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. 2021 Sep 23;16(9):e0257247. doi: 10.1371/journal.pone.0257247

Effect of advanced periodontal self-care in patients with early-stage periodontal diseases on endothelial function: An open-label, randomized controlled trial

Ayako Okada 1,2, Takatoshi Murata 1,*, Khairul Matin 3,4, Meu Ariyoshi 1,4, Ryoko Otsuka 1, Mamiko Yamashita 1, Masayuki Suzuki 5, Rumi Wakiyama 5, Ken Tateno 5,6, Megumi Suzuki 7, Hitomi Aoyagi 8, Hiromi Uematsu 8, Akiko Imamura 8, Miki Kosaka 1,9, Tomoko Mizukaki 10, Tsutomu Sato 11,12, Hiroshi Kawahara 5, Nobuhiro Hanada 1
Editor: Andrej M Kielbassa13
PMCID: PMC8459983  PMID: 34555048

Abstract

Although a significant association between periodontal disease and atherosclerotic cardiovascular disease has been reported, their cause-to-effect relationship remains controversial. This randomized controlled clinical trial aimed to investigate the effect of advanced self-care on atherosclerotic cardiovascular disease-related vascular function markers flow-mediated brachial artery dilatation (FMD) and serum asymmetric dimethylarginine (ADMA) level in patients with early-stage periodontal disease. The study was designed as a parallel group, 3-month follow-up, open-label, randomized controlled trial. The control group received standard care for periodontal diseases, whereas the test group additionally applied disinfectant using a custom-fabricated prescription tray for advanced self-care twice a day. Overall, 110 patients provided data for FMD and serum ADMA level. No significant improvements in FMD were observed in the control (mean increase, −0.1%; 95% confidence interval [CI], −1.0–0.8; P = 0.805) or test (mean increase, −0.3%; 95% CI, −1.1–0.4; P = 0.398) group. No significant changes in serum ADMA levels were observed (mean reduction, 0.01 μmol/L; 95% CI, −0.00–0.02; P = 0.366 and mean reduction, 0.00 μmol/L; 95% CI, −0.01–0.01; P = 0.349, respectively). No significant between-group differences were found in FMD (mean difference, −0.2%; 95% CI, −1.4–0.9; p = 0.708) or serum ADMA levels (mean difference, 0.01 nmol/L; 95% CI, −0.00–0.03; p = 0.122). Significant improvements in the average probing pocket depth were observed in the control and test groups. The bleeding on probing score in the test group was significantly reduced, while that in the control group was reduced, although not significantly. Periodontal care for a 3-month duration did not provide better endothelial function although improvements of periodontal status in patients with early-stage periodontal diseases. This trial is registered in UMIN Clinical Trials Registry (www.umin.ac.jp/ctr/; ID: UMIN000023395).

Introduction

Although a significant association between periodontal disease and atherosclerotic cardiovascular disease (ACVD) has been reported, the cause-to-effect relationship between them remains controversial [1]. The entry of oral bacteria, including periodontal pathogens and/or their products into the bloodstream, is common regardless of the periodontal status [2]. The immune response following persistent bacteremia from periodontal lesions may lead to ACVD [3]. Furthermore, it has also been suggested that inflammatory mediators attributable to periodontal diseases are associated with ACVD [4–6]. However, the verification of the hypothesis and identification of periodontal care effectiveness against ACVD require well-designed interventional studies.

Endothelial dysfunction occurs in the early stages of atherosclerosis and can be assessed by measuring the flow-mediated dilatation (FMD) of the brachial artery [7, 8]. Nitric oxide (NO), which is synthesized by vascular endothelial cells, is a primary contributor to FMD [7]. Noninvasively-measured FMD is considered a marker of vascular damage and predictor of future cardiovascular events [7–9]. Therefore, FMD appears to be an appropriate surrogate marker of periodontal care effectiveness against ACVD.

Observational studies have shown that periodontal diseases are associated with FMD [10]. Furthermore, some interventional studies without controls reported that the treatment of severe periodontitis improved FMD [11–14]. Intensive periodontal treatment significantly improved FMD in a subject without systemic diseases in one randomized controlled clinical trial (RCT) [15]; whereas, no significant effects were observed in a patient with stable coronary artery disease in another RCT [16]. It was reported that intensive nonsurgical periodontal treatment significantly improved FMD in patients with hypertension in a recent RCT [17]. The relationship between the periodontal condition and FMD appears to be affected by complex factors, including severity of the periodontal condition, systemic condition, and contents of the treatment. Further RCTs are required to indicate the causative association between periodontal diseases and specific ACVD events and the usefulness of periodontal care to improve endothelial function.

Periodontal diseases are prevalent worldwide, with an estimated large majority of cases ranging in severity from the initial stages to more advanced conditions [18]. Bacteremia, which can induce ACVD, routinely occurs even in the early stage of periodontal disease [2, 19]. Research findings covering the severity may make a major impact on patient behavior for ACVD prevention. Daily self-care and professional care are important for periodontal treatment and prevention. Previous studies have shown that advanced periodontal self-care using a custom-made tray application of a disinfectant effectively decreases periodontal tissue inflammation [20–22]. Therefore, we hypothesized that advanced self-care improves endothelial function. This randomized clinical trial aimed to investigate the effect of advanced self-care on ACVD-related vascular function markers. Our null hypothesis was that there was no difference in FMD between advanced self-care and standard care in patients with early-stage periodontal disease.

Materials and methods

Trial design

This parallel group (1:1), 3-month follow-up, open-label, RCT was conducted at the Tsurumi University Dental Hospital in Yokohama, Japan, and the Ariyoshi Dental Clinic in Tokyo, Japan, between August 2016 and April 2018 by a single team composed of dentists, dental hygienists, and nurses. The dental hospital and clinic were well equipped to provide standard care for the initial periodontal preparations. All participants chose the trial site at their convenience.

This study was conducted in accordance with the ethical principles of the Declaration of Helsinki. The study protocol was approved by the Ethics Committee of the Tsurumi University School of Dental Medicine (No. 1413) and registered in the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR: UMIN000023395). All participants provided written consent before the study began.

Participants

Participants were recruited between August 2016 and November 2016 from various sources, including advertisements in social networking services, the Tsurumi University Dental Hospital, and two private dental clinics in Tokyo, Japan. Between November 2016 and February 2017, 150 volunteers were assessed for eligibility in accordance with the inclusion and exclusion criteria. The inclusion criteria were an age range of 20–70 years, having ≥20 functioning teeth, and diagnosis of chronic periodontitis with ≥2 sites with bleeding on probing (BOP) or a probing pocket depth (PPD) of ≥4 mm at ≥1 site. The exclusion criteria were difficulties traveling alone to the institution, the need for periodontal surgery and/or prosthodontic treatment, the presence of an untreated carious cavity, the presence of a partially impacted tooth, having used systemic antibiotics within the previous 3 months, the regular use of medications for any other chronic diseases, the presence of diseases requiring higher-priority treatment than periodontitis, and participation in another clinical trial during the study period.

Interventions

All periodontal patients received standard care for periodontal diseases under local anesthesia if required, including same-day full-mouth scaling between April 2017 and January 2018. Patients who underwent standard care alone were assigned to the control group. Eleven care providers (Authors: R.O., M.S., H.A., H.U., A.I., Non-authors: refer to Acknowledgments; T.K., E.S., N.A., Y.U., M.E., J.T.) with >5 years of experience in dental practice discussed and standardized the procedures of the care before the trial began. Patients were randomly assigned to each care provider who conducted their care in a similar fashion using an ultrasonic scaler (Varios970; Nakanishi, Tochigi, Japan), hand instruments (FP scaler; Feed, Yokohama, Japan), and dental floss (Reach No-waxed; Johnson & Johnson, Tokyo, Japan) with a dental plaque-disclosing agent (Merssage PC Pellet Blue; Shofu, Kyoto, Japan) to visualize and remove dental plaque biofilms. After the complete removal of the plaque, all teeth surfaces were polished with a polishing paste (PTC Paste Fine/PTC Paste Regular; GC, Tokyo, Japan) using a rotating rubber cup (FP rubber; Feed, Yokohama, Japan) and/or rotating brush (FP profy brush; Feed, Yokohama, Japan). Complete plaque removal was confirmed with the repetitive application of the disclosing agent. No time restriction was imposed on the procedure. One dentist (T.M.) who was not a part of the care-providing team confirmed the fulfillment of the standardized care program and subsequently provided nutritional and exercise guidance in addition to basic oral hygiene instructions. Each patient was given the same toothpaste (Check-up standard; Lion, Tokyo, Japan) and toothbrush (Ci 202 premium; Ci Medical, Hakusan, Japan) every month during the trial and was instructed to perform standard self-care at least twice (in the morning and at bedtime) a day.

For patients assigned to the test group, the use of hypochlorous acid water, an electrolyzed disinfectant for dental use (MeDeSPro; Medoc International, Tokyo, Japan), with a custom-made prescription tray was introduced as an extra intervention in addition to standard self-care. The patients were instructed on the use of these materials after standard care for periodontal diseases under the guidance of one dentist (T.M.). The process of manufacture of the tray have been described previously [23] and is described below.

The inside of the tray was covered with gauze. Each patient was instructed to fill the tray with approximately 3 mL of disinfectant and then apply it to the tooth and gingival surfaces for 3 min after each standard self-care (twice a day). They were instructed not to eat or drink within 30 min after using disinfectants. We considered a 70% implementation rate as successful advanced periodontal self-care.

We asked all patients in both groups to keep a record of the frequency of oral self-care, general health condition, and drug being taken in a diary throughout the trial.

