Clinical and microbiological effects of the use of a cetylpyridinium chloride dentifrice and mouth rinse in orthodontic patients: a 3-month randomized clinical trial

Clinical and microbiological effects of the use of a cetylpyridinium chloride dentifrice and... Summary Objective To assess the clinical, microbiological, and patient-based effects of using a cetylpyridinium chloride (CPC) toothpaste and mouth rinse in orthodontic patients. Design Parallel randomized controlled, triple-blinded trial (participants, examiners, outcomes’ assessors). A computer-generated list was used to allocate treatments. Central allocation was used for concealment. Participants Thirty-one placebo (10 males, 21 females; mean age 15.2 ± 2.1) and 32 test patients (15 males, 17 females; mean age 15.0 ± 1.8) with fixed orthodontic appliance were included in the study. Interventions Patients were randomly assigned to both brush and rinse with placebo or with CPC-based products. Products were purposely prepared in white opaque bottles. After screening and professional prophylaxis, patients received a baseline examination, and started to use the assigned products. Patients were monthly assessed during a 3-month period. Main outcome measures Plaque (PlI) and gingival (GI) indexes. Secondary outcome variables: Calculus index, tooth staining, subgingival microbiological samples, patient-based variables (questionnaire, compliance, and remaining mouth rinse), side-effects (debonded braces or mucosal injuries). Results Sixty-three patients were randomized, 13 patients were excluded from analysis because of early dropout, leaving 50 patients for intention to treat analysis. PlI in the upper jaw diminished (0.18; SD = 0.82) after 1 month in the test group, while it increased (0.26; SD = 0.62) in the placebo group (P = 0.024). Statistically significant higher GI values were observed in the placebo group at 1 month (mean increase = 0.05; SD = 0.33 versus mean decrease = 0.03; SD = 0.38) and 3 months (mean = 0.05; SD = 0.43 versus mean = 0.02; SD = 0.34). The taste of the test product was rated higher than the placebo at 2- and 3-month visits (P < 0.05). Non-significant changes were observed in microbiological parameters, overgrowth of opportunistic species or remaining secondary outcome variables, including side-effects. Conclusion The use of CPC-based toothpaste and mouth rinse in orthodontic patients had limited effect in reducing plaque accumulation and gingival inflammation. Effects were little and highly variable. The use of the test products was not associated with relevant adverse effects. Trial registration Trial registration: Local internal reference: P07/133. Introduction The control of supragingival biofilm by means of mechanical devices, such as toothbrushes, dental floss, or interdental brushes, is a key element in the prevention of caries and periodontal diseases. The presence of orthodontic fixed appliances, however, makes this mechanical control more difficult, facilitating plaque retention, gingivitis (1, 2), and initial caries or white spots lesions (3). Enhanced plaque accumulation may also be associated with an increase in the colonization of periodontal pathogens, such as Prevotella intermedia (2, 4, 5, 6). Moreover, many orthodontic patients, especially children and adolescents, fail to floss because they find this procedure time-consuming and tedious in the presence of orthodontic arch wires (7). Studies assessing compliance in these patients have reported that their level of cooperation varies considerably depending on their age and gender, personality, perception of the malocclusion, parental influence, and socioeconomic factors (8). As a result of these facts, adolescents or young adults wearing fixed orthodontic appliances tend to accumulate more plaque and hence, they are at a higher risk of developing gingivitis. A common strategy to improve mechanical plaque removal in these patients is the addition of a chemical antimicrobial agent with anti-plaque or anti-caries activity in dentifrices, mouth rinses, or both (9). Ideally, these agents should have antimicrobial activity, for reducing plaque accumulation and gingival inflammation (10); anti-caries activity for decreasing the decalcifications usually occurring during orthodontic treatment and thus preventing the development of caries or white spots (11–13) and anti-inflammatory activity to help in the healing of traumatic mucosal injuries caused by the appliances (14). Since these products are aimed for long-term use compliance is a critical factor and therefore, they should have a pleasant flavour and lack of side effects, such as staining or de-bonding of the braces and bands. The effectiveness of different active ingredients, such as chlorhexidine (CHX) (15–17), essential oils (18), amine/stannous fluoride (19), or sanguinarine (20) in the form of mouth rinses, tooth pastes, or gels, has been evaluated in clinical studies. Most of these clinical studies have reported significant benefits in the adjunctive use of these products, although the magnitude of these reported benefits might not have a clear clinical relevance. In addition, the use of some of the formulations was associated with adverse effects (such as staining with the use of CHX). In recent years, the use of the chemical antimicrobial agent cetylpyridinium chloride (CPC) has attracted some attention, due to the advent of formulations with improved bioavailability or increased concentration, which have shown increased clinical benefits and since their adverse effects are very limited (tooth staining, ulcers, gingival irritation), its use may be indicated for longer periods (21,22). CPC has demonstrated in a number of clinical trials to reduce plaque formation (23–26) and gingivitis (24, 26–29). When used as a mouth rinse, a systematic review has shown heterogeneous results (30), although a significant effect on plaque has been demonstrated at different concentrations, such as 0.07 or 0.05 per cent (21,22,28). Recently, a new CPC-based mouth rinse and toothpaste were specifically formulated and marketed for orthodontic patients (VITIS Orthodontic, Dentaid, Cerdanyola, Spain). This formulation includes other active ingredients, such as allantoin, aloe vera, and sodium fluoride. Allantoin is widely used in dermatology since it promotes cell proliferation and stimulates epithelisation (31). In dentistry, it has been added in formulations for the treatment of aphthous stomatitis (14). Similarly, aloe vera has been added in formulations for the treatment of oral mucositis in cancer patients (32) and for aphthous stomatitis (14). This new formulation, aimed to be used by orthodontic patients, has not been evaluated in home-use randomised clinical trials. It was, therefore, the objective of this investigation to assess the clinical and microbiological efficacy of the combined use of a CPC-based toothpaste and mouth rinse formulations, adjunctive to mechanical plaque control in patients wearing fixed orthodontic appliances. In addition, compliance and the occurrence of adverse effects were evaluated. Subjects and methods/methods Patient population A consecutive sample of subjects undergoing treatment with fixed orthodontic appliances was screened during 12 months at the Department of Orthodontics in the Faculty of Odontology of the authors’ institution. The recruitment period for this study was from September 2013 to September 2014. During the screening visit, subjects were examined for fulfilment of the inclusion criteria and received a comprehensive explanation on the aims of the investigation. They were then asked to participate by signing an informed consent previously approved by the Ethical Review Committee (Comité ético de Investigación Clínica, Hospital Clínico San Carlos. Internal reference: P07/133. Date of approval: May 2007). Study participants were selected according to the following inclusion criteria: Healthy subjects between 12 and 25 years old. Participants were excluded in the presence of systemic diseases that might interfere with the study or if in need of chronic analgesic or anti-inflammatory medication. Undergoing orthodontic therapy with fixed appliances both in the upper and lower arch with monthly follow-up appointments, Clinical evidence of gingivitis (visually at screening, then confirmed with the Gingival Index). Participants were excluded in presence of periodontitis (clinical attachment loss > 4 mm) or gingival overgrowth (pseudopockets > 4 mm). Subjects were also excluded if they showed evidence of dental negligence. Study design The study was designed as a randomized, parallel, triple-blind, 3-month clinical trial. Participants, examiners, and those assessing the outcomes were blinded to group assignment. The following visits were scheduled: At the screening visit, all recruited subjects had a professional prophylaxis followed by specific oral hygiene instructions. They were then appointed for the baseline visit within 7–15 days. At the baseline visit, the clinical parameters were recorded and microbiological samples were retrieved. At this visit, each participant was randomly assigned by the study supervisor to one of the study groups through a computer-generated list (a block randomization with blocks of six). Block size was not disclosed to ensure concealment. Each subject was given a unique number, which was associated with the assigned product. Participants and investigators were unaware of the treatment assignment due to the central allocation, managed by the study supervisor. Allocation concealment was ensured, as the supervisor did not release by phone the randomisation code until the patient had been recruited into the trial. The assigned products included both a mouthwash and dentifrice, together with a standard toothbrush (VITIS Orthodontic, Dentaid, Cerdanyola, Spain) and dental floss (VITIS non-wax dental floss, Dentaid, Cerdanyola, Spain). Products were purposely prepared in white opaque bottles coded as A or B. Patients were requested to return the unused mouthrinse at the following visit and were instructed to fill in a compliance diary where the home use of the products was recorded. The content of the product’s assignment list was only known by the study supervisor who was not involved in the screening or in the subject’s evaluation. At the 1-month visit, all the clinical parameters were again recorded together with a questionnaire that was filled by the participants reporting on their subjective assessment of the used products. At this visit, the compliance diaries were collected as well as any remaining mouth rinse, which was later quantified in ml. New products (mouthwash and dentifrice) and new diaries were provided. The 2-month visit was identical to the 1-month visit. At the final 3-month visit, clinical parameters were evaluated, microbiological samples were taken, participants’ questionnaires were filled and compliance diaries and remaining mouth rinse were collected. At this time, a professional prophylaxis was carried out and oral hygiene instructions were reinforced. Treatment groups In the test group, subjects used the experimental mouthwash (VITIS Orthodontic®, Dentaid, Cerdanyola, Spain) containing 0.05 per cent CPC, 0.33 per cent sodium fluoride, allantoin and aloe vera, together with the experimental toothpaste (VITIS Orthodontic®, Dentaid, Cerdanyola, Spain) containing the same active ingredients. In the control group, subjects used a placebo mouth rinse and dentifrice that were identical to the test formulations, but without the active components (CPC, sodium fluoride, aloe vera, and allantoin). Both the test and the placebo formulations contained 1% xylitol. Subjects in both groups were instructed to use 15 ml of the mouthwash twice a day during 30 seconds, after brushing their teeth with the assigned tooth paste in the morning and in the evening. Outcome variables Clinical outcome variables The primary outcome variables of this study were the Plaque and gingival indexes. – Plaque index (PlI) was evaluated using the Turesky modification (33) of the Quigley and Hein index after disclosing dental plaque with erythrosine (Plac Control®, Dentaid, Cerdanyola, Barcelona, Spain). – Gingival index (GI) was assessed using the Lobene modification (34) of the Löe-Silness index, at six sites per tooth (24). As secondary variables, we recorded: – Calculus accumulation measured with the Volpe-Manhold calculus index (35) at the mid-lingual surfaces of the four lower incisors. The scores ranged from 0, no calculus; 1, one mm of calculus; 2, two mm of calculus to 3, three mm of calculus. – Tooth staining: Standardized digital photographs of the buccal side of the upper and lower incisors and canines (occluding edge to edge) were taken using a Canon EOS 450 digital camera mounted with a ring-flash and with a 105-mm Macro lens, with an aperture of F22 and shutter speed of 1/125. Two blinded, independent and calibrated judges evaluated the photographs using a standard template of different tooth staining degrees, as previously described by Gadhia et al. (36). They scored both the area and the intensity of tooth staining using a numerical scale from 0 to 3. The area was graded as: 0 (absence of staining); 1 (staining in the 1/3 of the clinical crown free of bracket; 2 (staining in the 2/3 of the clinical crown free of bracket); and 3 (staining in the 3/3 of the clinical crown free of bracket). The intensity was graded as: 0 (absence of staining); 1 (slight staining); 2 (moderate staining); and 3 (severe staining). A total of 193 pictures were assessed. The values of all the upper front teeth were added and compared to the sum of the other visits, and the same was done with the lower teeth. Values were expressed as the mean of the evaluation of both judges. – Orthodontic appliances. The presence of any bracket or band being de-bonded was recorded. Similarly, the occurrence of any soft tissue injury and its duration was recorded. Microbiological outcome variables Microbiological samples were taken using two sterile standard sized paper points (Maillefer, Ballaigues, Switzerland) that were inserted consecutively in each selected site after the removal of supragingival plaque (37). Samples were taken from the mesial-buccal site of upper first molars and the distal-buccal site of lower lateral incisors. At these sites, different clinical variables were recorded, including probing pocket depth (PPD), bleeding on probing (BOP), and plaque presence. Before the insertion of the paper points, sites were isolated with cotton rolls to avoid saliva contamination and the area was dried with an air syringe. Paper points were kept in place for 10 seconds and they were then pooled in a screw top vial containing 1.5 ml of reduced transport fluid (RTF) (38). Samples were transferred to the laboratory within 2 hours where they were homogenized by vortex vibration for 30 seconds (39) and sequentially diluted in phosphate buffer solution (PBS). Samples were cultivated on agar-blood medium (enriched with haemine and menadione) and incubated for 15 days in jars with an anaerobic atmosphere; and on selective medium Dentaid-1, incubated for 3–5 days in 5% carbon dioxide (40). Bacterial species identification was carried out by the assessment of the colony morphology and confirmed by the application of biochemical standard tests. In addition to the conventional evaluation of the plates, the possible overgrowth of opportunistic species, both in blood agar and selective plates, was investigated in order to detect possible undesired microbiological adverse effects. The main microbiological outcome variables included total anaerobic counts and the presence, counts, and proportions of different bacterial species, including both pathogenic and opportunistic species. Patient-reported outcomes In every follow-up visit, patients filled up a questionnaire reporting their subjective evaluation of the assigned products. The questionnaire used was custom-made for this investigation, including following questions, with three/four closed answers available: How do you feel rinsing with this mouth rinse’ (answers: great; good; unpleasant; very unpleasant) The taste of this mouth rinse is…’ (answers: very good; good; fair; bad) Did you use the mouth rinse everyday’ (answers: yes, twice a day; no, I forgot 1 day; no, I forgot 2 days; no, I forgot more than 2 days) Have you used any mouth rinse before’ (answers: yes, almost every day; yes, sometimes; yes, but very rarely; no) How do you feel your teeth after using this mouth rinse’ (answers: cleaner and smoother; rough and unpolished; I don’t feel any change) After using the mouth rinse, your breath feels…’ (answers: better than before; same as before; worse than before) Compliance Compliance with the assigned products was assessed by the information provided by the participants on their filled diaries and by the remaining mouth rinse (if any) returned after each follow-up visit, which was measured in ml. using a calibrated vase. The measured compliance was expressed by the number of patients that fulfilled with the study protocol at each visit and by the times that a patient failed to use the mouth rinse, according to the daily diaries. Percentage of failures was calculated in relation to the total available times. Patients were monthly assessed for clinical, patient-reported and compliance variables. Subgingival microbiological samples were taken at baseline and after 3 months. Statistical analysis The sample size calculation was based on the results from a 1-month clinical trial using a similar CPC-based mouth rinse (41). For statistical purposes, and in order to avoid adjusting for multiplicity analysis, we considered only the reduction in the mean Plaque Index as the primary outcome. To detect a reduction in the mean plaque index of 0.42 (SD 0.40) with a two-sided 5% significance level and a power of 80%, a sample size of 25 patients per group was necessary, taken into account an anticipated dropout rate of 10%. For entering the results, a database was designed in a spread sheet (Microsoft® Excel), which was then analysed