Efficacy of nano-silver small intestine submucosa repair of osteochondral defect in rabbit model by the AMPK-mTOR-ULK1 pathway

OBJECTIVE: This study evaluated the regenerative potential of nano-silver small intestine submucosa (NSSIS) scaffolds with a 4-D porous structure for repairing osteochondral defects in rabbit knee joints. METHODS: NSSIS scaffolds were prepared using nanosilver, fresh pig-derived small intestinal submucosa, and chondrocytes. Biocompatibility was assessed by methyl thiazolyl tetrazolium (MTT) assays measuring bone marrow stromal cell (BMSC) proliferation at 24, 48, and 72 h. A rabbit model of intercondylar groove cartilage defects was established and randomized into three groups (n = 12 each): NSSIS, NSSIS + BMSCs, and a control group. Scaffold morphology and cell growth were evaluated in vitro using H&E staining after 24 h. Following implantation, cartilage repair was assessed at 24, 48, and 72 h using ICRS macroscopic scoring and histological staining (H&E, Safranin O-fast green, toluidine blue). After 12 weeks, ELISA measured growth factor expression (PDGF, VEGF, TGF-β, IGF-1, FGF, EGF), and qRT-PCR and Western blotting assessed autophagy-related gene and protein expression (AMPK, ULK1, mTOR, and Beclin-1). RESULTS: Both NSSIS groups demonstrated significantly greater BMSC ingrowth compared with controls, with the NSSIS + BMSCs group exhibiting the most robust repair. This group showed significantly elevated growth factor expression at 12 weeks (p < 0.05), downregulation of AMPK, ULK1, and Beclin-1, and upregulation of mTOR (p < 0.01). Histological analysis revealed enhanced chondrocyte formation, thicker cartilage layers, increased chondroblast proliferation, and abundant extracellular matrix deposition in the NSSIS + BMSCs group, whereas the NSSIS-only group showed less cellular and collagen development. CONCLUSIONS: NSSIS scaffolds demonstrate good biocompatibility and promote BMSC ingrowth, chondrocyte development, and osteochondral repair. The addition of BMSCs further enhances these effects by facilitating in situ chondrogenic differentiation, stimulating BMSC and chondrocyte migration, and initiating tissue regeneration. These findings highlight the potential of NSSIS, particularly when combined with BMSCs, as a promising biomaterial for cartilage and subchondral bone repair, with potential clinical applications in regenerative medicine.