Macrophage Derived TGF-β Recruits BMSCs for Initiation of Bone Formation During Fracture Healing

Impaired fracture healing remains a major clinical challenge, with approximately 10% of fractures progressing to nonunion or delayed union. Although numerous studies have focused on strategies to promote fracture healing, the mechanisms governing the initiation phase of repair remain poorly understood. This study aimed to investigate the contribution of macrophage-derived TGF-β1 to the initiation of early fracture repair. A murine femoral osteotomy model was used to study fracture healing. We examined the temporal relationship among bone healing, callus maturation, pSmad2/3 positive cells, and osteoprogenitors. TGF-β1 signaling was systemically inhibited using a neutralizing antibody. Macrophages were selectively depleted in LysM-iDTR mice by diphtheria toxin administration. The source and expression levels of TGF-β1 at the fracture site were determined by pSmad2/3 staining and RT-qPCR. Fracture healing was assessed using micro-CT, histomorphometry, and immunohistochemistry. TGF-β1 signaling peaked during the inflammatory phase and preceded osteoprogenitor recruitment. Systemic inhibition of TGF-β1 or selective macrophage depletion before fracture establishment severely disrupted mesenchymal stem cell (MSC) recruitment and bone formation. In contrast, late-stage macrophage depletion did not substantially affect bone healing. CD86+ macrophages were identified as a key early source of TGF-β1. Importantly, local delivery of TGF-β1 via hydrogel microspheres during the early stage rescued osteogenic repair in macrophage-deficient mice. CD86+ macrophage-derived TGF-β1 is essential for initiating fracture repair by recruiting MSCs and promoting osteogenesis. Targeting this pathway may represent a novel therapeutic strategy to enhance bone regeneration in cases of impaired healing.