Effects of probiotics on the oral health of patients undergoing orthodontic treatment: a systematic review and meta-analysis

Abstract Background and objective The effect of probiotics on oral health maintenance in orthodontic patients remains controversial. The aim of the study is to systematically review and assess the effects of probiotics on the oral health and microbiome of patients undergoing orthodontic treatment. Search methods and selection criteria Databases including PubMed, Web of Science, Cochrane Library, ClinicalTrials.gov, and ProQuest Dissertations & Theses Global databases were searched from their inception until June 2022. Randomised controlled trials that assessed the effects of probiotics on clinical and microbial outcomes in patients undergoing orthodontic treatment were included. Data collection and analysis Data screening and collection were performed, and the risk of bias (RoB) was assessed using the Cochrane RoB 2 tool. The meta-analysis evaluated the effects of probiotics on Streptococcus mutans (S. mutans) and Lactobacillus counts. The quality of the evidence from the meta-analyses was assessed with Grading of Recommendations Assessment, Development and Evaluation (GRADE). Results A total of 405 records were identified, of which 15 studies were included in the qualitative synthesis and 4 in the meta-analysis. The patients in all the included studies were treated with fixed orthodontic appliances. Results regarding clinical outcomes were controversial; four out of five studies reported no significant changes in plaque in the probiotic group (P > .05), and two out of three studies reported no significant changes in the gingival index (P > .05). Regarding microbial outcomes, the meta-analysis results revealed that probiotics significantly increased the likelihood of reducing the abundance of S. mutans to below 105 CFU/ml (risk ratio: 2.05 [1.54, 2.72], P < .001) and reduced the likelihood of increasing the abundance of S. mutans to beyond 106 CFU/ml (risk ratio: 0.48 [0.28, 0.83], P = .009). However, the quality of evidence according to the GRADE was moderate. Conclusions and implications There is insufficient evidence to determine the clinical benefits of probiotics as a supplement for the oral health of patients undergoing orthodontic treatment. However, probiotics may have benefits in reducing the salivary S. mutans counts in orthodontic patients. Registration PROSPERO (CRD42022366650).


Introduction
Orthodontic treatment aims to correct occlusal anomalies and enhance facial aesthetics.However, the use of brackets, elastics, and archwires in fixed orthodontic treatment or the full coverage of teeth by clear aligners pose a significant challenge to maintaining oral hygiene.These orthodontic tools create retentive areas on the tooth surface, favourable for microorganisms and food accumulation.If not timely removed, enamel demineralisation and gingivitis would be developed [1,2].Furthermore, multiple studies have demonstrated that patients undergoing fixed orthodontic treatment are susceptible to gingival inflammation and enamel demineralisation [3][4][5].Streptococcus mutans (S. mutans) and Lactobacillus acidophilus are the most common colonisers responsible for these consequences [6,7].Oral hygiene strategies, including fluoride application, antimicrobial oral rinses, and dietary modifications, have been proposed to prevent the hazards of orthodontic treatment on the tooth structures and gingival tissues [8,9].Nevertheless, patients undergoing orthodontic treatment still have a high risk of dental caries and gingivitis.
Probiotics have been proposed as a novel method for oral health maintenance.They refer to 'live microorganisms, when administered in sufficient quantities, provide health benefits to the host', which are available in various forms, such as lozenges, tablets, mouthwashes, and yoghurt [10].The functions of probiotics are to modulate immunoinflammatory responses by producing bioactive substances, such as bacteriocins or organic acids, and competing with pathogenic bacteria after adhering to the oral cavity [11].Lactobacillus and Bifidobacterium are the most commonly used probiotic genera in orthodontic treatment [12].
Studies have shown that probiotics are beneficial in preventing caries, gingivitis, and halitosis [13][14][15].For example, Pahumunto et al. reported that the probiotic milk containing Lactobacillus paracasei SD1 significantly decreased the development of caries and the number of S.mutans in preschoolers as compared to the placebo [16].Vicario et al. assessed the clinical effect of Lactobacillus reuteri Prodentis (GUM, Sunstar, Switzerland) in the treatment of periodontitis and found that the mean bleeding on probing and pocket probing depths significantly decreased by 26% and 4.8 mm in the probiotic group, respectively [17]. Lee et al. evaluated the effects of Weissella cibaria Chonnam Medical University (CMU) (oraCMU; OraPharm, Inc., Seoul, South Korea) on halitosis and found that the volatile sulphur compounds level significantly decreased by 4.81 in the probiotic group [18].
The effects of probiotics on patients undergoing orthodontic treatment remain controversial due to the varied results reported by multiple studies [19][20][21][22].Two published systematic reviews have evaluated the effects of probiotics on the oral health of individuals undergoing orthodontic treatment [23,24].Hadj-Hamou et al. systematically reviewed the clinical effects of probiotics on the inflammation of the gingival tissues and the decalcification of the enamel in orthodontic patients.The review included four studies and concluded that supplementation of probiotics did not affect the development of inflammation in the gingivae or decalcification in the enamel [23].In another review, Pietri et al. assessed nine studies and qualitatively concluded that probiotics had antimicrobial activity against oral pathogenic bacteria [24].However, neither review employed quantitative syntheses (i.e.meta-analysis) to comprehensively analyse the clinical and microbial effects.Recently, a few more randomised controlled trials (RCTs) assessing the effects of probiotics on patients undergoing orthodontic treatment have emerged as the latest evidence, warranting an updated summary of all the evidence [25][26][27].The current study aimed to systematically synthesise data from the available literature to assess the clinical and microbial effects of probiotics on patients undergoing orthodontic treatment.Specifically, the study analysed clinical outcomes including white spot lesions (WSLs) and periodontal indexes (gingival index [GI] and plaque index [PI]), as well as the microbial outcome of salivary bacterial counts.

