ORIGINAL ARTICLE

Effects of 2 bracket and ligation types on plaque

retention: A quantitative microbiologic analysis
with real-time polymerase chain reaction
ge Baka,a Faruk Ayhan Basciftci,b and Ugur Arslanc
Zeliha Mu
Konya, Turkey

Introduction: The aim of this study was to evaluate the effects of self-ligating brackets and conventional
brackets ligated with stainless steel ligatures on dental plaque retention and microbial ora. Methods: Twenty
boys (mean age, 14.2 6 1.5 years) underwent bonding with self-ligating bracket systems and conventional
standard edgewise bracket systems ligated with stainless steel ligatures with a split-mouth design. Clinical
measurements, including plaque index, probing pocket depth, and bleeding on probing, were obtained before
bonding, 1 week after bonding, and 3 months after bonding. Supragingival plaque samples were obtained at
baseline and 3 months after bonding for the detection of bacteria. A quantitative analysis for Streptococcus
mutans, Streptococcus sobrinus, Lactobacillus casei, and Lactobacillus acidophilus was performed using
real-time polymerase chain reaction. The Mann-Whitney U test and the Hotelling T2 multivariate test were
used for statistical comparisons of the groups. Results: The numbers of S mutans, S sobrinus, L casei, and
L acidophilus were not statistically different between self-ligating brackets and conventional brackets ligated
with stainless steel ligatures (P .0.05). The 2 archwire ligation techniques showed no statistically signicant
differences in plaque index, bleeding on probing, and probing pocket depth values of the bonded teeth
(P .0.05). All clinical parameters and the numbers of all microorganisms showed statistically signicant
increases from baseline to 3 months after bonding in both groups (P \0.001). Conclusions: Self-ligating
brackets and conventional brackets ligated with stainless steel ligatures do not differ with regard to dental plaque
retention. (Am J Orthod Dentofacial Orthop 2013;144:260-7)

rthodontic appliances have a negative impact on

oral hygiene.1 Orthodontic bands, brackets, and
archwires used during xed orthodontic treatment impede oral hygiene procedures and cause the
accumulation of microbial dental plaque by creating
new retention areas.2,3 Microbial dental plaque is the
main etiologic factor in the development of dental
caries and periodontal diseases.4 Enamel demineralization occurs around the brackets because of a decrease
in the pH level caused by increases in the number and
From Selcuk University, Konya, Turkey.
a
Postgraduate student, Department of Orthodontics, Faculty of Dentistry.
b
Professor and chair, Department of Orthodontics, Faculty of Dentistry.
c
Associate professor, Department of Microbiology, Faculty of Medicine.
All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conicts of Interest, and none were reported.
Based on the thesis of Zeliha M
uge Baka for the PhD degree; supported by Selcuk
University Research Projects (11102023).

Reprint requests to: Zeliha M

uge Baka, Selcuk Universitesi,
Dishekimligi
Fak
ultesi, Ortodonti AD, Selcuklu-42079, Kamp
us/Konya, Turkey; e-mail,
mugen97@hotmail.com.
Submitted, January 2013; revised and accepted, March 2013.
0889-5406/$36.00
Copyright 2013 by the American Association of Orthodontists.
http://dx.doi.org/10.1016/j.ajodo.2013.03.022

260

volume of acid-producing bacteria, mainly Streptococcus mutans, Streptococcus sobrinus, and lactobacilli, and metabolization of sugars by these cariogenic
bacteria.5,6 Enamel demineralization, termed white
spot lesions, is a common side effect of orthodontic
treatment. White spot lesions can be seen in
approximately 50% of patients after xed orthodontic
treatment.7-9
Many studies have reported increases in the amounts
of cariogenic microorganisms, including S mutans and
lactobacilli, in the dental plaque and saliva of patients
after the bonding of orthodontic appliances.10-15
During xed orthodontic treatment, gingival
inammation occurs,1,16,17 and the pathologic changes
in patients treated with xed orthodontic appliances
have been reported as mostly gingivitis, gingival
bleeding, gingival enlargement, and increased
periodontal pocket depth.18 The ligation method of
the orthodontic archwires is an additional factor to be
taken into account for microbial dental plaque retention. Elastic and stainless steel ligatures are used to tie
stainless steel wires into the brackets and are often
linked to the risk of dental caries in orthodontic

Baka, Basciftci, and Arslan

patients.13 Many studies have evaluted the effects of

xed orthodontic appliances on dental plaque retention
and microbial ora.2,11,12,14,15,19-27 However, few
studies have evaluated the effect of the ligation
method.13,28-33
In previous studies, although various techniques have
been used for the assessment of microbial ora, the
microbiologic culture technique was the most widely
used.15,29,32,34-38 However, the laboratory procedures
for this technique can be faulty, time-consuming, and
laborious. Recently, to overcome these limitations, polymerase chain reaction (PCR) has been used. PCR is a simple, fast, and accurate method for the identication and
detection of microorganisms; in this method, specic
DNA fractions are used, and small numbers of pathogens
can be detected in the sample.39,40
Recently, the effects of self-ligating brackets on oral
hygiene have been investigated, and a few studies are
available on this topic. The hypothesis that we investigated was that self-ligating brackets have an advantage
in terms of the accumulation of plaque because of the
absence of ligatures. To our knowledge, no study has
compared the effects of self-ligation and stainless steel
wire ligation on dental plaque retention and microbial
ora with real-time PCR. Therefore, our aim was to evaluate the effects of self-ligating brackets and conventional brackets ligated with stainless steel ligatures on
dental plaque retention and microbial ora using realtime PCR and a split-mouth design.
MATERIAL AND METHODS

