| Objective:To isolate, culture and identificate canine bone marrow-derived endothelial progenitor cells (endothelial progenitor cells, EPCs), and evaluate the effect of EPCs on the bone forming ability of tissue engineered bone in vivo. Methods: Use density gradient centrifugation to extract mononuclear cells from canine bone marrow,through different methods to culture EPCs and osteogenesis-induced bone marrow mesenchymal stem cells, EPCs were observed and identified with Dil-ac-LDL, FITC-UEA-I fluorescence double labeling and flow cytometry; canine allogeneic demineralized bone matrix (demineralized bone matrix, DBM) was prepared and constructed with EPCs and osteogenesis-induced BMSCs in vitro, the composites were implanted under the canine latissimus dorsi muscle fascia,vascularization and bone formation level of tissue engineered bone were observed by radiological and histological methods at different time. The data were analyzed by paired t test with SPSS13.0 package. Results:Primary EPCs were colony grow, cells were "vascular-like"grow and turned from spindle into the typical "cobblestone"-like after cultured for 12 days.Dil-ac-LDL, FITC-UEA-I were positive for fluorescent double staining, Flow cytometry showed cells were positive for CD34 and CD133.4,8,12 weeks after animal experiment X ray showed that the experimental group had a higher bone mineral density, bone mineral density between the two groups was significantly different (P<0.05); histological evaluation showed that the experimental group had a larger vascular area and new bone area than the control one, which were both significantly different (P<0.05). Conclusion:The utility of density gradient centrifugation and adherent culture method can isolate and culture canine bone marrow mononuclear cells which can differentiate into EPCs after induced, EPCs can be applied in bone tissue engineering and promote its ability of vascularization, enventually accelerate bone formation. |
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