Flexible nanopillars to regulate cell adhesion and movement

Abstract

Flexible polymer nanopillar substrates were used to systematically demonstrate cell alignment and migration guided by the directional formation of focal adhesions. The polymer nanopillar substrates were constructed to various height specifications to provide an extensive variation of flexibility; a rectangular arrangement created spatial confinement between adjacent nanopillars, providing less spacing in the horizontal and vertical directions. Three polymer nanopillar substrates with the diameter of 400 nm and the heights of 400, 800, and 1200 nm were fabricated. Super-resolution localization imaging and protein pair-distance analysis of vinculin proteins revealed that Chinese hamster ovary (CHO) cells formed mature focal adhesions on 1200 nm high nanopillar substrates by bending adjacent nanopillars to link dot-like adhesions. The spacing confinement of the adjacent nanopillars enhanced the orthogonal directionality of the formation tendency of the mature focal adhesions. The directional formation of the mature focal adhesions also facilitated the organization of actin filaments in the horizontal and vertical directions. Moreover, 78% of the CHO cells were aligned in these two directions, in conformity with the flexibility and nanotopographical cues of the nanopillars. Biased cell migration was observed on the 1200 nm high nanopillar substrates.

High‐Aspect‐Ratio Nanostructured Surfaces as Biological Metamaterials

“…One potential advantage of the nanostructures discussed here is that they are attached to a surface. While freestanding silicon nanowires readily undergo cellular uptake by cells, SEM studies show their ability to strongly deform or bend nanostructures . Very few reports discuss detachment of patterned nanostructures from the surface, except where by explicit design .…”

“…Simultaneously, nanopatterning materials can fundamentally alter their mechanical properties (indeed this is one approach to creating mechanical metamaterials) . Hence, our perception of materials being hard or stiff is often inaccurate at the nanoscopic level, as illustrated by the many examples of cells deforming nanostructures made from macroscopically stiff materials . Even diamond nanoneedles undergo large elastic deformations at the nanoscale .…”

“…Super‐resolution microscopy techniques are less common, but are becoming increasingly useful to visualize the localization of subcellular components . Chien et al used stochastic optical reconstruction microscopy (STORM, which uses the intermittent blinking of fluorophores to exceed normal resolution limits) to show how cells can form mature focal adhesions by deforming polymer nanopillars . Structured‐illumination microscopy has also been used to visualize the formation of lamin A (a structural protein) rings in the nuclear membrane, around silicon nanoneedles …”

“…While freestanding silicon nanowires readily undergo cellular uptake by cells, [36] SEM studies show their ability to strongly deform or bend nanostructures. [95,150,224] Very few reports discuss detachment of patterned nanostructures from the surface, except where by explicit design. [101] Specifically, studies of nanoneedle-mediatedinduced endocytosis do not see the phagocytosis of nanoneedle structures, [83] although some authors have described the engulfment of cells of peptide-functionalized-gold-mushroom-shaped nanoelectrodes by neuronal cells as phagocytosis-like.…”

“…[14] Hence, our perception of materials being hard or stiff is often inaccurate at the nanoscopic level, as illustrated by the many examples of cells deforming nanostructures made from macroscopically stiff materials. [69,95,128,131,150,224,476] Even diamond nanoneedles undergo large elastic deformations at the nanoscale. [477] While stiffnessrelated effects have been studied extensively, [478] and shown to Adv.…”