Enhance Vascularization In The Process Of Repair Of Bone Defect By Using Of Two-gene Modified Artificial Bone

Large osseous defects are difficult to treat, and there is still no good means to tackle this problem. Recently, with the progress in tissue engineering, the artificial bone derived form bone tissue engineering has become a promising way to solve this difficult problem. However, the deficient blood supply in the defected area limits the repair ability of the artificial bone. So how to facilitate enough blood supply has become a bottleneck in the bone tissue engineering field. In this study, a new type of artificial bone was explored in order to repair large osseous defects by achieving sufficient blood supply with the help of gene therapy.1.Establishment of recombinated adenovirus simultaneously encoding VEGF and Ang-1Aim: To establish recombinated adenovirus simultaneously encoding VEGF and Ang-1, which will become effective tools in constructing new gene-modified artificial bone. Method: Molecular biologic technology was used to clone the genes of VEGF and Ang-1, and PCR was used to amplify the DNA sequence of IRES (internal ribozyme entry site) from pIRES2-EGFP. The genes of VEGF, IRES and Ang-1 were subcloned to ptrack-CMV one by one to get the plasmid named ptrack-CMV-VIA, which contained all the three genes. Then AdEasyTM system was used to recombinate and to pack adenovirus. Results: VEGF and Ang-1 genes were successfully cloned, and DNA sequencing results showed the genes acquired were exactly in the same sequence as that reported in Genebank. The DNA sequence of IRES was also successfully amplified. VEGF, IRES and Ang-1 were subcloned to vector ptrack-CMV and the plasmid which simultaneously encoded VEGF and Ang-1 was acquired. With the help of the AdEasyTM system, the recombinated adenovirus simultaneously encoding VEGF and Ang-1 was acquired and named pAd-VIA. In the similar way, the recombinated adenovirus encoding VEGF and the recombinated adenovirus encoding Ang-1 were also acquired and named pAd-VEGF and pAd-Ang-1 respectively. Conclusion:The recombinated adenovirus pAd-VIA was successfully established, thus providing convenient gene transfer tools for constructing the new gene-modified artificial bone.2. Exploration of transducted MSCs'ability to improve vascularization in vitro.Aim: To explore transducted MSCs'ability to improve vascularization in vitro. Method: MSCs were isolated and cultured, the third passage cells were differentiated into the osteogenic cell lineage, and the multiplicity of infection (MOI) was identified. MSCs were transducted by adenoviruses and the expressions of VEGF and Ang-1 were also identified by using RT-PCR and ELISA. Human endothelia progenitor cells(EPCs) were isolated from peripheral blood, 7 days after the cultivation, the EPCs were co-cultured with transducted MSCs, and then the MSCs'ability of vascularization was determined by the proliferation and migration of EPCs. Results: MSCs and EPCs were successfully isolated and cultured; MOI=100 was suitable for adenovirus pAd-VIA to infect MSCs. The MSCs transducted with adenovirus pAd-VIA expressed VEGF and Ang -1 in up to 20 days, and the peak of expression appeared on days 8-9. The results of proliferation of EPCs showed that two-gene transducted MSCs had the most satisfactory ability to accelerate the proliferation of EPCs and the strongest effect in migration assay. Conclusion: two-gene transducted MSCs have the power to accelerate vascularization in vitro.3. The ability of MSCs transducted with VEGF and Ang-1 to increase neovascularization in ischemic flaps.Aim : To investigate the feasibility of transplanted MSCs transducted with genes VEGF and Ang-1 to increase neovascularization and to augment the survival areas . Methos: MSCs were Isolated and cultured, and transducted by adenovirus pAd-VIA . Ischemic skins were made on the backs of SD rats, which were randomly divided into 3 groups. Group A: skin flaps transplanted with MSCs transducted with VEGF and Ang-1; Group B: skin flaps transplanted with untransducted MSCs; Group C: DMEM medium as the control. All the cells transplanted into skin flaps were dyed with CM-DiI. 4 days after the transplantation, peduncles of the skin flaps were cut. 7days later, the survival rates of skin flap were measured and the blood perfusion was observed with laser Doppler flowmetry. 14days after the flap transplantation, the density of capillary arteries were observed with microcirculation microscope.7 and 11 days after the skin flaps were made, three skin flap specimens were taken for histological examination to detect the density of capillary arteries by CD34 immunohistochemistry and to observe the proliferation of MSCs with fluorescence microscopy. Results: The skin flap survival rates of the three groups were 95.12%, 47.28% and 34.12% respectively with significant difference between any 2 groups (all P < 0.05). Compared with the other two groups, Group A had better survival quality and blood perfusion. The capillary density levels decreased progressively in the order of groups A, B, and C, with significant difference between any 2 groups ( all P <0.05) . Images under fluorescence microscopy showed Group A had more MSCs than Group B, and no MSCs were detected in Group C. Conclusion: MSCs transducted with VEGF and Ang-1 gene could increase the neovascularization in ischemic skin flaps and augment their survival rate, which means MSCs transducted with VEGF and Ang-1 gene may help to vascularize in vivo.4. Construction of two-gene modified artificial bone and exploration of the feasibility to repair segmental radial defects in rabbitsAim: To construct the two-gene modified artificial bone and to evaluate its repairing effect on segmental radial defects in rabbits. Method: Scaffold PLGA-TCP was made, and collagen I was used to modify the structure of PLGA-TCP by means of crosslinking. SEM(scanning electron microscope) was used to observe the microstructures of the scaffolds of PLGA-TCP and collagen I-modified PLGA-TCP. The hydrophilicity of the scaffolds was also assayed. MSCs were cultured and transducted with VEGF and Ang-1. After the transduction, MSCs were seeded into the scaffolds and co-cultured with scaffolds in vitro, and cell proliferation and gene expression were assayed. Established models of segmental radial defects and all the rabbits were randomly divided into 4 groups: A: two-gene transducted MSCs + collagen I-PLGA-TCP ; B: untransducted MSCs + collagen I- PLGA-TCP; C: collagen I- PLGA-TCP; D: the blank control. 1, 4, 8 weeks after the transplantation, X ray and histological assays were used to identify the bone repair. Results: Scaffold PLGA-TCP had porous structure, with the pore diameter about 300μm-350μm. After the crosslinkage with collagen I, the net made of collagen I appeared in the scaffolds. The modified scaffolds had better hydrophilicity than unmodified ones, so they obtained more cells after cell-seeding. The cells grew fast in the modified scaffolds compare with those in the unmodified scaffolds, and the gene could be highly expressed when transducted cells were co-cultured with modified scaffolds. The models of segmental radial defects were established, and progress of transplantation was successful. 1 week after the transplantation, the histological assay showed that Group A had many cartilage lacuna, and Group B had a few cartilage lacuna , which were fewer than Group A. Group C and Group D had no cartilage lacuna at all. 4 weeks after the transplantation, X-ray and histological assay showed that Group A and Group B had bone formation, and the amount of Group A was much higher than that of Group B. Groups C and Group D showed no sign of bone formation. 8 weeks after the transplantation, X-ray and histological assay showed that the new bone had completely filled up the defects in Group A, and there appeared sign of reconstruction of new bone in Group B and part of the defects had been filled up. Yet Group C had a little bone formation in the transplantation area. Group D had no bone formation. With the help of MicroCT, a direct-viewing three-dimensional image was established. And the results of MicroCT showed regular styloid bone structure had formed in Group A. In other Groups, there had been a little or no bone formation. Conclusion: New two-gene modified artificial bone has been successfully established. And this new kind of artificial bone can repair segmental radial bone defects in situ.