Effect of Mechanical Stress on Rheumatoid Arthritis of the Temporomandibular Joint: a Morphological and Histological Evaluation

Rheumatoid arthritis of the temporomandibular joint (TMJ-RA) has been reported to have a larger incidence range than systemic rheumatoid arthritis (RA). The presence or absence of mechanical stress (MS) is considered a factor in this. In this study, we hypothesized that TMJ-RA develops or worsens when excessive MS is applied to the temporomandibular joint of RA mouse models. We aimed to clarify the relationship between TMJ-RA and MS through morphological and histological evaluation.


Results
In the CAIA MS model, a three-dimensional analysis of the temporomandibular joint by microcomputer tomography showed a crude change in the surface of the mandibular condyle. Histological examination revealed a decrease in the chondrocyte layer width and an increase in the number of osteoclasts in the mandibular condyle. T cell accumulation was observed, and γδ T cell involvement was con rmed.

Conclusions
In the CAIA model, the TMJ was less sensitive to the initiation of RA. However, the results suggested that it was exacerbated by MS, and that γδ T cells may be involved in TMJ-RA.

Background
Rheumatoid arthritis (RA) is an unexplained autoimmune disease with a 1% prevalence worldwide.
Chronic in ammation of the joints and synovial hyperplasia, known as pannus, are observed, as well as cartilage and bone destruction by in ammatory cytokines. The detailed cause of RA has not yet been fully elucidated (1). The most common sites of RA are the peripheral limb joints (knees, elbows, ngers) and the temporomandibular joint (TMJ). Previous reports have shown that 4-85% of RA patients develop RA in the TMJ (2,3,4). In this way, the morbidity range is wide; unlike RA in the limb joints, the pathogenic mechanism of RA in the TMJ (TMJ-RA) is still unknown.
TMJ-RA rst causes degradation of proteoglycan and softening and degeneration of the mandibular condylar cartilage, followed by destruction of the subchondral bone and bone resorption by osteoclasts (2). In ammatory cells, such as macrophages, in ltrate the synovial tissue and form pannus. They then release a chemical mediator, destroying the joint and causing pain (2,3). In particular, tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) are associated with RA etiology (4,5,6). They cause excessive production and secretion of proteolytic enzymes such as matrix metalloproteinase (MMP) and A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) in synovial broblasts, and deform the mandibular condyle cartilage. These degenerative changes can cause joint dysfunction, brous and bony ankylosis, occlusal-facial malformations, and occlusal inconsistencies. Therefore, early diagnosis and treatment are required (7).
Several RA animal models have been established for the analysis (8). In particular, collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) mice share many morphological similarities with human RA, such as the production of autoantibodies to Type II collagen (9), and are therefore often used as RA models. However, most studies have focused on the knee and hind limb joints, and TMJ-RA has not yet been reported in detail. RA models and human RA clinical symptoms suggest that limb joint RA is exacerbated by overloading (10,11), and it has been reported that overloading the mandibular condyle also causes osteoarthritis-like cartilage resorption (12,13).
Unlike the joints of the extremities, made of hyaline cartilage, which is constantly loaded, the TMJ contains brocartilage, a tissue that is loaded only during functions such as mastication (14). Therefore, the TMJ may be more vulnerable to overload than the limb joints. In this study, we devised a method for overloading by pushing the mandibular condyle posteriorly according to this hypothesis. Therefore, the purpose of this study was to clarify the effect of load on the TMJ on the onset of RA by using the CAIA mouse model and to elucidate the causes of TMJ-RA.

Mice
Animal experiments were approved by the Ethics Committee of Tokyo Dental College (Ethics Application Number: 203102). Male DBA/1JNCrlj mice were bred until 7-8 weeks of age (Charles River, Yokohama, Japan) under standard environmental conditions and were given free access to solid feed and tap water. Tokyo) was bonded to the posterior surface of the maxillary portal teeth with dental composite resin to a basal thickness of 2 mm to induce an imbalanced occlusion. After anesthesia, the control and CAIA groups did not wear the device. Fourteen days after the start of the experiment, after induction of anesthesia with an inhalant anesthetic (sevo urane), the mice were euthanized by intraperitoneal overdose of 150 mg/kg pentobarbital sodium, and samples were collected (Fig. 1A, B).

Evaluation of arthritis
The severity of arthritis was blindly scored on a scale of 0 to 4 as follows: 1 (mild swelling con ned to the ankle or tarsal joint), 2 (mild swelling extending to the center of the foot), 3 (moderate swelling over the metatarsal joints), and 4 (severe swelling including the ankles, feet, and ngers). The scores of all four feet were summed to generate an arthritis score with a maximum value of 16 (16).

