The study was undertaken to investigate the stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin) at distinct elapsed storage durations. The glutaraldehyde-fixed counterpart was used as a control. Porcine pericardia procured from a slaughterhouse were used as raw materials. After fixation, the fixed tissues were sterilized in a graded series of ethanol solutions and thoroughly rinsed in phosphate buffered saline for 1 day, and then stored in a jar containing sterilized water. The samples were taken out and tested for their stability during the durations of 1day through 6 months after storage. The stability of each study group was tested by measuring its tensile strength, free-amino-group content, and denaturation temperature. Additionally, the cytotoxicity of each test sample and its corresponding storage solution were investigated in vitro using 3T3 fibroblasts. The results were examined using a microscope and 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was found that the stability of the genipin-fixed tissue during storage was superior to its glutaraldehyde-fixed counterpart. The differences in stability between the genipin- and glutaraldehyde-fixed tissues during storage may be caused by their differences in crosslinking structure. There was no apparent cytotoxicity for both the genipin-fixed tissue and its corresponding storage solution throughout the entire course of the study, whereas significant cytotoxicity was observed for both the glutaraldehyde-fixed tissue and its storage solution. However, the cytotoxicity of the glutaraldehyde-fixed tissue decreased with increasing elapsed storage duration, whereas that of its corresponding storage solution increased. This suggested that the toxic residues remaining in the glutaraldehyde-fixed tissue leached out slowly into its corresponding storage solution during the course of storage.

In an attempt to overcome the cytotoxicity problem of the glutaraldehyde-fixed tissues, a naturally occurring crosslinking agent (genipin) was used by our group to fix biological tissues. The study was intended to investigate the rate of tissue fixation by genipin. Glutaraldehyde was used as a control. In addition, the degrees of tissue fixation by genipin at different pHs (pH 4.0, pH 7. 4, pH 8.5, or pH 10.5), temperatures (4 degrees C, 25 degrees C, 37 degrees C, or 45 degrees C), and initial fixative concentrations (0.250%, 0.625%, or 1.000%) were examined. The results obtained revealed that the rate of tissue fixation by glutaraldehyde was significantly faster than that by genipin. The degree of tissue fixation by genipin may be controlled by adjusting its fixation duration or fixation conditions. The order in degree of tissue fixation by genipin at different pHs, from high to low, was: at nearly neutral pH (pH 7.4 or pH 8.5) > at basic pH (pH 10.5) > at acidic pH (pH 4.0). The degrees of tissue fixation by genipin at different temperatures were about the same, except for that at 4 degrees C. In contrast, the initial fixative concentration did not seem to affect the degree of tissue fixation by genipin, if only the amount of genipin in the fixation solution was sufficient to complete tissue fixation. The concentrations of genipin in the aqueous solutions at different pHs, temperatures, and initial fixative concentrations tended to decrease with time with or without the occurrence of tissue fixation. This indicated that genipin was not stable in the aqueous solution. The instability of aqueous genipin was more remarkable with increasing pH or temperature. The results obtained in this study may be used to optimize the fixation process for developing bioprostheses fixed by genipin.

Bioadhesives have been used in surgery as hemostatic and wound healing agents. GRF (gelatin + resorcinol + formaldehyde) glue, composed of a mixture of gelatin and resorcinol polymerized by the addition of formaldehyde, has been used for this purpose. Widespread acceptance of the GRF glue, however, has been limited by reports of cytotoxicity due to its release of formaldehyde upon degradation. It has been suggested by Wertzel et al. that the cytotoxicity problem of GRF glue may be overcome by changing its cross-linking method. The study was, therefore, undertaken to assess the feasibility of using a water-soluble carbodiimide or genipin to cross-link gelatin as new bioadhesives to close skin wound lesions in a rat model. Formaldehyde-cross-linked counterpart (GRF glue) and a resorbable suture were used as controls. It was noted that the tensile strength of the skin across each wound treated by either application of test glues or suture increased consistently with time during the healing process. Also, the wounds repaired by test glues or suture caused no calcification. The suture used in the study was completely resorbed at the wound area in about 6 days postoperatively. However, the durations required to completely resorb the carbodiimide- or genipin-cross-linked glues were approximately the same (9 days), while it took about 14 days to completely resorb the formaldehyde-cross-linked glue. The healing process for the suture wound repaired was more rapid than those treated by test glues. Of the test glues, the wounds treated by the carbodiimide- or genipin-cross-linked glues induced less inflammatory response and recovered sooner than that treated by the formaldehyde-cross-linked glue. This indicated that the biocompatibility of the carbodiimide- or genipin-cross-linked glues was superior to the formaldehyde-cross-linked glue. The results of this study may serve as a preliminary experimental model for the further investigation of both the carbodiimide- and genipin-cross-linked glues when applied to human skin closure.