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ORIGINAL Graziani ARTICLE et al Dental Plaque, Gingival Inflammation and Tooth Discolouration with Different Commercial ...

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ORIGINAL Graziani ARTICLE et al

Dental Plaque, Gingival Inflammation and Tooth Discolouration with Different Commercial Formulations of 0.2% Chlorhexidine Rinse: A Double-blind Randomised Controlled Clinical Trial Filippo Graziania/Mario Gabrieleb/Francesco D’Aiutoc/Jeanie Suvand/ Matteo Tonellie/Silvia Ceif

Purpose: To investigate the efficacy of various formulations of chlorhexidine 0.2% (CHX) in terms of plaque and gingival bleeding control compared to each other and to saline rinse (CTRL) over a 35-day rinsing period. Materials and Methods: Seventy subjects were randomly allocated to one of 4 groups rinsing twice daily for 35 days. The different groups used CHX 0.2% rinse with alcohol (CHX1) and without alcohol (CHX2), with an antidiscolouration system (CHX3) or saline rinse (CTRL). Clinical examinations to evaluate full-mouth plaque scores (FMPS) and periodontal parameters were performed at baseline, 7, 21 and 35 days. Tooth discolouration (TD) was measured at each time point using digital photographs and spectrophotometric analysis. Results: At 35 days, CTRL showed the highest levels of plaque. The mean changes in FMPS from baseline were 69.8% ± 6.8 for CHX1, 57.5% ± 9.8 for CHX2, 43.7% ± 9.8 for CHX3 and 25.8% ± 7.7 for CTRL. Statistically significant differences were demonstrated between CHX1 and CHX3 (p = 0.02), CHX2 vs CHX3 (p ≤ 0.05) and CHX1/CHX2 vs CHX3 (p < 0.05). In contrast, CHX3 appeared more effective in reducing inflammatory indexes. TD increased over time in 60% to 70% of participants, although lighter staining was found in the CHX3 group. Greater FMPS reduction was observed in participants with staining vs without staining (26.0% ± 12.3, p = 0.04). Conclusion: Conventional CHX appeared more effective in terms of plaque reduction. Interestingly, the newest formulation showed a higher control of gingival inflammation. Staining was associated with lower plaque levels. Key words: chlorhexidine, inflammation, mouthrinses, plaque, staining Oral Health Prev Dent 2015;13:101-111 doi: 10.3290/j.ohpd.a32827

a

Assistant Professor, Unit of Oral Surgery, Department of Surgical and Medical Pathology, University of Pisa, Pisa, Italy; Assistant Professor, Periodontology Unit, Eastman Clinical Investigation Centre, UCL Eastman Dental Institute, London, UK.

b

Chairman, Unit of Oral Surgery, Department of Surgical and Medical Pathology, University of Pisa, Pisa, Italy.

c

Associate Professor, Periodontology Unit, Eastman Clinical Investigation Centre, UCL Eastman Dental Institute, London, UK.

d

Clinical Trials Coordinator, Periodontology Unit, Eastman Clinical Investigation Centre, UCL Eastman Dental Institute, London, UK.

e

Hospital Consultant, Department of Surgical and Medical Pathology, Unit of Oral Surgery, University of Pisa, Pisa, Italy.

f

Assistant Professor, Unit of Oral Surgery, Department of Surgical and Medical Pathology, University of Pisa, Pisa, Italy.

Correspondence: Dr. Filippo Graziani, Department of Surgical and Medical Pathology, Unit of Dentistry and Oral Surgery, University of Pisa, Via Roma 67, 56126, Pisa, Italy. Tel: +39-050-99-2939, Fax: +39-050-555-232. Email: fi[email protected]

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Submitted for publication: 10.04.13; accepted for publication: 13.06.13

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hlorhexidine (CHX), a bisguanide, represents the gold standard of chemical dental plaque control due to its efficacy in control of plaque formation and gingivitis prevention (Löe et al, 1970; Jones, 1997). Its action is due to its capacity to link anionic groups present on bacterial surfaces, showing both bactericidal and bacteriostatical properties (Davies, 1973; Hennessey, 1973). Nevertheless, anionic groups are also shown on oral tissues. The efficacy of this electrochemical link allows the CHX to remain on the tissues for hours after rinsing, therefore increasing the inhibitory action on plaque (Jenkins et al, 1994). Clinically, CHX has demonstrated a consistent antiplaque effect in long-term clinical trials, show-

