Therefore the improved bone healing may also be attributed to a better angiogenesis caused by EPO. In the present study, we systemically examined the roles of EPO in regulating chondrogenesis and angiogenesis in vitro and in vivo. The promotive effects of EPO during the chondrogenic and angiogenic phases of femur fracture Ro 64-5229 repair highlight its therapeutic RG 102240 potential in skeletal regenerative medicine. Current procedures to promote skeletal regeneration include the applications of autografts, allografts, biocompatible implants, bioactive factors and mesenchymal stem cell-based therapy. However, the above approaches also face significant limitations due to insufficient supply, potential disease transmission, immunorejection, or less ability of functional engraftment. Thus, discovery of novel therapeutic approaches to improve skeletal repair and healing remains a great demand and clinical challenge in Orthopaedic regenerative medicine. In the present study, we demonstrated that local administration of EPO enhanced the consolidation and mechanical competence of the newly formed bone by promoting chondrogenic and angiogenic responses following injury, which can be explained by direct promotion effects of EPO on chondrocyte proliferation and differentiation, and on endothelial growth as shown in the in vitro studies. These findings expand the extent of EPO��s tissue-specific functions and suggest that EPO may serve as a promising therapeutic agent for skeletal regeneration. Previous study reported that EPOR was restrictedly expressed in hypertrophic chondrocytes during the cartilaginous callus stage of bone healing, and proposed that improved early endochondral ossification were mediated by EPO/EPOR signaling. In our study, knockdown of EPOR in primary chondrocytes resulted in a dramatic reduction of their responses to exogenous EPO in terms of cell proliferation and chondrogenic marker genes expression upon chondrogenic differentiation. These evident reduction in EPO��s effects on chondrocytes upon EPOR knockdown clearly demonstrated that EPO regulated the proliferation and differentiation of chondrocytes at least partially through EPOR. Interestingly, EPO was also found to be present in chondrocytes in developing bones of new born mice as well as in newly formed cartilaginous callus of the healing bone in our study. Blockage of the endogenous EPO in primary chondrocytes using EPO block peptide impaired the chondrogenic differentiation, suggesting that endogenous EPO may function as an autocrine or paracrine factor for chondrocytes in normal bone development.