Application of stem cells, tissue engineering scaffolds and neurotrophic factors in the treatment of spinal cord injury

doi:10.3969/j.issn.2095-4344.2085

Abstract

Abstract:

BACKGROUND: Stem cells have multi-directional differentiation potential. Tissue engineering scaffolds can provide support for them. Neurotrophic factors can promote the differentiation of stem cells. The combination of the three therapies to promote the functional recovery of spinal cord injury is one of the current research hotspots.

OBJECTIVE: To review the research progress of stem cells, tissue engineering scaffolds, neurotrophic factors and their combined transplantation in the treatment of spinal cord injury in recent years.

METHODS: The authors searched PubMed, ScienceDirect, Medline, and CNKI for original articles from January 2000 to October 2019. The key words were “spinal cord injury, mesenchymal stem cells, issue engineering, neurotrophic factor” in English and Chinese, respectively. Totally 712 studies were retrieved. After strict screening, 79 studies that met the requirements were classified and reviewed.

RESULTS AND CONCLUSION: Stem cell transplantation alone cannot reconstruct the complex structure and stability of the spinal cord. Biological materials or neurotrophic factors alone cannot replace the loss of neurons during spinal cord injury. Therefore, combined transplantation is the research direction of spinal cord injury treatment. However, due to the differences in the location of spinal cord injury, the tissue of injury and the nutritional requirements of its repair, the characteristics of various stem cells, tissue engineering scaffolds and neurotrophic factors are different. How to optimize the combination of the three is still a huge challenge.

Key words:

spinal cord injury, stem cells, mesenchymal stem cells, tissue engineering, scaffold, neurotrophic factor

CLC Number:

Ji Hangyu, Gu Jun, Xie Linghan, Wu Xiaotao.

Application of stem cells, tissue engineering scaffolds and neurotrophic factors in the treatment of spinal cord injury [J]. Chinese Journal of Tissue Engineering Research, 2020, 24(25): 4088-4093.

