Introduction
The aims of this study were to evaluate the effect of a self-etching adhesive system containing an antibacterial monomer on periodontal health and subgingival microbiologic composition in orthodontic patients and to compare it with a conventional adhesive system.
Methods
A split-mouth design was chosen, and 15 patients were included in the study. Brackets in contralateral quadrants were bonded with either a conventional adhesive system (control) or a self-etching adhesive system that contained an antibacterial monomer. Clinical periodontal parameters including plaque index, gingival index, probing depths, and bleeding on probing were determined. Subgingival plaque samples were collected before bracket placement (T0) and at the 6-month follow-up (T1). The real-time TaqMan polymerase chain reaction assay was used to determine the subgingival counts of Porphyromonas gingivalis , Tannerella forsythensis , Prevotella intermedia , Aggregatibacter actinomycetemcomitans , Fusobacterium nucleatum , and Campylobacter rectus . For clinical periodontal parameters, analysis of covariance (ANCOVA) and, for bacterial counts, Wilcoxon tests were used for statistical comparisons at the P <0.05 level.
Results
Clinical periodontal parameters were not changed, and they were not different between the groups from T0 to T1. T forsythensis and F nucleatum increased during the treatment period in both groups ( P <0.05). The majority of the bacteria were T nucleatum at T0 and T1 in both groups. Changes in bacterial load from T0 to T1 were not different between groups except for T forsythensis and F nucleatum ( P <0.05).
Conclusions
The use of an antibacterial monomer did not have an additional positive effect on clinical periodontal parameters. When used in bonding orthodontic brackets, the antibacterial monomer failed to reduce periodontopathogenic bacteria when compared with the conventional adhesive system during a 6-month treatment period.
Different types of adverse reactions to orthodontic treatment have been described. White spot lesions, caries, and periodontal diseases can occur. Microbial dental plaque is reported to be the most important etiologic factor in periodontal disease progression. Poor oral hygiene in conjunction with orthodontic therapy could result in undesirable effects on the periodontium. The numbers of microorganisms are significantly increased by fixed appliances during orthodontic treatment. Bacterial plaque per se, regardless of the specific bacteria, was associated with disease. Also, periodontal disease results from the action of at least 1 specific pathogenic species and is often associated with a relative increase in the numbers of these organisms found in plaque. Porphyromonas gingivalis , Tannerella forsythensis , Prevotella intermedia , Aggregatibacter actinomycetemcomitans , Fusobacterium nucleatum , and Campylobacter rectus are peridontopathogenic microbiota predominantly found in subjects with periodontal disease.
In the attempt to control bacterial plaque around the fixed appliances, a constant level of fluoride was tried to be maintained in the oral cavity followed by oral hygiene instructions and dietary control. Also, combined use of antimicrobials and agents containing fluoride has been suggested for plaque control.
A self-etching adhesive system containing an antibacterial monomer,12-methacryloxy dodecylprydinium bromide, has antibacterial activity against oral streptococci, and this monomer can be active even after being immobilized as 1 component of a cured composite. Based on the results obtained for this material, a new single-bottle primer containing 5% of this antibacterial monomer was developed, and this 2-step mild self-etching and fluoride-releasing adhesive system with this primer was marketed.
The bonding ability of the self-etching adhesive system containing an antibacterial monomer has been evaluated in vivo, and its antibacterial effects have been demonstrated by in-vitro studies. Although its antibacterial effects against cariogenic bacteria are well documented, no study has investigated the efficacy of this material on periodontopathogenic microbial composition around the teeth with orthodontic brackets.
The aims of this study were to evaluate the effect of a self-etching adhesive system containing an antibacterial monomer on periodontal health and subgingival microbiologic composition in orthodontic patients and to compare it with a conventional adhesive system. Thus, the null hypothesis of this study was that there were no statistically significant differences among periodontal indexes and subgingival microbiologic composition of teeth bonded with either the self-etching adhesive system containing an antibacterial monomer or the conventional adhesive system.
Material and methods
This study was approved by the ethical committee on research of the Gulhane Military Medical Academy in Ankara, Turkey.
Fifteen randomly selected orthodontic patients (7 boys, 8 girls; mean age, 14.40 ± 0.71 years) participated in the study. All participants required fixed orthodontic treatment for their misaligned teeth. They were in good health; none had taken antibiotics for 3 months before the baseline or during treatment, and none had clinical signs of gingival inflammation. Patients who had undergone periodontal treatment within 3 months before the baseline and those with a systemic disorder that could influence periodontal and microbiologic conditions or the response to treatment were excluded. All subjects provided written informed consent.
This study was organized with a split-mouth design with contralateral antagonistic quadrants receiving the experimental material and the control. Stainless steel orthodontic brackets (3M Unitek, Monrovia, Calif) were bonded by the same operator (M.A.) in the first quadrant, and the second quadrant was the contralateral side.