with the statistical program Statgraphic Plus® for Windows (5.1 edition, New Jersey, New Jersey, USA). In all cases, ITT (intention to treat) analysis was performed. Calibration of the examiners One clinician undertook all the clinical assessments and was calibrated for PI and GI recordings before the start of the trial. Two orthodontic patients wearing fixed appliances were enrolled for this purpose. Duplicated measurements (n = 328) were collected with an interval of 30 min between the first and the second recording. Intraclass correlation coefficient (ICC) of total agreement was used to test the intra-examiner reliability for PlI and GI. The Intra-examiner reliability was very good for PI (ICC = 0.923; 95% CI = 0.902–0.953) and GI (ICC = 0.904; 95% CI = 0.891–0.922) assessment. For the evaluation of tooth staining, the two judges who evaluated the photographs were previously calibrated by repeating twice the assessment of ten pictures and achieving a Cohen’s kappa index of 0.813. For the clinical variables, the averages were calculated per patient and visit, and then per group. Once the distribution of the data was confirmed (using the Kolmogorov–Smirnov and Shapiro–Wilk tests), if the variables fulfilled a normal distribution, ANCOVA (with baseline values as covariate) was initially used as primary statistical test. Then, paired t-tests were used for evaluating intra group changes and unpaired t-tests for inter group comparisons (at each visit, and in changes between baseline-follow up visits). Clinical indexes were calculated for the full-mouth and then stratified for the upper and lower jaw and for proximal and free surfaces, considering as primary result the ‘all sites’ evaluation. The values of intensity and area of tooth staining were compared between groups, between consecutive visits and between the baseline and final visits using the Chi-squared test. The data were grouped into upper arch and lower arch to make comparisons. Total anaerobic counts were log transformed in order to achieve a normal distribution, and were then statistically analysed in a similar way as the clinical variables. Counts and proportions of pathogens were compared by means of signed-rank tests, and frequencies of detections by means of Chi-squared test. Answers to questionnaires were compared between groups using the chi-square test or Fisher’s exact test when necessary. To get a more realistic estimation of the trend of those answers where differences were statistically significant, a transformation of categorical variables into semi-quantitative variables was made. 2 × 2 tables were constructed to analyse the number of patients that fulfilled with the study protocol at each visit and number of failures in mouth rinse intake. Fisher’s exact test was used to compare the results. The statistical significance was set at the level of P < 0.05. Results Demographic data Thirty-one patients were randomized to the placebo group (10 males, 21 females; mean age 15.2 ± 2.1, range 11.75–20.25). Twenty-eight attended the 1-month visit, 24 the 2-month visit and 23 the 3-month visit. After 1 month, three patients left the study: one patient did not comply with the appointments, one patient did not want to continue in the study for personal reasons, and the other patient finished the orthodontic treatment earlier than expected. After 2 months, three additional patients left the study, two for lack of compliance with the appointments, and the other for personal reasons. One additional patient missed the 2-month visit, but later attended the final visit. After 3 months, two additional patients missed the evaluation due to an excessive delay in that visit (more than 1 month) (Table 1). Table 1. Demographic data of the patients at baseline. Placebo group Test group N 31 (49.2%) 32 (50.8%) n males 10 (15.9%) 15 (23.8%) n females 21 (33.3%) 17 (27%) Age (years) 15.2* (2.1) 15.0* (1.8) Plaque index (mean, SD) 2.59* (0.88) 2.57* (0.88) Gingival index (mean, SD) 1.07* (0.36) 0.92* (0.31) Bleeding on probing (mean, SD) 0.79* (0.23) 0.78* (0.25) Probing pocket depth (mm) 3.09* (0.39) 3.04* (0.46) Recessions (mm) 0* (0) 0* (0) Staining intensity upper 0.35* (0.73) 0.08* (0.29) Staining intensity lower 0.85* (1.99) 1.25* (2.18) Placebo group Test group N 31 (49.2%) 32 (50.8%) n males 10 (15.9%) 15 (23.8%) n females 21 (33.3%) 17 (27%) Age (years) 15.2* (2.1) 15.0* (1.8) Plaque index (mean, SD) 2.59* (0.88) 2.57* (0.88) Gingival index (mean, SD) 1.07* (0.36) 0.92* (0.31) Bleeding on probing (mean, SD) 0.79* (0.23) 0.78* (0.25) Probing pocket depth (mm) 3.09* (0.39) 3.04* (0.46) Recessions (mm) 0* (0) 0* (0) Staining intensity upper 0.35* (0.73) 0.08* (0.29) Staining intensity lower 0.85* (1.99) 1.25* (2.18) * Data are means (SD) or numbers (%). View Large Table 1. Demographic data of the patients at baseline. Placebo group Test group N 31 (49.2%) 32 (50.8%) n males 10 (15.9%) 15 (23.8%) n females 21 (33.3%) 17 (27%) Age (years) 15.2* (2.1) 15.0* (1.8) Plaque index (mean, SD) 2.59* (0.88) 2.57* (0.88) Gingival index (mean, SD) 1.07* (0.36) 0.92* (0.31) Bleeding on probing (mean, SD) 0.79* (0.23) 0.78* (0.25) Probing pocket depth (mm) 3.09* (0.39) 3.04* (0.46) Recessions (mm) 0* (0) 0* (0) Staining intensity upper 0.35* (0.73) 0.08* (0.29) Staining intensity lower 0.85* (1.99) 1.25* (2.18) Placebo group Test group N 31 (49.2%) 32 (50.8%) n males 10 (15.9%) 15 (23.8%) n females 21 (33.3%) 17 (27%) Age (years) 15.2* (2.1) 15.0* (1.8) Plaque index (mean, SD) 2.59* (0.88) 2.57* (0.88) Gingival index (mean, SD) 1.07* (0.36) 0.92* (0.31) Bleeding on probing (mean, SD) 0.79* (0.23) 0.78* (0.25) Probing pocket depth (mm) 3.09* (0.39) 3.04* (0.46) Recessions (mm) 0* (0) 0* (0) Staining intensity upper 0.35* (0.73) 0.08* (0.29) Staining intensity lower 0.85* (1.99) 1.25* (2.18) * Data are means (SD) or numbers (%). View Large Thirty-two patients were allocated to the test group (15 males, 17 females; mean age 15.0 ± 1.8, range 11.67–20.17). Twenty-nine, 28, and 27 patients attended the 1, 2, and 3-month visits, respectively. Three patients left the study before the 1-month visit, due to a lack of compliance with the assigned appointments. One patient dropped out before the 2-month visit due to pregnancy. One additional patient missed the last visit due to an excessive delay in the appointment. Plaque index The baseline plaque levels were similar in both treatment groups. After 1 month, the mean PlI increased in the placebo group (0.16), while it decreased in the test group (0.08). This increase was statistically significant in the placebo group in the upper jaw (P = 0.032). Differences between groups in changes from baseline to 1 month were statistically significant (P = 0.024) only in the maxilla (mean: 0.18, SD = 0.82 versus mean: −0.26, SD = 0.62). Plaque increased after 2 months in both groups, being statistically significant in the placebo group for all sites (P = 0.031) and in the upper jaw (P = 0.033). At 3 months, plaque levels were also higher when compared with baseline in both groups, and the increase was statistically significant in the placebo group for all sites (P = 0.028) and in the upper jaw (P = 0.004). No significant inter group differences were detected (Table 2). Table 2. Plaque index (PlI) at each visit and changes (negative values mean increase, positive values mean reduction) expressed as mean, standard deviation (SD) and 95% confidence intervals (95% CI). Placebo group Test group Plaque index n Mean SD 95% CI n Mean SD 95% CI All sites Baseline 31 2.59 0.88 2.26 2.91 32 2.57 0.88 2.25 2.88 1 month 28 2.71 0.90 2.36 3.06 29 2.45 1.02 2.07 2.84 2 months 24 2.85 0.87 2.48 3.21 28 2.75 0.78 2.45 3.05 3 months 23 2.73 0.86 2.36 3.11 28 2.73 0.88 2.39 3.07 Upper jaw Baseline 31 2.36 0.95 2.01 2.71 32 2.48 0.96 2.14 2.83 1 month 28 2.57 0.95 2.20 2.94 29 2.29 1.17 1.85 2.74 2 months 24 2.64 0.96 2.24 3.05 28 2.61 0.86 2.27 2.94 3 months 23 2.62 0.91 2.23 3.02 28 2.61 1.05 2.21 3.02 Lower jaw Baseline 31 2.83 0.95 2.48 3.18 32 2.65 0.89 2.33 2.97 1 month 28 2.87 0.93 2.51 3.23 29 2.61 0.94 2.26 2.97 2 months 24 3.06 0.84 2.71 3.41 28 2.89 0.76 2.59 3.18 3 months 23 2.84 0.85 2.47 3.21 28 2.86 0.81 2.55 3.18 PlI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base 1 month 28 −0.17 0.58 −0.39 0.05 29 0.09 0.68 −0.17 0.35 Base 2 months 24 −0.30 0.64 −0.57 −0.03 28 −0.24 0.63 −0.48 0.01 Base 3 months 23 −0.25 0.52 −0.48 −0.03 27 −0.21 0.67 −0.48 0.06 Upper jaw Base 1 month 28 −0.26 0.62 −0.51 −0.02 29 0.18 0.82 −0.13 0.50 Base 2 months 24 −0.31 0.67 −0.59 −0.03 28 −0.16 0.63 −0.41 0.08 Base 3 months 23 −0.38 0.57 −0.62 −0.13 27 −0.16 0.71 −0.44 0.12 Lower jaw Base 1 month 28 −0.08 0.84 −0.40 0.25 29 −0.01 0.75 −0.29 0.27 Base 2 months 24 −0.28 0.78 −0.61 0.05 28 −0.31 0.81 −0.63 0.00 Base 3 months 23 −0.11 0.61 −0.38 0.15 27 −0.28 0.88 −0.63 0.07 Placebo group Test group Plaque index n Mean SD 95% CI n Mean SD 95% CI All sites Baseline 31 2.59 0.88 2.26 2.91 32 2.57 0.88 2.25 2.88 1 month 28 2.71 0.90 2.36 3.06 29 2.45 1.02 2.07 2.84 2 months 24 2.85 0.87 2.48 3.21 28 2.75 0.78 2.45 3.05 3 months 23 2.73 0.86 2.36 3.11 28 2.73 0.88 2.39 3.07 Upper jaw Baseline 31 2.36 0.95 2.01 2.71 32 2.48 0.96 2.14 2.83 1 month 28 2.57 0.95 2.20 2.94 29 2.29 1.17 1.85 2.74 2 months 24 2.64 0.96 2.24 3.05 28 2.61 0.86 2.27 2.94 3 months 23 2.62 0.91 2.23 3.02 28 2.61 1.05 2.21 3.02 Lower jaw Baseline 31 2.83 0.95 2.48 3.18 32 2.65 0.89 2.33 2.97 1 month 28 2.87 0.93 2.51 3.23 29 2.61 0.94 2.26 2.97 2 months 24 3.06 0.84 2.71 3.41 28 2.89 0.76 2.59 3.18 3 months 23 2.84 0.85 2.47 3.21 28 2.86 0.81 2.55 3.18 PlI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base 1 month 28 −0.17 0.58 −0.39 0.05 29 0.09 0.68 −0.17 0.35 Base 2 months 24 −0.30 0.64 −0.57 −0.03 28 −0.24 0.63 −0.48 0.01 Base 3 months 23 −0.25 0.52 −0.48 −0.03 27 −0.21 0.67 −0.48 0.06 Upper jaw Base 1 month 28 −0.26 0.62 −0.51 −0.02 29 0.18 0.82 −0.13 0.50 Base 2 months 24 −0.31 0.67 −0.59 −0.03 28 −0.16 0.63 −0.41 0.08 Base 3 months 23 −0.38 0.57 −0.62 −0.13 27 −0.16 0.71 −0.44 0.12 Lower jaw Base 1 month 28 −0.08 0.84 −0.40 0.25 29 −0.01 0.75 −0.29 0.27 Base 2 months 24 −0.28 0.78 −0.61 0.05 28 −0.31 0.81 −0.63 0.00 Base 3 months 23 −0.11 0.61 −0.38 0.15 27 −0.28 0.88 −0.63 0.07 View Large Table 2. Plaque index (PlI) at each visit and changes (negative values mean increase, positive values mean reduction) expressed as mean, standard deviation (SD) and 95% confidence intervals (95% CI). Placebo group Test group Plaque index n Mean SD 95% CI n Mean SD 95% CI All sites Baseline 31 2.59 0.88 2.26 2.91 32 2.57 0.88 2.25 2.88 1 month 28 2.71 0.90 2.36 3.06 29 2.45 1.02 2.07 2.84 2 months 24 2.85 0.87 2.48 3.21 28 2.75 0.78 2.45 3.05 3 months 23 2.73 0.86 2.36 3.11 28 2.73 0.88 2.39 3.07 Upper jaw Baseline 31 2.36 0.95 2.01 2.71 32 2.48 0.96 2.14 2.83 1 month 28 2.57 0.95 2.20 2.94 29 2.29 1.17 1.85 2.74 2 months 24 2.64 0.96 2.24 3.05 28 2.61 0.86 2.27 2.94 3 months 23 2.62 0.91 2.23 3.02 28 2.61 1.05 2.21 3.02 Lower jaw Baseline 31 2.83 0.95 2.48 3.18 32 2.65 0.89 2.33 2.97 1 month 28 2.87 0.93 2.51 3.23 29 2.61 0.94 2.26 2.97 2 months 24 3.06 0.84 2.71 3.41 28 2.89 0.76 2.59 3.18 3 months 23 2.84 0.85 2.47 3.21 28 2.86 0.81 2.55 3.18 PlI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base 1 month 28 −0.17 0.58 −0.39 0.05 29 0.09 0.68 −0.17 0.35 Base 2 months 24 −0.30 0.64 −0.57 −0.03 28 −0.24 0.63 −0.48 0.01 Base 3 months 23 −0.25 0.52 −0.48 −0.03 27 −0.21 0.67 −0.48 0.06 Upper jaw Base 1 month 28 −0.26 0.62 −0.51 −0.02 29 0.18 0.82 −0.13 0.50 Base 2 months 24 −0.31 0.67 −0.59 −0.03 28 −0.16 0.63 −0.41 0.08 Base 3 months 23 −0.38 0.57 −0.62 −0.13 27 −0.16 0.71 −0.44 0.12 Lower jaw Base 1 month 28 −0.08 0.84 −0.40 0.25 29 −0.01 0.75 −0.29 0.27 Base 2 months 24 −0.28 0.78 −0.61 0.05 28 −0.31 0.81 −0.63 0.00 Base 3 months 23 −0.11 0.61 −0.38 0.15 27 −0.28 0.88 −0.63 0.07 Placebo group Test group Plaque index n Mean SD 95% CI n Mean SD 95% CI All sites Baseline 31 2.59 0.88 2.26 2.91 32 2.57 0.88 2.25 2.88 1 month 28 2.71 0.90 2.36 3.06 29 2.45 1.02 2.07 2.84 2 months 24 2.85 0.87 2.48 3.21 28 2.75 0.78 2.45 3.05 3 months 23 2.73 0.86 2.36 3.11 28 2.73 0.88 2.39 3.07 Upper jaw Baseline 31 2.36 0.95 2.01 2.71 32 2.48 0.96 2.14 2.83 1 month 28 2.57 0.95 2.20 2.94 29 2.29 1.17 1.85 2.74 2 months 24 2.64 0.96 2.24 3.05 28 2.61 0.86 2.27 2.94 3 months 23 2.62 0.91 2.23 3.02 28 2.61 1.05 2.21 3.02 Lower jaw Baseline 31 2.83 0.95 2.48 3.18 32 2.65 0.89 2.33 2.97 1 month 28 2.87 0.93 2.51 3.23 29 2.61 0.94 2.26 2.97 2 months 24 3.06 0.84 2.71 3.41 28 2.89 0.76 2.59 3.18 3 months 23 2.84 0.85 2.47 3.21 28 2.86 0.81 2.55 3.18 PlI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base 1 month 28 −0.17 0.58 −0.39 0.05 29 0.09 0.68 −0.17 0.35 Base 2 months 24 −0.30 0.64 −0.57 −0.03 28 −0.24 0.63 −0.48 0.01 Base 3 months 23 −0.25 0.52 −0.48 −0.03 27 −0.21 0.67 −0.48 0.06 Upper jaw Base 1 month 28 −0.26 0.62 −0.51 −0.02 29 0.18 0.82 −0.13 0.50 Base 2 months 24 −0.31 0.67 −0.59 −0.03 28 −0.16 0.63 −0.41 0.08 Base 3 months 23 −0.38 0.57 −0.62 −0.13 27 −0.16 0.71 −0.44 0.12 Lower jaw Base 1 month 28 −0.08 0.84 −0.40 0.25 29 −0.01 0.75 −0.29 0.27 Base 2 months 24 −0.28 0.78 −0.61 0.05 28 −0.31 0.81 −0.63 0.00 Base 3 months 23 −0.11 0.61 −0.38 0.15 27 −0.28 0.88 −0.63 0.07 View Large Gingival index Higher GI baseline levels were observed in the placebo group (1.07) compared to the test group (0.92), although the differences were not significant (P = 0.085). After 1 month, GI increased in the placebo group (mean = 0.05; SD = 0.33), and decreased in the test group (mean = −0.03; SD = 0.38), with significant differences at all sites (P = 0.025) and lower jaw (P = 0.007). At the 2-month visit, no inter-group differences were detected, and both groups demonstrated an increase in GI. After 3 months, minor and comparable increases were observed in both groups, although a significantly higher level of gingivitis was observed in the placebo group (mean = 0.05; SD = 0.43 versus Mean = 0.02; SD = 0.34) for the full-mouth index (P = 0.050) and lower jaw (P = 0.020) (Table 3). Table 3. Gingival index (GI) at each visit and changes (negative values mean increase, positive values mean reduction) expressed as mean, standard deviation (SD) and 95% confidence intervals (95% CI). Placebo group Test group Gingival index n Mean SD 95% CI n Mean SD 95% CI all sites Baseline 31 1.07 0.36 0.94 1.21 32 0.92 0.31 0.81 1.04 1 month 28 1.09 0.34 0.95 1.22 29 0.89 0.31 0.77 1.00 2 months 24 1.06 0.40 0.89 1.23 28 1.07 0.41 0.92 1.23 3 months 23 1.13 0.36 0.98 1.29 28 0.94 0.34 0.80 1.07 Upper jaw Baseline 31 0.98 0.39 0.84 1.13 32 0.82 0.36 0.69 0.94 1 month 28 0.96 0.37 0.82 1.11 29 0.80 0.38 0.65 0.94 2 months 24 0.95 0.47 0.75 1.15 28 0.98 0.49 0.79 1.17 3 months 23 1.02 0.41 0.84 1.20 28 0.85 0.40 0.69 1.00 Lower jaw Baseline 31 1.17 0.37 1.03 1.31 32 1.03 0.34 0.90 1.15 1 month 28 1.22 0.35 1.08 1.36 29 0.97 0.31 0.85 1.09 2 months 24 1.17 0.40 1.00 1.34 28 1.17 0.36 1.03 1.31 3 months 23 1.25 0.36 1.10 1.41 28 1.02 0.32 0.89 1.14 GI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base-1m 28 −0.05 0.33 −0.17 0.08 29 0.03 0.38 −0.11 0.18 Base-2m 24 −0.01 0.46 −0.21 0.18 28 −0.16 0.51 −0.36 0.03 Base-3m 23 −0.05 0.45 −0.25 0.14 27 −0.02 0.34 −0.16 0.11 Upper jaw Base-1m 28 −0.01 0.38 −0.15 0.14 29 0.01 0.47 −0.17 0.19 Base-2m 24 0.02 0.56 −0.21 0.26 28 −0.18 0.64 −0.43 0.07 Base-3m 23 −0.02 0.55 −0.25 0.22 27 −0.06 0.45 −0.23 0.12 Lower jaw Base-1m 28 −0.08 0.35 −0.21 0.06 29 0.05 0.35 −0.08 0.19 Base-2m 24 −0.04 0.40 −0.21 0.13 28 −0.15 0.45 −0.32 0.03 Base-3m 23 −0.07 0.40 −0.25 0.10 27 0.01 0.32 −0.12 0.14 Placebo group Test group Gingival index n Mean SD 95% CI n Mean SD 95% CI all sites Baseline 31 1.07 0.36 0.94 1.21 32 0.92 0.31 0.81 1.04 1 month 28 1.09 0.34 0.95 1.22 29 0.89 0.31 0.77 1.00 2 months 24 1.06 0.40 0.89 1.23 28 1.07 0.41 0.92 1.23 3 months 23 1.13 0.36 0.98 1.29 28 0.94 0.34 0.80 1.07 Upper jaw Baseline 31 0.98 0.39 0.84 1.13 32 0.82 0.36 0.69 0.94 1 month 28 0.96 0.37 0.82 1.11 29 0.80 0.38 0.65 0.94 2 months 24 0.95 0.47 0.75 1.15 28 0.98 0.49 0.79 1.17 3 months 23 1.02 0.41 0.84 1.20 28 0.85 0.40 0.69 1.00 Lower jaw Baseline 31 1.17 0.37 1.03 1.31 32 1.03 0.34 0.90 1.15 1 month 28 1.22 0.35 1.08 1.36 29 0.97 0.31 0.85 1.09 2 months 24 1.17 0.40 1.00 1.34 28 1.17 0.36 1.03 1.31 3 months 23 1.25 0.36 1.10 1.41 28 1.02 0.32 0.89 1.14 GI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base-1m 28 −0.05 0.33 −0.17 0.08 29 0.03 0.38 −0.11 0.18 Base-2m 24 −0.01 0.46 −0.21 0.18 28 −0.16 0.51 −0.36 0.03 Base-3m 23 −0.05 0.45 −0.25 0.14 27 −0.02 0.34 −0.16 0.11 Upper jaw Base-1m 28 −0.01 0.38 −0.15 0.14 29 0.01 0.47 −0.17 0.19 Base-2m 24 0.02 0.56 −0.21 0.26 28 −0.18 0.64 −0.43 0.07 Base-3m 23 −0.02 0.55 −0.25 0.22 27 −0.06 0.45 −0.23 0.12 Lower jaw Base-1m 28 −0.08 0.35 −0.21 0.06 29 0.05 0.35 −0.08 0.19 Base-2m 24 −0.04 0.40 −0.21 0.13 28 −0.15 0.45 −0.32 0.03 Base-3m 23 −0.07 0.40 −0.25 0.10 27 0.01 0.32 −0.12 0.14 View Large Table 3. Gingival index (GI) at each visit and changes (negative values mean increase, positive values mean reduction) expressed as mean, standard deviation (SD) and 95% confidence intervals (95% CI). Placebo group Test group Gingival index n Mean SD 95% CI n Mean SD 95% CI all sites Baseline 31 1.07 0.36 0.94 1.21 32 0.92 0.31 0.81 1.04 1 month 28 1.09 0.34 0.95 1.22 29 0.89 0.31 0.77 1.00 2 months 24 1.06 0.40 0.89 1.23 28 1.07 0.41 0.92 1.23 3 months 23 1.13 0.36 0.98 1.29 28 0.94 0.34 0.80 1.07 Upper jaw Baseline 31 0.98 0.39 0.84 1.13 32 0.82 0.36 0.69 0.94 1 month 28 0.96 0.37 0.82 1.11 29 0.80 0.38 0.65 0.94 2 months 24 0.95 0.47 0.75 1.15 28 0.98 0.49 0.79 1.17 3 months 23 1.02 0.41 0.84 1.20 28 0.85 0.40 0.69 1.00 Lower jaw Baseline 31 1.17 0.37 1.03 1.31 32 1.03 0.34 0.90 1.15 1 month 28 1.22 0.35 1.08 1.36 29 0.97 0.31 0.85 1.09 2 months 24 1.17 0.40 1.00 1.34 28 1.17 0.36 1.03 1.31 3 months 23 1.25 0.36 1.10 1.41 28 1.02 0.32 0.89 1.14 GI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base-1m 28 −0.05 0.33 −0.17 0.08 29 0.03 0.38 −0.11 0.18 Base-2m 24 −0.01 0.46 −0.21 0.18 28 −0.16 0.51 −0.36 0.03 Base-3m 23 −0.05 0.45 −0.25 0.14 27 −0.02 0.34 −0.16 0.11 Upper jaw Base-1m 28 −0.01 0.38 −0.15 0.14 29 0.01 0.47 −0.17 0.19 Base-2m 24 0.02 0.56 −0.21 0.26 28 −0.18 0.64 −0.43 0.07 Base-3m 23 −0.02 0.55 −0.25 0.22 27 −0.06 0.45 −0.23 0.12 Lower jaw Base-1m 28 −0.08 0.35 −0.21 0.06 29 0.05 0.35 −0.08 0.19 Base-2m 24 −0.04 0.40 −0.21 0.13 28 −0.15 0.45 −0.32 0.03 Base-3m 23 −0.07 0.40 −0.25 0.10 27 0.01 0.32 −0.12 0.14 Placebo group Test group Gingival index n Mean SD 95% CI n Mean SD 95% CI all sites Baseline 31 1.07 0.36 0.94 1.21 32 0.92 0.31 0.81 1.04 1 month 28 1.09 0.34 0.95 1.22 29 0.89 0.31 0.77 1.00 2 months 24 1.06 0.40 0.89 1.23 28 1.07 0.41 0.92 1.23 3 months 23 1.13 0.36 0.98 1.29 28 0.94 0.34 0.80 1.07 Upper jaw Baseline 31 0.98 0.39 0.84 1.13 32 0.82 0.36 0.69 0.94 1 month 28 0.96 0.37 0.82 1.11 29 0.80 0.38 0.65 0.94 2 months 24 0.95 0.47 0.75 1.15 28 0.98 0.49 0.79 1.17 3 months 23 1.02 0.41 0.84 1.20 28 0.85 0.40 0.69 1.00 Lower jaw Baseline 31 1.17 0.37 1.03 1.31 32 1.03 0.34 0.90 1.15 1 month 28 1.22 0.35 1.08 1.36 29 0.97 0.31 0.85 1.09 2 months 24 1.17 0.40 1.00 1.34 28 1.17 0.36 1.03 1.31 3 months 23 1.25 0.36 1.10 1.41 28 1.02 0.32 0.89 1.14 GI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base-1m 28 −0.05 0.33 −0.17 0.08 29 0.03 0.38 −0.11 0.18 Base-2m 24 −0.01 0.46 −0.21 0.18 28 −0.16 0.51 −0.36 0.03 Base-3m 23 −0.05 0.45 −0.25 0.14 27 −0.02 0.34 −0.16 0.11 Upper jaw Base-1m 28 −0.01 0.38 −0.15 0.14 29 0.01 0.47 −0.17 0.19 Base-2m 24 0.02 0.56 −0.21 0.26 28 −0.18 0.64 −0.43 0.07 Base-3m 23 −0.02 0.55 −0.25 0.22 27 −0.06 0.45 −0.23 0.12 Lower jaw Base-1m 28 −0.08 0.35 −0.21 0.06 29 0.05 0.35 −0.08 0.19 Base-2m 24 −0.04 0.40 −0.21 0.13 28 −0.15 0.45 −0.32 0.03 Base-3m 23 −0.07 0.40 −0.25 0.10 27 0.01 0.32 −0.12 0.14 View Large Calculus index Baseline calculus levels were similar and low in both groups. A slow increase was observed throughout the study, reaching similar results in both groups at 3 months. The change between baseline and 3 months was statistically significant (P = 0.041) only in the test group. None of the intergroup comparisons were significant. Tooth staining Intensity of staining As seen in Table 4, it is clear that the scores of intensity in the upper jaw were low in both groups and showed only minor changes during the study, although somehow higher in the placebo group. No statistically significant differences were detected. The intensity of staining in the lower jaw was higher than in the upper jaw, and slightly higher in the test group at baseline (Table 4). Minor changes were observed in the placebo group, but a tendency to decrease was observed in the test group, with an exception between 1 and 2 months. None of the comparisons were statistically significant. Table 4. Mean values and standard deviation (SD) in staining (surface, intensity), as evaluated by two researchers. Surface Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.48 1.14 0.13 0.43 0.96 2.29 1.75 3.39 1 month 0.46 1.27 0.04 0.19 0.67 1.25 0.44 0.87 2 months 0.33 0.71 0.10 0.40 0.50 1.04 0.96 2.22 3 months 0.45 1.01 0.12 0.44 0.73 1.39 0.48 1.23 Baseline 1 month 0.02 1.09 0.09 0.53 0.29 1.94 1.31 3.72 Baseline 3 months 0.03 1.56 0.01 0.61 0.23 0.67 1.27 3.00 Intensity Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.35 0.73 0.08 0.29 0.85 1.99 1.25 2.18 1 month 0.35 0.94 0.04 0.19 0.67 1.25 0.37 0.77 2 months 0.33 0.71 0.08 0.31 0.50 1.04 0.71 1.47 3 months 0.40 0.90 0.12 0.44 0.73 1.39 0.44 1.12 Baseline 1 month 0.00 0.79 0.05 0.35 0.18 1.54 0.88 2.06 Baseline 3 months −0.05 1.18 −0.04 0.41 0.12 0.58 0.81 1.33 Surface Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.48 1.14 0.13 0.43 0.96 2.29 1.75 3.39 1 month 0.46 1.27 0.04 0.19 0.67 1.25 0.44 0.87 2 months 0.33 0.71 0.10 0.40 0.50 1.04 0.96 2.22 3 months 0.45 1.01 0.12 0.44 0.73 1.39 0.48 1.23 Baseline 1 month 0.02 1.09 0.09 0.53 0.29 1.94 1.31 3.72 Baseline 3 months 0.03 1.56 0.01 0.61 0.23 0.67 1.27 3.00 Intensity Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.35 0.73 0.08 0.29 0.85 1.99 1.25 2.18 1 month 0.35 0.94 0.04 0.19 0.67 1.25 0.37 0.77 2 months 0.33 0.71 0.08 0.31 0.50 1.04 0.71 1.47 3 months 0.40 0.90 0.12 0.44 0.73 1.39 0.44 1.12 Baseline 1 month 0.00 0.79 0.05 0.35 0.18 1.54 0.88 2.06 Baseline 3 months −0.05 1.18 −0.04 0.41 0.12 0.58 0.81 1.33 Positive changes mean decrease. View Large Table 4. Mean values and standard deviation (SD) in staining (surface, intensity), as evaluated by two researchers. Surface Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.48 1.14 0.13 0.43 0.96 2.29 1.75 3.39 1 month 0.46 1.27 0.04 0.19 0.67 1.25 0.44 0.87 2 months 0.33 0.71 0.10 0.40 0.50 1.04 0.96 2.22 3 months 0.45 1.01 0.12 0.44 0.73 1.39 0.48 1.23 Baseline 1 month 0.02 1.09 0.09 0.53 0.29 1.94 1.31 3.72 Baseline 3 months 0.03 1.56 0.01 0.61 0.23 0.67 1.27 3.00 Intensity Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.35 0.73 0.08 0.29 0.85 1.99 1.25 2.18 1 month 0.35 0.94 0.04 0.19 0.67 1.25 0.37 0.77 2 months 0.33 0.71 0.08 0.31 0.50 1.04 0.71 1.47 3 months 0.40 0.90 0.12 0.44 0.73 1.39 0.44 1.12 Baseline 1 month 0.00 0.79 0.05 0.35 0.18 1.54 0.88 2.06 Baseline 3 months −0.05 1.18 −0.04 0.41 0.12 0.58 0.81 1.33 Surface Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.48 1.14 0.13 0.43 0.96 2.29 1.75 3.39 1 month 0.46 1.27 0.04 0.19 0.67 1.25 0.44 0.87 2 months 0.33 0.71 0.10 0.40 0.50 1.04 0.96 2.22 3 months 0.45 1.01 0.12 0.44 0.73 1.39 0.48 1.23 Baseline 1 month 0.02 1.09 0.09 0.53 0.29 1.94 1.31 3.72 Baseline 3 months 0.03 1.56 0.01 0.61 0.23 0.67 1.27 3.00 Intensity Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.35 0.73 0.08 0.29 0.85 1.99 1.25 2.18 1 month 0.35 0.94 0.04 0.19 0.67 1.25 0.37 0.77 2 months 0.33 0.71 0.08 0.31 0.50 1.04 0.71 1.47 3 months 0.40 0.90 0.12 0.44 0.73 1.39 0.44 1.12 Baseline 1 month 0.00 0.79 0.05 0.35 0.18 1.54 0.88 2.06 Baseline 3 months −0.05 1.18 −0.04 0.41 0.12 0.58 0.81 1.33 Positive changes mean decrease. View Large Surface of staining The scores of surfaces of the upper jaw were low in both groups, and again higher in the placebo group. A tendency towards a decrease was observed in both groups, although changes were very small and not significant (Table 4). The results in surface of staining confirmed the finding in intensity. Higher values were observed in the lower jaw, and they were also higher in the test group at baseline. In the placebo group, minor changes were observed, with a tendency to a decrease. However, in the test group, a clear trend to a reduction was observed, with the exception of the period between the 1- and the 2-month visits. De-bonded bands and/or braces The number of de-bonded braces was checked at each visit. Means of de-bonded brackets per visit (baseline, 1-, 2-, 3-month visits) were 0, 0.23, 0, and 0.14, respectively, for the placebo group; and 0.36, 0.5, 0.43, and 0.39, respectively, for the experimental group. No significant differences were found among visits within each group (P = 0.251) or considering visits and groups together (P = 0.782). The same result was found for the number of loose bands. Means of number of loose bands were 0.05, 0.05, 0, and 0, respectively, for the placebo group; and 0.04, 0.04, 0.14, and 0.04, respectively, for the experimental group. There were no significant differences between groups and visits. Oral mucosa injuries Mean values of mucosa injuries were lower in the experimental group than in the control group, except at the last visit. The evolution of the estimated means of mucosal injuries at each visit (baseline, 1-, 2-, 3-month visits) was 0.23, 0.41, 0.27, and 0.05, respectively, for the control group; and 0.11, 0.25, 0.25, and 0.21, respectively, for the experimental group. Differences were not statistically significant (P = 0.133). Microbiological results In the placebo group, samples from 29 out of 31 patients were available at baseline, while after 3 months, samples were available from the 23 patients attending the final visit. Pair comparisons were available from 21 patients. In the test group, 30 out of 32 patients provided baseline samples, while final samples were available from the 27 patients, allowing for 27 pair comparisons. Two microbiological samples at baseline in each group could not be processed for technical reasons. Comparable findings were observed at baseline for PlI and GI at sampled sites. Non-significant changes were observed in microbiological parameters and overgrowth of opportunistic species. The evaluated products can be considered as safe from a microbiological point of view. Additional supporting information may be found in the online version of this article: Supplementary Text 1 and Supplementary Table 1. Patient-reported outcomes At the 1-, 2- and 3-month visits, participants filled up a questionnaire. In the control group, 96.4, 95.8, and 100 per cent of patients completed the questionnaire after 1, 2, and 3 months, respectively. In the experimental group, the corresponging percentages were 96.6, 100, and 96.3 per cent. No statistical differences were found between groups. Answers were compared between groups at the 1-, 2-, and 3-month visits. After 1 month, no differences were observed between groups. Most of the subjects (69.1%) liked rinsing with the provided mouth rinse, 67.3 per cent of them liked the taste, 72.7 per cent of them noticed their teeth cleaner and smoother and 81.8 per cent had a pleasant and fresh breath feeling after rinsing. After 2 months, statistically significant differences (P ≤ 0.05) were only found for the question related to the taste of the product (Figure 1). In the control group, the percentage of patients considering the taste as ‘good’ was higher than in the experimental group (87 and 39.3 per cent, respectively. However, in the experimental group, the percentage of patients that considered it as ‘very good’ was higher (35.7 vs. 4.3 per cent). After converting the categorical outcome variable (very good, good, fair, bad) into a semi quantitative variable (4, 3, 2, 1, respectively) and comparing the average value between groups, patients rated the experimental mouth rinse higher than the control mouth rinse. Figure 1. View largeDownload slide Frequency distribution (in %) of answers given to the second question (‘The taste of this mouth rinse is…’) at 1, 2, and 3 months. Key for answers: A. very good, B. good, C. fair, D. bad. Figure 1. View largeDownload slide Frequency distribution (in %) of answers given to the second question (‘The taste of this mouth rinse is…’) at 1, 2, and 3 months. Key for answers: A. very good, B. good, C. fair, D. bad. After 3 months, no differences were found between groups regarding the frequency of responses to each question, except again for the question concerning the taste of mouth rinse with the same trend as in visit 2 (Figure 1). Eighty-seven per cent of control patients noticed ‘good’ taste for their mouth rinse and 46.2 per cent of experimental patients thought that the taste was ‘very good’ (related to the total of patients in each group). After noticing the significant differences, the categorical variable was transformed into a semi-quantitative variable, and it was observed that the average of responses to this question in the experimental group was higher. Compliance Compliance was first assessed by analysing the number of patients that fulfilled with the study protocol at each visit, according to the diary cards. Percentages decreased with time, but no differences were found between groups or among visits (91.3 vs. 96.3% at 1 month, 69.6 vs. 85.2% at 2 months and 56.5 vs. 77.8% at 3 months for placebo and test groups, respectively). A second analysis was performed, by counting the number of occasions that a patient failed to use the mouth rinse according to the daily diaries. Percentages of failed intakes were 16, 13, and 21 per cent for each visit. In the test group, slightly lower values were found (12, 15, and 11 per cent for each visit). Although this percentage reached 21 per cent in the control group after 3 months, no significant differences were found between groups or among visits. As an indirect variable to test for compliance, the ml of mouth rinse left in the last bottle used (two 500 ml bottles were provided at each visit) was measured at the end of each visit. Comparisons between groups at each visit did not show statistically significant differences, although the amount of test product left was increasingly higher (mean: 284.19, SD: 173.75 vs. mean: 282.20, SD: 134.01 at 1 month; mean: 286.46, SD: 138.31 vs. mean: 316.21, SD: 161.38 at 2 months; mean: 289.31, SD: 146.66 vs. mean: 375.54, SD: 159.92 at 3 months for placebo and test groups, respectively). The amount of test product left at the 3-month visit was statistically higher than the amounts left at 1- and 2-month visits (P = 0.036). Discussion In the present study, the adjunctive use of the test mouth rinse and dentifrice had a modest effect on plaque and gingivitis reduction. The use of this adjunctive treatment was not associated with an increase in tooth staining, although the compliance during this 3-month period with the study protocol, in both test and control groups, was limited. In addition, the test products were able to control and even reduce some periodontal pathogens, without an overgrowth of opportunistic species. As it is customary in oral hygiene studies, the main outcome variables used were plaque accumulation and gingivitis. Plaque levels were high at baseline, and after 1 month, increased in the placebo group (significantly in the maxilla) and decreased in the test group, achieving significant inter-group differences (P = 0.024) at the maxilla. However, this effect was small, only in the upper jaw and not clinically significant. The size effect used for sample size calculation (mean 0.42, SD = 0.40) was far from what has been observed in the study. We were expecting to detect a standardized effect size of 1.05 (obtained from dividing mean/SD), but the largest effect size we actually found in the test group was 0.22. This means that, despite the fact of finding statistically significant differences when comparing the test and the control groups, no clinical significance can be drawn from these results. Moreover, it is not clear why plaque levels raised in the subjects from the control group when they were provided with oral hygiene instruction, continued to brush their teeth and presumably they suffered the Hawthorn effect since they were participating in a trial in which their oral hygiene would be assessed. After 2 and 3 months, plaque levels tended to increase in both groups, but with a lower magnitude in the test group. These findings have also been observed in previous oral hygiene studies in similar populations, evaluating either a mouth rinse with essential oils (18) or a tooth paste and a mouth rinse with stannous and amine fluoride (19). In contrast, when CHX formulations have been utilized, significant plaque reductions were maintained throughout the study. In a study using a CHX mouth rinse (16), significant reductions were observed in the test group after 3 months, while the control group, despite initial reductions after 1 and 2 months, baseline levels were reached after 3 months. In another study, comparing CHX dentifrices (with and without sodium fluoride) with a sodium fluoride dentifrice (15), a clear reduction was observed from baseline to the 6-week visit in all groups. In the present study, even though the gingivitis levels were higher at baseline in the placebo group, after 1 month, the test group showed a slight reduction, while there was a slight increase in the placebo group, which resulted in significant intergroup differences at 1-month for all sites (P = 0.025), as well as for proximal, buccal and lingual surfaces and mandibular sites. Differences were maintained at the 3-month visit, for all sites, proximal and in the mandibular sites. As discussed for plaque levels, a similar trend was observed in two studies assessing an essential oils mouth rinse (18) or the combination of a dentifrice and a mouth rinse with amine and stannous fluoride (19). It has also been suggested that effect of essential oils on gingivitis is more pronounced than on plaque formation due to anti-inflammatory effects of the phenolic components (42). The studies evaluating CHX formulations (15,16) also demonstrated a more significant and maintained effect on gingivitis, although the presence of adverse effects, specifically tooth staining, may limit their long-term use. In this investigation, the gingivitis reduction was also modest, following the same trend as the plaque levels. This study carefully evaluated the occurrence of side effects, such as tooth staining as well as adverse effects, as de-bonded appliances or mucosal injuries. Although few studies have reported allergic reactions associated to the topical use of aloe vera, it seems that they are mostly due to anthra-quinones, such as aloin and barbolin (43). None of our patients reported redness, burning or stinging sensation. A recent systematic review on the effectiveness of aloe vera in the management of oral mucosal diseases did not find any adverse effects (44). In contrast with the use of other formulations and active agents used in young orthodontic patients that have shown increased tooth staining (45), the subjects using the tested products did not have significantly higher staining when compared with the placebo group. In fact, a tendency towards reduced tooth staining was observed in both test and placebo groups, being slightly higher in the test group. Ulceration, erosion, and desquamation have also been associated to wearing fixed orthodontic appliances (46), especially during the initial phases of treatment. In our study, oral mucosal injuries were less frequent in the test group, although differences were not significant. However, their incidence was low in both groups, which could be explained by the fact that most of these patients had been wearing the orthodontic appliances for some time before the study started. In the present study, the baseline microbiological samples were taken when the patients already had their brackets