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Currently available crosslinking agents used in fixing bioprostheses are all highly (or relatively highly) cytotoxic, which may induce an adverse inflammatory reaction in vivo. It is therefore desirable to provide a crosslinking agent that is of low cytotoxicty and may form stable and biocompatible crosslinked products. To achieve this goal, a naturally occurring crosslinking agent-genipin-was used by our group to fix biological tissues. Genipin may be obtained from its parent compound, geniposide, which may be isolated from the fruits of Gardenia jasminoides Ellis. In our previous studies, it was found that the cytotoxicity of genipin is significantly lower than both glutaraldehyde and an epoxy compound. Also, it was shown that genipin can form stable and biocompatible crosslinked products. The present study further investigates the crosslinking characteristics and mechanical properties of a genipin-fixed bovine pericardium. Fresh and glutaraldehyde- and epoxy-fixed counterparts were used as controls. It was found that the denaturation temperatures of the glutaraldehyde- and genipin-fixed tissues were significantly greater than the epoxy-fixed tissue, although their fixation indices were comparable. The mechanical properties of fresh bovine pericardium are anisotropic. However, fixation tended to eliminate tissue anisotropy. The tendency in the elimination of tissue anisotropy for the genipin-fixed tissue was more remarkable than for the glutaraldehyde- and epoxy-fixed tissues. In addition, the genipin-fixed tissue had the greatest ultimate tensile strength and toughness among all the fixed tissues. Distinct patterns in rupture were observed in the study: The torn collagen fibers of the genipin- and glutaraldehyde-fixed tissues appeared to be bound together, while those of fresh and the epoxy-fixed tissues stayed loose. The results obtained in the study suggests that tissue fixation in glutaraldehyde, epoxy compound, and genipin may produce distinct crosslinking structures. The differences in crosslinking structure may affect the crosslinking characteristics and mechanical properties of the fixed tissues.

It was suggested in our previous studies that carbodiimide-and genipin-crosslinked gelatin hydrogels could be used as bioadhesives to overcome the cytotoxicity problem associated with formaldehyde-crosslinked gelatin hydrogels. In this study, we investigated the crosslinking structures of carbodiimide-and genipin-crosslinked gelatin hydrogels. We found that crosslinking gelatin hydrogels with carbodiimide or genipin could produce distinct crosslinking structures because of the differences in their crosslinking types. Carbodiimide could form intramolecular crosslinks within a gelatin molecule or short-range intermolecular crosslinks between two adjacent gelatin molecules. On the basis of gel permeation chromatography, we found that the polymerization of genipin molecules could occur under the conditions used in crosslinking gelatin hydrogels via a possible aldol condensation. Therefore, besides intramolecular and short-range intermolecular crosslinks, additional long-range intermolecular crosslinks could be introduced into genipin-crosslinked gelatin hydrogels. Crosslinking a gelatin hydrogel with carbodiimide was more rapid than crosslinking with genipin. Therefore, the gelation time for the carbodiimide-crosslinked gelatin hydrogels was significantly shorter than that of the genipin-crosslinked gelatin hydrogels. However, the cohesive (interconnected) structure of the carbodiimide-crosslinked gelatin hydrogels was readily broken because, unlike the genipin-crosslinked gelatin hydrogels, there were simply intramolecular and shortrange intermolecular crosslinks present in the carbodiimidecrosslinked hydrogel. In the cytotoxicity study, the carbodiimide-crosslinked gelatin hydrogels were dissolved into small fragments in the cultural medium within 10 min. In contrast, the genipin-crosslinked gelatin hydrogels remained intact in the medium throughout the entire course of the study. Again, this may be attributed to the differences in their crosslinking structures. The genipin-crosslinked gelatin hydrogels were less cytotoxic than the carbodiimidecrosslinked gelatin hydrogels.

Gelatin microspheres have been widely evaluated as a drug carrier. Nevertheless, gelatin dissolves rather rapidly in aqueous environments, making the use of the polymer difficult for the production of long-term delivery systems. This adverse aspect requires the use of a crosslinking agent in forming nonsoluble networks in microspheres. However, the use of crosslinking agents such as formaldehyde and glutaraldehyde can lead to toxic side effects owing to residual crosslinkers. In an attempt to overcome this problem, a naturally occurring crosslinking agent (genipin) was used to crosslink gelatin microspheres as a biodegradable drug-delivery system for intramuscular administration. Glutaraldehyde was used as a control. In the in vitro study, the morphology, dynamic swelling, and antienzymatic degradation of test microspheres were evaluated. In the in vivo study, the biocompatibility and degradability of test microspheres were implanted in the skeletal muscle of a rat model via intramuscular injection. The results obtained in the study suggested that crosslinking of gelatin microspheres with glutaraldehyde or genipin may produce distinct crosslinking structures. The water transport mechanism in both the glutaraldehyde- and genipin-crosslinked gelatin microspheres exhibit anomalous behavior ranging from Fickian to Case-II extremes. The increase of the swelling diameter for the genipin-crosslinked microspheres was significantly less than that observed for the glutaraldehyde-crosslinked microspheres. In the animal study, it was found that the degree in inflammatory reaction for tissues implanted with the genipin-crosslinked microspheres was significantly less than that implanted with the glutaraldehyde-crosslinked microspheres. Additionally, the degradation rate of the genipin-crosslinked microspheres was significantly slower than their glutaraldehyde-crosslinked counterparts. These results indicated that the genipin-crosslinked gelatin microspheres may be used as a long-acting drug carrier for intramuscular administration.