Rapid isothermal substrate microfabrication of a biocompatible thermoplastic elastomer for cellular contact guidance
Introduction
The production of living tissue engineered substitutes [1] requires adequate cell proliferation. Until now the most widely used cell culture surface has been flat polystyrene (PS). In order to mimic the organization of physiological tissue in tissue engineering cells have to grow in an oriented manner. This can be achieved via contact guidance, a principle by which cells align following physical cues, such as the silk fibers of spider webs [2] or surface topography, as used more commonly nowadays [3], [4], [5]. Tissue functionality is intimately related to tissue orientation, with such spatial three-dimensional organization giving the cornea its strength and transparence [6], [7], [8], tendons their mechanical properties [9], [10] and smooth muscle cell tissues from different organs their elasticity and compliance [11], [12]. For many tissue engineering and cell culture applications contact guidance provides an important stimulus to the cells and consequently dictates their physiological orientation [13], [14], [15].
The use of poly(dimethylsiloxane) (PDMS) in microfabrication has frequently been explored [16], [17], as its optical, mechanical and biocompatible properties allow imaging, stretching and, to some extent, cell culture. Unfortunately, PDMS has a long processing time and limited biocompatibility. In order to promote cell adhesion for tissue engineering purposes it is necessary to use exogenous protein, like collagen or fibronectin, which then create regulatory obstacles for clinical applications [18]. Hard thermoplastic polymers like polystyrene (PS) [19] and poly(methyl methacrylate) (PMMA) [20] have also been used as contact guidance substrates. Usually produced by hot embossing using silicon master molds fabricated by standard lithographic techniques and reactive ion etching (RIE), hard thermoplastic substrates are less time consuming to produce compared with PDMS elastomer substrates. However, thermoforming of such PS and PMMA materials, among others, requires solving some specific issues related to mold fabrication and demolding due to the overall thermal and mechanical constraint upon the molding process, thus major attention is needed to establish the stability and integrity of the master mold over embossing runs [21], [22]. The use of metallic molds for hard plastics can be considered, but their production is cost-effective only for production runs and requires fabrication processes and equipments that are not commonly found in laboratory set-ups, especially in tissue engineering and biology laboratories. Thus there is a need for new materials and fabrication techniques that could provide rapid and low cost microstructuration, while providing biocompatibility and long-term viability. In this study the possibility of using a robust and high throughput thermoforming method in order to microstructure a low cost thermoplastic elastomer (TPE) has been demonstrated. By using a low pressure (1.6 bars) and rapid isothermal (3 min) hot embossing approach a large number of styrene–(ethylene/butylene)–styrene (SEBS) substrates have been fabricated. Using a low cost and highly stable SU-8 master mold prepared by standard lithography we have replicated more than 50 substrates of 6 inch2 area having gratings of 8 μm period and 1.5 μm depth.
Prior to use the SEBS substrates were treated with oxygen plasma and sterilized with ethylene oxide gas. By using SEBS substrates without any exogenous protein coating we were able to grow smooth muscle cells (SMC) and show cell proliferation comparable with commercially available tissue culture PS. Contact guidance, providing cellular orientation, was evaluated and more than 95% of cells were within a 10° angle shift from the longitudinal axis of the substrate grating. Tissue sheets were also produced using SMC cultured on microstructured SEBS substrates and cellular alignment persisted throughout the experiment.
Section snippets
Substrate fabrication
A Versaflex TPE SEBS block co-polymer was purchased from GLS Corp. (McHenry, IL). Versaflex was used in the form of sheets (3.0 mm thickness, 160 mm width and >10 m length) prepared by extrusion using a Killion KL100 single screw extruder (Killion Laboratories Inc., Houston, TX).
The master mold for TPE thermoforming was fabricated by standard SU-8 photolithography. SU-8 GM1040 photo-resist (Gersteltec, Pully, Switzerland) was spin-coated on a 6 inch silicon wafer (Silicon Quest International Inc.,
SEBS substrate microfabrication and surface characterization
As SEBS is a thermoplastic elastomer it can be extruded in sheets of different thicknesses and rolled onto a mandrel for later use (Fig. 1). This process makes SEBS block co-polymers more convenient to use than PDMS, which requires a pre-compounding step and degassing for each replication cycle. Indeed, extruded sheets can be stored over long periods of time (at least 2 years) without any noticeable degradation, making use of the material possible on demand without any additional preparation
Discussion
Microstructured substrates used for contact guidance in cell culture and tissue engineering need to be easily processed via microfabrication methods, in order to facilitate mass production, as well as providing good surface biocompatibility to promote cell adhesion. Many microstructured biodegradable polymers display such important characteristics [26], [27], but for some applications, like fundamental studies in cell biology [28] and sheet-based tissue engineering [29], [30], [31], a
Conclusions
In this paper we have shown that SEBS block co-polymers are a suitable alternative to the use of hard polymers like PS and elastomer polymers like PDMS for tissue engineering. Unlike PDMS, TPE can be used in the form of extruded sheets that provide off the shelf availability without the necessity of performing any pre-compounding step. The demonstrated microfabrication capabilities for SEBS substrates seem to be an appealing alternative to standard hot embossing techniques. With our isothermal
Acknowledgements
The authors would like to acknowledge Robert Gauvin for help with manuscript preparation, and Cindy Perron and Helene Roberge for technical assistance. This work was supported jointly by the National Research Council of Canada and the Canadian Institute for Health and Research.
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Present address: Harvard–MIT Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.