In vitro evaluation of the genotoxicity of a naturally occurring crosslinking agent (genipin) for biologic tissue fixation

Abstract

The objective of the present study was to evaluate in vitro, using Chinese hamster ovary (CHO-K1) cells, the genotoxicity of genipin, a naturally occurring crosslinking agent. Glutaraldehyde, the most commonly used crosslinking agent for biologic tissue fixation, was employed as a reference chemical. The selected procedures for this evaluation were the micronucleus (MN) and sister chromatid exchange (SCE) assays with or without the addition of a metabolic activation system (S9 mix). Before starting the genotoxicity assays, the maximum noncytotoxic amounts of glutaraldehyde and genipin were determined using the MTT assay. The results obtained in the MTT assay revealed that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde with or without S9 mix. The frequencies of MN observed in the cases drugged with varying concentrations of glutaraldehyde or genipin were not statistically different from those seen in the negative controls (blank) in the presence or absence of S9 mix. However, it was noted that glutaraldehyde significantly inhibited the cell-cycle progression while the cells drugged with genipin did not result in cell-cycle delay. In the SCE assay, the numbers of SCE per cell observed in the cases drugged with varying concentrations of glutaraldehyde were significantly greater than those found in the negative controls with or without S9 mix. Nevertheless, these numbers were still low compared to the numbers of SCE induced by the strong mutagens used as our positive control substances. This suggests that glutaraldehyde may produce a weakly clastogenic response in CHO-K1 cells. In contrast, the numbers of SCE per cell obtained in the cases drugged with genipin were comparable to those observed in the negative controls in those that were except drugged with the highest dose (50 ppm). This suggests that genipin does not cause clastogenic response in CHO-K1 cells provided its concentration is lower than 50 ppm. In conclusion, as far as cytotoxicity and genotoxicity are concerned, genipin is a promising crosslinking agent for biologic tissue fixation.

Compliance-induced adherens junction formation in epithelial cells and tissues is regulated by JNK

“…In agreement with previous studies demonstrating genipin biocompatibility (Tsai et al, 2000), we found that the crosslinked substrate could be used to generate bioengineered epidermis of similar thickness as control dermis. Mechanical measurements showed that crosslinking increased the dermal substrate stiffness by threefold.…”

Micro/nanoscale technologies for the development of hormone-expressing islet-like cell clusters

“…Crosslinkers such as glutaraldehyde have been used previously to increase the stability of natural polymer scaffolds in different tissue engineering applications; however, this practice has been postulated to have negative repercussions (in vitro and in vivo) in some cases, including geno/cytotoxicity and graft failure (Heydarkhan-Hagvall et al 2008;Tsai et al 2000). The blending of synthetic and natural polymers has been viewed as a non-toxic alternative to the use of chemical cross-linkers (Heydarkhan-Hagvall et al 2008).…”

Collagen- vs. Gelatine-Based Biomaterials and Their Biocompatibility: Review and Perspectives

“…In reaction mechanism between gelatin and tannin are involved hydrogen bonds between hydroxyl groups of tannin and polar groups of gelatin, and hydrophobic interactions between pyrrolidine ring of Pro and pentagalloyl glucose from tannin (Obreque-Slier et al, 2010;Pena et al, 2010). Anti-inflammatory properties are added values during genipin-induced cross-linking, showing to be 10, 000 times less cytotoxic then glutaraldehide which may produce weakly clastogenic responces in CHO-K1 cells (Tsai et al, 2000;Sisson et al, 2009). Moreover, the minimal calcium content of genipin-fixed tissue was detected (Chang, 2001).…”