Elsevier

Archives of Oral Biology

Volume 95, November 2018, Pages 79-88
Archives of Oral Biology

Experimental model of temporomandibular joint arthritis: Evaluation of contralateral joint and masticatory muscles

https://doi.org/10.1016/j.archoralbio.2018.07.003Get rights and content

Highlights

  • A single intra-articular injection of CFA was efficient to induce a persistent inflammatory process in the ipsilateral TMJ.

  • The articular inflammation reduced area and diameter of fibers of masticatory muscles.

  • Induced inflammation in TMJ caused morphological and molecular changes in the contralateral joint.

Abstract

Objective

To investigate morphological and biochemical changes in an experimental model of temporomandibular joint (TMJ) arthritis, as well as examine contralateral joint involvement and morphometric aspects of masticatory muscles in affected animals.

Design

Forty Wistar rats were allocated into three groups, as follows: a healthy control group (HG); and a group with arthritis induced in the left TMJ (IG); and a contralateral group with noninflamed right TMJ (CG). Arthritis was induced by intra-articular injection of 50 μL of Complete Freund's Adjuvant (CFA). Morphological analysis was performed using histological sections of TMJs stained with hematoxylin-eosin, picrosirius and toluidine blue. In addition, histomorphometric analyses of the articular cartilage, articular disc and masticatory muscles, were performed. Biochemical analysis of TMJ tissues included dosage of sulfated glycosaminoglycans (GAGs), zymography of the Matrix Metalloproteinases (MMPs) 2 and 9 and ELISA for the cytokines IL-6, TNF-α and IL-1β. The data were analyzed by paired and unpaired t-tests, with a p < 0.05.

Results

IG animals presented morphological and biochemical changes in their TMJ, including synovial membrane hyperplasia; chronic inflammatory infiltrate; collagen fiber disorganization in the disc; reduced proteoglycans in the cartilage; greater disc thickness; lower cartilage thickness; lower feed intake; higher activity of MMP-9 and MMP-2, and higher concentration of IL-6, TNF-α, and IL-1β, as compared to HG. CG animals showed posterior disc thickening; reduced proteoglycans in the cartilage; lower collagen fiber organization of the disc; active MMP-9, and higher concentration of IL-6 and TNF-α compared to HG animals. Articular inflammation reduced the area and diameter of fibers of the temporal (right), medial pterygoid (left), and lateral pterygoid (right and left) muscles as compared to HG animals.

Conclusion

TMJ arthritis reduced the area and diameter of masticatory muscle fibers and caused morphological and molecular changes in the contralateral joint.

Introduction

The temporomandibular joint (TMJ) is a bicondylar, biaxial and complex synovial joint that allows for wide jaw movements (Madeira, 2008; Texeira, Reher, & Reher, 2008). While its anatomical and histological organization provides great adaptive and repair ability, it is subject to a variety of potentially harmful molecular processes, as are other load-bearing joints, which can be triggered by various local or systemic factors (de Leeuw, 2010; Haskin, Milam, & Cameron, 1995).

The Temporomandibular Disorder (TMD) is the most common affecting the TMJ (Tanaka, Detamore, & Mercuri, 2008). The American Academy of Orofacial Pain (AAOP) defines TMD as a collective expression of various clinical issues involving masticatory muscles, TMJs, and associated structures (de Leeuw, 2010). Articular inflammation plays an important role in the pathogenesis of TMD and contributes to the development of painful symptoms in affected patients (Kellesarian et al., 2016; Ribeiro-Dasilva, Fillingim, & Wallet, 2017; Slade et al., 2011).

Inflammation may also be associated with other TMJ disorders, osteoarthritis, or systemic polyarthritis. Previous studies have shown that patients with articular inflammation exhibit a higher prevalence of TMJ symptoms, such as joint pain, movement limitation, and muscle pain, as compared to the general population (Abramson, 2004; Cordeiro et al., 2016; de Leeuw, 2010; de Souza, Bansal, & Galloway, 2016; Haskin et al., 1995; Keriş, Yaman, Demirağ, & Haznedaroğlu, 2016; Mercuri, 2008; Tanaka et al., 2008).

Different proinflammatory mediators have been associated with structural changes found in osteoarthritis or other arthritic conditions of the TMJ (Cordeiro et al., 2016; de Leeuw, 2010; Haskin et al., 1995; Tanaka et al., 2008). Such mediators include IL-1β, IL-6, IL-12, IL-17, TNF-α, TNF-β, VEGF, IFN-γ, NGF, and iNOS (Ghassemi-Nejad et al., 2011; Haskin et al., 1995; Kyrkanides et al., 2007; Tanaka et al., 2008; Vernal et al., 2008).

