**Temporomandibular Joint Osseous Morphology of Class I and Class II Malocclusions in the Normal Skeletal Pattern: A Cone-Beam Computed Tomography Study**

**Xiao-Chuan Fan 1,† , Lin-Sha Ma 1,† , Li Chen 2 , Diwakar Singh 3 , Xiaohui Rausch-Fan 3, \* and Xiao-Feng Huang 1, \***


**Abstract:** (1) Background—The aim of the present study was to evaluate the correlation between the temporomandibular joint (TMJ) osseous morphology of normal skeletal pattern individuals with different dental malocclusions by using cone-beam computed tomography (CBCT). (2) Methods—The CBCT images of bilateral TMJs in 67 subjects with skeletal class I and average mandibular angle (26 males and 41 females, age range 20–49 years) were evaluated in this study. The subjects were divided into class I, class II division 1, and class II division 2 according to the molar relationship and retroclination of the maxillary incisors. Angular and linear measurements of TMJ were evaluated and the differences between the groups were statistically analyzed. (3) Results—Intragroup comparisons showed statistical differences for articular eminence inclination, the width of the glenoid fossa, the ratio of the width of the glenoid fossa to the depth of the glenoid fossa, the condylar angle, and the intercondylar angle between the malocclusion groups. The measurements of the glenoid fossa shape showed no significant difference between the left and right sides. Females showed more differences in the morphological parameters of TMJ between the three malocclusion groups than the males. (4) Conclusion—The present study revealed differences in the TMJ osseous morphology between dental class I and class II malocclusions in the normal skeletal pattern.

**Keywords:** temporomandibular joint; cone-beam computed tomography; malocclusions; articular eminence inclination

#### **1. Introduction**

The temporomandibular joint (TMJ) is one of the most complex joints in the human body. It is formed by the condyle of the mandibular, the inferior component of the joint, and the glenoid fossa forming the superior component of the joint, which is located at the inferior aspect of the squamous part of the temporal bone [1,2]. The joint cavity is separated into the upper and lower compartments by the articular disk, which is made of avascular and aneural dense fibrous connective tissue [3]. The unique anatomy of the TMJ allows for the hinging movement of the mandible and is therefore considered a ginglymoid joint. It can also provide gliding movements and is therefore also an arthrodial joint; thus, it is technically considered a compound joint.

Form and function are considered to be closely linked, and it follows that the osseous morphology of the TMJ might be related to the dynamic balance of mandibular functions in three dimensions. During the mandibular movement, the condyle-disk complex process slides over the posterior slope of the articular eminence. The inclination of articular eminence dictates the path of condylar movement, as well as the degree of rotation of

**Citation:** Fan, X.-C.; Ma, L.-S.; Chen, L.; Singh, D.; Rausch-Fan, X.; Huang, X.-F. Temporomandibular Joint Osseous Morphology of Class I and Class II Malocclusions in the Normal Skeletal Pattern: A Cone-Beam Computed Tomography Study. *Diagnostics* **2021**, *11*, 541. https:// doi.org/10.3390/diagnostics11030541

Academic Editor: Luis Eduardo Almeida

Received: 7 February 2021 Accepted: 16 March 2021 Published: 18 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the articular disk over the condyle [4,5]. For patients with a steeper articular eminence, the condyle is forced to move more inferior and the disk rotates more prominent when protruding or opening. This may lead the mandible to move more vertically during the functional movement [6]. It is reported that a patient with steeper articular eminence is more likely to develop internal dysfunctions, such as anterior disk displacements, than a patient with a flatter articular eminence [7,8].

The articular eminence is sometimes described as the anterior limit of the glenoid fossa. The quantitative evaluation of the articular eminence morphology can be assessed using the inclination, length, and height, where the articular eminence inclination (AEI) is defined as the angle formed by the articular eminence and the horizontal reference plane, which may be the Frankfort horizontal (FH) plane, the true horizontal line, the anterior nasal spine to the posterior nasal plane (ANS–PNS), or the occlusal plane [4,9–11]. The normal value of the AEI in adults has been reported to be 30 to 60 degrees. This angle is not only related to physiological factors, such as age, gender, tooth inclination, dental arch morphology, and the facial growth pattern [4,12–14], but also pathological factors, including occlusion change, TMJ osteoarthritis, internal derangements, and posterior tooth loss [15–18].

A thorough understanding of the morphology and anatomical features of the TMJ is crucial such that we can distinguish the normal condition from the abnormal variant. It is reported that the surface of the structural features of the glenoid fossa may take part in remodeling and reconfiguring following the mechanical and functional conditions to which the adjacent structures are subjected [19]. Some authors suggested that changing the relationship between the upper and lower dentition may lead to right-to-left-side differences in masticatory muscles, which affect the relative relationship of the condyle and glenoid fossa [20,21]. The effect of occlusal factors on the morphology of the temporomandibular joint remains to be clarified. Based on this context, we hypothesized that the discrepancy of the occlusion relationship may be an independent factor that affects the morphology of the TMJ. Thus, the purpose of the present study was to evaluate the correlation between the TMJ osseous morphology of the normal skeletal pattern individuals with different dental malocclusion by using cone-beam computed tomography (CBCT).

#### **2. Materials and Methods**

#### *2.1. Data Collection and Grouping*

The present study was performed at the Department of Stomatology, Beijing Friendship Hospital, Capital Medical University, and it was approved by the Ethical Committee of Beijing Friendship Hospital (approval number 2021-P2-008-01, updated on 1 February 2021). High-resolution CBCT imaging volumes were obtained from examinations that were previously conducted for orthodontic purposes between January 2019 to December 2020; therefore, they had no connection to the present study.

The age of the patients in the sample selected for the study needed to be no less than 20 years old. The sagittal skeletal relationship was defined using the ANB angle (ANB), Frankfurt horizontal–mandibular plane angle (FH–MP), and sella–nasion to gnathion– gonion angle (SN–GnGo), which were measured from the lateral cephalograms that were automatically reconstructed and generated using the QR-NNT Viewer version 5.6 software program (Quantitative Radiology, Verona, Italy). The participants included were limited to skeletal class I with an average mandibular angle, which was defined as 0.7◦ ≤ ANB ≤ 4.7◦ , 21.2◦ ≤ FH–MP ≤ 33.4◦ , 27.3◦ ≤ SN–GnGo ≤ 37.7◦ [22]. The exclusion criteria were as follows: (1) evidence of temporomandibular disorders (TMDs) in a clinical or imaging examination; (2) previous history of orthodontics or TMJ treatment; (3) craniofacial syndrome or anomalies, such as cleft lip and palate; (4) systemic diseases, such as rheumatic arthritis and rheumatoid arthritis; (5) deciduous or missing teeth, except third molars; (6) asymmetric molar relationship or class III molar relationship from the plaster models before treatment; (7) fracture or other pathologies in the region of the TMJs, such as anomalies, tumors, ankylosis, or degenerative changes; (8) poor image quality. After applying the in-

clusion and exclusion criteria, 67 patients (26 males and 41 females, age range 20–49 years) were included and recorded bilaterally; in total, 134 TMJs were evaluated.

The included samples were divided into three groups: class I (CI), class II division 1 (CII-1), and class II division 2 (CII-2). The assignment to each group was done based on the molar relationship on the patient's plaster models before treatment, and the class II groups were then divided according to the retroclination of the maxillary incisors. The interval between the model making and CBCT taking should be less than 1 week. Each study group was then subdivided according to gender and left or right side.

#### *2.2. Simple Size Calculation*

Based on our preliminary data, we got a minimum detectable difference value of 5, and we calculated the sample size using the Power Analysis and Sample Size software version 11.0 (PASS, NSCC, LLC, Kaysville, Utah, USA) in the present study. Considering a study with a two-tailed hypothesis, for an α value of 0.05, a β value of 0.2, and a statistical power of 80%, the minimum sample size was computed to be 38 subjects per group.

#### *2.3. Acquisition of CBCT Images*

The CBCT scans were performed using a New Tom 5G version FP (Quantitative Radiology, Verona, Italy) flat-panel-based CBCT machine with a field of view of 18 × 16 cm. The scanner operated with a maximum output of 110 kV and 5 mAs, exposure time of 3.6 s, and a voxel size of 0.15 mm. The patients with teeth in the maximum intercuspation position were placed in a horizontal position according to the laser indicators and we ensured that the Frankfort horizontal plane was perpendicular to the flat panel of the device in order to obtain a consistent orientation of sagittal images. All CBCT scans were obtained under the same scanning conditions by the same experienced oral radiologist with the same device.

#### *2.4. Measurements*

The CBCT examination results were analyzed using the QR-NNT Viewer version 5.6 software program, which was the proprietary software of the New Tom 5G CBCT system. Before the quantitative evaluation, a secondary calibration was performed to ensure the Frankfort plane was held parallel to the horizontal plane on the sagittal reference view. The CBCT data were also spatially oriented by aligning the anterior and posterior nasal spine on the axial reference view. Digital reconstruction was then conducted in the TMJ regions.

On the axial view, the slice of the condylar processes that had the widest mediolateral extent on both sides of TMJ was used to measure the angulation of the condyles, which involved two variables:

(1) Condylar angle (CA): the angle between the long axis (the line passing through the medial and lateral pole of the condyle) of the left or right condyle and the midsagittal plane in the axial view (Figure 1).

(2) Intercondylar angle (IA): the angle between the long axis of the right and left condyles in the axial view (Figure 2).

**Figure 1.** The measurement of condylar angle (CA) on the axial slice. The angle between the long axis of the condyle (yellow line) and the midsagittal plane (blue line) was measured.

**Figure 2.** The measurement of intercondylar angle (IA) on the axial slice. The angle between the long axis of the left and right condyle (yellow line) was measured.

The chosen slice was also used as the reference view for the secondary reconstruction of the sagittal slices [13], on which a line parallel to the long axis of the condylar process was drawn and sagittal images were reconstructed with a 0.5 mm slice interval and a 0.5 mm thickness. The following variables were calculated on the central sagittal slice of the TMJ:

(1) AEI using the best-fit line method (AEI-BFL): the angle between the tangent line drawn to the posterior slope of the articular eminence and a line parallel to the FH plane (Figure 3).

(2) AEI using the top-roof line method (AEI-TRL): the angle between the "top-roof line" of the articular eminence (the line connecting the crest point of the articular eminence and the roof of the glenoid fossa) and a line parallel to the FH plane (Figure 4).

(3) Width of the glenoid fossa (GFW): the distance between the crest point of the articular eminence and the posterior part of the glenoid process.

(4) Depth of the glenoid fossa (GFD): the perpendicular distance between the highest point of the glenoid fossa and the GFW line (the line passing through the crest point of the articular eminence and the posterior part of the glenoid process) (Figure 5).

(5) Ratio of the GFW to the GFD (GFW/GFD).

(6) Height of the articular eminence (AEH): the perpendicular distance between the highest point of the glenoid fossa and the line parallel to the FH plane through the crest point of the articular eminence (Figure 6). İ İ

**Figure 3.** The measurement of articular eminence inclination (AEI) using the best-fit line method (AEI-BFL) on the central sagittal slice. The angle between the tangent line drawn to the posterior slope of the articular eminence (yellow line) and a line parallel to the Frankfort horizontal (FH) plane (blue line) was measured.

**Figure 4.** The measurement of AEI using the top-roof line method (AEI-TRL) on the central sagittal slice. The angle between the "top-roof line (the line connecting the crest point of the articular eminence and the roof of the glenoid fossa)" (yellow line) and a line parallel to the FH plane (blue line) was measured.

**Figure 5.** The measurement of the width of the glenoid fossa (GFW) and depth of the glenoid fossa

(GFD) on the central sagittal slice. The distance between the crest point of the articular eminence and the posterior part of the glenoid process was measured as the GFW (yellow arrow) and the perpendicular distance between the highest point of the glenoid fossa and the GFW line was measured as the GFD (blue arrow).

**Figure 6.** The measurement of the height of the articular eminence (AEH) on the central sagittal slice. The perpendicular distance between the line parallel to the FH plane through the crest point of the articular eminence (blue line) and the highest point of the glenoid fossa was measured (yellow arrow).

The measurements and angles evaluated on both the axial and central sagittal slices were obtained according to the methods mentioned by ˙Ilgüy, Park, Sümbüllü, and Paknahad [13,23–25]. All the assessments were performed independently by two operators (X.-C.F. and L.-S.M.) and the mean of the results was used for the statistical analysis.

#### *2.5. Measurements Precision*

To test the reliability of the measurements, 30 joints (10 joints from each group) were randomly selected from the collected samples and measured twice with a 1-week interval by the same operators (X.-C.F. and L.-S.M.). The first and the second series of measurements were compared using a paired *t*-test to check for systematic error at a significance level

of *p* < 0.05. The random errors were assessed using the intraclass correlation coefficient (ICC) [26].

#### *2.6. Statistical Analysis*

All the variables were analyzed using the Statistical Package for Social Sciences software version 20.0 (SPSS, IBM, New York, NY, USA). The one-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparisons test was used to analyze the statistical differences between three malocclusion groups. A paired *t*-test and an independent sample *t*-test were applied to determine the possible differences between the left–right sides and the genders in the same malocclusion group, respectively. A *p*-value < 0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Error of the Study*

The paired *t*-test showed no statistically significant differences between the data obtained from the different operators and double measurements from the same operator at a significant level of 0.05. The ICC for intra-operators (operator 1: *r* = 0.981–0.987; operator 2: *r* = 0.875–0.912) and inter-operators (*r* = 0.871–0.901) showed excellent agreement and good reliability for all the measures analyzed.

#### *3.2. Descriptive Statistics of Age and Basic Measurements of the Skeletal Pattern*

A total of 67 high-resolution CBCT imaging volumes with skeletal class I (mean ANB angle of 3.44 ± 1.05◦ ) and average mandibular angle (mean FH–MP of 26.52 ± 3.76◦ with a mean SN–GnGo of 33.22 ± 3.35◦ ) were collected. The mean age of the participants of the present study was 27.91 ± 6.94 years. The means and standard deviations for age and the angular measurements of the skeletal pattern for the different malocclusion groups are presented in Table 1. The intergroup results showed that there were no statistically significant differences between the three malocclusion groups.


**Table 1.** Descriptive statistics of age and the basic measurements of the skeletal patterns.

ANB: ANB angle; FH–MP: Frankfurt horizontal–mandibular plane angle; SN–GnGo: sella-nasion to gnathion-gonion angle.

#### *3.3. Measurements of the Temporomandibular Joint According to Malocclusion*

The distributions of the TMJ osseous morphology measurements in the three malocclusion groups are summarized in Table 2. By using the one-way ANOVA, all the angular and linear measurements showed significant differences between the three groups, except for the GFD and AEH (*p* < 0.05). The Bonferroni multiple comparisons test further showed that the AEI found using the best-fit line method of class II division 2 was significantly higher than the class II division 1 (*p* = 0.017), followed by the class I AEI (*p* = 0.000). However, the difference was not obvious between the class II division 1 and class II division 2 (*p* = 1.000) for the AEI found using the top-roof methods. The widths of the glenoid fossa of the three groups were 17.37 ± 1.60 mm (C-I), 16.86 ± 1.40 mm (CII-1), and 16.59 ± 1.28 mm (CII-2). The indicators of the GFW and GFW/GFD only presented differences between the class I and the class II division 2 groups. As for the measurements of the condyle on the axial slice, the condylar and intercondylar angles of the class II division 2 group were lower than the other two groups (Table 3).


**Table 2.** Measurements of the temporomandibular joint osseous morphology according to the malocclusion.

AEI-BFL: AEI found using the best-fit line method; AEI-TRL: AEI found using the top-roof line method; GFW: width of the glenoid fossa; GFD: depth of the glenoid fossa; GFW/GFD: ratio of the GFW to the GFD; AEH: height of the articular eminence; CA: condylar angle; IA: intercondylar angle; \*: *p*-value < 0.05; \*\*: *p*-value < 0.01.

> **Table 3.** Bonferroni test results for the measurements of the temporomandibular joint for the three malocclusion groups.


CI: class I; CII-1: class II division 1; CII-2: class II division 2; AEI-BFL: AEI found using the best-fit line method; AEI-TRL: AEI found using the top-roof line method; GFW: width of the glenoid fossa; GFD: depth of the glenoid fossa; GFW/GFD: ratio of the GFW to the GFD; AEH: height of the articular eminence; CA: condylar angle; IA: intercondylar angle; \*: *p*-value < 0.05; \*\*: *p*-value < 0.01.

#### *3.4. Descriptive Statistics of the Measurements of the TMJ According to the Left and Right Side*

Table 4 lists the mean values and standard deviations of the TMJ morphology measurements for the left and right sides of the three malocclusion groups. According to the paired *t*-test, only the variables of GFW/GFD and CA in the class II division 1 group and CA in the class II division 2 group showed significant differences (*p* < 0.05).

**Table 4.** Descriptive statistics of the measurements of the temporomandibular joint according to the left and right sides for the three malocclusion groups.


AEI-BFL: AEI found using the best-fit line method; AEI-TRL: AEI found using the top-roof line method; GFW: width of the glenoid fossa; GFD: depth of the glenoid fossa; GFW/GFD: ratio of the GFW to the GFD; AEH: height of the articular eminence; CA: condylar angle; IA: intercondylar angle; \*: *p*-value < 0.05; \*\*: *p*-value < 0.01

#### *3.5. Descriptive Statistics of the Measurements of the TMJ According to Gender*

The differences between the male and female participants of the same occlusion pattern are shown in Table 5. No statistically significant differences were observed between both genders among three malocclusion groups except for the two angular variables of the condyle in the class I group and the GFW/GFD ratio of the class II division 1 group.

**Table 5.** Descriptive statistics of the measurements of the temporomandibular joint according to gender among the three malocclusion groups.


AEI-BFL: AEI found using the best-fit line method; AEI-TRL: AEI found using the top-roof line method; GFW: width of the glenoid fossa; GFD: depth of the glenoid fossa; GFW/GFD: ratio of the GFW to the GFD; AEH: height of the articular eminence; CA: condylar angle; IA: intercondylar angle; \*: *p*-value < 0.05; \*\*: *p*-value < 0.01.

> A comparison of the three malocclusion groups according to gender using one-way ANOVA followed by the Bonferroni multiple comparisons is illustrated in Table 6. The AEI evaluated using two methods presented significant differences between different malocclusion groups in both genders (*p* < 0.05). In addition, the indicators of GFW, GFW/GFD, CA, and IA showed more intergroup differences in females than in males.

**Table 6.** Statistical summary of the measurements of the temporomandibular joint according to malocclusion in different genders.


CI: class I; CII-1: class II division 1; CII-2: class II division 2; AEI-BFL: AEI found using the best-fit line method; AEI-TRL: AEI found using the top-roof line method; GFW: width of the glenoid fossa; GFD: depth of the glenoid fossa; GFW/GFD: ratio of the GFW to the GFD; AEH: height of the articular eminence; CA: condylar angle; IA: intercondylar angle; \*: *p*-value < 0.05; \*\*: *p*-value < 0.01.

#### **4. Discussion**

TMJ is a region with high anatomical complexity, whereas the clinical examination can only provide us with very limited information because it is hard to precisely reveal the internal environment. Taking this restriction into consideration, various radiographic methods were selected to evaluate the morphology of the TMJ in previous studies. Conventional two-dimensional radiographs, such as tomography or panoramic radiographs, were widely used in the early days. However, these modalities are inadequate for quantitative evaluation because of certain limitations, for example, they cannot reflect the three-dimensional shape accurately and may have image distortion and magnification [1,27]. Magnetic resonance imaging (MRI) can provide visualization in both osseous and soft tissue abnormalities, including the morphology of bone structures, the articular disk, and associated muscles and ligaments, in addition to evaluating the functional relationships between them [28]. It is considered the gold standard imaging diagnostic method for TMDs and is widely used in the qualitative evaluation of TMDs [28]. Unfortunately, it was difficult for us to use MRI for all participants included in the present study due to the limitations of the research conditions. The appearance of helical CT makes it possible to evaluate osseous components in three dimensions without superimposition or distortion. Nowadays, CBCT

has already replaced helical CT as a superior method in the stomatological area because of the high spatial resolution, lower radiation dose, shorter scanning time, and greater cost-effectiveness [24,25]. In this study, the CBCT was selected for angular and linear measurements of the TMJ osseous morphology.

The development stage of the articular eminence may influence the quantitative measurements of the TMJ. After reviewing the previous studies, the time to full development time of the articular eminence is still controversial. An autopsy study of Oberg reported that the tubercle and the fossa were well developed at the age of 14–15 years [29]. On the other hand, Katsavrias studied the dry skulls from Asiatic Indian individuals in 2002 and found that the articular eminence was 90–94% complete by the age of 20 years [4]. In order to minimize the influence of the growth on the experimental result, we limited the age of the patients in the sample selection to those that were at least 20 years old. Finally, the age range of the samples included in the present study was 20–49 years and the mean age was 27.91 ± 6.94 years. The sample size for understanding anatomical trends in patients should be as large as possible; however, the present study was just a pilot investigation that demonstrated the possibility of a trend existing. We calculated the sample size using the PASS software based on our preliminary data to increase the scientificity of the study, where the minimum sample size was computed to be 38 joints per group. It should be recognized that the present study aimed to access the association between the osseous morphology of the TMJ and the dental malocclusion. Therefore, the skeletal pattern of the individuals of the current study was strictly limited to skeletal class I with average mandibular angle by ANB, FH–MP, and SN–GnGo. After the statistical analysis of the age and basic measurement of the skeletal pattern, there was no statistical difference between the different malocclusion groups, which indicated that the samples of different malocclusion groups had excellent intergroup consistency for comparison.

The articular eminence is a small bone structure belonging to the cranium. The surface of its posterior slope is exposed to mechanical and functional load arising from biomechanical forces from other structures within the TMJ, where these loads influence the morphological characteristics of it [30]. It is crucial to choose a stable and comparable method for measuring the inclination of the articular eminence. The "best-fit line" method and the "top-roof line" method on the central sagittal slice of the TMJ are the two main methods described in previous studies [13]. The "best-fit line" method is considered as the functional inclination of the articular eminence because it is directly related to the movement direction of the condyle–disk complex and reflects the actual condylar path. In contrast, the "top-roof line" method is more concerned about the localization of the articular eminence in relation to the glenoid fossa and it largely depends on the development of the articular eminence. Therefore, it depicts the anatomical inclination of the articular eminence better. In the current study, the class II division 2 group showed the highest value of AEI-BFL, followed by the class II division 1 group, then the class I group, where the differences between the three groups were significant. For the AEI-TRL, class II division 2 also revealed the highest value. However, the statistical differences were only found between the dental class I and class II malocclusions. These results indicated that there might be some correlation between the AEI-TRL and the molar relationship. However, for the functional AEI, the angle was not only related to the molar relationship but was also affected by the inclination of the anterior teeth.

In previous studies, the fossa shapes were assessed in subjective ways and traditionally classified as triangular, trapezoidal, oval, and round [31]. In this study, the shapes of the fossa were studied quantitatively using their width and depth. Considering that the size of the fossa may have great variability in different individuals, we also introduced the variable of GFW/GFD to describe the relative relationship between the width and depth. The GFD and AEH were both used to analyze the vertical depth of the fossa; however, the GFD is focused more on describing the anatomical height of the glenoid fossa, regardless of the patient's head position. The AEH was highly related to the shape of the articular eminence, which reflected the vertical sliding space of the condyle in the normal

head position. Based on the results of this study, the difference in the fossa shapes only appeared in the GFW and its ratio to GFD between the class II division 2 and class I groups. There were no significant differences in the anatomic and functional fossa depths between different malocclusion groups. It indicated that the fossa shapes of class II divisions 1 and 2 were relatively similar, which was consistent with the findings obtained by Katsavrias and Halazonetis [32]. In addition, the height of the articular fossa might not be a specific index to distinguish between different malocclusions according to samples of the study. Moreover, Sümbüllü et al., C ˘glayan et al., and Poluha et al. [24,33,34] affirmed that the AEH and GFD were also not specific indicators to discriminate between the normal and TMD patients, though the opposite opinion was expressed by Paknahad et al. [25].

TMJ is the only diarthrodial joint with a bilateral linkage in human bodies. It can move synchronously during the symmetrical movement (open–close, protrusion–retrusion) or with its own movements on each side during the lateral movement. Several published papers only noted TMJ as an individual joint without taking into account the contralateral side [24,25]. In the present study, the left and right joints were measured separately and the differences between both sides were evaluated. Based on the results of the study, all the angular and linear measurements of the glenoid fossa showed no significant differences between the left and right sides. The findings of Shahidi et al. and Wu et al. also mentioned that the inclination of the left and right articular eminences did not display any significant differences, which is in agreement with the current study [1,11]. However, the condylar angle of the left joint in both class II division 1 and division 2 groups was significantly lower than that of the right, which was not seen in the class I group. This may indicate that the mandible of the class II patients revealed more asymmetry than that of the class I patients. The values of CA and IA also showed differences between different malocclusion groups. Compared with other types of malocclusion, the condyles of individuals in the class II division 2 group had a greater tendency to rotate inward.

The morphological discrepancies of TMJ due to differences in sex hormones and metabolic activity between male and female individuals have been reported in previous studies [35]. Beyond that, differences in the functional loading of TMJ according to gender can also cause changes in TMJ morphology [36]. Jasinevicius et al. [37] found a gender difference in AEI, which demonstrated a contrary result to the study of Sümbüllü et al. [24]. Based on our results, it was observed that the diversities of TMJ morphology between the two genders were only revealed in the CA and IA values of the class I group. As for the differences in the TMJ morphology variables between malocclusion groups that were separately analyzed according to gender, the AEI showed similar trends in different genders. However, the differences in other morphological parameters of both the glenoid fossa and condyle in female individuals between the three malocclusion groups mentioned in the current study were higher than those in males, which might be one of the possible reasons why TMJ dysfunctions occur more often in females than in males.

#### **5. Conclusions**

On the basis of our study, the following conclusions could be drawn:

1. The inclination of articular eminence displayed a great difference between class I and class II malocclusions in the normal skeletal pattern, and the individuals of class II division 2 showed the highest AEI.

2. The height of the glenoid fossa might not be a specific index to distinguish between different malocclusions.

3. The condyles of individuals in the class II division 2 group had a greater tendency to rotate inward.

4. The shape of the glenoid fossa showed no significant difference between the left and right sides.

5. The differences in morphological parameters of TMJ in female individuals between the three malocclusion groups were higher than those in males.

