Alizarin red s solution

The MC3T3-E1 cells were incubated in osteogenic medium for 21 days. Before staining, 4% paraformaldehyde was used to fix cells for 20 min. Incubation of cells was performed in an Alizarin red S (ARS) solution (Cyagen Biosciences) at pH 4.2 for 40 min. The images were taken after 3 times washing with ddH₂O. After that, the calcium nodules were treated with 5% perchloric acid solution at 37 °C for 30 min, and the optical density was assessed at 420 nm.

Alizarin red staining was applied to assess the mineral nodule formation ability as described previously [17 (link)]. The culture PDLSCs were fixed in 4% paraformaldehyde for 20 minutes at room temperature and were then stained by Alizarin Red S Solution (Cyagen, cat#ALIR-10001, Guangzhou, China) for 5 minutes at room temperature. Excessive dye was removed by several washes in deionized water. Then, the staining was imaged with an inverted light microscope and proceed to semiquantification. To quantify the matrix mineralization, the stain was eluted by 100 mM cetylpyridinium chloride for 10 minutes with gentle rotation and spectrophotometric absorbance at 562 nm was detected. ARS intensity relative to the control group was calculated after normalization to the total protein content.

Briefly, after three-week osteogenic differentiation induction, the fixed cells were stained with Alizarin Red S solution (Cyagen Biosciences) for 20 min at room temperature.

6-aminocaproic acid, L-hydroxyproline, L-alanine, L-phenylalanine, L-proline and L-lysine (99.0%, biochemical grade) were purchased from Hebei kairuijie amino co., Ltd. (Xintai, China). The pearl powder with 0.5–10 µm diameter was purchased from STS Biotech Co., Ltd. (Wuxi, China). Minimum essential medium eagle-alpha modification (α-MEM), fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin sulfate were purchased from Gibco (Grand Island, NY, USA); Cell counting kit-8 (CCK-8) was purchased from Nanjing Keygen Biotech Co., Ltd. (Nanjing, China); Glutaraldehyde (50.0%, AR) and formaldehyde (37.0–40.0%, AR) were purchased from Chengdu Chron Chemicals Co., Ltd. (Chengdu, China); Rhodamine-phalloidin was obtained from Servicebio Co., Ltd. (Wuhan, China), Dexamethasone (98.0%), ascorbic acid (99.0%), β-glycerophosphate sodium (99.0%) and hexadecylpyridinium chloride (99.0–102.0%) were purchased from Sigma-Aldrich Co., Ltd. (Saint Louis, MO, USA); Mouse Alkaline Phosphatase Activity (ALP) Enzyme-linked Immunosorbent Assay (ELISA) Kit was purchased from Shanghai MLBIO Biotechnology Co., Ltd., Alizarin Red S solution was purchased from Cyagen Biosciences Co., Ltd. (Guangzhou, China), and TRIZOL reagent from Ambion (Carlsbad, CA, USA).

Cells were cultured in 24-well plates at a density of 1×10⁵ cells per well and cultured in osteogenic medium for at least 21 days. During the late period of osteogenic differentiation, calcium deposition in the osteoblast was examined by alizarin red S staining. In brief, we used PBS to clean the cultured cells three times, fixed the cells for 10 min in 4% PFA, and stained them with 1% alizarin red S solution (Cyagen, United States) for 10 min. To remove non-specific staining, we used 50% ethanol to clean the stained cells thoroughly. Calcium deposits were represented by positive red staining, and representative images were taken.

The differentiation medium used in this study was human BMSC osteogenic differentiation basal medium purchased from Cyagen. Briefly, BMSCs were seeded at 5,000cells/100 μl in 96-well plates. For evaluating mineralization, cells were induced for 21 days, washed 1–2 times with PBS, and fixed with 4% paraformaldehyde for 30 min. This was supplemented with 50μl of freshly prepared alizarin red S solution (Cyagen), and cells were incubated for 3–5 min.