Polymer Preprints 2001, 42(1), 147

BIOADHESION STUDIES ON MICROTEXTURED SILOXANE ELASTOMERS

Wade R. Wilkerson1, Charles A. Seegert1, Adam W. Feinberg1, Lee C. Zhao1,

James A. Callow2, Maureen E. Callow2, Anthony B. Brennan1

1 Department of Materials Science & Engineering, University of Florida,

Gainesville, Florida 32611 2 University of Birmingham, School of Biosciences, Birmingham, B15 2TT,

W Midlands, England INTRODUCTION

There is significant need for surfaces that control cellular response and adhesion. Studies are in progress that investigate the affect of surface topography, surface chemistry, and bulk properties on the micro- and nano-scale to better understand cellular adhesion to different substrata. Biofouling is an example of a problem concerning cellular materials accumulating on surfaces such as the hulls of ships and water treatment facilities. The marine spore Enteromorpha is the most common macroalga that fouls ships and submarines. Reproduction is mainly through motile spores that swim until a suitable surface on which to settle and adhere is located.[1] Adhesion involves secretion of a glycoprotein adhesive that anchors the spore to the surface.[2] Cues for settlement include phototaxis, chemotaxis and thigmotaxis. Spore settlement in relation to surface topography has been examined. Previous anti-fouling coatings included biocides that did significant damage to marine life in harbors. Current research focuses on preventing adhesion forces able to withstand the shear forces during motion.[3,4] Many of these same principles can be used in biomedical applications to improve the biocompatibility of polymeric surfaces in the body.

One field in which surface topography already plays a role is vascular grafts. The luminal surface of conventional Dacron vascular grafts can be considered textured in a random roughness pattern. Studies in sheep have shown a distinct difference in the amount of cellular deposition on non-textured and textured surfaces after 1 week in vitro.[5]

Many studies over the past 10 years have shown significant control over cellular behavior based on surface characteristics. Recently, photolithography has allowed for highly uniform surface patterns in the form of grooves, pits, and pillars. Polymer films are cast on silicon wafers that have been etched with the surface geometry desired. PDMS based elastomers have been widely studied due to their ease of casting and biocompatibility. Polystyrene, PMMA, and PLA have also been used among others.[6-8]

Methods of producing the precise surface morphologies vary depending on the size of the pattern and the material on which the pattern is being replicated. The most common method used in these experiments for producing a pattern in a siloxane elastomer is micro-machining technology. First the pattern is produced on a silicon wafer with UV photolithography and then etched using reactive ion etching (RIE).

The grooves formed on these substrates have shown significant control over growth directions of cells. This phenomenon, commonly referred to as “contact guidance,” typically demonstrates cellular alignment along the grooves depending on the dimensions of the features. Current discussion focuses on the mechanisms behind the alignment of these cells to the surface topography. An area of concern is whether or not the actual geometries of the features are the defining factor, or the fact that there is a change in surface free energy due to edges and disruptions in the planar surface.[9] It has been demonstrated that parameters such as surface free energy and wettability influence cell growth, but not necessarily the shape or orientation of cells.[6] Typically, the more wettible the surface is, the more cell proliferation occurs. The wettability of a surface can be affected externally with a radio frequency glow discharge (RFGD) plasma treatment for example, and internally with the incorporation of chemical features in the form of trimethyl-terminated polydimethylsiloxane oils.

With the addition of silicone oils and surface texture, a hierarchical substrate system is examined to determine its effects on biological adhesion. EXPERIMENTAL Substrate production and characterization:

Textured surfaces were patterned using UV photolithography and a mask with four separate types of patterns shown below in Figure 1. All squares in the mask are divided into three sections, each with varying widths

of separation between features and size of features. The features include ridges and valleys and individual squares or “pillars.”

Figure 1 - Diagram of photomask pattern

Masked samples were developed and then etched by Unaxis USA, Inc.

using a RIE process to produce precise features and aspect ratios. Three depths of etching were examined: 5µ, 3µ, and 1µ, as well as flat surfaces with no features.

A negative of the silicon wafer master was made by solvent casting polystyrene in chloroform in a 1:6 g/mL ratio. The polystyrene copy was fixed to a glass surface for silicone replication.

The siloxane resin (Dow-Corning Silastic T-2) was mixed with its curing agent in a 10:1 g/g ratio as specified by the manufacturer. It was then degassed under vacuum for 20 minutes and cast on the polystyrene copies of the silicon wafer. A coverslip was placed on top of the elastomer and features. The samples were then cured for 24 hours before removing from the mold. The addition of the trimethyl-terminated PDMS oils to the resin at varying concentrations will be examined to determine the adhesion characteristics of chemically modified substrates.

