s7_Effects of Nanoimprinted Patterns in Tissue-Culture Polymer on Cell Behavior

Effects of Nanoimprinted Patterns in Tissue-Culture Polymer on Cell Behavior

S. W. Pang, W. Hu, R. M. Reano, A. F. Yee

University of Michigan, Ann Arbor, Michigan 48109-2122, USA

E. K. F. Yim, K. W. Leong, C. S. Chen

Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA

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 polymethylmethacrylate (PMMA) and polydimethylsiloxane (PDMS) surfaces comprising nanopatterned gratings with 350nm linewidth, 700nm pitch, and 350nm 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.

Tissue engineering seeks to develop functional tissues in a biomimetic environment in vitro. As the extracellular environment in vivo is composed of numerous nanostructures, fabrication of nanostructured substrates will be valuable for tissue engineering applications. A simple nanoimprint lithography (NIL) process to pattern nanostructures directly on tissue-culture polystyrene (TCPS) plates is reported. By repeating this NIL process, three-dimensional scaffolds consisting of multiple-layer nanostructures were also fabricated. Bovine pulmonary artery smooth muscle cells were cultured on imprinted gratings ranging from 350 nm to 10 µm. The smooth muscle cells attached and proliferated well on these imprinted substrates without additional surface treatment. Cell elongation and alignment were observed on the micro- and nanopatterns, with the effect significantly more pronounced on the nanostructures.

The surface morphology and fidelity of the nanopatterns were examined with scanning electron microscope. The gratings were about 350 nm wide on the nanoimprinted PMMA sample, as shown in Figure 1.

Figure 1. Scanning electron micrographs of nano-imprinted gratings on PMMA coating on SiO₂ wafer.

SMC cultured on the surface with nanogratings adapted with an elongated morphology and were mostly parallel to one another. In contrast, SMC cultured on unpatterned surfaces showed neither elongation nor orientation at both low and high cell densities. Figure 2 shows confocal micrographs of F-actin stained SMC on patterned and non-patterned PMMA.

Figure 2. Confocal micrographs of F-actin stained SMC on (a) nano-imprinted PMMA at high cell density, and (b) non-patterned PMMA. Bar = 100 µm for (a) and 50 µm for (b).

Figure 3 shows scanning electron micrographs of the TCPS nanostructures imprinted at a temperature of 150 °C and a pressure of 5 MPa for 10 min. At these imprint conditions, the imprinted gratings were well formed with high uniformity and low defects.

Figure 3. Nanoimprinted structure in thick tissue-culture polystyrene: (a) gratings with 0.5 µm half-pitch and 440 nm height and (b) gratings with 120 nm half-pitch and 290 nm height.

Figure 4 shows the 3D nanoscaffolds consisting of 100 nm gratings embedded on 1 µm wide grating structures with the top layer of gratings aligned perpendicular and parallel to the bottom layer.

Figure 4. 3D polystyrene nanostructures with multiple imprints: 100-nm-wide gratings imprinted on 1 µm half-pitch gratings with (a) perpendicular orientation (view at 45°) and (b) parallel orientation (top view).

The direction of SMCs on unpatterned TCPS plate was random and no cell elongation was observed. Cells show obvious alignment and elongation on the 2 µm, 1 µm, and 0.5 µm half-pitch TCPS gratings. Such results indicate that the imprinted micrometer and nanometer scale structures have strong influence on cell morphology.

Figure 5. Fluorescence micrographs of smooth muscle cells on (a) unpatterned TCPS, (b) 2 µm half-pitch gratings, (c) 1 µm half-pitch gratings, and (d) 0.5 µm half-pitch gratings. All gratings are along the horizontal direction.

References

Last Updated: November 19, 2007

E-Mail: pang@umich.edu

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