CN113509593A - 一种定向纳米纤维神经导管的制备方法 - Google Patents
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Abstract
本发明公开了一种定向纳米纤维神经导管的制备方法，包括以下步骤：(1)将聚乳酸和明胶共混溶于六氟异丙醇溶液中，配置成静电纺丝溶液；(2)将神经生长因子和黄芪多糖加入静电纺丝溶液中；(3)使用静电纺丝方法制备定向纳米纤维神经导管；(4)将所述神经导管加入到0.1‑2％的京尼平溶液中进行交联，洗涤后冷冻干燥，即得。该方法制备的神经导管具有优异的生物相容性、机械性能和降解性能，纳米纤维具有定向分布信号，可引导神经细胞轴突延伸。本发明实现了药物对促进神经再生的协同作用，大大提高了神经修复的速率，其中黄芪多糖还能为神经细胞的生长以及增殖分化提供良好的微环境，降低了炎症反应的发生。
Description
技术领域
本发明涉及生物医用材料领域，具体涉及一种定向纳米纤维神经导管的制备方法。
背景技术
周围神经损伤的修复是一个具有挑战性的医学问题，自体神经移植由于具有非免疫原性作用，因此是周围神经修复中的“金标准”，但也存在供体组织的供应有限，神经瘤形成风险，大小不匹配以及供体神经与损伤部位之间的轴突分布等问题。神经导管是人工制成的组织工程管状物，作为自体神经移植的替代治疗方法用于桥接神经断端。然而，缺乏有效的生物相容性和生物活性是大多数人工神经导管的主要缺点。
神经导管常用的制备方法包括浇铸成型法、溶液浸渍法、熔融挤出法、铺膜法和静电纺丝法等。其中，静电纺丝法制备的导管具有纳米级尺寸纤维，而且比表面积高，孔隙率大，可模仿天然细胞外基质的结构，而且，电纺是在常温常压下进行，可以在加工过程中保持生长因子的活性。
根据来源，神经导管材料可以分为天然材料与人工合成材料两大类，二者各有优缺点。天然材料有高度仿生性质与固有生物活性，但材料的提取和利用过程中易改变其自然结构。合成材料结构和性能灵活且具有更好的定制属性，但缺乏天然材料的仿生性和生物相容性。聚乳酸(Polylactic acid，PLA)是一种合成的可降解类材料，其无毒、可塑性强、机械性能和生物相容性良好，但降解速率较慢，且降解产物呈酸性，易引发周围组织炎症，一般需要复合其它材料。明胶是胶原蛋白的第一级降解产物，它可以根据化学交联法来调节明胶的降解速率或者改变明胶聚合物的侧链基团来调节明胶的物理和化学特性，在这一阶段，明胶已被广泛应用。一般研究学者会加一些细胞生长因子或特定的活性官能团在明胶上，明胶与其他组织工程支架材料相比拥有更优秀的生物相容性，可以加快神经细胞生长分化。
研究发现黄芪多糖可以增加NGF蛋白的表达，从而促进神经再生。血管生成是指在机体生长发育过程中或创伤修复过程中微血管内皮细胞经过生芽、迁移、增殖及基质重塑等形成新毛细血管的过程。黄芪多糖能直接或间接活化CD34+细胞，促进神经内血管新生，改善损伤神经的微循环，增加对神经再生物质的供给、加快轴浆流的恢复。陈图刚等研究发现黄芪能增加外周血内皮组织细胞数量，促进内皮组织细胞的增殖能力、迁移能力和体外血管形成能力，并随黄芪多糖和作用时间的增加而增加。
发明内容
本发明的目的在于提供一种定向纳米纤维神经导管的制备方法，该方法制备的神经导管具有优异的生物相容性、机械性能和降解性能，纳米纤维具有定向分布信号，可引导神经细胞轴突延伸，以及神经生长因子按照纳米纤维的空间信号分布，能促进受损的周围神经有效再生，更好地治疗周围神经损伤。
为了解决上述技术问题，本发明采用如下技术方案：
一种定向纳米纤维神经导管的制备方法，包括以下步骤：
(1)将聚乳酸和明胶共混溶于六氟异丙醇溶液中，配置成静电纺丝溶液；
(2)将神经生长因子和黄芪多糖加入静电纺丝溶液中；
(3)使用静电纺丝方法制备定向纳米纤维神经导管；
(4)将所述神经导管加入到0.1-2％的京尼平溶液中进行交联，洗涤后冷冻干燥，即得。
优选地，步骤(1)中，所述聚乳酸与明胶的质量比为0.5-5:1。
