Primary osteogenic cells were seeded in six-well plates (1 × 105 cells per well) and incubated for 24 h. Cells cultured in the medium without the addition of CCN were used as the control. CCN were added to the cells at increasing concentrations (5, 10, 20, 50, and 100 mL/L). After 24 h, the cells were fixed with 4% paraformaldehyde and stained with a 2% Alizarin red solution (Merck, Warsaw, Poland) [28 (link)]. Cell morphology was recorded using an optical microscope (TL-LED, Leica Microsystems, Wetzlar, Germany).
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