STABILIZED, STERILIZED COLLAGEN SCAFFOLDS WITH ACTIVE ADJUNCTS ATTACHED

Abstract

Bioimplants and methods of making the bioimplants are provided. The bioimplants comprise biological tissues having conjugated thereto adjunct molecules. The biological tissues are sterilized with a chemical sterilizing agent, such as a water soluble carbodiimide. The processes of making the bioimplants include a process in which an adjunct molecule is conjugated to a biological tissue during the sterilization process.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of embodiments of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 shows three different chemical reaction schemes for preparing a bioimplant of the present invention.

FIG. 2 shows two alternate chemical reaction schemes for preparing a bioimplant of the present invention.

FIG. 3 shows three additional reaction schemes according to the present invention.

FIG. 4 shows two additional alternate reaction schemes according to the present invention.

FIGS. 5A-5C are light microscopic pictures of Control (FIG. 5A), partial GAG-modified (FIG. 5B) and complete GAG-modified (FIG. 5C) pericardium. The tissue sections were stained with PAS-Alcian Blue; GAGs stain blue and collagen stains pink. These figures show the successful attachment of GAG to crosslinked and sterilized pericardial tissue by methods according to the present invention.

FIGS. 6A and 6B are low- and high-magnification images of Type 1 collagen sponge in which GAG has been attached to the collagen sponge during sterilization. As can be seen in FIGS. 6A and 6B, attachment of GAG to collagen sponge during sterilization leads to diffuse attachment of the GAG to the collagen sponge.

FIG. 7A is a picture of a histological section of cancellous bone having GAG attached thereto.

FIG. 7B is a picture of a histological section of Type I collagen sponge having GAG attached thereto during cross-linking by a method according to the present invention.

FIGS. 8A and 8B are pictures of histological sections of collagen sponge having hyaluronic acid attached thereto by a method according to the present invention.

FIG. 8C is a picture of a histological section of pericardium having hyaluronic acid attached thereto by a method according to the present invention.

FIG. 8D is a graph of results of a reverse ELISA assay for IGF-1 bound to collagen sponge by a method according to the present invention. Decreased ELISA signal in the IGF FX/STER assay versus the control assay indicates that anti-IGF antibody has bound to IGF in the IGF FX/STER sample, thus yielding a depressed ELISA signal in the anti-IGF antibody solution that has contacted the IGF FX/STER sample as compared to the signal in the anti-IGF antibody solution that has contacted the control sample.

FIG. 9 is a graph of results from a cell-viability assay. Control and chondroitin-sulfate attached collagen sponges were seeded with primary chondrocytes and incubated. Human chondrocyte-seeded collagen sponge was evaluated on day 7, while bovine chondrocyte-seeded collagen sponge was evaluated on day 14. The results of a cell viability assay (MTT) demonstrate the increased cell viability in chondroitin sulfate-attached collagen sponge (red) versus control (untreated) collagen sponge (blue).

FIGS. 10A, 10B and 10C are pictures of MTT assays of histological sections of chondrocyte-seeded GAG-attached collagen sponge according to the present invention. FIGS. 10A and 10B are low- and high-magnification images of MTT-treated GAG-attached collagen sponge seeded with chondrocytes. Viable cells are stained purple and the presence of newly synthesized matrix is seen around the cells as a thin fibrinous layer. FIG. 10C is a high-magnification image of the chondrocyte-seeded GAG-attached collagen sponge showing the appearance of newly synthesized matrix.

FIGS. 11A and 11B are histological sections of GAG-attached cellulose sponge. Selected samples of GAG-attached collagen sponge were implanted subcutaneously into rats. Explants were retrieved 4 weeks later and sections were stained with PAS-Alcian Blue (FIG. 11A) or hematoxylin and eosin (H&E; FIG. 11B). As can be seen in FIG. 11A, GAG remains attached to the cellulose matrix even after 4 weeks, as evidenced by the blue staining. The absence of overt and active inflammation and the appearance of new matrix and blood vessels between the collagen strands of the tissues (FIG. 11B) indicate a biocompatible response from the host.

Claims

1. A bioimplant comprising a chemically sterilized biological tissue and at least one adjunct, wherein the adjunct is covalently conjugated to the biological tissue.

