Bone repair involves bone resorption through osteoclastogenesis and the stimulation of neovascularization and osteogenesis by endothelial progenitor cells (
EPC
s). However, the role of
EPC
s in osteoclastogenesis is unclear. In this study, we assess the effects of
EPC
‐derived exosomes on the migration and osteoclastic differentiation of primary mouse bone marrow‐derived macrophages (
BMM
s) in vitro using immunofluorescence, western blotting,
RT
‐
PCR
and Transwell assays. We also evaluated the effects of
EPC
‐derived exosomes on the homing and osteoclastic differentiation of transplanted
BMM
s in a mouse bone fracture model in vivo. We found that
EPC
s cultured with
BMM
s secreted exosomes into the medium and, compared with
EPC
s, exosomes had a higher expression level of Lnc
RNA
‐
MALAT
1. We confirmed that Lnc
RNA
‐
MALAT
1 directly binds to miR‐124 to negatively control miR‐124 activity. Moreover, overexpression of miR‐124 could reverse the migration and osteoclastic differentiation of
BMM
s induced by
EPC
‐derived exosomes. A dual‐luciferase reporter assay indicated that the integrin
ITGB
1 is the target of miR‐124. Mice treated with
EPC
‐derived exosome‐
BMM
co‐transplantations exhibited increased neovascularization at the fracture site and enhanced fracture healing compared with those treated with
BMM
s alone. Overall, our results suggest that
EPC
‐derived exosomes can promote bone repair by enhancing recruitment and differentiation of osteoclast precursors through Lnc
RNA
‐
MALAT
1.
Background/Aims: Bone resorption mediated by osteoclasts plays an important role in bone healing. Endothelial progenitor cells (EPCs) promote bone repair by stimulating neovascularization and osteogenesis. However, the role of EPCs in osteoclast formation and function is not well defined. The aim of this study was to elucidate mechanisms of EPCs in osteoclast formation and function. Methods: In this study, we examined the effects of EPCs on the proliferation, migration and osteoclastic differentiation of primary mouse bone marrow-derived macrophages (BMMs) in a co-culture system in vitro. We also evaluated the effects of EPC co-transplantation on the homing and osteoclastic differentiation of transplanted BMMs in a mouse bone fracture model in vivo. The technology of immunofluorescence, immunohistochemical, western blot, Rt-PCR, cell co-culture and Transwell were used in this study. Results: EPCs secreted TGF-β1 in the EPC-BMM co-culture medium and increased Talin-1 expression in the co-cultured BMMs. Treatment with a TGF-β1 neutralizing antibody or Talin-1 silencing in BMMs completely inhibited BMM osteoclastic differentiation in the co-culture system. These results indicated that the osteoclastogenic effects of EPCs were mediated by TGF-β1-mediated Talin-1 expression in BMMs. In the femur fracture model, BMMs co-transplanted with EPCs exhibited enhanced engraftment into the fracture site and osteoclastic differentiation compared with those transplanted alone. Mice treated with EPC-BMM co-transplantation exhibited increased neovascularization at the fracture site and accelerated fracture healing compared with those treated with BMMs alone. Conclusion: Taken together, the results suggest that EPCs can promote bone repair by enhancing recruitment and differentiation of osteoclast precursors.
The present study indicates that EPC-dependent prevascularization contributes to bone healing with structural reconstruction and functional recovery and may improve the understanding of correlation between angiogenesis and osteogenesis.
Integration of tissue-engineered bone grafts with the host bone is vital for the healing of critical-size bone defects. An important aspect of this process is bone resorption, which must be carried out by osteoclasts derived from the host. However, the mechanism underlying recruitment of host osteoclast precursors to graft sites remains unclear. Endothelial progenitor cells (EPCs) mobilize from the bone marrow into the circulation and home to sites of angiogenesis such as tissue remodeling. Since EPCs express SDF-1, and C/EBPβ is known to regulate SDF-1α expression, we hypothesized that EPCs may recruit CXCR4-expressing host osteoclast precursors to the repair area and that this recruitment may be mediated through C/EBPβ signaling. Using an inflammatory EPC model we showed that EPCs upregulate protein levels of both SDF-1α and C/EBPβ. A luciferase assay confirmed that C/EBPβ acts on the SDF-1α promoter in these cells, and that binding is increased under conditions of inflammation, while silencing of C/EBPβ reduces expression of SDF-1 α and C/EBPβ. Using RAW264.7 cells as a model of osteoclastic monocyte precursors, we investigated their responses to migratory factors in EPC conditioned medium. We showed that RAW264.7 cells migrate towards conditioned medium from EPCs treated with IL-1β, an effect which could be abolished by silencing C/EBPβ in EPCs, and was almost completely blocked by silencing CXCR4 in RAW264.7 cells. These findings show that EPCs respond to inflammatory stimuli by signaling to osteoclast precursors via SDF-1, and that C/EBPβ mediates this response.
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