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.
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.
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