Co-expression of nerve growth factor and p75NGFR in the inferior alveolar nerve after mandibular distraction osteogenesis

During mandibular distraction osteogenesis (DO), the inferior alveolar nerve (IAN) is damaged during distractor activation, but spontaneously recovers during consolidation. Although many neurotrophic factors are known to play critical roles, there have been few studies on the mechanism of peripheral nerve recovery after DO. The aim of this study was to observe the expression pattern of p75NGFR (low-affinity receptor of NGF) and to detect autocrine growth activity in IANs following mandibular DO. Unilateral mandibular distractions (0.5mm each, twice per day for 10 days) were conducted on eight mongrel dogs. Two each were killed at 7, 14, 28 and 56 days after completing distraction. The distracted IAN and contralateral control nerve were harvested. Immunohistochemical staining was performed to determine p75NGFR expression, and double immunofluorescent staining to detect NGF and p75NGFR co-expression. Levels of p75NGFR expression were found to be significantly elevated at 7 and 14 days in Schwann cells located in the outer layer of axon, but were almost undetectable at 28 and 56 days. In double immunofluorescent images, the co-expression of NGF and p75NGFR was also detected at 7 and 14 days. p75NGFR plays an important role in remyelination due to its abundant expression in Schwann cells of damaged nerves, and NGF is an autocrine growth factor present in distracted IANs during the early consolidation period after mandibular DO.     

The role of nerve growth factor and brain-derived neurotrophic factor in inferior alveolar nerve regeneration in distraction osteogenesis

Distraction osteogenesis (DO) has become the mainstay of treatment of mandibular hypoplasias. Despite the clinical acceptance of the technique in the last decade, little is known of the biological mechanism of bone and soft tissue regeneration. The biological response of peripheral nerves to distraction has not been well documented. This study examined the role of two neurotrophic molecules, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), in DO on nerve regeneration of the inferior alveolar nerve (IAN) in an ovine mandible model. Twelve animals were randomly divided into three groups and distracted at 5, 10, and 15 days using a mandibular osteotomy and uniaxial external distractor. The mental nerves and the IAN from the distracted site were harvested at the end of the distraction period and examined for the presence of NGF and BDNF using immunohistochemistry. Nerve growth factor expression was increased at both sites, whereas BDNF was only expressed at the mental nerve on the distracted side. Nerve growth factor and BDNF are involved in the response of the peripheral nerves to injury. Mechanical force applied to the IAN by distraction may lead to detachment of Schwann cells from their axons, leading to segmental degeneration. The resulting myelin sheath debris may serve as a trigger for higher expression of NGF and BDNF, facilitating Schwann cell proliferation and remyelination of the affected segment. Distraction of the mandible may serve as a source of subacute injury to the IAN and influence NGF and BDNF.     

Response of Schwann cells in the inferior alveolar nerve to distraction osteogenesis: an ultrastructural and immunohistochemical study

The biological mechanisms of nerve adaptation to distraction osteogenesis have not yet been elucidated. This study observed response of Schwann cells in the inferior alveolar nerve (IAN) following mandibular lengthening by electron microscopy and immunohistochemistry of S-100 protein, a specific marker of Schwann cells. Unilateral mandibular distraction (10mm elongation) was performed in nine young adult goats. Three animals were sacrificed at 7, 14 and 28 days after completion of distraction, respectively. The distracted IAN specimens and control nerves (from the contralateral sides) were harvested and processed for histological, ultrastructural and immunohistochemical examinations. Wallerian degeneration was observed in the distracted IAN, and Signs of axonal regeneration, as well as many activated Schwann cells were seen in the lengthened nerves. The expression of S-100 protein increased significantly at early stage of distraction osteogenesis, but almost returned to the normal level at 28 days after distraction. This study suggests that Wallerian degeneration caused by mechanical stretching may stimulate Schwann cells to enter a proliferated and activated state. Schwann cells and S-100 protein appear to play crucial roles in axonal regeneration that contributes to nerve adaptation to gradual distraction. Therefore, the IAN injury caused by mandibular gradual distraction was not serious; it seems to recover totally through a complicated repair mechanism.     

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.     

