A 3D Co‐Culture System Inspired by Fracture Healing Cell Interactions for Bone Tissue Engineering

Abstract Peri‐bone fibroblasts play a crucial role in regulating bone regeneration during early fracture healing. Inspired by the synergy between osteoblasts and fibroblasts at fracture sites, a biomimetic three‐dimensional (3D) indirect co‐culture system is developed, comprising a 3D scaffold and co‐cultured cells. To mimic cellular interactions in the fracture healing zone, the scaffold features an inner–outer ring structure with communication channels that support indirect cell co‐culture. This setup provides fibroblasts and osteoblasts with a 3D culture environment resembling the in vivo extracellular matrix, enhancing intercellular signaling while minimizing risks of direct contact. Mechanically tunable bioinks are formulated by incorporating hyaluronic acid methacrylate (HAMA) hydrogel into gelatin methacryloyl (GelMA) hydrogel to construct the scaffold. The optimal co‐culture ratio is established in vitro, where fibroblasts are found to regulate the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) via zinc ion transport mechanisms. In vivo validations are conducted, including ectopic bone formation in nude mice and bone regeneration in rat cranial defect and tooth extraction socket models. This 3D indirect co‐culture system enhances osteogenesis by promoting functional fibroblast–osteoblast interactions, offering a novel platform for co‐culture studies and a promising strategy for clinical bone regeneration applications.