Papers

AVS1996 Session BI-TuM: Cell-Surface Interactions

Tuesday, October 15, 1996 9:00 AM in Room 203A

Tuesday Morning

Start	Invited?	Item
9:00 AM		BI-TuM-3 Fabrication and Analysis of Neuronal Networks J. Hickman, K. Bateman, D. Jung (Science Applications International Corporation); A. Schaffner, Y. Li, J. Barker (National Institutes of Health) We are fabricating defined but primitive circuits of living hippocampal neurons and studying their cellular interactions. We use high resolution deep UV lithography of self-assembled monolayers (SAMs) to create the circuit template patterns on Si\sub 3\N\sub 4\ or SiO\sub 2\. We can control the intrinsic and geometric properties of the growth surface by forming a template to guide neuronal adhesion and neurite outgrowth. The neurons are dissociated from the developing hippocampus of 19-day old rat embryos and cultured on SAMs in serum-free media to create a defined in vitro system. The ability to control surface composition allows a deeper understanding of cell-surface interactions. We are using patch-clamp techniques to record electrophysiological signals produced by neurons in response to stimuli. The patterns are analyzed prior to and following cell culture by high-resolution imaging XPS spectroscopy. We will report on our latest results.
9:20 AM		BI-TuM-4 Preferential Adhesion of Glial Cells to Microcontact Printed Surfaces P. St. John, M. Isaacson, H. Craighead (Cornell University); L. Kam (Rensselaer Polytechnic Institute); W. Shain (Wadsworth Center and School of Public Health); J. Turner (Rensselaer Polytechnic Institute) We are studying cell adhesion to chemically and topographically modified surfaces to be used in the fabrication of neural prostheses. We describe a new process for patterning surfaces for preferential cell adhesion using microcontact printing on silicon with an amine ethoxysilane as the ink. The ink forms a self-assembled monolayer (SAM) on the native oxide substrate and creates an attractive medium to which cells adhere. Using confocal microscopy to observe the stained cell membranes on various micron-size patterns, we show preferential adhesion of astrocytes from rats and LRM55 astroglial cells to the nitrogen containing monolayers, compared to both bare silicon and long chain hydrocarbon monolayers such as octadecyltrichlorosilane (OTS). We have also observed differing cell morphologies upon attachment to chemically modified surfaces. Glial cells attached to the amine SAMs resemble a focal-type contact: Cells are spread out in multiple directions and in some cases, bridge to neighboring cells. Cells attached to the OTS and bare Si substrates appear rounded and resemble point-type contacts. This work was supported by ARPA and NIH grant RR R01 10957
9:40 AM		BI-TuM-5 Electrical Characterization of Uromyces Germ Tubes Grown on Integrated Circuit Substrates M. Kozicki, R. Roberson, H. McNally, S. Kersey, T. Whidden (Arizona State University) We report on the latest results of an investigation into the directed growth and subsequent electrical characterization of fungal germ tubes on custom-designed integrated test circuits. The purpose of this study is to create functional bio-electronic systems in which the biological moiety, with its unique structure and characteristics, is an active component. A number of applications have been proposed for such systems, particularly in the area of bio-sensors. The integrated circuit element, produced by standard microelectronic fabrication techniques, forms the electrical interface between the biological element and the systems of the outside world. The biological element in this work is Uromyces appendiculatus, an organism which has been shown to exhibit precise and unique topographical signal recognition. Germination results in the formation of a germ tube from the spore which grows out to touch the surface of the circuit. This narrow, cytoplasm-filled structure is readily guided to self-connect with contacts on the integrated circuit by the use of topographical vectoring elements etched into the final layer of metallization. The vectoring structures have been shown to be capable of steering the hyphae to intended connection/termination points through a wide range of angles. Since the vectoring structures are part of the metallization pattern, they may also be biased to control ion movement in the tube. In this paper, we present current voltage (I-V) and current-time (I-t) measurement results for both dc and ac stimulation of cells which have been preserved using a variety of techniques and subjected to a number of chemical treatments. The magnitude of the current through the cell for constant voltage depends on which section of the germ tube is tested, (the vacuole region producing a higher value than the apical section), and the nature of the previous chemical exposure. The effect of preserved cell aging will also be discussed.
10:00 AM		BI-TuM-6 Oligo(ethylene Oxide) Effects on Bacterial Attachment to Synthetic Solid Surfaces L. Ista, H. Fan, G. L\aa o\pez (University of New Mexico) Self-assembled monolayers (SAMs) formed from \omega\-substituted alkanethiols on transparent gold films were used to study the attachment of bacteria to solid surfaces under defined culture and flow conditions. SAMs terminated with a number of organic functional groups were studied for their abilities to adhere bacteria of both marine (\italic Deleya marina\) and medical (\italic Staphylococcus epidermidis\) importance. Over the range of experimental conditions, SAMs terminated with oligo(ethylene oxide) were found to be uniformly resistant to bacterial attachment. Studies of mixed monolayers of oligo(ethylene oxide)- and methyl- terminated alkanethiols were also conducted. This study indicates that surfaces incorporating high densities of oligo(ethylene oxide) are good candidates for surfaces that resist bacterial adhesion.
