Crosslinking of biological tissues using genipin and/or carbodiimide

The study was to investigate the crosslinking characteristics, mechanical properties, and resistance against enzymatic degradation of biological tissues after fixation with genipin (a naturally occurring crosslinking agent) and/or carbodiimide. Fresh tissue was used as a control. It was found that both genipin and carbodiimide are effective crosslinking agents for tissue fixation and genipin crosslinking is comparatively slower than carbodiimide crosslinking. Additionally, tissue fixation in genipin and/or carbodiimide may produce distinct crosslinking structures. Carbodiimide may form intrahelical and interhelical crosslinks within or between tropocollagen molecules, whereas genipin may further introduce intermicrofibrillar crosslinks between adjacent collagen microfibrils. The stability (denaturation temperature and resistance against enzymatic degradation) of the fixed tissue is mainly determined by its intrahelical and interhelical crosslinks. In contrast, intermicrofibrillar crosslinks significantly affect the mechanical properties (tissue shrinkage during fixation, tensile strength, strain at break, and ruptured pattern) of the fixed tissue. Moreover, the degree of enzymatic degradation of the fixed tissue may be influenced by three factors: the availability, to the enzyme, of recognizable cleavage sites, the degree of crosslinking, and the extent of helical integrity of tropocollagen molecules in tissue.