placed, what prevents any inference on the possible impact of the brackets on the subgingival microbiota, since high prevalence of recognised periodontal pathogens were detected in both groups. The microbiological results did not find significant differences, but provided evidence on the safety of the evaluated products in terms of absence of microbiological adverse effects (namely, the overgrowth of opportunistic species). Additional supporting information may be found in the online version of this article: Supplementary Text 1 In regards to the recorded patient-reported outcomes, the test products showed a better-rated taste when compared with the control. Significant differences were demonstrated at the 2- and 3-month visit, but not in the 1-month visit. Analyzing the frequency of the given answers, patients in the test group either liked it very much or disliked it, but control patients had a less extreme appreciation of the taste. The number of patients who liked the test product ‘very much’ increased with time. It may be speculated that test product had a higher intensity in flavour, compared to the control. Repeated exposure to this strong flavour may have an effect in the increase of the patient’s preferences (47). However, a limitation of this study could be the use of a questionnaire custom-made for this investigation, instead of one previously validated. Compliance with the use of the tested products was evaluated by diary cards. Both groups showed a relatively high level of compliance during the first month of the study, since only 3 out of the 50 patients failed to fulfill the study protocol. At the end of the study, however, this number raised to 16 patients (10 in the control and 6 in the experimental group), what may have affected the clinical results of the study. Ay et al. (48) evaluated different oral hygiene motivation methods for orthodontic patients with fixed appliances based on verbal information and reported improvements after 4 weeks, but not for longer periods. A recently published 6-month study on the efficacy of three different motivational techniques on oral hygiene in orthodontic patients, however, concluded that all the used methods were equally effective in controlling gingival health provided periodic reinforcement was applied irrespective of the technique (49). Our tested sample demonstrated a low oral hygiene motivation, probably due to the specific population attending a public university clinic. Differences have been observed between public and private orthodontic clinics, being patients in public institutions less motivated than in private ones (50). This study has shown a limited efficacy in terms of plaque and gingivitis reduction when patients used their assigned test product in comparison with a placebo, being these differences of small magnitude. This fact can be explained in part by the heterogeneity in terms of compliance with mechanical plaque control demonstrated by the subjects participating in this study, as well as in the differences in the orthodontic therapy, since the size of the arch wires was not standardized, what may have influenced the subject’s mechanical plaque control. This low compliance in mechanical plaque control may however, represent the reality affecting young patients undergoing fixed orthodontic therapy. Moreover, this is a short-term study (3 months), where subjects usually will be more likely to adhere to the provided instructions, and therefore, with longer term evaluations, it is expected the efficacy of the recommended regime will decline. In conclusion, the present study indicates that the adjunctive use of a CPC-based mouth rinse and dentifrice had limited effect in controlling plaque accumulation and gingivitis levels, and no adverse microbiological effects. The size of the effects was small and highly variable, and multiple comparisons performed in the secondary statistical tests could have increased the risk of a Type I error (false positive findings). Within the limitations of this study, it could also be concluded that the use of the test products was not associated with relevant adverse effects (including tooth staining or debonded appliances) and young orthodontic patients liked the taste of a CPC-based mouth rinse, which may have help to comply with the assigned oral hygiene regimen. Supplementary material Supplementary material is available at European Journal of Orthodontics online. Funding Research contract between Dentaid (Cerdanyola, Barcelona, Spain) and the University Complutense of Madrid. This work was partially supported by a research contract between Dentaid (Cerdanyola, Barcelona, Spain) and the University Complutense of Madrid. Conflict of Interest Statement None to declare. References 1. Levin , L. , Samorodnitzky-Naveh , G.R. and Machtei , E.E . ( 2008 ) The association of orthodontic treatment and fixed retainers with gingival health . Journal of Periodontology , 79 , 2087 – 2092 . Google Scholar Crossref Search ADS PubMed 2. Ristic , M. , Vlahovic Svabic , M. , Sasic , M. and Zelic , O . ( 2007 ) Clinical and microbiological effects of fixed orthodontic appliances on periodontal tissues in adolescents . Orthodontics and Craniofacial Research , 10 , 187 – 195 . Google Scholar Crossref Search ADS PubMed 3. Artun , J. and Brobakken , B.O . ( 1986 ) Prevalence of carious white spots after orthodontic treatment with multibonded appliances . European Journal of Orthodontics , 8 , 229 – 234 . Google Scholar Crossref Search ADS PubMed 4. Naranjo , A.A. , Triviño , M.L. , Jaramillo , A. , Betancourth , M. and Botero , J.E . ( 2006 ) Changes in the subgingival microbiota and periodontal parameters before and 3 months after bracket placement . American Journal of Orthodontics and Dentofacial Orthopedics , 130 , 275.e17 – 275.e22 . Google Scholar Crossref Search ADS 5. Lee , S.M. , Yoo , S.Y. , Kim , H.S. , Kim , K.W. , Yoon , Y.J. , Lim , S.H. , Shin , H.Y. and Kook , J.K . ( 2005 ) Prevalence of putative periodontopathogens in subgingival dental plaques from gingivitis lesions in Korean orthodontic patients . Journal of Microbiology (Seoul, Korea) , 43 , 260 – 265 . Google Scholar PubMed 6. Diamanti-Kipioti , A. , Gusberti , F.A. and Lang , N.P . ( 1987 ) Clinical and microbiological effects of fixed orthodontic appliances . Journal of Clinical Periodontology , 14 , 326 – 333 . Google Scholar Crossref Search ADS PubMed 7. Alexander , S.A . ( 1993 ) The effect of fixed and functional appliances on enamel decalcifications in early Class II treatment . American Journal of Orthodontics and Dentofacial Orthopedics , 103 , 45 – 47 . Google Scholar Crossref Search ADS PubMed 8. Mehra , T. , Nanda , R.S. and Sinha , P.K . ( 1998 ) Orthodontists’ assessment and management of patient compliance . The Angle Orthodontist , 68 , 115 – 122 . Google Scholar PubMed 9. Ainamo , J . ( 1977 ) Control of plaque by chemical agents . Journal of Clinical Periodontology , 4 , 23 – 35 . Google Scholar Crossref Search ADS PubMed 10. Gunsolley , J.C . ( 2006 ) A meta-analysis of six-month studies of antiplaque and antigingivitis agents . Journal of the American Dental Association (1939) , 137 , 1649 – 1657 . Google Scholar Crossref Search ADS PubMed 11. Boyd , R.L . ( 1993 ) Comparison of three self-applied topical fluoride preparations for control of decalcification . The Angle Orthodontist , 63 , 25 – 30 . Google Scholar PubMed 12. Alexander , S.A. and Ripa , L.W . ( 2000 ) Effects of self-applied topical fluoride preparations in orthodontic patients . The Angle Orthodontist , 70 , 424 – 430 . Google Scholar PubMed 13. Kalha , A . ( 2004 ) Some evidence that fluoride during orthodontic treatment reduces occurrence and severity of white spot lesions . Evidence-Based Dentistry , 5 , 98 – 99 . Google Scholar Crossref Search ADS PubMed 14. Garnick , J.J. , Singh , B. and Winkley , G . ( 1998 ) Effectiveness of a medicament containing silicon dioxide, aloe, and allantoin on aphthous stomatitis . Oral surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics , 86 , 550 – 556 . Google Scholar Crossref Search ADS PubMed 15. Olympio , K.P. , Bardal , P.A. , de M Bastos , J.R. and Buzalaf , M.A . ( 2006 ) Effectiveness of a chlorhexidine dentifrice in orthodontic patients: a randomized-controlled trial . Journal of Clinical Periodontology , 33 , 421 – 426 . Google Scholar Crossref Search ADS PubMed 16. Anderson , G.B. , Bowden , J. , Morrison , E.C. and Caffesse , R.G . ( 1997 ) Clinical effects of chlorhexidine mouthwashes on patients undergoing orthodontic treatment . American Journal of Orthodontics and Dentofacial Orthopedics , 111 , 606 – 612 . Google Scholar Crossref Search ADS PubMed 17. Brightman , L.J. , Terezhalmy , G.T. , Greenwell , H. , Jacobs , M. and Enlow , D.H . ( 1991 ) The effects of a 0.12% chlorhexidine gluconate mouthrinse on orthodontic patients aged 11 through 17 with established gingivitis . American Journal of Orthodontics and Dentofacial Orthopedics , 100 , 324 – 329 . Google Scholar Crossref Search ADS PubMed 18. Tufekci , E. , Casagrande , Z.A. , Lindauer , S.J. , Fowler , C.E. and Williams , K.T . ( 2008 ) Effectiveness of an essential oil mouthrinse in improving oral health in orthodontic patients . The Angle Orthodontist , 78 , 294 – 298 . Google Scholar Crossref Search ADS PubMed 19. Øgaard , B. , Alm , A.A. , Larsson , E. and Adolfsson , U . ( 2006 ) A prospective, randomized clinical study on the effects of an amine fluoride/stannous fluoride toothpaste/mouthrinse on plaque, gingivitis and initial caries lesion development in orthodontic patients . European Journal of Orthodontics , 28 , 8 – 12 . Google Scholar Crossref Search ADS PubMed 20. Hannah , J.J. , Johnson , J.D. and Kuftinec , M.M . ( 1989 ) Long-term clinical evaluation of toothpaste and oral rinse containing sanguinaria extract in controlling plaque, gingival inflammation, and sulcular bleeding during orthodontic treatment . American Journal of Orthodontics and Dentofacial Orthopedics , 96 , 199 – 207 . Google Scholar Crossref Search ADS PubMed 21. Witt , J. , Ramji , N. , Gibb , R. , Dunavent , J. , Flood , J. and Barnes , J . ( 2005 ) Antibacterial and antiplaque effects of a novel, alcohol-free oral rinse with cetylpyridinium chloride . The Journal of Contemporary Dental Practice , 6 , 1 – 9 . Google Scholar PubMed 22. Witt , J.J. , Walters , P. , Bsoul , S. , Gibb , R. , Dunavent , J. and Putt , M . ( 2005 ) Comparative clinical trial of two antigingivitis mouthrinses . American Journal of Dentistry , 18 , 15A – 17A . Google Scholar PubMed 23. Rawlinson , A. , Pollington , S. , Walsh , T.F. , Lamb , D.J. , Marlow , I. , Haywood , J. and Wright , P . ( 2008 ) Efficacy of two alcohol-free cetylpyridinium chloride mouthwashes - a randomized double-blind crossover study . Journal of Clinical Periodontology , 35 , 230 – 235 . Google Scholar Crossref Search ADS PubMed 24. Mankodi , S. , Bauroth , K. , Witt , J.J. , Bsoul , S. , He , T. , Gibb , R. , Dunavent , J. and Hamilton , A . ( 2005 ) A 6-month clinical trial to study the effects of a cetylpyridinium chloride mouthrinse on gingivitis and plaque . American Journal of Dentistry , 18 Spec No , 9A – 14A . Google Scholar PubMed 25. Allen , D.R. et al. ( 1998 ) Efficacy of a mouthrinse containing 0.05% cetylpyridinium chloride for the control of plaque and gingivitis: a 6-month clinical study in adults . Compendium of Continuing Education in Dentistry (Jamesburg, N.J.: 1995) , 19 , 20 – 26 . Google Scholar PubMed 26. Silva , M.F. , dos Santos , N.B. , Stewart , B. , DeVizio , W. and Proskin , H.M . ( 2009 ) A clinical investigation of the efficacy of a commercial mouthrinse containing 0.05% cetylpyridinium chloride to control established dental plaque and gingivitis . The Journal of Clinical Dentistry , 20 , 55 – 61 . Google Scholar PubMed 27. Albert-Kiszely , A. , Pjetursson , B.E. , Salvi , G.E. , Witt , J. , Hamilton , A. , Persson , G.R. and Lang , N.P . ( 2007 ) Comparison of the effects of cetylpyridinium chloride with an essential oil mouth rinse on dental plaque and gingivitis - a six-month randomized controlled clinical trial . Journal of Clinical Periodontology , 34 , 658 – 667 . Google Scholar Crossref Search ADS PubMed 28. Jenkins , S. , Addy , M. and Newcombe , R.G . ( 1994 ) A comparison of cetylpyridinium chloride, triclosan and chlorhexidine mouthrinse formulations for effects on plaque regrowth . Journal of Clinical Periodontology , 21 , 441 – 444 . Google Scholar Crossref Search ADS PubMed 29. Kozak , K.M. , Gibb , R. , Dunavent , J. and White , D.J . ( 2005 ) Efficacy of a high bioavailable cetylpyridinium chloride mouthrinse over a 24-hour period: a plaque imaging study . American Journal of Dentistry , 18 , 18A – 23A . Google Scholar PubMed 30. Haps , S. , Slot , D.E. , Berchier , C.E. and Van der Weijden , G.A . ( 2008 ) The effect of cetylpyridinium chloride-containing mouth rinses as adjuncts to toothbrushing on plaque and parameters of gingival inflammation: a systematic review . International Journal of Dental Hygiene , 6 , 290 – 303 . Google Scholar Crossref Search ADS PubMed 31. Hsu , S . ( 2005 ) Green tea and the skin . Journal of the American Academy of Dermatology , 52 , 1049 – 1059 . Google Scholar Crossref Search ADS PubMed 32. Worthington , H.V. , Clarkson , J.E. and Eden , O.B . ( 2006 ) Interventions for preventing oral mucositis for patients with cancer receiving treatment . The Cochrane Database of Systematic Reviews , 19 , CD000978 . 33. Turesky , S. , Gilmore , N.D. and Glickman , I . ( 1970 ) Reduced plaque formation by the chloromethyl analogue of victamine C . Journal of Periodontology , 41 , 41 – 43 . Google Scholar Crossref Search ADS PubMed 34. Lobene , R.R. , Weatherford , T. , Ross , N.M. , Lamm , R.A. and Menaker , L . ( 1986 ) A modified gingival index for use in clinical trials . Clinical Preventive Dentistry , 8 , 3 – 6 . Google Scholar PubMed 35. Volpe , A.R. , Manhold , J.H. and Hazen , S.P . ( 1965 ) In vivo calculus assessment. I. A method and its examiner reproducibility . The Journal of Periodontology , 36 , 292 – 298 . Google Scholar Crossref Search ADS PubMed 36. Gadhia , K. , Shah , R. , Swaminathan , D. , Wetton , S. and Moran , J . ( 2006 ) Development of a stain shade guide to aid the measurement of extrinsic dental stain . International Journal of Dental Hygiene , 4 , 98 – 103 . Google Scholar Crossref Search ADS PubMed 37. Wikström , M. , Renvert , S. , Dahlén , G. and Johnsson , T . ( 1991 ) Variance in recovery of periodontitis-associated bacteria caused by sampling technique and laboratory processing . Oral Microbiology and Immunology , 6 , 102 – 106 . Google Scholar Crossref Search ADS PubMed 38. Syed , S.A. and Loesche , W.J . ( 1972 ) Survival of human dental plaque flora in various transport media . Applied Microbiology , 24 , 638 – 644 . Google Scholar PubMed 39. Dahlén , G. , Renvert , S. , Wikström , M. and Egelberg , J . ( 1990 ) Reproducibility of microbiological samples from periodontal pockets . Journal of Clinical Periodontology , 17 , 73 – 77 . Google Scholar Crossref Search ADS PubMed 40. Alsina , M. , Olle , E. and Frias , J . ( 2001 ) Improved, low-cost selective culture medium for Actinobacillus actinomycetemcomitans . Journal of Clinical Microbiology , 39 , 509 – 513 . Google Scholar Crossref Search ADS PubMed 41. Rioboo , M. , García , V. , Serrano , J. , O’Connor , A. , Herrera , D. and Sanz , M . ( 2012 ) Clinical and microbiological efficacy of an antimicrobial mouth rinse containing 0.05% cetylpyridinium chloride in patients with gingivitis . International Journal of Dental Hygiene , 10 , 98 – 106 . Google Scholar Crossref Search ADS PubMed 42. Sekino , S. and Ramberg , P . ( 2005 ) The effect of a mouth rinse containing phenolic compounds on plaque formation and developing gingivitis . Journal of Clinical Periodontology , 32 , 1083 – 1088 . Google Scholar Crossref Search ADS PubMed 43. Moore , T.E . ( 2001 ) The M and M’s of aloe vera–is it for dentistry ? Journal - Oklahoma Dental Association , 91 , 30 – 1, 36 . Google Scholar PubMed 44. Nair , G.R. , Naidu , G.S. , Jain , S. , Nagi , R. , Makkad , R.S. and Jha , A . ( 2016 ) Clinical effectiveness of aloe vera in the management of oral mucosal diseases- a systematic review . Journal of Clinical and Diagnostic Research , 10 , ZE01 – ZE07 . Google Scholar PubMed 45. Cosyn , J. and Verelst , K . ( 2006 ) An efficacy and safety analysis of a chlorhexidine chewing gum in young orthodontic patients . Journal of Clinical Periodontology , 33 , 894 – 899 . Google Scholar Crossref Search ADS PubMed 46. Baricevic , M. , Mravak-Stipetic , M. , Majstorovic , M. , Baranovic , M. , Baricevic , D. and Loncar , B . ( 2011 ) Oral mucosal lesions during orthodontic treatment . International Journal of Paediatric Dentistry , 21 , 96 – 102 . Google Scholar Crossref Search ADS PubMed 47. Liem , D.G. and de Graaf , C . ( 2004 ) Sweet and sour preferences in young children and adults: role of repeated exposure . Physiology and Behavior , 83 , 421 – 429 . Google Scholar Crossref Search ADS PubMed 48. Ay , Z.Y. , Sayin , M.O. , Ozat , Y. , Goster , T. , Atilla , A.O. and Bozkurt , F.Y . ( 2007 ) Appropriate oral hygiene motivation method for patients with fixed appliances . The Angle Orthodontist , 77 , 1085 – 1089 . Google Scholar Crossref Search ADS PubMed 49. Acharya , S. , Goyal , A. , Utreja , A.K. and Mohanty , U . ( 2011 ) Effect of three different motivational techniques on oral hygiene and gingival health of patients undergoing multibracketed orthodontics . The Angle Orthodontist , 81 , 884 – 888 . Google Scholar Crossref Search ADS PubMed 50. Tervonen , M.M. , Pirttiniemi , P. and Lahti , S . ( 2011 ) Development of a measure for orthodontists to evaluate patient compliance . American Journal of Orthodontics and Dentofacial Orthopedics , 139 , 791 – 796 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The European Journal of Orthodontics Oxford University Press

Clinical and microbiological effects of the use of a cetylpyridinium chloride dentifrice and mouth rinse in orthodontic patients: a 3-month randomized clinical trial

