Bone regeneration is mediated by macrophage extracellular vesicles.

Multiple local and systemic factors including inflammation influence bone regeneration. Several lines of evidence demonstrate that macrophages contribute to the immunological regulation of MSC and osteoblast function during bone regeneration. Recent studies demonstrate that macrophage polarization influences this regulatory process. In this manuscript, we investigated the paracrine functional role of naïve (M0), M1 and M2 polarized macrophage derived EVs in bone repair. Treatment of rat calvaria defects with no EVs, M0 EVs, M1 EVs, or M2 EVs revealed polarization-specific control of bone regeneration by macrophage EVs at 3 and 6 weeks. M0 and M2 EVs promoted repair/regeneration and M1 EVs inhibited bone repair. Pathway-specific studies conducted in cell culture showed that M1 EVs negatively regulated the BMP signaling pathway, specifically BMP2 and BMP9. In parallel, miRNA sequencing studies showed similar miRNA cargo in M0 and M2 EVs and different miRNA cargo in M1 EVs. Functional examination of M1 macrophage EV-enriched miR-155 demonstrated that miR-155 mimic treatment reduced MSC osteogenic differentiation as measured by reduced BMP2, BMP9 and RUNX2 expression when compared to controls. Conversely, treatment of MSCs with the M2 macrophage EV-enriched miR-378a mimic increased MSC osteoinductive gene expression when compared to controls. These functional studies implicate polarized macrophage EV miRNAs in the positive or negative regulation of bone regeneration that was observed in vivo. Overall, the results presented in this study indicate that macrophage polarization influences EV cargo and related EV function in the paracrine regulation of bone regeneration.