Gelatinases in soft tissue biomaterials. Analysis of different crosslinking agents

Chemical modification of pericardium-based cardiac valves tends to reduce the relatively high degree of biodegradation and calcification of the implanted bioprostheses. We analysed the tissue properties of pericardium from young calves and pigs after crosslinking with different agents (glutaraldehyde. diphenylphosphorylazide (DDPA), 1-ethyl-3,3-dimethyl-aminopropyl-carbodiimide (EDAC)) and when exposed to anticalcification treatments (chloroform/methanol or ethanol) prior to glutaraldehyde (GA) crosslinking. Protein extraction after tissue homogenisation in the presence of detergents showed that crosslinking using GA or DPPA was much more effective. The amounts of protein extracted from these two groups of chemically modified pericardium were significantly lower: the other modified tissues presented only a slight reduction when compared with untreated tissue. Matrix metalloproteinases- (MMP) 2 and 9 were detected in native pericardium from calf and pig by zymography. While the MMP-9/MMP-2 activity ratio was close to 1 in pig pericardium, it was 8.5-fold higher in bovine tissue. Crosslinking with GA and with DPPA almost completely abolished gelatinase activities, even when equal amounts of solubilised protein were loaded onto the zymograms. Anticalcification treatments followed by GA crosslinking or treatment with EDAC were not as effective in reducing gelatinase activities; but, interestingly, a relative reduction of MMP-9 versus MMP-2 was detected. The presence of these gelatinase activities in pericardium may contribute to the in vivo degradability of pericardium-based cardiac valves.