Protocol and registration
This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines.The study protocol has been registered in the PROSPERO database (No.: CRD42022366650).A detailed PRISMA checklist has been appended in Supplementary Table 1.

Eligibility criteria
The study followed the PICOS format, with the following criteria: (i) Population: healthy patients undergoing orthodontic treatment; (ii) Intervention: the use of probiotics; (iii) Comparison: placebo or alternative treatment or no intervention; (iv) Outcomes: clinical outcomes, which comprise periodontal-related indexes (GI and PI) and incidence of WSL, as well as microbial outcomes, which include bacterial counts; and (v) Study design: RCT.Thus, the overall study objective based on the PICOS format was as follows: what are the clinical and microbial effects of probiotics on the oral health of patients undergoing orthodontic treatment?The detailed inclusion and exclusion criteria are listed in Table 1.

Information sources and search strategy
The following electronic databases were searched by two authors (JR and WC) for the relevant literature published from the inception of each database until June 2022: PubMed, Web of Science, Cochrane Library, ClinicalTrials.gov,and ProQuest Dissertations & Theses Global.Furthermore, three major orthodontic journals (American Journal of Orthodontics and Dentofacial Orthopedics, Angle Orthodontist, and European Journal of Orthodontics) from inception to June 2022 and the reference lists of the selected articles were also hand searched.The following search terms were used: 'probiotic OR probiotics OR Lactobacillus OR Bifidobacterium' AND 'orthodontic OR orthodontics OR bracket OR brackets OR brace OR braces OR fixed appliance OR fixed appliances OR aligners OR aligner OR Invisalign'.

Study selection and data collection
The shortlisted studies were screened based on the titles and abstracts independently by two authors (WC and JL) to identify the available studies.Full-text papers were retrieved for additional evaluation when the titles and abstracts of the papers lacked adequate information.Disagreements were resolved through discussion with a third author (YL).The Kappa coefficient of agreement between the two reviewers was 0.80 for the screening of titles and abstracts and 0.89 for the screening of full texts.Data from the selected studies were extracted into specific extraction tables, and the following terms were recorded for each study: author names and year of publication, study design, baseline participant characteristics (age, sex ratio, number of participants, and type of orthodontic treatment), study groups, probiotic microorganisms and usage, clinical and microbiological parameters, follow-up duration, and main conclusions.

