【作者】 周诺；

【导师】 黄光武；

【作者基本信息】 广西医科大学 ， 耳鼻咽喉科学， 2012， 博士

【摘要】 目的牵张成骨(Distraction Osteogenesis,DO)技术是利用骨痂的愈合机理产生新骨,是一种内源性的骨组织工程学技术。自其开始应用于颅颌面领域以来,大量基础和临床研究逐渐开展,随着基础研究的深入以及该技术的改进和成熟,牵张成骨在颌面部整形、肿瘤的术后重建以及牙槽种植修复等方面展现出了广阔的应用前景,成为口腔颌面外科领域的研究热点。虽然DO有许多传统手术不可比拟的优势,已被广泛地应用于各种颅颌面的畸形以及四肢短小畸形的矫治,但其较长的牵张和固定时间,限制了其在临床上的广泛应用。因此如何提高DO的新骨形成的速度,缩短其固定时间,减少其并发症的发生和保证新骨形成质量,正成为目前该领域的研究热点。本课题利用hBMP-2作为促进因子,将基因工程和组织工程的技术结合起来,先把hBMP-2转染入兔BMSCs,构建稳定表达hBMP-2的BMSCs,再与组织工程支架材料Pluronic F-127复合,注射入牵张区内,使hBMP-2能在牵张区内稳定持续地起作用,拟让其能促进兔下颌骨的牵张成骨新骨生成。从组织学水平、蛋白水平、基因水平观察和检测牵张间隙内新骨的形成以及改建的情况,为牵张成骨技术在治疗各种颌面部畸形或骨缺损等疾患上的广泛应用奠定基础。方法1.兔骨髓间充质干细胞体外培养以及成骨定向诱导分化:取成年健康新西兰大白兔,穿刺双侧胫骨抽取骨髓,通过密度梯度离心法收集细胞后培养,原代培养8-10d后传代培养,按照1:3传代,每3-4天可传代1次,传到第3代时用地塞米松,β-甘油磷酸钠,抗坏血酸配制而成的成骨诱导分化液进行成骨诱导14至21天,并分别用茜素红染色,ELISA以及免疫组化的方法检测其矿化结节的形成和ALP、BGP的表达。2.脂质体介导的hBMP-2转染兔BMSCs:取第3代BMSCs,用脂质体介导携带hBMP-2基因的pcDNA3.1质粒转染入BMSCs中,观察GFP表达的情况,MTT法检测转染后细胞的增殖和生长的能力,通过RT-PCR检测瞬时转染后hBMP-2 mRNA的表达。转染后再用G418筛选2周,筛选出稳定转染的阳性细胞,收集备用。将经过筛选后的BMSCs培养4周后,对其进行BMP-2的免疫组织化学以及Western Blot检测。3.支架材料Pluronic F-127凝胶与兔BMSCs复合培养:利用Pluronic F-127低温下为水溶液,室温可凝固的特点,冰浴中制备凝胶并消毒后与转染hBMP-2的BMSCs复合,调整细胞浓度,使细胞浓度达到2.5×107个/ml,MTT法检测Pluronic F-127的细胞毒性。4.兔下颌骨牵张成骨动物模型的建立:取成年健康新西兰大白兔6只,依据其下颌骨的解剖结构设计牵张器。利用传统单线骨皮质切开的术式,在右侧下颌骨第二和三磨牙间行骨切开术,固定牵张器。术后潜伏期为5天,第6天开始牵张,每天牵张2次,每次0.5mm,共牵张7mm,随后固定。于牵张固定2周、6周后分别摄X线片以观察新生骨愈合以及改建情况。5.Pluronic F-127凝胶复合hBMP-2基因修饰BMSCs促进兔下颌骨牵张成骨新骨形成的实验研究:取健康新西兰白兔48只随机分为4组,每组12只,于固定期第2天进行干预。实验分组:A组:牵张间隙内注射200 u 1 hBMP-2基因修饰的BMSCs/Pluronic F-127凝胶复合物;B组:注射200μ1 hBMP-2基因修饰的BMSCs液;C组:注射200μ1 BMSCs液;D组:注射200μ1生理盐水。分别于牵张结束固定2w、6w各组处死动物6只,通过大体病理、摄X线片、扫面电镜、HE染色组织切片、免疫组化等手段检测新骨形成和改建情况。结果1.兔骨髓间充质干细胞体外培养以及成骨定向诱导分化结果:BMSCs培养8-10d后即可长满瓶底,并可传代,传代后细胞生长状态良好,可一直维持BMSCs的典型形态以及生长状态,细胞呈现长梭形形状,旋涡式的排列并可成功向成骨方向诱导。其中钙结节茜素红染色见阳性结节染色。ALP含量逐渐升高,并在2周后达到最高,与未诱导组有显著差异(p<0.05)。BGP免疫组化也呈阳性染色。2.脂质体介导的hBMP-2转染兔BMSCs的结果:利用脂质体介导携带hBMP-2的pcDNA3.1质粒成功转染BMSCs,转染效率约30%;转染24h能观察到弱荧光表达,48h后荧光强度增强;MTT法显示转染后的BMSCs生长增殖能力无明显变化;RT-PCR也检测到了hBMP-2 mRNA的表达。用G418成功筛选出稳定转染的细胞,该细胞培养4周可用免疫组织化学以及Western Blot方法检测到BMP-2蛋白地稳定表达。3.支架材料Pluronic F-127凝胶与兔BMSCs复合培养的结果:Pluronic F-127在4℃时为水溶液,37℃下为凝胶状,其与转染hBMP-2基因的BMSCs复合后,MTT检测结果见复合物中的BMSCs生长增殖能力无明显变化(p>0.05)。4.兔下颌骨牵张成骨动物模型建立的结果:所有的实验动物都成功地完成了手术,术区与口内无穿通,无感染,并能顺利牵张。实验动物全部存活至预定时间,体重皆有不同程度地增加。从手术至取材,应用于体内的牵张器均固位良好,无断裂以及脱钉现象。X线片见新骨形成在时间段有明显变化,符合临床上牵张成骨的成骨规律。动物模型构建成功。5.Pluronic F-127凝胶复合hBMP-2基因修饰BMSCs促进兔下颌骨牵张成骨新骨形成的实验研究结果:通过大体病理、X线、扫描电镜和HE染色组织切片观察,新骨形成质量由高到低排列为:A组>B组>C组=D组;通过免疫组化观察并经过灰度值统计软件分析,在固定期第2周和6周A组牵张区骨质量明显高于B组(p<0.05),B组牵张区骨质量明显高于C组与D组(p<0.05),在各期C、D两组间无明显差异(p>0.05)。结论1.密度梯度离心法简便、易行,可大量扩增、纯化兔骨髓间充质干细胞,所获细胞经过诱导后可向成骨细胞分化。2.通过脂质体介导的真核转染方法能成功将外源hBMP-2基因导入BMSCs,并使其获得基因及蛋白的表达。3.组织工程支架材料Pluronic F-127具有低温下为水溶液,室温可凝固的特点,细胞相容性良好。4.兔子不仅性情温顺,饲养方便,对手术耐受性好,易于操作,而且术后愈合效果好,是一个稳定可靠的实验模型动物,可适合较大样本的实验研究。5.hBMP-2基因修饰BMSCs复合可注射骨组织工程支架材料Pluronic F-127移植能有效促进兔下颌骨牵张成骨新骨形成,为临床上缩短牵张成骨治疗周期提供了一定的理论依据以及技术参考。更多还原

【Abstract】 ObjectiveDistraction Osteogenesis(DO)is an endogenous bone tissue engineering technique in which new bone is regenerated by the mechanism of callus healing.Numerous basic experimental and clinical researches have been carried out since its application in craniofacial field.With the proceeding development of basic study of this technique,DO shows its extensive application prospect in maxillofacial plastic surgery,bone reconstruction following tumor resection and implant restoration in alveolar bone,and has been one of the research hotspots in the field of oromaxillofacial surgery.Although its significan advantages over conventional surgical procedures,DO has been widely applied in the correction of craniofacial anomalies and extremity defects.However,the prolonged distraction and consolidation periods do considerably limit its wider clinical application.Therefore,how to increase the rate of bone regeneration,to shorten consolidation period,to reduce the complication and to enhance regeneration quality have become