L ascorbic acid

The osteogenic and adipogenic differentiation capacity of AL-MSCs and HD-MSCs was evaluated at passage P2/P3 [24 (link)]. For osteogenic differentiation, the induction medium was αMEM, 10% FBS, 10⁻⁷ M dexamethasone, 50 mg/mL L-ascorbic acid, and 5 mM β-glycerol phosphate (all from Sigma Aldrich, St. Louis, MO, USA); for adipogenic differentiation, the induction medium was αMEM, 10% FBS, 10⁻⁷ M dexamethasone, 50 mg/mL L-ascorbic acid and 5 mM β-glycerol phosphate, 100 mg/mL insulin, 50 mM isobutyl methylxanthine (Sigma-Aldrich), and 0.5 mM indomethacin (MP Biomedica, Illkirch, France). In both protocols, differentiation was evaluated after 21 days. In vitro osteogenic differentiation was evidenced by phosphatase alkaline activity stained in blue/violet by BCIP/NBT and calcium deposition stained by Alizarin Red S (both from Sigma-Aldrich). In vitro adipogenic differentiation was evidenced by the appearance of fat droplets stained with Oil Red O (Bio Optica, Milan, Italy).

The FRAP assay was conducted as recommended by Benzie and Strain [46 (link)] with slight adjustments. Firstly, the fresh blue FRAP reagent was prepared by mixing 30 mL of acetate buffer, 3 mL of 2,4,6-tris [2-pyridyl]-s-triazine (TPTZ) (Merck, Cat no. T1253) with 3 mL of FeCl₃ solution and 6.6 mL of distilled water. Then, an L-ascorbic acid (Sigma-Aldrich^®, Cat no. A5960) standard series of 50 μM, 100 μM, 200 μM, 500 μM, and 1000 μM was prepared from a 1 mM of L-ascorbic acid stock solution in distilled water. Lastly, in a clear 96-well plate, 300 μL of the FRAP reagent was added to 10 μL of L-ascorbic acid working standard solutions and EO sample (2.0 mg/mL) in triplicate (n = 3). Gallic acid was used as a positive control. For the blank, the phosphate buffer (pH 3.6) was added instead of the sample. The total volume of the assay was 310 μL. The absorbance of TPTZ-Fe (II) in the samples was read at 593 nm at 37 °C for 30 min. The results were calculated using the linear regression (R² = 0.9965) of the L-ascorbic acid (AA) standard series concentrations (μM) and absorbance signals expressed as mean (±SD) of triplicate measurements in μmol L-ascorbic acid equivalents per litre of the sample tested (μmol AAE/L).

The generation and culturing of human NPCs has been previously described (37 (link)). NPCs were grown in N2B27 medium (DMEM-F12/Neurobasal at 50:50 supplemented with 1% penicillin/streptomycin/glutamine, 1% B27 supplement without vitamin A, and 0.5% N2 supplement) containing 3 μM CHIR99021 (Cayman), 150 μM L-ascorbic acid (Sigma-Aldrich), and 0.5 μM Smoothened Agonist (SAG) (Cayman). For differentiation, the medium was replaced with N2B27 containing 1 ng/ml BDNF (Miltenyi Biotec), 0.2 mM L-ascorbic acid, 1 μM retinoic acid (Sigma-Aldrich), 1 ng/ml glial cell line-derived neurotrophic factor (GDNF) (Miltenyi Biotec), and 0.5 μM SAG. On day 8, the medium was changed for inducing neural maturation to N2B27 containing 5 ng/ml activin A (Miltenyi Biotec), 0.1 mM dbcAMP (Sigma-Aldrich), 2 ng/ml BDNF, 0.2 mM L-ascorbic acid, 1 ng/ml TGFβ-3 (Peprotech), and 2 ng/ml GDNF. On day 10, the cells were seeded on a four-well plate for immunofluorescence and 2.5 μM N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) (Sigma-Aldrich) and activin were added to the maturation medium. After 2 d, DAPT and acitivin were removed and the cells were further grown in the maturation medium for neural maturation until day 30.

