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Background: The incidence of osteochondral defects caused by trauma, arthritis or tumours is increasing annually, but progress has not been made in terms of treatment methods. Due to the heterogeneous structure and biological characteristics of cartilage and subchondral bone, the integration of osteochondral repair is still a challenge. Results: In the present study, a novel bilayer hydrogel scaffold was designed based on anatomical characteristics to imitate superficial cartilage and subchondral bone. The scaffold showed favourable biocompatibility, and the addition of an antioxidant nanozyme (LiMn2O4) promoted reactive oxygen species (ROS) scavenging by upregulating antioxidant proteins. The cartilage layer effectively protects against chondrocyte degradation in the inflammatory microenvironment. Subchondral bionic hydrogel scaffolds promote osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) by regulating the AMPK pathway in vitro. Finally, an in vivo rat preclinical osteochondral defect model confirmed that the bilayer hydrogel scaffold efficiently promoted cartilage and subchondral bone regeneration. Conclusions: In general, our biomimetic hydrogel scaffold with the ability to regulate the inflammatory microenvironment can effectively repair osteochondral defects. This strategy provides a promising method for regenerating tissues with heterogeneous structures and biological characteristics. [ABSTRACT FROM AUTHOR]