【作者】 来庆国；

【导师】 徐欣；

【作者基本信息】 山东大学 ， 口腔临床医学， 2010， 博士

【摘要】 牵张成骨(distraction osteogenesis, DO)技术最早源于矫形外科,用来矫正肢体长度缺陷,后来用于治疗颅颌面的先天畸形。牵张成骨成功引入口腔颌面外科为常规技术难以矫正的骨缺损和畸形提供了新的方法和思路,它的临床实践效果不仅突破了传统外科理论而且解决了许多传统外科手段所无法解决的临床难题。牵张成骨过程中形成新骨的形态与、结构和大小接近周围原骨,无需植骨,避免了骨移植术的各种并发症；骨周围的软组织,如肌肉、皮肤、神经和血管,可同期同步扩张；此外,牵张成骨手术创伤小。但牵张成骨的某些局限性如治疗周期长、牵张过快过长导致新骨形成不良等问题限制了其在临床上的进一步推广应用。因此,如何促进牵张新骨形成与矿化,从而缩短牵张成骨治疗周期正成为众多学者关注的热点。目前,许多方法被尝试用于促进牵张成骨过程中新骨和骨痂形成,缩短DO固定期。这些方法包括应用脱矿骨基质、无机盐、低强度超声、直流电、电磁场刺激、高压氧、干细胞和细胞因子等。虽然上述方法获得了一定的效果,但这些手段均未获得广泛的临床应用。很多生长因子和细胞因子已被证实促进体内骨再生,研究最多的是骨形成蛋白(bone morphogenetic proteins, BMPs)家族,在不同动物模型上都表明可以促进牵张间隙异位成骨,其中BMP-2和BMP-7已被美国FDA批准应用于骨不连的病人。其他诱导骨生长和分化细胞因子,如转化生长因子-β1 (transforming growth factor-betal, TGF-β1)、碱性成纤维细胞生长因子(basic f ibroblastgrowth factor, bFGF)、胰岛素样生长因子-1(insulin-like growthfactor-1,IGF-1)等也对新骨生成有一定促进作用。但这些外源性应用的生长因子数量要远远高于生理剂量才能发挥成骨的作用,可能与其半衰期短和导入途径有关。此外,细胞因子超生理剂量的应用不仅费用高昂,限制了临床广泛的应用,而且导致临近的非骨组织异位成骨。鉴于这些并发症,基于生长因子过表达策略的基因治疗成为促进成骨的一种新的策略。以BMP-2、BMP-7和bBGF为目的基因的基因治疗已用于实验动物并成功的促进牵张成骨新骨形成。但是这种使骨生长因子过表达的基因治疗策略,对周围非骨组织的旁分泌导致的复杂的释放动力学和无法调控的异位成骨阻碍了这一方法在临床的应用。此外,牵张成骨中新骨形成是一个非常复杂的过程,是多因素、多细胞因子共同参与的过程,而让多种生长因子同时高表达有一定困难。因此,我们设想了从众多生长因子、细胞因子和动力传导的中枢和靶向,即转录因子调控的水平寻找牵张成骨基因治疗的一种新方法,以避免基于骨生长因子基因治疗的一些弊端。转录因子,包括Runx2, Smads, Dlx-3, Dlx-5, MSX-2, AP-1和Osterix(OSX)都在骨愈合过程中表达,但在这些成骨分化的转录因子中,Runx2和Osterix是多潜能骨髓基质干细胞向成骨细胞分化的必不可少的关键性转录因子。Runx2属于runt结构域转录因子家族,在成骨部位高表达,在骨形成中发挥着重要作用。Runx2过表达在成骨早期能够促进成骨细胞分化,但可能抑制成骨细胞的晚期分化。OSX是一种具有锌指基序结构域的转录因子,位于Runx2的下游,OSX的过表达能诱导有成骨潜能的细胞向成骨细胞分化,补充成骨细胞来源,抑制软骨细胞形成,促进新骨的成熟和矿化,避免了Runx2抑制成骨细胞的晚期分化的问题。而国际上也尚未查见关于OSX基因治疗用于牵张成骨的报道。因此,在本研究中选择OSX为目的基因,首先建立兔单侧下颌骨牵张成骨动物模型,构建重组质粒pEGFP-OSX,脂质体介导下重组质粒pEGFP-OSX瞬时转染MSCs,将OSX修饰的MSCs导入到兔下颌牵张间隙中；应用影像学、组织学、组织形态计量学等手段与对照组比较,评价OSX局部基因治疗对牵张成骨中新骨的形成和矿化的作用,并检测BSP体内表达情况。本研究结果将为OSX基因治疗策略促进牵张成骨新骨形成和缩短牵张成骨疗程提供科学的实验依据,为最终的临床应用1.兔单侧下颌骨牵张成骨动物模型的建立：选择新西兰大白兔为实验动物,4～5个月龄,3.0～3.5 kg,雌雄不限。使用改进的内置式牵张器,由螺旋牵张杆、导向滑动杆、固定臂、旋转柄以及连结旋转柄的方向关节等组成,每旋转一圈,牵张距离为0.4mm.用戊巴比妥钠在兔耳缘静脉注射麻醉后,在下切牙与后牙之间约1公分的区域内选择截骨部位,骨切开后用4个钛螺钉固定牵张成骨器。6天的延迟期后,进行牵张,速度为0.8mm／天(上午8：00.,4mm,下午8：0,0.4mm),连续10天,共牵张延长8mm,牵张结束后固定牵张器。分别于固定期开始的第2、6周处死一半动物。动物处死后下颌骨标本行X线检查和组织学HE染色观察。2. pEGFP-OSX的构建：PCR扩增获得目的基因,经过纯化、酶切、酶切产物回收与纯化制备目的基因。载体进行酶切消化后将酶切产物回收、纯化,使载体线性化。然后将获得的目的基因连接到载体,制备感受态、转化,将构建的新质粒PCR扩增,测序鉴定。3.兔自体骨髓基质干细胞(BMSCs)的培养和传代,采用密度梯度离心法,取生长良好第3代细胞用于基因转染和治疗。4.脂质体介导的pEGFP-OSX质粒体外转染MSCs和基因治疗：取与每只兔相对应的第3代骨髓MSCs,消化计数,并传代至6孔板内,用以作基因转染。细胞传代24h后,细胞达到80%融合,此时采用脂质体法进行pEGFP-OSX:基因转染操作,通过荧光显微镜下检测转染效率来观察转染后OSX基因的表达。分别用pEGFP-OSX修饰的MSCs(A组,18只),MSCs(B组,18只)和生理盐水(C组,18只),在牵张结束后立即对兔牵张间隙多点注射治疗。5.成骨效果检测：于牵张结束第2周和第6周随机处死各9只动物,标本获取后立即行X线检查,然后检测牵张区骨密度。每个标本制作不连续切片16张,10张用以HE染色,在光镜下观察并进行形态计量学分析；6张用以免疫组织化学检测骨涎蛋白(BSP)表达。骨组织形态计量学采用图像分析软件进行分析。每一标本均测量10张不连续切片,测量结果的均数,为该标本的测量参数值。测量参数包括新生骨量和新生骨小梁厚度。6.统计学分析：实验中牵张间隙骨密度、骨组织形态计量学参数均用均数士标准差来表示。对每一时间点各指标的组间差异应用统计软件SPSS 11.0进行单因素的方差分析。P＜0.05差异具有显著性。1.活体观察实验动物,均耐受手术及术后牵张,饮食大小便正常,牵张结束后存在偏颌现象。大体标本发现牵张侧下颌骨延长。标本X线检查和组织学观察证实动物模型牵张间隙成骨良好,下颌骨按照预定目标延长。2.通过基因重组技术,成功构建了重组质粒pEGFP-OSX,测序分析证实了pEGFP-OSX质粒的正确性。3.应用密度梯度离心法成功分离兔骨髓MSCs,脂质体介导的pEGFP-OSX瞬时转染骨髓MSCs,荧光显微镜下观察48小时的转染效率为40-45%。4.X线、组织学和免疫组织化学检测结果显示,OSX基因修饰的自体骨髓MSCs治疗组(A组)比MSCs治疗组(B组)显示出更好的成骨和矿化,自体骨髓MSCs治疗组也比生理盐水注射组(C组)表现出更好的骨痂形成。骨密度和组织形态计量学检测后,统计分析结果与上述一致,A组显著优于B组(P<0.05),B组显著优于C组(P<0.05)。5.免疫组织化学检测表明在牵张结束第2周时A组中多数间充质细胞、成骨细胞和新生的骨基质均有BSP强染色,B组BSP染色较浅,C组染色最浅。1.内置式牵张器能够满足兔单侧下颌骨延长的要求,牵张成骨的延迟期成骨与骨折愈合过程类似,但是牵张期和固定期骨再生具有特征性的组织学变化。2.本实验成功建立了稳定的兔单侧下颌骨牵张成骨的动物模型,重复性高,适合大批量动物实验,为细胞和基因治疗提供了良好的实验平台。3.密度梯度离心法所获MSCs具有很强的体外增值活性,是进行细胞治疗和基因治疗的理想种子细胞。荧光显微镜下观察,脂质体介导的瞬时转染可以使pEGFP-OSX基因在MSCs中成功表达,但转染效率很低。4.X线、组织学和免疫组织化学观察都证实,与单纯MSCs及生理盐水比较,pEGFP-OSX修饰的MSCs可更有效促进兔下颌牵张成骨过程中新骨形成和矿化,提示OSX基因治疗是促进下颌骨牵张成骨行之有效的手段。5.免疫组织化学检测结果表明,OSX基因治疗提高了成骨标志性蛋白-BSP的表达水平,进一步提示OSX基因修饰的MSCs在体内发挥了促进新骨形成和矿化的作用。6.应用OSX基因体外转染MSCs进行基因治疗的方法,能够促进牵张成骨中骨再生和缩短治疗周期,为牵张成骨临床治疗提供了一个新的策略。更多还原

