不同时机转染基因对兔下颌骨牵引区新骨生成的影响
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摘要
背景：自1992年McCarthy首次将牵引成骨术(distraction osteogenesis, DO)用于下颌骨畸形患者的治疗以来,DO在颅颌面外科领域取得了显著的进展。随着经验的日益积累,人们对下颌骨DO的认识也在进一步深化。在临床应用中发现,尽管DO为许多常规外科手术难以矫治的颅颌面骨严重发育不足畸形及骨缺损的整复提供了一种新的矫治方法,然而,较长的疗程及牵张装置长时间留置面部或口腔内所引发的各种问题(如固定螺钉松脱、伤口感染、骨折等)成为临床运用和推广该技术的瓶颈和主要障碍。因此,如何促进新骨生成、缩短疗程、减少并发症,已成为目前颅颌面外科研究的热点和难点。
目的：本研究在前期研究的基础上,以新西兰大白兔双侧下颌骨牵引为动物模型,选择在术后即刻(潜伏期),牵引开始(牵引期)和牵引结束(固定期)利用电穿孔技术介导重组质粒pIRES-hBMP2-hVEGF 165转染下颌骨牵引区,观察不同时机进行基因转染对下颌骨牵引区新骨生成的影响,探索基因导入的最佳转染时间,以获得更好的治疗效果。
方法：新西兰大白兔48只,全麻下沿双侧下颌骨截骨后,在下颌骨断开处安放内置式下颌骨牵引器,钛钉固定,分层缝合切口,牵引器旋转杆外置。将兔随机分成4组：A组：于术后即刻,双侧牵引区注射2μg(0.1μg/μl)重组质粒pIRES-hBMP2-hVEGF165;B组：于术后3天,牵引开始时双侧牵引区注射2μg(0.1μg/μl)重组质粒pIRES-hBMP2-hVEGF165;C组：于牵引结束时,双侧牵引区注射2μg(0.1μg/μl)重组质粒pIRES-hBMP2-hVEGF165;三组均予电脉冲刺激(电脉冲参数：电压200V,电容10μF,频率0.2Hz,平均脉冲宽度2.5ms,单脉冲刺激,6个脉冲,3个脉冲后更换正负极)；D组：单纯牵引不行基因转染：各组于术后3天开始以lmm/d,1次/d速率进行牵引,连续牵引10天；各组分别于牵引结束后固定1、2、4、8周处死3只兔子,切取左侧下颌骨行x线检测下颌骨愈合情况、QCT测量牵引区新生骨组织密度后,将4、8周的标本加工成长20mm、宽10mm、厚3mm的标准试件行三点弯曲试验,测量牵引区新生骨组织的骨强度、弹性模量及最大破坏能量；切取右侧下颌牵引区新生组织行组织学检测和形态计量学分析。
结果：各组牵引间隙新生骨骨密度与骨强度随着固定期的延长逐渐增高；固定1周时A、B、C三组新生骨骨密度无显著差异性,但均高于D组：B组固定2、4、8周时骨密度高于A、C、D组,固定4、8周时生物力学各项指标高于A、C、D组；组织学检查和形态计量学分析发现,B组与A、C、D组比较间隙内有更多的新生血管、成骨细胞和间充质细胞等成分,各时点新生骨量与新生骨小梁宽度明显高于后者。
结论：在牵引开始时(牵引期)进行基因转染较其他时间转染促进新骨生成作用明显,能够获得最佳的促进新骨生成的效果,提示牵引期是下颌骨基因治疗的最佳时机。
Background:Since McCarthy reported that correction the human mandible malformation with distraction osteogenesis in 1992, this technology has been achieved remarkable progresses in craniomaxillofacial surgery. With the clinical application experience accumulation, we have known more and more about distraction osteogenesis. Clinically, many people realized that the technology provided a new method to treat severe craniomaxillifacial deformities and bone defects, which are difficult to manage with common operations, however, there are some problems may occur during the long time for distraction, such as loose of the fixation, infection, as well as bone fracture, all those because of the long period of the instruments placed introral or extroral, these would be the main hamper to further clinical application. Therefore, how to promote new bone formation, shorten treatment period and reduce the complications, has become a hot spot and challenge in craniomaxillofacial surgery research.
Objective:Based on previous research, We employed New Zealand rabbits bilateral mandibular distraction model and used electroporation mediate recombinant plasmid pIRES-hBMP2-hVEGF165 transfect into the distraction gap at the beginning of latency period、distraction period and consolidation period respectively, to explore the optimal time for gene therapy and obtain a better effect.
