The Experimental Study On Guided Bone Regeneration With Acellular Bovine Pericardium Combined With BMP-2Transfected Bone Mesenchymal Stem Cells

Guided bone regeneration (GBR) has developed in the last two decades which is to promote bone regeneration and healing as a new technology. It refers to guiding specific cell adhesion and proliferation to the lesion site, in which membrane barrier is used to block the proliferation of other tissue cells which grows rapidly in the lesion area from surroundings, and to eliminate competitive inhibition, thus promoting lesion repair. In recent years, the composite film is more effective in guided bone regeneration due to the seed cells and bioactive factor. Recently, there is few study about composite function GBR membrane preparation based on naturally derived bovine pericardium, and our study is divided into four parts. First, naturally derived GBR membrane is made from natural bovine pericardium through acellular preparation process and crosslinking treatment, according to the technical requirements of guided bone regeneration; second, bone marrow mesenchymal stem cells (BMSCs) are cultured and induced differentiation in vitro and BMP-2gene transfection experiments are conducted for the following composite GBR membrane research; in the third part, naturally derived acellular bovine pericardium is recombinated with BMP-2gene transfected BMSCs in vitro, testing the biocompatibility and cell proliferation, etc., laying foundation for the following animal experiments; fourth, the preliminary animal experiments are conducted using naturally derived acellular bovine pericardium compounding BMP-2gene transfected BMSCs for guiding bone regeneration, discussing the feasibility of clinical application of the acellular bovine pericardium compounding BMP-2gene transfected BMSCs in guiding bone regeneration and repair of bone defects.Part I The preparation of naturally derived acellular bovine pericardiumObjective:To find the best way for decellularization and cross-linking of naturally derived bovine pericardium, laying foundation for preparing GBR membrane which can meet the clinical demand.Methods:1.Under sterile conditions, fresh bovine pericardium were selected and decellularized using trypsin+detergent method (Ⅰ), trypsin+enzymatic nucleic acid method (Ⅱ), trypsin+detergent+enzymatic nucleic acid method (Ⅲ), freeze-thaw+detergent method (Ⅳ), freeze-thaw+enzymatic nucleic acid method (Ⅴ), or freeze- thaw+detergent+enzymatic nucleic acid method (VI), and then the best way of decellularization was screened by examining the acellular bovine pericardium by means of optical microscope, scanning electron microscope, cell toxicity test, the determination of the mechanical properties, and the measurement of the chemotactic properties.2. The naturally derived acellular bovine pericardium were cross-linked using glutaraldehyde or genipin, and the best acellular bovine pericardium cross-linking method was screened by optical microscopy, scanning electron microscopy, thickness test, mechanical properties test, cross-linking index determination, in vitro degradation and cell toxicity test.Results:1. Decellularization results showed that:freeze-thaw+detergent+enzymatic nucleic acid method, with bovine pericardium fibers arranged densely and orderly, structure well preserved, and low cytotoxicity after treatment, was the best method of decellularization.2. Cross-linking results showed that:Genipin crosslinking method, by which acellular bovine pericardium was crosslinked had shown better biocompatibility, was the best of cross-linking.Conclusions:Freeze-thaw+detergent+enzymatic nucleic acid method was an ideal method for preparation of acellular bovine pericardium, and genipin crosslinking method was an ideal method for crosslinking of acellular bovine pericardium.Part Ⅱ The experimental study on induced differentiation of bone marrow mesenchymal stem cells(BMSCs) and BMP-2gene transfection on BMSCsObjective:To explore the method of isolating rabbit BMSCs and induce differention of them, studying the multipotential differention of BMSCs; and to observe results of BMP-2gene transfection on BMSCs for further GBR studies.Methods:1.Rabbit BMSCs, isolated and cultured by adherent culture, were observed by the phase contrast microscope; and the second generation of BMSCs in good status were induced to differentiation in adipocytes, chondrocytes, and osteoblasts culture media respectively, detecting the differentiation ability of BMSCs.2. BMSCs was subcultured with plasmid vectors by electroporation transfection of BMP-2gene, and then the following identification was made:green fluorescent protein expression was detected by fluorescence microscopy; BMP-2mRNA expression in transfected cells by RT-PCR; BMP-2expression at the protein level by Westeblot; and osteogenic properties were evaluated by ALP quantitative detection.Results:1.The primary and subcultrued BMSCs from rabbit grew well; oil red staining showed that large amounts of fat droplets were bright red in cytoplasm, the nucleus were in blue and purple; alizarin red staining showed a large amount of red positive clones in the calcified nodules; immunohistochemistry detected the expression of collagen I and aggrecan, showing large yellowish-brown granules in cytoplasm.2. The transfection efficiency of BMSCs was41.2±1.1%; RT-PCR results showed the positive expression of BMP-2RNA; apoptotic Westernblot results show ed that BMP-2protein can be effectively expressed; ALP activity of BMSCs was significantly higher after BMP-2transfection.Conclusions:1. Rabbit BMSCs, which can differentiate into adipocytes, chondrocytes, osteoblasts in particular induction conditions, have multilineage differentiation potential.2. BMSCs, transfected with BMP-2gene, can successfully express BMP-2protein. Part III Acellular bovine pericardium combined with BMP-2transfected BMSCs guided bone regeneration in vitroObjective:To study the biocompatibility and cytotoxicity of acellular bovine pericardium combined with BMP-2gene transfected BMSCs, and BMSCs’adhesion, growth, proliferation and osteogenic differentiation on bovine pericardium.Methods:For cellular bovine pericardium combined with BMP-2transfected BMSCs, its adhesion, growth, proliferation on acellular bovine pericardium was assessed by scanning electron microscopy, the cytotoxicity of membrane on transfected cells assessed by MTT test, BMP-2content measured by ELISA, and cell adhesion capability detected by crystal violet staining, osteogenic properties evaluated by ALP activity.Results:1. The scanning electron microscopy results showed that:BMP-2gene transfected BMSCs, when combined with acellular bovine pericardium, can adhere to the pericardium successfully, and proliferate well.2. Cytotoxicity test results showed no significant difference in the survival rate of cells in each group. 3. Cell adhesion test results showed that:the cell adhesion capability of BMP-2transfected BMSCs was enhanced by acellular bovine pericardium.4. ALP activity detection, Westernblot detection and RT-PCR analysis showed that:the ability of BMP-2transfected BMSCs transforming into osteoblasts was enhanced by acellular bovine pericardium.Conclusions:Acellular bovine pericardium combined with BMP-2gene transfected BMSCs, with its good cell compatibility, can be used as GBR membrane.Part IV Acellular bovine pericardium combined with BMP-2transfected BMSCs guided bone regeneration in vivoObjective:To study the GBR effect of acellular bovine pericardium combined with BMP-2gene transfected BMSCs on the repair of rabbit bone defects.Methods:After New Zealand rabbits bilateral mandibular bone defect model was prepared, acellular bovine pericardium combined with BMP-2gene transfected BMSCs(group A) and acellular bovine pericardium only(group B) was implanted into the defects respectively, and group C was the blank control group.4weeks,8weeks, and16weeks after the operation, the gross specimen were observed, X-ray and histologic detection was made to study the bone regeneration.Results:General observation, X-ray and histologic detection showed that: There was significant difference in healing of bone defects after8weeks and16weeks between experimental group(group A, B) and blank control group(group C), with group A better than group B (p<0.05).Conclusions:There is a positive impact of acellular bovine pericardium combined with BMP-2gene transfected BMSCs on rabbit experimental bone defect repair.