**2. Results**

### *2.1. IL-1*β*RA*−/− *Mice Showed Early Signs of Condylar Cartilage Damage*

To investigate the role of IL-1β in TMJ damage, we assessed whether young mice that lack IL1-RA develop arthritis in the TMJ. Discs were barely visible in sections of mouse TMJ. Because of the similar histological appearance of fossa and disc tissue in both wild-type (WT) and IL-1RA−/− mice, only the

condyles were quantified. Safranin O staining was more intense in IL-1RA−/− condyles compared to WT condyles (Figure 1A,B). In addition, the most superficial layer of the cartilage in IL-1RA−/− condyles was positive for Safranin O staining (Figure 1B), which was not the case in WT mice (Figure 1A). The IL-1RA−/− TMJ samples had a significantly higher Mankin score compared to the joints of the WT mice (*p* < 0.01) (Figure 1C). The IL-1RA−/− condyles contained 11-fold more empty lacunae than the WT mice (*p* < 0.001) (Figure 1D).

**Figure 1.** Histologic assessment of the temporomandibular joint (TMJ) of IL-1 receptor antagonist (IL-1RA−/−) and wild-type (WT) mice. Sagittal section of the condyles of IL-1RA−/− and WT mice stained with Safranin O. (**A**) WT TMJ, original magnification 10×. The condyle cartilage can be divided into the fibrous, proliferative, and hypertrophic zones, indicated in the figure as I, II, III, respectively. In the WT sample the modest red staining is limited to zone III. (**B**) The IL-1R−/− mice condyle showed a higher level of Safranin O staining in comparison to WT. In the IL-1R−/− mice, Safranin O staining was not limited to the hypertrophic and the proliferative zone of the condyle but extended to the fibrous layer. Empty lacunae were frequently seen (arrows). (**C**) The Mankin score of the IL-1RA−/− mice was higher than the WT. (**D**) The number of empty lacunae in the condyles of the IL-1RA−/− mice was higher than in the WT. \*\* Significant difference between IL-1RA−/− and WT mice, *p* < 0.01; \*\*\* significant difference between IL-1R−/− and WT mice, *p* < 0.001, a *t*-test is used.

### *2.2. Cells from the Fossa, Disc, and Condyle Expressed IL-1 Receptors*

The ability of the cells isolated from porcine fossa, disc, and condyle cartilaginous structures to react to IL-1β was assessed by measuring gene expression of receptors for IL-1β. All cells from the three types of TMJ cartilage displayed similar gene expression levels for IL-1RI as well as of the mock receptor of IL-1β, IL-1RII (Figure 2A,B). The ratio of IL-1RI to IL-1RII gives a rough indication of the

effectiveness of IL-1β to elicit downstream signaling. The three cartilaginous structures displayed similar IL-1RI/IL-1RII ratios (Figure 2C). Expression of IL-1RA and IL-1β was in most cases undetectable, and therefore no statistical analysis could be performed.

**Figure 2.** Relative gene expression of IL-1 receptor (IL-1R)I, IL-1RII, disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4 and ADAMTS5 by porcine fossa, condyle and disc cells. (**A**) IL-1RI and (**B**) IL-1RII expression of the cells from fossa, disc, and condyle. All cells expressed IL-1RI and RII gene at similar levels. (**C**) The ratio between *IL-1RI* and *IL-1RII*. The ratio IL-1RI/IL-1RII was comparable for all cells. (**D**) *ADAMTS4* expression in the cells from the fossa, disc, and condyle. IL-1β incubation for 6 h enhanced ADAMTS4 expression in condyle cells. After 24 h of incubation with 10 ng/mL IL-1β, both fossa and discs showed an increase in ADAMTS4 expression in comparison to the vehicle-treated cells. (**E**) *ADAMTS5* expression in the cells from the fossa, disc, and condyle. Six hours of 10 ng/ml IL-1β treatment enhanced ADAMTS5 gene expression in condyle cells. After 24 h of 10 ng/mL IL-1β, the fossa cells showed an increased *ADAMTS5* expression. \* Significant effect of treatment with IL-1β relative to vehicle, *p* < 0.05.

### *2.3. IL-1*β *Increased ADAMTS4 and ADAMTS5 Gene Expression*

IL-1β at 10 ng/mL enhanced ADAMTS4 gene expression by 5-fold after 6 h in cells from the fossa (*p* < 0.01) (Figure 2D). After 24 h incubation, fossa cells showed a 13-fold increased expression of ADAMTS4 in response to 10 ng/mL IL1β (*p* < 0.01) (Figure 2D).

