| Objective:The technique of bone tissue engineering is a promising substitute for clinical treatment of bone defect. Autogenic mesenchymal stem cells(Auto-BMSCs) has been reported to construct tissue engineering bone and generate bone tissue and repair bone defect in samll mammals. However, it is still controversial if allogeneic BMSCs (Allo-BMSCs) can successfully construct tissue engineering bone, and how about the immunogenicity of Allo-BMSCs after transplantation.This study was firstly desighed to explore the potential of allogeneic bone mesenchymal stem cells to construct tissue engineering bone combined with composite biological scaffold in the back of beagle dogs. Therefore, the osteogenis area and scaffold area was compared between the Allo-BMSCs and Auto-BMSCs to explore the ability of the ectopic and orthotopic osteogenesis, and immunogenicity of allogeneic of BMSCs was detected.Meanwhile, an animal model of critical skull defect was built in Beagle dogs to evaluate the feasibility of allogeneic BMSCs to repair skull defect in large mammals combined with beta tricalcium phosphate (beta-TCP).Methods:1. BMSCs was purified from canine bone marrow blood, and then induced into osteoblast, chondroblast, adipose cell. The potential of three direction differentiation of bone mesenchymal stem cells was identified. Then, osteoinduced BMSCs of the second generation was labeled by CM-Dil to expore how long would the BMSCs would survive in vivo. The proliferation of CM-Dil labeled BMSCs was detected by MTT method. The osteogenic differentiation of the labeled BMSCs was detected by RT-PCR of collagen I, osteonectin, BMP-2, and osteocalcin. CM-Dil labeled BMSCs combined with beta TCP was transplanted into Beagle’s back subcutaneously.8weeks later, outcomes of BMSCs was traced by the fluorescence microscope. Meanwhile, ectopic osteogenesis of the labeled BMSCs-TCP complex was detected by histological observation. 2. The osteoinduced BMSCs of the second generation was composited with beta TCP in vitro, and then was subcutaneously transplanted to the autogenic or allogeneic canines respectively.β-TCP implantation alone was served as control group. Fluid cytology examination was performed on the day of surgery and3,7,14,28,56day postoperatively among the three groups. Systemic immune response was assessed through the change of peripheral blood T lymphocyte subsets.24weeks postoperatively, the tissue engineered bone was harvested and HE staining was performed. The osteogenesis of the three months was compare quantitatively by the histology measurement.3. The animal model of full-thickness critical skull defect (diameter:2cm) was built in Beagle dogs bilaterally. Allogeneic or autologous BMSCs-TCP complex was constructed to repair the above bone defect. Scaffold alone group was served as control.4,12,24and36weeks postoperatively, the repair effect of skull defect among the three groups was compaired quantitatively by3D-CT.36weeks postoperatively, the quality of the bone repaired among the three groups was evaluated by gross observation, micro-CT, biomechanical detect and histological observation respectively.Results1. BMSCs isolated from bone marrow blood of canines have the capacity to differentiate to osteoblast, chondroblast and adipose cells. The morphology was consistent between the CM-Dil labeled BMSCs and unlabeled cells. There was no significant difference of the proliferation rate between the two groups (P>0.05); RT-PCR results revealed the expression of Col-1, BMP-2, BGLAP, SPARC in the labeled BMSCs, which suggested that CM-Dil has little effect on the osteogenetic differentiation of BMSCs. Tissue engineering bone constructed with labeled BMSCs was implanted in Beagle dogs subcutaneously.8weeks later, CM-Dil labeled tissue engineering bone still could stimulate red fluorescence under fluorescence microscopy. HE staining confirmed that ectopic osteogenesis by labeled BMSCs in vivo.2. Ectopic osteogenesis happened both in allogeneic and autologous BMSCs-TCP complexs in vivo.24weeks postoperatively there is no significant difference between the allogeneic and autologous group in the osteogenesis percentage histologically (P>0.05), which was significantly higher than beta-TCP control group (P<0.001), Fluid cytology examination showed that CD4+T cells of allogeneic group at3,7day postoperatively is statistically higher than the other time points (P<0.05). However there is no obvious difference in CD4+T cell count, CD8+T cell count, the percentage of CD4+/CD8+T lymphocytes between the three groups (P>0.05). With time period, the percentage of CD4+/CD8+T cells firstly rosen up and then went down in the above three groups.3.3D-CT reconstruction and measurement analysis showed that bone mineral density of allogeneic and autologous bone tissue engineering bone gradually reduced at4,12weeks, but remained stable from24to36weeks. There is no significant difference in bone mineral density between the two groups (P>0.05). Bone mineral density of the control group gradually reduced as time goes by, which is significantly lower than the above two groups at12,24and36weeks (P<0.001). At36weeks, gross observation showed allogeneic and autologous tissue engineering bone still can maintain the integrity of the skull. Micro-CT and biomechanic examination showed no significant difference between the two groups (P>0.05). Histology showed that there was osteoblast and osteocyte in allogeneic and autologous groups, and osseous connection was formed. However in the control group,β-TCP degradated mostly and the defect was composed of fibrous tissue.Conclusions1. BMSCs of canines have osteoblast, chondroblast and adipocyte differentiation capacity. There is no obvious influence of CM-Dil on the growth, proliferation, osteo-differentiation of bone marrow stromal stem cells. Ectopic bone formation can be achieved by tissue engineering bone constructed by labeled cells and beta-TCP in vivo. Bone marrow stromal stem cells can survive as long as eight weeks post-implantation.2. Ectopic bone formation can be achieved by the BMSCs-TCP-constructed tissue engineering bone. There is no obvious difference in ectopic osteogenesis between the allogeneic and autologous group at24weeks after transplantation. There is no significant difference in systemic immune response between the two groups postoperatively, which suggests that allogeneic BMSCs caused no obvious immune rejection.3. The critical skull defects of beagle dogs can be repair by the orthotopic allogenic tissue engineering bone. The osteogenic rate at an early age (Week12) was slower than the autogenic group however there is no obvious difference between the two groups eventually (Week36). |
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