Preliminary Study Of Electroporation-mediated Gene Therapy For Mandibular Distraction Osteogenesis Impact

Distraction osteogenesis is a method of producing new bone directly from the osteotomy site by gradual traction of the divided bone fragments. The benefits of distraction include the avoidance of bone grafting and donor-site morbidity, and the concurrent expansion of the soft-tissue envelope. Since the first clinical report on the use of distraction osteogenesis to lengthen the human mandible in 1992, distraction osteogenesis has become a widely accepted approach in the treatment of severe craniofacial deformities and bone defects. However, one of the major disadvantages of this technique is the lengthy course of treatment required for distraction and consolidation, which may result in pin tract soft-tissue infection, bone infection, and psychological problems. In addition, fibrous nonunion still occurs from inadequate neovascularization (a significant problem in irradiated bone) or from unstable fixation resulting from secondary bone infection or device malposition. Consequently, both surgeons and patients would welcome any technical improvement to speed the treatment process.New Zealand rabbits bilateral mandibular DO model was employed in our studies. This study evaluates the effect of electroporation mediated transfecting recombinant plasmid on mandibular distraction at normal distraction rates. At first, we observed the effect of electroporation mediated transfecting recombinant plasmid pIRES-hVEGF165-EGFP on angiogenesis of the distraction area during early mandibular DO, and investigate the effect of electroporation mediated recombinant plasmid pIRES-hBMP2-VEGF165 transfect on osteogenesis of mandibular DO.PART I: The Effect of Electroporation Mediated Transfecting Recombinant Plasmid pIRES-hVEGF165-EGFP on Angiogenesis of The Distraction Area During Early Mandibular Distraction OsteogenesisObjective: To explore the effect of electroporation mediated transfecting recombinant plasmid pIRES-hVEGF165-EGFP on angiogenesis of the distraction area during early mandibular DO, for further study of distraction osteogenesis. Methods : Thirty New-Zeland rabbit employed submandible extraoral incisions to osteotomy and place the distaction devices. A mechanical burr supplemented by saline irrigation, can be used to interrupt the buccal cortex as well as the superior and inferior cortical borders of the mandible, The osteotomy is completed by inserting and rotation an osteotome to demonstrate separation of the bony segments. The arms of distractors are placed percutaneously outside the oral. Chose bicortical or moncortical bone screws to fix the distractor accoding to the rockiness and thickness of mandible. The arms kept outside through the anterior incision. The wound was irrigated with saline and closed with interrupted suture respectively. The latency period was 3 days. Activation of the device was commenced after the latency period and proceeds at the rate of 0.8mm per day for 7days. After the completion of activation, the device was maintained in position, and then the rabbits were randomly divided into 5 groups: group A: recombinant plasmid pIRES-VEGF165-EGFP was injected into the distraction area, after the completion of activation; group B: recombinant plasmid pIRES-VEGF165-EGFP was injected into the distraction area ; group C: normal saline (NS) group NS was injected into the distraction area. Afer injection group A and group C employed electroporation, but group B without electroporation, then the rabbits were sacrificed at 1d, 3d, 7d and 14d after injection respectively. Harvest the distraction area tissue for histological examination and electron microscope observe, immunohistochemical stain for CD34 and detection microvessel density. Results: Under electron microscope, generation of vascular endothelial cell(VEC) of group A and group B are active, VECs are spindle and have many ecptoma, basal membrane are integrity. Intracytoplasm, there are many ellipse or round chondriosome, majority of VEC in group C take on early change of cataplasia and apoptosis. Immunohistochemistry detection The immunochemical stain for CD34 showed it expressed weakly at the first day after transfection, from 3 to 14 days after transfection, CD34 of VECs in the distraction area expressed positively, it was stronger in group B than that of group C, and that in group A was the strongest(p<0.05), there are remarkable difference among three groups and different time respectively. Compare to each other, CD34 of VECs expressed continue positively with a tendency to rise in group A and B. But it fluctuate at the level of the frist day in group C. Conclusion: Electroporation-mediated transfecting recombinant plasmid could promote angiogenesis during early stage of mandibular DO. It could promote loca vascular proliferation and penetration, increase the blood flow of broken ends of fractured bone. It indicate that genen therapy can stimulate bone matrix synthesis and induce cell generation differentiation. Thus, it play an important role in regulate and promote growth and reparative process of bone.PART II: Primary Study of the Effect of Electroporation Mediated Gene Therapy on Rabbit Mandibular Distraction OsteogenesisObjective: To explore the effect of electroporation mediated gene therapy on the new bone formation in distraction area, and to find one potential approach to accelerating bone regeneration, to shorten the cycle of distraction. Methods:Thirty New-Zeland rabbit employed submandible extraoral incisions to osteotomy and place the distaction devices. A mechanical burr supplemented by saline irrigation, can be used to interrupt the buccal cortex as well as the superior and inferior cortical borders of the mandible, The osteotomy is completed by inserting and rotation an osteotome to demonstrate separation of the bony segments. The arms of distractors are placed percutaneously outside the oral. Chose bicortical or moncortical bone screws to fix the distractor accoding to the rockiness and thickness of mandible. The arms kept outside through the anterior incision. The wound was irrigated with saline and closed with interrupted suture respectively. The latency period was 3 days. Activation of the device was commenced after the latency period and proceeds at the rate of 0.8mm per day for 7days. After the completion of activation, the device was maintained in position, and then the rabbits were randomly divided into 5 groups: group A: recombinant plasmid 2μg ( 0.1μg/μl )pIRES-hVEGF165-hBMP2,was injected into the distraction area, after the completion of activation; group B: recombinant plasmid pIRES-hBMP2 was injected into the distraction area ; group C: recombinant plasmid pIRES-hVEGF165 was injected into the distraction area; group D: pIRES was injected into the distraction area, and group E: normal saline (NS) group NS was injected into the distraction area. After injection every group employed electroporation. then the rabbits were subjected to be examined by X ray and quantitative computed tomography (QCT), chose the distraction area as regions of interest(ROI), bone mineral density(BMD) of newly formed bone was detected at 1 week, 2 weeks, 4 weeks and 8 weeks after gene transfected. After examined the rabbits were sacrificed and harvest the distraction area tissue for detection crushing strength of three points of the newly formed bone. At the same time, all the specimen were taken for histology examination and morphometry analysis.Results: BMD of newly formed bone of the distraction area in group A, group B and group C were remarkable higher than those of group D and group E. The crushing strength of three points of the newly formed bone in group A, group B and group C were also remarkable higher than those of group D and group E. The findings of histology examination and morphometry analysis showed that compare to group D and group E, ther are more new vessels osteoblast and mesenchymal cells in group A, group B and group C, the volume of newly formed bone and the width of newly bone trabecular also higher than those of group D and group E. Conclusion: Electroporation-mediated transfecting recombinant plasmid pIRES-hVEGF165-hBMP2 could obtain satisfactory proceeding of osteogenesis and calcification, which surpassed that of control group. It indicate that VEGF and BMP may act as the molecule target of gene therapy for mandibular DO, combination of VEGF and BMP may be make osteogenesis and angiogenesis come true at the same time, further more, it may be magnify the effect of single growth factor, and promote growth and reparative process of bone.