Cell shape and regulation of biological processes such as proliferation and differentiation are to a large degree connected. Investigation of the possible relationship between cell shape and function is therefore important for developing new material concepts for medical applications as well as developing novel cell based sensors. Cell spreading requires a firm contact with the underlying substrate, with focal contacts (FC) being the primary sites of adhesion. They consist of a large number of clustered transmembrane proteins (integrins). FC integrins connect the cell cytoskeleton with the cell substratum. It has been demonstrated that cell spreading increases osteoblast differentiation in pre-osteoblastic progenitors. The gradual process of osteogenesis can be followed by different proteins being expressed at various time points, comprising early (e.g., bone-specific alkaline phosphatase (bALP)) and late genes (e.g., osteocalcin (OC)). In the present study we have used immunohistochemistry and RT-PCR to determine osteogenic differentiation of human bone cells (HBC). For online monitoring, fluorescently-tagged actin and vinculin were used for transfection of HBCs. Transfection of HBCs with an OC promoter gene construct allowed us to online monitor the gradual process of osteogenesis. We found distinct changes in cell architecture upon osteogenic differentiation thus providing evidence for the connection between cell shape and functional state.