Reticulocalbin‐2–Programmed Macrophage Exosomes Restore Immuno‐Osteogenic Coupling to Promote Calvarial Bone Regeneration

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.