| Background: Distraction osteogenesis(DO) is recognized as the best type of in vivotissue engineering in vivotechnology，it has been widely used in clinical and correctlimbs and many complex craniomaxillofacial deformity and bone defect which cannot be corrected by conventional orthognathic surgery operation. In spite of this,there are drawbacks. The major disadvantage relates to the prolonged consolidationphase. During this time period many complications can occur, including localinfections, distractor device or pin loosening, and refractures. As well, patients andfamily members can be subject to inconveniences and psychosocial burden due to theretained distractor device. Therefore, how to regulate cytokines in the process ofdistraction and promote new bone formation in distraction gap has become a hot spotin current research.Objective: On the basis of previous studies, the study transfected recombinantplasmid pIRES-hBMP2-hVEGF165into mandibular distraction gap mediated byelectroporation. is on the basis of previous research through electroporationmediated the area of mandible traction,to explore the changes of expression ofosteogenic factors following gene transfecting at different time in order to find out themolecular mechanism to promote new bone formation generated by the optimal timeof gene transfected into distraction gap.Method: Forty-eight6months age,healthy New Zealand rabbits were employed inthis study, Bilateral mandibular osteotomies were performed in all rabbits undergeneral anesthesia, implanted special distraction device in rabbit mandible, distractorrotating rod was set externally, incision was sutured layer by layer.We randomlydivided the experimental animal into four groups with12in each group. After alatency of3days, the mandibles were elongated using distractors with a rate of0.8 mm/day onece a day for10days. Gene transfection were performed at different time,group A, B and C were injected the recombinant plasmid pIRES-hBMP2-hVEGF1652μg （0.1μg/μl） in the bilateral mandible distractiongap immediatly compltedoperation(latency period),4days after operation(distraction period),14days ofsurgery(consolidation period), respectively. the three groups, were treated byelectroporation.(The electric pulse parameters: voltage is200V, the capacitance is10μ F, frequency is0.2Hz, the average pulse width is2.7ms, the single pulse stimulationfor a total of6pulse, replacing the positive and negative after3pulse). Control groupis Group D without gene transfection. Four animals were sacrificed at7thday,14thday28thday of consolidation period of different group, respectively. The lengthenedmandibles were harvested and processed for immunohistochemical examinations, themean optic densities (MOD) and Integral optical density (IOD) of BMP-2andTGF-β1positive cells were measured by CMIAS-2001A computerized imageanalyzer, the data were analysed with SPSS.Result: Histological examination revealed, the seventh day of consolidation period,early bridging of the osteotomized site occurred with formation of soft callus andcartilage tissue and with the presence of chondrocytes and fibroblastic cells; at14thday, chondrocytes and elongated fibroblastic cells were identified morphologically inthe distracted gap and appeared to be growing in the direction of distraction; at28thday, bridging of the distal and proximal mandibular bone fragments took place, withvisible bone and calcified tissue deposition in the distracted gap. Compared withgroup A, C and D, there were more new blood vessels, osteoblasts and mesenchymalcells in the distraction gap of group B.Immunohistochemistry showed that;1. BMP-2expression mainly located in cellcytoplasm. At7thday of consolidation period, BMP-2intensely expressed in themesenchymal cells, fibroblast, inflammatory cells (such as monocytes, mesenchymalcells), osteoblasts and chondrocytes in the middle of the distraction zones and thesurrounding connective tissue. At later time points,14th day, BMP-2staining wasmainly found in the cytoplasm of hypertrophic chondrocytes and osteoblastic cellsrimming the trabecular bones arranged along the stretching direction within the distraction regenerate callus. At28thday, BMP-2was observed mainly in theosteoblasts and fibroblasts although the population of cells decreased gradually. Thesemi quantitative analysis revealed that expression of BMP-2at peaked at7thday ofconsolidation period in all groups. Compare to other three groups, groupB(0.31±0.04，0.68±0.06) was the strongest, difference was significant among them(P <0.01, P <0.05),there is still a significant difference among group A and C, D,(P<0.05), compared with group D and C, there were significant differences (P <0.05).At day14, the number of positive staining cells declined, there was significantdifference among group B and A, C, D (P <0.05). The expression of BMP-2at28thday was weak, BMP-2expression is very weak, there was no significant differenceamong group A, B,C and D (P>0.05).2. TGF-β1expression correlated closely with BMP-2expression at all time points.At7th day of consolidation period, TGF-β1expression was prominent within thenewly formed callus, localizing to fibroblast, osteocytes, osteoblasts and chondrocytesalong the osteotomy edges. Furthermore, there was significant immunostaining of lessclearly defined mesenchymal cells positve staining. The pattern of staining forTGF-β1at14and28days of consolidation period was similar to that of BMP-2. Atday14after gene transfection, many specimens demonstrated evidence of ossification,TGF-β1immunostaining was particularly manifest along the borders of the islands ofossification and found in hypertrophic chondrocytes, osteocytes and osteoblastswithin the gap regenerate,as well as mesenchymal cells. At day28of consolidationperiod, the population of cells that stained for TGF-β1decreased, immunostainingpredominantly within osteoblasts and osteoclasts actively participating in matrixmineralization and remodeling. The expression of TGF-β1peaked at day7ofconsolidation period in each group. Group B(0.37±0.07，0.90±0.06) is strongestfollowed by group C(0.22±0.02，0.69±0.02) and group A(0.16±0.05，0.41±0.03),there was significant difference among them (P <0.01, P <0.05). At day14and28ofconsolidation period, the statistical analysis expression of TGF-β1in different groupswas similar to that of BMP-2. At14thday of consolidation period, the number ofpositive staining cells decreased, there was significant differences among group B and A, C, D (P <0.05), At28thday of consolidation period, the expression of TGF-β1isvery weak, there was no significant difference among them (P>0.05).Conclusion: Local gene transfection in distraction gap can up-reagulate theexpression of osteogenic mediators (BMP-2and TGF-β1), the effect was differentbecause of transfection gene at different time. the expression of BMP-2and TGF-β1of transfection gene at distraction period is stronger than those of latency period andconsolidation period, which may promote new bone formation in distraction gap via aseries of biological effects. |
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