10:20 AM		BI-TuM-7 Endothelial Cell Interactions with Model Surfaces: Effect of Surface Chemistry and the Adsorbed Protein Layer C. Tidwell, A. Belu (University of Washington); B. Tarasevich (Battelle Pacific Northwest Laboratory); D. Allara (Pennsylvania State University); B. Ratner (University of Washington) The role of surface chemistry and the effect of adsorbed proteins in promoting cell growth is an active area of investigation, with contradictory results often reported. In this study, a series of terminally functionalized alkanethiol self-assembled monolayers (SAMs) and their binary mixtures were used as model surfaces to probe the role of specific chemical functional groups in mediating interfacial biological interactions, specifically serum protein adsorption and endothelial cell growth. Homogenous SAMs of X(CH\sub 2\)\sub 15\SH on gold with X = -CH\sub 3\, -CH\sub 2\OH, -CO\sub 2\CH\sub 3\, -CO\sub 2\H and -SO\sub 4\H were studied as well as mixtures (3:1, 1:1, 1:3 targeted surface concentration) of the SAMs with functional groups -CH\sub 3\-CO\sub 2\H, -OH/-COOH. TOF-SIMS, ESCA, contact angle, IR and ellipsometry confirm the presence of the expected functionalities on the SAM surfaces. The growth of bovine aortic endothelial (BAE) cells and the adsorption and retention of two serum proteins (albumin and fibronectin) was investigated. Protein interaction studies demonstrated variations in protein adsorption and retention in response to SAM terminal functional group. Correlations between cell growth and protein interaction characteristics were observed. For the homogeneous SAMs, BAE cell growth varied significantly with surface chemical functionality. Cell growth increased in the following order: -CH\sub 2\OH < CH\sub 3\ < CO\sub 2\CH\sub 3\ << -COOH. Homogeneous SAM cell growth levels were lower than on tissue culture polystyrene suggesting that multiple chemical functionalities are a likely requirement for cell growth. The observation of significantly increased cell growth on mixed SAM surfaces compared with the homogeneous SAMs confirmed this hypothesis. Significantly, mixed SAM surfaces composed primarily of poor cell growth components (i.e., 3:1 CH\sub 2\OH /COOH) exhibited high cell growth levels.
10:40 AM		BI-TuM-8 Cell-Surface Interactions on Thiol Modified Gold Surfaces- an In-vivo Study in Mice and with Human Whole Blood In-vitro S. Kanagaraja (University of G\um o\teborg, Sweden); J. Lausmaa (Chalmers University of Technology, Sweden); H. Nygren (University of G\um o\teborg, Sweden) The process of non-self recognition of foreign materials begins immediately on implantation into a living organism. Water, salts and plasma proteins would initially react with the surface followed by cellular reactions. The sequence of reactions occuring at foreign surfaces is complex, ranging from seconds to years. The biocompatibility of materials has been traditionally assessed by the degree of the elicited inflammatory response to the surfaces. However, attempts have been made in predicting the long term performance of biomaterials by studying the events occuring initially at the surface interfaces. In this respect, gold surfaces modified by alkane thiols have been used as well defined surfaces with highly specific physical and chemical characteristics in the analysis of surface biochemical events. In this study two thiol modified surfaces with known surface characteristics and distinct differences in protein(albumin, fibrinogin IgG) adsorption, contact activation of coagulation and complement system activating patterns, have been evaluated in-vivo in mice. The biocompatibility of these surfaces as represented by the degree of cellular inflammatory response and the corelation with surface characteristics and events preceding cell recruitment has been studied. Normal mice and mice depleted of fibrinogen and complement were used. Cell adhesion was quantified by SEM and computer aided image analysis after fluorescence staining of cell nuclei. Cell spreading and activation and the cell-surface interface was analysed using TEM, respiratory burst(NBT)test and expression of selectin(CD62) by specific antibody binding. Results will be presented on tests done on Gold and Gold surfaces modified with Glutathione and 3-Mercapto-1,2-propanediol.
