Oil red o

For osteogenic and adipogenic differentiation, CBti MSCs at the end of P4 were seeded at a density of 4,000 cells/cm2 on cell culture coverslips (Thermo Fisher Scientific) and arranged in 24-well plates (Falcon®, Corning, Corning, NY, USA) in the presence of standard growth medium. At 70–80% cell confluence, the medium was replaced with specific differentiation media, then renewed every 3–4 days for 21 days. To induce adipogenic differentiation, cells were evaluated using the StemPro® Adipogenic Differentiation Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. The presence of intracellular lipid droplets was detected by standard staining with Oil Red O (Diapath, Bergamo, Italy). In parallel, cells were also grown using the StemPro® Osteogenic Differentiation Kit (Thermo Fisher Scientific). The presence of calcium deposits representing osteogenic differentiation were evaluated by von Kossa staining (Sigma-Aldrich). Cells were fixed with 10% formalin for 5 min at room temperature, incubated with 1% silver nitrate solution for 15 min and exposed to ultraviolet light for 2 h. Coverslips were rinsed with distilled water and 5% sodium thiosulfate to remove unreacted silver.