Figures/Tables 1

2 结果 Results 2.1 干细胞移植目前，细胞移植的治疗方法已经成为促进脊髓损伤后功能恢复的一种潜在方案，但其有效性与长期的安全性还需要进一步证实[11]。目前实验中所选用的移植细胞有很多种类，主要包括非干细胞和干细胞，前者包括嗅鞘细胞、许旺细胞、成纤维细胞等，后者包括成体干细胞和非成体干细胞[10，12]。胚胎干细胞和神经干细胞在早期的基础研究中曾有着极其重要的应用，但是由于存在着伦理问题、排斥反应以及来源受限等缺点，从而限制了其发展。目前干细胞中研究最多的是骨髓间充质干细胞和脂肪间充质干细胞[13]。 2.1.1 干细胞治疗脊髓损伤的机制干细胞治疗脊髓损伤的机制目前还不是很清楚，通常认为有以下几种可能：①移植后的干细胞可以分化为神经元细胞和神经胶质细胞，与正常的神经元细胞建立新的连接；②下调炎症与凋亡相关基因，上调具有神经保护的基因；③促进轴突周围髓鞘的形成；④促进轴突再生和突触形成[14-16]。 2.1.2 干细胞移植的方法干细胞移植的方法很多，目前较为常用的有髓内、鞘内、血管内等方法，其中髓内注射的方法最有效，但侵袭性最大，而血管内注射的方法侵袭性最小，但效果最差[17]。GEFFNER等[18]通过鞘内注射与血管内注射的方法将骨髓间充质干细胞移植到脊髓损伤患者体内，所有患者均获得了脊髓功能的部分恢复，而且移植后没有明显的不良反应。然而OSAKA等[19]使用神经干细胞与神经祖细胞采用鞘内移植的方法治疗小鼠脊髓损伤时，发现移植细胞存活率低，而且小鼠功能没有明显恢复。 2.1.3 干细胞移植的时机合适的移植时机对于干细胞的存活与受损神经系统的再生具有至关重要的作用。ANTONIC等[20]认为脊髓损伤后产生的神经毒素会对移植细胞的存活、增殖与分化造成不利的影响，所以合适的时机应该是脊髓损伤后的1，2周。NAKAJIMA等[21]则认为应该在脊髓损伤后立即进行细胞移植，因为脊髓损伤后的炎症对移植的干细胞影响并不大，而且移植后的细胞可以改变局部不利的微环境，从而减轻周围神经元的二次损害。 2.2 组织工程支架干细胞移植在脊髓损伤模型中的应用已被证明了有效性，但仍然存在着很多问题[22]，于是组织工程支架技术被引入了干细胞移植领域。支架可以提供一个三维的空间，为干细胞提供有效支持，从而促进干细胞在体内的黏附、迁移和分化[23]。研究表明，在动物模型中通过使用以生物材料为基础的支架[24]，可以有限地实现神经轴突及神经元的再生，通常来说组织工程支架需要具备以下标准[25]：生物相容性、生物降解性、机械强度。目前，根据不同的标准可以把支架分为不同的类型，根据材料来源可以分为天然支架和人工合成支架[26]，根据成分可以分为单组分支架和复合组分支架[27]，根据不同的状态可以分为固体支架和液体支架[28]。与单一组分支架相比，包含多个组分的复合支架可能更好，因为它可以避免单一组分支架的缺点；此外，与人工合成支架相比，天然成分的支架可能更好[29]，因为天然支架具有明显的优点：生物相容性、生物降解性、低毒性、渗透性、高孔隙度、可塑性、亲水性[30]，但是天然支架也存在着缺点：成本高、机械稳定性低[31]。目前用于脊髓组织工程的天然聚合物主要有壳聚糖、琼脂糖、藻酸盐、胶原蛋白、纤维蛋白和明胶[32]。在实验中应用比较广泛的是壳聚糖[33]，它作为一种天然支架材料，具有良好的生物相容性、生物降解性以及低生物毒性，但同时也存在着一定的缺点如降解性快、溶胀率高等。近年来，为了提供良好的机械支持以及递送细胞、药物和生长因子，设计了各种复杂结构的支架[34]，如水凝胶支架、三维多孔纳米纤维支架和脱细胞脊髓支架等。水凝胶为三维多孔结构[35]，其特征是与软组织具有相似的高含水量，可注射水凝胶作为一种微创技术在脊髓损伤再生领域得到了广泛关注[36]，其表面可以涂上天然的或合成的基质，这些基质注射到脊髓后会迅速从液体转化为凝胶[37]。水凝胶支架的优点在于：①可以填充损伤区域；②作为干细胞的载体，为脊髓损伤组织再生创造良好的环境；③具有更强的抵抗力；④更少的免疫原性[38]。三维多孔纳米纤维支架则具有更高的表面积，植入后可以促进干细胞的黏附、增殖和分化[39]。支架内的纳米纤维则通过引导细胞生长显著影响再生过程。此外，纳米纤维支架既可以单独使用，也可以与水凝胶一起使用[40]。脱细胞脊髓支架是脊髓组织工程的一种替代支架[41]。它们是通过化学提取获得天然脱细胞基质，这些基质由组织的非细胞部分组成，主要成分是蛋白质，如胶原蛋白、层粘连蛋白、纤维连接蛋白和胶原蛋白等。这种支架柔软而灵活，含有贯穿其全长的线性导向孔[42]。尽管支架在组织工程中具有治疗潜力，但是很多研究的支架都存在着一定的缺陷，如与移植物相关的生物材料的细胞毒性、移植物结构的部分或全部塌陷以及移植物在体内的降解率不足[43]。 2.3 间充质干细胞联合组织工程支架间充质干细胞由于具有其他干细胞所没有的优势而得到了广泛的研究，其优点在于[44]：①不存在伦理问题；②易于分离；③具有很强的再生能力和免疫调节特性，不存在免疫排斥的风险；④可以对抗炎症和凋亡等有害事件，为神经元再生创造了有利的微环境[45]。与单独的细胞移植或单独的工程支架移植相比，各种间充质干细胞联合组织工程支架移植表现出了更好的疗效[46]。 