Peripheral nerve injury and regeneration: application and progress of novel nerve scaffolds

doi:10.3969/j.issn.2095-4344.2017.06.025

Abstract

Abstract:

BACKGROUND: Autologous nerve grafts are the gold standard for large segment peripheral nerve injury, but in view of its limitations, scientists are researching for novel nerve guidance conduits.
OBJECTIVE: To overview the new view in peripheral nerve injury, key cells associated with axonal regeneration, and application of nerve guidance conduits.
METHODS: The first author retrieved PubMed database for relevant articles about tissue-engineered nerve scaffold published from 2001 to 2016 using the English keywords of “peripheral nerve injury, peripheral nerve regeneration，nerve scaffold”. Finally, 76 articles were enrolled based on the inclusion and exclusion criteria.
RESULTS AND CONCLUSION: Peripheral nerve repair mainly focuses on how to make the regenerative nerve grows “fast” and “accurate”. In the future, we should focus on not only developing better biocompatible materials, but also optimizing the design to simulate the nerve regeneration microenvironment at molecular, cell and tissue levels. The nerve scaffolds used for nerve regeneration not only exert a supporting mechanical effect, but also act as conduct electricity and sustained release of cytokine gradient. Bionics design should be further expanded from the morphology to the spatial changes of cytokines and bioelectric stimulation.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Peripheral Nerves, Nerve Regeneration, Tissue Engineering

CLC Number:

R318

Quan Qi, Chang Biao, Liu Ruo-xi, Sun Xun, Wang Yu, Lu Shi-bi, Peng Jiang, Zhao Qing.

Peripheral nerve injury and regeneration: application and progress of novel nerve scaffolds

[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(6): 962-968.

