Adhesion of cells to polystyrene surfaces.

The surface treatment of polystyrene, which is required to make polystyrene suitable for cell adhesion and spreading, was investigated. Examination of surfaces treated with sulfuric acid or various oxidizing agents using (a) x-ray photoelectron and attenuated total reflection spectroscopy and (b) measurement of surface carboxyl-, hydroxyl-, and sulfur-containing groups by various radiochemical methods showed that sulfuric acid produces an insignificant number of sulfonic acid groups on polystyrene. This technique together with various oxidation techniques that render surfaces suitable for cell culture generated high surface densities of hydroxyl groups. The importance of surface hydroxyl groups for the adhesion of baby hamster kidney cells or leukocytes was demonstrated by the inhibition of adhesion when these groups were blocked: blocking of carboxyl groups did not inhibit adhesion and may raise the adhesion of a surface. These results applied to cell adhesion in the presence and absence of serum. The relative unimportance of fibronectin for the adhesion and spreading of baby hamster kidney cells to hydroxyl-rich surfaces was concluded when cells spread on such surfaces after protein synthesis was inhibited with cycloheximide, fibronectin was removed by trypsinization, and trypsin activity was stopped with leupeptin.

Hurricane Allen's Impact on Jamaican Coral Reefs.

Coral reefs of north Jamaica, normally sheltered, were severely damaged by Hurricane Allen, the strongest Caribbean hurricane of this century. Immediate studies were made at Discovery Bay, where reef populations were already known in some detail. Data are presented to show how damage varied with the position and orientation of the substraturn and with the shape, size, and mechanical properties of exposed organisms. Data collected over succeeding weeks showed striking differences in the ability of organisms to heal and survive.

Nuclear localization of HIV-1 tat functionalized gold nanoparticles.

The impermeable nature of the cell plasma membrane limits the therapeutic uses of many macromolecules and there is therefore a growing effort to circumvent this problem by designing strategies for targeted intracellular delivery. During the last decade several cell penetrating peptides, such as the HIV-1 tat peptide, have been shown to traverse the cell membrane, where integral protein transduction domains (PTDs) are responsible for their cellular uptake, and to reach the nucleus while retaining biological activity. It has since been discovered that PTDs can enable the cellular delivery of conjugated biomolecules and even nanoparticles, but nuclear delivery has remained problematic. This present study focuses on the development of water soluble, biocompatible gold nanoparticles of differing size functionalized with the HIV-1 tat PTD with the aim of producing nuclear targeting agents. The particles were subsequently tested in vitro with a human fibroblast cell line, with results demonstrating successful nanoparticle transfer across the plasma membrane, with 5 nm particles achieving nuclear entry while larger 30 nm particles are retained in the cytoplasm, suggesting entry is blocked via nuclear pores dimensions.

Human fibroblast and human bone marrow cell response to lithographically nanopatterned adhesive domains on protein rejecting substrates.

The separate influence of topographical and chemical cues on cell attachment and spreading are well documented; however, that of duel-cue substrates is less so. In this study graft copolymers that sterically stabilize biological surfaces were employed alongside nanotopographical features fabricated by colloidal lithography. This resulted in the production of a range of substrates whereby the effect of chemistry and or topography on both on human fibroblast and bone marrow cell adhesion and spreading could be observed. The current studies indicate an enhancement of cell response as a consequence of modifications in material topography, whereas the current selected chemical cues inhibited cell function. Critically, in combination, topography modulated the effects of chemical environment.

Morphological and microarray analysis of human fibroblasts cultured on nanocolumns produced by colloidal lithography.

The environment around a cell during in vitro culture is unlikely to mimic those in vivo. Preliminary experiments with nanotopography have shown that nanoscale features can strongly influence cell morphology, adhesion, proliferation and gene regulation, but the mechanisms mediating this cell response remain unclear. In this study a well defined nanotopography, consisting of 100 nm wide and 160 nm high cylindrical columns, was used in fibroblast culture. In order to build on previously published morphological data that showed changes in cell spreading on the nanocolumns, in this study gene regulation was monitored using a 1718 gene microarray. Transmission electron microscopy, fluorescent observation of actin and Rac and area quantification have been used to re-affirm the microarray observations. The results indicate that changes in cell spreading correlate with a number of gene up- and down-regulations as will be described within the manuscript.

An in vivo microfabricated scaffold for tendon repair.

A new type of in vivo tissue engineering system for tendon repair in situ after cut or crush of a flexor tendon is described. The system is based on the topographical reaction, alignment, migration and perhaps proliferation of tendon cells on micrometrically grooved substrates made in a biodegradable polymer. Macrophage trapping in the structure may also help to prevent inflammation. Tendon damage including crush and section injury is a fairly frequent occurrence. The conventional treatment is surgical repair, however frequently this leads, especially in hand wounds, to attachment of the tendon surface to the surrounding synovium, which is very undesirable. We present an approach based on using a biodegradable device to ensure that the healing of severed or crushed flexor tendons is aided, synovial adhesion prevented and the final result anatomically correct. The biodegradable sheath carries microgrooves fabricated into the polymer by embossing that orient and guide the cells towards each other from either side of the region of damage. After six weeks an apparently normal functional tendon is reformed.