优选地，步骤(2)中，所述神经生长因子的加入量为20-60ng，所述黄芪多糖的载入量为5-15mg。
优选地，步骤(3)中，所述所述静电纺丝参数为喷头与接收板之间距离为15cm，电压9-12kV，负电压3KV，流速1.0ml/h。
优选地，步骤(4)中，所述京尼平溶液的浓度为1.5％。
优选地，步骤(4)中，所述交联的温度为35-45℃，交联时间为10-60min。
根据本发明的一个实施例，其中一个最佳的制备方法如下：
(1)取聚乳酸0.3g，将其与明胶按1:1的质量比共混后溶于10ml六氟异丙醇溶液中，配置成静电纺丝溶液；
(2)向静电纺丝溶液中加入40ng神经生长因子和10mg黄芪多糖，搅拌后超声脱气；
(3)使用静电纺丝方法制备定向纳米纤维神经导管；
(4)将所得神经导管加入到1.5(重量)％的京尼平溶液中，37℃交联30min，洗涤后冷冻干燥，即得。
与现有技术相比，本发明的有益效果如下：
1.本发明提供的神经导管有定向排序的结构，其中纳米纤维的定向排序有利于细胞突触的定向生长，为神经再生提供正确的方向引导，神经导管内神经生长因子与黄芪多糖协同作用促进了神经细胞的生长分化，有助于促进神经生长和神经损伤的修复。本发明实现了药物对促进神经再生的协同作用，大大提高了神经修复的速率，其中黄芪多糖还能为神经细胞的生长以及增殖分化提供良好的微环境，降低了炎症反应的发生。
2.本发明制备方法简单，可操作性强，所制备的神经导管具备优异的生物相容性、机械性能和降解性能，具有很好的应用前景。
附图说明
图1为步骤1)制备的神经导管的SEM图。
图2为步骤2)制备的神经导管横截面SEM图。
图3为神经导管中黄芪多糖的释放曲线。
图4为神经导管在京尼平交联前后的降解失重比较结果。
具体实施方式
为了更好地理解本发明，下面结合实施例进一步阐明本发明的内容，但本发明的内容不仅仅局限于下面的实施例。
实施例1
一种定向纳米纤维神经导管的制备方法，包括以下步骤：
1)采用静电纺丝技术制备定向纤维神经导管。取0.3g明胶，与聚乳酸按质量比1:1混合，溶于10ml六氟异丙醇中制成纺丝液，然后加入40ng神经生长因子NGF和10mg黄芪多糖，磁力搅拌0.5-2h后超声脱气10min；将上述纺丝液加入10ml注射器，连接21G不锈钢喷头，设定喷头与接收板之间距离为15cm，以电压9-12kV，负电压3KV，流速1.0ml/h，接受速度为2400r/min为条件进行电纺。所制得的样品真空干燥24h排出残留有机溶剂。
图1为制备的神经导管的SEM图，图中纤维定向有序排布，其尺寸为纳米级，可诱导神经定向生长。
2)神经导管的交联
神经导管长度为15mm，将1.5％的京尼平交联液预热至37℃，将制备的神经导管放入无水乙醇中，即刻取出，再放入交联液中，37℃交联30min，每10min翻动一次。取出后先用无水乙醇洗涤5min，再用蒸馏水洗涤3次，每次10min，取出后冷冻干燥。材料分装于自封袋中，灭菌后常温保存备用。
图2为制备的导管横截面SEM图，本发明制备的神经导管长度为15mm，内径约为2mm，外径约为2.2mm。
京尼平交联前神经导管的最大载荷为22.4-27.5N，断裂载荷为11.7-15.5N。交联后最大载荷为38.9-42.9N，断裂载荷为21.3-28.8N，交联前后差异显著(P<0.05)。本发明制备的神经导管具有一定的力学强度，用于神经导管支架可起到很好的支撑作用。
经检测，京尼平交联前神经导管的透气率为(66.2±2.3)％，交联后神经导管的透气率为(72.5±3.1)％，交联后的透气率略有增加。良好的透气性可使神经导管与周围组织液进行交换，为导管内营造良好的微环境。
试验例
1.细胞生物相容性试验
1.1试验方法
1.1.1细胞的培养
RSC96细胞培养液：高糖DMEM(89％),双抗(1％),胎牛血清(10％)；
PC12细胞培养液：RPMI1640(81.5％),双抗(1％),胎牛血清(2.5％),马血清(15％)；
1.1.2细胞的传代培养
