**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.