Introduction

Occlusal force and masticatory performance deteriorate mainly due to tooth loss [1], while reductions in tongue pressure are primarily associated with aging [2]. The impact of a decline in oral function extends beyond the oral cavity itself and can include serious health problems such as aspiration pneumonia, frailty, and malnutrition [3, 4]. A number of recent studies have reported on the relationship between oral function and systemic condition. Indeed, a previous study defined oral frailty as a condition with poor oral health, comprising tooth loss, and declines in occlusal force, tongue pressure, and masticatory ability [5]. Subjects with oral frailty were found to be more than twice as likely to have disability or mortality after 4 years, compared to subjects without oral frailty.

In addition to aging and tooth loss, the attenuation of perioral muscle characteristics is also considered to be a cause of oral function deterioration. Tongue thickness is positively associated with masticatory performance in healthy young dentate subjects [6], and with tongue pressure in healthy elders [7] and patients with neuromuscular disease [8]. The cross-sectional area of the geniohyoid muscle is also associated with masticatory performance [9] and jaw opening force [10] in community-dwelling subjects.

Ultrasonic apparatus can be also used to evaluate muscles, and has been shown to be a valid and reliable measure of both muscle quantity and quality [11]. Muscle quality is assessed by echo intensity; the whiter the muscle in the image, the more non-contractile tissue in the muscle and the lower the quality (Fig. 1) [12]. In limb muscles, the relationship between muscle characteristics (e.g., muscle quantity, muscle quality), muscle strength, and physical function has already been demonstrated [13]. A previous report has shown that sarcopenic patients have attenuated muscle quantity and quality [14]. Ultrasonic evaluation has also been demonstrated to be a practically convenient tool for perioral muscles (e.g., the masseter muscle), as there is no need for the subject to remove their clothes or change their posture during the assessment.

Fig. 1
figure 1

Evaluation of the masseter muscle using an ultrasonic diagnostic apparatus. a Probe position for masseter muscle measurement. b Measurement of masseter muscle thickness (double headed arrow) and masseter muscle echo intensity (dashed line). c Lower quality masseter muscle

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Perioral muscle characteristics vary with aging and tooth loss. In a study investigating the relationship between aging and quality of the suprahyoid muscle, as assessed by computed tomography, a deterioration due to increased intramuscular fat and atrophy was observed in an elderly group, compared to healthy adults [15]. Tooth loss has also been shown to affect MMT. Bhoyar et al. [16] documented a significantly thinner MMT among edentulous subjects aged 45–55 years, compared to healthy dentate adults. Nevertheless, no studies have compared masseter muscle characteristics (quantity and quality) among both adults and elders, and it remains unclear as to whether aging and tooth loss are independent factors associated with MMT and MMEI.

Therefore, the purpose of this study was to compare masseter muscle characteristics by sex, among both adults and elders, and to investigate whether aging and tooth loss are independent predictors of these characteristics. The null hypothesis was that masseter muscle characteristics deteriorate with aging and tooth loss regardless of sex.

Methods

Participants

There were 112 participants, comprising 49 adults (24 males and 25 females in their 20s to 40s) and 63 elders (24 males and 39 females aged 65 years and over). Recruitment was conducted between November 2017 and May 2019. All adults were recruited from the Tokyo Medical and Dental University. Elderly participants were either recruited from patients of the Gerodontics clinic of the Dental Hospital at Tokyo Medical and Dental University who visited for the purpose of dental treatment or from participants of a health survey conducted in Koiwa city (Tokyo, Japan). Inclusion criteria comprised individuals who were independent in activities of daily living and were able to follow verbal commands. Exclusion criteria included participants with (1) diseases (e.g., cerebrovascular disorders with paralysis, neuromuscular diseases) that could affect muscle function; (2) a lack of molar occlusal support, whether from natural teeth or dentures; or (3) significant symptoms of temporomandibular disorder. A total of 124 participants were initially recruited, with two participants being excluded owing to lack of occlusal support in the molar region upon examination. An additional participant was excluded because she had temporomandibular symptoms (severe pain during jaw opening), and nine participants were excluded due to missing data. The final analyses were based on the data obtained from 112 participants. All the adults were classified as Eichner A. None of the participants in this study were undergoing orthodontic treatment and none had severe malocclusion. This study was approved by the Tokyo Medical and Dental University Ethics Committee (ref. D2014-047). The study protocol was explained to all participants, both verbally and in writing, and written informed consent was obtained.

