Abstract
Introduction: The aim of this study was to determine the associations of time spent away from bed with whole-body muscle mass and swallowing function in older adults with low activities of daily living (ADL). Methods: This cross-sectional study was conducted at Tokyo Medical and Dental University and included adults over 65 years of age who underwent a medical intervention at their residence. Data regarding age, sex, body height and weight, activity status, medical history, time spent away from bed, and Functional Oral Intake Scale (FOIS) were collected. We calculated the body mass index, Charlson Comorbidity Index, whole-body muscle mass, the appendicular skeletal muscle mass index (ASMI), and the trunk muscle mass index (TMI). According to the time spent away from bed, the subjects were grouped as follows: <4 but ≥0 h (S), <6 but ≥4 h (M), and ≥6 h (L). These variables were analyzed using the one-way analysis of variance, the Kruskal-Wallis test, and the χ2 test, then differences among the three groups were examined. To adjust for confounding factors, we performed multiple regression analysis with ASMI and TMI as the dependent variables and ordinal logistic regression analysis with FOIS as the objective variable. Results: Ninety subjects (male: n = 42; female: n = 48; mean age = 82.9 ± 8.8 years, and groups S: n = 23; M: n = 30; L: n = 37) were analyzed. Group L had a significantly higher ASMI, TMI, and FOIS score than groups S and M, while group M had a significantly higher ASMI and FOIS score than group S. After adjusting for confounding factors, the significant explanatory factors for ASMI were sex, activity status, time spent away from bed, and TMI. The factors for TMI were activity status, time spent away from bed, ASMI, and FOIS. The factors for FOIS were time spent away from bed and TMI. Conclusion: Spending 4 or more hours away from the bed is related to appendicular skeletal muscle mass and FOIS, while spending 6 or more hours is related to appendicular skeletal muscle mass, trunk muscle mass, and FOIS in this population. These findings highlight factors that can prevent a decline in swallowing function in the daily life of older adults with low ADL who have difficulty performing exercises to preserve swallowing function.
Introduction
The relationship between time spent away from bed, muscle mass, and swallowing function in older adults remains unclear. Swallowing function in older adults aged ≥65 years is related to many factors such as tongue pressure and grip strength [1, 2]. Moreover, good posture during eating could be an important factor for good swallowing function [3, 4]. The necessary muscles for maintaining good posture are the appendicular skeletal muscles and trunk muscles, such as the back and neck muscles. We validated evidence of their relationship and reported that swallowing function is affected by trunk muscle mass and strength, such as the back muscles and neck circumference [5‒7].
For healthy older individuals, performing exercises to preserve swallowing function and prevent the decline of muscles related to swallowing function, as well as muscles in the trunk and neck, contributes to the prevention and improvement of dysphagia [8]. However, older individuals with low activities of daily living (ADL) do not perform any exercise to preserve swallowing function as per the recommended protocol because they cannot perform it due to a decline in their physical capacity. Therefore, it is advisable that older people who have difficulty performing exercises to preserve swallowing function find ways to prevent a decline in swallowing function. Thus far, most studies on dysphagia have analyzed factors related to swallowing function or the verification of training effects on swallowing muscles; only a few studies have focused on the daily life of older individuals with low ADL.
Inactivity due to long-term bed rest causes muscle atrophy due to disuse and loss of strength of the appendicular skeletal and trunk muscles [9, 10]. Antigravity muscles such as the transversus abdominis muscles are affected more by sarcopenia due to aging than by disuse due to inactivity [10]. When stroke patients spend long hours away from bed, their ADL has a good recovery rate because of the increase in their physical activity [11]. To prevent muscle disuse, spending more than 4 h in a seated position or performing activities for more than 2 h each day is recommended [12].
Our previous study reported the relationship between the time spent away from bed and swallowing function in older adults with low ADL [13]. However, it remains unclear whether the amount of time spent away from bed is related to the mass of appendicular skeletal muscles and trunk muscles in older adults with low ADL, and we did not have specific criteria for how much time is required to maintain skeletal muscle mass or swallowing function.
Considering the above background information, we hypothesized that swallowing function and skeletal muscle mass are affected by the amount of time spent away from bed in older adults with low ADL. This study aimed to identify factors related to the preservation of swallowing function and skeletal muscle mass in the daily life of older adults with low ADL and difficulty performing exercises.
