The effect of crosslinking concentration, time, temperature and pH on the characteristic of genipin-crosslinked small intestinal submucosa

In an attempt to prepare genipin-crosslinked collagen scaffolds with different crosslinking characteristics for experimental and clinical applications, the porcine small intestinal submucosa (SIS) was crosslinked with genipin under different conditions (concentrations, duration times, temperatures, pHs), and their crosslinking degree, macroscopic morphology, microstructure, swelling ratio, shrinkage temperature, degradation degree, maximum stress and cytotoxicity were determined respectively. SIS was efficiently crosslinked with all genipin concentrations, yielding genipin-crosslinked small intestinal submucosa (GP-SIS) with a high crosslinking degree (≥60%) and shrinkage temperature (≥80 °C), as well as a low swelling ratio (≤85%) and degradation degree (≤5%). However, only the concentration greater than or equal to 0.12% did significantly increase the maximum stress. Additionally, the fixation of different concentrations of genipin did not introduce cytotoxicity in SIS. As for the effect of duration times, enough crosslinking time was required for high crosslinking efficiency. When the crosslinking time was less than 24 h, GP-SIS had a crosslinking degree of less than 60%, and there was no significant decrease in swelling ratio in comparison to native SIS. When the crosslinking time was less than 24 h, the maximum stress did not reach 7 MPa. Only 3 h-fixed GP-SIS had a shrinkage temperature of less than 75 °C and a degradation degree of more than 6%. GP-SIS prepared at all temperatures and pHs, except for 4 °C, 25 °C and pH 4.0, had a crosslinking degree greater than 80%, a swelling ratio less than 50%, a shrinkage temperature greater than 80 °C, a degradation degree less than 4%, and a maximum stress greater than 8 MPa. Notably, only the fixation at alkaline conditions can increase crosslinking degree and maximum stress of SIS to 90% and 11 MPa respectively.