**Author Contributions:** Conceptualization, X.-C.F. and X.R.-F.; data curation, X.-C.F. and L.-S.M.; formal analysis, L.C.; funding acquisition, X.-F.H.; investigation, X.-C.F. and L.-S.M.; methodology, X.-C.F. and D.S.; project administration, X.-C.F., X.R.-F., and X.-F.H.; supervision, X.R.-F. and X.-F.H.; writing—original draft, X.-C.F.; writing—review and editing, L.C., X.R.-F., and X.-F.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Natural Science Foundation of Beijing Municipality (grant no. 7202036) and the Capital Health Research and Development of Special Funding (grant no. 2018-2-1102).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethical Committee of Beijing Friendship Hospital (approval number 2021-P2-008-01, updated on 1 February 2021).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

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### *Article* **SPECT/CT Correlation in the Diagnosis of Unilateral Condilar Hyperplasia**

**Diego Fernando López 1, \* , Valentina Ríos Borrás 2 , Juan Manuel Muñoz 3 , Rodrigo Cardenas-Perilla 3 and Luis Eduardo Almeida 4**


**Abstract:** Objective: To evaluate the correlation between metabolic bone activity measured by single photon emission computed tomography (SPECT) and the anatomic condylar characteristics acquired by computed tomography (CT), in patients with unilateral condylar hyperplasia (UCH). Method and Materials/Patients: Observational, descriptive study in a group of 71 patients with clinical diagnosis of UCH and indication of SPECT/CT. Bone SPECT images obtained in a gamma-camera GE Infina and processed in a station Xeleris 3 with the program Volumetrix MI Evolution for bone. CT images acquired in a PET/CT Biograph mcT20 equipment (Siemens) processed in a station Osirix V 7.5.1 (Pixmeo, Bomex, Switzerland). Results: The sample included 24 men (33.8%) and 47 women (66.2%). Active state UCH was detected in 40 (56.3%) cases (over 55% uptake in the affected condyle) and 38 (53.5%) presented mandibular deviation to the right side. No significant differences related to sex, age, or mandibular deviation side were found. Mandibular deviation was the only morphologic feature related to active/inactive UCH (*p* = 0.003). The likelihood of active CH was significantly higher in patients with mandibular deviation higher than 6 mm compared with <6 mm (odds ratio (OR): 3.51, confidence interval (CI) 95%: 1.27–9.72). Conclusion: There is a significant correlation between the magnitude of mandibular deviation quantified on CT and metabolic findings obtained by SPECT in patients with UCH. The risk of active UCH is 3.5 times higher in patients with a mandibular deviation ≥6 mm.

**Keywords:** bone scintigraphy; computed tomography; condylar hyperplasia; SPECT; 99mTc-MDP

#### **1. Introduction**

Condylar hyperplasia (CH) is a progressive and self-limiting pathology affecting the mandibular condyle growth and compromising the temporomandibular joint (TMJ) anatomy [1–3].

The functional, occlusal, and esthetic effects of CH in patients demand a multidisciplinary intervention to confirm a clinically suspected diagnosis and establish the therapeutic approach [4]. Early diagnosis and adequate treatment are important to avoid complicated sequelae [5].

UCH is effectively diagnosed by measurement of bone metabolic hyperactivity in SPECT mandibular TMJ 3D images [1,6,7]. Recent studies show that 3D SPECT images are superior to planar images [8]. Uptake radioactive values equal or higher than 55% for the suspected condyle or a percentage side difference over 10% are commonly accepted as positive results indicating hyperactivity (active disease) of the mandibular condyle [9,10]. However, the functional SPECT images are not adequate to show in detail the anatomic

**Citation:** López, D.F.; Ríos Borrás, V.; Muñoz, J.M.; Cardenas-Perilla, R.; Almeida, L.E. SPECT/CT Correlation in the Diagnosis of Unilateral Condilar Hyperplasia. *Diagnostics* **2021**, *11*, 477. https://doi.org/ 10.3390/diagnostics11030477

Academic Editor: Lioe-Fee de Geus-Oei

Received: 9 February 2021 Accepted: 2 March 2021 Published: 8 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

structures in the region of interest (ROI). Therefore, it is recommended to combine SPECT with CT images to characterize the pathology by both its anatomy and metabolism [11,12].

The correlation of metabolic and anatomic findings by a team of experts in the management and interpretation of SPECT/CT techniques allows the establishment of clear-cut parameters of the pathology to precisely indicate the extension of the altered region. This approach is a recent breakthrough in the procedures of diagnosis and treatment of UCH [12,13].

The authors' hypothesis from the existing literature is that the fusion of images and information obtained from SPECT/CT to diagnose UCH improves the precision and specificity of the diagnostic tests and, consequently, allows better therapeutic decisions [11,12,14–16]. Considering that there are few published studies and they provide information from poorly representative populations, the objective of the present study was to correlate the metabolic bone activity of the condyle measured by SPECT with the anatomic information provided by CT images, in patients with active or inactive UCH.

#### **2. Materials and Methods/Patients**

This is a retrospective observational study with no intervention or manipulation of variables from the patients. Therefore, it is a no risk investigation and was approved by the Institutional Ethics Committee (Approval number CEI-403) and conducted following all the regulations of the Declaration of Helsinki, last version.

A population of 153 image sets from patients tested by SPECT/CT (Figure 1), performed in the Nuclear Medicine deparment of a High Complexity Center, between January 2015 and January 2020, was evaluated for the study. The patients had been sent to Nuclear Medicine by the clinical specialists owing to facial asymmetry and suspected UCH. Following the classification of Lopez et al. 2019 [17], the patients were classified by types of facial asymmetry, obtaining 71 cases with UCH diagnosis. Taking into account the information from clinical records, patients with antecedents of TMJ trauma or fracture, previous orthognatic surgery, dentofacial syndromes, and arthritis were excluded. When the SPECT/CT information was not complete, the set of images was excluded as well.

The mandibular bone SPECT procedure was carried out 2 h after the endovenous administration of a dose of 15 mCi 99mTc-MDP for patients over 18 year and normalized according to the EANM Pediatric Dosage Card for patients under 18. The images were obtained using a double head gamma-camera GE Infinia (Chicago, IL, USA), with low energy collimators, and a 128 × 128 high resolution matrix to obtain 45 images, 18 s of exposure, for each 180◦ of detection.

The data were reconstructed in the processing station Xeleris 3 (General Electric, Chicago, IL, USA) using the program "Volumetrix MI Evolution for Bone", with the ordered subsets expectations maximization (OSEM) algorithm for iterative reconstruction, applying four interactions and eight subsets with a Butterworth 0.45 filter and Power 12, plus correction of resolution recovery. From the reconstruction, five transaxial images for quantification in the condyles were obtained, extracting the total counts for a fixed-size ROI (1.76 cm<sup>2</sup> ) [18].

The SPECT report provided quantitative information expressed as radionuclide percentage uptake in the condyles. The counts observed within the selected ROI were used to calculate the % uptake using the following equations:

% right condyle uptake = Maximum counts in the right condyle × 100

Right side counts + left side counts

% left condyle uptake = Maximum counts in the left condyle × 100.

Right side counts + left side counts

A difference in percentage uptake between condyles = or >10% was interpreted as a positive result indicating active pathology [3].

CT cranial images were acquired in a PET/CT Biograph mCT20 (Siemens, Erlangen, Germany) equipment without contrast enhanced, from vertex to sternal notch, applying the following parameters: section thickness: 1.0 mm, pitch: 1.0, and a 512 × 512 cubic matrix with isotropic voxel (size 0.75 × 0.75 × 0.75 mm) to avoid image distortion in the different planes. The same parameters were applied to adult and pediatric patients. The CT images were reconstructed using a B26F homogeneous low dose filter for anatomic localization. All the patients were positioned with a head fixing device to avoid artifacts owing to movement and facilitate the image fusion.

The set of DICOM images was processed in a work station Osirix V 7.5.1 (Pixmeo, Bernex, Switzerland) obtaining linear measurements in sagittal and frontal planes. The description of measurements taken in the 3D bone tissue reconstruction is described in Table 1.


**Table 1.** Description of craniofacial measurements taken in the 3D bone tissue reconstruction, frontal, and sagittal planes.

**Figure 2.** Bone tissue CT, sagittal view. (**A**) Condylar length. (**B**) Mandibular ramus length.

**Figure 3***.* Bone tissue CT, axial view. (**A**) Anteroposterior condylar length. (**B**) Midlateral condylar **Figure 3.** Bone tissue CT, axial view. (**A**) Anteroposterior condylar length. (**B**) Midlateral condylar length.

**Figure 4.** Bone tissue CT, 3D reconstruction. Coronal view. Mandibular deviation magnitude and deviation side.

The tomographic measurements were taken by a trained and calibrated operator. Each data set was simultaneously revised and classified according to the craniofacial characteristics of the asymmetry [17], together by the operator and a specialist with experience in diagnosis and treatment of patients with facial asymmetry.

To assess the reproducibility of the measurements, a duplicate reading was taken by the same observer on a subsample of 20 patients with a four-week interval between the two measurements. The correlation coefficients (Rho) indicate an agreement higher than 0.90 for all of the variables (Table 2).

**Table 2.** Intraobserver agreement for craniofacial measurements obtained in frontal and sagittal views.


Difference between first and second measurement \*. SD: standard deviation. Rho: Spearman correlation coefficient.

#### *Statistical Analysis*

The data were processed by one operator expert in the software management. All statistical analyses were carried out in the software Stata13® (StataCorp, College Station, TX, USA). Normality of distribution was tested by the method of Shapiro–Wilk and, according to it and the kind of variable, the results are expressed as average ± standard deviation, median, inter-quartile range, and absolute/relative frequencies. The Chi square test or Fisher test was applied for bivariate analysis of qualitative variables and either Student's t-test or U-test for quantitative variables, according to the distribution normality. Correlations were evaluated by the Spearman coefficient rho. The level of significance was *p* < 0.05.

Intraobserver agreement was evaluated by the intraclass correlation coefficient of Lin.

Receiver operating characteristics (ROC) curves were determined to establish the best cut-off value of mandibular deviation to classify the hyperplasia as active or inactive. ROC curves were obtained from estimated sensitivity, specificity, and positive and negative predictive values calculating their 95% confidence intervals.

#### **3. Results**

Data from 71 SPECT/CT files were analyzed. The sample included 47 (66.2%) women and 24 (33.8%) men, with a median age of 19 years. From the total number of patients, 40 (56.3%) presented active UCH (≥55% uptake in the affected condyle) and 38 (53.5%) presented right side deviation. No significant differences in the frequency of active UCH were detected in relation to age, (*p* = 0.1), sex (*p* = 0.22), or side of mandibular deviation (*p* = 0.99) (Table 3).


**Table 3.** General characteristics of the patients with active/inactive condylar hyperplasia. SPECT, single photon emission computed tomography.

Median (P25–P75); *n* (%); average (standard deviation). \* (≥10% active stage).

#### *Morphologic Data and Active Hyperplasia*

The measurements obtained in CT images of the patients were related to the active or inactive state of UCH. The results are presented in Table 4 and Figure 5. A statistically significant difference was found only for the amount of mandibular deviation, which was higher in active cases of UCH (6.3 ± 3.4 mm) compared with inactive cases (4.1 ± 2.2) (*p* = 0.003). ≥

**Table 4.** Comparison of morphologic measurements in active/inactive unilateral condylar hyperplasia (UCH) cases.


Average (standard deviation); \*\* *p* < 0.005.

**Figure 5.** Mandibular deviation 95% confidence intervals (CIs) in active an **Figure 5.** Mandibular deviation 95% confidence intervals (CIs) in active and inactive UCH patients.

The ability of mandibular deviation to classify the state of UCH as active or inactive was studied by ROC analysis. The area under ROC curve (AUC) was 0.695 (CI 95%: 0.57–0.82), indicating acceptable ability to distinguish the states, as the area is >0.5 (Figure 6).

**Figure 6.** Receiver operating characteristics (ROC) curve (sensitivity vs. 1—specificity) for condylar hyperplasia activity detection by mandibular deviation.

Two cut-off values of mandibular deviation were selected. The first was a 6 mm value because it is more specific, that is, it detects inactive UCH with 55% sensitivity and 74.19% specificity, providing a positive predictive value (PPV) of 73.3% and negative predictive value (NPV) of 56.1%. The other cut-off value was 4 mm, which shows the best sensitivity, that is, it detects more active cases of UCH with a sensitivity of 75% and 54.8% specificity, PPV of 68–18%, and NPV of 63% (Table 5).

**Table 5.** Diagnostic performance for two criteria (cut-off values: MD = 6 mm and MD = 4) to classify UCH as active/inactive based upon MD.


MD: mandibular deviation; TP (true positive); TN (true negative); FP (false positive); FN (false negative); PPV (positive predictive value); NPV (negative predictive value); LR+ (likelihood ratio positive); LR− (likelihood ratio negative).

The likelihood of having active UCH in patients with mandibular deviation equal or higher than 6 mm was 3.5× higher than the likelihood associated with mandibular deviations <6 mm (OR: 3.51, CI 95%: 1.27–9.72).

When the cut-off value was set to 4 mm, the likelihood of inactive UCH was 73% (OR: 0.27, CI 95%: 0.10–0.75).

#### **4. Discussion**

Image fusion for diagnostic purposes, as in the case of SPECT/CT, is known as a co-register or hybrid technique and it is used to improve the diagnostic precision and, therefore, to aid in the development of a better treatment plan positively determined by the prognosis [19]. In nuclear medicine, the use of hybrid tests increases the diagnostic precision by about 30% in skeletal conditions, as well as in tumors and inflammatory processes, owing to a better correction of attenuation, higher specificity, and a more accurate description of the disease location and possible compromise of the adjacent tissues [20,21].

In connection with this, Jacene et al. [11] postulated that the hybrid SPECT/CT image compared with SPECT alone provides additional interpretative information because the CT data indicate the anatomic location of abnormal findings.

The radioactive uptake in bone SPECT depends on the blood circulation and the absorption by the structure of hydroxyapatite crystals. The areas of high uptake of the radioactive tracer are correlated with hyperemia and more metabolic bone activity and, additionally, identify activity at the molecular level. Therefore, nuclear medicine images are highly sensitive for early detection of lesions, very much earlier than X-ray or tomographic images. Bone SPECT is very useful and has been validated for early diagnosis of UCH [1,6,7,22], because this is a condition that could be active during growth and development, but may be self-limited and finally expressed only by sequelae of the pathology [2,23]. Although the diagnosis is strictly clinical, based on intraoral and extraoral findings and tomographic or radiographic images, the evaluation of bone metabolism by SPECT is very useful to differentiate the active/inactive stages [17].

The hybrid SPECT/CT technique for the diagnosis of UCH provides detailed morphologic information about the mandibular condyles and other craniofacial structures that may be compromised in the pathology. This information is associated with the data of bone metabolic activity in the condyles [12], obtained by the comparative lateral uptake of 99mTc-MPD. In this context, Suh et al. [24] point out the need to have a standardized value for the radiopharmaceutical uptake and the CT data to evaluate temporomandibular disorders.

In the present study, the fusion of data from SPECT/CT to classify UCH conditions as active or inactive detected a significant difference in the magnitude of mandibular deviation (MD) associated with active cases. This finding is concordant with the results of Wang et al. [25], postulating that only MDs exceeding 5 mm are unacceptable according to the patient perception and demand for surgical treatment.

Regarding the diagnostic added value of SPECT/CT compared with SPECT alone to evaluate UCH, Fokoue et al. [26] indicate that this image fusion is superior to detect the hyperplasic area. Agarwal et al. [15] also evaluated, in 21 patients, the diagnostic improvement obtained by the SPECT/TCT fusion compared with SPECT alone, which is more sensitive (80%), but SPECT/CT is more specific (100%) and accurate (85.5%), while planar scintigraphy had the lowest diagnostic performance. However, Theerakulpisut et al. [14], in a study of 61 scintigraphies, concluded that the diagnostic specificity is not improved by fused tests and, as the radiation is increased, did not recommend its use. In the same sense, Verhelst et al. [27] reported that the anatomic changes detected by CT in the hybrid test are evident only in 50% of the patients, adding a minimum benefit, and Liu et al. [16] concluded that ROI delimitation in the drawing of condylar outline was not superior when SPECT/CT was used.

Taking into account these observations, the authors of the present study postulate that the specificity of the SPECT test is improved by the clinical and tomographic pre-diagnostic findings [17], and by the technique applied. The ROI selection; the number of trans-axial sections; and the quantification of radioactive uptake, either by total counts or mean counts, are critical aspects having an influence on the results of the test [18].

International studies indicate that, in different populations, the prevalence of UCH is higher in women than in men [13], as was found in the present study (66.2% women). However, the difference in number of active/inactive cases of the pathology was not significantly sex-dependent. Additionally, in the present study, the difference in laterognathia was not statistically significant, in agreement with previous reports [1,28].

Regarding the age distribution of UCH, the average age in the active UCH group was similar to that of inactive UCH (19 and 17, respectively), both including ranges of residual growth [29]. Although the early detection of UCH reduces the sequelae and invasiveness of the treatment, the fact that that only 10% of changes in bone metabolism appear as positive uptake in SPECT deserves consideration, but the anatomic changes detected by CT are able to indicate the compromise of a higher percentage of bone density [22]. Therefore, in very young patients or in patients that at the moment of examination have initial development of the pathology, the SPECT/CT correlation may not be positive because the pathology is not sufficiently expressed.

The difference between the average DM (6.3 mm) in active states of UCH and the average for inactive conditions (4.1 mm) is statistically significant (*p* = 0.003) and clinically relevant.

Therefore, a significant outcome of the study is the demonstration that a mandibular deviation >6 mm is able to classify the UCH condition as active or inactive, because the AUC in the ROC curve was 0.695. López et al. [30] recently evaluated the ability of mandibular deviation to differentiate the hemi-mandibular elongation (the most common form of condylar hyperplasia [17]) from the asymmetric mandibular prognathism, determining that MDs >5.1 mm are more frequent in hemi-mandibular elongation cases.

The present study provides data from a sample higher than other studies published to study the correlation SPECT/CT in UCH patients. However, a limitation of the study is that hybrid equipment was not used, but rather image fusion. There is no correction for attenuation in this case. Additionally, the use of two separate techniques generates more radiation and is more expensive than the SPECT alone, but the hybrid special scanning system is not yet available in development countries, except in a limited number of research institutions. It is also important to mention that volumetric assessment of the mandible and the articular surfaces provides information on the entire structure under study [31]; although, for this research, what was taken into account were linear measurements, including those of the active condylar surfaces such as the medial–lateral pole and the anterior–posterior pole, which represent the functional area, it is recommended that volumetric assessment of the articular structures be carried out in future studies.

#### **5. Conclusions**

The correlation between the magnitude of mandibular deviation measured in CT images and the percentage uptake obtained by SPECT is statistically significant (*p* = 0.003) and ROC analysis established that a mandibular deviation >6 mm is a risk factor for active UCH (OR: 3.51; CI 95%: 1.27–9.72).

**Author Contributions:** Conceptualization, D.F.L.; Formal analysis, R.C.-P. and J.M.M.; Investigation, D.F.L. and V.R.B.; Methodology, R.C.-P. and J.M.M.; Supervision, D.F.L., R.C.-P. and L.E.A.; Validation, J.M.M.; Visualization, V.R.B.; Writing—original draft, V.R.B., R.C.-P. and J.M.M.; Writing—review & editing, D.F.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** This is a study with no intervention or manipulation of variables from the patients. Therefore, it is a no risk investigation and was approved by the Institutional Ethics Committee (Approval number CEI-403) and conducted following all the regulations of the Declaration of Helsinki, last version.

**Informed Consent Statement:** Without patient consent due, this is a study with no intervention or manipulation of variables from the patients, therefore, it is a no risk investigation.

**Acknowledgments:** The authors wish to express their sincere acknowledge to the Imbanaco Medical Center, Research Institute staff for their support during the development of this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Case Report* **Chemical Diagnosis of Calcium Pyrophosphate Deposition Disease of the Temporomandibular Joint: A Case Report**

**Masahiko Terauchi 1, \* , Motohiro Uo 2 , Yuki Fukawa 3 , Hiroyuki Yoshitake 1 , Rina Tajima 1 , Tohru Ikeda 3 and Tetsuya Yoda 1**


**Abstract:** Calcium pyrophosphate dihydrate (CPPD) deposition disease is a benign disorder characterized by acute gouty arthritis-like attacks and first reported by McCarty. CPPD deposition disease rarely occurs in the temporomandibular joint (TMJ), and although confirmation of positive birefringence by polarized light microscopy is important for diagnosis, it is not reliable because other crystals also show birefringence. We reported a case of CPPD deposition disease of the TMJ that was diagnosed by chemical analysis. A 47-year-old man with a chief complaint of persistent pain in the right TMJ and trismus was referred to our department in 2020. Radiographic examination revealed destruction of the head of the mandibular condyle and cranial base with a neoplastic lesion involving calcification tissue. We suspected CPPD deposition disease and performed enucleation of the white, chalky masses. Histopathologically, we confirmed crystal deposition with weak birefringence. SEM/EDS revealed that the light emitting parts of Ca and P corresponded with the bright part of the SEM image. Through X-ray diffraction, almost all peaks were confirmed to be CPPD-derived. Inductively coupled plasma atomic emission spectroscopy revealed a Ca/P ratio of nearly 1. These chemical analyses further support the histological diagnosis of CPPD deposition disease.

**Keywords:** calcium pyrophosphate dihydrate deposition disease; pseudogout; temporomandibular joint; X-ray diffraction; inductively coupled plasma atomic emission spectroscopy

#### **1. Introduction**

McCarty was the first to report a case of calcium pyrophosphate dihydrate (CPPD) crystal deposition disease, a rare benign crystalline arthropathy also known as pseudogout [1,2]. This disease is characterized by the accumulation of CPPD crystals in various intra-articular and periarticular tissues [3]. Unfortunately, its etiology is unknown, but the disease has been associated with metabolic disorders such as hyperparathyroidism, hypothyroidism, hypomagnesemia, and hyperphosphatemia [4–6]. Diabetes mellitus is associated with a greater incidence of CPPD deposition disease [1,7]. CPPD deposition disease predominantly involves relatively large joints such as the knee, shoulder, hip, wrist, and pubic symphysis; small joints such as the temporomandibular joint (TMJ) are rarely affected [4,8,9]. Pritzker et al. were the first to describe pseudogout in the TMJ in 1976 [10]. Almost all previously reported cases of CPPD deposition disease of the TMJ were diagnosed using a polarized microscope to find positive birefringence. However, we consider this modality insufficient for diagnosis because, in addition to those in CPPD and gout, many

**Citation:** Terauchi, M.; Uo, M.; Fukawa, Y.; Yoshitake, H.; Tajima, R.; Ikeda, T.; Yoda, T. Chemical Diagnosis of Calcium Pyrophosphate Deposition Disease of the Temporomandibular Joint: A Case Report. *Diagnostics* **2022**, *12*, 651. https://doi.org/10.3390/ diagnostics12030651

Academic Editor: Luis Eduardo Almeida

Received: 16 February 2022 Accepted: 5 March 2022 Published: 7 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

birefringent crystals such as those of calcium oxalate, synthetic steroids, and ethylenediaminetetraacetic acid are present in the joint fluid, joint tissue, and bone [11]. Herein, we describe a case of CPPD deposition disease of the TMJ diagnosed using chemical analyses, scanning electronic microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS), XRD, and inductively coupled plasma atomic emission spectroscopy (ICP-AES).

#### **2. Case Presentation**

#### *2.1. Clinical Summary*

A 47-year-old man with a chief complaint of persistent pain in the right TMJ and trismus was referred to our department in 2020. He experienced a traffic accident approximately 25 years ago, which damaged his liver and pancreas and caused wrist and left shoulder bone fractures. His clinical history appeared related to the accident, in which he had also bruised his right TMJ but had not sought treatment for it. Since the accident, the patient experienced discomfort with irregular sudden pain in the right TMJ. This pain resolved after the use of analgesics at every episode. He visited a local hospital when the frequency and intensity of pain increased in 2020. He was then referred to our department when surgical management was anticipated.

Clinical examination revealed bilateral symmetry of the face. His mouth opening was limited, and there was limited lateral excursion to the left. The maximal mouth opening was 28 mm and accompanied by pain in the right TMJ. His uric acid level was normal.

The panoramic radiograph showed an unclear right mandibular condyle with a cloudlike mass (Figure 1). Computed tomography (CT) revealed that the right mandibular condyle was destroyed, and that mottled-like hard tissues had formed around the condyle as viewed on the axial plane (Figure 2A). Similarly, it was confirmed on the coronal plane that the mandibular fossa and cranial base were destroyed. Furthermore, calcified opacity was observed in the bone resorption fossa (Figure 2B). Proton density-weighted imaging showed no disc dislocation in the right TMJ, and the area corresponding to the upper and lower joint space was filled with uneven hypointensity, and the joint space appeared dilated. Additionally, the high signal inside and granular low-signal images were scattered inside the mandibular condyle and fossa (Figure 3). The left TMJ showed no abnormal findings. Based on these findings, we suspected CPPD deposition disease as a clinical diagnosis and excised the lesion under general anesthesia. The right TMJ was exposed using a preauricular approach. During surgery, we confirmed and removed the white chalk-like masses (Figure 4). These masses were present in the articular capsule, articular eminence, mandibular condyle, the upper and lower joint cavities, and articular disc. The maximum size of the masses was 16 × 5 × 5 mm, although various sizes were extracted. CT images were obtained after surgery, and we confirmed that the masses were extracted from the right temporomandibular joint (Figure 5). The postoperative healing was uneventful. This was six months post-surgery, and although the pain in the right TMJ was persistent when opening the mouth, the maximal mouth opening had improved to 42 mm.

#### *2.2. Pathological Findings*

Histologically, the masses consisted of chondroid tissue with island-like or nodular deposition of basophilic crystals (Figure 6A). A foreign body granulomatous reaction was observed in some areas around the crystal deposition (Figure 6B). The crystals appeared rhombus or needle shaped and showed weak birefringence under the polarized light microscopy (Figure 6C,D).

**Figure 1.** Panoramic radiograph from the first visit. The ill-defined calcification around the mandibular condyle is shown (yellow arrows).

**Figure 2.** Preoperative computed tomography (CT) images. (**A**) Axial CT images showing the intra-articular localized, non-corticated, and ill-marginated calcified lesion that abuts the articular surface of the glenoid fossa around the right mandibular condyle. (**B**) Coronal CT images of the right temporomandibular joint revealed resorption of part of the mandibular condyle and cranial base.

**Figure 3.** Magnetic resonance image. Proton density-weighted image of the sagittal plane.

**Figure 4.** Intraoperative photograph. The whitish calcified tumorou **Figure 4.** Intraoperative photograph. The whitish calcified tumorous mass was enucleated from the right infratemporal fossa.

