Abstract

Calvarial defect repair is frequently limited by immune dysregulation and insufficient coupling with bone regeneration. This study investigated the regulatory role of reticulocalbin-2 (RCN2) in macrophages and evaluated a biomimetic periosteum-bone bilayer scaffold (PB-BLS) designed to deliver RCN2-modified M2 macrophage-derived exosomes (RCN2-Exos). A photocrosslinkable periosteum-like hydrogel was prepared from decellularized periosteal matrix (DPM), methacrylic anhydride-modified DPM, and gelatin methacryloyl (GelMA), and was loaded with RCN2-Exos. A zinc-substituted tricalcium phosphate (Zn-TCP)/chitosan (CS) bone-like scaffold was fabricated by three-dimensional printing and integrated with the periosteum-like layer to form PB-BLS. Material characterization confirmed favorable mechanical properties and controllable exosome release. In vivo studies using calvarial defect models and RCN2-knockout controls showed that RCN2 deficiency impaired bone repair and aggravated inflammatory responses. PB-BLS promoted M2 macrophage polarization, enhanced skeletal stem cell (SSC) migration and osteogenic differentiation in vitro, and significantly increased bone volume fraction (BV/TV) and bone mineral density (BMD) in vivo. Single-cell RNA sequencing and cell-cell communication analyses indicated that PB-BLS remodeled the local immuno-osteogenic microenvironment and enhanced key signaling axes, including SPP1-CD44 and CXCL12-CXCR4. These findings identify RCN2 as a pivotal mediator linking macrophage immune phenotype with osteogenic fate and support RCN2-Exos-loaded PB-BLS as a promising strategy for bone defect repair.