , wherein the composition comprises a mixture of particles of the bioactive hydrogel matrix and particles of the osteoinductive or osteoconductive material. 
     
     
         5 . The connective tissue regenerative composition of  claim 1 , wherein the 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 composition. 
     
     
         6 . The connective tissue regenerative composition of  claim 1 , wherein at least a portion of the bioactive hydrogel matrix is in crosslinked form. 
     
     
         7 . The connective tissue regenerative composition of  claim 6 , wherein the bioactive hydrogel matrix comprises gelatin crosslinked to oxidized dextran. 
     
     
         8 . The connective tissue regenerative composition of  claim 6 , wherein the dehydrated form is a freeze-dried form. 
     
     
         9 . The connective tissue regenerative composition of  claim 6 , wherein the bioactive hydrogel matrix has been dehydrated using a method that maintains the connective tissue regenerative properties of the bioactive hydrogel matrix, said method comprising a first step wherein the hydrogel matrix is frozen at a temperature below the eutectic point of the hydrogel. 
     
     
         10 . The connective tissue regenerative composition of  claim 9 , wherein the first step in the dehydration method is carried out at a temperature of less than or equal to −30° C. 
     
     
         11 . The connective tissue regenerative composition of  claim 10 , wherein the dehydrating method further comprises a second, primary drying step that is carried out at a temperature greater than used in the first step 
     
     
         12 . The connective tissue regenerative composition of  claim 1 , wherein the bioactive hydrogel matrix comprises at least one additional 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). 
     
     
         13 . The connective tissue regenerative composition of  claim 1 , wherein the polypeptide derived from tissue selected from the group consisting of collagens, gelatins, keratin, decorin, aggrecan, and glycoproteins. 
     
     
         14 . The connective tissue regenerative composition of  claim 1 , wherein the long chain carbohydrate is a polysaccharide or a sulfated polysaccharide. 
     
     
         15 . The connective tissue regenerative composition of  claim 14 , wherein the long chain carbohydrate is a glycosaminoglycans or glucosaminoglycans. 
     
     
         16 . The connective tissue regenerative composition of  claim 14 , wherein the long chain carbohydrate is selected from the group consisting of dextran, dextrin, heparan, heparin, hyaluronic acid, chondroitin, alginate, agarose, carageenan, amylopectin, amylose, glycogen, starch, cellulose, chitin, chitosan, heparan sulfate, chondroitin sulfate, dextran sulfate, dermatan sulfate, keratan sulfate, and combinations thereof. 
     
     
         17 . The connective tissue regenerative composition of  claim 1 , wherein the bioactive 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. 
     
     
         18 . The connective tissue regenerative composition of  claim 17 , wherein the bioactive 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. 
     
     
         19 . The connective tissue regenerative composition of  claim 17 , wherein the bioactive hydrogel matrix comprises ethylenediaminetetraacetic acid or a salt thereof. 
     
     
         20 . The connective tissue regenerative composition of  claim 1 , wherein the polypeptide is gelatin and the long chain carbohydrate is dextran. 
     
     
         21 . The connective tissue regenerative composition of  claim 20 , 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. 
     
     
         22 . The connective tissue regenerative composition of  claim 20 , wherein the dextran has a molecular mass of about 200,000 to about 800,000 Da and the polydispersity of the molecular mass of the dextran is about 1 to about 3. 
     
     
         23 . A connective tissue regenerative composition comprising: a bioactive hydrogel matrix comprising a polypeptide and a long chain carbohydrate; and an osteoinductive or osteoconductive material comprising hydroxyapatite; wherein the composition is in a cast form adapted for integration into a bone defect. 
     
     
         24 . A method of treating a bone defect, comprising:
 (i) hydrating a dehydrated connective tissue regenerative composition, the dehydrated composition comprising: a dehydrated bioactive hydrogel matrix comprising a polypeptide and a long chain carbohydrate; and an osteoinductive or osteoconductive material comprising hydroxyapatite; and   (ii) administering the hydrated connective tissue regenerative composition to the site of the bone defect.   
     