Custom-made tray

Maxillary and mandibular dentition impressions were taken from each patient, and a dental working model using dental plaster (New Plastone II; GC, Tokyo, Japan) was prepared. The custom-made trays were manufactured at a contract dental laboratory (Shiken, Tokushima, Japan or Oral Bio Design, Shiga, Japan). Gauze and polypropylene sheets stacked in pairs were vacuum-adapted to each model using a vacuum-forming machine (Biostar; Scheu Dental, Iserlohn, Germany). Each tray was trimmed to an extension of approximately 5.0 mm above the maximum convexity of the jawbone, and inner gauze was trimmed to an extension of approximately 2.0 mm above the gingival margin (S1 Fig).

Outcome

The primary outcome was the occurrence of changes in FMD between baseline and 3 months after the start of the intervention. The change in serum asymmetric dimethylarginine (ADMA; an endogenous NO synthase inhibitor) levels was considered a secondary outcome. Elevated serum ADMA levels are thought to impair endothelial function and, promoting atherosclerosis as a result [24].

Sample size

We employed a priori power analysis for sample size calculation. Based on a previous study [15], we calculated that a minimum sample size of 90 was needed to detect a 1% difference in FMD between the two groups, with a standard deviation of the mean difference of 1.67% at a 2-sided α error of 0.05 and 80% power. Assuming a 20% dropout rate, 110 subjects were enrolled.

Randomization

The eligible patients were allocated randomly to the test group or control group via the envelope method stratified by the smoking habit, which is a common risk factor for ACVD and periodontal disease. Almost all participants wished to be allocated to the test group. Therefore, we used the following procedures for the allocation. We prepared 110 brown, standard, and non-labeled envelopes (29 for smokers and 81 for nonsmokers) containing a label with an Arabic number (1 to 29 for smokers and 1 to 81 for nonsmokers). All the researchers confirmed that no one could identify the number from the outside of each sealed envelope. We explained to all patients during eligibility assessment that the odd- and even-numbered labels were allocated to the control and test groups, respectively, and the custom-made tray would be manufactured for all control patients who desired one after their own trial. After eligibility assessment, each patient selected as a study subject made an appointment for random allocation at their convenience. Patients drew the envelope by themselves from the “smoker” or “nonsmoker” labeled box as required, and opened the envelope in front of us. Balloting was done on a first-come, first-served basis. The allocated Arabic number was particular for each subject, and the number was never used again. No one declined trial participation because of the allocation.

Periodontal examination

A single examiner (A.O.) collected the following clinical data from six sites (mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid- lingual, and disto-lingual) around each tooth using a color-coded periodontal probe (PO-9; Nippon Shiken, Tokyo, Japan). PPD was measured from the free gingival margin to the base of the pocket. The BOP was considered positive if a site bled within 20 s after gentle probing (25 g probing force). The average PPD and percentage of BOP sites (BOP score = number of sites with BOP/total number of sites × 100) were calculated for each patient.

FMD of the brachial artery

FMD was assessed using a high-resolution ultrasonography system (Unexef18G; UNEX, Nagoya, Japan) in an air-conditioned room. Each patient was instructed to refrain from physical activity, caffeine-containing foods, and smoking for at least 2 h before the examination. Blood pressure and heart rate were measured before measuring FMD. The examination began after a 10-min rest with the patient in the supine position and the arm placed comfortably. The left brachial artery was scanned with an ultrasound probe at 10 MHz for longitudinal and transverse windows. After recording the baseline image, the cuff that was placed distal to the ultrasound probe was inflated to 50 mmHg above systolic blood pressure for 5 min and then deflated. The post-deflation arterial image was recorded for 2 min. FMD was defined as the maximal percentage change in vessel diameter from the baseline value. All measurements were performed by a single operator (A.O.).

ADMA measurement

Right antecubital vein blood samples were placed into collection tubes containing serum separator gel and immediately centrifuged. The supernatants were frozen rapidly at -30°C for later analysis. Serum ADMA levels were measured at a contract laboratory (SRL, Tokyo, Japan) by high-performance liquid chromatography equipped with a fluorescence detector for excitation at 348 nm and emission at 450 nm with an octadecyl-silica column using derivatization with o-phthalaldehyde for fluorescent determination.

Statistical analysis

All analyses were performed based on the intention-to-treat principle. Missing data were managed using the baseline-observation-carried-forward approach in accordance with the existing guidelines [25]. A per-protocol analysis was also performed without imputation. The patients who took antibiotics during the trial were excluded from per-protocol analysis. Data are expressed as means and 95% confidence intervals (CIs) unless otherwise specified. The distribution of each continuous variable was checked for normality using the Shapiro–Wilk test. Levene’s test was performed for homogeneity of the variances when normality was confirmed. Paired statistical tests (paired Student’s t-test or Wilcoxon signed-rank test) were used to compare the difference in parameters obtained at baseline and endpoint in the same group, whereas unpaired tests (Student’s t-test, Welch’s t-test or Mann–Whitney U test) were used to compare differences between the control and test groups. The correlation between two parameters was assessed using Spearman’s rank correlation coefficient. All tests were 2-tailed, and values of p < 0.05 were considered statistically significant. All analyses were performed using JMP Ver. 12 (SAS Institute, Cary, NC, USA).

Results

Participant flow

The flow diagram of the trial is presented in Fig 1. Forty volunteers were excluded from the trial. The remaining 110 patients were allocated randomly to the control (n = 56) and test (n = 54) groups. The baseline characteristics of the patients were similar between the groups (Table 1). Forty-three patients in the test group achieved ≥70% advanced self-care implementation (mean, 85.1%; minimum, 34.7%; maximum, 100%). Control patients received the intervention immediately after allocation, whereas the test group began the intervention 2 weeks after allocation because of the need for custom-made tray manufacture. No severe adverse events were observed during the trial.

Fig 1. Flow diagram.

Fig 1

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IQR, interquartile range; TUH, Tsurumi University Dental Hospital; ADC, Ariyoshi Dental Clinic; ITT, intention-to-treat; PP, per-protocol.

Table 1. Baseline characteristics of the patients.

Characteristic Control (n = 56) Test (n = 54)
Age* (years), median (IQR) 37 (29–43) 38 (32–45)
Male sex, No. (%) 30 (54) 36 (67)
Smoker, No. (%) 14 (25) 13 (24)
Existing teeth (No.), median (IQR) 28 (27–30) 28 (28–29)
Average PPD (mm), median (IQR) 2.1 (1.9–2.4) 2.0 (1.8–2.1)
BOP score (%), median (IQR) 10.8 (5.9–20.0) 9.5 (4.6–15.9)
Systolic BP (mmHg), mean ± SD 119.6 ± 10.9 120.4 ± 14.7
Diastolic BP (mmHg), mean ± SD 70.4 ± 9.3 73.4 ± 11.6
Brachial artery diameter (mm), mean ± SD 3.4 ± 0.8 3.6 ± 0.7
FMD (%), mean ± SD 5.9 ± 3.0 5.8 ± 2.9
ADMA (nmol/L), median (IQR) 0.36 (0.32–0.39) 0.34 (0.30–0.37)
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IQR, interquartile range; No., number; PPD, periodontal pocket depth; BOP, bleeding on probing; SD, standard deviation; FMD, flow-mediated dilatation of the brachial artery; ADMA, serum asymmetric dimethylarginine level.

Vascular function

An improvement in FMD of at least 1% difference was not achieved between the groups. Table 2 presents the results of FMD and serum ADMA levels obtained at the endpoint and improvement by the intervention in the intention-to-treat analysis. The change in FMD at endpoint did not differ significantly between the control and test groups. No significant improvements in FMD were observed in the control or test groups. No significant between-group difference was detected (mean difference, −0.2%; 95% CI, −1.4–0.9; p = 0.708, unpaired t-test) (S2 Fig). The change in serum ADMA levels at endpoint differed significantly between the control and test groups. No significant improvements in serum ADMA levels were observed in either control group or test group, with no significant between-group difference observed (mean difference, 0.01 nmol/L; 95% CI, −0.00–0.03; p = 0.122, Mann–Whitney U test) (S2 Fig). The result in the per-protocol analysis was also similar (S1 Table and S3 Fig). FMD was not correlated with serum ADMA levels (r = 0.095, p = 0.172).

Table 2. Assessment of vascular function at 3 months after the start of the intervention.

Endpoint
Control
(n = 56)		 Test
(n = 54)		 Mean difference
(95% CI)		 p-value
FMD (%), mean ± SD 5.8 ± 2.4 5.5 ± 2.3 −0.3 (−1.2–0.5) 0.436
ADMA (nmol/L), median (IQR) 0.36 (0.33–0.38) 0.33 (0.31–0.38) −0.03 (−0.05–−0.01) 0.008
Improvement
Control
(n = 56)		 Test
(n = 54)
Mean difference
(95% CI)		 p-value Mean difference
(95% CI)		 p-value
FMD (%) −0.1 (−1.0–0.8) 0.805 −0.3 (−1.1–0.4) 0.398
ADMA (nmol/L) 0.01 (−0.00–0.02) 0.366 −0.00 (−0.01–0.01) 0.349
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P-values were calculated using the unpaired t-test (FMD) or the Mann–Whitney U test (serum ADMA level) for group differences at endpoint and the paired Student’s t-test (FMD) or Wilcoxon’s signed-rank test (serum ADMA level) for changes from baseline. FMD, flow-mediated dilatation of the brachial artery; ADMA, serum asymmetric dimethylarginine level; SD, standard deviation; IQR, interquartile range; CI, Confidence interval.

Periodontal condition

Table 3 presents the results of periodontal status obtained at endpoint and improvement by the intervention in the intention-to-treat analysis. The change in Mean PPD or BOP at endpoint did not differ significantly between the control and test groups. Significant improvements in the average PPD were observed in the control and test groups. The BOP score in the test group was significantly reduced, while that in the control group was reduced, although not significantly. No significant between-group differences were found in the improvement of the mean PPD (mean difference, −0.0 mm; 95% CI, −1.1–0.1; p = 0.820, Mann–Whitney U test) or BOP score (mean difference, −0.1%; 95% CI, −3.1–2.9; p = 0.955, Mann–Whitney U test). The result in the per-protocol analysis was also similar (S2 Table).

