CN108653807A - 一种表层包埋干细胞可持续诱导软骨分化的高强度水凝胶的制备方法 - Google Patents
一种表层包埋干细胞可持续诱导软骨分化的高强度水凝胶的制备方法 Download PDFInfo
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- CN108653807A CN108653807A CN201810616914.9A CN201810616914A CN108653807A CN 108653807 A CN108653807 A CN 108653807A CN 201810616914 A CN201810616914 A CN 201810616914A CN 108653807 A CN108653807 A CN 108653807A
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- hydrogel
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Abstract
本发明提供了一种表层包埋干细胞可持续诱导软骨分化的高强度水凝胶的制备方法。首先在干细胞表面包裹一层磷酸钙外壳。将硅酸钠、丙烯酰胺、交联剂和海藻酸钠溶解于水中得到铸膜液A，将包裹磷酸钙外壳的干细胞和生长因子分散到铸膜液A中得到铸膜液B。在A玻璃板上用缠绕铜丝的玻璃棒将铸膜液A刮均匀，在B玻璃板上用缠绕铜丝的玻璃棒将铸膜液B刮均匀，然后立即将玻璃板B倒扣在玻璃板A上。紫外引发丙烯酰胺聚合，经钙离子交联和葡萄糖酸‑δ‑内酯水溶液浸泡，将受保护的细胞释放出来，洗脱生长因子后，利用印迹孔穴和位点实现生长因子的识别和缓释，得到表层包埋干细胞可诱导软骨分化的高强度水凝胶。
Description
技术领域
本发明涉及一种表层包埋干细胞可持续诱导软骨分化的高强度水凝胶的制备方法，属于生物材料和组织工程领域。
背景技术
骨关节炎已成为世界上头号致残疾病，但是由于软骨中没有血管提供营养，软骨细胞迁徙能力差，关节软骨损伤后无法自行修复。目前临床上关节软骨损伤主要以手术治疗为主，包括骨切开术、关节镜修复术、自体软骨移植术和关节固定术等。这些方法仅能缓解疼痛，不能替代软骨的生物特性，更不能促进软骨再生。传统疗法费用昂贵，会出现炎症和排异反应等，长期疗效备受争议。目前关节软骨组织工程修复的研究尚处于实验阶段，仍存在支架材料机械性能差、降解不可控、材料缺乏生物活性、昂贵的生长因子易突释失活、不能持续诱导细胞软骨分化等问题。
水凝胶是由高分子三维网络与水组成的多元体系，其与许多组织和细胞外基质非常接近，表现出良好的生物相容性，放入体内不会引发排异反应，同时为营养物质运输和代谢废物的排出提供良好通道。水凝胶可作为永久软组织损伤替代物，或作为细胞培养基质材料。Chaudhuri等将含有软骨细胞的水凝胶用于替代损伤软骨【Nature Materials，2017，16：1243-1251】。常江等制备了可注射的复合水凝胶，该水凝胶可以促进大鼠骨髓间充质干细胞成骨分化【Acta Biomaterialia，2013，9(11)：9107-9117】。但是通常的水凝胶强度较低，限制了其在关节软骨修复中的实际应用。