不同速率牵张下颌后下齿槽神经的组织学和超微结构改变

目的 研究不同速率牵张延长下颌骨后下齿槽神经的组织学和超微结构改变,为临床上确立合理而安全的牵张速率提供实验依据。方法 ８只山羊随机分为Ａ、Ｂ、Ｃ三组,Ａ、Ｂ组各３只,Ａ组１ｍｍ／ｄ,Ｂ组以２ｍｍ／ｄ牵张,Ｃ组２只动物为对照。牵张延长下颌骨１０ｍｍ,固定２ｗ处死。取下齿槽神经行组织学,透射电镜观察。结果 牵张动物的下齿槽神经均发生了Ｗａｌｌｅｒ变性,以２ ｍｍ／ｄ牵张组神经退行性病变严重而广泛。超微结构病变主要发生于粗大的有髓神经纤维,而细小的有髓神经及无髓神经纤维未见异常。结论 ２ｍｍ／ｄ牵张会对下齿槽神经造成严重损伤,而１ｍｍ／ｄ牵张速率为较适宜而安全的下颌牵张速率。     

Temporospatial expression of vascular endothelial growth factor and basic fibroblast growth factor during mandibular distraction osteogenesis.

OBJECTIVE: Distraction osteogenesis is a vascular-dependent process. This study investigated expression patterns of two major angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), in the distracted calluses following mandibular lengthening in a goat model. MATERIAL AND METHODS: Bilateral mandibular osteotomies were performed in 15 young adult goats. After a latency of 7 days, the mandibles were elongated using custom-made distractors with a rate of 1 mm/day for 10 days. Three animals each were sacrificed at the end of the delay phase, at 0, 7, 14, and 28 days after completion of distraction, respectively. The lengthened mandibles were harvested and processed for histological and immunohistochemical examinations. RESULTS: Elevated cellular expression of VEGF and bFGF, with neovascularization in the distraction gap, was observed following mandibular lengthening. VEGF staining was noted in the endothelial cells and osteoblasts. bFGF staining was seen in the fibroblast-like cells, osteoblasts and immature osteocytes. Their strongest expression was found 0-7 days after the end of distraction, and declined with maturation of the newly formed bone. CONCLUSION: A temporal and spatial expression pattern of VEGF and bFGF was found during distraction osteogenesis in goat mandibles. It suggests that distraction forces can stimulate the production of VEGF and bFGF, which contribute to neovascularization and new bone formation during gradual distraction of the mandible. Application of angiogenic factors may be considered as a potential method to enhance angiogenesis and osteogenesis in osteodistraction, especially in sites without enough vascularization.     

下颌骨牵张成骨对下牙槽神经影响的实验研究

目的　观察狗双侧下颌骨牵张成骨术中不同时间点的下牙槽神经的电生理功能及组织学结构的变化。方法　实验组 :12只狗行保留下牙槽神经的双侧下颌骨截断术 ,装上自制的口内牵张器 ,于术后第 8天开始牵张 ,1mm/d ,共 10d ,牵张长度约 10mm。分别在牵张中第 6天、牵张完毕后固定 2周及 8周时各处死 4只动物。分别在术前及术后的不同时期用电生理方法检测双侧下牙槽神经的传导功能 ,并于处死前取出双侧下牙槽神经进行组织病理学及超微结构组织学检查。对照组 :4只狗 ,其中 2只狗作为对照组 ,未行手术 ;另 2只狗为手术对照组 ,进行与实验组相同的骨切开截断术 ,上牵张器 ,但未行牵张和固定 ,于操作完毕时取出双侧下牙槽神经作为术后的组织学结构标本。结果　实验组动物的下牙槽神经在光镜下显示部分神经纤维稍肿胀变粗 ;电镜下显示部分神经纤维髓鞘的板层松解及脱髓鞘现象 ;电生理结果显示 ,神经传导的速度于截断术后的不同时期均低于术前 ,但仅术后第 8天 (即牵张前 )的传导速度与术前的传导速度差异有显著性 (P <0 .0 1)。结论　对狗的双侧下颌骨进行牵张成骨时 ,可一过性引起下牙槽神经损伤。其损伤的主要原因是手术过程所致。在适当的牵张速率、牵张长度和牢固的固定条件下 ,下颌骨的牵张成骨术对下牙槽 还原。     