**Figure 5.** Postoperative computed tomography (CT) images. (**A**) Axial CT images and (**B**) Coronal CT images revealed that the masses were extracted from the right temporomandibular joint.

**Figure 6.** Histopathological examination. Representative specimen from the upper joint cavity showed the histopathological features of calcium pyrophosphate dihydrate deposition disease. (**A**) Chondroid metaplasia forms around basophilic islands of crystalline deposits. (**B**) A foreign body granulomatous reaction with multinucleated giant cells phagocytosing the crystals. (**C**) Deposited crystals appeared rhombus or needle shaped. (**D**) Under polarized light, these crystals demonstrated weak birefringence.

#### *2.3. Elemental Analysis Using SEM/EDS*

The two large deposits extracted from the upper and lower joint cavities were chosen as representative specimens for the chemical analysis. SEM/EDS microanalysis was performed to evaluate the calcified mass. Each deposit was fixed with 10% paraformaldehyde solution and washed with distilled water. Thereafter, it was dehydrated in a series of alcohol baths of increasing concentration and dried using vacuum drying. SEM was performed to observe the fine structure around the deposit surface. A carbon coat was formed on these surfaces and observed using SEM (TM4000Plus, Hitachi High-Tech Corporation, Tokyo, Japan) at an acceleration voltage of 15 kV. The elemental distribution around the interface was estimated using EDS (Quantax75 (Oxford Instruments, Oxford, England). The elemental distribution images of the interface were acquired with a resolution of 256 × 200 pixels with an integration time of 200 µs per point. The results are shown in Figure 7. The calcified mass from the upper joint cavity consisted of needle-like crystals, rhomboid masses, and soft tissue that lacked the crystal. However, the specimens from the lower joint cavity consisted of needle-like crystals. Both crystals were the same size with no more than 1 µm thickness and a length of approximately 10 µm. The elemental distribution images and spectrum are shown in Figure 8. The same specimen used in Figure 6A (upper joint cavity) was analyzed. The light emitting parts of Ca and P corresponded with each other. Figure 8C shows the elemental distribution diagram: Ca, P, O, and C were detected. The specimen in Figure 7B (lower joint cavity) was also analyzed, and the same results were obtained (data not shown). × 200 pixels with an integration time of 200 μs per point. The results are shown in with no more than 1 μm thickness and a length of approximately 10 μm.

**Figure 7.** Scanning electronic microscopic images of the masses from the joint cavities. Masses were extracted from the (**A**) upper and (**B**) lower joint cavities (30×). (**C**,**D**) present the 2000× high power fields of (**A-a**) and (**A-b**), respectively.

**Figure 8.** Elemental distribution images of (**A**) Ca and (**B**) P. (**C**) The EDS spectrum for the entire specimen (from Figure 6A) obtained by SEM/EDS.

#### *2.4. Crystal Phase Analysis Using XRD*

The calcified specimens extracted from the upper and lower joint cavities (Figure 6A,B) were washed several times with distilled water, dried at 180 ◦C for 1 h, and ground into powder using an agate mortar. The crystal phases of the powder specimens were analyzed using XRD (Miniflex, Rigaku Cooporation, Tokyo, Japan) under the following conditions: 40 kV, 15 mA, and 2 ◦/min.

Most diffraction peaks of both crystals were assigned to those of CPPD, and a few small peaks were assigned to those of hydroxyapatite (HAp). Therefore, the main crystal was CPPD (Figure 9).

**Figure 9.** X-ray diffraction. The blue and red wavelengths represent the specimens extracted from the upper and lower joint cavities, respectively. The circles and triangles indicate the intrinsic peaks of calcium pyrophosphate dihydrate (CPPD) and hydroxyapatite (HAp), respectively.

μ

#### *2.5. Quantitative Elemental Analysis for ICP-AES*

The tissue concentrations of Ca and P were quantitatively evaluated using ICP-AES. The specimens of the deposits were washed several times with distilled water and weighed while wet (upper: 0.0322 g, lower: 0.0582 g). The specimens were then dissolved in concentrated nitric acid (HNO3; 38 *w*/*v*%, UltraPur100, Kanto Chemical Co. Ltd., Tokyo, Japan) overnight at 90 ◦C. The trace element concentrations in the solutions were quantitated using ICP-AES (Spectro Arcos, Hitachi High-technologies, Tokyo, Japan). Multi-element (100 ppm, XSTC-22, Seishin Trading Co. Ltd., Kobe, Japan) and Sr standard solutions (1000 ppm, Nacalai Tesque, Kyoto, Japan) were used for ICP-AES analyses. The measurement results are presented in Table 1. In the upper cavity specimen, 11.20 wt% Ca and 9.20 wt% P were detected. In the lower cavity specimen, 9.12 wt% Ca and 6.75 wt% P were found (Table 1). Fe, K, Mg, Na, Zn, and Sr were also detected as the trace elements present in the specimens, while the other elements could not be detected or the detection limit or less by this method. In other words, it was clearly composed of elements of biological origin. Accordingly, a Ca/P molar ratio of 0.94 and 1.04 was obtained in the upper and lower cavity specimens, respectively. CPPD is a calcium phosphate that has a Ca/P molar ratio of 1.0. Therefore, the elemental analyses with ICP-AES further supported the histological diagnosis of CPPD deposition disease.


**Table 1.** ICP-AES for quantitative analysis of elements.

#### **3. Discussion**

McCarty's diagnostic criteria for CPPD deposition disease are based on the following: (1) the validation of the specimen by reliable methods such as XRD or chemical analysis or (2) the presence of typical calcific deposition and the detection of crystals suggestive of calcium pyrophosphate deposition through a polarized microscope [1]. The crystal deposits in CPPD deposition disease had a rhomboid structure and were positively birefringent under polarized light, whereas those in gout exhibited negative birefringence. Therefore, birefringence is an important differential diagnostic criterion for gout and CPPD [3,12]. In our case, these crystals clearly demonstrated a rhomboid and rod-shaped appearance, and they exhibited birefringence under a polarized microscope (Figure 6D). Based on these findings, CPPD deposition disease was suspected. However, definitive diagnosis of CPPD can be difficult because not only are these crystals small and often show weak birefringence, but there are also many other birefringent crystals such as those of calcium oxalate, synthetic steroids, and ethylenediaminetetraacetic acid, present in the joint fluid, joint tissue, and bone [11,13]. Therefore, because other quantitative and chemical analyses are required for definitive diagnosis of CPPD deposition disease, we performed SEM/EDS, XRD, and ICP-AES.

Asghar et al. described how crystals demonstrate peaks corresponding to Ca and P in SEM/EDS; therefore SEM/EDS is a rapid and effective method for diagnosing CPPD [3]. In elemental analysis using EDS, only Ca, P, and O derived from CPPD and C and O derived from soft tissue were observed, and the distribution of Ca and P was the same as the bright part of the SEM image (Figures 7A and 8). These results suggest that

the specimens contained CPPD. Most previous reports of CPPD deposition disease describe the detection of Ca and P using SEM/EDS or the diagnosis of CPPD based on a Ca/P ratio of approximately 1 on a rough composition analysis using EDS [3–5]. However, these diagnostic methods are considered inappropriate for the following reasons: (1) Since there are innumerable calcium phosphate compounds such as HAp, tricalcium phosphate (TCP), octacalcium phosphate, and dibasic calcium phosphate anhydrous, it is not possible to determine the exact calcium phosphate compound present despite the detection of Ca and P (Table 2), so accurate Ca and P concentrations should be determined to distinguish calcium phosphate compounds; and (2) most EDS composition analyses have a "standardless method," and their accuracy is lower than that of other analyses calibrated with the concentration standard specimens. Therefore, additional analyses are required to definitively diagnose the precipitation as CPPD.


**Table 2.** A list of the major calcium phosphate compounds.

XRD is a powerful method for the crystal phase and structure analyses of inorganic compounds. The basic method for the crystal identification of inorganic compounds through a database is XRD, and if the results are combined with the identification of major elements using EDS elemental analysis, the elements can be identified with high reliability [14]. XRD revealed that all diffraction peaks were consistent with those of CPPD. Even small peaks were thought to be derived from hydroxyapatite, and the main crystals were strongly considered to be derived from CPPD (Figure 9). XRD can help distinguish crystal phase identification and form, but cannot correctly quantify the chemical composition. This method uses a "standardless method," but SEM/EDS can be used for pseudo-analysis. Thus, the accuracy of the numerical value is questionable.

In this study, we focused on ICP-AES analysis to further accumulate evidence. Bones and teeth are not purely composed of calcium phosphate and often contain divalent cations of Mg, Sr, and Zn instead of Ca (for example, Sr exists at a concentration of one hundred to several hundred parts per million) [15]. Additionally, ICP-AES can help reliably quantify the Ca/P ratio and confirm CPPD based on the chemical composition of the specimen. In CPPD, the Ca/P ratio was 1, which was lower than that of HAp and TCP (Table 1). In our results, the Ca/P ratio in the upper and lower joint cavities was 0.94 and 1.04, respectively. The analysis value retention Ca/P ratio obtained through ICP-AES was approximately 1. These results indicate that there is no possibility that other calcium phosphate compounds are present, which supports the diagnosis of CPPD deposition from the perspective of the chemical composition. In addition, only cations contained in the human organism were detected in our case. In other words, heavy metals and other substances are unlikely to accumulate or be the cause of the problem. Assuming that all the aforementioned Ca values were associated with CPPD deposits, the weight ratio of CPPD in the tissue was estimated to be 40.6 wt% on the upper side and 33.0 wt% on the lower side. Considering this number as wet weight, most of the tissue was CPPD, which corresponds reasonably

well with the SEM observations. Thus, we diagnosed CPPD deposition disease of the right TMJ. The diagnosis of CPPD deposition disease by chemical analysis is not simple considering the special equipment and the number of specimens required for analysis. For this reason, in this study, we preoperatively suspected CPPD, consulted with pathologists and engineers, and used chemical analysis for postoperative diagnosis. Collaborating with pathologists and engineers on preoperatively suspected CPPD deposition disease was effective in obtaining a more reliable diagnosis.

#### **4. Conclusions**

In summary, the diagnosis of CPPD deposition disease of the TMJ is based on the presence of rhomboid positively birefringent crystals; however, because it is considered as a weak diagnostic criterion, performing chemical analyses such as SEM/EDS, XRD, and ICP-AES offers a reliable method for the diagnosis of CPPD deposition disease.

**Author Contributions:** Conceptualization, M.T. and M.U.; Surgeon, H.Y.; Data curation, M.T., R.T., Y.F. and M.U.; Funding acquisition: M.T.; Writing—original draft, M.T.; Writing—editing: M.U., H.Y., T.I. and T.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Informed consent was obtained from the patient to publish this paper.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We are grateful to Kaoru Kobayashi (Tsurumi University, Kanagawa, Japan) for his advice on the image readings.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Synovial Tissue Proteins and Patient-Specific Variables as Predictive Factors for Temporomandibular Joint Surgery**

**Mattias Ulmner 1,2, \* , Rachael Sugars 2 , Aron Naimi-Akbar 2,3 , Nikolce Tudzarovski 2 , Carina Kruger-Weiner 2,4 and Bodil Lund 2,5,6**


**Abstract:** Our knowledge of synovial tissues in patients that are scheduled for surgery as a result of temporomandibular joint (TMJ) disorders is limited. Characterising the protein profile, as well as mapping clinical preoperative variables, might increase our understanding of pathogenesis and forecast surgical outcome. A cohort of 100 patients with either disc displacement, osteoarthritis, or chronic inflammatory arthritis (CIA) was prospectively investigated for a set of preoperative clinical variables. During surgery, a synovial tissue biopsy was sampled and analysed via multi-analytic profiling. The surgical outcome was classified according to a predefined set of outcome criteria six months postoperatively. Higher concentrations of interleukin 8 (*p* = 0.049), matrix metalloproteinase 7 (*p* = 0.038), lumican (*p* = 0.037), and tissue inhibitor of metalloproteinase 2 (*p* = 0.015) were significantly related to an inferior surgical outcome. Several other proteins, which were not described earlier in the TMJ synovia, were detected but not related to surgical outcome. Bilateral masticatory muscle palpation pain had strong association to a poor outcome that was related to the diagnoses disc displacement and osteoarthritis. CIA and the patient-reported variable TMJ disability might be related to an unfavourable outcome according to the multivariate model. These findings of surgical predictors show potential in aiding clinical decision-making and they might enhance the understanding of aetiopathogenesis in TMJ disorders.

**Keywords:** temporomandibular joint; surgery; synovial tissue; synovitis; interleukin; lumican; matrix metalloproteinases; tissue inhibitor of metalloproteinases; cytokine; biomarker

#### **1. Introduction**

Temporomandibular joint (TMJ) diseases might be painful and restrictive by nature, hampering dietary intake and with a negative impact on psychosocial well-being [1]. Surgery is often not considered before a substantial period of failing non-invasive treatments has been tried. From this perspective, the demands on surgery from the affected patients are higher, which accounts for the long duration of symptoms and it is regarded as chronic at this timepoint. Arthroscopy is a minimally invasive surgical alternative that is often used in cases of disc displacement (DD), osteoarthritis (OA), and chronic inflammatory arthritis (CIA) [2–4]. Discectomy is an open surgical procedure that is mainly used for DD [5,6]. The outcome of arthroscopy or discectomy, when applied to patients with DD, OA, and CIA, has been reported to be 60 to 88%, where open joint surgery seems to be slightly superior when compared to arthroscopy in a meta-analysis [2–7]. Inferior surgical

**Citation:** Ulmner, M.; Sugars, R.; Naimi-Akbar, A.; Tudzarovski, N.; Kruger-Weiner, C.; Lund, B. Synovial Tissue Proteins and Patient-Specific Variables as Predictive Factors for Temporomandibular Joint Surgery. *Diagnostics* **2021**, *11*, 46. https://doi.org/10.3390/ diagnostics11010046

Received: 12 December 2020 Accepted: 28 December 2020 Published: 30 December 2020

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

outcome can be prevented by examining the patient in an organised fashion and applying strict diagnostic criteria in search of the correct diagnosis [8,9]. This is the foundation for the surgical decision, but, since TMJ DD, as well as TMJ OA, still lack formal explanatory grounds, a better understanding of the aetio-pathogenesis might shed new light on both diagnostic criteria and best-practice treatment. Characterising the synovial tissue profile and identifying patient-specific predictive factors is a possible approach for enhancing the surgical outcome. This will benefit the patient, as well as regulatory authorities, since a good surgical outcome often prevents further treatment, reduces medication, and minimises sick leave.

Potential predictive factors for TMJ surgery, such as age, TMJ pain, maximal interincisal opening (MIO), psychiatric co-morbidity, and masticatory muscle palpation pain, have been investigated [2,10–13]. In addition, cytokines have been identified in the TMJ synovial fluid, and high concentrations of interleukin (IL) 10 have been proposed for predicting a successful surgical outcome [14]. Studies have already highlighted cytokine localisation in synovial tissue as a valuable biomarker and predictor for treatment in rheumatoid arthritis [15,16]. However, this has not been assessed for the TMJ and diagnoses that are associated with TMJ disease or disorder.

The primary aim of the present study was to investigate synovial tissue protein concentrations and relate them to surgical outcome. The hypothesis was that the concentrations of pro-inflammatory cytokines were higher in patients with inferior outcome, whilst the anti-inflammatory cytokines were higher in patients with superior surgical outcomes. The secondary aim was to control for recorded objective and subjective patient variables, and their relation to surgical outcome. Identifying clinical variables or synovial tissue proteins that might influence surgical outcome could be a valuable contribution to oral- and maxillofacial surgeons decision-making. To our knowledge, this is the first attempt to investigate TMJ synovial tissue proteins in relation to surgical outcome.

#### **2. Materials and Methods**

#### *2.1. Study Design*

A prospective cohort study was performed at the Unit of Cranio- and Maxillofacial Surgery, Karolinska University Hospital, Stockholm, Sweden. The Regional Ethics Review Board approved the study (registration number 2014/622-31/1, approved on 23 April 2014. The patients referred due to DD with reduction (DDwR), DD without reduction (DDwoR), OA together with arthralgia, and CIA were eligible for inclusion. The patients were enrolled from December 2014 to January 2017 and written informed consent was mandatory before inclusion. The study was designed, and the article written, in accordance with the STROBE statement.

#### *2.2. Study Population*

TMJ diagnoses were set according to the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD), except for CIA diagnosis, where the requirement was rheumatic diagnosis that was set by a rheumatologist [8]. Criterions for surgery and inclusion were that the patient had one of the diagnoses DDwR, DDwoR, OA, or CIA, had tried noninvasive therapy for at least 3–6 months, visual analogue scale (VAS) value of ≥4 for TMJ functional pain or TMJ disability, and that DDwoR patients had a MIO of ≤35 mm. The patients were excluded if they had prior open TMJ surgery, were unable to give informed consent, or were younger than 18 years.

#### *2.3. Clinical Examination*

Patient-specific data were registered preoperatively, one and six months postoperatively, while using a standardised case record form. Patient inclusion and data gathering were performed by M.U., A.N.-A., C.K.-W, and B.L., who were calibrated for patient classification and clinical examination. The anamnestic variables collected included present illnesses, medication, prior jaw trauma, ongoing tinnitus/ear fullness affected side, dura-

tion of present TMJ symptoms, and subjective grading on a 0–10 graded VAS of TMJ pain, TMJ disability, psychosocial impact of TMJ problems, and global pain [17]. Joint mobility was measured with the Beighton scoring system, and a value of ≥4 was regarded to be indicative of general joint hypermobility [18]. Positive findings of palpation pain of the masticatory muscles and the TMJ's, and measurements of MIO, lateral excursion, and protrusion were registered in accordance with DC/TMD [8]. A calibration exercise preceded Wilks classification and two of the researchers (M.U. and B.L.) subsequently individually performed the grading [19]. Divergent conclusions were discussed, and consensus was reached. ≥

Surgical outcome was based on four parameters: MIO, TMJ pain, TMJ disability, and TMJ psychosocial impact registered at the last planned visit six months after surgery, and classified as either successful, good, intermediate, or deteriorated. The criteria for successful treatment were objective measurement of MIO ≥ 35 mm, and all subjective VAS scoring of TMJ pain, TMJ disability, and TMJ psychosocial impact of ≤3 or ≥40% improvement. A good surgical outcome was defined as MIO ≥ 35 mm and whether one or two of the VAS values of pain, functional disabilities, and psycho-social impact showed ≥40% improvement or a VAS value of ≤3. If the above-mentioned criteria got obviously worse, then the outcome was deemed to be deteriorated. With minor or no improvements, the result was classified as intermediate. ≥ ≤ ≥ ≥ ≥ ≤

#### *2.4. Surgical Procedure and Collection of Tissue Samples*

According to the departments' research-based guidelines, patients with DDwR were scheduled for discectomy, and patients with DDwoR, OA, or CIA had arthroscopic lysis and lavage generally. One surgeon performed all of the operations (M.U.). Two synovial tissue biopsies were taken from the posterior bilaminar zone in the superior joint compartment. The triangulation technique was used in order to collect biopsies under direct visualisation during arthroscopy (Figure 1) [20]. Biopsy forceps (Karl Storz SE & Co, Tuttlingen, Germany) were used, resulting in approximately 4 mm<sup>2</sup> tissue samples. Synovial tissue samples that were destined for protein extraction were placed in RNAlater (ThermoFisher Scientific, Waltham, MA, USA) and then refrigerated for 24 h. RNAlater was then removed and the samples stored at −80 ◦C. −

**Figure 1.** Photographs showing the synovial sample procedure. (**A**) In the triangulation technique, the instrument canal closest to the patient's ear contained the optic and the second instrument canal the biopsy forceps. (**B**) A synovial tissue sample from the posterior bilaminar zone in the superior temporomandibular joint (TMJ) compartment was taken with the biopsy forceps.

#### *2.5. Analysis of Synovial Tissues*

Synovial tissue was ground in liquid nitrogen in order to disrupt the tissue piece. The proteins were extracted in ice-cold cell lysis buffer NP40, prepared according to the manufacturer's instructions (ThermoFisher Scientific) [21]. 50 µL cell lysis buffer per 10 mg of tissue was used. The mixtures were centrifuged at 20,000× *g* at 4 ◦C for 10 min., and the supernatant stored at −80 ◦C until analysis.

The total protein concentration in each tissue sample was determined while using the Qubit Protein Assay Kit (ThermoFisher Scientific) and the Qubit Fluorometer (ThermoFisher Scientific). Magnetic bead panels HTMP2MAG-54K, HMMP2MAG-55K, and HCYTOMAG-60K (Merck Millipore, Burlington, MA, USA), and LXSAHM-20 (R&D Systems, Bio-Techne Corp., Minneapolis, MN, USA), were used in order to determine the levels of synovial tissue proteins with multi-analytic profiling while using a Luminex 200 system (Luminex, Austin, TX, USA) and xMAP technology. Attained data were analysed by xPONENT 3.1 software (Luminex). HCYTOMAG-60K and LXSAHM-20 were customised and contained the following proteins: a disintegrin and metalloproteinase with a thrombospondin type 1 motif member 13 (ADAMTS13), aggrecan, bone morphogenetic protein (BMP) 2, 4, and 9, collagen 1-α1, collagen 4-α1, epidermal growth factor (EGF), eotaxin, fibroblast activation protein (FAP), fibroblast growth factor 2 (FGF-2), fibronectin, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage (GM) CSF, hepatocyte growth factor receptor (HGFR), intercellular adhesion molecule 1 (ICAM-1), IL-1β, IL-1ra, IL-6, IL-7, IL-8, IL-10, IL-17, interferon gamma-induced protein 10 (IP-10), lumican, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, neural cell adhesion molecule (NCAM), osteoprotegerin (OPG), osteonectin, platelet-derived growth factor (PDGF) AA, PDGF-AB/BB, regulated on activation normal T-cell expressed and secreted (RANTES), syndecan-1, syndecan-4, tenascin C, transforming growth factor α (TGF-α), tumour necrosis factor α (TNF-α), TNF-β, triggering receptor expressed on myeloid cells 1 (TREM-1), and vascular endothelial growth factor (VEGF). In addition, HTMP2MAG-54K and HMMP2MAG-55K contained matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-7, MMP-9, MMP-10, tissue inhibitor of metalloproteinase 1 (TIMP-1), TIMP-2, TIMP-3, and TIMP-4. Fifty-one proteins were analysed in total.

#### *2.6. Statistical Analyses*

Stata version 15 (StataCorp, Collage Station, TX, USA) and IBM SPSS version 25.0 (IBM Corp, Armonk, NY, USA) were used to analyse the data. The descriptive statistics were calculated as mean ± SD for all continuous data and as a number and percentage for bivariate data. Data on patient characteristics were analysed with Student's T-test for continuous data and Fisher's exact test for categorical data. For the statistical analyses of synovial tissue proteins, the surgical outcome groups intermediate and deteriorated were merged into one group, since there was only one patient in the deteriorated group. The concentration of specified proteins (pg/mL) was used in the statistical analyses. The surgical outcome was the dependent variable and ordered logistic regression were used for both univariate and multivariate computations. The multivariate regression model was tested with Akaike's information criterion (AIC) in order to estimate the performance of the model. The best performance was reached by including the specific protein and the potential confounders CIA, TMJ disability, masticatory muscle palpation, and the interaction of CIA and positive finding of masticatory muscle palpation pain. Masticatory muscle palpation was dichotomised, and no finding of palpation pain was merged with unilateral positive sign, since it made the model perform better according to AIC. A *p*-value of ≤0.05 was regarded as significant.

#### **3. Results**

#### *3.1. Patient Demographics and Patient-Specific Clinical Variables*

One-hundred patients had followed the protocol at study closure (Figure 2). The 27 patients who were excluded or did not participate had a mean age of 40.4 years (SD 15.3)

and 81% were women. No differences were found when comparing participating patients to the non-participating with regards to sex and age. In six patients out of the hundred included, it was not possible to harvest any synovial tissue; therefore, their data were only included in the clinical parameter analyses. Table 1 compiles demographic data and preoperative patient-specific symptoms and signs. The outcome of surgery, as well as measured mean differences before and after surgery are displayed in Table 2. Patients in the diagnosis-group OA were significantly older when compared to the other groups (*p* = 0.022) and they had more co-morbidities, classified as "other diseases" (*p* = 0.041). Both OA (*p* = 0.003) and DDwR patients (*p* < 0.001) had larger MIO as compared to the rest of the cohort. The group with DDwR had significantly lower TMJ pain VAS-value (*p* = 0.008) and fewer patients with both palpation pain of the masticatory muscles (*p* < 0.001) and the TMJ (*p* < 0.001). On the other hand, the CIA-group had significantly more patients with palpation pain on muscles (*p* = 0.006) and TMJ (*p* < 0.001). Patients with DDwoR had significantly lower TMJ psychosocial impact (*p* = 0.003), as well as lower global pain (*p* = 0.050), when compared to the other three diagnoses.

**Figure 2.** Flow chart illustrating patients´ eligibility for inclusion into the study, reasons for not participating, and TMJ diagnoses. CIA, chronic inflammatory arthritis; DDwoR, disc displacement without reduction; DDwR, disc displacement with reduction; OA, osteoarthritis; *n*, number; ndd, no diagnosis defined; ST, synovial tissue.


**Table 1.** Preoperative registration of demographic data, anamnestic information, objective and subjective measurements of included patients.

Bilat, bilateral; CIA, chronic inflammatory arthritis; DDwoR, disc displacement without reduction; DDwR, disc displacement with reduction; lat, lateral; M, men; MIO, maximum interincisal opening; mos., months; *n*, number; na, not applicable; OA, osteoarthritis; palp, palpation; SD, standard deviation; TMJ, temporomandibular joint; VAS, visual analogue scale; W, women.

> All of the registered diagnoses and patient-specific factors were tabulated and those showing signs of association to outcome were further analysed in a univariate fashion. TMJ palpation pain (coef., 0.89; *p* = 0.044) and masticatory muscle palpation pain (coef., 1.97; *p* < 0.001) were both positively associated to a worse outcome (Figure 3). The four subjective VAS variables all had a linear association with surgical outcome, which was significant for TMJ disability (coef., 0.29; *p* = 0.011), TMJ psychosocial impact (coef., 0.15; *p* = 0.032), and global pain (coef., 0.13; *p* = 0.043), but not for TMJ pain (coef., 0.16; *p* = 0.073) (Figure 4). Tinnitus, sex, age, MIO, psychiatric disorder, TMJ pain, and the TMJ diagnoses showed no significant correlation to outcome.


**Table 2.** Outcome of surgery for the total cohort and for the different TMJ diagnoses, comparing mean differences of preoperative and postoperative values using paired samples t-test.

