CN107281548B - Y型含细胞的神经导管制备方法 - Google Patents
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Abstract
本发明涉及Y型含细胞的神经导管制备方法。该方法可实现多种材料和多结构集成加工,直接利用生物3D打印、细胞电纺丝和近场直写相复合的工艺制备神经导管。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,采用3D打印工艺制备出神经导管内层结构体;(2)配制可降解高分子聚合物2,高温蒸汽消毒后,旋转由(1)制备的内层结构体,同时采用3D打印工艺在其表面制备中间层;(3)配制可降解高分子聚合物3,进行高温蒸汽消毒;将细胞悬液加入聚合物3材料里面;旋转由(2)制备的结构体,采用细胞电纺丝和近场直写复合工艺制备神经导管外层。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Y型含细胞的神经导管的制备方法,属于组织工程神经导管制备与神经缺损修复领域。
背景技术
生物3D打印、细胞电纺丝、近场直写是近年来兴起的制备支架的3种不同工艺。生物3D打印工艺可以制备复杂形状的宏观3D结构体,无法制备微观结构,不满足细胞生长的微环境要求;细胞电纺丝工艺制备的纳米纤维支架与天然的细胞外基质形态相近,有利于细胞在支架上的粘附、识别和功能维持,且该工艺条件下细胞的存活率较高且分布均匀。但是,细胞电纺丝工艺制备的支架为无纺布形态,在力学性能方面存在较大的局限性;近场直写工艺可以制备出单根纤维或具有图案化的结构纤维层,其有序的微纳纤维结构,不仅模拟了细胞外基质有利于细胞的生长,而且有利于引导细胞按照一定的方向生长促进其向目标组织分化生长,但是其结构体无力学性能。
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。因此将生物3D打印、细胞电纺丝、近场直写工艺进行综合能够有效克服单项工艺的局限是一个行之有效的方法。
发明内容
本发明的目的是针对以上问题,提供Y型含细胞的神经导管制备方法,利用多工艺复合制备,着力于构建具有生物活性且细胞均匀分布的神经导管,为医学上的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:
Y型含细胞的神经导管制备方法,神经导管由可降解的高分子聚合物和细胞组成,利用生物3D打印挤出成形和细胞电纺丝、近场直写复合工艺制备,包括以下步骤:
1)称取15-20克明胶材料,在搅拌情况下缓缓加入到80-100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,将消毒后的材料装入喷头二中备用;
2)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;
3)第4代人的细胞用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,将其装入喷头三中备用;
4)称取10-20克聚对二氧环己酮(PPDO)颗粒置于喷头一(内层神经导管挤出喷头)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。喷头一与平台的距离为1-2mm,将材料以350-380ul/min稳定流量由微量泵提供,进行连续挤出。接收平台按照所构建的Y型神经导管模型沿X/Y方向进行规律运动,材料迅速固化成形,完成内层神经导管制备;
5)将固化成形后的内层神经导管安装在旋转装置上,运动到喷头二(中间层神经导管挤出喷头)工位,喷头与内层神经导管的外表面距离为0.4-0.7mm,将材料以390-410ul/min稳定流量由微量泵提供,进行连续挤出。旋转装置一边旋转一边移动,明胶材料包裹住PPDO并固化成形,完成中间层神经导管;
6)旋转装置运动到喷头三(外层神经导管喷头),喷头三上加载6-8KV的直流电压,喷头和神经导管之间的距离为3-5mm,负极加载在接收平台上。将材料以150-180ul/min稳定流量由微量泵提供,旋转装置一边旋转一边移动,纺丝1-3个小时,在中间层神经导管上沉积一层含细胞的纳米纤维膜,完成神经导管的整个制备。
所述步骤3)的细胞为神经干细胞或神经元细胞。
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:
本发明的神经导管呈Y形状,具有三层结构,每层结构由不同的可生物降解材料制备;内层和中间层神经导管具有宏观结构,外层神经导管具有纳米纤维结构;细胞均匀分布在外层神经导管上,加速了神经缺损的修复。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为神经导管内层结构的制备***示意图。
图2为神经导管中间层与外层结构的制备***示意图。
图3为神经导管内层结构示意图。
图4为神经导管内层和中间层成形结构示意图。
图5为神经导管内层和中间层以及外层成形结构示意图。
