Masticatory dysfunction in patients with diabetic neuropathy: A cross-sectional study.

INTRODUCTION: Chewing well is essential for successful diet therapy and control of blood glucose level in patients with diabetes. In addition, long-term hyperglycemia is a risk factor for microvascular complications, which are the main cause of morbidity and mortality in these patients. Hence, it is plausible that masticatory disorder may be relevant to diabetic microvascular complications which is caused by long-term hyperglycemia. The aim of this study was to investigate whether masticatory disorders are relevant to diabetic microvascular complications.
METHODS: This cross-sectional study included 172 patients with type 2 diabetes who underwent educational hospitalization in the Department of Endocrinology and Diabetic Medicine, Hiroshima University Hospital, from April 2016 to March 2020. Masticatory efficiency was determined quantitatively by using the GLUCO SENSOR GS-Ⅱ. Multivariable linear regression models were constructed to examine which factors were related to masticatory efficiency. Statistical significance was defined as a two-sided p value of < 0.05.
RESULTS: According to the bivariable analysis, masticatory efficiency was significantly correlated with duration of diabetes (p = 0. 049), number of remaining teeth (p < 0.0001), the number of moving teeth (p = 0.007) and condition of diabetic neuropathy (p < 0.0001). Moreover, the number of remaining teeth (p < 0.0001) and diabetic neuropathy (p = 0.007) remained significantly correlated with masticatory efficiency in the multivariable analysis.
CONCLUSIONS: For the first time, we demonstrated that patients with type 2 diabetes who developed diabetic neuropathy had significantly reduced masticatory efficiency. Effective mastication is an important factor in successful diet therapy for diabetes. To prevent the progression of diabetic complications, especially in patients with diabetic neuropathy, it may be necessary to combine individualized therapies from dentists and nutritionists with consideration for the level of masticatory dysfunction.

1. Introduction

The oral cavity is the first line of the gastrointestinal tract and a portal of entry for microbes and foods. Since the oral cavity is constantly exposed to a plethora of external factors (e.g., microorganisms, mechanical and chemical damage from food intake, and dietary and airborne antigens), its function is frequently disrupted. Dysfunction of the oral cavity by bacterial infection, inflammation, tooth loss or pain has been suggested to negatively affect general health [1, 2]. Periodontitis is a representative disorder of the oral cavity that is characterized by a tissue-destructive chronic inflammatory disease caused by periodontal pathogenic microorganism infection. Previous studies have clearly demonstrated the correlation between periodontitis and diabetes; inflammation developed due to periodontitis has been associated with insulin resistance and poor glycemic control [3-6]. In addition, improvement of local inflammation after periodontal treatment positively affected insulin resistance and hemoglobin A1c (HbA1c) in diabetes patients [7-9].

Periodontitis is also characterized by alveolar bone destruction and subsequent tooth mobility or loss, leading to masticatory dysfunction [10-12]. It is widely accepted that tooth loss is associated with diabetes [13, 14]. Substantial mastication induces histamine production in the hypothalamus and insulin secretion from the pancreas, thereby preventing overeating and increasing blood glucose levels [15-18]. In previous studies, patients who lost their teeth suffered from malnutrition and preferred soft meals mainly composed of fats and carbohydrates [19-25]. An epidemiological study indicated that chewing slowly and steadily reduced the risk of diabetes [26]. Taken together, these results suggest that chewing sufficiently is essential for the success of diet therapy for diabetes and that long-term masticatory disorders is associated with the progression of diabetic conditions. Nonetheless, there are few studies investigating masticatory efficiency quantitatively in diabetes patients.

On the other hand, long-term hyperglycemia is well known as the major risk factor for vascular complications, which are the major cause of morbidity and mortality in diabetic patients [27]. Both microvascular and macrovascular complications affect the quantity and quality of life of diabetes patients. In particular, microvascular complications are responsible for neuropathy, retinopathy and nephropathy in diabetic patients. In addition, glycemic control delays the onset and progression of microvascular complications [28, 29]. Here, as described above, it is plausible that masticatory disorders may be associated with the progression of diabetic conditions. Moreover, it is true that uncontrolled diabetes causes subsequent microvascular complications. Thus, we speculate that masticatory disorder may be relevant to diabetic microvascular complications, although no study has verified this relationship.

