**3. Discussion**

The TMJ is frequently affected in patients with chronic inflammation, which can result in permanent damage to the joint, especially in young patients. Since biological sampling of the TMJ of children for research purposes is unethical, the role of specific inflammatory factors in the degradation of the TMJ of young individuals remains elusive. In the present study, we made use of relatively young mice and juvenile porcine TMJs to investigate the effect of the inflammatory cytokine IL-1β on its three

cartilaginous structures. Our findings strongly sugges<sup>t</sup> that excess IL-1β induces degradation of TMJ cartilage. Young mice deficient for IL-1RA showed early histological signs of TMJ degradation, an e ffect preferentially found in the condyle. In culture, porcine cells isolated from the three cartilaginous structures expressed di fferent catabolic enzymes in response to IL-1β, e.g., IL-1β at 10 ng/mL induced the expression of ADAMTS4 and ADAMTS5 by cells from the fossa, while cells isolated from the condyle responded to IL-1β with an increased expression of MMP-9, and MMP-13. Mechanical loading reduced MMP-13 expression in IL-1β-treated condylar fibrochondrocytes.

Horai et al. previously demonstrated that IL-RA−/− mice developed spontaneous arthritis due to unopposed excess of IL-1 signaling. In this systemic arthritis model, between 5–20% of the front paws developed arthritis, which depended on, for instance, the microbiological status of the animal facility [20]. We used these mice to investigate whether an excess of IL-1 signaling could result in TMJ damage. We did indeed find some remarkable changes in the condyle. A high level of staining for proteoglycans was seen around the condyles and also in the fibrous areas of the condyle. This area normally does not contain proteoglycans. Condyles of the IL-RA−/− mice showed, overall, more clustering of cells, more intense proteoglycan staining, and higher Mankin score in comparison to WT mice. Over-production of proteoglycans and cluster formation of chondrocytes may represent signs of local repair of articular cartilage, an indication of the onset of the cartilage degradation process. Proteoglycans are unlike collagen in a continuous turnover [20], therefore overshoot in matrix synthesis might occur more easily with proteoglycans. Other studies have also found an increased level of proteoglycans in the early phases of condyle cartilage degradation [21–23]. In these studies, at later stages, a gradual loss of proteoglycans occurred together with cleaving of collagen fibrils. This pattern of degeneration implies that there may be a common chain of molecular events underlying degeneration [21]. Further studies in older IL-1RA−/− mice should indicate whether these mice will undergo loss of proteoglycans together with cleaving of the collagen fibrils in their TMJ by, for instance, MMP-13, which was upregulated in the porcine model. Taken together, our results with IL-1RA−/− mice sugges<sup>t</sup> that overactive IL-1β signaling induces damage in the fibrocartilage tissue of the condyle of the TMJ.

We assumed initially that the fossa and disc cells would not express the genes of the receptors related to IL-1β signaling, since these cartilage parts seemed to be una ffected in the inflamed joint of JIA patients [14]. However, we found that the cells from the fossa and disc expressed mRNA for these receptors, and cells from the fossa responded to IL-1β with an enhanced expression of ADAMTS4 and ADAMTS5. This shows that the receptors are present and functional in the fossa and disc, even though these structures are damaged to a lesser extent than the condyle in JIA patients. Increased ADAMTS5 expression in response to IL-1β in combination with lack of tissue damage was also observed in articular cartilage from knees of Sox9 knockout animals [24]. In addition, very limited numbers of proteoglycans are present in the fossa and disc. Therefore, with ADAMTS4 and ADAMTS5 being the catalytic enzymes that degrade proteoglycans, damage by these aggrecanases would be limited in comparison to the condyle.

Condylar cells responded to IL-1βby increasing the expression of the catabolic enzymes ADAMTS5, MMP-9 and MMP-13. These cells also expressed constitutively active MMP-2. These enzymes are able to cleave the matrix proteins of the condylar cartilage. The aggrecanases ADSMTS5, MMP-13 and MMP-2 are capable of cleaving proteoglycans [25,26], and both MMP-13 and MMP-2 are able to unwind and cleave collagen fibrils [27]. The resulting fragments will form an excellent substrate for the gelatinase MMP-2. This enzyme is also able to cleave the pro-MMP-13, thereby activating this collagenase [28]. We found that IL-1β enhanced MMP-13 expression in cells isolated from the porcine condyle. The isolated cells constitute a mix of more fibroblast-like cells from the upper layer of the condyle and chondrocyte-like cells from the deeper layers. It is possible that only one of these subtypes of cells responds to IL-1β with increased MMP-13 expression. We found in a limited set of histological slides that MMP-13 protein was mostly expressed by chondrocyte-like cells of the deeper layers of mouse condyles (data not shown). It is thus possible that the response to IL-1β was most pronounced

