*4.7. Statistical Analysis*

Prism (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analyses. Mean values and standard errors of the mean (SEM) were calculated and depicted in the figures throughout the manuscript. Differences in pericellular staining of extracellular matrix (proteoglycans), chondrocyte arrangement, and structural appearance of the articular cartilage between four wild-type and four IL-1RA−/− mice were tested using the Mann–Whitney *U* test (*n* = 4). For each parameter measured, the null hypothesis was that cartilage damage-related changes scored equally in TMJs from WT and IL-1RA−/− mice. Experiments with pig cells were performed at three separate occasions. Data obtained at one separate occasion were considered *n* = 1 (thus, total *n* = 3). At each separate occasion, a *new* cell pool (derived from three pig heads) per anatomical region was created. Cell pools derived from the condyle, fossa, and disc were kept separate, and all were treated with or without IL-1β. To determine whether IL-1β (0.1, 1, or 10 ng/mL) significantly affected gene expression of ADAMTS4, ADAMTS5, MMP-2, MMP-9 and MMP-13, as well as protein expression for MMP-13, and activity of MMP-2 and MMP-9, compared to vehicle, in TMJ-derived cell populations, Dunnett's multiple comparison test was performed. Bonferoni correction was applied, as three tests were performed per parameter (one for fossa, one for disc and one for condyle) at the 6 h and at the 24 h time point, separately. At a *p* < 0.017, the null hypothesis, i.e., IL-1β (at either 0.1, 1, or 10 ng/mL) did not affect gene or protein expression of the catabolic factor of interest in pig TMJ cells, was rejected. To test the effect of mechanical strain on IL-1β-induced gene expression by pig condylar fibrochondrocytes, one-way ANOVA was performed. Differences were regarded significant at values of *p* < 0.05.

**Author Contributions:** H.T. performed most of the practical work, and supervised A.V.t.L. B.F.B. was involved in experiments concerning mechanical loading, B.Z.-D. was involved in the protein analysis and zymography, C.d.S.C. performed immunohistochemistry. M.I.K. contributed on the IL1-RA−/− part, T.J.d.V. and A.D.B. were the daily PhD supervisors of H.T., F.L. and V.E. the formal PhD supervisors. All work was discussed on a weekly basis between H.T., T.J.d.V., A.D.B., F.L. and V.E. H.T. initiated writing, all authors have contributed to the writing of the submitted version.

**Funding:** This research was funded by research institute MOVE.

**Acknowledgments:** We thank Jolanda Hogervorst for her assistance with the qPCR; Rebecca Rogier and Debbie Roeleveld for collecting the mouse heads; Regina Maria Catarino for help with MMP-13 immunostaining; and Ben Nelemans and Manual Schmitz for their assistance with the Axio-zoom.

**Conflicts of Interest:** The authors declare no conflict of interest. Hessam Tabeian received funding from the MOVE Research Institute Amsterdam, Amsterdam, The Netherlands. The funding source had no other role in this study.