2.3.1 骨髓间充质干细胞联合组织工程支架 ITOSAKA等[47]在脊髓半切损伤的大鼠模型中，与单独植入骨髓间充质干细胞相比，联合纤维蛋白支架植入的干细胞显示出更高的存活率，并增强了向神经元分化的能力，同时他们观察到在移植后第14，21，28天大鼠的运动功能有显著改善。另一种方法则是将2种不同的生物材料结合在一个独特的支架上，以降低单一材料的缺点，这样不仅可以种植细胞，还可以传递神经营养因子[48]。此外，还有一种包含神经营养因子3的支架，支架表面覆盖纤维蛋白凝胶(NF-GS)，将该支架植入脊髓横切的大鼠和犬的损伤部位，以填补缺口，4周后在脊髓损伤处附近的头侧和尾侧可以观察到神经丝蛋白阳性的细胞数量明显增加，这可能意味着神经纤维出现了再生[49]。 2.3.2 脂肪间充质干细胞联合组织工程支架首先需要在体外将脂肪间充质干细胞诱导分化成许旺细胞，然后将其种植到胶原支架中，共同移植到脊髓半切损伤大鼠模型中观察[50]，与不接受干细胞移植组和接受骨髓间充质干细胞分化的许旺细胞移植组相比，接受细胞联合支架移植的大鼠在感觉与运动功能方面得到了明显改善。这些结果表明，干细胞联合支架的移植效果更好[51]。 2.3.3 脐血间充质干细胞联合组织工程支架 CARON等[52]将人脐血间充质干细胞种植在海绵状HG-RGD支架上，发现干细胞在体内存活时间更长，在植入8周后观察到细胞+支架组较单独植入支架组运动恢复更为明显。JIAO等[53]将复合支架与人脐血间充质干细胞联合使用也观察到同样结果，同时还观察到神经元标记物MAP2、NeuN和Nestin阳性细胞比例增加，这表明接种的脐血间充质干细胞向神经元转化的趋势在增加，见图1。 2.3.4 子宫内膜间充质干细胞联合支架 EBRAHIMI- BAROUGH等[54]用Ly294002在体外培养人子宫内膜间充质干细胞，然后将这些干细胞植入由聚己内酯/胶原电纺丝纤维构成的复合支架上，观察到支架上的干细胞向运动神经元样细胞分化更明显。 2.3.5 牙髓间充质干细胞联合支架口腔源性间充质干细胞是一种有广泛前景的干细胞来源，也可应用于脊髓损伤。据相关研究表明，与其他来源的间充质干细胞相比，来自口腔组织的间充质干细胞具有向神经元样细胞分化的天然倾向，这可能是由于它们起源于神经嵴[55]。为此，ZHANG等[56]将牙髓间充质干细胞接种于壳聚糖支架上，在特定的神经源性培养基中培养14 d，诱导细胞向神经元表型分化。值得注意的是，壳聚糖支架可以提高牙髓间充质干细胞的分化率，增加少突胶质细胞标记物CNPase、星形胶质细胞标记物GFAP和神经元标记物MAP-2的阳性细胞数量。在神经分化后，这些细胞被移植到慢性脊髓损伤大鼠模型中(脊髓损伤后1周)，与只接受牙髓间充质干细胞没有支架的对照组相比，神经营养因子如脑源性神经营养因子、胶质细胞源性神经营养因子、β-神经生长因子、神经营养因子3水平明显增加。 2.4 神经营养因子在脊髓损伤动物模型中，神经营养因子的作用越来越受到重视，一系列实验证明它们可以提高神经元的存活率，促进轴突的再生。在中枢与周围神经系统的细胞存活、分化与轴突生长的发育过程中，神经营养因子均发挥着至关重要的作用。它主要包括神经生长因子、脑源性神经生长因子、神经营养因子3、神经营养因子4/5等[57]。 2.4.1 脑源性神经营养因子脑源性神经营养因子是脊髓损伤实验中最常用的生长因子，据BROCK等[58]报道外源性的脑源性神经营养因子可以减少皮质萎缩，可以增加损伤部位轴突的数量、髓鞘的形成并诱导损伤部位广泛的再生。当然也不是所有的轴突都对脑源性神经营养因子有反应，MENEI等[59]发现它对本体感觉没有明显的作用，而BROCK等[58]则发现它对皮质脊髓束没有明显作用。但是，HAN等[60]将脑源性神经营养因子与胶原蛋白结合可以增加皮质脊髓束以及外周的轴突再生。 2.4.2 神经营养因子3 神经营养因子3在实验研究中也得到了广泛的应用，它大多在损伤后短时间内被递送到受损的脊髓及其毗邻区域。KADOYA等[61]通过改变神经营养因子3的浓度、周围微环境等方法，观察到了脊髓功能的改善。有研究认为神经营养因子3对髓鞘的形成有着积极的影响[62]，将神经营养因子3与其他神经营养因子如脑源性神经营养因子联合应用时则可以减少红核脊髓束中神经元的萎缩与死亡[63]。 2.4.3 神经生长因子和神经营养因子4/5 这2种神经营养因子在实验中的应用相对较少，有研究发现它们可以改善受损处轴突的生长情况[64]，也有研究发现神经生长因子可以降低T9挫伤后脊髓神经元内质网诱导的凋亡，从而改善运动功能[65]。 2.4.4 成纤维细胞生长因子成纤维细胞生长因子可与膜相关蛋白多糖相互作用，并与多种受体亚型结合，对损伤区域的组织有着明显的改善。GOLDSHMIT等[66]发现成年小鼠皮下注射成纤维细胞生长因子2可以减少炎症、小胶质细胞和巨噬细胞的激活。LEE等[67]研究发现在脊髓损伤后1 h内给予成纤维细胞生长因子2可以减少损伤范围，然而损伤后3 h再给予成纤维细胞生长因子2时，则不会出现上述的变化，说明移植时机对神经营养因子也很重要。 "

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