CN113730658A - 一种仿生骨软骨一体化修复支架及其制备方法 - Google Patents

一种仿生骨软骨一体化修复支架及其制备方法 Download PDF

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CN113730658A
CN113730658A CN202111104356.6A CN202111104356A CN113730658A CN 113730658 A CN113730658 A CN 113730658A CN 202111104356 A CN202111104356 A CN 202111104356A CN 113730658 A CN113730658 A CN 113730658A
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胡银春
丁慧秀
黄棣
魏延
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Taiyuan University of Technology
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Abstract

本发明涉及生物医学工程骨软骨修复支架领域，仿生骨软骨一体化修复支架，由表面软骨修复层、中间过渡层和软骨下骨修复层组成，所述表面软骨修复层为核壳结构的聚己内酯纳米纤维膜，内核为载有氨基葡萄糖的透明质酸钠，外壳为生物相容性良好的聚己内酯；软骨下骨修复层为矿化聚己内酯串晶纳米纤维膜；中间过渡层为核壳结构的聚己内酯串晶纳米纤维膜，为二者的有效结合提供支撑，实现骨软骨一体化修复。本发明还涉及该仿生骨软骨一体化修复支架的制备方法。

Description

一种仿生骨软骨一体化修复支架及其制备方法
技术领域
本发明涉及一种仿生骨软骨一体化修复支架及其制备方法，属于生物医学工程骨软骨修复支架领域，在骨软骨修复方面具有广泛的应用价值。
背景技术
骨软骨损伤是临床上常见的关节疾病。骨软骨组织包括关节软骨和软骨下骨：关节软骨，负责减少运动中的摩擦和冲击；以及软骨下骨，负责提供机械支持。骨软骨缺陷通常涉及关节软骨和软骨下骨的病变，这些病变是由创伤、疾病或衰老引起的。临床上主要采用自体移植、异体移植、显微骨折术和马克赛成形术等手段进行治疗，但是供体来源有限、手术复杂和免疫排斥反应等问题限制了这些技术的应用。由于骨软骨界面的复杂性，针对骨软骨损伤的临床治疗局限性较大，组织工程学为解决这一难题提供了新思路。
为了实现软骨和软骨下骨的同时再生，生物支架的设计通常有以下几种：（1）单相支架；（2）双相支架；（3）多相支架。单相支架不能满足成骨细胞和软骨细胞生长所需的微环境。双相支架存在界面连接不良等问题。多相支架是在表面软骨修复层和软骨下骨修复层之间增加中间过渡层使其结合紧密，更符合骨软骨支架的要求。静电纺丝技术是常用的制备组织工程支架的技术，且生产的纤维能够有效模仿骨或软骨组织天然细胞外基质的形态。
虽然静电纺丝技术在组织工程中具有广阔的应用前景，但用于制备静电纺丝的材料仍需慎重选择。不同材料构建的支架在微观结构、表面形貌及力学性能等方面各有优劣。聚己内酯具有良好的生物相容性和力学性能, 在完成降解后产生的产物无毒。然而，聚己内酯疏水性强、细胞黏附性差等缺点限制了其进一步的应用。氨糖能促进人体粘多糖的合成，提高关节滑液的粘性，改善关节软骨的代谢，促进软骨的再生。
发明内容
