*4.4. Cell Proliferation*

THP-1 MOM cells seeded in 96-well plates at 2 × 10<sup>5</sup> cells/well, and confluent chondrocytes and synoviocytes, were cultured in their respective growth media and supplemented with different concentrations of YP and HAP. Cell viability was determined at appropriate time points using the CellTiter 96 cell proliferation assay (Promega, Madison, WI, USA), following manufacturer's instructions.

### *4.5. Cartilage Collection and Tissue Explants Preparation*

Knee articular cartilage was obtained from osteoarticular cuts performed on the femoral and tibial sides, from eight patients (4 male and 4 female, aged 71.5 ± 5.9 years) who had undergone arthroplasty surgeries at Hospital Particular do Algarve (Faro, Portugal). This study was approved by the ethics committee of the hospital, and written informed consent was obtained from all the participants. All principles of the Declaration of Helsinki of 1975, as revised in 2000, were followed. Macroscopically normal full-depth cartilage slices were removed in sterile conditions using a scalpel, collected in complete Adv DMEM media, and incubated for 24 h, at 37 ◦C, in a humidified atmosphere containing 5% CO2, for tissue equilibration before preparation of tissue explants. After equilibration, 2 mm diameter and 1.71 ± 0.70 mm thickness cartilage explants, were obtained using a 2 mm biopsy punch (Integra-Miltex). Samples of the initial cartilage explant tissues were fixed in 4% PFA for histological evaluation.

### *4.6. Cartilage Explants Assays*

Cartilage explants (8–10 per well), were plated in a 12 well plate and cultured at 37 ◦C, in a humidified atmosphere containing 5% CO2, in 1 mL of complete Adv DMEM media supplemented with 10 μM of YP or 2 μM DXM for 24 h, and then treated with HAP (750 μg/mL), for further 72 h. As controls, explants were cultured without treatment. At the end of each experiment, cartilage explants were collected, washed twice with PBS, immediately processed for RNA extraction as described below, and the cell culture media collected for ELISA assays.
