CN115444983A - 一种鲟鱼软骨脱细胞基质丝胶蛋白生物墨水及其制备方法 - Google Patents

一种鲟鱼软骨脱细胞基质丝胶蛋白生物墨水及其制备方法 Download PDF

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CN115444983A
CN115444983A CN202211124034.2A CN202211124034A CN115444983A CN 115444983 A CN115444983 A CN 115444983A CN 202211124034 A CN202211124034 A CN 202211124034A CN 115444983 A CN115444983 A CN 115444983A
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sericin
acellular matrix
sturgeon cartilage
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刘海蓉
孟小琳
周征
戴瑶
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Abstract

本发明涉及一种鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水及其制备方法，甲基丙烯酸酐修饰丝胶蛋白上的羟基和氨基，得甲基丙烯酸酐改性丝胶蛋白；甲基丙烯酸缩水甘油酯修饰鲟鱼软骨脱细胞基质上的羟基和氨基，得甲基丙烯酸缩水甘油酯改性鲟鱼软骨脱细胞基质；甲基丙烯酸酐改性丝胶蛋白、甲基丙烯酸缩水甘油酯改性鲟鱼软骨脱细胞基质与光引发剂溶液混合制得鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水。本发明创新性将水生生物鲟鱼软骨脱细胞基质作为生物墨水原材料引入到组织工程中，并通过与丝胶蛋白材料进行复合，通过紫外光照射，使甲基丙烯酸酐或甲基丙烯酸缩水甘油酯的碳碳双键产生自由基发生聚合；形成的水凝胶具有可3D打印、性能可控、生物相容性好、可生物降解等优点，制备条件温和可控，可应用于组织工程材料等生物医用材料领域。为3D打印在组织工程中的应用提供新材料。

Description

一种鲟鱼软骨脱细胞基质丝胶蛋白生物墨水及其制备方法
技术领域
本发明涉及属于3D打印生物墨水及生物材料组织工程支架开发技术领域，尤其是涉及一种鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水及其制备方法。
背景技术
结合细胞与材料的3D生物打印技术是一种新兴增材制造技术，在健康科学相关领域取得了长足的发展，如组织工程领域的耳朵重建、皮肤再生、血管连接、骨和软骨再生等。
生物墨水在3D生物打印过程中作为承载细胞进行打印的载体，是细胞、生物材料和生物活性物质的混合物，决定了打印细胞的成活率、打印后的细胞生长情况、打印组织器官的精度及打印的方法。
因此生物墨水的基质材料的选择对于保证组织工程支架的生物相容性、印刷适性和机械性能来说是至关重要的。3D打印水凝胶材料由于其良好的生物相容性、生物降解性和可打印性强等优点，已成为生物墨水最适合的基质材料之一。
然而，目前可打印的生物墨水材料种类有限，力学性能和生物学性能参差不齐，难以适合快速发展的3D打印对材料功能化的要求。
鲟鱼软骨源生物材料除了水生生物来源生物材料无免疫原性风险、无疾病传播等独特优点外，同时富含胶原蛋白和硫酸软骨素，并且最令人感兴趣的是存在一些生物活性因子用于维持软骨组织表型，从而使鲟鱼体内骨架仅由白色软骨组成，具有不易钙化的独特生物学性质。这一特性可利于促进软骨再生，生成稳定的软骨组织。