Twenty boys were randomly selected from patients

about to start orthodontic treatment with maxillary and
mandibular xed appliances in the orthodontic department of Selcuk University in Konya, Turkey. Their mean
age was 14.2 6 1.5 years (range, 11.0-16.7 years). This
study was approved by the ethics committee of Selcuk
University Meram Medical School (number 2011/233),
and written informed consent was obtained from the patients or their parents. We evaluated the clinical index examinations and the supragingival plaque samples from
these subjects at different times during the study. Inclusion criteria were minimal or moderate crowding, nonextraction xed orthodontic therapy, permanent dentition,
adequate oral hygiene, and use of the right hand while
brushing the teeth. Exclusion criteria were impacted or
missing teeth (except molars), systemic disease, and use
of antibiotics within the previous 3 months.
After the initial examination, all patients underwent
supragingival scaling and polishing and were given instructions on dental hygiene. They were instructed to
brush their teeth thrice a day. They were provided standardized toothpastes and toothbrushes and asked not to

261

use any other oral-care products during the study. Also,

they were asked to maintain their routine eating habits.
No additional information about oral hygiene was given
during the 3 months. Three weeks after the initial examinations, the patients were given appointments for the
sampling and bonding processes.
This investigation was designed as a split-mouth
study. The patients were randomly assigned to 2 groups:
in the rst group, bonding was performed with selfligating brackets (Damon Q; Ormco, Orange, Calif) in
the maxillary right and mandibular left dentitions and
conventional edgewise brackets (Roth-equilibrium 2,
722-341; Dentaurum, Pforzheim, Germany) in the
maxillary left and mandibular right dentitions. In the
second group, bonding was performed using conventional edgewise brackets in the maxillary right and
mandibular left dentitions and self-ligating brackets in
the maxillary left and mandibular right dentitions,
both with 0.022-in slots. The conventional edgewise
brackets were ligated with 0.010-in conventional stainless steel ligature wires. A 0.014-in copper-nickeltitanium archwire was used for the initial leveling.
During the study period, no additional materials, such
as chains, coil springs, or gure-8 ligatures, which could
have adversely affected oral hygiene, were used. Clinical
periodontal measurements were obtained before
bonding, 1 week after bonding, and 3 months after
bonding. Supragingival plaque samples were obtained
before bonding and 3 months after bonding.
Clinical periodontal measurements, including plaque
index, probing pocket depth, and bleeding on probing,
were obtained before bonding, 1 week after bonding,
and 3 months after bonding. Plaque index, probing
pocket depth, and bleeding on probing values were recorded for all bonded teeth, except for the molars, at 3
sites per tooth. The periodontal evaluation was carried
out by the same trained clinician (Z.M.B) using a periodontal probe (Hu-Friedy, Chicago, Ill).
Supragingival plaque samples were obtained before
bonding and 3 months after bonding. The microbiologic
samples were collected before the clinical periodontal
evaluation by the same clinician (Z.M.B.). The sampling
process was conducted in the morning, and the patients
were asked to abstain from eating or toothbrushing on
the days of their appointments. At each appointment,
the ligatures and archwires were carefully removed.
The sampling sites were isolated from water and saliva
with cotton rolls and gently air dried. Sterilized curettes
were used to obtain microbial samples from the labial
surfaces of the lateral incisors. The samples from the
maxillary right lateral incisor and the mandibular left
lateral incisor were pooled, and the samples from the
maxillary left lateral incisor and the mandibular right

American Journal of Orthodontics and Dentofacial Orthopedics

August 2013
Vol 144
Issue 2

Baka, Basciftci, and Arslan

262

Table I. Primer and probe sequences for real-time PCR

Bacteria
S mutans

S sobrinus

L casei

L acidophilus

Primer- probe
Forward
Reverse
Probe
Forward
Reverse
Probe
Forward
Reverse
Probe
Forward
Reverse
Probe

