AVS2000 Session BI-WeP: Poster Session
Wednesday, October 4, 2000 11:00 AM in Room Exhibit Hall C & D
Wednesday Morning
Time Period WeP Sessions | Topic BI Sessions | Time Periods | Topics | AVS2000 Schedule
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BI-WeP-1 Hydroxy-containing Amino Acid Derivatives Adsorbed on Gold
J. Svensson, A. Borgh, K. Uvdal, B. Liedberg, P. Konradsson (Linköping University, Sweden) More than 10% of the proteins in the body are involved in phosphorylations. As a phosphate group is attached to serine, threonine or tyrosine, the protein changes its conformation. This change in conformation is often ascribed to neutralization of charges, i.e. by electrostatic interactions. The attachment also effects the surrounding water structure and this change in water structure could be the reason to conformational changes. We describe here the preparation of a series of model surfaces, based on self-assembly on gold, in order to study the surrounding water structure. Surface characterization is performed using infrared reflection-absorption spectroscopy (IRAS), ellipsometry, contact angle goniometry and X-ray photoelectron spectroscopy. The molecules are found to form highly ordered monolayers and the thicknesses of the monolayers are in good agreement with expected values from space filling models. Water structure studies are performed using IRAS and temperature programmed desorption. |
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BI-WeP-2 Template Stripped Gold Surfaces for Advanced Biological Applications
M. Hasselblatt, F. Zaugg, P. Wagner (Zyomyx, Inc.) Template Stripped Gold (TSG) surfaces have been used extensively as a source for ultra-flat substrates. Thin Gold films evaporated on a freshly cleaved mica surface at elevated temperatures are glued onto a substrate to allow the removal of the mica. The gold films accessible this way exhibit sub-nanometer roughness over areas larger than micrometers. Here, we review methods for preparing these surfaces. Also, we present a novel bonding technique, in which Indium solder is used. Such sandwiches resist all common organic solvents and aqueous buffers typically used for biologically relevant experiments. |
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BI-WeP-3 Macroporous TiO2 Films Prepared on Ti Surfaces with Predefined, Micron-Sized Pores
F.A. Akin, L. Hanley (University of Illinois at Chicago) Surface morphology is critical to the biocompatibility of hard tissue implants, such as those used for dental and orthopedic applications. Porous bioceramics are commonly used for some applications due to their ability to encourage bone ingrowth, but they lack the physical strength required for load bearing applications. A new method is described for preparing thick, macroporous TiO2 films on Ti surfaces with pre-defined, micron-sized pores. Scanning electron microscopy shows that these pores 1) are controlled to a narrow size range by the synthetic process, 2) can be prepared from 0.5 to ~100 µm diameter, and 3) are interconnected within the film. The films are ~0.1 mm thick and strongly adherent to the Ti surface. X-ray photoelectron spectra indicate that these films can be prepared as elementally pure TiO2. X-ray diffraction indicates that the films are monophasic as either anatase or rutile TiO2. Scanning electron microscopy following immersion in Ringer's solution for several days shows that the films are not expected to undergo resorption following implantation. The effect of pore size is examined for fibroblast proliferation and spreading in vitro. These macroporous TiO2 films should allow applications that combine the favorable mechanical properties of Ti metal implants with the tissue engineering enhancements possible with porous bioceramics. |
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BI-WeP-4 Photovoltaic Characteristics of bR/p-Silicon Heterostructures using Surface Photovoltage Spectroscopy
L.S. Li, T. Xu, J. Jin, Y.J. Zhang, T.J. Li (Jilin University, P.R. China); B.S. Zou, J.P. Wang (Georgia Institute of Technology) In bR-based photoelectric devices, the highly efficient electric response can be obtained only when the bR molecules have a nonrandom orientation. LB technique enables molecular-order organization that can be used to incorporate bR into devices. As a prototype molecular electronic device, it will be more significant to deposit oriented bR films on a silicon substrate. In this paper, orientated bR films were deposited on the hydrophilic and hydrophobic silicon substrates using the LB technique. The cytoplasmic (CP) or extracelluar (CE) surface of bR face the silicon directly, giving oriented patterns of Si/CP-EC and Si/EC-CP, respectively. The photovoltaic features and interfacial charge separation of p-Si/bR/ITO heterostructure are studies by surface photovoltage spectroscopy (SPS). The different photovoltage response values obtained are due to the nonrandom orientation of bR in the LB films on the hydrophilic versus hydrophobic silicon substrates. The photovoltage response value versus external potential of the p-Si/CP-EC/ITO heterostructure shows an obviously rectifying behavior. Compared with the p-Si/ITO heterostructure, the response value of SPS increases more rapidly in the case of the positive external potential. |