CIA, chronic inflammatory arthritis; DDwoR, disc displacement without reduction; DDwR, disc displacement with reduction; GJH, general joint hypermobility; LTR, lateral excursion; M, men; MIO, maximum interincisal opening; mm, millimetre; mos., months; *n*, number; OA, osteoarthritis; PTR, protrusion; SD, standard deviation; TMJ, temporomandibular joint; VAS, visual analogue scale; W, women. <sup>a</sup> The patient with deteriorated outcome was transferred to the intermediate group for statistical analyses. \* *p* ≤ 0.05, \*\* *p* ≤ 0.005.

− − − − − − − − −

− −

≤ ≤

≤ ≤ **Figure 3.** Line charts illustrating the preoperative TMJ and muscle palpation variables related to surgical outcome groups. The intermediate outcome group also contains the single deteriorated patient. Palpation of the lateral aspect of the TMJ and palpation of the masseter and temporal muscle was performed in accordance with the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD). Positive palpation findings were recorded as being unilateral or bilateral. Negative palpation findings were recorded as none registered. (**A**) The line chart shows the significant linear association of increased positive findings of TMJ palpation pain related to a worse outcome (*p* ≤ 0.05). (**B**) Masticatory muscle palpation pain had a strong association to surgical outcome in a similar manner as TMJ palpation pain (*p* ≤ 0.005). DC/TMD, diagnostic criteria for temporomandibular disorders; TMJ, temporomandibular joint.

**Figure 4.** Box plot showing the relation between preoperative patient-reported 0–10 VAS values of TMJ pain, TMJ disability, TMJ psychosocial, and global pain according to surgical outcome groups. The top of the box indicates the 75th percentile, and the bottom the 25th percentile. The line within the box shows the median and the cross indicates the mean. The whiskers show the 10th and 90th percentile and points outside the 10th and 90th percentile shows outliers. All four preoperative VAS values were higher in relation to a worsened outcome, but only TMJ pain were not significant. TMJ, temporomandibular joint; VAS, visual analogue scale. \* *p* < 0.05.

−

≤

−

#### *3.2. Synovial Tissue Analysis, Univariate Analysis*

When examining the proteins in the multi-analytic profiling system, some of the proteins were identified as being below or above the standard limits, as defined by the manufacturers. Those samples that were below the lowest standard were set at the lowest standard value and those above the highest standard were set at the highest standard value. The processed tissue samples with protein measurements out of the assay's precision and recovery were treated as the missing values.

ADAMTS13, BMP-9, HGFR, IL-7, MMP-10, NCAM, osteonectin, syndecan-1 and 4, TIMP-4, and TREM-1 were found with detectable concentrations in most patients. These proteins have not been previously described in the human TMJ.

All of the analysed proteins were related to outcome in a univariate ordered logistic regression model. Higher concentrations of both eotaxin (coef., 2.89 <sup>×</sup> <sup>10</sup>−<sup>3</sup> ; *p* = 0.038) and syndecan-1 (coef., 1.11 <sup>×</sup> <sup>10</sup>−<sup>4</sup> ; *p* = 0.024) significantly changed the outcome in a negative direction. None of the other proteins had any significant correlation to outcome.

#### *3.3. Multivariate Analysis of Synovial Tissue and Potential Confounders*

The significant results from univariate analyses with respect to patient-specific variables were tested in a multivariate model. The tested variables were TMJ disability (coef., 0.23; *p* = 0.054), TMJ psychosocial impact (coef., 0.06, *p* = 0.424), global pain (coef., 0.07, *p* = 0.352), and masticatory muscle palpation pain (coef., 1.69; *p* = 0.001). Table 3 presents multivariate ordered logistic regression analyses of association between the outcome of TMJ surgery and the specific proteins, including potential confounders and the interaction between CIA and positive bilateral masticatory muscle palpation pain. Higher concentrations of IL-8, lumican, MMP-7, and TIMP-2 were all associated to an inferior outcome in a significant way. ADAMTS13, BMP-4, eotaxin, NCAM-1, and TIMP-1 were close to significant, with *p*-values of ≤ 0.075. Patients with the interaction CIA and bilateral masticatory muscle palpation pain showed a significant association to a positive surgical outcome in the analysis of ADAMTS13, IL-1β, and TNF-β (Table 3). All of the analyses of the interaction variable showed a negative coefficient, indicating that positive bilateral muscle palpation pain does not predict a poor surgical outcome in patients that are suffering from CIA.


**Table 3.** Ordered logistic regression relating the dependent variable surgical outcome (successful, good, intermediate/deteriorated) to analysed proteins, potential confounders and the interaction of CIA and positive jaw muscle palpation tenderness.


**Table 3.** *Cont.*

ADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif member 13; BMP, bone morphogenetic protein; CIA, chronic inflammatory arthritis; Coef., coefficient; EGF, epidermal growth factor; FAP, fibroblast activation protein; FGF, fibroblast growth factor; G-CSF, granulocyte-colony stimulating factor; GM, granulocyte-macrophage; HGFR, hepatocyte growth factor receptor; ICAM, intercellular adhesion molecule; IL, interleukin; IP, interferon gamma-induced protein; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; MMP, matrix metalloproteinase; NCAM, neural cell adhesion molecule; No., number; obs., observations; OPG, osteoprotegerin; palp., palpation; PDGF, platelet-derived growth factor; TIMP, tissue inhibitors of metalloproteinases; TGF, transforming growth factor; TNF, tumour necrosis factor; TREM, triggering receptor expressed on myeloid cells; VEGF, vascular endothelial growth factor. <sup>a</sup> The ordered logistic regression was modelled from successful outcome in three steps down to intermediate/deteriorated outcome as the worst outcome. A positive coefficient thereby indicating that the higher the specific protein concentration, the worse the outcome. <sup>b</sup> A positive coefficient shows that the diagnosis or variable affects the outcome in a negative way. <sup>c</sup> Describes the interaction between CIA and positive jaw muscle palpation related to outcome. A negative coefficient indicates a positive correlation to outcome. <sup>d</sup> No observations in the sample. <sup>e</sup> Omitted because of collinearity. <sup>f</sup> Too few observations why calculations could not be done. \* *<sup>p</sup>* <sup>≤</sup> 0.05, \*\* *<sup>p</sup>* <sup>≤</sup> 0.005.

#### **4. Discussion**

The success rates in TMJ surgery have been reported as variable and they often not better than 80%. Identifying patient-specific predictors might be a valuable tool for surgeons, patients, and health-care providers to improve outcome.

The investigation of TMJ synovial fluid proteins potentially reflecting surgical outcome has to our knowledge been done twice before, where higher concentrations of IL-10 were significantly associated with a positive outcome of arthrocentesis, and TMJ pain was associated with higher concentrations of IL-6 and IL-8 indicating a negative outcome [14,22]. The TMJ synovial tissue proteins have not been investigated in relation to outcome earlier. In this study, four proteins—IL-8, lumican, MMP-7, and TIMP-2—were found to be associated with an impaired surgical outcome in a concentration dependent matter in multivariate analyses. The chemokine IL-8 exerts effects on cells, such as fibroblasts, neutrophils, and synovial cells during normal function and with an inflammatory state [23]. Higher levels of IL-8 have been associated with a higher severity of disease in rheumatoid arthritis and when comparing DDwR to DDwoR [23,24]. In oral squamous cell carcinoma, IL-8 was reported to up-regulate the production of MMP-7 via the IL-8 receptor β [25]. MMP´s are a group of proteases with the ability to degrade components of extracellular matrix (ECM) [26,27]. MMP-7 has been found to act on several collagens and proteoglycans directing to its role in joint degradation [26–28]. The main endogenous inhibitors of MMPs are TIMPs that bind MMPs in a 1:1 ratio [29]. TIMP-2 has been proposed to serve as a continuous ECM protector. Some of the studies have suggested that its mRNA expression does not respond to different stimuli during basal or inflammatory activity in joints, whereas other studies have detected mRNA in response to osteopontin or relaxin levels [30–32]. The small, leucine-rich, proteoglycan lumican has been associated with wound healing and found to be increased in degenerated TMJ discs when compared to normal discs [33,34]. All four proteins with a negative correlation to outcome are related to tissue turnover and remodeling, where lumican and TIMP-2 are suggested to promote TMJ healing, whilst IL-8 and MMP-7 possibly have degenerative properties. Therefore, they may potentially be useful as individual markers for a negative outcome and, if they are also demonstrated to relate to each other, they might provide a protein pattern that is indicative of biomarker quality.

In the univariate statistical analyses, higher concentrations of eotaxin and syndecan-1 showed a correlation to a suboptimal surgical outcome. Eotaxin is a chemokine that has been shown to increase osteoclast activity in bone inflammation, while syndecan-1 might be associated with attempted cartilage repair [35,36]. Fibrocartilage stem-cells (FCSC) with chondrogenic differentiation abilities have been identified in the human TMJ cartilage [37]. The association between the transmembrane proteoglycan syndecan-1 and FCSC reparative traits is unknown, but it deserves attention.

The age of the patient and preoperative MIO have previously been described as predictive factors for TMJ surgical outcome [2,13,38,39]. In the current study, this could not be confirmed for MIO and age. Univariate analysis revealed that TMJ disability, TMJ psychosocial impact, global pain, masticatory muscle, and TMJ palpation pain were significantly related to outcome. In the multivariate analysis, only masticatory muscle palpation pain remained significant. TMJ disability and CIA were included in the multivariate model, because they, according to AIC, strengthened the model. CIA showed a significant negative correlation to outcome in 10 of the 51 multivariate analyses of specified proteins, and TMJ disability in six of 51. This might imply that the diagnosis CIA and the variable TMJ disability individually can be valid predictors for a negative outcome of TMJ surgery.

The association between masticatory muscle palpation pain and negative outcome has earlier been presented by our group in two different patient cohorts [2,13]. Considering that the variable was significant in all but four multivariate calculations advocates its potential as a predictive factor. In contrast, CIA patients with the presence of bilateral muscle palpation pain did not seem to have any additional negative impact on outcome. The result strongly suggests that bilateral masticatory muscle palpation pain is an important predictive factor, which is why DDwR-, DDwoR-, and OA-patients with these findings should alert the clinician to consider a new round of non-invasive therapy.

A shortcoming of the study was the loss of 27 eligible patients, who did not participate for different reasons. Potential bias might be considered because the only variables possible to analyse for the non-participant group were sex, age, and TMJ diagnosis. A relatively short follow-up period was used, which might also implicate a bias in some of the diagnostic groups. Because only four out of 51 proteins correlated with outcome, there was a risk that these associations are by chance. This suggests that further investigations should be made to verify these findings.

To conclude, preoperative bilateral palpation pain of the masticatory muscles was found to be a predictor for negative surgical outcome, and it might alert the surgeon to consider non-invasive interventions that have not yet been tried before scheduling surgery. However, in patients diagnosed with CIA, bilateral masticatory muscle pain did not indicate a negative surgical outcome when compared to the other included TMJ diagnoses. TMJ disability was the only outcome measure that showed potential as predicting factor. IL-8, lumican, MMP-7, and TIMP-2 were individually shown to have a positive correlation to worse outcome. Altogether, the results demonstrate that the clinical variable bilateral masticatory muscle palpation pain seems to be a more robust predictor for surgical outcome when compared to any of the investigated proteins. Description of protein alterations due to diagnosis, severity, and progress of TMJ disease, but also in relation to different treatment modalities, has to continue. Further mapping will possibly reveal more of the potential multi-factorial pathogenesis.

**Author Contributions:** Conceptualization, M.U., R.S., C.K.-W., B.L.; Methodology, M.U., R.S., C.K.- W., B.L.; Validation, M.U., R.S., B.L.; Formal Analysis, M.U., R.S., A.N.-A., B.L.; Investigation, M.U., R.S., N.T., B.L.; Resources, M.U., R.S., A.N.-A., N.T., B.L.; Data Curation, M.U., R.S.; Writing—Original Draft Preparation, M.U., B.L.; Writing—Review & Editing, M.U., R.S., A.N.-A., N.T., C.K.-W., B.L.; Visualization, M.U., R.S., A.N.-A., B.L.; Supervision, R.S., B.L.; Project Administration, M.U., R.S., B.L.; Funding Acquisition, M.U., B.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by grants from the Swedish Dental Society, Karolinska Institutet funding, University of Bergen and HelseVest funding, Haukeland University Hospital, Bergen, Norway.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Regional Ethics Review Board in Stockholm (registration number 2014/622-31/1, approved on 23 April 2014).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors wish to thank the technical support of Janne Elin Reseland, Safiyye Suslu, and Aina Mari Lian at Oral Research Laboratory, Institute of Clinical Dentistry, University of Oslo.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Proteomic Expression Profile in Human Temporomandibular Joint Dysfunction**

**Andrea Duarte Doetzer 1, \*, Roberto Hirochi Herai 1 , Marília Afonso Rabelo Buzalaf <sup>2</sup> and Paula Cristina Trevilatto 1**


**Abstract:** Temporomandibular joint dysfunction (TMD) is a multifactorial condition that impairs human's health and quality of life. Its etiology is still a challenge due to its complex development and the great number of different conditions it comprises. One of the most common forms of TMD is anterior disc displacement without reduction (DDWoR) and other TMDs with distinct origins are condylar hyperplasia (CH) and mandibular dislocation (MD). Thus, the aim of this study is to identify the protein expression profile of synovial fluid and the temporomandibular joint disc of patients diagnosed with DDWoR, CH and MD. Synovial fluid and a fraction of the temporomandibular joint disc were collected from nine patients diagnosed with DDWoR (*n* = 3), CH (*n* = 4) and MD (*n* = 2). Samples were subjected to label-free nLC-MS/MS for proteomic data extraction, and then bioinformatics analysis were conducted for protein identification and functional annotation. The three TMD conditions showed different protein expression profiles, and novel proteins were identified in both synovial fluid and disc sample. TMD is a complex condition and the identification of the proteins expressed in the three different types of TMD may contribute to a better comprehension of how each pathology develops and evolutes, benefitting the patient with a focus–target treatment.

**Keywords:** temporomandibular joint; protein expression; temporomandibular joint dysfunction

#### **1. Introduction**

Temporomandibular dysfunction (TMD) is a disorder of the masticatory system and it is characterized by pain, loss of function of one or both articulations, and impairment of the masticatory system. TMD impacts not only jaw function, but the life quality of affected patients, increasing their treatment costs and work absence [1]. According to the National Institute of Health [2], TMD management in the USA costs approximately 4 billion dollars per year. A diagnostic protocol developed for research named Research Diagnostic Criteria for TMD (RDC/TMD), classifies TMD as myalgia, arthralgia, condylar pathologies, disc displacement, osteoarthrosis, osteoarthritis, degenerative joint disease and subluxation [3]. TMD has a multifactorial etiology, the most common being trauma, psychological alterations, hormone, inflammatory diseases, parafunction, and genetics [1,4]. TMD usually requires a panorex, and depending on the TMD type, magnetic resonance imaging, scintigraphy and tomography, besides a thorough clinical evaluation [5,6].

Depending on the TMD type, it can be classified as condylar hyperplasia (CH), disc displacement without reduction (DDWoR) and mandibular dislocation (MD). DDWoR is the most common TMD disorder [7], and along with CH, its etiology's understanding is still unclear. MD is a condition that is probably caused by physical alterations [8], and since it is less likely to have hormone contribution, it is a good TMD condition to compare the results with the other pathologies. DDWoR is caused by an abnormal positional association between the disc and the condyle, where the disc is permanently anteriorly displaced

**Citation:** Doetzer, A.D.; Herai, R.H.; Buzalaf, M.A.R.; Trevilatto, P.C. Proteomic Expression Profile in Human Temporomandibular Joint Dysfunction. *Diagnostics* **2021**, *11*, 601. https://doi.org/10.3390/ diagnostics11040601

Academic Editor: Gustavo Baldassarre

Received: 28 February 2021 Accepted: 24 March 2021 Published: 28 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in relation to the condyle, causing limited range of mouth opening, pain and may lead to temporomandibular joint (TMJ) degeneration [9]. Disc displacement corresponds to 41% of TMD intra-articular disorders [7], and it is considered a multifactorial disease, with overlapping conditions contributing to its modulation including stress, parafunction, behavioral pattern, emotional status, and genetic background [3]. Among its different types of treatment, clinical handling is firstly employed (splint therapy, medication, physiotherapy) and when unsuccessful, surgery is indicated [6,10]. MD is an involuntary forward movement of the condyle beyond the articular eminence, mostly associated with trauma or excessive mouth opening, impairing its essential functions (speaking, chewing), and it accounts for 3% of all documented dislocations [11]. It usually needs mechanical manipulation to return to its normal position, and recurrent dislocations require surgical treatment [8]. Between these TMD types, CH is the rarest pathology that manifests a head condyle overgrowth, causing facial asymmetry, deformity, malocclusion and sometimes pain and dysfunction [12]. It is a self-limiting condition, more prevalent in female teenagers, but it usually requires surgical treatment to limit facial asymmetry progression and condyle continuous elongation [13]. Studies suggest it has a genetic involvement on its development, but its main etiology is still poorly understood [14].

Despite the etiological differences between CH, DDWoR and MD, current studies have limited understanding of the molecular variations that differentiates these TMD diseases. Condylar hyperplasia, mandibular dislocation and disc displacement have been the aim of many studies, due to their difficulty in targeting the proper treatment to each disease [9]. The employment of specific treatment, which may be improved with the unveiling of its specific etiology factors, will allow us to diminish treatment time and costs.

At the proteomic level, current studies focus only on individual mandibular dysfunctions, without comparing different TMD types to show the proteomic variability that could drive novel biomarkers as targets for disease diagnostic and treatment [15,16]. Proteomic analysis is a gold standard approach to analyze all identifiable proteins in a certain tissue, investigating its abundance, variety of proteoforms, and their stable or transient protein– protein interactions. This approach is especially beneficial in the clinical setting when studying proteins involved in different pathologies [17]. To date, there are very few studies investigating human TMD samples through proteomic output, and these studies analyzed only synovial fluid, focusing on specific target proteins [15,16]. Therefore, analyzing all proteins present in the synovial fluid and disc sample of different types of TMD may potentially lead TMD treatments towards a new reality.

In this research, a high throughput proteomic investigation of the three TMD pathologies CH, DDWoR and MD, was performed. Using state-of-the-art sample extraction procedures, biological samples of synovial fluid and TMJ discs were collected from distinct patients diagnosed with these conditions. The samples were processed, subjected to protein extraction and mass spectrometry proteomic identification. Generated proteomic data were analyzed using bioinformatics methods, and a per-sample protein identification and annotation were performed. The clinical phenotypes were then used to correlate the proteomic profile of each TMD condition.

#### **2. Materials and Methods**

#### *2.1. Sample Selection*

The sample was composed of 9 disc and synovial fluid specimens from female patients, with a mean age of 31.22 years (18–52). The patients presented different TMJ conditions, with three samples being composed of TMJ displaced disc without reduction (*n* = 3), two mandibular dislocation (*n* = 2) and four patients with condylar hyperplasia (*n* = 4) (Table 1). The specimens were collected from patients treated at the Evangelic University Hospital of Curitiba, Brazil. The study was approved by the Ethical Committee on Research at Pontifical Catholic University of Paraná, Brazil, according to Resolution 196/96 of the National Health Council and approved on 6 May of 2016 under registration number 1.863.521.


**Table 1.** Baseline characteristics of the sample, showing age and pathology of each female patient.

Subjects did not present any of the following criteria: use of orthodontic appliances; chronic usage of anti-inflammatory drugs; history of diabetes, hepatitis, HIV infection; immunosuppressive chemotherapy; history of any disease known to compromise immune function; pregnancy or lactation; major jaw trauma; previous TMJ surgery; and previous steroid injection in the TMJ.

Subjects answered a personal medical history questionnaire and signed a consent form after being advised of the nature of the study. All patients were clinically examined by one experienced oral and maxillofacial surgeon. The clinical examination consisted of palpating the TMJ region, analyzing the occurrence of painful or limitation/excessiveness of mouth opening/closing, and the observation of facial asymmetry. Regarding complementary exams, all patients had a panorex and patients with disc displacement were submitted to a magnetic resonance image. The patients who were considered to be affected with disc displacement were treated surgically when they presented painful clinical signs of disc displacement after unsuccessful non-surgical treatment for at least 6 months [18]. Patients presenting pain related only to muscular spasms were not included in this research. Patients with condylar hyperplasia were diagnosed through clinical evaluation, panorex and when presenting a positive condylar growth in scintilography, a high condylectomy was indicated and performed [19]. Patients with recidivist mandibular dislocation (more than four episodes in six months) were treated with eminectomy [8].

#### *2.2. Sample Acquisition*

During access to the TMJ to perform the needed surgery [20], a 21-gauge needle was inserted into the upper TMJ space, then 1 mL of saline was injected into the joint space, which was aspirated thereafter by a second adapted syringe. This procedure was repeated five times to obtain a synovial fluid sample as described previously by Alstergren [21]. For each type of surgery performed, TMJ disc recontouring and repositioning was needed [16], therefore, first the displaced disc was freed, repositioned and sutured to the latero-posterior side of the condyle with a Mitek bone-cleat. The suture was then placed between the posterior and intermediate bands, and recontouring the thickened disk with a scalpel was necessary (this posterior debrided cartilage constituted the disc sample). Synovial fluid was spun down at 300× *g* to remove debris, and stored at −80◦C until use or analysis, and the disc samples rinsed in phosphate-buffered saline (PBS), and either snap frozen in liquid nitrogen and stored at −80◦C.

#### *2.3. Proteomic Analysis*

The microcentrifuge tubes containing the synovial fluid and TMJ discs were removed from the −80 ◦ C freezer, and after defrosting, the discs were cut into small pieces with the aid of sterile scissors, centrifuged, and the supernatants were collected and pooled according to each pathology group. The preparation of the samples for proteomic analysis was carried out as previously reported [22]. The analysis of the tryptic peptides was performed in the nanoACQUITY UPLC system (Waters, Milliford, CT, USA) coupled to the Xevo Q-TOF G2 mass spectrometer (MS) (Waters, Milliford, CT, USA). For this purpose,

the UPLC nanoACQUITY system was equipped with a column of type HSS T3 (Acquity UPLC HSS T3 column 75 mm × 150 mm; 1.8 µm, Waters), previously balanced with 7% of the mobile phase B (100% ACN + 0.1% formic acid). The peptides were separated through a linear gradient of 7%–85% of the mobile phase B over 70 min with a flow of 0.35 µL/min and the column temperature maintained at 45 ◦C. The MS was operated in positive ion mode, with a 75 min data acquisition time. The obtained data were processed using ProteinLynx GlobalServer (PLGS) version 3.03 (Waters, Milliford, CT, USA). Protein identification was obtained using the ion counting algorithm incorporated into the software. The collected data were searched in the database of the species *Homo sapiens* downloaded from the catalog of the UniProt [23] in September of 2020. The identified proteins for the groups DDWoR, MD, and CH of synovial fluid and TMJ disc were classified and attributed by biological function, origin, and molecular interaction with the program Genemania [24]. The overlapping proteins between the groups were clustered by using an automatic Venn diagram generator.

#### **3. Results**

In this qualitative study, our aim was to explore, for the first time, a comparative analysis of the proteomic profile of three distinct TMD diseases. Although a statistical analysis was not performed, we were able to identify and describe the function of the proteins, including overlapping proteins between the investigated samples (DDWoR, MD and CH, and between both synovial fluid and disc samples).

In the synovial fluid samples, a total of 225 proteins (351 counting the repeated proteins in all groups) were successfully identified: 190 in the group DDWoR, 154 in the group MD and seven in the group CH. We also compared these three groups to identify shared or condition-specific proteins. We found 114 shared proteins between groups DDWoR and MD, and six proteins were shared by all groups (Table 2).

In the disc sample, 379 proteins were identified (697 counting the repeated proteins in all groups), with 235 proteins in group DDWoR, 196 in group MD and 266 in group CH. These three groups were also compared to identify shared or condition-specific proteins. There were nine shared proteins between groups DDWoR and MD, 28 shared proteins between groups DDWoR and CH, 17 shared proteins between groups MD and CH, and 132 shared proteins by all groups (Table 3).

Regarding the proteins in common in both synovial fluid and disc in the same sample groups, DDWoR presented two common proteins, MD presented three proteins, group CH had no protein in common, and the three groups together had six proteins in common (Table 4).

All synovial fluid and disc samples presented proteins involved in DNA repair, muscle and neural regeneration.

A selective pool of proteins was chosen to be studied according to the pathology group and protein function for synovial fluid and disc sample (Tables 5 and 6).

The synovial fluid sample presented the following proteins functions for each group (Table 5): the DDWoR group presented proteins involved in inflammatory process, apoptosis, hearing, interleukine-6 cascade, and protection against oxidative stress; the MD group showed proteins involved in inflammatory process, apoptosis, hearing, interleukine-6 cascade, protection against oxidative stress, and immune response; in the CH group, the expression of alcohol degradation protein (ADH1) was identified. The group comprising the pathologies DDWoR and MD were mainly involved in inflammatory process inhibition, bone resorption, chondrogenesis, bone and cartilage formation, osteoarthrosis, and neuropathic pain. No proteins were observed in the groups DDWoR and CH, and MD and CH. The proteins expressed in all three groups (DDWoR, MD and CH) were mainly implicated with muscle regeneration.

**Table 2.** Gene code and name of the proteins expressed in synovial fluid of all groups (disc displacement without reduction (DDWoR), mandibular dislocation (MD), condylar hyperplasia (CH) and between the groups DDWoR and MD, DDWoR and CH, MD and CH and DDWoR, MD and CH.





**Table 2.** *Cont*.



**Table 2.** *Cont*.





**Table 3.** Gene code and name of the proteins expressed in temporomandibular joint (TMJ) discs of all groups (DDWoR, MD, CH) and between the groups DDWoR and MD, DDWoR and CH, MD and CH and DDWoR, MD and CH.



**Table 3.** *Cont*.


**Table 3.** *Cont*.


**Table 3.** *Cont*.


**Table 3.** *Cont*.


**Table 3.** *Cont*.


**Table 3.** *Cont*.


**Table 3.** *Cont*.


**Table3.***Cont*.



**Table3.***Cont*.


**Table 4.** Proteins expressed in both synovial fluid and TMJ disc samples of each group.

**Table 5.** Gene code, protein name and function for each sample of TMJ synovial fluid.



**Table 6.** Gene code, protein name and function for each sample of TMJ discs.





The disc sample presented the following protein functions for each group (Table 6): the DDWoR group expressed proteins involved in inflammatory process, neurogenesis, cartilage formation, extracellular matrix degradation, oxidative stress and apoptosis. The MD group presented proteins related to apoptosis, vascular growth, inflammatory inhibitors, immunologic factors and epithelial growth, and the CH group showed protein expression implicated in apoptosis, apoptosis inhibition, oxidative stress, bone formation, chondroitin, bone and cartilage formation. The group with DDWoR and MD samples had proteins involved in inflammatory process; the group with DDWoR and CH samples showed proteins with collagen formation and wound healing functions; the group with MD and CH was involved in wound healing; and the group containing DDWoR, MD and CH samples was involved with inflammatory cascade modulation, osteoclastogenesis, chondrogenesis, apoptosis, bone formation, vascular and tissue repair, antioxidative activity.