丝胶蛋白作为一种天然蛋白质类材料，具有优异的生物相容性、亲水性和生物降解性，且含有大量便于交联和修饰的氨基、羟基、羧基，是理想的生物墨水基质材料。
发明内容
本发明设计了一种鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水及其制备方法，以制备出能促进体外构建软骨生长、生物学功能和力学性能优良的组织工程支架。其解决的技术问题是现有3D生物打印的生物墨水种类有限，性能不可控、韧性表现差、生物相容性差等缺陷。
为了解决上述存在的技术问题，本发明采用了以下方案：
一种鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水的制备方法，包括以下步骤：甲基丙烯酸酐修饰丝胶蛋白上的羟基和氨基，得具有光聚合能力的甲基丙烯酸酐改性丝胶蛋白；甲基丙烯酸缩水甘油酯修饰鲟鱼软骨脱细胞基质上的羟基和氨基，得具有光聚合能力的甲基丙烯酸缩水甘油酯改性鲟鱼软骨脱细胞基质；甲基丙烯酸酐改性丝胶蛋白、甲基丙烯酸缩水甘油酯改性鲟鱼软骨脱细胞基质与光引发剂溶液混合制得鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水。
优选地，所述甲基丙烯酸酐改性丝胶蛋白的制备方法如下：
将丝胶蛋白溶于PBS中得到丝胶蛋白溶液；甲基丙烯酸酐溶于PBS得到甲基丙烯酸酐溶液；丝胶蛋白溶液与甲基丙烯酸酐溶液在碱性条件下反应获得碳碳双键修饰于丝胶蛋白羟基和氨基上的改性丝胶蛋白。
优选地，步骤1.1、将购买的蚕茧去除杂质，经Na2CO3 溶液煮沸，将初步得到的黄色液体透析3~10d，低温离心后将上清液冷冻干燥，将冷冻干燥后的粉末溶解于PBS后得到丝胶蛋白溶液；步骤1.2、将与步骤1.1中丝胶蛋白的质量比为0.2~2的甲基丙烯酸酐溶于PBS，获得质量分数为0.02~0.2 g/mL的甲基丙烯酸酐溶液；步骤1.3、向步骤1.1制得的丝胶蛋白溶液中逐滴加入步骤1.2中制得的甲基丙烯酸酐溶液，并实时加入5~8M氢氧化钠溶液来维持反应溶液pH8~9，在室温下反应8~24h后，透析3~7d，低温离心后将上清液冷冻干燥，得到碳碳双键修饰于丝胶蛋白羟基和氨基上的改性丝胶蛋白。
优选地，所述甲基丙烯酸缩水甘油酯改性鲟鱼软骨脱细胞基质制备方法如下：
使用鲟鱼软骨制得鲟鱼软骨脱细胞基质浆料溶液；甲基丙烯酸缩水甘油酯溶于PBS中得到甲基丙烯酸缩水甘油酯溶液；所述鲟鱼软骨脱细胞基质浆料溶液中加入甲基丙烯酸缩水甘油酯溶液得到碳碳双键修饰于鲟鱼软骨脱细胞基质氨基上的改性鲟鱼软骨脱细胞基质。
优选地，步骤2.1、鲟鱼初步解剖处理得到透明鲟鱼软骨，经过灭菌、破碎、循环冷冻、十二烷基硫酸钠（SDS）、核酸酶和匀浆处理后得到鲟鱼软骨脱细胞基质浆料；步骤2.2、将与步骤2.1中鲟鱼软骨脱细胞基质浆料的质量比为0.2~2的甲基丙烯酸缩水甘油酯溶于与步骤2.1的20%-50%鲟鱼软骨脱细胞基质浆料体积的PBS，获得质量分数为0.02~0.2g/mL的甲基丙烯酸缩水甘油酯溶液；步骤2.3、向步骤2.1制得的鲟鱼软骨脱细胞基质浆料溶液中逐滴加入步骤2.2中制得的甲基丙烯酸缩水甘油酯溶液，在室温下反应8~24h后，透析3~7d，低温离心后将底部沉淀冷冻干燥，得到碳碳双键修饰于鲟鱼软骨脱细胞基质氨基上的改性鲟鱼软骨脱细胞基质。
优选地，甲基丙烯酸酐改性丝胶蛋白和甲基丙烯酸缩水甘油酯改性鲟鱼软骨脱细胞基质的分别以5%-15%和0.5%-5%的浓度溶于含0.5%w/v的光引发剂LAP的磷酸盐缓冲溶液，再将细胞沉淀团块均匀分散在该混合溶液中，获得含细胞的生物墨水溶液。
本发明鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水，使用上述的制备方法制得。
