CN106563171A - 神经支架/导管材料及其制备方法 - Google Patents
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Abstract
本发明属于生物工程领域，具体涉及神经支架/导管材料及其制备方法。其包括具有神经修复作用的纳米纤维薄膜以及包含该纳米纤维薄膜的神经支架/导管材料。
Description
技术领域
本发明属于生物工程领域，具体涉及神经支架/导管材料及其制备方法。
背景技术
目前已在临床应用的可吸收神经再生导管材料，同自体神经移植相比其修复效果仍不理想。由于该类神经再生导管缺乏神经再生所需雪旺细胞和神经营养因子，限制其临床修复效果的进一步提高。组织工程的发展为提高周围神经损伤修复效果带来希望，基本要求是：种子细胞和生长因子对于组织工程神经的构建必不可少；制备具有组成成份，空间结构仿生以及满足对神经移植材料力学特性要求的组织工程神经。
根据周围神经的再生机制，包括雪旺细胞在内的多种细胞和其分泌多种神经营养因子共同发挥作用，引导轴突再生，使靶器官重新获得神经支配。大量研究表明组织工程神经支架内部添加雪旺细胞修复大鼠、犬、猕猴周围神经缺损效果明显强于单纯支架组。然而，雪旺细胞来源有限且异体雪旺细胞移植存在排斥反应等问题。此外，神经损伤局部应用外源神经营养因子存在因子活性容易失活需多次注射，而且在神经损伤修复过程中无法补充不同神经营养因子等缺点，限制其临床广泛应用。近年来研究表明，骨髓间充质干细胞(bone mesenchymal stem cells，BMSCs)可用于解决雪旺细胞来源和神经营养因子添加有限等不足而受到广泛关注。
在脑、心脏和肝脏等多种组织或器官损伤后发现干细胞归巢现象，干细胞归巢对组织或器官损伤修复发挥重要作用。神经系统损伤后同样存在干细胞归巢现象。最新研究表明，基质细胞衍生因子(stromal cell-derived factor 1，SDF-1)具有促进干细胞归巢的作用，而神经支架复合SDF-1能否募集BMSCs促进神经缺损修复尚未确定。该领域研究目前国内外尚未见有关应用SDF-1构建神经导管的报道。
发明内容
本发明的第一方面是提供一种用于神经修复的纳米纤维薄膜，其具有募集和趋化干细胞的作用，具体包括：生物相容的、多孔的聚合物基质；以及包含在聚合物基质中的具有趋化和/或募集干细胞作用的因子。
在另一个实施方案中，所述聚合物基质选自，包括但不限于，聚乳酸(PLA)、聚乙醇酸(PGA)、聚己内酯(PCL)、乳酸和乙醇酸的共聚物(PLGA)、乳酸和己内酯的共聚物、聚羟基丁酸和聚原酸酯、聚氨酯、聚氰基丙烯酸酯和聚碳酸亚丙酯(PPC)中的一种或多种；进一步地所述聚合物基质优选为PPC。
在又一个实施方案中，所述具有趋化和/或募集干细胞作用的因子选自，包括但不限于，LIF、脑源性神经营养因子(BDNF)、表皮生长因子受体(EGF)、成纤维细胞生长因子(bFGF)、FGF-6、胶质细胞衍生的神经营养因子(GDNF)、粒细胞集落刺激因子(GCSF)、肝细胞生长因子(HGF)、单核细胞趋化蛋白(MCP-1)、单核吞噬细胞集落刺激因子(M-CSF)、神经营养因子(NT3)、血管内皮生长因子(VEGF)、骨形态发生蛋白(BMP4)、干细胞因子(SCF)、间质细胞衍生因子-1(SDF-1)、血小板衍生生长因子-BB(PDGFBB)、转化生长因子β(TGFβ-1)和TGFβ-3中一种或多种；进一步地，所述因子为SDF-1。
在进一步地实施方案中，所述具有趋化和/或募集干细胞作用的因子与聚合物基质的重量比为1∶1000-1000000；例如，两者重量比可以为1∶1000，1∶10000，1∶20000，1∶30000，1∶50000，1∶70000或1∶100000。
本发明的第二方面是提供上述纳米纤维薄膜的制备方法，其包括：
1)将聚合物基质溶于氯仿中作为壳层溶液；将SDF-1和BSA溶于磷酸盐缓冲液中作为芯层溶液；
2)将上述两种溶液分别注入2个注射器中，进行静电纺丝；纺丝电压15～20kV，喷丝头与接地自转的接收金属棒之间的距离15～25cm，壳层溶液的流速与芯层溶液流速4～10mL/h，制备缓释SDF-1的PPC纳米纤维薄膜。
本发明的第三方面是上述纳米纤维薄膜在制备治疗神经损伤材料中的应用。
