Introduction
Fixed orthodontic appliances can alter the subgingival microbiota. Our aim was to compare the subgingival microbiota and clinical parameters in adolescent subjects at sites of teeth treated with orthodontic bands with margins at (OBM) or below the gingival margin (OBSM), or with brackets (OBR).
Methods
Microbial samples were collected from 33 subjects (ages, 12-18 years) in treatment more than 6 months. The microbiota was assessed by the DNA-DNA checkerboard hybridization method.
Results
Bacterial samples were taken from 83 OBR,103 OBSM, and 54 OBM sites. Probing pocket depths differed by orthodontic type ( P <0.001) with mean values of 2.9 mm (SD, 0.6) at OBSM sites, 2.5 mm (SD, 0.6) at OBM sites, and 2.3 mm (SD, 0.5) at OBR sites. Only Actinomyces israelii ( P <0.001) and Actinomyces naeslundii ( P <0.001) had higher levels at OBR sites, whereas Neisseria mucosa had higher levels at sites treated with OBSM or OBM ( P <0.001). Aggregatibacter actinomycetemcomitans was found in 25% of sites independent of the appliance.
Conclusions
Different types of orthodontic appliances cause minor differences in the subgingival microbiota ( A israelii and A naeslundii ) and higher levels at sites treated with orthodontic brackets. More sites with bleeding on probing and deeper pockets were found around orthodontic bands.
The primary etiology of gingivitis and periodontitis is a mixed bacterial infection in complex biofilm structures on supragingival and subgingival tooth surfaces and oral soft tissues. Treatment with fixed orthodontic appliances causes changes in the normal oral environment. This might disrupt the balance between the host and established microorganisms because of new oral environmental conditions that might favor pathogenic microbiota. Local clinical and microbiologic changes were induced on clinical and microbiologic parameters in subjects undergoing orthodontic therapy. During orthodontic therapy, there appears to be a shift in the subgingival microbiota. An increase in the total bacterial load, including spirochetes, fusiform bacteria, rods, and gram-negative anaerob species concomitant with a reduction in gram-positive and aerobic bacteria, has been reported.
Increased plaque accumulation, bleeding on probing (BOP), and probing pocket depth (PPD) have been reported shortly after placement of bonded and banded orthodontic appliances. Some of these studies have compared results in control subjects without orthodontic appliances with patients with various fixed orthodontic appliances. When comparing the effects of bonded and banded orthodontic appliances on clinical periodontal parameters, the banding of teeth seems to have a negative effect on at least 1 of these clinical parameters, with worse clinical effects at sites treated with orthodontic bands. The design and material characteristics of orthodontic brackets can affect bacterial profiles and periodontal parameters. It might however, require at least 6 months before the orthodontic appliances have effects on the supragingival and subgingival microbiota. Histologic specimens from human banded teeth have also shown the transition from chronic gingivitis to a periodontal lesion when an orthodontic band has been in place for 6 months.
The aims of this study were to compare the clinical parameters and subgingival microbiota in adolescents in fixed orthodontic therapy at subgingival sites from teeth treated either with orthodontic bands with margins at (OBM) or below the gingival margins OBSM) with sites treated with brackets in the supragingival position (OBR).
Material and methods
In this study, 33 subjects (ages, 12-18 years; mean age, 15.1 years; SD, 1.4) consented to participate. They had all been in treatment at the Department of Orthodontics, School of Dental Medicine, University of Berne in Switzerland. They met the following inclusion criteria: (1) good general health, (2) no prior history of periodontitis or periodontal therapy, (3) no systemic or local antibiotics during the preceding 6 months, (4) nonsmokers, (5) at least 1 first molar in 1 quadrant, and (6) in orthodontic therapy with fixed orthodontic appliances for at least for 6 months. In addition, they were all treated with the same type of prefabricated orthodontic band (Dentaform, Dentaurum, Ispringen, Germany), which had been cemented with Ketac Cem (3M ESPE, St Paul, Minn). The adjacent second premolars were treated with metal brackets (Mini-Mono bracket, Forestadent, Pforzheim, Germany) bonded on the buccal surfaces with Transbond XT (3M ESPE).