Nerve growth factor injected systemically improves the recovery of the inferior alveolar nerve in a rabbit model of mandibular distraction osteogenesis

Our aim was to find out if nerve growth factor (NGF) injected systemically could improve the recovery of the inferior alveolar nerve in a rabbit model of mandibular distraction osteogenesis. We used 48 New Zealand white rabbits that were treated with bilateral distraction osteogenesis at a rate of 0.5 mm/12 h for 10 days. Immediately postoperatively, NGF or sodium chloride 0.6 μg/day was injected intramuscularly for 20 days. At the end of distraction and after consolidation times of 1, 2, and 4 weeks, the inferior alveolar nerves were evaluated histologically and histomorphometrically. Histologically, at 2 and 4 weeks there was less myelin debris, and more regenerating axons were present, in the NGF than the control groups. The density of myelinated axons was significantly greater in groups with NGF than controls at 2 and 4 weeks (p < 0.05). NGF given systemically can accelerate the recovery of the inferior alveolar nerve in rabbits after mandibular distraction osteogenesis, and is a promising treatment option for neurological complications of mandibular distraction osteogenesis.     

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。     

HIF—1α在犬下颌骨持续牵张成骨中的表达及与局部血管形成的关系

目的初步探讨缺氧诱导因子-1α（HIF-1α）在犬下颌骨镍钛记忆合金持续牵张成骨中的表达．及HIF--1α在持续牵张成骨中的作用及与局部血管生成的关系。方法成年Beagle犬12只．实验组10只．双侧下颌骨制造1．5cm×1．0cm矩形缺损并在其近中形成相同大小的骨传松盘（采用保留骨松质的骨皮质切开术）．行镍钛记忆合金牵张器持续牵张术，于术后第1、4、7、10、15天取材．每组2只：对照组2只，下颌骨相同部位行相同大小的骨皮质切开术后未行骨牵张术，术后10d取材。RT-PCR法检测HIF-1α mRNA的表达．免疫组化SP法检测HIF—1α、VEGF的蛋白表达，CD34标记内皮细胞检测新生微血管密度（MVD），采用SPSS11．5统计软件分析。结果RT—PCR及免疫组化显示实验组HIF—1α mRNA和HIF-1α蛋白在术后第4、7、10、15天呈阳性表达，两者主要在牵张区的成骨细胞内表达．皆在第7d达最大值。免疫组化显示VEGF在术后第4、7、10、15天呈阳性表达．VEGF在牵张区内皮细胞和成骨细胞内表达，从术后第4天至第10天不断增加，术后第10天达最大值．新生微血管密度在牵张期逐渐升高、术后第7天达峰值（P〈0．05）。对照组HIF—1α mRNA及蛋白和VEGF皆未见明显表达。结论HIF—1α在犬下颌骨持续牵张成骨中高效表达，牵张末期是其发挥作用的关键时期．可能通过上调VEGF的表达，促进牵张成骨局部的血管生成过程。     

Effects of locally applied nerve growth factor to the inferior alveolar nerve histology in a rabbit model of mandibular distraction osteogenesis

Distraction osteogenesis (DO) is widely used in deformities and defects of the craniofacial bone. Accelerating inferior alveolar nerve (IAN) recovery would aid the process. Nerve growth factor (NGF) plays a vital role in peripheral nerve regeneration. In this study, the ability of locally applied human NGF beta (hNGFβ) to enhance the morphological recovery of the IAN in a rabbit model of mandibular DO was studied. Rabbits underwent bilateral DO with a rate of 0.5 mm per 12 h. Two doses of 40 μg hNGFβ in buffer were injected into callus at the beginning the of consolidation time. The contralateral side received injections of placebo. Rabbits were killed at 14 and 28 days. IAN specimens were subjected to histological and histomorphometric analysis. In both 14 and 28 days consolidation experiments, nerve histological analysis showed less degeneration and more regeneration in nerve fibers on the hNGFβ treated side than the control side. Histomorphometric analysis showed that the myelinated fiber density on the hNGFβ treated side was significantly higher than on the control side (p < 0.01). The data indicate that locally applied hNGFβ can accelerate the morphological recovery of the IAN and may play a role in reducing nerve injury in mandibular DO clinically.     