高强度水凝胶的研究近年来取得了一系列重要进展。龚剑萍等提出“双层网络”水凝胶的思想，在形成高交联度的刚性第一层网络的凝胶基础上，其内部合成交联度较低的柔性第二层网络【Advanced Materials.2014，26：436-442】。这种水凝胶在保持高含水量的同时也具有高的强度。但是该双化学网络交联水凝胶制备过程比较复杂。锁志刚等人用一步法制备了高弹性高韧性聚丙烯酰胺/海藻酸钙(PAM/CaAlg)双网络水凝胶【Nature，2012，489(7414)：133-136】。此水凝胶具有良好的生物相容性、优良的润滑性和耐磨性，可达到替代软骨组织的要求。Bakarich等用3D打印技术制备了纤维增强的PAM/CaAlg水凝胶人工关节软骨替代物【ACS Applied Materials&Interfaces，2014，6(18)：15998-16006】。但是在生理环境下，PAM/CaAlg双网络水凝胶中的交联离子被释放出来，使凝胶的力学性能迅速下降。
目前，构建在长期生理环境下具备高强度、高韧性和低溶胀的水凝胶仍存在很大挑战。柳明珠等将二氧化硅引入PAM/CaAlg水凝胶中，提高了该双网络凝胶的断裂应力和杨氏模量【Chemical Engineering Journal，2014，240(6)：331-337】。Kim等人利用介孔分子筛与聚合物之间存在的范德华力和氢键作用，得到了可在生理溶液中较长时间保持力学性能的PAM/CaAlg杂化水凝胶。Tiller等通过酶引发在双网络水凝胶中形成了均匀分散的纳米磷酸钙，使水凝胶的弹性模量达到了440MPa，远高于软骨【Nature，2017，543(7645)：407-410】。
但是由于水凝胶光滑的表面和高的亲水性，细胞在水凝胶表面不易粘附增殖。即使在水凝胶中加入了纤连蛋白促进细胞粘附，也难以控制贴壁细胞的密度。三维培养因细胞包埋过深影响养分和代谢物传输，引起细胞死亡。此外，高强度水凝胶在制备过程中的恶劣环境如反应热、单体毒性、聚合过程中的自由基等对细胞造成巨大损伤。为了减小聚合反应对细胞的损伤，唐睿康教授等采用层层自组装方法，在酵母菌表面包裹一层磷酸钙【Angewandte Chemie International Edition，2008，47，3560-3564】。
赋予高强度水凝胶诱导软骨分化的生物活性，防止“去分化”现象，是实现其软骨替代修复需要解决的另一科学难题。转化生长因子既是软骨分化最重要的启动者，又是软骨分化过程中的重要维持者。但是加入昂贵的转化生长因子存在以下问题：(1)外源性转化生长因子在体内容易失去活性；(2)生长因子易突释。因此，如何识别保护并缓慢释放生长因子是持续诱导软骨分化急需解决的关键问题。
分子印迹技术是制备在空间结构和结合位点上与模板分子相匹配，从而对模板分子具有专一识别特性的聚合物(分子印迹聚合物molecular imprinted polymer，MIP)的技术。相比天然抗体，MIP表现出稳定性高和使用寿命长的特点，可抗恶劣环境，且制备过程简单。Pan等采用分子印迹技术合成了对细胞粘附肽RGDS具有温度响应性识别能力的分子印迹水凝胶，可促进细胞粘附和增殖【Angewandte chemie 2013，52(27)：6907-6911】。分子印迹技术构建了与模板相互补的孔穴和结合位点，两者的协同作用可以实现对目标分子的控制释放。
发明内容
针对现有技术的不足，本发明拟解决的技术问题是水凝胶不能识别缓释生长因子，常规水凝胶机械性能差，水凝胶不能持续诱导软骨分化的问题。
本发明解决所述水凝胶不能识别缓释生长因子，常规水凝胶机械性能差，水凝胶不能持续诱导软骨分化的问题的技术方案是提供一种表层包埋干细胞可持续诱导软骨分化的高强度水凝胶的制备方法。