     
         25 . The method of  claim 24 , wherein the bioactive hydrogel matrix is in particulate form and the osteoinductive or osteoconductive material is dispersed within the bioactive hydrogel matrix. 
     
     
         26 . The method of  claim 24 , wherein at least a portion of the bioactive hydrogel matrix is in crosslinked form. 
     
     
         27 . The method of  claim 24 , wherein the bioactive hydrogel matrix comprises gelatin crosslinked to oxidized dextran. 
     
     
         28 . The method of  claim 24 , wherein the dehydrated form is a freeze-dried form. 
     
     
         29 . The method of  claim 24 , wherein the bioactive hydrogel matrix has been dehydrated using a method that maintains the connective tissue regenerative properties of the bioactive hydrogel matrix, said method comprising a first step wherein the hydrogel matrix is frozen at a temperature below the eutectic point of the hydrogel. 
     
     
         30 . The method of  claim 29 , wherein the first step in the dehydration method is carried out at a temperature of less than or equal to −30° C. 
     
     
         31 . The method of  claim 30 , wherein the dehydrating method further comprises a second, primary drying step that is carried out at a temperature greater than used in the first step 
     
     
         32 . The method of  claim 24 , wherein the polypeptide derived from tissue selected from the group consisting of collagens, gelatins, keratin, decorin, aggrecan, and glycoproteins. 
     
     
         33 . The method of  claim 24 , wherein the long chain carbohydrate is a polysaccharide or a sulfated polysaccharide. 
     
     
         34 . The method of  claim 33 , 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. 
     
     
         35 . The method of  claim 24 , wherein the bioactive 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. 
     
     
         36 . The method of  claim 35 , wherein the bioactive 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. 
     
     
         37 . The method of  claim 35 , wherein the bioactive hydrogel matrix comprises ethylenediaminetetraacetic acid or a salt thereof. 
     
     
         38 . The method of  claim 24 , wherein the composition further comprises cells selected from the group consisting of stem cells, progenitor cells, and mixtures thereof. 
     
     
         39 . The method of  claim 24 , wherein the polypeptide is gelatin and the long chain carbohydrate is dextran. 
     
     
         40 . The method of  claim 24 , wherein said hydrating comprises contacting the dehydrated connective tissue regenerative composition with water. 
     
     
         41 . The method of  claim 24 , wherein said hydrating comprises contacting the dehydrated connective tissue regenerative composition with bone marrow aspirate. 
     
     
         42 . The method of  claim 24 , wherein, prior to said administering, the method further comprises molding the hydrated connective tissue regenerative composition to a putty form. 
     
     
         43 . A method of preparing a connective tissue regenerative composition, comprising:
 dehydrating a bioactive hydrogel matrix comprising a polypeptide and a long chain carbohydrate; and   dispersing an osteoinductive or osteoconductive material comprising hydroxyapatite within the dehydrated bioactive hydrogel matrix.   
     
     
         44 . The method of  claim 43 , wherein said dehydrating step comprises a method that maintains connective tissue regenerative properties of the bioactive hydrogel matrix, said method comprising a first step wherein the hydrogel matrix is frozen at a temperature below the eutectic point of the hydrogel. 
     
     
         45 . The method of  claim 44 , wherein the first step in the dehydration method is carried out at a temperature of less than or equal to −30° C. 
     
     
         46 . The method of  claim 45 , wherein the dehydrating method further comprises a second, primary drying step that is carried out at a temperature greater than used in the first step. 
     
     
         47 . The method of  claim 43 , comprising particularizing the dehydrated bioactive hydrogel matrix. 
     
     
         48 . A method of preparing a connective tissue regenerative composition, comprising:
 dispersing an osteoinductive or osteoconductive material comprising hydroxyapatite within a bioactive hydrogel matrix comprising a polypeptide and a long chain carbohydrate; and   dehydrating the bioactive hydrogel matrix with the osteoinductive or osteoconductive material dispersed therein.