Functionalized multilayer hydrogels loaded with HDAC4-modified superficial and middle-deep zone chondrocytes for enabling precise zonal repair of osteochondral defects

A zoned osteochondral unit with scaffold zonation and cell zonation was constructed. • HDAC4 promotes osteochondral repair via improved function of SFZCs and MDZCs. • HDAC4 improves SFZCs/MDZCs function via activating oxidative phosphorylation. Achieving a physiological microenvironment in tissue engineering represents the ultimate objective. Current strategies for constructing osteochondral units focus primarily on biomimicking scaffold materials and often overlook the zonal distribution of seed cells. Studies indicate that chondrocytes, which are utilized as seed cells, are susceptible to reduced proliferation and dedifferentiation, which compromises regenerative outcomes. In this study, we biomimetically fabricated a three-layer hydrogel scaffold of zonal osteochondral units using gelatin methacryloyl and nanohydroxyapatite (GelMA/nHA), where the first and second layers were composed of superficial zone chondrocytes (SFZCs) and middle-deep zone chondrocytes (MDZCs), respectively. The lower layer contained a GelMA/nHA hydrogel loaded with bone marrow mesenchymal stem cells (BMSCs) derived from a rat osteochondral defect model. The SFZCs and MDZCs were modified with histone deacetylase 4 (HDAC4) to increase their viability. The results showed that the simultaneous zonation of cells and the scaffold effectively enhanced osteochondral defect repair. Moreover, delivering HDAC4 to chondrocytes in each layer further improved the repair outcomes. Omics and validation data suggest that HDAC4 optimizes chondrocyte function across zones by upregulating oxidative phosphorylation. This study highlights the importance of zonal cell biomimicry in engineered osteochondral constructs and defect repair. In addition, this study reveals the molecular mechanism by which HDAC4 regulates chondrocytes, providing new insights for osteochondral regeneration. Schematic illustration of the zonal osteochondral unit: fabrication, mechanism and in vivo repair. (A) Preparation of the osteochondral unit integrating scaffold and celluar zonation. (B) The role and mechanism of HDAC4 in the regulation of chondrocyte function. (C) In vivo osteochondral repair with cell and scaffold zonation. SFZ, superficial zone of cartilage; MDZ, middle–deep zone of cartilage; HDAC4, histone deacetylase 4; OXPHOS, oxidative phosphorylation; BMSCs, bone marrow mesenchymal stem cells; MF, molecular function; CC, cellular component; BP, biological process; NADH-DH, NADH dehydrogenase; IMMC, inner mitochondrial membrane protein complex. The graphical abstract was not generated by AI. Download: Download high-res image (129KB) Download: Download full-size image