The Study Of The Effect Of Electroporation Mediated Gene Transfecting Therapy On The Osteogenesis Of Onlay Iliac Graft

Part Ⅰ:The Effect of Electroporation Mediated Transfecting of Recombinant Plasmid pIRES-hVEGF165-EGFP on Revascularization at the Early Stage Following Onlay Iliac GraftingObjective:To investigate the feasibility of electroporation mediated transfecting of recombinant plasmid pIRES-hVEGF165-EGFP and its effect on revascularization at the early stage following onlay bone grafting.Methods:Forty eight New Zealand rabbits were randomly divided into3groups:Group A (electroporation with recombinant plasmid plRES-hVEGF165-EGFP), Group B (injection with recombinant plasmid plRES-hVEGF165-EGFP) and Group C (electroporation with normal saline). Submandibular incisions were performed on the rabbits to explore the mandible and autogenic iliac graft was onlay grafted on it. Following the bone grafting, pIRES-hVEGF165-EGFP was electroporated into the site of onlay bone graft in Group A, while pIRES-hVEGF165-EGFP was injected in Group B. As a control, NS was electroporated in Group C. The rabbits were sacrificed at3d,7d,14d and21d postoperatively. The gene expression level was assessed by the detection of green fluorescence protein (GFP) using fluorescence microscope. Liver and kidney function were tested (Extract blood from right ventricle to exam the value of ALT, AST, BUN, Scr). In addition, heart, liver and kidney tissue were harvested for histological examination. Finally, immunohistochemical analysis of vascular endothelial growth factor (VEGF) and CD34was also performed to assess the microvascular density.Results:There was no significant difference in the concentration of ALT, AST, BUN and Scr in serum of rabbits among the three groups (P>0.017). There was no significant destruction of normal organ structure and function of heart, liver and kidney in group A as shown by histological examination. Obvious expression of green fluorescent protein was seen in group A at day3. The peak value was detected at day7and the expression level started to decrease at day14, and became very low at day21. The GFP expression level was much stronger in group A than in group B at each time point while GFP was not detected in group C. Immunohistochemical analysis showed that VEGF was expressed in different types of cells, including neonatal vascular endothelial cells, osteoblasts, chondrocytes and interstitial cells, and the intensity of VEGF were most obvious in Group A. There was a significant difference in gray-scale value of VEGF among the three groups (P<0.017), but no significant difference was found between Group B and Group C (P>0.05). The VEGF protein content in Group A was apparently higher than that in Groups B and C at each time point. The microvascular density (MVD) was measured by CD34expression level. It showed that MVD in Group A was significantly higher than in Group B and C, with P value<0.017, but there was no significant difference was found between Group B and Group C (P>0.05). The VEGF expression level was positively correlated with MVD in the three groups at each time point (p<0.01).Conclusion:We demonstrated that electroporation of recombinant plasmid pIRES-VEGF165-EGFP into onlay iliac graft is safe and feasible. In addition, it obviously promoted revascularization the early stage following onlay iliac grafting.Part Ⅱ:the Effect of Electroporation Mediated Transfecting of Recombinant Plasmid pIRES-hVEGF165-EGFP on the Osteogenesis of Onlay iliac Graft.Objective:To explore the effect of electroporation mediated transfecting of recombinant plasmid pIRES-hVEGF165-EGFP on the osteogenesis of onlay iliac graft.Methods:Sixty New Zealand rabbits were randomly divided into3groups:Group A (electroporation with recombinant plasmid plRES-VEGF165-EGFP), Group B (injection with recombinant plasmid plRES-VEGF165-EGFP) and Group C (electroporation with normal saline). Submandibular incisions were performed on the rabbits to explore the mandible and autogenic iliac graft was onlay grafted on it. Following the bone grafting, pIRES-hVEGF165-EGFP was electroporated into the site of onlay bone graft in Group A, while pIRES-hVEGF165-EGFP was injected in Group B. As a control, NS was electroporated in Group C. The rabbits were sacrificed at3d,7d,14d,21d and70d postoperatively and the onlay bone graft was harvested together with the host bone for gross observation, histological examination and immunohistochemical stain with VEGF, OPN, OC, Col-I. The markers expressions were compared among three groups. The new bone formation of onlay bone graft was assessed. In addition, bone graft absorption rate were evaluated by measuring the volume of the bone before bone graft surgery and70d after surgery using a water displacement method.Results:At day70, the bone graft was found firmly attached to the host bone meanwhile bone absorption was observed in all groups. The bone volume of Group A was much larger than that of Group B and C. The absorption rate in Group A, B and C are55.25%、62.38%and64.25%respectively. Histological examination displayed that bone grafts survived completely at day70postoperatively in all three groups with no sign of sequestrum or absorptive vacuoles or osteoclasts. The integration at the interface between the graft and the host bone was more obvious in Group A than in Group B and C. The fiber connection at the interface was not obvious. Gray value evaluation based on the results of immunohistochemical stain showed that there was significant differences in the VEGF level between Group A and the other groups at day7,14and21; there was also significant differences in OC level between group A and the other groups at the day21; significant differences were also found in OPN level between Group A and the other groups at the day3,7,14; Col-I level in group A showed significantly different from Group B and C at day7,14and21(p<0.017). Immunohistochemical analysis showed that Group A displayed an earlier bone formation than Group B and C. The absorption ratio at day70in Group A is lower than that in Group B and C.Conclusion:Electroporation of recombinant plasmid pIRES-VEGF165-EGFP into onlay bone graft promoted the remodeling and reduced absorption of the bone graft.