Bioactive glasses, osteoproductive materials, have received considerable attention as bone graft substitutes in the treatment of bony defects. More recent strategies for achieving a predictable periodontal regeneration include the use of enamel matrix proteins, due to their role in the formation of bone tissue. The aim of our study is to examine the effects of these materials on the proliferation and differentiation of the mouse preosteoblastic cell line MC3T3-E1. Cells were cultured up to 28 days in contact with three types of granules: Bioglass 45S5 granules (BG), 45S5 granules coated with enamel matrix proteins (Emdogain) (BG/EMD), and a less reactive glass used as a control (60S). Phase contrast microscopic observations have shown that all substrates supported the growth of osteoblastic cells. Zones of differentiation were observed at an earlier stage in cultures of BG and BG/EMD. TEM observations revealed ultrastructural features very close to what is observed in vivo during intramembranous ossification with a direct bone apposition on the bioactive glasses. Total protein production was higher in the cultures with BG and BG/EMD. Northern Blot analysis revealed a stimulation of the transcription factor Cbfa1/Runx2 at day 13 in cultures of BG when compared to the two other cultures. Bone sialoprotein (early marker of differentiation) and osteocalcin (marker of late-stage differentiation) expression was increased in cultures with BG and BG/EMD when compared to 60S. Taken together, our findings indicate that Bioglass alone or combined with Emdogain, have the ability to support the growth of osteoblast-like cells in vitro and to promote osteoblast differentiation by stimulating the expression of major phenotypic markers. In addition, we noticed that the bioactive granules coated with Emdogain revealed significantly higher protein production than the bioactive granules alone at day 20.