Open Journal of Stomatology, 2013, 3, 323-328 OJST
http://dx.doi.org/10.4236/ojst.2013.36054 Published Online September 2013 (http://www.scirp.org/journal/ojst/)
The effect of chlorhexidine on plaque index and mutans
streptococci in orthodontic patients: A pilot study
Gisele Faria1, Milton Santamaria Jr.2, Bianca Mota dos Santos3, Izabel Yoko Ito4,
Janete Cinira Bregagnolo2, Maria Bernadete Sasso Stuani2*
1Department of Restorative Dentistry, School of Dentistry of Araraquara, University of the State of São Paulo (UNESP), Araraquara,
Brazil
2Department of Pediatric Clinics, Preventive and Social Dentistry, School of Dentistry of Ribeirao Preto, University of São Paulo
(USP), Ribeirão Preto, Brazil
3Private Practice, Aracaju, Brazil
4Department of Clinical Analysis, Toxicology and Bromatology, School of Pharmaceutical Sciences of Ribeirao Preto, University of
São Paulo (USP), Ribeirão Pret o, Brazil
Received 17 February 2013; revised 18 March 2013; accepted 17 April 2013
Copyright © 2013 Gisele Faria et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Aim: The purpose of this study was to assess chlor-
hexidine effects on plaque index and salivary levels of
mutans streptococci (MS) when used as the immer-
sion solution for removable orthodontic appliances
and added to their acrylic resin composition. Methods:
Forty-five patients (6 to 12 years old) were randomly
assigned into three groups with 15 patients each.
Group I (control)—without orthodontic appliances
disinfection; Group II—removable orthodontic ap-
pliances which had been immersed in 0.12% chlor-
hexidine digluconate overnight (8 hours), and Group
III—orthodontic appliances in which 0.12% chlor-
hexidine digluconate solution had been incorporated
into their resin composition. Saliva was collected for
quantification of MS and evaluation of plaque index
was performed before and after installation of ortho-
dontic appliance at 0, 2, 4, 6, 8, and 10 weeks. Data
were analyzed by using analysis of variance. Results:
Number of MS colonies in saliva and plaque index
showed no statistically differences among groups at
the different periods (p > 0.05). Conclusions: It could
be concluded that chlorhexidine incorporation into
the acrylic resin of removable orthodontic appliances
at 0.12% concentration and immersion of the appli-
ance into 0.12% chlorhexidine solution were not ef-
fective in reducing plaque index and the number of
MS in saliva.
Keywords: Chlorhexidine; Self-Polymerising Acrylic;
Removable Ort ho d ont i c Ap pl i ances; Mut ans
Streptococci; Plaque Index
1. INTRODUCTION
The insertion of orthodontic appliances results in a num-
ber of new retention sites which favor local adherence
and growth of mutans streptococci (MS) [1-3]. These
microorganisms can promote biofilm formation on bra-
ckets [4,5], orthodontic wires [6], elastics [7], and acrylic
resin [8-10]. Results of studies have shown that place-
ment of fixed orthodontic appliance can alter the oral
microbiota, increasing plaque volume and levels of MS
in saliva [2,5,11-13]. The increased levels of MS may
subsequently become a factor favouring the higher inci-
dence of enamel demineralization seen in some patients
with fixed orthodontic appliances [14]. This stresses the
need of preventive strategies during the treatment pe-
riod, for example, use of antimicrobial agents [1] to aid
in the control of bacterial colonization because tooth-
brushes cannot completely remove microorganisms from
critical retentive sites of the fixed orthodontic appli-
ances [6].
Some disinfectants such as cetylpyridinium chloride,
sodium hypochlorite, and chlorhexidine have been sug-
gested for disinfection of orthodontic appliances and
removable prosthesis [2,11,15,16]. The choice of disin-
fectant should be made with regard to its effectiveness in
inactivating microorganisms without any adverse effects
on the materials [17] and human tissues [18].
In the last few years, chlorhexidine has been one of the
most studied antimicrobial substances. It is considered
*Corresponding a uthor.

G. Faria et al. / Open Journal of Stomatology 3 (2013) 323-328 324
the best choice among antiseptics for dental biofilm con-
trol, being effective for prevention of dental caries, gin-
givitis, and stomatitis [2,19-21 ]. Moreover, chlorhexidine
gluconate has shown great efficacy in disinfection of
toothbrushes [5,22], removable prosthesis [16-18,23],
and removable orthodontic appliances [9]. However, no
studies have addressed the effect/impact of chlorhexidine
disinfection protocols for removable orthodontic appli-
ances on the oral microbiota.