research hotspots in this field at present.The gene of human bone morphogenetic protein 2(hBMP-2)was transfected into bone mesenchymal stem cells(BMSCs)to construct expression-stable hBMP-2 modified BMSCs.Then the gene modifying cells were seeded in Pluronic F-127 hydrogel,forming cell/gel composite which was then injected to the distraction gap,aiming to achieve the stable and continuous expression of hBMP2 and accelerate bone regeneration with the combination of genetic engineering and tissue engineering techniques.Bone regeneration and remodeling in the distraction gap was observed and detected on the histologic,proteinic and genetic levels,respectively.This study was designed to establish foundation for the extensive application of DO in the treatment of various maxillofacial deformities and defects.Methods1.BMSCs’ in-vitro cultivation and directional osteogetic differentiation:Bone marrow was extracted from adult healthy New Zealand’s white rabbit by puncturing bilaterial tibia,and then cells were collected by density gradient centrifugation.After 8 to 10 days’ culture,primary cells could be digested for passage cultured at 1:3 and every passage cells could be cultured for 3 to 4 days.Cells of passage Ⅲ were harvested for osteoanagenesis inducing for 14 to 21 days using osteoblast differentiation medium containing dexamethasone,β-sodium glycerophosphate,ascorbic acid.Mineralization nodules,ALP and BGP in the BMSCs after induction was dectected by Alizarin Red staining,ELISA and IHC methods respectively.2.Liposome-mediated transfection of hBMP-2 gene into BMSCs:The pcDNA3.1 plasmids carrying liposome-mediated hBMP-2 gene were transfected into BMSCs.GFP was observed under fluorescence microscope.The proliferation and growth of tranfected cells was investigated by MTT method.The instantaneous expression of hBMP-2 mRNA was detected by RT-PCR.G418 was used for 2 weeks to screen the stable transfected cells and then the cells were gathered for further use.Immunohistochemistry and Western Blot was used to dectect the expression of BMP-2 in the cells that had been screened and cultured for 4 weeks.3.Cultivation of hBMP-2-BMSCs/Pluronic F-127 hydrogel composite:Pluronic F-127 was prepared in ice bath with its characteristics of which it is water-solutionable under low temperature but solid under room temperature.After sterilization,Pluronic F-127 hydrogel was seeded with hBMP2-BMSCs(2.5×107cells/ml).The cytotoxicity of Pluronic F-127 hydrogel was investigated by MTT method.4.Model establishment of rabbit mandibular distraction osteogenesis:A total of 6 adult healthy New Zealand’s white rabbits were used in this study.Distractors were custom-made according to the mandibular anatomy of rabbit.Conventional osteotomy was conducted between right mandibular second and third molar.After a 5-day’s latency,distracers were activated at a rate of 0.5 mm/time for twice per day for 7 consecutive days.X-ray images were taken on 2 and 6 weeks consolidations,respectively,to observe bone regeneration and remolding.5.Experimental study on promoting bone regeneration of rabbit mandibular distraction osteogenesis with the composite of Pluronic F-127 hydrogel and hBMP-2 gene modifying BMSCs:Forty-eight New Zealand’s white rabbits were ramdomized into four groups(A,B,C and D)with twelve in each.In group A,200μl of hBMP-2-BMSCs/Pluronic F-127 composite was injected into the distraction gap at the first day of consolidation period,while in groups B,C and D the same amount of hBMP2-BMSCs suspension,BMSCs suspension and saline was injected in the distraction gap,respectively.The operated mandibles were harvested on 2-and 6-W consolidations,respectively.The regenerated bone was evaluated by the means of general pathology,radiographics,scanning electron microscopy(SEM),HE staining,immunohistochemistry staining.Results1.Results of the