Las af lite v2

At 24 h of culturing, samples cultured with osteoblasts and fibroblasts were washed with PBS (VWR^®, Radnor, PA, USA). Cell fixation was performed using 4% formaldehyde solution for 10 min and stained with Phalloidin-iFluor 488 Reagent (ab176753, Abcam^®, The Netherlands) and Propidium iodide (P4179, Sigma-Aldrich^®, St. Louis, MO, USA). Fluorescence images were carried out at 493/517 nm and 535/617 nm wavelength using a Leica TCS SP5 confocal microscope (Leica Microsystems, Deerfield, IL, USA) coupled to LAS-AF LITE v2.0 software (Leica Microsystems, Deerfield, IL, USA).

After 7 days of incubation, discs with osteoblasts were washed 3 times with PBS (VWR^®, Radnor, PA, USA) to remove non-adherent cells. Cells were fixed using 2.5% glutaraldehyde (VWR^®, Radnor, PA, USA) for 1 h and then the discs were washed with PBS. Cell culture mineralization was evaluated using the OsteoImage^TM Mineralization Assay (PA-1503, Lonza, Morristown, NJ, USA). This assay consists of the fluorescent staining of extracellular mineral content deposited by cells, specifically hydroxyapatite. Mineralization-stained images were obtained at 492/529 nm excitation/emission wavelengths using a Leica TCS SP5 confocal microscope (Leica Microsystems, Deerfield, IL, USA) coupled to LAS-AF LITE v2.0 software (Leica Microsystems, Deerfield, IL, USA).