Temporomandibular disorder (TMD) is the second most frequent musculoskeletal condition after chronic back pain, and pain and dysfunction occur due to abnormalities in the temporomandibular joint and masticatory muscle system [1]. Among them, the masticatory muscles exert loading on the teeth and temporomandibular joint and play an essential role in regulating the position and movement of the mandible [2]. Myogenic TMD (TMD involving the masticatory muscles) can produce chronic and recurrent pain and, if left untreated, can severely restrict daily activities, and lower the quality of life associated with maintaining oral health, such as mastication [3]. Moreover, chronic persistent pain in the masticatory muscles can result in disused atrophy of those muscles [4]. Until now, when diagnosing TMD, no evaluation method has been proposed to assess the condition of the masticatory muscles, including the masseter muscle.

Recently, as the use of ultrasonography (US) has gained popularity in clinical practice, interest in the use of the US for TMD diagnosis is gradually increasing. The US provides several benefits, including the capacity to evaluate soft tissue including muscle, in radiation-free, real-time on a portable device [4]. The US is fundamentally a tomographic method, so the US image reveals the cross-sectional thickness of the examined tissue [5]. If the muscle is used intensively and maintained in an overactive state, the thickness of the masseter muscle may increase, and the maximum bite force (MBF) may increase [2,6]. However, some research suggests that masticatory muscle overload may cause MBF to be lower than normal in people with TMD [7,8].

In diagnosing TMD and establishing a treatment plan, measuring MBF has been suggested as a useful method for quantifying and comprehending masticatory muscle function [9]. The study on the US, surface electromyography (sEMG), and MBF demonstrated that the masseter muscle thickness of the US, sEMG activity of the masseter muscle, and MBF revealed a positive correlation [9]. The thickness of the masseter cross section can be assessed during both rest and activity when examining the masseter muscle using US. Therefore, the thickness of the masseter muscle at maximum occlusion can be evaluated through the activation state of the muscle, and the functional evaluation of the masseter muscle, which is the mandibular elevator muscle, can be conducted. This procedure allows for the evaluation of the masseter muscle’s functional status as the mandibular elevator muscle.

However, the drawback of the US is that it is highly dependent on the inspector. Furthermore, measurement errors may occur depending on the location and application angle of the US transducer [10]. Moreover, when evaluating the activation state of the masseter muscle, the thickness of the masseter muscle is mainly determined during jaw clenching, and the conditions for jaw clenching vary from study to study [4,11,12]. To our knowledge, no study has explained the conditions that can induce maximal occlusion in the test subject. As a result, the goal of this study is to develop a reproducible and reliable method for assessing the state of the masseter muscle related to MBF when measuring masseter muscle with the US.

1. Subjects

Nineteen healthy adults (9 males, 10 females) were the subjects of this research. They were informed of the study and submitted written informed consent. The inclusion criteria for the study were adults aged 20 years or older without missing teeth, except for the third molar. The exclusion criteria for this study were those who underwent dental treatment including orthodontic treatment, those who demonstrated clinical symptoms related to TMD, and those who had a history of TMD treatment, orthodontic treatment, or botulinum neurotoxin injection treatment. Additionally, those having a history of degenerative arthritis or aberrant connective tissue were disqualified from this study. The Institutional Review Board of Chosun University Dental Hospital approved this study (Approval no. CUDHIRB 1901 012-02).

2. US Scanning of the Masseter Muscle

One researcher (HJP) with more than three years of orofacial US imaging experience assessed the masseter muscles of all participants using US (LOGIQ P9 R3 USG, GE, Milwaukee, Wisconsin 53201, USA) with a 50-mm-wide linear-array transducer (4-15 MHz; ML6-15-RS, GE). The US scan depth was set at 3.3 cm and the measurement frequency was fixed at 10 MHz when US images were being taken. The masseter muscles’ US pictures were all captured bilaterally.