In general, many previous reports have assessed masticatory efficiency by occlusal bite force, masticatory movement, or the ability to comminute test food [30-32]. Especially, assessing the mandibular kinematic of the chewing patterns is useful for understanding the neuromuscular function [33]. However, it is difficult to assess the masticatory performance quantitatively by these previous methods. Hence, we focused on a new testing system that can quantitatively determine masticatory efficiency by measuring the eluted glucose from gummy jelly [34, 35]. Accordingly, to answer our clinical question, we designed a cross-sectional study to measure masticatory efficiency quantitatively by a new testing system and assess microvascular complications in patients with diabetes.

2. Materials and methods

2.1 Subjects and study design

Participants of this study were enrolled from patients with type 2 diabetes who underwent educational hospitalization in the Department of Endocrinology and Diabetic Medicine, Hiroshima University Hospital, from April 2016 to March 2020. All participants in this study provided written informed consent prior to enrollment, consistent with the Helsinki Declaration and guidelines of the institutional review board of Hiroshima University. Masticatory efficiency was measured by using a gummy jelly in the Center for Oral Clinical Examination, Hiroshima University, and periodontitis was assessed in the Department of Periodontics, Hiroshima University Hospital. This study was approved by the Ethical Committee for Epidemiology of Hiroshima University at November 19, 2015. The approval number was E-150.

2.2 Assessment of diabetes

All the subjects received biochemical evaluations after blood sample collection. Plasma HbA1c was assessed by high-performance liquid chromatography. Serum creatinine (Cre) levels were measured by clinical analyzers. The estimated glomerular filtration rate (eGFR) was calculated according to age and Cre as follows: men; 194 × Cre-1.094 × age-0.287, women; 194 × Cre-1.094 × age-0.287 × 0.739 [36]. Diabetic nephropathy was defined based on the clinical practice guidelines of the Japan Diabetes Society[Stage 2: UAE 30–299 mg/day (or ACR 30–299 mg/g Cre), Stage 3: UAE ≥300 mg/day (or ACR ≥300 mg/g Cre) or a protein creatinine ratio ≥0.5 g/g Cre, Stage 4: eGFR <30 mL/min/1.73 m2, and Stage 5: maintenance dialysis] [37]. Daily urinary albumin excretion (UAE) and the albumin–creatinine ratio (ACR) were measured by immunoturbidimetric assays from 24-hour urine samples. Diabetic neuropathy was defined by the presence of at least 2 of the 3 following factors: 1) subjective symptoms, 2) reduced Achilles tendon reflex, or 3) decreased vibration sensation on the internal malleolus. These abbreviated diagnostic criteria, which are frequently employed for the diagnosis in Japan, are commended by Diabetic Neuropathy Study Group in Japan because of their sensitivity (68%) and specificity (74%) obtained by evaluating nerve conduction study as a gold standard [38]. The presence of diabetic retinopathy was diagnosed by ophthalmologists.

2.3 Assessment of periodontitis

The periodontal inflammatory surface area (PISA) is an index to assess the overall periodontal inflammatory status [39]. The PISA enables a quantitative evaluation of the burden of periodontal inflammation as a continuous variable in individual patients. To calculate the PISA, all participants were assessed for probing pocket depth and bleeding on probing at 6 sites per tooth, and these values were entered into a spreadsheet that is freely accessible at http://www.parsprototo.info/docs/PISA_CAL.xls [39]. The number of moving teeth was also assessed using Miller’s mobility index, which is the most widely accepted method for the routine clinical examination of tooth mobility; class 0 represents no mobility, and classes 1–3 represent consecutive degrees of tooth mobility [40].

2.4 Measurement of masticatory efficiency

Masticatory efficiency was measured by using a masticatory ability testing system (GLUCO SENSOR GS-Ⅱ, GC Corporation, Tokyo, Japan). In brief, patients freely chewed 2 g of gummy jelly, which included approximately 100 mg of glucose, measuring approximately 14 mm (diameter) × 10 mm (height) in size and weighing approximately 2 g (Gurukoramu, GC Corporation) for 20 s. Subsequently, the patients rinsed with 10 mL of water and spat both the chewed gummy jelly and the water into a cup through a 2 mm mesh. The amount of eluted glucose in the filtrate was measured using a glucose measuring device (GLUCO SENSOR GS-Ⅱ). The procedure was performed twice, and the average value of the results was defined as the quantitative parameter of masticatory efficiency [34, 35]. The patient using removable prostheses were assessed with them in place.

