Whole proteome analysis of osteoprogenitor differentiation induced by disordered nanotopography and mediated by ERK signalling Fahsai Kantawong a, * , Karl E.V. Burgess b , Kamburapola Jayawardena b , Andrew Hart a , Richard J. Burchmore b , Nikolaj Gadegaard c , Richard O.C. Oreffo d , Matthew J. Dalby a a Centre for Cell Engineering, Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK b Sir Henry Welcome Functional Genomics Facility, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK c Centre for Cell Engineering, Department of Electronics and Electrical Engineering, Rankine Building, University of Glasgow, Glasgow G12 8QQ, UK d Bone and Joint Research Group, Centre for Human Development, Stem Cells & Regeneration, Institute of Developmental Sciences, DOHAD, University of Southampton, Southampton S016 6YD, UK article info Article history: Received 3 March 2009 Accepted 18 May 2009 Available online 26 June 2009 Keywords: Tissue engineering Disordered topography Extracellular-activated protein kinase Osteoprogenitor cells Proteomics abstract Topographic features can modulate cell behaviours such as proliferation, migration, differentiation and apoptosis. Biochemical mechanotransduction implies the conversion of mechanical forces (e.g. changes in cell spreading and morphology from changing surface topography) into biochemical signal via biomolecules. Still, little is known concerning which pathways may be directly involved in cell response to changes in the material surface. A number of pathways have been implicated using focused studies of ‘selected’ biomolecules rather than a global analysis of signal pathways. This study used a controlled disorder nanopit topography (NSQ50, fabricated by electron beam lithography) to direct osteoblast differentiation of progenitor cells. This topography is unique as it represents a middle route (from absolute order or random roughness) that allows osteoconversion with similar efficiency as dexa- methasone and ascorbate treatment. Two direct-comparison proteomics techniques, firstly gel-based and then chromatography-based, were used to analyse progenitor proteome changes in response to the nanotopography. Many of the changed proteins form part of the Extracellular Signal-regulated Kinase (ERK1/2) pathway. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction An osteogenic, nanodisordered topography (near square) has recently been described [1]. Until now, there have been two distinct approaches to surface topographical fabrication: production of highly regular surfaces by electron beam lithography and photoli- thography [2] and production of random surfaces by e.g. anodisa- tion, etching, grit blasting, etc. However, the introduction of controlled error using electron beam lithography (50 nm from absolute centre positioning of pits) produces far greater levels of bone marker production from skeletal progenitor cell populations and thus represents a superior, as yet unexplored, middle route. The topography detailed in this study is termed NSQ50 (near- square 50 nm, 120 nm diameter, 100 nm deep, pits in a square arrangement with 300 nm centre–centre spacing but with 50 nm error in X and Y positioning in this spacing). Originally fabricated by electron beam lithography, the most high-resolution top-down nanofabrication technique available (down to 10 nm in X and Y can be achieved) [3], the pattern is embossed into polycaprolactone, an FDA approved biodegradable material. This is achieved via a nickel (Ni) shim (similar to the process of CD and DVD manufacture) with down to 5 nm fidelity [4]. In order to test the differentiation capacity of the near-square nanosurface, primary human osteoprogenitor cells (OPGs) were used. These cells contain the heterogeneous cell mixture ortho- paedic implants would have to influence in vivo including skeletal stem cells, committed osteoprogenitor cells, preosteoblasts and osteoblasts [5]. The skeletal stem cells themselves have the ability to develop along the bone, cartilage, connective tissue and fat lineages [6,7]; although it is unclear if these cells can also differ- entiate along neural and muscle lineages [8]. In order to probe the progenitor population response to the near- square topography using global strategies, proteomics approaches were taken. Proteomics offers a superior approach to genomics as there are a number of variables between the production of mRNA and the production of protein including efficiency (i.e. sometimes only a low level of mRNA regulatory change is required to produce a large protein change) and modulations. Two approaches were * Corresponding author. Tel.: þ44 141 339 8855x0352; fax: þ44 141 330 3730. E-mail address: f.kantawong.1@research.gla.ac.uk (F. Kantawong). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.05.040 Biomaterials 30 (2009) 4723–4731