Alizarin red dye

The alizarin red dye (Sigma-Aldrich) was used to stain the mineralized matrix.
The plates were washed with PBS and the cells fixed with 10% formalin solution
for 24 hours and, after that period, dehydrated in increasing concentrations of
alcohol (30, 50, 70, 90, 96%), each solution being held in contact with cells
for 1 hour. After the last hour, the solution was removed and the plates held
semi-open until complete drying. They were then filled with a solution of 2%
alizarin red pH 4.2 for 8 minutes. Excess dye was removed by plentiful washing
of the material with double-distilled water and the plates were again held
semi-open until drying. The quantification of the staining was evaluated by
colorimetric method according to Gregory et al.¹⁵.

ASC were induced into osteocytes using the StemXVivo Mouse/Rat osteogenic/adipogenic supplement (R&D Systems). Briefly, 7.6 × 10⁴ cells were suspended in 1.5 ml of complete medium and plated in a 24-well dish. After 1 day, cells were at 60-70% confluency. Cells were purged 3 h in StemXVivo osteogenic/adipogenic base media. Osteogenic differentiation was induced with 1 ml of StemXVivo Osteogenic differentiation media and was changed every 3-4 days. After 21 days, the cells were fixed for 15 minutes using 10% formaldehyde in PBS at room temperature. The osteogenic differentiation was revealed using 40 mM alizarin red dye (which stains calcium deposition) pH 4.3 (Sigma-Aldrich) for 20 minutes at room temperature. Brightfield images were taken with an optic microscope with a ×20 magnification. The expression of the osteocyte-specific genes Dmp1 and Gdf15 was measured by RT-qPCR.

Alizarin red staining was used to recognize calcium deposits secreted by differentiating cells. Alizarin red dye (A5533, Sigma Aldrich, USA) at a concentration of 40 mM was prepared in ddH₂O and the pH was adjusted to 4.1 using 10% ammonium hydroxide (NH₄OH, 221228, Sigma Aldrich, USA). ADSCs were fixed with 4% paraformaldehyde (1.00496.9011, Sigma Aldrich, USA) for 15 min at RT. The fixative was then removed and cells were washed with ddH₂O. After water removal, cells were stained for 20 min with Alizarin red dye using orbital shaking. Dye was removed and cells were washed 5 times with ddH₂O. An Olympus IX73 microscope (Olympus, Shinjuku, Tokyo, Japan) was used for photographic documentation.

For osteogenic differentiation assays, 3x10⁴ culture-expanded MSCs (passage 3) from donor-matched IC-BM and VB-BM samples were cultured in osteogenic media. The osteogenic media was formed of low glucose DMEM (ThermoFisher Scientific Waltham, MA, USA) supplemented with 10% FCS (ThermoFisher Scientific), penicillin and streptomycin (Thermo Fisher Scientific), 100nM dexamethasone, 10mM β-glycerophosphate and 0.05mM ascorbic acid (all from Sigma-Aldrich). After 14 days of culture, the quantification of the calcium level was performed using colorimetric calcium kit (Calcium Liquid, Sentinel Diagnostics, Milan, Italy) as previously described [37 (link)]. To extract calcium, cultured MSCs were treated with 1M of HCl solution. The spectrophotometric reading was taken on MULTISCAN EX reader and analysed using Ascent software (Thermo Fisher Scientific). Additionally, the staining for calcium deposition and alkaline phosphatase (ALP) expression was performed using Alizarin Red dye and fast blue RR salt dye respectively (both from Sigma-Aldrich) as used previously [32 (link), 38 (link)]. The culture plates were scanned using an Epson 3590 flatbed scanner (Epson Ltd, Hertfordshire, UK).