CN118178734B - 一种3d装载干细胞的复合水凝胶的制备方法及应用 - Google Patents
一种3d装载干细胞的复合水凝胶的制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种3D装载干细胞的复合水凝胶的制备方法及应用，属于创伤修复水凝胶技术领域。制备方法包括：S1、采用微流控法制备GelMA微观水凝胶；S2、配置HAMA和GelMA的总质量浓度为3wt%‑8wt%的水溶液，向HAMA和GelMA的水溶液中加入10‑100个冷冻干燥的GelMA微观水凝胶，加入0.5wt%‑3wt%苯基（2,4,6‑三甲基苯甲酰基）磷酸锂盐，震荡混匀，获得复合溶液；S3、取100μL‑300μL浓度为1×106 mL‑1‑5×106 mL‑1干细胞悬液加至步骤S2获得的复合溶液中，震荡混匀；将溶液移至硅胶模具中，通过光固化形成3D装载干细胞的复合水凝胶。本发明一种3D装载干细胞的复合水凝胶的制备方法及应用，将GelMA微观水凝胶和干细胞装载于HAMA‑GelMA水凝胶中，通过GelMA微观水凝胶的降解调控干细胞在3D水凝胶环境中的生长。
Description
技术领域
本发明涉及创伤修复水凝胶技术领域，尤其涉及一种3D装载干细胞的复合水凝胶的制备方法及应用。
背景技术
水凝胶作为一种具有三维网络结构的组织工程支架能够模拟细胞外基质（ECM）为细胞的粘附和增殖提供合适的微环境。此外，水凝胶组织工程支架具有良好的生物相容性、吸收损伤组织的渗出液、透气性良好、作为药物缓释载体材料、亲水性良好等诸多优点，因此水凝胶已经成为组织工程研究的热点，被应用于如骨缺损、皮肤损伤、脊柱损伤、食管损伤、胃肠损伤等不同组织部位的伤口修复。
研究者将干细胞与水凝胶复合后，发现修复效果优于单纯的水凝胶，但由于设计的水凝胶细胞相容性较差、力学强度过高等问题，极大的限制了干细胞在水凝胶的3D环境中的良好生长。
发明内容
为解决上述问题，本发明提供一种3D装载干细胞的复合水凝胶的制备方法及应用，选用具有较好细胞相容性的类细胞外基质材料明胶和透明质酸，制备出软硬适中的宏观-微观复合水凝胶，微观水凝胶的降解可以更好的为干细胞提供动态生长空间，对干细胞后期的增殖和分化起重要作用。
为实现上述目的，本发明提供了一种3D装载干细胞的复合水凝胶，3D装载干细胞的复合水凝胶为装载GelMA微观水凝胶和干细胞的HAMA-GelMA宏观水凝胶。
一种3D装载干细胞的复合水凝胶的制备方法，包括如下步骤：
S1、采用微流控法制备GelMA微观水凝胶；
S2、配置甲基丙烯酸酐化透明质酸HAMA和甲基丙烯酸酐化明胶GelMA的总质量浓度为3wt%-8wt%的水溶液，向HAMA和GelMA的水溶液中加入10-100个冷冻干燥的GelMA微观水凝胶，加入0.5wt%-3wt%苯基（2,4,6-三甲基苯甲酰基）磷酸锂盐，震荡混匀，获得复合溶液；
S3、取100μL-300μL浓度为1×106 mL-1-5×106 mL-1干细胞悬液加至步骤S2获得的复合溶液中，震荡混匀；将溶液移至硅胶模具中，通过光固化形成3D装载干细胞的复合水凝胶。
优选地，步骤S1中制备GelMA微观水凝胶的具体步骤包括：
S1-1、向0.05-0.5g/mL 甲基丙烯酸酐化明胶溶液中加入0.5wt%-3wt%苯基（2,4,6-三甲基苯甲酰基）磷酸锂盐，将溶液移入微量注射泵的其中一个注射器中，记为水相；
S1-2、取20mL-80mL橄榄油（含体积比为25%-50%的Span80）移入微量注射泵的另一个注射器中，记为油相；
S1-3、分别以0.2mL/h-0.6mL/h和120mL/h-180mL/h的速度注射水相和油相使其交汇于注射针头T形通道，形成剪切力从而产生GelMA微凝胶；