优选地，甲基丙烯酸酐改性丝胶蛋白在磷酸盐缓冲溶液中质量体积比为5%~40%；光引发剂为2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮（I2959）或苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐（LAP），光引发剂在磷酸盐缓冲溶液中质量体积比为0.1%~1%。
本发明可3D打印鲟鱼软骨脱细胞基质/丝胶蛋白水凝胶的制备方法，包括以下步骤：利用三维建模软件设计打印结构的数字模型，再利用切片软件将该模型进行切片处理以便用于立体光刻打印；将上述鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水转移至立体光刻打印机的料槽中，按照预先设定数字模型的切片文件利用紫外光源逐层打印该溶液，最终获得鲟鱼软骨脱细胞基质/丝胶蛋白水凝胶。
本发明可3D打印鲟鱼软骨脱细胞基质/丝胶蛋白水凝胶，使用上述制备方法制得。
该鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水及其制备方法具有以下有益效果：
（1）本发明创新性将水生生物鲟鱼软骨脱细胞基质作为生物墨水原材料引入到组织工程中，并通过与丝胶蛋白材料进行复合，通过紫外光照射，使甲基丙烯酸酐或甲基丙烯酸缩水甘油酯的碳碳双键产生自由基发生聚合，为3D打印在组织工程中的应用提供新材料。。
（2）本发明形成的水凝胶具有可3D打印、性能可控、生物相容性好、可生物降解等优点，制备条件温和可控，可应用于组织工程材料等生物医用材料领域。
（3）本发明采用立体光刻打印制备水凝胶，该制备方法精度高、尺寸范围广，污染风险小、无针头堵塞风险和剪切力对细胞的损伤，且制备过程中不涉及有毒交联剂。
（4）本发明所制备生物墨水可通过调节双键改性度来调节其水溶解性能和水溶液的粘度。
（5）本发明所制备水凝胶可通过调节双键改性度来调节其力学性能和含水量。
（6）本发明所制备水凝胶可根据需要原位封装特定细胞和生长因子，并通过建立3D数字模型制备特定形状的水凝胶，以适用于个性化定制组织缺损修复。
（7）本发明利用改性丝胶蛋白和改性鲟鱼软骨脱细胞基质的碳碳双键通过紫外光照引发聚合，获得水凝胶。该生物墨水具有可3D打印、性能可控、韧性优异、生物相容性好、可生物降解等优点，制备条件温和可控，可应用于组织工程材料等生物医用材料领域。
附图说明
图1是本发明实施例1中制备的改性丝胶蛋白粉末的核磁共振氢谱图。
图2是本发明实施例1中制备的改性鲟鱼软骨脱细胞基质粉末的核磁共振氢谱图。
图3是本发明实施例1中制备的丝胶蛋白基鲟鱼软骨脱细胞基质墨水的3D打印结构图。
图4是改性丝素蛋白与改性鲟鱼软骨脱细胞基质水凝胶光照成型图，其中a为质量体积浓度为15%的改性丝素蛋白溶液，b为15%的丝素蛋白溶液与0.5%改性鲟鱼软骨脱细胞基质的混合溶液。
具体实施方式
下面结合图1至图4，对本发明做进一步说明：
实施例1：
本发明的鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水材料及其立体光刻打印水凝胶的制备，包括以下步骤：
步骤1、将提取得到的2~6g丝胶蛋白溶于100~300ml的pH为7~9的PBS中。
步骤2、将与步骤1中丝胶蛋白的质量比为0.4~2的甲基丙烯酸酐（MA）溶于PBS，获得质量分数为0.04g~0.2g/mL的甲基丙烯酸酐溶液。
步骤3、向步骤1制得的丝胶蛋白溶液中逐滴加入步骤2中制得的甲基丙烯酸酐溶液，并实时加入5~8M氢氧化钠溶液来维持反应溶液pH8-9，在室温下反应4~24h后，室温透析4~7d，低温离心，将上清液冷冻干燥，得到改性丝胶蛋白（Ser-MA）。
步骤4、鲟鱼初步解剖处理得到透明鲟鱼软骨，经过灭菌、破碎，置于液氮中循环冷冻5~10次，SDS震荡清洗4~8h,核酸酶降解12~24h,最后匀浆处理后得到鲟鱼软骨脱细胞基质浆料。