Six hours of IL-1β stimulation (10 ng/mL) also enhanced ADAMTS5 by 4-fold, but only in condylar cells (*p* < 0.01) (Figure 2E). After 24 h incubation with 10 ng/mL IL-1β, only fossa cells demonstrated enhanced ADAMTS5 gene expression (7-fold) in comparison to vehicle-treated cells (*p* < 0.017) (Figure 2E).

### *2.4. MMP-2 Activity Was Higher in Condyle Than Disc and Fossa Cells; MMP9 mRNA Upregulated in Condyle by IL-1*β

Six hours of IL-1β treatment did not a ffect MMP-9 gene expression in any of the TMJ-derived cell types (Figure 3B). After 24 h of stimulation with 10 ng/mL IL-1β, there was a 3-fold increase of MMP-9 gene expression by condyle cells (*p* < 0.01, Figure 3B). MMP-9 enzyme activity was undetectable by zymographic analysis of the conditioned medium of fossa, disc, and condyle cells, regardless of the IL-1β treatment (Figure 3C), suggesting that the mRNA for MMP-9 was not su fficiently converted into active protein. Though not statistically significant at the mRNA level (Figure 3A), MMP-2 enzyme activity appeared higher in condyle cells than in the disc and fossa (Figure 3C). IL-1β did not visibly affect the level of MMP-2 activity in any of the cells (Figure 3C).

**Figure 3.** Matrix metalloproteinase (MMP)-2 and MMP-9 gene expression and activity. ( **A**) IL-1β did not a ffect *MMP-2* expression by the cells from the fossa, disc, and condyle at any time point tested. (**B**) After 24 h of 10 ng/mL IL-1β incubation, the *MMP-9* gene expression of the disc and condyle cells were higher than that of the vehicle-treated samples. ( **C**) Zymogram of the conditioned medium from fossa, disc, and condyle cells after 24 h of incubation with IL-1β. There was no MMP-9 activity detected. The condyle showed strong MMP-2 activity, but no e ffect of IL-1β was apparent. \* Significant e ffect of treatment with IL-1β, relative to vehicle, *p* < 0.05. Results are shown from one out of three identical experimental replicates.

### *2.5. IL-1*β *Induced MMP-13 Expression by Condylar Cells Only*

After 6 and 24 h of 10 ng/mL IL-1β stimulation, MMP-13 gene expression by cells of the condyle was up-regulated by 3.4- and 9-fold, respectively (*p* < 0.001 and *p* < 0.0001, respectively) (Figure 4A). MMP-13 gene expression was almost undetectable in the cells from the disc and fossa and remained low after IL-1β incubation (Figure 4A).

**Figure 4.** MMP-13 gene and protein expression. (**A**) MMP-13 gene expression by the cells from the fossa, disc, and condyle. IL-1β for 6 h and 24 h at 10 ng/mL increased MMP-13 expression in condyle cells in comparison to vehicle. (**B**) Number of MMP-13-positive cells. Condylar cells incubated with 10 ng/mL IL-1β for 24 h showed the highest number of MMP-13-positive cells. (**C**) Image of MMP-13-positive cells after 24 h of 10 ng/mL IL-1β treatment. The green label indicates the presence of MMP-13, and the nuclei are red. \* Significant effect of treatment with IL-1β relative to vehicle treatment, *p* < 0.05. Scale bar represents 5 μm.

Next, we analyzed the number of cells expressing MMP-13 by immunostaining. Twenty-four hours of 10 ng/mL IL-1β incubation increased the percentage of MMP-13-positive condylar cells (3.5-fold increase, *p* < 0.001) (Figure 4B). The number of MMP-13-positive cells derived from the condyle compared to the fossa and disc was remarkably higher (Figure 4C).

### *2.6. Cyclic Tensile Strain Reduced IL-1*β*-Induced MMP-13 Expression*

Six percent cyclic tensile strain (CTS) reduced IL-1β-induced MMP-13 gene expression by 3-fold (*p* < 0.05) (Figure 5B). CTS neither affected expression of MMP-2, IL-12RI, IL-1RII nor the ratio of IL-1RI and IL-1RII in control condylar cells or in those incubated with IL-1β (Figure 5A,C,D).

**Figure 5.** Mechanical strain reduces MMP-13 expression of condylar cells incubated with IL1-β. Gene expression of (**A**) MMP-13, (**B**) MMP-2, (**C**) IL1-RI, and (**D**) IL1-RII. (**E**) Ratio between IL-1RI and IL-1RII by condylar cells. Mechanical loading reduced IL-1β-induced gene expression of MMP-13. \*\* Significant effect of mechanical loading, *p* < 0.01.