Materials and Methods
Participants
This cross-sectional study was conducted at Tokyo Medical and Dental University. It included older adults aged over 65 years who received medical care at their residence (i.e., at their house or nursing home) from members of Tokyo Medical and Dental University between September 2018 and March 2020. The inclusion criteria were as follows: (1) those who aged ≥65 years; (2) those who used nursing services because they could not stand up and walk alone or were almost bedridden; and (3) those who agreed to participate. The exclusion criteria were as follows: (1) those who had a regular diet and had no problem with swallowing function, and (2) those whose bioelectrical impedance analysis (BIA) could not be measured because of their use of a heart pacer. We did not exclude subjects based on nutritional intake method (oral/tube) or primary disease. All participants lived in standard middle-class neighborhoods and business districts of Japan. Participants include those who cannot perform cognitive function tests due to low ADL. However, all of them have enough cognitive function to recognize food through visual, olfactory, or oral sensory inputs. In addition, all of them had low ADLs, and therefore, their scores on the Lawton instrumental ADL scale was 0 [14].
Some participants were the same as those reported in past studies conducted by the authors. However, these reports did not examine the specific time spent away from bed that is likely to maintain muscle mass and swallowing function [13].
This study was approved by the Dental Research Ethics Committee of Tokyo Medical and Dental University (No. D2018-015). We explained the purpose of this study to the subjects or their delegates and obtained consent before their participation.
We collected data on the age, sex, body height and weight, activity status, medical history, number of medication types, and time spent away from the bed. We calculated body mass index (BMI) from their height and weight and obtained the Charlson Comorbidity Index (CCI) from their medical history [15]. Activity status was categorized as follows: (1) those who could not stand up and walk without assistance, (2) those who had extreme difficulty standing up and walking, even with assistance, and (3) those who were almost bedridden [13]. It was set by using the guideline to determine the appropriate level of long-term home care in Japan as a reference.
The mass of the appendicular skeletal and trunk muscles was measured by BIA using InBodyS10 (InBody Japan, Tokyo, Japan). We then calculated the appendicular skeletal muscle mass index (ASMI) and the trunk muscle mass index (TMI) from the measured values [5]. ASMI was calculated from the total muscle mass of the upper and lower limbs (appendicular skeletal muscle mass) corrected by squared height. Similarly, trunk muscle mass was corrected by height squared, they defined it as TMI.
We administered a questionnaire on time spent away from bed. To prevent systemic disuse among older adults, it is necessary to ensure that individuals spend time in a seated position for more than 4 h per day or perform activity for more than 2 h per day [12]. Therefore, according to the time spent away from bed, we divided the subjects into three groups: <4 but ≥0 h (short, [S]); <6 but ≥4 h (middle, [M]), and 6 h or more (long, [L]) [13]. Since the time spent away from bed differed from day to day due to differences in service interventions, we presented options with the time ranges as previously mentioned. The most frequent option in a week was recorded. Regarding how participants spent their time away from bed, we asked a family member/caregiver to fill out a form with a free description of the topic because many subjects could not answer the questionnaire by themselves due to low ADL. This study was conducted based on how time away from bed was part of daily life, including whether or not they were performed functional ADL away from the bed. Thus, we did not subdivide how they spend their time. In general, physical function and swallowing function are better preserved with physical therapy. We recorded the intervention of a physical therapist.
Swallowing function was evaluated using the Functional Oral Intake Scale (FOIS), which comprises seven levels (1 = no oral intake, 2 = tube-dependent with minimal/inconsistent oral intake, 3 = tube supplements with consistent oral intake, 4 = total oral intake of a single consistency, 5 = total oral intake of multiple consistencies requiring special preparation, 6 = total oral intake with no special preparation but must avoid specific foods or liquid items, and 7 = total oral intake with no restrictions) [16].
Statistical Analysis
Continuous variables such as age, BMI, CCI, number of medication types, ASMI, TMI, and FOIS were checked for normal distribution by the Shapiro-Wilk test and analyzed using the one-way analysis of variance and the Kruskal-Wallis test. We examined whether there were differences between the three groups. Categorical variables such as sex and activity status were analyzed using the χ2 test. For the post hoc test, the Games-Howell test and Steel-Dwass test were used. To adjust for confounding factors, we performed multiple regression analysis with ASMI as the dependent variable and age, sex, activity status, CCI, time spent away from bed, TMI, and FOIS as explanatory variables. In the same way, we performed multiple regression analysis with TMI as the objective variable. Before we conducted the multiple regression analysis, we transformed nonuniform inter-spatial rank data into dummy variables, e.g., time spent away from bed. FOISs adjacent levels with a few relevant participants were integrated for analysis and divided into two groups: below level 5 and above level 5 [17]. We performed ordinal logistic regression analysis with FOIS as the objective variable and age, sex, activity status, CCI, time spent away from bed, ASMI, and TMI as explanatory variables. Before conducting the ordinal logistic regression analysis, we integrated adjacent levels with a small number of relevant participants in nonuniform inter-spatial rank data, e.g., FOIS. According to previous studies, the FOIS was divided into five levels: 1–3, 4, 5, 6, and 7 [13]. The significance level was set at p < 0.05. IBM SPSS Statistics for Windows, version 25 (IBM Japan, Tokyo, Japan), was used for all statistical analyses.
