Cytokines

BMSC micromass pellets were cultured in NM or CM supplemented with Complete Mini Protease Inhibitor Cocktail with and without EDTA (Roche) for 7 days to analyse if the upregulation of miR-29b could be prevented when proteases like matrix metalloproteases (MMP) are inhibited. BMSC of 6 different donors were used (n = 6).
To investigate the influence of different cytokines on miR-29b expression, BMSC micromass pellets were stimulated for 7 days with the following cytokines (all from Peprotech, Hamburg, Germany) in chondrogenic medium: IL-1β (1 ng/ml), IL-6 (5 ng/ml), IL-8 (10 ng/ml) and IFN-γ (1 ng/ml, 10 ng/ml and 50 ng/ml). BMSC of 4–11 different donors were used (n = 4–11).

The MTT assay is a colorimetric test used to measure cellular metabolic activity to indicate cell viability, proliferation, and cytotoxicity. In the MTT assay, metabolic active cells transform yellow tetrazolium salt MTT into purple DMSO-soluble formazan crystals. This process is possible due to the activity of mitochondrial dehydrogenase enzyme in living cells, which causes salt transformation. Cells were seeded at 8 × 10³ in 96-well culture plates, cultured for a day, and then incubated with cytokines (Peprotech, Rocky Hill, NJ, USA) at 50 ng/ml concentration or cytokines with curcumin for the next 24 hours, followed by the MTT assay. The MTT solution was added to wells at a final concentration of 1 mg/ml for 3 hours in an incubator. Next, formazan dye was solubilized with 50 μl DMSO for 30 min. Absorbance was measured at 490 nm in a BioTek Well-plate Reader (Winooski, VT, USA). The control group absorbance was 100%, whereas treated samples' cell viability was counted using the formula: % = (A of experimental wells/A of the control wells) × 100%. After preliminary studies with different concentrations of curcumin, 5 and 10 μM concentrations were chosen for further experiments.

Culture media and complements were purchased from GIBCO (Gibco BRL, Paisley, UK). Fetal bovine serum (FBS) was purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). The test compounds were acquired from Merck (Madrid, Spain). Cytokines were purchased from Peprotech (London, UK). Other chemicals were purchased from Merck.