Abstract
Three-dimensional (3D) hydrogel models play a crucial role in tissue engineering for promoting tissue regeneration. A biomimetic microchannel network system in the 3D hydrogel model is necessary for optimal cellular function. This report describes the preparation of a biomimetic hydrogel scaffold with an internal microchannel network, using electrospinning techniques and the sacrificial template method for 3D cell culture. Microchannels and cavities were created within the gelatin methacryloyl (GelMA) hydrogel by sacrificing polyvinyl alcohol (PVA) electrospun fibers (>10 µm), resulting in the creation of microvessel-like channels. Mechanical characterizations, swelling properties, and biodegradation analysis were conducted to investigate the feasibility of a biomimetic microchannel network hydrogel scaffold for 3D cell culture applications. Compared to pure GelMA hydrogel, the hydrogel with microchannels promoted cell proliferation, adhesion, and endothelial tube formation. Moreover, the results confirmed that the biomimetic microchannel network scaffold had a major impact on the distribution and arrangement of human umbilical vein endothelial cells (HUVECs) and can enable the formation of artificial microvasculature by the culture of HUVECs and cell media perfusion.
摘要
目 的
三维(3D)水凝胶模型在组织工程中起到了促进组织再生的关键作用. 三维水凝胶模型中的仿生微通道网络系统是实现最佳细胞功能的必要条件. 本文旨在研究利用电纺技术和牺牲模板法制备具有内部微通道网络的仿生水凝胶支架, 用于三维内皮细胞培养形成功能性人工微血管.
创新点
1. 首次利用静电纺丝技术成功制备直径>10 μm的纤维材料, 作为形成微通道网络的牺牲材料; 2. 通过反复挤压的方式, 在高浓度甲基丙烯酰化明胶(GelMA)水凝胶内成功创建了微通道和空腔, 从而创建了微血管样通道; 3. 成功在微通道腔内形成内皮细胞单层, 并研究了3D水凝胶支架表面和支架内对人脐静脉内皮细胞(HUVECs)的活性、 增殖、 形态发生和血管再生的影响.
方 法
1. 以难挥发的去离子水和易挥发的六氟异丙醇(HFIP)为溶剂, 通过优化配比及电纺条件, 使其静电纺直径更大的纤维, 用于形成微通道结构的牺牲材料(图1); 2. 合成并研究高浓度的纯GelMA水凝胶和支架的理化性能和生物相容性. 通过力学性能、溶胀性能和降解性能等表征和对比, 筛选出最优性能的支架(图2~5); 3. 通过对支架表面和支架内的内皮细胞培养, 量化对比内皮细胞的形态发生以及细胞核铺展, 研究支架对人工微血管形成的影响(图6~8).
结 论
1. 在室温条件下, 聚乙烯醇(PVA)纤维能够被聚丁二酸丁二醇酯(PBS)溶液快速溶解; 2. 通过力学性能、溶胀性能和降解性能等测试, 与纯GelMA水凝胶相比, 含微通道网络的仿生GelMA水凝胶力学性能有所下降. 虽然微通道网络的存在降低了其各种性能, 但仍有利于HUVECs的生长和增殖, 且满足组织再生的周期, 特别适用于三维细胞培养和组织工程应用; 3. CCK-8毒性测试和对细胞的活-死染色结果显示, 三种支架均具有良好的生物相容性. 在培养7 d后, 材料表面细胞相互连接形成管状结构, HUVECs在浓度为25%的支架比15%和20%的支架细胞数量显著增加, 且有更明显的铺展和管状结构. 这表明其状态良好, 支持了含内部结构的GelMA水凝胶支架的生物相容性, 且此支架可作为生物医学领域的3D细胞培养, 应证了上述所提到的内含微结构的水凝胶亦适用于细胞附着与增殖. 4. 经过对细胞的4′,6-二脒基-2-苯基吲哚(DAPI)和微丝染色, 结果显示培养3 d后, 在支架通道内形成了稳定的内皮单细胞层, 细胞核分布均匀且沿着孔道主轴方向排布, 微丝清晰可见, 大多倾向于轴向分布. 其次, 微孔道通过接触引导影响HUVECs的细胞行为与形态, 从而促进HUVECs形成微血管. 发现微通道管腔3D微环境通过接触引导促进融合的内皮细胞单层的形成, 进一步表明了此支架作为体外微血管的再生的适用性. 最后, 通过量化对比, 微通道加速了微血管的再生.
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This work is supported by the National Natural Science Foundation of China (No. 31870934) and the Natural Science Foundation for Young Scientists of Shanxi Province (No. 202103021223100), China.
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Haoyu SUN, Yang LIU, Meiwen AN, Meiling WEN, and Li WANG designed the research. Haoyu SUN, Haiyang MA, Tian HOU, Wenjie TANG, and Qing YU processed the corresponding data. Haoyu SUN wrote the first draft of the manuscript. Meiling WEN helped to organize the manuscript. Meiwen AN revised and edited the final version.
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Haoyu SUN, Haiyang MA, Li WANG, Yang LIU, Tian HOU, Wenjie TANG, Qing YU, Meiwen AN, and Meiling WEN declare that they have no conflict of interest.
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Biomimetic microchannel network with functional endothelium formed by sacrificial electrospun fibers inside 3D gelatin methacryloyl (GelMA) hydrogel models
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Sun, H., Ma, H., Wang, L. et al. Biomimetic microchannel network with functional endothelium formed by sacrificial electrospun fibers inside 3D gelatin methacryloyl (GelMA) hydrogel models. J. Zhejiang Univ. Sci. A 25, 79–96 (2024). https://doi.org/10.1631/jzus.A23D0045
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DOI: https://doi.org/10.1631/jzus.A23D0045