*2.5. Cell Proliferation*

MC3T3-E1 cells were used to evaluate the in vitro bioactivity of the PVDF composite nanofibers by a cell proliferation assay in alpha minimum essential medium (α-MEM) containing 10% of fetal bovine serum (FBS) and 1% of penicillin–streptomycin at 37 ◦C with 5% CO2. Before assaying, the composite nanofibers were cut into circular shape with a diameter of 15 mm, followed by putting into 24-well tissue culture plate and fixing with glass ring (inner diameter = 11 mm). Then, they were sterilized with 70% ethanol and rinsed with DPBS and α-MEM. After drying, the composite nanofibers were once again sterilized under UV radiation for 3 h.

The proliferation of viable cells on the composite nanofibers was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. One milliliter of cell suspension (2 × 104 cells/well) was placed onto the sterilized samples and cells were cultured for different periods of time. After culturing for 1, 3, 5, 7, and 14 days, culture media were replaced with 0.2 mL of the MTT solution and further incubation of the cells was maintained for another 4 h. Next, we removed the remaining media and added 1 mL of DMSO to solubilize the precipitated formazan crystals. Finally, the resulting supernatant was transferred to 96-well plate with 0.2 mL per well. The absorbance was determined at 570 nm using a spectrophotometric plate reader (OPSYS-MR, Dynex Technology Inc., USA). Furthermore, after days 3 and 7 of cell culture, the cell-nanofibers were fixed in 4% glutaraldehyde for 1 h, and then dehydrated using different concentrations of ethanol (25, 50, 70, and 100%), followed by vacuum-drying. The morphologies of dried samples were observed using SEM after sputter coating with gold.