2.5 Statistical analysis

Statistical analyses were performed using JMP® 13 (SAS Institute Inc., Cary, NC, USA). Correlations between continuous variables [i.e., age; body mass index (BMI); duration of diabetes; HbA1c; number of remaining teeth; number of moving teeth; PISA] and masticatory efficiency were assessed by Spearman rank correlation coefficient. The effects of ordinal variables (i.e., the stage of diabetic nephropathy) or nominal variables (i.e., sex; current smoking; presence of diabetic neuropathy or diabetic retinopathy) on masticatory efficiency were also assessed by one-way ANOVA or the t-test. Multivariable linear regression models were constructed to examine which factors were related to masticatory efficiency. The average amount of eluted glucose was defined as an objective variable. The covariates were included the factor of interest (i.e., sex; age; duration of diabetes; the presence of diabetic neuropathy and retinopathy; the stage of diabetic nephropathy; the number of remaining teeth; the number of moving teeth; PISA). Here, there is a fact that oral function, including masticatory efficiency, is apparently decreased in a patient whose number of remaining teeth is less than 20 [41]. Thus, to avoid a skewed assessment by subjects with reduced teeth number, we stratified patients who had more than 20 teeth for the statistical analysis. More specifically, the subjects with lower than 20 teeth were eliminated even if removal prostheses were placed. Statistical significance was defined as a two-sided p value of < 0.05.

3. Results

3.1 Baseline characteristics

The participant characteristics are listed in Table 1. This cross-sectional study included 172 individuals who agreed to participate in this study, comprising 100 men and 72 women and their mean age was 61 ± 14. Their mean duration of diabetes was 11.1 ± 10.7 years and their mean HbA1c was 10.3 ± 2.1% (87 ± 21 mmol/mol). Among the diabetic complications, 122 patients complicated with diabetic neuropathy and 43 patients complicated with diabetic retinopathy. Diabetic nephropathy consisted of 91 patients with stage 1, 59 patients with stage2, 12 patients with stage 3 and 10 patients with stage 4. There were no patients with stage 5 of diabetic nephropathy. The removable prostheses were placed in 46 patients and no patients were treated with dental implants. The mean masticatory efficiency assessed by the amount of eluted glucose was 172.4 ± 60.0 mg/dL.

Table 1

Participant characteristics at baseline.

Parameter	Value
n	172
Male/female, n (%)	100 (58)/72 (42)
Age (years)	60.8 ± 15.4
BMI (kg/m2)	25.7 ± 6.2
Current smoking, n (%)	37 (22)
Duration of diabetes (years)	11.1 ± 10.7
Systolic blood pressure (mmHg)	129 ± 19
Diastolic blood pressure (mmHg)	78 ± 13
HbA1c [% (mmol/mol)]	10.3 ± 2.1 (87 ± 21)
UAE (mg/day)	20.6 (11.4, 60.6)
eGFR (ml/min/1.73 m2)	74.7 ± 30.3
Diabetic neuropathy, n (%)	122 (71)
Diabetic retinopathy, n (%)	43 (25)
Diabetic nephropathy, n (%)
Stage 1	91 (53)
Stage 2	59 (34)
Stage 3	12 (7)
Stage 4	10 (6)
Stage 5	0 (0)
Number of remaining teeth, n	23 ± 7
Number of moving teeth, n	2 ± 3
PISA (mm2)	539.4 ± 484.6
Masticatory efficiency (mg/dL)	172.4 ± 60.0

UAE is expressed as median (first quartile, third quartile). Other values are expressed as the mean ± standard deviation or n (%). Abbreviations: BMI, body mass index; HbA1c, hemoglobin A1c; UAE, daily urinary albumin excretion; eGFR, estimated glomerular filtration rate; PISA, periodontal inflammatory surface area.