There were proteins identified in both synovial fluid and TMJ disc samples, however, some of them in different pathology groups (Table 7).

**Table 7.** Name and function of expressed proteins in common between synovial fluid and TMJ disc sample, and the groups in each protein was expressed.



Different types of collagen were identified in discs of the MD group, CH group, DDWoR and CH group, and in the group with all pathologies together (DDWoR, MD and CH). Besides the known collagen type I present in TMJ discs, collagen type IV, VI, XII and XIV were also identified (Table 8).

**Table 8.** Types of collagen identified in each TMJ disc group.


All shared and group-specific proteins are indicated in a Venn diagram for the synovial fluid (Figure 1) and disc samples (Figure 2).

**Figure 1.** Venn diagram for synovial fluid: group 1—DDWoR, group 2—MD, group 3—CH.

**Figure 2.** Venn diagram for the TMJ disc: group 1—DDWoR, group 2—MD, group 3—CH.

The interactions between the proteins were analyzed with Genemania (https:// genemania.org—accessed on 5 September 2020), and its genetic network pointed out distinct protein cascades that might be modulating each pathology through the synovial fluid and disc samples. The physical and genetic interactions, co-expression and pathway of the proteins are shown in Figures 3 and 4.

**Figure 3.** Gene interactions between the main functional proteins of synovial fluid. (**A**) showing the gene interactions of the DDWoR group. (**B**) showing the gene interactions of the MD group. (**C**) showing the gene interactions of the CH group. (**D**) showing the gene interactions of the DDWoR and MD group. (**E**) showing the gene interactions of the DDWoR, MD and CH group.

**Figure 4.** Gene interactions between the main functional proteins of the TMJ disc. (**A**) showing the gene interactions of the DDWoR group. (**B**) showing the gene interactions of the MD group. (**C**) showing the gene interactions of the CH group. (**D**) showing the gene interactions of the CH group. (**E**) showing the gene interactions of the DDWoR and CH group. (**F**) showing the gene interactions of the MD and CH group. (**G**) showing the gene interactions of the DDWoR, MD and CH group.

The main proteins with important functions and networks that were identified in the synovial fluid sample were analyzed for each group (Figure 3). A brief description of these findings are: in the DDWoR group (Figure 3A) alpha-2-macroglobulin (A2M) involved in inflammatory process, amyloid P component (APCS) involved with apoptosis and complement factor H (CFH) that modulates inflammatory cascade were highlighted in the Genemania interaction figure; in the MD group (Figure 3B), hemopexin (HPX) involved in protection against oxidative stress was present; in the CH group (Figure 3C), alcohol dehydrogenase subunit alpha (ADH1) that is responsible for alcohol degradation and interacts with growth hormone receptor (GHR) was present. In the group of DDWoR and MD (Figure 3D), annexin A1 (ANXA1), decorin (DCN), and immunoglobulin heavy constant gamma 1 (IGHG1) involved in inflammatory process, annexin A2 (ANXA2) involved with bone resorption, asporin (ASPN), biglycan (BGN), cartilage intermediate layer protein (CILP), osteoglycin (OGN), transforming growth factor beta induced (TGFBI) involved in bone and cartilage formation, fibronectin 1 (FN1), lumican (LUM) and tenascin XB (TNXB) involved in tissue repair, and neurofilament medium (NEFM) and thrombospondin 4 (THBS4) involved in neuropathic pain were included in the net. The DDWoR and CH group, and MD and CH group had no protein to be analyzed. The group with the three pathologies (DDWoR, MD and CH) showed an interaction of enolase 2 (ENO2) and 3 (ENO3), involved in muscle regeneration (Figure 3E).

The disc sample presented the following protein interactions in Genemania (Figure 4): group DDWoR (Figure 4A) presented mainly the matrix metalloproteinase protein (MMP) family (1,2,3,6,8,10,13,15,16), integrin subunit alpha 6 (ITGA6) and phospholipase A2 group VII (PLA2G7) that are involved in inflammatory cascade. Additionally, thrombospondin 3 (THBS3) and 4 (THBS4) involved in tissue remodeling, and THADA armadillo repeat containing (THADA) involved in apoptosis were present. In the MD group (Figure 4B), A-kinase anchor protein 13 (AKAP13), Erbin (ERBIN) and uroplakin-3a (UPK3A) involved in apoptosis, collagen alpha-1(IV) chain (COL4A1) and GTPase Eras (ERAS) involved in disc matrix constitution, and liprin-alpha-1 (PPFIA1) and (PPFIA2) 2 responsible for cell interactions were identified in the Genemania network. In the CH group (Figure 4C), the present proteins were ADAM metallopeptidase domain 10 (ADAM10), that regulates apoptosis, collagen type I alpha 2 chain (COL1A2) and serpin family H member 1 (SERPINH1) involved in collagen formation, actinin alpha 4 (ACTN4), PDZ Additionally, LIM domain 4 (PDLIM4), transthyretin (TTR) and protein tyrosine phosphatase non-receptor type 13

(PTPN13) involved in apoptosis, hormone modulation and bone formation. In the group of DDWoR and MD (Figure 4D), the complement C4A (C4A) and complement C4B (C4B) proteins that mediates the inflammatory process were identified. In the DDWoR and CH group (Figure 4E), mainly the proteins aggrecan (ACAN), collagen type I alpha 1 chain (COL1A1) and collagen type IV alpha 6 chain (COL4A6) that constitutes disc matrix, and periostin (POSTN) involved in wound healing were identified. In the MD and CH group (Figure 4F), keratin 6A (KRT6A) involved in wound healing was identified. Additionally, in the group with all three pathologies (DDWoR, MD and CH) the proteins that interacted were annexin A1 (ANXA1), complement C3 (C3) and tenascin C (TNC) involved in inflammatory cascade modulation, annexin A2 (ANXA2) and transforming growth factor beta induced (TGFBI) involved in osteoclastogenesis, asporin (ASPN), biglycan (BGN), collagen type VI alpha 1 chain (COL6A1), osteoglycin (OGN) and vimentin (VIM) involved in chondrogenesis and osteogenesis, amyloid P component (APCS) and complement C3 (C3) in apoptosis and lumican (LUM) involved in tissue repair (Figure 4G).

#### **4. Discussion**

The different types of TMD may jeopardize patients' quality of life, masticatory function and have a great impact on health expenses. The identification of its multifactorial etiological components will enhance the employment of specific treatments, diminishing the hazard it causes in the TMJ. Therefore, the identification of the proteins expressed on each pathology group of this study (DDWoR, MD, and CH) might elucidate the cascades involved in the progression and severity of each TMD, leading to an assertive handling of TMD.

A total of 225 proteins were identified in the synovial fluid sample, and 379 in the TMJ disc sample (Table 2). It is important to highlight that the synovial fluid sample is very complex to obtain, therefore some proteins might not have been identified due to the technique that advocates the dilution of the synovial fluid. Nevertheless, the sample was collected according to worldwide employed standard methods previously described by other research groups [21,25]. Additionally, even though few proteins' expression might not have been observed, the expression of new proteins were identified for each pathology group, which enriches the global analysis of this study.

In our analysis, we found that all proteins expressed in the DDWoR group (synovial fluid and disc sample) (Tables 2 and 3) presented many proteins related to inflammatory process (MMP-3, -10, -27 in the disc sample) and apoptosis (mitogen-activated protein kinase 7—MAP3K7) and THADA in synovial fluid). Only the MMP-3 protein was previously associated with TMD [26,27]. These are proteins that highly impact the degeneration process in the TMJ of patients with DDWoR [26,28]. In the MD group, ERBIN protein was found in the disc sample, and it modulates TGFB, which was previously associated with TMJ degeneration [29]. Additionally, unprecedented proteins were seen in the synovial fluid associated with apoptosis (aldehyde dehydrogenase 1 family member L—ALDH1L1) and protection against oxidative stress (HPX), which probably helps diminish the mechanical overload consequences of the dislocation in the TMJ. Regarding CH proteins in the synovial fluid sample, ADH1 catalyzes the oxidation of alcohols to aldehydes, but as seen in Genemania (Figure 3C), it interacts with GHR, which might be involved with the condylar overgrowth. In a previous study, GHR has been injected in rabbits' TMJ to increase cartilage thickness [30], but it has not been studied as a possible etiology of condylar overgrowth yet.

Additionally, we also found a set of proteins to be common in both synovial fluid and disc samples (Table 4) in the groups DDWoR (chromodomain-helicase-DNA-binding protein 8 and myosin light chain 6B), MD (filamin A and liprin-alpha-1), and in the three groups (enolase 1, 2, 3, myosin heavy chain 16, ribosomal protein L7 like 1 and component of the shield in complex). These proteins were involved in cell matrix adhesion, cellular motor protein, reorganization of cytoskeleton, muscle development and regeneration. Additionally, another group of proteins were identified in both synovial fluid and disc

samples (Table 7), being prevalent in all groups of disc samples. In the DDWoR and MD groups of synovial fluid samples, proteins implicated in apoptosis, inflammatory process, bone formation and resorption, chondrogenesis, wound healing, tissue repair and protection against oxidative stress were found. CH disc samples and MD synovial fluid samples presented, as common proteins, HPX (protection against oxidative stress) and SERPINC1 (biosynthetic pathway of collagen).

LUM is associated with the regulation of collagen fibers and with cell migration. In this study, LUM was present in all disc samples, and it has been pointed out to be elevated when the disc is under stress, as it enhances tissue repair [31]. Ulmner [32] reported that higher levels of LUM in synovial tissue might diminish TMD surgical success. On the other hand, TNC was present in all disc samples and in DDWoR and MD synovial fluid sample, being an important protein in wound healing [33].

Temporomandibular joint discs are fibrocartilaginous discs composed mainly by collagen, glycosaminoglycan and proteoglycans [34]. Studies in human adults and fetuses showed the expression of mainly collagen type I and III in TMJ discs, with type I collagen observed in the posterior band of the articular disc and collagen type III on the inferior surface of the articular disc [35,36]. Moreover, collagen type II synthesis was expressed on the external layer of the TMJ disc [37]. In this study, collagen type IV was identified in MD and CH samples (Table 8), and a previous study observed the presence of collagen type IV in the middle part of fetuses' TMJ disc, indicating the development of blood vessels [38]. The TMJ disc is an avascular tissue, although under stress it may undergo metaplasia, forming a vascularized fibrous tissue. Collagen type VII was present in all samples, and along with collagen type IV, it has chondroprotective effects against inflammation [39]. Collagen type XII and XIV were present in the disc samples of this study, which have never been identified in this region before in humans. A study identified collagen type XII only in bovine disc samples, which helps maintain collagen type I integrity [40]. Nevertheless, collagen type XIV was also observed in all TMJ disc samples, and it plays an essential structural role in the integrity of collagen type I, mechanical properties, organization, and shape of articular cartilage, which has never been described in the TMJ disc before [41]. This is important information to understand the composition's strength and weakness of the TMJ disc.

#### **5. Conclusions**

In conclusion, many proteins were identified for the first time in the TMJ disc and synovial fluid of the groups DDWoR, MD and CH, leading to the enlightenment of each pathology's etiology, modulation and progression. Further studies with a greater sample are necessary to evaluate other proteins that might be present in these pathologies as well.

**Author Contributions:** Conceptualization: A.D.D., P.C.T., R.H.H.; methodology: A.D.D., P.C.T., R.H.H., M.A.R.B.; software: A.D.D., R.H.H., M.A.R.B.; validation: A.D.D., P.C.T., R.H.H., M.A.R.B.; formal Analysis: A.D.D., P.C.T., R.H.H., M.A.R.B.; investigation: A.D.D., P.C.T., R.H.H., M.A.R.B.; resources: A.D.D., P.C.T., R.H.H.; data curation: A.D.D., P.C.T., R.H.H., M.A.R.B.; writing—original draft preparation A.D.D., R.H.H.; writing—review and editing: A.D.D., P.C.T., R.H.H., M.A.R.B.; supervision: P.C.T., R.H.H., M.A.R.B.; project administration: A.D.D.; funding acquisition: A.D.D., P.C.T., R.H.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** P.C.T. is supported by the National Council for Scientific and Technological Development, Chamada MCTIC/CNPq Nº 28/2018—Universal, Process: 426505/2018-2 for this research. R.H.H. is supported by Fundação Araucária (grant FA#09/2016).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Pontifical Catholic University of Paraná, Brazil, according to Resolution 196/96 of the National Health Council and approved under registration number 1.863.521, on the 20 May 2016.

**Informed Consent Statement:** Written informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** We thank all individuals that were volunteers for agreeing to participate in this study. A.D.D. was supported by Fundação Araucária scholarship. P.C.T. is supported by the National Council for Scientific and Technological Development, Chamada MCTIC/CNPq Nº 28/2018—Universal, Process: 426505/2018-2 for this research. R.H.H. is supported by Fundação Araucária (grant FA#09/2016). We thank Alexandra Senegaglia and Paulo R. S. Brofman for the laboratory support at Pontifícia Universidade Católica do Paraná, Brazil.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Is There an Association between Temporomandibular Disorders and Articular Eminence Inclination? A Systematic Review**

**Xiao-Chuan Fan 1 , Diwakar Singh 2 , Lin-Sha Ma 1 , Eva Piehslinger 3 , Xiao-Feng Huang 1, \* and Xiaohui Rausch-Fan 2, \***


**Abstract:** (1) Background: In order to determine the correlation between the inclination of articular eminence (AEI) and the development of temporomandibular disorders (TMDs), a systematic review was performed. (2) Methods: A systematic literature research was conducted between 1946 and January 2020, based on the following electronic databases: PubMed, Cochrane Library, Embase, Medline, Scope, SciELO, and Lilacs. Observational studies, analytical case-control studies, and cohort studies written in English were identified. The articles were selected and analyzed by two authors independently. The PICO format was used to analyze the studies and the Newcastle-Ottawa Scale (NOS) was used to verify the quality of the evidence. (3) Results: Sixteen articles were included in this review, ten case-control studies and six cohort studies. Eight articles (50%) established a positive relation between AEI and TMDs and eight (50%) did not. The scientific quality was medium-low, mainly influenced by the exposure to the risk of bias and the lack of clinical methods with adequate consistency and sensitivity on the diagnosis of TMDs. (4) Conclusions: It is controversial to establish a causal relationship between the TMDs and the AEI in the field of stomatology, due to limited and inconclusive evidence. However, it is suggested that the AEI defined by some specific methods may be associated with some special pathological stages of TMDs. High-quality prospective studies are required to draw any definitive conclusions.

**Keywords:** temporomandibular disorders; inclination of articular eminence; temporomandibular joint; glenoid fossa

#### **1. Introduction**

The temporomandibular joint (TMJ) is one of the most complex articular systems in human beings, which is formed by the glenoid fossa of the temporal bone (the superior component of the joint), and the mandibular condyle (the inferior component of the joint) and the two are separated by the articular disk [1,2]. The anatomy of the TMJ can provide capacity in both hinging movement and gliding movements of the mandibular within the three planes of space. The TMJ is critical to the craniomandibular system because it can achieve the mandibular functions with a dynamic balance mechanism [3]. Over the years, numerous studies have focused on the relation of the change of anatomical and physiological characteristics to stomatognathic dysfunctions [4], especially in cases of joint disorders [5].

Temporomandibular disorders (TMDs) are one of the most prevalent pathologies, which are defined as a comprehensive term of disorders affecting the TMJ, the muscles involved in mastication and/or the related structures [6]. Epidemiological studies of nonpatient adult populations have shown that about 40–75% of patients have at least one sign

**Citation:** Fan, X.-C.; Singh, D.; Ma, L.-S.; Piehslinger, E.; Huang, X.-F.; Rausch-Fan, X. Is There an Association between Temporomandibular Disorders and Articular Eminence Inclination? A Systematic Review. *Diagnostics* **2021**, *11*, 29. https://dx.doi.org/10.3390/ diagnostics11010029

Received: 23 November 2020 Accepted: 23 December 2020 Published: 26 December 2020

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**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/).

of joint dysfunction, such as joint clicking, abnormal movement, and 33% of them have joint or facial pain [6]. Although the prevalence of TMDs in the population has attracted more attention from clinicians and researchers over the years, the etiology of TMDs is still poorly understood and remains to be elucidated [7,8].

Numerous factors that contribute to the development of TMDs have been proposed, such as traumatic injuries, occlusal disharmony, psychological factors, luxation of the joints, loss of posterior teeth, spine and postural alterations, and muscle hyperactivity [9–12]. Beside these factors, the features of the anatomic structure of the TMJ are also considered to be a local factor involved in the development of TMDs. During functional movements of the mandibular, the condylar process slides along the posterior slope of the articular eminence. A change of inclination of articular eminence might result in biomechanical variations of the TMJ because its characteristics determine the trajectory of functional movement [13]. Therefore, we speculate that articular eminence steepness and mandibular fossa morphology may have some connections with certain diseases that induce TMJ.

The relationship between the TMDs with the articular eminence inclination (AEI) has been investigated by previous studies. However, the associations between these two indicators have been found to be inconsistent and definitive conclusions cannot be drawn [14–20]. On the basis of these premises, a well-designed systematic review is needed to clarify this opening question. This study attempts to systematically review the literature to find out the correlation between the inclination of articular eminence and the development of TMDs, analyzing the quality of the methodological soundness of previous studies.

#### **2. Materials and Methods**

In order to answer the research question about the relationship between the AEI and TMDs, a systematic search of the medical literature was performed on 17 June 2019 and updated on 27 January 2020. Databases used were as follows: PubMed, Cochrane Library, Embase, Medline, Scope, SciELO, and Lilacs.

#### *2.1. Protocol*

This systematic review was reported following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) checklist [21].

#### *2.2. Types of Studies*

Observational studies, analytical case-control studies, or cohort studies aimed to determine the relationship of the inclination of articular eminence to the occurrence of TMDs.

#### *2.3. Language Studies*

The search was limited to articles in peer-reviewed journals and written in the English language.

#### *2.4. Types of Participants*

The studies selected for this review included subjects of both genders without the limitation of age.

#### *2.5. Intervention Type*

Studies without intervention in order to correlate AEI and TMDs.

#### *2.6. Type of Results*

The primary outcome was to determine the relationship between AEI and TMDs.

The secondary outcome was to determine AEI and the morphology of glenoid fossa related to the different pathological stages of TMDs.

#### *2.7. Data Collection*

For TMDs, the data were collected from studies that showed the diagnosis of TMDs with a clear reference to the concept and diagnosis of temporomandibular pathology in any method without limitation. Diagnostic criteria for TMD was based on research diagnostic criteria for temporomandibular disorders (RDC/TMD), diagnostic criteria for temporomandibular disorders (DC/TMD) [22,23], evaluation according to the American Academy of Orofacial Pain (AAOP) guide [6], radiology studies (including magnetic resonance imaging (MRI), computed tomography (CT), cone-beam computed tomography (CBCT), sagittal corrected tomography, arthrography, and other methods), Helkimo index, surveys' studies, and/or clinical examination based on signs and symptoms with reference to TMD and others.

For AEI, the data were collected from studies that showed a clear method for measuring the AEI in degrees. The AEI is defined as the angle between the articular eminence and the Frankfort horizontal (FH) plane or any other horizontal reference plane, such as the palatal plane, the occlusion plane, the anterior nasal spine to the posterior nasal plane (ANS-PNS), and other defined reference planes. Data were collected based on MRI, CT, CBCT, tomography, dry skulls, autopsy, and other methods.

#### *2.8. Databases Used*


#### *2.9. Search Strategy*

A systematic search of the computerized database was performed to identify and select the potentially eligible literature that examined the association between AEI and TMDs for this systematic review. The semantic field related to the term "TMDs" (temporomandibular disorders, TMJ dysfunction, disk displacement, muscular pain, clicking) was crossed search with the semantic field related to the term "AEI" (glenoid fossa, posterior slope, articular eminence). For details regarding the specific search terms and combinations, see Table 1.

#### *2.10. Study Selection*

For article selection or first approach, all potentially eligible articles were listed by title and abstract and evaluated by two researchers independently (X-C.F. and D.S.). Then, the full text of articles, which may meet the inclusion criteria based on the first stage of selection, was assessed independently by the same two researchers (X-C.F. and D.S.). When no agreement was found during the first and second stage of selection, the data was discussed with a third researcher (X.R.F.), to reach final decision for including it or not. When the full-text version of the study was not directly available, the paper was requested from the corresponding author by email. Articles that met all inclusion and exclusion criteria were selected in the review for the final analysis. The reasons for the exclusion of the articles were recorded in an adjacent column and presented in the results (Table 2).


**Table 1.** Search strategy and terms used for the search.

**Table 2.** Studies retrieved in full text and excluded from the review.


#### *2.11. Extracting Data from the Studies*

The methodological features of the selected articles were assessed according to a format, the PICO criteria, which enabled a structured summary of the analyzed articles in relation to four main issues, namely, population, intervention, comparison, and outcome. For each article, we defined the following analysis variables in detail: population (sample size, distribution by gender, mean age, and age range); intervention (type of method used for the diagnosis of TMDs, main variables to compare, statistical analysis); comparison (assessed the presence of any comparison groups); outcomes (the answer to the hypothesis, the presence of causal relationship between AEI and TMD). Some studies investigating more items were reported in two or more groups of correlation.

#### *2.12. Quality Assessment*

Critical appraisal of studies included in the review was determined by the Newcastle-Ottawa Scale (NOS), which was used to assess the quality of case-control and cohort studies [39]. To determine the quality of case-control studies, there were three categories with a level of evidence score ranging from 0 to 9 points as follows: (1) selection (four points), (2) comparability (two points), and (3) exposure (three points). For cohort studies, there were also three categories assigning a score ranging from 0 to 9 points as follows: (1) selection (four points), (2) comparability (two points), and (3) outcome (three points).

The quality was determined by the same two researchers (X-C.F. and D.S.) in charge of the search, where the highest quality achieved was obtained by those items that were assigned a maximum score of 9.

#### **3. Results**

In total, 1235 potentially eligible articles were examined in the first approach in the seven databases used (Table 1). However, 299 of these articles were excluded due to duplication. On the basis of the title and abstract of the remaining 936 studies, 904 of them were eliminated due to their lack of relevance. Of the 32 articles left, after reading the full text, a consensus decision was to eliminate 17 articles that did not fulfill the inclusion criteria for this systematic review. Table 2 reveals the list of excluded studies including the reason for exclusion. Search expansion strategies allowed including one additional paper, thus, accounting for a total of 16 studies that were analyzed in the review and prepared according to the PICO criteria (Tables 3–5). Figure 1 summarizes the search strategy and results described.


#### **Table 3.** Summary of findings from studies of TMDs and AEI with MRI.


M: male; F: female; a.r.: age range; m.a.: mean age; ANOVA: analysis of variance; DD: disk displacement; DDWR: disk displacement with reduction; DDWOR: disk displacement without reduction; AV: asymptomatic volunteer; RDC/TMD: research diagnostic criteria for temporomandibular disorders; AEI: articular eminence inclination.


#### **Table 4.** Summary of findings from studies of TMDs and AEI with CBCT or Helical CT.


**Table4.***Cont.*

M: male; F: female; a.r.: age range; m.a.: mean age; ANOVA: analysis of variance; DD: disk displacement; DDWR: disk displacement with reduction; DDWOR: disk displacement without reduction; NBC: no bone change; BCBC: bilateral condylar bone change; AEI: articular eminence inclination.


#### **Table 5.** Summary of findings from studies of TMDs and AEI with two-dimensional radiographs.


M: male; F: female; a.r.: age range; m.a.: mean age; ANOVA: analysis of variance; RDC/TMD: research diagnostic criteria for temporomandibular disorders; DDWR: disk displacement with reduction; DDWOR: disk displacement without reduction; ADD: anterior disk displacement; AEI: articular eminence inclination; DD: disk displacement; OC: osseous changes; DDN/DJD: disk displacement without reduction with degenerative joint disease; TMJ: temporomandibular joint.

**Figure 1.** Search method, identification, selection, and inclusion of articles.

#### *3.1. Characteristics of Studies*

– In all, 16 articles were included in this systematic review. ten case-control studies and six cohort studies were identified. According to the radiological methods, four are MRI studies, seven are CT or CBCT studies, and the other five articles used two-dimensional (2D) radiographs (sagittal corrected tomography or lateral oblique transcranial radiographs). Among these articles, two of them used both three-dimensional (3D) (CT or MRI) and twodimensional (2D) radiographs (lateral cephalogram or laminography) [45,48] (Tables 3–5). We selected these two articles for a single group, because the other radiological methods were not used to evaluate the AEI after reading of the full text.

#### *3.2. Characteristics of Participants*

The age range of patients was between 14 and 88 years; the average age was from 19.1 ± 4.7 to 40.92 years. Three studies did not specify the age of the participants clearly [3,41,48]. In relation to gender, four studies included only females in their sample [19,45–47].

#### *3.3. Quality Assessment*

Among the total 16 articles selected in this review, eight studies presented correlations between TMDs and the AEI, however, the other eight studies did not find any correlations between the TMDs and the AEI. None of the included studies obtained the highest score based on NOS. The range of scores was between two and six (Table 6).


**Table 6.** Studies retrieved in full text and excluded from the review.

NOS score, Newcastle-Ottawa Scale, three categories with a score of level of evidence ranging from 0 to 9 points to determine the quality of case-control and cohort studies.

#### **4. Discussion**

A review of literature can help us gain knowledge more effectively, however, it is necessary to carefully analyze the quality, to avoid erroneous conclusions from their results. The objective of this systematic review was to select and analyze the studies that verify the correlation between the inclination of articular eminence and specific TMD signs and symptoms, presenting real applicability to clinical practice.

According to the inclusion and exclusion criteria of this review, the search was conducted with the limitation of peer-reviewed English language papers, although this strategy may lead to the possibility that some publications in other languages and/or publications included in databases were unjustly excluded. However, it is a way to improve the methodological rigor and the conclusion drawn to a certain extent. Case reports and reviews are also excluded, as they do not have uniform standards that could increase the risk of bias.