Evaluation of in ammation by histological staining of the knee joint
The mice were euthanized 14 days after the injection of anti-Type II collagen antibody, and the knee joints were xed with 10% formaldehyde (Wako Pure Chemical Corporation, Japan) for 2 days. The knee joints were then decalci ed in 10% ethylenediaminetetraacetic acid (EDTA [MUTO PURE CHEMICALS CO, LTD. Japan]) at 4°C for 30 days and embedded in para n. The knee joint tissues were sliced into 4 µm sections and subjected to hematoxylin and eosin (HE) and Safranin O staining to evaluate the morphological changes in the femur and tibia and the staining of proteoglycans in the chondrocyte layer.

Measurement of in ammatory cytokines in blood
All mice were standardized by restricting their eating and drinking for three hours prior to blood collection. Blood was collected using a 5 mm Goldenrod Animal Lancet (MEDI Point NY, USA) and bled using the submandibular bleeding method. Blood was collected in BD Microtina microcentrifuge tubes (365967 Fisher Scienti c Pittsburgh, PA) with coagulant accelerator and serum separator and allowed to coagulate at 4°C for 30 minutes. It was then centrifuged at 13,000 rpm for 10 mins and the serum was collected and stored at -20°C. The Mouse IL-1β/IL-1F2 Immunoassay ELISA kit (Catalog Number MLB00C Quantikine ®ELISA MN, USA) was used to evaluate the serum IL-1β levels in mice. The assay was performed in the non-RA and RA groups (n = 9).

Morphological evaluation by micro-computed tomography
The dimensions of bone destruction were measured and analyzed using microcomputer tomography (µCT) imaging after euthanasia (R_mCT, RIGAKU, Tokyo, Japan). The sample was irradiated with X-rays with a tube voltage of 90 kV and a tube current of 150 mA. The shooting time was 2 mins, the shooting magni cation was 10 times, and the voxel size was 20 × 20 × 20 mm. For evaluation, µCT images were constructed three-dimensionally using the bone structure analysis software TRI/3D-BON (Ratoc System Engineering Co. Ltd., Japan), and the mandibular condyle length and width were measured (17) (Fig. 1.C). The percentage of the crude area was measured using the ImageJ software (National Institutes of Health, Bethesda, MD) from the ratio of the number of pixels in the crude area of the mandibular condyle to the number of pixels in the entire mandibular condyle image. The area of interest was from the crown of the mandibular condyle to the rearmost part (18).

Histopathological analysis of the mandibular condyle
After euthanasia, the heads were xed with 10% formaldehyde for 2 days. They were then decalci ed in 10% EDTA at 4°C for 30 days and embedded in para n. The TMJ tissues were sliced into 4 µm sections. The TMJ was stained with HE staining to evaluate mandibular condyle morphology and to measure the average TMJ condylar cartilage cell layer thickness in the mid-coronal portion of the mandibular condylar head of ve mice in each group. Additionally, the staining of proteoglycans in the mandibular condyle was evaluated by staining with Safranin O. Tartrate-resistant acid phosphatase (TRAP) staining was then performed to evaluate osteoclast differentiation in subchondral bone. TRAP activity was measured according to the method given by Shirakura M et al. (19), and TRAP-positive cells with three or more nuclei were counted as osteoclasts using a TRAP staining kit (Sigma, St. Louis, MO, USA).

Evaluation of immunohistochemistry of the mandibular condyle
After depara nizing the sections of each group, we performed antigen retrieval with the agent ImmunoSaver Antigen Retriever (Electron Microscopic Sciences, Hat eld, PA), and blocking with 1% bovine serum albumin. Immuno uorescence staining was performed using ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs); -5 rabbit polyclonal antibody (abcam, Cambridge, MA, USA), CD (cluster of differentiation) 3 rabbit polyclonal antibody (my biosource Inc., CA, USA), CD45 rat monoclonal antibody (my biosource Inc., CA, USA), and γδ TCR (T cell receptor) mouse monoclonal antibody (Santa Cruz Biotechnology, TX, USA) were used as the primary antibodies. Secondary antibodies were Alexa Fluor 546 Donkey Anti-Rabbit IgG (Thermo Fisher Scienti c, US) to detect ADAMTS-5, and CD45, γ δTCR, and Alexa Fluor 647 Goat Anti-Rat IgG (Thermo Fisher Scienti c, US) to detect CD3. Nuclear staining was performed using stain solution Hoechst 33342 (Thermo Fisher Scienti c, US). The number of cells was measured using the ImageJ software.
Statistical analysis SPSS 17.0 (SPSS Inc. CHI, USA) was used for statistical analyses. For in-group comparisons, multiple comparisons were performed using the Tukey-Kramer test. For comparisons with the control group, multiple comparisons were performed using Dunnett's test. Comparisons between two groups were made using Student' s t test. The level of signi cance was set at P < 0.05 (*).