The purpose of this study was to assess the effect of
chlorhexidine on plaque index and salivary levels of MS
when used as the immersion solution for removable or-
thodontic appliances and added to their acrylic resin
composition.
2. MATERIAL AND METHODS
This research project was approved by the local Research
Ethics Committee, and written informed cons ent was ob-
tained from parents or legal representatives. Fifteen pa-
tients of both genders aged from 6 to 12 years old, who
had been referred to Preventive Orthodontic Clin ic o f th e
Faculty of Dentistry of Ribeirão Preto, University of São
Paulo, for treatment with removable orthodontic appli-
ances, were enrolled in this study. Patients, who were
using antimicrobial mouthwashes, presented any sys-
temic disease, had used antibiotics within the previous 3
months and with untreated caries lesions were excluded
from the trial. By using a table of random numbers, the
children were assigned to one of the 3 groups:
Group I: fifteen patients that did not make disinfec-
tion of orthodontic appliances, thus representing con-
trols;
Group II: fifteen patients instructed to immerse their
appliances into 0.12% chlorhexidine gluconate solution
(Periogard, Colgate-Palmolive Ind. Brasileira, Osasco,
SP, Brazil) overnight (8 hours), with solution being re-
placed every three days.
Group III: fifteen patients wearing removable ortho-
dontic appliances made from acrylic resin with chlor-
hexidine gl uco nat e.
Orthodontic appliances were constructed on cast mod-
els from each patient by using Hawley arch in the ante-
rior region and drop-shaped hook in th e posterior region,
whereas baseplates were fabricated by using self-po-
lymerising acrylic resin (Orto Class, Clássico Artigos
Odontológicos Ltda, São Paulo, SP, Brazil) according to
manufacturer ’s recommendations regarding monomer-to-
polymer ratio, standard acrylisation, trimming, and fin-
ishing/polishing techniques. The Group III baseplates
were made with 14.4 g powder and 5.8 g liquid, includ-
ing addition of 0.12 ml of chlorhexidine digluconate so-
lution at concentration of 20% (Natural Pharma, São
João Clímaco, São Paulo, Brazil) to monomer, thus
yielding acrylic resin with final concentration of 0.12%
chlorhexidine. The amount of antimicrobial agent added
to the acrylic resin was determined on the basis of their
concentrations in mouth rinses largely used in dentistry
clinic, that is, 0.12% chlorhexidine digluconate [12,24].
The patients were requested to wear the orthodontic ap-
pliances full time, except during sleep and meals. The
children were instructed to brush their teeth using the
Bass technique 4 times a day, and the parents or guardi-
ans were instructed to brush the orthodontic appliances
once a day at bedtime by using the same toothbrush
(Classic Infantil, Colgate-Palmolive Ind. Brasileira,
Osasco, SP, Brazil) and fluoride-containing dental cream
(Colgate, Colgate-Palmolive Ind. Brasileira, Osasco, SP,
Brazil) received by each patient in the beginning of the
study.
Saliva collection and plaque index were evaluated on
6 occasions: before the placement of orthodontic appli-
ances and weeks 2, 4, 6, 8 and 10 following initial col-
lection. Two ml of non-stimulated saliva were collected
into properly labeled 15 × 100 mm sterilised tubes con-
taining 4 to 5 glass beads and then processed. Saliva
samples were vortexed during 2.5 minutes for dispersion
and then submitted to tenfold serial dilution . Next, 50 ml
of each dilution was plated equidistantly on modified
SB20 culture medium and incubated under the candle-jar
system at 37˚C for 2 to 3 days. The modified SB20 cul-
ture medium, which is selective for MS, was prepared.
Colonies/biofilms were evaluated by fermentation of
mannitol, sorbitol, raffinose, and mellibiose; hydrolysis
of arginine and sculin; production of hydrogen peroxide;
and sensitivity to 2.0 IU bacitracin [25,26].
The plaque index score was obtained after saliva col-
lection. Determination of plaque index and material col-
lection for microbiological examination were performed
at 9 - 10 a.m., that is, about 2 hours after breakfast.