本发明的第四方面是提供一种神经导管，所述神经导管外壁采用上述纳米纤维薄膜形成的中空管状结构，且管内具有三维定向多孔生物可降解支架和有序性纳米纤维构成。
在一个实施方案中，所述生物可降解支架由ECM进行制备的。所述ECM包括至少一种ECM材料。根据本发明，所述ECM材料可来自各种哺乳动物组织来源，包括但不限于小肠、大肠、胃、肺、肝、肾、胰、胎盘、心脏、神经组织、膀胱和前列腺。例如，神经组织来源可以是人、猪、牛、羊、犬等动物的周围神经和脊髓马尾。
本发明的第五方面是提供上述神经导管的制备方法，其包括：
1)制备上述纳米纤维薄膜；
2)制备ECM支架：将ECM干粉用稀盐酸配成溶液，注入模具中，垂直放置在-40℃预冷的金属板上，然后转移到冷冻干燥机中干燥；支架从模具中取出波长258nm紫外线交联2～8h，然后再EDAC与NHS无水乙醇溶液中4℃下交联；0.1mol/LNa2HPO4清洗，无菌PBS缓冲液中浸泡，三蒸水漂洗，冷冻干燥后制得ECM取向支架；
3)将步骤(1)的材料包裹步骤(2)制备的材料上，即得。
本发明的第六方面是上述神经导管在制备治疗神经损伤材料中的应用。
本发明的有益效果是，具有缓释SDF-1促干细胞归巢的神经导管其显著优势包括：避免获取供区自体神经；防止吻合口处神经乳突外露；同传统神经移植手术相比缩短手术时间，避免神经损伤局部应用外源神经营养因子需多次注射，而且可避免神经损伤修复过程中无法补充不同神经营养因子等缺点，将对提高临床神经损伤修复效果起到积极的推动作用。
附图说明
图1 PPC纳米纤维薄膜扫描电镜图
图2缓释SDF-1“壳-芯”结构PPC-ECM支架显微镜图，A为横截面，B为纵截面
图3倒置显微镜下观察背根神经节在取向性纤维薄膜的生长情况
具体实施方式
下面将进一步的详细说明本发明。需要指出的是，以下说明仅仅是对本发明要求保护的技术方案的举例说明，并非对这些技术方案的任何限制。本发明的保护范围以所附权利要求书记载的内容为准。
实施例1制备缓释SDF-1的PPC纳米纤维薄膜
1.1缓释SDF-1的PPC纳米纤维薄膜制作
首先配制壳层溶液为10％(w/v)PPC氯仿溶液；芯层溶液溶剂为磷酸盐缓冲液(pH＝7.2)，溶质为100ng/ml SDF-1和50μg/ml BSA(其为稳定剂)的静电纺丝溶液，之后分别注入2个注射器中，进行静电纺丝。调节静电纺丝参数：环境温度、湿度、纺丝电压15～20kV、喷丝头与接地自转的接收金属棒之间的距离15～25cm、以及壳层溶液的流速与芯层溶液流速4～10mL/h，制备缓释SDF-1的PPC纳米纤维薄膜，室温下在真空箱内干燥48h。
1.2扫描电镜观察
对缓释SDF-1的PPC纳米纤维薄膜样品临界点干燥，表面镀金处理，扫描电子显微镜观察样本纤维的平均直径和直径分布范围。扫描电镜观察示，缓释SDF-1的PPC纤维每根直径为800～1300nm，纤维分布较均匀，形成具有亚微米尺度的结构(图1)
实施例2制备缓释SDF-1“壳-芯”结构PPC-ECM支架
2.1神经源性ECM的制备
无菌条件下，取SD大鼠坐骨神经，蒸馏水冲洗3次，-20℃低渗冻融后剪碎，浸泡于0～4℃含蛋白酶抑制物0.35mmol/L苯甲基磺酰氟的PBS缓冲液中，加无菌1％TritonX-100的Tris-HCl缓冲液4℃下持续振荡24h去除残余细胞成分，加50U/mL DNase I与1U/mL RNaseA溶液，超微湿法粉碎搅拌、室温中酶消化过夜，差速离心收集沉淀，冷冻干燥制备成干粉4℃下保存。
2.2神经源性ECM取向支架的制备
将神经源性ECM干粉用pH3.2的稀盐酸配成3％溶液，注入模具中，垂直放置在-40℃预冷的金属板上，-40℃放置2h，然后转移到冷冻干燥机中干燥24-48h。支架从模具中取出波长258nm紫外线交联2～8h，然后再EDAC与NHS无水乙醇溶液中(浓度为：EDAC 50mmol/L；NHS 20mmol/L)4℃下交联24h。0.1mol/LNa2HP04(pH9.1)洗2h，无菌PBS缓冲液中浸泡2h，三蒸水漂洗，冷冻干燥后制得ECM取向支架。
2.3按照实施例1方法1.1中的步骤，以自转的ECM取向支架和金属棒为接收极，制备缓释SDF-1“壳-芯”结构PPC-ECM支架，室温下在真空箱内干燥48h备用。
