Nanopattern-induced changes in morphology and motility of smooth muscle cells

Abstract

Cells are known to be surrounded by nanoscale topography in their natural extracellular environment. The cell behavior, including morphology, proliferation, and motility of bovine pulmonary artery smooth muscle cells (SMC) were studied on poly(methyl methacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) surfaces comprising nanopatterned gratings with 350 nm linewidth, 700 nm pitch, and 350 nm depth. More than 90% of the cells aligned to the gratings, and were significantly elongated compared to the SMC cultured on non-patterned surfaces. The nuclei were also elongated and aligned. Proliferation of the cells was significantly reduced on the nanopatterned surfaces. The polarization of microtubule organizing centers (MTOC), which are associated with cell migration, of SMC cultured on nanopatterned surfaces showed a preference towards the axis of cell alignment in an in vitro wound healing assay. In contrast, the MTOC of SMC on non-patterned surfaces preferentially polarized towards the wound edge. It is proposed that this nanoimprinting technology will provide a valuable platform for studies in cell-substrate interactions and for development of medical devices with nanoscale features.

Fig. 1

Scanning electron micrographs of (A and B) nano-imprinted gratins on PMMA coating on SiO₂ wafer, (C) PDMS nanopatterned by replica molding and (D) collagen coated PDMS with nanopattern. Bar = 2 μm for A, C and D, bar = 500 nm for B.

Fig. 2

Confocal micrographs of F-actin stained SMC on (A) nano-imprinted PMMA at low cell density, (B) nano-imprinted PMMA at high cell density, (C) nanopatterned PDMS at low cell density, (D) nano-patterned PDMS at high cell density, (E) non-patterned PMMA and (F) glass cover slip. Scanning electron micrographs of SMC cultured on (G) nano-imprinted gratings on PMMA coated on SiO₂ wafer and (H) non-patterned PMMA coated on SiO₂ wafer. Bar = 50 μm for all except (B) Bar = 100 μm.

Fig. 3

The percentage of cell alignment and the elongation factor of SMC cultured on glass cover slip, nano-imprinted PMMA and nanopatterned PDMS. Cells were considered as aligned if the angle between the long axis of the cells and the gratings was less then 15 °. Factor E = (long axis/short axis)−1.

Fig. 4

(A) The percentage of SMC incorporated BrdU during 4 h of incubation cultured on plain non-patterned PMMA, nano-imprinted PMMA, plain non-patterned PDMS and nanopatterned PDMS. Fluorescence images showing BrdU incorporation into the nucleus of SMC cultured on (B) non-patterned PMMA and (C) nano-imprinted PMMA after immuno-fluorescent staining. Bar = 50 μm.

Fig. 5

MTOC polarization over the time course of wound closure for SMC. Fluorescent labeling of the MTOC and nucleus on the wounded SMC monolayer on (A) glass cover slip and (B) nanopatterned PDMS 1-h post wounding. Each cell at the wound edge was divided into four regions as shown in (A) and (B). The location of MTOC of one of the cell is indicated with a block white arrow in (A). The white dotted line outlines the wound edge. The direction of cell alignment, hence the gratings on the nanopatterned PDMS, is indicated with a white arrow in (B). The percentage of wound-edge cells with an MTOC orientated towards the wound edge and sideway of the edge at 0, 1, 2, 3 and 4 h post wounding for SMC cultured on (C) glass cover slip and (D) nanopatterned PDMS gratings. Bar = 20 μm. WE = wound edge, F = front quadrant, S = side quadrants.