Ascorbic acid phosphate

Modified eagle’s medium (α-MEM), Dulbecco’s modified eagle’s medium (DMEM), trypsin-EDTA, and fetal bovine serum (FBS) were obtained from Gibco-BRL (Grand Island, NY). Recombinant mouse M-CSF and RANKL were obtained from Peprotech (Rocky Hill, NJ, USA). Rabbit polyclonal antibodies specific for phospho-ERK, phospho-JNK, phospho-p38, phospho-PLCγ2, phospho-Syk, IκB, phospho-IKK (Ser176/180), phospho-STAT3 (Ser727), and phospho-Gab2 were purchased from Cell Signaling Technology (Danvers, MS, USA). Mouse monoclonal antibodies specific for NFATc1, TRAF6, cathepsin K, c-Fos, STAT3, TBP, and NF-κB were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-actin antibody, β-glycerophosphate disodium salt hydrate, MSM, ascorbic acid phosphate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and L-glutamine were purchased from Sigma Chemical Co. (St. Louis, MO). A bone resorption assay kit was purchased from COSMO BIO Co. (Tokyo, Japan). The electrophoretic mobility shift assay (EMSA) kit and oligonucleotide probes (NF-κB) were purchased from Panomics (Redwood City, CA). The RNeasy mini kit was purchased from Qiagen (Hilden, Germany) and enhanced chemiluminescence (ECL) plus detection kit from Amersham Pharmacia Biotech. (Piscataway, NJ).

To induce osteoblast differentiation of hMSCs or mMSCs, the culture medium was removed after cell reach confluency and replaced by osteogenic medium containing α-MEM with 10% FBS, 10 mM β-glycerol phosphate, 10 nM dexamethasone and 50 μg/ml ascorbic acid phosphate (Sigma-Aldrich, China). The medium was changed every 3 days for 2–4 weeks. At the end point of the assays, Alizarin-Red staining (Sigma, China) was used to detect the calcium deposition associated with osteoblasts. For Alizarin-Red staining, cells were fixed in 70% ethanol for 30 min and then subjected to 1% Alizarin-Red solution for 1 min. Images of the stained cells were scanned and captured. Alizarin Red staining were then dissolved using a solution of 20% methanol and 10% acetic acid in water and quantified by measuring its absorbance at 450 nm. Relative Alizarin Red staining was then calculated as a fold change of the control. For ALP staining, cells were fixed with 70% ethanol for 30 min and then incubated with the BCIP/NBT liquid substrate system (Sigma-Aldrich, China) at 37°C for 30 min. Images of the stained cells were scanned and captured. For quantitative analysis, the ALP stain was extracted with 10% cetylpyridinium chloride for 15 min and quantified by measuring its absorbance at 540 nm. Relative ALP staining was then calculated as a fold change of the control.

The cells were detached, counted and transferred into conical 15 mL tubes (250,000 cells/tube). After that, the cells were washed with DMEM medium without FBS, centrifuged at 600× g for 5 min and supernatant was changed to the chondrogenic medium (high glucose (4.5 g/L) DMEM medium) and incubated for 24 h. During this period, the cells formed 3D cell pellets. The next day, chondrogenic medium (high glucose (4.5 g/L) DMEM medium, 1% PS (Merck, Rahway, NJ, USA), 1% insulin-transferrin-selenium (ITS) (Gibco Life Technologies, Grand Island, NY, USA), 0.35 mM L-proline (Carl Roth, Karlsruhe, Germany), 10⁻⁷ M dexamethasone, 0.17 mM ascorbic acid phosphate (Sigma Aldrich, St. Louis, MO, USA) with or without 10 ng/mL of TGF-β3 (Thermo Fisher Scientific, Waltham, MA, USA) was added. The chondrogenic differentiation of each sample was evaluated comparing samples with and without growth factor TGF-β3. The chondrogenic medium was changed three times a week.
The effect of nifedipine and BayK8644 on chondrogenesis of MenSCs, BMMSCs and chondrocytes was investigated as follows: 1 μL/mL of DMSO (Control), 10 μM of nifedipine and 10 μM of BayK8644 were added to the cells in complete chondrogenic media and incubated for 21 days, changing chondrogenic medium as usual. After chondrogenic differentiation, the cell pellets were analyzed by RT-qPCR.