In vitro evaluation of cytotoxicity of a naturally occurring cross-linking reagent for biological tissue fixation

A recognized drawback of the currently available chemical cross-linking reagents used to fix bioprostheses is the potential toxic effects a recipient may be exposed to from the fixed tissues and/or the residues. It is, therefore, desirable to provide a cross-linking reagent which is of low cytotoxicity and may form stable and biocompatible cross-linked products. To achieve this goal, a naturally occurring cross-linking reagent - genipin - which has been used in herbal medicine and in the fabrication of food dyes, was used by our group to fix biological tissues. The study was to assess the cytotoxicity of genipin in vitro using 3T3 fibroblasts (BALB/3T3 CIA31-1-1). Glutaraldehyde, the most commonly used cross-linking reagent for tissue fixation, was used as a control. The cytotoxicity of the glutaraldehyde- and genipin-fixed tissues and their residues was also evaluated and compared. The observation in the light microscopic examination revealed that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde. Additionally, the results obtained in the MTT assay implied that genipin was about 10000 times less cytotoxic than glutaraldehyde. Moreover, the colony forming assay suggested that the proliferative capacity of cells after exposure to genipin was approximately 5000 times greater than that after exposure to glutaraldehyde. It was noted that the cells seeded on the surface of the glutaraldehyde-fixed tissue were not able to survive. In contrast, the surface of the genipin-fixed tissue was found to be filled with 3T3 fibroblasts. Additionally, neocollagen fibrils made by these fibroblasts were observed on the genipin-fixed tissue. This fact suggested that the cellular compatibility of the genipin-fixed tissue was superior to its glutaraldehyde-fixed counterpart. Also, the residues from the glutaraldehyde-fixed tissue markedly reduced the population of the cultured cells, while those released from the genipin-fixed tissue had no toxic effect on the seeded cells. In conclusion, as far as cytotoxicity is concerned, genipin is a promising cross-linking reagent for biological tissue fixation.