本发明的目的在于提供一种仿生骨软骨一体化修复支架及其制备方法，解决现有技术中聚己内酯亲水性差和骨软骨修复支架界面结合的问题。该支架以聚己内酯为基材，兼具良好的生物相容性及力学性能，并且具有成本低、操作简单等优点。利用静电纺丝技术和仿生矿化法制备具有串晶结构的聚己内酯纳米纤维膜作为软骨下骨修复层；将上述纳米纤维膜固定于静电纺丝接收器上，利用微溶胶静电纺丝和自诱导结晶技术制备具有核壳结构的聚己内酯串晶纳米纤维膜作为中间过渡层；最后利用微溶胶静电纺丝技术制备核壳结构的聚己内酯纳米纤维膜作为表面软骨修复层；矿化聚己内酯串晶、核壳结构的聚己内酯串晶及核壳结构的聚己内酯构成的三层纳米纤维膜仿生构建骨软骨一体化修复支架。
本发明所采用的技术方案：仿生骨软骨一体化修复支架，由表面软骨修复层、中间过渡层和软骨下骨修复层组成，所述表面软骨修复层为核壳结构的聚己内酯纳米纤维膜，内核为载有氨基葡萄糖的透明质酸钠，外壳为生物相容性良好的聚己内酯；软骨下骨修复层为矿化聚己内酯串晶纳米纤维膜；中间过渡层为核壳结构的聚己内酯串晶纳米纤维膜，为二者的有效结合提供支撑，实现骨软骨一体化修复。
骨软骨一体化修复支架的制备方法：利用静电纺丝技术和仿生矿化法制备矿化聚己内酯串晶纳米纤维膜作为软骨下骨修复层；将矿化聚己内酯串晶纳米纤维膜固定于静电纺丝接收器上，利用微溶胶静电纺丝技术和自诱导结晶法制备核壳结构的聚己内酯串晶纳米纤维膜作为中间过渡层；最后利用微溶胶静电纺丝技术制备核壳结构的聚己内酯纳米纤维膜作为表面软骨修复层；所述的矿化聚己内酯串晶、核壳结构的聚己内酯串晶及核壳结构的聚己内酯构成的三层纳米纤维膜仿生构建骨软骨一体化修复支架。
所述微溶胶静电纺丝的工艺参数为：电压为20 kV，喷丝头和接收板之间的距离为20 cm，推进泵的推进速度为0.001 mm/s。
所述自诱导结晶法为将聚己内酯稀溶液滴在核壳结构的聚己内酯纳米纤维膜表面进行自诱导结晶，其中聚己内酯稀溶液中聚己内酯的质量分数为0.1wt%，0.2wt%和0.5wt%，冰乙酸和去离子水的体积比为3:1，聚己内酯的分子量为80000。
将纳米纤维膜浸泡在矿化液中进行仿生矿化，所述矿化液为过饱和钙磷混合液，矿化时间为4d。
所述表面软骨修复层的制备方法具体包括：载有氨糖（1mg/ml）的透明质酸钠水溶胶（1.2 wt%）与Span-80和二氯甲烷的混合物在高速搅拌下得到油包水型乳液，随后将聚己内酯和N,N-二甲基甲酰胺溶解在上述乳液中配制成浓度为9 wt%的纺丝溶液，利用静电纺丝技术得到核壳结构的聚己内酯纳米纤维膜；所述中间过渡层的制备方法具体包括：将聚己内酯稀溶液滴加到上述纳米纤维膜表面，待溶剂挥发完后得到具有核壳结构的聚己内酯串晶纳米纤维膜；所述软骨下骨修复层的制备方法具体包括：将聚己内酯溶解在二氯甲烷和N,N-二甲基甲酰胺（V:V=7:3）的混合液中配置成浓度为15 wt%的纺丝溶液，通过静电纺丝技术得到聚己内酯纳米纤维膜。将聚己内酯稀溶液滴加到聚己内酯纳米纤维膜上，待溶剂挥发完全后，将纤维膜在饱和钙磷混合液中孵育4d后得到具有串晶结构的聚己内酯纳米纤维膜。
进一步的，所述表面软骨修复层制备方法中透明质酸钠水溶胶的质量分数为1.2wt%，氨糖水溶液与透明质酸钠水溶胶的体积比为1:4；所述透明质酸钠水溶胶与二氯甲烷的体积比为60.6，Span-80和二氯甲烷的质量比为1:400；所述二氯甲烷的体积比与N,N-二甲基甲酰胺的体积比为1.43。
进一步的，所述聚己内酯稀溶液是用冰乙酸和去离子水进行稀释的，其中冰乙酸和去离子水的体积比为3:1，聚己内酯的质量分数为0.1wt%，0.2wt%和0.5wt%。