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ing plaque score reductions of up to 80% more than placebo controls (Gusberti et al, 1988; Gunsolley, 2006). Moreover, CHX has been reported to have a preventive action on gingivitis (Lang et al, 1988). Overall, CHX is a very effective chemical agent with numerous indications (Addy and Moran, 1997). Indeed, a recent review had shown that CHX is effective in controlling both plaque and gingivitis (Van Strydonck et al, 2012). Despite these positive effects, CHX usage may be limited by its side effects, as it is the main cause of extrinsic tooth discolouration (TD) and oral mucosal pigmentation (Flotra et al, 1971). The mechanism underlying this is still not fully understood (Watts and Addy, 2001). Firstly, CHX may accelerate a non-enzymatic browning reaction catalysing the Maillard reaction, thus discolouring the acquired pellicle on teeth (Yates et al, 1993). CHX may also denature proteins of the acquired pellicle to form sulfur radicals able to react with metal ions, thus forming metal sulfide. Importantly, CHX may also precipitate chromogens, such as polyphenols, acquired from diet (Eriksen et al, 1985). Numerous attempts have been made to limit TD by adding chemical reagents to CHX such as M239144, monoperoxyphthalic acid, centylpiridium chloride, peroxyborate, polyvinyl pyrrolidone and most recently ascorbic acid and sodium metabisulfite (antidiscolouration system, ADS) (Wade et al, 1994; Addy and Wade, 1995; Charbonneau and Snider, 1997; Grundemann et al, 2000; Claydon et al, 2001; Bernardi et al, 2004). ADS-enriched CHX has shown efficacy equal to that of CHX without ADS in terms of reduction of gingival inflammation in post-operative patients (Cortellini et al, 2008). However, contradictory results have been found in terms of plaque control (Bernardi et al, 2004; Arweiler et al, 2006). Therefore, the benefit of adding ADS to CHX remains unclear. The aim of this randomised controlled clinical trial was to evaluate the efficacy of various commercial CHX formulations in terms of plaque and bleeding control and to assess possible side effects, such as TD.

MATERIALS AND METHODS Study design and patient selection This was a single-centre, double-blind, parallelgroup, randomised clinical trial with 70 participants carrying out 35 days of mouthrinsing with one of 3

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different 0.2% CHX mouthrinses or placebo control rinse with no efficacy in plaque and bleeding reduction (Fig 1A). Ethical approval was obtained from the ethics committee of the University Hospital of Pisa (Italy). The study was conducted in accordance with the principles outlined in the Declaration of Helsinki on experiments involving human subjects. Potential participants for this study were identified from individuals referred to the Unit of Dentistry and Oral Surgery of the University Hospital of Pisa for periodontal or dental pathologies. A complete dental examination including a medical and dental history, an intra-oral assessment and full-mouth periodontal probing was performed at the initial visit. Radiographic examination was performed by taking an orthopantomogramme (OPT). Every individual who fulfilled the inclusion criteria was provided with detailed information about the study and invited to participate. Subjects signed a written consent form. To be included, potential participants were required to have at least 20 teeth, with the 8 incisors free of restorations or prosthetic reconstructions and be in general good health. Subjects were excluded if they were: (i) suffering from any systemic illnesses, also including cardiovascolar, renal or liver diseases, (ii) smokers, (iii) undergoing any periodontal and/or dental treatment during the 35 days of the trial, (iv) pregnant or lactating females, (v) undergoing orthodontic treatment or (vi) not able to sign the consent form. Patients were randomly allocated to one of the following four groups: H2O/NaCl (CTRL); an alcoholcontaining 0.2% CHX gluconate (CHX1) (Corsodyl 0.2%, GlaxoSmithKline; Milan, Italy); an alcoholfree 0.2% CHX gluconate (CHX2) (Dentosan 0.2%, Johnson & Johnson; Rome, Italy); an alcohol-free 0.2% CHX gluconate with ADS (CHX3) (Curaden Healthcare; Saronno, Italy).

