Reconstruction Of Orbital Defects By Implantation Of Antigen-free Bovine Cancellous Bone Scaffold Combined With Bone Marrow Mesenchymal Stem Cells In Rats

Part I The Preparation of Antigen-free Bovine Cancellous Bone Carrier and The Structural AnalysisObjective:To prepare the antigen-free bovine cancellous bone (BCB) carrier and evaluate the structural analysis of BCB.Methods:The fresh bovine proximal humerus cancellous bone was cuts into8×5×5mm3and rinsed with water. Particles were immersed in0.5%Triton X-100and10%sodium chloride to remove cells. After immersion in the mixture of equal volume of methanol and chloroform to degrease, they were deproteinized with30%hydrogen peroxide. Then they were dunked in0.5M hydrochloric acid for partial decalcification. BCB particles were sterilized by Co-60gamma irradiation and stored at-80℃. The macroscopical structure, microstructure by scanning electron microscopy, the pore diameter and the porosity of BCB particles were observed and analyzed.Results:The macroscopical structure of BCB scaffolds presented uniformly distributed, interconnected reticular network. SEM analysis showed clear porous structure and trabecular structure of BCB scaffolds. The average value of the BCB porosity was (58.1±11.4)%. The average value of The BCB pore diameter was (374.2±69.2) μm.Conclusion:The antigen-free bovine cancellous bone has the interconnected reticular structure and meets the demands of the porosity and the pore diameter as scaffold material of bone tissue engineering.Part Ⅱ The Biocompatibility of Antigen-free Bovine Cancellous BoneObjective:To study the biocompatibility of the antigen-free bovine cancellous bone.Methods:The five experiments were performed with antigen-free bovine cancellous bone in vivo. Acute toxicity test:antigen-free bovine cancellous bone extracts were injected into the abdominal cavity of mice. Pyrogen test:antigen-free bovine cancellous bone extracts were injected into the ear vein of the rabbits. Hemolysis test: the mixed blood of rabbits were added into the antigen-free bovine cancellous bone extracts, sodium carbonate (positive control), and normal saline (negative control). Intracutaneous stimulation test:antigen-free bovine cancellous bone extracts were injected into subcutaneous tissue of rabbits. The situation was recorded at the instant,6h,24h,48h,72h after injection and primarilystimulation index and average primarily stimulation index were calculated. Muscle embedding test:antigen-free bovine cancellous bone particles were implanted into the thigh muscle of rats.Results:antigen-free bovine cancellous bone did not induce acute toxic reaction, pyrogen reaction, hemolysis reactions, hemolytic reaction, skin irritation, and not lead to rejection reaction after it was implanted into the thigh muscle pouches of rats.Conclusion:Antigen-free bovine cancellous bone has the property of good biocompatibility.Part Ⅲ The Cytocompatibility of Antigen-free Bovine Cancellous Bone Combined with Bone Marrow Mesenchymal stem cells of Green Fluorescent Protein Transgenic Rats in VitroObjective:To test the cytocompatibility of antigen-free bovine cancellous bone (BCB) with induced bone marrow msenchymal stem cells of green fluorescent protein transgenic rats (GFP-BMSCs) in vitro and estimate the feasibility of the establishment of GFP-BMSCs/BCB composites.Methods:BMSCs of GFP-rats were isolated, purified, cultured, identified and multidirectional induced differentiation was performed. Third-passage BMSCs were digested, Cells suspension was dropped into BCB scaffold and cocultured for12days. The morphology, attachment, growth and proliferation of BMSCs were observed. MTT assay were performed to decide the toxicity of BCB extracts to BMSCs.Results:BMSCs adhered onto the surface of BCB showed the normal morphology and adhered, grew and proliferate vigorously. There was not a statistically significant difference in the OD value between BMSCs cocultured with BCB extracts and control groups (P>0.05). The cytotoxicity of BCB to BMSCs was grade0-1.Conclusion:BCB scaffolds having no cytotoxicity to BMSCs have the good cytocompatibility to BMSCs. BCB can be applied as practicable scaffolds for BMSCs in tissue engineering for the reconstruction of orbit defect.Part IV Reconstruction of Segmental Orbital Defects by Implantation of Antigen-Free Bovine Cancellous Bone Scaffolds Combined with Bone Marrow Mesenchymal Stem Cells in RatsObjective:To reconstruct the segmental orbital rim defects with antigen-free bovine cancellous bone (BCB) scaffolds combined with bone marrow mesenchymal stem cells (BMSCs) in rats.Methods:BCB was prepared by removement of cells, degreasing, deproteinization and partly decalcification. BMSCs isolated fromGFP-rats were osteogenically induced and seeded onto BCB scaffolds to construct induced BMSCs/BCB composites. An8mm full-thickness defect on the inferior-orbit rim of40rats was established. Induced BMSCs/BCB composites cultured for5days were implanted into the orbital defects as experimental group. Noninduced BMSCs/BCB group, BCB group and exclusive group were set. General condition of the animals after surgery was recorded daily. Spiral CT and3D orbital reconstruction were performed at2,4,8,12weeks after implantation. Histological analysis and histomorphometric analysis were performed at12weeks after implantation.Results:The diet, activity and eyeball motility of all animals were normal and no infection, implants dislocation or adverse tissue reactions appeared through all the process after operation. In induced BMSCs/BCB group, CT and3D reconstruction showed perfect orbital repair situation. Histological analysis indicated BCB was biodegraded mostly; newly-formed bone and complete synostosis were observed. The percentage of newly-formed bone was (57.12±6.28)%.Conclusions:Antigen-free bovine cancellous bone has the moderate mechanical strength and good osteoconduction. BCB combined with BMSCs is a promising composite for tissue engineering and can effectively reconstruct the orbit rim defects in rats.