S1-4、用含油相的小玻璃瓶接收微凝胶，于365nm、150mW/cm2-250mW/cm2紫外光固化10s-60s，静置，吸去上层油相，用丙酮清洗多次，得到GelMA微观水凝胶。
优选地，步骤S3中，光固化在405nm、5mW/cm²-12mW/cm²的蓝光下照射固化10s-60s。
本发明一种3D装载干细胞的复合水凝胶的制备方法及应用具有以下有益效果：
（1）与2D平面培养相比，3D立体培养可以为干细胞提供更真实的生长微环境，利于细胞铺展、增殖和分化，最终可以加速损伤组织的修复；
（2）采用光固化方法通过双键自由基聚合方式形成化学键合的微观和宏观互穿网络结构的水凝胶，制备的水凝胶结构软硬适中，适合干细胞长期生存；
（3）微凝胶引入宏观水凝胶中，通过微凝胶自身降解形成的动态空间对干细胞的生长起到了重要作用；以小鼠颅骨缺损修复为例进行研究，组织学评价结果表明宏观-微观水凝胶/干细胞组修复效果优于空白组和宏观水凝胶/干细胞组。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图说明
图1为实施例一中的宏观水凝胶和实施例二中的GelMA微观水凝胶的形貌图；其中，A为HAMA-GelMA宏观水凝胶的外观形貌；B为GelMA微观水凝胶的光镜图；
图2为实施例三中3D装载bMSCs的宏观-微观复合水凝胶和对比例一中装载bMSCs的宏观水凝胶中不同时期的细胞生长情况；其中，A为装载bMSCs的宏观水凝胶中荧光分布图，B为3D装载bMSCs的宏观-微观复合水凝胶中的荧光分布图；
图3为小鼠颅骨缺损修复2个月的HE染色结果。
具体实施方式
为了使本发明实施例公开的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明实施例进行进一步详细说明。应当理解，此处描述的具体实施例仅用以解释本发明实施例，并不用于限定本发明实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本申请保护的范围。所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。
需要说明的是，术语“包括”和“具有”以及他们的任何变形，意图在于覆盖不排他的包含，例如，包含了一系列步骤或单元的过程、方法、系统、产品或服务器不必限于清楚地列出的那些步骤或单元，而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
相似的标号和字母在下面的附图中表示类似项，因此，一旦某一项在一个附图中被定义，则在随后的附图中不需要对其进行进一步定义和解释。
实施例一
制备HAMA-GelMA宏观水凝胶支架，包括：
（1）配制浓度为5wt%的双键透明质酸（HAMA）和50wt%的双键明胶（GelMA），将其配制为HAMA和GelMA的总质量浓度为6wt%的水溶液，温度为40℃。
（2）向上述溶液中加入2wt%苯基（2,4,6-三甲基苯甲酰基）磷酸锂盐（LAP），涡旋震荡使溶液混合均匀，将溶液滴于模具中，于405nm，10mW/cm²的蓝光下照射固化30s，形成HAMA-GelMA宏观水凝胶支架。
实施例二
制备GelMA微观水凝胶支架和宏观-微观复合水凝胶支架，包括：
S1、制备GelMA微观水凝胶支架
（1）去离子水配制0.5g/mL GelMA溶液，加入3wt%苯基（2,4,6-三甲基苯甲酰基）磷酸锂盐，将溶液移入微量注射泵的其中一个注射器中，记为水相。
取40mL橄榄油（含体积比为35%的Span80）移入微量注射泵的另一个注射器中，记为油相。
