Crosslinking of tissue-derived biomaterials in 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)

Abstract

In contrast to bifunctional reagents such as glutaraldehyde or polyfunctional reagents such as polyepoxides, carbodiimides belong to the class of zero-length crosslinkers which modify amino acid side-groups to permit crosslink formation, but do not remain as part of that linkage. The authors have compared the effects of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and glutaraldehyde (the de facto industrial standard crosslinker) on the hydrothermal, biochemical, and uniaxial mechanical properties of bovine pericardium. EDC crosslinking was optimized for maximum increase in collagen denaturation temperature using variables of pH, concentration, and ratio of EDC to N-hydroxysuccinimide (NHS): a reagent for formation of activated esters. EDC and glutaraldehyde crosslinked materials were subjected to hydrothermal denaturation tests, biochemical degradation by enzymes (collagenase, trypsin) and CNBr, amino acid analysis for unreacted lysine, and to high strain rate mechanical tests including: large deformation stress-strain studies (0.1 to 10 Hz), stress relaxation experiments (loading time 0.1 s) and small deformation forced vibration (1 and 10 Hz). A protocol for EDC crosslinking was developed which used 1.15% EDC (2:1 EDC:NHS) at pH 5.5 for 24 h. The increase in denaturation temperature for EDC (from 69.7±1.2°C to 86.0±0.3°C) was equivalent to that produced by glutaraldehyde (85.3±0.4°C). Both treatments equivalently increased resistance to collagenase and CNBr degradation; however, after denaturation, the EDC-treated tissue was slightly more resistant to collagenase, and markedly more resistant to trypsin. EDC-treated materials were more extensible and more elastic than glutaraldehyde-treated materials. Despite the differences in crosslinking mechanism, EDC and glutaraldehyde-treated materials are very similar. Subtle but intriguing differences in biochemical structure remain to be investigated.