Sequence
5'-CCGGTGACGGCAAGCTAA-3'
5'-TCATGGAGGCGAGTTGCA-3'
FAM-5'-CTCTGAAAGCCGATCTCAGTTCGGATTG-TAMRA-3'
5'-TTCAAAGCCAAGACCAAGGCTAGT-3'
5'-CCAGCCTGAGATTCAAGCTTGT-3'
FAM-5'-CCTGCTCCAGCGACAAAGGCAGC-TAMRA-3'
5'-CTATAAGTAAGCTTTGATCCGGAGATTT-3'
5'-CTTCCTGCCGGTACTGAGATGT-3'
FAM-5'-ACAAGCTATGAATTCACTATGC-TAMRA-3'
5'-GAAAGAGCCCAAACCAAGTGATT-3'
5'-CTTCCCAGATAATTCAACTATCGCTTA-3'
FAM-5'-TACCACTTTGCAGTCCTACA-TAMRA-3'

lateral incisor were pooled; thus, the results were bracket

specic, not site specic. The samples were immediately
placed in sterile Eppendorf tubes (Greiner Bioone,
Austria) containing 500 mL of a sterilized phosphatebuffered saline solution and stored at 80 C for the
real-time PCR analysis.
Bacterial DNA was extracted from the supragingival
plaque samples using an extraction kit (DNeasy Blood
& Tissue kit; Qiagen, Hilden, Germany), according to
the manufacturers instructions.
The primers and probes used for the detection and
quantication of the cariogenic microorganisms are
shown in Table I. The uorescent dyes at the 50 and 30
ends of the probe were FAM (6-carboxyuorescein; reporter) and TAMRA (6-carboxytetramethylrhodamine;
quencher), respectively. The species-specic probe and
primer sets were designed based on the variable regions
of the 16S ribosomal RNAs of S mutans,41 S sobrinus,42
L casei,43 and L acidophilus,44 as previously described. A
universal bacterial primer pair was used to detect DNA
from all eubacterial species in the samples. All primers
and probes were checked for possible crosshybridization with bacterial genes using a database similarity search program.
A quantitative assay was achieved by cloning plasmids containing the amplied region of each target bacteria with cloning procedures (Topo-XL PCR Cloning;
Invitrogen, Carlsbad, Calif). Each PCR amplicon for S
mutans, S sobrinus, L casei, and L acidophilus was individually inserted into a separate plasmid vector; the recombinant vectors were transformed into One Shot
Chemically Competent Esherichia coli (Invitrogen).
The plasmids were puried using a plasmid purication
kit (Plasmid DNA Purication; Macherey-Nagel, D
uren,
Germany). Quantication of the target DNA was
achieved with serial 10-fold dilutions from 10x to 10y
of the plasmid copies from the previously quantied
standards, specically from 102 to 106 for L casei and

August 2013
Vol 144
Issue 2

Replicated base pairs

114

232

132

391

L acidophilus, and from 103 to 106 for S mutans and

S sobrinus. The plasmid standards and clinical samples
were run in duplicate, and average values were used
for calculating the bacterial loads.
Real-time PCR reactions were performed using
Lightcycler TaqMan master mix (Roche Applied Science,
Mannheim, Germany). The samples were assayed in
duplicate in a 20-mL reaction mixture containing 5 mL
of template DNA, 4 mL of master mix at 5 times concentration, 10 pmol of forward primer and reverse primer,
and 5 pmol of the probe (Synthesis Report; Metabion,
Martinsried, Germany). The cycling conditions used
were as follows: 95 C for 10 minutes, followed by 40
cycles at 95 C for 30 seconds, 60 C for 1 minute,
40 C for 40 seconds each, and extension at 72 C for
1 minute. The results were analyzed on the thermal
cycler instrument software (Light Cycler, version 1.2;
Roche Applied Science) by quantitatively analyzing the
uorescence emissions. All PCRs were performed in
duplicate.
Statistical analysis

The data were statistically analyzed using statistical

software (version 17.0; SPSS, Chicago, Ill). The log 10
transformation was applied to the microbiologic data
for normalizing the distribution and stabilizing the variance. The Shapiro-Wilks test for normality and the Levene test for variance homogeneity were applied to the
periodontal and microbiologic data. Nonparametric tests
were used because of the nonnormal distribution and
the lack of sufcient data. Descriptive statistics were
calculated for each group. Statistically signicant differences for the microbiologic and periodontal data
between the groups were determined using the MannWhitney U test. The Wilcoxon signed rank test was
used for the microbiologic data, and the Friedman test
(Bonferroni adjustment, a 5 0.017) was used for the
periodontal data to determine the differences in the

American Journal of Orthodontics and Dentofacial Orthopedics

Baka, Basciftci, and Arslan

263

Table II. Means, standard deviations, and statistical comparisons of the bacterial counts (log 10)
T0
Group
Self-ligating
Steel ligature
Self-ligating
Steel ligature
Self-ligating
Steel ligature
Self-ligating
Steel ligature

S mutans
S sobrinus
L casei
L acidophilus

n
20
20
20
20
20
20
20
20

Mean
4.44
4.55
2.36
2.45
2.99
2.82
2.10
1.79

T2
SD
0.94
1.21
0.33
0.36
0.83
0.74
0.88
0.39

Mean
6.15
6.39
3.05
3.34
4.45
4.23
3.09
2.78

Signicance between
SD
1.05
1.00
0.84
0.67
1.56
1.53
1.03
1.11

T0-T2
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000

T0, Before bonding; T2, 3 months after bonding.