细胞复苏：首先将细胞从液氮罐中取出，37℃解冻，在细胞操作台上将其置于5mL细胞培养液中，离心，吸取上清液，加入5mL新鲜的培养液，反复轻轻吹打，转移至细胞培养瓶中，放置于CO2饱和湿度为5％的细胞培养箱中培养，隔天换一次新鲜培养液。
细胞传代：在倒置显微镜下观察细胞生长状况，约长满细胞瓶底时，加入胰酶进行消化，时间为30s-l分钟，将细胞培养瓶中的细胞液转移至15mL离心管中，离心，吸取上清液，用细胞计数器计数，并稀释，将其放置细胞培养箱中继续培养。
1.1.3细胞毒性试验
CCK-8的原理是WST-8在电子耦合作用下被细胞线粒体中的脱氢还原酶还原，生成橙黄色物质，颜色越深说明其细胞增殖效果越好，活细胞数量较多，反之，颜色越浅，活细胞数量越少，毒性越高。
选取RSC96与PC12两种细胞对神经导管进行毒性评价，一共4组:定向纳米纤维载神经生长因子神经导管组、定向纳米纤维载黄芪多糖神经导管组、定向纳米纤维载神经生长因子与黄芪多糖神经导管组、空白对照组。
材料的细胞毒性试验根据国标进行，将各试验材料置于超净工作台紫外光照射12小时灭菌，用生理盐水浸泡10分钟，然后将培养至2-3代的细胞用胰酶消化，稀释细胞浓度为lxlO4/mL，向各孔加入100μL细胞悬液，每组设6个复孔，培养5天，隔天换一次新鲜培养液。于第5天进行毒性检测，先在倒置荧光显微镜下观察细胞生长状态，然后向每孔加入10μLCCK-8，轻轻晃动使其混合均匀，在细胞培养箱培养4小时，取出培养板，弃去上清液，酶标仪上检测其OD值，根据OD值判断细胞毒性。
1.2实验结果
表1为RSC96细胞的细胞毒性试验结果，表2为PC12细胞的细胞毒性试验结果。从表中可知，定向纳米纤维载神经生长因子与黄芪多糖神经导管组OD值最高，说明其对RSC96和PC12细胞增殖的促进作用最强。
表1
| 组别 | OD450 |
| 空白对照组 | 1.62 |
| 定向纳米纤维载神经生长因子神经导管组 | 1.71 |
| 定向纳米纤维载黄芪多糖神经导管组 | 1.66 |
| 定向纳米纤维载神经生长因子与黄芪多糖神经导管组 | 1.83 |
表2
| 组别 | OD450 |
| 空白对照组 | 1.53 |
| 定向纳米纤维载神经生长因子神经导管组 | 1.64 |
| 定向纳米纤维载黄芪多糖神经导管组 | 1.58 |
| 定向纳米纤维载神经生长因子与黄芪多糖神经导管组 | 1.70 |
2.药物释放度检测
2.1试验方法
取15mm神经导管置于蒸馏水中，置于37℃条件下恒温振荡器上；在0.5h、lh、2h、6h、12h、24h、48h、72h和96h时间点取样并测定其在278nm处的吸光度，根据标准曲线计算出各时间点的黄芪多糖含量，绘制出黄芪多糖从其中释放的缓释曲线。
2.2实验结果
图3为神经导管中黄芪多糖的释放曲线。黄芪多糖在前2天释放速度较快，在第3天趋于平稳，累积缓释率为80.12％。
3.神经导管的降解试验
3.1试验方法
将材料置于pH＝7.4的磷酸缓冲液中模拟体内温度环境进行恒温降解,考察京尼平交联前后神经导管的降解性能，考察指标：神经导管各时间点质量变化。将同等大小的交联前和交联后神经导管切片，干燥后称重，质量记为Wo,置于离心管中，并加入等量的PBS(pH＝7.4)溶液，模拟体液环境恒温降解。固定时间点取出各样品，并用蒸馏水冲洗材料表面，采用滤纸吸干表面多余的水分，干燥至恒重并称重记为W1,计算质量失重率为：(W0-W1)/W0×l00％。
3.2实验结果
图4为本发明制备的神经导管在京尼平交联前后的降解失重对比图，图中失重变化现象说明，在第4周降解实验结束后，交联组失重率只能达到8.6％,而未交联的神经导管降解率达到22.5％,说明京尼平降低了神经导管的降解速度。
实施例2
一种定向纳米纤维神经导管的制备方法，包括以下步骤：
1)采用静电纺丝技术制备定向纤维神经导管。
取0.3g聚乳酸，与明胶按质量比2:1混合，溶于10ml六氟异丙醇中制成纺丝液，然后加入30ng神经生长因子NGF和15mg黄芪多糖，磁力搅拌0.5-2h后超声脱气10min；将上述纺丝液加入10ml注射器，连接21G不锈钢喷头，设定喷头与接收板之间距离为15cm，以电压9-12kV，负电压3KV，流速1.0ml/h，接受速度为2400r/min为条件进行电纺。所制得的样品真空干燥24h排出残留有机溶剂。