2. The bioimplant of claim 1, wherein the chemically sterilized biological tissue is sterilized with a carbodiimide, such as EDC, optionally in the presence of an alkanol, such as a C2-C4 alkanol, especially isopropanol.

3. The bioimplant of claim 1, wherein the chemically sterilized biological tissue is crosslinked with a carbodiimide, optionally in the presence of a bifunctional crosslinking agent.

4. The bioimplant of claim 1, wherein the biological tissue comprises native tissue, processed tissue in native form, processed tissue in non-native form, a composite or a complex composite.

5. The bioimplant of claim 4, wherein: (1) said native tissue comprises bone, tendon, ligament, dermis, fascia, pericardium, and combinations thereof, including bone-connective tissue combinations, such as bone-tendon combinations and bone-ligament-bone combinations; (2) said processed tissue in native form comprises crosslinked tissue, decellularized crushed bone fragments, decellularized collagen or other decellularized and/or defatted bone, tendon, ligament, fascia or bone-connective tissue combinations, such as bone-ligament-bone or bone-tendon combinations; (3) said processed tissue in non-native form comprises solubilized or purified collagen from connective tissue, gelatin from mammals or fish or demineralized bone; (4) said composites comprise combinations of native tissues, processed tissues in native form and/or processed tissues in non-native form, such as pericardium with gelatin, bone with gelatin, purified collagen with gelatin or demineralized bone with solubilized or purified collagen; and (5) said complex composite comprises native tissue, processed tissue in native form, processed tissue in non-native form or a composite of native tissue, processed tissue in native form and/or processed tissue in non-native form with a biocompatible material such as a hydrogel, an alginate and/or chitosan.

6. The bioimplant of claim 1, wherein the adjunct is a protein, a small peptide, a ribonucleic acid, a deoxyribonucleic acid, a polysaccharide, glycosaminoglycan (GAG) or an antibiotic.

7. The bioimplant of claim 6, wherein the adjunct comprises: (1) one or more proteoglycans, glycosaminoglycans, growth factors, including any member of the transforming growth factor (TGF) superfamily and proteoglycans; (2) a deoxyribonucleic acid; (3) ribonucleic acid, such as a small interfering RNA or microRNA; (4) an antibiotic selected from aminoglycosides, the amphenicols, the ansamycins, the β-lactams, the lincosamides, the macrolides, the polypeptide antibiotics, the tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, the nitroftirans, the quinolones, the sulfonamides, the sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibernol.

8. The bioimplant of claim 1, wherein the adjunct comprises: (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) a deoxyribonucleic acid selected from genes, gene fragments and antisense DNA; (4) a ribonucleic acid such as a small interfering RNA (siRNA) or a microRNA; or (5) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, β-lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibemol.

9. The bioimplant of claim 1, wherein the biological tissue comprises collagen, purified collagen or solubilized collagen.

10. The bioimplant of claim 1 in the form of a suture, a sheet, an implantable valve, an implantable sponge or an implantable paste.

11. A process of making the bioimplant of claim 1, comprising: (a) contacting a biological tissue with an adjunct to form a combination; and(b) contacting the combination with a chemical sterilizing agent to form the bioimplant.

12. The process of claim 11, wherein the sterilizing agent is a carbodiimide, such as EDC, optionally in the presence of an alkanol, such as a C2-C4 alkanol, especially isopropanol.

13. The process of claim 11, wherein the wherein the biological tissue comprises native tissue, processed tissue in native form, processed tissue in non-native form, a composite or a complex composite.

14. The process of claim 11, wherein: (1) said native tissue comprises bone, tendon, ligament, dermis, fascia, pericardium, said native tissue comprises bone, tendon, ligament, dermis, fascia, pericardium, and combinations thereof, including bone-connective tissue combinations, such as bone-tendon combinations and bone-ligament-bone combinations; (2) said processed tissue in native form comprises crosslinked tissue, decellularized crushed bone fragments, decellularized collagen or other decellularized and/or defatted bone, tendon, ligament, fascia or bone-connective tissue combinations, such as bone-ligament-bone or bone-tendon combinations; (3) said processed tissue in non-native form comprises solubilized or purified collagen from connective tissue, gelatin from mammals or fish or demineralized bone; (4) said composites comprise combinations of native tissues, processed tissues in native form and/or processed tissues in non-native form, such as pericardium with gelatin, bone with gelatin, purified collagen with gelatin or demineralized bone with solubilized or purified collagen; and (5) said complex composite comprises native tissue, processed tissue in native form, processed tissue in non-native form or a composite of native tissue, processed tissue in native form and/or processed tissue in non-native form with a biocompatible material such as a hydrogel, an alginate and/or chitosan.