Results
Comparison of Subject Characteristics among the Three Groups
We excluded subjects with imprecise data measurements using InBodyS10 due to body movement. Finally, 90 subjects (42 men [46.7%] and 48 women [53.3%]) were included in the analysis. The mean age was 82.9 ± 8.8 years. Subjects were divided into three groups according to the amount of time spent away from bed, as follows: group S (n = 23; 25.6%), group M (n = 30; 33.3%), and group L (n = 37; 41.1%). We checked if the continuous variable was normally distributed. Age, BMI, number of medication types, ASMI, and TMI were normally distributed. FOIS and CCI were not normally distributed. The subjects’ characteristics are shown in Table 1. As for how they spent time away from bed, other than ADL, such as eating, they sat and watched television, enjoyed the interaction with their grandchildren, and went out to their favorite places in wheelchairs. Most of them had interventions by physical therapists (83.3%). Those without physical therapist intervention had the opportunity to move their bodies passively with the help of family members or caregivers.
There were significant differences in the sex, activity status, CCI, ASMI, TMI, and FOIS (p < 0.05, Table 1). More specifically, the more time they spent away from bed, the higher the ASMI, TMI, and FOIS scores were. There were no significant differences in age, BMI, or the number of medication types between groups.
We performed a post hoc test for items with a significant difference. Group L had a significantly higher ASMI and TMI than groups S and M, and group M had a significantly higher ASMI than group S (Fig. 1). Group L had a significantly higher FOIS score than groups S and M, and group M had a significantly higher FOIS score than group S (Fig. 2). Those with the FOIS score of 7 ate regular food but drank thickened water. In other words, they were patients with mild dysphagia. In CCI, no significant difference was observed between groups S and M, groups S and L, and groups M and L (p = 0.97, p = 0.55, and p = 0.35).
Comparison of the body muscle mass. The left graph shows the results of the analysis concerning any significant differences in the ASMI between the three groups: <4 but ≥0 h (short [S]), <6 but ≥4 h (middle [M]), and ≥6 h (long [L]). The right graph shows the results of the analysis concerning any significant differences in the TMI between the three groups. The data were analyzed using the one-way analysis of variance and a post hoc test using the Games-Howell test. These graphs show the median value and interquartile range. *p< 0.05, **p< 0.01.
Comparison of the body muscle mass. The left graph shows the results of the analysis concerning any significant differences in the ASMI between the three groups: <4 but ≥0 h (short [S]), <6 but ≥4 h (middle [M]), and ≥6 h (long [L]). The right graph shows the results of the analysis concerning any significant differences in the TMI between the three groups. The data were analyzed using the one-way analysis of variance and a post hoc test using the Games-Howell test. These graphs show the median value and interquartile range. *p< 0.05, **p< 0.01.
Comparison of FOIS scores. The graph shows the results of the analysis concerning any significant differences in the FOIS scores between the three groups: <4 but ≥0 h (short [S]), <6 but ≥4 h (middle [M]), and ≥6 h (long [L]). Data were analyzed using the Kruskal-Wallis test and a post hoc test using the Steel-Dwass test. The graph shows the median value and interquartile range. *p< 0.05, **p< 0.01.
Comparison of FOIS scores. The graph shows the results of the analysis concerning any significant differences in the FOIS scores between the three groups: <4 but ≥0 h (short [S]), <6 but ≥4 h (middle [M]), and ≥6 h (long [L]). Data were analyzed using the Kruskal-Wallis test and a post hoc test using the Steel-Dwass test. The graph shows the median value and interquartile range. *p< 0.05, **p< 0.01.
