A5533

The calcific deposition in DPSCs was evaluated by alizarin red staining (Sigma-Aldrich, A5533). After 3 weeks of DPSC differentiation with osteogenic media, as described above, the cells were fixed with 4% paraformaldehyde for 30 min, and stained with 2% alizarin red solution for 30 min. ImageJ was used to measure the relative alizarin red-positive area.

We used flow cytometry to analyze cell surface
marker expressions by the ASCs culture
prior to ZnO-NPs treatment. The cells were
characterized according to a set of cell surface
markers characteristic for ASCs that included
cluster of differentiation (CD) CD73 (sc-14682,
Santa Cruz Biotechnology Inc., USA), CD105
(sc-20632, Santa Cruz Biotechnology Inc.,
USA), CD45 (sc-25590, Santa Cruz Biotechnology
Inc., USA) and CD34 (sc-7045, Santa
Cruz Biotechnology Inc., USA).The differentiation
potentials of the ASCs were checked in
specific media. For adipocyte differentiation,
cells were cultured in 1 μmol/l dexamethasone
(D4902, Sigma, USA), 60 μmol/l indomethacin
(I7378, Sigma, USA), and 450 μl 3-isobutyl-
1-methylxanthine (I5879, Sigma, USA). Adipocytes
were characterized by oil red-O (O0652,
Sigma, USA) staining under a light microscope.
For differentiation into osteoblasts, culture medium
was supplemented with 0.1 μmol/l dexamethasone,
10 mmol/l β-glycerophosphate
(G9422, Sigma, USA) and 60 mmol/l ascorbate
(A0157, Sigma, USA). Osteoblasts were characterized
by alizarin red (A5533, Sigma, USA)
staining and microscopic examination (16 (link)).

The cell cultures were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Three more washes were performed. The cell cultures were stained with a 40 mM alizarin red staining solution (pH 4.2, A5533 Sigma-Aldrich) for 20 min at 37 °C [36 (link)]. Washings were performed until complete removal of the dye. After the acquisition of photomicrographs, an extraction solution, composed of 10% acetic acid and 20% methanol, was used for 40 min under stirring at room temperature, followed by the absorbance reading at 490 nm using the EnSpire^® spectrophotometer.

Osteogenesis of MSCs was conducted using a kit (A10072-01, STEMPRO, Thermo Fisher Scientific) following the manufacturer's instructions. In brief, MSCs (19,000) were seeded per well into a 12-well cell culture plate at a density of 0.5 × 10⁴ cells/cm² in MSCs medium at 37°C in a humidified atmosphere at 5% CO₂. After 3 h, media were replaced with prewarmed complete osteogenesis differentiation medium and continued incubation (day 0). MSCs continued to undergo limited expansion as they differentiated under osteogenic conditions. Media were replaced every 3 days. On day 20, the cells were stained with Alizarin Red S and photographed.
Alizarin Red S Staining. For osteogenesis, the deposition of calcium phosphate is an indication of MSCs differentiation into osteoblast and hence in vitro bone formation. Medium in the cell culture wells was removed; cells were washed with Ca⁺⁺-Mg⁺⁺-free D-PBS and then fixed in 2 mL 10% NBF (40 min). Cells were washed with dH₂O, stained with Alizarin Red S (2%, pH 4.3; A5533, Sigma-Aldrich; 2-3 min), and washed with dH2O (3x). Stained cells were photographed while being still under water using Zeiss Discovery V8 Stereomicroscope.

For all animal models, the dyes were prepared as follows: calcein (0.9 mg/ml; C0875, Sigma-Aldrich) and alizarin red S complex (4.5 mg/ml; A5533, Sigma-Aldrich). Solutions were prepared in 2% NaHCO₃ (71628, Honeywell Fluka) in MilliQ water, filtered using a syringe filter 22 μm polyethersulfone membrane (99722, TPP) under sterile conditions and stored at 4°C in the dark until its use (up to 1 month). The volume and ratios of injections for the different animal models are specified in table S1.