3.2 Correlation between masticatory efficiency and diabetic microvascular complications

We first performed bivariable analyses to assess associations between participant characteristics and masticatory efficiency (Fig 1). Masticatory efficiency was significantly negatively correlated with duration of diabetes (p = 0.049; Fig 1C) and the number of moving teeth (p = 0.007; Fig 1F) and positively correlated with the number of remaining teeth (p < 0.0001; Fig 1E), whereas age, BMI, HbA1c and PISA did not have significant associations with masticatory efficiency (Fig 1A, 1B, 1D and 1G). Then, we investigated the correlation between masticatory efficiency and diabetic conditions, including neuropathy, retinopathy, and nephropathy (Fig 2). Masticatory efficiency was significantly reduced in patients with diabetic neuropathy (p < 0.0001; Fig 2A). On the other hand, diabetic nephropathy and retinopathy were not associated with the level of masticatory efficiency (Fig 2B and 2C). Moreover, sex and current smoking were not correlated with masticatory efficiency (Fig 2D and 2E).

Fig 1

Spearman rank correlation.

Correlations between masticatory efficiency and (A) age, (B) BMI, (C) duration of diabetes, (D) HbA1c, (E) number of remaining teeth, (F) number of moving teeth and (G) PISA were assessed. Spearman rank correlation coefficients (r) and p values (p) are shown. Statistical significance was defined as a two-sided p value of < 0.05. Abbreviations: BMI, body mass index; HbA1c, hemoglobin A1c; PISA, periodontal inflammatory surface area.

Fig 2

One-way ANOVA and the t-test.

Effects of (A) diabetic neuropathy, (B) stage of diabetic nephropathy, (C) diabetic retinopathy, (D) sex and (E) current smoking on masticatory efficiency were assessed. Statistical significance was defined as a two-sided p value of < 0.05.

In the multivariable linear regression analysis, masticatory efficiency was positively associated with the number of remaining teeth (p < 0.0001) and negatively associated with the complication of diabetic neuropathy (p = 0.007) (Table 2). In contrast, duration of diabetes and the number of moving teeth, which were statistically significant in the bivariable analysis, was not associated with masticatory efficiency in the multivariable analysis (Table 2).

Table 2

Multivariable analysis.

Parameter	95% CI, upper/lower	p value
Sex	2.672/-13.418	0.189
Age	0.962/-0.221	0.218
Duration of diabetes	0.716/-1.075	0.693
Diabetic neuropathy	21.522/3.476	0.007
Diabetic retinopathy	10.041/-10.111	0.995
Diabetic nephropathy
Stage 2–1	14.161/-20.341	0.724
Stage 3–2	25.404/-40.981	0.644
Stage 4–3	41.145/-47.214	0.892
Number of remaining teeth	5.384/2.984	< 0.0001
Number of moving teeth	2.601/-3.297	0.816
PISA	0.009/-0.029	0.308

Statistical significance was defined as a two-sided p value of < 0.05. Abbreviations: CI, confidence interval; PISA, periodontal inflammatory surface area.

3.3 Stratified analysis of the patients with more than 20 remaining teeth

Since the remaining number of teeth less than 20 reduces the oral function [41], in order to avoid a skewed assessment, we stratified the patients with more than 20 remaining teeth. As a result, 124 patients had more than 20 remaining teeth, of which 12 patients used removable prostheses. The presence of diabetic neuropathy did not affect the number of remaining teeth (data not shown). Nevertheless, the complication of diabetic neuropathy showed lower mastication (Fig 3). Furthermore, the multivariable linear regression analysis indicated a significant correlation between masticatory efficiency and the number of remaining teeth (p = 0.0002); diabetic neuropathy (p = 0.0004) (Table 3).

Fig 3

Effect of diabetic neuropathy on masticatory efficiency in patients with more than 20 remaining teeth.

Statistical significance was defined as a two-sided p value of < 0.05.

Table 3

Multivariable analysis after stratification.

Parameter	95% CI, upper/lower	p value
Sex	4.840/-13.596	0.349
Age	1.288/-0.079	0.082
Duration of diabetes	1.106/-1.371	0.832
Diabetic neuropathy	28.792/8.606	0.0004
Diabetic retinopathy	18.473/-6.202	0.327
Diabetic nephropathy
Stage 2–1	26.450/-14.048	0.545
Stage 3–2	45.093/-36.560	0.836
Stage 4–3	49.857/-60.392	0.850
Number of remaining teeth	10.646/3.396	0.0002
Number of moving teeth	4.172/-3.501	0.170
PISA	0.010/-0.031	0.291

Statistical significance was defined as a two-sided p value of < 0.05. Abbreviations: CI, confidence interval; PISA, periodontal inflammatory surface area.