Factors Affecting Muscle Mass
The significant explanatory factors for ASMI were sex (B = 0.52, 95% CI for B = 0.19 to 0.84, p = 0.002), activity status (B = −0.34, 95% CI for B = −0.50 to −0.18, p < 0.001), time spent away from bed (B = 0.83, 95% CI for B = 0.42 to 1.23, p < 0.001 for trend), and TMI (B = 0.83, 95% CI for B = 0.68 to 0.99, p < 0.001). ASMI was not significantly associated with age, CCI, or FOIS (Table 2). We could read standard partial regression coefficient in Table 2 as follows: ASMI was most strongly associated with TMI, followed by time spent away from bed and sex.
The significant explanatory factors for TMI were activity status (B = −0.29, 95% CI for B = −0.44 to −0.13, p < 0.001), time spent away from bed (B = 0.46, 95% CI for B = 0.06 to 0.86, p = 0.024 for trend), ASMI (B = 0.71, 95% CI for B = 0.58 to 0.84, p < 0.001), and FOIS (B = 0.30, 95% CI for B = 0.01 to 0.60, p = 0.046). TMI was not significantly associated with age, sex, or CCI (Table 3). We could read standard partial regression coefficient in Table 3 as follows: TMI was most strongly associated with ASMI, followed by time spent away from bed, and FOIS.
Factors Affecting FOIS
The significant explanatory factors for FOIS were time spent away from bed (B = 3.19, 95% CI for B = 1.71 to 4.67, p < 0.001 for trend), and TMI (B = 0.82, 95% CI for B = 0.03 to 1.62, p = 0.041). FOIS was not significantly associated with age, sex, activity status, CCI, or ASMI (Table 4). The partial regression coefficients (B) in Table 4 shows a shift in the time spent away from bed from 0–4 h to 4–6 h results in an increased FOIS score of 1.58, and a shift from 0–4 h to ≥6 h results in increased FOIS score of 3.19. One unit increase in TMI results in an increased FOIS score of 0.82. The longer the time spent away from bed and the more trunk muscle mass, the higher the FOIS score.
Discussion
Time Spent Away from Bed in Older Adults with Low ADL
This study showed that compared with older adults who spent less than 4 h away from bed, those who spent more than 4 h away from bed showed greater appendicular skeletal muscle mass and swallowing function. Moreover, more than 6 h away from bed was associated with much trunk muscle mass and good swallowing function.
After adjusting for confounding factors, the better the activity status and the longer the time spent away from bed, the greater the appendicular skeletal muscle mass and trunk muscle mass. There was a sex difference in appendicular skeletal muscle mass and a difference in trunk muscle mass between those below level 5 of FOIS and those above level 5. In this study, age was not found to be related to muscle mass as expected. Factors related to swallowing function were trunk muscle mass and time spent away from bed.
For older adults with low ADL, we consider that spending more than 4 h away from bed may help maintain whole-body muscle mass related to swallowing function. If individuals can spend more time away from bed, more than 6 h is ideal for swallowing function.
Maintaining Muscle Mass and Types of Muscle Fibers
The different types of muscle fibers (type 1 and type 2 fibers) are important in considering total body muscle mass changes in older adults. Type 1 fibers undergo atrophy mainly due to disuse, while type 2 fibers undergo atrophy mainly due to aging [18, 19]. The major appendicular skeletal muscles have a higher percentage of type 2 fibers than the trunk muscles (62% in biceps brachii muscle and 24% in erector spinae muscle) [20]. In this study, there was no significant association between appendicular skeletal muscle mass and age, which may be because the participants were older adults, and all of them were well after the effects of aging. In a previous study examining muscle biopsy samples of thighs from healthy individuals in the 20s–70s, women had a higher percentage of type 2 fibers.
Furthermore, there is a clear sex difference in the rate of decrease in muscle fibers with age (men’s type 1: 15%, type 2: 19%, women’s type 1: 25%, type 2: 45%) [21]. Our study’s sex difference in appendicular skeletal muscle mass may reflect this difference in the percentage and rate of muscle fibers decrease. Trunk muscles are mostly antigravity muscles for postural maintenance, and antigravity muscles have a high percentage of type 1 fibers with high endurance [20]. The appendicular skeletal muscles also contain antigravity muscles, e.g., soleus muscle, which supports postural changes during ADL. It is likely that time spent away from bed, and the activities with assistance help resist gravity and prevent the atrophy of type 1 fibers in the antigravity muscles. Therefore, the better the activity status and the longer the time spent away from bed, the greater the muscle mass.