本发明提供了一种表层包埋干细胞可持续诱导软骨分化的高强度水凝胶的制备方法。首先在干细胞表面包裹一层磷酸钙外壳。将硅酸钠、丙烯酰胺、交联剂和海藻酸钠溶解于水中得到铸膜液A，将包裹磷酸钙外壳的干细胞和生长因子分散到铸膜液A中得到铸膜液B。在A玻璃板上用缠绕直径为dA的铜丝的玻璃棒将铸膜液A刮均匀，在B玻璃板上用缠绕直径为dB的铜丝的玻璃棒将铸膜液B刮均匀，然后立即将玻璃板B倒扣在玻璃板A上。紫外引发丙烯酰胺聚合，经钙离子交联和葡萄糖酸-δ-内酯水溶液浸泡，将受保护的细胞释放出来，洗脱生长因子后，利用印迹孔穴和位点实现生长因子的识别和缓释，得到表层包埋干细胞可持续诱导软骨分化的高强度水凝胶。
本发明提供了一种表层包埋干细胞可持续诱导软骨分化的高强度水凝胶的制备方法，其特征是包括以下步骤：
a)首先采用层层自组装的方法在干细胞表面矿化沉积一层磷酸钙外壳，将干细胞保护起来以减小聚合反应过程对干细胞的伤害；称取0.005-2g硅酸钠，5-15g丙烯酰胺，0.5-2g海藻酸钠，丙烯酰胺质量百分比0.03％-0.30％的N，N′-亚甲基双丙烯酰胺，一起溶于50-100ml去离子水中，搅拌溶解均匀，静置消泡后得到铸膜液A，然后将生长因子和包裹磷酸钙外壳的干细胞分别溶解及分散于铸膜液A中，得到铸膜液B，将铸膜液A放置于无菌容器中备用，含有干细胞的铸膜液B现配现用；
b)配制质量百分比为0.2％-30％的可溶性钙盐水溶液，灭菌消毒，置于无菌容器中备用；
c)在无菌条件下，向步骤a)制备的铸膜液A和铸膜液B中分别加入丙烯酰胺质量百分比0.1％-5％的过硫酸铵，丙烯酰胺质量百分比0.1％-5％的亚硫酸氢钠和丙烯酰胺质量百分比0.01％-2％的四甲基乙二胺，搅拌分散均匀后，立即将铸膜液A倒入干燥清洁的玻璃板上，用厚度为10-1000μm的刮膜棒刮成厚度均匀的液膜，然后尽快在该液膜上面倒入铸膜液B，马上用厚度为100-2000μm的刮膜棒刮平；在N2保护下紫外照射1-20min引发丙烯酰胺聚合，得到化学交联的凝胶膜；
d)在无菌条件下，将步骤c)得到的化学交联的凝胶膜和玻璃板一起浸泡到步骤b)得到的可溶性钙盐水溶液中，浸泡0.1-24h，在浸泡过程中将凝胶膜从玻璃板上揭下来，可溶性钙盐与海藻酸钠反应生成离子交联的海藻酸钙水凝胶交联网络，同时可溶性钙盐与硅酸钠反应在聚丙烯酰胺/海藻酸钙水凝胶中原位生成硅酸钙纳米粒子；
e)在无菌条件下，配制质量百分比浓度为0.1％-10％的葡萄糖酸-δ-内酯水溶液，将步骤d)得到的含硅酸钙的凝胶膜浸泡到葡萄糖酸-δ-内酯水溶液中0.1-12h，葡萄糖酸-δ-内酯水解释放出氢离子，氢离子与硅酸钙反应，在硅酸钙纳米粒子表面形成介孔硅胶结构，同时氢离子与包裹干细胞的磷酸钙反应将干细胞释放出来，介孔硅胶与海藻酸钙和聚丙烯酰胺发生氢键相互作用，再加上纳米粒子的增强效应，提高了聚丙烯酰胺/海藻酸钙水凝胶在生理环境下的力学稳定性和抗溶胀性；
f)在无菌条件下，将步骤e)得到的水凝胶膜用洗脱溶液洗脱掉生长因子后，得到表层包埋干细胞可持续诱导软骨分化的高强度水凝胶，利用洗脱生长因子后在水凝胶中留下的互补空穴和印迹位点实现对生长因子的缓释，缓释的生长因子促进干细胞软骨分化，已分化的软骨细胞自分泌生长因子，这些自分泌生长因子部分被吸附到含互补空穴和印迹位点的水凝胶上，缓释后继续诱导干细胞软骨分化，从而达到持续诱导软骨分化的目的。
本发明所述的干细胞为骨髓间充质干细胞、脐带血干细胞、羊膜干细胞、脂肪干细胞的任意一种；所述的可溶性钙盐水溶液为硝酸钙、氯化钙、磷酸二氢钙、硫酸钙水溶液中的任意一种或两种以上混合物；所述的生长因子为转化生长因子-β1、转化生长因子-β2、转化生长因子-β3、胰岛素样生长因子的任意一种；所述的洗脱溶液为磷酸盐缓冲溶液、Tris-HCl缓冲溶液、生理盐水、SDS-醋酸缓冲溶液、胃蛋白酶水溶液中的任意一种。
具体实施方式
下面介绍本发明的具体实施例，但本发明不受实施例的限制。
实施例1.
