Effects of clear aligners and traditional removable appliances on oral microbiome in mixed dentition: a comparative study

Abstract

Background

This prospective study aims to investigate the comparative effects of clear aligners (CA) and traditional removable appliances (RA) on the cariogenic risk of patients in mixed dentition, focusing on the oral microbiome.

Methods

25 children were included and assigned into CA and RA groups. Supragingival plaque and saliva samples were collected, and clinical parameters including Decay-missing-filled teeth index (DMFT), Plaque Index (PI) and Gorelick Index (GI) were recorded before treatment (T₀) and after 6-month follow-up (T₁). DNA was extracted from supragingival plaque and saliva and analyzed via 16S rDNA gene sequencing.

Results

Clinical parameters showed no statistically significant difference between groups at each time point or within group over time (p > 0.05). In both RA and CA groups, saliva exhibited significantly higher alpha diversity compared to supragingival plaque at T₁, as indicated by the significantly higher Chao1 and Shannon indexes (p < 0.05). Regarding beta diversity, significant difference was observed in saliva and supragingival plaque samples between T₀ and T₁ within group RA (p < 0.05, Adonis), whereas no such significance was noted in the CA group (p > 0.05, Adonis). At the genus level, Lactobacillus exhibited a statistically significant increase in saliva and supragingival plaque of group RA from T₀ to T₁ (p < 0.05), and an increasing trend in the group CA without statistical significance (p > 0.05). At T₁, Lactobacillus levels were comparable between groups, whereas species-level analysis revealed distinct cariogenic species.

Conclusion

Both clear aligners and traditional removable appliances resulted in elevated cariogenic risk of patients in mixed dentition at the microbial level. Distinct alterations in cariogenic species were observed to be induced by various orthodontic appliances.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12903-024-05063-2.

Background

Children with malocclusion are prone to poor oral hygiene and orofacial trauma, both of which would exert adverse effect on oral function and orofacial growth. Interceptive orthodontic treatment is therefore always adopted to reduce the severity of malocclusion and thus provide appropriate oral environment for growth. Conventionally, large-volume removable orthodontic appliances like Twin-block (TB) and Frankel Regulator have been utilized in interceptive treatments. However, their extensive coverage and interference with intra-oral mucosal tissues would potentially cause discomfort and hamper their practical application [1–4]. In recent years, clear aligners have emerged as a popular choice among orthodontic patients, with characteristics of comfort, aesthetics, and easy-to-clean [5]. Functional clear aligners have demonstrated effective therapeutic outcomes in interceptive treatments, particularly for Class II malocclusion, presenting a viable alternative to traditional Class II functional appliances [4, 6–9].

A common complication of interceptive orthodontic treatment among children is dental caries. This condition, characterized by the phasic demineralization and remineralization of dental hard tissues, can significantly damage the tooth crown and thus compromise the masticatory function and even impact the quality of life [10]. The risk of caries is intricately linked to oral microenvironment, while orthodontic appliances have the potential to inevitably affect the oral environment. Notably, the oral microbiome plays a pivotal role in the genesis and progression of caries [11]. For instance, cariogenic bacteria including Streptococcus mutans, Scardovia wiggsiae, Lactobacillus salivarius, Propionibacterium sp. strain FMA5, Parascardovia denticolens, and Bifidobacterium demonstrated positive correlation with early childhood caries (ECC) [12–16]. As demonstrated in previous researches, bacterial species, including Kingella oralis, Rothia dentocariosa, and Gemella sp. may elevate the risk for caries development [17–19]. Furthermore, various species of Lactobacillus have been reported to exhibit a complex and diverse manifestation in the advancing front of dentinal carious lesions, demonstrating a strong correlation with progression of dentine caries, such as L. salivarius, L. gasseri, L. fermentum, and L. casei [20–22].

The oral microenvironment undergoes different alterations depending on different types of appliances. The existing literatures predominantly concentrate on the comparison between the fixed and removable appliances owing to their significant divergence in removable nature which profoundly impacts oral hygiene maintenance strategies. These studies have indicated a heightened degree of microbiome regulation associated with clear aligners [23–26], and superior maintenance of microscopic oral hygiene levels, as evidenced by Streptococcus mutans and Lactobacilli colony forming unit (CFU) in removable positioner and clear aligners, in comparison to the fixed appliances [27]. However, despite the considerable distinctions in material compositions and structural designs, research focusing on the effects of clear aligners and traditional removable appliances on oral hygiene is lacking. For instance, compared with traditional removable appliances, clear aligners tightly encase around the teeth, thereby hindering the anti-bacteria, pH buffering, re-mineralization, and cleaning capability of saliva [28]. Considering the different shapes, materials, and configurations, microbiome colonization patterns may also vary accordingly.