Sample size calculation, randomisation procedures and allocation concealment A sample size of 12 individuals per group, with at least three subjects to compensate for dropouts, with 3 treatment arms and one control group would provide any-pair power of 80% (α = 0.05) (KruskalWallis Test) for comparing each treatment mean with the control mean (standard deviation of within group differences of 1). A computer-generated random sequence conducted by a research fellow not directly involved in the experiments was used to assign participants to one of the four treatment

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groups. Allocation to the treatment was concealed as a code number identifying the allocated group. It was sealed in an opaque envelope which was opened at the baseline visit after completion of the clinical measurements, photographs, supragingival scaling and polishing.

Inclusion Baseline FMPS + FMBS + GI Photographic and spectrophotometric assessment OH instruction Supragingival scaling and polishing

Baseline Randomisation

At the baseline visit, standardised clinical, digital photographs of maxillary and mandibular central incisors were taken. Following the clinical examination, coronal scaling and polishing was performed and oral hygiene instructions were provided. Oral hygiene instructions were given as follows: interdental brushing with appropriate size interdental brushes (TePe Munhygienprodukter; Malmö, Sweden) and/or dental floss when interdental embrasures did not allow interdental brushing. Interdental cleaning was followed by electric toothbrushing (Oral-B Braun, Procter & Gamble; Rome, Italy). A sodium lauryl sulfate-free toothpaste was provided to each participant (Oral-B Braun). At the end of the baseline visit, the randomisation envelope was opened and mouthrinse bottles were dispensed to the participants according to the allocated group by a research fellow not directly involved in the reFig 1  Study design (A), and flow of subjects throughout the study (B).

CTRL

CHX 1

CHX 2

CHX 3

7 days FMPS + GI Photographic and spectrophotometric assessment 21 days

FMPS + GI Photographic and spectrophotometric assessment

35 days

FMPS + FMBS + GI Photographic and spectrophotometric assessment

Supragingival scaling and polishing A

Assessed for eligibility (N=91) Excluded (N=19) Did not meet inclusion criteria (N=14) Refused to participate (N=5) Randomised (N=70)

Allocated to CTRL (N=15)

Allocated to CHX 1 (N=15)

Allocated to CHX 2 (N=21)

Allocated to CHX 3 (N=19)

Followed up at 7 days (N=15) 21 days (N=14) 35 days (N=9)

Followed up at 7 days (N=15) 21 days (N=14) 35 days (N=11)

Followed up at 7 days (N=20) 21 days (N=19) 35 days (N=18)

Followed up at 7 days (N=19) 21 days (N=16) 35 days (N=15)

Analysed (N=15)

Analysed (N=15)

Analysed (N=21)

Analysed (N=19)

B

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search. Participants were instructed to rinse for 35 days for 1 min twice a day with 10 ml solution and to refrain from tea, coffee and red wine intake for at least 1 h after rinsing. A diary was also provided to participants to record the frequency of rinsing (0, 1 or 2) and the intake of coffee, tea and red wine on a daily basis.

Clinical examinations Clinical examinations were performed at baseline, then repeated at 7, 21 and 35 days after baseline by an examiner unaware of the group allocation (Fig 1B). At each time point, the full-mouth plaque score (FMPS) and gingival index (GI) were recorded. FMPS was recorded by assigning a binary score (plaque present or absent) to each site and calculating the percentage of total tooth surfaces that revealed the presence of plaque detected by the use of a periodontal probe (O’Leary et al, 1972). GI was measured on four sites per tooth, then an overall mean for the patient was calculated (Löe, 1967). A full-mouth bleeding score (FMBS) was also recorded at baseline and after 35 days, after dichotomously assessing the presence or absence of bleeding on probing from the bottom of the pocket upon gently probing (Ainamo and Bay, 1975). A UNC-15 periodontal probe was used by a blinded, calibrated and experienced examiner at 6 sites/ tooth, excluding third molars.

