Degradation behavior is a key fundamental topic in the field of silk-based biomaterials. In this study, genipin-crosslinked silk fibroin films with varying crosslinking degrees were generated to investigate the effect of the crosslinking degree on the degradation behavior. Higher crosslinking degrees resulted in increased inter-intramolecular network crosslinking density through covalent bonds, which restricted enzymatic attacks and the release of enzyme-degraded polypeptides from silk fibroin molecular networks, providing greater resistance to enzyme degradation. Furthermore, genipin crosslinking induced the conformational transition from random coil to β-sheet due to the structural rearrangement of the chains to form covalent bonds. High β-sheet content contributes to a more crystalline structure that also enhances resistance to enzyme degradation. Consequently, changing the crosslinking degree had a substantial impact on the degradation rate. The high-crosslinking films (with a crosslinking degree greater than 90%) showed a similar degradability to 75% ethanol-treated films, which were barely degraded by collagenase IA and subcutaneous implantation in SD rats. However, decreasing the crosslinking degree from 90% to 78% significantly increased the degradation ratio from 4 wt% to 18 wt% after 28 days in vivo degradation, respectively. In particular, the degradability was strongly correlated to the crosslinking degree in the low-crosslinking (lower than 90%) films. These results reveal that the crosslinking process can efficiently control molecular structures and regulate the crystallization of silk fibroin materials, in turn providing control of the degradation rate.
