A synergistically freeze-enzymatically crosslinked silk fibroin hydrogel for sustained kartogenin delivery and enhanced cartilage regeneration

Directional freezing and enzymatic crosslinking formed a hierarchical SF network. • EF-SF achieved 1.67 MPa strength, <10% swelling, and 40-day stability. • PLGA nanoparticles reduced initial KGN release to 9.4% and sustained delivery. • EF-SF/PK enhanced BMSC chondrogenesis without progressive cytotoxicity. • EF-SF/PK improved cartilage integration and matrix deposition in rabbits. Articular cartilage repair remains challenging because of limited intrinsic regeneration and the need for sustained mechanical and biological support. We developed a silk fibroin (SF) scaffold combining directional freezing with HRP/H2O2-mediated enzymatic crosslinking. The resulting EF-SF hydrogel exhibited high compressive strength, limited swelling, aligned porosity, and delayed degradation. Kartogenin (KGN)-loaded PLGA nanoparticles were incorporated to form EF-SF/PK. Compared with direct KGN loading, PLGA encapsulation markedly reduced early release and prolonged KGN delivery. EF-SF/PK supported BMSC viability, proliferation, and chondrogenic gene expression, while LDH and residual peroxide measurements showed no progressive in vitro cytotoxicity. In a rabbit femoral cartilage-defect model, EF-SF/PK improved macroscopic, imaging, and histological repair over 12 weeks. These findings support a hierarchical SF scaffold that integrates mechanical stability with controlled local delivery for cartilage regeneration. Download: Download high-res image (62KB) Download: Download full-size image