CN106166306B - 一种复合干细胞的仿生神经导管及其制作方法 - Google Patents

一种复合干细胞的仿生神经导管及其制作方法 Download PDF

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CN106166306B
CN106166306B CN201610725035.0A CN201610725035A CN106166306B CN 106166306 B CN106166306 B CN 106166306B CN 201610725035 A CN201610725035 A CN 201610725035A CN 106166306 B CN106166306 B CN 106166306B
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周佳
厉民
王峥
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Zhejiang Provincial Peoples Hospital
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Abstract

本发明涉及一种复合干细胞的仿生神经导管，包括编织管侧壁，所述编织管侧壁围成圆管，在编织管侧壁内设有凝胶和干细胞。本发明具有以下的特点和有益效果：神经导管所用材料为高分子可降解材料，材料易得，具备一定的强度且生物相容性好；通过编织得到的神经导管侧壁具有较多孔隙、具有一致的方向性，可以通过编织方法的不同，调节导管侧壁的致密疏松程度，利于营养物质的运输和交换；常压等离子体射流处理增加了神经导管编织纤维表面积，提高导管的亲水性和细胞亲和力；凝胶在导管内呈半凝固状态，形成支持界面和物质交换界面，有利于干细胞均匀分布在导管内部并定植、分化，有利于再生神经轴突生长、髓鞘形成，有利于长段神经修复。