Table 3. Periodontal status at 3 months after the start of the intervention.

Endpoint
Control
(n = 56)		 Test
(n = 54)		 Mean difference
(95% CI)		 p-value
Mean PPD (mm), median (IQR) 1.9 (1.8–2.1) 1.9 (1.8–2.0) −0.1 (−0.2–−0.0) 0.072
BOP (%), median (IQR) 9.7 (3.7–17.2) 8.2 (3.5–15.4) −2.2 (−6.1–1.7) 0.686
Improvement
Control
(n = 56)		 Test
(n = 54)
Mean difference
(95% CI)		 p-value Mean difference
(95% CI)		 p-value
Mean PPD (mm) 0.2 (0.1–0.2) <0.001 0.2 (0.1–0.2) <0.001
BOP (%) 1.8 (−0.8–4.4) 0.052 1.7 (0.1–3.2) 0.041
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The Mann–Whitney U test was used for comparisons between groups at endpoint. The Wilcoxon signed-rank test† was used for comparison with baseline. PPD, periodontal pocket depth; BOP, bleeding on probing; IQR, interquartile range; CI, Confidence interval.

Harms

No study-related serious adverse events occurred in any of the study participants. Nonsurgical periodontal treatment is basically low-risk. None of the participants required any dental therapy during the study.

Discussion

Advanced periodontal self-care for three months did not significantly improve FMD as compared to standard care. The findings were similar in the intention-to-treat and the per-protocol analysis. Therefore, our null hypothesis ‘there was no difference in FMD between advanced self-care and standard care in patients with early-stage periodontal disease’ was not rejected.

The immune response following persisting bacteremia from periodontal lesions reportedly leads to ACVD regardless of the severity of the periodontal condition [2, 3, 19]. In addition, the production of inflammatory mediators attributable to periodontal diseases might be associated with ACVD [4–6]. Reduced FMD is associated with ACVD risk and improves with risk-reduction therapy [26]. Consequently, the endothelial function has been defined as an “excellent barometer” of vascular health [27]. FMD assessing vascular endothelial function might be ideal for exploring factors associated with the improvement of vascular health. Based on these suggestions, several studies have described the efficacy of periodontal care for FMD [11–14]. However, a limited number of RCTs have been conducted. A few RCTs reported the effectiveness of intensive periodontal treatment [15, 17], whereas others did not identify significant changes [16]. The discrepancy among RCTs might be attributable to differences in systemic characteristics between patients. The characteristics were population-specific, such as the presence of coronary artery disease or hypertension or the absence of systemic disease. In addition, the severity of the periodontal disease and the contents of the treatment also appeared to differ. We set the inclusion and exclusion criteria such that they emphasized the systemic health and periodontal status of the subjects. Therefore, we considered the study population to have good systemic health and early-stage periodontal disease requiring no surgical periodontal treatment. Despite the improved periodontal conditions observed in both groups, no significant increase in FMD was demonstrated in this trial. Periodontal treatment might be effective for improving FMD in patients with more severe periodontal disease than in patients with early-stage periodontal diseases. The main contents of standard care at the clinic are biofilm removal. Employment of more effective methods for the biofilm removal may affect the improving FMD through delayed biofilm regrowth rates [28].

We also selected serum ADMA levels as a secondary outcome. ADMA is a naturally occurring endogenous inhibitor of NO synthase [29–31]. It reduces NO production; consequently, it can lead to endothelial dysfunction and cardiovascular events [24, 32–34]. We hypothesized that the inhibition of the entry of oral bacteria into the bloodstream through periodontal care would lead to FMD improvement through the inhibition of ADMA synthesis. However, no significant decrease in serum ADMA levels from baseline was observed in spite of significant difference between the groups at endpoint. No significant correlation between FMD and serum ADMA levels was demonstrated in this trial; however, an inverse association between FMD and plasma ADMA levels has been reported in young Finns [35] and in subjects at low cardiovascular risk [36]. The reason for the discrepancy between the trials may be the underlying factors characterizing the study population which may be affecting FMD and/or plasma ADMA levels. Further investigations may be required to identify the factors in healthy subjects.

When mentioning the association between the periodontal condition and ACVD, appropriate periodontal parameters should be selected carefully to ensure a precise evaluation of the periodontal status. PPD and BOP represent the degree of swelling in the gingiva and the existence of a lesion at the periodontal pocket base, respectively. Changes in PPD and the number of BOP sites directly reflect changes in the periodontal status. Therefore, the mean PPD and number of BOP sites appeared to be appropriate indices for similar studies to portray an improvement and/or a deterioration of the periodontal status. The mean PPD and number of BOP sites improved in both study groups in this trial. On statistical analysis, the improvement in the mean PPD was significant in both groups when comparing the data before and after the intervention. Given that significant between-group differences were not found in the average PPD or number of sites with BOP, we could not conclude that advanced self-care was superior to standard care alone in this trial. Health behavior changes appeared to have a major role in the improvements in addition to periodontal treatment in the hospital or clinic. Gentle instructions and training with an adequate explanation given by oral care providers would probably result in health behavior changes and sincere efforts to continue self-care on a daily basis among almost all patients.

Limitations

This trial had some limitations. The first limitation was the lack of blinding. Given the nature of the intervention and control, we could not mask care providers and subjects to treatments; therefore, our results may reflect expectation bias. Second, the assessment of adherence to advanced self-care depended on a self-reported individual diary. Although we considered 70% implementation as successful advanced periodontal self-care in this trial, the selection of the minimal care frequency should remain a consideration. Third, the 3-month follow-up period was chosen based on a previous study [12–15] and selected in view of the burden on subjects. A longer period, which enables further improvement in bacteremia, might be required to alter endothelial function in patients with mild-to-moderate periodontal diseases.

Conclusion

Advanced periodontal self-care for three months did not significantly improve FMD as compared to standard care in patients with early-stage periodontal diseases. Neither care significantly improved FMD despite its effectiveness in improving the periodontal status.

Supporting information

S1 Checklist. CONSORT non-pharmacologic treatment extension checklist.

(DOCX)

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S1 File. Trial protocol.

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S1 Fig. Custom-manufactured tray.

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S2 Fig. Effect of periodontal care on vascular function in the intention-to-treat analysis.

Box and whisker plot of FMD (A) and serum ADMA (B) levels by group. The box contains values between the 25th and 75th percentiles (central line, median). Vertical lines represent the minimum and maximum. P-values were calculated using the paired Student’s t-test (FMD) or Wilcoxon’s signed-rank test (serum ADMA level) for changes from baseline and the unpaired t-test (FMD) or the Mann–Whitney U test (serum ADMA level) for group differences. FMD, brachial artery dilatation; AMDA, asymmetric dimethylarginine.

(TIF)

Click here for additional data file. (156.9KB, tif)
S3 Fig. Effect of periodontal care on vascular function in the per-protocol analysis.

Box and whisker plot of FMD (A) and serum ADMA levels (B) by group. The box contains values between the 25th and 75th percentiles (central line, median) of serum ADMA levels. Vertical lines represent the minimum and maximum. P-values were calculated using the paired Student’s t-test (FMD) or Wilcoxon’s signed-rank test (serum ADMA level) for changes from baseline and the unpaired t-test (FMD) or the Mann–Whitney U test (serum ADMA level) for group differences. FMD, brachial artery dilatation; AMDA, asymmetric dimethylarginine.

(TIF)

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S1 Table. Assessment of vascular function in the per-protocol analysis.

(DOCX)

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S2 Table. Periodontal status in the per-protocol analysis.

(DOCX)

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Acknowledgments

The authors are grateful to the staff of the Ariyoshi Dental Clinic and the Kioicho Plaza Clinic for their invaluable help with the recruitment process. The authors would also like to thank Ms. Tomomi Kurashita, Ms. Eri Sato, Ms. Nozomi Aiba, Ms. Yurie Uno., Ms. Mitsuko Endo, and Ms. Juli Tomitani for their oral health care provision to the patients and Dr. Junko Kobayashi, Ms. Masumi Kawauchino, Ms. Chiemi Honda, and Ms. Hideko Aratani for their contribution in collecting data on serum ADMA levels.

Data Availability

The data contain potentially sensitive patient information. The Ethics Committee of the Tsurumi University School of Dental Medicine prohibits the preservation of any data set in a public repository (even if the data are de-identified), the preservation of hard copy data in an unlocked cabinet, the preservation of electronic data in a computer connected to the internet. All the subjects provided informed consent on the condition of our adherence to IRB guidelines. The datasets generated and/or analyzed during the current study are available from Dr. Hidenori Yamada (yamada-h@tsurumi-u.ac.jp) or Dr. Ayako Okada (okada-a@tsurumi-u.ac.jp) on reasonable request. The Ethics Committee of the Tsurumi University School of Dental Medicine 2-1-3, Tsurumi, Yokohama, 230-8501 Japan Email Address: kyoken@tsurumi-u.ac.jp.

Funding Statement

This work was supported by the Japan Society for the Promotion of Science (JSPS) [Grant numbers 16K20704 (MA), 17K12031 (AO), 17K11688 (KM), 18K09926 (NH), 19K10471 (TM), 19K19339 (RO)] and the SECOM Science and Technology Foundation [Grant number 2018.09.10 No. 1 (NH)]. Japan Society for the Promotion of Science: https://www.jsps.go.jp/ SECOM Science and Technology Foundation: https://www.secomzaidan.jp/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Andrej M Kielbassa

2 Jul 2021

PONE-D-21-11565

Effect of advanced periodontal self-care in patients with early-stage periodontal diseases on endothelial function: An open-label, randomized controlled trial

PLOS ONE

Dear Dr. Murata,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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[Dr. Khairul Matin and Dr. Nobuhiro Hanada received research grant from Medoc International Co. Ltd. Dr. Nobuhiro Hanada received a research grant from Shiken Corp.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Abstract

- Remember to provide as much as information as possible here. Plos One accepts 300 words with this section, and you should enrich your information.

