Keratin-Incorporated Gradient Hydroxyapatite-Reinforced GelMA/OCMC Hydrogel Scaffolds for Rotator Cuff Tendon-to-Bone Repair

Background: Suboptimal tendon-to-bone interface (TBI) healing after rotator cuff repair is associated with fibrous scar formation and limited restoration of the graded tendon–fibrocartilage–bone transition. This study developed a keratin-incorporated, gradient hydroxyapatite (gHAP)-reinforced gelatin methacryloyl (GelMA)/oxidized carboxymethyl cellulose (OCMC) hydrogel scaffold, designated K+gHAP@GC, to provide spatial mineral cues, keratin-associated bioactivity, and local mechanical support for TBI repair. Methods: K+gHAP@GC comprised a GelMA/OCMC/keratin matrix with a layer-by-layer assembled HAP gradient and was stabilized by Schiff-base reaction and ultraviolet photocrosslinking. Scaffold morphology, chemical composition, HAP distribution, mechanical and rheological properties, swelling, degradation, and in vivo retention were characterized. Its effects on bone marrow mesenchymal stem cell (BMSC) viability, osteogenic differentiation, fibrocartilage-like matrix deposition, endothelial angiogenic activity, and in vivo tendon tobone healing were evaluated. Results: The scaffold exhibited an interconnected porous architecture, an scanning electron microscopy– energy-dispersive X-ray spectroscopy(SEM-EDS)-verified HAP gradient, improved gel stability, and enhanced compressive, rheological, and cyclic compression performance. In vitro, the scaffold sustained BMSC viability and proliferation, promoted osteogenic differentiation, and enhanced COL2A1-related matrix expression, with enhanced YAP1 nuclear localization in HAP-containing groups. Keratincontaining hydrogels further enhanced endothelial cell migration, tube formation, and angiogenesis-related gene expression. In vivo, K+gHAP@GC improved bone-side reconstruction, Safranin O-Fast Green (SO-FG)-positive fibrocartilage-like matrix formation, collagen organization, local VEGF positive staining, and biomechanical performance. Conclusions: K+gHAP@GC provides an integrated mineral–protein gradient hydrogel strategy that combines spatial mineral cues, keratin-associated bioactivity, and local mechanical support to improve rotator cuff TBI healing. These findings support its potential for TBI repair, although long-term efficacy and causal mechanisms require further validation.