步骤5、将与步骤4中鲟鱼软骨脱细胞基质浆料的质量比为0.4~1.4的甲基丙烯酸缩水甘油酯（GMA）溶于PBS，获得质量分数为0.2~2g/mL的甲基丙烯酸缩水甘油酯溶液。
步骤6、向步骤4制得的鲟鱼软骨脱细胞基质浆料溶液中逐滴加入步骤5中制得的甲基丙烯酸缩水甘油酯溶液，在室温下反应8~12h后，透析4~7d，低温离心后将底部沉淀冷冻干燥，得到改性鲟鱼软骨脱细胞基质（Stur-GMA）。
步骤7、将步骤3制得的改性丝胶蛋白和步骤6制得的改性鲟鱼软骨脱细胞基质分别与以15%~40%和0.5%~2%的质量体积浓度混合溶解至质量体积分数为0.5%~2%的光引发剂LAP的PBS中，联合细胞（细胞密度为1kw/mL）均匀混合得到15%~40%的丝胶蛋白和0.5%~2%质量体积分数的鲟鱼软骨脱细胞基质生物墨水溶液。
步骤8、利用三维建模软件设计打印结构的数字模型星型，再利用切片软件将该模型进行切片处理以便用于立体光刻打印。
步骤9、将步骤7制得的溶液转移至立体光刻打印机的料槽中，按照预先设定数字模型的切片文件利用405nm二维光源逐层打印该溶液，最终获得鲟鱼软骨脱细胞基质/丝胶蛋白水凝胶。
本实施例制备的改性丝胶蛋白的核磁共振氢谱图如图1所示，MA修饰后在化学位移为6.10 ppm和5.60 ppm处出现了两个新的信号峰，这是-C=CH2中两个氢原子核振动所产生的信号;在化学位移为1.90ppm处出现了明显的甲基（-CH3）信号，证明丝胶蛋白改性成功。
本实施例制备的改性鲟鱼软骨脱细胞基质的核磁共振氢谱图如图2所示，GMA 修饰后在化学位移为6.22 ppm和5.65 ppm处出现了两个新的信号峰，这是-C=CH2中两个氢原子核振动所产生的信号;在化学位移为1.80 ppm处出现了明显的甲基（-CH3）信号，证明鲟鱼软骨脱细胞基质改性成功。
通过立体光刻打印机打印星型载软骨细胞丝胶蛋白基鲟鱼软骨脱细胞基质生物墨水，结果如图3所示，打印结构形状完整、星型保真度高。
改性的鲟鱼软骨脱细胞基质不仅可以与实施例中制备的丝胶蛋白复合，也可与丝素蛋白复合，室温下光固化情况如图4所示。
上面结合附图对本发明进行了示例性的描述，显然本发明的实现并不受上述方式的限制，只要采用了本发明的方法构思和技术方案进行的各种改进，或未经改进将本发明的构思和技术方案直接应用于其它场合的，均在本发明的保护范围内。

Claims (10)

1.一种鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水的制备方法，包括以下步骤：
甲基丙烯酸酐修饰丝胶蛋白上的羟基和氨基，得具有光聚合能力的甲基丙烯酸酐改性丝胶蛋白；
甲基丙烯酸缩水甘油酯修饰鲟鱼软骨脱细胞基质上的羟基和氨基，得具有光聚合能力的甲基丙烯酸缩水甘油酯改性鲟鱼软骨脱细胞基质；
甲基丙烯酸酐改性丝胶蛋白、甲基丙烯酸缩水甘油酯改性鲟鱼软骨脱细胞基质与光引发剂溶液混合制得鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水。
2.根据权利要求1所述的鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水的制备方法，其特征在于：
所述甲基丙烯酸酐改性丝胶蛋白的制备方法如下：
将丝胶蛋白溶于PBS中得到丝胶蛋白溶液；
甲基丙烯酸酐溶于PBS得到甲基丙烯酸酐溶液；
丝胶蛋白溶液与甲基丙烯酸酐溶液在碱性条件下反应获得碳碳双键修饰于丝胶蛋白羟基和氨基上的改性丝胶蛋白。
3.根据权利要求2所述的鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水的制备方法，其特征在于：
步骤1.1、将购买的蚕茧去除杂质，经Na2CO3 溶液煮沸，将初步得到的黄色液体透析3~10d，低温离心后将上清液冷冻干燥，将冷冻干燥后的粉末溶解于PBS后得到丝胶蛋白溶液；
