Enzymatic interfacial engineering of silk fibroin scaffolds using functionalized chitosan oligosaccharides for enhanced in situ biomimetic mineralization

Phosphorylation and phenolic modification enhanced chitosan oligosaccharide reactivity. • Enzymatic grafting of modified chitosan oligosaccharides stabilized silk fibroin. • Modified silk fibroin scaffolds promoted efficient biomimetic mineralization. • Mineralized scaffolds enhanced mechanics and osteoblast differentiation. Silk fibroin (SF) is widely used in biomedical materials owing to its biocompatibility and biodegradability. However, natural SF scaffolds lack sufficient calcium-binding motifs, particularly carboxylate groups derived from acidic amino acid residues, thereby hindering efficient nucleation and oriented growth of hydroxyapatite (HAp) during biomimetic mineralization, and ultimately compromising the mechanical properties of the resulting scaffold. To address this, we functionalized chitosan oligosaccharide (COS) with phosphate and phenolic hydroxyl groups to yield a highly reactive modified COS (mCOS), which was site-specifically grafted onto SF chains via laccase-catalyzed oxidative coupling, followed by in situ mineralization. The resulting SF- g -mCOS hybrid scaffold exhibited enhanced calcium affinity, improved structural stability, and enabled controlled HAp deposition under alternating biomimetic mineralization conditions. The compressive stress of scaffolds increased from 7.13 kPa (SF) to 19.76 kPa (SF- g -mCOS), and further to 79.14 kPa after cyclic mineralization, accompanied by elevated phosphorus incorporation and significantly improved crystallinity and orientation of HAp. In vitro assays demonstrated that the objective scaffold showed superior cytocompatibility and accelerated osteogenic differentiation compared to native SF. This work provides a sustainable strategy for modifying SF with polysaccharides, achieving nanoscale interfacial mineralization and macroscale mechanical enhancement, thus offering a highly translatable platform for SF-based bone regeneration materials.