Lentiviral Vector Mediated HBMP-2 Gene Transfected Bone Marrow Derived Mesenchymal Stem Cells For Repair Of Bone Defects

Partâ… isolation culture and identification of rat Mesenchymal stem cells(rMSCs)Experiment 1 isolation and culture of rat Mesenchymal stem cells(rMSCs) Objective:to isolate and culture rat Mesenchymal stem cells(rMSCs).To grasp the techniques of rMSCs culture and investigate the biological characters of rMSCs. Materials and Methods:4-month-old SD rats were used,follow the methods of Friedenstein,because of the characters of rMSCs adhereing to tissue culture flask,(as rMSCs is adherent to tissue petri dish,) monocytes were isolated by gradient centrifugation first;then pure adherent rMSCs were obtained by the property that rMSCs is adherent to plastic petri dish.Results:rMSCs are rapidly adherent,grow in clone,the morphology of cell are homogeneous fusiform,within 24hs fibrous like cells were seen sporadic adherent,some cell clony were seen in 2-3 days,and they increased rapidly in 7-10 days,gradually grow into mixture. As the cell clony grown,they became monolayer at last.After passage,rMSCs no longer growed in clony but in homogeneous distribution,rMSCs number of primary culture is about 10⁵,and passage 10 is about 10¹².Conclusions:isolation and culture of rMSCs are feasible,and have powerful ability of cell proliferation.Experiment 2 identification of rat bone marrow Mesenchymal stem cells (rMSCs)Objective:surface antigen was used to identify the property of cultured cells. to prepare the seed cells of next step.methods and materials:to detect the surface antigen of rat bone marrow MSCs with flow cytometry,include CD29,CD44,CD90,CD105,CD34,CD45,CD11b.results:in contrast to the control group, the surface antigen CD29,CD44,CD90,CD105 of rat bone marrow rMSCs were positive; surface antigen CD34,CD45,CD11b were negative,and major MHC-â… ,â…¡(major histocompatibility complex-â… ,â…¡) were negative too.Conclusions:rat bone marrow rMSCs surface markers are multiplicity,which express the surface markers of interstitial cells,endotheliocytes and epidermic cells,generally CD29(the member of integrin family),adhesion molecule CD44,CD105 and CD166,surface marker CD90 of T lymphocytes in thoracic gland and periphery circulation and so on were important markers of bone marrow rMSCs.partâ…¡directional differentiation of rMSCs infected by lentiviral vector with hBMP-2Objectives:to observe the change of rMSCs induced by lentivral vector,to detect if rMSCs transform to osteoblast as expectation.Materials and methods:after lentiviral vector induced rMSCs,in the 0,3,6,9,12,15,18 and 21 day,alkaline phosphatase(ALP)activity is evaluated;to determin calcium ion concentration with quantitative assay;immunohistochemistry is used to detect the expression of typeâ… collagen.Results:as the induced time prolonging,in rMSCs after induction, alkaline phosphatase(ALP) activity is gradually increasing,but ALP activity of rMSCs that were not induced had no obvious change after 21 days culture.After induced,Calciumion concentration is continuous increasing,reached climax after 15 days,then get in stationary phase.Calcium deposition had no marked change in the group that were not induced;typeâ… collagen expression increased obviously in induced rMSCs and staining is positive,in contrast,which had no change in the control.Conclusions:with immunohistochemistry and histochemistry methods we revealed that induced rMSCs can produce typeâ… collagen,ALP and had the ability of mineralization,which confirmed rMSCs can be induced to differentiate into osteoblast by lentivral vector.partâ…¢osteoblast cultivated with TCP in vitroObjective:To investigate osteoblast and MSCs grewing and adhension in calciumphosphate bioceram.Materials and methods:Four porous ceramics 8mm×1mm divided into experimental and control groups randomly.The experimental group was soaked in 100g/ml recombinant human fibronectin under negative pressure over night.Then both groups were cultivated in vitro simultaneously with osteoblasts derived from bone marrow MSCs and MSCs of rats respectively for ten days.The morphogenetic pattern and number of the anchored cells were observed under scanning electron microscope.Results:There was statistically significant difference of cell numbers between the experimental and the control groups P＜0.01. Many secretory collagen fiber bundles and mineralized nods were observed in the experimental group under high magnifier.Conclusions:It seems that the fibronectin surface coating is beneficial to osteoblast anchorage and its osteogenic pheno type.Partâ…£Repair of critical size rat calvarial defects with autogenous osteoblasts loading onβ-TCPObjective:To evaluate osteogenetic effectiveness of lentiviral vector mediated hBMP-2 gene(Lev-hBMP-2) transfected bone marrow derived mesenchymal stem cells for repair of bone defect mixed withβ-TCP in the repair of critical size rat calvarial defects.Materials and Methods:Forty two male adult SD rats(weight: 250-350g) were obtained bone marrow from the left femurs and tibias under general anesthesia and sterile condition.BMSCs were cultured and transfected by Lev-hBMP-2 in vitro.The BMSCs were collected from bone marrow of rats,cultured and transfected by Lev-hBMP-2 in vitro for 14 days.The cells were then seeded and subcultured for another 10 days inβ-TCP that had been subjected to surface porous calcium phosphate ceramic that had been subjected to surface modification via soaking in recombinant human fibronectin.The cells and cell-ceramic compound were used to repair critical size(diameterof 8 mm) calvarial defects in the corresponding rat in the experimental group.The rat were divided into six groups at randomly:1.Lev-hBMP-2 transfected BMSCs+β-TCP(n=10);2.BMSCs+β-TCP(n=10); 3.β-TCP(n=10);4.Lev-hBMP-2 transfected BMSCs(n=4);5.BMSCs(n=4);6. control(n=4).The skull defect reconstruction was observed histomorphometrically at 4th,8th,12th,20th week after operation.Results:tissue slicing showed that the whole defected areas can found new bone grewing during different stages in the experimental group while osteogenic tissue only appeared in the margin of ceramic in the control group. Conclusions:The tissue-engineered bone made by Lev-hBMP-2 transfected BMSCs mixed withβ-TCP can repair critical size rat calvarial defects...