Risk-of-bias assessment
The risk of bias (RoB) was independently assessed by two authors (WC and YL) using the Cochrane RoB tool for randomised trials (RoB 2.0).The quality assessment criteria spanned five domains: randomisation process, deviations from intended interventions, missing outcome data, outcome measurement, and reported result selection.The RoB in a study was classified as 'low' if all the domains were judged to have a low-risk bias; 'some concerns' if at least one domain was judged to have some concern bias but not a high RoB for any domain; and 'high RoB' if at least one domain was judged to have a high RoB or included some concerns for multiple domains.

Summary measures and quantitative synthesis of the results
Using the RevMan software (Review Manager version 5.3; Copenhagen: the Nordic Cochrane Centre, the Cochrane Collaboration, 2014), a meta-analysis of dichotomous outcomes was performed to compare the number of probiotictreated patients with those in the control group with high (>10 6 CFU/ml) and low (<10

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Lactobacillus counts after treatment.Studies with a 'high RoB' were excluded from the meta-analysis.The risk ratios and 95% confidence intervals were calculated and displayed in forest plots.A P value < .05indicated statistical significance.The I 2 test was performed to assess the heterogeneity of the studies.The fixed effects model was applied if the I 2 was < 50, whereas the random effects model was used if the I 2 was ≥ 50.Data that could not be analysed quantitatively were evaluated descriptively.Furthermore, due to insufficient data and variations in the included studies, subgroup analyses, analyses for 'small-study effects', and assessment of publication bias could not be carried out.The quality of the evidence from the meta-analyses was assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) method [28].

Study selection
The study flow is depicted in Fig. 1.The systematic search identified 579 records and 405 studies were screened after excluding duplicates.A total of 350 studies were excluded after screening for titles and abstracts.The remaining 55 studies were further evaluated through full-text screening for eligibility.Finally, a total of 15 studies were included in the qualitative synthesis (Fig. 1).

General characteristics of the included studies
The general characteristics of the 15 studies are presented in Table 2.All of these studies were RCTs that were published between 2009 and 2022 and used a double-blind (10 RCTs), parallel-group (13 RCTs), and cross-over (2 RCTs) design.The participant age ranged between 8 and 35 years, and the sample size ranged between 24 and 85.All of the included studies had test and control groups for comparison.Although we had no restrictions in terms of the type of orthodontic appliance used, all of the included studies used fixed orthodontic appliances.

Characteristics related to probiotic administration
The durations of the probiotic interventions ranged from 14 days to 17 ± 6.8 months, and the most typical intervention period was 14 days [22,25,29,33,36].The measurements were taken at the beginning before probiotic administration and immediately after administration.Four studies examined the effects of probiotic treatment by measuring participants both before and after treatment, as well as at follow-up periods ranging from 28 days to 3 months after treatment cessation [20,26,30,31].

Clinical changes
The clinical outcome parameters used to assess the effects of probiotics on the oral health of the patients undergoing orthodontic treatment were PI, GI, and incidence of WSLs.Due to the high level of heterogeneity in the clinical examinations and parameters used, quantitative synthesis of the clinical changes could not be conducted.
Of the five studies that recorded the PI, four reported no significant changes between the probiotic and placebo groups (P > .05)[20,26,29,30].In contrast, Shah et al. [21] reported a considerably decreased PI in the probiotic group compared to the control group without intervention (P < .05).Specifically, the mean PI decrease was 0.6 in the probiotic group, which was considerably greater than the 0.03  [20,31] found that probiotics did not affect the GI significantly (P > .05);however, Shah et al. [21] reported a significant reduction of 0.87 in GI of the probiotic group (P < .05).In terms of the incidence of WSLs, Gizani et al. [32] found no significant differences between the probiotic and placebo groups.At debonding, no new lesion was found in 22 out of 42 patients in the probiotic group and 26 out of 43 in the placebo group.