15. The process of claim 11, wherein the adjunct is a protein, a small peptide, a ribonucleic acid, a deoxyribonucleic acid, a polysaccharide, glycosaminoglycan (GAG) or an antibiotic.

16. The process of claim 15, wherein the adjunct comprises: (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) a deoxyribonucleic acid selected from genes, gene fragments and antisense DNA; (4) ribonucleic acid such as a small interfering RNA (siRNA) or a microRNA; or (5) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, β-lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibernol.

17. The process of claim 11, wherein the adjunct is (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) a deoxyribonucleic acid selected from genes, gene fragments and antisense DNA; (3) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, β-lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibemol.

18. The process of claim 11, wherein the biological tissue comprises collagen, purified collagen or solubilized collagen.

19. The process of claim 11, further comprising shaping or forming the biological tissue into the form of a suture, a sheet, an implantable valve, an implantable sponge or an implantable paste.

20. A bioimplant made by a process comprising contacting a biological tissue with an adjunct molecule in the presence of a sterilizing agent.

21. A process of making a sterilized biological implant, comprising: (a) contacting a starting tissue with an adjunct to form an intermediate;(b) freezing the intermediate product of (a) to produce a frozen intermediate;(c) lyophilizing the frozen intermediate from (b) to produce a lyophilized intermediate; and(d) contacting the lyophilized intermediate with a sterilizing solution comprising a carbodiimide sterilizing agent to produce the biological implant.

22. The process of claim 21, wherein said sterilizing agent is a carbodiimide, such as EDC, optionally in the presence of an alkanol, such as a C2-C4 alkanol, especially isopropanol.

23. The process of claim 21, wherein the starting tissue is a native tissue, a processed tissue in native form or a composite tissue.

24. The process of claim 23, wherein the starting tissue is: (1) a native tissue comprising bone, tendon, ligament, dermis, fascia, pericardium, and combinations thereof, including bone-connective tissue combinations, such as bone-tendon combinations and bone-ligament-bone combinations; (2) said processed tissue in native form comprises crosslinked tissue, decellularized crushed bone fragments, decellularized collagen or other decellularized and/or defatted bone, tendon, ligament, fascia or bone-connective tissue combinations, such as bone-ligament-bone or bone-tendon combinations; or (3) a composite comprising combinations of native tissues, processed tissues in native form and/or processed tissues in non-native form, such as pericardium with gelatin, bone with gelatin, purified collagen with gelatin or demineralized bone with solubilized or purified collagen.

25. The process of claim 21, wherein the adjunct is a protein, a small peptide, a ribonucleic acid, a deoxyribonucleic acid, a polysaccharide, glycosaminoglycan (GAG) or an antibiotic.

26. The process of claim 25, wherein the adjunct comprises: (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) a deoxyribonucleic acid selected from genes, gene fragments and antisense DNA; (4) ribonucleic acid such as a small interfering RNA (siRNA) or a microRNA; or (5) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, β-lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibernol.

27. The process of claim 21, wherein the adjunct is (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, β-lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibemol.

28. The process of claim 21, wherein the biological tissue comprises collagen, purified collagen or solubilized collagen.

29. The process of claim 21, further comprising shaping or forming the biological tissue into the form of a suture, a sheet, an implantable valve, an implantable sponge or an implantable paste.

30. A bioimplant produced by the process of claim 21.

31. A process of making a sterilized biological implant, comprising: (a) preparing a composition comprising a starting tissue;(b) freezing the composition from (a) to form a frozen composition;(c) lyophilizing the frozen composition from (b) to form a lyophilized composition; and(d) contacting the lyophilized composition from (c) with a sterilizing solution comprising a sterilizing agent and an adjunct to produce the biological implant.

32. The process of claim 31, wherein said sterilizing agent is a carbodiimide, such as EDC, optionally in the presence of an alkanol, such as a C2-C4 alkanol, especially isopropanol.

