Study On Repairmen Of Rabbit Radial Bone Defects Using BMP-2and BMSCs Combined With N-HA/n-LDIG Composite Material

Chapter1Separation, Culture, Identification of Rabbit Bone Marrow Mesenchymal Stem Cells and Their Induced Differentiation to OsteoblastsObjective:To explore the separation, culture, identification of rabbit bone marrow mesenchymal stem cells (BMSCs) and their induced differentiation to osteoblasts, as well as the capacity of BMSCs in proliferation in vitro and directed differentiation.Method:The bone marrow washing fluid of rabbit femur was taken for separation, culture, purified rabbit of BMSCs by Percoll separation medium density gradient centrifugation and adherence screening method, the growth conditions and morphological changes of passaged cells were observed under light microscope; the cell proliferation was determined by MTT assay so as to fit growth kinetic curves; the rabbit BMSCs surface markers were observed by HE staining morphology and identified by flow cytometry antibodies; the P3BMSCs were induced in vitro to differentiate into the osteoblasts, the induced BMSCs were stained by Gomori Gomori ALP staining and alizarin Red calcium mineralized nodules to identify their capacity to differentiate into osteoblasts.Results:The cells for separated culture were highly homogeneous, and grew along the long axis of cell body, presented swirly, mesh, fusiform, radial, and formed typical BMSCs. The growth curve of P3BMSCs was roughly S-shaped, their7～11d cells were in a period of rapid growth, the number of cells here rapidly increased; the HE staining BMSCs morphology presented long fusiform or long triangle, the nucleus cytoplasm ratio was high, the nuclear was blue-stained, the cytoplasm was abundantly red-stained. The flow cytometry detection for the cell surface markers showed the expression percentage of the BMSCs CD44was98.63%, positive, while the expression of CD45presented negative. At3weeks after the P3BMSCs were induced to osteoblasts in vitro, the Gomori calcium cobalt staining ALP was positive, the cytoplasm showed gray and black particles or brown-black or black precipitates. Alizarin Red staining was positive, the intercellular space appeared round or oval dense mineralized nodules.Conclusion:The rabbit BMSCs can be separated and cultured through the combination of Percoll separated medium density gradient centrifugation and adherence screening method which is an ideal method to obtain rabbit BMSCs; rabbit P3BMSCs have good purity, good proliferative capacity, and biological characteristics of directed differentiation to osteoblasts, which is a kind of ideal seed cells for tissue engineering and also lays a solid foundation for follow-up bone defect repair experiments. Chapter2Rreparation of n-HA/n-LDIG composites and biology assessment for the cells compoundly cultured from bmscs in vitroObjective:The lysine salt, glycerine and n-HA were selected as the main raw materials to prepare a new n-HA/n-LDIG composite, analyzing its compatibility to the cells compoundly cultured from BMSCs in vitro so as to explore its feasibility to be a tissue engineering scaffold.Methods:The lysine salt and glycerol were selected as raw materials, through organic synthesis, polymerization, nanotechnology, supercritical anti-solvent crystallization, ultrasonic dispersion technologies, they were processed to prepare a n-HA/n-LDIG composite. By electron microscopy, its structure and porosity was observed and calculated, then the cell adhesion of it was determined by cell counting method, and its influence on cell proliferation was determined by MTT assay, next its influence on cell differentiation was determined by ALP activity and calcium content determination.Results:The n-HA/n-LDIG composite visually likes scatteredly white, relatively uniform powdery solid. The3D porous network structure similar to natural cancellous bone was observed under the scanning of electron microscopy, and its irregular arrangement of whiskers and pores could be found under transmission electron microscopy. The cells were cultured within2h to10h, with the time increased, the cell adhesion also increased, and the cell adhesion rate at6h was significantly higher than that at4h, the cell adhesion rate at8h was significantly higher than that at6h, their differences were statistically significant (P<0.05). MTT assay results showed:from2d to16d of culture, with the time increased, the cell counts on composite also increased, especially rapidly increased from6d (the cells were in the exponential growth phase), on12d, the cells developed to a growth plateau with slow proliferation. From4d, the ALP relative activity and calcium relative content quickly raised; the ALP relative activity and calcium relative content on8d were significantly higher than those on4d, and the ALP relative activity and calcium relative content on12d were significantly higher than those on8d, their differences were statistically significant (P<0.05); from12d, the growth rate of ALP relative activity and calcium relative content slowed down, their difference was not statistically significant (P>0.05).Conclusions:The prepared n-HA/n-LDIG composite has suitable porosity and pore size, and its composition properties are similar to those of natural bone structures, having good biological characteristics, so it is a new nano-bone repair material. The n-HA/n-LDIG composite have good cell compatibility and can act as microenvironment for the growth, proliferation, matrix secretion of cells on its surface. As a premium bone repair alternative material, it laid a foundation for rapid bone repair and bone tissue engineering scaffold material development. Chapter3Experimental study on repairmen of rabbit radial bone defects using BMP-2and BMSCs combined with n-HA/n-LDIG composite materialObjective:To build the rabbit radial bone defect model, and explore the repair effects of rabbit radial bone defects using BMP-2and BMSCscombined with n-HA/n-LDIG composite material.Method:The New Zealand white rabbits were used to build bone defects models, the experiment group was implanted BMP-2and BMSCs combined with n-HA/n-LDIG composite material, and the control group was implanted the BMSCs combined with n-HA/n-LDIG composite material, and the blank group was not implanted. The rabbits were randomly sacrificed after2,4,8and12weeks and taken the general observation, X-ray examination and Lane-Sandhu scoring to observe the healing of the bone defect, and through the histological examination and Lane-Sandhu histological scoring to observe the repair of the bone defect.Results:All experimental animal radius bone defect models were successfully built, subcutaneous and muscle tissue congestion, edema, exudate and infection were not observed. From the general observation, X-ray examination and histological examination, we can found that, with the time going, the repair effects of the experiment group were better than that of the control group, and the repair effects of the control group were better than that of the blank group. The area percentage of newborn bone of bone defect, Lane-Sandhu radiological scores and the Lane-Sandhu histological scores after3,6and9weeks of the experiment group were significantly higher than of the control group, and the control group were better than the blank group, and the differences were statistically significant (P<0.05).Conclusion:The BMP-2can significantly promote the early osteogenesis, shorten the osteogenesis time and accelerate the regeneration and calcification of the bone tissue at bone defect area; the BMP-2and BMSCs combined with n-HA/n-LDIG composite material has good bone defect repair effects, can significantly promote the early osteogenesis, shorten the osteogenesis time and accelerate the regeneration and calcification of the bone tissue at bone defects area, provides a theoretical basis and new idea for clinical application, and has good prospects for clinical application.