From a methodological point of view, all the articles selected in this systematic review were retrospective observational studies with or without control groups verifying the correlation between AEI and TMDs. The scientific quality of evidence of the analyzed studies included in the present review was medium-low, mainly influenced by the exposure to the risk of bias and the lack of clinical methods with adequate consistency and sensitivity used for the diagnosis of TMDs. One of the methods created with the purpose of clinical and epidemiological research used for the diagnosis of evidence-based TMDs is the RDC/TMD, and the other method is the DC/TMD, which results in an evidence-based system with greater validity for clinical use [23]. The RDC/TMD criteria and the DC/TMD criteria are emphasized as the international standard for examination of patients, which have existed since 1992 or 2014. Therefore, the qualities of all studies before that time are evaluated as weak. All of the selected articles in this systematic review, except for one (Panmekiate, 1991 [49]), were published after 1992. The types of method used for the diagnosis of TMDs of the selected articles were shown in the "intervention" part of Table 3. However, only four studies, included in the review, diagnosed TMDs and classified samples according to RDC/TMD criteria [19,40,43,44], two studies diagnosed TMDs based on Helkimo index [14,18], and others diagnosed TMDs only by clinical sign and symptoms or then further

confirmed by MRI or arthrography. That means the inclusion criteria of the papers are not consistent. The lack of introduction of uniform diagnostic criteria, such as RDC/TMD or DC/TMD defining the different categories of TMDs, decreases the level of consistency, resulting in a low quality of studies, and therefore comparisons between different studies could not be established. Without consistency, may imply that the observed correlations between two variables appeared because of chance or error [50]. Furthermore, TMDs are considered to be a heterogeneous group of different diseases involving the craniomandibular system, other than a single pathology [51]. It is difficult to control for all of the other variables when evaluating the relative importance of single risk factors for disorders with a multifactorial etiology [52,53]. Some studies that still seem to continue to use "TMDs" as a collective term of all TMD signs and symptoms during the clinical examinations, pooled them in a unique dependent variable in the statistical analysis and the results [3,43,44]. Nevertheless, the evaluation of the multifactorial complex pathologies, such as TMDs, should use multivariate statistical analyses, as univariate models may overestimate some resulting associations and possibly produce misleading conclusions [54,55]. This could be shown from the study of Rabelo KA et al. [17], who found an important correlation among the type of disk displacement of the AEI (*p* < 0.001), but there was no statistical correlation between the presence and absence of disk displacement of AEI measurements (*p* > 0.05). Similarly, the AEI was steeper in the no condyle bone change group than in those of the bilateral condylar bone change (centre section *p* < 0.05, lateral section *p* < 0.01). However, these differences were only seen in the joints with osteophyte (all three sections *p* < 0.05) but not with erosion (all three sections *p* > 0.05), based on the study of Yamada K et al. [46].

Many radiographic methods have been selected to measure the AEI in previous studies. In the early days, conventional radiographs, such as tomography or arthrography, were used for diagnosing the morphology of TMJ, but these modalities proved to have certain limitations [1] and were replaced by helical CT, which evaluates osseous components in 3D without superimposition or distortion. The CBCT, which has high dimensional accuracy in measuring maxillofacial structures including TMJ, is considered to be one of the preferred ways to evaluate bone structure in the stomatological area [3,14,18]. Nowadays, CBCT was selected rather than helical CT because of lower radiation dose, better spatial resolution, shorter scanning time, and more cost effective [56]. The MRI also allows a tridimensional analysis of the TMJ, and this technology can provide hard tissue and also soft tissue imaging, such as articular disk, related muscles, and ligaments. It has already been considered to be the gold standard imaging method for the diagnosis of internal derangement and the disk displacements with or without reduction. The radiographic methods are very important factors for angular and linear measurements as it influences the results. The articles included in this systematic review involve five imaging methods, from two-dimensional methods to three-dimensional methods. Using 3D imaging, the steepness of the eminence may be influenced by the location of the image (more laterally, centrally, more medially), whereas the 2D images show a summarization of the whole articular eminence as a three-dimensional structure. It is hard for us to establish comparisons of the values of AEI between different studies with different imaging methods because the consistencies of them still need more studies to support.

The AEI is defined as the angle formed by one of the lines that passes through the articular eminence and the horizontal reference plane [57]. In previous articles, two main methods have been described for evaluating the AEI, i.e., the "top-roof line" method and the "best-fit line" method, which are reliable and have already been used in studies. The "toproof line" is obtained by connecting the crest point of the articular eminence and the roof of the mandibular fossa (Figure 2). The angle between the "top-roof line" and the horizontal reference plane is related to the height of articular eminence, which focuses on the localization of the tubercle in relation to the mandibular fossa and depicts the morphology of articular eminence better. The "best-fit line" method was defined as the angle between the tangent line drawn to the posterior slope of the articular eminence and the horizontal reference plane, which is directly related to the movement direction of the condyle-disk

complex and reflects the actual condylar path (Figure 3) [26,30,31,41,57,58]. Five of 16 articles selected in this systematic review used the "top-roof line" method [14,17,18,43,49]; eight articles used the "best-fit line" method [19,20,40–42,45,46,48]; two articles used both the "top-roof line" method and the "top-roof line" method to evaluate the AEI [3,47], and the other article used the angle between tangent line from the uppermost point of the glenoid fossa and the true horizontal line as AEI [44]. Although the three mentioned methods all represent the inclination of the articulator eminence, the features they focus on are different. Therefore, they should be considered separately.

**Figure 2.** Representative images of "top-roof line" method, the articular eminence inclination (AEI) defined as the angle between the line connecting the crest point of the articular eminence and the roof of the mandibular fossa and the horizontal reference plane.

**Figure 3.** Representative images of the "best-fit line" method, the articular eminence inclination (AEI) defined as the angle between the tangent line drawn to the posterior slope of the articular eminence and the horizontal reference plane.

The horizontal reference plane is the other important factor affecting the AEI, which determines the degree of the angle directly. At the stage of the literature selection, the reference planes used were not limited, which can be FH plane, palatal plane, occlusion plane, and other defined reference planes. Except for three studies (one study used the true horizontal line [44], one study used the line tangent to the anterior and posterior articular eminences [19], one study used the line tangent to the curve of articular eminence and the point of squamotympanic fissure [47]), and the remaining 13 studies included in this review all used the FH plane as horizontal reference planes. It has been generally recognized as an important reference plane and has proved to be of great value in cephalometric analysis and the three dimensions measurement since the Frankfurt agreement concluded

in Germany in 1884, which is defined by a line drawn from the lowest point on the inferior orbital margin (Or) to the most superior point of the outline of the external auditory meatus (Po) [59,60]. A stable and comparable horizontal reference plane is very essential, and the FH plane seems to be a relatively ideal reference plane for evaluating AEI because the landmarks of the FH plane are independent of TMJ structure, which are not affected by the changes of mandibular fossa and articular eminence. However, although FH is well defined, the external auditory meatus changes its shape looking on a more lateral, central, or medial slice of MRI, CT, or CBCT, which may also influence the steepness of the articular eminence.

Another important confounding factor in the analysis of the correlation between TMDs and AEI may be represented by the selection of the samples. Some of the studies were based on orthodontic patients [45,46], who may be alerted to the potential role of malocclusion as a risk factor of TMD. The control group or the asymptomatic volunteers of some studies was selected from the dental students [42], who can be aware of the risk factors of the TMDs and avoid them. In such cases, the samples selected may hardly represent the general population. The genders of the samples included also have such a problem. The groups of symptomatic patients in most studies included in this systematic review contain more female than male, which has a significant difference in gender distribution from the general population [3,14,17,18,20,42,45,48,49]. However, TMDs affect approximately 40% to 75% of the general adult population, 80% of which seeking for TMD are females. Milano et al. reported that disk displacements of TMJ appeared considerably more often in females than in males because of altered collagen metabolism associated with joint laxity of genetic origin [61]. Peroz et al. also found that females present a greater correlation with disk displacements than males [62]. According to Warren and Fried, estrogen may influence the development and metabolism of the TMJ and associated structures (include bone, cartilage, and articular disk), and it may also influence the pain regulation mechanism [63]. The evidence in the previous articles suggested that the pathogenesis of TMDs may have a possible link with estrogen and that TMDs is more prevalent in the female. Therefore, we also included four articles containing only female subjects [19,45–47].

The development stage of the articulation may also influence the AEI. According to the previous studies [64,65], from newborn to infancy, the articular surface was largely flat and the articular eminence was poorly developed. From the stage of the end of the primary dentition to mixed dentition, the fossa and the articular eminence had clearly developed and completed approximately 45% of its development, but the articular eminence was still fairly flat. Around the age of 10 years old, articular eminence completed approximately 70–72% of its development. The fully developed time of the articular eminence is still controversial. From the study by Katsavrias and Dibbets [65], articular eminence was 90–94% complete by the age of 20 years. However, based on the autopsy study published in 1971 [64], tubercle and the fossa were well developed at the age of 14–15 years. This review presents a high variability in the age range of 14–88 years. A poorly developed fossa may show a flatter tendency, which may possibly produce misleading conclusions.

This systematic review retrieves and analyzes the medical literature about the relationships between the TMDs and the AEI published in seven databases over the past 74 years, 50% of the studies showed a positive correlation between the TMDs and AEI, but the evidence is not in high quality. In relation to the findings in this review, the following suggestions can be drawn:


4. More quality and carefully designed prospective studies are required by future researchers to determine the causal relationship between TMDs and AEI.

#### **5. Conclusions**

Definitive conclusions cannot be drawn based on the quality of evidence available, since the definition and clinical methods were very heterogeneous and presented a high risk of bias. The insufficient number of articles considered of high methodological quality is another factor that hinders the acceptance or denial of this correlation. However, it is suggested that the AEI defined by some specific methods may be related to some special pathological stages of TMDs to a certain extent. Well-designed prospective studies are required to draw any further definitive conclusions.

**Author Contributions:** Literature search, X.-C.F., D.S. and X.R.-F.; conceptualization, X.-C.F. and L.-S.M.; methodology, X.-C.F., D.S. and E.P.; formal analysis, X.-C.F., X.-F.H. and X.R.-F.; data curation, E.P. and L.-S.M.; writing—original draft preparation, X.-C.F. and L.-S.M.; writing—review and editing, X.-F.H. and X.R.-F.; supervision, X.-F.H. and X.R.-F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Natural Science Foundation of Beijing Municipality (grant No. 7202036) and the Capital Health Research and Development of Special Funding (grant No. 2018-2-1102).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** No new data were created or analyzed in this study. Data sharing is not applicable to this article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Temporomandibular Disorders: Current Concepts and Controversies in Diagnosis and Management**

**Dion Tik Shun Li and Yiu Yan Leung \***

Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China; diontsli@hku.hk

**\*** Correspondence: mikeyyleung@hku.hk; Tel.: +852-28890511

**Abstract:** Temporomandibular disorders (TMD) are a group of orofacial pain conditions which are the most common non-dental pain complaint in the maxillofacial region. Due to the complexity of the etiology, the diagnosis and management of TMD remain a challenge where consensus is still lacking in many aspects. While clinical examination is considered the most important process in the diagnosis of TMD, imaging may serve as a valuable adjunct in selected cases. Depending on the type of TMD, many treatment modalities have been proposed, ranging from conservative options to open surgical procedures. In this review, the authors discuss the present thinking in the etiology and classification of TMD, followed by the diagnostic approach and the current trend and controversies in management.

**Keywords:** temporomandibular disorders; temporomandibular joint; TMD; facial pain; craniomandibular disorders

#### **1. Introduction**

The diagnosis and management of the most common cause of non-dental pain in the maxillofacial region, namely temporomandibular disorders (TMD), remains a challenge for clinicians to this day, despite extensive clinical research into the topic. This is because TMD is a broad term comprising of different conditions with complex etiologies, with symptoms that vary in intensity. Intriguingly, some signs and symptoms resolve spontaneously even without treatment, whereas others persist for years despite all treatment options having been exhausted. More perplexing is that while some may have a recognizable physical basis, many cases of TMD also involve a significant biopsychosocial component [1–3] with various associated psychological symptoms, such as depression and anxiety [4–6]. Numerous treatment modalities have been proposed over the years, with some becoming obsolete while others are gaining in popularity. Nevertheless, it seems that there is no single solution for every case as many different symptoms are included in TMD. Controversies exist in the literature regarding the diagnosis and the management protocol for TMD, hence the selection of treatment modality may often be largely influenced by the expertise of the treating healthcare provider.

In general, TMD is believed to affect anywhere between 5 and 15% of adults in the population [7–10], yet TMD related symptoms have been reported to be present in up to 50% of adults [11]. Interestingly, there is evidence that the prevalence of TMD appears to be on the rise in recent years [12–16]. A recent systematic review and meta-analysis in 2021 concluded that the prevalence of TMD was 31% for adults and 11% for children and adolescence [17]. The fact that TMD encompasses a broad assortment of clinical diseases is partially responsible for the wide range of prevalence rate estimates among studies, as the classification of different types of TMD, the distinction between disease and non-disease, as well as whether to include those with inactive disease as having TMD, may all be subject to the partialities of the assessing clinical researchers. In addition, studies that are questionnaire-based might over-estimate the prevalence of TMD, as the symptoms of

**Citation:** Li, D.T.S.; Leung, Y.Y. Temporomandibular Disorders: Current Concepts and Controversies in Diagnosis and Management. *Diagnostics* **2021**, *11*, 459. https:// doi.org/10.3390/diagnostics11030459

Academic Editor: Luis Eduardo Almeida

Received: 17 February 2021 Accepted: 5 March 2021 Published: 6 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

many other conditions, such as headache not caused by TMD, dental pain, neuropathic conditions, and otological diseases, can mimic the presentation of TMD.

TMD represents a significant and complex health problem, with opinions regarding the appropriate course of management often equivocal. In this review, we discuss the current concepts in the etiology and diagnosis of TMD, followed by an up-to-date management approach from a surgeons' perspective.

#### **2. Etiologies and Classifications**

As an umbrella term for pain and dysfunction of the temporomandibular regions, TMD encompasses a wide variety of clinical conditions. The etiologies of TMD are multifactorial and can be attributed to both physical and psychosocial factors [18–20]. The physical causes can broadly be divided into arthrogenous, and the more common myogenous origins. Many believe that TMD symptoms of arthrogenous origin may be related to internal derangement of the TMJ, which can be defined as a disruption of the internal aspect of the joint, and usually pertains to an articular disc that has been displaced. Although internal derangement does not necessarily lead to pain, it is generally believed that internal derangement precedes degenerative joint diseases, namely osteoarthritis [21]. Osteoarthritis is associated with pain and functional impairment of the TMJ, and is characterized by subchondral bony changes such as cortical erosion and marginal lipping, secondary to pathological changes of the cartilaginous articular disc [22]. Note that the term "osteoarthrosis" has been used as a synonym of osteoarthritis, but also has been used to describe degenerative joint changes of non-inflammatory cause [22]. The severity of internal derangement has been classified by Wilkes into five stages with relations to pain, mouth opening, disc location and anatomy [21]. The classification ranges from painless clicking of the joint (Stage I) to severe pain of the joint with severe degenerative bony changes (Stage V), which has served as an aid to guide treatment options in the management of arthrogenous TMD.

While structural anomalies of the TMJ may predispose the patients to symptoms of TMD [23], it should be noted that not all those with structural abnormalities suffer from the same level of clinical symptoms. Apart from physical causes, the association between biopsychosocial factors and TMD has been described by many [1–4,19,24]. Similar to other chronic pain conditions, such as back pain and headache, it appears that there are those in the population who are at risk for developing symptomatic TMD, who also share a certain psychological profile and dysfunction [25,26]. Higher levels of depression and somatization are associated with TMD of arthrogenous and myogenous origins [27]. Moreover, in those with pre-existing TMD, symptoms may be exacerbated during times of stressful events. For example, recent studies have suggested that the during periods of lockdown and social isolation due to the ongoing COVID-19 pandemic, an impact was found on the prevalence of depressive symptoms, stress, as well as pain related to TMD [28,29]. The finding that psychological variables are closely tied to the development of TMD has been confirmed by the Orofacial Pain: Prospective Evaluation and Risk Assessment (OPPERA) study, which found that TMD onset was strongly associated with somatic symptoms, while previous life events, perceived stress and negative affect were also associated with the incidence of TMD [30].

What makes the diagnosis and classification of TMD complicated at times is that many patients present with multiple diagnoses of TMD simultaneously, and it is impossible to isolate the condition to a single particular cause. When discussing about TMD, most clinical researchers refer to those pain conditions that are most commonly seen. However, one must not forget that disorders related to the TMJ include those that are less routinely encountered. Importantly, the presentation of these uncommon conditions of the TMJ may initially mimic those of the more common TMD, yet the management approach may be completely different. For example, a patient who presents with ankylosis of the TMJ may initially present with signs and symptoms similar to closed-lock due to disc displacement, but the standard treatment for ankylosis is surgical release of ankylosis, while conservative

or minimally invasive options, such as arthrocentesis, are usually indicated for closed-lock of the TMJ due to disc displacement.

The crude classification of the most common diagnoses of TMD into arthrogenous, myogenous, or of mixed origin is helpful in steering the clinician into the appropriate path in the initial phases of management. However, more specific diagnoses are usually required, especially if the management progresses beyond conservative options. In the past, classification was often confusing, with many different terminologies referring to similar entities. Today, the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) is the most widely accepted and standardized tool for assessment and classification of TMD, with sensitivity and specificity established for the most common diagnoses of TMD [31]. Recognizing that TMD contains a structural as well as a biopsychosocial component, the DC/TMD consists of two Axes in its assessment. Axis-I contains a protocol for a prescribed physical examination to arrive at specific physical diagnoses of TMD with regard to the joint and musculature, while Axis-II contains several instruments to assess the psychological state of the patient.

There are 12 most common diagnoses of TMD described in Axis-I of the DC/TMD, which are divided into painful conditions (myalgia, local myalgia, myofascial pain, myofascial pain with referral, arthralgia, headache attributed to TMD) and non-painful conditions (disc displacement with reduction, disc displacement with reduction with intermittent locking, disc displacement without reduction with limited opening, disc displacement without reduction without limited opening, degenerative joint disease, subluxation) [31] (Table 1). Note that in many cases, multiple diagnoses are present at any timepoint in a single patient, and that diagnoses may change as the disease progresses or resolves. For example, a patient with complaints of joint clicking with pain in the TMJ and masseter muscle, and headache during mouth opening may be diagnosed with having local myalgia, arthralgia, disc displacement with reduction, and headache attributed to TMD. The classification of TMD also includes those that are less common, but clinically important diseases [32]. Some of these less common diagnoses include fractures of the TMJ, manifestations of systemic diseases, as well as rare conditions such as neoplasms and developmental disorders (Table 2) [32]. However, when these diagnoses do not fit the clinical symptoms, other conditions should also be considered.


**Table 1.** Common diagnoses of temporomandibular disorders (TMD) and their clinical findings.


Modified from Schiffman et al., 2014 [31].

**Table 2.** Some less common diagnoses of temporomandibular disorders (TMD).


1. Fibromyalgia/widespread pain

#### **III. Associated Structures**

A. Coronoid hyperplasia

Modified from Peck et al., 2014 [32].

#### **3. Diagnostic Approach**

The signs and symptoms of TMD may mimic other orofacial pain conditions. Although precise physical diagnosis into the type of TMD is helpful in developing an appropriate

treatment plan, it might not be straight forward in every case. Taking a patients' history is an important part of diagnosing the TMJ condition. The acquisition of history follows the usual format. Apart from the chief complaint, inquiries should be made regarding any history of trauma or previous episodes, aggravating factors, such as eating, talking, yawning or spontaneous background pain, and any previous investigations or treatment. The severity of pain should also be graded using a visual analogue scale (VAS), so treatment progress can be quantitatively monitored. A past and current medical history, including a full medications list, may reveal any comorbidities that may be related to TMD. The clinician should note any habits such as smoking, drinking and recreational drug use, and any history of clenching or bruxism as complained by the patients' bed partner. Additionally, the clinician should ask questions regarding stress and level of life satisfaction, and whether there are any recent life events, such as change of job or loss of a loved one. Although most clinicians treating TMD may be experienced with acquiring a clinical history, some may not be comfortable with taking a psychological history. If desired, the clinician may employ the numerous psychosocial instruments available to aid in their diagnosis, such as those in Axis-II of DC/TMD [31]. When necessary, the patient may be referred for a psychological assessment.

Most clinicians who treat orofacial pain believe clinical examination is the most crucial process of diagnosing TMD. The location of pain, and whether the pain is localized, remains within or spreads beyond the confines of the muscle, should be confirmed with palpation, which is done at rest and during mandibular function. Clicking or crepitus upon mandibular function might be quite obvious in some cases, and the detection might be aided by the use of a stethoscope. Intriguingly, the presence or location of clicking detected by the clinician might be different from that reported by the patient, and this should be documented. The range of mouth opening measured should include pain-free maximum mouth opening, maximum unassisted mouth opening, and maximum assisted mouth opening. Any deviation of the mandible may indicate differential obstruction of the movement of the mandibular condyle in rotation and/or translation. An intra-oral examination is performed to rule out any mucosal pathologies of the oral cavity and oropharyngeal region, as well as to assess the state of the dentition.

#### *3.1. Imaging and Other Investigations*

Imaging is considered to be a useful adjunct in the diagnosis of TMD. Although the diagnostic information provided by plain radiographs like orthopantomogram is limited, they are convenient, simple and serve to rule out some of the differential diagnoses of the bony TMJ, such as fractures, ankylosis, growth disturbances, as well as neoplasms. For the most common types of TMD which clinical presentation is typical, many units might not routinely employ additional imaging. This is due to availability and cost, and that additional imaging might not alter the initial management plan. However, when further information is desired, magnetic resonance imaging (MRI) is the gold standard for TMJ imaging, and is useful in assessing the status of the osseous, as well as the non-osseous structures of the TMJ, such as the masticatory muscles, ligaments and the cartilaginous disc [33] (Figure 1). Classification systems, such as Wilkes [21], combine clinical and MRI findings to stage the extent of internal derangement in order to guide treatment protocol. MRI is therefore considered mandatory prior to any surgical intervention.

While MRI is the most commonly used diagnostic imaging for the common diagnoses of TMD, other imaging modalities are also employed for specific indications. Cone-beam computed tomography (CBCT) has been used to further assess the osseous structure of the TMJ [34–36]. This may be desirable in cases of TMJ ankylosis, benign bony neoplasms or overgrowth, or for the planning of osseous surgery, such as for eminectomy for recurrence TMJ dislocation. However, for most other diagnoses of TMJ, the value of CBCT is not well-established since the information provided in terms of soft tissues is limited [36]. Moreover, the use of ultrasound as a diagnostic tool for TMD has been suggested [15,37,38]. Ultrasound has the advantages of being non-invasive, cheap, and widely available in many

health institutions, yet the effectiveness as a diagnostic method remains to be confirmed [15]. For some inflammatory conditions of the TMJ, such as osteoarthritis and joint inflammation, bone scintigraphy may be of value as a diagnostic tool [39–43]. Moreover, bone scintigraphy has been proposed as a method for the evaluation of active TMJ condylar growth, but it has been shown that both the sensitivity and specificity are low for this indication [44].

**Figure 1.** Magnetic resonance imaging (MRI) showing anteriorly displaced disc in both the close and open mouth position in a patient presented with lock jaw.

Apart from the different imaging modalities available, other investigations are not commonly done for most diagnoses of TMD, except in specific indications. For example, blood investigations may be done for TMD related to systemic conditions, such as rheumatoid arthritis or gout. In the case of uncertain diagnoses of rare diseases or neoplasms, tissue biopsies might be taken, which may be done by fine-needle aspiration, arthroscopic or open joint approach.

#### *3.2. Diagnosis of TMD*

Recognizing the causes of pain and dysfunction related to TMD is important in order to guide treatment decisions. For instance, different treatment options are often employed for the treatment of myogenous versus arthrogenous TMD. Moreover, in those patients who present with TMD symptoms without an obvious physical cause, who also suffer from psychological comorbidities, may be best treated by counselling and psychological intervention.

The most important part of the diagnosis of TMD is to differentiate the common diseases from those clinically significant, but unusual conditions, as well as conditions that are more serious which urgent attention is needed. For example, some neoplasms, such as chondrosarcoma of the TMJ may initially share signs and symptoms as some of the common diagnoses of TMD, such as pain at the preauricular region and limited opening. Another example that requires urgent attention is temporal arteritis, which is an inflammatory condition of the temporal vessels with some TMD-like symptoms, such as headache, pain in the temporal region, and limited mouth opening. However, temporal arteritis is a medical emergency which may cause permanent blindness if not treated promptly. Some of the differential diagnoses of orofacial pain that may mimic TMD are listed in Table 3 [45].


**Table 3.** Differential diagnosis of temporomandibular disorders (TMD)**.**

Modified from Kumar et al. (2013) [45].

#### **4. Treatment Modalities—A Change in Paradigm?**

The goals of treatment for TMD include reduction of pain and improvement of jaw function. Additionally, treatment with the goal of behavioural change may be important in the reduction of tension and parafunction. Currently, physically restoring the disc position in the case of internal derangement is not the primary treatment objective as it may not be relevant to clinical improvement [46,47], unless of course if there is inflammation related to disc displacement then it should be addressed. Symptoms of TMD should be addressed promptly, as chronic pain becomes more difficult to manage due to psychological deterioration and somatization [2,19]. Since conservative options are less likely to cause any harm, they are usually indicated in the early stages of treatment. This is especially true when definitive diagnosis is difficult to ascertain and treatment is performed empirically. However, there is no agreement on how long conservative treatment should be attempted before progressing to other options when clear benefits are not observed. Although the treatment of TMD has shifted away from open procedures which were once popular,

the demonstrated success of minimally invasive options may indicate that they may be considered as an early option for those cases refractory to conservatory approaches.

#### *4.1. Conservative Options*

The initial management of TMD may include various medications, such as analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), anxiolytics, and anti-depressants. Occlusal appliances of various designs are routinely prescribed, which represent a noninvasive option with minimal risks (Figure 2). The use of occlusal splint therapy has been shown to reduce pain intensity and increase maximal mouth opening [48]. However, whether the effect of an occlusal splint is due to the placebo effect has been questioned, and that the evidence of its efficacy remains to be low [49,50]. A systematic review in 2018 by Alkhutari et al. has suggested that the use of occlusal splint may improve patient-centred treatment outcomes, which may be more than merely a placebo effect [51]. Multiple designs are available, such as hard, soft, and anterior repositioning splint. At present, there is no consensus on which design is superior, as results from different studies are equivocal in terms of the efficacy of different designs of occlusal splints [50,52].