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。     

下颌骨牵引成骨术对下牙槽神经功能影响的实验研究

目的　研究下颌骨牵引成骨术对下牙槽神经功能的影响。方法　对青年恒河猴 16只 ,全麻下行下颌角部骨截开术及牵引器置入术 ,术后第 5天开始以 0 5mm× 2次 /d的速度牵引 ,共15d。采用八导肌电图仪 ,表面电极在颏孔处刺激 ,针电极在卵圆孔附近记录 ,分别在术前、牵引完成后 0、2、4、6、9、12周时行感觉神经动作电位测试。结果　牵引完成时潜伏期较术前增加 2 2 18% ,其后逐渐降低 ,至牵引完成后 12周时仍较术前高 10 70 %。牵引完成时波幅降至术前的 2 8 5 4% ,牵引后 12周时恢复至术前的 99 84%。结论　下颌骨牵引成骨术对下牙槽神经的功能会产生暂时性影响 ,随着神经髓鞘和轴突的再生 ,神经功能可逐渐恢复正常。     

转化生长因子β_1在下颌牵张成骨过程中的表达及意义

目的:研究转化生长因子B1(TGF-B1)在下颌牵张成骨过程中的表达及意义。方法:对15只山羊行双下颌骨皮质切开术,安置口外牵张器,经7 d间歇期后,按1 mmPd的速率延长下颌骨10 mm。在间歇期末、牵张结束当天及牵张结束后第7、14和28天分别处死3只动物,取牵张区骨痂用免疫组化方法检测不同时间内TGF-B1表达水平的变化。结果:TGF-B1在下颌牵张成骨过程中呈高水平表达,主要定位于间充质细胞和成骨细胞的胞浆中,其中以牵张结束当天和第7天的表达最强,以后逐渐下降,第28天时仅有微弱表达。结论:TGF-B1可能在牵张成骨过程中, 特别是调控组织细胞应力应变信号传递中扮演十分重要的角色。     

神经生长因子在大鼠牙髓炎症中的作用的实验研究

目的 探讨神经生长因子（NGF）在牙髓炎症中的作用。方法 建立大鼠磨牙内毒素炎症模型后,进行NGF的免疫组化染色。结果 经开髓和内毒素处理后12h－3d,冠髓内牙髓细胞表达NGF明显增加,5d反恢复正常。结论 NGF参与牙髓炎症过程,可能与牙髓炎的激发痛症状相关。     

下颌骨牵引成骨过程中VEGF及其受体的表达

目的:通过建立兔下颌骨牵引成骨实验动物模型,研究下颌骨牵引成骨过程中血管内皮生长因子(vascular endothelial growth factor,VEGF)及其受体的表达及意义。方法:选用新西兰白兔30只,随机选取6只动物作空白对照组,24只动物右侧下颌骨植入外置式颌骨牵引器。经7d延迟期后,按1mm/d速率延长下颌骨7d,固定期8周。在延迟期第7d,牵引期第2、4、7d,固定期第1、3、6、8周分别处死3只动物,采用免疫组化方法,检测VEGF及其受体FLT-1(fms-like tyrosine kinase-1)、FLK-1(fetal liver kinese-1)的表达变化。结果:VEGF及其受体FLT-1、FLK-1的表达贯穿下颌骨牵引成骨全过程。下颌骨牵引成骨过程中不同时相多种组织细胞参与表达VEGF及其受体FLT-1、FLK-1,但在表达的变化上存在一定的差异。结论:VEGF通过与其受体特异性结合,在下颌骨牵引成骨血运重建及新骨生成过程中发挥重要作用。     

