Papers

AVS2004 Session BI1-WeM: Cell-Surface Interactions

Wednesday, November 17, 2004 8:20 AM in Room 210D

Wednesday Morning

Start	Invited?	Item
8:20 AM		BI1-WeM-1 Study of Confluent Cell Culture Monolayers by XPS and SIMS M. Greenfeld, H.E. Canavan, X. Cheng, B.D. Ratner, D.G. Castner (University of Washington) Adhered cells transform the surfaces on which they are cultured by excreting and remodeling the underlying extracellular matrix (ECM) proteins. As the ECM is known to play a vital role in the processes of differentiation, motility, and proliferation, the characteristics and identity of the ECM proteins excreted by different cells, or by the same cells throughout its lifetime, are of a great deal of interest in biology and surface science alike. Until now, traditional high vacuum techniques such as X-ray Photoelectron Spectroscopy (XPS) and Secondary Ion Mass Spectrometry (SIMS) have played only a minor role in the analysis of the ECM, as traditional cell removal techniques are destructive to both the cells and the underlying ECMâ?"in effect damaging the structure of analytical interest. Recently, poly(n-isopropylacrylamide) (pNIPAM) treated tissue culture polystyrene (TCPS) has been developed as a method to non-destructively harvest intact cell monolayers. Using the low-temperature liftoff technique, cell sheets may be non-destructively removed from surfaces and re-deposited atop new surfaces, achieving multilayer structures of different cell types, such are used for tissue engineering. To date, studies of cells harvested via this method have primarily utilized traditional biological techniques to track the morphology of the cells and the location of their ECM proteins. Previously, we have used XPS and SIMS to examine culture surfaces after cell liftoff and identify the ECM proteins retained. In this work, we present the first application of high vacuum techniques to an examination of cell monolayers harvested by low-temperature liftoff. We find that the presence of proteins in the basal surface of the ECM is easily detected via XPS and SIMS. We then compare the identities and relative amounts of ECM proteins at the apical and basal surfaces of the cell sheet to those retained by the underlying surface.
8:40 AM		BI1-WeM-2 Modeling of Bacterial Attachment Using Lewis Acid-Base Models of Colloidal Adhesion L.K. Ista, K. Artyushkova, T.M. Madrid, J.E. Fulghum, G.P. Lopez (The University of New Mexico) Understanding the processes involved in primary bacterial adhesion to solid surfaces is an important step in development of surfaces on which biofilm formation can be controlled. The relationship of the interfacial tensions between the attaching organism, the liquid medium and the solid substratum determines whether or not attachment can proceed. Control of bacterial attachment is most easily addressed, therefore, by control of substratum surface energy. The relationship between surface energy and attachment can be described qualitatively using colloidal models of adhesion, although a definitive quantitative model is still elusive. Most attempts at modeling bacterial attachment have been made using data generated from attachment to commercially available substrata or their derivatives, many of which are chemically ill-defined. Investigation of substratum physicochemistry on the attachment of a marine bacterium is described. Model solid substrata were generated using mixed self-assembled monolayers of ω-terminated alkanethiolates on gold. These substrata varied systematically in Lewis acid-base, dispersive and polar characteristics, while controlling for other surface factors that may affect bacterial adhesion. The test bacterial strain was the gram negative, marine bacterium Cobetia marina. The surface energetics of this organism were determined by partition into organic solvents that differed in their surface energy . Attachment of this organism to SAM surfaces was then quantified and modeled using standard Lewis acid base models of colloidal attachment and multivariate analysis.
9:00 AM		BI1-WeM-3 Deconstruction the Cell-Biomaterial Interface J.Y. Wong (Boston University) Cells respond to three main categories of physicochemical cues: chemical, topographical, and mechanical. While surface chemistry and topography have been studied extensively, substrate mechanics has only recently been appreciated. Recent technologies of creating surfaces with well-defined chemistry and topography combined with sensitive surface characterization techniques have unquestionably deepened our understanding of surface chemical and topographical effects on cell behavior. In contrast, much less is known about substrate mechanics effects on cell behavior. This talk discusses the types of substrata and characterization methods that have been used to investigate substrate mechanics effects on cell behavior. We also speculate on the relationships between changes in substrate elasticity that occur naturally in vivo (e.g. wound healing) and cellular response. We present recent developments in creating substrata with well-defined mechanical properties in our own laboratory and the major challenges and issues of determining whether substrate mechanics effects are a material-independent phenomenon. We also discuss the effects of combining multiple physicochemical cues on cell behavior. The use of model systems in which chemistry, topography, and mechanics can be independently controlled will facilitate the quest for design principles and material selection rules to control cell response.