Description

一种复合干细胞的仿生神经导管及其制作方法
技术领域
本发明涉及一种神经导管及其制作方法，尤其涉及一种复合干细胞的仿生神经导管及其制作方法。
背景技术
神经纤维缺损常由于创伤、挤压、手术等原因导致患者神经纤维直接或者间接受到损伤而造成。典型表现包括运动障碍、感觉障碍和自主神经功能障碍。神经纤维损伤后的修复与多种因素有关，但普遍存在着时间长、预后差等多种问题，甚至部分神经纤维的损伤、断裂、缺损根本无法修复，严重者将导致终身残疾。故神经纤维损伤后的再生和功能恢复一直是神经科学领域的热门课题。设计具有良好的生物相容性、生物可降解性和力学性能的神经导管是临床医学的重大需求。
目前，自体神经移植作为神经纤维损伤的金标准，是桥接修复神经缺损的经典方法，但这种方法存在一定缺陷，除供体来源有限，会造成供体部位部分功能丧失及永久性神经损伤，还受可修复长度、瘫痕形成等问题的限制。当前研究着重于发展能够通过设计仿生物理结构促使再生神经通过损伤区的神经导管。
具备单孔状截面结构的人工神经导管，在神经纤维再生过程中，可提供足够的力学支撑，以确保再生神经纤维不被塌陷或受压迫的导管结构以及体内组织所阻挡。但由于神经纤维是神经元轴突的集合，每个神经元要正确执行各自的功能，需要通过雪旺细胞在轴突表面形成的髓鞘，为其提供支持和营养并传导神经冲动。缺损的神经纤维的再生过程中，神经断端的雪旺细胞可不断增殖、移行至神经纤维缺损局部。但是在长段神经缺损情况下，由于局部炎症反应及纤维疤痕组织的长入，雪旺细胞的增殖、移行速度往往无法满足神经轴突修复的需要。
近年来研究发现，成年动物哺乳的组织中仍然存在干细胞，具有多向分化的能力，经过20－30个培养周期仍能保持其多向分化潜能。在适当的条件下可分化为骨骼肌细胞、血管内皮细胞、中枢神经元和神经胶质细胞等。干细胞在组织工程研究中有来源广泛、取材方便、自体取材、不存在伦理问题等优势，是组织工程研究、功能细胞再生以及动物创伤模型研究中的理想种子细胞。
既往的神经导管多采用可降解材料注模制备。然而高分子材料亲水性差、细胞吸附力弱，在制作细胞与支架材料复合物时，往往效果不甚理想。若在制备过程中加入造孔剂，残留物存在潜在细胞毒性，影响神经生长的危险；采用制备条件控制法造孔又存在孔隙不连续，缺乏方向性的缺点，因此导管的高度仿生化是制备人工神经导管的目标。
为了提供缺损神经纤维的良好修复再生条件，需要神经导管具备一定的强度、通透性以及较好的细胞亲和力、亲水性和较大的表面积，从而在保持神经轴突生长通道通畅、方便营养物质自由扩散的同时，为细胞提供良好的支持和生长界面；神经导管内部亦需要有大量雪旺细胞为再生神经轴突提供营养、支持并形成髓鞘结构，以便于神经轴突再生以及神经功能的恢复。故而需要设计既能提高神经纤维的结构修复程度又能提高神经纤维的修复速度的神经导管。
发明内容
本发明的目的是克服现有技术中的不足，提供一种由高分子丝线采用编织工艺制作成可降解的编织管，并通过常压等离子体处理后，将培养扩增获得的哺乳动物干细胞制成凝胶悬液，将编织管与干细胞凝胶悬液复合组装而成仿生神经导管。具有较好的仿生结构并能获得较快的神经纤维修复速度，提高了神经纤维修复的效果。
为实现上述技术目的，本发明采用了以下技术方案：这种复合干细胞的仿生神经导管，包括编织管侧壁，所述编织管侧壁围成圆管，在编织管侧壁内设有凝胶和干细胞。
作为优选：所述编织管侧壁的编织纱为2-4股长丝并捻而成，捻度为200-300捻/m。长丝平均细度6-12tex，为2-3股复丝组成，每根复丝中包含6-12根单丝，单丝直径10-18μm。
作为优选：所述编织管侧壁围成的导管的长度为5-100mm，内径为3-5mm，壁厚为0.5-0.8mm。
这种复合干细胞的仿生神经导管的制备方法，包括如下步骤：
1)导管编织所用的高分子丝线连接于编织装置，采用赫格利斯编织工艺或二维三轴向编织工艺，制作编织导管；
2)编织机锭数：8-16锭，编织环境：温度20±2℃，相对湿度65±2％；编织完毕，需要编织后定型，定型温度65-75℃，定型时间：10-20min；
3)对定型后的编织导管进行常压等离子体处理，所用气体为氦气或氧气，气体温度为90-110℃，流量为6-18L/min，处理时长为3-15min；
4)将干细胞，在37℃、5％CO2、95％～98％湿度的条件下扩增，通过传代纯化；收集干细胞后，在4℃条件下用ECM凝胶制成浓度为5×106～1×108cells/ml的悬液；
5)在25℃～28℃条件下，将干细胞凝胶悬液缓缓注入编织导管中，注射速度1-1.5ul/min。
作为优选：所述步骤1)中，制作单丝采用聚羟基丁酸-羟基戊酸共聚物PHBV，聚己内酯PCL，聚乙二醇PEG、聚乳酸PLA，聚羟基乙酸PGA，聚乳酸-羟基乙酸共聚物PLGA中的一种或几种材料。
作为优选：所述步骤1)中，编织导管所用的编织纱为2-4股长丝并捻而成，捻度为200-300捻/m；长丝平均细度6-12tex，为2-3股复丝组成，每根复丝中包含6-12根单丝，单丝直径10-18μm。
作为优选：所述步骤1)中，制作编织导管采用赫格利斯编织工艺或二维三轴向编织工艺，管壁孔隙率40％-65％。
本发明具有以下的特点和有益效果：1)神经导管所用材料为高分子可降解材料，材料易得，具备一定的强度且生物相容性好；2)通过编织得到的神经导管侧壁具有较多孔隙、具有一致的方向性，可以通过编织方法的不同，调节导管侧壁的致密疏松程度，利于营养物质的运输和交换；3)常压等离子体射流处理增加了神经导管编织纤维表面积，提高导管的亲水性和细胞亲和力；4)凝胶在导管内呈半凝固状态，形成支持界面和物质交换界面，有利于干细胞均匀分布在导管内部并定植、分化，有利于再生神经轴突生长、髓鞘形成，有利于长段神经修复；5)本发明的制备方法步骤简单，制作成本低，经济效益高。
附图说明
图1为本发明的结构示意图；
图2为本发明的截面结构示意图；
图3为再生神经的功能恢复比较图；
附图标记说明：编织管侧壁1、凝胶2、干细胞3。
具体实施方式
下面结合实施例对本发明做进一步描述。下述实施例的说明只是用于帮助理解本发明。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
实施例1
1)编织线的制备:制作直径12μm的单丝，8根单丝制作成复丝，2股复丝制作成长丝，将2股平均细度6tex的长丝，在温度25℃、相对湿度70％的大气条件下，按200捻/m将2股长丝并捻成编织线；
2)编织管的制作:将编织线在10锭立式锭子编织机上，温度20℃，相对湿度65％环境下，采用赫格利斯编织工艺进行编织，编织角为45°，套管内径为3mm，壁厚为0.5mm；
3)常压等离子体处理:所用气体为氦气，气体温度为90℃，流量为6L/min，处理时长为8min；
4)从自体或异体哺乳动物获得的骨髓基质干细胞，并置于37℃、5％CO2、95％湿度的培养箱中扩增，通过传代纯化；
5)在4℃的环境中，将收集的干细胞悬浮于ECM凝胶制成悬液，悬液的浓度为5×106cells/ml；
6)神经导管的组装：在25℃的环境中，以1ul/min的注射速度，将干细胞凝胶悬液缓缓注入编织导管中，组装形成仿生神经导管。
实施例2
1)编织线的制备:制作直径14μm的单丝，6根单丝制作成复丝，2股复丝制作成长丝，将3股平均细度8tex的长丝，在温度25℃、相对湿度70％的大气条件下，按250捻/m将2股长丝并捻成编织线；
2)编织管的制作:将编织线在12锭立式锭子编织机上，温度20℃，相对湿度65％环境下，采用二维三轴向编织工艺，编织角为55°，套管内径为4mm，壁厚为0.6mm；
3)常压等离子体处理:所用气体为氦气，气体温度为100℃，流量为10L/min，处理时长为6min；
4)从自体或异体哺乳动物获得的胚胎干细胞，并置于37℃、5％CO2、95％湿度的培养箱中扩增，通过传代纯化；
5)在4℃的环境中，将收集的干细胞悬浮于ECM凝胶制成悬液，悬液的浓度为1×107cells/ml
6)神经导管的组装：在28℃的环境中，以1.5ul/min的注射速度，将干细胞凝胶悬液缓缓注入编织导管中，组装形成仿生神经导管。
实施例1-2的神经导管促进再生神经功能恢复的比较如下：
再生神经的功能恢复比较如图3所示。不同导管修复大鼠神经缺损6周后，用行走足印对比法来计算坐骨神经指数(SFI)并进行比较。导管+干细胞组与单纯导管组及自体组SFI值均存在明显差异(P<0.05)。导管+干细胞组SFI值虽不及自体组(P<0.05)，但要优于与单纯导管组神经修复组(P<0.05)。

