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张健, 李超超, 孟繁琪, 管延军, 杨博尧, 张铁元, 任致奇, 刘修志, 赵杰, 杨凯, 王玉, 彭江. 微组织修复大鼠坐骨神经缺损的早期实验研究[J]. 解放军医学院学报, 2022, 43(1): 87-94. DOI: 10.3969/j.issn.2095-5227.2022.01.017
引用本文: 张健, 李超超, 孟繁琪, 管延军, 杨博尧, 张铁元, 任致奇, 刘修志, 赵杰, 杨凯, 王玉, 彭江. 微组织修复大鼠坐骨神经缺损的早期实验研究[J]. 解放军医学院学报, 2022, 43(1): 87-94. DOI: 10.3969/j.issn.2095-5227.2022.01.017
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ZHANG Jian, LI Chaochao, MENG Fanqi, GUAN Yanjun, YANG Boyao, ZHANG Tieyuan, REN Zhiqi, LIU Xiuzhi, ZHAO Jie, YANG Kai, WANG Yu, PENG Jiang. Early repair effect of tissue-engineered microtissue on rat sciatic nerve defect model[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL, 2022, 43(1): 87-94. DOI: 10.3969/j.issn.2095-5227.2022.01.017
Citation: ZHANG Jian, LI Chaochao, MENG Fanqi, GUAN Yanjun, YANG Boyao, ZHANG Tieyuan, REN Zhiqi, LIU Xiuzhi, ZHAO Jie, YANG Kai, WANG Yu, PENG Jiang. Early repair effect of tissue-engineered microtissue on rat sciatic nerve defect model[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL, 2022, 43(1): 87-94. DOI: 10.3969/j.issn.2095-5227.2022.01.017
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微组织修复大鼠坐骨神经缺损的早期实验研究

Early repair effect of tissue-engineered microtissue on rat sciatic nerve defect model

    摘要
  • 摘要:
      背景  微组织是种子细胞在细胞-细胞或细胞-细胞外基质(extracellular matrix, ECM)作用下聚集而成的微型组织，这种三维培养方式有利于提高细胞活性，提高组织修复效果，然而神经组织工程中微组织的研究却很少。
      目的  将脂肪来源间充质干细胞(adipose-derived mesenchymal stem cells，ASCs)构建成微组织，探索微组织对大鼠坐骨神经缺损模型的早期修复效果。
      方法  提取ASCs进行鉴定；利用悬浮液滴法和低黏附细胞培养板法构建微组织，并对其性质进行研究；将微组织与大鼠背根神经节(dorsal root ganglia，DRG)进行直接和间接共培养，观察微组织对DRG轴突生长的影响；利用静电纺丝机构建聚己内酯(polycaprolactone，PCL)神经导管，并验证其取向性；最后取SD大鼠24只，随机分为空导管组、2D细胞组、微组织组和自体神经移植组，每组6只。构建坐骨神经1 cm缺损模型，分别采用PCL导管桥接后注射培养基、PCL导管桥接后注射2D ASCs、PCL导管桥接后注射微组织和自体神经反向移植。术后4周取出神经移植物进行组织学评价。
      结果  成功提取ASCs，能够表达间充质干细胞表面标记蛋白CD73、CD90、CD105。微组织构建后，对其形态和直径进行连续7 d的动态观察，微组织的直径与细胞数量呈正相关，且随着培养时间延长，微组织呈现“压实”的状态，直径逐渐变小。在微组织和DRG间接共培养体系中，微组织组的轴突长度明显长于2D细胞组(P＜0.05)和单纯培养基组(对照组)(P＜0.05)。在微组织和DRG直接共培养体系中，DRG发出的轴突与附近的微组织紧密相连。PKH26标记的ASCs在PCL薄膜上呈取向性排列。动物实验结果表明，微组织组轴突生长明显优于2D细胞组(P＜0.05)和空导管组(P＜0.05)。
      结论  微组织可促进DRG轴突生长，有效促进坐骨神经缺损大鼠模型的神经再生。

     

    Abstract:
      Background  Microtissue is formed by the aggregation of seed cells under the action of cell-cell or cell-ECM (extracellular matrix). This 3D-cultured method is conducive to improve cell activity and tissue repair effect. However, microtissue is rarely used in neural tissue engineering.
      Objective  To construct adipose-derived mesenchymal stem cells (ASCs) into microtissues and explore the early repair effect of microtissues on rat sciatic nerve defect model.
      Methods  ASCs were extracted and identified. The microtissue was constructed by hanging-drop method or low-adhesion cell culture plate, and its properties were studied. The microtissue and the dorsal root ganglion (DRG) were co-cultured directly or indirectly, and the effect of microtissue on the growth of DRG axons was observed. Polycaprolactone (PCL) nerve conduit was constructed by electrospinning mechanism and its orientation was verified. Finally, 24 SD rats were randomly divided into hollow conduit group (hollow), 2D cells group (2D), microtissues group (microtissue) and autologous nerve graft group (ANG), with 6 rats in each group. The 1 cm defect model of sciatic nerve was constructed. The ASCs culture medium was injected after PCL conduit bridging, 2D ASCs were injected after PCL conduit bridging, microtissues were injected after PCL conduit bridging, and autologous nerve reverse transplantation were performed for four groups. The nerve grafts were removed for histological evaluation at 4 weeks after transplantation.
      Results  ASCs were successfully extracted and expressed mesenchymal stem cell surface marker proteins CD73, CD90 and CD105. After 7 days of dynamic observation, the diameter of microtissue was positively correlated with the number of cells, and with the extension of culture time, the microtissue showed a "compacted" state, and the diameter gradually decreased. In the indirect co-culture system of microtissue and DRG, the axon length of microtissue group was significantly longer than that of 2D group (P＜0.05) and control group (P＜0.05). In the direct co-culture system of microtissue and DRG, the axons emitted by DRG were closely connected with the nearby microtissues. PKH26 labeled ASCs were aligned on PCL film. The results of animal experiments showed that the axon growth of microtissue group was significantly better than that of 2D group (P＜0.05) and hollow group (P＜0.05).
      Conclusion  Microtissue can promote peripheral nerve regeneration and achieve good repair effect in the rat model of sciatic nerve defect.

     

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