Composite Artificial Bone Artificial Bone Allograft Bone Marrow Stromal Stem Cells To Repair Bone Defect In Rabbit Of Experimental Research

Part one Isolation and cultivation of rabbit bone marrow stromal stem cells in vitroObjective:To grasp the cultural method of rabbit bone marrow stromal stem cells and lay the foundation for subsequent experiments.Methods:The whole bone marrow was obtained from rabbit femoral condyle. Then bone marrow stromal stem cells(BMSCs) were purified and cultured with 0.25% trypsin enzyme digesting technique. MTT assay was tested to observe the proliferative ability of BMSCs which were positively expressed CD44.Results:Rabbit marrow stromal cells were isolated and cultured through whole marrow culture method. The cells were Confirmed nucleus pulposus cells by immunocytochemistry staining which were positively expressed CD44, and then the activity of BMSCs were validated normal by MTT assay.Conclusion:Normally proliferated MSCs could be successfully primary cultured and subcultured with 0.25% trypsin enzyme digesting technique.Part two The expression and significance of rabbit bone marrow stromal stem cells after GFP trasfectionObjective:To observe the expression differences and transfection efficiency of green fluorescent protein(GFP) in nomal rabbit bone marrow stromal stem cells(BMSCs).Methods:bone marrow stromal stem cells(BMSCs) were transfected by Green fluorescent protein(GFP) which was carried by plasmid and mediated by liposomal. Cells were observed under the phase contrast microscope and fluorescence microscope. In addition, GFP transfection efficiency was detected.Results:GFP transfection efficiency could reach 40%, after further screening, even reach 95%. The grow curve characteristics between nomal rabbit BMSCs and GFP transfected BMSCs had no significant differences(p>0.05).Conclusion:Using immunocytochemistry and flow cytometer, our experiment demonstrated that to lead GFP into rabbit bone marrow stromal stem cells which were cultured in vitro could not affect their biological activity. GFP could act as a testing tool to exam the biological activity of BMSCs.Part three The observation of preparation and degradation in vivo of tissue-engineered artificial boneObjective:To discuss the effects on osteoblast biological behavior in vitro circumstance of the artificial bone materials which is composited with Marrow stromal stem cells and polylactic acid.Methods:Polylactic acid artificial bone and rabbit osteoblasts were co-cultured in vitro. Inverted phase contrast microscope, scanning electron microscope, transmission electron microscope and fluorescence microscope were used to observe the adhesive capacity and biological behavior of rabbit osteoblast on Nacre-polylactic acid artificial bone.Results:By means of pseudopods, osteoblast attached to the surface of polylactic acid artificial bone 5 days after co-cultured. Transmission electron microscope showed that osteoblast eumorphism, their were abundant rough surfaced endoplasmic reticulum and mitochondria in cytoplasm, and, their were Connections between cells.Conclusion:Composite artifical bone had the strongest effect on the attachment, proliferation and differentiation of osteoblast and could promote osteogenic potential in vitro.Part four The study of Composite artificial bone in repairing bone defect in rabbitsObjective:To study the mechanism of osteogenesis and degradation of artificial bone Composited with rabbit bone marrow stromal stem cells in rabbit radius defects models.Methods:Implanting artificial bone between radius defects of rabbits in experimental group, another taking the control group. Then the rabbits were sacrificed after 6,12,18 weeks and taken out needed tissues to detect bone growth based on imaging test, scanning electron microscopy test and histological inspection.Results:ALP positive cells in experimental group were more than that in control group after cultured 7 days and 14 days. After implantation in the body, composite artificial bone degraded gradually and were replaced by bone tissue. New bone tissue grew in the pore of artificial bone material formed during the process of polylactic acid degradation. Artificial bone materials and rabbit radius bone defects fused generally at 18 weeks. The materials in control group were replaced by new born woven bone and osteoid tissues.Conclusion:Composite artificial bone was degradable in vivo and had good bone conduction function. Artificial bone implanted into body and adsorbed proteins and other substances around to form a fiber fonnatio reticularis and prompt it to calcify, which was called calcium deposition.