Extracellular vesicles from mechanical loading stimulated-macrophages favor fracture healing through targeting Adrb2 of osteoblasts

• Appropriate mechanical stimulation favors fracture healing. • PIEZO1 of macrophage is essential for mechanical stimulation-induced bone repair. • PIEZO1-activated macrophages secrete extracellular vesicles to promote osteogenesis. • Extracellular vesicles enriched microRNAs regulate adrenergic receptor signaling. • Adrb2 of osteoblast could be a target to promote fracture healing. Appropriate mechanical loading (ML) accelerates fracture healing. Macrophages, being the primary mechanosensitive immune cells, play a pivotal role in the regulation of bone formation. However, the exact mechanism through which macrophages respond to ML during the fracture healing process remains unclear. The current study revealed that conditional knockout of Piezo1 in macrophages impairs the infiltration and alternative activation of macrophages during fracture healing. In addition, ML activates PIEZO1 in macrophages to release small extracellular vesicles (sEVs) to foster osteogenesis. sEVs deriving from ML-treated macrophages (ML-Mφ-sEVs) enriched miRNAs in regulating adrenergic receptor signaling pathway. We next discovered that selected miRNAs within ML-Mφ-sEVs could target the expression of Adrb2 , thereby impeding β2 -adrenergic receptor signaling in osteoblasts during fracture healing. Conditional knockout of Adrb2 in osteoblasts increased mineralization in the callus, indicating that ML-Mφ-sEVs promote fracture healing through confining ADRB2 of osteoblasts. Based on these discoveries, we introduced engineered sEVs in the treatment of bone fracture, revealing that the osteoblasts targeting sEVs accelerated bone formation and ameliorated mechanical unloading-induced fracture delayed union. Collectively, our results reveal the relationship between ML-induced Mφ-sEVs and osteogenesis, which provides a potential therapeutic strategy for fracture management via targeting Adrb2 in osteoblasts.