AbstractMacrophages play an important role in material-related immune responses and bone formation, but the functionality of macrophage-derived extracellular vesicles (EVs) in material-mediated bone regeneration is still unclear. Here, we evaluated intracellular communication through small extracellular vesicles (sEVs) and its effects on endogenous bone regeneration mediated by biomimetic intrafibrillarly mineralized collagen (IMC). After implantation in the bone defect area, IMC generated more neobone and recruited more mesenchymal stem cells (MSCs) than did extrafibrillarly mineralized collagen (EMC). More CD63⁺CD90⁺ and CD63⁺CD163⁺ cells were detected in the defect area in the IMC group than in the EMC group. To determine the functional roles of sEVs, extracellular vesicles from macrophages cultured on different mineralized collagen were isolated, and they showed no morphological differences. However, macrophage-derived sEVs in the IMC group showed an enhanced Young’s modulus and exerted beneficial effects on the osteogenic differentiation of bone marrow MSCs by increasing the expression of the osteoblastic differentiation markers BMP2, BGLAP, COL1, and OSX and calcium nodule formation. Mechanistically, sEVs from IMC-treated macrophages facilitated MSC osteogenesis through the BMP2/Smad5 pathway, and blocking sEV secretion with GW4869 significantly impaired MSC proliferative, immunomodulative and osteogenic potential. Taken together, these findings show that macrophage-derived sEVs may serve as an emerging functional tool in biomaterial-mediated endogenous bone regeneration.