CN109125803A - 一种导电型分区式聚丙烯腈脊髓导管的制备方法 - Google Patents
一种导电型分区式聚丙烯腈脊髓导管的制备方法 Download PDFInfo
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Abstract
本发明公开了一种导电型分区式聚丙烯腈脊髓导管的制备方法,选用聚丙烯腈为原料,利用取向冷冻技术联合高温碳化制备宏观构型与脊髓组织结构匹配、微观结构有序多孔的导电型分区式脊髓导管;选用外源性电刺激作为物理因子和特定生物活性因子,协同调控复合神经干细胞的定向分化,实现NSCs在同一脊髓导管内不同分区分化为与脊髓灰质、白质对应的功能细胞。研究制备参数对脊髓导管微观结构、导电性能的影响;揭示导管微观结构与细胞粘附、迁移的关联效应;探究物理/生物活性因子协同调控NSCs在脊髓导管内的分化规律,为组织工程化神经支架的设计构建提供实验依据和理论指导。
Description
技术领域
本发明属于医用材料领域,具体涉及一种导电型分区式聚丙烯腈脊髓导管的制备方法。
技术背景
随着社会经济水平的发展,脊髓损伤发生率呈现逐年增高的趋势。改善局部再生微环境以有效支持神经再生成为目前脊髓损伤修复研究的关键策略之一。自体或异体移植可修复损伤脊髓的部分功能,但效果并不理想;而组织工程化神经支架则为修复脊髓损伤提供了新的思路。理想的神经支架可为神经断端架起一座桥梁并提供合适的微环境,为轴突再生及神经结构的重塑提供仿生结构支持和导向信息。初期神经支架修复脊髓损伤的思路来源于已发展成熟的外周神经修复,但组织学结构的差异,使得脊髓修复的效果并不理想。因此,针对脊髓结构特征设计相匹配的脊髓导管构型是组织工程化神经支架构建的重点。
神经干细胞(Neural Stem Cell,NSCs)是一种多潜能的神经母细胞,能够自我更新、复制,具有分化为神经元、星形胶质细胞及少突胶质细胞的能力NSCs的存活和分化受到多因素调控作用,比如内源性基因调控,外源性生物、化学因子调控等。物理因子刺激(电刺激、机械力、超声波等)是调控NSCs分化的另一重要途径。将NSCs分化为神经元重建损伤两端功能性连接,以及分化为少突胶质细胞促进再生髓鞘化对脊髓损伤修复十分重要。因此,在分区式脊髓导管内复合NSCs并调控其在相应分区内分化为合适的神经细胞是脊髓损伤修复的关键。
NSCs的存活和分化受到多因素调控作用,比如内源性基因调控,外源性生物、化学因子调控等。物理因子刺激(电刺激、机械力、超声波等)是调控NSCs分化的另一重要途径。电刺激因具有使用方便、无创和副作用小等优点,越来越受到研究者的青睐;它能有效促进体内、体外神经再生,并且能够调控干细胞的粘附、迁移、增殖和分化
聚丙烯腈(polyacrylonitrile,PAN)经预氧化、高温热处理得到的碳化PAN可作为CNT的替代物用于组织工程。高温碳化过程中,N元素以N2形式大量脱出,使碳元素富集,PAN聚合物结构向多晶碳结构转变,最终形成类似于CNT的石墨结构。高温碳化过程中无需引入催化剂,使得碳化PAN具有良好的生物相容性。其次,通过改变高温碳化的条件可有效调控碳化PAN的形貌、强度及导电活性。体外研究表明,碳化PAN纤维能支持、引导神经纤维再生;将其植入横断的大鼠脊髓内,它能支持轴突的生长,减少脊髓残端空洞的形成,具有良好的组织相容性。
因此,本发明以静电纺丝聚丙烯腈一维纳米纤维为原料,通过高速搅拌,定向冷冻,冷冻干燥,预氧化,碳化等工艺过程,成功制备了一种具有多孔有序结构的导电型分区式聚丙烯腈脊髓导管。
发明内容
本发明目的在于提供一种导电型分区式聚丙烯腈脊髓导管的制备方法。材料生物相容性好,制备方法简单易操作,利于脊髓分区生长兼有优良的导电活性,适用于脊髓损伤修复。
为解决上述技术问题,本发明提出导电型分区式聚丙烯腈脊髓导管的制备方法如下:
1) 在搅拌下,配制一定浓度的聚丙烯腈(PAN)纺丝液;
