Bone Morphogenetic Proteins2 Gene Therapy For Mandibular Distraction Osteogenesis In Rabbits

Objective:1. Bone Morphogenetic Protein 2 (BMP2) is one of members of Transforming Growth Factor (3 (TGF p) superfamily and one of growth factors. The major biological role of BMP2 is to induce undifferentiated mesenchymal cell into cartilage and newly formed bone. There is enormous potential value in clinical application. We shall raise a modus operandi to amplificate and purify recombinant adenovirus vectors containing BMP2 gene and to accredit it's biological activity. That is foundation to bone morphogenetic proteins 2 gene therapy for mandibular distraction osteogenesis in rabbits.2. Distraction Osteogenesis (DO) is a valuable clinical technology developed in recent years, which have a great potential in clinical applications and gave us a new way to study the basic mechanisms of bone regeneration. It requires a long consolidation period and has a low but real failure rate. Bone Morphogenetic Proteins have effectiveness on promoting fracture union and stage bone coloboma union. But the study of their effectiveness on mandibular distraction osteogenesis is initial. In this study, we were to applicate ad-hBMP2 topically on the basis of the established DO animal model in rabbits and to detect the action in bone regeneration of DO, in order that we might find a new way to improve bone regeneration of DO.Methods:1.The cell 293 ,a human embryonic kidney cell line as a packaging cellwas used for culture of recombinant adenovirus vectors containing hBMP2 gene and recombinant adenovirus vectors containing EGFP gene, adenovirus vectors were reproduced and purified after 2 rounds of cscl centrification, biological activity identification was carried out using infectious dose 50. 6 adult male New Zealand rabbits weighting between 3.0-3.5 kilograms were used in this study, the bilateral Muscle of thigh were injected respectively with lOOul ad-hBMP2 and Ad-EGFP. Other treatment was identical. Three rabbits were killed randomly at the 14th and 28th day after operation. All the specimens were X-rayed and histologically examinated.2. 36 adult male New Zealand rabbits weighting between 3.0-3.5 kilograms were used in this study. Under general anesthesia, an incision on the skin along the inferior margin of mandible was performed. The soft tissue was separated and the surface of bone was exposed. Then mandibular osteotomy was done on the edentulous area about 1 mm before the first premolar. In operation, periosteum, bone marrow and the inferior alveolar artery should be carefully protected. After good hemostasia, the cut bone was fixed with a self-prepared bilateral extra-oral bone-supported distractor. Then periosteum was sutured and the wound was closed seriously. The bone stumps were fixed in situ for 7 days after operation(latency period), then distractors were activated at a speed of 0.5 millimeter every 12 hours for 7 days before they were fixed(consolidation period). At The 15th day after operation one side of all the animals'distraction gaps were injected lOOul ad-hBMP2, 18 sides of other sides were injected lOOul ad-EGFP and 18sides received no treatment. 12 rabbits including 12 Ad-BMP2-treated sides, 6 Ad-EGFP control sides and 6 blank sides were killed randomly at the 7th day, the 14th and 28th day of consolidation period separately. Specimens were observed, and inspections were taken with X-ray, histomorphology and immunohistochemical techniques. Results:I. We have succeed to culture cell 293, recombinant adenovirus vectors containing hBMP2 and purify adenovirus. The purified recombinant adenovirus vectors containing hBMP2 was obtained titer of 1.58xlOnpfu/ml in this study. Skeletogenous cells and cartilage cells were observed in the sides of Ad-hBMP2 treatment and there were not density change of the two sides two weeks after operation. Four weeks after operation new bone had formed, trabeculae of bone, cavitas medullaris bone marrow and small amounts lymphocytes around of circum- muscle were observed, there were obviously density change in the sides of Ad-hBMP2 treatment and no change of the control sides.2. we prolonged all animals' mandibles successfully, bone density and the quantity of new bone formation in the distraction gaps of Ad-hBMP2 treatment were higher than the control sides at the 7th day, the 14th and 28th day of consolidation period. The expression of BMP2 was found both in the Ad-hBMP2 treatment and control sides. The staining intensity of side of Ad-hBMP2 treatment was higher than that of the control side. Conclusions:1. We have succeed to culture cell 293, recombinant adenovirus vectors containing hBMP2 and purify adenovirus. We had checked that Ad-rhBMP2 could induce aberrance bone formation. It's very important to the further animal experiment.2. The animal model of bilateral mandibular distraction osteogenesis in rabbits was well established, which can be easily repeated, in this experiment. two different interventions were applied on both sides of the mandible in this model. The rate of the new bone formation and the density of the new bone of the sides treated by Ad-hBMP2 were superior to the control sides, which demonstrated that local application of Ad-hBMP2 could accelerate bone formation during mandibular distraction osteogenesis in rabbits.