Calcium colorimetric assay

hPDLSCs (P3) were seeded in 12-well plates at 5 × 10⁴ cells/well and cultured until 80% confluence. Then, hPDLSCs were cultivated in osteogenic differentiation medium consisting of α-MEM containing 10⁻⁸ M dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 10 mM β-glycerophosphate (Sigma-Aldrich), 50 ng/mL ascorbic acid (Sigma-Aldrich), 10% FBS, and 1% penicillin–streptomycin. The osteogenic medium was changed every 2 d.
Induction of hPDLSCs was completed with various concentrations of TGF-β3 (0, 62.5, 250, and 500 ng/mL). After cultivation for 3 and 7 d, hPDLSCs were stained with an alkaline phosphatase (ALP) staining kit (Beyotime Institute of Biotechnology, Shanghai, China) to test their early osteogenic differentiation capacity. After cultivation for 14 d, the hPDLSCs were stained with alizarin red (Cyagen Biosciences, CA, USA) to test their late osteogenic differentiation capacity.
Four concentrations of TGF-β3 (0, 62.5, 250, and 500 ng/mL) were loaded in CS. After cultivation for 3, 7, and 14 d, the ALP activity of hPDLSCs on TGF-β3/CS was determined using the ALP staining kit. To detect and quantify osteocalcin in hPDLSCs at the late stage of each group of materials, a calcium colorimetric assay (Sigma-Aldrich) was applied after cultivation for 14, 21, and 28 d.

Calcium levels were measured using a calcium colorimetric assay purchased from Sigma-Aldrich. Ileal contents were diluted 1:200 in PBS to fit into the linear range of the assay. Briefly, 90μL of the chromogenic reagent was added to each well. 50μL of either sample or diluted calcium standard were added to each well. 60μL of calcium assay buffer are then added to each well and samples were incubated for 5–10 minutes at room temperature and absorbance was measured at 575nm in a Spectramax M2 microplate reader (Molecular Devices). Each sample was measured in triplicate.