Enhancing in situ biomimetic mineralization of silk fibroin scaffolds via synergistic phosphorylation and laccase-catalyzed cross-linking.

Silk fibroin (SF) is widely investigated in bone tissue engineering owing to its biocompatibility, enzymatic biodegradability, and ability to promote osteoblast adhesion. However, native SF scaffolds lack sufficient calcium-binding sites for effective hydroxyapatite (HAp) nucleation and inadequate mechanical strength for load-bearing applications. To address both limitations, this study developed a strategy combining chemical phosphorylation and laccase-catalyzed cross-linking. Specifically, phosphorylation of primary amine and hydroxyl groups on SF chains introduced negative charges, thereby chelating calcium ions and promoting the in situ formation of HAp during the alternating biomimetic mineralization process, while enzymatic cross-linking improved the mechanical properties of the resulting SF scaffolds. Following the combined treatment, surface electronegativity decreased, and the molecular weight of SF increased, enabling rapid, uniform deposition of carbonate nanocrystalline HAp with a bone-like calcium-to‑phosphorus ratio. The compressive strength was noticeably increased from 8.67 kPa to 45.68 kPa. In vitro evaluations confirm the excellent cytocompatibility of the engineered scaffolds and accelerated osteogenic commitment compared to pristine SF. By bridging nano-level interfacial mineralization with macro-scale mechanical enhancement, this eco-friendly and sustainable strategy provides a highly translatable platform for clinically relevant bone regeneration.