2)神经导管的合成与交联
神经导管长度为15mm，将0.7％的京尼平交联液预热至37℃，将制备的神经导管放入无水乙醇中，即刻取出，再放入交联液中，37℃交联30min，每10min翻动一次。取出后先用无水乙醇洗涤5min，再用蒸馏水洗涤3次，每次10min，取出后冷冻干燥。材料分装于自封袋中，灭菌后常温保存备用。
实施例3
一种定向纳米纤维神经导管的制备方法，包括以下步骤：
1)采用静电纺丝技术制备定向纤维神经导管。
取0.3g聚乳酸与明胶按质量比4:1混合，溶于10ml六氟异丙醇中制成纺丝液，然后加入60ng神经生长因子NGF和8mg黄芪多糖，磁力搅拌0.5-2h后超声脱气10min；将上述纺丝液加入10ml注射器，连接21G不锈钢喷头，设定喷头与接收板之间距离为15cm，以电压9-12kV，负电压3KV，流速1.0ml/h，接受速度为2400r/min为条件进行电纺。所制得的样品真空干燥24h排出残留有机溶剂。
2)神经导管的合成与交联
定向纤维神经导管长度为15mm，然后将2％的京尼平交联液预热至37℃，将制备的神经导管放入无水乙醇中，即刻取出，再放入交联液中，37℃交联30min，每10min翻动一次。取出后先用无水乙醇洗涤5min，再用蒸馏水洗涤3次，每次10min，取出后冷冻干燥。材料分装于自封袋中，灭菌后常温保存备用。
实施例4
一种定向纳米纤维神经导管的制备方法，包括以下步骤：
1)采用静电纺丝技术制备定向纤维神经导管。
取0.3g聚乳酸，与明胶按质量比0.6:1混合，溶于10ml六氟异丙醇中制成纺丝液，然后加入50ng神经生长因子NGF和6mg黄芪多糖，磁力搅拌0.5-2h后超声脱气10min；将上述纺丝液进行电纺。所制得的样品真空干燥24h排出残留有机溶剂。
2)神经导管的合成与交联
定向纤维神经导管长度为15mm，然后将0.5％的京尼平交联液预热至37℃，将制备的神经导管放入无水乙醇中，即刻取出，再放入交联液中，37℃交联30min，每10min翻动一次。取出后先用无水乙醇洗涤5min，再用蒸馏水洗涤3次，每次10min，取出后冷冻干燥。材料分装于自封袋中，灭菌后常温保存备用。
按照试验例1的方法对实施例2-4的细胞相容性进行检测，试验所用的细胞为RSC96细胞，检测时间为共培养后的第5天，结果测得的OD值如下：
从试验结果的比较中可以看出，实施例1制备的定向纳米纤维神经导管其对细胞的生物相容性高于实施例2-4。
Claims (6)
1.一种定向纳米纤维神经导管的制备方法，其特征在于包括以下步骤：
(1)将聚乳酸和明胶共混溶于六氟异丙醇溶液中，配置成静电纺丝溶液；
(2)将神经生长因子和黄芪多糖加入静电纺丝溶液中；
(3)使用静电纺丝方法制备定向纳米纤维神经导管；
(4)将所述神经导管加入到0.1-2％的京尼平溶液中进行交联，洗涤后冷冻干燥，即得。
2.如权利要求1所述定向纳米纤维神经导管的制备方法，其特征在于：步骤(1)中，所述聚乳酸与明胶的质量比为0.5-5:1。
3.如权利要求1所述定向纳米纤维神经导管的制备方法，其特征在于：步骤(2)中，所述神经生长因子的加入量为20-60ng，所述黄芪多糖的载入量为5-15mg。
4.如权利要求1所述定向纳米纤维神经导管的制备方法，其特征在于：步骤(3)中，所述静电纺丝参数为喷头与接收板之间距离为15cm，电压9-12kV，负电压3KV，流速1.0ml/h。
5.如权利要求1所述定向纳米纤维神经导管的制备方法，其特征在于：步骤(4)中，所述京尼平溶液的浓度为1.5％。
6.如权利要求1所述定向纳米纤维神经导管的制备方法，其特征在于：步骤(4)中，所述交联的温度为35-45℃，交联时间为10-60min。
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