33. The process of claim 31, wherein the starting tissue is a processed tissue in native form or a complex composite.

34. The process of claim 33, wherein the starting tissue is: (1) a processed tissue in native form comprising crosslinked tissue, decellularized crushed bone fragments, decellularized collagen or other decellularized and/or defatted bone, tendon, ligament, fascia or bone-connective tissue combinations, such as bone-ligament-bone or bone-tendon combinations; or (2) complex composite comprising native tissue, processed tissue in native form, processed tissue in non-native form or a composite of native tissue, processed tissue in native form and/or processed tissue in non-native form with a biocompatible material such as a hydrogel, an alginate and/or chitosan.

35. The process of claim 31, wherein the adjunct is a protein, a small peptide, a ribonucleic acid, a deoxyribonucleic acid, a polysaccharide, glycosaminoglycan (GAG) or an antibiotic.

36. The process of claim 35, wherein the adjunct comprises: (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) a deoxyribonucleic acid selected from genes, gene fragments and antisense DNA; (4) ribonucleic acid such as a small interfering RNA (siRNA) or a microRNA; or (5) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, β-lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibernol.

37. The process of claim 31, wherein the adjunct is (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) a deoxyribonucleic acid selected from genes, gene fragments and antisense DNA; (3) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, β-lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibernol.

38. The process of claim 31, wherein the biological tissue comprises collagen, purified collagen or solubilized collagen.

39. The process of claim 31, further comprising shaping or forming the biological tissue into the form of a suture, a sheet, an implantable valve, an implantable sponge or an implantable paste.

40. A biological implant produced by the process of claim 31.

41. A process of making a sterilized biological implant, comprising: (a) contacting a starting tissue with a crosslinking agent to at least partially crosslink the starting tissue to produce a crosslinked tissue; and(b) contacting the crosslinked tissue from (a) with a sterilizing solution comprising a sterilizing agent and an adjunct to produce the biological implant.

42. The process of claim 41, wherein said sterilizing agent is a carbodiimide, such as EDC, optionally in the presence of an alkanol, such as a C2-C4 alkanol, especially isopropanol.

43. The process of claim 41, wherein the starting tissue is a native tissue or a processed tissue in native form.

44. The process of claim 43, wherein the starting tissue is: (1) a native tissue comprising bone, tendon, ligament, dermis, fascia, pericardium, or combinations thereof, including bone-connective tissue combinations, such as bone-tendon combinations and bone-ligament-bone combinations; or (2) a processed tissue in native form comprising crosslinked tissue, decellularized crushed bone fragments, decellularized collagen or other decellularized and/or defatted bone, tendon, ligament, fascia or bone-connective tissue combinations, such as bone-ligament-bone or bone-tendon combinations.

45. The process of claim 41, wherein the adjunct is a protein, a small peptide, a ribonucleic acid, a deoxyribonucleic acid, a polysaccharide, glycosaminoglycan (GAG) or an antibiotic.

46. The process of claim 45, wherein the adjunct comprises: (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) a deoxyribonucleic acid selected from genes, gene fragments and antisense DNA; (4) ribonucleic acid such as a small interfering RNA (siRNA) or a microRNA; or (5) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, β-lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibernol.

47. The process of claim 41, wherein the adjunct is (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) a deoxyribonucleic acid selected from genes, gene fragments and antisense DNA; (4) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, β-lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibemol.

48. The process of claim 41, wherein the biological tissue comprises collagen, purified collagen or solubilized collagen.

49. The process of claim 41, further comprising shaping or forming the biological tissue into the form of a suture, a sheet, an implantable valve, an implantable sponge or an implantable paste.

50. A bioimplant produced by the process of claim 41.

51. A process of making a sterilized biological implant, comprising: (a) contacting a starting tissue with a crosslinking agent and an adjunct to at least partially crosslink the starting tissue to produce a crosslinked tissue adjunct-conjugated tissue; and(b) contacting the crosslinked tissue from (a) with a sterilizing solution comprising a sterilizing agent to produce the biological implant.

52. The process of claim 51, wherein said sterilizing agent is a carbodiimide, such as EDC, optionally in the presence of an alkanol, such as a C2-C4 alkanol, especially isopropanol.