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BI-WeP-5 Patterning Hybrid Surfaces of Proteins and Supported Lipid Bilayers
L.A. Kung, L.C. Kam, J.S. Hovis, S.G. Boxer (Stanford University) Two methods for patterning surfaces with supported lipid bilayers and immobilized protein are described. First, proteins are used to fabricate corrals for supported lipid bilayers. Poly(dimethylsiloxane) (PDMS) stamps are used to deposit arbitrarily-shaped patterns of thin layers of immobilized protein onto glass surfaces. This is followed by formation of supported lipid bilayers via vesicle fusion into the regions that are not coated with proteins. Second, supported bilayer membranes are blotted to remove patterned regions of the membrane,1 and the blotted regions are filled in (or caulked) with protein from solution. In both cases, the lipid bilayer regions exhibit lateral fluidity, but each region is confined or corralled by the protein. These two methods can be combined and used iteratively to create arrays with increasing lateral complexity in both the fixed protein and mobile supported membrane regions for biophysical studies or cell-based assays. |
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BI-WeP-6 Individually Addressable Solid Supported Membranes Formed by Micromolding in Capillaries
S. Kuenneke, A. Janshoff, H. Fuchs (WWU Muenster, Germany) The formation of spatially individually addressable, patterned biomaterial on surfaces is of paramount interest for the development of biosensors, combinatorial libraries, and high-throughput systems for pharma screening. Particularly, the combination of high resolution scanning devices with lithographically structured biomolecules is advantageous if the amount of biomaterial is limited or if the number of surface reactions is vast. The most versatile matrix for embedding and immobilizing natural and artificial receptor molecules such as functionalized lipids or proteins are solid supported membranes. Here we present a new type of microstructured membrane compartments, which are individually addressable by the operator on a common substrate on a nanometer to micrometer scale. The membrane segments are designed to be accessible to all available microscopic techniques and surface analysis tools. We developed a general procedure to generate patterned lipid bilayers by using a three dimensional network of capillaries as provided by microfluidic networks. The fluidic network (elastomer stamp) was formed from polydimethylsiloxane (PDMS) using an appropriate master displaying the inverted desired structure, which can be conveniently obtained by optical lithography of silicon wafers. Lipid bilayers were deposited by fusing unilamellar vesicles on the hydrophilic glass substrate. Visualization of the liposome flow in the capillaries and the formed planar bilayers was performed using a confocal laser scanning microscope. The planar bilayers were subsequently imaged by means of scanning force microscopy revealing a typical height of 4-6 nm. |
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BI-WeP-7 Vesicle -> Supported Bilayer Transformation Kinetics; Influence of Support Material, Vesicle Size and Temperature
E. Reimhult, F. Höök, B. Kasemo (Chalmers University of Technology, Sweden) Supported phospholipid bilayers (SPB) on a solid surface are biologically functional components of high current interest, e.g., for biosensors, tissue engineering, and basic science (Sackman, Science 271:43 (1996); Stelzle et al., J. Phys. Chem. 97:2974 (1993)). We have recently reported the kinetics of SPB formation from sonicated, unilamellar vesicles (SUV) of average size 25 nm, on a SiO2 support, using the quartz crystal microbalance - dissipation (QCM-D) technique (Keller and Kasemo, Biophysical Journal 75:1397 (1998); Keller et al., Phys. Rev. Lett. 84:5443 (2000)). Several interesting questions arose from the latter results; how does the vesicle -> bilayer transformation kinetics depend on the vesicle size, on temperature, and on the support. In the present study we are addressing these questions, whose answers are important for future sensor, biomaterial and micro-patterning applications. Already obtained results reveal a (vesicle) size-dependent kinetics, where also the end result (the final bilayer) may have different properties for different sizes of extruded unilamellar vesicles (EUV). The dependence on temperature is currently studied and will be reported. An exploratory study shows a strong temperature dependence for the vesicle -> bilayer transition. Different surfaces also cause different kinetics. So far, SiO2 surfaces have been the dominating support used to promote complete bilayer formation. Vesicle adsorption, but no bilayer formation, is observed for oxidized Ti and Au surfaces , while partial bilayer formation may occur on Pt. The above results constitute a platform from which more complex functional supported bio-membranes can be constructed (Höök et al, to be published). |