It has been shown in the literature that these proinflammatory mediators may induce the synthesis of proteolytic enzymes, which are responsible for extracellular matrix degradation, such as matrix metalloproteinases - MMPs (Abramson, 2004; Brooks, 2003; Ghassemi-Nejad et al., 2011; Zhang et al., 2016). Accordingly, recent studies have shown that IL-1β induces the synthesis and activity of MMP-2, MMP-3, and MMP-9 (Cevidanes et al., 2014; Chu et al., 2017; Zhang et al., 2016). On the other hand, these MMPs may also have a direct or indirect effect on the activity of proinflammatory cytokines, such as IL-1β itself, thereby increasing the inflammatory response (Parks, Wilson, & López-Boado, 2004). Consistently with these findings, several studies have shown increased activity of MMPs, especially MMP-2 and MMP-9, in TMJ tissues of patients with osteoarthritis (Almeida et al., 2015; Cevidanes et al., 2014; Chu et al., 2017; Mizui, Ishimaru, Miyamoto, & Kurita, 2001; Srinivas et al., 2001; Yoshida et al., 2006; Zhang et al., 2016).

Given the clinical importance of TMJ inflammatory disorders, the development and improvement of articular inflammation experimental models are key to better understand the molecular mechanisms and morphological impact underlying such conditions, ultimately enabling the establishment of more effective therapies. Therefore, intra-articular injection of Freund’s Complete Adjuvant - CFA (dry antigen of Mycobacterium tuberculosis suspended in mineral oil) is capable of inducing an intense inflammatory response mediated by the release of pro-inflammatory cytokines (Spears, Dees, Sapozhnikov, Bellinger, & Hutchins, 2005; Wang, Kou, Mao, Gan, & Zhou, 2012), e.g. IL-1β, which has shown positive results in the induction of persistent (chronic) inflammation of the TMJ (George et al., 2013; Harper, Kerins, McIntosh, Spears, & Bellinger, 2001; Kramer, Kerins, Schneiderman, & Bellinger, 2010; Kuroki et al., 2011). Furthermore, previous studies in our laboratory have shown the high activity of MMP-2 and MMP-9 for up to 3 weeks upon CFA injection (Lemos, Rissi, Pimentel, & Palomari, 2015, 2016).

Functionally, the right and left TMJs are interconnected by the jaw and interdependent. With separate (albeit simultaneous) movements on either side, the TMJs are characterized as a single bilateral joint (de Leeuw, 2010; Madeira, 2008; Okeson, 2008; Oliveira, 2008) whose movements are determined by the action of masticatory muscles (de Leeuw, 2010; Texeira et al., 2008). However, to date, there have been no studies addressing the involvement of this group of muscles as well as the contralateral TMJ after induction of joint inflammation.

In our study, we investigated the morphological and biochemical changes in an experimental model of temporomandibular joint arthritis, as well as examined the contralateral joint involvement and morphometric aspects of masticatory muscles. Muscle evaluation is highly important as the presence of articular disorders may reflect in muscle morphophysiology, which makes it an important functional parameter of induced inflammation and therapy development.

Section snippets

Sample calculation

The sample size was determined based on the following formula (Sokal & Rohlf, 2012; Zar, 2010):n = 1 + [2C x (s / d) 2], where C = (zα + zβ) 2A statistical power of 90% (error β), significance level p = 0.05 (error type I), maximum deviation (s) of 20% and minimum difference (d) of 50%, were adopted. The value of 10.5 was used for the formula (zα + zβ) 2, which is applied to two-tailed tests, with a statistical power of 90% and a significance level of 0.05 (Fontelles, Simões, Almeida, &

Temporomandibular joint

Animals of the healthy group (HG) showed normal morphology of the condyle, temporal bone, articular disc, and periarticular tissues (Fig. 2A1-3). Animals of the CG showed morphology similar to that of the HG (Fig. 2B1-3). However, the histomorphometric analysis showed that the posterior region of the articular disc in the CG was statistically thicker as compared to that of the HG (Table 1).

We observed marked morphological changes in the TMJ components in the IG, such as increased articular disc

Discussion

Considering the limitations of this study, our results indicate that a single intra-articular injection of CFA into the ipsilateral TMJ is able to induce a chronic inflammatory process in the period of 27 days after injection. These results are consistent with previous studies showing greater effectiveness of CFA compared to other irritants in inducing articular inflammation in various periods of time (Kramer et al., 2010; Lemos et al., 2016, 2015; Spears et al., 2005; Wang et al., 2017a, 2017b

Conclusion

Considering the morphological and biochemical findings presented herein, we conclude that temporomandibular joint arthritis reduced the area and diameter of masticatory muscle fibers, with the lateral pterygoid being the most affected muscle. In addition, TMJ arthritis led to morphological and molecular changes in the contralateral joint, including increased thickness of the articular disc (posterior region), MMP-9 activity and increased concentration of the active pro-inflammatory cytokines

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