The MPCs underwent multi-lineage differentiation analysis to determine their osteo/chondro/adipo-genic capacity^{30 (link),34 (link)}.
Osteogenesis: For each replicate, 5 × 10⁵ cells were seeded into each well in a 24-well plate and then placed into DMEM/F-12 media that contained Dexamethasone (final concentration (FC): 100 nM) (Sigma), L-Ascorbic Acid (FC: 50 μg/mL) (Sigma), β-Glycerolphosphate (FC: 10 mM) (Sigma).
Adipogenesis: For each replicate, 5 × 10⁵ cells were seeded into each well in a 24-well plate and then placed into DMEM/F-12 media that contained Dexamethasone (FC: 1 μM) (Sigma), Insulin (FC: 10 μM) (Sigma), Indomethacin (FC: 200 μM) (Sigma), and Isobutylmethylxanthine (FC: 500 μM) (Sigma).
Chondrogenesis: For each replicate, 5 × 10⁵ cells were pelleted through centrifugation and placed into DMEM/F-12 media that contained Dexamethasone (FC: 10 nM) (Sigma), L-Ascorbic Acid (FC: 50 μg/mL) (Sigma), MEM Non-Essential Amino Acids (FC: 1%) (MEM-NEAA Gibco), Transforming growth factor (TGF)-β3 (FC: 10 ng/mL) (Peprotech), Bone morphogenetic protein (BMP)-2 (FC: 500 ng/mL) (Peprotech), Insulin transferrin selenium (FC: 1%) (Lonza- BioWhittaker), and sodium pyruvate (FC: 1%) (ThermoFisher). Media was adjusted to neutral pH (7.0–7.6).
After 21 days, differentiation was assayed using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and histological staining.

Human VICs plated in 48-well plates were cultured in control normal medium (termed NM) containing DMEM with 5% FBS, or osteogenic medium (termed OM) containing, DMEM with 5% FBS, 10 nmol/L dexamethasone (MP Biomedicals, Santa Ana, CA), 10 mmol/L β-glycerophosphate (EMD Millipore, Burlington, MA), and 50 μg/mL L-ascorbic acid (Sigma-Aldrich), or pro-calcifying medium (termed PM) containing, DMEM with 5% FBS, 2 mmol/L NaH₂PO₄ (Sigma-Aldrich) (pH 7.4) and 50 μg/mL L-ascorbic acid) for 28 days. To assess TNAP calcification dependency, 1 μmol/L TNAP inhibitor (EMD Millipore) or 0.01% DMSO vehicle control as added to OM and PM. Calcium deposition was stained using 2% Alizarin red (Lifeline Cell Technology, Frederick, MD). Briefly, cells were fixed by 10% formalin for 15 minutes and washed twice with distilled water. After adding Alizarin red solution, cells were stained for 15 minutes at room temperature. Excess stain was washed twice with distilled water. Alizarin red stain was quantified by extracting the stain with 100 mmol/L cetylpyridinium chloride (bioWORLD, Dublin, OH) and measuring the absorbance at 540 nm.

The FRAP assay was conducted as recommended by Benzie and Strain [68 (link)] with slight adjustments. Firstly, the fresh blue FRAP reagent was achieved by mixing 30 mL of acetate buffer, 3 mL of 2,4,6-tris[2-pyridyl]-s-triazine (TPTZ) (Merck, Cat no. T1253) with 3 mL of FeCl₃ solution and 6.6 mL of distilled water. Then, an L-ascorbic acid (Sigma-Aldrich^®, Cat no. A5960) standard series of 50 μM, 100 μM, 200 μM, 500 μM, and 1000 μM was prepared from a 1 mM of L-ascorbic acid stock in distilled water. Lastly, in a clear 96-well plate, 300 μL of the FRAP reagent was added to 10 μL of L-ascorbic acid working standard solutions and EO sample (2.0 mg/mL) in triplicate (n = 3). Gallic acid was used as a positive control. For the blank, the phosphate buffer (pH 3.6) was added instead of the sample. The total volume of the assay was 310 μL. The absorbance of TPTZ-Fe (II) in the samples was read at 593 nm at 37 °C for 30 min. The results were calculated using the linear regression (R² = 0.9965) of the L-ascorbic acid (AA) standard series concentrations (μM) and absorbance signals expressed as mean (±SD) of triplicate measurements in μmol L-ascorbic acid equivalents per liter of the sample tested (μmol AAE/L).

The antioxidant capacity of the PGBE complex was investigated using ABTS cation radical scavenging activity assay, as described by Cai et al [13 (link)]. ABTS radical cations generated by reacting 7.2 millimolar (mM) ABTS and 2.6 mM potassium persulfate for 24 h in a dark room were diluted with phosphate-buffered saline (pH 7.4) so as to give 20% to 30% inhibition of the blank absorbance at 734 nm with a spectrophotometer (Biotek Instruments, Inc., Winooski, VT, USA). Subsequently, 10 μL of PGBE complex or L-ascorbic acid was added into 190 μL of ABTS solution to get a final PGBE concentration of 0.16% to 0.01% (vol/vol) or L-ascorbic acid (1.0, 0.1 and 0.01 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) in a 96-well plate (Thermo Fisher, USA), and reacted for 10 min in a dark room. The optical density (OD) of the resulting mixture was measured at 734 nm, and radical scavenging activity was calculated using the following formula: 1 – (OD blank – OD sample)/OD blank.