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Oxford University Press
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© The Author(s) 2017. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0141-5387
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10.1093/ejo/cjx096
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Abstract

Summary Objective To assess the clinical, microbiological, and patient-based effects of using a cetylpyridinium chloride (CPC) toothpaste and mouth rinse in orthodontic patients. Design Parallel randomized controlled, triple-blinded trial (participants, examiners, outcomes’ assessors). A computer-generated list was used to allocate treatments. Central allocation was used for concealment. Participants Thirty-one placebo (10 males, 21 females; mean age 15.2 ± 2.1) and 32 test patients (15 males, 17 females; mean age 15.0 ± 1.8) with fixed orthodontic appliance were included in the study. Interventions Patients were randomly assigned to both brush and rinse with placebo or with CPC-based products. Products were purposely prepared in white opaque bottles. After screening and professional prophylaxis, patients received a baseline examination, and started to use the assigned products. Patients were monthly assessed during a 3-month period. Main outcome measures Plaque (PlI) and gingival (GI) indexes. Secondary outcome variables: Calculus index, tooth staining, subgingival microbiological samples, patient-based variables (questionnaire, compliance, and remaining mouth rinse), side-effects (debonded braces or mucosal injuries). Results Sixty-three patients were randomized, 13 patients were excluded from analysis because of early dropout, leaving 50 patients for intention to treat analysis. PlI in the upper jaw diminished (0.18; SD = 0.82) after 1 month in the test group, while it increased (0.26; SD = 0.62) in the placebo group (P = 0.024). Statistically significant higher GI values were observed in the placebo group at 1 month (mean increase = 0.05; SD = 0.33 versus mean decrease = 0.03; SD = 0.38) and 3 months (mean = 0.05; SD = 0.43 versus mean = 0.02; SD = 0.34). The taste of the test product was rated higher than the placebo at 2- and 3-month visits (P < 0.05). Non-significant changes were observed in microbiological parameters, overgrowth of opportunistic species or remaining secondary outcome variables, including side-effects. Conclusion The use of CPC-based toothpaste and mouth rinse in orthodontic patients had limited effect in reducing plaque accumulation and gingival inflammation. Effects were little and highly variable. The use of the test products was not associated with relevant adverse effects. Trial registration Trial registration: Local internal reference: P07/133. Introduction The control of supragingival biofilm by means of mechanical devices, such as toothbrushes, dental floss, or interdental brushes, is a key element in the prevention of caries and periodontal diseases. The presence of orthodontic fixed appliances, however, makes this mechanical control more difficult, facilitating plaque retention, gingivitis (1, 2), and initial caries or white spots lesions (3). Enhanced plaque accumulation may also be associated with an increase in the colonization of periodontal pathogens, such as Prevotella intermedia (2, 4, 5, 6). Moreover, many orthodontic patients, especially children and adolescents, fail to floss because they find this procedure time-consuming and tedious in the presence of orthodontic arch wires (7). Studies assessing compliance in these patients have reported that their level of cooperation varies considerably depending on their age and gender, personality, perception of the malocclusion, parental influence, and socioeconomic factors (8). As a result of these facts, adolescents or young adults wearing fixed orthodontic appliances tend to accumulate more plaque and hence, they are at a higher risk of developing gingivitis. A common strategy to improve mechanical plaque removal in these patients is the addition of a chemical antimicrobial agent with anti-plaque or anti-caries activity in dentifrices, mouth rinses, or both (9). Ideally, these agents should have antimicrobial activity, for reducing plaque accumulation and gingival inflammation (10); anti-caries activity for decreasing the decalcifications usually occurring during orthodontic treatment and thus preventing the development of caries or white spots (11–13) and anti-inflammatory activity to help in the healing of traumatic mucosal injuries caused by the appliances (14). Since these products are aimed for long-term use compliance is a critical factor and therefore, they should have a pleasant flavour and lack of side effects, such as staining or de-bonding of the braces and bands. The effectiveness of different active ingredients, such as chlorhexidine (CHX) (15–17), essential oils (18), amine/stannous fluoride (19), or sanguinarine (20) in the form of mouth rinses, tooth pastes, or gels, has been evaluated in clinical studies. Most of these clinical studies have reported significant benefits in the adjunctive use of these products, although the magnitude of these reported benefits might not have a clear clinical relevance. In addition, the use of some of the formulations was associated with adverse effects (such as staining with the use of CHX). In recent years, the use of the chemical antimicrobial agent cetylpyridinium chloride (CPC) has attracted some attention, due to the advent of formulations with improved bioavailability or increased concentration, which have shown increased clinical benefits and since their adverse effects are very limited (tooth staining, ulcers, gingival irritation), its use may be indicated for longer periods (21,22). CPC has demonstrated in a number of clinical trials to reduce plaque formation (23–26) and gingivitis (24, 26–29). When used as a mouth rinse, a systematic review has shown heterogeneous results (30), although a significant effect on plaque has been demonstrated at different concentrations, such as 0.07 or 0.05 per cent (21,22,28). Recently, a new CPC-based mouth rinse and toothpaste were specifically formulated and marketed for orthodontic patients (VITIS Orthodontic, Dentaid, Cerdanyola, Spain). This formulation includes other active ingredients, such as allantoin, aloe vera, and sodium fluoride. Allantoin is widely used in dermatology since it promotes cell proliferation and stimulates epithelisation (31). In dentistry, it has been added in formulations for the treatment of aphthous stomatitis (14). Similarly, aloe vera has been added in formulations for the treatment of oral mucositis in cancer patients (32) and for aphthous stomatitis (14). This new formulation, aimed to be used by orthodontic patients, has not been evaluated in home-use randomised clinical trials. It was, therefore, the objective of this investigation to assess the clinical and microbiological efficacy of the combined use of a CPC-based toothpaste and mouth rinse formulations, adjunctive to mechanical plaque control in patients wearing fixed orthodontic appliances. In addition, compliance and the occurrence of adverse effects were evaluated. Subjects and methods/methods Patient population A consecutive sample of subjects undergoing treatment with fixed orthodontic appliances was screened during 12 months at the Department of Orthodontics in the Faculty of Odontology of the authors’ institution. The recruitment period for this study was from September 2013 to September 2014. During the screening visit, subjects were examined for fulfilment of the inclusion criteria and received a comprehensive explanation on the aims of the investigation. They were then asked to participate by signing an informed consent previously approved by the Ethical Review Committee (Comité ético de Investigación Clínica, Hospital Clínico San Carlos. Internal reference: P07/133. Date of approval: May 2007). Study participants were selected according to the following inclusion criteria: Healthy subjects between 12 and 25 years old. Participants were excluded in the presence of systemic diseases that might interfere with the study or if in need of chronic analgesic or anti-inflammatory medication. Undergoing orthodontic therapy with fixed appliances both in the upper and lower arch with monthly follow-up appointments, Clinical evidence of gingivitis (visually at screening, then confirmed with the Gingival Index). Participants were excluded in presence of periodontitis (clinical attachment loss > 4 mm) or gingival overgrowth (pseudopockets > 4 mm). Subjects were also excluded if they showed evidence of dental negligence. Study design The study was designed as a randomized, parallel, triple-blind, 3-month clinical trial. Participants, examiners, and those assessing the outcomes were blinded to group assignment. The following visits were scheduled: At the screening visit, all recruited subjects had a professional prophylaxis followed by specific oral hygiene instructions. They were then appointed for the baseline visit within 7–15 days. At the baseline visit, the clinical parameters were recorded and microbiological samples were retrieved. At this visit, each participant was randomly assigned by the study supervisor to one of the study groups through a computer-generated list (a block randomization with blocks of six). Block size was not disclosed to ensure concealment. Each subject was given a unique number, which was associated with the assigned product. Participants and investigators were unaware of the treatment assignment due to the central allocation, managed by the study supervisor. Allocation concealment was ensured, as the supervisor did not release by phone the randomisation code until the patient had been recruited into the trial. The assigned products included both a mouthwash and dentifrice, together with a standard toothbrush (VITIS Orthodontic, Dentaid, Cerdanyola, Spain) and dental floss (VITIS non-wax dental floss, Dentaid, Cerdanyola, Spain). Products were purposely prepared in white opaque bottles coded as A or B. Patients were requested to return the unused mouthrinse at the following visit and were instructed to fill in a compliance diary where the home use of the products was recorded. The content of the product’s assignment list was only known by the study supervisor who was not involved in the screening or in the subject’s evaluation. At the 1-month visit, all the clinical parameters were again recorded together with a questionnaire that was filled by the participants reporting on their subjective assessment of the used products. At this visit, the compliance diaries were collected as well as any remaining mouth rinse, which was later quantified in ml. New products (mouthwash and dentifrice) and new diaries were provided. The 2-month visit was identical to the 1-month visit. At the final 3-month visit, clinical parameters were evaluated, microbiological samples were taken, participants’ questionnaires were filled and compliance diaries and remaining mouth rinse were collected. At this time, a professional prophylaxis was carried out and oral hygiene instructions were reinforced. Treatment groups In the test group, subjects used the experimental mouthwash (VITIS Orthodontic®, Dentaid, Cerdanyola, Spain) containing 0.05 per cent CPC, 0.33 per cent sodium fluoride, allantoin and aloe vera, together with the experimental toothpaste (VITIS Orthodontic®, Dentaid, Cerdanyola, Spain) containing the same active ingredients. In the control group, subjects used a placebo mouth rinse and dentifrice that were identical to the test formulations, but without the active components (CPC, sodium fluoride, aloe vera, and allantoin). Both the test and the placebo formulations contained 1% xylitol. Subjects in both groups were instructed to use 15 ml of the mouthwash twice a day during 30 seconds, after brushing their teeth with the assigned tooth paste in the morning and in the evening. Outcome variables Clinical outcome variables The primary outcome variables of this study were the Plaque and gingival indexes. – Plaque index (PlI) was evaluated using the Turesky modification (33) of the Quigley and Hein index after disclosing dental plaque with erythrosine (Plac Control®, Dentaid, Cerdanyola, Barcelona, Spain). – Gingival index (GI) was assessed using the Lobene modification (34) of the Löe-Silness index, at six sites per tooth (24). As secondary variables, we recorded: – Calculus accumulation measured with the Volpe-Manhold calculus index (35) at the mid-lingual surfaces of the four lower incisors. The scores ranged from 0, no calculus; 1, one mm of calculus; 2, two mm of calculus to 3, three mm of calculus. – Tooth staining: Standardized digital photographs of the buccal side of the upper and lower incisors and canines (occluding edge to edge) were taken using a Canon EOS 450 digital camera mounted with a ring-flash and with a 105-mm Macro lens, with an aperture of F22 and shutter speed of 1/125. Two blinded, independent and calibrated judges evaluated the photographs using a standard template of different tooth staining degrees, as previously described by Gadhia et al. (36). They scored both the area and the intensity of tooth staining using a numerical scale from 0 to 3. The area was graded as: 0 (absence of staining); 1 (staining in the 1/3 of the clinical crown free of bracket; 2 (staining in the 2/3 of the clinical crown free of bracket); and 3 (staining in the 3/3 of the clinical crown free of bracket). The intensity was graded as: 0 (absence of staining); 1 (slight staining); 2 (moderate staining); and 3 (severe staining). A total of 193 pictures were assessed. The values of all the upper front teeth were added and compared to the sum of the other visits, and the same was done with the lower teeth. Values were expressed as the mean of the evaluation of both judges. – Orthodontic appliances. The presence of any bracket or band being de-bonded was recorded. Similarly, the occurrence of any soft tissue injury and its duration was recorded. Microbiological outcome variables Microbiological samples were taken using two sterile standard sized paper points (Maillefer, Ballaigues, Switzerland) that were inserted consecutively in each selected site after the removal of supragingival plaque (37). Samples were taken from the mesial-buccal site of upper first molars and the distal-buccal site of lower lateral incisors. At these sites, different clinical variables were recorded, including probing pocket depth (PPD), bleeding on probing (BOP), and plaque presence. Before the insertion of the paper points, sites were isolated with cotton rolls to avoid saliva contamination and the area was dried with an air syringe. Paper points were kept in place for 10 seconds and they were then pooled in a screw top vial containing 1.5 ml of reduced transport fluid (RTF) (38). Samples were transferred to the laboratory within 2 hours where they were homogenized by vortex vibration for 30 seconds (39) and sequentially diluted in phosphate buffer solution (PBS). Samples were cultivated on agar-blood medium (enriched with haemine and menadione) and incubated for 15 days in jars with an anaerobic atmosphere; and on selective medium Dentaid-1, incubated for 3–5 days in 5% carbon dioxide (40). Bacterial species identification was carried out by the assessment of the colony morphology and confirmed by the application of biochemical standard tests. In addition to the conventional evaluation of the plates, the possible overgrowth of opportunistic species, both in blood agar and selective plates, was investigated in order to detect possible undesired microbiological adverse effects. The main microbiological outcome variables included total anaerobic counts and the presence, counts, and proportions of different bacterial species, including both pathogenic and opportunistic species. Patient-reported outcomes In every follow-up visit, patients filled up a questionnaire reporting their subjective evaluation of the assigned products. The questionnaire used was custom-made for this investigation, including following questions, with three/four closed answers available: How do you feel rinsing with this mouth rinse’ (answers: great; good; unpleasant; very unpleasant) The taste of this mouth rinse is…’ (answers: very good; good; fair; bad) Did you use the mouth rinse everyday’ (answers: yes, twice a day; no, I forgot 1 day; no, I forgot 2 days; no, I forgot more than 2 days) Have you used any mouth rinse before’ (answers: yes, almost every day; yes, sometimes; yes, but very rarely; no) How do you feel your teeth after using this mouth rinse’ (answers: cleaner and smoother; rough and unpolished; I don’t feel any change) After using the mouth rinse, your breath feels…’ (answers: better than before; same as before; worse than before) Compliance Compliance with the assigned products was assessed by the information provided by the participants on their filled diaries and by the remaining mouth rinse (if any) returned after each follow-up visit, which was measured in ml. using a calibrated vase. The measured compliance was expressed by the number of patients that fulfilled with the study protocol at each visit and by the times that a patient failed to use the mouth rinse, according to the daily diaries. Percentage of failures was calculated in relation to the total available times. Patients were monthly assessed for clinical, patient-reported and compliance variables. Subgingival microbiological samples were taken at baseline and after 3 months. Statistical analysis The sample size calculation was based on the results from a 1-month clinical trial using a similar CPC-based mouth rinse (41). For statistical purposes, and in order to avoid adjusting for multiplicity analysis, we considered only the reduction in the mean Plaque Index as the primary outcome. To detect a reduction in the mean plaque index of 0.42 (SD 0.40) with a two-sided 5% significance level and a power of 80%, a sample size of 25 patients per group was necessary, taken into account an anticipated dropout rate of 10%. For entering the results, a database was