a)首先采用层层自组装的方法在骨髓间充质干细胞表面矿化沉积一层磷酸钙外壳，将骨髓间充质干细胞保护起来以减小聚合反应过程对骨髓间充质干细胞的伤害；称取0.005g硅酸钠，5g丙烯酰胺，0.5g海藻酸钠，丙烯酰胺质量百分比0.03％的N，N′-亚甲基双丙烯酰胺，一起溶于50ml去离子水中，搅拌溶解均匀，静置消泡后得到铸膜液A，然后将转化生长因子-β1和包裹磷酸钙外壳的骨髓间充质干细胞分别溶解及分散于铸膜液A中，得到铸膜液B，将铸膜液A放置于无菌容器中备用，含有骨髓间充质干细胞的铸膜液B现配现用；
b)配制质量百分比为0.2％的硝酸钙水溶液，灭菌消毒，置于无菌容器中备用；
c)在无菌条件下，向步骤a)制备的铸膜液A和铸膜液B中分别加入丙烯酰胺质量百分比0.1％的过硫酸铵，丙烯酰胺质量百分比0.1％的亚硫酸氢钠和丙烯酰胺质量百分比0.01％的四甲基乙二胺，搅拌分散均匀后，立即将铸膜液A倒入干燥清洁的玻璃板上，用厚度为10μm的刮膜棒刮成厚度均匀的液膜，然后尽快在该液膜上面倒入铸膜液B，马上用厚度为100μm的刮膜棒刮平；在N2保护下紫外照射1min引发丙烯酰胺聚合，得到化学交联的凝胶膜；
d)在无菌条件下，将步骤c)得到的化学交联的凝胶膜和玻璃板一起浸泡到步骤b)得到的硝酸钙水溶液中，浸泡0.1h，在浸泡过程中将凝胶膜从玻璃板上揭下来，硝酸钙与海藻酸钠反应生成离子交联的海藻酸钙水凝胶交联网络，同时硝酸钙与硅酸钠反应在聚丙烯酰胺/海藻酸钙水凝胶中原位生成硅酸钙纳米粒子；
e)在无菌条件下，配制质量百分比浓度为0.1％的葡萄糖酸-δ-内酯水溶液，将步骤d)得到的含硅酸钙的凝胶膜浸泡到葡萄糖酸-δ-内酯水溶液中0.1h，葡萄糖酸-δ-内酯水解释放出氢离子，氢离子与硅酸钙反应，在硅酸钙纳米粒子表面形成介孔硅胶结构，同时氢离子与包裹骨髓间充质干细胞的磷酸钙反应将骨髓间充质干细胞释放出来，介孔硅胶与海藻酸钙和聚丙烯酰胺发生氢键相互作用，再加上纳米粒子的增强效应，提高了聚丙烯酰胺/海藻酸钙水凝胶在生理环境下的力学稳定性和抗溶胀性；
f)在无菌条件下，将步骤e)得到的水凝胶膜用磷酸盐缓冲溶液洗脱掉转化生长因子-β1后，得到表层包埋骨髓间充质干细胞可持续诱导软骨分化的高强度水凝胶，利用洗脱转化生长因子-β1后在水凝胶中留下的互补空穴和印迹位点实现对转化生长因子-β1的缓释，缓释的转化生长因子-β1促进骨髓间充质干细胞软骨分化，已分化的软骨细胞自分泌转化生长因子-β1，这些自分泌转化生长因子-β1部分被吸附到含互补空穴和印迹位点的水凝胶上，缓释后继续诱导骨髓间充质干细胞软骨分化，从而达到持续诱导软骨分化的目的。
实施例2.
