Fibroblast reaction to island topography: changes in cytoskeleton and morphology with time

Abstract

In order to develop next-generation tissue engineering materials, the understanding of cell responses to novel material surfaces needs to be better understood. Topography presents powerful cues for cells, and it is becoming clear that cells will react to nanometric, as well as micrometric, scale surface features. Polymer-demixing of polystyrene and polybromostyrene has been found to produce nanoscale islands of reproducible height, and is very cheap and fast compared to techniques such as electron beam lithography. This study observed temporal changes in cell morphology and actin and tubulin cytoskeleton using scanning electron and fluorescence microscopy. The results show large differences in cell response to 95 nm high islands from 5 min to 3 weeks of culture. The results also show a change in cell response from initial fast organisation of cytoskeleton in reaction to the islands, through to lack of cell spreading and low recruitment of cell numbers on the islands.

Introduction

Tissue engineering utilises scaffold materials that elicit specific cell reactions. Such desired reactions may involve cell phenotype; for example in some orthopaedic applications, where an ideal material would encourage osteoprogenitor cells to commit to the osteoblastic lineage. Another type of reaction that may be desirable could be increased tissue production from cells, thus giving greater wound healing capacity; such an effect may speed up post-operative recovery times, or may allow the formation of new body parts around a scaffold. A third type of application does exist, however, and that is the reduction of cell adhesion to materials. In the body, materials in this classification could prevent unwanted cell adhesion, for example the adhesion of tendons to implant materials and the bio-fouling of artificial heart valves and stents.

The aim of tissue engineering, is to augment, replace, or restore complex human tissue function by combining synthetic and living components in correct environmental conditions [1], one such environmental condition may be substrate shape. Thus, the idea of adding topography, or three-dimensionality, to material surfaces may be of great value in the future of materials for medical applications. It was proposed in 1964 that cells reacted to the shape (topography) of their environment [2]. Since that time, it has become well documented that many cell types react strongly to micrometric topography [3], [4], [5], [6], [7], and more recently, it has been demonstrated that cells can respond to nanometric cues in vitro [8], [9], [10], [11].

Cells recognise surface features and react to them, resulting in contact guidance. Fibroblastic cells probe the substrate using filopodia presented on the cell lamellae. When a suitable site for adhesion has been detected, focal adhesions and mature actin fibres are formed; tubulin microtubules are then recruited, stabilising the contact [12].

The ability of the substrate to promote the formation of focal contacts and the development of the cell cytoskeleton are important for the performance of the material. Integrins located within the adhesions, and actin cytoskeleton linked to integrins, are involved in signal transduction pathways (as reviewed by Burridge and Chrzanowska-Wodnick, 1996 [13]). The signal transductive events locating from focal contacts can affect the long-term cell differentiation [14], [15], [16].

The study of cell reaction to topography with nanometric dimensions is dependent on the fabrication methods available. Very precise methods of manufacture have been developed, with electron beam lithography giving sub-nanometer resolutions (down to 5 Å) vertically and 5 nm laterally [17]. The problem with these techniques is that whilst precise and reproducible, they are time consuming and expensive; especially when patterning large areas. Thus, there is an incentive to develop useful topographies which can be quickly produced over large areas.

One of the several methods under investigation is polymer demixing, for example blends of polystyrene (PS, hydrophobic) and poly(4-bromostyrene) (PBrS, hydrophilic) spontaneously undergo phase separation during spin casting onto silicon wafers [18]. By controlling the polymer concentration and the proportions of the polymers, different topographies can be produced; these can be pits, islands, or ribbons of varying height or depth. The ratio of the polymers used varies the topography shape, and the concentration of polymer in the casting solution changes the feature sizes [18], [19], [20]. X-ray photoelectron spectroscopy (XPS) and static secondary ion mass spectrometry (SIMS) have been used to determine the surface composition of the blends [18], [19], [20], and in consensus with neutron reflectrometry experiments [21], showed that PS segregates to the material surface on annealing the films. Thus, despite the topography being formed by polymer blends, after the films are annealed the cells only interact with a single chemistry, in this case PS. It is important to know the chemistry of the material surface, as it affects material interaction with the extracellular matrix, and hence the materials interaction with the cells [22], [23], [24], [25]. The PS/PBrS polymer blend was chosen as the features produced by changing blend composition have been previously characterised [18], [19], [20].

Initial research with these materials showed large changes in cellular response dependant in island scale. The range of sizes used in these studies are from 13 to 95 nm in height (300 nm to 2.5 μm laterally). Fibroblastic [26] and endothelial-like cells [27] reacted to the islands with increasing cellular response to 13 nm islands and reduced cellular response to 95 nm islands, compared to flat controls. These results showed that small changes in nanotopography can produce significant changes in cellular response. The experiments did not, however, reveal why the cells showed increased or reduced adhesion and growth on the different islands.

This report investigates the influence of a model non-adhesive surface using 95 nm high demixed islands on the cell cytoskeleton over time. The cytoskeletal responses are compared and contrasted with scanning electron microscopical (SEM) images of cell morphology at selected time points.