Evaluation of arthritis in CAIA
All mice injected with the collagen antibody cocktail developed in ammatory arthritis after LPS administration. The arthritis score was 0 in the non-RA group, and 12 in the RA group on the 6th day of administration, and thereafter maintained a stable score of about 14 until sacri ce ( Fig. 2A, B). The localization of in ammatory cells in knee osteoarthritis and their effect on the chondrocyte layer were investigated using HE and Safranin O staining. As a result, in the RA group, in ltration of in ammatory cells was observed in the joint cavity, and a defect in the surface layer of the femur and proteoglycan erosion in the chondrocyte layer were observed, compared with the non-RA group (Fig. 2C). In the RA group, the systemic concentration of the pro-in ammatory cytokine IL1-β increased approximately 1.5fold compared to the non-RA group (Fig. 2D).

Evaluation of mandibular condyle morphology in µCT
A morphological evaluation of the mandibular condyle was performed using µCT. The mandibular condyle length and width were measured as shown in Fig. 1C, but no signi cant change was found in the width diameter of each group (Fig. 3A). The morphological evaluation of each group revealed changes in the posterior part of the mandibular condyle and signs of bone destruction in the CAIA MS group (Fig.  3B). Moreover, the crude area increased approximately 1.5-fold in the CAIA MS group compared with the control group (Fig. 3C).

Histological evaluation of mandibular condyle morphology
The mandibular condyle HE staining showed no clear morphological changes in each group compared to the control group ( Fig. 4A(a-d)). In the HE staining, the CAIA group did not show abnormal synovial proliferation or accumulation of in ammatory cells (Fig. 4A(c)). In the CAIA MS group, accumulation of in ammatory cells in the TMJ cavity was observed (Fig. 4A(d), Fig. 4B). Additionally, the mean TMJ condyle cartilage cell layer thickness in the CAIA MS group was signi cantly thinner than that in the control group (Fig. 4C). Safranin O staining showed decreased staining of proteoglycans in the MS and CAIA MS groups compared to the control group (Fig. 4A(e-h)). The number of TRAP-positive cells in the subchondral bone was higher in each group compared with the control group. (Fig. 4A(i-l)). The total number of osteoclasts measured by TRAP-positive cell counting increased signi cantly in all experimental groups compared to the control group, especially in the CAIA MS group, in which the number of osteoclasts increased by approximately 2-fold compared to the control group (Fig. 4D). The expression of ADAMTS-5, a chondrocyte-destroying enzyme, was also higher in the chondrocyte layer of the CAIA MS group (Fig. 4A(n-p)).

Con rmation of lymphocyte localization in TMJ by immuno uorescent staining
The localization of lymphocytes was con rmed by immuno uorescence staining. Accumulated B cell expression was observed in the subchondral bone of each group. Accumulated T cell expression was hardly observed in the control and MS groups. However, in the CAIA and CAIA MS groups, the expression of accumulated T cells in the subchondral bone was observed (Fig. 5A). There was no signi cant difference in the expression of B cells in each group (Fig. 5B). The localization of T cells in the CAIA MS group was approximately 2-fold higher than that in the control group (Fig. 5C).

Con rmation of localization of γδ T cells in TMJ by immuno uorescent staining
There was little localization of γδ T cells in the control, MS, and CAIA groups. However, in the CAIA MS group, γ δT cells were mostly localized in the subchondral bone (Fig. 6A). The localization of γ δT cells in the CAIA MS group was approximately 6-fold higher than that in the control group (Fig. 6B).