a)首先采用层层自组装的方法在脐带血干细胞表面矿化沉积一层磷酸钙外壳，将脐带血干细胞保护起来以减小聚合反应过程对脐带血干细胞的伤害；称取2g硅酸钠，15g丙烯酰胺，2g海藻酸钠，丙烯酰胺质量百分比0.30％的N，N′-亚甲基双丙烯酰胺，一起溶于100ml去离子水中，搅拌溶解均匀，静置消泡后得到铸膜液A，然后将转化生长因子-β2和包裹磷酸钙外壳的脐带血干细胞分别溶解及分散于铸膜液A中，得到铸膜液B，将铸膜液A放置于无菌容器中备用，含有脐带血干细胞的铸膜液B现配现用；
b)配制质量百分比为30％的氯化钙水溶液，灭菌消毒，置于无菌容器中备用；
c)在无菌条件下，向步骤a)制备的铸膜液A和铸膜液B中分别加入丙烯酰胺质量百分比0.1％-5％的过硫酸铵，丙烯酰胺质量百分比0.1％的亚硫酸氢钠和丙烯酰胺质量百分比0.01％的四甲基乙二胺，搅拌分散均匀后，立即将铸膜液A倒入干燥清洁的玻璃板上，用厚度为1000μm的刮膜棒刮成厚度均匀的液膜，然后尽快在该液膜上面倒入铸膜液B，马上用厚度为2000μm的刮膜棒刮平；在N2保护下紫外照射20min引发丙烯酰胺聚合，得到化学交联的凝胶膜；
d)在无菌条件下，将步骤c)得到的化学交联的凝胶膜和玻璃板一起浸泡到步骤b)得到的氯化钙水溶液中，浸泡24h，在浸泡过程中将凝胶膜从玻璃板上揭下来，氯化钙与海藻酸钠反应生成离子交联的海藻酸钙水凝胶交联网络，同时氯化钙与硅酸钠反应在聚丙烯酰胺/海藻酸钙水凝胶中原位生成硅酸钙纳米粒子；
e)在无菌条件下，配制质量百分比浓度为10％的葡萄糖酸-δ-内酯水溶液，将步骤d)得到的含硅酸钙的凝胶膜浸泡到葡萄糖酸-δ-内酯水溶液中12h，葡萄糖酸-δ-内酯水解释放出氢离子，氢离子与硅酸钙反应，在硅酸钙纳米粒子表面形成介孔硅胶结构，同时氢离子与包裹脐带血干细胞的磷酸钙反应将脐带血干细胞释放出来，介孔硅胶与海藻酸钙和聚丙烯酰胺发生氢键相互作用，再加上纳米粒子的增强效应，提高了聚丙烯酰胺/海藻酸钙水凝胶在生理环境下的力学稳定性和抗溶胀性；
f)在无菌条件下，将步骤e)得到的水凝胶膜用Tris-HCl缓冲溶液洗脱掉转化生长因子-β2后，得到表层包埋脐带血干细胞可持续诱导软骨分化的高强度水凝胶，利用洗脱转化生长因子-β2后在水凝胶中留下的互补空穴和印迹位点实现对转化生长因子-β2的缓释，缓释的转化生长因子-β2促进脐带血干细胞软骨分化，已分化的软骨细胞自分泌转化生长因子-β2，这些自分泌转化生长因子-β2部分被吸附到含互补空穴和印迹位点的水凝胶上，缓释后继续诱导脐带血干细胞软骨分化，从而达到持续诱导软骨分化的目的。
实施例3.
