Effects of protein conformational transition accompanied with crosslinking density cues in silk fibroin hydrogels on the proliferation and chondrogenesis of encapsulated stem cells

Silk fibroin (SF) hydrogels possess excellent biocompatibility and biomimetic properties of the extracellular matrix. Among them, the mild chemical crosslinked SF hydrogels show great application potential in the fields of 3D cell culture and tissue repairing and thus have attracted widespread attention. However, the mobility of hydrophobic chain segments of SF molecules in these chemical crosslinked hydrogels can easily cause the molecules to undergo a self-assembly process from random coil to <i>β</i>-sheet conformation due to its lower energy state, thus inducing an inevitable conformational transition process. This process further leads to dynamic changes of important material features, such as the hydrogel pore size and mechanical properties, which can probably bring some non-negligible and unknown impacts on cell behaviors and their biomedical applications. In this study, a typical mild crosslinking system composed of horseradish peroxidase and hydrogen peroxide was chosen to prepare SF hydrogels. A feasible protein conformational transition rate controlling strategy based on hydrogel crosslinking density regulation was also proposed. Our results demonstrate that the lower the hydrogel crosslinking density, the faster the conformational transition rate. Subsequently, SF hydrogels with different conformational transition rates were successfully constructed to investigate the impact of the protein conformational transition rate accompanied with initial crosslinking density on the proliferation and chondrogenic differentiation of encapsulated stem cells. Results comprehensively illustrated that the conformational transition process could effectively regulate cell behavior. The hydrogel with an appropriate conformational transition rate obviously promoted the proliferation and chondrogenesis of encapsulated stem cells, while too fast or too slow transition processes slowed down these cell activities. These findings are hopefully to provide valuable guidance for the development and efficient usage of SF hydrogels in the fields of 3D cell culture and tissue engineering.