摘要
目的：为了加速四肢长骨牵张成骨(DO)过程中新骨的形成、矿化及塑型改建,将具有特异性促进成骨分化及矿化功能的NELL1因子应用于大鼠股骨牵张成骨模型,通过创新性的外固定架设计及动态Micro-CT观察,研究NELL1因子对股骨牵张成骨的影响,从而为临床上治疗四肢大段骨缺损寻找一种快速有效的治疗方法。方法：构建同时携有绿色荧光蛋白(GFP)报告基因和NELL1基因的重组腺病毒载体Ad-GFP-NELL1,经扩增、纯化后转染大鼠骨髓基质细胞(BMSCs)观察其体外对BMSCs的影响。进一步应用自行研制的大鼠股骨DO外固定架系统将30只SD大鼠随机分为3组,每组于术后14天分别于骨延长局部注射等量Ad-GFP-NELL1、Ad-GFP、生理盐水,术后21、28、42、56天麻醉后行X线及活体Micro-CT检查,并于术后28天,各组取1只采用小动物活体荧光成像系统检测NELL1及GFP表达情况。其余动物于术后56天取材行行生物力学、组织学及免疫组化检测。结果：①成功构建出Ad-GFP-NELL1重组腺病毒,纯化后获得滴度高达1×1011pfu／ml。②用构建好的病毒转染鉴定过的大鼠BMSCs后,NELL1蛋白及GFP均阳性表达,转染后对细胞的生长无明显影响。③Micro-CT动态观察大鼠股骨DO模型显示：在静息期及延长期,延长区无明显新骨形成,延长区内主要为由纤维样组织组成的假性生长板结构。在巩固期前期(术后21～42天)主要为骨量的增加,表现为骨矿含量增加,骨小梁数量增多；巩固后期(术后42～56天),骨量仍持续增加,同时骨的质量也开始明显提高,表现为骨小梁增厚,已矿化组织矿化度提高。在术后56天,延长区内仍处于成骨和骨结构的改建中,假性生长板基本消失,未骨化的软骨组织依然存在。术后56天,DO侧股骨的最大扭矩及失效能量均小于健侧股骨。④Ad-GFP-NELL1局部应用于大鼠DO模型,注射后14天可见延长区NELL1蛋白表达。X线片显示,术后56天,Ad-GFP-NELL1组9只延长区均达到骨性愈合,对照Ad-GFP、生理盐水组分别为4只、5只达到骨性愈合。生物力学测试Ad-GFP-NELL1组明显优于两对照组,与正常股骨的抗扭强度相当。Micro-CT数据显示：巩固期前期3组数据无明显差异；巩固后期Ad-GFP-NELL1组骨痂增速减缓,骨体积分数(BVF)增速由前3周每周10%下降到6%。而两对照组增加到13～14%。到术后56天,Ad-GFP-NELL1组骨体积分数(BVF)、矿化组织密度(BMD)均低于两对照组。Micro-CT三维重建显示：到巩固期后期,Ad-GFP-NELL1组生成的骨痂以皮质骨为主,松质骨较少,骨髓腔贯通,形成了类似长骨管状骨的结构,两对照组形成了较多的骨痂,以松质骨为主,改建较差。组织学切片显示：两对照组延长区中央于术后56天仍有软骨组织结构,而Ad-GFP-NELL1组未见明显软骨组织存留,均为骨痂结构。免疫组化结果显示：3组RUNX2、BMP2、BMP7表达无明显差异,Ad-GFP-NELL1组骨钙素及骨桥蛋白表达高于两对照组。结论：①构建并纯化后的重组腺病毒载体Ad-GFP-NELL1可高效转染大鼠BMSCs,并稳定表达NELL1和GFP两种蛋白；②在大鼠股骨DO延长结束后应用可透X线的高分子材料固定牵引针可以获得稳定的固定并便于采用Micro-CT活体动态观察及评价DO过程中新骨的形成；③长骨牵张成骨中骨量的增加自延长结束后开始,持续5周以上,骨质量的改建在延长结束后2-3周才开始,二者的发生具有时相性,提示相应的干预手段应在不同的时期进行；④NELL1具有促进股骨DO过程中皮质骨生成的作用。在牵张成骨的环境下,NELL1与已应用于临床的BMPs相比,并不是单纯的促进局部新骨形成的量,而是促进缺损局部新骨的形成及骨痂的矿化、改建,形成较少但结构及生物力学强度更佳类似于长骨管状骨结构的骨痂结构,从而加速牵张成骨的愈合。这一结果改变了过去认为NELL1只在颅骨、下颌骨等神经脊来源骨组织中具有促进成骨作用的结论。提示我们,NELL1因子应用于临床可以缩短四肢牵张成骨术后患者携带外固定架的时间,有望使牵张成骨这种治疗大段骨缺损的方法得到更广泛的应用,具有一定的实用价值。
Objective:NELL1 is a cranisynostosis-associated molecule directly regulated by Runx2, the master molecule in controlling osteoblastic differentiation. NELL1 has exhibited potent osteoinductive activity for bone regeneration in several animal models. However, its capacity for promoting repair of long-bone defects remains unknown, In this study, we investigated the osteogenic effects of NELL1 on femoral distraction osteogenesis using adenoviral gene delivery and multiple approaches of in vivo analysis. Materials and Methods:We subcloned a 2.3-kb full-length human NELL1 cDNA and inserted into the pAdxsi vector to package the recombinant adenovirus vector carrying NELL1 and GFP (Ad-GFP-NELL1). Rat bone-marrow-derived stromal cells (BMSCs) were infected with various doses of adenovirus to establish the optimal multiplicity of infection (MOI). NELL1 expression was verified by RT-PCR, and the co-expression of NELL1 with GFP was confirmed by fluoreScent immunocytochemistry. Femur distraction osteogenesis model were made on 30 Sprague-Dawley (SD) rats. After 7 days latency, the femurs were distracted at a speed of 0.25mm every 12 hours for 14 days. At the mid-distraction period (Day 14), each animal received a single dose of 0.2ml