The effect of removable orthodontic appliances on oral microbiota: systematic review

Background Removable orthodontic appliances may favour plaque accumulation and oral microbe colonization. This might be associated with intraoral adverse effects on enamel or periodontal tissues. The proposed systematic review was carried out to evaluate qualitatively and quantitatively the microbiological changes occurring during orthodontic therapy with removable orthodontic appliances. Methods PubMed, Cochrane, EMBASE, Web of Science, Scopus, Ovid Medline, Dentistry & Oral Sciences Source and Vita-Salute San Raffaele University databases were searched. The research included every article published up to December 2018 . The Preferred Reporting Items for Reporting Systematic reviews and Meta Analyses (PRISMA) protocol and the ‘Swedish Council on Technology Assessment in Health Care Criteria for Grading Assessed Studies’ (SBU) method were adopted to conduct this systematic review. Results The current study has a moderate evidence, demonstrating that removable appliances do inuence the oral microbiota. Signicant alterations occur just 15 days after the beginning of therapy, independently from the type of appliance. Furthermore, the levels of oral pathogens decrease signicantly or even returned to pre-treatment levels several months later the therapy end. Conclusions This review suggests that orthodontic treatment with removable appliances induces changes to the oral microora, but these alterations might not be permanent. Protocol : PROSPERO database registration number CRD42019121762.


Background
Physiologically the human's oral micro ora is consists of a mixture of organisms, which are common also to other anatomical districts. This bacterial charge is extremely complex, being composed of over 700 different species of bacteria [1][2][3][4][5]. Humans are not randomly colonized and the diverse community that makes up the oral microbiome is nely tuned by nature to protect against disease, and it is of great importance to maintain its natural diversity. This particular composition depends on numerous factors, some non-modi able such as genetics, age, sex, change of dentition [6] and some modi able, including stress, nutrition, dental treatment and diet of the individual [7][8][9]. The placement of removable orthodontic appliances creates a favorable environment for the accumulation of microbiota components and food residues, which, in time, may cause caries or exacerbate any pre-existing periodontal disease [10][11][12][13]. The appliances, both xed and removable ones [14,15], may interfere with oral hygiene practice and cover considerable parts of the tooth surfaces, so an increase of the total microbial population as well as an altered micro ora have been reported in relation to orthodontic treatment [16].
Once dysbiosis occurs, the goal of treatment should be to restore the lost harmonic balance by maintaining good oral hygiene and modifying lifestyle factors such as diet and smoking. The indiscriminate use of antibiotics for the treatment of oral diseases should be avoided to safeguard the bene cial oral microbiota and avoid resistance to antibiotics. For the control of caries, in addition to the use of topical uoride, measures should be taken to promote the elimination from the acidic environment, through the reduction of the amount and frequency of consumption of sucrose and acidic drinks (even if the latter are sugar-free), together with an integration with agents that can reduce the production of acid and/or promote the generation of alkali in dental plaque. For periodontal diseases, therapeutic strategies should aim to mechanically reduce accumulated bio lm by mechanically removing plaque to levels compatible with oral health. This would reduce the in ammation and ow of GCF and promote a favorable microenvironment to support the formation of a balanced microbiome. The role of the oral microbiome is important not only in the disease, but also in the response of the oral tissues.
Patients need to understand and be aware of the implications for their oral health when undergoing recommended orthodontic treatment. On the other hand, when the patient accepts to undergo orthodontic treatment, including those using removable orthodontic devices, he should be reminded that it entails a commitment to a higher regimen of attention towards oral hygiene and health in patient's home care [17,18].
The purpose of this review is to investigate the available evidence regarding the association between removable orthodontic appliances and both qualitative and quantitative changes of oral microbiota.
Thus, the clinical research questions proposed are: Do removable orthodontic appliances in uence the quality and quantity of oral microbiota?
Which are the effects of removable orthodontic appliances on the different bacterial species in the oral cavity?