in-vitro cultivation of BMSCs and osteogenic differentiation:8 to 10 days after cultivation,primary BMSCs were overgrowthing for passage cultured.After subculturing,in addition to the preservation of typical morphological feature and growth state of BMSCs,it could be osteogenicly induced successfully.Under microscope,spindle-shaped cells and vortex arrangement were observed.After induction,positive dying of calcium nodules was observed.There was significant difference(p<0.05)in the contents of ALP between uninduced group and induced one in which the content reached highest on 2-week consolidation.In addition,positive staining of BGP was observed by immunohistochemistry.2.Results of the transfection of hBMP-2 gene into BMSCs mediated by liposome:The transfection efficiency of plasmid-carrying hBMP-2 gene was approximately 30%.The fluorescence intensity was rather poor on post-transfection 24h but became stronger after 48h.The result of MTT revealed that the proliferation capacity of transfected BMSCs did not change significantly.And the gene expression of hBMP-2 could be detected.Stable transfected cells were obtained and its stable expression of BMP-2 was dectected by immunohistochemistry and Western Blot after 4 weeks’cultured.3.Results of the cultivation of hBMP-2-BMSCs/Pluronic F-127 composite:Pluronic F-127 was solutive at 4℃ but solid at 37℃.There was no significant change in the proliferation activity of BMSCs in the composite,which was invested by MTT method(p>0.05).4.Results of model establishment of rabbit mandibular distraction osteogenesis:All the experimental animals have survived the whole process without any severe complication.Moreover,distraction proceeded on smoothly.During the whole process,the retention of bone fragment was stable without breakage of distractors or loosening of screws.X-ray images showed that there was significant translucency change of regenerated bone at various consolidays,which corresponded to the clinical principle of distraction osteogenesis.The rabbit model of mandibular distraction osteogenesis was constructed successfully.5.Results of experimental study on promoting bone regeneration of rabbit mandibular distraction osteogenesis with the compound of Pluronic F-127 hydrogel and hBMP-2 gene modifying BMSCs:The results of general pathology,X-ray,SEM and HE staining showed that the quality of regenerated bone of the groups ranked as follows:group A>group B>group C=group D.The gray value of the results of IHC showed that the quality of regenerated bone of group A was obviously higher than that of group B(p<0.05),and group B was higher than group C and D(p<0.05),but there was no significant difference between group C and D(p>0.05).Conclusions1.Rabbit bone mesenchymal stem cells(BMSCs)could be cultivated,amplified and purified by the method of density gradient centrifugation which was convenient and practical.The BMSCs could be directionally induced to osteoblasts.2.Extragenous hBMP-2 gene could be successfully transfected into rabbit BMSCs and got rather stable in-vivo gene and BMP-2 protein expression by the mean of liposome-mediated plasmid transfection.3.The scaffold material of pluronic F-127 had the characteristics of excellent cell compatibility and temperature-sensitivity which was solutionable under low temperature but solid under room temperature.4.Rabbit could be an optimal model animal for large-sample experimental study for its docile disposition,easy raising,favourable operation tolerance,simple management and excellent postoperative healing process.5.The transplantation of the composite of human BMP-2 gene modifying BMSCs and injectable scaffold material Pluronic F-127 could effectively promote bone regeneration in rabbit mandibular distraction,which would provide theoretical and technical foundation for shortening the consolidation period.更多还原