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
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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
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***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***示意图。其中:控制***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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