In the control group, teeth in contralateral quadrants were etched for 15 seconds with 37% ortho-phosphoric acid (3M Dental Products, St Paul, Minn), rinsed with water from a 3-in-1 syringe for 15 seconds, and dried with an oil-free source for 15 seconds. Before bracket placement, Transbond XT primer (3M Unitek) was applied to the etched surface in a thin, uniform coat. The primer was cured for 10 seconds with a light-emitting diode unit (Ortholux XT, 3M Unitek). Adhesive paste (Transbond XT, 3M Unitek) was applied to the bracket base, and the bracket was positioned on the facial surface and pressed firmly into place. The excess adhesive around the bracket was carefully removed with a scaler.
In the experimental group, teeth in the remaining quadrants were etched similarly to the control group for 15 seconds. First, Clearfil Protect Bond self-etch primer (Kuraray Medical, Okayama, Japan) was applied to the etched surface for 20 seconds and sprayed with a mild airstream to evaporate the solvent. Then Clearfil Protect Bond was applied, gently air dried, and light cured for 10 seconds. Finally, a thin layer of Transbond XT adhesive paste was applied to the base of the bracket and immediately pressed into the adhesive on the tooth surface.
Seven days before the baseline examinations, the subjects had oral hygiene instructions. During the treatment, the subjects did not receive prophylaxis procedures. A full periodontal examination, including plaque index, gingival index, probing depth, and bleeding on probing, was performed before bonding (T0) and 6 months after the beginning of orthodontic treatment (T1). Probing depth and bleeding on probing were obtained at 6 sites per tooth; the plaque and the gingival indexes were determined for the labial and lingual sites separately. Periodontal evaluation was carried out by the same trained clinician (S.E.) in all patients using a marked periodontal probe (Hu-Friedy, Chicago, Ill). This clinician was blinded to the group allocations. Probing depth was measured to the nearest millimeter on the scale.
In every recording, microbiologic samples were collected before the clinical procedures by the same clinician (S.E.). Subgingival plaque samples were collected from the mesiobuccal gingival crevices of the second premolars and the lateral incisors in each quadrant (maxillary right second premolars and lateral incisors, and mandibular left second premolars and lateral incisors for 1 group, and teeth in the contralateral quadrants for the other group).
Sampling sites were isolated with sterile cotton rolls and dried by a gentle airstream. Then a sterile curette was gently inserted to the bottom of the test periodontal pocket, and subgingival material was removed with a single stroke; pooled samples were stored in Eppendorf tubes (Eppendorf AG, Hamburg, Germany) at −80°C that contained 400 μL of distilled water.
Before DNA extraction, the deep-frozen samples were thawed and dispersed by vortexing for 15 seconds. DNA was extracted from the clinical sample material by using an alkali phenol-chloroform-isoamyl alcohol procedure. Briefly, 120 μL of a specimen was placed in 12 μL of proteinase K solution (20 mg/mL) (Sigma-Aldrich, St Louis, Mo) and 500 μL of potassium buffer for 60 minutes at 55°C. After centrifugation at 10,000 × g for 10 minutes at 12°C, DNA was extracted from the supernatant by using a mixture of 250 μL of alkali phenol and 250 μL of chloroform-isoamyl alcohol, and then precipitated by using 500 μL of isopropyl alcohol. DNA was washed in 75% ethyl alcohol at 10.000 g for 5 minutes at 4°C, air dried at 37°C, and dissolved in 200 μL of distilled water.
The TaqMan fluorogenic real-time polymerase chain reaction detection system was used to determine infectious agent counts. The TaqMan system uses species-specific primers and probes that are dually labeled with a fluorescent reporter and a quencher dye. The Taq polymerase cleaves the TaqMan probe during the polymerase chain reaction amplification process, separating the reporter from the quencher dye, which increases the intensity of the reporter fluorescence proportionally to the starting copy counts of the target DNA. Table I lists the nucleotide sequence of the polymerase chain reaction primers and probes (Oligoware version 3.0, Medical Microbiology Department, Gulhane Military Medical Academy, Ankara, Turkey). The primers and probes were synthesized by Metabion International (Planegg-Martinsried, Germany). Infectious agents included in the study were P gingivalis , T forsythensis , P intermedia , A actinomycetemcomitans , F nucleatum , and C rectus . For each infectious agent studied, polymerase chain reaction amplification was performed as an individual assay. Amplification, data acquisition, and all analyses were carried out by using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif). For each infectious agent tested, the TaqMan polymerase chain reaction assay was performed with a final volume of 25 μL of reaction mixture, containing 5 μL of extracted clinical sample, 12.5 μL of TaqMan universal polymerase chain reaction master mix (Applied Biosystems), 5 pmol of primers, and 3 or 4 pmol of TaqMan probe. The polymerase chain reaction cycling program included 2 minutes at 50°C to eliminate carryover contamination, 10 minutes at 95°C to activate the hot-start Taq DNA polymerase (AmpliTaq Gold DNA Polymerase, Applied Biosystems), and then 40 cycles, with each cycle consisting of 2 steps at 60°C for 1 minute and 1 step at 95°C for 15 seconds. Bacterial copy numbers were multiplied by 100 to adjust for sample dilution in the polymerase chain reaction assay. Polymerase chain reaction quantification standards included plasmids containing bacterial amplicons, which were cloned by using a cloning kit (TOPO-TA; Invitrogen, Carlsbad, Calif). The 6 tested bacteria showed no cross-reactivity with even closely related species, such as prevotellanigrescens. Also, a blast search (National Center for Biotechnology Information, Bethesda, Md), to check the specificity of the primers and probes used, showed no genomic cross-reactivity with other mammalian viruses, bacteria, or cells. The dynamic range of quantification of the TaqMan polymerase chain reaction assay was determined by serial dilution of the plasmid-generated standards in the range of 109 to 101 copies per milliliter.
