Il 1β

Rat chondrocytes were collected following previous steps [21 (link)]. Isolated chondrocytes were cultured in DMEM/F12 containing 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin and streptomycin (Sigma-Aldrich) at 37 ℃ with 5% CO₂. For IL-1β induction, chondrocytes were subjected to IL-1β (10 ng/ml, R&D System) for 24 h.

The cytokine concentrations used were based on our previous experiments on human islets [18 (link)]. Islets and EndoC-βH1 cells were stimulated either with a cytokine cocktail consisting of IL-1β (5 ng/ml) and IFN-γ (50 ng/ml) (cytokine cocktail I) or with a more potent cytokine cocktail consisting of IL-1β (5 ng/ml), IFN-γ (50 ng/ml), IL-17 (100 ng/ml) and TNF-α (10 ng/ml), all from R&D Systems (Minneapolis, MN, USA) (cytokine cocktail II), with or without MANF (100 ng/ml). Recombinant human MANF protein was produced in CHO cells (no. P-101-100; Icosagen, Tartumaa, Estonia). The cytokine stimulations were performed in Ham’s-F10 medium supplemented with 0.5% (vol./vol.) BSA using the cytokine concentrations described above. RNA samples were collected after 48 h and cell death was analysed after 72 h of exposure to cytokines.

Synovial fluid was aseptically drawn from the knee without any lavage at the beginning of the arthroscopic surgery. Synovial samples were firstly collected in tubes containing EDTA, immediately centrifuged at 3000 × g to remove cellular debris, and the supernatant was stored at -80°C until assayed [13 (link), 21 (link)–23 (link)]. The levels of cytokines TNF-α, IL-1β, IL-6, IL-8, and IL-10 were measured in the synovial fluid using specific sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (TNF-α, IL-1β, and IL-10 were from R&D Systems, Minneapolis, MN; IL-6 and IL-8 were from eBioscience, San Diego, CA, U.S.A.).

Primary human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (batch p1032 and p1028) and were cultured in endothelial cell medium (ScienceCell, Carlsbad, CA, USA, 1001), supplemented with endothelial cell growth supplement (ECGS) (ScienceCell, Carlsbad, CA, USA, 1052), penicillin/streptomycin (ScienceCell, Carlsbad, CA, USA, 0503), and 5% fetal bovine serum (ScienceCell, Carlsbad, CA, USA, 0025). Primary HUVECs were cultured between passage 1 and 5 for experiments. Induction of EndMT in HUVECs was performed with IL-1β (10 ng/mL, R&D Systems, Minneapolis, MN, USA, 201-LB) and TGF-β2 (10 ng/mL, R&D Systems, 302-B2-002) for 72 h. EndMT conditions are defined as the HUVECs treated with IL-1β and TGF-β2. Hek293T cells were acquired from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher, Waltham, MA, USA, 31966021), supplemented with 10% FCS, 1% Pyruvate, 1% D-glucose, 1% penicillin/streptomycin and 1% minimum essential media with non-essential amino acid mix (Sigma-Aldrich, St. Louis, MI, USA, M7145). Hek293T and primary HUVECs were cultured at 37 °C with 5% CO₂. Cells were counted with the Countess II cell counter (Thermo Fisher). All cell types tested negative for mycoplasma.

For monoculture experiments, chondrocytes and synoviocytes were thawed and seeded in 24-well plates at high density (2 × 10⁵/cm² and of 6 × 10⁴/cm², respectively) for 3 days in DMEM (SIGMA-ALDRICH) with 10% FCS. Successively, cells were treated with IL-1β (5 ng/mL, R&D SYSTEMS, Minneapolis, MN) for 18 h to mimic an inflammatory environment. Then the IL-1β containing medium was substituted with fresh 10% FBS—DMEM containing 10 μg/mL of sEV (sEV group) or without sEV (CTR group). In detail, the effects of three different purified sEV preparations were evaluated on pooled chondrocytes and pooled synoviocytes in either monoculture or co-culture. Co-cultures were set up by seeding 2 × 10⁵/cm² chondrocytes in the lower chamber of a 24 well plate and 6 × 10⁴/cm² synoviocytes in the transwell (0.4 μm pore size, CORNING, Toledo, OH) for 3 days in DMEM (SIGMA-ALDRICH) with 10% FCS. Successively, cells were treated with IL-1β (5 ng/mL, R&D SYSTEMS, Minneapolis, MN) for 18 h to mimic an inflammatory environment. The following day, the co-cultures were assembled, the medium containing IL-1β was substituted with fresh DMEM + 10% FBS containing 10 μg/mL of sEV or without sEV. All evaluations were carried out at 4 and 15 h after the addition of sEV. Chondrocytes and synoviocytes were used at passage 2 and 3, respectively.