The effects of cross‐link density and chemistry on the calcification potential of diamine‐extended glutaraldehyde‐fixed bioprosthetic heart‐valve materials

Abstract

Despite indications that GA (glutaraldehyde)-crosslinked tissues remain prone to long-term degradation and calcification, it is still the reagent of choice in the fixation of bioprosthetic heart valves. We have shown previously that increased GA concentrations and diamine extension of cross-links with lysine incorporation lead to mitigated in vivo calcification, mainly of porcine aortic-wall tissue. The present study was performed to assess the correlation between the cross-link density of all three commonly used tissue types [PW (porcine aortic wall), PL (porcine aortic leaflet) and BP (bovine pericardium)] and tissue calcification in the subcutaneous rat model after GA treatment with or without lysine. The effect of lysine enhancement, and increased GA concentration in the presence of lysine, resulted in significant increases in tissue cross-linking in all three tissue types. Although increased GA concentration on its own resulted in decreased calcification without an increase in cross-link density, overall positive correlations were found between denaturation temperature and RPD (resistance towards protease degradation) [correlation coefficient (rho) values: rhoPW =0.922, rhoPL =0.783 and rhoBP =0.955], whereas negative correlations existed between RPD and calcification (rhoPW=-0.836, rhoPL=-0.929 and rhoBP=-0.579). The combination of lysine enhancement and an increase in GA concentration from 0.2 to 3% resulted in 79, 44 and 56% decreases in calcification in PW, PL and BP. In the case of BP, a decrease in calcification of 81% could be achieved merely by adding lysine extension to low-concentration (0.2 %) GA cross-linking. Thus it is concluded that the increase in cross-link density achieved by lysine incorporation, and by increased GA concentration in the presence of lysine, results in significant and marked decreases in calcification of all three types of tissues commonly used in bioprosthetic heart valves.

A novel crosslinking method for improved tear resistance and biocompatibility of tissue based biomaterials

“…Schiff-base crosslinking [24, 31] is shown to be reversible and susceptible to hydrolysis [31, 32]. Though the reaction can be made more irreversible by using reducing agents such as sodium tetraborate [33]; such treatments are not used for clinical BHVs.…”

The performance of cross-linked acellular arterial scaffolds as vascular grafts; pre-clinical testing in direct and isolation loop circulatory models

“…Thermograms of ETVG samples (5-10mg, n=3) in sealed aluminum sample pans were obtained at a heating rate of 10°C/min (DSC 7; Perkin Elmer) and the onset temperature of the denaturation endotherm recorded as the denaturation temperature [20]. …”

Effects of dialdehyde starch on calcification of collagen matrix

“…The resistance of the sample toward protease degradation assay was used to assess the degree of crosslinking. The protocol was adapted from the method as previously reported 22, 23. Briefly, stock protease solution was prepared by dissolving 10.8 mg of Pronase E and 10.8 mg of CaCl 2 .2H 2 O in 20 mL of HEPES saline buffer consisting of 0.9% (w/v) NaCl and 0.01 M HEPES with added 0.1 M glycine.…”

“…The protocol was adapted from the method as previously reported. 22,23 Briefly, stock protease solution was prepared by dissolving 10.8 mg of Pronase E and 10.8 mg of CaCl 2 .2H 2 O in 20 mL of HEPES saline buffer consisting of 0.9% (w/v) NaCl and 0.01M HEPES with added 0.1M glycine. A known mass (3-6 mg) of lyophilized sample was incubated at 37 C for 30 h in 0.5 mL of the protease solution.…”