4. Discussion

The present study revealed, for the first time, that patients with type 2 diabetes who have diabetic neuropathy have significantly lower masticatory efficiency. Besides, stratified analysis for the patients with more than 20 remaining teeth demonstrated that the negative effect of diabetic neuropathy remained. Taken together, these findings suggested the association between masticatory efficiency and diabetic neuropathy. Since masticatory dysfunction is an obstacle to medical diet therapy, masticatory efficiency in diabetic patients with diabetic neuropathy may be a useful clinical indicator for successful diabetes therapy. On the other hand, unexpectedly, neither diabetic retinopathy nor diabetic nephropathy correlated with masticatory efficiency. These results imply that diabetic neuropathy could be the cause of the resultant masticatory disorder in diabetes patients. We discuss the plausible mechanisms of impairment for mastication below.

First, the possible factor responsible for masticatory dysfunction in diabetic neuropathy patients may be sarcopenia. It is widely accepted that type 2 diabetes is associated with sarcopenia [42-44]. Furthermore, diabetic neuropathy is one of the important risk factors for the occurrence of sarcopenia in diabetic patients [45-47]. Notably, sarcopenia induces masseter muscle atrophy and chewing dysfunction [48, 49]. Although this present clinical study did not assess the motor neuronal function, diabetic neuropathy was correlated with masticatory disorder. Consequently, it is plausible that sarcopenia associated with diabetic neuropathy may cause masticatory muscle mass reduction, which results in the development of masticatory dysfunction. Since this present clinical study lacked the data evaluating sarcopenia, including such as muscle strength, physical function and muscle mass, we cannot show the precise association of sarcopenia for masticatory disorder in patients with diabetic neuropathy. To clarify this tentative hypothesis, additional studies evaluating the relation between sarcopenia/diabetic motor neuropathy and masticatory dysfunction will be needed. More specifically, in addition to our mastication efficacy testing system, recording the mandibular kinematics and muscular activation during chewing will strengthen such promising future study [50].

The other plausible factor of impairment for mastication could be trigeminal nerve dysfunction due to diabetic neuropathy. Although most of the facial muscles are innervated by the facial nerve, the muscles involved in mastication, consisting of the masseter, temporal, medial pterygoid, and lateral pterygoid muscles, are innervated by the trigeminal nerve. When these mandibular motor fibers are affected, the patients may complain of weakness in chewing [51]. Thus, masticatory dysfunction in the patients with diabetic neuropathy in this study may be attributed to impaired trigeminal nerve function. Oculomotor, abducens, facial, and trochlear nerve palsies are frequently observed as diabetic mononeuropathies; however, there are no reports demonstrating trigeminal nerve dysfunction in patients with diabetic neuropathy. However, pure trigeminal motor neuropathy, caused by viral or bacterial infection [52, 53], tumors [54] or unknown causes [55], can lead to masticatory muscle dysfunction. Accordingly, it is highly conceivable that masticatory dysfunction due to trigeminal nerve palsy occurs in diabetic neuropathy. More importantly, compared to the well-reported prognosis of diabetic mononeuropathy, which can be treated by controlling blood glucose levels, the prognosis of trigeminal nerve palsy may be relatively poor. As described above, masticatory disorder caused by trigeminal nerve dysfunction may disrupt healthy eating habits and thereby worsen blood glucose control.

PISA was not associated with masticatory dysfunction in this study; however, previous studies have demonstrated a correlation between periodontitis and masticatory disability [10-12]. Despite this, our results may not contradict those of previous reports. The PISA reflects the inflamed surface area of periodontal pockets. In brief, as the number of remaining teeth decreased, the PISA decreased. Accordingly, severe periodontitis patients who lose their teeth and develop masticatory disorder have a decreased PISA.

In this study, diabetic neuropathy was diagnosed by abbreviated diagnostic criteria, which are generally employed in Japan. These abbreviated diagnostic criteria are easy and reliable for the diagnosis but not quantitative. Thus, to investigate the association between the objective degree of diabetic neuropathy and masticatory disorder, a quantitative assessment such as a nerve conduction test will be required. Moreover, a future study testing the association between diabetic neuropathy and neuromuscular control of mastication will clarify the effect of diabetic neuropathy on masticatory dysfunction in more detail. To this end, in addition to the masticatory efficiency testing system, monitoring of the mandibular kinematic and muscular activity should be employed.

