CN109420199B - 细胞定向平行排列与微孔结构的仿生神经支架制备方法 - Google Patents

细胞定向平行排列与微孔结构的仿生神经支架制备方法 Download PDF

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CN109420199B
CN109420199B CN201710825015.5A CN201710825015A CN109420199B CN 109420199 B CN109420199 B CN 109420199B CN 201710825015 A CN201710825015 A CN 201710825015A CN 109420199 B CN109420199 B CN 109420199B
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陈海萍
袁邦兴
谢世坤
周太平
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Abstract

本发明涉及细胞定向平行排列与微孔结构的仿生神经支架制备方法。该方法可实现多种材料和多结构集成加工，直接利用热熔挤出和细胞电纺丝相复合的工艺制备仿生神经支架。具体步骤为：(1)用加热装置将可降解高分子聚合物1热熔后，利用热熔挤出工艺制备出神经支架壳层；(2)配制可降解高分子聚合物2，进行高温蒸汽消毒；将细胞悬液加入聚合物2材料里面；利用细胞电纺丝工艺制备神经支架芯层。本发明整个制备方法简单易行，对神经缺损快速修复具有重大的现实意义。

Description

细胞定向平行排列与微孔结构的仿生神经支架制备方法
技术领域
本发明涉及微孔结构与细胞定向平行排列的仿生神经支架制备方法，属于神经支架的制备与神经缺损修复领域。
背景技术
生物热熔挤出、细胞电纺丝是近年来兴起的制备支架的2种不同工艺。生物热熔挤出工艺可以实现支架外形的可控制造，制造出的支架力学性能好、结构稳定，但作为神经细胞的附着场所还存在很大的局限性，不能模拟纳米级别的神经纤维、不能给神经轴突提供合适的三维微环境生长，致使在神经支架的制备方面还存在难以逾越的技术壁垒；细胞电纺丝工艺制备的纳米纤维支架力学强度太低、细胞易受外力袭扰、细胞在支架上分布杂乱无序，这不利于组织的均衡生长和快速修复；纳米纤维排列无序，成形结构非定向，易导致神经轴突迂曲生长。
人体组织内的细胞都处于三维空间结构中，接受着周围的信号。该环境为细胞的生长提供了极为有利的条件，均衡的营养和物质能量的交换，使细胞不断增殖和分泌自身的外基质。在体外如何构建这一微环境，实际上就是要解决支架对材料、结构及力学性能等多方面的要求。因此将结合生物热熔挤出、细胞电纺丝两种技术的不同优势，研究和探索一种能够制备细胞定向平行排列与5-30μm微孔结构的仿生神经支架复合制备方法。
发明内容
本发明的目的是针对以上问题，提供细胞定向平行排列与5-30μm微孔结构的仿生神经支架制备方法，利用多工艺复合制备，着力于构建具有生物活性且细胞定向分布的微结构神经支架，为缺损神经快速修复提供更为先进的技术支持。
为了实现上述目的，本发明采用如下技术方案：
仿生神经支架制备方法，神经支架由可降解的高分子聚合物和细胞组成，利用生物热熔挤出成形和细胞电纺丝复合工艺制备，包括以下步骤：
1)称取10-20克聚对二氧环己酮(PPDO)颗粒置于喷头1(神经壳层挤出喷头)加热装置中，将加热装置的温控器温度设为100-120℃，对PPDO进行加热，当温度达到设定值时保温2分钟。
2)喷头1上加载11-13KV的直流电压，与旋转接收装置的距离为30-50mm，负极加载在旋转装置上。将材料以350-380ul/min稳定流量由微量泵提供，进行连续挤出。旋转接收装置1按照所构建的神经支架壳层模型沿X/Y方向进行规律运动，材料迅速固化成形，完成5-30μm微孔结构的神经支架壳层制备；
3)称取1.5-2.0克聚乙烯醇(PVA)材料，在搅拌情况下缓缓加入到15-20克的去离子水中，至充分溶胀后升温至80-100℃左右加速溶解，并保温3小时制得均匀的高分子溶液再经100℃高温蒸汽消毒、冷却；
4)第4代大鼠细胞用0.25％的胰蛋白酶消化后收集在50mL离心管，1000r/min离心5min，弃上清液，将细胞悬液移至消毒后的PVA材料搅拌均匀得到含细胞的PVA溶液，将其装入喷头2中备用；
5)喷头2上加载6-8KV的直流电压，喷头和旋转接收装置之间的距离为6-8mm，负极加载在旋转装置2上。将材料以150-180ul/min稳定流量由微量泵提供，纺丝1-3个小时，得到含有细胞定向平行排列的纳米纤维；
6)剪下纳米纤维，捆扎成平行排列的纤维束作为神经支架芯层，将纤维束穿进神经支架壳层中，完成神经支架的整个制备。
所述步骤4)的细胞为雪旺细胞或神经元细胞。
本发明与现有技术相比较，具有如下显而易见的突出实质性特点和显著优点：
本发明的神经支架具有结构和性能上的仿生特性：支架壳层和芯层分别有宏微观结构相结合；5-30μm的微孔导管结构的支架壳层；支架芯层为平行排列的纳米纤维束，细胞定向分布在纳米纤维上，加速了神经缺损的修复；每层结构由不同的可生物降解材料制备。该方法具备工艺简单、可控性好及效率高等优点。
附图说明
图1为制备神经支架系统示意图。
图2为热熔挤出工艺示意图。
图3为细胞电纺丝工艺示意图。
图4为神经支架壳层结构示意图。
图5为神经支架结构示意图。
具体实施方式
本发明的优选实施案例，结合附图详述如下：
如图1所示为制备细胞定向平行排列与微孔结构的仿生神经支架系统示意图。其中：控制系统1连接控制器2，控制器2连接高压电源6和微量泵3，微量泵3连接喷头4，高压电源6的正极加载在喷头4的针头上，负极加载在接收平台5上，旋转装置7安装在平台5上。实施例1
细胞定向平行排列与微孔结构的仿生神经支架制备方法，神经支架由可降解的高分子聚合物和细胞组成，利用热熔挤出和细胞电纺丝复合工艺制备，包括以下步骤：
1)称取10克聚对二氧环己酮(PPDO)颗粒置于喷头(神经支架壳层挤出喷头)加热装置中，将加热装置的温控器温度设为110℃，对PPDO进行加热，当温度达到设定值时保温2分钟。
2)喷头上加载12KV的直流电压，喷头与旋转接收平台的距离为40mm，负极加载在旋转装置上。将材料以370ul/min稳定流量由微量泵提供，进行连续挤出。同时旋转接收平台按照所构建的神经支架壳层模型沿X/Y方向进行规律运动，并迅速固化成形，完成5-30μm微孔结构的神经支架壳层制备；
3)称取1.5克PVA材料，在搅拌情况下缓缓加入到15克的去离子水中，至充分溶胀后升温至80-100℃左右加速溶解，并保温3小时制得均匀的高分子溶液，再经100℃高温蒸汽消毒、冷却；
4)第4代神经干细胞用0.25％的胰蛋白酶消化后收集在50mL离心管，1000r/min离心5min，弃上清液，将细胞悬液移至消毒后的PVA材料搅拌均匀得到含细胞的PVA溶液，将其装入无菌注射器中备用；
5)在注射器上加载7KV的直流电压，喷头和旋转接收装置之间的距离为7cm，负极加载在旋转接收装置上。将材料以170ul/min稳定流量由微量泵提供，纺丝1.5个小时，得到含有细胞定向平行排列的纳米纤维；
6)剪下纳米纤维，捆扎成平行排列的纤维束作为神经支架芯层，将纤维束穿进神经支架壳层中；
7)仿生神经支架制备结束后，将其放进培养皿中加入培养液再放入培养箱中进行培养。
实施例3
本实例与实例1基本相同，不同之处在于：步骤4)所采用的细胞为雪旺细胞。