- With your conclusions in mind, please revise carefully, and see comments given below.

Intro

- Aims and objectives have been satisfyingly elaborated.

- Please note that this section must not provide what you have done (but, instead, what you were going to do). Phrases like "In an RCT, we determined the effect (...)." or "We attempted to advance (...)." must be carefully revised.

- Please provide a reasonable null hypothesis. Remember that H0 must be deducible from the foregoing thoughts.

Meths

- What is meant when referring to "whole-mouth scaling and root planning"? Performed in one visit? Please clarify.

- Please refer to the CONSORT statement with your full text.

- Additionally, please stick to the SQUIRE guidelines. You are reporting new knowledge about how to improve healthcare, right?

- This was an intention-to-treat study, right? Please refer to the respective analysis.

- "ultrasonic scaler", "hand instruments", "dental plaque-disclosing agent", "polishing paste", "rotating rubber cup", "rotating brush", and so on: Please note with ALL materials (including chemicals) and methodologies (including statistical software), please use general names with your text, followed by (brand name; manufacturer, city, STATE (abbreviated, if US), country) in parentheses. Stick to semicolon. Revise thoroughly throughout your text.

- Remember that reproducibility is the cornerstone of scientific advancement. Outputs like exact methodology protocols empower researchers to go one step further in contextualizing their work to ensure it remains replicable. This section has not been satisfying elaborated.

- Do not use legal terms with your text. Delete "Corporation", "Corp.", "Co. Ltd.", "Co.", "Ltd.", "Inc.",

- "A single examiner collected (...)." Please provide initials in parentheses.

- "−30°C" must read "-30 °C". Use minus/hyphen instead of dash. The unit is "°C", and must be separated from the number.

- Report exact p-values for all values greater than or equal to 0.001 (note the 3-digit basis). P-values less than 0.001 may be expressed as p < 0.001. Remember to use lowercase letter p. See Authors' Guidelines, and revise thoroughly.

Results

- Again, double check and revise p values.

Disc

- Stick to H0 when staring this section. Remember that H0 can be rejected or not rejected.

- This section would seem perfectible, please add more discursive thoughts on your outcome.

Concl

- You aimed to "determine the effect of advanced periodontal self-care in patients with early-stage periodontal diseases on endothelial function". Additionally, you "attempted to advance periodontal self-care in patients with early-stage periodontal disease to determine any effect on ACVD-related vascular function markers FMD and ADMA." Hence, please adapt your conclusion ("Advanced periodontal self-care, in addition to standard care, did not result in better vascular function in patients with mild-to-moderate periodontal diseases in this trial.") to your aims. Do not simply repeat your results, but provide a reasonable extension of your outcome.

In total, this submitted draft would seem interesting, is considered easily intelligible, and should be worth following after revision. This paper is ready for external review.

Reviewer #2: In the manuscript entitled: “Effect of advanced periodontal self-care in patients with early-stage periodontal diseases on endothelial function: An open-label, randomized controlled trial”, the authors identified the effects of advanced periodontal selfcare on endothelial function in patients with early-stage periodontal disease. The study was designed as a parallel group, 3-month follow-up, open-label, randomized controlled trial for evaluating the effectiveness of periodontal care against atherosclerotic cardiovascular disease.

The authors found that both groups demonstrated improved periodontal status. No significant improvements in FMD were observed in the control or test group. No significant changes in serum ADMA levels were observed. No significant between-group differences were found in FMD or serum ADMA levels.

The authors concluded that periodontal care for a 3-month duration did not provide better endothelial function in patients with early-stage periodontal diseases.

Major comments:

In general, the idea and innovation of this study, regards analysis of eriodontal self-care in patients with early-stage periodontal diseases on endothelial function is interesting, because the role of these factors in dentistry are validated but further studies on this topic could be an innovative issue in this field could be open a creative matter of debate in literature by adding new information. Moreover, there are few reports in the literature that studied this interesting topic with this kind of study design.

The study was well conducted by the authors; However, there are some concerns to revise that are described below.

The introduction section resumes the existing knowledge regarding the important factor linked with periodontal inflammation.

However, as the importance of the topic, the reviewer strongly recommends, before a further re-evaluation of the manuscript, to update the literature through read, discuss and must cites in the references with great attention all of those recent interesting articles, that helps the authors to better introduce and discuss the role of periodontitis and related biomarkers as cause of implant failure (Galectin, NLRP3): 1) Isola G, Polizzi A, Alibrandi A, Williams RC, Lo Giudice A. Analysis of galectin-3 levels as a source of coronary heart disease risk during periodontitis. J Periodontal Res. 2021 Jun;56(3):597-605. doi: 10.1111/jre.12860. 2) Isola G, Polizzi A, Santonocito S, Alibrandi A, Williams RC. Periodontitis activates the NLRP3 inflammasome in serum and saliva. J Periodontol. 2021 May 19. doi: 10.1002/JPER.21-0049. 3) Isola G, Lo Giudice A, Polizzi A, Alibrandi A, Murabito P, Indelicato F. Identification of the different salivary Interleukin-6 profiles in patients with periodontitis: A cross-sectional study. Arch Oral Biol. 2021 Feb;122:104997. doi: 10.1016/j.archoralbio.2020.104997.

The authors should be better specified, at the end of the introduction section, the rational of the study and the aim of the study. In the material and methods section, should better clarify randomization and periodontal examination. Moreover, please more specifiy the clinicians involved in the different stages of the stusy.

The discussion section appears well organized with the relevant paper that support the conclusions, even if the authors should better discuss the relationship between periodontitis and endotelial dysfunction. The conclusion should reinforce in light of the discussions.

In conclusion, I am sure that the authors are fine clinicians who achieve very nice results with their adopted protocol. However, this study, in my view does not in its current form satisfy a very high scientific requirement for publication in this journal and requests a revision before a futher re-evaluation of the manuscript.

Minor Comments:

Abstract:

- Better formulate the abstract section by better describing the aim of the study

Introduction:

- Please refer to major comments

Discussion

- Please add a specific sentence that clarifies the results obtained in the first part of the discussion

- Page 16 last paragraph: Please reorganize this paragraph that is not clear

Reviewer #3: 1. Sample size calculation needs more details. What is the mean (SD) of each group at each time point? What test was used?

2. Linear mixed models (repeated ANOVA) may be considered for analyzing the outcomes. If data/model residuals follow normal distributions, it is more powerful to use parametric methods.

3. Results:

a. Table 1: add p values. Specify methods used for comparison in the footnote.

b. Periodontal condition: move this section after “Vascular function” as they are secondary outcomes. Show median (IQR) for each group at each time point, not just improvement. Add all p values for within-group comparisons and between-group comparisons.

c. Vascular function: add a table with detailed statistics, e.g. mean (SD) /median (IQR) for each group at each time point. Add all p values for within-group comparisons and between-group comparisons. Need to perform a consistency analysis for ITT and per-protocol also.

d. Figure 2: using boxplot for both outcomes. Specify clearly which methods were used for generating the p values.

e. Line 275 is not a complete sentence.

Consort checklist:

1) Outcomes are clearly defined.

2) Sample size needs more details for mean (SD) in each group, not mean difference (SD). What test was used?

3) Method used to generate random allocation sequence was not mentioned.

4) Allocation concealment was clearly described.

5) Blinding is not available.

6) Details of outcomes and estimation were not provided. Need means (SD) for each group at each time point and mean different (with 95% CI). A table is necessary. Effect size was not provided either.

7) Important harms or unintended effects were not discussed.

8) The registry and the registration number were reported.

9) The trial protocol is attached.

10) Sources of funding was described.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Sep 23;16(9):e0257247. doi: 10.1371/journal.pone.0257247.r002

Author response to Decision Letter 0

22 Jul 2021

Response to Reviewers

We are grateful to each reviewer for the valuable feedback and critical comments, which have helped us improve our manuscript considerably. As indicated in the responses that follow, we have taken all these comments and suggestions into account in the revised version of our manuscript.

Point-by-point responses to Reviewer #1

Abstract

Comment:

- Remember to provide as much as information as possible here. Plos One accepts 300 words with this section, and you should enrich your information.

- With your conclusions in mind, please revise carefully, and see comments given below.

Response:

Based on this comment, we have added the sentences of the background and aim of the study in the revised manuscript (Lines 33–41). The word count of the Abstract is now 295 words.

We have taken all the Reviewer #1's comments and suggestions into account in the revised version of our manuscript.

Intro

Comment:

- Aims and objectives have been satisfyingly elaborated.

Response:

Thank you for your positive evaluation.

Comment:

- Please note that this section must not provide what you have done (but, instead, what you were going to do). Phrases like "In an RCT, we determined the effect (...)." or "We attempted to advance (...)." must be carefully revised.

Response:

Based on this comment, we have replaced “Therefore, advanced self-care may contribute to improving FMD. In an RCT, we determined the effect of advanced periodontal self-care in patients with early-stage periodontal diseases on endothelial function. FMD was set as the primary outcome, while serum asymmetric dimethylarginine (ADMA; an endogenous NO synthase inhibitor) level was the secondary outcome. Elevated serum ADMA is thought to impair endothelial function and, thus, promote atherosclerosis [20]. We attempted to advance periodontal self-care in patients with early-stage periodontal disease to determine any effect on ACVD-related vascular function markers FMD and ADMA.” in the original manuscript (lines 82–90) with “Therefore, we hypothesized that advanced self-care improves endothelial function. This randomized clinical trial aimed to investigate the effect of advanced self-care on ACVD-related vascular function markers.” in the revised manuscript (Lines 89–91).