a)首先采用层层自组装的方法在羊膜干细胞表面矿化沉积一层磷酸钙外壳，将羊膜干细胞保护起来以减小聚合反应过程对羊膜干细胞的伤害；称取1g硅酸钠，10g丙烯酰胺，1g海藻酸钠，丙烯酰胺质量百分比0.20％的N，N′-亚甲基双丙烯酰胺，一起溶于60ml去离子水中，搅拌溶解均匀，静置消泡后得到铸膜液A，然后将转化生长因子-β3和包裹磷酸钙外壳的羊膜干细胞分别溶解及分散于铸膜液A中，得到铸膜液B，将铸膜液A放置于无菌容器中备用，含有羊膜干细胞的铸膜液B现配现用；
b)配制质量百分比为5％的磷酸二氢钙水溶液，灭菌消毒，置于无菌容器中备用；
c)在无菌条件下，向步骤a)制备的铸膜液A和铸膜液B中分别加入丙烯酰胺质量百分比2％的过硫酸铵，丙烯酰胺质量百分比2％的亚硫酸氢钠和丙烯酰胺质量百分比1％的四甲基乙二胺，搅拌分散均匀后，立即将铸膜液A倒入干燥清洁的玻璃板上，用厚度为200μm的刮膜棒刮成厚度均匀的液膜，然后尽快在该液膜上面倒入铸膜液B，马上用厚度为400μm的刮膜棒刮平；在N2保护下紫外照射1-20min引发丙烯酰胺聚合，得到化学交联的凝胶膜；
d)在无菌条件下，将步骤c)得到的化学交联的凝胶膜和玻璃板一起浸泡到步骤b)得到的磷酸二氢钙水溶液中，浸泡8h，在浸泡过程中将凝胶膜从玻璃板上揭下来，磷酸二氢钙与海藻酸钠反应生成离子交联的海藻酸钙水凝胶交联网络，同时磷酸二氢钙与硅酸钠反应在聚丙烯酰胺/海藻酸钙水凝胶中原位生成硅酸钙纳米粒子；
e)在无菌条件下，配制质量百分比浓度为5％的葡萄糖酸-δ-内酯水溶液，将步骤d)得到的含硅酸钙的凝胶膜浸泡到葡萄糖酸-δ-内酯水溶液中8h，葡萄糖酸-δ-内酯水解释放出氢离子，氢离子与硅酸钙反应，在硅酸钙纳米粒子表面形成介孔硅胶结构，同时氢离子与包裹羊膜干细胞的磷酸钙反应将羊膜干细胞释放出来，介孔硅胶与海藻酸钙和聚丙烯酰胺发生氢键相互作用，再加上纳米粒子的增强效应，提高了聚丙烯酰胺/海藻酸钙水凝胶在生理环境下的力学稳定性和抗溶胀性；
f)在无菌条件下，将步骤e)得到的水凝胶膜用胃蛋白酶水溶液洗脱掉转化生长因子-β3后，得到表层包埋羊膜干细胞可持续诱导软骨分化的高强度水凝胶，利用洗脱转化生长因子-β3后在水凝胶中留下的互补空穴和印迹位点实现对转化生长因子-β3的缓释，缓释的转化生长因子-β3促进羊膜干细胞软骨分化，已分化的软骨细胞自分泌转化生长因子-β3，这些自分泌转化生长因子-β3部分被吸附到含互补空穴和印迹位点的水凝胶上，缓释后继续诱导羊膜干细胞软骨分化，从而达到持续诱导软骨分化的目的。
实施例4.
