The role of surface microtopography in combination with different surface wettability for rat calvaria cell differentiation was examined. Mineralization and alkaline phosphatase (ALP) activity of rat calvaria cells on flat polydimethylsiloxane (PDMS) or PDMS contained pyramids which were either hydrophilic or hydrophobic were compared. ALP expressing cells were more frequent on hydrophilic PDMS contained pyramids. ALP activity, peaked at day 9, was highest for hydrophilic pyramids followed by hydrophobic pyramids and flat hydrophilic PDMS surfaces. A similar pattern was obtained with respect to mineralized nodules. These observations showed that micro-sized surface features promote differentiation of rat calvaria cells. Further, hydrophilic surfaces are more prone to stimulate differentiation in comparison with hydrophobic surfaces. The results suggest that both material surface chemistry and topography affect osteoblast differentiation.