下齿槽神经缺失大鼠下颌骨牵张成骨牵张期蛋白质组学分析

目的：探讨用蛋白质组学iTRAQ技术分析下齿槽神经缺失大鼠下颌骨牵张成骨牵张期新生组织蛋白表达的改变.方法：6只大鼠随机分为2组,实验组为下齿槽神经缺失大鼠下颌骨牵张成骨,对照组为正常大鼠下颌骨牵张成骨,均进行单侧下颌骨牵张,速率：0.2mm/12h,牵张期为10d,下颌骨牵张成骨牵张期第10d取材.将取材的新生骨组织标本进行理化性分析、蛋白质提取及蛋白质定量检测.应用iTRAQ技术对蛋白质样本进行检测,寻找及鉴定差异蛋白.结果：应用iTRAQ技术质谱鉴定出置信度95％的蛋白315种,共鉴定出差异蛋白146个,其中上调≥1.5倍的39个,下降≤0.8倍的58个.结论：感觉神经系统在牵张成骨的成骨过程中起到一定调控作用.筛选出多种下齿槽神经缺失下颌骨牵张成骨牵张期新骨形成相关的差异蛋白,为进一步验证感觉神经缺失对下颌骨牵张成骨新骨形成相关蛋白质奠定了基础.     

大鼠下颌骨牵张成骨模型的改进

目的：建立一个新的可行性和重复性俱佳的大鼠下颌骨牵张成骨模型?方法：对30只雄性大鼠从升支前缘中部至下颌下缘行全层骨切开，并安放特制的纯钛牵张器．用螺钉固定5d延迟期后．以0．2mm／次、2次／d的速度进行牵张，共8d；随后进入固定期：分别于固定期第2、4、8周分3批处死大鼠，进行大体标本观察和放射学、组织学检测。结果：实验过程被所有大鼠很好的耐受，切口感染率低(6．7％)．无牵张器脱落。大体标本观察表明，在牵张间隙形成了很好的骨痂组织．牵张间隙达到了预期的长度：新生骨组织放射学和组织学表现与大动物的表现相似。结论：我们成功建立了一个新的可行性和重复性俱佳的大鼠下颌骨牵张成骨模型；该模型有助于牵张成骨分子机制的进一步深入研究。     

下颌骨牵张成骨中牵张器拆除时机的实验研究

目的:探讨下颌骨牵张成骨中牵张器的最佳拆除时机。方法:12只山羊随机分为3组,每组各4只,在对动物右下颌骨行骨皮质切开术后进行牵张,第1组动物在牵张后固定期第6周处死,第2组动物在固定期第8周处死,第3组动物在固定期第10周处死,随机选取实验组4只动物的未手术侧下颌骨作为对照组,将各组新骨组织和对照组下颌骨组织分别进行压缩实验和三点弯曲实验,观察新骨生物力学强度随固定期时间延长的变化规律。结果:随着固定期时间的延长,新生骨抗压缩和抗弯曲实验各项力学指标值逐渐增大;固定期6周组和固定期8周组与对照组相比各指标有显著性差异(P<0.05),而10周组与对照组之间各个指标对应均数无显著性差异(P>0.05)。结论:牵张后固定期10周时新生骨的机械强度已接近于正常下颌骨骨组织,固定期10周可能是较为适宜的牵张器拆除时机。     

不同牵张速率对山羊下颌骨牵张成骨中新骨形成的影响

目的:探讨山羊下颌骨牵张成骨中不同牵张速率对术后新骨形成的影响。方法:12只山羊随机分为3组,每组各4只,在对动物右下颌骨行骨皮质切开术后进行牵张,第1组动物以0.8mm/d的牵张速率进行牵张,第2组动物以1.6mm/d的牵张速率进行牵张,第3组动物以2.0mm/d的牵张速率进行牵张,随机选取实验组4只动物未手术侧正常下颌骨作为对照组。将各组新骨组织和对照组下颌骨组织分别进行骨密度检测和三点弯曲测试,对采用不同速率进行骨牵张后动物下颌骨新骨的生物力学强度和骨密度进行了对比观察。结果:0.8mm/d牵张组新骨骨密度值显著高于其余各牵张组,0.8mm/d牵张组新生骨三点弯曲实验指标均大于另2牵张组。结论:采用0.8mm/d的牵张速率进行牵张能最快促进新骨形成,提高成骨质量。     

