Effects of 2 bracket and ligation types on plaque retention: A quantitative microbiologic analysis with real-time polymerase chain reaction

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 flora.

Methods

Twenty boys (mean age, 14.2 ± 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 T 2 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 significant 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 significant 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.

Orthodontic appliances have a negative impact on oral hygiene. Orthodontic bands, brackets, and archwires used during fixed orthodontic treatment impede oral hygiene procedures and cause the accumulation of microbial dental plaque by creating new retention areas. Microbial dental plaque is the main etiologic factor in the development of dental caries and periodontal diseases. Enamel demineralization occurs around the brackets because of a decrease in the pH level caused by increases in the number and volume of acid-producing bacteria, mainly Streptococcus mutans , Streptococcus sobrinus , and lactobacilli, and metabolization of sugars by these cariogenic bacteria. 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 fixed orthodontic treatment.

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. During fixed orthodontic treatment, gingival inflammation occurs, and the pathologic changes in patients treated with fixed orthodontic appliances have been reported as mostly gingivitis, gingival bleeding, gingival enlargement, and increased periodontal pocket depth. 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 patients. Many studies have evaluted the effects of fixed orthodontic appliances on dental plaque retention and microbial flora. However, few studies have evaluated the effect of the ligation method.

In previous studies, although various techniques have been used for the assessment of microbial flora, the microbiologic culture technique was the most widely used. 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 identification and detection of microorganisms; in this method, specific DNA fractions are used, and small numbers of pathogens can be detected in the sample.

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 flora 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 flora using real-time 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 fixed appliances in the orthodontic department of Selçuk University in Konya, Turkey. Their mean age was 14.2 ± 1.5 years (range, 11.0-16.7 years). This study was approved by the ethics committee of Selçuk 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 fixed 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 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 first group, bonding was performed with self-ligating 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-nickel-titanium archwire was used for the initial leveling. During the study period, no additional materials, such as chains, coil springs, or figure-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 lateral incisor were pooled; thus, the results were bracket specific, not site specific. The samples were immediately placed in sterile Eppendorf tubes (Greiner Bioone, Austria) containing 500 μL of a sterilized phosphate-buffered 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 manufacturer’s instructions.

The primers and probes used for the detection and quantification of the cariogenic microorganisms are shown in Table I . The fluorescent dyes at the 5′ and 3′ ends of the probe were FAM (6-carboxyfluorescein; reporter) and TAMRA (6-carboxytetramethylrhodamine; quencher), respectively. The species-specific probe and primer sets were designed based on the variable regions of the 16S ribosomal RNAs of S mutans , S sobrinus , L casei , and L acidophilus , 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 cross-hybridization with bacterial genes using a database similarity search program.

Table I
Primer and probe sequences for real-time PCR
Bacteria Primer- probe Sequence Replicated base pairs
S mutans Forward 5′-CCGGTGACGGCAAGCTAA-3′ 114
Reverse 5′-TCATGGAGGCGAGTTGCA-3′
Probe FAM-5′-CTCTGAAAGCCGATCTCAGTTCGGATTG-TAMRA-3′
S sobrinus Forward 5′-TTCAAAGCCAAGACCAAGGCTAGT-3′ 232
Reverse 5′-CCAGCCTGAGATTCAAGCTTGT-3′
Probe FAM-5′-CCTGCTCCAGCGACAAAGGCAGC-TAMRA-3′
L casei Forward 5′-CTATAAGTAAGCTTTGATCCGGAGATTT-3′ 132
Reverse 5′-CTTCCTGCCGGTACTGAGATGT-3′
Probe FAM-5′-ACAAGCTATGAATTCACTATGC-TAMRA-3′
L acidophilus Forward 5′-GAAAGAGCCCAAACCAAGTGATT-3′ 391
Reverse 5′-CTTCCCAGATAATTCAACTATCGCTTA-3′
Probe FAM-5′-TACCACTTTGCAGTCCTACA-TAMRA-3′

A quantitative assay was achieved by cloning plasmids containing the amplified 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 purified using a plasmid purification kit (Plasmid DNA Purification; Macherey-Nagel, Düren, Germany). Quantification of the target DNA was achieved with serial 10-fold dilutions from 10 x to 10 y of the plasmid copies from the previously quantified standards, specifically from 10 2 to 10 6 for L casei and L acidophilus , and from 10 3 to 10 6 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-μL reaction mixture containing 5 μL of template DNA, 4 μL 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 fluorescence 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 sufficient data. Descriptive statistics were calculated for each group. Statistically significant differences for the microbiologic and periodontal data between the groups were determined using the Mann-Whitney U test. The Wilcoxon signed rank test was used for the microbiologic data, and the Friedman test (Bonferroni adjustment, α = 0.017) was used for the periodontal data to determine the differences in the 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 T 2 ). The Pearson correlation test was performed to correlate the clinical and microbiologic parameters. The significance 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 significant 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 significant 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 significant ( P >0.05).

Table II
Means, standard deviations, and statistical comparisons of the bacterial counts (log 10)
Group n T0 T2 Significance between
Mean SD Mean SD T0-T2
S mutans Self-ligating 20 4.44 0.94 6.15 1.05 0.000
Steel ligature 20 4.55 1.21 6.39 1.00 0.000
S sobrinus Self-ligating 20 2.36 0.33 3.05 0.84 0.000
Steel ligature 20 2.45 0.36 3.34 0.67 0.000
L casei Self-ligating 20 2.99 0.83 4.45 1.56 0.000
Steel ligature 20 2.82 0.74 4.23 1.53 0.000
L acidophilus Self-ligating 20 2.10 0.88 3.09 1.03 0.000
Steel ligature 20 1.79 0.39 2.78 1.11 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
Microorganism n Self-ligating Steel ligature Intergroup comparison
Mean SD Minimum Maximum Mean SD Minimum Maximum P value
S mutans 20 1.72 1.15 −0.03 3.26 1.84 1.37 −0.06 4.19 0.787
S sobrinus 20 0.69 0.67 0.16 2.52 0.89 0.65 0.15 2.97 0.104
L casei 20 1.47 1.34 0.09 4.82 1.41 1.25 0.04 4.4 0.978
L acidophilus 20 0.99 0.56 0.18 1.88 0.99 1.01 0.17 4.24 0.386

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 significant increases in both groups, and these increases continued throughout the study ( P <0.001). An intergroup comparison of the difference in the 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 significant between the 2 groups ( P >0.05).

Table IV
Means, standard deviations, and statistical comparisons of the periodontal measurements
Group n T0 T1 T2 Significance between
Mean SD Mean SD Mean SD T0-T1 T1-T2 T0-T2
Plaque index Self-ligating 20 1.11 0.30 1.94 0.30 2.27 0.34 0.000 0.000 0.000
Steel ligature 20 1.11 0.25 2.09 0.31 2.48 0.26 0.000 0.000 0.000
Bleeding on probing Self-ligating 20 48.17 16.31 68.50 13.83 86.00 7.30 0.000 0.000 0.000
Steel ligature 20 51.83 14.37 68.33 14.24 89.83 7.05 0.000 0.000 0.000
Probing pocket depth Self-ligating 20 2.00 0.43 2.44 0.44 2.73 0.49 0.000 0.000 0.000
Steel ligature 20 2.02 0.37 2.40 0.51 2.71 0.48 0.000 0.001 0.000
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Apr 7, 2017 | Posted by in Orthodontics | Comments Off on Effects of 2 bracket and ligation types on plaque retention: A quantitative microbiologic analysis with real-time polymerase chain reaction
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