Feasibility study of a natural crosslinking reagent for biological tissue fixation

Bioprostheses derived from biological tissues must be chemically modified and subsequently sterilized before they can be implanted in humans. Various crosslinking reagents, including formaldehyde, glutaraldehyde, dialdehyde starch, and epoxy compound, have been used to chemically modify biological tissues. However, these synthetic crosslinking reagents are all highly (or relatively highly) cytotoxic. It is therefore desirable to provide a crosslinking reagent suitable for use in biomedical applications that is of low cytotoxicity and that forms stable and biocompatible crosslinked products. This study evaluates the feasibility of using a naturally occurring crosslinking reagent--genipin--to chemically modify biological tissues. Genipin and its related iridoid compounds, extracted from gardenia fruits, have been used in traditional Chinese medicine for the treatments of jaundice and various inflammatory and hepatic diseases. In this feasibility study, the cytotoxicity of genipin and the crosslinking characteristics of genipin-fixed biological tissues were investigated. Fresh porcine pericardia procured from a slaughterhouse were used as raw materials. Glutaraldehyde and an epoxy compound (ethylene glycol diglycidyl ether), which has been used extensively in developing bioprostheses, were used as controls. It was found that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde and the epoxy compound. The amino acid residues in the porcine pericardium that may react with genipin were lysine, hydroxylysine, and arginine. Additionally, the genipin-fixed tissue had a mechanical strength and resistance against enzymatic degradation comparable to the glutaraldehyde-fixed tissue. This suggests that genipin can form stable crosslinked products. The results of this in vitro study demonstrate that genipin is an effective crosslinking reagent for biological tissue fixation.