Multi-step Connective Tissue Stabilization Method and Stabilized Tissue Formed Thereby

• US 20150087611A1
• Filed: 09/24/2014
• Published: 03/26/2015
• Est. Priority Date: 09/24/2013
• Status: Active Grant

• Current Assignee: Clemson University
• Original Assignee: Clemson University
• Inventors: Vyavahare, Naren, Tam, Hobey
• Granted Patent: US 9,795,573 B2
• Time in Patent Office: Days
• US Class Current: 514/39
• CPC Class Codes: A01N 1/0231 Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/15 Oximes (>C=N—O—); Hydrazines (>N—N<); Hydrazones (>N—N=) ; Imines (C—N=C)
• CPC Class Codes: A61K 31/17 having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine isoureas, isothioureas A61K31/155; sulfonylureas A61K31/64
• CPC Class Codes: A61K 31/7028 Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
• CPC Class Codes: A61K 31/7036 having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
• CPC Class Codes: A61K 35/34 Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes vascular smooth muscle A61K35/44
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Abstract

A multi-step stabilization method for connective tissue is described. Stabilized tissues can exhibit increased resistance to degradation due to enzyme activity, fatigue and storage. The multi-step method includes a first step during which the tissue can be incubated with a glycosaminoglycanase inhibitor, a second step during which the tissue can be incubated with a crosslink activator and/or a phenolic compound, and a third step during which the tissue can be incubated with a second crosslink activator.

Claims

1. A method for stabilizing connective tissue comprising:

a first step including contacting the connective tissue with a glycosaminoglycanase inhibitor, the connective tissue including collagen, elastin, and one or more glycosaminoglycans;

a second step carried out subsequent to the first step, the second step including contacting the connective tissue with a first solution comprising a first crosslink activator and/or a phenolic compound comprising at least one phenolic group and a hydrophobic core;

a third step carried out subsequent to the second step, the third step including contacting the connective tissue with a second crosslink activator.

2. The method of claim 1, wherein the connective tissue is a component of a cardiac structure.

3. The method of claim 2, wherein the connective tissue is a component of a heart valve.

4. The method of claim 3, wherein the connective tissue is a component of a valve leaflet.

5. The method of claim 1, wherein the connective tissue is a component of dura mater, a tendon, a ligament, a dermal structure, a blood vessel, umbilical material, vein, pericardium, fascia, or submucosa.

6. The method of claim 1, wherein the glycosaminoglycan inhibitor is a component of a second solution and at a concentration of about 0.2 mM or greater within the second solution.

7. The method of claim 1, wherein the contact of the first step is carried out for a period of time of about 30 minutes or more.

8. The method of claim 1, wherein the glycosaminoglycan inhibitor is neomycin or a salt of neomycin.

9. The method of claim 8, wherein the glycosaminoglycan inhibitor is a sulfate salt of neomycin.

10. The method of claim 1, wherein the first crosslink activator is a carbodiimide.

11. The method of claim 10, wherein the carbodiimide is 1-ethyl-3-(3 dimethyl-aminopropyl)carbodiimide.

12. The method of claim 1, wherein the first solution comprises the crosslink activator in a concentration of about 10 millimolar or greater.

13. The method of claim 1, wherein the phenolic compound is a tannic acid or a derivative thereof.

14. The method of claim 1, wherein the phenolic compound is pentagalloylglucose.

15. The method of claim 1, wherein the first solution comprises the phenolic compound in a concentration of about 3 millimolar or greater.

16. The method of claim 1, wherein the first solution further comprises a crosslinking agent.

17. The method of claim 1, wherein the contact of the second step is carried out for a period of time of about 12 hours or more.

18. The method of claim 1, wherein the second crosslink activator is the same as the first crosslink activator.

19. The method of claim 1, wherein the second crosslink activator is a carbodiimide.

20. The method of claim 1, wherein the second crosslink activator is 1-ethyl-3-(3 dimethyl-aminopropyl)carbodiimide.

21. The method of claim 1, wherein the second crosslink activator is in a third solution at a concentration of about 10 millimolar or greater.

22. The method of claim 1, wherein the contact of the third step is carried out for a period of time of about 12 hours or more.

23. The method of claim 1, the third step further comprising contacting the connective tissue with a crosslinking agent.

24. The method of claim 1, wherein the connective tissue is in vivo tissue.

25. The method of claim 1, wherein the connective tissue is ex vivo or in vitro tissue.

26. A stabilized connective tissue comprising a glycosaminoglycanase inhibitor and a phenolic compound, the stabilized connective tissue exhibiting about 5% or less weight loss following contact with collagenase for 48 hours.

27. The stabilized connective tissue of claim 26, the tissue exhibiting about 10% or less weight loss following contact with elastase for 24 hours.

28. The stabilized connective tissue of claim 27, the tissue exhibiting a glycosaminoglycan concentration of greater than about 150 micrograms per 10 milligrams of the tissue following treatment with a glycosaminoglycanase.

29. The stabilized connective tissue of claim 26, the tissue exhibiting a glycosaminoglycan concentration of greater than about 150 micrograms per 10 milligrams of the tissue following treatment with a glycosaminoglycanase.

30. The stabilized connective tissue of claim 26, the tissue exhibiting a calcium content of about 10 parts per million calcium per milligram of dry tissue or less 30 days following implantation.

31. The stabilized connective tissue of claim 26, the tissue exhibiting a phosphorous content of about 3 parts per million phosphorous per milligram of dry tissue or less 30 days following implantation.

32. The stabilized connective tissue of claim 26, the tissue exhibiting a peak stress of about 60 kiloPascals or greater.

33. The stabilized connective tissue of claim 26, the tissue exhibiting a peak strain of about 1.1 or greater.

34. The stabilized connective tissue of claim 26, wherein the glycosaminoglycanase inhibitor is neomycin or a salt of neomycin.

35. The stabilized connective tissue of claim 26, wherein the phenolic compound is a tannic acid or a derivative thereof.

36. The stabilized connective tissue of claim 35, wherein the phenolic compound is pentagalloylglucose.

37. The stabilized connective tissue of claim 26, wherein the tissue is a component of a bioprosthetic implant.

38. The stabilized connective tissue of claim 37, wherein the bioprosthetic implant is a heart implant.

39. The stabilized connective tissue of claim 37, wherein the heart implant is a bioprosthetic heart valve.

40. The stabilized connective tissue of claim 26, wherein the tissue is a component of a heart, dura mater, a tendon, a ligament, a dermal structure, a blood vessel, umbilical material, fascia, or submucosa.