Alizarin red s staining

Alizarin red S staining (G3280, Solarbio, China) was used to detect calcium deposition. Three-micrometer sections of rat arterial tissues and RASMCs were fixed in 10% formalin and incubated with 2% alizarin red S solution for 15 min, McGee-Russell for 5 s, and hematoxylin for 5 min before washing with deionized distilled water for 5 min. Arterial tissue slices were then sealed with neutral gum and imaged under a microscope. The RASMCs in the six-well plates were photographed using a camera (6 D, Canon, Japan).

To identify ADSC surface markers, ADSCs at passage 3 were collected in tubes at 4 × 10⁵/tube. Next, anti-CD31-PE, anti-CD45-PE, anti-CD44-FITC, anti-CD29-FITC, anti-CD73-PE, anti-CD90-FITC, and anti-CD105-PE (all from BD Biosciences, San Jose, CA, USA) antibodies were incubated. Then, ADSCs in solution were identified by a FACS Calibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).
Trilineage differentiation was performed to show the differentiation potency of ADSCs. In brief, ADSCs were seeded into 6-well plates (5.0 × 10⁵ cells/well) with normal medium until cell confluence reached approximately 70%. The nonadherent cells were removed by replacing the medium, and the attached cells were cultured until confluence. The cells were then grown for 21 days in the adipogenic, osteogenic, and chondrogenic medium (Cyagen, Guangzhou, China). Alizarin red S staining (ARS, Solarbio, Beijing, China) was performed to assess osteogenic differentiation. Adipogenic differentiation was visualized using Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) staining. Alcian Blue (Sigma-Aldrich, St. Louis, MO, USA) staining was used to assess chondrogenic differentiation.

Cells were seeded in a 6-well culture plate at a density of 2 × 10⁵ cells/well, containing the aforementioned conditioned medium. The alkaline phosphatase (ALP) activity of the cells was assessed using the ALP activity colorimetric assay kit (BioVison, Milpitas, CA, USA) and BCA protein kit assay (Thermo Fisher Scientific, Waltham, MA, USA) following the protocol used in our previous investigation [21 (link)]. ALP staining (Promega, Madison, WI, USA) was conducted on day 7 to confirm the results of the ALP activity assay. The mineralization of rMSCs was assessed on day 21 by Alizarin Red S (ARS) staining (Solarbio, Beijing, China). Briefly, the cells were fixed with 4% paraformaldehyde for 15 min and stained with 2% Alizarin Red S solution for 30 min at 37 °C, followed by washing with PBS twice. Then, the stained calcium nodules were observed under an optical microscope.

MC3T3-E1 cells were cultured in osteogenic differentiation medium containing 100 nM dexamethasone, 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate. The medium was changed every 2 days, and osteoblast differentiation was assessed by alkaline phosphatase (ALP) staining (Solarbio, Beijing, China) and Alizarin Red S (ARS) staining (Solarbio) according to the manufacturer’s instructions on Day 7 and Day 21, respectively. Images were acquired using an inverted microscope (Olympus).