Figures/Tables 1

2.1 周围神经损伤与再生 2.1.1 瓦勒氏变性周围神经损伤，通常是指轴突损伤，轴突是神经元的巨大细胞结构，单维持其本身正常生理功能就是一个挑战[3]。当其受到如挤压、撕裂、甚至完全断裂等损伤后的几分钟内，其末端便开始形成营养不良性小泡，这种早期的变性即为急性轴突损伤[4]，当损伤变得不可逆的时，残端轴突将会在24-72 h发生崩解，之后便是许旺细胞的增殖与巨噬细胞的清理这一不可逆的过程。这一现象在1850年被Augustus Volney Waller发现并描述，也就是人们既熟悉又陌生的瓦勒氏变性。熟悉是因为距这一概念的提出已有165年了，陌生是因为时至今日，对远端轴突发生的瓦勒氏变性的机制仍然不清晰[5]，遥想十几年前，人们还简单的认为瓦勒变性是由于缺乏来自胞体的营养，而发生的营养不良性变性，直到Wlds的出现，才让人们意识到，缺乏营养的远端轴突在一些状态下也可保持基本形态长达数周[6-7]。Wlds的发现，对于轴突的认识从传统的组织学、生理学、药理学研究拓展到分子生物学与生物信息学[8]。急性轴突退变被认为在损伤开始的几分钟内发生，这一过程包括第二信使的改变、细胞骨架结构的变化、程序化死亡的开启(凋亡)。过去认为在损伤的那一刻，钙离子的内流与分布改变，诱导了细胞骨架的变化，不能维持正常结构的轴突只能发生瓦勒氏变性[9]。而近期研究表明，钙离子或许不是早期的瓦勒氏变性的起始点[10]。作者通过对Schmidt- Lanterman clefts这一轴突微结构的周围钙离子分布进行研究，发现在轴突损伤的前4 h内，钙离子分布并无显著变化。而这4 h也是明显长于通常定义的急性轴突退变时限。细胞的自噬并不是单纯意义上的死亡，或电镜下出现吞噬泡这一碗型结构，自噬对于细胞起着双重作用，一方面，自噬在清除降解异常蛋白，恢复正常细胞代谢和功能上有保护作用；另一方面，如果这种反应过大，达到一定阈值后，细胞器便会开始损伤，造成对细胞的伤害，这一过程中的主要受害者便是有着能量工厂之称的线粒体。线粒体在神经轴突中如何担负起供能任务，曾经一度被认为是解决瓦勒氏变性的关键一环。因为ATP在细胞浆中有限的扩散能力(特别对于轴突来说，这简直是万里长征)，线粒体—可移动的能量工厂—对于保障突触末端递质释放时瞬时大量ATP的消耗就显得至关重要。那瓦勒氏变性重要原因，是因为线粒体在轴突运输的障碍吗？正常情况下，线粒体在轴突中通过Syntaphilin的锚定作用，及时准确地转移到需要部位，静态情况下线粒体常固定在最需要能量的部位，如突触前膜，正常状态下参与线粒体固定运输的酶有很多，如Klf5、trak1、trak2等，其中以Klf5最为人所熟知[11]。Klf5分为A/B/C三型，在神经特异性表达的是A/C型，如果阻滞其作用，可以观测到的线粒体密度分布异常，慢性退行性轴突病变被证明与Kif5a相关[12]。在一些少见的人类相关疾病中，由Kif5Aa突变所导致的线粒体密度改变，常导致轴突的变性[13]，这一变性除了许旺细胞与巨噬细胞迁移、增殖、分化外，其余病理改变与瓦勒氏变性类似[14]。但这一过程往往是需要较长时间完成的，相较于定义4 h左右的急性轴突退变还是明显滞后许多，现有的实验数据不支持线粒体分布异常在急性期引发了瓦勒氏变性。但就其是否导致了远端轴突突触结构功能的丧失，还不明朗。可由于线粒体分布密度的巨大变化，确实可导致轴突变性，究其根本，似乎可归因于能量供应出现的问题。 2.1.2 由WLDs得出新的认识当发现WLDs小鼠后，改变了人们一些固有的思考。其一，相较于正常情况下损伤远端轴突基本结构维持1到2 d，在WLDs模型中，残端轴突可维持几周[7]。这一发现使科学家意识到，在一些情况下，作为一个巨大细胞结构的轴突，即使缺少胞体的调控，也可维持很长的一段时间。其二，缺少胞体的支持，在能量及营养缺乏的情况下，轴突在相当长的一段时间内也可维持正常的形态与功能[15]。其三，与其说瓦勒氏变性是一个被动的日渐降解的病理过程，不如说是一个类似于凋亡，主动衰减的过程[16-17]。Wlds不仅可以在哺乳动物中发挥作用，也在以果蝇或斑马鱼的实验模型中发挥作用[6，18]。Wlds由Nmnat1和ube4b两段基因编码，其中Nmnat主要发挥抗瓦勒氏变性的作用[19]。