| Objective:To solve the problem of slow vascularization of tissue engineered bone and new bone growth retardation, not only Osteogenesis potential of stem cells need to be implanted, but also endothelial progenitor cells to accelerate the formation of blood vessels was required. In this study, tissue engineered bone was constructed by co-cultured mesenchymal stem cells and endothelial progenitor cells with partially deproteinised biologic bone and implanted in animal model of bone defect. Osteogenesis and immune rejection after transplant was explored.Methods:(1)24New Zealand rabbits,12in each group, bilateral and unilateral tibial defect model were created. One day, three days, one week, two weeks, four weeks,12weeks after creating bone defect model, CT examination was conformed to compare bone defect model reliability and feasibility.(2)48New Zealand rabbits were divided into four groups,12in each group. Tissue engineered bones constructed by co-cultured autologous BMSCs and autologous EPCs with PDPBB and allogeneic BMSCs and allogeneic EPCs with PDPBB were implanted in animal of group A and group B respectively, while, animals in group C were implanted with PDPBB. Group D was control group.3days,7days,2weeks,4weeks,8weeks after, CD3CD4and CD3CD8was detected in order to compare CD4/CD8value of each group, at same time, IL-1, IL-6, GM-CSF, M-CSF, TNF content in peripheral blood was also detected by enzyme-linked immunosorbent assay. Histological section observation was conformed at4weeks,8weeks and12weeks.(3)36New Zealand rabbits were divided into three groups,12in each group. Tissue engineered bone constructed by co-cultured autologous BMSCs and autologous EPCs with PDPBB and allogeneic BMSCs and allogeneic EPCs with PDPBB was implanted in animal in group A and group B respectively, while, animal in group C was implant with PDPBB. Local anatomy, CT examination, tissue sections stained by HE and Masson was conformed after4weeks,8weeks,12weeks. The rate of bone ingrowth was obtained by image analysis system of LeicaQwin for HE sections of bone tissue measurement. The amount of collagen was marked with the magic wand tool of adobe photoshop7.0softwere, the histogram function was applied, carried out morphometric analysis of collagen, the bone area was represented in pixels.Results:(1) There were11successful cases in the unilateral bone defects group, which no healing of bone defects. While, the failed cases in bilateral bone defect group were11and the reasons were unilateral or bilateral bone defect deformation shift, concentrating in the five days, especially two days after surgery, malunion occurred up to4-8weeks after.(2) The ulceration of about0.5cm in surgery area of one animal in group B was found at7days after transplantation, swelling, and the bone graft was connected to the ulceration area. The secretions culture was negative and this animal died14days after transplantation. The other animals were normal after transplantation. CD4/CD8value of group B was higher than those in the control group at each time point (P<0.05). CD4/CD8value of group A was higher than those in the control group3days and7days after transplantation (P<0.05) and similar to the control group at other time point (P>0.05). CD4/CD8value of group C was similar to those in the control group at each time point (P>0.05). The content of IL-1, IL-6, GM-CSF, M-CSF, TNF were higher than those in the control group at each time point (P<0.05) CD4/CD8value of group A was higher than those in the control group3days and7days after transplantation (P<0.05) and similar to the control group at other time point (P>0.05). The content of IL-1, IL-6, GM-CSF, M-CSF, TNF of group C was similar to those in the control group at each time point (P>0.05). Lot of interstitial lymphocytes and monocytes were found in the tissue sections stained by HE of group B and were not found in other groups.(23) The anatomical observation12weeks after implantation:In the group A, the color and texture in the repair area were similar to the normal bone and bone defect was healled well. The pores occurred in the bone defect area in the group B and osteogenic was worse than that in the group A. Lots of pores were found in the group C. CT findings:In group A, bone defect has been blurred, filled with more bone formation8weeks after implantation.12weeks after, the boundary of the bone defect had been unable to identify and the bone tissue remodeling. Smooth surface could be seen, with similar outlook to normal tibia from different side. The area of bone defect was filled with uneven density of the new bone, which was higher than surrounding area. In group C, the bone defect was still visible12weeks after implantation. Histopathology finding:Lot of bone trabecula was found in which blood vessel could be seen. There were many interstitial lymphocytes and monocytes and little angiogenesis. There were little bone trabecula and angiogenesis in group C.4weeks,8weeksand weeks after implantation, the rate of bone ingrowth of group A was higher significantly than that of other group (P<0.01) and the rate of bone ingrowth was higher than group C (P<0.01). Collagen content was higher significantly than that of other group at each group, with significant increasing4-8weeks after transplantation.Conclusions:(1) The feasibility and stability of rabbit unilateral tibial defect model were good, can be used as the experimental animal models; rabbit bilateral tibial defect model is not fit to the experimental study.(2) There was no significant rejection after repairing rabbit tibial defect with autologous BMSCs and autologous EPCs composite PDPBB, while rejection was found with allogeneic BMSCs and allogeneic EPCs composite PDPBB. PDPBB can be used as a bone tissue engineering scaffold, which was not cause immune rejection.(3) The effect of repair bone defects with tissue engineered bone constructed by autologous BMSCs and autologous EPCs with PDPBB was significantly better than that with allogeneic stem cells. |
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