Study Of Tissue Engineering Bone Modified By BMP-2,VEGF Gene

Bone defect is sclerotin loss for the reasons of trauma,infection and tumor.In osteology clinic,Bone defect is the common and hard treated disease. Autogeneic bone transplantation,artifical synthesis surrogate transplantation as well as vascular anatomizes bone transplantation are the traditional strategies used at present.Following the development of tissue engineering,a new way to heal the bone defect is found.The basic method is to culture in vivo the living cells into the scaffolds,formating a compound of cells and scaffolds. Then,the compound is transplanted to the position of bone defect,to repair and reconstructi the defect.Seed cells,scaffolds and cell factors are the three critical elements of the tissue engineering bone.The compound of cells and scaffolds constructed in vitro has to get the nutrition and oxygen by blood and intracellular fluid after implantation. Poor blood supply of planting areas or long revascularization would result in nutrition deficiency of the seed cells grow on the scaffold,which leads to disorders in cell metabolism,proliferation,differentiation and secretion, at last, devitalizing of the cells. the transplanted bone would just act as a pure bone transduction material and lost the osteogenesis ability. In recent,the important of revascularization of tissue engineering bone is thought highly. Fast revascularization of combination after tissue engineering bone transplantation is the premise for keeping the vitality and activity.For this reason, the revascularization has been considered as the quartus element of tissue engineering bone.BMSCs(bone mesenchymal stem cells)is one of good seed cells of tissue engineering bone,have the potential ability of ossific differentiation.In the specified environment, BMSCs can be induce to the osteoblast,encourage the reparative process of bone defect.For this reason,BMSCs are generally used on the field of bone tissue engineering.The utilization of many differentiation factors and inducing factor in region has the effect of promoting the osteanagenesis.The compound of BMSCs and scaffolds is not good at repairing the bone defect. The utilization of cell factors in region has the shortages such as short time leng,repeate administration,too espensive.In the view of endogenous bone growth factor,the gene treatment is gradually used on the field of bone tissue engineering,unfolding the enormous potentiality.Now we can introduce the gene of code cell factors into BMSCs,complex the scaffolds ,implant the compound to the bone defect.The cell will keep on producing the cell factors in region.With the autocrine and paracrine,the factors induce BMSCs to bone formation,promote the reparation of bone defect.In the numerous bone growth factors, bone morphogenetic protein(BMP) is one of the most important,it is regarded as the most fortis osteoinductive factor.VEGF is not only to promote the hyperplasy of endothelial cell and vasculogenesis,but also evident osteogenesis. Therefore,BMP-2 and VEGF gene transfer into BMSCs,and use the cells to construct the tissue engineering bone,this method will promote the osteogenesis,and accelerate the vascular production in region at the same time.It is the better to meet the demand of the clinical.The nanometer materials is the new biomaterial appeared recently.The nano-HA is regarded as a kind of ideal scaffold,because the structure of nano-HA is similar with nature bone.The nano-HA can not use as scaffold alonely for it's high fragility.The compound of nano-HA and high molecular polymer improve the high fragility of nano-HA,and compensate the descend of PH that cause by the degradation of high molecular polymer, conduce to prevent aseprtic inflammation.This research approach the relation of BMP-2 and VEGF,and osteoplastic effect.We hope to find a way that can construct a kind of tissue engineering bone which have the ability of ossify and accomplish vessel.This experiment was composed of four portions.The main method,consequence and conclusion decribed as follows:1.Isolation,culture and identification of rabbit BMSCs(bone mesenchymal stem cells).Isolation,culture of rabbit BMSCs is get from the tibia of wistar rat 5 days after birth according adherent.The BMSCs were certified by observation of morphology,determination of generation cycle,detection of surface marker.The cells have the favourable reproductive activity. With the appreciation,the cells can be induced to poly-directions.2.Consturction of eukaryotic expression vectorThe plasmids of pEGFP-N1/VEGF, pEGFP-N1/BMP-2 and pEGFP-N1/VEGF-BMP2 were constructed with gene recombinant technique by extraction of RNA. Through the sequencing ,compared with Genebank, identited by restriction enzyme,we certificated that the plasmid was right.3.Expression of BMSCs after cells transfected BMP-2,VEGF gene in VitroThese gene plasmids(pEGFP-N1/BMP-2,pEGFP-N1/VEGF,pEGFP-N1/BMP2-VEGF) were transfected into BMSCs by liposome in Vitro, fluorescence microscope observe eells culture after BMSCs transfected.The expression of purpose gene mRNA was detected by RT-PCR.The protein of cells transfected was detected by ELISA. 4.Construction of the gene modified tissue engineering bone in vitroThe nano-HA/PLGA scaffold was co-cultured with BMSCs and cells transfected.The compt tissue engineering bone was constructed.Cells was able to culture well with scaffold,conformed by digestion and canning electron microscope(SEM). The status that inorganic sedimentum was found in the scaffolds defferent degree. Bone salts deposited on the scoffild by the effect of cells.Compt tissue engineering bone was constructed invitro.