Participants sat with their backs leaning against the dental chair and maintained an upright posture, and the headrest was adjusted so that the head was in a neutral position [4]. To examine the masseter muscl’s anterior-posterior border, subjects were instructed to clench their teeth. The participant’s face’s cheilion and otobasion inferius line served as the upper limit for US scanning (Fig. 1A) [13]. After a sufficient amount of gel was applied to the US transducer, it was placed perpendicular to the skin during US scanning to obtain cross-sectional images of the masseter muscles. Then, the US transducer was gently shifted upward from the inferior margin of the mandible to the superior boundary within the anterior-posterior border of the masseter muscle (Fig. 1B). The masseter muscle’s thickest section was used to create the image during US scanning.

To evaluate changes in thickness during masseter function, US images were taken of the participant’s masseter muscle at rest and during clenching. In this research, US scanning was performed using two methods to compare the difference in masseter muscle thickness determined when inducing MBF.

1) A conventional method

In the resting state, participants were told to keep their heads in a neutral position, lightly press their upper and lower lips, keep their teeth from making contact and relax other body parts [2]. Participants were instructed to bite their teeth as tightly as they could under their centric occlusion in the jaw-clenching condition.

2) A modified method using dental articulation paper

The method of measuring the thickness of the masseter muscle during resting and jaw clenching using articulation paper was done the day after the examination using the traditional method. We used dental articulation paper (The AccuFilm 80 µm; Parkell, Inc., Edgewood, NY, USA) cut into 15×15 mm in size and used in this investigation. Before the US evaluation, while the subject was sitting in the dental chair, the examiner placed dental articulation papers on the occlusal surfaces of the first molar teeth on both sides of the participants. The measurement procedure during rest and jaw clenching is the same as the traditional approach once the articulation paper has been placed.

The examiner repeated all of the US scannings three times to measure the examiner’s reliability.

3. Data Analysis of US Images

US images were sent to the picture archiving and communication system (PACS) (ZETTA PACS; Taeyoung Soft Co., Anyang, Korea). Muscle thickness was determined as the maximum distance between the external and internal fascia of the masseter on the image (Fig. 2) [4]. Digital ruler that the PACS provided was used to take the measurements. Three measurements were taken, and the average was used to conduct the study.

4. Statistical Analysis

All statistical analyses were conducted using IBM SPSS Statistics for Windows, Version 26.0 (IBM Co., Armonk, NY, USA). For all variables measured in this study, normality verification was conducted using the Shapiro-Wilk test. Intra-examiner reliability was measured using the intraclass correlation coefficient (ICC), with its 95% confidence interval. To compare the variations in masseter muscle thickness between the two measuring techniques, a separate t-test was performed. Differences between left and right measures and between the sexes were evaluated using independent t-tests.

All 19 subjects completed US scanning of the masseter muscle at rest and during clenching under the conventional method and the articulating paper method. Subjects’ ages ranged from 21 to 40 years, and the mean age was 23.5±0.71. The Shapiro-Wilk test was used to ensure the normality of all the variables examined in this study. The ICC for the intra-examiner agreement was 0.959 (p<0.05). This value showed that the examiner had excellent reproducibility and reliability of US scanning of the masseter muscle. The changes in masseter muscle thickness between the left and right during US scanning were compared using an independent t-test. It was verified that there was no statistically significant difference. As a result, we integrated the left and right measurements into one variable without dividing them. Thus, 38 masseters of 19 participants were the subjects of this study.

Table 1 depicts the average thickness of the masseter muscle according to the measurement technique. In the masseter muscle’s resting state, the masseter muscle’s thickness revealed no difference between the measurement methods. However, when the masseter muscle was clenched, the thickness of the muscle determined using the articulating paper was thicker than that determined using the conventional method, and this difference was statistically significant.

Table 2 illustrates the average measured values of the masseter muscle according to sex. The mean values of masseter muscle thickness evaluated by US during rest and clenching demonstrated statistically significant differences between the sexes.

