In this work, we demonstrate the efficiency of substrate-mediated electrical stimulation of micropatterned polypyrrole/polyurethane (PPy/PU) composites for enhancing the osteogenesis in osteoblast-like cells. The PPy/PU substrates were obtained by dispersing electrically conductive PPy nanograins within a mechanically resistant PU matrix. Spin-coated PPy/PU layers were micropatterned with predefined 3D geometries by ultrashort laser ablation. Then they were conformally coated by Matrix Assisted Pulsed Laser Evaporation, in order to restore their chemical and electrical integrity. The chemical structure of the laser-processed PPy/PU substrates was investigated by 2D and 3D mapping of the laser-processed areas, via Raman microspectroscopy. In vitro studies revealed that the micropatterned PPy/PU substrates facilitated the topological and electrical communication of the seeded osteoblasts. Specifically, we demonstrated the cells attachment on the predefined 3D micropatterns. More importantly, we found evidence about the cells mineralization inside the 3D micropatterns by investigating the calcium deposits by Energy-Dispersive X-Ray Spectroscopy (EDS) and Alizarin Red staining. We found that the substrate-mediated electrical stimulation of the PPy/PU substrates induced a twofold increase of the Ca deposits in the cultured cells. (C) 2017 Elsevier B.V. All rights reserved.