The Experiment Study On Repairing Critical-Sized Bone Skull Defect With Symphyseal Gene Transfected BMSCs

BackgroundCranio-maxillo-facial bone defect is common in daily life and still remains clinical problem for orthopaedic surgeons. Tissue engineered bone has been advancing rapidly in recent years and has become a research focus in the treatment of bone defect. Bone Morphogenetic Proteins (BMPs) play a critical role in the differentiation of osteoprogenitor cells and the bone formation process. Vascular Endothelial Growth Factor (VEGF) is a special mitogen in vascular endothelial cell,accelerating the growth of new vessels. The expression of BMPs and VEGF exists mutual influence relations, leading to a synergetic effect on bone reconstruction. Construction of BMPs and VEGF modified tissue-engineered bone become a novel method to repair bone defect.Objectives(1)To explore the possibility of utilizing symphyseal gene transfect bone marrow mesenchymal stem cells in vivo to repaire critical-sized bone skull. (2)To validate the synergistic interaction between VEGF165 and BMP2 during bone reconstruction.(3) To explore the differences between symphyseal gene (BMP2 and VEGF165) and single gene (BMP2 or VEGF165) transfect bone marrow mesenchymal stem cells to repair critical-sized bone skull defect.Methods(1) Ovine bone marrow mesenchymal stem cells (BMSCs) were isolated and expanded in vitro, then were identified by flow cytometer.(2) The cultured ovine bone marrow mesenchymal stem cells (BMSCs) were co-transfected with BMP2 and VEGF165, or each alone(BMP2 group and VEGF165 group), or pIRES alone with cation liposome. The mRNA expression of VEGF165 and BMP2 genes in each group was detected by reverse transcription PCR (RT-PCR) after transfection. And the protein expression of VEGF 165 and BMP2 genes in each group was detected by Western-blot after transfection.(3) BMSCs of each group were collected and seeded on theβ-tricalcium phosphate (β-TCP) scaffolds. Theβ-TCP scaffolds combining BMSCs were randomly chosen for scanning electron microscopy (SEM) observation at 7day after cell seeding. Cell proliferation of each group was determined every day by a hoechst assay for up to 2 weeks after cell seeding.(4) Theβ-TCP scaffolds combining BMSCs were implanted to the defect area after critical-sized bone skull model was made.The reconstruction of cranium bone defect of each group was detected by gross observation,X-ray examination and histological test at the end of 12th weeks and 24 th weeks.Results(1)The cultured ovine BMSCs were transfected successfully with vector containing combinations of condensed plasmid DNA encoding for BMP2andVEGF165 mediated by cation liposome.(2)After seeding BMSCs onto the scaffolds,SEM observation showed that BMSCs grew and proliferated well in each group at 7 days.There was no significant difference among all the groups in hoechst DNA assay for cell proliferation for 14 days after cell seeding.(3)For defects treated with autogenous mesenchymal stem cell implants, no adverse host response could be detected at any time-point.(4) Macroscopically,Histologically and Radiographically, by 24th weeks, there were obvious signs of osteogenesis in the group with symphyseal gene and BMP2transfected BMSCs/β-TCP complex implants, combined use of BMP2 and VEGF165 gene can produce better osteogenesis than BMP2 single gene alone,and no ectopic osteogenesis was found in the other groups.Conclusions(1)Symphyseal gene transfected bone marrow mesenchymal stem cells can repair critical-sized bone skull defect. Combined use of BMP2 and VEGF165 gene can produce better osteogenesis in vivo than BMP2 single gene alone.Single application of VEGF165 can not induce the bone formation of the cell and scaffold complex in vivo.(2)BMP2 and VEGF 165 can be expressed in the transfected BMSCs by constructed vector system(plRES-BMP2-VEGF165,pIRES-BMP2,pIRES-VEGF165),further to promote the reconstruction of canium bone defect.(3) The cation liposome vector system with low cell toxicity system has no influence on the cell proliferation and osteogenic differentiation of BMSCs on scaffolds.(4) The constructed pIRES-BMP2-VEGF165 can be successfully transfected into ovine BMSCs by bangosome.So this way can faciliate the bone ossification in tissue engineering.we can also advance our reserch from vitro study to vivo study primarily with this cation liposome-mediated transfection method. VEGF165 and BMP2 have synergistic interaction in ossification.