2) 通过静电纺丝法,制备PAN纳米纤维;
3) 将步骤2)的PAN纳米纤维分散于去离子水中,高速搅拌后得到均匀分散的分散液;
4) 将步骤3)得到的分散液注入模具,运用单面接触法进行定向冷冻,随后真空冻干,制备形成分区式聚丙烯腈脊髓导管;
5) 在空气条件下程序升温,将步骤4)得到的导管进行预氧化,得到具有含氧官能团的亲水性预氧化分区式聚丙烯腈脊髓导管;
6) 利用程序升温过程,将步骤5)所得的预分区式聚丙烯腈脊髓导管进行碳化,得到导电型分区式聚丙烯腈脊髓导管。
本发明步骤1)中,聚丙烯腈纺丝液所用的溶剂为N,N-二甲基甲酰胺,掺杂2-5%的对苯二甲酸(PTA),聚丙烯腈纺丝液的质量浓度为5-10%
本发明步骤2)中,静电纺丝工艺参数为:电压为10-15 kV,所用纺丝容器为针孔直径为0.5 mm的10 mL塑料注射器,流速为0.05-0.15 mm/min,接收距离为10-20 cm。
本发明步骤3)中,对聚丙烯腈纳米纤维的高速搅拌借助型号IKA T10的匀浆仪实现,搅拌速度为15000-21000 r/min,搅拌时间为10-30 min。
本发明步骤4)中,模具为根据人脊髓水平结构,设计的三维仿真脊髓导管,由3D打印制备而成,材料为有机玻璃(PMMA)。
本发明步骤5)中所述的程序升温,升温速率为2-5℃/min,升至250-300℃后保持1-2 h。
本发明步骤6)中,预氧化分区式聚丙烯腈脊髓导管在氮气氛围中程序升温,以2-5℃/min 的升温速率从室温升至800-1200℃,保持1-2h,然后自然降至室温。
本发明制备的导电型分区式聚丙烯腈脊髓导管,生物相容性高,导电性能好,制备方法简单易操作,能与脊髓灰质、白质构型匹配的分区式脊髓导管利于脊髓分区生长,适用于脊髓损伤修复。
附图说明
图1为实施例产物的扫描电镜图片。
具体实施方式
为了使本发明易于理解,将通过下面的实施例进一步阐述本发明,应理解,下面实施例仅用于说明本发明而不用于限制本发明的范围,下面实施例中未提及的具体实验方法,通常按照常规实验方法进行。
实施例1,本实施例包括以下步骤:
称取0.5 g对苯二甲酸(PTA)粉末混入10 mL DMF中,于室温下搅拌10 min至完全溶解。称取1 g聚丙烯腈(PAN)粉末溶于溶液,于80℃加热搅拌3 h得到颜色微黄的聚丙烯腈纺丝溶液。通过静电纺丝技术将上述溶液纺成纳米纤维,如图1。具体参数为:电压为12 kV,所用纺丝容器为真空直径20 mm的10 mL塑料注射器,流速为0.08 mm/min,接收距离为15 cm,纺丝时间为10 h。将沉积在收集器上的聚丙烯腈纳米纤维放入60℃烘箱放置24 h。
将2 g上述聚丙烯腈纳米纤维分散于50 mL去离子水中,在18000 r/min的搅拌速度下高速搅拌10 min,得到均匀分散的悬浮液。将该混合悬浮液取适量倒入冰模板模具中,定向冷冻后在冷冻干燥机中冷冻干燥48 h,得到分区式聚丙烯腈脊髓导管。
随后,将上述分区式聚丙烯腈脊髓导管进行预氧化,具体程序为:以2℃/min的升温速率从室温升至280℃,并保持2 h后自然降至室温。然后对样品进行碳化,具体程序为:在氮气的保护下,以5℃/min的升温速率从室温升温至800℃,保持2 h后自然降至室温。
实施例2,本实施例包括以下步骤:
称取0.3 g对苯二甲酸(PTA)粉末混入10 mL DMF中,于室温下搅拌10 min至完全溶解。称取1.2 g聚丙烯腈(PAN)粉末溶于溶液,于70℃加热搅拌3 h得到颜色微黄的聚丙烯腈纺丝溶液。通过静电纺丝技术将上述溶液纺成纳米纤维,具体参数为:电压为10 kV,所用纺丝容器为真空直径20 mm的10 mL塑料注射器,流速为0.05 mm/min,接收距离为10 cm,纺丝时间为8 h。将沉积在收集器上的聚丙烯腈纳米纤维放入60℃烘箱放置24 h。
