L-leucine-incorporated 3D-printed SilMA hydrogel scaffolds promote calvarial defect repair with PLOD2-associated collagen remodeling

Effective repair of bone defects requires scaffolds that not only match defect geometry and provide mechanical support, but also support both osteogenic and angiogenic processes during regeneration. Here, a photocurable hydrogel ink integrating methacrylated silk fibroin (SilMA) with L-leucine (L-Leu) is developed for 3D printing of architected scaffolds with programmable pore structures. L-Leu incorporation enhances the compressive performance and surface wettability of the hydrogel without compromising print fidelity, thereby improving the interfacial microenvironment for cell attachment and tissue remodeling. In vitro, SilMA@L-Leu exhibits good cytocompatibility, promotes bone marrow–derived mesenchymal stem cell proliferation and osteogenic differentiation, and enhances the angiogenic activity of human umbilical vein endothelial cells. Among the tested formulations, SilMA@L-Leu containing 300 ng mL -1 L-Leu showed the most balanced osteogenic and angiogenic performance and was therefore selected for mechanistic and in vivo evaluation. In a rat critical-size calvarial defect model, this scaffold markedly promotes new bone formation, collagen deposition, and mineralized tissue development. Mechanistically, siRNA-mediated silencing of procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) attenuated the scaffold-induced osteogenic effects, suggesting the involvement of PLOD2-associated collagen matrix remodeling in the pro-osteogenic activity of SilMA@L-Leu. These findings identify SilMA@L-Leu as a 3D-printable, small-molecule-incorporated scaffold with promise for bone defect repair.