Tooth loss evaluation

A dentist and two dental hygienists categorized tooth loss using the Eichner classification [17]. Both the dentist and the dental hygienists have more than 8 years of experience.

Ultrasound measurements of the masseter muscle

The masseter muscle was evaluated with an ultrasonic diagnostic apparatus (SonoSite M-turbo; Fujifilm, Tokyo, Japan), by two dentists (both were experienced in the assessment method). We have previously reported high intraclass correlation coefficients and high inter-rater reliability for ultrasonic measurements of the masseter muscle [6]. During the measurements, participants were instructed to relax while looking straight ahead in a sitting position. They were also instructed not to bite together.

A linear probe with a 6–13 MHz broadband frequency was used for masseter muscle measurement, with gain, depth, and frequency of echo being kept constant at appropriate levels. As based on a previous study, the probe was positioned approximately halfway between the zygomatic arch and the mandibular margin, parallel to the mandibular margin and perpendicular to the skin (Fig. 1) [18]. At the time of measurement, the probe was covered with a water-soluble transmission gel.

MMT and MMEI were measured using ImageJ (version 1.49, National Institutes of Health, Bethesda, MD, USA). The average of the two measurements was taken as the measurement value. MMT was measured from the surface layer of the masseter muscle to the mandibular ramus, with the thickest region (without fascia and bone) being recorded (Fig. 1) [18].

At the time of echo intensity measurement, the range of interest (ROI) included the maximum muscle without fascia and bone (Fig. 1) [19]. The intraclass correlation coefficient (ICC) was calculated to evaluate intra- and inter-examiner reliability for ultrasonic measurement of the masseter muscle in this study. The measurements were performed on eight volunteers. The intra-rater reliability was evaluated by calculating the ICC from two measurements performed by one examiner on different days. For the intra-rater reliability, the ICC of MMT was 0.97, and the ICC of MMEI was 0.83. The inter-rater reliability was evaluated by calculating the ICC from measurements by two examiners. For the inter-rater reliability, the ICC of MMT was 0.93 and that of MMEI was 0.84. All ICC values exceeded 0.8, indicating high reliability in ultrasonic measurements of the masseter muscle.

Statistical analysis

We calculated the sample size using G*Power3.1 software (Kiel University, Kiel, Germany). The required sample size was calculated to be 36 participants, based on an α of 0.05, power of 0.8, and an effect size of 0.35 reported in a previous study [19].

The participants were classified as either adults or elders. The elderly group was further divided into three groups (Eichner A, B, or C) based on the degree of tooth loss. Thus, there were a total of four groups for both males and females (adults, elders with Eichner A, elders with Eichner B, and elders with Eichner C). The normality of each outcome measure was confirmed by the Shapiro–Wilk test. Comparisons for each outcome measure were made with the one-way analysis of variance and Kruskal–Wallis test. To determine which groups had significant differences in MMT and MMEI, a t test with Bonferroni correction was performed. A multiple regression analysis was carried out for each sex to examine the relationship between masseter muscle characteristics (MMT, MMEI) and potential explanatory variables (aging, weight, tooth loss). Both aging (adult group or elder group) and tooth loss (Eichner A, B, or C) were entered into the regression model as categorical variables. To avoid multicollinearity, the correlation between each dependent variable and explanatory variable was confirmed by Pearson’s correlation coefficient and Spearman’s rank correlation coefficient. The level of statistical significance was set at p < 0.05. For all statistical analyses, the Japanese version of SPSS for Windows (version 25J, IBM Inc., Tokyo, Japan) was used.