a)首先采用层层自组装的方法在脂肪干细胞表面矿化沉积一层磷酸钙外壳，将脂肪干细胞保护起来以减小聚合反应过程对脂肪干细胞的伤害；称取1.5g硅酸钠，6g丙烯酰胺，1.5g海藻酸钠，丙烯酰胺质量百分比0.10％的N，N′-亚甲基双丙烯酰胺，一起溶于80ml去离子水中，搅拌溶解均匀，静置消泡后得到铸膜液A，然后将胰岛素样生长因子和包裹磷酸钙外壳的脂肪干细胞分别溶解及分散于铸膜液A中，得到铸膜液B，将铸膜液A放置于无菌容器中备用，含有脂肪干细胞的铸膜液B现配现用；
b)配制质量百分比为1.0％的硫酸钙水溶液，灭菌消毒，置于无菌容器中备用；
c)在无菌条件下，向步骤a)制备的铸膜液A和铸膜液B中分别加入丙烯酰胺质量百分比1％的过硫酸铵，丙烯酰胺质量百分比1％的亚硫酸氢钠和丙烯酰胺质量百分比1％的四甲基乙二胺，搅拌分散均匀后，立即将铸膜液A倒入干燥清洁的玻璃板上，用厚度为100μm的刮膜棒刮成厚度均匀的液膜，然后尽快在该液膜上面倒入铸膜液B，马上用厚度为150μm的刮膜棒刮平；在N2保护下紫外照射5min引发丙烯酰胺聚合，得到化学交联的凝胶膜；
d)在无菌条件下，将步骤c)得到的化学交联的凝胶膜和玻璃板一起浸泡到步骤b)得到的硫酸钙水溶液中，浸泡6h，在浸泡过程中将凝胶膜从玻璃板上揭下来，硫酸钙与海藻酸钠反应生成离子交联的海藻酸钙水凝胶交联网络，同时硫酸钙与硅酸钠反应在聚丙烯酰胺/海藻酸钙水凝胶中原位生成硅酸钙纳米粒子；
e)在无菌条件下，配制质量百分比浓度为1.0％的葡萄糖酸-δ-内酯水溶液，将步骤d)得到的含硅酸钙的凝胶膜浸泡到葡萄糖酸-δ-内酯水溶液中6h，葡萄糖酸-δ-内酯水解释放出氢离子，氢离子与硅酸钙反应，在硅酸钙纳米粒子表面形成介孔硅胶结构，同时氢离子与包裹脂肪干细胞的磷酸钙反应将脂肪干细胞释放出来，介孔硅胶与海藻酸钙和聚丙烯酰胺发生氢键相互作用，再加上纳米粒子的增强效应，提高了聚丙烯酰胺/海藻酸钙水凝胶在生理环境下的力学稳定性和抗溶胀性；
f)在无菌条件下，将步骤e)得到的水凝胶膜用SDS-醋酸缓冲溶液洗脱掉胰岛素样生长因子后，得到表层包埋脂肪干细胞可持续诱导软骨分化的高强度水凝胶，利用洗脱胰岛素样生长因子后在水凝胶中留下的互补空穴和印迹位点实现对胰岛素样生长因子的缓释，缓释的胰岛素样生长因子促进脂肪干细胞软骨分化，已分化的软骨细胞自分泌胰岛素样生长因子，这些自分泌胰岛素样生长因子部分被吸附到含互补空穴和印迹位点的水凝胶上，缓释后继续诱导脂肪干细胞软骨分化，从而达到持续诱导软骨分化的目的。
Claims (5)
1.一种表层包埋干细胞可持续诱导软骨分化的高强度水凝胶的制备方法，其特征是包括以下步骤：
a)首先采用层层自组装的方法在干细胞表面矿化沉积一层磷酸钙外壳，将干细胞保护起来以减小聚合反应过程对干细胞的伤害；称取0.005-2g硅酸钠，5-15g丙烯酰胺，0.5-2g海藻酸钠，丙烯酰胺质量百分比0.03％-0.30％的N，N′-亚甲基双丙烯酰胺，一起溶于50-100ml去离子水中，搅拌溶解均匀，静置消泡后得到铸膜液A，然后将生长因子和包裹磷酸钙外壳的干细胞分别溶解及分散于铸膜液A中，得到铸膜液B，将铸膜液A放置于无菌容器中备用，含有干细胞的铸膜液B现配现用；