【Abstract】 Distraction osteogenesis (DO) is a well-established technique, originally developed in orthopedic surgery for correction of limb length discrepancies and later also used to treat hereditary malformations in the craniomaxillofacial region. The application of Distraction osteogenesis (DO) to oral and maxillofacial surgery provides a new method dealing with difficult bone defects and deformity. and its effectiveness in clinical practice not only breaks through the traditional surgery theory, but also solve many of the clinical problems traditionally hard to solve.The size, shape and structure of the new bone during distraction osteogenesis are close to the original bone,and bone grafting is unnecessary which avoids many complications.Besides, the soft tissue (such as muscle, skin, nerves, blood vessels) around the distracted region is expanded during the bone distraction without biological changes.however, the long treatment period and the potential of fibrous union or nonunion under some circumstances remain major limitations that hamper further clinical application of DO. Therefore, how to promote the formation and mineralization of the new bone and shorten the treatment period during distraction osteogenesis is becoming the focus of attention of many scholars.Many attempts have been made to improve the technique to accelerate osteogenesis in the distraction gap, including the application of precursor stem cells, demineralized bone matrix, and other inorganic salts, low-intensity ultrasound, direct current or electromagnetic stimulation and hcyperbaric oxygen therapy. The above methods may have certain effect on osteogenesis, but none of these means have been put into a wide range of clinical application.Various growth factors and cytokines have been investigated for their capacity to promote bone regeneration in vivo. Among these, recombinant human bone morphogenetic proteins (rhBMPs) induce robust ectopic bone formation and healing of orthotopic bone defects in several different animal models. Formulations based on bone morphogenetic protein-2 (BMP-2) and BMP-7 have been approved by the FDA for the treatment of severe orthopedic conditions such as spinal fusion and skeletal nonunion. Other growth factors and cytokines, such as transforming growth factor-β1, basic fibroblast growth factor and insulin-like growth factor-1 also have a certain role in promoting new bone formation. However, the doses of recombinant protein required to accelerate healing in humans are significantly higher than the levels expressed during normal bone repair, likely due to suboptimal delivery vehicles and rapid in vivo protein degradation. These supraphysiologicconcentrations are cost-prohibitive to widespread clinical usage and may be problematic if the non-selective targeting of neighboring non-osseous tissues leads to ectopic bone formation, In view of these complications, gene therapy based on over-expression of growth factor became a new strategy strategy for promoting osteogenesisGene therapy with BMP-2, BMP-7, and bBGF as target genes has been used in experimental animals and successfully promoted the new bone