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、2所示为制备Y型含细胞的神经导管***示意图。其中:图1为生物3D打印***图:计算机控制***1连接控制器2,控制器2连接微量泵3,微量泵3连接喷头4,5为接收平台。图2包含细胞电纺丝与近场直写***。计算机控制***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
Y型含细胞的神经导管制备方法,神经导管由可降解的高分子聚合物和细胞组成,利用生物3D打印挤出成形和细胞电纺丝、近场直写复合工艺制备,包括以下步骤:
1)称取15克明胶材料,在搅拌情况下缓缓加入到80克的去离子水中,至充分溶胀后升温至80℃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,将消毒后的材料装入喷头二中备用;
2)称取1.5克聚乙烯醇(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℃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;
3)第4代人的神经干细胞用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,将其装入喷头三中备用;
4)称取10克聚对二氧环己酮(PPDO)颗粒置于喷头一(内层神经导管挤出喷头)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。喷头一与接收平台的距离为1.5mm,将材料以370ul/min稳定流量由微量泵提供,进行连续挤出。接收平台按照所构建的Y型神经导管模型沿X/Y方向进行规律运动,并迅速固化成形,完成内层神经导管制备;
5)将固化成形后的内层神经导管安装在旋转装置上,运动到喷头二(中间层神经导管挤出喷头)工位,喷头与内层神经导管的外表面距离为0.5mm,将材料以400ul/min稳定流量由微量泵提供,进行连续挤出。旋转装置一边旋转一边移动,明胶材料包裹住PPDO并固化成形,完成中间层神经导管;
6)旋转装置运动到喷头三(外层神经导管喷头)工位,喷头三上加载8KV的直流电压,喷头和神经导管之间的距离为5mm,负极加载在接收平台上。将材料以180ul/min稳定流量由微量泵提供,旋转装置一边旋转一边移动,纺丝3个小时,在中间层神经导管上沉积一层含细胞的纳米纤维膜,完成神经导管的整个制备。最后得到含细胞的纳米纤维神经导管。
实施例2
本实例与实例1基本相同,不同之处在于:步骤3)所采用的细胞为神经元细胞。
Claims (2)
1.Y型含细胞的神经导管制备方法,神经导管由可降解的高分子聚合物和细胞组成,利用生物3D打印挤出成形、细胞电纺丝和近场直写复合工艺制备,包括以下步骤:
1)称取15克明胶材料,在搅拌情况下缓缓加入到80克的去离子水中,至充分溶胀后升温至80-100℃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,将消毒后的材料装入喷头二中备用;
2)称取1.5克聚乙烯醇(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℃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;
3)第4代人的细胞用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溶液搅拌均匀得到含细胞的PVA溶液,将其装入喷头三中备用;
4)称取10克聚对二氧环己酮(PPDO)颗粒置于喷头一加热装置中,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,喷头一与接收平台的距离为1.5mm,将材料以370μ L /min由微量泵提供的稳定流量,进行连续挤出,同时接收平台按照所构建的Y型神经导管模型沿X/Y方向进行规律运动,PPDO迅速固化成型,完成内层神经导管制备;
5)将固化成形后的内层神经导管安装在旋转装置上,运动到喷头二工位,喷头与内层神经导管的外表面距离为0.5mm,将材料以400μ L /min由微量泵提供的稳定流量,进行连续挤出,旋转装置一边旋转一边移动,明胶材料包裹住PPDO并固化成形,完成中间层和内层神经导管制备;
6)旋转装置运动到喷头三工位,喷头三上加载8k V的直流电压,喷头和神经导管之间的距离为5mm,负极加载在接收平台上,将材料以180μ L /min由微量泵提供的稳定流量,进行连续挤出,旋转装置一边旋转一边移动,纺丝3个小时,在中间层神经导管上沉积一层含细胞的纳米纤维膜,完成神经导管的整个制备。
2.根据权利要求1所述的Y型含细胞的神经导管的制备方法,其特征在于:所述步骤3)的细胞为神经干细胞或神经元细胞。
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