而ube4b由N16与N70组成，则被认为与Wlds作用位点有关[20]。Nmnat1是一种核内的NAD+合成酶，但仅仅Nmnat1的单独作用不能阻止瓦勒氏变性[21-22]。虽然目前大家仍然没有找到阻止瓦勒氏变性的根本方法，但基于现有研究，认为瓦勒氏变性是与轴突能量供应紧密相关的，最近的研究表明SARM1是激活轴突变性的起点[23-24]。大量研究表明，调控急性与慢性轴突变性的机制没有明显区别，也就是说，在减少轴突变性上，急性与慢性神经损伤上的策略很可能是一致的[19，25]。以往认为损伤后的轴突坏死归因于胞体能量供应的不足，但通过对损伤后轴突细胞自噬机制的研究，发现也许轴突自身的调节发挥着更为重要的作用[26]，而这种影响并不单来自胞体营养的缺乏[27]。通过对周围神经瓦勒氏变性机制更加深入的研究，也许可使人们接近神经焊接这一梦想，但现阶段还言之过早。 2.1.3 Bungner氏带的形成与轴突再生神经细胞轴突的再生与上述过程同时发生，由许旺细胞与巨噬细胞组成的“清道夫”开始进行“打扫”工作[28]。提别是许旺细胞还同时形成Bungner氏带。这条由许旺细胞形成的高速通道连接了损伤神经的两端[29]。为了方便理解，神经的再生可以分为5个时期[30]，水肿渗出期，基质形成期，细胞迁移期，轴突再生期，髓鞘形成期。简单的概括周围神经轴突再生的特点：损伤的神经组织渗出富含细胞外基质及神经营养因子的渗出液，一般损伤后3-6 h渗出达峰。第一、二期形成重要的纤维索，之后再生的许旺细胞沿着纤维索形成Bungner氏带[31]，轴突的再生随之开始，与此同时，再生型许旺细胞开始分化，形成成熟的髓鞘型许旺细胞，在轴突到达远端神经支配区域后，纤维索便开始降解，最后再生的轴突外形成髓鞘[2，32]。 2.1.4 周围神经轴突再生过程中的关键细胞许旺细胞起着支持并诱导再生轴突的重要功能[33]。由许旺细胞构建的再生微环境[34]，不仅对新生的轴突起着力学支撑的作用[29]，并且由其分泌的多种细胞因子，包括神经生长因子、脑源性神经生长因子、胰岛素样因子，均诱导并促进了轴突的再生[35-36]。干细胞如诱导多能干细胞[37]、神经干细胞、间充质干细胞等均被证明可促进神经的再生[38-39]，其中以诱导多能干细胞最为引人瞩目[40-41]，其在中枢神经与周围神经中均得到了成功运用。并且，由诱导多能干细胞诱导形成的许旺细胞生理学功能与天然环境下的许旺细胞相似。但复合干细胞的支架也面临着巨大挑战，如干细胞植入人工支架后，不受控制的增殖与分化[42]；耦合细胞的支架在植入体内后引起异常疼痛等问题[43]。支架复合许旺细胞：用于定植于支架上的许旺细胞可分为同基因型与非同基因型，无论是哪种，都可提高轴突的再生[28]。但总体来讲同基因型来源的许旺细胞修复效果优于异基因型，这并不难理解，同基因型，代表着更少的炎症反应、无排斥反应、与再生过程中的其他细胞配合默契。而对于非同基因型的则需要免疫抑制剂来保障植入细胞的存活，常用的免疫抑制剂有经典的环孢素A，他克莫司、JNJ460(FK506的衍生物)。实验表明，免疫抑制剂的介入有利于异基因型许旺细胞发挥功能，并且有助于周围神经再生[36，44-45]。同基因型许旺细胞来源有限，准备周期时间长，保存困难，这些限制都是广泛应用的巨大障碍。还有少部分许旺细胞可来自许旺细胞系，有证据表明这种来源的细胞也表现出令人满意的效果[46]。确定来源的许旺细胞可直接与支架共培养，与神经修补材料形成整体，丰富修补材料的功能[47]。 2.2 新型神经修补支架 2.2.1 材料自体神经移植一直是治疗大段神经缺损的金标准，但这一治疗方法并不完美，存在着供体区域感觉功能丧失、来源有限、运动与感觉不匹配及瘢痕等问题[30, 48-50]。因此科学家从未放弃探索新型的神经修补材料以取代自体神经移植。长段神经缺损的修复与再生存在着两大难题。第一，神经修补不是简单的“修补”，而是要通过修补材料架起的“桥梁”引导近端神经纤维快速长向靶器官，减少因失神经引起的靶器官萎缩；第二，绝大部分的周围神经是感觉与运动的混合支，因此须使感觉、运动神经纤维准确到达相应靶器官，才能准确实现神经再支配。简而言之，新型神经修补材料的构建主要集中在使再生神经长得“快”与长得“准”这两点上。