Alizarin red

2,7-Dichlorofluorescein diacetate (DCFH-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alizarin red, and ALP staining and activity kits were obtained from Beyotime (Shanghai, China). All primary and secondary antibodies and inhibitors were purchased from CST (Danvers, MA, United States). All chemicals and other agents were purchased from Sigma (St. Louis, MO, United States).

DFCs, iDFCs and dDFCs were seeded into a six-well plate (1 *10⁵ cells/well) separately and further cultured overnight for eight hours.
To induce osteogenic differentiation, cells were infected with AdBMP9, and the medium was replaced with StemPro Osteogenesis Differentiation Kit (Gibco) for two weeks. Then the differentiated cells were fixed with 4% polyoxymethylene for 20 min, followed by Alkaline phosphatase (Beyotime) staining and Alizarin Red (Beyotime) staining to assess mineral deposition. Cells infected with AdGFP were used as control.
To induce chondrogenic differentiation, cells were infected with AdBMP9, and the medium was replaced with Chondrogesis Differentiation medium (Biowit) for two weeks. The induced cells were fixed with 4% polyoxymethylene for 20 min, followed by alcian blue staining. Cells infected with AdGFP were used as control.
To induce adipogenic differentiation, cells were infected with Ad PPARγ2, and the medium was replaced with Adipogesis Differentiation medium (Biowit) for one week. The differentiated cells were fixed with 4% polyoxymethylene for 20 min and then stained with 0.3% Oil Red O (Sigma) solution to evaluate adipogenesis. Cells infected with AdRFP were used as control.

Cells were cultured in mineralization-inducing medium for 2 weeks, fixed with 75% ethanol, and stained with 2% Alizarin red (Beyotime Institute of Biotechnology, Shanghai, China). Alizarin red was destained using 10% cetylpyridinium chloride (CPC) in 10 mM sodium phosphate for 30 min at room temperature for quantitative estimation of calcium mineral content. The concentration was evaluated by measuring the absorbance at 562 nm on a 96-well plate reader^{34 (link), 35 (link)}.

To detect mineralization, cells were induced for 2 weeks, fixed with 70% ethanol, and stained with 2% alizarin red (Beyotime Institute of Biotechnology). To quantify the calcium mineral density, alizarin red was destained with 10% cetylpyridinium chloride (CPC) in 10 mmol/L sodium phosphate for 30 min at room temperature. Calcium concentration was determined by measuring the absorbance of the test sample at 562 nm on a multiplate reader. A standard calcium curve was prepared with calcium dilutions in the same solution. The final calcium level in each group was normalized to the total protein concentration detected in duplicate plates.

Cells from each group were seeded in 24-well plates at a density of 2 × 10^4 cells per well, maintaining consistent culture conditions and cell densities. When the cell confluence reached 80–90%, cells were cultured with MC3T3-E1 cell osteogenic differentiation induction medium for a period of 14 days. The experimental group received daily irradiation with an energy density of 5.3 J/cm². After 14 days, all groups were stained with Alizarin Red (Beyotime Institute of Biotechnology, Shanghai, China).