本发明的有益效果是：本发明根据骨软骨的层次结构，从仿生骨软骨天然结构设计了一种骨软骨一体化修复支架，该支架以聚己内酯为基材，兼具良好的生物相容性和力学性能，并且具有成本低、操作简单等优点。通过在表面软骨修复层中纳米纤维核内载氨糖促进软骨细胞增殖；软骨下骨修复层引入磷酸钙，修复复杂的大面积骨缺损，支撑表面软骨层修复；中间过渡层的串晶结构仿生胶原纤维的微结构，改善聚己内酯纳米纤维膜的亲水性和力学性能，为表面软骨修复层和软骨下骨修复层的有效结合提供支撑。
附图说明
图1是核壳结构的聚己内酯纳米纤维的SEM图；
图2是核壳结构的聚己内酯纳米纤维的直径分布图；
图3是聚己内酯纳米纤维的SEM图；
图4是聚己内酯纳米纤维的直径分布图；
图5是核壳结构的聚己内酯串晶（SKMGPCL02）纳米纤维膜的SEM图；
图6是矿化聚己内酯串晶（MSKPCL02）纳米纤维膜的SEM图；
图7是MC3T3-E1细胞在不同纳米纤维材料上的增殖情况；
PCL：聚己内酯纳米纤维膜；SKPCL01：实施例1中聚己内酯串晶纳米纤维膜；SKPCL02：实施例2中聚己内酯串晶纳米纤维膜；SKPCL05: 实施例3中聚己内酯串晶纳米纤维膜；MPCL：矿化聚己内酯纳米纤维膜；MSKPCL01：实施例1中矿化聚己内酯串晶纳米纤维膜；MSKPCL02：实施例2中矿化聚己内酯串晶纳米纤维膜；MSKPCL05: 实施例3中矿化聚己内酯串晶纳米纤维膜。
具体实施方式
为使本发明的技术方案和优点更加清楚，下面将结合附图和具体实施例对本发明提出的骨软骨一体化支架进一步阐述。所述的实施例仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
1.表面软骨修复层的制备：将透明质酸钠溶于去离子水中，得到浓度为1.2wt% 透明质酸钠水溶胶。将40 µl 氨糖水溶液(1 mg/ml)混合到200 µl透明质酸水溶胶中，随后加入12.12ml 二氯甲烷和0.04g Span-80的混合物并高速搅拌获得油包水型乳化液。加入2g聚己内酯和8.44ml N,N-二甲基甲酰胺，室温下充分搅拌得到纺丝液。静电纺丝过程是在纺丝电压20kv，针头和收集器间的距离20cm，推进速度0.001mm/s的条件下进行的，纺丝结束后得到核壳结构的聚己内酯纳米纤维膜。图1为核壳结构的聚己内酯纳米纤维膜的扫描电镜图片，可以看到光滑连续的纳米纤维结构，用ImageJ 软件分析纳米纤维直径为140.94±36.35nm (图2).
2.中间过渡层的制备：将聚己内酯溶于冰乙酸和去离子水(V:V=3:1)的混合溶剂中，70℃下磁力搅拌得到质量分数为0.1wt%的稀溶液。将聚己内酯稀溶液滴在上述纳米纤维膜表面进行自诱导结晶，之后真空干燥24h，去除残留溶剂，得到核壳结构的聚己内酯串晶（SKMGPCL01）纳米纤维膜.
3.软骨下骨修复层的制备：将聚己内酯溶于二氯甲烷和N,N-二甲基甲酰胺的混合液（V:V=7:3）中制备浓度为15wt%的静电纺丝溶液，将纺丝液吸入在注射器中进行静电纺丝，纺丝电压为20kv，喷丝头和接收板之间的距离为20cm，推进泵的推进速度为0.003mm/s，制备成聚己内酯纳米纤维膜。将聚己内酯稀溶液(0.1wt%)滴在聚己内酯纳米纤维膜表面进行自诱导结晶，之后真空干燥24h，去除残留溶剂，得到聚己内酯串晶（SKPCL01）纳米纤维膜。将纳米纤维膜浸泡在饱和氢氧化钙溶液中预矿化1h，去离子水漂洗后，37℃恒温浸泡于过饱和钙磷混合液中4d。矿化结束后，样品用去离子水漂洗，真空干燥后得到矿化聚己内酯串晶（MSKPCL01）纳米纤维膜。图3为聚己内酯纳米纤维膜扫描电镜图片，可以看到光滑的纳米纤维结构，用ImageJ 软件分析纳米纤维直径为199.3±43.8nm (图4).