采用倒置显微镜观察初步观察支架空间结构，结果显示缓释SDF-1的PPC-ECM支架内部ECM孔呈取向性多孔蜂窝状结构，孔径分布尚均匀(图2)；纵截面观孔道部分呈部分平行排列的管道结构。
实施例3缓释SDF-1的PPC纳米纤维薄膜在体外募集BMSCs情况
使用Transwell共培养体系将支架和BMSC隔开，具体分组为：
对照组Transwell上室添加100μl BMSC细胞悬液(1×106个/ml)，下室添加500μl无血清培养基；
实施例1组Transwell上室添加100μl BMSC细胞悬液(1×106个/ml)，下室添加500μl浸泡SDF-1的PPC纳米纤维薄膜的无血清培养基；
对比例1组Transwell上室添加100μl BMSC细胞悬液(1×106个/ml)，下室添加500μl浸泡BDNF的PPC纳米纤维薄膜的无血清培养基；
对比例2组Transwell上室添加100μl BMSC细胞悬液(1×106个/ml)，下室添加500μl浸泡bFGF的PPC纳米纤维薄膜的无血清培养基(对比例1和2的制备方法同实施例1，区别仅在于使用的细胞因子存在不同)。
37℃孵育24小时，PBS洗3次，4％多聚甲醛固定10min，用棉签擦去上室未迁移过膜的细胞，DAPI染色30min，PBS洗3次，荧光显微镜下随机选择5个视野，拍照计数。相同实验重复3次，具体结果如下：
| 细胞计数(个) | |
| 对照组 | - |
| 实施例1组 | 23.2±4.4** |
| 对比例1组 | 8.1±2.1 |
| 对比例2组 | 5.4±1.6 |
**表示与对比例组相比P＜0.01
实施例4缓释SDF-1的PPC纳米纤维薄膜体外诱导BMSC定向分化神经细胞的研究
分别将实施例1、对比例1和对比例2制备的纳米纤维薄膜置于不同的培养皿中，加入1ml包含1×106个/ml BMSC的培养基(10％FBS/DMEM)，对照组中不添加纳米纤维薄膜，37℃孵育8小时后进行免疫细胞化学检测，分别采用nestin和GFAP抗体作为一抗，FITC标记的羊抗小鼠IgG为二抗，计算各组细胞表达阳性率，每组平行试验3次，具体结果如下：
| Nestin阳性率(％) | GFAP阳性率(％) | |
| 对照组 | - | - |
| 实施例1组 | 84.2±6.7** | 69.4±4.9** |
| 对比例1组 | 46.8±3.9 | 32.5±3.1 |
| 对比例2组 | 61.3±4.6 | 44.7±3.6 |
**表示与对比例组相比P＜0.01
实施例5背根神经节和缓释SDF-1的PPC薄膜体外共培养
下取出新生1～2d SD大鼠的脊柱并纵行剖开，体式显微镜下暴露椎间孔，见椎体外侧隐窝处有圆形透亮背根神经节，显微镊取出背根神经节并剥除其表面结缔组织，然后置于DMEM/F12培养基备用。将背根神经节接种至缓释SDF-1的PPC薄膜上，置于含10％胎牛血清和50ng/mL NGF的DMEM/F12培养基的6孔板中，于37℃、5％CO2培养箱中孵育，隔天半量换液。于培养5d取出接种背根神经节的缓释SDF-1的PPC薄膜，倒置显微镜下观察背根神经节在取向性纤维薄膜的生长情况(结果见图3)。
实施例6缓释SDF-1的PPC-ECM仿生支架修复大鼠坐骨神经缺损效果
取成年雄性SD大鼠，体重200～250克，10％水合氯醛腹腔麻醉后，显露左侧坐骨神经，于梨状肌下缘切除12mm坐骨神经，神经收缩后制备坐骨神经15mm缺损动物模型。大鼠术后2周取各组神经移植物，冰冻纵行切片，神经微丝NF-200免疫荧光染色观察，荧光显微镜下测量近端吻合口至荧光标记微丝的最远距离，测定各组周围神经再生距离。具体结果如下：
| 周围神经再生距离(mm) | |
| 对照组 | 1.3±0.5 |
| 实施例1组 | 5.9±1.4** |
| 对比例1组 | 2.8±0.9 |
| 对比例2组 | 3.7±1.1 |
| 对比例3组 | 3.2±1.2 |
**表示与对比例组相比P＜0.01
对比例3为不包裹SDF-1的PPC纳米纤维薄膜的ECM支架，制备方法同实施例2
本发明内容仅仅举例说明了要求保护的一些具体实施方案，其中一个或更多个技术方案中所记载的技术特征可以与任意的一个或多个技术方案相组合，这些经组合而得到的技术方案也在本申请保护范围内，就像这些经组合而得到的技术方案已经在本发明公开内容中具体记载一样。
