Erythropoietin stimulates bone formation, cell proliferation, and angiogenesis in a femoral segmental defect model in mice

The glycoprotein erythropoietin (EPO) has been demonstrated to stimulate fracture healing. The aim of the present study was to investigate the effect of EPO treatment on bone repair in a femoral segmental defect model. Bone repair was analyzed in mice which were treated by EPO (500IE/kg/d intraperitoneally; n=38) and in mice which received the vehicle for control (n=40). Two and 10 weeks after creating a 1.8mm femoral segmental defect, bone repair was studied by micro-CT, histology, and Western blot analysis. At 10 weeks, micro-CT and histomorphometric analyses showed a significantly higher bridging rate of the bone defects in EPO-treated animals than in controls. This was associated by a significantly higher bone volume within the segmental defects of the EPO-treated animals. At 2 weeks, Western blot analyses revealed a significantly higher expression of vascular endothelial growth factor (VEGF) in EPO-treated animals compared to controls. Accordingly, the number of blood vessels was significantly increased in the EPO group at 2 weeks. At 10 weeks, we found a significantly higher expression of proliferating cell nuclear antigen (PCNA) in EPO-treated animals when compared to controls. Western blot analyses showed no significant differences between the groups in the expression of the endothelial and inducible nitric oxide synthases (eNOS and iNOS) and the angiopoietin receptor Tie-2. Immunohistochemistry confirmed the results of the Western blot analyses, demonstrating a significantly higher number of VEGF- and PCNA-positive cells in EPO-treated animals than in controls at 2 and 10 weeks, respectively. We conclude that EPO is capable of stimulating bone formation, cell proliferation and VEGF-mediated angiogenesis in a femoral segmental defect model.