in the chondrocyte-like cells within our mix of isolated condyle cells. The importance of MMP-13 in cartilage degradation in arthritis was demonstrated in transgenic mice overexpressing MMP-13 [29] and elevated levels of MMP-13 were found in synovial fluid of arthritic patients [30]. Therefore, MMP-13 can be considered as one of the prime suspects in the degradation of condylar cartilage in JIA. Taken together, we found that IL-1β enhances the expression of catabolic enzymes by TMJ-derived cells, thereby possibly explaining cartilage damage as observed after overactive IL-1 signaling.

One limitation of this study is that we cannot be certain that histological changes indicative of degeneration in the condylar fibrocartilage of the TMJ of IL-1R−/− mice can be attributed to MMP-13 over-expression. Studies using IL-1R−/− mice treated with MMP-13 inhibitors could provide clarity, but such experiments were beyond the scope of the current study. In addition, our in vitro studies showing the e ffect of IL-1β on MMP-13 expression in condyle-derived fibrochondrocytes were performed with cells from pig TMJs but not mice, and species di fferences can occur. We have performed immunohistochemistry for MMP-13 on sections of mouse TMJs, but the resulting quality prevented accurate quantitative assessment, though roughly 60% of the condylar cells seemed positive in wildtype animals and nearly 100% in IL-RA knock-out mice (data not shown), which indicates that the e ffects of overactive IL-1β with regards to MMP-13 expression is similar between pig and mouse. Another limitation is the selection of only one mechanical loading regime of tensile forces, whereas compressive forces are also occurring in the moving jaw.

Since MMP-13 plays an important role in many biological processes, including growth and development [31], inhibition of activity of this enzyme could have severe, undesirable side-e ffects in the children with JIA that are still growing. This important role of MMP-13 in many biological processes [31] requires a *direct* inhibition. Pharmaceutical intervention should therefore be based on tempering IL-1β's destructive e ffects [32,33]. A potential non-invasive, non-pharmaceutical approach to inhibit inflammation-induced MMP-13 expression is exercise or physical therapy of inflamed joints. We found that 6% cyclic tensile strain exerted on the condylar cells significantly reduced the IL-1β-induced MMP-13 gene expression, similar to our previous finding that tensile strain exerted on condylar cells significantly reduced TNF α-induced MMP-13 gene expression [34]. These findings are in line with several other studies, in which the anti-catabolic capacity of cyclic strain was analyzed [34–36]. In our study, the cells maintained their pericellular matrix when they were embedded in an agarose gel, thereby allowing proper transmission of mechanical forces to the cells. The condylar cartilage undergoes considerable tensile forces due to compression and shear [37]. For this reason, we used 6% cyclic tensile strain. This percentage was calculated by using the following literature data. Deschner et al. used 20% of strain to stimulate rat disc cells [35], but Chain et al. calculated that the maximal tensile strain that the condyle cartilage would experience would be 3.7-fold lower than the disc [38]. Further in vivo studies are needed to assess whether 6% tensile strain is e ffective in downregulating catabolic enzymes induced by inflammation.

In conclusion, overactive IL-1 signaling can induce changes in condyle cartilage metabolism indicative of degeneration, and cells from the three cartilaginous structures of the TMJ react to exposure to the inflammatory cytokine IL-1β, whereby the condyle seems particularly sensitive in terms of catabolic enzyme expression. This might explain why only the condyle is disproportionately degraded in children with JIA. MMP-13 induced by IL-1β might be a prime suspect in causing degradation of the condyle in JIA patients, and mechanical loading could inhibit expression. Future studies should confirm whether a direct link exists between JIA, IL-1β and MMP-13 over-expression, and whether controlled exercise can reduce MMP-13 expression in the condyle of the TMJ in vivo. These are important future steps with high clinical relevance because controlled physical exercise could provide a therapeutic intervention in children with JIA, potentially preventing serious e ffects of TMJ inflammation such as pain, dysfunction, and even malformations. Non-invasive studies, for instance using MRI, could be useful to monitor the e ffect of motion on the progression of JIA.

### **4. Materials and Methods**