Methods:48 New-Zealand rabbits were employed, mandibles were osteotomy bilaterly under general anaesthesia, the distraction devices were fixed by titanium screws at the ends of bone gap. The arms of distractors are placed percutaneously outside the oral and the wound was closed layer by layer. The rabbits were randomly divided into 4 groups:Group A:2μg (0.1μg/μl) recombinant plasmids pIRES-hBMP2-hVEGF165 were injected into distraction area instantly after operation; Group B:2μg (0.1μg/μl) recombinant plasmids pIRES-hBMP2-hVEGF165 were injected into distraction area at the beginning of distraction; Group C:2μg (0.1μg/μl) recombinant plasmids pIRES-hBMP2-hVEGF165 were injected into distraction area after the completion of distraction. Each group was employed electroporation 5 minutes after the injection of plasmids (electric impulses parameters:200V in voltage,10μF in capacitance,0.2Hz in frequency,2.5ms in average pulse width,6 single pulses, change the positive and negative poles after 3 pulses). Group D:only distracted without gene transfection. In each group, after a 3 days of latency period, the device were activated at the rate of 1mm per day and rhythm of once per day for 10 days. Three rabbits of each group were sacrificed at lwk,2wk,4wk and 8wk of consolidation respectively. The mandibles were harvested and the left subject to X-ray examination for bone healing, and quantitative computed tomography (QCT) dectection for the density of newly formed bone in the distraction gap. The biomechanical properties of the new generation bone at 4th and 8th week of consolidation of each group were detected by three point bending test. The newly formed tissue in the distraction area of the right mandible was harvested for histology examination and histomorphometry analysis.
Results:The bone mineral density and the biomechanical strength of newly formed bone increased with the pass of the consolidation time in each group. After 1 week of consolidation, there is no significant difference of BMD among group A, group B, group C; But the BMD of group A, B and C is higher than that of group D. After 2wk,4wk and 8wk of consolidation, the BMD of group B is significantly higher than those of group A, C, and D. The biomechanical parameters are also higher in group B than those of group A, C and D after four and eight weeks of consolidation. The amounts of newly formed vessel、osteoblast and mesenchymal cell of group B were greater than those of group A、group B and group D. The new bone volume and width of bone trabecula of distraction area in group B were higher than those of group A、group B and group D at 1wk,2wk,4wk and 8wk of consolidation respectively.
Conclusion:It is better to transfect gene at the beginning of traction (distraction period) than at other stages of DO, in this way, we can obtain more remarkable effect on new bone formation. It suggests that the distraction stage is the optimal time for gene therapy.
目的：本研究在前期研究的基础上,以新西兰大白兔双侧下颌骨牵引为动物模型,选择在术后即刻(潜伏期),牵引开始(牵引期)和牵引结束(固定期)利用电穿孔技术介导重组质粒pIRES-hBMP2-hVEGF 165转染下颌骨牵引区,观察不同时机进行基因转染对下颌骨牵引区新骨生成的影响,探索基因导入的最佳转染时间,以获得更好的治疗效果。
方法：新西兰大白兔48只,全麻下沿双侧下颌骨截骨后,在下颌骨断开处安放内置式下颌骨牵引器,钛钉固定,分层缝合切口,牵引器旋转杆外置。将兔随机分成4组：A组：于术后即刻,双侧牵引区注射2μg(0.1μg/μl)重组质粒pIRES-hBMP2-hVEGF165;B组：于术后3天,牵引开始时双侧牵引区注射2μg(0.1μg/μl)重组质粒pIRES-hBMP2-hVEGF165;C组：于牵引结束时,双侧牵引区注射2μg(0.1μg/μl)重组质粒pIRES-hBMP2-hVEGF165;三组均予电脉冲刺激(电脉冲参数：电压200V,电容10μF,频率0.2Hz,平均脉冲宽度2.5ms,单脉冲刺激,6个脉冲,3个脉冲后更换正负极)；D组：单纯牵引不行基因转染：各组于术后3天开始以lmm/d,1次/d速率进行牵引,连续牵引10天；各组分别于牵引结束后固定1、2、4、8周处死3只兔子,切取左侧下颌骨行x线检测下颌骨愈合情况、QCT测量牵引区新生骨组织密度后,将4、8周的标本加工成长20mm、宽10mm、厚3mm的标准试件行三点弯曲试验,测量牵引区新生骨组织的骨强度、弹性模量及最大破坏能量；切取右侧下颌牵引区新生组织行组织学检测和形态计量学分析。
结果：各组牵引间隙新生骨骨密度与骨强度随着固定期的延长逐渐增高；固定1周时A、B、C三组新生骨骨密度无显著差异性,但均高于D组：B组固定2、4、8周时骨密度高于A、C、D组,固定4、8周时生物力学各项指标高于A、C、D组；组织学检查和形态计量学分析发现,B组与A、C、D组比较间隙内有更多的新生血管、成骨细胞和间充质细胞等成分,各时点新生骨量与新生骨小梁宽度明显高于后者。
结论：在牵引开始时(牵引期)进行基因转染较其他时间转染促进新骨生成作用明显,能够获得最佳的促进新骨生成的效果,提示牵引期是下颌骨基因治疗的最佳时机。
Background:Since McCarthy reported that correction the human mandible malformation with distraction osteogenesis in 1992, this technology has been achieved remarkable progresses in craniomaxillofacial surgery. With the clinical application experience accumulation, we have known more and more about distraction osteogenesis. Clinically, many people realized that the technology provided a new method to treat severe craniomaxillifacial deformities and bone defects, which are difficult to manage with common operations, however, there are some problems may occur during the long time for distraction, such as loose of the fixation, infection, as well as bone fracture, all those because of the long period of the instruments placed introral or extroral, these would be the main hamper to further clinical application. Therefore, how to promote new bone formation, shorten treatment period and reduce the complications, has become a hot spot and challenge in craniomaxillofacial surgery research.