Table III. Statistical comparisons of the differences in the bacterial counts (log 10) between groups
Self-ligating
Microorganism
S mutans
S sobrinus
L casei
L acidophilus

n
20
20
20
20

Mean
1.72
0.69
1.47
0.99

SD
1.15
0.67
1.34
0.56

Minimum
0.03
0.16
0.09
0.18

Steel ligature
Maximum
3.26
2.52
4.82
1.88

mean changes within each group. The total effects of the

periodontal or microbiologic data in each group and between the groups were determined with multivariate
analysis of variance (Hotelling T2). The Pearson correlation test was performed to correlate the clinical and
microbiologic parameters. The signicance for all statistical tests was predetermined at P \0.05.
RESULTS

The mean values of the bacterial counts and clinical

periodontal measurements before bonding were not statistically signicant between the 2 groups. The descriptive statistics and an intragroup comparison of the
bacterial counts for both groups are shown in Table II.
After the bonding of the orthodontic brackets, the
numbers of S mutans, S sobrinus, L casei, and L acidophilus showed statistically signicant increases in
both groups (P \0.001). An intergroup comparison of
the difference in the bacterial counts is shown in
Table III. The increases in the bacteria were similar in
both groups, and the differences were not statistically
signicant (P .0.05).
The descriptive statistics and an intragroup comparison of the periodontal measurements for both groups
are shown in Table IV. After the bonding of the orthodontic brackets, the initial plaque index, bleeding on
probing, and probing pocket depth values showed statistically signicant increases in both groups, and these increases continued throughout the study (P \0.001). An
intergroup comparison of the difference in the

Mean
1.84
0.89
1.41
0.99

SD
1.37
0.65
1.25
1.01

Intergroup comparison

Minimum
0.06
0.15
0.04
0.17

Maximum
4.19
2.97
4.4
4.24

P value
0.787
0.104
0.978
0.386

periodontal measurements is shown in Table V. The

increases in plaque index, bleeding on probing, and
probing pocket depth values were similar in both groups,
and the differences were not statistically signicant between the 2 groups (P .0.05).
When the total effects of the periodontal or microbiologic data within and between the groups were determined by multivariate analysis of variance, there were
no statistically signicant differences between the
groups (P .0.05). There were statistically signicant increases in the periodontal or microbiologic data within
the groups (P \0.001). There was a signicant positive
correlation between the plaque index and bleeding on
probing (r 5 0.527; P \0.001). The other correlations
were not statistically signicant.
DISCUSSION

The effects of xed orthodontic appliances on dental

plaque retention and microbial ora have been evaluated in many studies.2,11,12,14,15,19-27 However, the
effect of the ligation method on these factors has been
evaluated only in a few studies.13,28-33 These studies
have usually focused on elastomeric rings. Therefore,
in this study, we aimed to evaluate the effects of selfligating brackets and conventional brackets ligated
with stainless steel ligatures on the clinical and microbiologic parameters using real-time PCR and a splitmouth design.
The development of microbial dental plaque varies
among patients and is inuenced by dietary habits,

American Journal of Orthodontics and Dentofacial Orthopedics

August 2013
Vol 144
Issue 2

Baka, Basciftci, and Arslan

264

Table IV. Means, standard deviations, and statistical comparisons of the periodontal measurements
T0

Plaque index
Bleeding on probing
Probing pocket depth

Group
Self-ligating
Steel ligature
Self-ligating
Steel ligature
Self-ligating
Steel ligature

n
20
20
20
20
20
20

Mean
1.11
1.11
48.17
51.83
2.00
2.02

T1
SD
0.30
0.25
16.31
14.37
0.43
0.37

Mean
1.94
2.09
68.50
68.33
2.44
2.40

T2
SD
0.30
0.31
13.83
14.24
0.44
0.51

Mean
2.27
2.48
86.00
89.83
2.73
2.71

Signicance between
SD
0.34
0.26
7.30
7.05
0.49
0.48

T0-T1
0.000
0.000
0.000
0.000
0.000
0.000

T1-T2
0.000
0.000
0.000
0.000
0.000
0.001

T0-T2
0.000
0.000
0.000
0.000
0.000
0.000

T0, Before bonding; T1, 1 week after bonding; T2, 3 months after bonding.

Table V. Statistical comparison of the difference in the clinical periodontal measurements between groups
Self-ligating
Clinical periodontal index
Plaque index
Bleeding on probing
Probing pocket depth

n
20
20
20

Mean
1.16
37.84
0.72

SD
0.38
15.87
0.31

Minimum
0.43
0.00
0.06

Steel ligature
Maximum
2.03
60.00
1.23

Mean
1.37
38.00
0.68

SD
0.34
14.53
0.32

Minimum
0.76
13.33
0.07

Intergroup comparison
Maximum
1.96
63.33
1.20

P value (T0-T2)
0.091
0.871
0.882

T0, Before bonding; T2, 3 months after bonding.

