Ascorbic acid

Osteogenic induced medium contained complete culture medium supplemented with 100 nM dexamethasone (Solarbio, Beijing, China), 10 mM β-glycerophosphate (Solarbio) and 50 mg/l ascorbic acid (Solarbio). Adipogenic medium contained complete culture medium supplemented with 1 μM dexamethasone (Solarbio), 0.2 mM indomethacin (Solarbio), 0.01 g/l insulin (Solarbio) and 0.5 mM isobutyl-methylxanthine (Solarbio). Cells were cultured in 24-well plates with osteogenic differentiation medium or adipogenic differentiation medium. The medium was replaced every other day. Osteogenic differentiation was evaluated with alkaline phosphatase (ALP) and alizarin red (AR) staining (Sigma-Aldrich). Adipogenic differentiation was evaluated with Oil Red O staining (Solarbio). All methods followed protocols recommended by the manufacturers.

The metabolites contents of AsA-GSH cycle, including AsA and GSH, and their oxidized forms, DHA and GSSG were determined using Ascorbic Acid or Glutathione Colorimetric Assay Kit purchased from Solarbio Science and Technology (Beijing, China), as per the manufacturer’s protocol. The assays of hydrogen peroxide (H₂O₂) content were conducted according to the method published previously (Velikova et al., 2000 (link)). The membrane damage was determined with regard to thiobarbituric acid- reactive substances (TBARS) content, a product of lipid peroxidation (Hodges et al., 1999 (link)). Briefly, the soybean root samples (0.5 g) were extracted with 10 mL 0.1% (w/v) trichloroacetic acid (TCA), and then the homogenate was centrifuged at 10,000 g for 10 min at 4°C. The supernatant was used for the determination of H₂O₂ and TBARS contents. The total reaction volume of H₂O₂ assay was 2 mL containing 0.5 mL of the supernatant, 1 mL 1M potassium iodide and 0.5 mL 10 mM potassium phosphate buffer (pH 7.0). The intensity was measured at 390 nm. The total reaction volume of TBARS assay was 2 mL containing 0.5 mL of the supernatant and 1.5 mL 0.5% (w/v) thiobarbital acid in 15% TCA. The absorbancy of supernatant was read at both 532 and 600 nm.

To mimic the bone matrix, MC3T3-E1 cells were induced for osteogenic differentiation in MEM-α media containing 10% of FBS, 10 mM β-glycerophosphate (Solarbio), and 50 µg/ml of ascorbic acid (Solarbio) for 9 days. Subsequently, the cultured MC3T3-E1 cells were treated with 20 mM NH₄OH (Sigma, USA) and 0.5% Triton X-100 (Solarbio) for 5 min to form the bone matrix layer. GFP expressing breast cancer cells (2 × 10⁵ cells/well) were added into each bone matrix layer and incubated for 15 min. After aspirating floating cells, the adhering cells were washed with PBS and counted under a fluorescent microscope.

A Cu electrode, Ag/AgCl electrode, and Pt electrode were purchased from Aidahengsheng, Tianjin, China. Ascorbic acid (99%, analytical pure) was purchased from Solarbio, Beijing, China. Lactose (99%, analytical pure) was purchased from Sinopharm Chemical Reagent, Beijing, China. Dopamine (99%, analytical pure) and uric acid (99%, analytical pure) were purchased from Aladdin, Shanghai, China. All agents were used without further purification.

PLGA (Medical grade, LA: GA = 75:25) was purchased by Regenovo Biotechnology Co., Ltd., Hangzhou, China. 1,4‐Dioxane was obtained in Aladdin Biochemical Technology Co., Ltd., Shanghai, China. DBM‐MPs were autonomously synthesized by the laboratory. MH‐NPs with an average particle size of 50 nm were purchased from Zhongke Leiming Technology Co., Ltd., Beijing, China. Fetal bovine serum (FBS), α‐MEM, DMEM, penicillin–streptomycin, and trypsin were purchased from Gibco Life Technologies Co. Grand Island, USA. Dexamethasone, ascorbic acid, and β‐Sodium Glycerol 3‐phosphate were purchased from Solarbio Science&Technology Co., Ltd., Beijing, China. PBS, Tween‐20, and bovine serum albumin (BSA) were bought from Sigma–Aldrich Products (USA).