Sequences of primers for qRT-PCR.

Matrix vesicles (MtVs) are one of the extracellular vesicles (EVs) secreted by osteoblasts. Although MtVs have a classically-defined function as an initiator of ossification and recent findings suggest a role for MtVs in the regulation of bone cell biology, the effects of MtVs on bone repair remain unclear. In the present study, we employed collagenase-released EVs (CREVs) containing abundant MtVs from mouse osteoblasts. CREVs were administered locally in gelatin hydrogels to damaged sites after a femoral bone defect in mice. CREVs exhibited the characteristics of MtVs with a diameter <200 nm. The local administration of CREVs significantly promoted the formation of new bone with increases in the number of alkaline phosphatase (ALP)-positive cells and cartilage formation at the damaged site after the femoral bone defect. However, the addition of CREVs to the medium did not promote the osteogenic differentiation of ST2 cells or the ALP activity or mineralization of mouse osteoblasts in vitro. In conclusion, we herein showed for the first time that MtVs enhanced bone repair after a femoral bone defect partly through osteogenesis and chondrogenesis in mice. Therefore, MtVs have potential as a tool for bone regeneration.