3D-printed bilayer hydrogel scaffolds incorporating HAP and KGN@Lip for osteochondral regeneration

Abstract

Osteochondral defects require the simultaneous regeneration of cartilage and subchondral bone, posing a major challenge for current biomaterial strategies. Here, we present a radially oriented 3D-printed bilayer hydrogel scaffold composed of a mechanically reinforced GelMA/HAP osteogenic layer and a compliant, KGN@Lip-loaded GelMA chondrogenic layer, to achieve coordinated osteochondral repair. Structural and physicochemical characterization confirmed the formation of a functionally graded architecture with uniform HAP and KGN@Lip distribution, while mechanical, rheological, swelling, and degradation analyses revealed a layer-dependent gradient that resembles certain structural features of the native osteochondral interface. In vitro, the GelMA/HAP layer promoted robust osteogenic differentiation through ion-mediated activation of osteogenic pathways, whereas the GelMA/KGN@Lip layer sustained chondrogenic stimulation and markedly enhanced SOX9, Col2a1, and Acan expression. The radially oriented microchannels provided an interconnected structure that may support nutrient transport and cell infiltration. In vivo implantation demonstrated substantial subchondral bone regeneration and the formation of cartilage-like tissue with improved matrix organization, along with enhanced integration at the osteochondral interface, confirming the scaffold's ability to provide spatially coordinated biochemical and structural cues. Overall, this anisotropic bilayer hydrogel scaffold offers a promising strategy for integrated and functional osteochondral regeneration.