S tatistical Analysis
The original data, measured as colony forming unit
(CFU), were transformed into log10 (CFU) for statistical
analysis and expressed as log CFU. The results were
analysed statistically by ANOVA and Tukey test by using
GraphPad Prism® statistical software (GraphPad Soft-
ware Inc., San Diego, CA, USA) at 5% significance
level.
3. RESULTS
Statistical analysis of the means of MS (log CFU) before
treatment showed that the 3 groups were similar (p >
0.05). Evaluation of the number of MS (log cfu) in the
saliva in the different phases of the study showed that
there were no significant differences between the group I
(control) and groups II and III (p > 0.05) or at the 6 col-
lection times (p > 0.05) in each group. In other words,
the quantity of MS (log CFU) in the saliva remains stable

G. Faria et al. / Open Journal of Stomatology 3 (2013) 323-328 325
during treatment in 3 groups and that the amout of MS in
the saliva at each collection time during treatment is
similar to quantity before treatment in 3 groups. The
means and standard deviation of mutans streptococci (log
CFU) in saliva at each collection time are shown in Ta-
ble 1.
Table 2 shows index plaque at each collection time
according to studied groups. Analysis of the plaque index
before orthodontic treatment by ANOVA showed that the
3 groups had similar mean values (p > 0.05). The plaque
index was found to be statistically smaller at weeks 6, 8,
and 10 in Group I (control) as well as at weeks 4, 6, 8,
and 10 in Groups II and III compared to those values
obtained before treatment. Although, the results showed
no evidence of statistical difference between the three
groups at each experimental period (p > 0.05), that is,
there was no dif fe rence between the three grou ps.
4. DISCUSSION
Chlorhexidine has been employed as spray [9,27], as im-
mersion solution [11,17,18,28], or added to acrylic resin
[29,30] for disinfection of prosthesis and removable or-
thodontic appliances. In our experiment, we have used
the immersion technique like others [31] and added
chlorhexidine into the composition of acrylic resin. The
statistical analysis showed that incorporation of chlor-
hexidine to acrylic resin of removable orthodontic appli-
ances in a final concentration of 0.12% or immersion
solution at 0.12% concentration for eight hours had no
effect on amount of MS in saliva and plaque index dur-
ing the orthodontic treatment. Studies of high and low
concentrations of CHX have been reported to reduce the
number of MS in plaque and saliva, and investigators
have concluded that the use of 0.12% CHX mouth rinses
could be beneficial for orthodontic patients in achieving
improved oral hygiene. Evidence of the efficacy of CHX
in biofilms was reported by Pratten et al. [32]. The lit-
erature suggests that the use of 1% CHX gel significantly
decreases MS levels [2,11,33]. Moreover, chlorhexidine
used as the immersion solution did not induce adverse ef-
fects on acrylic resin at the concentration of 2% for 10
minutes [11] or at 4% for 7 days [17].
Advantages in adding chlorhexidine into the composi-
tion of acrylic resin of orthodontic appliance are that
such approach does not depend on the patient’s co-op-
eration and is a method for extending the duration of
effective chlorhexidine therapy [34,35]. Chlorhexidine
diffuses out of acrylic resin in vivo at amounts sufficient
to prevent or cure palatal candidosis for up to three
weeks when mixed with acrylic p owder in the proportion
of 7.5% (w/w) [29].
Chlorhexidine can be added to acrylic resin as powder
[31,34] or liquid [30]. Samples of acrylic resin contain-
ing chlorhexidine at the concentration of 4% incorpo-
rated at liquid form released the substance over a period
of 72 days, with peak release observed in the first 20
days. Release from samples containing 1% and 2%
chlorhexidine was observed for 10 days and was much
lesser. The reason for this release pattern is probably due
to the fact that chlorhexidine, under these circumstances,
is a surface phenomenon [30]. Acrylic samples impreg-
nated with chlorhexid ine acetate at co ncentratio ns of 2%,
5% and 10% by mixing polymer powder released chlor-
hexi- dine into water for more than 100 days [31]. How-
ever, the physical properties of the resins were compro-
mised due to the presence of chlorhexidine particles,
Table 1. Means and standard deviations of mutans MS (log CFU) in saliva at each collection time according to studied groups.
Before tr eatm ent 2 weeks 4 weeks 6 weeks 8 weeks 10 weeks
Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD
Group I 5.672 0.697 5.760 0.630 5.482 1.012 5.433 1.027 5.776 0.735 5.954 0.703
Group II 5.007 1.226 5.300 2.066 5.302 1.705 5.381 1.717 5.494 1.003 5.440 1.040
Group III 5.753 1.296 5.024 2.970 5.690 1.940 4.282 2.839 4.559 1.345 5.572 1.365
SD = standa rd deviation.