2型糖尿病大鼠下颌骨的蛋白质组学研究

目的:通过蛋白质组学研究,检测2型糖尿病大鼠下颌骨与正常大鼠下颌骨表达差异的蛋白。方法:选取8周龄Wistar大鼠和2型糖尿病大鼠Goto-Kakizaki(GK)大鼠,分别作为对照组和实验组。测量2组大鼠的体重和血糖,同期处死后切取下颌骨。提取各组下颌骨蛋白后,经双向凝胶电泳(2-DE)及MALDI-TOF/TOF质谱分析,鉴定筛选出2组大鼠下颌骨组织差异表达的蛋白。采用SPSS15.0软件包对数据进行统计学分析。结果:2组大鼠体重无显著差异,GK大鼠血糖显著高于Wistar大鼠(P<0.05)。经蛋白质组学研究,成功鉴定出20个2组间差异3倍以上的蛋白,其中5个蛋白点差异倍数在20倍以上。5个蛋白主要涉及代谢、蛋白结合以及信号转导3大功能。结论:2型糖尿病大鼠与正常大鼠下颌骨组织间存在的差异蛋白,有助于探讨2型糖尿病对下颌骨的影响机制。     

兔下颌骨牵张成骨中神经生长因子对骨痂钙化的作用

目的:观察局部注射神经生长因子(NGF)对下颌骨牵张成骨中新生骨痂钙化的作用。方法:对20只新西兰白兔实施牵张速率为1mm/d的双侧下颌骨牵张成骨。从牵张结束时开始,每只兔的一侧下颌骨新生骨区接受人NGFβ溶液注射(40μg/次,2次),另一侧注射生理盐水作为对照。在固定期第14和28d,新生骨痂经X线侧位片和外径测量后,进行骨沉积速度和钙化面积比定量分析。应用SPSS10.0软件包进行配对t检验,分析NGF处理侧与对照侧之间上述2个钙化指标的差异。结果:与对照侧比较,NGF处理侧的钙化面积比和固定期第1～11d内的骨沉积速度均显著提高(P<0.05),骨痂钙化得到了促进。结论:局部注射人NGFβ溶液能促进兔下颌骨牵张成骨中新生骨痂的钙化,为临床上解决固定期过长的问题提供了一种新的思路。     

失感觉神经支配大鼠下颌骨牵张成骨动物模型的建立

目的：建立一个新的可行性和重复性俱佳的失感觉神经支配大鼠下颌骨牵张成骨模型。方法：24只大鼠随机分为2组,实验组大鼠先自下颌孔至颏孔切除下齿槽神经后,从升支前缘至下颌骨下缘行全层骨切开,用螺钉固定特制的钛牵张器,对照组为保留下齿槽神经的大鼠下颌骨牵张成骨,5d延迟期后,均进行单侧下颌骨牵张,速率：0．2mm／12h,牵张期为lOd,随后进入固定期。分别于固定期第14d、28d处死大鼠,进行大体标本观察和组织学检测。结果：实验过程被所有24只大鼠很好的耐受,切口感染率低,无牵张器脱落。大体标本观察表明,在牵张间隙形成了很好的骨痂组织,牵张间隙达到了预期的长度。感觉神经缺失对牵张成骨具有负面调节作用。结论：成功建立了一个新的可行性和重复性俱佳的失感觉神经支配大鼠下颌骨牵张成骨模型,该模型有助于感觉神经对牵张成骨影响的分子机制的进一步深入研究。     

牵张成骨修复兔下颌骨缺损中血管生成素-1的表达及意义

目的探讨血管生成素(Ang)-1在牵张成骨修复兔下颌骨缺损中的时空表达及生物学意义。方法对24只大白兔行单侧下颌骨缺损牵张成骨术,分别在延迟期末、牵张中期、牵张期末、固定期第12、、35、、7周末各处死3只动物,取牵张区骨痂,采用组织学和免疫组化法观察微血管生成以及Ang-1的表达变化。结果下颌骨牵张区主要以膜内成骨方式成骨,在牵张区内有明显的血管生成及较明显的Ang-1的表达。在新生血管管周前成骨细胞和成骨细胞中可见Ang-1的表达。非应力区(缺损区)以软骨内成骨为主,在肥大的软骨细胞中存在Ang-1弱表达。结论牵张力所产生的机械刺激可以导致牵张区中微血管和骨组织的生成,而Ang-1可能在牵张区新生血管的形成及稳定和新骨形成中起重要作用。     