5. Conclusion

In conclusion, we revealed a significant association between diabetic neuropathy and masticatory dysfunction. Additional assessment for a mandibular kinematic and muscular activity will deepen our understanding of the neuropathic effects of diabetes on the craniofacial disorder.

For diabetic patients with masticatory disorder, it may be difficult to implement a dietary treatment regimen. To control hyperglycemic conditions in diabetic neuropathy patients, diet therapy considering a patient’s masticatory function by dentists and nutritionists could be helpful.

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Reviewer #1: While the paper entitled “Masticatory dysfunction in patients with diabetic neuropathy” is interesting and insightful, some revisions of the text are needed.

Materials and Methods: since the paper focuses on diabetic neuropathy, the fact that it was exclusively diagnosed by means of clinical examination, without the use of any of the available quantitative tests, is problematic. Moreover, it precludes the possibility to investigate the association between the objective degree of neuropathy and the reduction in masticatory efficiency.

Patient description is also problematic: a major bias is due to the fact that no mention is made of the occlusal condition of the patients the presence of prosthodontic rehabilitation, which is very likely to be present given the age and condition of the subjects, and which is certainly capable of altering masticatory efficiency. No information is given as to whether the masticatory efficiency test was carried out with or without removable prostheses in place. Moreover, the presence of fixed prosthodontic rehabilitation, either on natural teeth or implants, should be noted and taken into account. No attempt was made to stratify the subjects according to the number of teeth still present, even though it is well attested that masticatory efficiency sharply decreases if the number of remaining teeth decreases below 20. In the context of this research, a few subject with an extremely compromised dentition may have skewed the analysis exaggerating the effect of tooth loss. At the very least, the presence of prosthodontic rehabilitation should be discussed.

Results: the meaning of line 160-161 is unclear. Line 183-184 is unclear in the context as well, and needs to be clarified (positively or negatively associated?). the same goes for line 210 (…were associated with reduced masticatory efficiency).

Discussion: in the context of this paper’s research, to hypothesize a role for diabetic neuropathy in masticatory dysfunction is not of itself implausible (although it should probably be quite advanced to manifest on the trigeminal nerve), but it really is not well tested in the context of this research. The only way to do it is to separate the confounding factor or remaining teeth (a bias in this case) and investigate the effects of neuropathy on neuromuscular control of mastication, not on masticatory efficiency. This distinction, that admittedly reduces the claim of the paper, should be included and discussed.

Reviewer #2: The manuscript is well written, the statistical data are satisfactory and self explanatory. The figures provided in the manuscript lacks visual clarity and can be made more prominent. There are some background correction to be made in each figure for clarity. Thanks

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Reviewer #1: Yes: Maria Grazia Piancino

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13 Apr 2022

Response to Reviewer Comments

Reviewer #1

1. While the paper entitled “Masticatory dysfunction in patients with diabetic neuropathy” is interesting and insightful, some revisions of the text are needed.

Response: We are delighted to hear that the reviewer thinks our manuscript is interesting, albeit with some drawbacks. To increase the quality of our manuscript, we followed the suggestions described below.

2. Materials and Methods: since the paper focuses on diabetic neuropathy, the fact that it was exclusively diagnosed by means of clinical examination, without the use of any of the available quantitative tests, is problematic. Moreover, it precludes the possibility to investigate the association between the objective degree of neuropathy and the reduction in masticatory efficiency.

Response: We completely agree with this reviewer’s insightful criticism. In this present study, diabetic neuropathy was diagnosed using the abbreviated diagnostic criteria. These criteria are frequently employed for the diagnosis in Japan and are commended by Diabetic Neuropathy Study Group in Japan because of their sensitivity (68%) and specificity (74%) obtained by evaluating nerve conduction test as a gold standard (Yasuda H et al., Diabetes Res Clin Pract, 2007). Thus, we believe our present study using the abbreviated diagnostic criteria can support our conclusion. However, as the reviewer pointed out, the quantitative analysis such as the nerve conduction test should be helpful to assess the association between the objective degree of neuropathy and the reduction in masticatory efficiency. This point is discussed in the revised discussion section.