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BI-WeP-8 Use of Bacterial Adhesion Related and Collagen Related Peptides to Bind and Orient Fibronectin on Surfaces
U. Klueh, D.L. Kreutzer, J.D. Bryers (University of Connecticut, Schools of Medicine and Dentistry) Although small molecular weight proteins and peptides have been bound and oriented on surfaces, little is known about orienting large molecular weight proteins, (e.g. FN) on surfaces. Recently two classes of peptides have been shown to bind to FN in vitro, i.e. Collagen Related Peptides (CRP) and Bacterial Adhesion Related Peptides (BARP). We hypothesized that if these peptides could be used to not only bind, but also orient FN on surfaces. We further hypothesized that antibodies to specific regions of the FN molecule can be used to demonstrate the orientation of the peptide bound FN. CRP and BARP peptides were synthesized commercially and immobilized on polystyrene surfaces. FN was nonspecifically bound to polystyrene by physisorbtion. FN binding to the immobilized peptides was quantitated using monomeric/functional 125I-FN and polyclonal antibodies to FN. Orientation of the bound FN was demonstrated using antibodies specific to the amino (anti-N) and carboxyl (anti-C) termini of FN. Polystyrene immobilized CRP and BARP bound 125 ng/cm2 and 94 ng/cm2 of FN. Little FN bound to control (non-peptide containing) surfaces 5 ng/cm2. FN bound to CRP and BAR peptide bound anti-FN and anti-C antibodies but did not bind significant levels of anti-N antibodies, compared to randomly bound FN (i.e. physisorbed FN). Additionally, we demonstrated that although CRP did inhibit FN binding to immobilized collagen, BRAP did not. Finally we demonstrated that the uses of monomeric/functional FN was critical in establishing FN monolayers on CRP or BARP coated surfaces. Our results not only demonstrate the ability of CRP and BAR peptide to specifically bind and orient fibronectin in monolayers, but also underscore the usefulness of specific polyclonal and monoclonal antibodies to characterize the binding and orientation of FN on these surfaces. |
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BI-WeP-9 Conformational Changes of the Extracellular-matrix Protein Fibronectin Induced by Force Spectroscopy
Y. Oberdoerfer, H. Fuchs, A. Janshoff (WWU Muenster, Germany) Since its invention, force spectroscopy by SFM became a powerful instrument to study the structure, mechanism and behaviour of polymers. Especially for biopolymers it is important to be able to perform these studies in a native environment, an advantage which is provided by SFM. In this work we studied conformational changes of the extracellular-matrix protein fibronectin and provide direct proof for the presence of the protein on the cantilever verifying whether it was pulled on the polymer or not. Fibronectin is a modular protein consisting of three different FN-domains: FN I, FN II and FN III. These three domains differ in the number of their appearance in one single fibronectin polymer and also in the number of amino-acids. Extending fibronectin during a force-measurement results in unfolding events of single domains can be distinguish in a force-curve due to the elongation of the polymer itself and also the absolute number of unfolded domains. In this way it is possible to determine whether a FN I-, FN II- or FN III-domain was unfolded. Another topic that should be presented is the possibility to make an elemental mapping of a cantilever surface by means of SIMS and SNMS to determine if the investigated polymer was attached to the probe. With this kind of measurement one can verify if unfolding events occurring in a force-curve arise from the substance itself or any kind of contamination. |
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BI-WeP-10 Observation of Bone Fracture Healing Processes by Atomic Force Microscopy
V. Baranauskas, I.G. Freitas, Z. Jingguo, M.A. Cruz-Hofling (Universidade Estadual de Campinas, Brazil) Atomic Force Microscopy (AFM) was used to study the healing process of bone surgical fracture in rats. We used young male adult rats (Wistar), with corporal masses between 250 and 300 grams. Each fracture was provoked by drilling a 2 mm diameter hole in one cortical tibia surface. The healing course was monitored at 8, 15 and 22 days after the fracturing. AFM images, at different magnifications, allowed to the identification of the time dependence of the osteoblastic activity, measured by the increase in the primary bone trabeculae surface and the increase in the synthesis and organization of collagen fibers of the bone matrix. Characterization of the natural recovery of the damaged bone tissue by AFM is potentially of great importance because it allows the comparison of natural recovery and the recovery induced by medicines or other cures.1
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BI-WeP-11 Dielectric Characterisation of Aminal Bone