将3 g上述聚丙烯腈纳米纤维分散于50 mL去离子水中,在18000 r/min的搅拌速度下高速搅拌30 min,得到均匀分散的悬浮液。将该混合悬浮液取适量倒入冰模板模具中,定向冷冻后在冷冻干燥机中冷冻干燥48 h,得到分区式聚丙烯腈脊髓导管。
随后,将上述分区式聚丙烯腈脊髓导管进行预氧化,具体程序为:以3℃/min的升温速率从室温升至300℃,并保持2 h后自然降至室温。然后对样品进行碳化,具体程序为:在氮气的保护下,以5℃/min的升温速率从室温升温至1000℃,保持1 h后自然降至室温。
实施例3,本实施例包括以下步骤:
称取0.5 g对苯二甲酸(PTA)粉末混入10 mL DMF中,于室温下搅拌10 min至完全溶解。称取1.2 g聚丙烯腈(PAN)粉末混入10 mL DMF中,于80℃加热搅拌3 h得到颜色微黄的聚丙烯腈纺丝溶液。通过静电纺丝技术将上述溶液纺成纳米纤维,具体参数为:电压为15 kV,所用纺丝容器为真空直径20 mm的10 mL塑料注射器,流速为0.10 mm/min,接收距离为20 cm,纺丝时间为8 h。将沉积在收集器上的聚丙烯腈纳米纤维放入60℃烘箱放置24 h。
将2 g上述聚丙烯腈纳米纤维分散于50 mL去离子水中,在21000 r/min的搅拌速度下高速搅拌30 min,得到均匀分散的悬浮液。将该混合悬浮液取适量倒入冰模板模具中,定向冷冻后在冷冻干燥机中冷冻干燥48 h,得到分区式聚丙烯腈脊髓导管。
随后,将上述分区式聚丙烯腈脊髓导管进行预氧化,具体程序为:以5℃/min的升温速率从室温升至300℃,并保持1 h后自然降至室温。然后对样品进行碳化,具体程序为:在氮气的保护下,以5℃/min的升温速率从室温升温至1200℃,保持1 h后自然降至室温。
Claims (7)
1.一种导电型分区式聚丙烯腈脊髓导管的制备方法,其特征在于具体步骤如下:
1)在搅拌下,配制聚丙烯腈(PAN)纺丝液;
2)通过静电纺丝法,制备PAN纳米纤维;
3)将步骤(2)的PAN纳米纤维分散于去离子水中,高速搅拌后得到均匀分散的分散液;
4)将步骤(3)得到的分散液注入模具,运用单面接触法进行定向冷冻,随后真空冻干,制备形成分区式聚丙烯腈脊髓导管;
5)将步骤(4)得到的导管进行预氧化,得到具有含氧官能团的亲水性预氧化分区式聚丙烯腈脊髓导管;
6)利用程序升温过程,将步骤(5)所得的预分区式聚丙烯腈脊髓导管进行碳化,得到导电型分区式聚丙烯腈脊髓导管。
2.根据权利要求1所述的导电型分区式聚丙烯腈脊髓导管的制备方法,其特征在于步骤(1)所述的聚丙烯腈纺丝液所用的溶剂为N,N-二甲基甲酰胺,掺杂2-5%的对苯二甲酸(PTA),聚丙烯腈纺丝液的质量浓度为5-10%。
3.根据权利要求1所述的导电型分区式聚丙烯腈脊髓导管的制备方法,其特征步骤(2)中,静电纺丝工艺参数为:电压为10-15 kV,所用纺丝容器为针孔直径为20 mm的10 mL塑料注射器,流速为0.05-0.15 mm/min,接收距离为10-20 cm。
4.根据权利要求1所述的导电型分区式聚丙烯腈脊髓导管的制备方法,其特征在于步骤(3)中,搅拌速度为15000-21000 r/min,搅拌时间为10-30 min。
5.根据权利要求1所述导电型分区式聚丙烯腈脊髓导管的制备方法,其特征在于步骤(4)中,模具为根据人脊髓水平结构,设计的三维仿真脊髓导管,由3D打印制备而成,材料为有机玻璃(PMMA)。
6. 根据权利要求1所述的导电型分区式聚丙烯腈脊髓导管的制备方法,其特征在于步骤(5)中升温速率为2-5℃/min,升至250-300℃后保持1-3 h。
7.根据权利要求1所述的导电型分区式聚丙烯腈脊髓导管的制备方法,其特征在于步骤(6)中,预氧化分区式聚丙烯腈脊髓导管在氮气氛围中程序升温,具体参数如下:以2-5℃/min 的升温速率从室温升至800-1200℃,保持1-2h,然后自然降至室温。
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