designed in a spread sheet (Microsoft® Excel), which was then analysed with the statistical program Statgraphic Plus® for Windows (5.1 edition, New Jersey, New Jersey, USA). In all cases, ITT (intention to treat) analysis was performed. Calibration of the examiners One clinician undertook all the clinical assessments and was calibrated for PI and GI recordings before the start of the trial. Two orthodontic patients wearing fixed appliances were enrolled for this purpose. Duplicated measurements (n = 328) were collected with an interval of 30 min between the first and the second recording. Intraclass correlation coefficient (ICC) of total agreement was used to test the intra-examiner reliability for PlI and GI. The Intra-examiner reliability was very good for PI (ICC = 0.923; 95% CI = 0.902–0.953) and GI (ICC = 0.904; 95% CI = 0.891–0.922) assessment. For the evaluation of tooth staining, the two judges who evaluated the photographs were previously calibrated by repeating twice the assessment of ten pictures and achieving a Cohen’s kappa index of 0.813. For the clinical variables, the averages were calculated per patient and visit, and then per group. Once the distribution of the data was confirmed (using the Kolmogorov–Smirnov and Shapiro–Wilk tests), if the variables fulfilled a normal distribution, ANCOVA (with baseline values as covariate) was initially used as primary statistical test. Then, paired t-tests were used for evaluating intra group changes and unpaired t-tests for inter group comparisons (at each visit, and in changes between baseline-follow up visits). Clinical indexes were calculated for the full-mouth and then stratified for the upper and lower jaw and for proximal and free surfaces, considering as primary result the ‘all sites’ evaluation. The values of intensity and area of tooth staining were compared between groups, between consecutive visits and between the baseline and final visits using the Chi-squared test. The data were grouped into upper arch and lower arch to make comparisons. Total anaerobic counts were log transformed in order to achieve a normal distribution, and were then statistically analysed in a similar way as the clinical variables. Counts and proportions of pathogens were compared by means of signed-rank tests, and frequencies of detections by means of Chi-squared test. Answers to questionnaires were compared between groups using the chi-square test or Fisher’s exact test when necessary. To get a more realistic estimation of the trend of those answers where differences were statistically significant, a transformation of categorical variables into semi-quantitative variables was made. 2 × 2 tables were constructed to analyse the number of patients that fulfilled with the study protocol at each visit and number of failures in mouth rinse intake. Fisher’s exact test was used to compare the results. The statistical significance was set at the level of P < 0.05. Results Demographic data Thirty-one patients were randomized to the placebo group (10 males, 21 females; mean age 15.2 ± 2.1, range 11.75–20.25). Twenty-eight attended the 1-month visit, 24 the 2-month visit and 23 the 3-month visit. After 1 month, three patients left the study: one patient did not comply with the appointments, one patient did not want to continue in the study for personal reasons, and the other patient finished the orthodontic treatment earlier than expected. After 2 months, three additional patients left the study, two for lack of compliance with the appointments, and the other for personal reasons. One additional patient missed the 2-month visit, but later attended the final visit. After 3 months, two additional patients missed the evaluation due to an excessive delay in that visit (more than 1 month) (Table 1). Table 1. Demographic data of the patients at baseline. Placebo group Test group N 31 (49.2%) 32 (50.8%) n males 10 (15.9%) 15 (23.8%) n females 21 (33.3%) 17 (27%) Age (years) 15.2* (2.1) 15.0* (1.8) Plaque index (mean, SD) 2.59* (0.88) 2.57* (0.88) Gingival index (mean, SD) 1.07* (0.36) 0.92* (0.31) Bleeding on probing (mean, SD) 0.79* (0.23) 0.78* (0.25) Probing pocket depth (mm) 3.09* (0.39) 3.04* (0.46) Recessions (mm) 0* (0) 0* (0) Staining intensity upper 0.35* (0.73) 0.08* (0.29) Staining intensity lower 0.85* (1.99) 1.25* (2.18) Placebo group Test group N 31 (49.2%) 32 (50.8%) n males 10 (15.9%) 15 (23.8%) n females 21 (33.3%) 17 (27%) Age (years) 15.2* (2.1) 15.0* (1.8) Plaque index (mean, SD) 2.59* (0.88) 2.57* (0.88) Gingival index (mean, SD) 1.07* (0.36) 0.92* (0.31) Bleeding on probing (mean, SD) 0.79* (0.23) 0.78* (0.25) Probing pocket depth (mm) 3.09* (0.39) 3.04* (0.46) Recessions (mm) 0* (0) 0* (0) Staining intensity upper 0.35* (0.73) 0.08* (0.29) Staining intensity lower 0.85* (1.99) 1.25* (2.18) * Data are means (SD) or numbers (%). View Large Table 1. Demographic data of the patients at baseline. Placebo group Test group N 31 (49.2%) 32 (50.8%) n males 10 (15.9%) 15 (23.8%) n females 21 (33.3%) 17 (27%) Age (years) 15.2* (2.1) 15.0* (1.8) Plaque index (mean, SD) 2.59* (0.88) 2.57* (0.88) Gingival index (mean, SD) 1.07* (0.36) 0.92* (0.31) Bleeding on probing (mean, SD) 0.79* (0.23) 0.78* (0.25) Probing pocket depth (mm) 3.09* (0.39) 3.04* (0.46) Recessions (mm) 0* (0) 0* (0) Staining intensity upper 0.35* (0.73) 0.08* (0.29) Staining intensity lower 0.85* (1.99) 1.25* (2.18) Placebo group Test group N 31 (49.2%) 32 (50.8%) n males 10 (15.9%) 15 (23.8%) n females 21 (33.3%) 17 (27%) Age (years) 15.2* (2.1) 15.0* (1.8) Plaque index (mean, SD) 2.59* (0.88) 2.57* (0.88) Gingival index (mean, SD) 1.07* (0.36) 0.92* (0.31) Bleeding on probing (mean, SD) 0.79* (0.23) 0.78* (0.25) Probing pocket depth (mm) 3.09* (0.39) 3.04* (0.46) Recessions (mm) 0* (0) 0* (0) Staining intensity upper 0.35* (0.73) 0.08* (0.29) Staining intensity lower 0.85* (1.99) 1.25* (2.18) * Data are means (SD) or numbers (%). View Large Thirty-two patients were allocated to the test group (15 males, 17 females; mean age 15.0 ± 1.8, range 11.67–20.17). Twenty-nine, 28, and 27 patients attended the 1, 2, and 3-month visits, respectively. Three patients left the study before the 1-month visit, due to a lack of compliance with the assigned appointments. One patient dropped out before the 2-month visit due to pregnancy. One additional patient missed the last visit due to an excessive delay in the appointment. Plaque index The baseline plaque levels were similar in both treatment groups. After 1 month, the mean PlI increased in the placebo group (0.16), while it decreased in the test group (0.08). This increase was statistically significant in the placebo group in the upper jaw (P = 0.032). Differences between groups in changes from baseline to 1 month were statistically significant (P = 0.024) only in the maxilla (mean: 0.18, SD = 0.82 versus mean: −0.26, SD = 0.62). Plaque increased after 2 months in both groups, being statistically significant in the placebo group for all sites (P = 0.031) and in the upper jaw (P = 0.033). At 3 months, plaque levels were also higher when compared with baseline in both groups, and the increase was statistically significant in the placebo group for all sites (P = 0.028) and in the upper jaw (P = 0.004). No significant inter group differences were detected (Table 2). Table 2. Plaque index (PlI) at each visit and changes (negative values mean increase, positive values mean reduction) expressed as mean, standard deviation (SD) and 95% confidence intervals (95% CI). Placebo group Test group Plaque index n Mean SD 95% CI n Mean SD 95% CI All sites Baseline 31 2.59 0.88 2.26 2.91 32 2.57 0.88 2.25 2.88 1 month 28 2.71 0.90 2.36 3.06 29 2.45 1.02 2.07 2.84 2 months 24 2.85 0.87 2.48 3.21 28 2.75 0.78 2.45 3.05 3 months 23 2.73 0.86 2.36 3.11 28 2.73 0.88 2.39 3.07 Upper jaw Baseline 31 2.36 0.95 2.01 2.71 32 2.48 0.96 2.14 2.83 1 month 28 2.57 0.95 2.20 2.94 29 2.29 1.17 1.85 2.74 2 months 24 2.64 0.96 2.24 3.05 28 2.61 0.86 2.27 2.94 3 months 23 2.62 0.91 2.23 3.02 28 2.61 1.05 2.21 3.02 Lower jaw Baseline 31 2.83 0.95 2.48 3.18 32 2.65 0.89 2.33 2.97 1 month 28 2.87 0.93 2.51 3.23 29 2.61 0.94 2.26 2.97 2 months 24 3.06 0.84 2.71 3.41 28 2.89 0.76 2.59 3.18 3 months 23 2.84 0.85 2.47 3.21 28 2.86 0.81 2.55 3.18 PlI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base 1 month 28 −0.17 0.58 −0.39 0.05 29 0.09 0.68 −0.17 0.35 Base 2 months 24 −0.30 0.64 −0.57 −0.03 28 −0.24 0.63 −0.48 0.01 Base 3 months 23 −0.25 0.52 −0.48 −0.03 27 −0.21 0.67 −0.48 0.06 Upper jaw Base 1 month 28 −0.26 0.62 −0.51 −0.02 29 0.18 0.82 −0.13 0.50 Base 2 months 24 −0.31 0.67 −0.59 −0.03 28 −0.16 0.63 −0.41 0.08 Base 3 months 23 −0.38 0.57 −0.62 −0.13 27 −0.16 0.71 −0.44 0.12 Lower jaw Base 1 month 28 −0.08 0.84 −0.40 0.25 29 −0.01 0.75 −0.29 0.27 Base 2 months 24 −0.28 0.78 −0.61 0.05 28 −0.31 0.81 −0.63 0.00 Base 3 months 23 −0.11 0.61 −0.38 0.15 27 −0.28 0.88 −0.63 0.07 Placebo group Test group Plaque index n Mean SD 95% CI n Mean SD 95% CI All sites Baseline 31 2.59 0.88 2.26 2.91 32 2.57 0.88 2.25 2.88 1 month 28 2.71 0.90 2.36 3.06 29 2.45 1.02 2.07 2.84 2 months 24 2.85 0.87 2.48 3.21 28 2.75 0.78 2.45 3.05 3 months 23 2.73 0.86 2.36 3.11 28 2.73 0.88 2.39 3.07 Upper jaw Baseline 31 2.36 0.95 2.01 2.71 32 2.48 0.96 2.14 2.83 1 month 28 2.57 0.95 2.20 2.94 29 2.29 1.17 1.85 2.74 2 months 24 2.64 0.96 2.24 3.05 28 2.61 0.86 2.27 2.94 3 months 23 2.62 0.91 2.23 3.02 28 2.61 1.05 2.21 3.02 Lower jaw Baseline 31 2.83 0.95 2.48 3.18 32 2.65 0.89 2.33 2.97 1 month 28 2.87 0.93 2.51 3.23 29 2.61 0.94 2.26 2.97 2 months 24 3.06 0.84 2.71 3.41 28 2.89 0.76 2.59 3.18 3 months 23 2.84 0.85 2.47 3.21 28 2.86 0.81 2.55 3.18 PlI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base 1 month 28 −0.17 0.58 −0.39 0.05 29 0.09 0.68 −0.17 0.35 Base 2 months 24 −0.30 0.64 −0.57 −0.03 28 −0.24 0.63 −0.48 0.01 Base 3 months 23 −0.25 0.52 −0.48 −0.03 27 −0.21 0.67 −0.48 0.06 Upper jaw Base 1 month 28 −0.26 0.62 −0.51 −0.02 29 0.18 0.82 −0.13 0.50 Base 2 months 24 −0.31 0.67 −0.59 −0.03 28 −0.16 0.63 −0.41 0.08 Base 3 months 23 −0.38 0.57 −0.62 −0.13 27 −0.16 0.71 −0.44 0.12 Lower jaw Base 1 month 28 −0.08 0.84 −0.40 0.25 29 −0.01 0.75 −0.29 0.27 Base 2 months 24 −0.28 0.78 −0.61 0.05 28 −0.31 0.81 −0.63 0.00 Base 3 months 23 −0.11 0.61 −0.38 0.15 27 −0.28 0.88 −0.63 0.07 View Large Table 2. Plaque index (PlI) at each visit and changes (negative values mean increase, positive values mean reduction) expressed as mean, standard deviation (SD) and 95% confidence intervals (95% CI). Placebo group Test group Plaque index n Mean SD 95% CI n Mean SD 95% CI All sites Baseline 31 2.59 0.88 2.26 2.91 32 2.57 0.88 2.25 2.88 1 month 28 2.71 0.90 2.36 3.06 29 2.45 1.02 2.07 2.84 2 months 24 2.85 0.87 2.48 3.21 28 2.75 0.78 2.45 3.05 3 months 23 2.73 0.86 2.36 3.11 28 2.73 0.88 2.39 3.07 Upper jaw Baseline 31 2.36 0.95 2.01 2.71 32 2.48 0.96 2.14 2.83 1 month 28 2.57 0.95 2.20 2.94 29 2.29 1.17 1.85 2.74 2 months 24 2.64 0.96 2.24 3.05 28 2.61 0.86 2.27 2.94 3 months 23 2.62 0.91 2.23 3.02 28 2.61 1.05 2.21 3.02 Lower jaw Baseline 31 2.83 0.95 2.48 3.18 32 2.65 0.89 2.33 2.97 1 month 28 2.87 0.93 2.51 3.23 29 2.61 0.94 2.26 2.97 2 months 24 3.06 0.84 2.71 3.41 28 2.89 0.76 2.59 3.18 3 months 23 2.84 0.85 2.47 3.21 28 2.86 0.81 2.55 3.18 PlI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base 1 month 28 −0.17 0.58 −0.39 0.05 29 0.09 0.68 −0.17 0.35 Base 2 months 24 −0.30 0.64 −0.57 −0.03 28 −0.24 0.63 −0.48 0.01 Base 3 months 23 −0.25 0.52 −0.48 −0.03 27 −0.21 0.67 −0.48 0.06 Upper jaw Base 1 month 28 −0.26 0.62 −0.51 −0.02 29 0.18 0.82 −0.13 0.50 Base 2 months 24 −0.31 0.67 −0.59 −0.03 28 −0.16 0.63 −0.41 0.08 Base 3 months 23 −0.38 0.57 −0.62 −0.13 27 −0.16 0.71 −0.44 0.12 Lower jaw Base 1 month 28 −0.08 0.84 −0.40 0.25 29 −0.01 0.75 −0.29 0.27 Base 2 months 24 −0.28 0.78 −0.61 0.05 28 −0.31 0.81 −0.63 0.00 Base 3 months 23 −0.11 0.61 −0.38 0.15 27 −0.28 0.88 −0.63 0.07 Placebo group Test group Plaque index n Mean SD 95% CI n Mean SD 95% CI All sites Baseline 31 2.59 0.88 2.26 2.91 32 2.57 0.88 2.25 2.88 1 month 28 2.71 0.90 2.36 3.06 29 2.45 1.02 2.07 2.84 2 months 24 2.85 0.87 2.48 3.21 28 2.75 0.78 2.45 3.05 3 months 23 2.73 0.86 2.36 3.11 28 2.73 0.88 2.39 3.07 Upper jaw Baseline 31 2.36 0.95 2.01 2.71 32 2.48 0.96 2.14 2.83 1 month 28 2.57 0.95 2.20 2.94 29 2.29 1.17 1.85 2.74 2 months 24 2.64 0.96 2.24 3.05 28 2.61 0.86 2.27 2.94 3 months 23 2.62 0.91 2.23 3.02 28 2.61 1.05 2.21 3.02 Lower jaw Baseline 31 2.83 0.95 2.48 3.18 32 2.65 0.89 2.33 2.97 1 month 28 2.87 0.93 2.51 3.23 29 2.61 0.94 2.26 2.97 2 months 24 3.06 0.84 2.71 3.41 28 2.89 0.76 2.59 3.18 3 months 23 2.84 0.85 2.47 3.21 28 2.86 0.81 2.55 3.18 PlI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base 1 month 28 −0.17 0.58 −0.39 0.05 29 0.09 0.68 −0.17 0.35 Base 2 months 24 −0.30 0.64 −0.57 −0.03 28 −0.24 0.63 −0.48 0.01 Base 3 months 23 −0.25 0.52 −0.48 −0.03 27 −0.21 0.67 −0.48 0.06 Upper jaw Base 1 month 28 −0.26 0.62 −0.51 −0.02 29 0.18 0.82 −0.13 0.50 Base 2 months 24 −0.31 0.67 −0.59 −0.03 28 −0.16 0.63 −0.41 0.08 Base 3 months 23 −0.38 0.57 −0.62 −0.13 27 −0.16 0.71 −0.44 0.12 Lower jaw Base 1 month 28 −0.08 0.84 −0.40 0.25 29 −0.01 0.75 −0.29 0.27 Base 2 months 24 −0.28 0.78 −0.61 0.05 28 −0.31 0.81 −0.63 0.00 Base 3 months 23 −0.11 0.61 −0.38 0.15 27 −0.28 0.88 −0.63 0.07 View Large Gingival index Higher GI baseline levels were observed in the placebo group (1.07) compared to the test group (0.92), although the differences were not significant (P = 0.085). After 1 month, GI increased in the placebo group (mean = 0.05; SD = 0.33), and decreased in the test group (mean = −0.03; SD = 0.38), with significant differences at all sites (P = 0.025) and lower jaw (P = 0.007). At the 2-month visit, no inter-group differences were detected, and both groups demonstrated an increase in GI. After 3 months, minor and comparable increases were observed in both groups, although a significantly higher level of gingivitis was observed in the placebo group (mean = 0.05; SD = 0.43 versus Mean = 0.02; SD = 0.34) for the full-mouth index (P = 0.050) and lower jaw (P = 0.020) (Table 3). Table 3. Gingival index (GI) at each visit and changes (negative values mean increase, positive values mean reduction) expressed as mean, standard deviation (SD) and 95% confidence intervals (95% CI). Placebo group Test group Gingival index n Mean SD 95% CI n Mean SD 95% CI all sites Baseline 31 1.07 0.36 0.94 1.21 32 0.92 0.31 0.81 1.04 1 month 28 1.09 0.34 0.95 1.22 29 0.89 0.31 0.77 1.00 2 months 24 1.06 0.40 0.89 1.23 28 1.07 0.41 0.92 1.23 3 months 23 1.13 0.36 0.98 1.29 28 0.94 0.34 0.80 1.07 Upper jaw Baseline 31 0.98 0.39 0.84 1.13 32 0.82 0.36 0.69 0.94 1 month 28 0.96 0.37 0.82 1.11 29 0.80 0.38 0.65 0.94 2 months 24 0.95 0.47 0.75 1.15 28 0.98 0.49 0.79 1.17 3 months 23 1.02 0.41 0.84 1.20 28 0.85 0.40 0.69 1.00 Lower jaw Baseline 31 1.17 0.37 1.03 1.31 32 1.03 0.34 0.90 1.15 1 month 28 1.22 0.35 1.08 1.36 29 0.97 0.31 0.85 1.09 2 months 24 1.17 0.40 1.00 1.34 28 1.17 0.36 1.03 1.31 3 months 23 1.25 0.36 1.10 1.41 28 1.02 0.32 0.89 1.14 GI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base-1m 28 −0.05 0.33 −0.17 0.08 29 0.03 0.38 −0.11 0.18 Base-2m 24 −0.01 0.46 −0.21 0.18 28 −0.16 0.51 −0.36 0.03 Base-3m 23 −0.05 0.45 −0.25 0.14 27 −0.02 0.34 −0.16 0.11 Upper jaw Base-1m 28 −0.01 0.38 −0.15 0.14 29 0.01 0.47 −0.17 0.19 Base-2m 24 0.02 0.56 −0.21 0.26 28 −0.18 0.64 −0.43 0.07 Base-3m 23 −0.02 0.55 −0.25 0.22 27 −0.06 0.45 −0.23 0.12 Lower jaw Base-1m 28 −0.08 0.35 −0.21 0.06 29 0.05 0.35 −0.08 0.19 Base-2m 24 −0.04 0.40 −0.21 0.13 28 −0.15 0.45 −0.32 0.03 Base-3m 23 −0.07 0.40 −0.25 0.10 27 0.01 0.32 −0.12 0.14 Placebo group Test group Gingival index n Mean SD 95% CI n Mean SD 95% CI all sites Baseline 31 1.07 0.36 0.94 1.21 32 0.92 0.31 0.81 1.04 1 month 28 1.09 0.34 0.95 1.22 29 0.89 0.31 0.77 1.00 2 months 24 1.06 0.40 0.89 1.23 28 1.07 0.41 0.92 1.23 3 months 23 1.13 0.36 0.98 1.29 28 0.94 0.34 0.80 1.07 Upper jaw Baseline 31 0.98 0.39 0.84 1.13 32 0.82 0.36 0.69 0.94 1 month 28 0.96 0.37 0.82 1.11 29 0.80 0.38 0.65 0.94 2 months 24 0.95 0.47 0.75 1.15 28 0.98 0.49 0.79 1.17 3 months 23 1.02 0.41 0.84 1.20 28 0.85 0.40 0.69 1.00 Lower jaw Baseline 31 1.17 0.37 1.03 1.31 32 1.03 0.34 0.90 1.15 1 month 28 1.22 0.35 1.08 1.36 29 0.97 0.31 0.85 1.09 2 months 24 1.17 0.40 1.00 1.34 28 1.17 0.36 1.03 1.31 3 months 23 1.25 0.36 1.10 1.41 28 1.02 0.32 0.89 1.14 GI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base-1m 28 −0.05 0.33 −0.17 0.08 29 0.03 0.38 −0.11 0.18 Base-2m 24 −0.01 0.46 −0.21 0.18 28 −0.16 0.51 −0.36 0.03 Base-3m 23 −0.05 0.45 −0.25 0.14 27 −0.02 0.34 −0.16 0.11 Upper jaw Base-1m 28 −0.01 0.38 −0.15 0.14 29 0.01 0.47 −0.17 0.19 Base-2m 24 0.02 0.56 −0.21 0.26 28 −0.18 0.64 −0.43 0.07 Base-3m 23 −0.02 0.55 −0.25 0.22 27 −0.06 0.45 −0.23 0.12 Lower jaw Base-1m 28 −0.08 0.35 −0.21 0.06 29 0.05 0.35 −0.08 0.19 Base-2m 24 −0.04 0.40 −0.21 0.13 28 −0.15 0.45 −0.32 0.03 Base-3m 23 −0.07 0.40 −0.25 0.10 27 0.01 0.32 −0.12 0.14 View Large Table 3. Gingival index (GI) at each visit and changes (negative values mean increase, positive values mean reduction) expressed as mean, standard deviation (SD) and 95% confidence intervals (95% CI). Placebo group Test group Gingival index n Mean SD 95% CI n Mean SD 95% CI all sites Baseline 31 1.07 0.36 0.94 1.21 32 0.92 0.31 0.81 1.04 1 month 28 1.09 0.34 0.95 1.22 29 0.89 0.31 0.77 1.00 2 months 24 1.06 0.40 0.89 1.23 28 1.07 0.41 0.92 1.23 3 months 23 1.13 0.36 0.98 1.29 28 0.94 0.34 0.80 1.07 Upper jaw Baseline 31 0.98 0.39 0.84 1.13 32 0.82 0.36 0.69 0.94 1 month 28 0.96 0.37 0.82 1.11 29 0.80 0.38 0.65 0.94 2 months 24 0.95 0.47 0.75 1.15 28 0.98 0.49 0.79 1.17 3 months 23 1.02 0.41 0.84 1.20 28 0.85 0.40 0.69 1.00 Lower jaw Baseline 31 1.17 0.37 1.03 1.31 32 1.03 0.34 0.90 1.15 1 month 28 1.22 0.35 1.08 1.36 29 0.97 0.31 0.85 1.09 2 months 24 1.17 0.40 1.00 1.34 28 1.17 0.36 1.03 1.31 3 months 23 1.25 0.36 1.10 1.41 28 1.02 0.32 0.89 1.14 GI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base-1m 28 −0.05 0.33 −0.17 0.08 29 0.03 0.38 −0.11 0.18 Base-2m 24 −0.01 0.46 −0.21 0.18 