| Infectious agents (GenBank accession number) | Amplicon size (bp) | |
|---|---|---|
| Porphyromonas gingivalis ( AB261608.1 ) | 194 | |
| Forward | 5′-TGGGACTTGCTGCTCTTGCTATG-3′ | |
| Reverse | 5′-GATGGCTTCCTGCTGTTCTCCA-3′ | |
| Probe | FAM-5′-CAAAGACAACGAGGCAGAACCCGTTA-TAMRA-3′ | |
| Tannerella forsythia ( DQ344918.1 ) | 149 | |
| Forward | 5′-GCGTATGTAACCTGCCCGCA-3′ | |
| Reverse | 5′-CCGTTACCTCACCAACTACCTAATG-3′ | |
| Probe | FAM-5′-AGGGATAACCCGGCGAAAGTCGGA-TAMRA-3′ | |
| Prevotella intermedia ( AY689226.1 ) | 105 | |
| Forward | 5′-AGACGGCCTAATACCCGATGTTG-3′ | |
| Reverse | 5′-TTACCCGCACCAACAAGCTAATCAG-3′ | |
| Probe | JOE-5′-TGGCATCTGACGTGGACCAAAGATTC-TAMRA-3′ | |
| Aggregatibacter actinomycetemcomitans (AF35951.1) | 288 | |
| Forward | 5′-CGGTTACCGTTATGACCGTGTGA-3′ | |
| Reverse | 5′-GCCCGGAATGCTTTGCTATATTTC-3′ | |
| Probe | FAM-5′-AGGCAAGACGGGAAGCTAACGCAAA-TAMRA-3′ | |
| Fusobacterium nucleatum ( EF089177.1 ) | 175 | |
| Forward | 5′-GCGGAACTACAAGTGTAGAGGTG-3′ | |
| Reverse | 5′-GTTCGACCCCCAACACCTAGTA-3′ | |
| Probe | JOE-5′-AATGCCGATGGGGAAGCCAGCTTA-TAMRA-3′ | |
| Campylobacter rectus (AF0351193.1) | 132 | |
| Forward | 5′-CACCCGATAACCCTACTCCTCCTA-3′ | |
| Reverse | 5′-GATCCGTTCCATCAGTACCCACTA-3′ | |
| Probe | FAM-5′-CCGGTACCGAATCCTGAGGAACCA-TAMRA-3′ | |
Statistical analysis
All statistical analyses were performed by using the Statistical Package for the Social Sciences (version 15.0; SPSS, Chicago, Ill). The Shapiro-Wilks normality test and the Levene variance homogeneity test were applied to the data. Clinical periodontal parameters showed normal distribution; there was homogeneity of variances between the groups. The effects of the different adhesive systems on the groups were evaluated with analysis of covariance (ANCOVA).
Counts of bacteria were recorded by conversion to logarithmic values, but they did not show normal distribution; there was no homogeneity of variances between groups. Intragroup and intergroup comparisons were evaluated by using the nonparametric Wilcoxon test. The statistical significance level was set at P <0.05.
Results
Because of the oral hygiene instructions given 1 week before the placement of fixed appliances, the patients were good in periodontal health. In the experimental and the control groups, after the bonding of brackets, the results of ANCOVA analyses showed that the different adhesive systems did not have significant effects on the groups during the study ( P >0.05) ( Table II ).
| Clinical periodontal index | Experimental group | Control group | F | P | ||
|---|---|---|---|---|---|---|
| Mean | SD | Mean | SD | |||
| Gingival index | 0.331 | 0.046 | 0.322 | 0.042 | 0.268 | 0.609 |
| Plaque index | 0.430 | 0.051 | 0.440 | 0.051 | 0.315 | 0.579 |
| Bleeding on probing | 18.400 | 3.521 | 18.467 | 3.314 | 0.031 | 0.862 |
| Probing depth | 1.272 | 1.870 | 1.264 | 0.096 | 0.600 | 0.808 |
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