Results

Table 1 shows the background characteristics of the participants. Among males, there were 24 participants in the adult group, 6 participants in the elder group with Eichner A, 13 participants in the elder group with Eichner B, and 5 participants in the elder group with Eichner C. Among females, there were 25 participants in the adult group, 11 participants in the elder group with Eichner A, 18 participants in the elder group with Eichner B, and 10 participants in the elder group with Eichner C. All participants in the adult group were categorized as having an Eichner A classification. Age, weight, MMT, and MMEI were significantly different between the four groups among males. Similar results were observed among females, with the exception that the weight was not significantly different.

Table 1 Participant characteristics
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Figure 2 shows the results of comparisons of MMT and MMEI for each group among males and females. Among males, the MMT was significantly different between the adult group and the elder group with Eichner B, and the MMEI was significantly different between the adult group and the elder groups with Eichner A and Eichner B. Among females, the MMT was significantly different between the adult group and the elder group with Eichner C, and the MMEI was significantly different between the adult group and all elder groups. Table 2 shows the correlation between aging (adult group or elder group), weight, tooth loss, MMT, and MMEI for each sex. The highest correlation was for aging and tooth loss (r = 0.76, p < 0.01) in males. No correlations exhibited a r > 0.8. MMT and MMEI were highly correlated in both males (r = − 0.7, p < 0.01) and females (r = − 0.58, p < 0.01).

Fig. 2
figure 2

Masseter muscle thickness and masseter muscle echo intensity in each group. a Masseter muscle thickness in each group of males. b Masseter muscle echo intensity in each group of males. c Masseter muscle thickness in each group of females. d Masseter muscle echo intensity in each group of females. *p < 0.05, **p < 0.01. MMT, masseter muscle thickness; MMEI, masseter muscle echo intensity

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Table 2 Correlation matrix table of parameters for all study participants
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Table 3 shows the results of multiple regression analyses by sex. In males, aging was the only significant explanatory variable for MMT. When MMEI was the dependent variable, aging and weight were significant explanatory variables. In females, tooth loss and weight were significant explanatory variables for MMT. When MMEI was the dependent variable, aging and weight were significant explanatory variables. All explanatory variables had a VIF < 2.5. In an analysis of MMEI among both males and females, multiple correlation coefficients (R) exceeded 0.7, indicating a high degree of association.

Table 3 Multiple regression analysis values with MMT and MMEI as the dependent variables
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Discussion

In this study, MMT and MMEI in males were related to aging, while in females, MMT was related to tooth loss, and MMEI was related to aging (Table 3). Thus, these results suggested that the attenuation of masseter muscle quantity and quality differs based on sex. These results may be specific to the perioral muscles (e.g., the masseter muscle), as it has been previously demonstrated that both quantity and quality of limb muscles decrease with aging.

MMT was found to be associated with aging in males and tooth loss in females (Table 3). In terms of the relationship between MMT and aging, a previous study compared MMT among the following five age groups: group I (7–12 years old), group II (13–20 years old), group III (21–40 years old), group IV (41–60 years old), and group V (61–80 years old) [20]. Among males, MMT gradually increased from group I to group IV, but it was thinner in group V compared to group IV. In females, the average value of MMT in group III was lower than that in group IV, and the differences between these two age groups was not as profound as that observed in males. Notably, this study was limited to fully dentate participants, and no adjustments were made for systemic variables such as weight. Nevertheless, these results provide support for the present study, which found an association of MMT with aging for males, but not for females.

This study showed an association between MMT and tooth loss in women (Table 3). A previous study reported that the MMT of edentulous subjects was significantly thinner when compared to dentate subjects [16]. In addition, when edentulous subjects wore complete dentures, a significant increase in MMT was observed 3 months after denture insertion. While recovery of MMT may be achieved with prosthodontic treatments such as dentures, the maintenance of occlusal support with natural teeth is the most effective way of maintaining MMT.