Protocol
The present study was conducted by the Department of Dentistry at Vita-Salute San Raffaele University of Milan. This systematic review was performed in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [19,20]. The analysis' methods and inclusion criteria were speci ed in advance. No funding was given for the realization of the present review.

Search Strategy
The following electronic databases were searched from their respective sources: PubMed, Cochrane Library, Embase, Web of Science, Scopus, Ovid Medline, Dentistry & Oral Sciences Source and Cochrane Library. In addition, the databases within the library of the Vita-Salute San Raffaele University of Milan were searched.
To create an appropriate research question and review of the literature the PICOS strategy was used [21,22].
The key words and combinations used in searching the databases were "(Functional appliance OR removable orthodontic appliance OR Frankel appliance OR Bionator OR LM activator OR Twin Block) AND oral microbiology".
Articles published up to 20 December, 2018 were included without language and initial date restriction.

Eligibility criteria
Initially, all articles were selected by title and abstract. Articles present in different databases were considered only once.
In a second moment inclusion and exclusion criteria were applied.
The inclusion criteria were: The microbial analysis had to focus on the quality and quantity of changes in the mouth and not on the appliance and the statistical analysis of the studies had to be adequate [23,24].
Only in vivo, in humans, studies were considered.
Statistical analysis of results.

The exclusion criteria:
Patients with systemic diseases or under any condition that could in uence oral microbiota or periodontal support tissues; Antibiotic therapy within three months before or during the study; No standardization and training in oral hygiene.
Case reports, case series, reviews and author opinions

Study selection
To minimize bias, two review authors, with experience in Oral Microbiology (MM) and Functional Orthodontics (LA), analyzed each selected paper and extracted data independently. If data were not clear enough, an attempt was made to contact the author by e-mail. Any disagreement between the two reviewers was resolved by discussion or consultation with a third experienced author (PM).
The selection of articles was processed according with PRISMA protocol (Fig. 1).

Data collection
The following data was then collected from each included study: author/year of publication, study design, sample size, sample/age/sex, type of appliance, collection time, collection method of analysis, microbial analysis outcome and quality of the study (Table 1).]

Risk of bias and quality analysis
The methodological quality is 'the extent to which the design and conduct of a study are likely to have prevented systematic errors (bias)'. Different quality criteria can explain variation in the results of studies included in a systematic review. More rigorously designed (better 'quality') trials are more likely to reach results that are closer to the 'truth' [33]. The 'Swedish Council on Technology Assessment in Health Care Criteria for Grading Assessed Studies'(SBU) method was adopted to report the level of evidence of this systematic review. To minimize the risk of bias during the inclusion of studies in the analysis, the two reviewers (LA and LA) applied independently the SBU criteria. When there was any disagreement concerning the relevance of an article, it was solved by the intervention of a third reviewer (PM). This Protocol organized the articles in three grades according to their methodological quality [34], as Table 2 shows.
Based on the grade of quality, four evidence levels were used (Table 3).

Quality of evidence
Five of the eight chosen articles presented a moderate methodological quality [10,35,37,38,40]: the major concern regarding these studies is the lack of blinded outcome assessment, diagnostic reliability tests and reproducibility tests.
One article had a high quality [36] and the remaining two were classi ed as having a low quality [39,41]. Due to the absence of homogeneity in the study formulation, a meta-analysis could not be performed though,a systematic review was realized.
When organizing the data according to pathogens, the following results were obtained