Claims (10)
1.一种用于神经修复的纳米纤维薄膜，其具有募集和趋化干细胞的作用，具体包括：生物相容的、多孔的聚合物基质；以及包含在聚合物基质中的具有趋化和/或募集干细胞作用的因子。
2.根据权利要求1所述的纳米纤维薄膜，其特征在于，所述聚合物基质选自，包括但不限于，聚乳酸(PLA)、聚乙醇酸(PGA)、聚己内酯(PCL)、乳酸和乙醇酸的共聚物(PLGA)、乳酸和己内酯的共聚物、聚羟基丁酸和聚原酸酯、聚氨酯、聚氰基丙烯酸酯和聚碳酸亚丙酯(PPC)中的一种或多种；进一步地所述聚合物基质优选为PPC。
3.根据权利要求1所述的纳米纤维薄膜，其特征在于，所述具有趋化和/或募集干细胞作用的因子选自，包括但不限于，LIF、脑源性神经营养因子(BDNF)、表皮生长因子受体(EGF)、成纤维细胞生长因子(bFGF)、FGF-6、胶质细胞衍生的神经营养因子(GDNF)、粒细胞集落刺激因子(GCSF)、肝细胞生长因子(HGF)、单核细胞趋化蛋白(MCP-1)、单核吞噬细胞集落刺激因子(M-CSF)、神经营养因子(NT3)、血管内皮生长因子(VEGF)、骨形态发生蛋白(BMP4)、干细胞因子(SCF)、间质细胞衍生因子-1(SDF-1)、血小板衍生生长因子-BB(PDGFBB)、转化生长因子β(TGFβ-1)和TGFβ-3中一种或多种；进一步地，所述因子为SDF-1。
4.根据权利要求1所述的纳米纤维薄膜，其特征在于，所述具有趋化和/或募集干细胞作用的因子与聚合物基质的重量比为1∶1000-1000000；例如，两者重量比可以为1∶1000，1∶10000，1∶20000，1∶30000，1∶50000，1∶70000或1∶100000。
5.一种权利要求1所述的纳米纤维薄膜的制备方法，其包括：
1)将聚合物基质溶于氯仿中作为壳层溶液；将SDF-1和BSA溶于磷酸盐缓冲液中作为芯层溶液；2)将上述两种溶液分别注入2个注射器中，进行静电纺丝；纺丝电压15～20kV，喷丝头与接地自转的接收金属棒之间的距离15～25cm，壳层溶液的流速与芯层溶液流速4～10mL/h，制备缓释SDF-1的PPC纳米纤维薄膜。
6.权利要求1-4任一项所述纳米纤维薄膜在制备治疗神经损伤材料中的应用。
7.一种神经导管，所述神经导管外壁具有权利要求1-4任一项所述纳米纤维薄膜形成的中空管状结构，且管内具有三维定向多孔生物可降解支架。
8.根据权利要求7所述的神经导管，其特征在于，所述生物可降解支架由ECM进行制备的。
9.一种制备权利要求7所述神经导管的方法，其包括：
1)制备上述纳米纤维薄膜；
2)制备ECM支架：将ECM干粉用稀盐酸配成溶液，注入模具中，垂直放置在-40℃预冷的金属板上，然后转移到冷冻干燥机中干燥；支架从模具中取出波长258nm紫外线交联2～8h，然后再EDAC与NHS无水乙醇溶液中4℃下交联；0.1mol/LNa2HPO4清洗，无菌PBS缓冲液中浸泡，三蒸水漂洗，冷冻干燥后制得ECM取向支架；
3)将步骤(1)的材料包裹步骤(2)制备的材料上，即得。
10.权利要求7所述神经导管在制备治疗神经损伤材料中的应用。
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| CN108503768A (zh) * | 2018-04-26 | 2018-09-07 | 代清燕 | 一种可降解聚酯生物材料及其在神经修复套接管中的应用 |
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| CN108503768A (zh) * | 2018-04-26 | 2018-09-07 | 代清燕 | 一种可降解聚酯生物材料及其在神经修复套接管中的应用 |
| CN115337458A (zh) * | 2022-08-16 | 2022-11-15 | 尧舜泽生物医药(南京)有限公司 | 一种修复周围神经损伤的细胞基质神经移植物及其制备方法 |
| CN115337458B (zh) * | 2022-08-16 | 2023-08-22 | 尧舜泽生物医药(南京)有限公司 | 一种修复周围神经损伤的细胞基质神经移植物及其制备方法 |
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