Microbial changes
Of the 11 studies that assessed S. mutans counts in the saliva or plaque of patients, five [21,22,[34][35][36] revealed that probiotic use can significantly reduce S. mutans counts (P < .05).For instance, Cildir et al. [36] demonstrated that the percentage of subjects with high S. mutans counts dropped from 63% to 21% after 14 days consumption of probiotic yoghurt.Similarly, Shah et al. [21] observed a significant decrease in S. mutans counts from 7.1 × 10 4 CFU/ml to 1.1 × 10 3 CFU/ml after 28 days of using probiotic mouthwash.Of the six studies [27,30,[32][33][34]36] that evaluated Lactobacillus counts in the saliva or plaque of patients, Alp and Baka [34] and Gizani et al. (32) found significant reductions in Lactobacillus counts (P < .05).Specifically, Alp and Baka [34] reported that the percentage of subjects with a Lactobacillus level of 10 6 or greater at the beginning of the study decreased from 13.3% to 0% in the probiotic toothpaste group.In the study by Gizani et al., [32] such proportion decreased from 40.5% to 21.4% in the probiotic lozenge group.Goyal et al. [19] reported that the use of probiotic mouthwashes significantly decreased the levels of Porphyromonas gingivalis by 1.6 × 10 6 CFU/ml (P < .05).Conversely, Habib [31] assessed the levels of other periodontal pathogens (P.intermedia, C. rectus, and F. nucleatum) in the subgingival plaque and found no significant changes (P > .05).

Meta-analysis
Of the 15 studies, three studies classified as having a 'high RoB' were excluded from the meta-analysis [19,21,29].Finally, four studies [22,32,34,36] were included in a dichotomous meta-analysis as information on the proportion of patients with low, medium, or high counts of salivary S. mutans or Lactobacillus could be extracted.These RCTs used a chair-side test for evaluating the levels of S. mutans or Lactobacillus.In the first meta-analysis for low S.mutans counts (<10 5 CFU/ml), the results showed that 79 (69.3%) of the 114 individuals who used probiotics had low S.mutans counts (<10 5 CFU/ml), compared to 38 (33.6%) of the 113 participants in the placebo group.This finding indicates that probiotics significantly increased the likelihood of reducing the abundance of S. mutans to <10 5 CFU/ml (RR: 2.05 [1.54, 2.72]; P < .001;I 2 = 33%) (Fig. 3a).In the second metaanalysis for high S.mutans counts (>10 6 CFU/ml), 13 (11.4%) of the 114 individuals in the probiotic group exhibited high S.mutans counts (>10 6 CFU/ml), compared to 29 (25.7%) of the 113 patients in the control group.The supplementation of probiotics significantly reduced the likelihood of increasing this abundance to >10 6 CFU/ml (RR: 0.48 [0.28, 0.83]; P = .009;I 2 = 5%) (Fig. 3b).In the third and fourth meta-analyses for low and high Lactobacillus counts, patients that used (n = 47) and did not use (n = 37) probiotics exhibited low Lactobacillus counts (<10 5 CFU/ml), and 11 patients in the probiotic group and 17 patients in the control group showed high Lactobacillus counts (>10 6 CFU/ml).No significant differences were observed in the abundance of Lactobacillus at either <10 5 CFU/ml (RR: 1.28 [0.93, 1.77]; P = .13;I 2 = 0%) (Fig. 4a) or >10 6 CFU/ml (RR: 0.67 [0.34,1.30];P = .24;I 2 = 0%) (Fig. 4b).Quantitative analyses for other outcome parameters could not be conducted due to insufficient data and the diverse measuring methods.The quality of evidence on the microbial measures according to the GRADE approach was moderate due to limited sample sizes (Supplementary Table 2).

