Abstract
Osteochondral defects present a formidable clinical challenge due to the intricate structural and functional interdependence of cartilage and subchondral bone. Conventional scaffolds, characterized by single-scale, disconnected pores, inherently restrict cell-cell communication and nutrient diffusion, thereby impeding osteoblast-to-osteocyte transformation and matrix mineralization. Herein, a 3D-printed topologically hierarchical mechanical hydrogel (THMH) scaffold was developed featuring a biomimetic bilayer architecture that recapitulates the native osteochondral microenvironment. THMH integrates a nanoporous cartilage-mimetic layer and a macroporous osteogenic layer, interconnected via gradient pores to facilitate nutrient transport, vascularization, and cellular crosstalk. In vitro evaluations revealed that the THMH has excellent biocompatibility and the ability to promote multidirectional differentiation of BMSCs. RNA-seq results indicated that cartilage-like layer of THMH may promote the chondrogenic differentiation of BMSCs by its mechanical properties and KGN to activate integrin-PI3K-AKT signaling axis. Osteogenic layer of THMH may promote the osteogenic differentiation of BMSCs due to its hierarchically porous structure for improving the internal hypoxic environment. In vivo implantation in a rat osteochondral defect model achieving near-complete osteochondral regeneration. This study establishes THMH as a multifunctional platform that bridges biomechanical robustness with biofunction fidelity, offering a transformative paradigm for addressing complex osteochondral regeneration challenges.