Comment:

- Please provide a reasonable null hypothesis. Remember that H0 must be deducible from the foregoing thoughts.

Response:

Based on this comment, we have added the following sentence:

“Our null hypothesis was that there was no difference in FMD or serum ADMA level between advanced self-care was compared and standard care in patients with early-stage periodontal disease” (Lines 91–92).

Meths

Comment:

- What is meant when referring to "whole-mouth scaling and root planning"? Performed in one visit? Please clarify.

Response:

We would like to mean “whole-mouth scaling in a single visit”. Based on this comment, we have replaced “whole-mouth scaling” in the original manuscript (Line 121) with “same-day full-mouth scaling” in the revised manuscript (Line 123).

The “same-day full-mouth root planning” was used in the title of a clinical trial report*.

We have deleted “root planning”. No subjects needed to receive root planning because of their low severity of periodontal diseases.

* Apatzidou DA and Kinane DF. Quadrant root planing versus same-day full-mouth root planing. I. Clinical findings. J Clin Periodontol. 2004; 31: 132-140. doi: 10.1111/j.0303-6979.2004.00461.x.

Comment:

- Please refer to the CONSORT statement with your full text.

Response:

We have reviewed overall manuscript to adjust it according to the CONSORT statement and replaced with “S1 CONSORT NPT Extension Checklist”.

Comment:

- Additionally, please stick to the SQUIRE guidelines. You are reporting new knowledge about how to improve healthcare, right?

Response:

Thank you for your advice. However, we are not focusing the improvement of the quality of healthcare. The effectiveness of oral care has not been established on the improvement of endothelial function. Therefore, we must clarify the effectiveness of oral care prior to the evaluation of quality improvement in this study. The reason for setting the standard care group as the control group is due to ethical decision. Given that all the subjects were diagnosed with early-stage periodontal diseases, they have a right to receive standard care. We would like you to understand that we are not reporting the improvement of the quality of healthcare.

Comment:

- This was an intention-to-treat study, right? Please refer to the respective analysis.

Response:

Based on this comment, we have added revised S1 Table, S2 Table, and S3 Figure showing per-protocol analysis on vascular function and periodontal status in the revised manuscript, respectively.

Comment:

- "ultrasonic scaler", "hand instruments", "dental plaque-disclosing agent", "polishing paste", "rotating rubber cup", "rotating brush", and so on: Please note with ALL materials (including chemicals) and methodologies (including statistical software), please use general names with your text, followed by (brand name; manufacturer, city, STATE (abbreviated, if US), country) in parentheses. Stick to semicolon. Revise thoroughly throughout your text.

- Remember that reproducibility is the cornerstone of scientific advancement. Outputs like exact methodology protocols empower researchers to go one step further in contextualizing their work to ensure it remains replicable. This section has not been satisfying elaborated.

Response:

Based on this comment, we have reviewed overall the manuscript and added the required information about the equipment.

“ultrasonic scaler (Varios970; Nakanishi, Tochigi, Japan)” (Line 129).

“hand instruments (FP scaler; Feed, Yokohama, Japan)” (Line 129).

“dental floss （Reach No-waxed; Johnson & Johnson, Tokyo, Japan)” (Line 130)

“dental plaque-disclosing agent (Merssage PC Pellet Blue; Shofu, Kyoto, Japan)” (Line 131).

“polishing paste (PTC Paste Fine/PTC Paste Regular; GC, Tokyo, Japan)” (Line 133)

“rotating rubber cup (FP rubber; Feed, Yokohama, Japan)” (Lines 133–134).

“rotating brush (FP profy brush; Feed, Yokohama, Japan)” (Line 134).

“dental plaster (New Plastone II; GC, Tokyo, Japan)” (Line 158).

“vacuum-forming machine (Biostar; Scheu Dental, Iserlohn, Germany:)” (Lines 161–162).

Comment:

- Do not use legal terms with your text. Delete "Corporation", "Corp.", "Co. Ltd.", "Co.", "Ltd.", "Inc.",

Response:

Based on this comment, we have gone through the entire manuscript and deleted the legal terms.

Comment:

- "A single examiner collected (...)." Please provide initials in parentheses.

Response:

Based on this comment, we have added the “(A.O.)” in the revised manuscript (Line 195).

Comment:

- "−30°C" must read "-30 °C". Use minus/hyphen instead of dash. The unit is "°C", and must be separated from the number.

Response:

Based on this comment, we have replaced “−30°C” in the original manuscript (Line 201) with “-30 ℃” in the revised manuscript (Line 218).

Comment:

- Report exact p-values for all values greater than or equal to 0.001 (note the 3-digit basis). P-values less than 0.001 may be expressed as p < 0.001. Remember to use lowercase letter p. See Authors' Guidelines, and revise thoroughly.

Response:

Based on this comment, we have reviewed overall the manuscript and revised the appropriate p-values.

Results

Comment:

- Again, double check and revise p values.

Response:

Based on this comment, we have reviewed overall the manuscript and revised the appropriate p-values.

Disc

Comment:

- Stick to H0 when staring this section. Remember that H0 can be rejected or not rejected.

Response:

Based on this comment, we have added the following sentence:

Advanced periodontal self-care for three months did not significantly improve FMD as compared to standard care (Lines 303–304).

Comment:

- This section would seem perfectible, please add more discursive thoughts on your outcome.

Response:

Thank you for your positive evaluation. Based on this comment, we have added the following sentences in the revised manuscript:

Reduced FMD is associated with the risk of ACVD and improves with risk-reduction therapy. Consequently, endothelial function has been defined as an “excellent barometer” of vascular health. FMD assessing vascular endothelial function might be ideal for exploring factors associated with the improvement of vascular health (Lines 308–311).

Concl

Comment:

- You aimed to "determine the effect of advanced periodontal self-care in patients with early-stage periodontal diseases on endothelial function". Additionally, you "attempted to advance periodontal self-care in patients with early-stage periodontal disease to determine any effect on ACVD-related vascular function markers FMD and ADMA." Hence, please adapt your conclusion ("Advanced periodontal self-care, in addition to standard care, did not result in better vascular function in patients with mild-to-moderate periodontal diseases in this trial.") to your aims. Do not simply repeat your results, but provide a reasonable extension of your outcome.

Response:

Based on this comment, we have replaced “Advanced periodontal self-care, in addition to standard care, did not result in better vascular function in patients with mild-to-moderate periodontal diseases in this trial” in the original manuscript (Lines 329–330) with “Advanced periodontal self-care for three months did not significantly improve FMD as compared to standard care in patients with early-stage periodontal diseases. Neither care significantly improved FMD despite its effectiveness in improving periodontal status. (Lines 362–364).

Comment:

In total, this submitted draft would seem interesting, is considered easily intelligible, and should be worth following after revision. This paper is ready for external review.

Response:

Thank you for your positive evaluation. We hope that our explanations and revisions are satisfactory.

 

Point-by-point responses to Reviewer #2

In the manuscript entitled: “Effect of advanced periodontal self-care in patients with early-stage periodontal diseases on endothelial function: An open-label, randomized controlled trial”, the authors identified the effects of advanced periodontal selfcare on endothelial function in patients with early-stage periodontal disease. The study was designed as a parallel group, 3-month follow-up, open-label, randomized controlled trial for evaluating the effectiveness of periodontal care against atherosclerotic cardiovascular disease.

The authors found that both groups demonstrated improved periodontal status. No significant improvements in FMD were observed in the control or test group. No significant changes in serum ADMA levels were observed. No significant between-group differences were found in FMD or serum ADMA levels.

The authors concluded that periodontal care for a 3-month duration did not provide better endothelial function in patients with early-stage periodontal diseases.

Major comments:

In general, the idea and innovation of this study, regards analysis of eriodontal self-care in patients with early-stage periodontal diseases on endothelial function is interesting, because the role of these factors in dentistry are validated but further studies on this topic could be an innovative issue in this field could be open a creative matter of debate in literature by adding new information. Moreover, there are few reports in the literature that studied this interesting topic with this kind of study design.

The study was well conducted by the authors; However, there are some concerns to revise that are described below.

Comment:

The introduction section resumes the existing knowledge regarding the important factor linked with periodontal inflammation. However, as the importance of the topic, the reviewer strongly recommends, before a further re-evaluation of the manuscript, to update the literature through read, discuss and must cites in the references with great attention all of those recent interesting articles, that helps the authors to better introduce and discuss the role of periodontitis and related biomarkers as cause of implant failure (Galectin, NLRP3): 1) Isola G, Polizzi A, Alibrandi A, Williams RC, Lo Giudice A. Analysis of galectin-3 levels as a source of coronary heart disease risk during periodontitis. J Periodontal Res. 2021 Jun;56(3):597-605. doi: 10.1111/jre.12860. 2) Isola G, Polizzi A, Santonocito S, Alibrandi A, Williams RC. Periodontitis activates the NLRP3 inflammasome in serum and saliva. J Periodontol. 2021 May 19. doi: 10.1002/JPER.21-0049. 3) Isola G, Lo Giudice A, Polizzi A, Alibrandi A, Murabito P, Indelicato F. Identification of the different salivary Interleukin-6 profiles in patients with periodontitis: A cross-sectional study. Arch Oral Biol. 2021 Feb;122:104997. doi: 10.1016/j.archoralbio.2020.104997.

Response:

Thank you for your critical advice. As mentioned by Reviewer #2, periodontal inflammation may induce atherosclerotic cardiovascular disease through not only bacteremia but also signal transduction involved in inflammatory mediators.

Based on this comment, we have added the following sentence with citations of all the introduced references in the revised manuscript:

“Furthermore, it has also been suggested that inflammatory mediators attributable to periodontal diseases are associated with ACVD” (Lines 61 –62).