Muscle Mass, Time Spent Away from Bed, and ADL
Individuals who spend 4 h away from bed are more likely to have independent eating and voiding behaviors [22]. Eating involves using utensils, wherein eating requires control of an individual’s upper limbs, and using utensils requires support from the lower limbs [23]. Individuals must be able to transport themselves to a toilet seat using the lower limbs, and coordination of both the upper and lower limbs is important for voiding not using a catheter [24]. In this study, individuals whose time spent away from bed was more than 4 h (groups M and L) had a higher appendicular skeletal muscle mass than those whose time spent away from bed was less than 4 h (group S).
Moreover, those who spent away from bed was long were more likely to have independent dressing and bathing behaviors [25]. Dressing and bathing behaviors require trunk flexion and posture control while raising the upper arms [26, 27]. For the transfer to a bathtub, standing on one leg is needed in some cases, and this ability is related to the trunk muscles and hip abductor muscles [28, 29]. Therefore, dressing and bathing behaviors require both appendicular skeletal muscle mass and trunk muscle mass; muscles are used when they perform those movements with assistance. In this study, individuals whose time spent away from bed was more than 6 h (group L) had higher appendicular skeletal muscle mass and trunk muscle mass than those whose time spent away from bed was less than 6 h (groups S and M). Thus, there is a connection between time spent away from bed and ADL and between the time and body muscle mass.
The cooperation of a caregiver is essential for their daily living activities. According to the Japanese government’s previous survey, there is a yearly increase in the percentage of households with an older adult in need of care, and with the ratio of those whose caregiver was their spouse who was over 65 years of age was 51.2%. Compared with male caregivers, female caregivers assist with more ADL and spend more time in care [30]. Therefore, the spouses of female caregivers, in other words, men, may have had more opportunities to spend time away from bed in this study.
Time Spent Away from Bed and Muscle Mass
After adjusting for the effect of ADL, the time spent away from bed and the muscle mass were significantly related. One study compared the muscle weight and muscle fiber type composition ratio of soleus muscle in mice model of disuse muscle atrophy by excluding gravity loading of the hind limbs and varying the frequency of loading with body weight. The muscle weight and the percentage of type 1 fibers were maintained in the loaded group, even though no special exercise load was applied. Load frequency was more effective twice daily than once and daily than every other day [31, 32]. When the average muscle fiber diameter of soleus muscle in the same kind of model mice was compared between the groups loaded with 50% and 100% of body weight, the muscle fiber diameter was larger in both groups than the group without weight. However, the percentage of muscle fibers that underwent necrosis was higher in the 100% weight group. In other words, the disuse atrophic muscles had a low tolerance to exercise load [33]. This mouse model approximates the prolonged bed rest on humans since hindlimb muscle activity and substance metabolism are not restricted except for body weight loading [34]. Previous studies examine the soleus muscle as a typical antigravity muscle because mice are quadrupedal. The atrophy was observed in units of muscle fibers. The same phenomenon may have occurred in antigravity muscles of the human limb and trunk except for the soleus muscle. Regarding the frequency and strength of gravity load, applying load multiple times a day, every day from previous studies is ideal. Therefore, although rehabilitation once a week is important, spending time away from bed every day for daily activities may have led to the maintenance of muscle mass. In addition, appendicular skeletal muscle mass and trunk muscle mass were related to each other. When applying exercise load, it is necessary to apply load to each of the limbs and trunk. When spending time away from bed, it is possible to apply gravity load to the antigravity muscles of the limbs and trunk at once. Therefore, it is likely that many of them maintained both appendicular muscle mass and trunk muscle mass.
Trunk Muscle Mass and Swallowing Function
We examined factors related to the preservation of swallowing function in the daily life of older adults with low ADL. Our study shows that the factor influencing swallowing function is not the appendicular skeletal muscle but the trunk muscle. In addition, those eating meals that required chewing corresponding to FOIS 5–7 had greater trunk muscle mass. Chewing movement, which is important for bolus formation, requires the action of chewing muscles, neck muscles, and upper trunk muscles [35]. Tongue pressure related to bolus formation and the transmission of bolus from the mouth to the pharynx is affected by the trunk muscles, such as the back muscles [5, 7]. The jaw-opening force is performed by one of the suprahyoid muscles and is related to raising the hyoid bone for sending the bolus from the pharynx to the esophagus. The jaw-opening force is related to the neck circumference and TMI [5, 6]. Trunk stabilization and optimal posture maintenance make swallowing easier [4]. In addition, posture plays an important role in sending the bolus from the esophagus to the stomach. In older people, the stomach is often compressed in a hunched position. High intragastric pressure interferes with the peristaltic action of the esophagus [36]. Thus, trunk stability is important in each process involved in swallowing.
