This journal is c The Royal Society of Chemistry 2012 Integr. Biol., 2012, 4, 531–539 531 Cite this: Integr. Biol., 2012, 4, 531–539 Surface mobility regulates skeletal stem cell differentiation Cristina Gonza´lez-Garcı´a, a David Moratal, a Richard O. C. Oreffo, b Matthew J. Dalby* c and Manuel Salmero´n-Sa´nchez* ad Received 14th October 2011, Accepted 11th February 2012 DOI: 10.1039/c2ib00139j A family of polymer substrates which consists of a vinyl backbone chain with the side groups –COO(CH 2 ) x H, with x = 1, 2, 4, was prepared. Substrates with similar chemical groups but decreasing stiffness, characterized by their elastic modulus at 37 1C, as well as surface mobility, characterized by the glass transition temperature, were obtained. We have investigated whether these subtle variations in polymer chemistry lead to alterations in fibronectin (FN) adsorption and mesenchymal stem cell response. The same FN density was adsorbed on every substrate (B450 ng cm À2 ) although the supramolecular organization of the protein at the material interface, as obtained with AFM, was different for x = 1 and the other two surfaces (x = 2, 4). Consequently, this allows one to investigate the effect of physical properties of the matrix on stem cell differentiation after ruling out any influence of protein activity. Cell adhesion was quantified by calculating the size distribution of focal adhesions. Mesenchymal stem cell differentiation to the osteoblastic lineage was determined by quantifying protein levels for osteocalcin, osteopontin and Runx2, in the absence of any additional osteogenic soluble factors in the culture media, but as a direct effect of material properties. The findings indicate the potential to modulate skeletal progenitor cell commitment to the osteoblastic lineage through surface mobility of the underlying material surface. Introduction Mesenchymal or skeletal stem cells are able to differentiate along the stromal lineage to give rise to osteoblasts, chondrocytes and adipocytes through the application, typically, of chemical cues and specific factors. 1,2 There is also a wealth of emergent data that mesenchymal or skeletal stem cells are highly sensitive to their environment and, when cultured on synthetic substrates, these cells respond to cues provided by chemistry, stiffness, surface topography and dimensionality (2D vs. 3D), which are able to direct skeletal stem cell lineage differentiation. 3–10 Cell–material interactions occur through a layer of matrix proteins including fibronectin (FN), vitronectin, fibrinogen and laminin that interface living cells and synthetic surfaces. 11–15 The concentration, distribution, and motility of the adsorbed protein layer on a surface play a fundamental role in the biofunctionality of a synthetic material. Thus it may be possible to manipulate these parameters to augment the biological a Center for Biomaterials and Tissue Engineering, Universitat Polite`cnica de Vale`ncia, 46022 Valencia, Spain. E-mail: masalsan@fis.upv.es; Fax: +34 963877276; Tel: +34 963877275 b Bone and Joint Research Group, University of Southampton Medical School, Southampton, UK c Center for Cell Engineering, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK. E-mail: m.dalby@bio.gla.ac.uk; Tel: +44 (0)141 330 3550 d CIBER de Bioingenierı´a, Biomateriales y Nanomedicina (CIBER-BBN), Valencia, Spain Insight, innovation, integration This work identifies surface mobility as a novel mechanism able to regulate the differentiation of skeletal stem cells. Moreover, it points out the importance of investigating the intermediate layer of proteins at the cell–material interface before discussing the effect of any physical properties of the matrix (e.g. stiffness) on cell differentiation. To do that, we have synthesized a family of polymers with subtle variations in chemistry and qualitatively different stiffness and surface mobility, which lead to alterations in fibronectin (FN) adsorption and human mesenchymal stem cell behavior. Our findings identify surface mobility as a key factor able to regulate skeletal stem cell differentiation with wider implications therein for the modulation and manipulation of stem, progenitor and adult populations in hard and soft tissue regeneration. This paper integrates technology and biology in two ways: the preparation of material surfaces able to direct cell differentiation making use of a novel surface property (mobility), as well as the methodology developed to quantify cell differentia- tion through image analysis of secreted proteins. Integrative Biology Dynamic Article Links www.rsc.org/ibiology PAPER