Chris D. W. Wilkinson

Topographical control of cells

We review the literature on the reaction of cells to their surrounding topography. The topography may be that of surrounding cells, intercellular materials or biomaterials. The reactions include cell orientation, rates of movement, and activations of the cells. We concentrate on those papers where quantitative measurements of the reactions have been made and largely ignore those on subjective impressions. A wide range of topographies are considered but special attention is given to results on groove-ridge topographies. The question of whether the cells are reacting to the topography directly or to patterned substratum chemistry formed on the topography is discussed. The review ends with a summary of the types of prosthesis where advantage has been taken of the ability to fabricate topography.

Nantotechniques and approaches in biotechnology

Nanotechnology has enabled the development of an amazing variety of methods for fabricating nanotopography and nanopatterned chemistry in recent years. Some of these techniques are directed towards producing single component particles, as well as multi-component assembly or self-assembly. Other methods are aimed at nanofeaturing and patterning surfaces that have a specific chemistry or topography. This article concentrates mainly on surface-directed nanobiotechnologies because they are nearer to commercial realisation, such as use in tissue engineering, control of biofouling and cell culture, than those directed at producing nanoparticles.

Nucleus alignment and cell signaling in fibroblasts: response to a micro-grooved topography

Cellular response to scaffold materials is of great importance in cellular and tissue engineering, and it is perhaps the initial cell contact with the scaffold that determines development of new tissue. Material surface morphology has strong effects on cell cytoskeleton and morphology, and it is thought that cells may react to the topography of collagen and surrounding cells during tissue embryology. A poorly understood area is, however, gene-level responses to topography. Thus, this paper used microarray to probe for consistent gene changes in response to lithographically produced topography (12.5 x 2-microm grooves) with time. The results showed many initial gene changes and also down-regulation of gene response with time. Cell and nucleus morphology were also considered, with nuclear deformation linked to cell signaling.

Reactions of cells to topography.

Though contact guidance has been known since the very early days of cell culture very little quantitative examination of the reaction of cells to topography has been made. Exceptions to this subjective approach are given prominence below. Yet if we are to understand how cells react and if we are to be able to design ideal substrata for particular cells we need this information. Precision and quantitation are required both of the methods of examination of the cells but also in the definition of that topography. Recently it has become clear that the these reactions occur at the nanometric scale and have importance for use in cellular engineering and tissue repair. Topography appears to provide a set of very powerful signals for cells.

An Extracellular microelectrode Array for monitoring electrogenic cells in culture

This paper describes a planar array of microelectrodes developed for monitoring the electrical activity of cells in culture. The device allows the incorporation of surface topographical features in an insulating layer above the electrodes. Semiconductor technology is employed for the fabrication of the gold electrodes and for the deposition and patterning of an insulating layer of silicon nitride. The electrodes have been tested using a cardiac cell culture of chick embryo myocytes, and the physical beating of the cultured cells correlated with the simultaneous extracellular voltage measurements obtained. It was found that extracellular stimulation of the cells was possible via the same electrodes used for recording.

Interaction of animal cells with ordered nanotopography

Animal cells live in a complex and diverse environment where they encounter a vast amount of information, a considerable amount of which is in the nanometer range. The surface topography that a cell encounters has a role to play in influencing cell behavior. It has been demonstrated widely that surface shape can directly influence the behavior of cells. In this paper, we discuss the interactions of animal cells with engineered nanotopography, fabricated in quartz and reverse embossed into polycaprolactone, fibroblast cells show reduced adhesion to the ordered nano pits. We show that the area of cells spreading on a structured nanotopography is reduced compared with that on a planar substrate. Furthermore, cytoskeletal organization is disrupted as indicated by a marked decrease in number and size of focal contacts.

Nanotopographical stimulation of mechanotransduction and changes in interphase centromere positioning

We apply a recently developed method for controlling the spreading of cultured cells using electron beam lithography (EBL) to create polymethylmethacrylate (PMMA) substrata with repeating nanostructures. There are indications that the reduced cell spreading on these substrata, compared with planar PMMA, results from a reduced adhesivity since there are fewer adhesive structures and fewer of their associated stress fibres. The reduced cell spreading also results in a reduced nuclear area and a closer spacing of centrosomes within the nucleus, suggesting that the tension applied to the nucleus is reduced as would be expected from the reduction in stress fibres. In order to obtain further evidence for this, we have used specific inhibitors of components of the cytoskeleton and have found effects comparable with those induced by the new substrata. We have also obtained evidence that these subtrata result in downregulation of gene expression which suggests that this may be due to the changed tension on the nucleus: an intriguing possibility that merits further investigation. J. Cell. Biochem. 100: 326–338, 2007.

Arrays of nano-dots for cellular engineering

Efficient patterning of large areas with nanometre features is required for cellular engineering applications. The final product must be made at an economic cost. 100 nm diameter pits on a 300 nm pitch have shown to be non-adhesive to a range of different mammalian cells. A strategy is described which uses an electron beam lithographic tool with focussed Gaussian beam to define shapes directly. Features as small as 20 nm in diameter on 100 nm pitch were fabricated. It was found that focus of 20 nm spot size is critical. The effect of deflection aberrations across the writing field was investigated. Various resists have been examined. Patterns were successfully embossed in poly(caprolactone). The overall cost of a die in terms of machine time has been significantly reduced.

Nanomechanotransduction and interphase nuclear organization influence on genomic control.

The ability of cells to alter their genomic regulation in response to mechanical conditioning or through changes in morphology and the organization of the interphase nuclei are key questions in cell biology. Here, two nanotopographies have been used as a model surfaces to change cell morphology in order to investigate spatial genomic changes within the nuclei of fibroblasts. Initially, centromeres for chromosome pairs were labeled and the average distance on different substrates calculated. Further to this, Affymetrix whole genome GeneChips® were used to rank genomic changes in response to topography and plot the whereabouts on the chromosomes these changes were occurring. It was seen that as cell spreading was changed, so were the positions along the chromosomes that gene regulations were being observed. We hypothesize that as changes in cell and thus nuclear morphology occur, that this may alter the probability of transcription through opening or closing areas of the chromosomes to transcription factors. J. Cell. Biochem. 102: 1234–1244, 2007.

The role of microtopography in cellular mechanotransduction.

Mechanotransduction is crucial for cellular processes including cell survival, growth and differentiation. Topographically patterned surfaces offer an invaluable non-invasive means of investigating the cell response to such cues, and greater understanding of mechanotransduction at the cell-material interface has the potential to advance development of tailored topographical substrates and new generation implantable devices. This study focuses on the effects of topographical modulation of cell morphology on chromosomal positioning and gene regulation, using a microgrooved substrate as a non-invasive mechanostimulus. Intra-nuclear reorganisation of the nuclear lamina was noted, and the lamina was required for chromosomal repositioning. It appears that larger chromosomes could be predisposed to such repositioning. Microarrays and a high sensitivity proteomic approach (saturation DiGE) were utilised to identify transcripts and proteins that were subject to mechanoregulated changes in abundance, including mediators of chromatin remodelling and DNA synthesis linked to the changes in nucleolar morphology and the nucleoskeleton.