age, oral hygiene, salivary factors, systemic disease, and

host factors. In orthodontic patients, the types of appliances and malpositioned teeth can also affect plaque
accumulation.13 In this study, variations in subject age
were minimized, and subjects of the same sex were
included, taking into account that toothbrushing habits
might change with age, and the effect of orthodontic
treatments on the gingiva can differ with age and sex.
In general, people tend to brush their contralateral sides
(the left side in right-handed people) more than their
ipsilateral sides.45 To standardize the effects of brushing,
only right-handed patients were included in our study.
The subjects were randomly assigned to 1 of 2 groups
to receive self-ligating brackets and conventional
brackets ligated with stainless steel ligatures in half of
each arch, either the right or left side. It is difcult to
achieve standardization in hygiene studies because it depends on many variables. Therefore, this was designed as
a split-mouth study to minimize the variations and intragroup differences.
The acid-producing bacteria around brackets, mainly
S mutans, S sobrinus, and lactobacilli, lead to enamel
demineralization and usually change the appearance of
enamel surfaces.5,6 Mizrahi46 reported that the highest
prevalence of enamel opacity was on the maxillary and
mandibular rst molars, maxillary lateral incisors, and
mandibular lateral incisors and canines. Gorelick et al7
found that the labiogingival area of the maxillary lateral
incisors had the highest incidence of white spot lesions.
This nding might be associated with the proximity of
the maxillary lateral incisor bracket to the gingiva. In

August 2013
Vol 144
Issue 2

this study, the lateral incisors were selected as the

plaque-sampling areas because they are the most
affected teeth and because of their esthetic importance
in the anterior dentition.
In a scanning electron microscopic study, Sukontapatipark et al28 evaluated the accumulation of bacterial
plaque adjacent to orthodontic brackets and reported
the formation of abundant plaque on the bonded teeth
within 1 week. Peros et al15 evaluated the salivary levels
of S mutans and lactobacillus species in 23 patients undergoing xed orthodontic treatment; the saliva samples
were obtained before placement of the appliances and at
weeks 6, 12, and 18. They reported signicant increases
in the salivary levels of S mutans and lactobacillus species, and a major peak at week 12 of the xed orthodontic treatment. Gorelick et al7 also reported that the
length of treatment had little impact on the incidence
of white spot lesions. Based on these studies, clinical
periodontal measurements were obtained before
bonding, 1 week after bonding, and 3 months after
bonding. The supragingival plaque samples were obtained before bonding and 3 months after bonding.
The ligation method of the orthodontic archwire is an
additional factor to be considered for retention of microbial dental plaque. Elastic and stainless steel ligatures,
used to tie the stainless steel wires to the brackets, are
often linked with the risk of dental caries in orthodontic
patients.13 Self-ligating brackets usually have hinged
covers and facilitate clinical study by eliminating the necessity of tying with elastic or stainless steel ligatures.22,23 Recently, a few authors have evaluated the

American Journal of Orthodontics and Dentofacial Orthopedics

Baka, Basciftci, and Arslan

effects of self-ligating brackets on oral hygiene. However, to our knowledge, no studies have compared the differences in retention of microbial dental plaque between
self-ligating brackets and stainless steel ligatures; it is
necessary to compare the 2 ligation systems, as
mentioned by Pellegrini et al.31
Forsberg et al13 evaluated microbial colonization in
12 patients undergoing xed orthodontic treatment
and found that the numbers of S mutans and lactobacilli
in the saliva increased signicantly after the insertion of
xed appliances, and that the maxillary lateral incisors
ligated to the archwires with elastomeric rings had
more bacteria in the plaque than did the incisors ligated
with steel ligatures. They recommended avoiding the use
of elastomeric rings in patients with poor oral hygiene
because elastomeric rings can signicantly increase the
microbial accumulation on the tooth surfaces adjacent
to the brackets, leading to a predisposition for the development of dental caries and gingivitis. In contrast,
Sukontapatipark et al28 used scanning electron microscopy to evaluate the effect of elastomeric rings and steel
ligatures on bacterial colonization at 1, 2, and 3 weeks
after bonding and reported that the ligation method
did not appear to inuence the bacterial morphotypes
on either the composite or the enamel surface. However,
an archwire was not used because the study design
included only 1 bonded tooth in each quadrant. Similarly, Turkkahraman et al29 reported that the differences
between elastomeric rings and steel ligatures were not
signicant and could be ignored. They also reported
that teeth ligated with elastomeric rings were more
prone to bleeding; therefore, elastomeric rings were
not recommended for patients with poor oral hygiene,
as stated by Forsberg et al.13 However, this experimental
design was different from that used by Forsberg et al. In
addition, Pellegrini et al31 compared self-ligating and
elastomeric orthodontic brackets in terms of plaque
retention using adenosine triphosphate-driven bioluminescence and reported that in most patients, teeth
bonded with self-ligating attachments had fewer bacteria in the plaque than did teeth bonded with elastomeric
brackets at 1 and 5 weeks after the bonding. In contrast,
Pandis et al32 investigated the effect of bracket type on
the levels of S mutans and total bacterial counts in
whole saliva of orthodontic patients 2 to 3 months after
appliance bonding and found no difference between
conventional brackets ligated with elastomeric rings
and self-ligating brackets. In our study, a steel ligature
was used instead of an elastomeric ring because an elastomeric ring has been clearly shown to cause more
plaque accumulation in several studies. In an in-vitro
study, Garcez et al33 evaluated biolm retention around
orthodontic brackets involved in the 3 ligation methods