Comment:

The authors should be better specified, at the end of the introduction section, the rational of the study and the aim of the study.

Response:

Based on this comment, we have added the following sentence in the revised manuscript:

Therefore, we hypothesized that advanced self-care improves endothelial function. The aim of this randomized clinical trial was to investigate the effect of advanced self-care on ACVD-related vascular function markers.” (Lines 89–91).

Comment:

In the material and methods section, should better clarify randomization* and periodontal examination**.

Response:

*Based on this comment, we have added the following sentences in the revised manuscript:

“All the researchers confirmed that no one could identify the number” (Lines 184–185).

“After eligibility assessment, each patient selected as a study subject made an appointment for random allocation at their convenience.” (Lines 188–189).

“Balloting was done on a first-come, first-served basis. The allocated Arabic number was particular for each subject, and the number was never used again” (Lines 190–192).

** Based on this comment, we have replaced “A single examiner collected the following clinical data from six sites around each tooth” in the original manuscript (Line 182) with “A single examiner (A.O.) collected the following clinical data from six sites (mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid- lingual, and disto-lingual) around each tooth using a color-coded periodontal probe (PO-9; Nippon Shiken, Tokyo, Japan) in the revised manuscript (Lines 195–197).

Comment:

Moreover, please more specific the clinicians involved in the different stages of the study.

Response:

Based on this comment, we have provided initials of each clinician or qualified medical personnel in parentheses in each of the different stages in the revised manuscript:

Authors: R.O., M.S., H.A., H.U., A.I., Non-authors: refer to Acknowledgments; T.K., E.S., N.A., Y.U., M.E., J.T. (Lines 125–126),

T.M. (Line 136, 146)

A.O. (Line 195, 214).

Comment:

The discussion section appears well organized with the relevant paper that support the conclusions, even if the authors should better discuss the relationship between periodontitis and endothelial dysfunction.

Response:

Based on this comment, we have added the following sentence with a citation concerning ACVD through inflammatory mediators in the revised manuscript:

“In addition, production of inflammatory mediators attributable to periodontal diseases might be associated with ACVD (Lines 306–308).

Comment:

The conclusion should reinforce in light of the discussions.

Response:

Based on this comment, we have replaced “Advanced periodontal self-care, in addition to standard care, did not result in better vascular function in patients with mild-to-moderate periodontal diseases in this trial” in the original manuscript (Lines 329–330) with “Advanced periodontal self-care for three months did not significantly improve FMD as compared to standard care in patients with early-stage periodontal diseases. Neither care significantly improved FMD despite its effectiveness in improving the periodontal status.” in the revised manuscript (Lines 362–364).

Comment:

In conclusion, I am sure that the authors are fine clinicians who achieve very nice results with their adopted protocol. However, this study, in my view does not in its current form satisfy a very high scientific requirement for publication in this journal and requests a revision before a further re-evaluation of the manuscript.

Response:

We have addressed all the comments by Reviewer #2. We hope that our explanations and revisions are satisfactory.

Minor Comments

Abstract

Comment:

- Better formulate the Abstract section by better describing the aim of the study

Response:

Based on this comment, we have added the following sentence in the Abstract:

This randomized clinical trial aimed to investigate the effect of advanced self-care on ACVD-related vascular function markers flow-mediated brachial artery dilatation (FMD) and serum asymmetric dimethylarginine (ADMA) levels in patients with early-stage periodontal disease (lines 37–41)

Introduction

Comment:

- Please refer to major comments

Response:

Based on the major comment above, we have added the following sentence with citation in the revised manuscript:

Furthermore, it has also been suggested that inflammatory mediators attributable to periodontal diseases are associated with ACVD (Lines 61–62).

Discussion

Comment:

- Please add a specific sentence that clarifies the results obtained in the first part of the discussion

Response:

Based on this comment, we have added the following sentences in the revised manuscript:

Advanced periodontal self-care for three months did not significantly improve FMD as compared to standard care. (Lines 303–304).

Comment:

- Page 16 last paragraph: Please reorganize this paragraph that is not clear

Response:

Based on this comment, we have added “than in patients with early-stage periodontal diseases” in the revised manuscript (Line 324).

 

Point-by-point responses to Reviewer #3

Comment:

1. Sample size calculation needs more details. What is the mean (SD) of each group at each time point? What test was used?

Response:

We did not have enough data prior to the trial. Therefore, we referred to previous similar trials (Tonetti MS, et al. Treatment of periodontitis and endothelial function. N Engl J Med. 2007; 356: 911-920.) as described in the original manuscript (Lines 164–167) and the revised manuscript (Lines 173–176). The authors set: effect size; 1%, standard deviation of the mean difference: 1.67%.

We have added the following sentence “We employed a priori power analysis for sample size calculation” in the revised manuscript (Line 173).

Comment:

2. Linear mixed models (repeated ANOVA) may be considered for analyzing the outcomes. If data/model residuals follow normal distributions, it is more powerful to use parametric methods.

Response:

Thank you for your advice. There are two time points in this trial (baseline and endpoint). Therefore, the paired t-test is used for comparison between baseline and endpoint. Repeated ANOVA should be used in cases where there are more than three time points.

Comment:

3. Results:

a. Table 1: add p values. Specify methods used for comparison in the footnote.

Response:

We show the baseline comparison in Table 1. As described in “How to Report Statistics in Medicine (2nd ed.) (Thomas A. Lang, Michelle Secic. American College of Physicians Philadelphia 2006, pp 207-2081), the p-values for baseline comparisons would not be necessary in randomized trials. Therefore, we did not report the p-values for baseline comparison. We have removed the description of the baseline comparison on vascular function from the original manuscript (Lines 256–257).

1 It is not necessary to report the p-values for baseline comparisons in randomized trials. In such trials, any differences between groups in baseline variables will be the result of chance because participants were assigned to groups at random. Baseline comparisons do need to be made, however, to identify any statistical imbalances that may need to be adjusted for in the final multivariable model. If p-values are reported for baseline comparisons in a randomized trial, they should be interpreted only as measure of the strength of the imbalance between the groups, not as evidence of bias.

Comment:

b. Periodontal condition: move this section after “Vascular function” as they are secondary outcomes*. Show median (IQR) for each group at each time point, not just improvement**. Add all p values for within-group comparisons and between-group comparisons**.

Response:

* Based on this comment, the description of “Vascular function” is followed by that of “Periodontal condition” in the revised manuscript.

** Based on these comments, we have replaced Table 2 and S1 Table in the original manuscript with revised Table 3 (ITT) and S2 Table (per-protocol analysis) in the revised manuscript, respectively. The revised tables show the median (IQR) for each group at endpoint with p-values in addition to the mean improvement (95% CI). Baseline data have already been described in Table 1. The revised S2 Table includes baseline data because of no descriptions on baseline for per-protocol analysis in the manuscript. We have removed statistical results from the text to avoid duplicate descriptions.

Comment:

c. Vascular function: add a table with detailed statistics, e.g. mean (SD) /median (IQR) for each group at each time point. Add all p values for within-group comparisons and between-group comparisons. Need to perform a consistency analysis for ITT and per-protocol also.

Response:

Based on this comment, we have added revised Table 2 (ITT) and S1 Table (per-protocol analysis) in the revised manuscript. The revised tables show the mean (SD) or median (IQR) for each group at endpoint with p-values in addition to the mean improvement (95% CI). Baseline data have already been described in Table 1. The revised S1 Table includes baseline data because of the absence of descriptions on baseline for per-protocol analysis in the manuscript. We have removed statistical results from the text except mean improvement between group to avoid duplicate descriptions. We have deleted Fig. 2 in the original manuscript to avoid duplicate descriptions. However, figures are generally preferred to tables for presentations of comparisons2. Therefore, we have shown the revised figures as supporting information (S2 Fig and S3 Fig).

2 How to Report Statistics in Medicine (2nd ed.). Thomas A. Lang, Michelle Secic. American College of Physicians Philadelphia 2006, pp 328

“We recommend that every effort be made to present comparisons in figures rather than in tables, even when the amount of data is small.”

Comment:

d. Figure 2: using boxplot for both outcomes. Specify clearly which methods were used for generating the p values.

Response:

Based on this comment, we have replaced the original Figure 2 with revised S2 Fig, indicating the results with boxplots. The boxplots are presented as supporting information to avoid duplicate descriptions. We have also adjusted the corresponding captions. Statistical procedures were described in the captions.

Comment:

e. Line 275 is not a complete sentence.

Response:

Line 275 contains definitions of abbreviations and a part of Figure captions. We have undone the line break in the revised manuscript.

CONSORT checklist:

1) Outcomes are clearly defined.

Complete

2) Sample size needs more details for mean (SD) in each group, not mean difference (SD). What test was used?

Response:

As described above, we did not have enough data prior trial. Therefore, we referred to previous similar trials (Tonetti MS, et al. Treatment of periodontitis and endothelial function. N Engl J Med. 2007; 356: 911-920.) as described in the original (Lines 164–167) and revised (Lines 173–176) manuscripts. The authors set: effect size; 1%, standard deviation of the mean difference: 1.67%.

We have added the following sentence in the revised manuscript “We used a priori power analysis for sample size calculation” (Line 173).

3) Method used to generate random allocation sequence was not mentioned.

Response:

We have added the following sentences to clarify the random allocation sequence in the revised manuscript:

“All the researchers confirmed that no one could identify the number (Lines 184–185).

“After eligibility assessment, each patient selected as a study subject made an appointment for random allocation at their convenience.” (Lines 188–189).

“Balloting was done on a first-come, first-served basis. The allocated Arabic number was particular for each subject, and the number was never used again.” (Lines 190–192).

4) Allocation concealment was clearly described.

Complete

5) Blinding is not available.

Response:

This trial is open-labeled.