步骤1.2、将与步骤1.1中丝胶蛋白的质量比为0.2~2的甲基丙烯酸酐溶于PBS，获得质量分数为0.02~2 g/mL的甲基丙烯酸酐溶液；
步骤1.3、向步骤1.1制得的丝胶蛋白溶液中逐滴加入步骤1.2中制得的甲基丙烯酸酐溶液，并实时加入5~8M氢氧化钠溶液来维持反应溶液pH8~9，在室温下反应8~24h后，透析3~10d，低温离心后将上清液冷冻干燥，得到碳碳双键修饰于丝胶蛋白羟基和氨基上的改性丝胶蛋白。
4.根据权利要求1所述的鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水的制备方法，其特征在于：
所述甲基丙烯酸缩水甘油酯改性鲟鱼软骨脱细胞基质制备方法如下：
使用鲟鱼软骨制得鲟鱼软骨脱细胞基质浆料溶液；
甲基丙烯酸缩水甘油酯溶于PBS中得到甲基丙烯酸缩水甘油酯溶液；
所述鲟鱼软骨脱细胞基质浆料溶液中加入甲基丙烯酸缩水甘油酯溶液得到碳碳双键修饰于鲟鱼软骨脱细胞基质氨基上的改性鲟鱼软骨脱细胞基质。
5.根据权利要求4所述的鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水的制备方法，其特征在于：
步骤2.1、鲟鱼初步解剖处理得到透明鲟鱼软骨，经过灭菌、破碎、循环冷冻、十二烷基硫酸钠（SDS）、核酸酶和匀浆处理后得到鲟鱼软骨脱细胞基质浆料；
步骤2.2、将与步骤2.1中鲟鱼软骨脱细胞基质浆料的质量比为0.2~1.2的甲基丙烯酸缩水甘油酯溶于与步骤2.1中鲟鱼软骨脱细胞基质浆料体积比为20%-70%的PBS，获得质量分数为0.02~0.2g/mL的甲基丙烯酸缩水甘油酯溶液；
步骤2.3、向步骤2.1制得的鲟鱼软骨脱细胞基质浆料溶液中逐滴加入步骤2.2中制得的甲基丙烯酸缩水甘油酯溶液，在室温下反应8~24h后，透析3~10d，低温离心后将底部沉淀冷冻干燥，得到碳碳双键修饰于鲟鱼软骨脱细胞基质氨基上的改性鲟鱼软骨脱细胞基质。
6.根据权利要求1-5中任何一项所述的鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水的制备方法，其特征在于：
甲基丙烯酸酐改性丝胶蛋白和甲基丙烯酸缩水甘油酯改性鲟鱼软骨脱细胞基质的分别以5%-40%和0.5%-5%的浓度溶于含0.5%-5%w/v的光引发剂LAP的磷酸盐缓冲溶液，再将细胞沉淀团块均匀分散在该混合溶液中，获得含细胞的生物墨水溶液。
7.一种鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水，其特征在于：使用权利要求1-6中任何一项所述的制备方法制得。
8.根据权利要求7所述的鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水，其特征在于：甲基丙烯酸酐改性丝胶蛋白在磷酸盐缓冲溶液中质量体积比为5%~40%；光引发剂为2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮（I2959）或苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐（LAP），光引发剂在磷酸盐缓冲溶液中质量体积比为0.1%~2%。
9.一种可3D打印鲟鱼软骨脱细胞基质/丝胶蛋白水凝胶的制备方法，包括以下步骤：
利用三维建模软件设计打印结构的数字模型，再利用切片软件将该模型进行切片处理以便用于立体光刻打印；
将权利要求7或8所述鲟鱼软骨脱细胞基质/丝胶蛋白生物墨水转移至立体光刻打印机的料槽中，按照预先设定数字模型的切片文件利用紫外光源逐层打印该溶液，最终获得鲟鱼软骨脱细胞基质/丝胶蛋白水凝胶。
10.一种可3D打印鲟鱼软骨脱细胞基质/丝胶蛋白水凝胶，其特征在于：使用权利要求9所述制备方法制得。
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