265

using optical coherence tomography and microbiologic

sampling. They found that brackets ligated with elastomeric rings held more S mutans biolm than did the
other brackets, and that steel ligatures had less biolm
retention than did self-ligating brackets. In our study,
we found increases in the numbers of bacteria after
placement of orthodontic appliances, in accordance
with the ndings of previous studies, and these increases
were similar in both the self-ligating and the steel ligature groups. In contrast to the ndings of Garcez et al,
the differences between self-ligating brackets and steel
ligatures were not statistically signicant. The different
outcomes might be due to differences in study designs,
bracket types, and techniques used.
Several studies have reported increases in the
amounts of cariogenic microorganisms, including S mutans and lactobacilli, in the dental plaque and the saliva
of patients after the bonding of orthodontic appliances.10-15 S mutans and S sobrinus are the most
frequently isolated microorganisms from the human
oral cavity and are mainly responsible for dental
caries.47 We found a higher prevalence of S mutans
than of the other bacteria. The prevalence of S mutans
was about 2 times higher than that of S sobrinus, in
agreement with the ndings of Ahn et al.48 They evaluated S mutans and S sobrinus on incisor brackets by
PCR after debonding and reported that the prevalence
of S mutans was higher than that of S sobrinus on the
incisor brackets. Two species of lactobacilli, mainly L acidophilus and L casei, are often associated with dental
caries. Although the numbers of S sobrinus, L casei,
and L acidophilus were similar, the second most
frequently detected bacterium was L casei, the third
was S sobrinus, and the last was L acidophilus. The relatively high level of L casei can be explained because it is
the most common type of lactobacilli in caries lesions,
and orthodontic treatment might have increased the
colonization of lactobacilli after the 3-month period.
The bonding of the orthodontic brackets led to increases in plaque accumulation, bleeding on probing,
and probing pocket depth during the 3-month period.
The increases in the plaque index agreed with the
results of previous studies that xed orthodontic
appliances cause increases in the accumulation of
plaque.22-24,26,28,30 Huser at el22 evaluated the effect of
orthodontic band placement on the gingival tissues and
microbial composition of dental plaque before placement
and at 5, 7, 47, 72, and 90 days after placement of orthodontic appliances; they found that the plaque index and
bleeding on probing values were signicantly higher on
banded teeth than at the control sites, supporting our results. In this study, the probing pocket depth showed a
statistically signicant increase, in contrast to the ndings

American Journal of Orthodontics and Dentofacial Orthopedics

August 2013
Vol 144
Issue 2

Baka, Basciftci, and Arslan

266

of some studies that reported no signicant changes in

probing depth.22,26,27,29 Souza et al30 performed periodontal and microbiologic evaluations of the 2 ligation
methods and reported a signicant difference in probing
pocket depth 6 months after orthodontic appliance placement, in agreement with our ndings. However, the increases in the probing pocket depth were based on the
gingival enlargement seen during our clinical observations. The probing pocket depth values remained within
the normal values during the study. We also found a signicant increase in bleeding on probing. Increases in
bleeding on probing were also reported in several
studies.22,24-26,30 Turkkahraman et al29 found no signicant differences in bleeding on probing and plaque index
values between steel and elastic ligatures, and they reported that teeth ligated with elastomeric rings were
more prone to bleeding. In contrast, Souza et al found
that elastomeric rings were associated with higher scores
for bleeding on probing and plaque index than were steel
ligatures. In our study, no signicant differences were
found in clinical periodontal parameters between
self-ligating and steel ligatures, and all periodontal evaluations were carried out by the same trained clinician to
prevent interobserver differences. Our hypothesis that
self-ligating brackets have an advantage in terms of the
accumulation of plaque because of the absence of ligatures was not conrmed.
This study provides information on the retention of
dental plaque and the quantitative analyses of S mutans, S sobrinus, L casei, and L acidophilus between
self-ligating brackets and conventional brackets ligated
with stainless steel ligatures using real-time PCR. PCR
eliminates the difculties in the processes of bacterial
culture and can be used for detecting small numbers
of cariogenic bacteria in patients who are prone to developing enamel demineralization. Further studies
comparing different ages and sexes in larger samples
are required.
CONCLUSIONS

Considering the limitations of in-vivo studies, the

following conclusions can be made.
1.

2.

3.

Fixed orthodontic appliances signicantly increase

the colonization of S mutans, S sobrinus, L casei,
and L acidophilus. S mutans had a higher prevalence than did the other bacteria.
Plaque index, bleeding on probing, and probing
pocket depth values also signicantly increased during the rst 3 months of orthodontic treatment.
The results of this study indicate that self-ligating
brackets and conventional brackets ligated with
stainless steel ligatures exhibited similar changes

August 2013
Vol 144
Issue 2

4.

in the numbers of microorganisms and periodontal

parameters. The differences were not statistically
signicant.
Self-ligating brackets and conventional brackets
ligated with stainless steel ligatures do not differ
with regard to dental plaque retention.

