Paraformadedyde

The accumulated calcium deposition was analyzed using Alizarin Red S staining, following a method developed for a previous study [22 (link)]. In brief, the specimens were fixed with 4% paraformadedyde (Sigma-Aldrich) for 15 min and then incubated in 0.5% Alizarin Red S (Sigma-Aldrich) at pH of 4.0 for 15 min at room temperature. Then, the photographs were observed using an optical microscope (BH2-UMA; Olympus, Tokyo, Japan) equipped with a digital camera (Nikon, Tokyo, Japan) at 200 magnifications. After this, the scaffolds were washed with PBS and quantified using a solution of 20% methanol and 10% acetic acid in water. After 15 min, the liquid was transferred to a 96-well plate, and the quantity of Alizarin Red was determined using a spectrophotometer at 450 nm.

Calcium depositions after seven and 14 days of cell culture were analyzed using Alizarin Red S staining in accordance to methods previously described in Reference [13 (link)]. In brief, the cells were fixed for 15 min using 4% paraformadedyde (Sigma-Aldrich), and further cultured using 0.5% Alizarin Red S (Sigma-Aldrich) for 15 min at pH 4.0 room temperature, whilst undergoing 25 rpm of oscillation. Cells were subsequently washed thoroughly, and an optical microscope (BH2-UMA, Olympus, Tokyo, Japan) furnished with a digital camera (Nikon, Tokyo, Japan, 200X) was used to capture images of the stains. Furthermore, the specimens were soaked in 1.5 mL of 5% SDS in 0.5N HCl at room temperature for 30 min, then centrifuged at 5000 rpm for 10 min. The supernatant was transferred to a 96-well plate to measure for the absorbance at 405 nm wavelength (Infinite Pro M200). The above protocol was done to quantify for the amount of calcium depositions after the Alizarin Red S stains.

The Ca deposition was considered after two weeks by Alizarin Red S-stained as described in a previous study [28 (link),29 (link),30 (link)]. In briefly, hBMSCs were fixed by paraformadedyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 20 min and then incubated with 0.5% Alizarin Red S (pH 4.0, Sigma-Aldrich, St. Louis, MO, USA) for 20 min in the shaker (30 rpm). In addition, all specimens were immersed with 1.0 mL of 5% SDS in 0.5N HCl for 0.5 h at room temperature, following which the tubes were centrifuged at 6000 rpm for 15 min and the mixtures were transferred to the 96-well plate to quantify the calcified nodules.