*4.2. Cell Culture*

Primary human chondrocytes and synoviocytes were commercially acquired (chondrocytes, Lonza, Visp, Switzerland; synoviocytes, ECACC, Sigma-Aldrich, St. Louis, MO, USA) and obtained from human tissue explants using well-defined methodology [53,54]. Both cell types were cultured in Advanced Dulbecco's Modified Eagle's Medium (Adv DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (*v*/*v*) of heat-inactivated Fetal Bovine Serum (FBS, Sigma-Aldrich), 1 mM of l-glutamine (l-Gln, Invitrogen) and 1% (*v*/*v*) of penicillin-streptomycin (PS, Invitrogen). THP-1 cell line was kindly given by Dr. Santos (CBME, University of Algarve, Faro) and was cultured according to ATCC instructions in RPMI Growth Medium (RPMI 1640 with l-glutamine (Lonza)) containing 10% heat-inactivated FBS (invitrogen) and 1% PS. Differentiation into THP-1 macrophage (THP-1 MOM) cells was achieved by culturing THP-1 cells in 25 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma) in complete RPMI for 48 h. All cell cultures were maintained at 37 ◦C in a humidified atmosphere containing 5% CO2, and experiments were performed on confluent cells.

### *4.3. Inflammatory Assays in Monolayer Cells*

THP-1 MOM (1 × 10<sup>6</sup> cells/mL) were cultured in 500 μL of complete RPMI supplemented with different amentadione (YP) concentrations (2.5, 5 and 10 μM) in dose-dependence experiments and with 10 μM YP in subsequent experiments, or with 2 μM dexamethasone (DXM), during 24 h. After, 100 ng/mL of lipopolysaccharides (LPS) or synthetic hydroxyapatite nano-crystals (HAP) (Sigma) (750 μg/mL) were added to the culture media for another 24 h or 72 h, respectively. Confluent chondrocytes and synoviocytes were cultured in 1 mL of Adv DMEM supplemented with 10 μM of YP or 2 μM of DXM during 24 h, and further treated with: 10 ng/mL of interleukin-1β (IL-1β) for 3 and 6 h, or for 30 min in the assay for pIKBα content analysis; 750 μg/mL HAP for 6h. Control cells were cultured with respective media without any treatment. At determined time points, cell culture media were collected for ELISA analysis and cells harvested for RNA extraction.