TD measurements: digital image and spectrophotometric analysis TD was measured using digital photographic measurements. Standardised photographs of the central incisors were taken at each time point. Buccal incisors surfaces were divided into three areas: incisal, approximal and gingival (Lobene, 1968; Grundemann et al, 2000). Staining index (SI) was then recorded as the dichotomous presence or absence of staining in each area and calculated as the percentage of the total area showing staining on the 8 vestibular surfaces of the central incisors. Tooth colour was also measured according to the CIE-L*a*b* parameters (CIE, 1971). The value L* (lightness), a* (chroma along the green-red axis), b* (chroma along the yellow-blue axis) were obtained with digital image and spectrophotometric analysis. Images were acquired using photo analysing software (Adobe Photoshop, Adobe Systems;

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San Jose, CA, USA). The L*a*b* value was calculated in the darkest spot of the area on each of the three areas for each 8 vestibular surface by a blinded examiner. Spectrophotometric analysis was also performed for each of the 8 vestibular incisor surfaces with a commercially available spectrophotometer (Vita EasyShade Compact, VITA Zahnfabrik; Bad Säckingen, Germany). The spectrophotometer was positioned over the crown of the central incisors and spectrophotometric data (Spectr-L*a*b*) were recorded as the mean of three consecutive measures (Paul et al, 2002). SI, L*a*b* and SpectrL*a*b* were calculated at each time point.

Data management and statistics Data are presented as means and standard errors unless otherwise specified. At the completion of examination visits, the data collected from all participants were entered into an Excel spreadsheet and checked for data entry errors, then locked until final analysis. The analysis plan was designed prior to unblinding of the data. The primary outcome of the study was the change in mean FMPS between groups after 35 days. The mean change for each outcome studied was calculated as the relative difference between day 35 and baseline, divided by the baseline value and multiplied by 100. Secondary outcomes included differences and changes in FMBS, GI, SI and TD based on the L*a*b* system. Further colour differences among the 3 measurements were grouped into changes in Delta E (ΔE), calculated as follows: ΔE = [(ΔL)2 + (Δa)2 + (Δb)2]1/2 (Johnston and Kao, 1989). Spectrophotometric Delta E (spectr-ΔE) was calculated in the same way utilising spectr-L*a*b* values. Interproximal ΔE was calculated in the same fashion considering only digital image L*, a* and b* values of the interproximal areas. Due to the lack of normal distribution of most of the variables studied, a two-way non-parametric ANOVA (Friedman test) was used to compare differences in all primary and secondary outcomes between groups at various time points. Post-hoc comparisons were performed as previously described (Conover, 1980). Subgroup analyses were performed in those individuals who presented with staining at day 35 (binary variable, yes or no). Correlation analyses were performed with the Spearman rank correlation test. Data were analysed with a statistical package by a masked statistician (SPSS version 17.0, SPSS; Chicago, IL, USA). All analyses were two-tailed with significance set at α = 0.05. Oral Health & Preventive Dentistry

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Table 1 Average daily consumption of coffee, tea and red wine Group

CTRL

CHX1

CHX2

CHX3

p-value

Coffee (N cups/day)

0.8 ± 0.2

1.8 ± 0.4

1.6 ± 0.2

1.4 ± 0.4

0.112

Tea (N cups/day)

0.3 ± 0.2

0.1 ± 0.1

0.1 ± 0.1

0.2 ± 0.1

0.141

Wine (N glasses/day)

0.2 ± 0.1

0.3 ± 0.2

0.4 ± 0.2

0.5 ± 0.3

0.754

Table 2 Subject and clinical characteristics at baseline CTRL N = 15

CHX1 N = 15

CHX2 N = 20

CHX3 N = 20

Mean age (95%CI)

29.71 (24.0–35.5)

29.7 (23.4–35.9)

39.5 (31.6–35.9)

36.0 (30.3–37.2)

Females, %

40

60

48

47

Number of teeth (min, max)

29 (26, 32)

29 (28, 30)

26 (24, 28)

28 (24, 32)

Periodontitis diagnosed, %

17

50

39

44

Number of pockets ≥ 5 mm mean (95% CI)

5.5 (1.4–12.2)

17.3 (0.4–34.0)

11.2 (2.9–19.0)

17.2 (2.3–32.1)

% of pockets ≥ 5 mm mean (95% CI)

3.3 (0.9–7.5)

10.6 (0.2–21.3)

6.8 (1.9–11.7)

11.7 (0.3–23.4)

FMPS mean (95% CI)

32.3 (19.9–44.7)

44.3 (29.4–59.1)

40.0 (27.3–52.6)

41.4 (32.4–50.4)

FMBS mean (95% CI)

16.7 (4.7–28.7)

19.7 (7.4–32.1)

15.1 (7.1–23.1)

16.9 (9.8–24.0)

GI mean (95% CI)

1.5 (1.3–1.9)

1.8 (1.4–2.2)

1.6 (1.3–1.9)

1.8 (1.5–2.2)

Variable

RESULTS

over the study period did not differ substantially between the four study groups (Table 1).

