The role of glutaraldehyde-induced cross-links in calcification of bovine pericardium used in cardiac valve bioprostheses

Calcification is the principal cause of failure of tissue-derived cardiac valve replacements pretreated with glutaraldehyde (GLUT). The objective of this study was to determine the role of GLUT-induced cross-links in bovine pericardial tissue calcification. Various levels of ³H-GLUT incorporation were obtained by varying incubation pH, and protein modification was determined by amino acid analysis and resistance to collagenase digestion. Calcification of cross-linked tissue was studied using subdermal implants in rats. Low GLUT uptake (< 150 nm/mg) resulted in minimal calcification (Ca²⁺, 12.8 μg/mg) and stability (4% residual weight following digestion) due to a limited crosslinking (lysine + hydroxylysine = 26.1 residues/1000 amino acids [AA]). In contrast, higher GLUT uptake induced more cross-links (Lys + Hyl = 8.2 residues/1000 AA) and consequent higher stability (95% residual wt); such tissues calcified severely (Ca²⁺, 93.5 μg/mg). Incorporation of GLUT two or three times beyond a critical level did not further enhance calcification. It is concluded that the amount of GLUT incorporated controls the extent of cross-links, which in turn directly determines tissue stability and calcification.