*4.1. Mice*

IL-1RA-deficient (IL-1RA−/−) mice on a BALB/c background were kindly supplied by Martin Nicklin (She ffield, UK). Wild-type control mice were 8–10 weeks old and were purchased from Charles River (Sulzfeld, Germany). Before the age of 12 weeks, mice were sacrificed, heads were dissected and fixed in 4% formaldehyde for 6–12 weeks. Ethical permission was obtained in July 2013 at the Radboud University Nijmegen, RU-DEC 2013-096.

### *4.2. Histological Analysis of Murine TMJ*

Mouse heads (four mice per strain) were decalcified for 6 days in 10% formic acid and 10% sodium citrate solution. The heads were then dehydrated, embedded in para ffin, and 5 μm-thick sagittal sections were cut. Safranin O-fast green and hematoxylin and eosin staining were performed. Mouse TMJ cartilage was evaluated by a blinded observer based on pericellular staining, chondrocyte arrangement, and structural appearance of the articular cartilage, using a modified Mankin score [21] (Table 1).



### *4.3. Cell Isolation and Culture*

Because mouse TMJs only contain few cells, all in vitro studies were performed with cells isolated from pig TMJs. Heads of Dutch Landrace pigs (*Sus scrofa*), with a body weight in the range of 70–80 kg and aged 6–8 months old, were obtained from a local abattoir (Westford, Gorinchem, The Netherlands). Approval by the Animal Ethics Committee of the VU University was not required as the animals were not sacrificed for the purpose of the experiment. Within 4 h after sacrifice, the entire articular cartilage of the fossa and condyle and the whole disc were dissected. The cells were isolated as previously described [39,40]. The medium containing the cells was mixed 1:1 with 6% ultrapure low melting point agarose (Invitrogen, Carlsbad, CA, USA) to a final concentration of 1 × 10<sup>6</sup> cells/mL, 3% agarose, 1× DMEM supplemented with 50 μg/mL ascorbic acid (Merck, Darmstadt, Germany), 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin/streptomycin/fungizone (Invitrogen). The non-solidified gel was poured in a 2 mL syringe with a diameter of 8 mm of which the needle end was cut o ff, leaving a cylinder with an open needle-end. After gelation, the cell-gel

construct was gently pressed out using the plunger and was cut into slices with a 2 mm thickness and transferred into a 24-wells plate. The 3D constructs were cultured for 6 days (Table 2) using a previously described protocol that is suitable for culturing chondrocytes [41]. On day 6, the cells were incubated with vehicle (PBS) or with 0.1, 1, or 10 ng/mL (7) recombinant porcine IL-1β (R&D Systems, Minneapolis, MN, USA) for 6 or 24 h.


**Table 2.** Timetable of progressive substitution of serum for ITS in the cell-agarose construct.

For mechanical loading experiments, the cell-gel solution was poured on the silicone membrane of a Flexcell tissue train plate (Dunn Labortechnik, Asbach, Germany) on which two Velcro strips were glued. The Velcro strips ensured that the agarose cell-gel constructs would stick to the membrane of the tissue train plate. The rectangular shape of the cell-gel construct was confined by a 3D-printed mold to match the standard dimensions of gels on a tissue train plate. The cell-gel constructs were cultured as described in Table 2 for 6 days before performing tensile strain experiments. In the cyclic tensile strain experiments, the cells were incubated first for 12 h with IL-1β (10 ng/mL, R&D Systems), followed by 6% of sinusoidal mechanical strain at 0.5 Hz for 8 h, which was applied using the Flexcell system. Cells were post-incubated without strain for 24 h with IL-1β.

### *4.4. RNA Extraction and Real-Time Quantitative PCR*

After 6 and 24 h of incubation with IL-1β, the cell-gel constructs were snap-frozen, and the RNA was extracted according to the protocol developed by Bougault and co-workers [41], cDNA was made using SuperScript ® VILOTM cDNA Synthesis Kit according to the manufacturer's instructions (Life Technologies, Carlsbad, CA, USA). Real-time PCR reactions were performed according to the manufacturer's instructions in a LightCycler480 ® (Roche Diagnostics, Switzerland). The sequences of the primer pairs are presented in Table 3.


**Table 3.** Primers used for real-time PCR.


**Table 3.** *Cont.*

a Only primers with equal efficacy were used.