Table 2. Means and standa rd de via t i on s of d e nta l plaque index values before and after installation of removable appliances according
to each studied groups.
Before treatment2 weeks 4 weeks 6 weeks 8 weeks 10 weeks
Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD
Group I 0.958 0.029 0.744 0.253 0.692 0.145 0.566*0.269 0.62* 0.225 0.63* 0.30
Group II 0.928 0.072 0.758 0.403 0.540*0.211 0.436*0.227 0.42* 0.234 0.45* 0.25
Group III 0.925 0.049 0.631 0.308 0.510*0.348 0.333*0.242 0.38* 0.259 0.32* 0.21
S
D = standard devia ti on; *Statistically significant d ifference in relation to values obtained before the treatment at p < 0.05.

G. Faria et al. / Open Journal of Stomatology 3 (2013) 323-328 326
which then dissolved in the water and created porosity in
the polymer matrix [31,34]. Therefore, in the present
study, we added chlorhexidine to acrylic resin through
the monomer to obtain samples containing the substance
at the concentration of 0.12%. Incorporation of chlor-
hexidine into the monomer caused no visible structural
changes in the acrylic surface, as the same finishing and
polishing characteristics seen in appliances made without
chlorhexidine were also observed.
Chlorhexidine should be used in small amounts to
avoid the adverse effect on the physical properties of
acrylic resin and deleterious effects on the user of ortho-
dontic appliance such as tooth stain, loss of taste, and
loss of appetite [29]. The amount of chlorhexidine added
to acrylic resin to obtain a final concentration of 0.12%
was determined based on concentrations in formulations
of mouth rinses commonly used in dentistry clinic [24,
36].
Rigour in controlling the levels of MS within the oral
cavity of orthodontic patients is crucial for keeping their
oral health, reducing the occurrence of caries lesions
However, the use of 0.12% chlorhexidine as immersion
solution and add to self polymerizing acrylic resin did
not change the levels of MS in saliva and the plaque in-
dex, thus suggesting that these protocols to disinfection
of removable orthodontic appliances at this concentration
have no benefit in reducing the risk of caries in ortho-
dontic patients.
Although the salivary levels of MS and the plaque in-
dex have no reduction through the use of chlorhexidine
in this pilot study, this does not mean that disinfection of
orthodontic appliances is not necessary. Controlling the
microbial contamination of removable orthodontic ap-
pliances by means of disinfection is of crucial impor-
tance to preserve the patient’s general health, since not
only healthy patients are treated in the dentistry office,
but also individuals with heart disease, diabetes mellitus,
immunodeficiency and other systemic changes. These
patients need ad ditional care as there is always the possi-
bility of occurrence of unwished bacteremia and infec-
tions. In addition, con trolling microbial contamination of
removable orthodontic appliances with strict disinfection
measures is important to prevent inter-patient microbi-
ological cross-contamination in the orthodontic setting
[9].
The lack of difference between control group and the
group that the chlorhexidine was added to acrylic resin
can be attributed not only to the chlorh exidine co ncen tra-
tion adopted in the present study, but also to the fact that
the use of removable appliances depended on the pa-
tient’s co-operation. On the other hand, for fixed ortho-
dontic appliances whose structure contains acrylic resin
(e.g. Hass expander), more effective microbial control
and lower plaque index could be achieved.
Control of microorganisms on acrylic surface through
immersion in disinfection solutions is well demonstrated
in the literature, but incorporation of chlorhexidine into
acrylic resin and its release into oral milieu require fur-
ther investigation to verify the effectiveness of this me-
thod. In this way, it is necessary to perform both in vitro
and in vivo studies using different concentrations of
chlorhexidine added to acrylic resin in order to assess its
effective antimicrobial action. A follow-up study with a
larger study population is warranted to confirm the re-
sults of the present study and further studies with other
antiseptic substances and methods of disinfection are
also required.
Based on the results of this pilot study, it was con-
cluded that incorporation of chlorhexidine into acrylic
resin of removable orthodontic appliances at final con-
centration of 0.12% or immers ion of such appliances into
0.12% chlorhexidine solution for eight hours was not
effective in decreasing plaque index and amount of MS
in saliva.
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