S. Mohiuddin (King Saud University, Saudi Arabia) The investigation deals with the electrical properties of bovine bones. Dielectric constant, dielectric loss, conductivity and resistivity are determined in bovine tissues are lossy dielectric. They are partly polarisable and partly conductive. The influence of calcium phosphate on the electrical behavior of bone is also studied. Data of electrical parameters of bovine scapula, rib and femur bones reveal the considerable variation in different bones samples and also in different specimens of the same bone, obtained from various parts of the bone. This may be attributed to the inhomogeneous deposition of calcium phosphate and water content of the bones. The sharp fractional changes in dielectric constant and resistivity with the water content of bone specimens suggests that the electrical parameters are very sensitive to free water present in bones in contrast to ultrasonic and mechanical properties. Influence of water on electrical behavior is specific to bone because of the fact that mineral content of the bone is found to be in different proportions which also effects the electrical make up of the bone. The three parameters namely water content, mineral content (calcium phosphate) and orientation of the collagen fibers with respect to the applied electric field, play an important role in influencing the electrical parameters conductivity of the bone tissue, when measured at the bulk, level. |
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BI-WeP-12 TOF-SIMS Characterization of Nucleic Acid Biosensors
H.F. Arlinghaus, M. Ostrop, O. Friedrichs, U. Gunst (Physikalisches Institut der Universität Münster, Germany) In recent years, biosensors consisting of immobilized oligodeoxynucleotides (ODN) have been a subject of growing interest for DNA sequencing and clinical diagnostics. We have used static TOF-SIMS and temperature-programmed SIMS (TP-SIMS) to examine in detail the immobilization process of ODNs, which were directly bound to Au- and Ag-surfaces by thyol-linkers. Protonated (M+H)+ and deprotonated (M-H)- signals of the different ODNs and bases as well as phosphate ions were used to monitor the ODN concentration. The influence of ODN concentration and immobilization time on the immobilization process was investigated. It was found that the maximum intensity for characteristic ODN peaks was obtained using a 1 µM solution and an immobilization time of 24 h. According to our estimate, the surface coverage under these conditions should be close to a monolayer. Measurements of how surface structure affects the process of immobilization showed a higher intensity of the characteristic ODN signals and a less homogeneous oligonucleotide layer with increasing surface roughness. TP-SIMS was used to measure the thermal stability of the immobilized layers. The data show that the characteristic ODN fragment ions start to decrease at a temperature of about 150° C, with differences in the point of onset for the different bases. It can be concluded that the combination of TOF-SIMS and TP-SIMS is a powerful technique to examine the complexity of the immobilization and hybridization processes of nucleic acid. |
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BI-WeP-13 Characterization of Multi-Component Adsorbed Protein Films by ToF-SIMS
M.S. Wagner, D.G. Castner (University of Washington) Characterization of the adsorbed protein films on biomaterial surfaces is needed for the rational design of biomaterial surfaces. Many surface analysis techniques, however, do not provide efficient means for analysis of these surfaces. Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is an excellent technique for the analysis of complex protein films due to its chemical specificity and surface sensitivity. The ToF-SIMS fragmentation pattern is highly complex due to the heterogeneity of the protein composition and the absence of unique, identifying peaks from protein to protein. Analysis of such complex spectra requires the use of multivariate analysis methods to effectively use the ToF-SIMS data. Therefore, we have utilized ToF-SIMS in conjunction with Partial Least Squares (PLS) regression to estimate the surface compositions of binary and ternary adsorbed protein films. Using only the pure component ToF-SIMS spectra, PLS was able to estimate the relative concentrations of the proteins on the surface. Furthermore, using a set of standard protein spectra, Principal Components Analysis (PCA) was able to provide insight into how the composition of a protein film adsorbed from 1% bovine plasma varied with adsorption time. PCA of the ToF-SIMS spectra revealed a shift from mostly fibrinogen to mostly immunoglobulins over the course of two hours. ToF-SIMS/PCA has the tremendous advantage that several proteins can be analyzed in a single experiment, given the appropriate standard spectra. The combination of multivariate analysis methods and ToF-SIMS greatly simplifies the analysis of adsorbed protein films. |
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BI-WeP-14 Correlation of Cell Health with Protein Layer Thickness on Modified Surfaces Characterized by XPS, XAS, and Morphological Analysis