Subject compliance and study schedule Ninety-one participants were assessed for their eligibility before entering the study. Of these, 19 were excluded, 14 because they did not meet the inclusion criteria (presence of a restoration on the vestibular surfaces of central incisors), while the remaining 5 chose not to participate (Fig 1B). Hence, 70 participants were recruited and randomly allocated to one of the four study groups. Unequal allocation to groups reflects the fact that recruitment was stopped before reaching the recruitment target and the random number sequence was not constructed using blocks. All participants received the allocated intervention and were followed throughout the study, as depicted in Fig 1B. Compliance with medication was self-reported. Percentage of rinses performed during the trial were 92.7% ± 8.1 for CHX1, 94.1% ± 4.6 for CHX2, 89.3% ± 10.3 for CHX3, and 99.8% ± 0.4 for CTRL. Average consumption of tea, coffee and wine

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Subject characteristics at baseline Baseline characteristics of all participants included in the study are displayed in Table 2. Generally, subjects were in their third decade of life with equal gender distribution, presented with mild periodontal involvement and periodontal inflammation limited to approximately a fifth of the entire dentition. No changes in systemic medications, diet or lifestyle were reported by any participants throughout the entire duration of the study.

FMPS Seven days after oral hygiene instruction and mouthrinsing, FMPS levels decreased dramatically in all 4 groups when compared to baseline (intra-

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Table 3 Variations of clinical parameters throughout the study (for significant differences, see text) Variable Mean (95%CI)

Baseline

7 days

21 days

35 days

CTRL

32.3 (19.9–44.7)

21.6 (11.8–31.5)

16.2 (10.3–22.0)

21.4 (13.5–29.3)

CHX1

44.3 (29.4–59.1)

14.2 (5.8–22.6)

14.1 (7.0–21.3)

11.5 (7.1–16.0)

CHX2

40.0 (27.3–52.6)

12.4 (8.2–16.5)

9.0 (5.5–12.5)

10.1 (7.1–13.2)

CHX3

41.4 (32.4–50.4)

16.3 (10.6–22.1)

16.5 (12.0–21.0)

FMPS 17.8 (13.5–22.2)

CTRL

1.5 (1.3–1.8)

1.1 (0.8–1.5)

0.9 (0.4–1.3)

0.8 (0.4–1.2)

CHX1

1.8 (1.4–2.2)

1.1 (0.5–1.6)

1.1 (0.5–1.6)

0.9 (0.5–1.2)

CHX2

1.6 (1.3–1.9)

1.20 (1.0–1.4)

0.9 (0.6–1.2)

0.6 (0.3–0.9)

CHX3

1.8 (1.5–2.2)

1.05 (0.8–1.3)

0.9 (0.6–1.3)

0.6 (0.3–0.8)

GI

CTRL









CHX1



3.6 (0.9–8.0)

12.9 (3.0–22.8)

31.8 (18.8–44.7)

CHX2



1.3 (0.2–2.9)

9.2 (4.2–14.2)

21.7 (14.3–29.1)

CHX3



0.8 (0.2–1.7)

5.9 (2.2–9.7)

15.4 (9.2–21.7)

SI

0

– 25

– 50

– 75

–100

CTRL CHX1 CHX2 CHX3

P = 0.04

Average change in FMPS at D35

Average change in FMPS at D35

0

– 50

– 100

P = 0.02

– 150

A

B

CTRL CHX1 CHX2 CHX3 No staining

Staining

P = 0.025

Average change in GI at D35

0

– 20

– 40

– 60

– 80

C

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CTRL CHX1 CHX2 CHX3

Fig 2  Average changes from baseline (%) of FMPS at 35 days. CHX1 and CHX2 changes were also statistically significantly lower than the CTRL group (p < 0.05) (A); average changes from baseline (%) of FMPS at 35 days stratified according to staining vs no staining (B); average changes from baseline (%) of GI at 35 days. Statistically significant differences were noted only between CHX3 and CTRL (C).