injection of either adenovirus Ad-GFP-NELL1 diluted in physiological saline, adenovirus Ad-GFP diluted in physiological saline, or physiological saline into the distraction site. After the distraction completed (Day 21), the steel external fixators were substituted by radio-transparent polymer splint material made fixators, and maintained for another 5 weeks for the distracted zone to consolidate. Healing was assessed with serial radiographs and micro-computed tomography (Micro-CT) of the living animals on Days 21,28,42,56. Animals were killed on Day 56 for biomechanical, histological, and histochemical analysis. Results: Recombinant adenoviral vector Ad-GFP-NELL1, which encodes a fusion protein of human NELL 1, was successfully constructed and amplified with titer of 1×1011 pfu/ml. An MOI of 200 pfu/cell produced optimal effects in transfer efficiency without excessive cell death in vitro. Exogenous NELL1 was expressed in the distracted gap for at least 14 days after Ad-GFP-NELL1 transfection. The bone union rate in the distracted gap was significantly higher with Ad-GFP-NELL1 than with Ad-GFP (9/9 vs. 4/9 rats) or saline alone (5/9 rats) at day 56. The serial 3-D micro-CT images and quantitation obtained with the development and application of radiolucent external fixators showed less callus but more mature cortical bones formed with Ad-GFP-NELL1 than with Ad-GFP transfection and saline administration during distraction osteogenesis. The biomechanical properties of femur samples with Ad-GFP-NELL1 transfection and unoperated femurs were similar. Histology revealed cartilaginous tissues in the middle of distraction gaps with Ad-GFP transfection and saline treatment but only bony bridges with Ad-GFP-NELL1 transfection at the final time point (day 56). Coincidently, the expression of Runx2, BMP2, and BMP7 did not differ among groups at day 56, whereas the expression of osteocalcin and osteopontin was slightly higher with Ad-GFP-NELL1 transfection. Conclusions:Recombinant adenovirus vector Ad-GFP-NELL1 can steady expressing both GFP and NELL1 protein after infected into rat BMSCs. It provided us a useful tool for real time trace the expression of NELL1 and investigate its'function in vitro and in vivo. We developed a method that could serially acquire Micro-CT data during rat femur distraction osteogenesis. By applying this method, we showed the increasing of bone volume began at the end of distraction, lasting at least 5 weeks, while the remodeling of new formed bone began 3 weeks after distraction ended. The increasing and remodeling of new formed bone was not concord. Sustained Ad-NELL1 protein delivery into a local area of a rat femoral distraction osteogenesis model remarkably improved regeneration of good-quality bones and accelerated bone union at high rate. Acquiring serial micro-CT data during rat femur distraction osteogenesis and regional adeno virus delivery of NELL1 may facilitate future in vivo studies.
引文
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