6) Details of outcomes and estimation were not provided*. Need means (SD) for each group at each time point and mean different (with 95% CI)**. A table is necessary**. Effect size was not provided either*.

Response:

* We have added the following sentences to the revised manuscript:

“An improvement in FMD of at least 1% difference was not achieved between the groups.” (Line 261).

We set the effect size at 1% difference in FMD as described in the original (Line 165) and revised (Line 174) manuscripts.

** We have added revised Table 2 (ITT) and S1 Table (per-protocol analysis) showing the mean ± SD/median (IQR) for each group at endpoint and mean difference (with 95% CI) in the revised manuscript.

7) Important harms or unintended effects were not discussed.

Response:

We have added the following sentence:

Harms

No study-related serious adverse events occurred in any of the study participants. Nonsurgical periodontal treatment is basically low-risk. None of the participants required any dental therapy during the study. (Lines 297–300).

8) The registry and the registration number were reported.

Complete

9) The trial protocol is attached.

Complete

10) Sources of funding was described.

Complete

Attachment

Submitted filename: 02 Response to Reviewers.docx

Click here for additional data file. (46KB, docx)

Decision Letter 1

Andrej M Kielbassa

27 Jul 2021

PONE-D-21-11565R1

Effect of advanced periodontal self-care in patients with early-stage periodontal diseases on endothelial function: An open-label, randomized controlled trial

PLOS ONE

Dear Dr. Murata,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Having intensively reviewed your revised draft, our external reviewers still differed with their final recommendations, at least to some extent. I have again double checked your revised version, to come to a more balanced decision (see R #1). All in all, our  identified shortcomings are considered reasonable with regard to both PLOS ONE’s quality standards and our readership's expectations. Therefore, we invite you to submit a carefully revised version of the manuscript that addresses EACH AND EVERY point raised during the current review process. Please note that a further non-convincing revision (not considered acceptable with regard to language, content, reviewers' constructive criticism, generalizable conclusions, and/or Authors' Guidelines) must lead to outright reject. 

Please submit your revised manuscript by Sep 10 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Andrej M Kielbassa

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: No doubt, this revised draft has been considerably increased. However, some minor aspects would still seem in need of further revisions, please see below.

- With reference to your Abstract section, do not provide a shortened literature review, please. Instead, please focus more intensely on your outcome.

- "The immune response following persistent bacteremia from periodontal lesions may lead to ACVD [3]. Furthermore, it has also been suggested that inflammatory mediators attributable to periodontal diseases are associated with ACVD [4-6]." Consequently, "bacteremia-induced ACVD routinely occurs even in the early stage of periodontal disease [2,19]". Undoubtedly, this would seem right. However, this would lead to the idea of a thorough eradication of all biofilms deemed responsible for infection, persistent bacteremia, and presence of inflammatory mediators (due to the biofilm). This refers to your "same-day full-mouth scaling, an ultrasonic scaler, along with hand instruments, and dental floss, and polishing paste with rotating rubber cup/brush", which is not deemed sufficient (even if widely established). Please refer to https://pubmed.ncbi.nlm.nih.gov/34269042/; there you will see that "(not only supragingival) biofilm removal by means of air polishing combined with low-abrasive erythritol has been shown to be more efficacious than the traditional polishing method". Thus, your applied methodology should be discussed more thoroughly, in particular since "thorough biofilm removal clearly results in delayed plaque regrowth rates", thus potentially showing a clear influence on inflammatory mediators and ACVD, in particular in combination with your "extra intervention in addition to standard self-care". Again, go to https://doi.org/10.3290/j.qi.b1763661, and discuss, both with regard to general effects and with regard to possible limitations.

- "We considered a 70% implementation rate as successful advanced periodontal self-care." Do you mean "successfully advanced periodontal self-care"?

- "(1–29 for smokers and 1–81 for nonsmokers)" should read "(1 to 29 for smokers and 1 to 81 for nonsmokers)".

- "All the researchers confirmed that no one could identify the number." This would not seem clear, please clarify.

- "(Unexef18G, UNEX, Nagoya, Japan)" must read "(Unexef18G; UNEX, Nagoya, Japan)". Double check thoroughly.

- With your results section, please add exact p values. (See, for example, "Significant improvements in the average PPD were observed in the control and test groups. The BOP score in the test group was significantly reduced, while that in the control group was reduced, although not significantly.").

- Again, with the first paragraph of your Disc section, please refer explicitly to H0, to provide a clear answer. "The findings were similar in the intention-to-treat and the per-protocol analysis." should read ""The findings were similar in the intention-to-treat and the per-protocol analysis; thus H0 was not rejected."

- "ADMA is a naturally occurring endogenous inhibitor of NO synthase." Reference(s) missing.

- Same with "It reduces NO production; consequently, it can lead to endothelial dysfunction and cardiovascular events." Please revise carefully, and double check your whole draft, to support statements.

- "Further investigations may be required in healthy subjects." To study what kind of effects? Please clarify.

- "A longer period might be required to alter endothelial function in patients with mild-to-moderate periodontal diseases." Please provide reasons why you think that prolonged follow-up periods would change the outcome.

- Regarding the reference list, again, revise for uniform formatting (see Guidelines). Format must be "Bürgers R, Eidt A, Frankenberger R, Rosentritt M, Schweikl H, Handel G, et al. The anti-adherence activity and bactericidal effect of microparticulate silver additives in composite resin materials. Arch Oral Biol. 2009; 54(6): 595-601. https://doi.org/10.1016/j.archoralbio.2009.03.004 PMID: 19375069". Please revise for spacebar use to separate year and volume. Provide issue numbers (if available). Revise for correct doi numbers, and provide PMID numbers.

Again, this study would seem worth following after revision. Compliments to the authors!

Reviewer #2: The authors have well addressed to all reviewer's comments. There are no futher issues in the present version of the manuscript.

Reviewer #3: (No Response)

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PLoS One. 2021 Sep 23;16(9):e0257247. doi: 10.1371/journal.pone.0257247.r004

Author response to Decision Letter 1

4 Aug 2021

Response to Reviewers

Point-by-point responses to Reviewer #1

Comment:

No doubt, this revised draft has been considerably increased. However, some minor aspects would still seem in need of further revisions, please see below.

Response:

We are grateful to Reviewer #1 for the second review and for providing advice. As indicated in the responses that follow, we have taken all these comments and suggestions into account in the revised version of our manuscript.

Comment:

- With reference to your Abstract section, do not provide a shortened literature review, please. Instead, please focus more intensely on your outcome.

Response:

Based on this comment, we have added the details of between-group differences in FMD and serum ADMA levels, and results of periodontal status in the Abstract.

For example,

“(mean difference, −0.2%; 95% CI, −1.4–0.9; p = 0.708)” (Line 46).

“(mean difference, 0.01 nmol/L; 95% CI, −0.00–0.03; p = 0.122)” (Lines 46–47).

“Significant improvements in the average probing pocket depth were observed in the control and test groups. The bleeding on probing score in the test group was significantly reduced, while that in the control group was reduced, although not significantly” (Lines 47–50).

Comment:

-"The immune response following persistent bacteremia from periodontal lesions may lead to ACVD [3]. Furthermore, it has also been suggested that inflammatory mediators attributable to periodontal diseases are associated with ACVD [4-6]." Consequently, "bacteremia-induced ACVD routinely occurs even in the early stage of periodontal disease [2,19]". Undoubtedly, this would seem right. However, this would lead to the idea of a thorough eradication of all biofilms deemed responsible for infection, persistent bacteremia, and presence of inflammatory mediators (due to the biofilm). This refers to your "same-day full-mouth scaling, an ultrasonic scaler, along with hand instruments, and dental floss, and polishing paste with rotating rubber cup/brush", which is not deemed sufficient (even if widely established). Please refer to https://pubmed.ncbi.nlm.nih.gov/34269042/; there you will see that "(not only supragingival) biofilm removal by means of air polishing combined with low-abrasive erythritol has been shown to be more efficacious than the traditional polishing method". Thus, your applied methodology should be discussed more thoroughly, in particular since "thorough biofilm removal clearly results in delayed plaque regrowth rates", thus potentially showing a clear influence on inflammatory mediators and ACVD, in particular in combination with your "extra intervention in addition to standard self-care". Again, go to https://doi.org/10.3290/j.qi.b1763661, and discuss, both with regard to general effects and with regard to possible limitations.

Response:

Thank you for bringing this recent paper to our attention. The paper shows that “polishing combined with low-abrasive erythritol” is more efficacious than “air polishing or rubber cups with prophylaxis paste” for biofilm removal and delayed biofilm regrowth rates within 24 hours. As mentioned by Reviewer #2, the outcomes may be affected by the method employed for biofilm removal.

Based on this comment, we have added the following sentence to the revised manuscript with an appropriate citation:

“The main contents of standard care at the clinic are biofilm removal. Employment of more effective methods for biofilm removal may affect the improving FMD through delayed biofilm regrowth rates [28].” (Lines 325–327).

Comment:

- "We considered a 70% implementation rate as successful advanced periodontal self-care." Do you mean "successfully advanced periodontal self-care"?

Response:

Yes, we do. We decided that a “70% implementation rate represented successful advanced periodontal self-care” before the trial began. The decision was made in consideration of the burden on the subject and our empirical tolerance level for "successfully advanced periodontal self-care.” Those subjects with a below “70% implementation rate” were excluded from per-protocol analysis.

Comment:

- "(1–29 for smokers and 1–81 for nonsmokers)" should read "(1 to 29 for smokers and 1 to 81 for nonsmokers)".

Response:

Based on this comment, we have replaced “1–29 for smokers and 1–81 for nonsmokers” with “1 to 29 for smokers and 1 to 81 for nonsmokers” in the revised manuscript (Line 182).

Comment:

- "All the researchers confirmed that no one could identify the number." This would not seem clear, please clarify.