Candida
All studies detected an increase of Candida spp. concentration during therapy with removable orthodontic therapy [10,[36][37][38][39]. According to Jabur et al. study, removable orthodontic appliances induced an increase of Candida level up to 13.3% after an average of 5 weeks and 20% after 4 months [10].
On the contrary, the increase in Candida was very low after 3 weeks [36] and 6 months [37]. In Addy's study, the Candida prevalence after three weeks from the beginning of treatment resulted to be 46% in the control group and 52% of removable appliance wearers [36].
In Arendorf et al. study, noted a prevalence of Candida of 57.6% for all study subjects, but the 39.4% of the sample was a prior Candida-carrier, so only 18.2% became carriers 5 months after starting the therapy. Results of Mc-Nemar test show a highly signi cant overall increase in Candida prevalence while patients were wearing the appliances (p < 0.001), especially in posterior and anterior palatal sites, respectively. However, it was followed by a highly signi cant fall in number after removal of the appliance (p < 0.001), in fact after 5 months form the end of the therapy, only 42.4% reported Candida colonies. This means that removable orthodontic appliances induced an increase of Candida colonies only of 3% [38].
An increasing number of microbiological counts of Candida albicans from baseline to one month, three months and six months after the beginning of therapy was observed, with a sudden increase in the number of Candida counts between the baseline and the end of the rst month, which was found to be statistically signi cant (p < 0.001) [39].
According to Khanpayeh et al. study, the Candida colonies isolated from saliva after 6 months from the beginning of the therapy with removable appliance belonged mainly to Albicans spp (25%). Though, salivary carrier of Candida species decreased with increasing duration of orthodontic treatment [37].

Streptococcus mutans
All three articles [35,39,41] which analysed S. mutans colonization of the mouth agreed that removable orthodontic appliances represent a promoting factor for the colonization of the oral cavity by this microorganism.
In Kundu's et al. article a statistically signi cant increase of S. mutans was recorded during orthodontic therapy with removable appliances, from the baseline to six months (p < 0.001). Furthermore, S. mutans bacterial counts were signi cantly higher than those of Lactobacillus spp. and Candida albicans at alltime intervals (1 -3 -6 months) [39].
The study that analyzed different interceptive removable appliances [41], demonstrated a constant increase of Lactobacillus and an increase of S. mutans after 15 days, followed by a progressive decrease after 30 and 60 days.
The numbers of S. mutans colonies showed a continuous increase during therapy from baseline to one month with statistical signi cance (p < 0.05) [35].

Lactobacillus
All the studies [10,39,41] which quantitatively and qualitatively evaluated the difference of frequency in Lactobacillus spp. demonstrated an increase in the microbiological counts. Kundu et al. and Topaloglu et al. studies both suggested that the microscopic counts of Lactobacillus spp. increased signi cantly during orthodontic treatment with removable appliances from the baseline (before appliance placement), to follow-up visits at 1 month, 3 months, and 6 months, these were found to be statistically signi cant (p < 0.05) [39,41]. Jabur et al. noted an increase (6.66%) in Lactobacillus spp. after 4 months of therapy, too [10].

Moraxella catharralis
According to Jabur et al. study, this pathogen was found in all the included patients, furthermore, its oral colonization incredibly increased with removable orthodontic appliances. After a mean of 5 weeks from the appliance use, Moraxella prevalence was of 73.33% and after 5 months it arrived to 100% [10].
3.5 Staphylococcus epidermidis S. epidermidis colonization of the mouth also appears to be in uenced by the use of removable appliances. As Jabur et al. stated, in patients using these devices the percentage increased up to 40% after an average of 5 weeks and arrived to 60% after 4 months [10].

