The topology and chemistry of the substrate was proofed to be relevant to the cell–substrate interactions [2-5]. During the last decade, the micro- and nanotechnology fields have been matured rapidly, resulting in the production of varied kinds of nanostructures. Scientists have developed nanostructures with different shape and tried to apply to life sciences.
Nealey and coworkers have created silicon substrata with grooves and ridges having pitch dimensions of 400-4000 nm using X-ray lithography. They have investigated the strength of cell-substratum adhesion on nanoscale topographic features of a similar scale to that of the native basement membrane. When human corneal epithelial cells (SV40-HCECs) cultured on ridges and grooves of nanoscale dimensions, cells migrated more extensively to the ridges than into the grooves. Cell shape was aligned and extended in the direction of the grooves, but the percentage of aligned cells was only increased with groove depth. Figure 1.1 presented that actin filaments and focal adhesions were aligned along the substrate topographies, too [6, 7].
In the latest paper published in 2006, this group found cells aligned preferentially in the direction perpendicular to nanoscale grooves and ridges in Epilife medium (Figure 1.2). This is in contrast to a previous study where cells cultured in DMEM/F12 medium with 10% FBS aligned in the direction parallel to nanoscale topographic features [6, 7]. Cells switched from perpendicular to parallel alignment when the pitch was increased from 400 to 4000 nm. There was a transition region (between 800 and 1600nm pitch) where both parallel and perpendicular alignments were favored compared to all other cellular orientations. Cells formed focal adhesions parallel to the substrate topographies in this transition region. On the nano- and microscale patterns, 400 and 4000nm pitch, focal adhesions were almost exclusively oriented obliquely to the topographic patterns [8].
Figure1. 1 Cells stained for actin (red), vinculin (green) and the nucleus (blue) and cultured on (A) 600 nm deep grooves and 70 nm wide ridges on a 400 nm pitch. (B) 600 nm deep grooves and 1900 nm ridges on a 4000 nm pitch. A reflection image of the substrates is included in the figure insets. (C) a smooth silicon oxide substrate [7].
Figure1. 2 SEM images of cells cultured on patterned substrates. (A) Perpendicularly aligned cell on 70nm wide ridges on a 400nm pitch. (B) Detail of previous cell. Filopodia were aligned perpendicularly to the patterns. (C) Parallel aligned cell on 1900nm ridges on a 4000nm pitch. (D) Filopodia were guided by the topographic pattern [8].
Meiners and coworkers have designed a synthetic nanofibrillar matrix that more accurately models the porosity and fibrillar geometry of cell attachment surfaces in tissues. The synthetic nanofibrillar matrices were composed of nanofibers prepared by electrospinning a polymer solution of polyamide onto glass coverslips (Figure 1.3). Scanning electron and atomic force microscopy showed that the nanofibers were organized into fibrillar networks reminiscent of the architecture of basement membrane, a structurally compact form of the extracellular matrix (ECM). They have inspected F-actin, vinculin, FAK (focal adhesion components), and fibronectin organization for NIH 3T3 fibroblasts (Figure 1.4) and found their morphology and characteristics displaying the counterparts in vivo. Normal rat kidney (NRK) cells and breast epithelial cells also showed similar result. Hence the synthetic nanofibrillar matrix could act as a physically and chemically stable three-dimensional surface for ex vivo growth of cells [9].
(A) (B)
Figure1. 3 (A) SEM and (B) AFM image of a glass coverslip coated with nanofibers [9].
Figure1. 4 A comparison of the F-actin network, focal adhesion components, and fibronectin organization for NIH 3T3 fibroblasts cultured on glass and nanofibers. Fluorescent image of phalloidin-Alexa Fluor staining on glass (A) and nanofibers (B). Indirect immunofluorescence of fibroblasts on glass (C,E,G) and nanofibers (D,F,H) stained with vinculin (C,D), FAK PY397 (E,F), and fibronectin (G,H) antibodies. Scale bar, 10 mm [9].
Dalby and coworkers have used polymer demixing of polystyrene and poly (4-bromostyrene) producing nanometrically high islands, and observed endothelial cell response to the islands (Figure 1.5A). They have proposed three island heights for investigation: 13, 35 and 95nm. For the two controls, PBrS and PS, the cells gave a significantly more spread morphology on the PBrS, possibly due to its more hydrophilic chemistry. A morphological feature common on the nanoislands was the arcuate, or curved, cell shapes. They supposed that the regular nanometric topography produced by the test
substrates might provide cues similar to those given by collagen, resulting in the cells having a more natural phenotype in vitro than is achieved on flat culture dishes. The SEM results also have shown many of the cells being seen to have filopodia in contact with the islands in preference to the dip. Observation of the actin cytoskeleton showed that the 13nm substrate was accelerating cell spreading. While tubulin cytoskeleton was seen to be well formed in cells on all the materials, it could be seen to be aligning around arcuate features on the nanotopography (Figure 1.6). In their conclusion, 13-nm-high islands produced highly spread cellular morphologies containing well-defined cytoskeleton, but larger islands produced a stepwise decrease in response [10].
The previous result made this group having great interest in the 13-nm-high islands. Thus they employed include scanning electron microscopy, fluorescent microscopy, and 1718 gene microarray to investigate cell response to 13-nm-high islands. In this study, the most worthy to discuss is gene microarray. They picked many genes relevant to cytoskeleton, extracellular matrix, cell replication and signaling. The genes involved in cell signaling and proteins of ECM modeling were upregulated. Rho, Rac, and Ras genes, the proteins which are involved in cell shape, production of filopodia and lamelapodia, and movement, were up-regulated, too.
Other growth hormones, ion channels, and receptor gene up-regulations were also noted.
These observations indicate that increased cell attachment and spreading is required for up-regulation proliferation and matrix synthesis [11]. In their review paper published in 2004, they made a statistics (Figure 1.5B) of cell respond to polymer islands with different heights.
Cells grown on 95-nm islands show reduced adhesion and cytoskeleton, but on 13-nm islands show the greatest respond in biological characterization [12].
(A) (B)
Figure1. 5 (A) Atomic force microscopical images of 35 nm high islands [10]. (B) Generalised cell responses to changes in island size [12].
Figure1. 6 Fluorescent images of HGTFN cytoskeletons on control and test materials. Bar=50 mm [10].
But there are still some problems for these nanostructures applying to biology. The limited resolution makes X-ray lithography unable to produce patterns with dimension under 50 nm.
In addition, the method of X-ray lithography cost expensive and spend a lot of time. In a view of nanotopography, nanofibers can’t be well defined in their dimensions and shape. Although the nanostructure formed nanofibers is most similar to the environment for in vivo growth of cells. The homogeneity of polymer islands is low. The range of their diameters and heights distribute widely. To breakthrough previous study about cellular response to nanotopography, we selected nanodot arrays as the substrate which presented by Ko et al [1]. Benefiting from characteristic of anodic aluminum oxide (AAO), Ko have use AAO as template to fabricate nanodot arrays on silicon substrate. Nanodot arrays made by AAO template are highly packed, uniformly distributed and easy to control their size. So we can define a series of different size nanodots and study more completely what physical topography affect cells. Moreover this method is not only convenient but also has high yield.
Figure1. 7 SEM image of tantalum oxide nanodots arrays [1].