Claims (4)

1.一种复合干细胞的仿生神经导管的制备方法，复合干细胞的仿生神经导管包括编织管侧壁(1)，所述编织管侧壁(1)围成圆管，在编织管侧壁(1)内设有凝胶(2)和干细胞(3)，其特征在于包括如下步骤：
1)导管编织所用的高分子丝线连接于编织装置，采用赫格利斯编织工艺或二维三轴向编织工艺，制作编织导管；
2)编织机锭数：8-16锭，编织环境：温度20±2℃，相对湿度65±2％；编织完毕，需要编织后定型，定型温度65-75℃，定型时间：10-20min；
3)对定型后的编织导管进行常压等离子体处理，所用气体为氦气或氧气，气体温度为90-110℃，流量为6-18L/min，处理时长为3-15min；
4)将干细胞，在37℃、5％CO2、95％～98％湿度的条件下扩增，通过传代纯化；收集干细胞后，在4℃条件下用ECM凝胶制成浓度为5×106～1×108cells/ml的悬液；
5)在25℃～28℃条件下，将干细胞凝胶悬液缓缓注入编织导管中，注射速度1-1.5ul/min。
2.根据权利要求1所述的复合干细胞的仿生神经导管的制备方法，其特征在于：所述步骤1)中，制作单丝采用聚羟基丁酸-羟基戊酸共聚物PHBV，聚己内酯PCL，聚乙二醇PEG、聚乳酸PLA，聚羟基乙酸PGA，聚乳酸-羟基乙酸共聚物PLGA中的一种或几种材料。
3.根据权利要求1所述的复合干细胞的仿生神经导管的制备方法，其特征在于：所述步骤1)中，编织导管所用的编织纱为2-4股长丝并捻而成，捻度为200-300捻/m；长丝平均细度6-12tex，为2-3股复丝组成，每根复丝中包含6-12根单丝，单丝直径10-18μm。
4.根据权利要求1所述的复合干细胞的仿生神经导管的制备方法，其特征在于：所述步骤1)中，制作编织导管采用赫格利斯编织工艺或二维三轴向编织工艺，管壁孔隙率40％-65％。
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