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A

B

C

Fig 3  Clinical images of subjects’ teeth at baseline (top row) and after 35 days (bottom row) of CHX rinsing, showing tooth discolouration for CHX1 (A), CHX2 (B) and CHX3 (C).

group comparison) (Table 3). At 21 and 35 days, FMPS showed only minor further decreases (scores at day 35 vs baseline were all statistically significantly lower, p < 0.01 for all groups). At day 35, FMPS was statistically significantly lower in CHX1 and CHX2 groups compared to the control (p < 0.005) (intergroup comparison) (Table 3). Mean FMPS changes from baseline were 69.8% ± 6.8 for CHX1, 57.5% ± 9.8 for CHX2, 43.7% ± 9.8 for CHX3 and 25.8% ± 7.7 for CTRL (Fig 2A). Mean FMPS changes were greater in CHX1 (p = 0.02) and CHX2 compared to CHX3 (p = 0.04). Similarly, mean FMPS changes (baseline to day 35) in CHX1 and CHX2 were greater than in the CTRL group. An almost statistically significant increase in FMPS from baseline to day 35 was observed for CHX3 compared to CTRL (p = 0.07). When all subjects were grouped, a greater reduction of FMPS was observed for those who presented with staining at day 35 (average difference of 26.0% ± 12.3, p = 0.04) compared to those without any staining (Fig 2B).

change of 34.6% ± 15.1, p = 0.025) (Fig 2C). Analysis of staining did not show any statistically significant differences.

Full-mouth bleeding score A significant reduction of FMBS scores by half was noted in all study groups between baseline and 35 days with a great variability in patient response. The control group decreased from 16.67% (95%CI 4.65– 28.68) to 13.47% (95%CI 1.04–25.89), showing no significant differences. On the other hand, statistically significant decreases in mean full-mouth scores of gingival inflammation were noted for all experimental study groups at day 35 compared to baseline. Between-group changes in FMBS, however, were statistically significant only when CHX2 (mean difference of 43.4 ± 22.4, p = 0.05) and CHX3 (means difference of 46.1 ± 23.1, p = 0.05) were compared to the CTRL group.

Tooth discolouration Gingival index Consistent reductions in GI were observed among all study groups (Table 3). Despite the fact that all three CHX groups presented greater reductions compared to CTRL, the only statistically significant difference in mean change in GI at 35 days was observed between CHX3 and CTRL groups (mean

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Discolouration was rare at 7 days. As the trial progressed, however, the number of participants showing TD increased in all three experimental groups. At 35 days, staining was frequent, reported in well above 60% of all cases rinsing with the three CHX formulations (Fig 3). The magnitude of staining differed between study groups (Table 3).

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CTRL CHX1 CHX2 CHX3

12

10

10 ΔE Interproximal

8

ΔE

8 6 4

6 4 2

2 0

0 Baseline

Day 7

Day 21

Day 35

A

B

Baseline

Day 7

Day 21

Day 35

Fig 4  Variations of ΔE (A) and ΔE_interproximal (B) throughout the trial.

Delta E and ΔE-interproximal increased throughout the trial in all three experimental groups (Fig 4). At 35 days, differences in ΔE were noted between CHX1 vs CHX2 and CHX3 (average differences between groups of 5.85 ± 0.67 and 6.52 ± 1.05 respectively, p < 0.05 for both). Spectrophotometric analyses showed similar values as spectr-ΔE increased at 21 and 35 days. No differences could be observed between CHX1, CHX2 and CHX3, whereas all three experimental groups showed a statistically significant increase compared to CTRL. In particular, L*a*b* analysis showed a clear trend of staining in all three test groups (Fig 5). L* after day 7 tended to group into two different clusters, as CHX1 and CHX2 showed similar higher values than CHX3 and CTRL. At day 35, the values of the CHX groups were statistically significantly lower than CTRL (mean differences of 5.28 ± 2.31 [p = 0.023] for group CHX1 and 4.46 ± 2.09 [p = 0.034] for group CHX2). Similarly, these differences were noted between the mean CHX1 and CHX2 values compared to the CHX3 group (mean differences of 6.33 ± 2.02 [p = 0.002] for the CHX1 group and 5.52 ± 1.76 [p = 0.002] for the CHX2 group). A similar trend, but in the opposite direction, was noted for the chroma a*. Indeed, at day 35, values for CHX1 and CHX2 were significantly higher than for CTRL (average differences of 3.90 ± 1.14 [p = 0.001] for the CHX1 group and 2.91 ± 1.03 [p = 0.005] for the CHX2 group) and CHX3 groups (average differences of 3.69 ± 1.00 [p < 0.0001] for the CHX1 group and 2.70 ± 0.87 [p = 0.002] for the CHX2 group). Values of chroma b* at day 35 were statistically significantly lower for all three experimental groups, CHX1 (average difference of