REFERENCES
1. Zachrisson BU. Cause and prevention of injuries to teeth and supporting structures during orthodontic treatment. Am J Orthod
1976;69:285-300.
2. Balenseifen JW, Madonia JV. Study of dental plaque in orthodontic patients. J Dent Res 1970;49:320-4.
3. Wites M, Panuszka J, Dyras M. Evaluation of oral and orthodontic
appliance hygiene in orthodontically treated patients. Przegl Lek
2003;60:126-36.
4. Sanders NL. Evidence-based care in orthodontics and periodontics: a review of the literature. J Am Dent Assoc 1999;130:
521-7.
5. Mitchell L. Decalcication during orthodontic treatment with xed
appliancesan overview. Br J Orthod 1992;19:199-205.
6. Beyth N, Redlich M, Harari D, Friedman M, Steinberg D. Effect of
sustained-release chlorhexidine varnish on Streptococcus mutans
and Actinomyces viscosus in orthodontic patients. Am J Orthod
Dentofacial Orthop 2003;123:345-8.
7. Gorelick L, Geiger AM, Gwinnett AJ. Incidence of white spot
formation after bonding and banding. Am J Orthod 1982;81:
93-8.
8. 
Artun J, Brobakken B. Prevalence of caries and white spots after
orthodontic treatment with multibonded appliances. Eur J Orthod
1986;8:229-34.
9. Akin M, Basciftci FA. Can white spot lesions be treated effectively?
Angle Orthod 2012;82:770-5.
10. Bloom RH, Brown LR. A study of the effects of orthodontic appliances on the oral microbial ora. Oral Surg Oral Med Oral Pathol
1964;17:658-67.
11. Sakamaki ST, Bahn AN. Effect of orthodontic banding on localized
oral lactobacilli. J Dent Res 1968;47:275-9.
12. Corbett JA, Brown LR, Keene HJ, Horton IM. Comparison of Streptococcus mutans concentrations in non-banded and banded orthodontic patients. J Dent Res 1981;60:1936-42.
13. Forsberg CM, Brattstr
om V, Malmberg E, Nord CE. Ligature wires
and elastomeric rings: two methods of ligation, and their association with microbial colonization of Streptococcus mutans and lactobacilli. Eur J Orthod 1991;13:416-20.
14. Rosenbloom RG, Tinanoff N. Salivary Streptococcus mutans levels
in patients before, during, and after orthodontic treatment. Am J
Orthod Dentofacial Orthop 1991;100:35-7.
15. Peros K, Mestrovic S, Anic-Milosevic S, Slaj M. Salivary microbial
and nonmicrobial parameters in children with xed orthodontic
appliances. Angle Orthod 2011;81:901-6.
16. Zachrisson S, Zachrisson BU. Gingival condition associated with
orthodontic treatment. Angle Orthod 1972;42:26-34.
17. Kloehn JS, Pfeifer JS. The effect of orthodontic treatment on the
periodontium. Angle Orthod 1974;44:127-34.
18. Atack NE, Sandy J, Addy M. Periodontal and microbiological
changes associated with the placement of orthodontic appliances.
A review. J Periodontol 1996;67:78-85.
19. Scheie AA, Arneberg PAL, Krogstad O. Effect of orthodontic treatment on prevalence of Streptococcus mutans in plaque and saliva.
Scand J Dent Res 1984;92:211-7.

American Journal of Orthodontics and Dentofacial Orthopedics

Baka, Basciftci, and Arslan

20. Pender N. Aspects of oral health in orthodontic patients. Br J Orthod 1986;13:95-103.

21. Sinclair PM, Berry CW, Bennett CL, Israelson H. Changes in gingiva
and gingival ora with bonding and banding. Angle Orthod 1987;
57:271-8.
22. Huser MC, Baehni PC, Lang R. Effects of orthodontic bands on
microbiologic and clinical parameters. Am J Orthod Dentofacial
Orthop 1990;97:213-8.
23. Chang HS, Walsh LJ, Freer TJ. The effect of orthodontic treatment
on salivary ow, pH, buffer capacity, and levels of mutans streptococci and lactobacilli. Aust Orthod J 1999;15:229-34.
24. Paolantonio M, Festa F, di Placido G, D'Attilio M, Catamo G,
Piccolomini R. Site specic subgingival colonization by Actinobacillus actinomycetemcomitans in orthodontic patients. Am J Orthod Dentofacial Orthop 1999;115:423-8.
25. Glans R, Larsson E, Ogaard B. Longitudinal changes in gingival
condition in crowded and noncrowded dentitions subjected to
xed orthodontic treatment. Am J Orthod Dentofacial Orthop
2003;124:679-82.
26. Naranjo AA, Trivi~
no ML, Jaramillo A, Betancourth M, Botero JE.
Changes in the subgingival microbiota and periodontal parameters
before and 3 months after bracket placement. Am J Orthod Dentofacial Orthop 2006;130:275.e17-22.
27. Liu H, Sun J, Dong Y, Lu H, Zhou H, Hansen BF, et al. Periodontal
health and relative quantity of subgingival Porphyromonas gingivalis during orthodontic treatment. Angle Orthod 2011;81:
609-15.
28. Sukontapatipark W, el-Agroudi MA, Selliseth NJ, Thunold K,
Selvig KA. Bacterial colonization associated with xed orthodontic
appliances. A scanning electron microscopy study. Eur J Orthod
2001;23:475-84.
29. Turkkahraman H, Sayin MO, Bozkurt FY, Yetkin Z, Kaya S, Onal S.
Archwire ligation techniques, microbial colonization, and periodontal status in orthodontically treated patients. Angle Orthod
2005;75:231-6.
30. Souza RA, Magnani MBBA, Nouer DF, Silva CO, Klein MI,
Sallum EA, et al. Periodontal and microbiologic evaluation of 2
methods of archwire ligation: ligature wires and elastomeric rings.
Am J Orthod Dentofacial Orthop 2008;134:506-12.
31. Pellegrini P, Sauerwein R, Finlayson T, McLeod J, Covell DA Jr,
Maier T, et al. Plaque retention by self-ligating vs elastomeric orthodontic brackets: quantitative comparison of oral bacteria and
detection with adenosine triphosphate-driven bioluminescence.
Am J Orthod Dentofacial Orthop 2009;135:426-7.
32. Pandis N, Papaioannou W, Kontou E, Nakou M, Makou M,
Eliades T. Salivary Streptococcus mutans levels in patients with
conventional and self-ligating brackets. Eur J Orthod 2010;32:
94-9.
33. Garcez AS, Suzuki SS, Ribeiro MS, Mada EY, Freitas AZ, Suzuki H.
Biolm retention by 3 methods of ligation on orthodontic
brackets: a microbiologic and optical coherence tomography analysis. Am J Orthod Dentofacial Orthop 2011;140:193-8.

