The Experimental Research Of The Treatment For Rabbit Tibia Bone Defect With BMSCs Composite GeneX/TGF-β2

Objective:GeneX is a kind of new artificial bone, which consists of β-tricalcium phosphate and calcium sulfate. It has microporous structure, injectable and has the advantages of strong bearing capacity after Frozen,and it also can be completely degradation. In order to find a way to show zhe effect,which could accelerate the repair of bone defects,we have designed this experiment.Implanting in the rabbit tibia defect parts with the composite materials which contains BMSCs, geneX, TGF-β2, and observing zhe ability of repair bone defect and then observing zhe influence of the quantity for VEGF and BMP-2on new bone tissue of rabbits. By this animal experiment we can explore the clinical treatment of bone defect.Method:1-. primary culture bone marrow stromal cells and continuous cell culture.Firstly pick up a rabbit and Stripping leg femur and tibia,then soaking bone marrow into culture dish. Secondly centrifuge for5minutes with1500rpm and collect cells. Took one generation of cells to the next, got the third generations cells and proved it has high purity.2、Obtain zhe composite of BMSCs/geneX/TGF-β22.1、Take the third generations cells, which the density is3×105/ml on culture dish and divid into two. One is purely-culture and another is mixed with TGF-β2,which zhe density is lng/mL.2.2、Open zhe geneX and linking injection syringe A and B. Then mix powder and normal saline,in order to get zhe pastd geneX.2.3、Squeeze geneX on the mould and prepare of artificial bone,which zhe size is about5mm to3mm, waiting for solidification.2.5、Divide the geneX into two. One will be purely-cultured and another will be mixed zhe BMSCs/TGF-β2. 3、Make the rabbit model of bone defectTake30rabbits and randomly divided into three groups. After narcosis.make zhe defect of tibia, which zhe size is15mm×5mm. A group’s wound was direct sutured; B group’s bone defect was put into geneX; C group’s bone defect was put into BMSCs/geneX/TGF-β2.4、Observed zhe osteogenic capability of three groupTake zhe X-ray、anatomy observation and tissue slice observation, when the time is4week、8wee、12week.5、Observed zhe quantity for VEGF and BMP-2、VEGF’s mRNAand BMP-2’s mRNA on new bone tissue of rabbitsThe experimental data using SPSS17.0statistical software for statistical analysis and P<0.05was statistically significant.Result:1、The normal activity of the21rabbits, no abnormal activities and Skin incision healed well.2、Imaging observation anatomy observation and tissue slice observation2.1、At4weeks to observe:in group A, there are a little of poromas on defect area and has few new blood vessels on it; in group B, there are a middle of poromas and fibroblast on defect area and fibroblast; in group C, there are a lot of poromas、 fibroblast and new cancellous bone on zhe defect area.2.2、At8weeks to observe:in group A, the bone defect became smaller, there are a lot of osteoblast in bone defect：in group B, there are a lot of poromas on defect area and that defect almost has been repaired; in group C. there are a lot of poromas on defect area and zhe bone defect was been repaired, but that traces.2.3、At12weeks to observe:in group A, the bone defect almostly repaired, but we still can find one rabbit,which still has a small bone defect among them and zhe size is about5mm×2mm; in group B zhe bone defect was repaired, but that traces. We can see zhe thick poroma around the bone defect; in group C. zhe bone defect has alreadv been repaired. 3、observe zhe quantity for VEGF and BMP-2、VEGF’s mRNAand BMP-2’s mRNA on new bone tissue of rabbits3.1、From zhe PCR:the BMP-2’s mRNA and VEGF’s mRNA of group C compared with group A when the time is2week has a significantly different (P<0.01); the BMP-2’s mRNA and VEGF’s mRNA of group C compared with group A and group B when the time is4week and8week have a significantly different (P<0.01), showed statistically significant.3.2、From zhe western:The BMP-2’s protein expression of group C compared with group A when the time is2week and4week have a significantly different (2week P <0.05;4weekP<0.01); The BMP-2’s protein expression of group C compared with group A and group B when the time is8week has a significantly different(P<0.01)。 The VEGF’s protein expression of group C compared with group A when the time is2week has a significantly different (P<0.05); the VEGF’s protein expression of group C compared with group A when the time is4week and8week have a significantly different (P<0.01); and zhe VEGF’s protein expression of group C compared with group B when the time is4week and8week have a significantly different (P<0.01)Conclusion1、GeneX is an ideal substitute for bone graft.2、geneX can enhance the level of BMP-2and VEGF protein and gene expression, and accelerate the speed of osteogenesis and angiogenesis, enhance the capacity of bone defect repair.3、BMSCs/geneX/TGF-β2can enhance the level of BMP-2and VEGF protein and gene expression,and significantly accelerate the speed of osteogenesis and angiogenesis observably, so we can speculate that.it has potential clinical application value of bone defect repairation.