European Journal of Orthodontics, 2017, 1–10

doi:10.1093/ejo/cjx096

Randomized controlled trial

Clinical and microbiological effects of the use

of a cetylpyridinium chloride dentifrice and
mouth rinse in orthodontic patients: a 3-month
randomized clinical trial
David Herrera1,2, Nayra Escudero1, Leire Pérez1, María Otheo3,
Elena Cañete-Sánchez3, Tania Pérez3, Bettina Alonso1,2, Jorge Serrano1,2,
Juan Carlos Palma3, Mariano Sanz1,2 and Conchita Martín2,3
1
Section of Graduate Periodontology, Faculty of Odontology, Complutense University, Madrid, Spain, 2Etiology and
Therapy of Periodontal Diseases (ETEP) Research Group, Complutense University, Madrid, Spain, and 3Section of
Orthodontics, Complutense University, Madrid, Spain

Correspondence to: Conchita Martin, Departamento de Estomatología IV, Facultad de Odontología, Plaza Ramón y Cajal s/n,
Ciudad Universitaria, 28040 Madrid, Spain. E-mail: conchitamartin@odon.ucm.es

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.

© The Author(s) 2017. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved.
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 2 European Journal of Orthodontics, 2017

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: Local internal reference: P07/133.

Introduction Recently, a new CPC-based mouth rinse and toothpaste were

specifically formulated and marketed for orthodontic patients
The control of supragingival biofilm by means of mechanical
(VITIS Orthodontic, Dentaid, Cerdanyola, Spain). This formula-
devices, such as toothbrushes, dental floss, or interdental brushes, is
tion includes other active ingredients, such as allantoin, aloe vera,
a key element in the prevention of caries and periodontal diseases.
and sodium fluoride. Allantoin is widely used in dermatology since
The presence of orthodontic fixed appliances, however, makes this
it promotes cell proliferation and stimulates epithelisation (31). In
mechanical control more difficult, facilitating plaque retention, gin-
dentistry, it has been added in formulations for the treatment of aph-
givitis (1, 2), and initial caries or white spots lesions (3). Enhanced
thous stomatitis (14). Similarly, aloe vera has been added in formu-
plaque accumulation may also be associated with an increase in the
lations for the treatment of oral mucositis in cancer patients (32)
colonization of periodontal pathogens, such as Prevotella intermedia
and for aphthous stomatitis (14). This new formulation, aimed to
(2, 4, 5, 6). Moreover, many orthodontic patients, especially children
be used by orthodontic patients, has not been evaluated in home-
and adolescents, fail to floss because they find this procedure time-
use randomised clinical trials. It was, therefore, the objective of this
consuming and tedious in the presence of orthodontic arch wires
investigation to assess the clinical and microbiological efficacy of the
(7). Studies assessing compliance in these patients have reported that
combined use of a CPC-based toothpaste and mouth rinse formula-
their level of cooperation varies considerably depending on their age
tions, adjunctive to mechanical plaque control in patients wearing
and gender, personality, perception of the malocclusion, parental
fixed orthodontic appliances. In addition, compliance and the occur-
influence, and socioeconomic factors (8). As a result of these facts,
rence of adverse effects were evaluated.
adolescents or young adults wearing fixed orthodontic appliances
tend to accumulate more plaque and hence, they are at a higher risk
of developing gingivitis. Subjects and methods/methods
A common strategy to improve mechanical plaque removal in
Patient population
these patients is the addition of a chemical antimicrobial agent with
A consecutive sample of subjects undergoing treatment with fixed
anti-plaque or anti-caries activity in dentifrices, mouth rinses, or
orthodontic appliances was screened during 12  months at the
both (9). Ideally, these agents should have antimicrobial activity, for
Department of Orthodontics in the Faculty of Odontology of the
reducing plaque accumulation and gingival inflammation (10); anti-
authors’ institution. The recruitment period for this study was from
caries activity for decreasing the decalcifications usually occurring
September 2013 to September 2014. During the screening visit,
during orthodontic treatment and thus preventing the development
subjects were examined for fulfilment of the inclusion criteria and
of caries or white spots (11–13) and anti-inflammatory activity to
received a comprehensive explanation on the aims of the investiga-
help in the healing of traumatic mucosal injuries caused by the appli-
tion. They were then asked to participate by signing an informed
ances (14). Since these products are aimed for long-term use compli-
consent previously approved by the Ethical Review Committee
ance is a critical factor and therefore, they should have a pleasant
(Comité ético de Investigación Clínica, Hospital Clínico San Carlos.
flavour and lack of side effects, such as staining or de-bonding of the
Internal reference: P07/133. Date of approval: May 2007).
braces and bands.
Study participants were selected according to the following inclu-
The effectiveness of different active ingredients, such as chlo-
sion criteria:
rhexidine (CHX) (15–17), essential oils (18), amine/stannous fluo-
ride (19), or sanguinarine (20) in the form of mouth rinses, tooth • Healthy subjects between 12 and 25 years old. Participants were
pastes, or gels, has been evaluated in clinical studies. Most of these excluded in the presence of systemic diseases that might interfere
clinical studies have reported significant benefits in the adjunctive with the study or if in need of chronic analgesic or anti-inflam-
use of these products, although the magnitude of these reported ben- matory medication.
efits might not have a clear clinical relevance. In addition, the use of • Undergoing orthodontic therapy with fixed appliances both in
some of the formulations was associated with adverse effects (such the upper and lower arch with monthly follow-up appointments,
as staining with the use of CHX). • Clinical evidence of gingivitis (visually at screening, then con-
In recent years, the use of the chemical antimicrobial agent firmed with the Gingival Index).
cetylpyridinium chloride (CPC) has attracted some attention, due
to the advent of formulations with improved bioavailability or Participants were excluded in presence of periodontitis (clinical
increased concentration, which have shown increased clinical ben- attachment loss > 4 mm) or gingival overgrowth (pseudopockets >
efits and since their adverse effects are very limited (tooth staining, 4 mm). Subjects were also excluded if they showed evidence of den-
ulcers, gingival irritation), its use may be indicated for longer peri- tal negligence.
ods (21,22). CPC has demonstrated in a number of clinical trials to
reduce plaque formation (23–26) and gingivitis (24, 26–29). When Study design
used as a mouth rinse, a systematic review has shown heterogeneous The study was designed as a randomized, parallel, triple-blind,
results (30), although a significant effect on plaque has been dem- 3-month clinical trial. Participants, examiners, and those assessing
onstrated at different concentrations, such as 0.07 or 0.05 per cent the outcomes were blinded to group assignment. The following visits
(21,22,28). were scheduled:

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 D. Herrera et al.
• 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 par-
ticipant 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 denti-
frice, together with a standard toothbrush (VITIS Orthodontic,
Dentaid, Cerdanyola, Spain) and dental floss (VITIS non-wax
dental floss, Dentaid, Cerdanyola, Spain). Products were pur-
posely prepared in white opaque bottles coded as A or B. Patients
were requested to return the unused mouthrinse at the follow-
ing 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 super-
visor 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 car-
ried out and oral hygiene instructions were reinforced.
Treatment groups
In the test group, subjects used the experimental mouthwash
(VITIS Orthodontic®, Dentaid, Cerdanyola, Spain) contain-
ing 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 al-
lantoin). 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.
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3
– Plaque index (PlI) was evaluated using the Turesky modifica-
tion (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 cal-
culus 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 buc-
cal 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, in-
dependent and calibrated judges evaluated the photographs using
a standard template of different tooth staining degrees, as previ-
ously 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 (stain-
ing 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 supragingi-
val 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 sec-
onds 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 vor-
tex vibration for 30 seconds (39) and sequentially diluted in phos-
phate 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 assess-
ment of the colony morphology and confirmed by the application of
biochemical standard tests. In addition to the conventional evalua-
tion 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
 4 European Journal of Orthodontics, 2017
and the presence, counts, and proportions of different bacterial spe-
cies, 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 question-
naire used was custom-made for this investigation, including follow-
ing questions, with three/four closed answers available:
1. ‘How do you feel rinsing with this mouth rinse’ (answers: great;
good; unpleasant; very unpleasant)
2. ‘The taste of this mouth rinse is…’ (answers: very good; good; fair;
bad)
3. ‘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)
4. ‘Have you used any mouth rinse before’ (answers: yes, almost
every day; yes, sometimes; yes, but very rarely; no)
5. ‘How do you feel your teeth after using this mouth rinse’ (answers:
cleaner and smoother; rough and unpolished; I  don’t feel any
change)
6. ‘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 infor-
mation 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 meas-
ured compliance was expressed by the number of patients that ful-
filled with the study protocol at each visit and by the times that
a patient failed to use the mouth rinse, according to the daily dia-
ries. 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 statis-
tical purposes, and in order to avoid adjusting for multiplicity ana-
lysis, 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 ne-
cessary, 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 cali-
brated 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 re-
cording. Intraclass  correlation coefficient (ICC) of total agree-
ment was used to test the intra-examiner reliability for PlI and GI.
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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 evalu-
ated 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 base-
line 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 differ-
ences 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 com-
pare 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).
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, respect-
ively. 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 add-
itional patient missed the last visit due to an excessive delay in the
appointment.
 D. Herrera et al. 5