Response:

Based on this comment, we have added “from the outside of each sealed envelope” (Line 183).

Comment:

- "(Unexef18G, UNEX, Nagoya, Japan)" must read "(Unexef18G; UNEX, Nagoya, Japan)". Double check thoroughly.

Response:

Based on this comment, we have replaced “(Unexef18G, UNEX, Nagoya, Japan)” with “Unexef18G; UNEX, Nagoya, Japan” in the revised manuscript (Lines 203–204).

Comment:

- With your results section, please add exact p values. (See, for example, "Significant improvements in the average PPD were observed in the control and test groups. The BOP score in the test group was significantly reduced, while that in the control group was reduced, although not significantly.").

Response:

We have added a revised Table 3 and S2 Table based on the other reviewer’s comment. We have shown the exact p values in the Tables. We have removed statistical results from the text to avoid duplicate descriptions in the revised manuscript.

Comment:

- Again, with the first paragraph of your Disc section, please refer explicitly to H0, to provide a clear answer. "The findings were similar in the intention-to-treat and the per-protocol analysis." should read ""The findings were similar in the intention-to-treat and the per-protocol analysis; thus H0 was not rejected.

Response:

Thank you for your observation. Based on this comment, we have added the following sentence:

“Therefore, our null hypothesis ‘there was no difference in FMD between advanced self-care and standard care in patients with early-stage periodontal disease’ was not rejected.” (Lines 304–305).

Comment:

- "ADMA is a naturally occurring endogenous inhibitor of NO synthase." Reference(s) missing.

Response:

Based on this comment, we have added the following three references:

29. Najbauer J, Johnson BA, Young AL, Aswad DW. Peptides with sequences similar to glycine, arginine-rich motifs in proteins interacting with RNA are efficiently recognized by methyltransferase(s) modifying arginine in numerous proteins. J Biol Chem. 1993; 268(14): 10501-10509. PMID: 7683681.

30. Tang J, Kao PN, Herschman HR. Protein-arginine methyltransferase I, the predominant protein-arginine methyltransferase in cells, interacts with and is regulated by interleukin enhancer-binding factor 3. J Biol Chem. 2000; 275(26): 19866-19876. https://doi.org/10.1074/jbc.M000023200 PMID: 10749851.

31. MacAllister RJ, Fickling SA, Whitley GS, Vallance P. Metabolism of methylarginines by human vasculature; implications for the regulation of nitric oxide synthesis. Br J Pharmacol. 1994; 112(1): 43-48. https://doi.org/10.1111/j.1476-5381.1994.tb13026.x PMID: 7518309.

Comment:

- Same with "It reduces NO production; consequently, it can lead to endothelial dysfunction and cardiovascular events." Please revise carefully, and double check your whole draft, to support statements.

Response:

Based on this comment, we have added the following four references:

24. Sibal L, Agarwal SC, Home PD, Boger RH. The role of asymmetric dimethylarginine (ADMA) in endothelial dysfunction and cardiovascular disease. Curr Cardiol Rev. 2010; 6(2): 82-90. https://doi.org/10.2174/157340310791162659 PMID: 21532773.

32. Achan V, Broadhead M, Malaki M, Whitley G, Leiper J, MacAllister R, et al. Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase. Arterioscler Thromb Vasc Biol. 2003; 23(8): 1455-1459. https://doi.org/10.1161/01.ATV.0000081742.92006.59 PMID: 12805079.

33. Kielstein JT, Impraim B, Simmel S, Bode-Böger SM, Tsikas D, Frölich JC, et al. Cardiovascular effects of systemic NO synthase inhibition with asymmetric dimethylarginine in humans. Circulation. 2004; 109(2): 172-177. https://doi.org/10.1161/01.CIR.0000105764.22626.B1 PMID: 14662708.

34. Fliser D. Asymmetric dimethylarginine (ADMA): the silent transition from an ‘uraemic toxin’ to a global cardiovascular risk molecule. Eur J Clin Invest. 2005; 35(2): 71-79. https://doi.org/10.1111/j.1365-2362.2005.01457.x PMID: 15667575.

Comment:

- "Further investigations may be required in healthy subjects." To study what kind of effects? Please clarify.

Response:

Based on this comment, we have replaced “Further investigations may be required in healthy subjects” with “The reason for the discrepancy between the trials may be the underlying factors characterizing the study population which may be affecting FMD and/or plasma ADMA levels. Further investigations may be required to identify the factors in healthy subjects.” (Lines 336–339).

Comment:

- "A longer period might be required to alter endothelial function in patients with mild-to-moderate periodontal diseases." Please provide reasons why you think that prolonged follow-up periods would change the outcome.

Response:

When a certain treatment does not give positive results in a short span of time, continuation of the treatment is one of the common options available to medical practitioners. A longer period of care may increase the effectiveness of treatment for persistent bacteremia from periodontal lesions in patients with mild-to-moderate periodontal diseases.

Based on this comment, we have added “, which enables further improvement in bacteremia,” (Line 364).

Comment:

- Regarding the reference list, again, revise for uniform formatting (see Guidelines). Format must be "Bürgers R, Eidt A, Frankenberger R, Rosentritt M, Schweikl H, Handel G, et al. The anti-adherence activity and bactericidal effect of microparticulate silver additives in composite resin materials. Arch Oral Biol. 2009; 54(6): 595-601. https://doi.org/10.1016/j.archoralbio.2009.03.004 PMID: 19375069". Please revise for spacebar use to separate year and volume. Provide issue numbers (if available). Revise for correct doi numbers, and provide PMID numbers.

Response:

Based on this comment, we have reviewed all the references and adjusted them according to the Guidelines.

Comment:

Again, this study would seem worth following after revision. Compliments to the authors!

Response:

Thank you for the encouragement. We hope that our explanations and revisions are satisfactory.

Attachment

Submitted filename: 04 Response to Reviewers.docx

Click here for additional data file. (29.1KB, docx)

Decision Letter 2

Andrej M Kielbassa

27 Aug 2021

Effect of advanced periodontal self-care in patients with early-stage periodontal diseases on endothelial function: An open-label, randomized controlled trial

PONE-D-21-11565R2

Dear Dr. Murata,

After having double checked your revised and re-submitted paper, I am pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards, congratulations and compliments, and stay healthy

Andrej M Kielbassa, Prof. Dr. med. dent. Dr. h. c.

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: After having updated, adapted, and polished their revised and re-submitted draft, this manuscript is ready to proceed.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Acceptance letter

Andrej M Kielbassa

15 Sep 2021

PONE-D-21-11565R2

Effect of advanced periodontal self-care in patients with early-stage periodontal diseases on endothelial function: An open-label, randomized controlled trial

Dear Dr. Murata:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Dr. med. dent. Dr. h. c. Andrej M Kielbassa

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Checklist. CONSORT non-pharmacologic treatment extension checklist.

    (DOCX)

    Click here for additional data file. (39.2KB, docx)
    S1 File. Trial protocol.

    (DOCX)

    Click here for additional data file. (286KB, docx)
    S1 Fig. Custom-manufactured tray.

    (TIF)

    Click here for additional data file. (3.8MB, tif)
    S2 Fig. Effect of periodontal care on vascular function in the intention-to-treat analysis.

    Box and whisker plot of FMD (A) and serum ADMA (B) levels by group. The box contains values between the 25th and 75th percentiles (central line, median). Vertical lines represent the minimum and maximum. P-values were calculated using the paired Student’s t-test (FMD) or Wilcoxon’s signed-rank test (serum ADMA level) for changes from baseline and the unpaired t-test (FMD) or the Mann–Whitney U test (serum ADMA level) for group differences. FMD, brachial artery dilatation; AMDA, asymmetric dimethylarginine.

    (TIF)

    Click here for additional data file. (156.9KB, tif)
    S3 Fig. Effect of periodontal care on vascular function in the per-protocol analysis.

    Box and whisker plot of FMD (A) and serum ADMA levels (B) by group. The box contains values between the 25th and 75th percentiles (central line, median) of serum ADMA levels. Vertical lines represent the minimum and maximum. P-values were calculated using the paired Student’s t-test (FMD) or Wilcoxon’s signed-rank test (serum ADMA level) for changes from baseline and the unpaired t-test (FMD) or the Mann–Whitney U test (serum ADMA level) for group differences. FMD, brachial artery dilatation; AMDA, asymmetric dimethylarginine.

    (TIF)

    Click here for additional data file. (159KB, tif)
    S1 Table. Assessment of vascular function in the per-protocol analysis.

    (DOCX)

    Click here for additional data file. (33.7KB, docx)
    S2 Table. Periodontal status in the per-protocol analysis.

    (DOCX)

    Click here for additional data file. (33.1KB, docx)
    Attachment

    Submitted filename: 02 Response to Reviewers.docx

    Click here for additional data file. (46KB, docx)
    Attachment

    Submitted filename: 04 Response to Reviewers.docx

    Click here for additional data file. (29.1KB, docx)

    Data Availability Statement

    The data contain potentially sensitive patient information. The Ethics Committee of the Tsurumi University School of Dental Medicine prohibits the preservation of any data set in a public repository (even if the data are de-identified), the preservation of hard copy data in an unlocked cabinet, the preservation of electronic data in a computer connected to the internet. All the subjects provided informed consent on the condition of our adherence to IRB guidelines. The datasets generated and/or analyzed during the current study are available from Dr. Hidenori Yamada (yamada-h@tsurumi-u.ac.jp) or Dr. Ayako Okada (okada-a@tsurumi-u.ac.jp) on reasonable request. The Ethics Committee of the Tsurumi University School of Dental Medicine 2-1-3, Tsurumi, Yokohama, 230-8501 Japan Email Address: kyoken@tsurumi-u.ac.jp.

    Articles from PLoS ONE are provided here courtesy of PLOS

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