CN106474560B - 一种用于3d生物打印的水凝胶材料及其制备方法与应用 - Google Patents
一种用于3d生物打印的水凝胶材料及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于生物材料技术领域，公开了一种用于3D生物打印的水凝胶材料及其制备方法与应用。本发明的水凝胶材料包括以下质量百分比组分：结冷胶和/或其衍生物0.5～10％，PEG和/或其衍生物0.1～20％，交联引发剂0～1％，生物活性组分0～15％，溶剂余量。本发明水凝胶材料为基于结冷胶和PEG的双网络水凝胶，生理环境下形成互穿的双网络结构，具有更好的结构和尺寸稳定性，具有可在生理条件下快速成胶、细胞相容性优良、免疫排斥小、细胞包封率高、力学强度可控、可生物降解等优点。将其应用于3D生物打印中，克服了固化速度慢、固化条件苛刻、力学性能有限、细胞相容性差等缺点，具有明显优势和良好的产业化前景。
Description
技术领域
本发明属于生物材料技术领域，特别涉及一种用于3D生物打印的水凝胶材料及其制备方法与应用。
背景技术
3D生物打印是在快速成形技术发展的基础上，结合细胞生物学、计算机辅助设计和生物材料学等多个领域的研究成果，发展而来的一种新型的组织工程技术，其最终目标是实现器官打印。3D生物打印技术能够克服传统组织工程技术的局限性，通过精确定位生物材料、生物化学分子和活细胞层间位置，可实现不同密度的种子细胞在不同支架材料中的三维精确定位，控制功能性成分的空间位置，用于制造三维结构。
水凝胶具有高含水率、良好的生物相容性、可降解性、亲水性以及包裹细胞的强大能力，是3D生物打印和人工器官制备的首选材料，水凝胶和三维生物打印技术的结合为开发可细胞接种/封装的组织工程支架提供了一种设计关联的可控解决方案。目前用于生物打印的水凝胶主要包括海藻酸钠、壳聚糖、胶原蛋白、琼脂等天然水凝胶，以及人工合成的聚多肽、聚乙二醇等化学水凝胶。但现有的水凝胶体系都很难同时满足三维生物打印的诸多要求，例如：生理条件下快速成胶、细胞相容性、力学强度、通透性、生物降解性等。因此开发新型的水凝胶用于3D生物打印仍然是该领域中的亟待解决的问题。
发明内容
为了克服上述现有技术的缺点与不足，本发明的首要目的在于提供一种用于3D生物打印的水凝胶材料。本发明水凝胶材料为基于结冷胶和PEG的双网络水凝胶，其具有可在生理条件下快速成胶、细胞相容性优良、力学强度可控、可生物降解等优点，且可形成一定微观结构和力学强度的自支撑水凝胶材料。
本发明另一目的在于提供一种上述用于3D生物打印的水凝胶材料的制备方法。
本发明再一目的在于提供上述水凝胶材料在3D生物打印中的应用。本发明的水凝胶材料克服了传统的3D打印墨水溶液固化速度慢、固化条件苛刻、力学性能有限、细胞相容性差等缺点，与现有的3D生物打印用水凝胶相比，具有明显优势和良好的产业化前景。
本发明的目的通过下述方案实现：
一种用于3D生物打印的水凝胶材料，包括以下质量百分比组分：结冷胶和/或其衍生物0.5～10％，PEG和/或其衍生物0.1～20％，交联引发剂0～1％，生物活性组分0～15％，溶剂余量。
本发明中，所述的结冷胶和/或其衍生物包括并不限于羧甲基化结冷胶，或采用双键、巯基、生物活性分子等修饰的结冷胶。
本发明中，所述的PEG和/或其衍生物包括并不限于PEG，或采用双键、炔基、巯基等修饰的PEG。
本发明中，所述的生物活性组分包括并不限于细胞生长因子、细胞诱导因子、多肽、药物等。这些生物活性成分不仅可以提供良好的生物学功能促进细胞和组织的再生，还可以用于调控细胞的增殖、分化。
本发明中，所述的交联引发剂包括并不限于水溶性自由基引发剂或光引发剂；其中具体可包括并不限于过硫酸铵、I2959、MBS、樟脑酮(CQ)中的一种。本发明的交联引发剂与双键等官能团作用产生自由基，使聚合物交联形成网络结构。
