*3.4. Cell Culture*

MC3T3-E1 pre-osteoblasts (Riken bank, Tsukuba, Japan) were grown in a 75 cm<sup>2</sup> cell-culture flask with modified Eagle's medium (DMEM, Biochrom, Berlin, Germany) containing 1 <sup>g</sup>·L−<sup>1</sup> glucose, 1% penicillin/streptomycin (P/S, Biochrom, Berlin, Germany) and 10% Fetal Bovine Serum (FBS, Biochrom, Berlin, Germany). The flask was placed in a 37 ◦C incubator under 95% humidified air and 5% CO2 conditions. Culture medium was changed every two days and, at a 60–70% confluence, cells were trypsinized with 0.05% trypsin-EDTA (Biochrom, Berlin, Germany). A 25 μL drop of cell suspension was added over each P(VDF-TrFE) sample with a density of 10 × 10<sup>4</sup> cells.mL−<sup>1</sup> for the proliferation assays (cell viability) and at a density of 50 × 10<sup>4</sup> cells·mL−<sup>1</sup> for the differentiation studies (cell viability, ALP and alizarin red). After that, the plates were incubated during 30 min for cell adhesion. The well volume was then completed with the growth medium (GM) and incubated once more for 24 h.

For proliferation assessment, cells were cultivated for 3 days, with 24 h and 72 h as timepoints. For the di fferentiation assays, the medium was exchanged by osteogenic di fferentiating medium (DM) after 24 h and cells were maintained up to 21 days, with 7, 14, and 21 days as timepoints. DM was composed of the GM supplemented with 0.1 μM dexamethasone (Sigma-Aldrich, Sintra, Portugal), 50 μg.mL−<sup>1</sup> of ascorbic acid (Sigma-Aldrich, city, state, country), and 10 mM of b-glycerophosphate (Sigma-Aldrich, Sintra, Portugal). Culture media were changed every two days.