4.骨软骨一体化修复支架的制备：通过逐层纺丝方法进行表面软骨修复层、中间过渡层和软骨下骨修复层仿生构建骨软骨一体化修复支架。
5.取上述实施例中表面软骨修复层和软骨下骨修复层的纳米纤维膜制成直径为1cm的圆片状并置于48孔板中，将软骨细胞和MC3T3-E1细胞制成细胞悬液。每个样品孔接种400微升细胞悬液，将细胞与材料共培养1、4、7天，不含纳米纤维膜为空白对照组。
实施例2
1.表面软骨修复层的制备：同实施例1
2.中间过渡层的制备：将聚己内酯溶于冰乙酸和去离子水(V:V=3:1)的混合溶剂中，70℃下磁力搅拌得到质量分数为0.2wt%的稀溶液。将聚己内酯稀溶液滴在上述纳米纤维膜表面进行自诱导结晶，之后真空干燥24h，去除残留溶剂，得到核壳结构的聚己内酯串晶（SKMGPCL02）纳米纤维膜.图5为SKMGPCL02纳米纤维膜的扫描电镜图, 可以明显看到纤维表面的片晶结构并且垂直于纤维轴周期排列。其中，直径较小的纤维的串晶结构较为规整。
3.软骨下骨修复层的制备：将聚己内酯溶于二氯甲烷和N,N-二甲基甲酰胺的混合液（V:V=7:3）中制备浓度为15wt%的静电纺丝溶液，将纺丝液吸入在注射器中进行静电纺丝，纺丝电压为20kv，喷丝头和接收板之间的距离为20cm，推进泵的推进速度为0.003mm/s，制备成聚己内酯纳米纤维膜。将聚己内酯稀溶液(0.2wt%)滴在上述纳米纤维膜表面进行自诱导结晶，之后真空干燥24h，去除残留溶剂，得到聚己内酯串晶(SKPCL02)纳米纤维膜。将纳米纤维膜浸泡在饱和氢氧化钙溶液中预矿化1h，去离子水漂洗后，37℃恒温浸泡于过饱和钙磷混合液中4d。矿化结束后，样品用去离子水漂洗，真空干燥后得到矿化聚己内酯串晶（MSKPCL02）纳米纤维膜。图6为MSKPCL02的扫描电镜图，可以看到磷酸钙涂层均匀覆盖MSKPCL02纳米纤维表面。
4.骨软骨一体化修复支架的制备：通过逐层纺丝方法进行表面软骨修复层、中间过渡层和软骨下骨修复层复合制备骨软骨一体化修复支架。
5.取上述实施例中表面软骨修复层和软骨下骨修复层的纳米纤维膜制成直径为1cm的圆片状并置于48孔板中，将软骨细胞和MC3T3-E1细胞制成细胞悬液。每个样品孔接种400微升细胞悬液，将细胞与材料共培养1、4、7天，不含纳米纤维膜为空白对照组。
实施例3
1. 表面软骨修复层的制备：同实施例1
2. 中间过渡层的制备：将聚己内酯溶于冰乙酸和去离子水的混合溶剂中(V:V=3:1)，70℃下磁力搅拌得到质量分数为0.5wt%的稀溶液。将聚己内酯稀溶液滴在上述纳米纤维膜表面进行自诱导结晶，之后真空干燥24h，去除残留溶剂，得到核壳结构的聚己内酯串晶（SKMGPCL05）纳米纤维膜.