All subjects were given standard oral hygiene instructions according to the protocol used at the orthodontic clinic. The subjects and their parents were informed about the rationale and study design. Both subject consent and written parental assent were obtained before the procedures, according to the requirements of the Ethics Committee (Kantonale Ethikkommission, Berne, Switzerland).
The subjects were periodontally evaluated. The second premolars and the first molars were assessed. The amount of supragingival plaque was evaluated according to the Silness-Löe plaque index (PI). The supragingival plaque was then carefully removed with a sterile curette and the tooth isolated to prevent salivary contamination. Microbial samples were obtained by inserting a sterile paper point (Absorbent Paper points 0.02, Dentsply, Maillefer, Switzerland) into the bottom of the periodontal sulcus for 10 seconds. The PPD was measured at the site sampled with a periodontal probe (model HH 12 DMS, A. Deppeler S.A., Rolle, Switzerland) after sampling the bacteria. The BOP at the site was also assessed.
Microbiologic samples were individually placed in labeled Eppendorf tubes containing 0.15 mL TE (10 mmol/L Tris-HCL, 1 mmol/L EDTA, pH 7.6) buffer; 0.5 mL sodiumhydroxide was added to the samples. The vials were stored at −20°C for 3 to 4 weeks and then processed by the checkerboard DNA-DNA hybridization technique. A total of 40 bacterial strains were included in the checkerboard panel ( Table ). Whole genomic DNA probes and sample DNA precipitations were obtained as described elsewhere. Briefly, bacterial DNA was extracted, concentrated on nylon membranes (Roche Diagnostics GmbH, Mannheim, Germany) and fixed by cross-linking with ultraviolet light (Stratalinker 1800, Stratagene, La Jolla, Calif). The membranes with fixed DNA were placed in a Miniblotter 45 (Immunetics, Cambridge, Mass). Signals were detected by chemiluminescence by using the Fluor-Imager (Storm 840, Amersham Biosciences, Piscataway, NJ) with a setup of 200 μ and 600 V. The digitized information was analyzed by a software program (ImageQuant, Amersham Pharmacia, Piscataway, NJ) allowing comparison of the density of the 19 sample lanes against the 2 standard lanes (10 5 or 10 6 cells). Signals were converted to absolute counts by comparisons with these standards. Total bacterial load was computed as the sum of bacterial load for each of 37 taxa and 40 species included in the panel ( Table ).
| Species | OBR (n = 83) | OBSM/OBM (n = 157) |
|---|---|---|
| 1a. Aggregatibacter actinomycetemcomitans (a) | 9.6 | 10.2 |
| 1b. Aggregatibacter actinomycetemcomitans (Y4) | 24.1 | 24.8 |
| 2. Actinomyces israelii | 21.7 | 6.4 |
| 3. Actinomyces naeslundii (types I + II) | 22.9 | 6.4 |
| 4. Actinomyces odontolyticus | 4.8 | 3.2 |
| 5. Campylobacter gracilis | 9.6 | 10.2 |
| 6. Campylobacter rectus | 28.9 | 33.3 |
| 7. Campylobacter showae | 16.9 | 16.6 |
| 8. Capnocytophaga gingivalis | 21.7 | 26.8 |
| 9. Capnocytophaga ochracea | 53 | 45.2 |
| 10. Capnocytophaga sputigena | 50.6 | 54.8 |
| 11. Eikenella corrodens | 12 | 27.4 |
| 12. Eubacterium saburreum | 19.3 | 15.9 |
| 13a. Fusobacterium nucleatum subsp. nucleatum | 65.1 | 58.6 |
| 13b. Fusobacterium nucleatum subsp. polymorphum | 50.6 | 44.6 |
| 13c. Fusobacterium nucleatum subsp. vincentii | 25.3 | 24.8 |
| 14. Fusobacterium periodonticum | 51.8 | 42.7 |
| 15. Lactobacillus acidophilus | 13.3 | 16.7 |
| 16. Leptotrichia buccalis | 77.8 | 72.2 |
| 17. Peptostreptococcus (Micromonas) micros | 14.5 | 19.5 |
| 18. Neisseria mucosa | 34.9 | 52.2 |
| 19. Prevotella intermedia | 20.5 | 21 |