Discussion
Probiotics suppress the growth of pathogenic microorganisms by producing antimicrobial compounds and competing for adhesion sites with pathogens [11], offering an alternative for the prevention and treatment of caries and gingivitis in patients undergoing orthodontic treatment [23,24].This systematic review and meta-analysis aimed to summarise the recently published data and evaluate whether supplementation with probiotics is beneficial to the oral health of patients undergoing orthodontic treatment.The outcome measurements include periodontal indexes, incidence of WSLs, and bacterial counts, which are the most commonly affected oral health-related measures during orthodontic treatment.
In general, the clinical effectiveness of probiotics for orthodontic patients is conflicting.Four studies reported no significant changes in PI in the probiotic group [20,26,29,30], and two demonstrated that probiotics did not significantly reduce GI [20,31] However, Shah et al. [21] reported that the probiotic group's build-up of plaque and gingival inflammation were significantly decreased.One study assessing the WSLs reported that there was no significant difference in the incidence of WSLs between the probiotic   and placebo groups at debonding [32].The variations in the probiotic administration vehicle, concentration, strains, and intervention duration could explain these controversial results.
Concerning the administration methods, probiotics were found to be positive in most studies when used in mouth rinses and dairy products, including yoghurt and curd [19,21,22,27,35,36].Dairy products such as milk and yoghurt are generally regarded as effective probiotic administration vehicles.These products include calcium phosphate, ammonia, and casein, improving enamel remineralisation, raising plaque-PH, and preventing bacterial cells from adhering to the teeth [37].According to a systematic review and meta-analysis, the dairy products used in 14 of the 20 trials substantially reduced the S. mutans levels [38].The use of mouth rinses is efficient since these can reach the tooth surface easily, including around the brackets and archwires, leading to the effective colonisation of the probiotic microorganisms in the oral cavity [21].By contrast, six studies that demonstrated no positive effects of probiotics used lozenges as the probiotic carrier, suggesting lozenges may not be effective vehicles in promoting oral health [20,26,[29][30][31][32].
The optimal strains and concentrations of probiotics for use in oral health maintenance are yet to be clarified.A large number of current dental research articles used probiotic doses ranging from 10 8 to 10 9 CFU [32,39,40].Most of the studies included in this systematic review used probiotic doses within this range, however, in one study, Ebrahim et al. used a concentration of 10 5 CFUs/lozenge and failed to observe any clinically detectable effects on plaque build-up [26].The authors considered that the lack of significant changes could be due to the low concentration of probiotics.Apart from variations in concentrations, there was also a diversity of probiotic bacterial species.Lactobacillus and Bifidobacterium species are the most common probiotic microorganisms used for oral health, with most of the studies in this review using these two species [19, 21, 22, 25-27, 29-33, 35, 36].Similar to probiotics used in the gastrointestinal system, a combination   [19,22,34].By contrast, the Lorodent probiotic complex, which contains five Lactobacillus strains, exhibited no positive effects in reducing plaque accumulation and S. mutans counts in patients undergoing orthodontic treatment [26,30,31].It is crucial to consider the unique orthodontic environment when determining the optimal probiotic concentration and strain for patients undergoing orthodontic treatment.Oral biofilms in these individuals tend to be thicker and more pathogenic compared to those in the general population, primarily due to the adverse effects of fixed orthodontic appliances on oral hygiene [42].Previous research has revealed that the S. mutans counts were four times higher in patients undergoing orthodontic treatment than in the general population [43].Therefore, future studies are still required to determine the optimal species and concentrations of probiotics to be used as a supplement for orthodontic patients.
In terms of the microbiological outcomes, five studies [21,22,[34][35][36] concluded that probiotic use can significantly reduce S. mutans counts in the saliva or plaque of patients, whereas the other six studies reported contradictory results [25-27, 30, 32, 33].Alp and Baka [34] and Gizani et al. [32] found significant decreases in the Lactobacillus counts in the saliva of patients in the probiotic group, whereas the other four RCTs showed no considerable alternations [27,30,33,36].The possible reasons for the contradictory microbiological outcomes could be the clinical heterogeneity factors mentioned before.An additional reason is the different methods used to assess the microorganism counts.There are three methods for assessing microorganism counts: traditional plate counting, real-time quantitative polymerase chain reaction, and chair-side test kits.The chair-side tests correspond well with traditional laboratory methods, are simple to handle in clinical settings, and are thus extensively used in evaluating bacterial levels [32,36,44].The four studies included in the meta-analysis used the chair-side test for evaluating the levels of S. mutans or Lactobacillus [22,32,34,36].A meta-analysis was conducted on these four studies that summarised the number of patients with different levels of S. mutans or Lactobacillus before and after probiotic use.The results showed that when the probiotic and control groups were compared after treatment, the probiotic group had more patients with low salivary S. mutans counts (<10 5 CFU/ml) and fewer patients with high counts (>10 6 CFU/ml); however, no such significant effects were observed for Lactobacillus.The results suggested that probiotics significantly increased the likelihood of reducing the abundance of S. mutans to below 10 5 CFU/ml and reduced the likelihood of increasing the abundance of S. mutans to beyond 10 6 CFU/ml.This result corroborates those of other meta-analyses conducted in the general population without orthodontic treatment: three meta-analyses concluded that probiotic therapy in the general population may decrease the S. mutans counts but have no effects on Lactobacillus [38,45,46].Based on these results, probiotics might have a potential preventive effect on dental caries by reducing the S. mutans counts in saliva.However, whether this reduction in S. mutans counts in orthodontic patients has any clinical significance remains to be elucidated.In addition, Lactobacillus counts were not significantly changed when comparing the probiotic and control groups at the end of the intervention.The reason underlying the nonsignificant differences in the Lactobacillus counts may be explained by the fact that some of the studies used Lactobacillus as the probiotic strain, which may have masked the ultimate bacterial counts [38].
It is worth noting that although the probiotic strains varied in the current analysis, previous research suggested that some observed probiotic effects are traits shared by multiple probiotic species rather than just a few well-studied strains [47,48].For instance, Caglar and colleagues conducted several studies examining the change in saliva S. mutans counts after the consumption of various probiotics, including Bifidobacterium lactis Bb-12, Lactobacillus reuteri ATCC 55730 and ATCC PTA 5289, Bifidobacterium DN-173010, using different delivery methods (chewing gum, yoghurt, water, and tablets) [49][50][51].The results demonstrated a significant decrease in S. mutans counts in all studies, independent of the probiotic strain used.Therefore, the current study focuses on the overall effects rather than differentiating between specific probiotic species, thereby encompassing a broader understanding of the collective impact of probiotics in orthodontic treatment.

