Heather J. H. Johnstone

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.

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.

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.

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.

Cell behaviour of rat calvaria bone cells on surfaces with random nanometric features

Cells encounter patterns around them in the form of fixed chemical and topographical patterns such as those of the extracellular matrix, or on artificial material, which can be of micro- or nanometric dimensions. In addition, nanometric surfaces are known to have distinct physicochemical properties from the bulk material. For us, these information serve as the reasons to deliberately pattern surfaces with nanometric features for biomedical applications. One of the possible methods to fabricate large areas consistently patterned with random nanofeatures is based on phase separation during spin casting of binary polymer mixes such as polystyrene and poly-bromostyrene. We present in this paper the reaction of rat calvaria bone cells to the same surfaces where we knew that endothelia and fibroblastic cells showed a differential reaction. The cells adhered to all surfaces in the same way after an initially increased adhesion on the rough surfaces (35 and 95 nm). The cells interacted with all surfaces in a similar way extending filopodia, spreading and extending after initial adhesion. The results illustrate that compared to either endothelia or fibroblasts, osteoblast cells proliferate less on the surface chemistry (PS) provided, and that the surface features used do not induce a changed behaviour after an initial increase in adhesion on the rougher surfaces.

Interactions of human blood and tissue cell types with 95-nm-high nanotopography

Two of the major concerns for tissue engineering materials are inflammatory responses from blood cells and fibrous encapsulation by the body in order to shield the implant from blood reaction. A further hurdle is that of vascularization. In order to develop new tissues, or to repair parts of the vascular system, nutrients need to be carried to the basal cell layers. If a material promotes tissue formation, but not vascularization, necrosis will be observed as multilayered cells develop. In this paper, polymer demixed island topography with a 95-nm Z axis was tested using human mononuclear blood cells, platelets, fibroblasts, and endothelial cells. The results showed no difference in blood response between the islands and the flat controls, suggesting that in vivo there would be negligible immunological difference. Fibroblasts reacted by changing morphology into a rounded shape with thick processes and poorly developed cytoskeleton. Retardation of fibroblast growth may be an advantageous, as it is this cell type that forms the fibrous capsule, preventing growth of the required tissue type. Finally, endothelial cells were seen to form arcuate, or curved, morphologies in response to the islands. This is the normal, in vivo, morphology for vascular endothelium. This result suggests that the nano-features are promoting a more phenotypically correct morphology.

Fibroblast signaling events in response to nanotopography: a gene array study

When considering the complicated nature of cell/tissue interactions with biomaterials, especially materials with nanometric surface features, observation of changes in one or two selected genes or proteins may not be sufficient. To get a fuller understanding of the scope of responses effected by nanotopography on cells, many genes need to be surveyed. Recent developments in molecular biology have lead to the commercial production of microarrays. Microarray presents a powerful tool by which many genes (up to many thousands) can be probed simultaneously. In this study, 1718 gene arrays have been used to measure human fibroblast response to 13-nm-high polymer demixed islands. The results have shown many changes in genes involved in signaling, cytoskeleton, extracellular matrix, gene transcription, and protein translation; these results have been used to build a more complete overview of fibroblast response to the islands. The use of microarray has expanded the range of observations possible using established microscopical and biochemical techniques.

Nanometric Surface Patterns for Tissue Engineering: Fabrication and Biocompatibility in Vitro

Three fundamentally different methods were used to fabricate nanometric surface features on polymers or fused silica. Phase separation of binary polymer mixes resulted in randomly distributed features whose depth and shape could be tightly controlled over large areas. Colloidal resist patterned large areas randomly and uniformly with very fine spikes. In contrast e-beam and reactive ion etching were used to create a set of regular spaced pillars on an orthogonal pattern. Some of the surfaces were replicated by in situ polymerization, solvent casting, embossing or melt molding onto polystyrene (PS) or e–poly caprolactone (e–PCL). Nanometric features down to 60nm were imprinted onto the polymers with high fidelity. Cells were seeded onto the nanometric surfaces and adhesion, morphology and cytoskeleton investigated. Cells respond to regular features of 170/80nm (width/depth) with reduced adhesion and changes in overall morphology and cytoskeleton. Small nanofeatures (13nm, 35nm depth) made by phase separation on the other hand increased adhesion and promoted cytoskeletal differentiation. The responses of the cells are indicative that nanometric surface features are useful modifications on scaffolds for tissue engineering or on medical implants.