Mathis O. Riehle

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.

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.

Cells react to nanoscale order and symmetry in their surroundings

Mammalian cells react to microstructured surfaces, but there is little information on the reactions to nanostructured surfaces, and such as have been tested are poorly ordered or random in their structure. We now report that ordered surface arrays (orthogonal or hexagonal) of nanopits in polycaprolactone or polymethylmethacrylate have marked effects in reducing cell adhesion compared with less regular arrays or planar surfaces. The pits had diameters of 35, 75, and 120 nm, respectively, with pitch between the pits of 100, 200, and 300 nm, respectively. The cells appear to be able to distinguish between different symmetries of array. We suggest that interfacial forces may be organized by the nanostructures to affect the cells in the same way as they affect liquid crystal orientations.

The use of materials patterned on a nano- and micro-metric scale in cellular engineering

Biological cells form distinct groupings in tissue depending on their function. This is as essential in the regrowth of damaged tissue as it is in the development of the mature animal from its egg. For a number of years we have been exploring the effect of surfaces patterned with a topographic relief pattern and with patterns of bound active bio-molecules. The response of cells to micron-sized features, the response of cells to nano-metric features and the effects of soft materials are discussed.

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.

An Integrative Study of Insect Adhesion: Mechanics and Wet Adhesion of Pretarsal Pads in Ants

Abstract Many animals that locomote by legs possess adhesive pads. Such organs are rapidly releasable and adhesive forces can be controlled during walking and running. This capacity results from the interaction of adhesive with complex mechanical systems. Here we present an integrative study of the mechanics and adhesion of smooth attachment pads (arolia) in Asian Weaver ants (Oecophylla smaragdina). Arolia can be unfolded and folded back with each step. They are extended either actively by contraction of the claw flexor muscle or passively when legs are pulled toward the body. Regulation of arolium use and surface attachment includes purely mechanical control inherent in the arrangement of the claw flexor system. Predictions derived from a ‘wet’ adhesion mechanism were tested by measuring attachment forces on a smooth surface using a centrifuge technique. Consistent with the behavior of a viscid secretion, frictional forces per unit contact area linearly increased with sliding velocity and the increment strongly decreased with temperature. We studied the nature and dimensions of the adhesive liquid film using Interference Reflection Microscopy (IRM). Analysis of ‘footprint’ droplets showed that they are hydrophobic and form low contact angles. In vivo IRM of insect pads in contact with glass, however, revealed that the adhesive liquid film not only consists of a hydrophobic fluid, but also of a volatile, hydrophilic phase. IRM allows estimation of the height of the liquid film and its viscosity. Preliminary data indicate that the adhesive secretion alone is insufficient to explain the observed friction and that rubbery deformation of the pad cuticle is involved.

Rapid fibroblast adhesion to 27 nm 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.

Tissue engineering: the biophysical background.

Tissue engineering is the construction, repair or replacement of damaged or missing tissue in humans and other animals. This engineering may take place within the animal body or as tissue constructs to be made in a bioreactor for later grafting into the animal. The minimal set of materials for this are the appropriate types of cell. Usually, however, non-living substrata are used as well. These substrata may be nothing more than materials that bulk up any voids in the damaged tissue and provide the mechanical strength that has been lost when the tissue is damaged or removed. They may serve a similar pair of functions in the bioreactor. They can do much more in terms of pattern formation. The orientations and morphology of the cells, the arrangement of intercellular material as it is laid down and the relationships between different cell types in the repairing or construct tissue are all of importance, for these should resemble the correct normal tissue as closely as possible. Most of these requirements are ones involving pattern formation. This review discusses the various ways in which tissue pattern can be engineered chiefly from a biophysical standpoint. Unpatterned cells are effectively not tissue. This engineering includes the use of topography on the substrata, chemical patterning of adhesive and other cues for the cells, mechanical force application to cause cell orientation and appropriate synthetic responses and electrical fields. The review also discusses the methods used to impart the appropriate cues to and through the materials which are often biodegradable polymers. The article gives particular attention to regions of research and practice where the involvement of the physicist or biophysicist is of importance.