The effect of chlorhexidine on plaque index and mutans streptococci in orthodontic patients: A pilot study

doi:10.4236/ojst.2013.36054

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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

Email: *[email protected]

Received 17 February 2013; revised 18 March 2013; accepted 17 April 2013

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.

OPEN ACCESS

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

OPEN ACCESS

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

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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