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



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.

Keywords: Micropatterning,  Vascular endothelial cell,  Cellular morphology,  Cell orientation, Cytoskeleton

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KRÜGER-GENGE A, BLOCKI A, FRANKE R P, et al. Vascular endothelial cell biology: an update. Int J Mol Sci,2019,20(18): 4411. doi: 10.3390/ijms20184411.

SUMPIO B E, RILEY J T, DARDIK A. Cells in focus: endothelial cell. Int J Biochem Cell Biol,2002,34(12): 1508–1512. doi: 10.1016/S1357-2725 (02)00075-4.

FALLON M E, HINDS M T. Single cell morphological metrics and cytoskeletal alignment regulate VCAM-1 protein expression. Biochem Biophys Res Commun,2021,555: 160–167. doi: 10.1016/j.bbrc.2021.03. 129.

STEWARD R, Jr, TAMBE D, HARDIN C C, et al. Fluid shear, intercellular stress, and endothelial cell alignment. Am J Physiol Cell Physiol,2015,308(8): C657–C664. doi: 10.1152/ajpcell.00363.2014.

CHEN J, ZHANG D, LI Q, et al. Effect of different cell sheet ECM microenvironment on the formation of vascular network. Tissue Cell, 2016,48(5): 442–451. doi: 10.1016/j.tice.2016.08.002.

STREICHAN S J, HOERNER C R, SCHNEIDT T, et al. Spatial constraints control cell proliferation in tissues. Proc Natl Acad Sci U S A, 2014,111(15): 5586–5591. doi: 10.1073/pnas.1323016111.

MILANO D F, NGAI N A, MUTHUSWAMY S K, et al. Regulators of metastasis modulate the migratory response to cell contact under spatial confinement. Biophys J,2016,110(8): 1886–1895. doi: 10.1016/j.bpj.2016. 02.040.

NELSON C M, JEAN R P, TAN J L, et al. Emergent patterns of growth controlled by multicellular form and mechanics. Proc Natl Acad Sci U S A,2005,102(33): 11594–11599. doi: 10.1073/pnas.0502575102.

LIU W, SUN Q, ZHENG Z L, et al. Topographic cues guiding cell polarization via distinct cellular mechanosensing pathways. Small,2022, 18(2): e2104328. doi: 10.1002/smll.202104328.

HAGEN M W, HINDS M T. Static spatial growth restriction micropatterning of endothelial colony forming cells influences their morphology and gene expression. PLoS One,2019,14(6): e0218197. doi: 10.1371/journal.pone.0218197.

Van Der PUTTEN C, BUSKERMOLEN A B C, WERNER M, et al. Protein micropatterning in 2.5D: an approach to investigate cellular responses in multi-cue environments. ACS Appl Mater Interfaces,2021, 13(22): 25589–25598. doi: 10.1021/acsami.1c01984.

PORRAS HERNÁNDEZ A M, TENJE M, ANTFOLK M. Cell chirality exhibition of brain microvascular endothelial cells is dependent on micropattern width. RSC Adv,2022,12(46): 30135–30144. doi: 10.1039/D2RA05434E.

HAGEN M W, HINDS M T. The effects of topographic micropatterning on endothelial colony-forming cells. Tissue Eng Part A,2021,27(3/4): 270–281. doi: 10.1089/ten.tea.2020.0066.

NAGAYAMA K. A loss of nuclear-cytoskeletal interactions in vascular smooth muscle cell differentiation induced by a micro-grooved collagen substrate enabling the modeling of an in vivo cell arrangement. Bioengineering (Basel),2021,8(9): 124. doi: 10.3390/bioengineering 8090124.

LI J, ZHANG K, XU Y, et al. A novel coculture model of HUVECs and HUASMCs by hyaluronic acid micropattern on titanium surface. J Biomed Mater Res A,2014,102(6): 1950–1960. doi: 10.1002/jbm.a.34867. PORRAS HERNÁNDEZ A M, BARBE L, POHLIT H, et al. Brain microvasculature endothelial cell orientation on micropatterned hydrogels is affected by glucose level variations. Sci Rep,2021,11(1): 19608. doi: 10.1038/s41598-021-99136-9.

LEVINA E M, KHARITONOVA M A, ROVENSKY Y A, et al. Cytoskeletal control of fibroblast length: experiments with linear strips of substrate. J Cell Sci,2001,114(Pt 23): 4335–4341. doi: 10.1242/jcs.114.23. 4335.

CHOI Y S, VINCENT L G, LEE A R, et al. The alignment and fusion assembly of adipose-derived stem cells on mechanically patterned matrices. Biomaterials,2012,33(29): 6943–6951. doi: 10.1016/j. biomaterials.2012.06.057.


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