**Figure 2.** Occlusal splint for the management of temporomandibular disorders (TMD) and bruxism.

Physiotherapy has been suggested to be an important part in the management of TMD [53,54], which may be particularly useful for myalgia or myofascial pain. Understanding the loading of the stomatognathic system, and the existence of any tension and parafunctions, is important in delivering physiotherapy such as muscle training and changing of behaviour. Evidence shows that physiotherapy is effective in treatment of TMD, in particular the headache symptoms associated with the condition; future research into this area will further ascertain these findings [54]. For myogenous TMD, Botox injection and dry-needling techniques have been suggested [55,56]. Note that Botox is not considered a standard treatment option for TMD, while dry-needling, or acupuncture, may be an effective method to reduce tension in some patients. Additionally, initial results regarding extracorporeal shock wave therapy for myogenous TMD appear to show positive results [57,58].

There has been increasing evidence demonstrating that psychosocial assessment serves as a powerful tool in terms of predicting treatment outcome [59,60]. For those patients with a significant psychosocial component, counselling seems to be a promising treatment adjunct [50,61–63], which might be most beneficial when included in a multimodal approach [50]. Other conservative treatment options for TMD include stress reduction

techniques and diet modification. In the past, a causative relationship between occlusion and TMD had been suggested, but it is now considered an outdated theory not supported by robust evidence, and occlusal adjustment is an irreversible treatment which is no longer supported by the recent literature [64–67].

#### *4.2. Minimally Invasive Options—Arthroscopy, Arthrocentesis and Intra-Articular Injections*

In the 1980s, the availability of MRI has led clinicians to acknowledge the structural anomalies related to TMD. This has resulted in a boom of open joint surgeries, which were unfortunately ineffective in the most part. For those cases of TMD that are arthrogenous and not responsive to conservative treatment, more focus has since been shifted to minimally invasive procedures which have shown promising clinical results.

Arthroscopy of the TMJ was initially pioneered by the Japanese in the 1970s [68,69], and later popularized by the Americans [70–72]. TMJ arthroscopy may involve lysis and lavage of the superior joint space, as well as operative procedures, such as repositioning of a displaced disc, arthroplasty, and removal of inflamed tissues and adhesions. The efficacy of arthroscopy has since been well-recognized [73–79], and has been found that the therapeutic effect was mainly due to lysis and lavage but not disc position [80]. It was due to this finding that a modification was made, where lysis and lavage was performed without arthroscopic view. This was termed arthrocentesis which was first described by Nitzan et al., in 1991 [81], with efficacy that has since been well-documented [46,82–94] (Figure 3).

**Figure 3.** Arthrocentesis performed under local anaesthesia.

In addition to the shift from open joint surgery to minimally invasive treatment for those cases not responsive to conservative treatment, recent literature seems to support that minimally invasive options may be attempted early for arthrogenous TMD [95,96], and this may represent a paradigm shift in the management protocol. A recent integrated review and meta-analysis performed by the authors of this article showed that arthrocentesis was beneficial, whether it was performed as an initial treatment, as an early or late treatment with regard to conservative treatment [97]. However, the best timing to perform arthrocentesis is still unclear due to the paucity of research on the topic, which warrants more future well-designed clinical trials [97].

Although both arthroscopy and arthrocentesis have been shown to be beneficial in the treatment of TMD, it is unclear which method produces better clinical results. In a systematic review and meta-analysis by Al-Moraissi, it was revealed that arthroscopy was superior to arthrocentesis in pain reduction and jaw function improvement, with similar complication rates for both methods [78]. However, other studies have shown comparable results with the two procedures [98,99]. Nevertheless, arthrocentesis has been suggested to be attempted first due to simplicity and cost-effectiveness, with a similar or potentially lower complication rate [99].

Several modifications have been suggested for the conventional arthrocentesis, which involves two puncture needles into the superior joint space guided by landmarks in relations to adjacent structures, followed by lavage with an irrigation solution. For example, single-puncture techniques employ specially designed devices, and may have both the inflow and outflow fluid going through a single cannula but with different ports. Although single-puncture techniques may appear more simple than double-puncture arthrocentesis, most studies to date have shown a similar clinical outcome between the two techniques [83,100–102]. In addition, ultrasound-guided arthrocentesis has been proposed to increase the accuracy of puncture into the superior joint space [103–106]. However, a recent systematic review by Leung et al. has shown that no additional benefit is seen with ultrasound-guided arthrocentesis compared to conventional arthrocentesis [107]. Furthermore, different pharmacological agents for intra-articular injection have been proposed, with the common ones including hyaluronic acid, corticosteroid, analgesics, and plateletrich plasma [93,96,108,109]. Although promising results are seen in some studies, there is currently no consensus regarding which intra-articular injection agent is superior over the others.

Despite the reported efficacy, arthroscopy is seldom required in TMD patients, even in cases of true arthrogenous disorders. Additionally, arthrocentesis is still considered to be a controversial procedure [87], despite the documented efficacy and low complication rates. The reasons for this controversy are as follows. Firstly, some cases of TMD improve with mere conservative options, or even without treatment. Additionally, many cases of TMD are due to multiple etiologies, which may require a multimodal approach before any clear clinical improvement can be appreciated. In addition, intra-articular injection of corticosteroids is a simple and very effective treatment, which may be attempted prior to arthrocentesis. In short, minimally invasive procedures may be the answer in those patients with true arthrogenous TMD not responsive to conservative treatment options, whose condition also lack a significant biopsychosocial component.

#### *4.3. Open Joint Surgery*

Open surgical treatment for TMD is now uncommon, and is reserved for specific indications as well as end-stage diseases. Though, surgery may be the only viable option in some conditions, such as ankylosis and neoplasms, which require release of ankylosis and removal of tumour, respectively. Pending on the availability of equipment and skills, there is now an option of arthroscopic surgery for procedures that were only performed with an open-joint approach in the past. These procedures include disc repositioning procedures, removal of osteophyte, removal of pathologic tissue, and biopsy of the TMJ. In recent years, much work has been done regarding replacement of the TMJ with alloplastic prosthesis [110–116] with an observed improvement in prognosis and longevity. Due to this success, it is likely that we will see a continuous increase in popularity of alloplastic replacement of the TMJ for conditions such as end stage arthritic conditions, ankylosis, post-tumour resection, and developmental anomalies of the TMJ.

#### **5. Conclusions**

TMD represents a divergent group of orofacial pain symptoms which shares similarities with other chronic pain conditions. The etiology of TMD is often multi-factorial, and precise causes for the symptoms may be difficult to pinpoint. In the past, focus has been placed on the physical origins of TMD, but an at least equally significant psychosocial factor is now well-recognized. Consequently, a multimodal approach, which might include counselling and psychological therapy, is being increasingly advocated. Most instances of

TMD are managed conservatively and empirically during the early phases of treatment, yet lingering in the conservative phase for an extended period when clinical improvement is unclear is not recommended. Though open joint surgery is rare nowadays and is reserved for specific situations, we may be in the midst of a changing paradigm which favours early minimally invasive procedures.

**Author Contributions:** Both authors are responsible for all parts of the work. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Comprehensive Management of Rheumatic Diseases Affecting the Temporomandibular Joint**

**Lauren Covert 1 , Heather Van Mater <sup>1</sup> and Benjamin L. Hechler 2,3, \***


**Abstract:** The temporomandibular joint (TMJ) is a synovial joint and thus is vulnerable to the afflictions that may affect other joints in the fields of rheumatology and orthopedics. Too often temporomandibular complaints are seen strictly as dental or orofacial concerns. Similarly, patients with known rheumatic disease may not have their TMJs included in routine screening and monitoring protocols. The purpose of this review is to highlight the rheumatic conditions likely to affect the TMJ and outline medical and surgical management in these patients with a focus on the need for continued patient reassessment and monitoring.

**Keywords:** temporomandibular joint; temporomandibular disorder; rheumatic disease; juvenile idiopathic arthritis; rheumatoid arthritis; inflammatory arthritis

#### **1. Introduction**

The temporomandibular joint (TMJ) is a synovial joint of high functional significance. Although TMJ disorders (TMD) are often thought of as dental or orofacial phenomena, we must not forget that true intracapsular, diacapitular TMJ disease is an arthropathy. When significant intracapsular TMJ damage and dysfunction are present, occlusal imbalance or myofascial pain are never the cause, and an underlying arthropathy must be investigated. Similarly, patients with known rheumatic diseases should be investigated for TMJ involvement. Furthermore, it should be kept in mind that the young to middle-aged female population is the typical patient population presenting with autoimmune disorders and TMD [1]. It is thus crucial that rheumatologists, dentists, oral and maxillofacial surgeons (OMS), and head and neck surgeons are able to independently understand the TMJ's place in manifesting rheumatic diseases.

One-fifth to one-fourth of Americans report a doctor-diagnosed arthritic condition [2], with the Centers for Disease Control and Prevention (CDC) confirming that approximately 25% of US adults suffer from arthritis [3]. The World Health Organization (WHO) has quantified the worldwide morbidity of some of the most common arthritic conditions, with 25% of those with osteoarthritis (OA) unable to perform major daily activities of living, and 50% of those with rheumatoid arthritis (RA) unable to perform full-time job activities within 10 years of disease onset [4]. Although certain autoimmune rheumatic diseases are less common, their individual patient morbidity can be significantly more serious. The terminology used to refer to these musculoskeletal conditions is often inconsistent and confusing. Joint diseases should collectively be referred to as "arthropathies", although in the English language the term "arthritis" has been used extensively and in disparate contexts. Throughout this manuscript, "rheumatic diseases" is used to refer primarily to inflammatory autoimmune conditions, unless otherwise indicated (e.g., osteoarthritis).

**Citation:** Covert, L.; Mater, H.V.; Hechler, B.L. Comprehensive Management of Rheumatic Diseases Affecting the Temporomandibular Joint. *Diagnostics* **2021**, *11*, 409. https://doi.org/10.3390/diagnostics 11030409

Academic Editor: Luis Eduardo Almeida

Received: 13 February 2021 Accepted: 25 February 2021 Published: 27 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Comprehensive management of the TMJ in rheumatic diseases is based upon the initial understanding of four important principles:

#### *1.1. All TMD Should Be Considered as Potentially Secondary to an Underlying Systemic Condition*

The first principle in understanding how to manage TMD in rheumatic diseases is simply the realization that TMD may be a manifestation of an underlying rheumatic condition. It can again not be overemphasized that true intracapsular, diacapitular TMJ disease is an arthropathy. Similar to how visualizing an oral lesion concerning for squamous cell carcinoma should prompt questions of weight loss or dysphagia, TMJ signs or symptoms should prompt questions of other joint involvement, constitutional symptoms, or synchronously or metachronously identified organ involvement.

#### *1.2. TMD Presents Differently in Rheumatic Diseases Than in Non-Rheumatic TMD*

Clinicians who frequently treat TMD in non-rheumatic patients are accustomed to a pattern of linear disease progression consistent with the Wilkes classification. Indeed, Wilkes commented on the "strong relation to the time course of the organic lesions present", with clinical, radiographic, and pathologic findings correlating well [5]. In rheumatic diseases—particularly autoimmune conditions requiring various medical interventions and those present in children and adolescents—the temporal progression of TMD may be unexpected, and a correlation between clinical and radiographic findings is not to be assumed.

#### *1.3. Temporomandibular Joint Disease Is a Continuum*

In science in general and medicine in particular, "cut offs" are often chosen by statistical optimization and therefore may not have strict clinical correlations. In reality, clinical continua are much more common. Accordingly, it is thus much more clinically relevant to follow parameters *within* an individual patient *across time* than to compare parameters *across* patients *at a single time point*. Patients who develop more severe systemic symptoms will be more likely to develop more severe TMJ symptoms.

#### *1.4. Rheumatic Diseases Can also Manifest as Parotid Abnormalities*

Although not the topic of this review, it should be realized that rheumatic conditions frequently affect not only the TMJ but the anatomically proximate parotid glands. Indeed, those with rheumatoid arthritis (RA) have been shown to have abnormal intra-parotid lymph nodes as compared to controls [6]. This gives further credence to principle one, that pre-auricular signs and symptoms should always be investigated in the context of known or potential rheumatic conditions. A corollary to this line of thought is that should a CT of the face be planned for evaluation of possible TMJ pathology, addition of IV contrast should be considered—assuming the patient's medical condition allows—in the event that TMJ, infratemporal fossa, or parotid soft tissue pathology is the true etiology (Figure 1).

‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ **Figure 1.** A 26-year-old female presented for evaluation with chief complaints of right TMJ popping and pre-auricular pain for one year. She reported a strong family history of both systemic lupus erythematosus and Sjogren Syndrome. C-reactive protein was elevated and anti-nuclear antibodies showed speckled and homogenous patterns in high titers. (**a**) Dotted line indicates location of pain per patient; (**b**) CT was intentionally obtained with contrast to evaluate for TMJ and soft tissue abnormalities. Note an intra-parotid lesion lying immediately lateral to the mandibular condyle; (**c**) Intra-parotid lesion removed and found to be a basal cell adenoma. The patient's symptoms resolved after treatment. ‐ ‐ ‐ ‐ ‐

#### **2. Rheumatic DISEASES Affecting the Temporomandibular Joint**

‐ ‐ ‐ ‐ As will be seen, many rheumatic diseases can affect the TMJ. Original reviews suggesting that those with rheumatoid arthritis (RA) will have bilateral, symmetric TMJ involvement while those with seronegative spondyloarthropathies (SNS) will have unilateral disease should be viewed with caution [7] (Figure 2). For example, a review of the currently reported TMJ ankylosis cases in ankylosing spondylitis (AS) patients in the English literature suggests that approximately half presented with bilateral ankylosis [8]. A corollary of this line of thought is the fact that no radiographic findings or clinical signs or symptoms are pathognomonic for a specific rheumatologic disease. ‐ ‐ ‐ ‐

**(a) (b) (c)** 

‐

 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ **Figure 2.** A 56-year-old female presented for evaluation with chief complaints of right TMJ pain and limited mandibular opening. Her history was most notable for long-standing RA refractory to multiple medications. (**a**) Bilateral toe involvement requiring Hoffman procedure; (**b**) Bilateral wrist involvement; (**c**) CT of the face showed early unilateral right TMJ ankylosis, lateral pannus formation, and heterotopic bone formation. The left TMJ was completely normal.

> ‐ ‐ It should also be noted that the more infrequent a specific set of conditions is reported in the literature (e.g., significant TMJ disease in patients with a specific rheumatologic diagnosis), the more anecdotal the reports become. For example, a cursory review of the literature on TMJ disease in rheumatic diseases reveals many case reports of TMJ ankylosis; however, the astute reader must realize the entire reason such reports are worthy of case reports is their overall infrequency amongst a given population.

An abbreviated reference of diagnostic criteria for each condition which can manifest with TMJ dysfunction is presented in Table 1.

**Table 1.** Abbreviated summary of diagnostic criteria for various rheumatic diseases affecting the TMJ. Criteria provided have been commonly used for clinical and/or research purposes. Abbreviations: **ACR**—American College of Rheumatology; **ANA**—anti-nuclear antibodies; **ARA**—American Rheumatism Association (predecessor of the ACR); **ASAS**—Assessment of Spondyloarthritis International Society; **axSpA**—axial spondyloarthritis; **CASPAR**—Classification of Psoriatic Arthritis Study; **CCP**—cyclic citrullinated peptide; **CRP**—C-reactive protein; **ESR**—erythrocyte sedimentation rate; **EULAR**—European League Against Rheumatism; **FM**—fibromyalgia; **ILAR**—International League of Associations for Rheumatology; **JIA**—juvenile idiopathic arthritis; **PsA**—psoriatic arthritis; **RA** rheumatoid arthritis; **RF**—rheumatoid factors; **SLE**—systemic lupus erythematosus; **SSc**—systemic sclerosis; **SSS**—symptom severity scale; **WPI**—widespread pain index.



#### *2.1. Juvenile Idiopathic Arthritis*

Juvenile idiopathic arthritis (JIA), formerly known as juvenile rheumatoid arthritis (JRA), is seen by many as the most concerning rheumatic condition associated with TMJ dysfunction given the risk of dentofacial deformity in the growing child. Consequently, it is the most studied rheumatic condition causing TMJ dysfunction. Its diagnosis per the International League of Associations for Rheumatology (ILAR) requires six weeks of arthritis in a patient under 16 years of age with the exclusion of other etiologic diagnoses [9]. Seven subcategories of the disease are recognized. It is reported that 17–87% of JIA patients will have TMJ involvement [10]. Of particular interest is that in JIA patients with acute TMJ arthritis up to 71% of cases may be asymptomatic and up to 63% may have normal findings on clinical exam [11]. Indeed, even when ultrasonic or MRI evaluation confirms joint effusion in these patients, the vast majority have been shown to be asymptomatic [12,13]. Because by definition the disease process begins in childhood or adolescence, the risk of dentofacial deformity is substantial.

#### *2.2. Systemic Sclerosis/Scleroderma*

Systemic sclerosis (SSc) is a heterogeneous group of disorders which like JIA can manifest in childhood (e.g., localized scleroderma) but is much more common in adults. For decades the mandible has been documented as a bone affected by the disease, both directly and indirectly [14,15]. Compared to the general population, even asymptomatic patients with SSc have decreased mandibular range of motion, although this can be confounded by soft tissue thickening resulting in micrognathia [16]. Although less common than other rheumatic diseases, SSc may be the rheumatic condition most associated with TMJ signs and symptoms, with multiple sources reporting >90% of SSc patients with TMJ signs and symptoms [17,18].

#### *2.3. Rheumatoid Arthritis*

Rheumatoid arthritis is characterized by polyarticular, erosive synovitis that is often relatively symmetrical and may present with significant extra-articular organ disease, a point made clear when it is recognized that those with RA have shorter lifespans than healthy controls [19]. It is reported that 5–86% of RA patients will have TMJ involvement, with 20–40% as a relatively consistent finding [20]. Similar to JIA, asymptomatic patients often have significant disease demonstrable on three-dimensional imaging. It has even been suggested that those with RA who are asymptomatic actually have a *higher* likelihood of TMJ degenerative disease detected on CT than symptomatic patients [21]. In contradistinction, symptoms may occur prior to overt TMJ signs, making disease monitoring via C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and number of involved systemic joints important [22,23]. The presence of anti-cyclic citrullinated protein anti-

bodies has been shown to be significantly associated with development of TMD in RA patients [24]. Cervical spine involvement also appears to increase the likelihood of TMJ disease [25]. A consistent finding in RA patients with TMJ involvement is the predominant sign and symptom being TMJ sounds [17,26], with disease severity (defined by number of edematous joints) associated with TMJ sounds [19].

#### *2.4. Systemic Lupus Erythematosus*

Systemic lupus erythematosus (SLE) has for decades been seen as the quintessential autoimmune disorder. Compared to the other rheumatic diseases affecting the TMJ, patients with SLE are less likely to have TMJ signs or symptoms, and there is conflicting data as to whether their signs and symptoms are different from control populations [17,20,27]. A now classic study by Jonsonn compared 37 SLE patients to 37 dental patients (controls) and found significantly worse signs, symptoms, and radiographic condylar flattening in the SLE patients; however, the majority of the SLE cohort had long-standing disease, all but one had systemic arthritis and arthralgia, and radiographic TMJ changes were significantly more common in patients with renal involvement, suggesting that the high frequency of TMJ complaints may represent an overall more active SLE population. [28]. A more recent study did indeed correlate more severe TMJ dysfunction in SLE with increased number of immunosuppressive medications, presumably a surrogate for disease activity [29]. SLE is one of the few conditions where "avascular" or "aseptic" necrosis of the TMJs is mentioned [30–33]; however, most of these reports come from single groups without histologic analysis of condylar specimens with the only assumption being that because patients have been on glucocorticoids avascular necrosis is likely. Conversely, what is much more likely is inflammatory arthritic destruction.

#### *2.5. Axial Spondyloarthritis (Ankylosing Spondylitis, Non-Radiographic Axial Spondyloarthritis)*

Both ankylosing spondylitis (AS) and non-radiographic axial spondyloarthritis (nraxSpA) are subcategories of the umbrella diagnosis of axial spondyloarthritis (axSpA). Both are considered SNS processes. As the name implies, axSpA primarily involves the axial skeleton, either with (AS) or without (nr-axSpA) plain radiographic evidence of disease. AS and nr-axSpA may be distinct disease phenotypes or simply the spectrum of a single underlying disease process, as over the course of five years 20% of nr-axSpA cases develop radiographic evidence of disease [34]. The majority of axSpA patients are HLA-B27 positive, although this test is not completely sensitive or specific for the disease [35]. The TMJ is reported to be involved in 3–22% of patients, with the literature mainly focusing on patients with AS [36]. A general pattern observed in the cases of TMJ ankyloses in axSpA patients is that (1) the rheumatologic diagnosis is often made many years prior to TMJ dysfunction and (2) essentially all patients developing TMJ ankylosis previously had developed cervical spine fusion [37].

#### *2.6. Psoriatic Arthrits*

Psoriatic arthritis (PsA) is also an SNS and is a disease process originally said to be found in 5–7% of patients with psoriasis [38] but now thought to occur in 15–25% given the increased awareness and diagnosis of the disease [39]. The clinical patterns of the arthritic component most specific to PsA, as originally described [40], include distal interphalangeal (DIP) arthritis and arthritis mutilans (destructive arthritis), although other patterns may be present with significant overlap to other conditions, most notably RA. Although the TMJ is an infrequently involved joint in PsA, it has indeed been described as the first joint involved in PsA [41]. Because of the relatively low number of reports of PsA affecting the TMJs, firm conclusions on prevalence are difficult to make [8], although a recent review has suggested approximately one-third of PsA patients have TMJ symptoms [42]. Review of reports to date, however, do suggest a tendency for those with PsA and subsequent TMD to have worse disease and a significant erosive component, possibly not surprising given the destructive arthritic pattern present in many with severe PsA [38,43].

### *2.7. Others*

### 2.7.1. Osteoarthritis

Unlike the disorders described thus far, osteoarthritis (OA) is not a primary autoimmune inflammatory condition but a disease process marked by mechanical breakdown in the setting of abnormal forces or abnormal response to normal forces, with or without the presence of inflammation. Abnormal forces can be of increased magnitude (microtrauma) or increased frequency (microtrauma) [44], or normal forces can be applied to impaired articular cartilage or an abnormal disc-condyle complex [45]. Consequently, unilateral TMJ OA is often associated with asymmetric anatomy, asymmetric masticatory forces, or previous unilateral injury [46]. Unlike the axial or appendicular skeleton, obesity and occupation are not necessarily associated with OA of the TMJ. The diagnosis of TMJ osteoarthritis should, however, mirror the American College of Rheumatology (ACR) classification criteria for OA of the knee and hip: pain should be a primary symptom; joint stiffness, limited mobility, and crepitus will likely be present; radiographic evidence of erosion, subchondral cysts, subchondral sclerosis, and osteophytes are common; and elimination of autoimmune or infectious causes should be ensured [47,48].

#### 2.7.2. Fibromyalgia

Although fibromyalgia (FM) is not a cause of intra-articular TMD, patients with FM often present with signs and symptoms concerning for inflammatory articular disease including pre-auricular pain, pain on mandibular function, limited mouth opening, and diurnal change in symptoms. At least one study has gone as far as to suggest that all patients with FM present with pain when the TMJs and retrodiskal tissues are palpated [49]. A recent systematic review revealed a strong association between FM and TMD; however, the overwhelming association was with regard to complaints of pain, particularly masticatory muscular pain [50]. In this way, the FM patient often has a higher symptom burden relative to any radiographic abnormality while the inflammatory arthritis patient is more likely to have a lower symptom burden relative to the degree of radiographic joint disease. It should be noted that TMJ arthritic disease can present in patients with FM, but FM is not the etiology.

#### 2.7.3. Idiopathic Condylar Resorption

Although not a rheumatic inflammatory disease, idiopathic condylar resorption (ICR) must be mentioned as it presents nearly exclusively in adolescent and young women and thus demographically overlaps the patient population represented by systemic rheumatic conditions. Indeed, the original discussions on this phenomenon highlighted similarity to autoimmune resorption [51], although further investigations also emphasized what is now generally accepted as the role of hormones such as estrogen, prolactin, and endogenous steroids in this process [52]. Although ICR is usually symmetric, unlike rheumatic diseases it is not autoimmune and usually not inflammatory in nature, evidenced by the typical lack of synovitis and joint effusion on MRI even in the setting of active condylysis [53]. One frequently propagated misconception is that ICR is usually asymptomatic [54], when surveys actually suggest that the majority of ICR patients present with TMJ pain and myofascial pain [55]. ICR thus becomes a diagnosis of exclusion when symmetric condylysis is appreciated in a female patient whose rheumatologic work-up is otherwise negative.

#### **3. Systemic Management of Rheumatic Diseases**

While there are different types of inflammatory arthritides, as described above, systemic management across these distinct conditions share a similar approach and classes of medication including non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, conventional and biologic disease-modifying antirheumatic drugs (DMARDs). Empirical practice with systemic treatment has beneficial effects on TMJ arthritis [56]. Goals of therapy for TMJ arthritis are similar to the treatment of arthritis in general—the cessation and prevention of joint damage, suppression of systemic disease, and eventual

remission off medications [57]. Treatments for inflammatory arthritis are individualized based on severity of disease, number of joints involved, physical limitations and potential for joint damage [53].

Most of the literature regarding treatment of inflammatory TMJ arthritis with systemic medication is specific to JIA with a focus on an approach to normalize mandibular growth, reduce MRI-verified inflammation, and preserve osseous TMJ morphology. Current biologic medications have significantly decreased the extent of disability and need for major surgeries and joint replacement in JIA [57]. A retrospective study of 38 patients with JIA involving the TMJ, who were receiving systemic therapy, showed less severe osseous deformity and maintained normal mandibular ramus growth at 2 year follow up compared to baseline MRI. This contrasted to cohort studies with corticosteroid TMJ injections, in which TMJ deformity deteriorated and mandibular ramus growth was impaired [58].

Generally, however, there has been little evidence to guide management for TMJ arthritis. Most randomized controlled trials of DMARDs have not included TMJ involvement as an outcome, and there is minimal prospective data on medical therapy [59]. Consensus on treatment is lacking. In 2014, an 87-center multinational survey of pediatric rheumatologists worldwide showed that first-line treatment of TMJ arthritis varied with NSAIDs in 33%, non-biologic DMARDs in 36%, anti-TNF medication in 5%, and intra-articular steroid injection in 26% [60]. Furthermore, a cross-sectional survey of 52 academic OMS in the US revealed that the majority (81%) of JIA patients were being treated on average with 1–2 systemic medications, 13% on 3–4 medications and only 5% on no systemic medications [61]. It is worth noting that even with optimal medical management for peripheral arthritis in JIA, the TMJ is the most common joint that does not respond to initial therapy. Retrospective studies suggest that response to medical therapy of the TMJ may lag behind that of other joints for unclear reasons [62]. Consensus on treatment of TMJ arthritis in JIA is currently in development amongst pediatric rheumatologists within the US based on expert opinion.

