The Construction Of Allogeneic Tissue-Engineered Bone And Repair Of Bone Defects In A Large Animal Model

Objective:Whether allogeneic bone mesenchymal stem cells (allo-BMSCs) arecompletely immune privilege and play a biological role in vivo or not is still a controversial issue. This experiment studied the ectopic osteogenesis capacity and immune response of recipient of allogenic tissue-engineered bone (Allo-TEB) in vivo by the subcutaneously transplantation of allo-BMSCs-TEB. At the same time, the feasibility of repairing canine critical-sized mandibular defects by allo-TEB was also been explored.Methods:(1)BMSCs was isolated from bone marrow of Beagles dogs and induced into osteoblast, chondroblast or adipocyte in vitro. The potential of triple differentiation of BMSCs was identified and the growth of BMSCs inside the TEB was observed by electronic microscope.(2)EGFP and CM-Dil were used, to label Beagle's BMSCs and the proliferation of the cells was determined by MTT. Then the labeled BMSCs-Coral complexes were transplanted subcutaneously to nude mice.4,8and12weeks later, the biological outcome of the transplanted BMSCs inside TEB was verified by fluorescence microscope and immunohistochemistry observation. The bone formations of the two groups were evaluated by histological examination at the end.(3)The gene type of MHC I of all the animal was detected by PCR. The experimental grouping was made according to the result of MHC I and MLC (Mixed Lymphocyte Culture) to make sure the genetic mismatch/match in allogeneic/autogenic groups respectively.(4) TEB were constructed by coral or β-TCP scaffold which inoculated with Beagle's BMSCs. After one week's osteogenic differentiation, the constructs were transplanted subcutaneously to autologous or allogeneic Beagle dogs. Scaffold only group was served as control. The histological observation and analysis was used to evaluate the ectopic osteogenesis capacity of each groups3,7,14,28and56days after the operation. The peripheral blood content of inflammatory markers interleukin (IL)-2and IL-10were detected at the same time.(5) Canine critical-sized mandibular defects (3cm) were built at the right side mandibular of Beagles dogs and repaired by BMSCs-coral TEB which was constructed with ether autologous or allogeneic BMSCs. Coral scaffolds only was served as negative control and autologous bone grafts served as positive control. The repair of the defects were evaluated by X-ray,3D-CT, Micro-CT, gross observation, biomechanical testing and histological analysis24weeks after the operation. The content of immune-related cytokines and lymphocyte typing of peripheral blood were detected3,7,14,28and56days after operation.Results:(1) The BMSCs which isolated from bone marrow of Beagles dogs have the capacity to be triple differentiated to osteoblast, chondroblast and adipocyte. The BMSCs inside the scaffold can adhere, proliferate and secret extracellular matrix in3-D environment.(2) There is no significant difference between EGFP/CM-Dil labeled BMSCs and unlabeled BMSCs on the respect of proliferation ability (P>0.05). Red fluorescence still could be observed in CM-Dil labeled TEB under fluorescence microscopy12weeks after operation. The immunohistochemical staining demonstrated that GFP positive cells presented in EGFP labeled TEB. The new bone formation could be found in both TEB groups.(3) Genetic mismatch between the different individuals:The Beagles dogs with different MHC I genes were selected. The result of MLC displayed that the proliferation rate of lymphocytes between different individuals was significantly higher than the autologous lymphocytes culture (P<0.05).(4) Ectopic construction of TEB by allogeneic BMSCs:There were new bone formations was found both in auto and-allo-TEB groups. The osteogenesis speed of auto-BMSCs-TEB groups was faster than allo-BMSCs-TEB groups before8weeks after transplantation (P<0.05). However there was no significant difference (P>0.05) between the two groups at the end (12weeks after.the transplantation, P>0.05). None of the new bone formation was found in empty control group.7days after operation, the levels of IL-2in peripheral blood was increased transiently in allo-TEB groups (P<0.05) compared with transiently reducing of IL-10(P<0.05). There was no significant difference between the two groups at any of the other time points (P>0.05).(5) Repair the bone defect by TEB:X-ray examinations after operation displayed that the radiopacity of auto and allo-TEB groups gradually increased with time period compared with the reverse tendency in control group. At24weeks, the continuity of the mandible was restored in auto and allo-TEB groups which were determined by gross view and CT examination. No significant difference on bone mineral density (BMD) and biomechanical stress measurement was found between auto and allo-TEB groups (P<0.05). The osteocytes, lacunae and osteo-intergration phenomenon were found in fracture site of autologous, allogeneic and autograft groups by histological examination. There was only fibrous tissue and fibrous union presented in scaffold only control group and most of the scaffold was absorbed.3days after operation, peripheral blood content of TNF-a and INF-y in allo-TEB group were transiently increased compared with auto-TEB group (P<0.05). The content of IL-2increased transiently (P<0.05) accompanied with transiently reducing of IL-10(P<0.05). There was no significant difference between the two groups at any of the other time points for TNF-α,INF-γ,IL-2and IL-10. There was also no significant difference between the two groups on lymphocyte typing during the whole time after transplantation.Conclusions:(1)Beagles BMSCs possess osteogenic, chondrogenic and adipogenic differentiation potential and owned a good compatibility with coral scaffolds(2)EGFP and CM-Dil labeled BMSCs maintain their proliferation and osteogenesis ability. The TEB constructed by which possess ectopic osteogenesis ability in vivo. BMSCs could survive over12weeks after transplantation in nude mice.(3)The ectopic osteogenesis rate of Beagles Allo-TEB group was lower than auto-TEB group at early stage and obtained the same bone formation effect at the end point (12weeks after operation) in vivo compared with auto-TEB group. The immune response of recipient of allo-TEB group was stronger than auto-TEB group on ectopic site at early stage (7days after operation) and tended to be the same level of auto-TEB group in the end.(4) Allo-BMSCs-coral constructs could be used to repair canine critical-sized mandibular defects. The immune response of recipient of allo-TEB group was stronger than auto-TEB group in situ at early stage (7days after operation) and tended to be the same level of auto-TEB group in the end.