Claims (2)

1.细胞定向平行排列与微孔结构的仿生神经支架制备方法，神经支架由可降解的高分子聚合物和细胞组成，利用热熔挤出和细胞电纺丝复合工艺制备，包括以下步骤：
1)称取10克聚对二氧环己酮(PPDO)颗粒置于喷头加热装置中，将加热装置的温控器温度设为110℃，对PPDO进行加热，当温度达到设定值时保温2分钟；
2)喷头上加载12KV的直流电压，喷头与旋转接收平台的距离为40mm，负极加载在旋转装置上，将材料以370ul/min稳定流量由微量泵提供，进行连续挤出，同时旋转接收平台按照所构建的神经支架壳层模型沿X/Y方向进行规律运动，并迅速固化成形，完成5-30μm微孔结构的神经支架壳层制备；
3)称取1.5克PVA材料，在搅拌情况下缓缓加入到15克的去离子水中，至充分溶胀后升温至80-100℃左右加速溶解，并保温3小时制得均匀的高分子溶液，再经100℃高温蒸汽消毒、冷却；
4)第4代大鼠细胞用0.25％的胰蛋白酶消化后收集在50mL离心管，1000r/min离心5min，弃上清液，将细胞悬液移至消毒后的PVA材料搅拌均匀得到含细胞的PVA溶液，将其装入无菌注射器中备用；
5)在注射器上加载7KV的直流电压，喷头和旋转接收装置之间的距离为7cm，负极加载在旋转接收装置上，将材料以170ul/min稳定流量由微量泵提供，纺丝1.5个小时，得到含有细胞定向平行排列的纳米纤维；
6)剪下纳米纤维，捆扎成平行排列的纤维束作为神经支架芯层，将纤维束穿进神经支架壳层中；
7)仿生神经支架制备结束后，将其放进培养皿中加入培养液再放入培养箱中进行培养。
2.根据权利要求1所述的神经支架的制备方法，其特征在于：所述步骤4)的细胞为雪旺细胞或神经元细胞。
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