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4.08 ± 1.89, p = 0.032), CHX2 (average difference of 4.17 ± 1.71, p = 0.015) and CHX3 (average difference of 3.52 ± 1.77, p = 0.043) compared CTRL. The differences in Spectr-L*a*b* had a pattern similar to that of the differences of Spectr-L* and Spectr-a* after 21 days between CHX1 and CHX2 on the one hand and CHX3 and CTRL on the other. Nonetheless, these differences were not statistically significant.

Side effects Side effects other than TD were rare. Tongue staining was noted in the following 5 subjects: 1 subject in CHX1 at 35 days; 2 subjects in CHX2, both at 21 days; and 2 subjects in CHX3 at 7 and 21 days. Taste alteration was only noted in 7 subjects: 2 subjects in CHX1, noted at 7 days; 4 subjects in CHX2 – 2 after 7 days and 2 after 21 days; and 1 subject in CHX3 complained of taste alteration after 35 days. One subject in CHX2 reported gastric acidity at day 7. No serious adverse events were reported.

DISCUSSION This trial was designed to assess the effect of various CHX formulations on dental plaque and gingival bleeding control and to measure the extent of TD after usage. Plaque control was definitely improved by using CHX. In particular, traditional CHX formulations appeared more effective than ADSenriched CHX. However, ADS-CHX achieved a higher control of gingival inflammation. TD was more

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CTRL CHX1 CHX2 CHX3

70

CTRL CHX1 CHX2 CHX3

12 10

65 a Total

L Total

8 6

60

4 2

0 Baseline

Day 7

Day 21

Day 35

A

0 Baseline

B

Day 7

Day 21

Fig 5  Variations of digital image analysis of L* (A) , a* (B) and *b (C) throughout the trial.

CTRL CHX1 CHX2 CHX3

24

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21 18 b Total

evident with the conventional CHX formulations, whereas ADS-CHX produced less intense staining, although the staining was distributed in the anterior teeth in the same manner as when conventional CHX products were used. CHX is extremely effective in plaque reduction and it is unequivocally considered the ‘gold standard’ of chemical plaque control (Jones, 2000). This clinical benchmark has been long established with the same alcohol-containing CHX that was used in this study. Controversial data are reported concerning the inclusion of alcohol in CHX solutions (Leyes Borrajo et al, 2002; Herrera et al, 2003). In this study, a similar clinical performance in plaque reduction was also noted for the tested alcohol-free CHX. This may have an important clinical impact as, despite the lack of epidemiological evidence of a higher risk for oral and pharyngeal cancer, some side effects such as mouth burning and drying of the mucosa may ultimately affect patient compliance (La Vecchia, 2009). Controversial results have been reported in terms of plaque control with CHX enriched with ADS. Trials measuring ADS-CHX efficacy vs CHX claimed equivalency in terms of plaque control in both healthy and periodontally affected subjects (Bernardi et al, 2004; Cortellini et al, 2008; Solis et al, 2011). Our results agree with the study of Arweiler et al (2006), showing in a 4-day plaque regrowth study that ADS rinsing did not achieve the same amount of plaque inhibition and bacterial reduction as the same alcohol-containing CHX used in our study. Speculation on these findings focuses on the ADS molecules: ascorbic acid and sodium metabisulfite. It is plausible that these compo-

Day 35

15 12

0

Baseline

Day 7

Day 21

Day 35

C

nents may interfere somewhat with CHX’s ability to selectively bind anionic charges such as the ones presented by oral bacteria and tissues (Arweiler et al, 2006). This may ultimately determine a lower efficacy of the CHX in terms of plaque control. On the other hand, it would seem logical to relate the efficacy of the rinse in terms of plaque control to gingivitis prevention. Plaque levels may also be variable, so that looking at the gingival response can provide useful information to the clinician. Interestingly, our data indicated that ADS yielded higher positive effects on bleeding reduction, as noted previously (Cortellini et al, 2008). The reason for the discrepancy between the lower anti-plaque effect and the same efficacy, if not better in terms of reduction of gingival inflammation, may only be speculated. One possible explanation is that ADSCHX may qualitatively alter dental plaque, acting more evidently on periopathogens. Thus, further microbiological research is needed for a better understanding of this phenomenon. Moreover, one