神经修补材料或神经修复导管，距今已有100多年的历史了，从1882年空心骨质导管成功修复犬坐骨神经缺损，到如今复杂内部空间结构并添加天然神经诱导因子的复合导管的研制[51]，大段神经缺损修复走过了激动人心的100年。随着人们对神经再生基础研究的深入，发现空心修复导管很难满足神经功能恢复的要求[52]。表1总结了国内外部分神经导管产品[49，53-55]。基础医学新的发现，引导着人们发展具有更加复杂内部空间结构的新型导管。理想的神经移植物应该具有如下特点：良好的生物相容性及温和的降解过程，避免引起炎性反应，已有实验证实，炎性反应不利于神经再生；管壁允许营养物质与细胞因子的交换，内壁可进行修饰或复合二级空间结构，达到形态仿生；释放与神经再生相关的生长因子；便于临床操作。根据神经导管的材料来源可分为天然与合成材料两类[56]。天然材料包括自体静脉、自体骨骼肌、异体或异种神经、藻酸盐、胶原[57]、壳聚糖等[58]；人工合成材料主要有硅胶、聚乳酸-羟基乙酸共聚物、聚乳酸等[59]。静脉、骨骼肌等天然生物活性材料存在管壁塌陷、瘢痕组织增生及粘连等问题且来源均比较有限，不适于大批量生产。人工合成材料主要是制备成中空管状结构的引导再生导管，但这种导管的缺点主要是渗透性差，无取向性，不利于近端轴突的生长。构建神经导管的方法包括化学去细胞法[60]、静电纺丝法[61]、3D打印、相分离技术等。应明确的是，这两大类材料间可相互混合，取长补短，形成复合材料，改善如亲水性在内的多种物理化学特性，如壳聚糖与聚羟基乙酸的混合就被证明有减轻炎症反应，促进血管生成的作用[62]。支架结构与形状：亚里士多德曾说过“形状是生命的灵魂”。支架结构和形状的设计，决定着支架的性能，从最简单的单纯管状结构到复杂的多管道且内部纤维排列方向与神经生长方向一致都影响着神经的再生。单纯管状结构：应用如电纺丝法制备管状模型的方法大体可有两类，应用模具方法或单纯应用薄膜通过卷曲的方式制造[33]。模具法可选用可溶型或不可溶型的模具。可溶型微管道模型材料选取可溶于水的有机材料，如聚乙烯醇、蚕丝蛋白/聚环氧乙烷、聚乙二醇等，依材料熔点不同选取特定温度，熔融状态下制成不同直径圆柱状模型，取出时溶于水中，得到管状神经支架。对于不溶型的模具可选用金属模具，模具外涂抹如聚乙二醇等物质，制备完成后，溶于水中，易于取下。复合型神经支架：为了更好地模拟天然神经结构，科学家在中空管道内引入了呈取向排列的纤维，试图模拟损伤神经在修复再生过程中纤维索的形成，引导许旺细胞的迁移、增殖及分泌形成细胞外基质，加速神经再生过程。纤维的材料可以是聚L-丙交酯、聚乳酸-羟基乙酸共聚物、聚乙醇酸等高分子聚合物，也可以是壳聚糖、蚕丝等天然材料[63]。在一项为期6个月的研究里，应用聚乳酸-羟基乙酸共聚物纤维与间充质干细胞修复犬50 mm坐骨神经损伤，动物神经功能的恢复明显高于低密度时，神经修复能力得到增强[65-66]。 2.2.3 国内在这一领域的进展近年来国内科学工作者在周围神经再生领域也取得了令人欣喜的成功。中山大学刘小林教授团队研发了全球第二个去细胞神经移植物(神桥)，目前已在全国90余家医院使用，取得了良好临床效果[67]。南通大学顾晓松院士、杨宇民教授团队发明的壳聚糖中和固定加工技术，制备壳聚糖/PGA神经移植物，率先将壳聚糖人工神经移植物应用于临床，并成功实现了35 mm长段周围神经缺损的修复，Science杂志曾专门刊文报道，后期研究在神经导管中添加蚕丝，引导再生轴突取向排列也取得了成功[54，62]。北京大学的姜保国教授、张殿英教授团队研发的体内可降解生物套管，在小间隙套接修复神经损伤修复实验中被证明优于传统神经外膜缝合技术，恒河猴实验更表明植入材料取得了优秀的修复效果[68]。由武汉理工大学戴红莲教授团队研发的仿生纳米复合可降解神经移植物，重塑神经再生过程中的纳米微环境，促进了NGF、BDNF的合成与表达，被证实可诱导神经再生[69-70]。解放军第四军医大学罗卓荆教授团队研发的导电神经修补材料，模拟了生理条件下生物电对再生轴突的刺激，体外实验表明有利于轴突再生[35]。"

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