大鼠舌下神经压榨伤后p38MAPK的活化及外源性神经生长因子促神经再生作用的研究

目的检测舌下神经压榨损伤前后及神经生长因子（NGF）干预后磷酸化p38MAPK的表达,探讨p38MAPK在大鼠舌下神经损伤及干预后的作用和NGF对大鼠舌下神经损伤后神经修复再生的作用。方法60只SD大鼠随机分为正常对照组（NC组）、实验对照组（NS组）和NGF治疗组（NGF组）,动物存活时间分别为1、3、5、7和14 d。分别于各时间点取脑干做免疫组化测定及Nissl染色,取神经干做透射电镜观察。结果舌下神经压榨损伤后,舌下神经核内磷酸化p38MAPK免疫阳性神经元数目和染色深度均增加（P<0.05）。NGF干预后舌下神经核内磷酸化p38MAPK免疫阳性神经元数目和染色深度均明显减少（P<0.05）。Nissl染色显示,术后7、14 d NGF组损伤侧舌下神经核内运动神经元存活率明显高于NS组。透射电镜观察NGF组神经干形态优于NS组。结论大鼠舌下神经压榨损伤后受损神经元p38MAPK的活性增强;外源性NGF能抑制大鼠舌下神经压榨损伤引起的舌下神经核运动神经元p38MAPK的激活;大鼠舌下神经压榨损伤后外源性NGF具有保护受损的舌下神经元及促进神经再生的作用。     

牵张成骨延长山羊下颌骨后下齿槽神经的组织学变化

目的 :研究下颌骨牵张成骨术后不同时间内下齿槽神经的组织学改变。方法 :对 8只成年山羊行双侧下颌体骨皮质切开术 ,经口外安置自行研制的下颌牵张器 ,以每天 1mm的速率向前牵引延长其中 6只山羊的下颌骨10mm。于牵张结束后第 2、4、8周各处死 2只动物 ,取双侧下齿槽神经作组织学检查 ,另 2只未牵张的山羊作对照。结果 :下齿槽神经受牵张力作用发生了一定程度的沃勒变性 ,主要表现为髓鞘肿胀、碎裂及轴索数目减少。但随着固定时间的延长 ,受损神经纤维逐渐得以再生。结论 :下颌骨牵张成骨术后下齿槽神经发生了轻度的退行性变 ,但这种退行性变在适宜的速率牵张下是可逆的     

兔下颌牵张后血管内皮生长因子在新骨组织中的定位表达

目的 研究血管内皮生长因子（VEGF)在兔下颌牵张成骨过程中的定位表达。方法 对12只大耳白兔行双侧下颌骨牵张成骨术，在牵张结束后当天及7、14和28d分别处死3只动物，取牵张区骨痂。采用组织学和免疫组化方法观察微血管生成和VEGF的表达变化。结果 下颌骨延长后牵张间隙内有强烈的血管生成反应及高水平的VEGF，表达。VEGF阳性信号主要定位于血管内皮细胞和增殖活跃的成骨细胞。结论 牵张力刺激可以导致牵张间隙中强烈的血管生成反应，VEGF可能在牵张后骨再生的血管生成和新骨形成过程中起非常重要的调控作用。     

Application of nerve growth factor by gel increases formation of bone in mandibular distraction osteogenesis in rabbits

The long period of bony consolidation is a concern in mandibular distraction osteogenesis (DO). We have previously shown that repeated local injections of human nerve growth factor beta (NGFβ) can appreciably improve bony consolidation in a rabbit model of DO. The present study was designed to test the effect of a single injection of human NGFβ delivered by collagen/nano-hydroxyapatite/kappa-carrageenan gels to sites of new bony formation in DO. Rabbits underwent mandibular DO at a rate of 0.75 mm/12 h for 6 days. At the end of the distraction period, the following injections were given percutaneously into the callus (n = 6 in each of the four groups): human NGFβ in the gel; human NGFβ in saline; the gels alone; and saline alone. Fourteen days after the end of distraction, mechanical testing, histological and histomorphometric variables of the new bone were evaluated. Histologically, the NGFβ group had more advanced consolidation than the other three groups. Both maximal load and bone volume/total volume in this group were significantly higher than in the other three (P < 0.05). In conclusion, the delivery of human NGFβ in the gels results in better acceleration of new bone formation than when it is given in saline, and may be a possible way to shorten the duration of craniofacial DO.