Discussion
Creating a CAIA mouse model In this experiment, CAIA was developed with reference to the report by Nandakumar et al. (15). In general, because of the action of female hormones, female mice may not be suitable for accurate studies on bone metabolism or joint in ammation and bone tissue destruction; therefore, male mice were used in this experiment (20,21). CAIA was used because it shares many morphological similarities with human RA, including the production of autoantibodies to Type II collagen. The CAIA group showed a signi cant increase in the arthritis score and swelling of the limb joints compared with the control group. Furthermore, HE staining showed in ammatory cell proliferation in the knee joint space. Safranin O staining showed a decrease in proteoglycans. This is consistent with a report that the CAIA model causes extensive in ltration of subsynovial tissue by in ammatory cells, cell in ltration into the joint space, and signi cant cartilage destruction (9). Furthermore, there was an increase in the concentration of IL-1β in the blood. These results con rmed the existence of systemic arthritis in ammation and proved that the CAIA mouse model was correctly generated.
Effects of excessive MS on the mandibular condyle in the CAIA mouse model In the RA model, limb joints show swelling, erythema, and synovial proliferation, but clinical signs in the TMJ are generally unclear; therefore, attempts to investigate the RA model of the TMJ have been made recently. In a study using a cartilage proteoglycan (PG)-induced arthritis (PGIA) mouse model, increased ADAMTS in the TMJ was observed, but structural damage was only observed in the TMJ of mice with severe arthritis symptoms (22). In another study using the K/BxN model of spontaneous in ammation, increased expression of vascular endothelial growth factor and IL-17, and decreased expression of osteoprotegerin were observed in the limb joints, but not in the TMJ (23). In the same study with the K/BxN mouse model, the enlargement of the upper joint cavity in arthritic mice was con rmed by Magnetic Resonance Imaging, and only cartilage detachment of the TMJ surface was observed (24). In the present study, although the CAIA group showed increased osteoclast differentiation, there was no signi cant thinning of the chondrocyte layer or localization of lymphocytes. These results suggest that the TMJ is a less primary target for in ammation than the limbic joints in the RA model. However, in the CAIA MS group, ADAMTS-5 was strongly observed, the chondrocyte layer was thinned, and lymphocytes increased in localization. Therefore, it can be inferred that overloading of the TMJ is a factor in the development of TMJ-RA. Functional occlusal loading on the TMJ (by forced unilateral or anterior occlusion) has been shown to lead to worsening of TMJ arthritis (25,26). Therefore, overloading the mandibular condyle may accelerate the degeneration of the mandibular condyle cartilage, and overloading the TMJ may be an important factor for the development of in ammation in the TMJ in the RA model.
The metal plate device used in the present study as a method of applying MS to the mandibular condyle applied excessive MS to the TMJ, demonstrating that it can induce TMJ-RA in CAIA mice. It has been reported that TMJ osteoarthritis-like changes occurred when a resin block was attached to a rats maxillary horn teeth and the mandible was pushed backward (19). Therefore, we designed a similar device for the mouse model. Unlike the TMJ, which is composed of hyaline cartilage, the mandibular condyle cartilage is composed of brocartilage derived from periosteal tissue, so the size and characteristics of the tissue are adjusted to adapt to changes in load (27). As the device we used in this study used a metal plate for the occlusal part, it did not wear because of occlusion, and its thickness remained constant during the device wearing period. Therefore, as it can be regarded that a stable overload was applied to the mandibular condyle, this method is considered to be very useful for establishing a load on the TMJ in mice.
Among all the experimental groups examined in this study, TMJ-RA showed worsening of pathology in the group that combined excessive MS and systemic in ammation (CAIA MS group). Therefore, we con rmed that excessive MS worsens the pathology of the TMJ in CAIA mice.

Involvement of γδ T cells in the mandibular condyle in CAIA mouse model
The CAIA mouse model is not affected by lymphocytes when in ammation develops. However, exacerbation of arthritis due to Type II collagen-reactive T cells in limb joints has been reported in CAIA (28,29). In this study, T cell localization was observed in the subchondral bone of the CAIA MS group. Furthermore, the localization of γδ T cells, which have the smallest number among T cells, was also observed. γδ T cells produce IL-17 and are known to be an important factor in cancer research (30). IL-17 is a cytokine that induces the expression of various pro-in ammatory cytokines and chemokines in a wide variety of cells (31). γδ T cells have also been shown to be associated with RA (32). It has been reported that the number of IL-17-producing cells in mouse femoral bone marrow also increases in CAIA mice (33). Furthermore, it has been reported that Vγ4 / Vδ4 + γδ T cells, one of the γ δT cell subsets, produce IL-17, with localization in the synovial membrane and peripheral blood in CIA mice (34). However, because there is no report that γδ T cells are involved in TMJ-RA, we investigated this and found that they were involved. Therefore, T cells increase in TMJ-RA by applying MS, and among them, γδ T cells increase. Moreover, it is suggested that this may be aggravated by the production of IL-17.
However, this experiment could not clarify why γδ T cells show increased localization in the TMJ.
Isopentenyl pyrophosphate (IPP) is a factor that activates γδ T cells (35). However, IPP is an intermediate product of the intracellular mevalonate pathway, which is di cult to quantify and has not been identi ed. Therefore, further research on quanti cation methods is needed.

Conclusion
The TMJ is less susceptible to in ammation in RA. However, MS exacerbates the disease. The ndings suggested that γδ T cells are involved in TMJ-RA as a causal factor. In the future, it is considered that new treatments targeting γδ T cells may be required for TMJ-RA.

Consent for publication
Not applicable.

Availability of data and materials
Relevant les of this work will be shared on request.

Competing interests
Not applicable.

Funding
The work was supported by the Department of Orthodontics, Tokyo Dental College.
Authors' contributions KN along with TI designed and performed the experiments and conducted data analysis. KN wrote the manuscript. TI and YN contributed in manuscript editing. All authors read and approved the nal manuscript.