Electroporation-mediated Gene Therapy Rabbit Mandibular Distraction Osteogenesis Bone Modeling And Identification

Distraction osteogenesis(DO) was initially used in orthopedic surgery by Codivilla in 1905, it was systematically developed and refined by Ilizarov. In 1992, McCarthy reported in the English literature the first application of distraction osteogenesis to lengthen the human mandible. This method is now used extensively at every level of the craniofacial skeleton. Despite the success of DO, a number of problems have driven research for methods to improve bone generation. One limitation is the long consolidation phase during which the patient must wear a cumbersome device. 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. Strategies to accelerate osteogenesis would therefore significantly benefit the distraction patient. We hypothesized that local delivery of plasmid would allow efficient transduction of bone-generating cells in the distraction zone and accelerate bone regeneration in mandibular DO. A series of experimental studies have been done to investigate the feasibility of electroporation mediated transfecting recombinant plasmid to mandibular distraction area of rabbit.PARTⅠ: The Establishment of Animal Model of Bilateral Mandibular Distraction Osteogenesis of RabbitsObjective: To estabilsh an animal model of bilateral mandibular distraction osteogenesis with good feasibility and repeatability of rabbits, and establish the foundation for further study of distraction osteogenesis. Methods: Twenty white New Zealand rabbits were divided into 5 groups randomly, 4 in each group. Osteotomy was performed on mandible bilaterally anterior to the first premolar. The mandibular segments were repositioned and fixed with special distractors for rabbits. After 3 days of latent period, the distraction started at a rate of 8mm/d for 10 days , then the distractors were hold in place. After X-ray and three dimension computer reconstruction examination were employed for the rabbits on the day of the end of latent period, the second week, the fourth week and the eighth week of the consolidation, and four rabbits were killed separately at the time point. All specimens of the distracted area were taken for histology examination. Results: All rabbits tolerated the experiment without complications except for one wound was infected. The distractors were enough stable to produce and sustain the distance that we needed. All mandibles were lengthened successfully. Radiological findings and histological examinations showed that bone formed gradually following distraction. Conclusion As one of the experimental models of mandibular distraction osteogenesis, this method is economical and has good feasibility and repeatability.PARTⅡ: The Establishment and Identification of Electroporation Mediated Gene Therapy Model for The Mandibular Distraction Osteogenesis of RabbitObjective:To evaluate the feasibility of Electroporation-mediated transfecting recombinant plasmid to mandibular distraction area of rabbit in vivo. 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 10days. After the completion of activation, the device was maintained in position, and then the rabbits were randomly divided into 3 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 3 hours, 1d, 3d, 7d and 14d after injection respectively. Harvest the distraction area tissue for frozen section and observed in fluorescence microscope for expression of green fluorescence protein(GFP). At the same time, parameters of liver and kidney function were detected in serum of rabbits, the tissue of heart, liver and kidney were checked histologically. Results: green fluorescence protein(GFP) was seen in the distraction area tissue of rabbits of group A and group B 3 hours after injection, enhanced at 1 day, it expressed most strongly at 3 days,and weakened at 14. but in group B obviously weaker than that in in group A. green fluorescence protein(GFP) expressed negatively at any other time in group C. There was no statistical difference in the concentration of ALT,AST,BUN and Scr in serum of rabbits among the three groups. Conclusion: Electroporation-mediated transfecting recombinant plasmid could enter intodistraction area tissue of rabbits , and there was no toxic side effect of liver and kidney in mandibulardistraction of rabbits. Electroporation could obviously improve transfectiing efficiency in vivo. Electroporation-mediated transfecting recombinant plasmid to distraction area tissue of rabbits is feasible.