Comment on "Nanorobotics control design: a collective behavior approach for medicine".

The limitations on nanorobot design and activity imposed by Brownian motion events, communication problems, and the nature of the intercellular space are discussed. It is shown that severe problems exist for a nanorobot designed to enter tissues for therapeutic purposes when it is smaller than about 1 microm in any one of its dimensions.

Graft rejection in sponges. Genetic structure of accepting and rejecting populations.

Graft rejection frequencies in a population of the sponge Ectyoplasia ferox are reported in relation to the complexity of the histocompatibility system in this species. The frequency of graft acceptance is high but we show that, despite assumptions in the literature to the contrary, this does not imply that sponges accepting each other's grafts are genetically identical. This paper reports that graft-accepting pairs of sponges can have dissimilar plasmalemmal proteins. In addition, a theoretical analysis is presented of the types of histocompatibility systems that would account for the present results and those of others. We concluded that there is no evidence that sponges have highly polymorphic histocompatibility systems. The paper also reports on the histology of graft rejection and discusses some of the ecological relevance of the findings.

Modulation of adhesion of lymphocytes to murine brain endothelial cells in vitro: relation to class II major histocompatibility complex expression.

Adhesion of lymphocytes to mouse brain endothelial cells was studied after treatment of the endothelium with 1000 U/ml gamma interferon (IFN-gamma) for 1 h to 2 days. Adhesion was not significantly different from controls after 1 h but at 4 h and thereafter, adhesion increased in a time-related manner. IFN-gamma also increased the expression of class II major histocompatibility complex (MHC) and murine intercellular adhesion molecule-1 (ICAM-1) molecules on the endothelial cells. The level of expression of class II MHC molecules was related to the length of exposure to IFN-gamma. MAb blocking studies suggested that class II molecules were responsible for the IFN-gamma-induced increase in lymphocyte-endothelial cell adhesion. Transfection of a murine lung endothelial cell line with cDNA for the class II MHC molecule also produced a significant increase in lymphocyte-endothelial cell adhesion, suggesting that the class II MHC molecule may have a role in adhesion which is distinct from antigen presentation.

The superoxide anion in lymphocyte transformation.

This work suggests that reactive oxygen intermediates can stimulate lymphocytes mitogenically. Incubation of human peripheral blood lymphocytes in solutions of potassium superoxide induced high levels of uptake of tritiated thymidine as did activation of monocyte production of superoxide ions using zymosan-A. Superoxide dismutase successfully reduced this stimulation but catalase and mannitol, scavengers of hydrogen peroxide and hydroxyl radicals, respectively, had no effect. The same cells are also shown to become more adhesive after superoxide treatment and we would suggest that these two phenomena may be linked.

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.

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.

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.

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.

Small is beautiful but smaller is the aim: review of a life of research.

Background and origins of research of Adam Curtis. One persisting theme has been the pursuit of different landscapes at different scales to discover the routes to explain how the body is built. His research life fell in a fortunate period during which techniques and concepts for investigating structure have improved year by year. His most fortunate encounter was with Michael Abercrombie and his views on the social behaviour of cells, aims for quantitation, and statistical testing. Adam worked in various environments--in turn Geology as an undergraduate, Biophysics Ph.D. in a Genetics department and various departments in turn from anatomy via zoology to Cell Biology. Adam started his Ph.D. work in cell adhesion, studying cell movement, trapping and reaggregation phenomena, having an early start from the physico-chemical viewpoint. He made quantitative measurements of cell adhesion by kinetic methods. Interference reflection microscopy (IRM) and related optical interference techniques were brought into the field of biology by him. In turn this led with Chris Wilkinson, a long term colleague, to the use of micro- and nanofabrication for biological research. Polscope and photoelastic measurements were introduced to biology recently in his laboratory. One long term theme has been to map the adhesion of cells to substrates to discover contact areas. Early data came from IRM and then TIRF (Total Internal Reflection Fluorescence Microscopy) and then from Forster Resonance Energy Microscopy (FRET). Another important theme was the time scale that needed to be measured--very short indeed in suspension. This was very difficult and has only become possible very recently but hydrodynamic calculation shows it must be very short. The attractions of the Derjagin-Landau-Verwey-Overbeek theory (DLVO theory) are that they explain many features of biological adhesion. The main test of this theory depends upon the energy of the adhesion at various different separation distances between cell and cell or cell and substrate. Problems with cell adhesion molecules are discussed. Contact guidance of cells by oriented structures and Paul Weiss--Tests with grating replicas suggested that topographic rather than biochemical explanations were applicable. It became clearer later that this was an area of research waiting for microfabrication. Albert Harris influenced me considerably to start thinking about mechanical forces produced by cells. Pulling at cells showed effects on the cytoskeleton and on cell cycle time. Such thoughts led to a microfabricated device for tendon repair. Recent photoelastic measurements with the Polscope have allowed much more detailed analysis of the forces between cells. The interesting results on microfabricated devices led to work on nanostructures. Results led the Glasgow group to consider dimensions of structures and how cells could sense such small objects and questions about why order and size may be important. Differential protein adsorption onto surfaces seems to provide defective explanations of the effects. The results will be discussed in terms of very recent theories of cell interaction and cell signals and possible future developments will be outlined.