b)配制质量百分比为0.2％-30％的可溶性钙盐水溶液，灭菌消毒，置于无菌容器中备用；
c)在无菌条件下，向步骤a)制备的铸膜液A和铸膜液B中分别加入丙烯酰胺质量百分比0.1％-5％的过硫酸铵，丙烯酰胺质量百分比0.1％-5％的亚硫酸氢钠和丙烯酰胺质量百分比0.01％-2％的四甲基乙二胺，搅拌分散均匀后，立即将铸膜液A倒入干燥清洁的玻璃板上，用厚度为10-1000μm的刮膜棒刮成厚度均匀的液膜，然后尽快在该液膜上面倒入铸膜液B，马上用厚度为100-2000μm的刮膜棒刮平；在N2保护下紫外照射1-20min引发丙烯酰胺聚合，得到化学交联的凝胶膜；
d)在无菌条件下，将步骤c)得到的化学交联的凝胶膜和玻璃板一起浸泡到步骤b)得到的可溶性钙盐水溶液中，浸泡0.1-24h，在浸泡过程中将凝胶膜从玻璃板上揭下来，可溶性钙盐与海藻酸钠反应生成离子交联的海藻酸钙水凝胶交联网络，同时可溶性钙盐与硅酸钠反应在聚丙烯酰胺/海藻酸钙水凝胶中原位生成硅酸钙纳米粒子；
e)在无菌条件下，配制质量百分比浓度为0.1％-10％的葡萄糖酸-δ-内酯水溶液，将步骤d)得到的含硅酸钙的凝胶膜浸泡到葡萄糖酸-δ-内酯水溶液中0.1-12h，葡萄糖酸-δ-内酯水解释放出氢离子，氢离子与硅酸钙反应，在硅酸钙纳米粒子表面形成介孔硅胶结构，同时氢离子与包裹干细胞的磷酸钙反应将干细胞释放出来，介孔硅胶与海藻酸钙和聚丙烯酰胺发生氢键相互作用，再加上纳米粒子的增强效应，提高了聚丙烯酰胺/海藻酸钙水凝胶在生理环境下的力学稳定性和抗溶胀性；
f)在无菌条件下，将步骤e)得到的水凝胶膜用洗脱溶液洗脱掉生长因子后，得到表层包埋干细胞可持续诱导软骨分化的高强度水凝胶，利用洗脱生长因子后在水凝胶中留下的互补空穴和印迹位点实现对生长因子的缓释，缓释的生长因子促进干细胞软骨分化，已分化的软骨细胞自分泌生长因子，这些自分泌生长因子部分被吸附到含互补空穴和印迹位点的水凝胶上，缓释后继续诱导干细胞软骨分化，从而达到持续诱导软骨分化的目的。
2.如权利要求1所述的一种表层包埋干细胞可持续诱导软骨分化的高强度水凝胶的制备方法，其特征是所述的干细胞为骨髓间充质干细胞、脐带血干细胞、羊膜干细胞、脂肪干细胞的任意一种。
3.如权利要求1所述的一种表层包埋干细胞可持续诱导软骨分化的高强度水凝胶的制备方法，其特征是所述的可溶性钙盐水溶液为硝酸钙、氯化钙、磷酸二氢钙、硫酸钙水溶液中的任意一种或两种以上混合物。
4.如权利要求1所述的一种表层包埋干细胞可持续诱导软骨分化的高强度水凝胶的制备方法，其特征是所述的生长因子为转化生长因子-β1、转化生长因子-β2、转化生长因子-β3、胰岛素样生长因子的任意一种。
5.如权利要求1所述的一种高强度高韧性蛋白质分子印迹杂化凝胶膜的制备方法，其特征是所述的洗脱溶液为磷酸盐缓冲溶液、Tris-HCl缓冲溶液、生理盐水、SDS-醋酸缓冲溶液、胃蛋白酶水溶液中的任意一种。
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