H.E. Canavan (The George Washington University); W.E. O'Grady (Naval Research Laboratory); J.J. Hickman, D.E. Ramaker (The George Washington University) The interactions of biomolecules with surfaces are of significant interest in the areas of biocorrosion, bio-implant rejection, and biological fluid interactions with MEMS devices. This interaction may dictate the health of nearby cells by either a) continuing cell function as normal, b) continuing cell function in an abnormal manner, or c) cell death. In the work presented here, the biomolecular interaction is altered via prior surface modification with Self-Assembled Monolayers (SAMs) of amines or fluorinated compounds. The surfaces are then introduced into cell culture using cardiac and other cells. The interaction of the derivatized surfaces with proteins and cells are investigated to see a differential response to the modifications. X-ray Photoelectron Spectroscopy (XPS) is used as an analytical technique to characterize the modified surfaces of the silanes prior to cell culture. XPS is also used characterize the protein deposition layer thickness which serves as an indication of cell function and health. Optical microscope images of cells grown on different substrates are compared to time-dependent XPS results to correlate cell morphology with protein layer thickness. Sulfur K-Edge X-Ray Absorption Spectroscopy (XAS) data are used to monitor the extent of S-C, S-O, and S-H bonds, which affect the character of the extruded proteins. |
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BI-WeP-15 Soft X-ray Spectromicroscopy of Protein Adsorption on Polyurethanes
C. Morin, A.P. Hitchcock, I.N. Koprinarov, R. Cornelius, J.L. Brash (McMaster University, Canada) New quantitative techniques for chemical microanalysis which allow detailed study of protein polymer interactions are required for biomaterial interface optimization. We are particularly interested in identification of possible preferences of first sites of protein attachment to polyurethane polymers used in blood contact medical applications. We are exploring Scanning Transmission X-Ray Microscopy (STXM) and Photoemission Electron Microscopy (PEEM) in this context. These techniques use near edge X-ray absorption spectroscopy (NEXAFS) for chemical identification. Both techniques have been used to map albumin and fibrinogen adsorbed on various polymer surfaces. As an example, STXM was used to study protein adsorption from a 0.1 mg/ml albumin solution onto a TDI-based, high-ether polyurethane film (~100 nm thick) which had submicron phase segregated regions of a highly aromatic polyisocyanate polyaddition product (PIPA) reinforcement material. Image sequences recorded throughout the C 1s and N 1s regions were used to generate composition maps by fitting the spectrum at each pixel to spectra of pure reference materials. The strong amide carbonyl resonance at 288.2 eV provides a sufficiently strong signature of protein to allow mapping down to monolayer levels even though the STXM results average over the full thickness of the polymer and protein sample. PEEM studies on similar materials provide greater surface sensitivity but are complicated by high sensitivity to topography as well as charging artefacts. Results from the two techniques will be compared to illustrate the strengths and weakness of these soft X-ray spectromicroscopy techniques when applied to biomaterials problems. X-ray microscopy is carried out at the Advanced Light Source (supported by DoE under contract DE-AC03-76SF00098), supported financially by NSERC (Canada). |
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BI-WeP-16 A Novel Approach to Studying Structures and Orientation at the Protein-Self Assembled Monolayer Interface
L.F. Pardo, T. Boland (Clemson University) Self-assembled monolayers (SAMs) have become an important tool in protein adsorption studies, partly because they represent chemically, well-defined model systems. However, specifics on how the structure and orientation of both the SAM and protein change during adsorption in situ remain unknown. The purpose of this study is to quantify these changes via analysis of protein adsorption onto SAMs by a novel technique using Evanescence Reflection Spectroscopy (ERS). This technique allows in situ characterization of the surface chemistry, providing quantitative information on structure and orientation at the interface only. In this study, model proteins, (polyserine and fibrinogen) were adsorbed onto, -OH, -COOH, and CH3 terminated SAMs of hexadecanethiols on gold. The surfaces were then characterized by FTIR and ellipsomentry. The IR measurements reveal significant differences were between protein structures in dry and wet states. The spectra of polyserine in the dry state show only amide II stretch (1507 cm-1) while the amide I stretch is absent implying that the polyserine lies flat on the SAM surface. Analysis of the IR spectra measured in PBS solution depicting peaks in the Amide I and II regions showed that polyserine extends away from the surface under aqueous conditions. Furthermore, information on the secondary structure of the solvated proteins adsorbed to the various SAMs was attained. The resulting of protein-SAM interfaces are dynamic and will undergo structural changes, desorption or surface reactions. The structural characterization of protein SAM interactions will be helpful when designing templates for tissue engineering applications. |