（2）分别以0.4mL/h和120mL/h的速度注射水相和油相，使其交汇于注射针头T形通道，形成剪切力从而产生GelMA微凝胶。
用含油相的小玻璃瓶接收微凝胶，于365nm，180mW/cm2紫外光固化30s。静置，吸去上层油相，用丙酮清洗多次，得到微观水凝胶即微凝胶，将微凝胶进行冷冻干燥，备用。
S2、罗丹明B红色荧光标记微凝胶GelMA
（1）0.15g罗丹明B溶于10mL N,N-二甲基甲酰胺中，随后依次加入0.2g N-羟基琥珀酰亚胺和0.2g 1-乙基-3-二甲基氨基丙基-碳二亚胺盐酸盐，搅拌溶解，室温反应4h，记为活化液。
（2）取1g GelMA微凝胶溶于20mL去离子水，水浴加热，磁力搅拌，加入3mL活化液，避光反应24h，加入80mL去离子水终止反应。将上述溶液装入透析袋中，去离子水透析（避光）。透析后的溶液离心去沉淀，将上清液转移至培养皿中，冷冻干燥，得到罗丹明标记的微凝胶。
S3、构建宏观-微观复合水凝胶支架
HAMA和GelMA的总质量浓度为6wt%的水溶液中加入50个冷冻干燥的GelMA微凝胶，加入2wt%LAP蓝光引发剂，震荡混匀，得到复合溶液，取该复合溶液滴于模具中，用注射针头轻微搅动混匀，使GelMA微凝胶均匀分散于制胶溶液中，在405nm，10mW/cm2的蓝光下照射固化30s，形成宏观-微观复合水凝胶支架。
实施例三
复合水凝胶3D装载骨髓间充质干细胞（bMSCs）
为了在三维水凝胶中追踪干细胞的生长和增殖情况，从兔子脊髓中原代提取骨髓间充质干细胞，并对扩增后的干细胞进行GFP转染使其带上绿色荧光，保证干细胞在增殖和分化阶段荧光不会淬灭。微凝胶用罗丹明红色荧光进行标记，可以在宏观水凝胶内部很好的追踪并区分干细胞（绿色）和微凝胶（红色）的动态变化情况。
用胰酶消化绿色荧光蛋白（GFP）转染的bMSCs，离心浓缩细胞浓度约为3×106 mL-1。取200μL浓缩细胞悬液，分散至实施例二步骤S3中的复合溶液中，震荡混匀。
用移液枪吸取上述溶液滴于硅胶模具中，在405nm，10mW/cm²的蓝光下照射固化30s，形成3D装载bMSCs的宏观-微观复合水凝胶。
对比例一
用胰酶消化绿色荧光蛋白（GFP）转染的bMSCs，离心浓缩细胞浓度约为3×106 mL-1。取200μL浓缩细胞悬液，分散至实施例一中加入了LAP的HAMA和GelMA水溶液中，震荡混匀。
用移液枪吸取上述溶液滴于硅胶模具中，在405nm，10mW/cm²的蓝光下照射固化30s，形成装载bMSCs的宏观水凝胶。
试验测试
（一）如图1所示，为实施例一中的宏观水凝胶和实施例二中的GelMA微观水凝胶的形貌图；图1中A为HAMA-GelMA宏观水凝胶的外观形貌；图1中B为GelMA微凝胶的光镜图。
采用微流控方法制备的微凝胶可以保持粒径均一，在粒径控制方面优于机械搅拌方法，粒径均一的优点是可以使微凝胶在宏观水凝胶中的降解等方面具有可重复性，研究时准确性和严谨性更高。
由图1可知，本发明成功制备出宏观水凝胶和微凝胶。
（二）将实施例三中3D装载bMSCs的宏观-微观复合水凝胶和对比例一中装载bMSCs的宏观水凝胶分别转移至盛有细胞培养液的孔板中，孔板放入二氧化碳培养箱中培养，隔天换液，不同时间点评价干细胞生长情况。
如图2所示，图2中A为装载bMSCs的宏观水凝胶中荧光分布图；可以看出7天到14天时，干细胞数量稍微增加，但是形状几乎无变化，仍然是圆形。14天到21天时，干细胞数量增加说明发生了增殖和分化，尤其少量干细胞的形状由圆形变为长梭形，说明随着培养时间的延长，宏观水凝胶的缓慢降解为干细胞的生长提供了少量空间。
