Rapid isothermal substrate microfabrication of a biocompatible thermoplastic elastomer for cellular contact guidance Maxime D. Guillemette a,b,c,1 , Emmanuel Roy a,⇑ , François A. Auger b,c , Teodor Veres a a Industrial Materials Institute, National Research Council, Boucherville, QC, Canada J4B 6Y4 b Department of Surgery, Faculty of Medicine, Laval University, Quebec City, QC, Canada G1V 0A6 c LOEX, Centre de Recherche FRSQ, CHA Universitaire de Quebec, Quebec City, QC, Canada G1S 4L8 article info Article history: Received 18 August 2010 Received in revised form 26 January 2011 Accepted 9 February 2011 Available online 15 February 2011 Keywords: Cell culture Cell proliferation Microstructure Elastomer Smooth muscle cell abstract The use of microstructured substrates to study and influence cell orientation, which plays an important role in tissue functionality, has been of great interest lately. Silicon and poly(dimethylsiloxane) substrates have typically been used, but long processing times and exogenous protein surface coating, required to enhance cell viability, limit their use as large-scale platforms. There is thus a need for a non-biodegrad- able biocompatible substrate that allows rapid and low cost microfabrication. In this paper a styrene– (ethylene/butylene)–styrene block co-polymer (SEBS) microstructured by a rapid replication technique using low pressure an isothermal hot embossing approach has been demonstrated. SEBS substrates were treated with oxygen plasma to enhance cell adhesion and sterilized using ethylene oxide gas. While cell adhesion to and proliferation on these substrates was as good as on tissue culture polystyrene, cellular alignment on microstructured SEBS was also very high (97.7 ± 0.5%) when calculated within a 10° angle variation from the longitudinal axis. Furthermore, tissue sheets on microstructured SEBS have been pro- duced and exhibited cellular alignment within the engineered tissue. In addition, these results were obtained without coating the material with exogenous proteins. Such substrates should be helpful in the culture of tissue engineered substitutes with an intrinsic orientation and to elucidate questions in cell biology. Ó 2011 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. 1. Introduction The production of living tissue engineered substitutes [1] re- quires adequate cell proliferation. Until now the most widely used cell culture surface has been flat polystyrene (PS). In order to mi- mic 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 [3–5]. Tissue func- tionality is intimately related to tissue orientation, with such spa- tial three-dimensional organization giving the cornea its strength and transparence [6–8], 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 impor- tant stimulus to the cells and consequently dictates their physio- logical orientation [13–15]. 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 process- ing 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 thermo- plastic polymers like polystyrene (PS) [19] and poly(methyl meth- acrylate) (PMMA) [20] have also been used as contact guidance substrates. Usually produced by hot embossing using silicon mas- ter molds fabricated by standard lithographic techniques and reac- tive 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 fabri- cation 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 1742-7061/$ - see front matter Ó 2011 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. doi:10.1016/j.actbio.2011.02.019 ⇑ Corresponding author. Tel.: +1 450 641 5394; fax: +1 450 641 5105. E-mail addresses: emmanuel.roy@imi.cnrc-nrc.gc.ca, Emmanuel.roy@cnrc- nrc.gc.ca (E. Roy). 1 Present address: Harvard–MIT Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Acta Biomaterialia 7 (2011) 2492–2498 Contents lists available at ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat