| Objective To explore the osteogenesis effect of bone marrow stromal stem cells-derived osteoblasts (OB) and vascular endothelial cells (VEC) compound with allogeneic freeze-dried partially bone(AFDPB) in repairing of segmental radial defect in rats, so as to provide the experimental strategies for the clinical application of vascular tissue engineered bone.Methods The BMSCs were isolated from bone marrow in SD rats about four-week-old. The BMSCs in 3rd generation were induced into osteoblasts and vascular endothelial cells. The typeâ… collagen and Alkaline phosphatase (ALP) of osteoblasts were detected by immuno- cytochemistry and ALP staining. The CD34 and CD31 proteins of vascular endothelial cells were examined by immunocytochemistry staining. The biocompatibility and cytotoxicity of OB and VEC compounded with AFDPB in vitro were detected by MTT and scanning electron microscope. The cell circle of OB and VEC compounded with AFDPB was detected by flow cytometry. Labeled osteoblasts and vascular endothelial cells with BrdU(5-bromo-2-deoxy Uridine) was detected in vitro and traced in vivo by immunohisto- chemistry.The 5mm segmental radial defect in rats model was created in thirty-six SD rats about eight-week-old which divided randomly into experimental (OB/VEC/allogeneic bone) group and control (allogeneic bone) group respectively. 16 animals in each group were sacrificed successively at 2, 4, 8, 12 week postoperatively. 4 animals were empty operation. The animal specimens were examined through gross and histological observation. Immunohistochemitry were used to determine the expression of OPG/OPGL protein and microvessel density(MVD) counting in new bone. Results 1. There was positive expression in alkaline phosphatase (ALP) , typeâ… collagen and CD31,CD34.2. The MTT test and scanning electron microscope showed that allogeneic freeze-dried partially bone had better biocompatibility and no cytotoxicity, and cells grew well and spread extensively and proliferated rapidly on AFDPB.3. Flow cytometry showed that allogeneic freeze-dried partially bone had no effect on the cell circle. There were normal diploid cells in experimental group and no significant difference between experimental group and control group (P>0.05).4. The model of segmental radial defect was confirmed in two groups. Compared with control group, gross and histological observation were better in experimental groups at different periods. There was positive expression in vivo labeled OB and VEC with BrdU by immunohisto- chemistry at 2, 4 week postoperatively.The expression of OPG protein in experimental groups was significantly higher than that of control groups at 2, 4, 8 week (P<0.05) and there was no significant difference between two groups at 12 week (P>0.05). The expression of OPGL protein in experimental groups was significantly higher than that of control groups at 2, 4 week (P<0.05) and there was no significant difference between two groups at 8, 12 week (P>0.05). The microvessel density (MVD) counting in experimental groups was significantly higher than that of control group at 2, 4, 8 week (P<0.05) and there was no significant difference between two groups at 12 week (P>0.05).Conclusion 1.Osteoblasts and vascular endothelial cells from BMSCs are optimal seed cells of bone formation and vascularization of bone tissue engineering.2. The allogeneic freeze-dried partially bone has good biocompatibility with low antigenicity and no cytotoxicity, which is optimal scaffold material as tissue engineering bone.3. The labeled OB and VEC with BrdU in vitro were traced in vivo by immunohistochemistry, and they can promote ossification and vasculari- zation.4. The bone Marrow stromal stem cells-derived OB and VEC compound with allogeneic freeze-dried partially bone can promote bone formation and vascularization on bone defect and accelerate the healing of bone defect.
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