3. 软骨下骨修复层的制备：将聚己内酯溶于二氯甲烷和N,N-二甲基甲酰胺的混合液（V:V=7:3）中制备浓度为15wt%的静电纺丝溶液，将纺丝液吸入在注射器中进行静电纺丝，纺丝电压为20kv，喷丝头和接收板之间的距离为20cm，推进泵的推进速度为0.003mm/s，制备成聚己内酯纳米纤维膜。将聚己内酯稀溶液(0.5wt%)滴在上述纳米纤维膜表面进行自诱导结晶，之后真空干燥24h，去除残留溶剂，得到聚己内酯串晶(SKPCL05)纳米纤维膜。将纳米纤维膜浸泡在饱和氢氧化钙溶液中预矿化1h，去离子水漂洗后，37℃恒温浸泡于过饱和钙磷混合液中4d。矿化结束后，样品用去离子水漂洗，真空干燥后得到矿化聚己内酯串晶（MSKPCL05）纳米纤维膜。
4. 骨软骨一体化修复支架的制备：通过逐层纺丝方法进行表面软骨修复层、中间过渡层和软骨下骨修复层复合制备骨软骨一体化修复支架。
5. 取上述实施例中表面软骨修复层和软骨下骨修复层的纳米纤维膜制成直径为1cm的圆片状并置于48孔板中，将软骨细胞和MC3T3-E1细胞制成细胞悬液。每个样品孔接种400微升细胞悬液，将细胞与材料共培养1、4、7天，不含纳米纤维膜为空白对照组。检测结果如图7所示，实施例2样品对MC3T3-E1细胞增殖的效果更显著。

Claims (6)

1.一种仿生骨软骨一体化修复支架，其特征在于，由表面软骨修复层、中间过渡层和软骨下骨修复层组成，所述表面软骨修复层为核壳结构的聚己内酯纳米纤维膜，内核为载有氨基葡萄糖的透明质酸钠，外壳为生物相容性良好的聚己内酯；软骨下骨修复层为矿化聚己内酯串晶纳米纤维膜；中间过渡层为核壳结构的聚己内酯串晶纳米纤维膜，为二者的有效结合提供支撑。
2.根据权利要求1所述骨软骨一体化修复支架，其特征在于，表面软骨修复层、中间过渡层和软骨下骨修复层实现骨软骨一体化修复。
3.根据权利要求1所述骨软骨一体化修复支架的制备方法，其特征在于：利用静电纺丝技术和仿生矿化法制备矿化聚己内酯串晶纳米纤维膜作为软骨下骨修复层；将矿化聚己内酯串晶纳米纤维膜固定于静电纺丝接收器上，利用微溶胶静电纺丝技术和自诱导结晶法制备核壳结构的聚己内酯串晶纳米纤维膜作为中间过渡层；最后利用微溶胶静电纺丝技术制备核壳结构的聚己内酯纳米纤维膜作为表面软骨修复层；所述的矿化聚己内酯串晶、核壳结构的聚己内酯串晶及核壳结构的聚己内酯构成的三层纳米纤维膜仿生构建骨软骨一体化修复支架。
4.根据权利要求3所述的骨软骨一体化修复支架的制备方法， 其特征在于，所述微溶胶静电纺丝的工艺参数为：电压为20kv，喷丝头和接收板之间的距离为20cm，推进泵的推进速度为0.001mm/s。
5.根据权利要求3所述的骨软骨一体化修复支架的制备方法，其特征在于，所述自诱导结晶法为将聚己内酯稀溶液滴在核壳结构的聚己内酯纳米纤维膜表面进行自诱导结晶，其中聚己内酯稀溶液中聚己内酯的质量分数为0.1wt%，0.2wt%和0.5wt%，冰乙酸和去离子水的体积比为3:1，聚己内酯的分子量为80000。
6.根据权利要求3所述的骨软骨一体化修复支架的制备方法，其特征在于，将纳米纤维膜浸泡在矿化液中进行仿生矿化，所述矿化液为过饱和钙磷混合液，矿化时间为4d。
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