本发明中，所述的溶剂可为水、PBS或细胞培养基等。
本发明还提供一种上述用于3D生物打印的水凝胶材料的制备方法，通过将各组分混合均匀即可，优选为在37℃下与生物活性组分混合。
本发明的用于3D生物打印的水凝胶材料为基于结冷胶和PEG的双网络水凝胶，在生理环境下能够形成互穿的双网络结构，具有更好的结构和尺寸稳定性，且具有可在生理条件下快速成胶、细胞相容性优良、力学强度可控、可生物降解等优点。
将本发明的水凝胶材料作为3D打印墨水，采用注射挤出的方式进行3D打印，即可形成一定微观结构和形状的材料，进一步利用紫外光照射固化，能够得到较高力学强度的自支撑水凝胶材料。克服了传统的3D打印墨水溶液固化速度慢、固化条件苛刻、力学性能有限、细胞相容性差等缺点，将其应用于3D生物打印中，与现有的3D生物打印用水凝胶相比，具有明显优势和良好的产业化前景。
本发明的水凝胶材料可通过多层打印实现厘米级的自支撑结构，支架材料具有较高的细胞包封率，能够成功实现活细胞的3D打印。且在成胶后，通过紫外光交联可进一步提高其力学性能。并可通过调整各组分的比例来调控水凝胶的力学性能，可制备与人体软组织或软骨力学性能相当的材料。本发明的水凝胶材料可添加细胞悬液进行3D打印，两者直接混合打印即可。
本发明中的水凝胶材料具有良好的生物相容性，免疫排斥小，生物降解性好，具有较高的细胞包封率，能够为细胞的生长提供支撑骨架和合适的生理环境，加入其中的活性物质也能够有效的促进细胞的增殖、分化和组织再生。
附图说明
图1为实施例1制备的水凝胶材料3D打印成型的支架外观。
图2为实施例1制备的水凝胶材料的SEM观察结果。
图3为实施例3中的水凝胶材料与骨髓间充质干细胞复合3D打印后继续培养5天(A)和7天(B)的激光共聚焦观察结果。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。
下列实施例中使用的材料均可从商业渠道获得。
实施例1
一种用于3D生物打印的水凝胶材料，由结冷胶、聚乙二醇丙烯酸酯(PEGDA)、溶剂、生物活性成分组成，本实施例中生物活性成分采用骨形态发生蛋白BMP-2，其制备方法如下：
(1)通过常规方法利用丙烯酰氯对PEG端基进行修饰，获得聚乙二醇丙烯酸酯；
(2)按质量分数，将0.75％的结冷胶，10％的PEGDA，0.1％的引发剂I-2959溶于80℃的PBS溶液中，搅拌均匀，再于37℃加入骨形态发生蛋白BMP-2，混合均匀，得到水凝胶材料；
(3)3D打印：将上述水凝胶材料与前体成骨细胞悬液混合均匀，细胞密度为5×105cells/mL，置于3D生物打印机墨水管内，打印成一定结构的含细胞支架，打印后将材料置于紫外反应仪中照射10min，紫外光波长312nm，辐照强度为50J/cm2，最终获得材料的外观如图1所示，通过3D打印该水凝胶能够形成厘米级具有联通孔洞的自支撑结构，水凝胶冻干后的SEM观察结果如图2所示；
(4)将上述含细胞支架在二氧化碳培养箱继续培养7天，隔天换液，细胞活死染色的结果显示培养期间细胞存活率达95％以上，说明该水凝胶均能够支持细胞的生长行为，保持细胞形态、存活、增殖等，并能为细胞的生长提供较理想的微环境。
实施例2
一种用于3D生物打印的水凝胶材料，由巯基化结冷胶、炔基化聚乙二醇、溶剂、生物活性成分组成，本实施例中生物活性成分采用小分子药物TWS119(TWS119是一种对定向诱导神经干细胞向神经元分化具有显著作用的小分子药物)，其制备方法如下：
(1)通过常规方法利用半胱氨酸对结冷胶进行修饰，获得巯基化结冷胶；
(2)通过常规方法利用炔丙基溴对PEG端基进行修饰，获得炔基化聚乙二醇；
(3)按质量分数，将0.6％的巯基化结冷胶，10％的炔基化聚乙二醇，0.1％的引发剂I-2959溶于80℃的去离子水中，搅拌均匀，再于37℃加入含有小分子药物TWS119的纳米粒子(TWS119用量0.5％)，混合均匀，得到水凝胶材料；