This study aims to develop a reproducible and reliable method to evaluate the masseter’s functional state by measuring the masseter muscle with the US. In the meantime, sEMG has been regarded as a reliable source of quantitative data regarding the state of function of the masticatory muscles [9]. However, experimental evidence for an association between masticatory muscle pain and activity and sEMG results is lacking [14]. On the other hand, US will be a more useful tool than sEMG in the clinical field because it is simple to assess muscle structure and the functional ability of masseter muscle contraction [9]. According to a review study on masseter muscle thickness, US is an accurate clinical tool for determining the masseter muscle’s thickness, although standardizing procedures and standardization of measurement parameters are still required [12].

Studies that have assessed masseter muscle thickness with US have reported that the thickness of the masseter muscle is more reproducible when it contracts than when it relaxes [15,16]. The masseter muscle is more sensitive to the pressure created by the US transducer during relaxation, which accounts for the low reproducibility and reliability of US readings during this muscle’s relaxation [12]. Factors that can decrease the reliability of US measurement results include the pressure applied to the US transducer in contact with the evaluation site, the direction of transducer movement, and the absence of an anatomical reference point for the evaluation site [16]. In this research, the examiner’s reliability for masseter muscle measurement by US was very high (ICC=0.959). To reduce the measurement error mentioned above, we tried to reduce the transducer’s pressure during US scanning. Additionally, we attempted to maintain the shifting location of the US transducer by establishing a reference point in the test region (Fig. 1). These efforts could result in high intra-examiner reliability in the measurement of this study.

Comparing the measured average thickness of the masseter muscle revealed a difference in the occlusal force generated between conventional jaw clenching and clenching using articulation papers (Table 1). Although the subject of this study did not have TMD-related symptoms, they did not generate the MBF when jaw clenching was instructed using the conventional method. Factors caused by the subject, such as posture during the examination and occlusal force applied by the participant, may also hinder US reliability [12]. In particular, the measurement values related to MBF may have different results depending on how maximally participants clench their teeth. Furthermore, when measuring masticatory function in patients with pain-related TMD, pain may prevent adequate performance of the clenching related to MBF. The inability to perform MBF-related tasks due to pain and the decreased MBF due to muscle atrophy are entirely different issues. These differences may affect the establishment of a successful treatment strategy for TMD. The articulation paper used in this study was 80 micrometers thick. It was assumed that it could induce MBF effectively during jaw clenching and would not induce activation of the jaw-opening muscles when resting the jaw. In this study, the results demonstrating no difference in masseter muscle thickness at rest and differences in masseter muscle thickness during jaw clenching reveal that our assumption was reasonable. As a result, when evaluating masticatory muscle function using US, our technique using the articulation paper would help determine the thickness of the masticatory muscle related to MBF.

In this study, there was a difference in masseter muscle thickness between the sexes when resting and clenching their jaws (Table 2). The association between masseter muscle thickness and sex is questionable, considering existing published studies [4,9,11,17-19]. It may be due to variations in the number of subjects, the age group covered, and the US measurement method.

These conflicting results may be due to differences in the number of subjects in the study groups, age ranges covered, and US measurement methods. As a result, along with the standardization of US measurement, research should be carried out targeting a large number of subjects of various ages. Masseter muscle thickness can also be affected by weight, height, and skeletal characteristics [4,11]. In this study, we did not examine the variables mentioned above. Follow-up studies that complement these limitations should be performed.

In conclusion, employing the US for masseter muscle examination can provide objective and functional information on the masseter muscle. A standardized US scanning method requires to be developed to realize reproducible and reliable information of the masseter muscle at rest and during clenching. In particular, when using the US for diagnosing and treating TMD, ensure that the MBF is produced when the participant clenches and US images are taken. Generating MBF using an articulation paper can be a reproducible and reliable method for determining the functional status of the masseter muscle.

No potential conflict of interest relevant to this article was reported.

Conceptualization: JWR. Data curation: YSS. Formal analysis: SKY. Funding acquisition: JWR. Methodology: HJP, SKY, JWR. Project administration: JWR. Visualization: HJP, JWR. Writing original draft: JWR, HJP. Writing review & editing: YSS, JWR.

We express our special thanks to Gi Hyeon Moon for providing the face and ultrasonographic images used in this paper.