Influence of Nanopillar Arrays on Fibroblast Motility, Adhesion, and Migration Mechanisms

Abstract

of cell mechanotransduction machinery [23] or controlling the geometry of in vitro neuronal networks. [24] Recently arrays of SU-8 nanopillars were employed to demonstrate that in contrast to previous measurements the highest cell traction forces are not generated at the cell periphery, but instead are associated with perinuclear adhesions. [25] In a different report an ultraflexible GaAs nanowire array was used as a nanomechanical biosensor to probe cell-induced forces by living cells with a resolution of 50 piconewton. [26] An attempt to control the geometry of in vitro cultivated neuronal cells revealed that the cytoskeleton dynamics at the axon shaft in primary hippocampal rat neurons were changed when cultivated on hard poly(dimethylsiloxane) nanopillar arrays, which in turn lead to enhanced formation of axon collateral branches. [27] An example of in vivo application is provided by Tang et al. who apply vertically oriented Au-TiO 2 nanowire arrays as artificial photoreceptors which restore functional and behavioral light sensitivity in blind mice. [28] Despite the range of applications, there is still much unknown about how cells are influenced by HARNs. High viability and reduced spreading area of adherent cells is often reported. [29] Arrays of nanowires have been shown to inhibit fibroblast migration with longer nanowires having a stronger effect, [30] while small patches of nanopillars inhibited neuronal cell migration. [31] Reduced spreading of cells on HARN arrays has been reported, [16,32] as well as altered expression of genes related to the cytoskeleton and cell adhesion. [33,34] It has also been demonstrated that the cell membrane is able to wrap tightly over HARNs while maintaining membrane integrity. [35][36][37] Careful investigations of the membrane integrity in cardiomyocyte-like HL-1 cells and Human embryonic kidney cells (HEK 293) on a variety of nanostructures by Dipalo et al. showed that vertical nanostructures can spontaneously penetrate the cellular membrane only in rare cases and under specific conditions. [35] Local membrane curvature can act as a biochemical signal for endocytic proteins, and HARNs which induced sites of positive membrane curvature were found to be hot spots for clathrin-mediated endocytosis (CME), as determined by preferential accumulation of CME-related proteins at these sites. [38,39] Nanopillars were also applied for controlled probing of nuclear mechanical properties. [40] In that report, the mechanism of nuclear deformation was linked to adhesive actin patches associating with the nanopillars, pulling the nucleus down, as well Surfaces decorated with high aspect ratio nanostructures are a promising tool to study cellular processes and design novel devices to control cellular behavior. However, little is known about the dynamics of cellular phenomenon such as adhesion, spreading, and migration on such surfaces. In particular, how these are influenced by the surface properties. In this work, fibroblast behavior is investigated on regular arrays of 1 µm high polym...

Nanoneedle Platforms: The Many Ways to Pierce the Cell Membrane

“…While the height and density of nanoneedles can influence the cell settlement morphology: dangling, semidangling, or engulfed state. [ 128,129 ] The sparse density of nanoneedle array can allow the cell membrane to engulf the nanoneedles. While with the increase of density, some part of cell will be propped up, allowing only sporadic contact with the underlying substrate, and eventually the whole cell are suspended over the nanoneedles.…”

“…[ 129 ] Regarding the effect of nanoneedle density on cell adhesion, Beckwith et al recently conducted a study where they investigated the morphology of fibroblasts on aligned polymer nanopillar arrays with different spacing distances. [ 128 ] From the high resolution Airyscan z ‐stacks of characteristic cells visualizing the F‐actin and plasma membrane profiles at different pitches, it was found that nanopillars with higher density could prop up the whole cell, allowing only sporadic contact with the underlying support, while in the case of sparse nanopillars, most of the nanopillars were engulfed by plasma membrane (Figure 17b).…”

Electron Beam Lithography Fabrication of SU-8 Polymer Structures for Cell Studies

“…SU-8 is stiff with a Young's modulus reported in the range from 0.9 GPa to 7.4 GPa [34,41], its processing is well established [42], it has high chemical stability [41] and it is known to be bio-compatible [43]. High resolution, high aspect ratio combined with rapid fabrication time due to very high sensitivity [44] makes SU-8 EBL fabrication a promising approach for further exploration as structural component in structured cellular substrates [11,45]. Furthermore, SU-8 is suitable for grey-scale electron beam lithography due to its low contrast [32].…”

Electron beam lithography fabrication of SU-8 polymer structures for cell studies

“…41 High resolution, high aspect ratio combined with rapid fabrication time due to very high sensitivity 42 makes SU-8 EBL fabrication a promising approach for further exploration as structural component in structured cellular substrates. 11,43 Furthermore, SU-8 is suitable for grey-scale electron beam lithography due to its low contrast. 44 Fabrication of high aspect ratio structures using a 100 keV beam has previously been performed, realising structures with heights up to 5 µm.…”

“…30 Bilenberg et al tested the resolution for nanofabrication of SU-8 using a 100 keV EBL system and showed that lines with widths of 24 nm can be written in a 99 nm thick resist. 44 In previous work, 11,43 our focus has been on exploring cell behaviour on substrates decorated with SU-8 structures, however in this work we focus on exploring the limitations to our fabrication process to understand which structures we are able to fabricate in this system. In this work we present a flexible approach for high-throughput fabrication of SU-8 nanostructures on glass using electron beam lithography.…”