9:40 AM		BI1-WeM-5 Supramolecular Structure of Adsorbed Collagen Layers and Influence on Endothelial Cells Behavior C.C. Dupont-Gillain, E. Gurdak, Z. Keresztes, P.G. Rouxhet (Universitat Catholique de Louvain, Belgium) The aim of this study is to examine the supramolecular organization of adsorbed collagen and to evaluate its influence on endothelial cells, thereby increasing our understanding of cell-material interactions. Collagen was adsorbed on polystyrene (PS) and plasma-oxidized PS (PSox) in different conditions, likely to affect the supramolecular structure of the adsorbed layers. The collagen layers and their mechanism of formation were examined using atomic force microscopy, quartz crystal microbalance, X-ray photoelectron spectroscopy and radiolabeling. On PS, the adsorbed collagen molecules leave protruding segments in solution, allowing fibril formation at the interface; this increases with concentration and with time. Dewetting of the collagen layer leads to the formation of discontinuous layers with a net-like nanopattern. On PSox, collagen mainly forms a felt of lying molecules. The adhesion of human umbilical vein endothelial cells (HUVEC) was studied on collagen layers adsorbed on PS or PSox and presenting a diversity of supramolecular structures. In presence of serum, HUVEC cells could not adhere to PS. After adsorption of a smooth collagen layer, cell adhesion became high, and increased with the adsorbed amount. However, the formation of fibrils at the interface provoked a decrease of cell spreading. The last trend was also observed on PSox. This may be related to the accessibility of recognition sites, which could be hidden once collagen forms fibrils. In contrast, the spreading of HUVEC cells was enhanced on discontinuous collagen layers compared to smooth, continuous ones. In this case, collagen association was triggered by dewetting, which could change the availability of recognition sites. Moreover, the discontinuous pattern could stimulate the organization of cell surface receptors, or allow coadsorption of proteins secreted by the cells. Further work includes antibody assays to assess the availability of recognition sites on adsorbed collagen.
10:00 AM		BI1-WeM-6 Using Thin Films of Fibrillar Type I Collagen to Investigate a Signaling Mechanism that Mediates Growth Arrest in Smooth Muscle Cells J.T. Elliott (National Institute of Standards and Technology) Smooth muscle cells (SMC) on fibrillar collagen activate different signaling pathways, have a minimally spread morphology and appear growth arrested compared to SMC cultured on non-fibrillar native collagen. Because studies suggest that SMC interact with both matrices through the same integrin receptors, it appears that it is the supramolecular fibrils that are responsible for the phenotypic response. We used thin films of fibrillar collagen assembled on hexadecanethiol monolayers to investigate which properties of the collagen fibrils control the proliferation signaling. The films are on average 30 nm thick and composed of collagen fibrils that are microns long and as large as several hundred nanometers in diameter. They also have optical properties that are ideal for both phase and fluorescence microscopy. When the fibrillar films are kept hydrated, they induce a growth arrest response in SMC that is similar to the response that is observed on fibrillar collagen gels. If the thin films are dried for several hours before rehydration, the SMC exhibit a well-spread proliferative phenotype and begin to proliferate. Atomic force microscopy (AFM) analysis of these fibrillar films indicates that they are nearly identical in topography, density of fibrils and size of fibrillar structures. These data suggest that the presence of collagen fibrils alone is not sufficient to induce the growth-arrested phenotype. AFM imaging of the fibrillar films under aqueous conditions suggest that the flexibility of the collagen fibrils is reduced during the drying process. We hypothesize that the mechanical properties of the fibrils are an essential determinant of the SMC growth-arrest response. We are currently using live-cell microscopy to understand how these cells interrogate the mechanical properties of collagen fibrils when deciding their phenotypic state.
