In this study, it was aimed to investigate the combinatory effect of biophysical and biochemical factors on human dental pulp stem cells' (hDPSCs) behavior. For this purpose, well-defined nanotopography of nanowells with two different pitch size of 109 nm and 341 nm were prepared on polyhydroxymethylsiloxane (PHMS) by using colloidal particles nanofabrication. The nanopatterned PHMS surfaces (PHMS/109 and PHMS/341) were subsequently used for fibronectin (Fn) adsorption. With this approach, nanotopographical details were combined with biochemical signals from Fn. Depending upon the size of cavities created by the nanowells, Fn molecules followed a site-selective adsorption. While they adsorbed both inside and outside the nanowells of PHMS/341, they preferred to adsorb outside the cavities of PHMS/109 surfaces. Human dental pulp stem cells were cultured on nanopatterned PHMS with or without Fn adsorption in the presence and absence of serum. Scanning electron microscopy and fluorescence microscopy analyses showed the interaction of cells was dependent on nanotopography size especially in serum-free medium. Furthermore, hDPSCs' morphology and cytoskeletal organization changed in correlation with preferential Fn adsorption. On Fn adsorbed PHMS/109 surfaces, cells displayed stretched bundles whereas, they showed extensive spreading and followed the Fn adsorbed sites inside the cavities of PHMS/341 surfaces. The observed effects are interpreted in terms of the preferential exposure of different Fn epitopes occurring on PHMS/109 and PHMS/341 as a consequence of the different hydrophilic/hydrophobic adsorbing surface. (C) 2014 Elsevier B.V. All rights reserved.