Cell Culture and TreatmentThe T/C-28a2 chondrocyte cell line (HSS Research Institute, New York, NY, USA) was maintained in Dulbecco´s Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS; Gibco), penicillin (100 U/ml)/streptomycin (100 μg/ml) (Gibco). PA, OL and PA/OL in a ratio of 1:2 stock solutions were prepared at 5 mM in 10% bovine serum albumin (BSA; Sigma Aldrich, St.Louis, MO,USA), as a vehicle for PA 200 mM and OL 700 mM (Sigma Aldrich) solutions. These solutions were incubated at 55° for 30 minutes (21). Then, for each condition of FA, the different test concentration solutions were prepared in DMEM without FBS. For the baseline condition (BC), DMEM without FBS was used, to which the corresponding BSA percentage was added.The experiments were performed after 12h of incubation with the FA.
Cell viability assay
Cell viability after the exposure to various concentrations of PA, OL and PA/OL mix (0.4, 0.7, and 1mM) was assessedusing the MTS assay (Promega). The incubation medium with tested compounds (12 hours) was changed to a medium with the MTS reagent at the end of exposure and cells were incubated for additional 3 h at 37°C. The absorbance of dissolved formazan was measured at 490 nm in a microplate reader. Data are displayed as a percentage of cells with basal condition (BC).
Quantification of superoxide anion(O2-) production
To measure superoxide anion (O2-) production, cells were incubated with 4 µg/µl Mitosox™ in Hank's Buffered Salt Solution (HBSS) (Thermo Fisher Scientific, Waltham, MA, USA) for 15 minutes at 37ºC and 5% CO2 in darkness. The pelleted cells were re-suspended to measure fluorescence by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).
Quantitative Real-Time PCR
Real-time PCR was performed with a LightCycler 480-II Instrument (Roche Diagnostics, Risch-Rotkreuz, Suiza) with TaqManUniversal Master Mix (Roche). Gene expression was calculated relative to the housekeeping gene (RPL13A). Sequence primers, probes and PCRconditions are shown in Additional file 1.Table S1.
Extracellular Flux Analysis
The Seahorse XFpExtracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA) was used to estimate the chondrocyte metabolic stress. Based on the presence of sensors sensitive to the concentration of oxygen and protons, this technique allows to measure simultaneously, in living cells, the two main cellular energy pathways:mitochondrial respiration and glycolysis.
- Mitochondrial stress testing
2x104 cells were seeded in XFp Cell Culture Miniplates (Agilent Technologies). Following incubation with the FA, the assay was carried out following the manufacturer's recommendations. The oxygen consumption rate (OCR) was determined in the presence of specific mitochondrial modulators successively added: 2mM oligomycin (OLG); 1mM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP); and finally a mixture of rotetone and antimycin A (Rot/AntA) 0.5mM. Each condition was performed in triplicate and the data obtained (normalized by cell number) wasused to calculate the Basal respiration, ATP synthesis, Proton leak, Maximal respiration, Spare respiratory capacity, and the Non-mitochondrial respiration, expressed as OCR (pmol/min)/103 cells; and Coupling efficiency as a percentage (Additional file 2.Table S2a).
- Glycolysis stress testing
2x104 cells were seeded in XFp Cell Culture Miniplates.Following incubation with the FA, the cells were incubated in medium without glucose or pyruvate and the extracellular acidification rate (ECAR) was measured. Then, the different modulators were separately injected: 10mM glucose, 2µM OLG and 50mM 2-deoxyglucose (2-DG). Each condition was performed in triplicate and the data obtained in the test was used to calculate the Glycolysis rate and the Glycolytic capacity, expressed as ECAR (mpH/min)/103cells; and Glycolytic reserve expressed as a percentage (Additional file 2.Table S2b).
Assessment of energy balance
Contribution of mitochondrial oxidative phosphorilation system (OXPHOS) and glycolysis to the production of ATP
Taking into account that, under normal conditions, the stoichiometry ratio of OCR and proton production rate (PPR) withthe ATP production is 5 and 1 respectively (22), from the OCR and PPR data obtained from the Seahorse XFp Extracellular Flux Analyzer, the rate of ATP production (pmol / min) due to OXPHOS and glycolysis was estimated.
Quantification of total and mitochondrial ATP by luminescence
To measure intracellular ATP, an ATP bioluminescence assay kit (Roche Applied Science)was used according to the manufacturer’s recommendations. T/C-28a2 chondrocytes were treated with FA before incubation for two hours with a solution of 156mM NaCl, 3mM KCl, 2mM MgSO4, 1.25mM KH2PO4, 2mM CaCl2 dihydrate, 20mM HEPES and 10mM glucose for the quantification of total ATP; or 5mM 2-DG and 5mM of sodium pyruvate for the quantification of mitochondrial ATP. The ATP levels were normalized to the protein concentration.
Mitochondrial DNA (mtDNA) copy number quantification
To confirm the level of mitochondrial DNA recovered after incubation with FA, the mtDNA copy number was determined by real time polymerase chain reaction as previously described (23). The targeted genes were the mitochondrial12S ribosomal gene (forward 5´-CCACGGGAAACAGCAGTGAT-3', reverse 5´-CTATTGACTTGGGTTAATCGTGTGA-3') and RNAseP (forward: 5´-GCACTGAGCACGTTGAGAGA-3'; reverse: 5´-CCAGTCGAAGAGCTCCAGA-3'). For determining mtDNA copy number, an independent standard curve was generated for each gene (12S-rRNA and RNAseP). The total mtDNA copy number was determined from the Ct values and was extrapolated into the external standard curve. The concentration for each gene was obtained in the analyzed samples. MtDNA copy number values were expressed by the ratio 12S rRNA/RNAseP. To normalize the values between all experiments, we established the mtDNA copy number using the BC condition as 100%.
Evaluation of lipid accumulation
104 cells were seeded in each well of a chamber slide (Thermo Fisher Scientific) and incubated with the different FA conditions described above. After fixation for 10 minutes with a 4 % paraformaldehyde solution, the following two stains were applied:
OilRed-O (1- [2,5-dimethyl-4- (2,5-dimethylphenylazo) phenylazo] -2-naphthol)staining: for 20 minutes, then counter stained with haematoxylin. The proportion of positively stained cells was analyzed and quantified using Image J, “Image Processing and Analysis in Java,” software (National Institutes of Health, Bethesda, MD,USA)
LD540 (4,4-Difluoro-2,3,5,6-bis-tetramethylene-4-bora-3a, 4a-diaza-indacene) staining: for 30 minutes at a 1:10,000 dilution, then counter stained with Hoechst 33258 solution (Sigma Aldrich). To quantify positivity of LD540 staining were analyzed by flow cytometry (Becton Dickinson).
Quantification of intracellular triglycerides
Following 12 h culture of T/C-28a2 cells with the different FA conditions, the cells were collected to determine their triglyceride content. A suspension of the intracellular content of the cellswas obtained by sonication. The samples were then centrifuged at 10,000xg for 10 min at 4°C. For the spectrophotometric quantification of triglycerides (TG) the Glycerol Phosphate Oxidase / Peroxidase method was used (Biosystems, Barcelona,Spain), following the manufacturer's instructions.
Statistical analysis
Appropriate statistical analyses were performed using GraphPad Prism v6 software. Differences between the mean of the groups were determined using unpaired and nonparametric t-test. The results are reported as mean ± SD. A p value less than 0.05 was considered significant.