10:40 AM		BI1-WeM-8 Engineering of Functional Three Dimensional Cell Structures by Inkjet Printing T. Boland, P. Kesari, T. Xu (Clemson University); D. Varghese (Southampton General Hospital) Tissues and organs exhibit distinct shapes and functions nurtured by vascular connectivity. In order to mimic and examine these intricate structure-function relationships, it is necessary to develop efficient strategies for assembling tissue-like constructs. Many of the top-down fabrication techniques used to build microelectromechanical systems including photolithography are attractive due to the similar feature sizes, but are not suitable for delicate biological systems or aqueous environments. A bottom-up approach using inkjet printers has been proposed to pattern functional cell structures in three dimensions. The freeform cell structures created by the inkjet method are viable and show mature character as exemplified by the contractile responses of smooth muscle cell tubes. These results show promise of the inkjet method for vascular tissue engineering and other applications.
11:00 AM		BI1-WeM-9 Nanofabrication of a Novel Cell Array on Ultrathin Hydrophilic Polymer Gels Utilizing Electron Beam Irradiation and UV Excimer Laser Ablation M. Yamato (Tokyo Women's Medical University, Japan) Many methods for surface patterning presented to date are based on lithography techniques and microfabrication onto silicon or glass substrates. Here, we show a novel method to prepare patterned surfaces on polystyrene substrates by grafting ultrathin cell-repellent polymer layers utilizing both electron beam (EB) polymerization and local laser ablation techniques for microfabrication. Polyacrylamide(PAAm) was grafted onto tissue culture polystyrene (TCPS) dishes using electron beam irradiation. Water contact angles for these PAAm-grafted TCPS (PAAm-TCPS) surfaces were less than 10° with grafted amounts of PAAm of 1.6 mg/cm² as determined by FT-IR/ATR method. UV excimer laser (ArF: 193 nm) ablation resulted in the successful fabrication of micropatterned surfaces by exposure of the basal polystyrene layers. Many cell types adhered only to the ablated domains after pretreatment of the patterned surfaces with optimized concentration of fibronectin solution. The ablated domain sizes have significant influence on the number of cells occupying each domain. Cell patterning functionality of the patterned surfaces was maintained for more than 2 months without losing pattern fidelity. Utilization of these surface fabrication techniques are also presented for basic cell biology as well as preparation of cell-based biosensors.
11:40 AM		BI1-WeM-11 Evaluation of PDMS as a Model Substrate to Investigate Effects of Substrate Compliance on Cell Behavior: Interplay of Surface Chemistry and Substrate Mechanics X.Q. Brown, J.Y. Wong (Boston University) Polydimethylsiloxane (PDMS) is an attractive model system for studying the effects of tissue mechanical properties on cell behavior, because the elastic modulus of PDMS can be tuned to achieve a physiologically-relevant range. However, it has been suggested that altering crosslink density can also modulate surface properties. Both the chemical and mechanical properties of a substrate can affect cell behavior: while the importance of surface chemistry and substrate mechanics have been studied independent of each other, few studies have considered their integrated effects. In this study, we characterized the mechanical and surface properties of PDMS substrata with different crosslink density and systematically investigated the effect of PDMS crosslink density on vascular smooth muscle cell (VSMC) attachment, spreading and proliferation. We find that after the same surface treatment, the water contact angle of PDMS decreases with decreased crosslink density, whereas the amount of protein adsorbed onto the material surface remains the same. We also find that in the absence of serum, there is a 39% decrease in cell attachment and a 42% decrease in projected cell area as the Youngâ?Ts modulus decreases from 1.79 to 0.05 MPa. Although these differences in VSMC adhesion are diminished in the presence of serum or adsorbed fibronectin, the rate of serum-stimulated cell proliferation is significantly lower on PDMS with higher crosslink density. We conclude that for the range of crosslink density we investigated, the surface properties of PDMS play a major role in controlling the initial attachment and spreading of VSMC, whereas the mechanical properties of PDMS influence the long term growth of VSMC.