Given the increasing popularity of clear aligner therapy in the mixed dentition, the present study aims to delve into the effects of clear aligners and traditional removable appliances (TB appliances) on saliva and supragingival plaque microbiome of mixed dentition patients receiving interceptive orthodontic treatment, compare the cariogenic risk induced by these two appliances at the microbial level, hoping to provide instruction for the selection of appliances for mixed dentition patients with a high caries risk and offer insights for the anti-bacterial material optimization of clear aligners and traditional removable appliances.

Methods and materials

Study participants

The study was approved by the ethics committee of the West China Stomatology Hospital of Sichuan University (Ethics No. WCHSIRB-D-2020-236) and written informed consent was obtained from the parents of all participating patients before the performance of the study following recruitment. The study included 25 patients who met the following criteria: (1) having mixed dentition and aged between 6 and 12 years old; (2) caries-free or caries-arrested (Nyvad system); (3) without orthodontic treatment history or systemic diseases; (4) free from antibiotics, hormones or mouthwash containing antibacterial drugs at least 1 month before sample collection. Patients with active caries, metal crown or other fixed prostheses, or lack of attendance were excluded from this study.

Based on the power analysis using G*Power version 3.1.9.7, a minimum required sample size of 24 participants was sufficient to reach a statistical power of 80%, with an alpha level of 0.05 and an effect size (f) of 0.25 [29]. Ultimately, a total of 25 participants were recruited. Among them, 14 underwent clear aligners (CA) treatment while 11 underwent traditional removable appliances (RA) treatment (i.e., TB appliance) as planned by an orthodontic professor, and all of them received interceptive orthodontic treatment between June 2020 and March 2022. Before their enrollment in the study and throughout the follow-up, the participants and their parents received oral hygiene instructions including brushing their teeth for 3 min three times daily using the standard Bass method and cleaning their appliance properly. Oral hygiene compliance was assessed upon each orthodontic visit.

Sample collection and clinical examination

Saliva and supragingival plaque samples were collected and clinical parameters including decay, missing, filled tooth (DMFT), Plaque Index (PI), and Gorelick Index (GI) were recorded before treatment (T₀) and at 6-month follow-up (T₁) [30–35]. Collection of Saliva samples and supragingival plaque samples was conducted by a single examiner at least 2 h after meals or oral hygiene procedures. 2 ml unstimulated saliva was collected in sterilized centrifuge tube, while supragingival plaque was scraped from dried surface of the upper first molars using a sterilized probe and dissolved in 1.5 ml of TE buffer. All samples were fast freeze in liquid nitrogen and then stored at -80℃ until DNA extraction. Participants were rinsed with plaque disclosing agent after samples collection to visualize the biofilm on their teeth for plaque evaluation.

DNA extraction and 16S rDNA sequencing

DNA from saliva and supragingival plaque samples were extracted using hexadecyltrimethylammonium bromide (CTAB). The bacterial 16S rDNA(V3-V4) gene was amplified with the primers 341F(5’-CCTACGGGNGGCWGCAG-3’) and 805R(5’-GACTACHVGGGTATCTAATCC-3’). The PCR products were confirmed with 2% agarose gel electrophoresis. The PCR products were purified by AMPure XT beads (Beckman Coulter Genomic, Danvers, MA, USA) and quantified by Qubit (Invitrogen, USA). The amplicon pools were prepared for sequencing and the size and quantity of the amplicon library were assessed on Agilent 2100 Bioanalyzer (Agilent, USA) and with the Library Quantfication Kit for Illumina (Kapa Biosciences, Woburn, MA, USA), respectively. The libraries were sequenced on NovaSeq PE250 platform, provided by LC-Bio Technology Co, Ltd, Hang Zhou, Zhejiang Province, China.