In addition, those with good swallowing function and who eat meals corresponding to FOIS 5 or higher have more opportunities to use their trunk muscles than those who eat meals corresponding to FOIS 4 or lower, which does not require chewing. The opportunities might help maintain their muscle mass. Although previous studies have investigated the relationship between the strength of swallowing-related muscles and the trunk muscles, this is the first study to clarify the relationship between specific swallowing function status and trunk muscle mass.
Time Spent Away from Bed and Swallowing Function
Individuals whose time spent away from bed was longer had good swallowing function. Especially those who spent more than 6 h away from bed (group L) were more likely to eat meals that require chewing, corresponding to FOIS 5–7.
This may be attributed to the fact that swallowing function is closely related to trunk muscle mass and arousal. When people are awake, they can pay attention to things around them, accept, and respond to information. Then sensory input from the five senses triggers the swallowing reflex [37, 38]. In addition, swallowing reflex elicitation, like salivary swallowing, could occur unconsciously, but chewing can only be performed in an awake state [39]. Previous studies have shown that older adults with low ADL who stay away from bed for more than 6 h a day have stable arousal, and it is known that cerebral activity increases when in sitting or upright positions [40‒42]. Therefore, it can be inferred that spending away from bed for more than 6 h and promoting arousal affected swallowing function.
How Can They Spend Time Away from Bed Longer?
From the free comments in the questionnaire, group S, which was not particularly active, included those who did not spend time away from bed during ADL such as eating and bathing, or who spent time away from bed only during such ADL. For those who did not spend time away from bed, it is recommended to incorporate spending time away from bed into ADL, such as eating in a wheelchair, taking a bath for a short time, as long as they are not tired, instead of eating and cleaning the body in bed. Group M included those who experience gravity load during their ADL. It is recommended to incorporate spending time away from bed into the enjoyment of life instead of watching TV or meeting visitors in bed. They can further increase the time spent away from bed by participating in events at home or in the facility and going out.
Limitations
This is a cross-sectional study; therefore, it is unclear whether longer time spent away from bed would maintain or improve whole-body muscle mass and swallowing function. In the future, subjects with different amounts of time spent away from bed need to be prospectively examined. The next phase of this study will examine whether time spent away from bed affects complications such as unscheduled hospitalization and aspiration pneumonia and whether extending the time in subjects with short time will improve swallowing function, appendicular skeletal muscle mass, and trunk muscle mass. Second, because the BIA was used to measure muscle mass, individuals with an implanted heart pacer were excluded from the study.
Conclusion
We verified the association between the time spent away from bed and whole-body muscle mass and swallowing function in older adults with low ADL. The results suggested that appendicular skeletal muscle mass and certain swallowing functions are preserved if older adults with low ADL spend more than 4 h away from bed. Moreover, trunk muscle mass and swallowing function in older adults are well maintained if they spend more than 6 h away from bed. Thus, we consider that 4 h, or 6 h if possible, spent away from bed can help maintain whole-body muscle mass and swallowing function in older adults with low ADL and difficulty in performing exercises to preserve swallowing function. These findings highlight factors that can prevent a decline in swallowing function in the daily life of these individuals.
Acknowledgment
We would like to thank Editage (www.editage.jp) for English language editing.
Statement of Ethics
This study was approved by the Dental Research Ethics Committee of Tokyo Medical and Dental University (No. D2018-015). We explained the purpose of this study to the subjects or their delegates and obtained their written informed consent before participation. The research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki.
Conflict of Interest Statement
There are no potential conflicts of interest to declare.
Funding Sources
This research did not receive any funding from agencies in the public, commercial, or not-for-profit sectors.
Author Contributions
Study concept and design: Miki Ishii and Haruka Tohara. Acquisition of data: Takuma Okumura, Shohei Hasegawa, Tomoe Tamai, Yuki Nagasawa, and Akira Yoshizawa. Analysis and interpretation of data: Kohei Yamaguchi and Ayako Nakane. Drafting of the manuscript: Miki Ishii, Kazuharu Nakagawa, and Kanako Yoshimi. Critical revision of the manuscript for important intellectual content: Kazuharu Nakagawa and Kanako Yoshimi.
Data Availability Statement
The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author (K.N.).