Objective:Based on previous research, We employed New Zealand rabbits bilateral mandibular distraction model and used electroporation mediate recombinant plasmid pIRES-hBMP2-hVEGF165 transfect into the distraction gap at the beginning of latency period、distraction period and consolidation period respectively, to explore the optimal time for gene therapy and obtain a better effect.
Methods:48 New-Zealand rabbits were employed, mandibles were osteotomy bilaterly under general anaesthesia, the distraction devices were fixed by titanium screws at the ends of bone gap. The arms of distractors are placed percutaneously outside the oral and the wound was closed layer by layer. The rabbits were randomly divided into 4 groups:Group A:2μg (0.1μg/μl) recombinant plasmids pIRES-hBMP2-hVEGF165 were injected into distraction area instantly after operation; Group B:2μg (0.1μg/μl) recombinant plasmids pIRES-hBMP2-hVEGF165 were injected into distraction area at the beginning of distraction; Group C:2μg (0.1μg/μl) recombinant plasmids pIRES-hBMP2-hVEGF165 were injected into distraction area after the completion of distraction. Each group was employed electroporation 5 minutes after the injection of plasmids (electric impulses parameters:200V in voltage,10μF in capacitance,0.2Hz in frequency,2.5ms in average pulse width,6 single pulses, change the positive and negative poles after 3 pulses). Group D:only distracted without gene transfection. In each group, after a 3 days of latency period, the device were activated at the rate of 1mm per day and rhythm of once per day for 10 days. Three rabbits of each group were sacrificed at lwk,2wk,4wk and 8wk of consolidation respectively. The mandibles were harvested and the left subject to X-ray examination for bone healing, and quantitative computed tomography (QCT) dectection for the density of newly formed bone in the distraction gap. The biomechanical properties of the new generation bone at 4th and 8th week of consolidation of each group were detected by three point bending test. The newly formed tissue in the distraction area of the right mandible was harvested for histology examination and histomorphometry analysis.
Results:The bone mineral density and the biomechanical strength of newly formed bone increased with the pass of the consolidation time in each group. After 1 week of consolidation, there is no significant difference of BMD among group A, group B, group C; But the BMD of group A, B and C is higher than that of group D. After 2wk,4wk and 8wk of consolidation, the BMD of group B is significantly higher than those of group A, C, and D. The biomechanical parameters are also higher in group B than those of group A, C and D after four and eight weeks of consolidation. The amounts of newly formed vessel、osteoblast and mesenchymal cell of group B were greater than those of group A、group B and group D. The new bone volume and width of bone trabecula of distraction area in group B were higher than those of group A、group B and group D at 1wk,2wk,4wk and 8wk of consolidation respectively.
Conclusion:It is better to transfect gene at the beginning of traction (distraction period) than at other stages of DO, in this way, we can obtain more remarkable effect on new bone formation. It suggests that the distraction stage is the optimal time for gene therapy.
引文
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49. Koh JT, Zhao Z, Wang Z, et al. Combinatorial gene therapy with BMP2/7 enhances cranial bone regeneration[J]. J Dent Res.2008,87(9):845-849.
50. Wu GP, He XC, Yang ZH, et al. Influence on the osteogenic activity of the human bone marrow mesenchymal stem cells transfected by liposome-mediated recombinant plasmid pIRES-hBMP2-hVEGF165 in vitro[J]. Ann Plast Surg, 2010,65(1):80-84.
51.吴国平,黎德平,胡纯兵,等.电穿孔介导的基因治疗对兔下颌骨牵引区新生骨骨密度与强度的影响[J].中华整形外科杂志,2010,26(3)：207-211.
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