Multi-step connective tissue stabilization method and stabilized tissue formed thereby
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 such as a sulfated oligosaccharide, one example of which being neomycin, a second step during which the tissue can be incubated with a crosslink activator such as a carbodiimide crosslink activator and/or a crosslinking agent such as a heterobifunctional crosslinking agent and/or a phenolic compound such as a tannin, examples of which include tannic acid and pentagalloylglucose, and a third step during which the tissue can be incubated with a second crosslink activator that can be the same or different as the first crosslink activator.
1. A method for stabilizing ex vivo or in vitro a cardiac/heart valve connective tissue comprising:
a first step including contacting the ex vivo or in vitro connective tissue with a neomycin trisulfate salt, 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 ex vivo or in vitro connective tissue with a solution comprising a first carbodiimide crosslinking reagent and pentagalloylglucose;
a third step carried out subsequent to the second step, the third step including contacting the ex vivo or in vitro connective tissue with a second carbodiimide crosslinking reagent.
2. The method of claim 1 , wherein the ex vivo or in vitro connective tissue comprises a heart valve leaflet.
3. The method of claim 1 , wherein the neomycin trisulfate salt is provided at a concentration of about 0.2 mM or greater.
4. 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.
5. The method of claim 1 , wherein the first carbodiimide crosslinking reagent comprises 1-ethyl-3-(3 dimethyl-aminopropyl) carbodiimide.
6. The method of claim 5 , the solution comprising the first carbodiimide crosslinking reagent and pentagalloylglucose further comprising N-hydroxysuccinimide.
7. The method of claim 1 , wherein the first carbodiimide crosslinking reagent is provided at a concentration of about 10 millimolar or greater.
8. The method of claim 1 , wherein the solution comprises the pentagalloylglucose at a concentration of about 3 millimolar or greater.
9. 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.
10. The method of claim 1 , wherein the second carbodiimide crosslinking reagent is the same as the first carbodiimide crosslinking reagent.
11. The method of claim 1 , wherein the second carbodiimide crosslinking reagent comprises 1-ethyl-3-(3 dimethyl-aminopropyl) carbodiimide.
12. The method of claim 1 , wherein the second carbodiimide-crosslinking reagent is provided at a concentration of about 10 millimolar or greater.
13. 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.
14. A stabilized ex vivo or in vitro connective heart valve tissue comprising a neomycin trisulfate salt and a pentagalloylglucose.
15. The stabilized ex vivo or in vitro connective tissue of claim 14 , the tissue having a glycosaminoglycan concentration of greater than about 150 micrograms per 10 milligrams of the tissue.
16. The stabilized connective tissue of claim 14 , wherein the tissue is a component of a bioprosthetic implant.
17. The stabilized connective tissue of claim 16 , wherein the bioprosthetic implant is a heart component implant.
18. The stabilized connective tissue of claim 17 , wherein the heart component implant is a bioprosthetic heart valve.