formation during distraction osteogenesis.Despite promising results, the clinical feasibility of these growth factor-based gene therapy approaches may be hampered by complex release kinetics and unregulated,ectopic bone formation caused by paracrine signaling to neighboring non-osseous tissues. In addition, the new bone formation during distraction osteogenesis is a very complex process,in which multi-factors and multi-cellular factors participate, while the high expression of several growth factors at the same time is difficult.Therefore, we assume to find a new method of gene therapy based on transcription factor regulation which is the hub and target of a large number of growth factors, cytokines, and power transmission to resolve the disadvantages of gene therapy based on bone growth factors.Many transcription factors are expressed during bone development and fracture healing including Runx2, Smads, Dlx-3,Dlx-5, MSX-2, AP-1, and Osterix. Among these,Runx2 and Osterix have been extensively characterized for their role in regulating the commitment of multipotent MSCs toward the osteoblastic lineage. Runx2 is an essential transcriptional regulator of chondrocyte hypertrophy, osteoblast differentiation, and bone formation. Runx2 can induce new bone formation in multipotential mesenchymal cells, this process might occur more efficiently and lead to bone matrix of better quality in vivo if Runx2 levels are high during the early differentiation stage and low during the osteoblast maturation stage. Osterix is a zinc-finger-containing transcription factor that acts downstream of Runx2 to induce the differentiation of osteoprogenitors into mature osteoblasts,which avoid the problem that Runx2 inhibits osteoblast differentiation of cells in the late stage.In addition, Osx gene therapy for distraction osteogenesis has not been reported to date.Therefore, in this study we select OSX gene for the target gene. Firstly the mandibular distraction osteogenesis model in rabbits was established and recombinant plasmid pEGFP-OSX was constructed in vitro. Then pEGFP-OSX was transiently transfected into rabbit bone marrow MSCs and OSX modified autologuous MSCs were thereafter introduced into rabbit mandibular distraction gap. The effect of OSX gene therapy on callus formation and mineralization were evaluated through radioghaphic,histological and histomorphometric examinations.The results of this study will provide valuable informations for OSX gene therapy to accelerate callus formation and shorten treatment period during distraction osteogenesis,and lay the foundation for eventual clinical application.【Methods】1.Creation of the mandibular distraction osteogenesis model in rabbits. Skeletally mature male New Zealand White rabbits were used in this study. The body weights ranged from 3.0 to 3.5 kg at the beginning of the experiment, without restriction on male and female. Inernal distractors were used which were composed of screw rod for traction, sliding rod, fixed arm, rotating handle as well as rotation joints linking to handle. The distraction distance is 0.4mm after each rotating circle. Anesthesia for all experimental procedures was achieved by intravenous injection of 3% pentobarbital (1 ml/kg), and a 2-cm incision was made along the inferior border of the right mandible. The periosteum and the masseter muscle were incised and carefully elevated. Under constant saline irrigation, the osteotomy was