| 20. Prevotella melaninogenica | 45.8 | 39.1 |
| 21. Prevotella nigrescens | 15.7 | 12.1 |
| 22. Porphyromonas gingivalis | 15.7 | 9.6 |
| 23. Propionibacterium acnes (types I + II) | 6 | 14.1 |
| 24. Selenomonas noxia | 27.7 | 1.7 |
| 25. Staphylococcus aureus | 7.1 | 4.5 |
| 26. Streptococcus anginosus | 4.8 | 1.9 |
| 27. Streptococcus constellatus | 1.2 | 0.6 |
| 28. Streptococcus gordonii | 26.5 | 29.3 |
| 29. Streptococcus intermedius | 6.1 | 5.7 |
| 30. Streptococcus mitis | 3.6 | 5.7 |
| 31. Streptococcus oralis | 2.6 | 3.2 |
| 32. Streptococcus sanguinis | 3.7 | 5.1 |
| 33. Streptococcus mutans | 2.4 | 4.5 |
| 34. Tannerella forsythia | 14.5 | 11.5 |
| 35. Treponema denticola | 12 | 17.3 |
| 36. Treponema socranskii | 20.5 | 9 |
| 37. Veillonella parvula | 47 | 38.9 |
Statistical analysis
The nonparametric Kruskal-Wallis analysis of variance (ANOVA) and Mann-Whitney U tests were used for analysis of data with no normal distribution or nonparametric characteristics, and independent t tests for parametric data. Because of the number of variables, the P value for a significant difference was defined at the P <0.001 level. The SPSS statistical package for Macintosh computers was used (version 16.0, SPSS, Chicago, IIl).
Results
Bacterial samples were taken from 240 sites in 33 subjects. Samples were taken from 83 OBR sites (34.6%), 103 OBSM sites (42.9%), and 54 OBM sites (22.5%). All orthodontic brackets were on second premolars, and the orthodontic bands were on first molars. The mean treatment time with orthodontic bands at data collection was 25.2 months (SD, 13.3). The mean treatment time with brackets was 18.0 months (SD, 7.9).
The overall mean PPD was 2.6 mm (SD, 0.6). The distribution of PPDs included 45% of sites with a PPD less than 2 mm, 50.8% with 3-mm PPD, 4.2% with a PPD more than 4 mm, and 1 site with a PPD of 5 mm. The PPDs differed statistically by orthodontic type and marginal location (1-way ANOVA, F = 26.7, P <0.001). The mean PPD values were 2.9 mm (SD, 0.6) at OBSM sites, 2.5 mm (SD, 0.6) at OBM sites, and 2.3 mm (SD, 0.5) at OBR sites. Thus, PPDs at OBSM sites were significantly greater than at OBM sites (mean difference, 0.4 mm; 95% CI, 0.2-0.6; P <0.001). PPDs at OBSM sites were also significantly greater than at OBR sites (mean difference, 0.6; 95% CI, 0.4-0.8; P <0.001), whereas no differences in PPDs were found between OBR and OBM sites. When the OBSM and OBM groups were pooled (OBSM/OBM), statistically higher PPDs were found at those sites compared with OBR sites ( t tests with equal variance not assumed and with a PPD mean difference, 0.4 mm; 95% CI, 0.3-0.7 mm; P <0.001).
Statistical analysis failed to identify differences in PI between OBR, OBM, and OBSM sites ( P = 0.41, Kruskal-Wallis ANOVA). BOP was found at 15.7% of teeth with OBR, 59.2% of teeth with OBM, and 59.1% of teeth with OBSM. The difference in BOP between the OBSM/OBM sites compared with BOP at the OBR sites was statistically significant ( P <0.001, Mann-Whitney U test).
The distribution of bacteria at OBSM/OBM sites is given in the Table . Thus, Leptotrichia buccalis was identified as the most prevalent species followed by Fusobacterium nucleatum sp. nucleatum , both at higher levels at either OBR or OBSM/OBM sites. Aggregatibacter actinomycetemcomitans (Y4 strain) was found in about 25% of both OBR and OBSM/OBM sites. Porphyromonas gingivalis was found at 15.7% of OBR and 9.6% of sites with OBSM/OBM. The distributions of Actinomyces israelii , Actinomyces naeslundii , P gingivalis , and Peptostreptococcus (Micromonas) micros are shown in a diagram based on type of orthodontic appliance ( Fig ).