Gender-based differences in MMT deterioration may possibly be due to changes in the endocrine system with aging. Muscle atrophy may be attributed to various factors such as aging and disuse, as well as hormones. Testosterone is an essential hormone for muscle repair and strengthening, but it decreases with aging [21]. Indeed, masseter muscle mass has been shown to be very sensitive to testosterone. In a study using rats, it was reported that masseter muscle mass was reduced by castration and increased by 38% with testosterone injection [22]. Notably, this increase was greater than that observed in limb muscles. Testosterone secretion is overwhelmingly higher in men than women [23, 24]. Thus, based on the above, the decrease in testosterone with aging may have a potentially significant effect on masseter muscle mass, especially in men.

MMEI was strongly related to aging, regardless of tooth loss in both males and females (Table 1, Table 3, Fig. 2). Muscle echo intensity represents non-contractible tissue in muscle such as intramuscular fat and fibrous tissue [12]. The interaction between muscle satellite cells and mesenchymal progenitor cells has been demonstrated as the mechanism of fat formation in muscles. Skeletal muscle has a very high regenerative ability, due to muscle satellite cells [25]. Muscle satellite cells create muscle fibers that prevent mesenchymal progenitor cells from differentiating into fat [26]. Mesenchymal progenitor cells promote muscle differentiation of muscle satellite cells [27], and this interaction is important in maintaining homeostasis of muscle tissue. Therefore, if muscle satellite cells decrease or muscle atrophy progresses, the inhibitory signal for adipose differentiation of mesenchymal progenitor cells is weakened and fat differentiation occurs. There are two types of muscle fibers (type 1 and type 2), with type 2 atrophy and a decrease in the number of type 2 muscle satellite cells being particularly associated with aging [28]. As type 2 fibers make up 40% of all muscle fibers in the masseter muscle [29], it was not unexpected that an increase in echo intensity was observed with aging, regardless of sex.

Observation with an ultrasound diagnostic apparatus is simple and non-invasive and can be used by paramedical stuff, such as nurses and therapists. Understanding masseter muscle characteristics using an ultrasonic diagnostic apparatus is useful because masseter muscle characteristics can be a predictor of systemic condition as well as oral condition. MMT has been previously related not only to occlusal force [30], but also to skeletal muscle mass [31], and grip strength [32]. Further, a 2-year observational study conducted in trauma patients over 65 years of age reported that masseter muscle mass could be an excellent indicator of sarcopenia [33]. A cross-sectional study of healthy elders found that masseter muscle echo intensity (MMEI) was independently associated with MMT, grip strength, and walking speed and suggested that MMEI may be a predictor of dynapenia [19]. Performing similar examinations not only for the masseter muscle but also for other peroral muscles such as the tongue could lead to an interdisciplinary approach to oral medicine.

Some limitations are acknowledged in this study. Firstly, since this was a cross-sectional study, longitudinal studies are required to determine cause-and-effect relationships between aging and changes in masseter muscle characteristics. Secondly, this study was limited in terms of the sample size employed, especially for males among Eichner groups A and C. Additional studies with larger samples sizes are required to confirm the results of this study.

Nevertheless, this was the first study to examine the relationships between MMT, MMEI, aging, and tooth loss among both males and females. In males, aging was associated with both MMT and MMEI, while tooth loss was not. This suggested that masseter muscle characteristics in males were more susceptible to the effects of aging than in females. Maintenance of masseter muscle characteristics in males may require not only the preservation of the natural dentition, but also resistance exercises to increase the amount of available testosterone [34]. However, in females, MMT was related to tooth loss, and MMEI was related to aging. Thus, good muscle quantity may equate to good muscle quality in the masseter muscle. Preservation of the natural molars or replacement of missing molars with prostheses is important for maintenance of good masseter muscle characteristics.