Endothelial cell responses to micropillar substrates of varying dimensions and stiffness

Abstract

In the vascular niche, the extracellular matrix (ECM) provides a structural scaffold with a rich ligand landscape of essential matrix proteins that supports the organization and stabilization of endothelial cells (ECs) into functional blood vessels. Many of the physical interactions between ECs and macromolecular components of the ECM occur at both the micron and submicron scale. In addition, the elasticity of the ECM has been shown to be a critical factor in the progress of the angiogenic cascade. Here, we sought to determine the effect of substrate topography and elasticity (stiffness) on EC behavior. Utilizing a unique SiO(2) substrate with an array of micropillars, we first demonstrate that micropillars with heights >3 μm significantly decrease EC adhesion and spreading. Fibronectin (Fn) patterning of 1 μm high micropillars enabled EC adhesion onto the micropillars and promoted alignment in a single-cell chain manner. We then developed a robust method to generate a soft micropillar substrate array made of polydimethylsiloxane (PDMS), similar to the SiO(2) substrate. Finally, we examined the kinetics of EC adhesion and spreading on the soft PDMS substrates compared to the stiff SiO(2) substrates. Culturing cells on the PDMS substrates demonstrated an enhanced EC elongation and alignment when compared to stiff SiO(2) with similar topographical features. We conclude that the elongation and alignment of ECs is coregulated by substrate topography and stiffness and can be harnessed to guide vascular organization.

Figure 1. SiO₂ micropillar substrates

(A) Light microscope image of representative SiO₂ micropillar substrate. SiO₂ micropillar substrates were fabricated using I-line lithography followed by a timed reactive ion etch to create a unique substrate design presenting micropillar diameters ranging from 1-5.6 μm, with spacing between pillars ranging from 0.6-15 μm. (B) SEM images of various locations of the SiO₂ micropillar substrate (indicated by stars), demonstrating wide array of design. (C) SEM images of SiO₂ substrate of 1 μm, 3 μm, 6 μm, and 8 μm micropillar heights. Scale bar is 1 mm in (A), 5 μm in (B), and 2 μm in (C).

Figure 2. EC adhesion onto Fn-coated micropillared SiO₂ substrates

(A) HUVEC (upper panel) and ECFC (lower panel) adhesion onto Fn-coated micropillar SiO₂ substrates with topographical feature heights ranging from 1-8 μm, cells stained with calcein (green-live staining). For clarification, some micropillar regions are outlined with a white box. (B) Quantification of percent of HUVEC (left) and ECFC (right) live cells on micropillar regions for each of the feature heights shows marked decreased percentage of live cells on substrates with topographical features with >3 μm heights. (C) HUVEC (upper panel) and ECFC (lower panel) expressing endothelial surface marker CD31 (red) have decreased cell spreading on substrates with topographical feature heights >1 μm as demonstrated by fluorescence microscope imaging (i) and cell spreading quantification (ii). Values shown are means ± SD. Scale bar is 1 mm in (A) and 100 μm in (C). Image analyses were performed on triplicate samples (n = 3).

Figure 3. EC adhesion and alignment on Fn-patterned SiO₂ subtrates

(A) ECs align on Fn patterns substrates after 2 days in culture. Despite large variances in topographical feature diameter and spacing, HUVECs align on Fn patterns throughout entire SiO₂ substrate as shown by fluorescence microscope imaging of VECAD (green), propidium iodide (red) (i), and remain restricted to Fn patterns (Fn = green, phalloidin = red) (ii). SEM images show the density of HUVEC adhesive protrusions (indicated by an arrow) differs based on topographical diameter and spacing (iii). (B) ECFCs also align on Fn patterns, however adhesion and alignment is limited to topographical features with diameters ≤ 2.0 μm as shown by fluorescent micropscope imaging of VECAD (green), propidium iodide (red). Scale bar is 1 mm in (Ai), 100 μm in (Aii), and 1 mm, 100 μm, and 10 μm in (Aiii). Scale bar is 1 mm in (B).

Figure 4. Development of PDMS micropillar substrates

Schematic describing the fabrication of PDMS “soft” micropillar substrates.

Figure 5. Characterization of PDMS micropillar substrates

SEM images of fabricated PDMS micropillar substrates. Scale bar = 5 μm.

Figure 6. EC elongation of PDMS micropillar substrates

(A) Light microscope images of HUVECs (i) and ECFCs(ii) demonstrate aligned and elongated morphologies after 24 h. (B) Flourescence microscope imaging of HUVECs stained with phalloidin (green) demonstrate successful patterning of PDMS micropillar substrates (Fn=red) and the preferential adhesion of HUVECs to Fn patterned regions. (C) Quantification of cell shape indicates significant HUVEC and ECFC elongation (a measure of circularity) on Fn-patterned SiO₂ and PDMS micropillars compared to cells cultured on flat substrates. Significance levels were set at: *p < 0.05, **p < 0.01, and ***p < 0.001. Values shown are means ± SD.