应用螺旋扩弓器牵引兔下颌中缝后新骨的OPG及RANKL的表达

目的:利用螺旋扩弓器进行下颌正中联合横向牵张成骨,扩展下颌间隙,并探讨在机械张力作用下新骨组织中骨保护素(Osteoprotegerin,OPG)、破骨细胞分化因子(Receptor or Activator of NF-KB Ligand,RANKL)表达规律.方法:建立兔下颌骨正中联合牵张成骨模型,用免疫组化方法检测牵张后1d、牵张后4d、固定后1w,固定后4w、固定后12w的牵张区新生骨组织中骨保护素(OPG)、破骨细胞分化因子(RANKL)不同时期的分布和表达.结果:下颌正中联合牵张成骨除一只外,其余实验动物均成功,牵张平均量为4mm,牵张成骨1d和牵张成骨4dOPG的阳性细胞数百分比逐渐增加,固定1w到12w组织中阳性细胞数百分比逐渐减少,RANKL阳性细胞数的百分比仅在牙缝关闭后与对照组有差异,其余各时间点均无明显差异.结论:下颌正中联合横向牵张成骨是扩展下颌间隙的一种行之有效的方法.机械牵张力能够促进OPG表达,OPG可能在牵张成骨的早期起一定的抑制破骨并促进调节新骨形成作用,RANKL则与骨改建有关,发挥一定的作用.     

山羊下颌骨三焦点牵张成骨的超微结构观察

目的：为探索三焦点牵张成骨的成骨方式及新骨改建过程提供实验依据。方法：取6只山羊下颌骨牵张成骨形成的新生骨组织及其邻近原骨组织,硬组织磨片,扫描电镜观察骨组织断面的超微结构,同时用硬组织磨片四环素荧光双标记技术分析。结果：牵张间隙超微结构观察显示大量新生骨小梁,骨质密度好,中央区新生类骨质沿牵张方向排列,新骨与原骨边界呈骨性融合,在牵张区内有大量的荧光结合,并与周围原有组织有明显的界限。结论：山羊下颌骨三焦点牵张成骨形成的新生骨段可逐渐改建为具有正常结构的骨组织。     

bFGF基因修饰的BMSCs促进大鼠下颌牵引成骨的研究

目的：探讨碱性成纤维生长因子（bFGF）基因修饰的自体骨髓间充质干细胞（BMSCs）促进大鼠下颌牵引成骨的作用。方法：36只雄性SD大鼠,随机分为实验组和对照组,同时对两组大鼠右下颌骨进行牵引成骨,牵引期最后1 d,实验组大鼠牵引间隙内注射转染重组质粒pcDNA-bFGF的BMSCs,对照组注射转染pcDNA空质粒的BMSCs。分别于固定期第2,4,8周分3批处死,并进行放射学检查、组织学检查及骨密度检测。结果：两组牵引间隙内均有新骨形成。各时间点实验组牵引间隙内新骨的形成和骨质密度均较对照组高（P〈0.05）。结论：bFGF基因修饰的BMSCs可有效促进DO新骨形成,为颌面部骨缺损的重建提供了一个新的修复方法。     

Rat mandibular distraction osteogenesis: latency, rate, and rhythm determine the adaptive response

Distraction osteogenesis is a well-established technique of endogenous tissue engineering. The biomechanical factors thought to affect the quality of the distraction regenerate include the latency, rate, rhythm, and consolidation period. In an effort to understand the impact of these parameters on regenerate bone formation, this study was designed to decipher the most adaptive response in a rat model of mandibular distraction osteogenesis. Ninety-six adult Sprague-Dawley rats were divided into 16 subgroups (n = 6 per subgroup) based on variations in the distraction parameters (i.e., latency, rate, and rhythm). After a 28-day consolidation period, the mandibles were harvested, decalcified, and sectioned. A standardized histologic ranking system was used to evaluate the effect of each protocol on the adaptive response of the regenerate bone. In this study, we have demonstrated that the latency period dramatically affects the success of distraction osteogenesis. Furthermore, distraction rates up to 0.50 mm per day stimulated excellent regenerate bone formation, whereas greater distraction rates produced a fibrous union. Finally, higher frequency distraction (i.e., increased rhythm) appeared to accelerate regenerate bone formation. We believe that defining the critical parameters of this model will improve future analysis of gene expression during rat mandibular distraction osteogenesis and may facilitate the development of biologically based strategies designed to enhance regenerate bone formation.     

rhBMP-2对兔下颌骨牵引成骨区骨保护素表达的影响

目的：通过动物实验,研究应用外源性rhBMP-2对兔下颌骨牵引成骨区骨保护素（osteoprotegerin,OPG）的影响。方法：在48只成年大耳白兔的一侧下颌骨前部行骨切开术,分别将空白胶原、rhBMP-2 1.5mg胶原复合物植入下颌骨切开处,用牵引器延长一侧下颌骨4mm,稳定期第1、3、7、14天,分别处死各组动物,取牵引区新生骨痂行组织学及OPG免疫组化染色。结果：下颌牵引延长后牵引间隙均有新骨形成,应用rhBMP-2 1.5mg效果好。免疫组化染色OPG主要定位于成骨细胞的胞浆中。在同一时间内,应用rhBMP-2组较对照组有显著性差异（P〈0.05）。结论：动物实验表明,rhBMP-2能促进兔下颌骨牵引成骨区新骨的生成。     

The molecular biology of distraction osteogenesis.