Plaque index Gingival index
The baseline plaque levels were similar in both treatment groups. Higher GI baseline levels were observed in the placebo group (1.07)
After 1  month, the mean PlI increased in the placebo group compared to the test group (0.92), although the differences were not
(0.16), while it decreased in the test group (0.08). This increase significant (P = 0.085). After 1 month, GI increased in the placebo
was statistically significant in the placebo group in the upper group (mean  =  0.05; SD  =  0.33), and decreased in the test group
jaw (P  =  0.032). Differences between groups in changes from (mean  =  −0.03; SD  =  0.38), with significant differences at all sites
baseline to 1  month were statistically significant (P  =  0.024) (P = 0.025) and lower jaw (P = 0.007). At the 2-month visit, no inter-
only in the maxilla (mean: 0.18, SD = 0.82 versus mean: −0.26, group differences were detected, and both groups demonstrated an
SD  =  0.62). Plaque increased after 2  months in both groups, increase in GI. After 3 months, minor and comparable increases were
being statistically significant in the placebo group for all sites observed in both groups, although a significantly higher level of gin-
(P  =  0.031) and in the upper jaw (P  =  0.033). At 3  months, givitis was observed in the placebo group (mean = 0.05; SD = 0.43
plaque levels were also higher when compared with baseline in versus Mean = 0.02; SD = 0.34) for the full-mouth index (P = 0.050)
both groups, and the increase was statistically significant in the and lower jaw (P = 0.020) (Table 3).
placebo group for all sites (P  =  0.028) and in the upper jaw
(P = 0.004). No significant inter group differences were detected Calculus index
(Table 2). Baseline calculus levels were similar and low in both groups. A slow
increase was observed throughout the study, reaching similar results
Table 1.  Demographic data of the patients at baseline. in both groups at 3  months. The change between baseline and
3  months was statistically significant (P  =  0.041) only in the test
Placebo group Test group
group. None of the intergroup comparisons were significant.
N 31 (49.2%) 32 (50.8%)
n males 10 (15.9%) 15 (23.8%) Tooth staining
n females 21 (33.3%) 17 (27%)
Intensity of staining
Age (years) 15.2* (2.1) 15.0* (1.8)
As seen in Table 4, it is clear that the scores of intensity in the upper
Plaque index (mean, SD) 2.59* (0.88) 2.57* (0.88)
Gingival index (mean, SD) 1.07* (0.36) 0.92* (0.31) jaw were low in both groups and showed only minor changes during
Bleeding on probing (mean, SD) 0.79* (0.23) 0.78* (0.25) the study, although somehow higher in the placebo group. No statis-
Probing pocket depth (mm) 3.09* (0.39) 3.04* (0.46) tically significant differences were detected. The intensity of staining
Recessions (mm) 0* (0) 0* (0) in the lower jaw was higher than in the upper jaw, and slightly higher
Staining intensity upper 0.35* (0.73) 0.08* (0.29) in the test group at baseline (Table 4). Minor changes were observed
Staining intensity lower 0.85* (1.99) 1.25* (2.18) 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
*Data are means (SD) or numbers (%). comparisons were statistically significant.

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

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 6 European Journal of Orthodontics, 2017

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

Table 4.  Mean values and standard deviation (SD) in staining (surface, intensity), as evaluated by two researchers.

Upper jaw Lower jaw

Placebo Test Placebo Test

Surface 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

Upper jaw Lower jaw

Placebo Test Placebo Test

Intensity 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.

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,

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 D. Herrera et al. 7

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 con-
trol group; and 0.11, 0.25, 0.25, and 0.21, respectively, for the experi-
mental 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, allow-
ing 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 sup-
porting 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 experi-
mental 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 ex-
perimental mouth rinse higher than the control mouth rinse.
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 ex-
perimental patients thought that the taste was ‘very good’ (related to
Figure  1.  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.
the total of patients in each group). After noticing the significant dif-
ferences, the categorical variable was transformed into a semi-quan-
titative 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 pla-
cebo 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 per-
centage 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

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 8 European Journal of Orthodontics, 2017
between groups at each visit did not show statistically significant dif-
ferences, 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 den-
tifrice 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 vari-
ables 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 signifi-
cant. 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 find-
ing 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 sub-
jects from the control group when they were provided with oral hy-
giene 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 mag-
nitude 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 stan-
nous fluoride (19). It has also been suggested that effect of essential
oils on gingivitis is more pronounced than on plaque formation due
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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 sen-
sation. A recent systematic review on the effectiveness of aloe vera
in the management of oral mucosal diseases did not find any ad-
verse 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 signifi-
cant. 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 microbio-
logical results did not find significant differences, but provided evi-
dence on the safety of the evaluated products in terms of absence of
microbiological adverse effects (namely, the overgrowth of oppor-
tunistic 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 con-
trol. 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 investiga-
tion, 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 compli-
ance 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 motiv-
ation 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
 D. Herrera et al.
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 peri-
odic 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 moti-
vated 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 mag-
nitude. 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 differ-
ences 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 undergo-
ing 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 con-
trolling 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 sta-
tistical 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.
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