The present study aimed to evaluate key parameters of in vitro osteogenesis on (1) commercially pure titanium (cpTi) discs with 20-200-microm-scale microtopography patterned with additional micron- and submicron-scale topography (0.5-20 microm; Plus surface, Dentsply Friadent), (2) control cpTi discs with 20-200-microm-scale microtopography (DPS, Deep Profile Surface, Dentsply Friadent), and (3) a machined surface. Using calvaria-derived osteogenic cultures, the following parameters were assessed: cell adhesion and spreading, growth curve and cell viability, alkaline phosphatase (ALP) activity and total protein content, immunolocalization of fibronectin, bone sialoprotein (BSP) and osteopontin (OPN), and bone-like tissue formation. The results showed no major differences between surfaces in terms of cell adhesion, growth curve, cell viability (days 4 and 11), ALP activity, or total protein content (days 11 and 17). At day 11, cultures grown on Plus exhibited small, well-defined nodular areas of calcified matrix, which were only rarely observed on DPS and absent on the machined surface. Such areas were larger at day 17 and were not associated with the typical mineralized bone-like nodules (with BSP- and OPN-positive osteoblastic cells on top). At day 17, the total mineralized area was significantly larger on DPS than on a Plus or machined surface (DPS>Plus>machined; Kruskal-Wallis test, P<0.05). Direct fluorescence allowed the straightforward observation of higher amounts of apoptotic bodies associated with mineralized nodules for Plus. The results suggested the occurrence of an additional, early pattern of matrix mineralization mostly for the Plus microstructured surface, which did not necessarily translate into larger bone-like tissue formation in vitro.