To investigate the directive importance of nanophysical properties on the morphological and protein expression responses of dibutyryladenosine cyclic monophosphate (dBcAMP)-treated cerebral cortical astrocytes in vitro. Elasticity and work of adhesion characterizations of culture surfaces were performed using atomic force microscopy and combined with previous surface roughness and polarity results. The morphological and biochemical differentiation of dBcAMP-treated astrocytes cultured on promising nanofibrillar scaffolds and comparative culture surfaces were investigated by immunocytochemistry, colocalization, super resolution microscopy and atomic force microscopy. The dBcAMP-treated astrocyte responses were further compared with untreated astrocyte responses. Nanofibrillar scaffold properties were shown to reduce immunoreactivity responses while poly-L-lysine-functionalized Aclar® (Ted Pella Inc., CA, USA) properties were shown to induce responses reminiscent of glial scar formation. The comparison study indicated that directive cues may differ in wound-healing versus quiescent situations.