Samples of the cured elastomer were characterized by a variety of methods. Mechanical properties were determined with DMS and tensile testing. Surface energy is measured by dynamic contact angle analysis and fidelity of replicated features will be examined by atomic force microscopy and SEM. Enteromorpha Studies:

Textured and non-textured siloxane elastomer samples were sent to Dr. Maureen Callow, University of Birmingham, UK, for spore accumulation studies. The samples were soaked in sterile seawater overnight and then cultured with Enteromorpha spores at ~2 x 106 spores/cc density for 1 hour. The surfaces were then rinsed, fixed, and examined using a Zeiss imaging system attached to a fluorescent microscope to determine the spore density on each surface. Endothelial Cell Culture:

The elastomer samples were treated with argon RFGD plasma to improve wettability and sterilized with ethylene oxide. Each pattern was separated and cultured in a dish of porcine endothelial cells. After 3 days, the samples were removed, fixed, and examined under light microscopy. RESULTS AND DISCUSSION

A SEM of a silicon wafer with 10 µm spacing is illustrated in Figure 2. Examination by SEM and AFM demonstrates good fidelity of features upon transfer to the polystyrene mold as well as the siloxane elastomer.

Polymer Preprints 2001, 42(1), 148

Figure 2 - SEM of silicon wafer with 5 µm wide ridges and 10 µm wide

valleys

The textured surfaces showed a significant increase in spore

accumulation compared to non-textured surfaces as seen in Figure 3. The flat elastomer surface provided the least favorable substrate in terms of spore adhesion, and the 5 µm width between features had the greatest spore accumulation. This increase in spore density on smaller widths also corresponds to the highest density of microfeatures, which may also be related to the length of the groove floor or sidewall. The spores appeared to congregate inside the grooves between the ridges and not as much on the ridges themselves.

Figure 3 - Spore attachment data for Enteromorpha on patterned siloxane elastomer

Preliminary endothelial cell (EC) studies show a decrease in confluence

on textured surfaces, and data on contact guidance will be presented. Figure 4 is a light microscope image of stained EC’s on textured siloxane substrate. The cells form a confluent layer in the smooth region, but are less dense on the ridges. Similar results are seen with pillar features on the substrates.

Figure 4 - Stained porcine vascular endothelial cells on textured siloxane

substrate.

CONCLUSIONS The overall goal of this project is to develop a deeper understanding of cellular response to the features of a siloxane elastomer. These features include topography, surface chemistry, and bulk properties. The surface chemistry and bulk properties of the elastomer are altered by the addition of non-functionalized PDMS oils. The topographical features are added using micro-machining technology to produce highly controlled and repeatable surfaces. Two main biological systems were studied: marine Enteromorpha spores and endothelial cells. The reaction of the marine spores to the microtextured surfaces seems to enhance density of adhesion as the space between features decreases and number of total features increases. The endothelial cells appear to prefer the smooth siloxane surface and the confluence of cell coverage is decreased when micro-topography is introduced. The complexity of biological systems requires a better fundamental understanding of the substrate/cell interface in order to engineer biomaterials for specific applications. REFERENCES 1. Callow, M.E., et al. Journal of Phycology. 1997, 33(6), 938-947 2. Stanley, M.S., M.E. Callow, and J.A. Callow. Planta. 1999, 210(1), 61-

71 3. Schultz, M.P., C.J. Kavanagh, and G.W. Swain. Biofouling. 1999, 13(4),

323-335 4. Swain, G.W., W.G. Nelson, and S. Preedeekanit. Biofouling. 1998, 12(1-

3), 257-269 5. Fujisawa, N., et al. Biomaterials. 1999, 20, 955-962 6. den Braber, E.T., et al. J. of Biomedical Materials Research. 1995, 29,

511-518 7. Matsuzaka, K., et al. Biomaterials. 1999, 20, 1293-1301 8. Walboomers, X.F., et al. J. Biomed Mater Res. 1999, 47, 204-212 9. von Recum, A.F. and T.G. Van Kooten. J. Biomater. Sci. Polymer Edn.

1995, 7(2), 181-198 ACKNOWLEDGEMENTS Special thanks are given to the research groups of Dr. C. Keith Ozaki and Dr. Edward R. Block, Malcom Randall VAMC, for providing and assisting with the porcine endothelial cells. This work was funded by a grant from the Office of Naval Research.