performed on the buccal (outer side) through the anterior part of the mandible (just anterior to the first molar) using a diamond disk. A custom-made distractor was placed and fixed to the mandible with 4 self-tapping screws After 6 days of latency, unilateral mandibular distraction was activated at a rate of 0.4 mm/12 hrs for 10 days, to produce a distraction gap of approximately 8 mm. After distraction was completed, the distractor was left in situ. Half of animals randomly chosen from each group were sacrificed with sodium pentothal injections at the end of week 2 and 6 throughout the consolidation stage respectively. The mandibular samples were harvested for Radiologic and histological examination2.Construction of recombinant plasmid pEGFP-OSX:The target gene was obtained through PCR amplification, purification, digestion, recovery and purification.Enhanced green fluorescent protein vector was Linearized after enzymy digestion, recovery and purification. Then the target gene was connected to the vector, and the new plasmid was identified by PCR amplification and DNA sequencing after competent cell preparation and transformation.3.Autologous bonemarrow stem cells (BMMSCs) were removed from the left tibia and isolated by density gradient centrifugation,and cells of passage 3 were used for the gene transfection in vitro and transplantation in vivo.4.Liposome-mediated in vitro transfection of plasmid pEGFP-OSX to bone marrowMSCs and gene therapy:The third generation of bone MSCs corresponding to each rabbit were obtained and after digestion and counting,passaged to 6-well plates. When MSCs were grown to 80% confluence, they were transfected with pEGFP-OSX using Lipofecta-mineTM2000. Expression of EGFP was observed under fluorescent microscopy to determine the transfection efficiency. Immediately after distraction, autologous bone marrow mesenchymal stem cells (MSCs) transfected with pEGFP-OSX, MSCs, and physiological saline were injected into the distraction gaps of the mandibles in groups A, B, and C,respectively.5. Osteogenic effect of detection:Half of animals from each group were euthanized at 2 and 6 weeks after completion of distraction. The distracted mandibles were removed and processed for radiographic, histological and immunohistochemical examinations. The density of distraction callus was then measured to represent its projectional bone mineral density (BMD).Sixteen serial sections of each specimen were made, ten for the HE staining and were observed in the light microscope t for morphometry analysis; six for the immunohistochemical detection of bone sialoprotein (BSP) expression.Image analysis software was used to analyze bone histomorphometry. Ten discrete sections were measured for each specimens, and the mean measurement results of the specimens were the parameter values.The bone volumes in the newly formed cortical bone area and in the cancellous bone area of the distraction zones were measured.6. Statistical analysis:All data are expressed as means±standard errors of the mean of 3 or more independent experiments.Statistical differences among groups were evaluated by one-way analysis of variance (ANOVA) and post hoc multiple comparison tests (Ducan’s multiple range test) were performed when significance was obtained. p<0.05 was considered statistically significant.