L proline

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.

Unless otherwise specified, all reagents and materials used were obtained without additional purification from commercial suppliers. Betulinic acid (BA) (≥98%), 1-octanol, and acetonitrile (for HPLC, gradient grade) were obtained from Sigma-Aldrich (Steinheim am Albuch, Germany). L-aspartic acid, L-isoleucine, L-leucine, L-methionine, L-proline, L-threonine, and L-valine of purity ≥98% were purchased from FluoroChem (Derbyshire, UK). L-serine (≥98.5%), L-phenylalanine (≥98.5%), L-proline (≥98.5%), and L-cysteine (≥98%) were provided by Carl Roth (Karlsruhe, Germany). L-alanine (>99.0%) was acquired from Bachem (Bubendorf, Switzerland) and L-lysine (≥98.0%, anhydrous) from Glentham Life Sciences (Corsham, UK). L-phenylalanine (99%) was provided by Alfa Aesar (Ward Hill, MA, USA). L-tryptophan (>98.5%) was purchased from TCI. Thionyl chloride (99.5%) was provided by Across Organic Geel (Geel, Belgium). Ammonium hydroxide solution 25% (NH₃.H₂O) of analytical grade was purchased from StanLab (Lublin, Poland). Ethanol (99.8%) was provided by Merck (Darmstadt, Germany). Methylene chloride and DMSO of high purity were provided by Chempur (Gliwice, Poland). Deuterated dimethyl sulfoxide (DMSO-d₆) (99.8%) was obtained from Deutero GMBH (Kastellaun, Germany).

Chondrogenesis was induced in chondrocytes and BMMSCs using standard protocol used by State Research Institute Center for Innovative medicine. Chondrogenic medium included high glucose (4.5 g/L) DMEM medium (Merck Millipore), 1% penicillin/streptomycin, 1% insulin-transferrin-selenium (all from Gibco Life Technologies), L-proline (350 nM) (Carl Roth, Karlsruhe, Germany), dexamethasone (100 nM) (Sigma Aldrich), ascorbic acid-phosphate (170 nM) (Sigma Aldrich) and TGF-β3 (10 ng/mL) (Gibco, Life Technologies). Incomplete chondrogenic medium (the same constituents without TGF-β3) was used as control.
Furthermore, control and TGF-β3 groups were subdivided into 3 subgroups: (1) with addition of DMSO, which is a solvent for nifedipine and BayK8644, (2) with nifedipine (10 μM), and (3) BayK866 (10 μM). In total, 6 subgroups of different stimulation conditions were applied for cell cultivation in pellets in 15 mL tubes (Gibco, Life Technologies) for 21 day. Each subgroup was made in three replicates. Extracellular matrix formation in pellets was assessed by histological methods.