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BI-WeP-17 Microbial Adhesion on Polymer: Role of Morphological and Chemical Properties in the Micro-organism Behaviour
M. Anderle, R. Canteri, E. Carli, S. Janikowska, A. Lui, C. Pederzolli, G. Speranza (ITC-irst, Italy); D. Maniglio, C. Della Volpe (Università di Trento, Italy) Infections caused by implanted polymeric devices (especially catheters) have an increasing importance in the medical routine (up to 40% of nosocomial infections). The critical event in the pathogenesis of foreign body infection is the adhesion of the micro-organisms to the biomaterial surfaces followed to the colonisation. In order to achieve a deeper understanding of the molecular-level interactions between catheters and biological system, the aim of this work is to study the physicochemical properties of the polymeric surface and their influence on the microbial adhesion and colonisation. Some common polymers produced by standard processing methods have been analysed by dynamic contact angle (DCA), X-ray Photoelectron Spectroscopy (XPS), Time of Flight Sims (TOF Sims) and Scanning electron microscope (SEM). Moreover biological tests were performed to determine the degree of gram+ and gram- bacterial adhesion on these surfaces. The results show relevant deviation of the contact angles from the expected values. These results are only partially explained by the XPS and the TOF Sims analysis. XPS and TOF Sims spectra revealed normally contaminated polymer surfaces and deviations from the nominal composition also after accurate cleaning, performed using different methods. In particular oxidation of the polymer surface occurring probably during the moulding process and other factors are able to introduce chemical functions which lead to a surface chemistry significantly different from the expected one. High value of contact angle and unexpected values of bacteria adhesion can be explained taking into account the presence of basic functions and the roughness of the surface. These elements decrease the differences expected on the basis of the acid, basic or dispersive characters of the examined polymers. |
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BI-WeP-18 Functionalised Plasma Polymers for Control of Cell Attachment
J.M. Kelly, R. Daw, R.D. Short (University of Sheffield, UK) Surface chemistry is known to be an important factor in mediating cell attachment and subsequent activity on materials whether in the context of an in vitro culture system, implanted biomaterial or tissue engineered construct. In our studies on alcohol and carbonyl containing surfaces, cell attachment increased almost linearly with functional group concentration (up to 20-30%) whilst on carboxyl containing surfaces a low threshold concentration (approximately 5%) promoted excellent levels of cell attachment above which further increase of carboxyl concentration does not appear to promote greater cell attachment. Using a binary mixture of functional monomer (acrylic acid, methyl vinyl ketone or allyl alcohol) with a diluent hydrocarbon monomer (octa-1,7-diene), thin layers of polymer have been produced by radio frequency (RF) plasma deposition. Polymerisation took place at a pressure of 4x10-2 mbar with the plasma supported by a 13.56 MHz, 2 W continuous wave power supply. X-Ray Photoelectron spectroscopy has shown the surfaces to contain hydrocarbon as well as hydroxyl, carbonyl and carboxyl functionality and that increasing the amount of functional monomer used in the plasma led to an increase of the corresponding functionality in the deposit. A rat osteosarcoma cell line (ROS 17/2.8) and human bone marrow cells were used for cell attachment studies. Cells were seeded at 3x104 cells/cm2 and incubated for 3 h and 24 h in serum containing media. Cell attachment was quantified by direct counting after staining with methylene blue. Cell attachment studies are being carried out in parallel on self assembled monolayers containing similar ranges of functionality to allow comparison of plasma polymerised deposits with model surfaces. |
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BI-WeP-19 Study of Protein Adsorption on Hydrophilic and Hydrophobic Polysiloxane Surfaces Modified by O2 Plasma Technique