图2中B为3D装载bMSCs的宏观-微观复合水凝胶中的荧光分布图。与图2中A相比，图2中B在原来基础上引入了红色标记的微凝胶，可见7天和14天时，图2中B和图2中A中干细胞数量和形状几乎相同，图2中B在14天时少量干细胞变为长梭形。21天时，图2中B干细胞数量明显增加并且大部分细胞已是长梭形，但是图2中A干细胞数量少且细胞铺展少，说明随着时间的增加，微凝胶逐渐降解从而为干细胞提供了动态生长空间。
（三）图3为小鼠颅骨缺损后水凝胶修复2个月的苏木素-伊红（HE）染色组织修复情况，样品组分别为空白组（未放样品小鼠自身修复），宏观水凝胶/干细胞组，宏观-微观水凝胶/干细胞组。
图中绿色箭头表明形成了骨膜，3组都出现了新生骨膜，骨膜有成骨趋势，最右侧含微凝胶组骨膜最厚。黑色箭头表明形成了松质骨骨小梁，3组都有骨小梁形成，含微凝胶组新生骨小梁面积最大。红色箭头是淋巴细胞，空白组仍有部分区域被大量淋巴细胞浸润，说明仍然有炎症存在，另外2组没有炎症。紫色箭头表明血管神经生长，只有含微凝胶组出现了此种情况。因此，HE染色的总体结果表明宏观-微观水凝胶/干细胞组促修复效果最好，宏观水凝胶/干细胞促修复效果次之，空白组促修复效果最差，说明微凝胶与干细胞协同作用优于单纯干细胞作用。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。对于实施例公开的装置而言，由于其与实施例公开的方法相对应，所以描述的比较简单，相关之处参见方法部分说明即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (2)
1.一种3D装载干细胞的复合水凝胶在调控干细胞在3D水凝胶环境中的生长中的应用，其特征在于，3D装载干细胞的复合水凝胶为装载GelMA微观水凝胶和干细胞的HAMA-GelMA水凝胶，GelMA微观水凝胶降解为干细胞生长提供动态空间；
3D装载干细胞的复合水凝胶的制备方法包括如下步骤：
S1、采用微流控法制备GelMA微观水凝胶；
S2、配置甲基丙烯酸酐化透明质酸HAMA和甲基丙烯酸酐化明胶GelMA的总质量浓度为3wt%-8wt%的水溶液，向HAMA和GelMA的水溶液中加入10-100个冷冻干燥的GelMA微观水凝胶，加入0.5wt%-3wt%苯基（2,4,6-三甲基苯甲酰基）磷酸锂盐，震荡混匀，获得复合溶液；
S3、取100μL-300μL浓度为1×106 mL-1-5×106 mL-1干细胞悬液加至步骤S2获得的复合溶液中，震荡混匀；将溶液移至硅胶模具中，通过光固化形成3D装载干细胞的复合水凝胶；
步骤S1中制备GelMA微观水凝胶的具体步骤包括：
S1-1、向0.05-0.5g/mL甲基丙烯酸酐化明胶溶液中加入0.5wt%-3wt%苯基（2,4,6-三甲基苯甲酰基）磷酸锂盐，将溶液移入微量注射泵的其中一个注射器中，记为水相；
S1-2、取20mL-80mL含体积比为25%-50%的Span80的橄榄油移入微量注射泵的另一个注射器中，记为油相；
S1-3、分别以0.2mL/h-0.6mL/h和120mL/h-180mL/h的速度注射水相和油相使其交汇于注射针头T形通道，形成剪切力从而产生GelMA微凝胶；
S1-4、用含油相的小玻璃瓶接收微凝胶，于365nm、150mW/cm2-250mW/cm2紫外光固化10s-60s，静置，吸去上层油相，用丙酮清洗多次，得到GelMA微观水凝胶。
2.根据权利要求1所述的一种3D装载干细胞的复合水凝胶在调控干细胞在3D水凝胶环境中的生长中的应用，其特征在于：步骤S3中，光固化在405nm、5mW/cm²-12mW/cm²的蓝光下照射固化10s-60s。
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