| Cranialofacial bone regeneration needs to be enhanced for repairing large bone defects secondary to tumor, congenital malformations or trauma and for treating fracture-delayed unions or nonunions. The approach of bone regeneration includes autograft, allogenic graft, and artificial substitutes graft. But But there are some shortage such as: limited source, secondary trauma, immunological rejection, poor plasticity. With the development of the molecular biology, cellular biology and gene engineer, bone tissue engineer have become the focus to resolve the problem. Bone tissue engimmer have three factors:1)mesenchymal stem cell or osteoprogenitor cells isolated and expanded; 2)growth factor; 3)scaffold. But the administration of growth factor had been limited by the half life span, low efficiency, easy lost, high cost, etc. So the gene modified tissue engineer have become the hot spot.Vascularization of the tissue is a key factor on the success of tissue engineering bone. VEGF, the best-characterized angiogenic factor, promotes the angiogenisis, increase the permeability of vessels. VEGF plays an indirect role in the bone development, bone fracture healing and bone repair by promoting the angionenisis.VEGF is one of the candidate factors in the vascularization of tissue engineer. But the effect of VEGF on the bone marrow mesenchymal stem cells (BMSCs) is not known clearly.BMSCs are the non hematopoietic stem cells in the bone marrow. The characteristic of BMSCs is multi-directional differentiation potential. BMSCs can differentiate into the osteoblasts, chondroblasts, myoblasts under the certain conditions. Due to its the easy isolation, abundance source, foreign gene import and expression easily BMSCs have become the promising seed cells in bone tissue engineer.Adenovirus vector(Ad Vector) is a promising gene transferring vehicle. Adenovirus is stable , without being integrated into the host genome , has no genotoxicity, and cannot induce insertion mutation. Adenovirus vector has little pathologic injury ,can transfect dividing cells and nondividing cells, and has high transfection efficiency. So adenovirus vector have been used in the researches. The duration of gene expression in the AdV is from weeks to months. It is fit to the bone repair and regeneration.On the basis of above, the aim of this study is to evaluate the effect of VEGF transfection on the rat BMSCs mediated by Ad vector in vitro. Firstly, rat BMSCs were isolated and expanded in vitro and cultured in conditioned medium to evaluate their differentiation potential. The rat BMSCs were isolated from the bone marrow of rat femurs and tibia by total bone marrow adherence. The cells were cultured in the L-DMEM medium containing 10% fetal calf serum(FCS), 100U/ml penicillin, 100μg /mL streptomycin. When the cells were confluent to 80-90%, the cells were digested with 0.25% trypsin and 0.02% EDTA and then subcultured. The cells were polygon, spindle in shape with 35 processes. With the culture went on, the cells were aligned as swirl, radiated and formed colony. The 3rd passage cells were cultured in the conditioned medium containing 50mg/L ascorbic acid, 10mmol/Lβ-glycerophosphate sodium and 10-8mol/L dexamethasone. After 21 days, the cells were stained with Alizarin Red. There are red nodules under the microscope. The 3rd passage cells were cultured in the conditioned medium containing 0.5mmol/L IBMX,10μmol/L dexamethasone,10μmol/L insulin and 200μmol/L indomethacin. After 14 days, the cells were stained with Oil Red O. The red lipid droplets were seen under the microscope. It can be concluded that the rat BMSCs were isolated from the bone marrow and expanded in vitro and can differentiate into osteoblasts and adipocytes. This will provide abundant cells for the following researchs.Secondly, AdCMV-EGFP was transfected into the rat BMSCs to detect the transfection efficiency and the effect of the AdCMV-EGFP transfection on the BMSCs. The 3rd passage rat BMSCs were transfected with AdCMV-EGFP in different multiplicity of infection(MOI: 0,100,200,400,600,800,1000particle/cell). With the increase of MOI, the transfection efficiency was increased. When MOI was lower than 400particles/cell, the morphology of the cells didn't change. When MOI was higher than 400particles/cell, the cells became circle and fell off the wells. There were GFP expressions in the cells 24hour after transfection. The intensity and amount of GFP became increasing as time passed. It reached its peak on the 5-7th day. There were still GFP in the cells on the 28th day after transfection. After the transfected cells were cultured in the conditioned medium (containing 50mg/L ascorbic acid, 10mmol/Lβ-glycerophosphate sodium, 10-8mol/L dexamethasone), there were some mineralized nodules with Alizarin Red staining. There wasn't significant difference between the AdCMV-EGFP transfected cells and untransfected cells. So, AdCMV-EGFP transfection with a range of MOI should not impact on the BMSCs proliferation and osteogenic differentiation potential.Thirdly, AdCMV-VEGF was transferred into rat BMSCs, then VEGF mRNA and protein expression was detected by RT-PCR and ELISA. The effect of AdCMV-VEGF transfection on the cells was evaluated by MTT assay. After transfection there were VEGF mRNA and protein expression in the cells. The OD value of the AdCMV-VEGF transfected cells is higher than the untransfected cells 3d after transfection (p<0.05). The results show that AdCMV-VEGF have been transferred into rat BMSCs successfully and VEGF gene is expressed, and that VEGF can promote the proliferation of the cells.Fourthly, AdCMV-VEGF was transferred into rat BMSCs with the MOI of 400particles/cell. Then the transfected cells were cultured in conditioned medium (containing 50mg/L ascorbic acid, 10mmol/Lβ-glycerophosphate sodium, 10-8mol/L dexamethasone). On 6, 9, 12d, ALP level of the transfected cells were higher than untransfected ones(p<0.05). OCN and COLI were detected in the supernatant. OCN on 12d and COLI on 6, 9, 12d of the transfected cells were higher than untransfected ones(p<0.05). COLI, OCN, RUNX2 and OSX were evaluated by real time PCR on 3, 7, 14 and 21d. On 3d COLI,OCN,RUNX2 and OSX of the transfected cells were higher than untransfected ones. The results show that AdCMV-VEGF transfection can promote the osteogenic differentiation of BMSCs.Finally, many growth factors involve in bone development and bone repairing. Both VEGF and BMP2 are important factors. AdCMV-VEGF and AdCMV-BMP2 were transfected BMSCs. VEGF and BMP2 expression were detected by RT-PCR and ELISA.The mineralized nodules of AdCMV-VEGF and AdCMV-BMP2 transfected ones were more than AdCMV-BMP2 transfected ones. The results show that the foreign gene can be expressed after transfection and AdCMV-VEGF transfection can promote the BMP2 induced osteogenic differentiation.Tissue engineering bone is a hot spot in the bone defect therapy.The innovation of this study is to study VEGF transfection mediated by adenovirus vector on the study of tissue engineering bone and the effect of VEGF on the osteogenic differentiation of BMSCs.
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