267

34. Br^etas SM, Macari S, Elias AM, Ito IY, Matsumoto MA. Effect of
0.4% stannous uoride gel on Streptococci mutans in relation to
elastomeric rings and steel ligatures in orthodontic patients. Am
J Orthod Dentofacial Orthop 2005;127:428-33.
35. Lessa FC, Enoki C, Ito IY, Faria G, Matsumoto MA, Nelson- Filho P.
In-vivo evaluation of the bacterial contamination and disinfection
of acrylic baseplates of removable orthodontic appliances. Am J
Orthod Dentofacial Orthop 2007;131:705.e11-7.
36. Lim BS, Lee SJ, Lee JW, Ahn SJ. Quantitative analysis of adhesion
of cariogenic streptococci to orthodontic raw materials. Am J Orthod Dentofacial Orthop 2008;133:882-8.
37. Magno AF, Enoki C, Ito IY, Matsumoto MA, Faria G, NelsonFilho P. In-vivo evaluation of the contamination of Super Slick
elastomeric rings by Streptococcus mutans in orthodontic patients. Am J Orthod Dentofacial Orthop 2008;133:104-9.
38. Pandis N, Vlachopoulos K, Polychronopoulou A, Madianos P,
Eliades T. Periodontal condition of the mandibular anterior dentition in patients with conventional and self-ligating brackets. Orthod Craniofac Res 2008;11:211-5.
39. Igarashi T, Yamamoto A, Goto N. Direct detection of Streptococcus
mutans in human dental plaque by polymerase chain reaction. Oral
Microbiol Immunol 1996;5:294-8.
40. Igarashi T, Yamamoto A, Goto N. PCR for detection and identication of Streptococcus sobrinus. J Med Microbiol 2000;49:
1069-74.
41. Childers NK, Osgood RC, Hsu KL, Manmontri C, Momeni SS,
Mahtani H, et al. Real-time quantitative polymerase chain reaction
for enumeration of Streptococcus mutans from oral samples. Eur J
Oral Sci 2011;119:447-54.
42. Choi EJ, Lee SH, Kim YJ. Quantitative real-time polymerase chain
reaction for Streptococcus mutans and Streptococcus sobrinus in
dental plaque samples and its association with early childhood
caries. Int J Paediatr Denti 2009;19:141-7.
43. Penders J, Thijs C, Mommers M, Stobberingh EE, Dompeling E,
Reijmerink NE, et al. Intestinal lactobacilli and the DC-SIGN
gene for their recognition by dendritic cells play a role in the
aetiology of allergic manifestations. Microbiology 2010;156:
3298-305.
44. Malinen E, Kassinen A, Rinttila T, Palva A. Comparison of real-time
PCR with SYBR Green I or 5'-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantication of selected faecal bacteria. Microbiology 2003;149:269-77.
45. Rugg-Gunn AJ, Macgregor ID. A survey of toothbrushing behaviour in children and young adults. J Periodontal Res 1978;13:
382-9.
46. Mizrahi E. Surface distribution of enamel opacities following orthodontic treatment. Am J Orthod 1983;84:323-31.
47. Hamada S, Slade HD. Biology, immunology, and cariogenicity of
Streptococcus mutans. Microbiol Rev 1980;44:331-84.
48. Ahn SJ, Lim BS, Lee SJ. Prevalence of cariogenic streptococci on
incisor brackets detected by polymerase chain reaction. Am J Orthod Dentofacial Orthop 2007;131:736-41.

American Journal of Orthodontics and Dentofacial Orthopedics

August 2013
Vol 144
Issue 2