【Results】1. The animals tolerated the surgical procedures and distraction well.Diet,feces and urine were normal,and mandibular deviation occurred at the end of the distraction. longer mandibles were found in distracted specimens. Radiological and histological study confirmed osteogenesis in gaps in animal models,and the mandibles were extended in accordance with the intended target.2. Through gene recombination technology, the recombinant plasmid pEGFP-OSX was successfully constructed and confirmed the correctness by sequencing analysis3. Bone marrow MSCs were successfully isolated with density gradient centrifugation method, Liposome-mediated transient transfection of pEGFP-OSX to bone marrow MSCs were observed under fluorescence microscope, with 40-45% transfection efficiency at 48 hours.4. Animals in group A showed greater bone formation and earlier mineralization in the distracted callus when compared with that in group B,and similarly increased callus formation was found in group B than group C under radiographic, histological and immunohistochemical examinations.Results of statistical analysis on bone mineral density and histomorphometry showed that group A was significantly better than the group B(P<0.05),group B was significantly better than the group C (P<0.05) in osteogenesis.5. BSP in the sections was detected using immunohistochemical staining using a BSP monoclonal antibody followed by counterstaining with hematoxylin. BSP expression is localized to osteoblasts and osteocytes within newly formed bone at 2 weeks after the end of distraction,and Strong BSP expression of was observed in group A, moderate in group B, and weak in group C.[Conclusion]1. Internal distractors were well designed to meet the requirements of unilateral extension of mandibles in rabbits.The osteogenesis in delayed phase of distraction was similar with fracture healing process, but the osteogenesis in distraction and retention period showed characteristic histological changes2. In this study, stable and highly repetitive rabbit models of unilateral mandibular distraction osteogenesis were successfully established which were suitable for high-volume animal experiments and would provide a good experimental platform for cell and gene therapy.3. MSCs obtained by density gradient centrifugation in vitro has a strong value-added activity, and may be the ideal seed cells for cell therapy and gene therapy. Observed under fluorescence microscope, liposome-mediated transient transfection can make pEGFP-OSX gene expression in MSCs successfully.4.pEGFP-OSX-modified MSCs can more effectively promote new bone formation and mineralization during mandibular distraction osteogenesis compared with MSCs alone under radiographic, histological and immunohistochemical examinations,suggesting that OSX gene therapy is an effective method to promote the mandibular distraction osteogenesis5.Immunohistochemical detection showed increased expression of BSP in cells and bone matrix of distraction gaps in group A,which suggest that the imported OSX gene were effectively expressed and OSX gene play an important role in promoting new bone formation and mineralization.6.Application of ex vivo gene therapy of OSX in autologous MSCs can effectively promote the callus formation and shorten the treatment cycle in distraction osteogenesis. The approach developed in this study may provide an alternative new method to facilitate clinical distraction osteogenesis treatment, especially for the patients whose osteogenic potentials are compromised.更多还原