#### *3.1. NSAIDs*

NSAIDs such as naproxen, ibuprofen, and indomethacin are commonly used as an initial therapy in inflammatory arthritis with or without TMJ involvement. NSAIDs inhibit cyclooxygenase (COX)-2 activity, reducing cytokine-induced destruction of the extracellular matrix of the TMJ [63]. While often part of a maintenance medication regimen, NSAIDs are only beneficial for reducing TMJ complaints in a minority of patients; more aggressive treatment with DMARDs is generally necessary [10]. In fact, NSAIDs are effective for TMJ arthritis for one-fourth to one-third of JIA patients but primarily in oligoarticular disease. They are often considered as adjunctive or bridge therapy to more definitive interventions for TMJ disease [57]. NSAIDs are usually well tolerated. Potential side effects include gastritis, gastrointestinal bleeding, headache, increased sun sensitivity, and hepatic and/or renal dysfunction [53].

#### *3.2. Conventional DMARDs*

Conventional DMARDs include sulfasalazine, leflunomide, and methotrexate. Methotrexate is the only medication with significant evidence in the treatment of TMJ arthritis [57] and is usually first line in practice for JIA with TMJ involvement. Weekly intramuscular injection of methotrexate has been shown to decrease cartilage degeneration in rabbits with antigen-induced arthritis but failed to eliminate arthritis completely [64]. Furthermore, in a cross-sectional study, Ince et al. demonstrated that methotrexate therapy may minimize TMJ destruction in polyarticular JIA. Methotrexate is a folic acid analog that inhibits dihydrofolate reductase, leading to inhibition of purine and thymine synthesis, a reduction in T and B cell activation, and antibody formation. The dosing range is 0.5 to 1 mg/kg weekly, or 15 mg/m<sup>2</sup> , with a maximum dose of 25 mg weekly. It can be given by mouth or subcutaneously. Over sixty percent of patients with JIA benefit significantly, though given its slower onset of action, effects are usually not apparent until 4–6 months after initiation. Serious toxicity is uncommon, but side effects including nausea, anorexia, stomatitis, transient aminotransferase level elevation, and malaise 24 hours after administration are relatively common. Folic acid supplementation has been shown to decrease these common side effects [53].

#### *3.3. Biologic DMARDs*

Biologic DMARDs used in the treatment of TMJ inflammatory arthritis include tumor necrosis factor (TNF) inhibitors such as adalimumab, etanercept, and infliximab. These medications are usually administered systemically via subcutaneous injection (etanercept and adalimumab) or intravenous infusion (infliximab). Local therapy with intra-articular injection of infliximab has been attempted but has failed to show efficacy in improving acute or chronic synovitis, or in changing maximal incisional opening [59]. TNF inhibitors are generally given in combination with methotrexate for TMJ arthritis that is refractory to methotrexate alone. The decision on whether to initiate systemic TNF blockade when severe disease is identified or to wait until after failure of initial methotrexate is currently based on expert opinion [10]. TNF inhibition has been shown to reduce TMJ pain and improve oral function in the literature for adults, however there is not strong evidence for juvenile TMJ arthritis. Other biologic DMARDS may also be considered including tocilizumab and abatacept. Current consensus is that non-systemic JIA responds well to TNF inhibition and methotrexate while systemic JIA responds well to IL-1 and Il-6 blockade with medications such as canakinumab and tocilizumab, respectively [65]. Overall, biologic DMARDs are generally well tolerated and require minimal lab monitoring. The main adverse effect is increased risk of infection.

#### *3.4. Timing of Systemic Therapy*

While those with isolated TMJ arthritis may start with isolated steroid injection or irrigation, patients with polyarticular arthritis, or more systemic disease activity, benefit from antirheumatic medications. Systemic medications are generally optimized and continued until all aspects of disease including arthritis, uveitis, and systemic symptoms are well controlled. Once remission on medications is obtained, in pediatrics, treatment usually continues for at least 12–24 months before attempting to taper off, assuming the treatments are well tolerated. Recent recommendations in orthopedic literature include stopping patients' biologic medications one dose before any planned joint replacement and waiting 14 days or until wound healing is complete until restarting the medications. New recommendations include continuing conventional DMARDs such as methotrexate during the perioperative period [62].

#### *3.5. Potential Side Effects of Other Systemic Therapy*

It is worth mentioning that some rheumatic disease systemic therapies, particularly bisphosphonates and corticosteroids, can be associated with TMJ disease. Bisphosphonates are potent inhibitors of osteoclastic bone resorption and are known for their use in treating osteopenia and osteoporosis but are also used in the management of chronic nonbacterial osteomyelitis (CNO, also known in the OMS literature as diffuse sclerosing osteomyelitis (DSO) or primary chronic osteomyelitis (PCO)), a rheumatic condition of inflammatory bone destruction. Jaw osteonecrosis is a potential risk of bisphosphonate use and should be considered in patients treated with bisphosphonates who present with TMJ complaints. Corticosteroids are used more widely across many rheumatic conditions as part of both acute and maintenance therapy. The side effect profile of corticosteroids will not be discussed in depth here, but it is worth noting that the risk of osteoporosis, osteopenia, and avascular necrosis is much greater when a patient is on chronic corticosteroids.

#### *3.6. Systemic Therapy for Non-Rheumatic Causes of TMJ Arthritis*

Traditional treatment of TMJ osteoarthritis mainly includes NSAIDs. De Souza et al [66] demonstrated equivalent pain reduction with diclofenac sodium compared with occlusal

splints as well as intra-articular injections of sodium hyaluronate or corticosteroid. Research more recently has investigated oral glucosamine as an adjunctive therapy for TMJ osteoarthritis treatment. In a double-blinded randomized controlled trial conducted by Yang et al [67], oral glucosamine hydrochloride added to hyaluronate sodium injection failed to have meaningful effect on pain at month 6 post-injection but did improve pain and function at month 12, suggesting possible efficacy after prolonged use.

Systemic treatment is not indicated for idiopathic condylar resorption (ICR), which was mentioned above as a diagnosis of exclusion and can be a mimicker of systemic rheumatic disease. While differentiating isolated TMJ JIA from ICR can be difficult, the distinction is crucial as systemic therapy is not warranted for ICR but a cornerstone of JIA management.

#### **4. Assessment of the Temporomandibular Joint in Rheumatic Disease**

As noted in the Introduction, a critically important distinction between TMJ disease presentation in rheumatic diseases and non-rheumatic TMD is the delay—or even complete absence—of clinical signs and symptoms relative to anatomic destruction in rheumatologic patients. Since providers who frequently treat non-rheumatic TMD patients often do not recommend imaging until significant signs or symptoms are present, a known rheumatic diagnosis should prompt the clinician to consider earlier application of imaging modalities in this patient population (Figure 3). This may alert the provider to situations where earlier initiation of non- or minimally-invasive treatments (conventional or biologic DMARD adjustment, arthrocentesis, intra-articular medicament application, etc.) may delay further joint destruction. The relapsing/remitting nature of some of these conditions, in concert with the use of DMARDs, NSAIDs, biologics, and the associated individual patient variations in response, complicate any expected association between signs, symptoms, and imaging findings which is usually more robust in the non-rheumatic patient.

#### *4.1. Patient History*

For the patient without a previous rheumatic diagnosis, new signs and/or symptoms of TMD should include a broad patient history including questions regarding constitutional symptoms, pain and dysfunction of other joints, back complaints, muscle weakness, and skin/nail lesions [8]. Questions specific to vasculitides, which may occur with rheumatic conditions, can also be helpful, particularly questions about new respiratory, ophthalmologic, mucosal, or renal abnormalities.

For all patients, with or without a previous rheumatic diagnosis, a more traditional history—one more pointed at orofacial musculoskeletal disease and osteoarthritis—still remains appropriate. Questions include those regarding headaches, earaches, recent or remote trauma, parafunctional habits, and bone and cartilage diseases. Patients should specifically be asked to quantify and qualify pain, clicking, crepitus, locking, dislocation, reduced opening, stiffness, change in diet, and sense of altered occlusion.

#### *4.2. Clinical Examination*

Although the TMJ clinical examination should always be comprehensive and is therefore not fundamentally different in patients with a known rheumatic disease, it does become helpful for the clinician to understand which metrics have been shown to be helpful in these patients.

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**Figure 3.** Basic framework for incorporating rheumatology referral and evaluation in patients presenting with signs and symptoms of a temporomandibular joint disorder. Patients found to have rheumatic diseases should undergo period TMJ imaging.

The Helkimo Clinical Dysfunction Index (Di) and Helkimo Anamnestic Index (Ai) are useful metrics for assessing and monitoring such patients [68]. While the Ai is technically subjective and thus truly part of the patient's history or subjective assessment, it is often recorded simultaneously during the clinical examination. Subjectively (Ai), the patient can be completely asymptomatic, mildly-moderately symptomatic (joint sounds, jaw fatigue, jaw stiffness), or severely symptomatic (trismus, locking, luxation, discoordination). Objectively (Di), mandibular range of motion, dysfunction with motion, and pain are measured, with significant weight being placed on end-stage pain and dysfunction (Table 2).


**Table 2.** Helkimo clinical dysfunction index (Di). The maximum score recorded from each domain is added to determine the total clinical dysfunction index score. Abbreviations: **MIO**—maximum incisal opening; mm—millimeter.


The Helkimo indices have been most rigorously studied in the TMJ OA population. It should be noted, however, that OA patients often present for evaluation because of pain, and thus the translatability of these results to the autoimmune population—many who either do not have pain initially or at least have a weaker association between pain and clinical and radiographic signs—should be considered cautiously. Said another way, the Helkimo index alone may underestimate the degree of damage in inflammatory rheumatic disease. Strong associations between the Helkimo index and bony changes (condylar head or fossa) but not soft tissue changes (joint space size) have been reported when using CT [69,70].

Juvenile SLE patients have been found to have significantly worse Di scores than healthy controls, with the discrepancy due primarily to TMJ dysfunction and not pain. Even more specifically, it appears that decreased laterotrusive movements may be the first signs of dysfunction in this population [29]. This has been demonstrated in the RA population as well, where worse Di scores were found to be primarily due to decreased mandibular mobility and not necessarily worse pain [71]. On the contrary, others have found that both the Ai and Di were significantly worse in RA patients than control patients, and the Helkimo indices performed significantly better at discriminating RA versus control patients than other indices [1]. As noted previously, the relapsing/remitting nature of these conditions, in concert with the use of DMARDs and NSAIDs—which frequently are not reported or controlled for as confounders in studies—complicates the association between symptoms, particularly pain, and overall cumulative TMJ damage. Accordingly, duration of autoimmune disease alone does not necessarily correlate with worsening Helkimo indices [72]. Studies generally agree that the Helkimo index as a whole helps to discriminate patients with significant arthritic disease from those without significant TMJ involvement [73,74]. A qualitative summary of studies to date finds that decreased mandibular mobility and pain on mandibular function are the most commonly reported findings.

In addition to routine use of the Helkimo indices, international consensus guidelines have been established for orofacial examination in patients with juvenile idiopathic arthritis and can be extrapolated to the rheumatic TMD population in general [75,76]. These guidelines have resulted in a minimum recommended "short screening protocol" that includes assessment of TMJ pain in open and closed positions, mandibular deviation on opening, maximum incisal opening (MIO), frontal facial asymmetry, and facial profile. While the Helkimo indices focus more on grades of pain and dysfunction, the consensus guidelines are meant to screen for and monitor diacapitular disease activity and resulting dentofacial deformity. Monitoring in the rheumatic population will be further discussed below.

#### *4.3. Imaging*

#### 4.3.1. Three-Dimensional Bone Imaging

Bony destruction is reliably associated with periods of more severe disease activity. Although erosions, cortical morphology, and subcortical changes can fluctuate over time, both two-dimensional and volumetric condylar changes appear to correlate with cumulative disease activity in the joint. In RA and JIA patients, CT and MRI reveal that condylar or ramal height, condylar volume, anteroposterior length, and mediolateral width are all

associated with disease severity [77], although findings are not specific to inflammatory diseases and thus cannot be used to diagnose autoimmune TMJ disease [13]. The most unifying finding in active rheumatic TMJ disease—regardless of whether the condyle, ramus, or both are affected—is asymmetry [78].

It must not be forgotten that conventional radiographs remain a reasonable screening examination in asymptomatic patients without dysfunction per the Helkimo index, with the possible exception of JIA patients. Even in this population, however, it has been suggested that condylar asymmetry on screening panoramic is specific for joint damage [79], but a concern remains for low sensitivity and reproducibility with this modality compared with three-dimensional imaging [80]. In both osteoarthritic and RA patients it has been suggested that CT may not add much to the bony changes visible on plain radiographs [81].

#### 4.3.2. Three-Dimensional Soft Tissue Imaging

MRI is the gold standard for soft tissue TMJ imaging including assessment of the articular disc, synovium, joint spaces, bone marrow, and surrounding musculature. This requires imaging protocols including fluid-sensitive (usually T2), pre-contrast T1 (usually fast spin echo, FSE), and post-contrast fat-saturated T1 sequences [82–84]. By far the most studied population is those with JIA, as this population is the most likely to lack signs and symptoms with significant disease activity. In a most extreme example, a study by Kellenberger et al. showed that 100% of control patients with joint effusions on MRI had pain while 0% of JIA patients with joint effusions had pain [85].

An enhancement ratio (ER) or enhancement value (EV), defined as the contrast enhancement of the superior joint space divided by that of a nearby muscle (often the longus capitis), has been described and validated in the JIA population [86,87]. Other semiquantitative MRI grading systems exist, with the OMERACT (Outcome Measures in Rheumatoid Arthritis and Clinical Trials) and EuroTMJoint (now TMJaw) research group having the most applicable systems [13]. These scoring systems evaluate inflammation (bone marrow edema, joint effusion, synovial thickening, joint enhancement) and damage (condylar flattening, erosions, and disc abnormality) and provide either a cumulative score that can be followed (OMERACT), similar to the Helkimo indices, or a progressive score (EuroTMJoint/TMJaw), similar to the Wilkes staging system.

#### 4.3.3. Nuclear Medicine Imaging

Bone scintigraphy is a nuclear medicine examination based upon the premise that high bone turnover and/or osteoblastic activity—indicative of hyperplasia, active growth centers, or inflammatory turnover, among others—increases local uptake of radiopharmaceuticals which mimic pyrophosphate [88]. Accordingly, studies show that those with active rheumatic conditions have increased condylar uptake allowing reasonable discrimination from healthy controls and those with non-inflammatory TMJ OA, which seems to mirror discrimination by inflammatory laboratory markers (e.g., ESR, CRP) [89]. Similar findings using positron emission tomography (PET), where avidity is based upon increased glucose uptake in inflammatory environments, have been noted [90]. Nuclear medicine studies have no role in identifying TMJ OA [91].

#### 4.3.4. Ultrasonography

In theory, ultrasonography (US) seems an ideal modality to evaluate the soft tissues of the TMJ, with the TMJ being relatively superficial and US being a continuous imaging modality conducive to dynamic imaging. Unfortunately, only a few studies have attempted to objectively compare US to the current gold standard for soft tissue imaging, MRI, in autoimmune TMJ disease [11,92,93]. Although some suggest that there is at least moderate correlation between US and MRI for the assessment of synovitis in childhood arthritis [93], a recent systematic review in the JIA population could not recommend US as a standard imaging modality in these patients [94].

#### **5. Interventions for Temporomandibular Joint Dysfunction in Rheumatic Disease**

Outcomes purportedly expected in the management of TMD in the rheumatic patient population should be reviewed with caution. A careful review of the available literature demonstrates that many authors reference studies involving non-rheumatic TMD patients. As the reader is already well aware, there are vast differences in presentation and outcomes in rheumatic and non-rheumatic TMD patients. Given the available evidence, an algorithm for management of rheumatic TMD patients is presented in Figure 4. Although this algorithm is based upon the TMJ Working Group's recommendations in the JIA patient [95], less emphasis is placed on the skeletal maturity of the patient and more emphasis is placed on the disease state and degree of patient dysfunction. The central role of the systemic rheumatologic management is also highlighted by this algorithm. ‐

‐ ‐ **Figure 4.** Recommended algorithm for treatment. Abbreviations: **CCG**—costochondral grafting; **DO**—distraction osteogenesis; **IACS**—intra-articular corticosteroids; mod—moderate; **PT**—physical therapy; **TJR**—total joint replacement.

#### *5.1. Conservative Interventions*

‐ ‐ ‐ ‐ ‐ ‐ In non-rheumatic TMD patients, "conservative" interventions typically convey ideas of joint rest, diet alteration, occlusal guards, physical therapy, NSAIDs, and muscle relaxants. Although these certainly may also be beneficial for the rheumatic patient [96,97], the foundation of conservative management in these patients is systemic management of their inflammatory disease, as described above. That being said, self-directed physical therapy has shown effective in improving mandibular function, TMJ related pain, or both in patients with RA and AS [98,99]. In FM patients, tactile stimulation has been shown to improve sleep quality, quality of life, and TMD symptoms [100]. Low-level laser therapy for TMJ inflammatory arthritis has only been preliminarily investigated in animal models [101], and thus no conclusions regarding efficacy should be made.

‐ Although many still propagate "occlusal equilibration" or "fixed prosthetics" for the treatment of TMD in general and TMJ OA or autoimmune diseases in particular [102,103], it should be made clear that no robust evidence supports these practices [104,105], and the senior author finds the continued use of these practices for this purpose highly misleading to patients. Although occlusal modification, including orthodontic treatment, can certainly improve facial appearance, masticatory function, and oral hygiene in these patients [106,107], it should not in any way be expected to improve rheumatic TMD.

#### *5.2. Minor Procedures*

#### Arthrocentesis and Intra-Articular Injection

It is well documented that arthrocentesis improves pain and dysfunction in patients with osteoarthritis, particularly Wilkes stages II, III, and IV [108,109]. Arthrocentesis with lysis and lavage alone likely improves pain and dysfunction in the rheumatic TMD population as well [110]; however, analysis is at times confounded by the fact that most rheumatic patients have traditionally also received intra-articular corticosteroid injection (IACS) at the conclusion of arthrocentesis [111]. A more recent study found that the IACS component does indeed improve the Helkimo index over arthrocentesis alone [112]. There is no question that TMJ IACS can at least temporarily improve symptoms in properly selected patients with active RA [111,113] or JIA [112], but concerns remain regarding long-term effects of IACS.

For example, multiple studies have specifically reported on the presence of heterotopic bone formation in JIA patients who have IACS, but a cause-and-effect relation has never been proven [83,114]. More recently, a retrospective review of JIA patients illustrated the complexity of the cause-and-effect relation, as the authors found that the total number of injections and time to first injection were associated with increased risk of heterotopic bone formation, yet they noted that children with more severe arthritis were likely to receive IACS [115]. Clearly, indiscriminate use of IACS should be avoided, and it should only be considered during active inflammation not responsive to medical management, preferably when confirmed by MRI [116]. Alternatively, consideration should be made for arthrocentesis with lysis and lavage *without* IACS, or with injection of hyaluronic acid [117].

More recently, intra-articular biologic injection (IAB) has been studied in the TMJ, with the first being a case report of IAB with infliximab in a patient with PsA unresponsive to both systemic infliximab and TMJ IACS [118]. Subsequent reports of IAB with infliximab in JIA patients show that although the injections appear safe, they do not affect jaw opening or improve inflammation or destruction as appreciated on MRI [119,120]. IAB with etanercept has been reported in rabbit [121] and rat [122] models of inflammatory TMJ arthritis and TMJ loading, respectively. The rabbit model showed that IAB with etanercept did not perform as well as systemic etanercept and performed no different than intra-articular saline injection. The rat model simply suggested that biochemical and biomechanical processes in the TMJ are likely driven in part by TNF-α. In conclusion, evidence to date does not support intra-articular biologic injection of the TMJs.

#### *5.3. Major Procedures*

#### 5.3.1. Open Arthroplasty and Associated Procedures

Synovectomy and discectomy, or possibly discectomy alone, have been shown to improve mandibular function [123] and pain [124] in patients with rheumatic TMJ disease, including RA, AS, and PsA patients. The effectiveness of these procedures should be taken into context, however, as many of these studies were performed before the application of biologic DMARDs for autoimmune rheumatic diseases. Additionally, overly aggressive attempts at or simply multiplicity of open arthroplasties may complicate eventual joint replacement, if this is foreseen in the patient's future.

#### 5.3.2. Orthognathic Surgery

Debate continues on the stability of orthognathic surgery results in patients with resorptive TMJ processes such as inflammatory rheumatic diseases and ICR. It should also be noted that this does not treat the underlying pathology but simply masks a subset of the orofacial manifestations. The optimistic hope is that if a patient's disease process is well controlled, the result will be stable. Unfortunately, this essentially can never be guaranteed, and therefore many "successes" end up being measured in the short term of months [125–128]. A patient with a process defined by condylar resorption electing to undergo orthognathic surgery alone must absolutely be informed that relapse is expected, TMJ pain and dysfunction are not expected to resolve, and only TMJ TJR will predictably

result in long-term stability [129]. Thus, the patient best suited for orthognathic surgery alone is one with stably quiescent disease with relatively mild deformities.

Condylotomy—which has evolved to its current day form of essentially a vertical ramus osteotomy—has been documented as a treatment in active inflammatory TMD [103], but this represents a lack of understanding of the disease process and should not be performed.

#### 5.3.3. Distraction Osteogenesis

Distraction osteogenesis (DO) of the mandibular rami has been reported in JIA patients. As would be expected for a treatment aimed primarily at altering the dentofacial abnormality without addressing the TMJ disease process itself, facial appearance and occlusal relationship were improved while long-term pain, mandibular mobility, and TMJ signs had either mixed results or continued progression [130,131]. It should also be noted that inclusion criteria in the only prospective study to date were unilateral TMJ involvement, inactive disease, and TMJs with "clinical and subjective good function" preoperatively [130]. Therefore, similar to the potential orthognathic patient, patients with a process defined by condylar resorption electing to undergo DO alone must absolutely be informed that relapse is expected, TMJ pain and dysfunction are not expected to resolve, and only TMJ TJR will predictably result in long-term stability.

#### 5.3.4. Total Joint Replacement

Although historically costochondral grafting (CCG) has been performed in patients with rheumatic TMD [132–134], and although debate continues on the application of autogenous or alloplastic procedures for TMJ TJR in non-rheumatic end-stage joint disease, the senior author agrees with the idea that inflammatory TMJ destruction is best treated with alloplastic methods [135].

Guidelines have been put forth to guide physicians when prosthetic TMJ TJR may be appropriate, including in inflammatory joint disease [136]. Not surprisingly, the superiority of alloplastic TMJ TJR in non-rheumatic end-stage joint disease patients has been found to translate to the autoimmune population as well [137]. Outcomes of alloplastic TMJ TJR in RA, PsA, AS, SSc, and JIA patients have been reported, showing consistent improvement in associated pain and dysfunction [138–147]. The literature nearly unanimously suggests that patients with appropriate indications for TMJ TJR have seen improved, durable outcomes.

A legitimate concern in open surgery—particularly those involving alloplastic implantation—is the immunosuppressive therapies that many patients will be taking, particularly those patients with disease severe enough to require such surgery [116]. Studies of TMJ TJR often do not comment on perioperative medication management, although this is vitally important to success. Although developed with the American Association of Hip and Knee Surgeons (AAHKS), the ACR has published perioperative guidelines for management of antirheumatic medications in those undergoing arthroplasty [148]. As mentioned previously, conventional DMARDs should generally be continued through surgery while surgery should occur at the end of biologic DMARD dosing cycles, and the biologic should not be resumed until 14 or more days post-operatively (assuming no post-operative infectious or wound healing complications).

#### **6. Monitoring of the Rheumatic Patient with Temporomandibular Joint Disease**

There are minimal evidence-based or consensus guidelines for monitoring in rheumatic patients with TMD, with most available data pertaining to the JIA population. Consensus assessment methods were reached by the Temporomandibular Joint Juvenile Arthritis (TMJaw) Working Group for monitoring of TMJ arthritis and involvement in JIA patients in 2019 [95]. These include MRI with contrast, 3D scans (which may include CBCT or medical grade CT as appropriate), clinical examination, and patient-reported outcome measures. Consensus could not be reached to recommend the use of MRI without contrast, plain radiographs, or ultrasound in the monitoring of TMJ arthritis in these patients. The TMJaw group also proposed a clinical evaluation protocol for regular assessment of the TMJ joint in

patients with JIA, which is applicable both for screening as well as following patients with a history of TMJ arthritis [75,76]. As discussed above, the components of the exam allow for a quick assessment of pain, range of motion, and dentofacial deformity and asymmetry, which when followed over time can assist in detecting subtle changes indicative of active or progressive disease. However, as previously stated, given the potential for active, erosive TMJ arthritis in asymptomatic or minimally symptomatic patients, there is also a need for imaging to both evaluate for initial disease, as well as to follow the course of TMJ arthritis during and after treatment. This is the case when following up after either TMJ arthrocentesis or initiation of systemic rheumatic medications. Given MRI with contrast is the gold standard for active synovitis, monitoring 6 months after a treatment is initiated or changed with an MRI is the most accurate for assessing whether there is ongoing disease activity that would warrant additional measures. ‐ ‐ ‐ ‐ 

A survey of academic American OMS practice patterns in managing and monitoring JIA patients suggests that once inflammatory arthritic patients are deemed to be in remission, most are monitored at 6 to 12 month intervals [61]. However, this study also revealed that the average OMS often relies more on symptoms and plain radiography rather than MRI when following this patient population. This highlights the potential benefit of ongoing discussions between rheumatology and OMS to determine the best imaging modality for individual patients. ‐ ‐ ‐ 

With regard to monitoring for disease activity and its effect on surgical treatment decisions, the TMJaw group recommends that a lack of progression over one year combined with contrasted MRI confirmation of quiescent disease serves as reasonable evidence to proceed with autologous reconstruction (e.g., costochondral grafting and/or orthognathic surgery). The unpredictability of the disease process, particularly in younger patients, should be considered however when deciding on surgical intervention. A suggested monitoring protocol is presented in Figure 5. ‐ ‐ ‐ ‐

**Figure 5.** Recommended monitoring protocol. Abbreviations: **q6m**—every 6 months; **q12m**—every 12 months; **PE** physical exam; **S/S**—signs and symptoms; **tx**—treatment.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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