Others
The following results revealed that the changes in oral microbiota during treatment with removable orthodontic devices involved also other bacterial species.
Petti et al. revealed that in supragingival and in subgingival plaque G+ cocci decreased after 6 -8 weeks and increased at 6-7 months, with nal values higher than baseline values. Gingivitis risk indices (bacterial count and G-rods) signi cantly increased progressively after 6-8 weeks. Among periodontitis risk indices, only supragingival m rods and subgingival Spirochetes signi cantly increased at 6-7 months. Aggregatibacter actinomycetemcomitans (Aa) prevalence was near zero [40].
The presence of anaerobic bacteria in the subgingival dental plaque was detected with the same density (n = 15.75%) at baseline (T0) and at three months (T1), while it increased at nine months (T2) (n = 17.85%), but not signi cantly (p > 0.05). The most important bacteria that cause periodontal tissue loss -Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf) and Prevotella nigrescens -were not detected in any patients [40].
Outcomes summary: Removable orthodontic appliances have the following effects on oral micro ora: Candida colonies increase, especially albicans species, during the rst month of therapy, followed by a decrease after few months mutans is the main microorganism to increase during the rst months of therapy and the main increment occurs in the rst 15 days Lactobacillus spp. microbiological count increases during the rst months of therapy Moraxella catharralis and epidermidis values increase signi cantly during the rst month of therapy Spirochaetes signi cantly increased during the rst 6-7 months Aa, Pg, Tf and Prevotella nigrescens were not detected in any patient during therapy.

Discussion
The present systematic review is based on a low number of selected articles (n=8) because of the reduced number of papers focused on this topic. Nevertheless, the lack of standardization between the studies, the disparities in the category of devices analyzed and the discrepancy in wearing-time made the choice very limited. Though, this would not represent a major concern for the review quality, because the study-selection process has been very accurate to try to achieve the most "reliable" results as possible.
Any appliance or device placed in the oral cavity creates new retentive surfaces, promoting plaque accumulation and alteration of oral micro ora. Consequently, the pH values and the buffering capacity of saliva signi cantly reduces during the therapy. This situation can promote the increment of cariogenic bacteria in dental plaque and saliva [35]. Statistically signi cant increases were recorded in the following bacteria: Streptococcus mutans, [35,39,41], Lactobacillus spp. [10,39,41], Staphylococcus epidermidis [10], Moraxella catharralis [10], and subgingival Spirochetes [40] It was interesting to notice that the initial microorganisms' increment was followed by a progressive decrease towards more physiological values [37]. The same happened to pH values, which seem to return to physiological levels after 6 months from the end of therapy [38].
It was also demonstrated that previous orthodontic therapies do not alter the response of the oral microbiota to removable orthodontic treatment. Indeed, patients who have already undergone orthodontic treatment [28,29] have the same alterations in microorganisms of those who never wore orthodontic appliances [24].
These results emphasize that removable orthodontic appliances, when inserted in the oral cavity, begin to accumulate plaque. However, it is not possible to understand whether the accumulation of plaque may depend on the material from which the device is made, because most studies in the literature do not specify it. The microorganisms load increment could be strictly related to the appliance surface roughness as well as the time spent in the oral cavity. Generally, removable orthodontic appliances are made of heat-setting plastic or acrylic resin, which are both microporous and rough materials. The introduction of smoother surface removable devices could be less resilient for microorganisms and more biocompatible.
Therefore, it is essential that both patients and healthcare professionals embrace the concept of a balanced oral microbiota and its importance to oral and systemic health. Treatment sessions should include prevention strategies that promote active maintenance of oral health, rather than disease management Oral health professionals can achieve this educating patients to appropriate life-style choice and an effective bio lm-formation control. This approach would maintain the bene cial properties of resident microbiota and would reduce Conclusions According to our systematic review, removable orthodontic appliances in uence oral micro ora qualitatively and quantitatively Moreover, the main changes seem to manifest during the rst 15 days, independently from the type of appliance. Nevertheless, after the end of the treatment, the concentration of pathogenic microorganisms seems to be leaning towards more physiological values.
Though, the oral microbial alterations promoted by orthodontic removable appliances do not seem to be permanent.

Declarations
Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable.
Availability of data and materials: all data generated or analysed during this study are included in this published article.
Competing interests: the authors declare that they have no competing interests.
Funding: Not applicable.
Authors' contributions: AL conceived the work. MP and AL collected, analysed and interpreted data regarding microbial oral disease and the removable oral appliances. EM and MM gave a critical revision of the article. MP and AL were the major contributors in writing the manuscript. All authors read and approved the nal manuscript.

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