Alizarin red s ar

At 1, 7 and 14 days, the hPDLSC–CPC disks were fixed with 10% formaldehyde for 30 minutes and stained with Alizarin Red S (ARS, Millipore, Billerica, MA) for 30 minutes (n = 6). The ARS stained the mineral deposits synthesized by the cells into a red color. After staining, the ARS solution was removed, and the disks were washed gently with PBS to remove any loose ARS. Then the specimens were photographed. The CPC disks were soaked in 10% cetylpyridinium chloride (Millipore) for 1 hour to extract the stained deposit.^{35 (link)} The ARS concentration was measured at optical density of 652 nm using the microplate reader (SpectraMax M5). Blank CPC scaffolds with the same treatments, but without cell seeding, were also measured. The value of blank CPC scaffolds was subtracted from the value of the cell-seeded scaffolds to calculate the mineral concentration synthesized by the cells.^{36 (link)} The concentration of control group at 1 day was used as the calibrator.

Adipogenic and osteogenic differentiation of AMSCs were induced according to a previously reported procedure (18 ). Briefly, for adipocytic differentiation we grew AMSCs for 2-4 weeks in 1 mM dexamethasone, 10 μg/ml insulin, and 0.5 mM isobutylxanthine (Sigma, Germany) in alpha MEM medium that contained 10% FBS. Oil red O staining was used for adipocytes according to standard techniques.
AMSCs were induced to osteogenic differentiation over a 3-4 week period in osteogenic medium that consisted of AMSCs growth medium supplemented with 100 nM dexamethasone, 0.05 mM ascorbic acid, 10 mM β-glycerophosphate, and 10 nM 1α,25-dihydroxyvitamin (Sigma, Germany). Alizarin red S (ARS, Millipore, Germany) staining was used for osteoblasts according to standard techniques.

Alizarin Red S (ARS) (Sigma-Aldrich) was used to quantify calcific nodule formation (33 (link), 34 (link)). Briefly, hydrogels were washed with 1× phosphate buffered saline (PBS, Omnipur^®, Baltimore, MD, USA) and fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) overnight at 4°C. A 40 mM ARS stain solution was added, and then rinsed with 1× PBS. Bright field images were taken using a Nikon Eclipse Ts2 at 20× magnification to visualize ARS-stained area. After imaging, a 10% acetic acid solution was used to release ARS and a 10% ammonium hydroxide solution was used to neutralize. Absorbance was measured with a plate reader at 405 nm and concentration was calculated using a standard curve (35 (link)).

VSa13 cells were cultured as described above and seeded in 24-well plates at a density of 5 × 10⁴ cells per well. Extracellular matrix (ECM) mineralization was induced in confluent cultures by supplementing culture medium with L-ascorbic acid (50 μg/mL), β-glycerophosphate (10 mM) and calcium chloride (4 mM). Extract and fractions dissolved in distilled water (AF), 50% ethanol (CE) or 100% ethanol (EAF) were added to the culture medium with final concentrations ranging from 0.05 to 250 μg/mL, as described above. After 17 days of culture, mineral deposition was revealed through alizarin red S (AR-S; Sigma-Aldrich) staining and quantified by spectrophotometry (48 (link)).

Mineralization of the MC3T3-E1 cells was assessed on day 14. The colonies were fixed with 70% ethanol for 10 min at room temperature, rinsed with water, and then stained with 40 mM of Alizarin red S (ARS) (Sigma-Aldrich). For quantification of the mineralized nodules, the stained ARS was extracted with 10% cethylpyridinium chloride, and its absorbance was read at 562 nm with a microplate reader (BIO-RAD).