| BackgroundTreatment of bone defects remain a clinical challenge in orthopedic surgery. The commonly used method for the bone defect is autogenous bone graft. But for the reason of inadequate donor bone, donor area dysfunction, and various complications caused by bone harvest,so its application is limited. The osteogenic ability of bone morphogenetic protein (BMP) has been widely admitted and vascular endothelial growth factors (VEGF) is special mitogen in vascular endothelial cell, which could promote new vessels growth. Studies have demonstrated that there is a mutual influence relation-ship between BMP and VEGF expression which leads to a synergetic effect on bone regeneration. Combination of bone formation and bone supply can be acquired by integrating VEGF and BMP genes into one vector,which can provide a novel method to repair bone defect in basic research and clinical therapy. It has become a hot subject to transfer the endogenous genes to seed cells of tissue-engineered bone through recombinant virus vector system which is combined with scaffolds to form gene-modified tissue engineered bone. Replication-defective HSV1vector system can infect most of none-dividing cells and owns the capacity to express exogenous efficiently, safely and stably.Objectives(l)Establish the rdHSV-1-VEGF165-BMP4repair vector system, and express the VEGF165and BMP4gene effectively in MSCs cells.(2)0bserve the collagen, osteocalcin and alkaline phosphatase activity of the rdHSV-1-VEGF165-BMP4and bioglass scaffolds complex, so as to identify the Osteogenic activity in vitro.(3)Identify the therapeutic effects of the rdHSV-1-VEGF165-BMP4and BG complex in the bone defect model in vivo.Materials and Methods(1)The VEGF165and BMP4gene DNA were cloned from human osteosarcoma cell line MG63. Reconstruct the HSV1, and establish the replication-defective HSV1vector system. The rdHSV-1-VEGF165-BMP4carrying the VEGF165and BMP4gene were constructed by employing the shuttle plasmid.(2)Rabbit bone marrow mesenchymal stem cells (MSCs) were isolated and expanded in vitro.(3)The former three experiments were divided into two groups, including rdHSV-1-VEGF165-BMP4group (VEGF165-BMP4group) and control group (CON group). The last part experiment was divided into four groups, including rdHSV-1-VEGF165-BMP4and BG complex group (VEGF165-BMP4group), single Bioglass group (BG group),(4)The proteins expression of human VEGF165and BMP4genes in each group were detected by IFA, western blot, immunohistochemistry and ELISA method at48hours after transfection.(5)Alkaline phosphatase activity (ALP) staining, ALP activity, collagen expression were detected in the two groups2weeks after the rdHSV-1-VEGF165-BMP4transfection.(6)In both group, the MSCs were collected and seeded on the BG material scaffolds to form the complex. The ALP activity and ALP and collagen were detected in tissue engineering bone complex after the rdHSV-1-VEGF165-BMP4transfection. (7)Establish the bone defect model on rabbits, and implant the complex constructed by tissue engineering techniques in the bone defect model. The implants were harvested and used for histological ananlysis, X ray detection, bone density measurement and biomechanical testing after4,8and12weeks after implantation.Results(1)The rdHSV-1-VEGF165-BMP4viral vector were constructed successfully.(2)The mRNA transcription level in VEGF165-BMP4group was higher significantly compared with the CON group after transfecting the MSCs.(3)The activity and amounts of ALP, expression of collagen and osteocalcin in the VEGF165-BMP4group were increased significantly compared with the CON gourp after transfecting the MSCs.(4)After integrating the rdHSV-1-VEGF165-BMP4infected MSCs and bioglass material together, the levels of ALP (activity), collagen, osteocalcin in VEGF165-BMP4were also higher than the CON gourp.(5)In the bone repair experiment, the X-ray results indicated that new callus in group VEGF165-BMP4and AB were significantly more evident than that in group BG and CON,8weeks after operation. Twelve weeks later, the bone defect was healed in group VEGF165-BMP4and AB, the medullary cavity was opened again. The bone defect in group BG and CON were closed up, and filled with many connective tissue.(6)The histologic investigatin showed:Bone cells in group VEGF165-BMP4was quite active, lots of osteoblast could be observed. The amont of new bones in group VEGF165-BMP4was more obvious than that in group BG4,8,12weeks after operation. Histologic assessment suggested that bone healing in group VEGF165-BMP4was superior than that in group BG. Statistical analysis showed that there was no significant difference between group VEGF165-BMP4and group AB (P>0.05), but group VEGF165-BMP4and BG were significantly different (P<0.01).(7)The biomechanical testing results indicated that the maximum bending load in the VEGF165-BMP4group were significantly higher than that in the group BG (P<0.05). Group AB was superior to group BG (P<0.01), whereas there was no significant difference between group VEGF165-BMP4and group AB (P<0.01).(8)The bone density measurement results showed that there were no significant differences of bone density between group VEGF165-BMP4and AB group, but both of them were higher significantly compared with the BG and CON group.ConclusionsIn this study, the tissue engineering bone was constructed successfully utilizing the bioglass combined with replicative-defected HSV infected MSCs. The VEGF165and BMP4gene were co-transfected to the MSCs cells by replicative-defected HSV1virus. A stable, safe and relatively long term expression of both genes can be obtained through the replicative-defected HSV1vector mediated transfection of both genes. VEGF-165-BMP4and bioactive glass complex bone behaved well at both osteoconduction and osteoinduction, and its effect to bone defect was significantly better than pure bioactive glass, and was almost similar to the autogenous bone. VEGF165and BMP4genes promoted the bioactive glass to become vascularization and osteogenesis, promoted the growth of chondrocyte and osteocyte, and thus improved the repairment of defect bones. So the tissue engineering bone could serve as a new method for treatment of bone defect. The thesis comprises18figures29... |
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