11:00 AM		BI-TuM-9 Influence of Self-assembled Monolayer Defect Structure on Cell Growth S. Golledge, B. Ratner, D. Castner, M. Garrison, C. Tidwell, A. Belu (University of Washington) The order characteristic of self-assembled monolayers (SAMs) allows the preparation of surfaces with well-defined chemistries. The chemistry may be modified to provide a surface with specific properties useful in any number of applications. SAMs are used in the present study as model substrates for probing biological interactions with surfaces. Although SAMs have been so utilized by a few other groups, to date no fundamental study on the effect of the unavoidable imperfections in the monolayer on biological interactions has been reported. In the initial stage of the present project, the effect of the gold substrate roughness on the SAM defect structure is being examined. Subsequently, the effect of the defects on cellular and protein interactions with the SAMs will be considered. Pulsed plasma, thermal, electron-beam, and RF-sputtering have been used to deposit the gold onto various substrates: mica, silicon, silicon-chromium, glass, and glass-chromium. In some cases the gold layer was also annealed; the effect of the anneal varied with deposition method. For example, the average roughness of the electron-beam deposited gold layer on silicon decreased from 2.0nm to 1.1nm, whereas for the RF sputtered-layer on silicon the roughness increased from 0.9nm to 2.8nm, as measured by Atomic Force Microscopy. Based on the entire AFM measurement set, six preparation method/substrate combinations have been selected for further study: electron-beam and RF-sputtering of gold onto mica substrates with and without anneal, and electron-beam deposition onto silicon and silicon-chromium substrates without anneal. These provide substrates with a range of roughnesses, which may lead to differing SAM defect structures. The C16 methyl- and hydroxyl-teminated SAMs are being characterized by ellipsometry, FTIR, XPS, and time-of-flight SIMS.
11:20 AM		BI-TuM-10 Differential Expression of Neurotransmitter Phenotypes in Cortical Cell Cultures Grown on Organosilane Surfaces W. Ma, M. Coulombe, J. Hickman (Science Applications International Corporation) Increasing evidence suggests that the composition of culture substratum is critical for cell adhesion, survival and differentiation in vitro. However, it is not well known if substratum affects cell phenotype expression in culture. In the rat cerebral cortex, two distinct types of neurons, pyramidal and non-pyramidal cells, have been identified. Pyramidal neurons use the excitatory transmitter glutamate, whereas most non-pyramidal neurons use the inhibitory neurotransmitter GABA. We have developed an in vitro system that combines the use of a defined serum-free medium with a chemically defined surface. This system allows for the study of effects of substratum on the expression of glutamatergic and GABAergic phenotypes in a well-defined environment. Cells were mechanically or enzymatically (papain) dissociated from the developing cerebral cortex of 14 and 16 day-old rat embryos and cultured substratum, and silica substrates modified with artificial surfaces composed of silane self-assembled monolayers (SAMs). Surface properties were examined by X-ray photoelectron spectroscopy measurements before and after cell cultures. Double-immunoflourescence staining for glutamate and glutamate decarboxylase (GAD) with a mixture of anti-glutamate (Incstar) and anti-GAD was carried out in sister cultures. Preliminary results showed that in cultures grown on poly-D-lysine, glutamatergic neurons represent approximately 30-40% of total cells, GABAergic neurons represent about 50-60% of total cells, and the ratio of glutamatergic to GABAergic cells was 0.63:1, which is consistent with findings of immunocytochemical studies in vivo. In cultures on a hydrophilic surface, this ratio was 0.60:1. In contrast, on hydrophobic surfaces, about 20-30% were glutamatergic, 50-60% expressed GAD, and the ratio of glutamatergic to GABAergic cells was 0.45:1. This suggests that a hydrophilic surface similar to poly-D-lysine supports both glutamatergic and GABAergic transmitter phenotypes, while hydrophobic surfaces decrease the number of glutamatergic cells more than GABAergic cells.
11:40 AM		BI-TuM-11 Porous Bioactive Glass (45S5) Templates for the In Vitro Synthesis of Bone-like Tissue E. Effah, P. Ducheyne, I. Shapiro (University of Pennsylvania) Our program focuses on bone tissue engineering. Specifically, we address the issue of optimum bioactive glass template characteristics to stimulate in vitro formation of bone and bone-like tissue. We also examine the mechanisms by which material surfaces elicit the in vitro response from cells of the osteoblastic phenotype. In previous studies we synthesized porous bioactive glass (BG) templates by hot pressing. These templates were found to enhance the formation of bone-like tissue by rat calvaria osteoblasts in vitro. A new processing technique for the efficient fabrication of porous BG templates is now presented. The templates are made by compacting BG granules with camphor followed by sintering at 590 \super o\C for 45 minutes. The additive, camphor, has a sublimation temperature of 204 \super o\C and is intended as a sacrificial agent. Thus, during firing, the sublimation of camphor leaves a network of pores throughout the templates. Using Scanning Electron Microscopy (SEM, JEOL JSM 6300FV), SEM-EDXA (JEOL JSM 6400) and Fourier transform infrared spectroscopy (FTIR, Nicolet Magna-IRTM 550 spectrometer), the templates were characterized upon sintering. The templates were also analyzed by X-Ray Diffraction (XRD, Automated Rigaku-Geigerflex) to detect any devitrification that might have occurred as a result of firing. Finally, the templates were seeded with osteoblasts and our results indicate no significant differences between these templates and those that were made by hot pressing.