Limitations
Despite a comprehensive search strategy, there is a shortage of high-quality RCTs with adequate sample size to make clinical recommendations.Current studies lack standardisation in their probiotic protocols, resulting in variations in strains, concentrations, intervention durations, and follow-up durations.Additionally, subgroup or correlation analyses were not possible due to the heterogeneities between the studies and the limited sample sizes.Owing to these methodological shortcomings, the conclusions should be generalised with caution.

Conclusion
There is insufficient evidence to determine the clinical benefits of probiotics on the oral health of patients undergoing orthodontic treatment.Probiotics may have potential benefits in reducing salivary S. mutans counts; however, whether this reduction in S. mutans counts has any clinical significance remains to be elucidated.Future studies should be conducted to determine the optimum delivery system, appropriate probiotic strains, and effective concentrations and should have a longer follow-up duration.Moreover, the effects of probiotics on the oral health of patients undergoing orthodontic treatment with other appliances, such as clear aligners and lingual appliances, should also be explored.

Figure 1 .
Figure 1.PRISMA flow diagram of the search results from the electronic databases.

Figure 2 .
Figure 2. Quality assessment of the RCTs (RoB 2.0).Symbol: green (+): low RoB; yellow (!): some concerns of bias; red (−): high RoB.Domains: D1: Bias arising from the randomisation process; D2: Bias due to deviations from intended intervention; D3: Bias due to missing outcome data; D4: Bias in measurement of the outcome; D5: Bias in selection of the reported result.

Table 1 .
Eligibility criteria for study selection.
Downloaded from https://academic.oup.com/ejo/advance-article/doi/10.1093/ejo/cjad046/7238860 by University of Hong Kong user on 10 August 2023 decrease in the control group.In terms of the GI, Benic et al. and Habib