Cell reactions with biomaterials: the microscopies.

The methods and results of optical microscopy that can be used to observe cell reactions to biomaterials are Interference Reflection Microscopy (IRM), Total Internal Reflection Fluorescence Microscopy (TIRFM), Surface Plasmon Resonance Microscopy (SPRM) and Förster Resonance Energy Transfer Microscopy (FRETM) and Standing Wave Fluorescence Microscopy. The last three are new developments, which have not yet been fully perfected. TIRFM and SPRM are evanescent wave methods. The physics of these methods depend upon optical phenomena at interfaces. All these methods give information on the dimensions of the gap between cell and the substratum to which it is adhering and thus are especially suited to work with biomaterials. IRM and FRETM can be used on opaque surfaces though image interpretation is especially difficult for IRM on a reflecting opaque surface. These methods are compared with several electron microscopical methods for studying cell adhesion to substrata. These methods all yield fairly consistent results and show that the cell to substratum distance on many materials is in the range 5 to 30 nm. The area of contact relative to the total projected area of the cell may vary from a few per cent to close to 100% depending on the cell type and substratum. These methods show that those discrete contact areas well known as focal contacts are frequently present. The results of FRETM suggest that the separation from the substratum even in a focal contact is about 5 nm.

Chloride channels and the reactions of cells to topography.

The reactions of rat epitenon cells to substratum topography on the micrometric and nanometric scale such as groove-ridge structures include cell extension, elongation and orientation reactions. In this paper we report that stretch-sensitive chloride channels may be involved in the earliest stages of these reactions in epitenon fibroblast-like cells. We report that rat epitenon-cells can develop appreciable lateral mechanical tension that could stretch both the force generating cells themselves and those nearby. We show that cells in medium in which more than 80% of the chloride has been replaced by nitrate show little reaction to topography. Spreading of the cells takes place but is much reduced along the direction of the groove-ridge topography but enhanced across the topography. The chloride channel inhibitors NPPB (5-Nitro-2- (3phenylpropylamino) benzoicacid) 4,4'-disothiocyanostilbene-2, 2' sulphonic acid (DIDS) and Chlorotoxin produce similar results which are further accentuated when these inhibitors are presented in low chloride medium. An antibody against ClC3, which has close homology to ClC5/6 also, blocked reaction to topography. These treatments have no significant effect on cell spreading on planar surfaces nor do they lead to changes in internal pH in the cells. There is a slight inhibition of rates of cell movement. Experiments using antisense oligoribonucleotides to ClC-5 or ClC-6 channel m-RNA also inhibit topographic reactions, which provides further confirmation of the hypothesis. Since the ClC-3,4 and 5 share considerable sequence similarities in the genes and in their proteins it has not been possible to make an unambigous determination of which precise chloride channel(s) is (are) involved.

Effective extra-cellular recording from vertebrate neurons in culture using a new type of micro-electrode array.

We describe the fabrication and use of a new type of extracellular micro-electrode array mounted on a flexible transparent polyimide substrate that can be rapidly moved from one part of a culture of vertebrate neurons (rat nodose) to another, which permits co-culture of glia under the neurons and is easily and rapidly replaceable in the event of damage. The array can be mounted on a micromanipulator and moved into place whenever and wherever recordings with or without stimulation are needed. The basic electrode system consists of 20-30 microm diameter gold electrodes, with or without platinisation, exposed to the cells through openings in the polyimide and joined to the recording or stimulating circuitry through gold tracks embedded in the polyimide. If rigid control over neuron placement has been achieved the patterns of electrodes can be matched to the neuron positions.

Simultaneous multisite recordings and stimulation of single isolated leech neurons using planar extracellular electrode arrays.

Planar extracellular electrode arrays provide a non-toxic, non-invasive method of making long-term, multisite recordings with moderately high spatial frequency (recording sites per unit area). This paper reports advances in the use of this approach to record from and stimulate single identified leech neurons in vitro. A modified enzyme treatment allowed identified neurons to be extracted with very long processes. Multisite extracellular recordings from the processes of such isolated neurons revealed both the velocity and direction of action potential propagation. Propagation in two cell types examined was from the broken stump towards the cell body (antidromic). This was true for spontaneous action potentials, action potentials produced by injecting current into the cell body and extracellular stimulation of the extracted process via a planar extracellular electrode. These results extend previous findings which have shown that the tip of the broken stump of extracted neurons has a high density of voltage-activated sodium channels. Moreover they demonstrate the applicability of extracellular electrode arrays for recording the electrical excitability of single 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.