In vitro mineralization and osteogenesis effects of the silk fibroin hydrogels mineralized with SBF and CaCl2/Na2HPO4 methods.

In vitro mineralization has been proposed as promising approach for fabricating mineralized scaffolds and repairing bone defects. In this study, various mineralized glycidyl methacrylate-modified silk fibroin (GMA-SF) hydrogels were developed using two mineralization techniques: calcium chloride/sodium phosphate (CaCl2/Na2HPO4) solution immersion and simulated body fluid (SBF) solution immersion. Compared to the SBF immersion method, the CaCl2/Na2HPO4 method produced a greater quantity of minerals on the surface of GMA-SF hydrogels within the same mineralization duration and cycle numbers. Moreover, the mineralized phase in CaCl2/Na2HPO4 group exhibited a more uniform distribution and tighter arrangement along the hydrogel surface compared to the SBF group. Although both EDS and XPS analyses confirmed the presence of calcium (Ca) and phosphorus (P), significantly higher contents of Ca and P were observed in the GMA-SF hydrogels treated with the CaCl₂/Na₂HPO₄ method compared to the SBF method. This was also accompanied by stronger compressive strength in the CaCl2/Na2HPO4 group. Furthermore, in vitro and in vivo studies demonstrated that the mineralized GMA-SF hydrogels from both groups promoted osteogenic differentiation and bone regeneration, with the CaCl₂/Na₂HPO₄ group showing superior efficacy. These findings highlight the advantages of the CaCl₂/Na₂HPO₄ mineralization method over the SBF method for bone regeneration applications using GMA-SF hydrogels.