Fixation of biological tissues with a naturally occurring crosslinking agent: Fixation rate and effects of pH, temperature, and initial fixative concentration

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°C, 25°C, 37°C, or 45°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°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. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 52, 77–87, 2000.