Mechanochemically zoned bioprinted bilayer scaffold with sustained kartogenin delivery and mineral reinforcement for osteochondral regeneration

Osteochondral defects require coordinated regeneration of both articular cartilage and subchondral bone, yet most scaffold systems fail to recapitulate the native depth-dependent mechanochemical microenvironment. Here, we developed a three-dimensional (3D) bioprinted bilayer hydrogel scaffold with mechanochemical zoning based on gelatin methacryloyl (GelMA) and hyaluronic acid methacrylate (HAMA) for spatially programmed osteochondral repair. The cartilage-mimetic upper layer incorporated microfluidically prepared kartogenin (KGN)-loaded poly(lactic- co -glycolic acid) (PLGA) microspheres to reduce initial burst release and provide sustained chondrogenic cues. The bone-mimetic lower layer was reinforced with nano-hydroxyapatite (nHA) to enhance stiffness and osteoconductivity. The printed constructs exhibited a well-defined bilayer interface with spatially separated components and distinct layer-specific mechanical properties, together with prolonged KGN release compared with free-drug hydrogels. In vitro , bone marrow mesenchymal stem cells remained viable in both layers and showed region-specific differentiation, with enhanced chondrogenic matrix formation in the upper layer and increased osteogenic commitment in the nHA-enriched lower layer. In a rabbit femoral trochlear groove osteochondral defect model, the functionalized bilayer scaffold improved defect filling and osteochondral repair, promoted subchondral trabecular bone regeneration, and partially improved the mechanical performance of the repaired cartilage, with values trending toward those of native cartilage. Histological analyses further indicated the formation of a proteoglycan-rich repair matrix with improved tissue integration and more organized collagen architecture. Overall, this modular bioprinting strategy provides a practical approach for engineering spatially programmed, drug-releasing hydrogel constructs for osteochondral regeneration.