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component of ADS is ascorbic acid (vitamin C), the levels of which have been shown to influence gingival bleeding tendency (Leggott et al, 1986). Tooth discolouration is the most common side effect of CHX and therefore the main limitation of its routine usage. Our data indicated that after 35 days of rinsing, all three CHX groups showed appreciable TD in more than the 60% of the subjects. The percentage of subjects exhibiting tooth staining did not differ among CHX1, CHX2 and CHX3 at 35 days. This indicates that, in the long-term, TD is almost inevitable with CHX usage, even when ADS is added, as already noted in vitro (Addy et al, 2005). In terms of TD characteristics, brown staining especially in the interproximal areas was particularly evident using conventional CHX, whereas TD was less intense in the group using ADS. Indeed, both digital image and spectrophotometric analyses indicated that tooth colour in the ADS (CHX3) group was bright and with the same tonality of chroma along the green-red axis compared to teeth in the control group. Moreover, the extent of discolouration was definitely lower, as appreciable staining was noted only after 3 weeks of usage (Bernardi et al, 2004; Cortellini et al, 2008). Interestingly, the greater the tooth discolouration, the higher the efficacy in terms of plaque control. Indeed, over the last three decades, an extensive amount of evidence has clearly indicated that inhibition of staining is associated with loss of activity (Wade et al, 1994; Addy et al, 1995; Moran et al, 1995; Claydon et al, 1996). Our study supports these data as, irrespective of the type of CHX used, post-hoc stratification for staining showed that subjects presenting TD had significantly higher plaque control than subjects whose teeth were not stained. There are several possible explanations for this finding. Plaque inhibition is dependent upon absorption of CHX onto the tooth surface (Davies, 1970). Similarly, discolouration after CHX usage is related to the chemical interaction of the molecule on the tooth surface (Watts and Addy, 2001). Most probably, the absence of staining would indicate a lack of interaction between the CHX molecule and the tooth surface, with subsequent loss of efficacy. This lack of interaction may in turn be related either to some individual variations in the type of toothpaste used and the timing of rinsing after toothbrushing, or some unknown factors derived from diet and, obviously, loss of patient compliance (Watts and Addy, 2001; Kolahi and Solari, 2006). In our study, sodium lauryl sulfate-free toothpaste was prescribed; further, compliance with rinsing reached 90%,

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whereas discolouration was present in about 60% to 70% of the samples. It is likely that in the remaining 20% to 30% of participants without stained teeth, some unknown factors may have impaired the interaction between CHX and the tooth surface. The authors are aware of the intrinsic limitations of this study. First, the long-term nature of a mouthrinse study determined a significant loss of follow-up. This was evident in the control group, as subjects were most probably conscious of not being part of the active testing and therefore did not always consider the adherence to the study necessary after three weeks, as already noted in similar trial (Leyes Borrajo et al, 2002). Second, control of dietary intake of polyphenols was performed only for the classic chromogens, such as tea, coffee and red wine. Dietary habits ove a 35-day period may significantly vary and other substances may have enhanced CHX staining. Nevertheless, strict diet control during long-term clinical trials may be difficult to realise.

CONCLUSION CHX appears to be highly efficacious in terms of plaque reduction. Anti-plaque activity was noted irrespective of the presence of alcohol. Interestingly, traditional CHX formulations appeared less effective in gingival inflammation control. Moreover, the clinical efficacy was related to extrinsic tooth discolouration, showing a significant range of variability.

ACKNOWLEDGEMENTS Johnson & Johnson supported this study with a grant to purchase the Vita EasyShade spectrophotometer and to perform the data analysis (Dr. D’Aiuto). UCLH/UCL received a proportion of funding from the Department of Health’s NIHR Biomedical Research Centres funding scheme. Dr. Graziani has received lecture fees from Johnson & Johnson and from Curaden Healthcare.

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