28 −0.16 0.51 −0.36 0.03 Base-3m 23 −0.05 0.45 −0.25 0.14 27 −0.02 0.34 −0.16 0.11 Upper jaw Base-1m 28 −0.01 0.38 −0.15 0.14 29 0.01 0.47 −0.17 0.19 Base-2m 24 0.02 0.56 −0.21 0.26 28 −0.18 0.64 −0.43 0.07 Base-3m 23 −0.02 0.55 −0.25 0.22 27 −0.06 0.45 −0.23 0.12 Lower jaw Base-1m 28 −0.08 0.35 −0.21 0.06 29 0.05 0.35 −0.08 0.19 Base-2m 24 −0.04 0.40 −0.21 0.13 28 −0.15 0.45 −0.32 0.03 Base-3m 23 −0.07 0.40 −0.25 0.10 27 0.01 0.32 −0.12 0.14 Placebo group Test group Gingival index n Mean SD 95% CI n Mean SD 95% CI all sites Baseline 31 1.07 0.36 0.94 1.21 32 0.92 0.31 0.81 1.04 1 month 28 1.09 0.34 0.95 1.22 29 0.89 0.31 0.77 1.00 2 months 24 1.06 0.40 0.89 1.23 28 1.07 0.41 0.92 1.23 3 months 23 1.13 0.36 0.98 1.29 28 0.94 0.34 0.80 1.07 Upper jaw Baseline 31 0.98 0.39 0.84 1.13 32 0.82 0.36 0.69 0.94 1 month 28 0.96 0.37 0.82 1.11 29 0.80 0.38 0.65 0.94 2 months 24 0.95 0.47 0.75 1.15 28 0.98 0.49 0.79 1.17 3 months 23 1.02 0.41 0.84 1.20 28 0.85 0.40 0.69 1.00 Lower jaw Baseline 31 1.17 0.37 1.03 1.31 32 1.03 0.34 0.90 1.15 1 month 28 1.22 0.35 1.08 1.36 29 0.97 0.31 0.85 1.09 2 months 24 1.17 0.40 1.00 1.34 28 1.17 0.36 1.03 1.31 3 months 23 1.25 0.36 1.10 1.41 28 1.02 0.32 0.89 1.14 GI changes n Mean SD 95% CI n Mean SD 95% CI All sites Base-1m 28 −0.05 0.33 −0.17 0.08 29 0.03 0.38 −0.11 0.18 Base-2m 24 −0.01 0.46 −0.21 0.18 28 −0.16 0.51 −0.36 0.03 Base-3m 23 −0.05 0.45 −0.25 0.14 27 −0.02 0.34 −0.16 0.11 Upper jaw Base-1m 28 −0.01 0.38 −0.15 0.14 29 0.01 0.47 −0.17 0.19 Base-2m 24 0.02 0.56 −0.21 0.26 28 −0.18 0.64 −0.43 0.07 Base-3m 23 −0.02 0.55 −0.25 0.22 27 −0.06 0.45 −0.23 0.12 Lower jaw Base-1m 28 −0.08 0.35 −0.21 0.06 29 0.05 0.35 −0.08 0.19 Base-2m 24 −0.04 0.40 −0.21 0.13 28 −0.15 0.45 −0.32 0.03 Base-3m 23 −0.07 0.40 −0.25 0.10 27 0.01 0.32 −0.12 0.14 View Large Calculus index Baseline calculus levels were similar and low in both groups. A slow increase was observed throughout the study, reaching similar results in both groups at 3 months. The change between baseline and 3 months was statistically significant (P = 0.041) only in the test group. None of the intergroup comparisons were significant. Tooth staining Intensity of staining As seen in Table 4, it is clear that the scores of intensity in the upper jaw were low in both groups and showed only minor changes during the study, although somehow higher in the placebo group. No statistically significant differences were detected. The intensity of staining in the lower jaw was higher than in the upper jaw, and slightly higher in the test group at baseline (Table 4). Minor changes were observed in the placebo group, but a tendency to decrease was observed in the test group, with an exception between 1 and 2 months. None of the comparisons were statistically significant. Table 4. Mean values and standard deviation (SD) in staining (surface, intensity), as evaluated by two researchers. Surface Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.48 1.14 0.13 0.43 0.96 2.29 1.75 3.39 1 month 0.46 1.27 0.04 0.19 0.67 1.25 0.44 0.87 2 months 0.33 0.71 0.10 0.40 0.50 1.04 0.96 2.22 3 months 0.45 1.01 0.12 0.44 0.73 1.39 0.48 1.23 Baseline 1 month 0.02 1.09 0.09 0.53 0.29 1.94 1.31 3.72 Baseline 3 months 0.03 1.56 0.01 0.61 0.23 0.67 1.27 3.00 Intensity Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.35 0.73 0.08 0.29 0.85 1.99 1.25 2.18 1 month 0.35 0.94 0.04 0.19 0.67 1.25 0.37 0.77 2 months 0.33 0.71 0.08 0.31 0.50 1.04 0.71 1.47 3 months 0.40 0.90 0.12 0.44 0.73 1.39 0.44 1.12 Baseline 1 month 0.00 0.79 0.05 0.35 0.18 1.54 0.88 2.06 Baseline 3 months −0.05 1.18 −0.04 0.41 0.12 0.58 0.81 1.33 Surface Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.48 1.14 0.13 0.43 0.96 2.29 1.75 3.39 1 month 0.46 1.27 0.04 0.19 0.67 1.25 0.44 0.87 2 months 0.33 0.71 0.10 0.40 0.50 1.04 0.96 2.22 3 months 0.45 1.01 0.12 0.44 0.73 1.39 0.48 1.23 Baseline 1 month 0.02 1.09 0.09 0.53 0.29 1.94 1.31 3.72 Baseline 3 months 0.03 1.56 0.01 0.61 0.23 0.67 1.27 3.00 Intensity Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.35 0.73 0.08 0.29 0.85 1.99 1.25 2.18 1 month 0.35 0.94 0.04 0.19 0.67 1.25 0.37 0.77 2 months 0.33 0.71 0.08 0.31 0.50 1.04 0.71 1.47 3 months 0.40 0.90 0.12 0.44 0.73 1.39 0.44 1.12 Baseline 1 month 0.00 0.79 0.05 0.35 0.18 1.54 0.88 2.06 Baseline 3 months −0.05 1.18 −0.04 0.41 0.12 0.58 0.81 1.33 Positive changes mean decrease. View Large Table 4. Mean values and standard deviation (SD) in staining (surface, intensity), as evaluated by two researchers. Surface Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.48 1.14 0.13 0.43 0.96 2.29 1.75 3.39 1 month 0.46 1.27 0.04 0.19 0.67 1.25 0.44 0.87 2 months 0.33 0.71 0.10 0.40 0.50 1.04 0.96 2.22 3 months 0.45 1.01 0.12 0.44 0.73 1.39 0.48 1.23 Baseline 1 month 0.02 1.09 0.09 0.53 0.29 1.94 1.31 3.72 Baseline 3 months 0.03 1.56 0.01 0.61 0.23 0.67 1.27 3.00 Intensity Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.35 0.73 0.08 0.29 0.85 1.99 1.25 2.18 1 month 0.35 0.94 0.04 0.19 0.67 1.25 0.37 0.77 2 months 0.33 0.71 0.08 0.31 0.50 1.04 0.71 1.47 3 months 0.40 0.90 0.12 0.44 0.73 1.39 0.44 1.12 Baseline 1 month 0.00 0.79 0.05 0.35 0.18 1.54 0.88 2.06 Baseline 3 months −0.05 1.18 −0.04 0.41 0.12 0.58 0.81 1.33 Surface Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.48 1.14 0.13 0.43 0.96 2.29 1.75 3.39 1 month 0.46 1.27 0.04 0.19 0.67 1.25 0.44 0.87 2 months 0.33 0.71 0.10 0.40 0.50 1.04 0.96 2.22 3 months 0.45 1.01 0.12 0.44 0.73 1.39 0.48 1.23 Baseline 1 month 0.02 1.09 0.09 0.53 0.29 1.94 1.31 3.72 Baseline 3 months 0.03 1.56 0.01 0.61 0.23 0.67 1.27 3.00 Intensity Upper jaw Lower jaw Placebo Test Placebo Test Mean SD Mean SD Mean SD Mean SD Baseline 0.35 0.73 0.08 0.29 0.85 1.99 1.25 2.18 1 month 0.35 0.94 0.04 0.19 0.67 1.25 0.37 0.77 2 months 0.33 0.71 0.08 0.31 0.50 1.04 0.71 1.47 3 months 0.40 0.90 0.12 0.44 0.73 1.39 0.44 1.12 Baseline 1 month 0.00 0.79 0.05 0.35 0.18 1.54 0.88 2.06 Baseline 3 months −0.05 1.18 −0.04 0.41 0.12 0.58 0.81 1.33 Positive changes mean decrease. View Large Surface of staining The scores of surfaces of the upper jaw were low in both groups, and again higher in the placebo group. A tendency towards a decrease was observed in both groups, although changes were very small and not significant (Table 4). The results in surface of staining confirmed the finding in intensity. Higher values were observed in the lower jaw, and they were also higher in the test group at baseline. In the placebo group, minor changes were observed, with a tendency to a decrease. However, in the test group, a clear trend to a reduction was observed, with the exception of the period between the 1- and the 2-month visits. De-bonded bands and/or braces The number of de-bonded braces was checked at each visit. Means of de-bonded brackets per visit (baseline, 1-, 2-, 3-month visits) were 0, 0.23, 0, and 0.14, respectively, for the placebo group; and 0.36, 0.5, 0.43, and 0.39, respectively, for the experimental group. No significant differences were found among visits within each group (P = 0.251) or considering visits and groups together (P = 0.782). The same result was found for the number of loose bands. Means of number of loose bands were 0.05, 0.05, 0, and 0, respectively, for the placebo group; and 0.04, 0.04, 0.14, and 0.04, respectively, for the experimental group. There were no significant differences between groups and visits. Oral mucosa injuries Mean values of mucosa injuries were lower in the experimental group than in the control group, except at the last visit. The evolution of the estimated means of mucosal injuries at each visit (baseline, 1-, 2-, 3-month visits) was 0.23, 0.41, 0.27, and 0.05, respectively, for the control group; and 0.11, 0.25, 0.25, and 0.21, respectively, for the experimental group. Differences were not statistically significant (P = 0.133). Microbiological results In the placebo group, samples from 29 out of 31 patients were available at baseline, while after 3 months, samples were available from the 23 patients attending the final visit. Pair comparisons were available from 21 patients. In the test group, 30 out of 32 patients provided baseline samples, while final samples were available from the 27 patients, allowing for 27 pair comparisons. Two microbiological samples at baseline in each group could not be processed for technical reasons. Comparable findings were observed at baseline for PlI and GI at sampled sites. Non-significant changes were observed in microbiological parameters and overgrowth of opportunistic species. The evaluated products can be considered as safe from a microbiological point of view. Additional supporting information may be found in the online version of this article: Supplementary Text 1 and Supplementary Table 1. Patient-reported outcomes At the 1-, 2- and 3-month visits, participants filled up a questionnaire. In the control group, 96.4, 95.8, and 100 per cent of patients completed the questionnaire after 1, 2, and 3 months, respectively. In the experimental group, the corresponging percentages were 96.6, 100, and 96.3 per cent. No statistical differences were found between groups. Answers were compared between groups at the 1-, 2-, and 3-month visits. After 1 month, no differences were observed between groups. Most of the subjects (69.1%) liked rinsing with the provided mouth rinse, 67.3 per cent of them liked the taste, 72.7 per cent of them noticed their teeth cleaner and smoother and 81.8 per cent had a pleasant and fresh breath feeling after rinsing. After 2 months, statistically significant differences (P ≤ 0.05) were only found for the question related to the taste of the product (Figure 1). In the control group, the percentage of patients considering the taste as ‘good’ was higher than in the experimental group (87 and 39.3 per cent, respectively. However, in the experimental group, the percentage of patients that considered it as ‘very good’ was higher (35.7 vs. 4.3 per cent). After converting the categorical outcome variable (very good, good, fair, bad) into a semi quantitative variable (4, 3, 2, 1, respectively) and comparing the average value between groups, patients rated the experimental mouth rinse higher than the control mouth rinse. Figure 1. View largeDownload slide Frequency distribution (in %) of answers given to the second question (‘The taste of this mouth rinse is…’) at 1, 2, and 3 months. Key for answers: A. very good, B. good, C. fair, D. bad. Figure 1. View largeDownload slide Frequency distribution (in %) of answers given to the second question (‘The taste of this mouth rinse is…’) at 1, 2, and 3 months. Key for answers: A. very good, B. good, C. fair, D. bad. After 3 months, no differences were found between groups regarding the frequency of responses to each question, except again for the question concerning the taste of mouth rinse with the same trend as in visit 2 (Figure 1). Eighty-seven per cent of control patients noticed ‘good’ taste for their mouth rinse and 46.2 per cent of experimental patients thought that the taste was ‘very good’ (related to the total of patients in each group). After noticing the significant differences, the categorical variable was transformed into a semi-quantitative variable, and it was observed that the average of responses to this question in the experimental group was higher. Compliance Compliance was first assessed by analysing the number of patients that fulfilled with the study protocol at each visit, according to the diary cards. Percentages decreased with time, but no differences were found between groups or among visits (91.3 vs. 96.3% at 1 month, 69.6 vs. 85.2% at 2 months and 56.5 vs. 77.8% at 3 months for placebo and test groups, respectively). A second analysis was performed, by counting the number of occasions that a patient failed to use the mouth rinse according to the daily diaries. Percentages of failed intakes were 16, 13, and 21 per cent for each visit. In the test group, slightly lower values were found (12, 15, and 11 per cent for each visit). Although this percentage reached 21 per cent in the control group after 3 months, no significant differences were found between groups or among visits. As an indirect variable to test for compliance, the ml of mouth rinse left in the last bottle used (two 500 ml bottles were provided at each visit) was measured at the end of each visit. Comparisons between groups at each visit did not show statistically significant differences, although the amount of test product left was increasingly higher (mean: 284.19, SD: 173.75 vs. mean: 282.20, SD: 134.01 at 1 month; mean: 286.46, SD: 138.31 vs. mean: 316.21, SD: 161.38 at 2 months; mean: 289.31, SD: 146.66 vs. mean: 375.54, SD: 159.92 at 3 months for placebo and test groups, respectively). The amount of test product left at the 3-month visit was statistically higher than the amounts left at 1- and 2-month visits (P = 0.036). Discussion In the present study, the adjunctive use of the test mouth rinse and dentifrice had a modest effect on plaque and gingivitis reduction. The use of this adjunctive treatment was not associated with an increase in tooth staining, although the compliance during this 3-month period with the study protocol, in both test and control groups, was limited. In addition, the test products were able to control and even reduce some periodontal pathogens, without an overgrowth of opportunistic species. As it is customary in oral hygiene studies, the main outcome variables used were plaque accumulation and gingivitis. Plaque levels were high at baseline, and after 1 month, increased in the placebo group (significantly in the maxilla) and decreased in the test group, achieving significant inter-group differences (P = 0.024) at the maxilla. However, this effect was small, only in the upper jaw and not clinically significant. The size effect used for sample size calculation (mean 0.42, SD = 0.40) was far from what has been observed in the study. We were expecting to detect a standardized effect size of 1.05 (obtained from dividing mean/SD), but the largest effect size we actually found in the test group was 0.22. This means that, despite the fact of finding statistically significant differences when comparing the test and the control groups, no clinical significance can be drawn from these results. Moreover, it is not clear why plaque levels raised in the subjects from the control group when they were provided with oral hygiene instruction, continued to brush their teeth and presumably they suffered the Hawthorn effect since they were participating in a trial in which their oral hygiene would be assessed. After 2 and 3 months, plaque levels tended to increase in both groups, but with a lower magnitude in the test group. These findings have also been observed in previous oral hygiene studies in similar populations, evaluating either a mouth rinse with essential oils (18) or a tooth paste and a mouth rinse with stannous and amine fluoride (19). In contrast, when CHX formulations have been utilized, significant plaque reductions were maintained throughout the study. In a study using a CHX mouth rinse (16), significant reductions were observed in the test group after 3 months, while the control group, despite initial reductions after 1 and 2 months, baseline levels were reached after 3 months. In another study, comparing CHX dentifrices (with and without sodium fluoride) with a sodium fluoride dentifrice (15), a clear reduction was observed from baseline to the 6-week visit in all groups. In the present study, even though the gingivitis levels were higher at baseline in the placebo group, after 1 month, the test group showed a slight reduction, while there was a slight increase in the placebo group, which resulted in significant intergroup differences at 1-month for all sites (P = 0.025), as well as for proximal, buccal and lingual surfaces and mandibular sites. Differences were maintained at the 3-month visit, for all sites, proximal and in the mandibular sites. As discussed for plaque levels, a similar trend was observed in two studies assessing an essential oils mouth rinse (18) or the combination of a dentifrice and a mouth rinse with amine and stannous fluoride (19). It has also been suggested that effect of essential oils on gingivitis is more pronounced than on plaque formation due to anti-inflammatory effects of the phenolic components (42). The studies evaluating CHX formulations (15,16) also demonstrated a more significant and maintained effect on gingivitis, although the presence of adverse effects, specifically tooth staining, may limit their long-term use. In this investigation, the gingivitis reduction was also modest, following the same trend as the plaque levels. This study carefully evaluated the occurrence of side effects, such as tooth staining as well as adverse effects, as de-bonded appliances or mucosal injuries. Although few studies have reported allergic reactions associated to the topical use of aloe vera, it seems that they are mostly due to anthra-quinones, such as aloin and barbolin (43). None of our patients reported redness, burning or stinging sensation. A recent systematic review on the effectiveness of aloe vera in the management of oral mucosal diseases did not find any adverse effects (44). In contrast with the use of other formulations and active agents used in young orthodontic patients that have shown increased tooth staining (45), the subjects using the tested products did not have significantly higher staining when compared with the placebo group. In fact, a tendency towards reduced tooth staining was observed in both test and placebo groups, being slightly higher in the test group. Ulceration, erosion, and desquamation have also been associated to wearing fixed orthodontic appliances (46), especially during the initial phases of treatment. In our study, oral mucosal injuries were less frequent in the test group, although differences were not significant. However, their incidence was low in both groups, which could be explained by the fact that most of these patients had been wearing the orthodontic appliances for some time before the study started. In the present