Enzymatic crosslinking of Bombyx mori silk fibroin biomaterials: An investigation of the gel point by dynamic rheology

Fibroin is the main polypeptidic component of the silk fibre generated by the larvae of the domesticated silk moth (Bombyx mori), and it has been extensively studied as a biomaterial with applications in tissue engineering and regenerative medicine. Due to their inherent practical advantages, the hydrogels constitute the preferred format for the B. mori silk fibroin (BMSF) biomaterials, which can conveniently be obtained by crosslinking processes. While the physically or chemically crosslinked BMSF hydrogels have been frequently described, the self-crosslinking of BMSF solutions induced by the catalytic effect of the enzyme horseradish peroxidase (HRP) has been barely reported. Following a previous preliminary study, where we demonstrated the advantages of using this enzyme for crosslinking BMSF, in the present work we investigated factors (amount of enzyme, initial fibroin concentration) that may affect the gelation. The measurement of dynamic moduli resulting from application of an oscillatory shear stress in a rheometer was our method to estimate the gelation time and to investigate the influence of certain factors on the process of crosslinking. It was found that both a higher initial concentration of the BMSF solution and a higher amount of the catalyst HRP induced a significant reduction of the gelation time.