Cetylpyridinium chloride monohydrate solution

BMSCs were fixed, and 500 μl 1% Alizarin Red S (ARS) was added to the plate wells. After 15 min, the staining solution was discarded, and the plate wells were washed with phosphate‐buffered saline (PBS) three times. The results were obtained with an optical microscope. After that, cetylpyridinium chloride monohydrate solution (Sigma‐Aldrich) was added to extract the dye, and the absorbance (562 nm) was measured with a multiscan spectrophotometer.

Mineralization or calcification ability of the tested CSMs on hPDLSCs were evaluated by Alizarin Red S staining after 21 days of culturing with undiluted (1:1) cement-conditioned medium, as follows: (A) control (DMEM), (B) Osteodiff, (C) Ceraputty, (D) ERRM, and (E) Biodentine. After the culture period, the samples were rinsed with fetal bovine serum and fixed with 70% ethanol for 1 h. Then, samples were stained with 2% Alizarin Red solution (Sigma Aldrich) for 30 min in controlled conditions (dark ambient and room temperature) and solubilized using 10% cetylpyridinium chloride monohydrate solution (Sigma-Aldrich). Finally, Synergy H1 multi-mode microplate reader (BioTek, Winooski, VT, USA) was used to measure the absorbance values of the samples at 570 nm. The methodology for Alizarin Red S staining assay was based on a previous similar study [21 (link)].

Alizarin Red S staining (ARS) was conducted to evaluate hPDLSC calcified nodule formation in contact with the tested sealers (BrF, AHPbcs, and AHP) to measure of their biomineralization ability, as performed in similar studies [43 (link), 44 (link)]. Twenty thousand hPDLSCs per well were seeded onto 12-well plates (n = 3) and allowed to proliferate until confluency was attained.
For this assay, both a negative control (hPDLSCs cultured in unconditioned growth medium (DMEM; Gibco, USA)) and a positive control (hPLDSCs cultured in osteogenic medium (OsteoDiff; Miltenyi Biotec, Germany) were included for reference.
The cells were transferred into undiluted (1:1) sealer-conditioned medium and cultured for 21 days. Following the culture period, the samples were rinsed with foetal bovine serum and fixed with 70% ethanol for 1 h. The fixed samples were then stained with a 2% Alizarin Red solution (Sigma Aldrich, USA) for 30 min under controlled conditions (dark ambient and room temperature) and solubilized using a 10% cetylpyridinium chloride monohydrate solution (Sigma-Aldrich, USA). Finally, a Synergy H1 multi-mode microplate reader (BioTek, Winooski, VT, USA) was used to measure the absorbance values of the samples at 405 nm.