study, the baseline microbiological samples were taken when the patients already had their brackets placed, what prevents any inference on the possible impact of the brackets on the subgingival microbiota, since high prevalence of recognised periodontal pathogens were detected in both groups. The microbiological results did not find significant differences, but provided evidence on the safety of the evaluated products in terms of absence of microbiological adverse effects (namely, the overgrowth of opportunistic species). Additional supporting information may be found in the online version of this article: Supplementary Text 1 In regards to the recorded patient-reported outcomes, the test products showed a better-rated taste when compared with the control. Significant differences were demonstrated at the 2- and 3-month visit, but not in the 1-month visit. Analyzing the frequency of the given answers, patients in the test group either liked it very much or disliked it, but control patients had a less extreme appreciation of the taste. The number of patients who liked the test product ‘very much’ increased with time. It may be speculated that test product had a higher intensity in flavour, compared to the control. Repeated exposure to this strong flavour may have an effect in the increase of the patient’s preferences (47). However, a limitation of this study could be the use of a questionnaire custom-made for this investigation, instead of one previously validated. Compliance with the use of the tested products was evaluated by diary cards. Both groups showed a relatively high level of compliance during the first month of the study, since only 3 out of the 50 patients failed to fulfill the study protocol. At the end of the study, however, this number raised to 16 patients (10 in the control and 6 in the experimental group), what may have affected the clinical results of the study. Ay et al. (48) evaluated different oral hygiene motivation methods for orthodontic patients with fixed appliances based on verbal information and reported improvements after 4 weeks, but not for longer periods. A recently published 6-month study on the efficacy of three different motivational techniques on oral hygiene in orthodontic patients, however, concluded that all the used methods were equally effective in controlling gingival health provided periodic reinforcement was applied irrespective of the technique (49). Our tested sample demonstrated a low oral hygiene motivation, probably due to the specific population attending a public university clinic. Differences have been observed between public and private orthodontic clinics, being patients in public institutions less motivated than in private ones (50). This study has shown a limited efficacy in terms of plaque and gingivitis reduction when patients used their assigned test product in comparison with a placebo, being these differences of small magnitude. This fact can be explained in part by the heterogeneity in terms of compliance with mechanical plaque control demonstrated by the subjects participating in this study, as well as in the differences in the orthodontic therapy, since the size of the arch wires was not standardized, what may have influenced the subject’s mechanical plaque control. This low compliance in mechanical plaque control may however, represent the reality affecting young patients undergoing fixed orthodontic therapy. Moreover, this is a short-term study (3 months), where subjects usually will be more likely to adhere to the provided instructions, and therefore, with longer term evaluations, it is expected the efficacy of the recommended regime will decline. In conclusion, the present study indicates that the adjunctive use of a CPC-based mouth rinse and dentifrice had limited effect in controlling plaque accumulation and gingivitis levels, and no adverse microbiological effects. The size of the effects was small and highly variable, and multiple comparisons performed in the secondary statistical tests could have increased the risk of a Type I error (false positive findings). Within the limitations of this study, it could also be concluded that the use of the test products was not associated with relevant adverse effects (including tooth staining or debonded appliances) and young orthodontic patients liked the taste of a CPC-based mouth rinse, which may have help to comply with the assigned oral hygiene regimen. Supplementary material Supplementary material is available at European Journal of Orthodontics online. Funding Research contract between Dentaid (Cerdanyola, Barcelona, Spain) and the University Complutense of Madrid. This work was partially supported by a research contract between Dentaid (Cerdanyola, Barcelona, Spain) and the University Complutense of Madrid. Conflict of Interest Statement None to declare. References 1. Levin , L. , Samorodnitzky-Naveh , G.R. and Machtei , E.E . ( 2008 ) The association of orthodontic treatment and fixed retainers with gingival health . Journal of Periodontology , 79 , 2087 – 2092 . Google Scholar Crossref Search ADS PubMed 2. Ristic , M. , Vlahovic Svabic , M. , Sasic , M. and Zelic , O . ( 2007 ) Clinical and microbiological effects of fixed orthodontic appliances on periodontal tissues in adolescents . Orthodontics and Craniofacial Research , 10 , 187 – 195 . Google Scholar Crossref Search ADS PubMed 3. Artun , J. and Brobakken , B.O . ( 1986 ) Prevalence of carious white spots after orthodontic treatment with multibonded appliances . European Journal of Orthodontics , 8 , 229 – 234 . Google Scholar Crossref Search ADS PubMed 4. Naranjo , A.A. , Triviño , M.L. , Jaramillo , A. , Betancourth , M. and Botero , J.E . ( 2006 ) Changes in the subgingival microbiota and periodontal parameters before and 3 months after bracket placement . American Journal of Orthodontics and Dentofacial Orthopedics , 130 , 275.e17 – 275.e22 . Google Scholar Crossref Search ADS 5. Lee , S.M. , Yoo , S.Y. , Kim , H.S. , Kim , K.W. , Yoon , Y.J. , Lim , S.H. , Shin , H.Y. and Kook , J.K . ( 2005 ) Prevalence of putative periodontopathogens in subgingival dental plaques from gingivitis lesions in Korean orthodontic patients . Journal of Microbiology (Seoul, Korea) , 43 , 260 – 265 . Google Scholar PubMed 6. Diamanti-Kipioti , A. , Gusberti , F.A. and Lang , N.P . ( 1987 ) Clinical and microbiological effects of fixed orthodontic appliances . Journal of Clinical Periodontology , 14 , 326 – 333 . Google Scholar Crossref Search ADS PubMed 7. Alexander , S.A . ( 1993 ) The effect of fixed and functional appliances on enamel decalcifications in early Class II treatment . American Journal of Orthodontics and Dentofacial Orthopedics , 103 , 45 – 47 . Google Scholar Crossref Search ADS PubMed 8. Mehra , T. , Nanda , R.S. and Sinha , P.K . ( 1998 ) Orthodontists’ assessment and management of patient compliance . The Angle Orthodontist , 68 , 115 – 122 . Google Scholar PubMed 9. Ainamo , J . ( 1977 ) Control of plaque by chemical agents . Journal of Clinical Periodontology , 4 , 23 – 35 . Google Scholar Crossref Search ADS PubMed 10. Gunsolley , J.C . ( 2006 ) A meta-analysis of six-month studies of antiplaque and antigingivitis agents . Journal of the American Dental Association (1939) , 137 , 1649 – 1657 . Google Scholar Crossref Search ADS PubMed 11. Boyd , R.L . ( 1993 ) Comparison of three self-applied topical fluoride preparations for control of decalcification . The Angle Orthodontist , 63 , 25 – 30 . Google Scholar PubMed 12. Alexander , S.A. and Ripa , L.W . ( 2000 ) Effects of self-applied topical fluoride preparations in orthodontic patients . The Angle Orthodontist , 70 , 424 – 430 . Google Scholar PubMed 13. Kalha , A . ( 2004 ) Some evidence that fluoride during orthodontic treatment reduces occurrence and severity of white spot lesions . Evidence-Based Dentistry , 5 , 98 – 99 . Google Scholar Crossref Search ADS PubMed 14. Garnick , J.J. , Singh , B. and Winkley , G . ( 1998 ) Effectiveness of a medicament containing silicon dioxide, aloe, and allantoin on aphthous stomatitis . Oral surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics , 86 , 550 – 556 . Google Scholar Crossref Search ADS PubMed 15. Olympio , K.P. , Bardal , P.A. , de M Bastos , J.R. and Buzalaf , M.A . ( 2006 ) Effectiveness of a chlorhexidine dentifrice in orthodontic patients: a randomized-controlled trial . Journal of Clinical Periodontology , 33 , 421 – 426 . Google Scholar Crossref Search ADS PubMed 16. Anderson , G.B. , Bowden , J. , Morrison , E.C. and Caffesse , R.G . ( 1997 ) Clinical effects of chlorhexidine mouthwashes on patients undergoing orthodontic treatment . American Journal of Orthodontics and Dentofacial Orthopedics , 111 , 606 – 612 . Google Scholar Crossref Search ADS PubMed 17. Brightman , L.J. , Terezhalmy , G.T. , Greenwell , H. , Jacobs , M. and Enlow , D.H . ( 1991 ) The effects of a 0.12% chlorhexidine gluconate mouthrinse on orthodontic patients aged 11 through 17 with established gingivitis . American Journal of Orthodontics and Dentofacial Orthopedics , 100 , 324 – 329 . Google Scholar Crossref Search ADS PubMed 18. Tufekci , E. , Casagrande , Z.A. , Lindauer , S.J. , Fowler , C.E. and Williams , K.T . ( 2008 ) Effectiveness of an essential oil mouthrinse in improving oral health in orthodontic patients . The Angle Orthodontist , 78 , 294 – 298 . Google Scholar Crossref Search ADS PubMed 19. Øgaard , B. , Alm , A.A. , Larsson , E. and Adolfsson , U . ( 2006 ) A prospective, randomized clinical study on the effects of an amine fluoride/stannous fluoride toothpaste/mouthrinse on plaque, gingivitis and initial caries lesion development in orthodontic patients . European Journal of Orthodontics , 28 , 8 – 12 . Google Scholar Crossref Search ADS PubMed 20. Hannah , J.J. , Johnson , J.D. and Kuftinec , M.M . ( 1989 ) Long-term clinical evaluation of toothpaste and oral rinse containing sanguinaria extract in controlling plaque, gingival inflammation, and sulcular bleeding during orthodontic treatment . American Journal of Orthodontics and Dentofacial Orthopedics , 96 , 199 – 207 . Google Scholar Crossref Search ADS PubMed 21. Witt , J. , Ramji , N. , Gibb , R. , Dunavent , J. , Flood , J. and Barnes , J . ( 2005 ) Antibacterial and antiplaque effects of a novel, alcohol-free oral rinse with cetylpyridinium chloride . The Journal of Contemporary Dental Practice , 6 , 1 – 9 . Google Scholar PubMed 22. Witt , J.J. , Walters , P. , Bsoul , S. , Gibb , R. , Dunavent , J. and Putt , M . ( 2005 ) Comparative clinical trial of two antigingivitis mouthrinses . American Journal of Dentistry , 18 , 15A – 17A . Google Scholar PubMed 23. Rawlinson , A. , Pollington , S. , Walsh , T.F. , Lamb , D.J. , Marlow , I. , Haywood , J. and Wright , P . ( 2008 ) Efficacy of two alcohol-free cetylpyridinium chloride mouthwashes - a randomized double-blind crossover study . Journal of Clinical Periodontology , 35 , 230 – 235 . Google Scholar Crossref Search ADS PubMed 24. Mankodi , S. , Bauroth , K. , Witt , J.J. , Bsoul , S. , He , T. , Gibb , R. , Dunavent , J. and Hamilton , A . ( 2005 ) A 6-month clinical trial to study the effects of a cetylpyridinium chloride mouthrinse on gingivitis and plaque . American Journal of Dentistry , 18 Spec No , 9A – 14A . Google Scholar PubMed 25. Allen , D.R. et al. ( 1998 ) Efficacy of a mouthrinse containing 0.05% cetylpyridinium chloride for the control of plaque and gingivitis: a 6-month clinical study in adults . Compendium of Continuing Education in Dentistry (Jamesburg, N.J.: 1995) , 19 , 20 – 26 . Google Scholar PubMed 26. Silva , M.F. , dos Santos , N.B. , Stewart , B. , DeVizio , W. and Proskin , H.M . ( 2009 ) A clinical investigation of the efficacy of a commercial mouthrinse containing 0.05% cetylpyridinium chloride to control established dental plaque and gingivitis . The Journal of Clinical Dentistry , 20 , 55 – 61 . Google Scholar PubMed 27. Albert-Kiszely , A. , Pjetursson , B.E. , Salvi , G.E. , Witt , J. , Hamilton , A. , Persson , G.R. and Lang , N.P . ( 2007 ) Comparison of the effects of cetylpyridinium chloride with an essential oil mouth rinse on dental plaque and gingivitis - a six-month randomized controlled clinical trial . Journal of Clinical Periodontology , 34 , 658 – 667 . Google Scholar Crossref Search ADS PubMed 28. Jenkins , S. , Addy , M. and Newcombe , R.G . ( 1994 ) A comparison of cetylpyridinium chloride, triclosan and chlorhexidine mouthrinse formulations for effects on plaque regrowth . Journal of Clinical Periodontology , 21 , 441 – 444 . Google Scholar Crossref Search ADS PubMed 29. Kozak , K.M. , Gibb , R. , Dunavent , J. and White , D.J . ( 2005 ) Efficacy of a high bioavailable cetylpyridinium chloride mouthrinse over a 24-hour period: a plaque imaging study . American Journal of Dentistry , 18 , 18A – 23A . Google Scholar PubMed 30. Haps , S. , Slot , D.E. , Berchier , C.E. and Van der Weijden , G.A . ( 2008 ) The effect of cetylpyridinium chloride-containing mouth rinses as adjuncts to toothbrushing on plaque and parameters of gingival inflammation: a systematic review . International Journal of Dental Hygiene , 6 , 290 – 303 . Google Scholar Crossref Search ADS PubMed 31. Hsu , S . ( 2005 ) Green tea and the skin . Journal of the American Academy of Dermatology , 52 , 1049 – 1059 . Google Scholar Crossref Search ADS PubMed 32. Worthington , H.V. , Clarkson , J.E. and Eden , O.B . ( 2006 ) Interventions for preventing oral mucositis for patients with cancer receiving treatment . The Cochrane Database of Systematic Reviews , 19 , CD000978 . 33. Turesky , S. , Gilmore , N.D. and Glickman , I . ( 1970 ) Reduced plaque formation by the chloromethyl analogue of victamine C . Journal of Periodontology , 41 , 41 – 43 . Google Scholar Crossref Search ADS PubMed 34. Lobene , R.R. , Weatherford , T. , Ross , N.M. , Lamm , R.A. and Menaker , L . ( 1986 ) A modified gingival index for use in clinical trials . Clinical Preventive Dentistry , 8 , 3 – 6 . Google Scholar PubMed 35. Volpe , A.R. , Manhold , J.H. and Hazen , S.P . ( 1965 ) In vivo calculus assessment. I. A method and its examiner reproducibility . The Journal of Periodontology , 36 , 292 – 298 . Google Scholar Crossref Search ADS PubMed 36. Gadhia , K. , Shah , R. , Swaminathan , D. , Wetton , S. and Moran , J . ( 2006 ) Development of a stain shade guide to aid the measurement of extrinsic dental stain . International Journal of Dental Hygiene , 4 , 98 – 103 . Google Scholar Crossref Search ADS PubMed 37. Wikström , M. , Renvert , S. , Dahlén , G. and Johnsson , T . ( 1991 ) Variance in recovery of periodontitis-associated bacteria caused by sampling technique and laboratory processing . Oral Microbiology and Immunology , 6 , 102 – 106 . Google Scholar Crossref Search ADS PubMed 38. Syed , S.A. and Loesche , W.J . ( 1972 ) Survival of human dental plaque flora in various transport media . Applied Microbiology , 24 , 638 – 644 . Google Scholar PubMed 39. Dahlén , G. , Renvert , S. , Wikström , M. and Egelberg , J . ( 1990 ) Reproducibility of microbiological samples from periodontal pockets . Journal of Clinical Periodontology , 17 , 73 – 77 . Google Scholar Crossref Search ADS PubMed 40. Alsina , M. , Olle , E. and Frias , J . ( 2001 ) Improved, low-cost selective culture medium for Actinobacillus actinomycetemcomitans . Journal of Clinical Microbiology , 39 , 509 – 513 . Google Scholar Crossref Search ADS PubMed 41. Rioboo , M. , García , V. , Serrano , J. , O’Connor , A. , Herrera , D. and Sanz , M . ( 2012 ) Clinical and microbiological efficacy of an antimicrobial mouth rinse containing 0.05% cetylpyridinium chloride in patients with gingivitis . International Journal of Dental Hygiene , 10 , 98 – 106 . Google Scholar Crossref Search ADS PubMed 42. Sekino , S. and Ramberg , P . ( 2005 ) The effect of a mouth rinse containing phenolic compounds on plaque formation and developing gingivitis . Journal of Clinical Periodontology , 32 , 1083 – 1088 . Google Scholar Crossref Search ADS PubMed 43. Moore , T.E . ( 2001 ) The M and M’s of aloe vera–is it for dentistry ? Journal - Oklahoma Dental Association , 91 , 30 – 1, 36 . Google Scholar PubMed 44. Nair , G.R. , Naidu , G.S. , Jain , S. , Nagi , R. , Makkad , R.S. and Jha , A . ( 2016 ) Clinical effectiveness of aloe vera in the management of oral mucosal diseases- a systematic review . Journal of Clinical and Diagnostic Research , 10 , ZE01 – ZE07 . Google Scholar PubMed 45. Cosyn , J. and Verelst , K . ( 2006 ) An efficacy and safety analysis of a chlorhexidine chewing gum in young orthodontic patients . Journal of Clinical Periodontology , 33 , 894 – 899 . Google Scholar Crossref Search ADS PubMed 46. Baricevic , M. , Mravak-Stipetic , M. , Majstorovic , M. , Baranovic , M. , Baricevic , D. and Loncar , B . ( 2011 ) Oral mucosal lesions during orthodontic treatment . International Journal of Paediatric Dentistry , 21 , 96 – 102 . Google Scholar Crossref Search ADS PubMed 47. Liem , D.G. and de Graaf , C . ( 2004 ) Sweet and sour preferences in young children and adults: role of repeated exposure . Physiology and Behavior , 83 , 421 – 429 . Google Scholar Crossref Search ADS PubMed 48. Ay , Z.Y. , Sayin , M.O. , Ozat , Y. , Goster , T. , Atilla , A.O. and Bozkurt , F.Y . ( 2007 ) Appropriate oral hygiene motivation method for patients with fixed appliances . The Angle Orthodontist , 77 , 1085 – 1089 . Google Scholar Crossref Search ADS PubMed 49. Acharya , S. , Goyal , A. , Utreja , A.K. and Mohanty , U . ( 2011 ) Effect of three different motivational techniques on oral hygiene and gingival health of patients undergoing multibracketed orthodontics . The Angle Orthodontist , 81 , 884 – 888 . Google Scholar Crossref Search ADS PubMed 50. Tervonen , M.M. , Pirttiniemi , P. and Lahti , S . ( 2011 ) Development of a measure for orthodontists to evaluate patient compliance . American Journal of Orthodontics and Dentofacial Orthopedics , 139 , 791 – 796 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

The European Journal of OrthodonticsOxford University Press

Published: Sep 28, 2018

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