Materials and Methods section: Page 6, line 118-121

Discussion section: Page 14, line 285-289

3. Patient description is also problematic: a major bias is due to the fact that no mention is made of the occlusal condition of the patients the presence of prosthodontic rehabilitation, which is very likely to be present given the age and condition of the subjects, and which is certainly capable of altering masticatory efficiency. No information is given as to whether the masticatory efficiency test was carried out with or without removable prostheses in place. Moreover, the presence of fixed prosthodontic rehabilitation, either on natural teeth or implants, should be noted and taken into account. No attempt was made to stratify the subjects according to the number of teeth still present, even though it is well attested that masticatory efficiency sharply decreases if the number of remaining teeth decreases below 20. In the context of this research, a few subject with an extremely compromised dentition may have skewed the analysis exaggerating the effect of tooth loss. At the very least, the presence of prosthodontic rehabilitation should be discussed.

Response: We sincerely appreciate this reviewer’s keen eye. Prosthodontic rehabilitation and the number of remaining teeth should be considered in more detail. First of all, the removable protheses were used in 46 patients, whereas no dental implant was observed in all subjects. Besides, the measurement of masticatory efficiency was conducted with the removable prostheses in place. These points are described in the revised Materials and Methods and Results section.

In addition, as the reviewer pointed out, it is true that the reduced number of remaining teeth, less than 20, should affect oral function, including masticatory efficiency. Including such subjects may have skewed our analysis. Accordingly, encouraged by this reviewer’s constructive comment, we conducted a stratified analysis of the patients with more than 20 remaining teeth. As a result, we found that diabetic neuropathy decreased mastication efficacy. This finding can support our conclusion that the complication of diabetic neuropathy is associated with masticatory disfunction.

Materials and Methods section: Page 7, line 143-144

Page 7, line 158-162

Results section: Page 8, line 173-174

Page 11, line 223- Page 12, line 235

New Figure 3

New Table 3

Discussion section: Page 13, line 245-248

4. Results: the meaning of line 160-161 is unclear. Line 183-184 is unclear in the context as well, and needs to be clarified (positively or negatively associated?). the same goes for line 210 (…were associated with reduced masticatory efficiency).

Response: In accordance with the reviewer’s suggestion, we have amended these unreadable sentences.

Results section: Page 8, line 169-171

Page 10, line 195

Page 10, line 211-213

5. Discussion: in the context of this paper’s research, to hypothesize a role for diabetic neuropathy in masticatory dysfunction is not of itself implausible (although it should probably be quite advanced to manifest on the trigeminal nerve), but it really is not well tested in the context of this research. The only way to do it is to separate the confounding factor or remaining teeth (a bias in this case) and investigate the effects of neuropathy on neuromuscular control of mastication, not on masticatory efficiency. This distinction, that admittedly reduces the claim of the paper, should be included and discussed.

Response: We sincerely appreciate this insightful suggestion. We agree entirely that this present study is still enough to draw the precise association between diabetic neuropathy and masticatory function, even though a new stratified analysis can solve the remaining teeth number problems. Following the reviewer’s constructive comments, we will conduct the next study assessing the neuromuscular control of mastication. This point regarding the future study is discussed in the revised Discussion section.

Discussion section: Page 14, line 289-291

Reviewer #2

1. The manuscript is well written, the statistical data are satisfactory and self explanatory. The figures provided in the manuscript lacks visual clarity and can be made more prominent. There are some background correction to be made in each figure for clarity. Thanks.

Response: We sincerely appreciate these positive and encouraging comments on our manuscript. Following this reviewer’s constructive suggestion, we have amended the incorrect notation in Figure 1. Besides, Figures 1 and 2 are revised to be more visible.

Revised Figure1 and 2

Submitted filename: Response to Reviewers.docx

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28 Apr 2022

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Reviewer #1: (No Response)

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Reviewer #1: No

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Reviewer #1: The revised manuscript has been improved, but some points are still lacking. Considering the significant limitations of the masticatory ability testing system as methodology for the evaluation of a complex pathology such as diabetes in this study, this paper needs to be careful about the strength of the conclusions. Association does not imply a cause/effect relationship, and demonstrating one will require experimental evidence, which is, by its very design, not presented by the study. Nevertheless, it is useful and inherently interesting to speculate on the possible mechanism linking diabetes and reduced mastication, given the high prevalence of both conditions and the relevance of the topic.