(4)3D打印：将上述水凝胶材料与神经干细胞悬液混合均匀，细胞密度为1×106cells/mL，将上述混合物置于3D生物打印机墨水管内，打印成一定结构的含细胞支架，打印后将材料置于紫外反应仪中照射10min，紫外光波长312nm，辐照强度为50J/cm2，通过3D打印该水凝胶能够形成特定的结构的水凝胶支架；
(5)将上述含细胞支架在二氧化碳培养箱继续培养14天，隔天换液，细胞分化的统计结果显示，神经干细胞分化成神经元的比例为85％，证明水凝胶系统对神经干细胞有定向诱导分化为神经元的功能。
实施例3
一种用于3D生物打印的水凝胶材料，由巯基化结冷胶、聚乙二醇、溶剂、生物活性成分组成，本实施例中生物活性成分采用碱性成纤维细胞生长因子(bFGF)，其制备方法如下：
(1)通过常规方法利用半胱氨酸对结冷胶进行修饰，获得巯基化结冷胶；
(2)按质量分数，将1.5％的结冷胶，1％的PEG-6000，溶于80℃的去离子水中，搅拌均匀，再于37℃加入100ng/mL碱性成纤维细胞生长因子(bFGF)，混合均匀，得到水凝胶材料；
(3)3D打印：向上述水凝胶材料中加入骨髓间充质干细胞悬液，细胞密度为1×105cells/100μL，置于3D生物打印机墨水管内，打印出一定结构的含细胞支架，打印后将材料立即置于骨髓间充质干细胞培养基中；
(4)含细胞支架在二氧化碳培养箱中继续培养5～7天，隔天换液，骨髓间充质干细胞在水凝胶中的荧光染色照片如图3所示，细胞在该水凝胶中能够正常增殖、分化，该体系显示出良好的细胞包封效果和理想的生物相容性。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (6)
1.一种用于3D生物打印的水凝胶材料，其特征在于包括以下质量百分比组分：结冷胶和/或其衍生物0.5～10％，PEG和/或其衍生物0.1～20％，交联引发剂0～1％，生物活性组分0～15％，溶剂余量；
所述的结冷胶和/或其衍生物包括羧甲基化结冷胶，或采用双键、巯基、生物活性分子修饰的结冷胶中的至少一种；
所述的PEG和/或其衍生物包括PEG，或采用双键、炔基、巯基修饰的PEG中的至少一种；
所述的交联引发剂包括过硫酸铵、I2959、MBS和樟脑酮中的至少一种。
2.根据权利要求1所述的用于3D生物打印的水凝胶材料，其特征在于：所述的生物活性组分包括细胞生长因子、细胞诱导因子、多肽和药物中的至少一种。
3.根据权利要求1所述的用于3D生物打印的水凝胶材料，其特征在于：所述的溶剂包括水、PBS和细胞培养基中的至少一种。
4.一种权利要求1～3任一项所述的用于3D生物打印的水凝胶材料的制备方法，其特征在于通过将各组分混合均匀得到。
5.权利要求1～3任一项所述的用于3D生物打印的水凝胶材料在3D打印中的应用。
6.根据权利要求5所述的应用，其特征在于具体为将所述的用于3D生物打印的水凝胶材料作为3D打印墨水，采用注射挤出的方式进行3D打印，形成微观结构和形状的材料，进一步利用紫外光照射固化，得到较高力学强度的自支撑水凝胶材料。
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| CN115120779B (zh) * | 2021-03-23 | 2023-11-03 | 吴东蔚 | 一种可用于3d打印的水凝胶泡沫材料及其制备方法与应用 |
| CN113679884A (zh) * | 2021-07-27 | 2021-11-23 | 华南理工大学 | 促进细胞迁移的组织工程水凝胶支架及其制备方法、3d打印浆料及其制备方法 |
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