The response of fibroblasts to hexagonal nanotopography fabricated by electron beam lithography Matthew J. Dalby, 1 Nikolaj Gadegaard, 2 Chris D.W. Wilkinson 1 1 Centre for Cell Engineering, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow, G12 8QQ, Scotland, United Kingdom 2 Centre for Cell Engineering, Department of Electronics and Electrical Engineering, Rankine Building, University of Glasgow, Glasgow, G12 8QQ, Scotland, United Kingdom Received 2 August 2006; revised 15 February 2007; accepted 20 March 2007 Published online 23 July 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31409 Abstract: It has been known for many years that cells will react to the shape of their microenvironment. It is more recently becoming clear that cells can alter their morphology, adhesions, and cytoskeleton in response to their nanoenvironment. A few studies have gone further and measured cellular response to high-adhesion nano- materials. There have, however, been practical difficulties associated with genomic studies focusing on low-adhesion nanotopographies. Because of advancement in fabrication techniques allowing the production of large area of struc- ture and the ability to amplify mRNA prior to microarray hybridization, these difficulties can be overcome. Here, electron beam lithography has been used to fabricate arrays of pits with 120 nm diameters, 100 nm depth and 300 nm center to center spacing in hexagonal arrangement. Electron and fluorescent microscopies have been used to observe morphological changes in fibroblasts cultured on the pits. 1.7k gene microarray was used to gauge genomic response to the pits. The results show reduction in cellular adhesion, decrease in spreading, and a broad genomic down-regulation. Also noted was an increase in endocy- totic activity in cells on the pits. Ó 2007 Wiley Periodicals, Inc. J Biomed Mater Res 84A: 973–979, 2008 Key words: nanotopography; nanobioscience; fibroblast; microarray INTRODUCTION It has been known for many years that cells will respond to their topographical environment, 1,2 with effects such as contact guidance to grooves. 3–8 As technology has progressed, engineering has allowed the fabrication of continually smaller structures; this is largely driven by the development of faster micro- chips and the miniaturization required to fit more transistors on a chip. Hence, techniques such as elec- tron beam lithography (EBL) have become available to biologists. 9–11 This and other techniques such as chemical phase separation 12 and colloidal lithogra- phy, 13 have allowed the production of differentially adhesive surfaces. Highly ordered EBL, square arrangement nanoar- rays tend to have properties of low cell adhesion. 14,15 It has been postulated that this is due to changes in surface energy at the fluid/solid interface resulting from nanotopography. Texture and topography can influence the hydrophobicity and hydrophilicity of a surface and can indeed produce ‘‘superhydrophobic’’ and ‘‘superhydrophilic’’ surfaces depending on fea- ture shape and aspect ratio. 16 Here, we consider hydrophobic surfaces. If a droplet of water is placed on these surfaces, it will remain rounded and roll off rather than spreading into a thin film. This phenom- enon has been termed the ‘‘Lotus effect’’ after the water repellent leaves of the lotus plant (Nelumbo nucifera). 17 These leaves exhibit a double-structured roughness, where submicrometric wax crystals cover a larger micrometric structure. Increasing the hydro- phobicity of a material surface will reduce protein interaction and hence reduce cellular adhesion. 18 EBL and subsequent etching can be used to fabri- cate surfaces with an accuracy of 10 nm in X, Y, and Z axes. 11 Here, rather than testing square arrange- ments of pits as have been observed previously, we use 120 nm diameter, 100 nm deep pits with a 300 nm pitch in a hexagonal (HEX) arrangement. Human fibroblasts, as a model of a typical tissue cell, have Correspondence to: M.J. Dalby; e-mail: m.dalby@bio.gla. ac.uk Contract grant sponsor: MJD’s BBSRC David Phillips Fellowship ' 2007 Wiley Periodicals, Inc.