Investigating the limits of filopodial sensing: a brief report using SEM to image the interaction between 10 nm high nano-topography and fibroblast filopodia.

Having the ability to control cell behaviour would be of great advantage in tissue engineering. One method of gaining control over cell adhesion, proliferation, guidance and differentiation is use of topography. Whilst it has be known for some time that cells can be guided by micro-topography, it is only recently becoming clear that cells will respond strongly to nano-scale topography. The fact that cells will take cues from their micro- and nano-environment suggests that the cells are in some way 'spatially aware'. It is likely that cells probe the shape of their surroundings using filopodia, and that this initial filopodia/topography interaction may be critical to down-stream cell reactions to biomaterials, or indeed, the extracellular matrix. One intriguing question is how small a feature can cells sense? In order to investigate the limits of cell sensing, high-resolution scanning electron microscopy has been used to simultaneously view cell filopodia and 10 nm high nano-islands. Fluorescence microscopy has also been used to look at adhesion formation. The results showed distinct filopodial/nano-island interaction and changes in adhesion morphology.

Rapid fibroblast adhesion to 27nm high polymer demixed nano-topography.

It is well known that many cell types react strongly to micro-topography. It is rapidly becoming clear than cells will also react to nano-topography. Polymer demixing is a rapid and low-cost chemical method of producing nano-topography. This manuscript investigates human fibroblast response to 27nm high nano-islands produced by polymer demixing. Cell spreading, cytoskeleton, focal adhesion and Rac localisation were studied. The results showed that an initial rapid adhesion and cytoskeletal formation on the islands at 4 days of culture gave way to poorly formed contacts and vimentin cytoskeleton at 30 days of culture.

Cell response to nano-islands produced by polymer demixing: a brief review.

This review looks at the present literature available regarding cell response to nano-islands produced by nanotopography. Polymer demixing is a chemical method of fabricating large areas of nanotopography quickly and cheaply, making it ideal for cell testing and thus allowing it to be one of the first well-researched methods in cell engineering. The review shows that cells respond strongly to the islands (cell types observed include endothelial cells, fibroblasts, osteoblasts, leucocytes and platelets). Such changes include differences in adhesion, growth, gene expression and morphology.

Nonadhesive nanotopography: fibroblast response to poly(n-butyl methacrylate)-poly(styrene) demixed surface features.

It is becoming clear that cells do not only respond to micrometric scale topography, but may also respond to topography at the nanometric scale. Nano-fabrication methods such as electron beam lithography are, however, expensive and time consuming. Polymer demixing of poly(styrene) and poly(4-bromostyrene) has been found to produce nano-scale islands of reproducible height, and the islands have been previously shown to effect cell events such as adhesion, spreading, proliferation, and differentiation. This study uses demixed poly(styrene) and poly(n-butyl methacrylate) to produce nano-islands with closer packing and narrower widths compared with those previously studied. Observations have been made of morphological and cytoskeletal changes in human fibroblasts interacting with 10- and 50-nm-high islands. The methods used included scanning electron microscopy, fluorescent microscopy, and optical microscopy. The results indicated that the cells do not respond differently to the 10-nm islands compared with planar samples but, in contrast, the 50-nm islands are nonadhesive.

Endothelization and adherence of leucocytes to nanostructured surfaces.

We analyse the leucocyte and endothelial cell response to polybromostyrene-polystyrene (PS/PBrS) and the poly-n-butylmethacrylate-polystyrene (PnBMA/PS) systems, both in flat form or nanostructured surfaces consisting of nanohills with increasing hill height (13-95nm). Experiments were carried out first with blood leucocytes alone, endothelial cells (of three different types) alone, and finally, using blood cells and endothelized nanosurfaces. Blocking monoclonal antibodies specific for CD11, CD29, CD31, CD54, CD166 were used to analyse whether and to what extent adhesion molecules could be involved in the adherence of both blood leucocytes and endothelial cells to different nanosurfaces. Expression of CD29 (beta-1 integrin), CD54 (ICAM-1) and CD166 (ALCAM) on blood leucocytes was dependent on the hill height, being most prominent with 13nm (PS/PBrS) and 45nm hill (PnBMA/PS) nanosurfaces. Adherence of a human microvascular endothelial cell line and umbilical primary endothelial cells was also related to hill height, being most prominent with 13nm hill height. An indirect correlation was observed between the extent of endothelization and the degree of leucocyte adherence. In cases of low to medium extent of endothelization, the adherence of monocytes and granulocytes was mediated by the expression of CD166, CD29 and CD11a (alpha-L integrin), CD29, CD31 (PECAM-1), respectively. Scanning electron microscopy studies showed the predominant emission of pseudopodia at the holes of the surfaces and the focal contacts with the nanosurfaces. Our studies emphasize the relevance of testing functional properties in co-culture experiments in the development and optimization of nanosurfaces for biomedical application.

