Exploring cellular contact guidance using gradient nanogratings

Abstract

Nanoscale surface features that mimic extracellular matrix are critical environmental cues for cell contact guidance and are vital in advanced medical devices in order to manipulate cell behaviors. Among them, nanogratings (line-and-space gratings) are common platforms to study geometric effects on cell contact guidance, especially cell alignment, but generally are one pattern height per platform. In this study, we developed a strategy to fabricate controlled substrates with a wide range of pattern shapes and surface chemistries and to separate surface chemistry and topography effects. As a demonstration of this strategy, six nanograting platforms on three materials were fabricated and applied to examine and differentiate the effects of surface topography and surface chemistry on cell contact guidance of murine preosteoblasts. All of the six platforms contained the same gradient in pattern height (0 to ≈350 nm). They were prepared using nanoimprint lithography and annealing for thermoplastic materials (low molecular weight polystyrene (PS) and polymethylmethacrylate (PMMA)) and photoimprint for a thermoset material (a cross-linked dimethacrylate (DMA)). Each material contains two platforms that are only different in line-and-space pitch (420 or 800 nm). The DMA nanogratings had a reverse line-and-space profile to those of the PS and PMMA nanogratings. Using these platforms, a full range of cell alignment, from randomly orientated to completely parallel to the grating direction was achieved. Results from focal adhesion assays and scanning electronic microscopy indicated a change in cell-substrate contact from a noncomposite state (full contact) to a composite state (partial contact between cell and substrate) as pattern height increased. These gradient platforms allowed for the separation of surface chemistry and surface topography to provide insight into the mechanisms responsible for cell contact guidance on nanopatterned surfaces.

Figure 1

(a) Fabrication of gradient platforms with inverse line and space profiles. PS gratings were produced by NIL followed by annealing on a temperature gradient. Some PS gratings were subsequently used as a mold to form DMA gratings with the inverted pattern structure through photopolymerization. (b) Pattern height as a function of position (x) for PS and DMA grating platforms determined by AFM for Λ = 420 nm and 800 nm. Lines are drawn to guide the reader’s eyes.

Figure 2

Cross-sectional shape profile of grating platforms at different H. (a) and (b) are SEM images for PS and DMA platforms, respectively, at H ≈ 300 nm. Scale bars = 500 nm; (c) and (d) are AFM images for PS and DMA platforms, respectively, at H ≈ 250 nm; (e) and (f) are AFM cross-sectional measurements for PS and DMA platforms, respectively, at 5 different positions.

Figure 3

Cell alignment on grating platforms. Fluorescent images of cells on PS platforms taken at (a) high H (≈ 300 nm) and (b) low H (≈ 10 nm). Scale bars = 100 μm. Cells were elongated on the hydrophobic PS patterns. Cells on low H regions of the relatively more hydrophilic PMMA substrate were more spread (image not shown) (c) Percentage of aligned cells as a function of H on PS, PMMA, and DMA platforms (Λ = 420 nm and 800 nm). Inset plots the same data in a semi-log format to identify the critical height for cell alignment. (d) FE-SEM images of a representative cell and cell extension on DMA platforms illustrate that cell extensions follow the surface of the gratings and penetrate at least partially into the troughs.

Figure 4

(a) Fluorescent images of focal adhesion complexes for cells on PS grating (H ≈ 300 nm). The nuclei are in blue, the actin is in red, and the focal adhesion is in green. A box is drawn to enclose a region with a high concentration of focal adhesion complexes. The green channel in the box was subsequently analyzed via FFT. (b) Image generated by FFT of the boxed region. (c) Image by FFT of a control grating at high H without cells (DMA, real space image provided as inset). (d) Intensity versus q plot for focal adhesion complexes on PS gratings at high and low H, and on the control gratings without cells. Solid and dashed arrows correspond to the first and second order diffraction peaks, respectively.