| dc.description.abstract | Bioprostheses derived from biological tissues can be used to repair or replace defect tissues or organs in the clinical applications. These biological tissues, however, must be chemically modified by a crosslinking agent before they can be implanted in humans. Various synthetic crosslinking agents have been used in modifying biological tissues. Nevertheless, these synthetic crosslinking agents are all highly cytotoxic. It is therefore desirable to provide a crosslinking agent for use in biomedical applications that is of low cytotoxicity and that forms stable and biocompatible crosslinked products. In an attempt to achieve this goal, a naturally occurring crosslinking agent (genipin) was used by our group to fix biological tissues. Genipin and its related iridoid glucosides extracted from the fruits of Gardenia jasminoides ELLIS have been widely used as an antiphlogistic and cholagogue in herbal medicine. In this dissertation, we present the results obtained in our feasibility, cytotoxicity, genotoxicity, and biocompatibility (cellular tissue, acellular tissue, and valved conduit) studies of this naturally occurring crosslinking agent for biological tissue fixation.
In the study, we successfully purified a naturally occurring crosslinking reagent (genipin) from gardenia fruits, a traditional Chinese medicine. The purity of genipin obtained was approximately 98%.
In the feasibility study, the crosslinking characteristics of the genipin-fixed biological tissues was investigated. It was found that the amino acid residues in the porcine pericardium that may react with genipin were lysine, hydroxylysine, and arginine. Additionally, the genipin-fixed tissue had comparable mechanical strength and resistance against enzymatic degradation as the glutaraldehyde-fixed tissue. This suggested that genipin can form stable crosslinked products. The results of this in vitro study demonstrated that genipin is an effective crosslinking agent for biological tissue fixation.
In the cytotoxicity study, the cytotoxicity of genipin was assessed in vitro using 3T3 fibroblasts (BALB/3T3 C1A31-1-1). Glutaraldehyde, the most commonly used crosslinking agent 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 results of the study demonstrated that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde. Also, the cellular compatibility of the genipin-fixed tissue was superior to its glutaraldehyde-fixed counterpart. Finally, 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.
The genotoxicity study was to evaluate the genotoxicity of genipin in vitro using Chinese hamster ovary (CHO-K1) cells. Glutaraldehyde 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 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 study revealed that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde with or without the addition of metabolic activation system (S9 mix). Additionally, a weakly clastogenic response of glutaraldehyde in CHO-K1 cells was found in the genotoxic assays. In contrast, no clastogenic effect from genipin in CHO-K1 cells was observed, except that drugged with the highest dose (50 ppm).
In the biocompatibility study, a genipin-fixed porcine pericardium was implanted subcutaneously in a growing rat model. Fresh, and the glutaraldehyde- and epoxy-fixed counterparts were used as controls. It was noted that the inflammatory reaction of the genipin-fixed tissue was significantly less than its glutaraldehyde- and epoxy-fixed counterparts. Also, the genipin-fixed tissue had comparable tensile strength and resistance against in vivo degradation as the glutaraldehyde-fixed tissue.
In the In the acellular tissue study, the biocompatibility of cellular and acellular bovine pericardia fixed with genipin were evaluated in implanted subcutaneously in a growing rat model and to investigate the cell migration rate into cellular and acellular bovine pericardia fixed with genipin. The glutaraldehyde-fixed counterparts were used as controls. The results indicated that the inflammatory reactions of the cellular and acelluar genipin-fixed tissues were significantly less than their glutaraldehyde-fixed counterparts. The acellular tissues provided a scaffold that be used for cell migration for tissue regeneration. The tissue regenerating rate for the acellular genipin-fixed tissue was faster than its glutaraldehyde-fixed counterpart.
In the valved conduit study, the biocompatibility of bovine jugular vein fixed with genipin was to reconstruct the right ventricular outflow tract implanted in a canine model. The glutaraldehyde-fixed counterparts were used as controls. It was noted that the inflammatory reaction of the genipin-fixed valved conduit was significantly less than its glutaraldehyde-fixed counterpart. Also, in the blood flow surface of the genipin-fixed valved conduit was covered by flattened endothelial cells. In contrast, in flow surface of glutaraldehyde-fixed valved conduits, no endothelial cell were observed.
The results obtained in the aforementioned studies indicated that genipin is an effective crosslinking agent for biological tissue fixation. Additionally, as far as cytotoxicity, biocompatibility, and genotoxicity are concerned, genipin is a promising crosslinking agent for biological tissue fixation. | en_US |