Distraction osteogenesis has become a mainstay in bone tissue engineering and has significantly improved our armamentarium for reconstructive craniomaxillofacial procedures. However, although the biomechanical, histological, and ultrastructural changes associated with distraction osteogenesis have been widely described, the molecular mechanisms governing the formation of new bone in the interfragmental gap of gradually distracted bone segments remain largely unclear. Recently, a rat model of mandibular distraction was described that provides an excellent environment for deciphering the molecular mechanisms that mediate distraction osteogenesis. This article presents the hypotheses and current research that have furthered knowledge of the molecular mechanisms that govern distraction osteogenesis.Recent studies have implicated a growing number of cytokines that are intimately involved in the regulation of bone synthesis and turnover. The gene regulation of numerous cytokines (transforming growth factor-beta1, -beta2, -beta3, bone morphogenetic proteins, insulin-like growth factor-1, fibroblast growth factor-2) and extracellular matrix proteins (osteonectin, osteopontin) during distraction osteogenesis have been best characterized and are discussed in this article. It is believed that understanding the biomolecular mechanisms that mediate membranous distraction osteogenesis may guide the development of targeted strategies designed to improve distraction osteogenesis and accelerate bone healing.     

Expression of vascular endothelial growth factor and its receptors after mandibular distraction osteogenesis

During distraction osteogenesis, angiogenic activity is essential for new bone formation. This study examined the expression of vascular endothelial growth factor (VEGF) and two of its receptors, Flt-1 (VEGFR-1) and Flk-1 (VEGFR-2), in cellular components after mandibular distraction osteogenesis. Unilateral mandibular distraction (0.5 mm twice per day for 10 days) was performed in six mongrel dogs. Two animals each were killed on days 7, 14 and 28 after completion of distraction. The distracted mandibular segments and contralateral undistracted control segments were harvested and processed for immunohistochemical examination. Seven days after distraction, there was a significant increase in the expression levels of VEGF and its receptors in the osteoblasts, osteocytes and immature fibroblast-like cells compared to control specimens. These levels were maintained for 14 days after distraction in the osteoblasts and fibroblast-like cells. Twenty-eight days after distraction, VEGF and VEGFR-1 were expressed only moderately/weakly in the osteoblasts, and no VEGFR-2 expression was detected in the cellular component of the distracted bone. Throughout the observation period, VEGFR-1 expression was stronger than that of VEGFR-2. The expression patterns of VEGF and its receptors suggest that it plays an important role in osteogenesis, and that osteoblasts and immature fibroblast-like cells of the distracted bone may have an autocrine growth effect during distraction osteogenesis.     

The BMP signaling and its Smads in mandibular distraction osteogenesis

Aim:  The aim of the present study was to clarify the mechanism of signal transduction of bone morphogenetic proteins (BMPs) through their specific down signaling molecules Smads inducing bone formation in response to mechanical stimulus during distraction osteogenesis (DO) in the rat mandible.
Materials and methods:  Osteotomy of the left mandible was performed in 45 rats. Thirty rats underwent mandibular distraction (protocol; 5 days latency, 8 days distraction, and 2 weeks consolidation) while 15 rats served as non-distracted (fracture healing) group. The expression of BMPs-2,-4 and Smads 1, 5, and 8 were evaluated in the new regenerate area using immunohistochemistry.
Results:  Expressions of BMPs-2,-4 and Smads 1, 5, and 8 were moderate during latency, significantly increased during distraction and decreased towards consolidation period.
Conclusions:  The enhanced expression of BMPs and its Smads during distraction compared to the non-distracted group suggests the possible role of BMP signaling pathway in translation of mechanical forces into biological results during DO.     

内置式牵张成骨术在矫治火器伤性下颌骨畸形中的应用

目的:探讨内置式牵张成骨术矫治火器伤性下颌骨畸形临床应用的可行性。方法:为3例火器伤性下颌骨畸形患者延期(伤后2～3个月)行内置式牵张成骨术。术中常规截骨,制作骨转移盘,固定内置式骨牵张器,术后1周以每日1 mm的速度行骨牵引(8～22 mm)。固定期拍曲面断层片观察新骨生成及畸形矫治状况。结果:治疗按计划完成,无感染等并发症,骨牵引顺利完成;固定期3个月时,新生骨骨质、骨量良好,下颌骨畸形矫治效果满意,咬合关系良好。结论:在早期合理清创基础上,延期牵张成骨术可有效地矫治火器伤性下颌骨畸形,值得推广。