Ars solution

The ADSCs were seeded on nanofibrous scaffolds with different concentrations of MgO nanoparticles, as described earlier. After 21 days, the ARS staining assay was used to evaluate the calcium mineralization on samples. Briefly, after removing the media, the scaffolds were washed three times in PBS. The cells were fixed by immersion in 10% formalin for 30 minutes at room temperature, followed by washing with distilled water. Then, the cells were stained with ARS solution (40 mM, pH 4.2, Sigma) at room temperature for 30 minutes with gentle shaking. The plates were then washed thoroughly in distilled water, and samples were incubated with 10% cetylpyridinium chloride for 15 minutes. After that, to determine the levels of Ca deposit in the scaffolds, the absorption of supernatant solutions was measured at 405 nm.

The cells in all groups were incubated in osteogenic medium for 3 weeks and then rinsed twice with PBS and fixed in 4% paraformaldehyde for 30 min. The cells were washed with distilled water (DW), treated with 2% ARS solution (Sigma-Aldrich, USA) for 5 min and then washed 3–5 times with DW to remove unbound ARS. The stained plates were air-dried and examined under a light microscope (Olympus IMT-2, Tokyo, Japan) and photographed with a digital camera (Canon EOS 550D, Tokyo, Japan). For the quantification of ARS staining, the cells were desorbed with 10% (w/v) cetylpyridinium chloride (Sigma-Aldrich, St. Louis, MO, USA); then the absorbance was measured at 562 nm by a SpectraMax M3 microplate reader.

Osteoblast MC3T3-E1 cells were seeded in 24-well plates at a density of 5.0 × 10⁴ cm⁻² and treated with an osteogenic inductive medium including 100 nM dexamethasone, 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate sodium, and different concentrations of RbCl (low: 1.6 mM; high: 3.2 mM). The medium was refreshed every 2 days until the 21st day. Cells were then fixed in 4% PFA for 15 min at 37°C before being stained with 1% ARS solution (Sigma-Aldrich) for 50 min. PBS was used to wash the non-specific staining, and representative images were taken by optical microscopy. For the quantitative assay, 10% cetylpyridinium chloride in 10 mM sodium phosphate was administered to stained cells, followed by the determination of optical density at 620 nm (OD₆₂₀).

After 21-day treatment of PRP, cells were fixed in 4% paraformaldehyde for 30 min and stained with 2% ARS solution (Sigma-Aldrich) for 5 min. The stained cells were desorbed with 10% cetylpyridinium chloride (Sigma-Aldrich), observed under an inverted optical microscope and photographed using a digital camera.

Following a 21-day period of osteogenic differentiation, the cells were fixed using 4 % paraformaldehyde for 30 min. Subsequently, the cells were subjected to incubation in a 40 mM ARS solution (Sigma-Aldrich) at room temperature for 30 min. Digital images were acquired utilizing an Olympus IX51 microscope. For quantitative analysis, a 5 % perchloric acid solution (Sigma-Aldrich) was introduced to dissolve the stain present within the calcified layer, and the absorbance was measured at a wavelength of 420 nm using a PowerWave XS spectrophotometer (BioTek).