53. The process of claim 51, wherein the starting tissue is a processed tissue in non-native form.

54. The process of claim 53, wherein the starting tissue is a processed tissue in non-native form comprising solubilized or purified collagen from connective tissue, gelatin from mammals or fish or demineralized bone.

55. The process of claim 51, wherein the adjunct is a protein, a small peptide, a ribonucleic acid, a deoxyribonucleic acid, a polysaccharide, glycosaminoglycan (GAG) or an antibiotic.

56. The process of claim 55, wherein the adjunct comprises: (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) a deoxyribonucleic acid selected from genes, gene fragments and antisense DNA; (4) ribonucleic acid such as a small interfering RNA (siRNA) or a microRNA; or (5) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, #lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibernol.

57. The process of claim 51, wherein the adjunct is (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) a deoxyribonucleic acid selected from genes, gene fragments and antisense DNA; (3) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, β-lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibernol.

58. The process of claim 51, wherein the biological tissue comprises collagen, purified collagen or solubilized collagen.

59. The process of claim 51, further comprising shaping or forming the biological tissue into the form of a suture, a sheet, an implantable valve, an implantable sponge or an implantable paste.

60. A bioimplant produced by the process of claim 51.

61. A process of making a sterilized biological implant, comprising: (a) contacting a starting tissue with a sterilizing solution comprising a sterilizing agent to produce a sterilized intermediate;(b) contacting the sterilized intermediate from (a) with an adjunct and a conjugating agent to produce an implant; and(c) optionally subjecting the implant to another sterilization step; whereby a sterilized biological implant is produced.

62. The process of claim 61, wherein said sterilizing agent is a carbodiimide, such as EDC, optionally in the presence of an alkanol, such as a C2-C4 alkanol, especially isopropanol.

63. The process of claim 61, wherein the starting tissue is a processed tissue in non-native form.

64. The process of claim 63, wherein the starting tissue is: (1) a native tissue, such as bone, tendon, ligament, dermis, fascia, pericardium, and combinations thereof, including bone-connective tissue combinations, such as bone-tendon combinations and bone-ligament-bone combinations; (2) a processed tissue in non-native form, such as solubilized or purified collagen from connective tissue, gelatin from mammals or fish or demineralized bone; or (3) combinations of native tissues, processed tissues in native form and/or processed tissues in non-native form, such as pericardium with gelatin, bone with gelatin, purified collagen with gelatin or demineralized bone with solubilized or purified collagen.

65. The process of claim 61, wherein the adjunct is a protein, a small peptide, a ribonucleic acid, a deoxyribonucleic acid, a polysaccharide, glycosaminoglycan (GAG) or an antibiotic.

66. The process of claim 65, wherein the adjunct comprises: (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) a deoxyribonucleic acid selected from genes, gene fragments and antisense DNA; (4) ribonucleic acid such as a small interfering RNA (siRNA) or a microRNA; or (5) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, β-lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibernol.

67. The process of claim 61, wherein the adjunct is (1) one or more proteoglycans or glycosaminoglycans, (2) one or more proteins, such as: (a) any member of the Transforming Growth Factor (TGF) superfamily, such as BMP-2, BMP-4 and BMP-7, transforming growth factor-β (TGF-β); (b) platelet derived growth factor (PDGF); (c) fibroblast growth factor (FGF); (d) insulin-like growth factors (IGF); (e) cartilage-derived growth factors (CDGF); (3) a deoxyribonucleic acid selected from genes, gene fragments and antisense DNA; (4) an antibiotic, such as one or more aminoglycosides, amphenicols, ansamycins, β-lactams, lincosamides, macrolides, polypeptide antibiotics, tetracyclines, cycloserine, mupirocin, tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides, sulfones, clofoctol, hexedine, methenamine, nitroxoline, taurolidine and xibemol.

68. The process of claim 61, wherein the biological tissue comprises collagen, purified collagen or solubilized collagen.

69. The process of claim 61, further comprising shaping or forming the biological tissue into the form of a suture, a sheet, an implantable valve, an implantable sponge or an implantable paste.

70. A bioimplant produced by the process of claim 61.

71. A bioimplant of claim 1, wherein the adjunct retains at least some of its native activity after it has been conjugated to the biological tissue.

72. A bioimplant of claim 1, wherein the adjunct is adapted to be released in vivo and the adjunct, once release in vivo possesses at least some of its native activity.