Multiskan spectrum spectrometer

The enzymatic activity of ALP during osteogenic differentiation was measured for all four donors using standard procedures. Cells were grown for 7, 14, or 28 days in 12-well plates at 3500 cells/cm² with 0.75 mL OM. For the Day 0 (control) samples, cells were collected from BGM prior to culturing. After the relevant culture period, cells were washed with phosphate-buffered saline (PBS) and then lysed for protein extraction with 0.02% Triton-100 (Sigma) in PBS. A liquid p-nitrophenyl phosphate solution was then added at a 1:1 ratio and incubated at 37 °C for 30 min in the dark before being measured using a Multiskan Spectrum spectrometer (Thermo Scientific, Helsinki, Finland). The ALP activity in terms of the conversion of p-nitrophenyl phosphate into p-nitrophenol was calculated using Equation (1): $$ALP activity\left[\frac{\mathrm{nmol}}{\min }\right]=\frac{optical density}{18.8\left[{\mathsf{\mu }\mathrm{mol}}^{-1}\right]\times time \left[\min \right]}\times 1000\left[\frac{\mathrm{nmol}}{\mathsf{\mu }\mathrm{mol}}\right]$$

Mineralization during osteogenic differentiation was determined by staining using Alizarin Red S indicator (Sigma-Aldrich) according to standard procedures. Cells for Alizarin Red staining were seeded at 3500 cells/cm² in a 12-well plate and grown for up to 28 days with 0.75 mL OM, with the Day 0 samples cultured in BGM. After sampling, cells were washed with PBS and incubated for 15 min with paraformaldehyde. They were then washed again with deionized water before 2% Alizarin Red S solution (pH 4.1–4.3) was added. The samples were stained for 20 min on a rotating shaker then washed with deionized water and dried overnight. After drying, the samples were rehydrated by adding 1 mL of deionized water and rehydrated overnight. After rehydration, 1 mL of 10% cetylpyridinium chloride solution was added to the cells and incubated for 15 min at 37 °C on a rotating shaker. The cell layer was then scraped, centrifuged for 10 min at 16,100× g and 24 °C, and the resulting supernatant was measured for optical density in a Multiskan Spectrum spectrometer (Thermo Scientific) at 562 nm.