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江文丽, 钟健, 欧阳智, 等. 矩形水凝胶微凹槽的空间约束调控人脐静脉内皮细胞的形态和排列[J]. 四川大学学报(医学版), 2024, 55(1): 87-94. DOI: 10.12182/20240160402
引用本文: 江文丽, 钟健, 欧阳智, 等. 矩形水凝胶微凹槽的空间约束调控人脐静脉内皮细胞的形态和排列[J]. 四川大学学报(医学版), 2024, 55(1): 87-94. DOI: 10.12182/20240160402
JIANG Wenli, ZHONG Jian, OUYANG Zhi, et al. Spatial Constraints of Rectangular Hydrogel Microgrooves Regulate the Morphology and Arrangement of Human Umbilical Vein Endothelial Cells[J]. Journal of Sichuan University (Medical Sciences), 2024, 55(1): 87-94. DOI: 10.12182/20240160402
Citation: JIANG Wenli, ZHONG Jian, OUYANG Zhi, et al. Spatial Constraints of Rectangular Hydrogel Microgrooves Regulate the Morphology and Arrangement of Human Umbilical Vein Endothelial Cells[J]. Journal of Sichuan University (Medical Sciences), 2024, 55(1): 87-94. DOI: 10.12182/20240160402

矩形水凝胶微凹槽的空间约束调控人脐静脉内皮细胞的形态和排列

Spatial Constraints of Rectangular Hydrogel Microgrooves Regulate the Morphology and Arrangement of Human Umbilical Vein Endothelial Cells

  • 摘要:
    目的 本研究拟构建微米级矩形水凝胶凹槽,探究人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)在三维空间约束条件下的形态和排列规律。
    方法 使用四臂-聚乙二醇-丙烯酸酯水凝胶制备宽度为60 μm、100 μm、140 μm的矩形微凹槽,检测其尺寸和纤连蛋白(fibronectin, FN)黏附情况;在FN包被的微凹槽中接种HUVECs,培养48 h后检测细胞的形态和取向,利用鬼笔环肽标记细胞骨架和激光共聚焦显微镜观察水凝胶微凹槽中HUVECs细胞骨架取向,以无微图案平面为对照。
    结果 构建的水凝胶微凹槽形态均一、结构完整,边缘清晰,宽度误差<3.5%,不同宽度水凝胶微凹槽的深度差异较小,FN黏附均匀,为细胞提供了微图案化的生长界面。对照组中细胞排列杂乱,取向随机,细胞取向角为(46.9±1.8)°,而水凝胶微凹槽中细胞取向角显著减小(P<0.001),但随着水凝胶微凹槽宽度增加而增大,60 μm、100 μm、140 μm水凝胶微凹槽中细胞取向角分别为(16.4±2.8)°、(24.5±3.2)°、(30.3±3.5)°;与对照组相比较(35.7%),不同宽度水凝胶微凹槽中取向角<30°的细胞数量增多(P<0.001),但随着水凝胶微凹槽宽度增加,取向角<30°的细胞数量逐渐减少(79.9%、62.3%、54.7%),而取向角60°~90°的细胞数量逐渐增加(P<0.001)。微凹槽中细胞胞体变小、变圆,细胞沿微凹槽方向排列,细胞骨架排列发生相应改变,对照组中细胞骨架纤维丝排列方向随机,取向角为(45.5±3.7)°,各取向角内骨架纤维数量分布均匀,但60 μm、100 μm、140 μm的水凝胶微凹槽中的细胞骨架蛋白纤维取向角显著降低,分别为(14.4±3.1)°、(24.7±3.5)°、(31.9±3.3)°,不同宽度水凝胶微凹槽中取向角<30°的骨架纤维数量明显增加(P<0.001),但随着水凝胶微凹槽宽度增加,取向角<30°的骨架纤维数量逐渐减少,而取向角60°~90°的骨架纤维数量逐渐增加(P<0.001)。
    结论 水凝胶微凹槽可调控HUVECs形态与取向,在一定程度上可模拟血管内皮细胞的在体微环境,为研究血管内皮细胞的独特生理功能提供了更符合生理条件的实验模型,但三维空间约束影响血管内皮细胞形态和组装的分子机制有待进一步研究。

     

    Abstract:
    Objective To construct microscale rectangular hydrogel grooves and to investigate the morphology and alignment of human umbilical vein endothelial cells (HUVECs) under spatial constraints. Vascular endothelial cell morphology and alignment are important factors in vascular development and the maintenance of homeostasis.
    Methods A 4-arm polyethylene glycol-acrylate (PEG-acrylate) hydrogel was used to fabricate rectangular microgrooves of the widths of 60 μm, 100 μm, and 140 μm. The sizes and the fibronectin (FN) adhesion of these hydrogel microgrooves were measured. HUVECs were seeded onto the FN-coated microgrooves, while the flat surface without micropatterns was used as the control. After 48 hours of incubation, the morphology and orientation of the cells were examined. The cytoskeleton was labelled with phalloidine and the orientation of the cytoskeleton in the hydrogel microgrooves was observed by laser confocal microscopy.
    Results The hydrogel microgrooves constructed exhibited uniform and well-defined morphology, a complete structure, and clear edges, with the width deviation being less than 3.5%. The depth differences between the hydrogel microgrooves of different widths were small and the FN adhesion is uniform, providing a micro-patterned growth interface for cells. In the control group, the cells were arranged haphazardly in random orientations and the cell orientation angle was (46.9±1.8)°. In contrast, the cell orientation angle in the hydrogel microgrooves was significantly reduced (P<0.001). However, the cell orientation angles increased with the increase in hydrogel microgroove width. For the 60 μm, 100 μm, and 140 μm hydrogel microgrooves, the cell orientation angles were (16.4±2.8)°, (24.5±3.2)°, and (30.3±3.5)°, respectively. Compared to that of the control group (35.7%), the number of cells with orientation angles <30° increased significantly in the hydrogel microgrooves of different widths (P<0.001). However, as the width of the hydrogel microgrooves increased, the number of cells with orientation angles <30° gradually decreased (79.9%, 62.3%, 54.7%, respectively), while the number of cells with orientation angles between 60°-90° increased (P<0.001). The cell bodies in the microgrooves were smaller and more rounded in shape. The cells were aligned along the direction of the microgrooves and corresponding changes occurred in the arrangement of the cell cytoskeleton. In the control group, cytoskeletal filaments were aligned in random directions, presenting an orientation angle of (45.5±3.7)°. Cytoskeletal filaments were distributed evenly within various orientation angles. However, in the 60 μm, 100 μm, and 140 μm hydrogel microgrooves, the orientation angles of the cytoskeletal filaments were significantly decreased, measuring (14.4±3.1)°, (24.7±3.5)°, and (31.9±3.3)°, respectively. The number of cytoskeletal filaments with orientation angles <30° significantly increased in hydrogel microgrooves of different widths (P<0.001). However, as the width of the hydrogel microgrooves increased, the number of cytoskeletal filaments with orientation angles <30° gradually decreased, while the number of cytoskeletal filaments with orientation angles between 60°-90° gradually increased (P<0.001).
    Conclusion Hydrogel microgrooves can regulate the morphology and orientation of HUVECs and mimic to a certain extent the in vivo microenvironment of vascular endothelial cells, providing an experimental model that bears better resemblance to human physiology for the study of the unique physiological functions of vascular endothelial cells. Nonetheless, the molecular mechanism of spatial constraints on the morphology and the assembly of vascular endothelial cell needs to be further investigated.

     

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