C. Satriano (University of Catania, Italy); F. Höök (Chalmers University of Technology and Göteborg University, Sweden); G. Marletta (University of Catania, Italy); B. Kasemo (Chalmers University of Technology, Sweden) Thin films of a poly(hydroxymethyl)siloxane have been chemically modified by using the O2 plasma technique at increasing treatment times ranging from 15 seconds to 10 minutes, with the applied power of 100 Watts and a residual gas pressure of 0.25 torr. The plasma-induced surface chemical modifications were investigated in situ by means of X-Ray Photoelectron Spectroscopy (XPS) for both the in situ samples and the samples aged in air and/or in water. By means of Static Contact Angle measurements the wettability properties of the unmodified and the O2 plasma-exposed surfaces were investigated. The XPS results show that the compositional modification involves a dramatic decrease in the carbon content and the formation of [SiO4] clusters, indicating a successive transition with treatment time from the original [SiO3C] structure to [SiOxCy] phases. The contact angle measurements indicate that the plasma treatment changes the initial hydrophobic surface (θ ~ 90°) of the polymer into a completely wettable surface, with θ = 0-10° contact angle. After aging in water the surface is still completely wettable, while aging in ambient atmosphere produces a mild recovery in the contact angle values. These surfaces are currently subject to measurements of protein adsorption and of vesicle to supported membrane transformations, using QCM-D and other techniques. The results of these measurements and how they correlate with the XPS and wetting angle measurements will be presented. |
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BI-WeP-20 Colloidal Lithographic Methods for Cell Culture Experiments
A.S. Andersson, D.S. Sutherland, P. Hanarp, B. Kasemo (Chalmers University of Technology, Sweden) The interaction of cells with surfaces can be modulated by the surface topography and chemistry on the micrometer and submicron length scale. Relatively recent evidence has shown that topography and chemistry on the nanometre scale can influence the funct ional behaviour of both protein molecules and selected cells. It is likely that the structural properties of a surface from the micron down to the nanometre and molecular scale are able to influence cellular behaviour, either directly or via an adsorbed hydrated protein layer. In order to systematically study the influence of surface properties on cellular behaviour methods to fabricate surfaces with defined and varied chemical and topographic architectures on a range of different length scales are requi red. A prime requirement of fabrication for cell culture experiments is that (by the standards of nanofabrication) extremely large areas of surface can be quickly fabricated. Colloidal lithographic methods have been developed to systematically fabricate nanometer features with defined size, shape and distribution over large areas in a single fabrication process. These methods utilise individual colloidal particles as a mask material for lithographic processing and have been used to create surfaces with a single type of topographic or chemical feature of defined size (available size in the range 10-200nm in all spatial dimensions). In combination with traditional photolithographic methods micrometer sized strips have been patterned with the nanometre-sized features to create surfaces with hierarchical chemical and topographic structures. These surfaces have been used in range of exploratory cell culture experiments. |
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BI-WeP-21 Endothelial Cell Organization on Micropatterned Protein Surfaces
R. Daw, T.N. Wight (University of Washington); R.B. Vernon (Hope Heart Institute); P.S. Stayton (University of Washington) We have employed microcontact printing to investigate how spatial control of adhesive domains can direct the development of endothelial cell tubes for applications in tissue engineering and array-based sensors. Previous studies by Dike and co-workers showed that controlling adhesive geometries can dramatically affect endothelial cell fate and tube formation.1 Initial studies were directed toward comparing bovine aortic endothelial cell adhesion and activation on 5, 15 and 30 µm lanes of fibronectin (FN) versus laminin (LM) in the presence of 1 ng/ml of VEGF. Cells on LM tracks were able to migrate into intervening spaces of 20 µmm after 24 h. When the spaces between the lanes were increased from 20 to 80 µm cells remained adherent to the LM tracks except for those on the 5 µm tracks. Here, cells could spread between adjacent lanes. Endothelial cells were adherent to FN lanes throughout the range of pattern dimensions. A higher concentration of VEGF (10 ng/ml) stimulated migration off the patterned FN lines. FN lanes of 5, 15 or 30 µm were selected for subsequent studies directed toward defining the dimensionality of endothelial cell organisation into tubes. TEM showed that cells on tracks of 5 µm exhibited a significant arc of curvature and single endothelial cells encircled an organised fibrillar material to form tubes. Single tubes were also observed on 15 µm tracks but at this lane width, 2-3 cells organised together and circled a larger central fibrillar tube. These studies suggest that the composition of matrix proteins may play an important role in controlling endothelial cell development in confined geometries and that the organisation of endothelial cells into tube structures can be readily manipulated by controlling adhesive pattern dimensions. |