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

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.

Micro and nano-structured surfaces.

The study of cell reaction to micro and nanotopography is dependent on the method of manufacture available. Several methods of manufacture have been developed: polymer demixing, embossing and photolithography. Surfaces obtained with these different techniques, having micro and/or nanodomains, have been studied toward the same type of cells, i.e. human endothelial cells (HGTFN) and mouse fibroblasts (3T3). Polymer demixing of polystyrene (PS) and poly(4-bromostyrene) (PBrS) producing nanometrically islands of 18, 45 and 100 nm height, polycarbonate (PC) and polycaprolactone (PCL) grooved with grooves 450 nm wide and 190 high, the natural polysaccharide hyaluronic acid (Hyal) and its sulfated derivative (HyalS) photoimmobilized on silanized glass as grooves 250 nm high and 100, 50, 25 or 10 microm wide have been obtained. The morphology and polarization of the cells has been studied by optical microscopy and scanning electron microscopy. Cells respond in different way to the topography of the materials, but the surface chemistry is dominant in inducing different cell behavior.

In vitro reaction of endothelial cells to polymer demixed nanotopography.

The introduction of topography to material surfaces has been shown to strongly affect cell behaviour, and the effects of micrometric surface morphologies have been extensively characterised. Research is now starting to investigate the reaction of cells to nanometric topography. This study used polymer demixing of polystyrene and poly(4-bromostyrene) producing nanometrically high islands, and observed endothelial cell response to the islands. Three island heights were investigated; these were 13, 35 and 95 nm. The cells were seen to be more spread on the manufactured topographies than that on flat surfaces of similar chemistry. Other morphological differences were also noted by histology, fluorescence and scanning electron microscopy, with many arcuate cells noted on the test surfaces, and cytoskeletal alignment along the arcuate features. Of the nanotopographies, the 13 nm islands were seen to give the largest response, with highly spread cell morphologies containing well-defined cytoskeleton.

Polymer-demixed nanotopography: control of fibroblast spreading and proliferation.

Cell response to nanometric scale topography is a growing field. Nanometric topography production has traditionally relied on expensive and time-consuming techniques such as electron beam lithography. This presents disadvantages to the cell biologist in regard to material availability. New research is focusing on less expensive methods of nanotopography production for in vitro cell engineering. One such method is the spontaneous demixing of polymers (in this case polystyrene and polybromostyrene) to produce nanometrically high islands. This article observes fibroblast response to nanometric islands (13, 35, and 95 nm in height) produced by polymer demixing. Changes in cell morphology, cytoskeleton, and proliferation are observed by light, fluorescence, and scanning electron microscopy. Morphological features produced by cells in response to the materials were selected, and cell shape parameters were measured with shape-recognition software. The results showed that island height could either increase or reduce cell spreading and proliferation in relation to control, with 13-nm islands producing cells with the greatest area and 95 nm islands producing cells with the lowest areas. Interaction of filopodia with the islands could been seen to increase as island size was increased.

Phase Separation and Dewetting of Weakly Incompatible Polymer Blend Films.

The phase separation and dewetting of thin films of blends of deuterated polystyrene (dPS) and poly(p-methylstyrene) (PpMS) were investigated during annealing. The surface morphology, obtained from atomic force microscopy and phase measurement interference microscopy, the density profile, determined by X-ray reflectivity in the region of total external reflection, and the surface composition obtained from static secondary ion mass spectroscopy, are reported. This system is only weakly incompatible. The interaction of the components with substrate and air during phase separation leads to a bilayer formation with a broad polymer-polymer interface. PpMS segregates to the air interface. The bilayer structure is unstable and defines the starting point for the dewetting of PpMS on top of the dPS layer. In the final dewetting state a homogeneous layer of dPS on top of the substrate is covered with an ultrathin layer of PpMS as well as with quite thick mesoscopic drops of PpMS.