Introduction

Please mention the actual possibility of recording the mandibular kinematic of the chewing patterns during chewing that gives information on the peripheral effects of neuromuscular motor control that would be useful for understanding the neuropathic effects of diabetes(doi: 10.1093/ejo/cjr109.).

Discussion

Line 267 please add that to properly consider the role of sarcopenia during mastication it is necessary to record the mandibular kinematics and muscular activation during chewing (doi: 10.1016/j.archoralbio.2016.03.013.). To this end methods other than masticatory ability testing system are needed.

Please add at the end of the discussion as limitation of the study that the methodology used i.e. the ability testing system, is not refined enough for the study of a complex pathology such as diabetes and that the future direction will be the recording of the mandibular kinematic together with the muscular activation to establish a deeper understanding of the topic.

Abstract and Conclusion

Please, add a reference to the methodological limitation of the study.

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12 May 2022

Response to Reviewer Comments

Reviewer #1

1. The revised manuscript has been improved, but some points are still lacking. Considering the significant limitations of the masticatory ability testing system as methodology for the evaluation of a complex pathology such as diabetes in this study, this paper needs to be careful about the strength of the conclusions. Association does not imply a cause/effect relationship, and demonstrating one will require experimental evidence, which is, by its very design, not presented by the study. Nevertheless, it is useful and inherently interesting to speculate on the possible mechanism linking diabetes and reduced mastication, given the high prevalence of both conditions and the relevance of the topic.

Response: We appreciate this reviewer #1, who acknowledged that our first revision responding to the previous questions was improved and our study could be potentially interesting. Besides, we agree with the new critiques suggesting the effectiveness of the other examinations. To increase the quality of our manuscript, we followed such constructive suggestions described below.

2. Introduction: Please mention the actual possibility of recording the mandibular kinematic of the chewing patterns during chewing that gives information on the peripheral effects of neuromuscular motor control that would be useful for understanding the neuropathic effects of diabetes(doi: 10.1093/ejo/cjr109.).

Response: We completely agree that assessing the mandibular kinematic is helpful in clarifying the neuromuscular motor function. Thus, this point was mentioned in the revised introduction section with appropriate reference (Piancino et al., 2021, European Journal of Orthodontics).

Introduction section: Page 3 line 85-87

3. Discussion: Line 267 please add that to properly consider the role of sarcopenia during mastication it is necessary to record the mandibular kinematics and muscular activation during chewing (doi: 10.1016/j.archoralbio.2016.03.013.). To this end methods other than masticatory ability testing system are needed.

Please add at the end of the discussion as limitation of the study that the methodology used i.e. the ability testing system, is not refined enough for the study of a complex pathology such as diabetes and that the future direction will be the recording of the mandibular kinematic together with the muscular activation to establish a deeper understanding of the topic.

Response: We sincerely appreciate this insightful comment. Monitoring the mandibular kinematics and muscular activation should be helpful in clarifying the relationship between sarcopenia and masticatory function. Following this reviewer’s suggestion, we introduced this idea with a proper reference (Piancino et al., 2016, Archives of Oral Biology) in the revised manuscript. Besides, at the end of the discussion section, we have referred that the additional examinations recording mandibular kinematic and muscular activity will strengthen our findings in this present study.

Discussion section: Page 13, line 268- Page 14, line 271. Page 15, line 301-303

4. Abstract and Conclusion: Please, add a reference to the methodological limitation of the study.

Response: In accordance with the reviewer’s comment, we have referred to the necessity of the mandibular kinetics and muscle activity examinations in the revised conclusion section. On the other hand, the abstract section, having a character limit, simply referred to the results demonstrating that diabetic patients who developed diabetic neuropathy had reduced masticatory efficiency. Thus, we think the abstract in this paper may not need to be amended from its current form.

Conclusion section: Page 16, line 307-309.

Submitted filename: Response to Reviewers.docx

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25 May 2022

Masticatory dysfunction in patients with diabetic neuropathy: A cross-sectional study

PONE-D-22-02324R2

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Reviewer #1: All comments have been addressed

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Reviewer #1: Yes

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27 May 2022

PONE-D-22-02324R2

Masticatory dysfunction in patients with diabetic neuropathy: A cross-sectional study

Dear Dr. Hamamoto:

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