Osteogenic differentiation

Cells at 1.5 × 10⁴ cells/cm² were plated in 24-well plates, and after 24 hours of culture in complete medium washed with PBS and cultivated with StemPro Osteogenesis Differentiation Kit medium (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The medium was refreshed every two days. After three weeks in culture, the cells were washed twice with PBS and fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), for 15 minutes.

After washing the cells in PBS, they were incubated with 0.1% alizarin red solution (Sigma, USA) in Tris-HCl pH 8.3, at 37 ^oC for 30 minutes. After two washes with PBS, cells were observed and photographed under a phase-contrast inverted microscope (Axiovert 40C, Carl Zeiss, Oberkochen, Germany). Cells stained with alizarin red were incubated for 15 minutes with 20% methanol and 10% acetic acid solution. The supernatant photometric absorbance at 450 nm was determined in the Epoch Microplate Spectrophotometer (BioTek, Vermont, USA).