Statistical analysis

Paired-end reads was assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Paired-end reads were merged using FLASH. Quality filtering on the raw reads were performed under specific filtering conditions to obtain the high-quality clean tags according to the fqtrim (v0.94). Chimeric sequences were filtered using Vsearch software (v2.3.4). After dereplication using DADA2, we obtained feature table and feature sequence. Alpha diversity and beta diversity were calculated by normalized to the same sequences randomly. Then according to eHOMD classifier, feature abundance was normalized using relative abundance of each sample. Alpha diversity was applied in analyzing complexity of species diversity for a sample through 2 indices, namely Chao1 and Shannon index, and both indices were calculated with QIIME2. Beta diversity was calculated by QIIME2, the graphs were drawn by R package. Blast was used for sequence alignment, and the feature sequences were annotated with eHOMD database for each representative sequence. Other diagrams were implemented using the R package.

Gender and caries prevalence were analyzed using Fisher’s exact test. Age was analyzed using unpaired t-test. Plaque Index and Gorelick Index were analyzed using one-way Analysis of Variance (ANOVA). DMFT were analyzed using Kruskal Wallis test. The significance level was determined at p < 0.05. Alpha diversity was analyzed by Wilcoxon rank sum test, and beta diversity was analyzed by anosim analysis. The microbial differences between each subgroup were analyzed by Wilcoxon test (p < 0.05 and q < 0.05). The microbial differences in saliva or supragingival plaque between two groups at T₁ were analyzed by Linear discriminant analysis Effect Size (LEfSe).

Results

Characteristics of the study participants

Age, gender, and status of caries of the clear aligner group (group CA) or traditional removable appliance group (group RA) were shown as Table 1. The 25 participants were classified by gender and statistical analyses of clinical parameters including PI, GI, and DMFT were conducted (Additional file 1: Table. S1). No statistically significant differences were found in all parameters between genders at either T₀ or T₁ (p > 0.05), and thus the results from male and female participants were pooled for analysis. During the 6-month follow-up period, no incidence of new caries lesions was observed in group RA, whereas six patients in group CA developed new caries lesions albeit without statistical significance in DMFT values (p > 0.05). No obvious increase of plaque accumulation or demineralization on tooth surface was noticed, as evidenced by non-significant differences in PI and GI scores between T₀ and T₁ within each group (p > 0.05). Also, no statistically significant difference was found in all the clinical parameters between the two groups at either T₀ or T₁ (p > 0.05) (Fig. 1).

Table 1.

Demographic and clinical characteristics of participants. The data are presented as mean ± standard deviation (SD). T₀, before treatment; T₁, after 6-month follow-up

		Clear aligners (CA)	Traditional removable appliances (RA)
Age		8.86	8.64
Gender(F/M)		5/9	8/3
Non-caries/caries-arrested		3/11	4/7
T0	DMFT	3.14 ± 3.14	1.64 ± 1.87
	PI	3.88 ± 0.44	3.54 ± 0.53
	GI	1.15 ± 0.11	1.17 ± 0.27
T1	DMFT	3.57 ± 3.22	1.64 ± 1.87
	PI	3.84 ± 0.51	3.74 ± 0.41
	GI	1.14 ± 0.15	1.14 ± 0.17

Fig. 1.

Plaque Index (PI) and Gorelick Index (GI) of the participants. (a) Plaque Index. (b) Gorelick Index. Box represent 25th and 75th percentile, while whisker represents 5th and 95th percentile. The data of GI are presented as mean ± standard deviation (SD). T₀, before treatment; T₁, after 6-month follow-up; ns, not significant, p>0.05

Sequencing data

The collected saliva and supragingival plaque samples from both groups were analyzed via 16S rDNA sequence. A total of 8,276,019 raw reads were obtained from 50 saliva and 50 supragingival plaque samples of 25 participants (T₀ and T₁), and 6,550,594 clean reads were obtained for analysis after trimming and filtering. Notably, the length of 99.9% clean reads fell within 400–500 base pairs range. Amplicon Sequence Variant (ASV) analysis showed that 34,717 ASVs were obtained with average counts of 368 at T₀ and 326 at T₁. As shown in the dilution curve, with increasing sequencing depth, observed operational taxonomic units (OTUs) count gradually stabilized and the dilution curve flattened (Additional file 2: Fig. S1), indicating adequacy of sequencing depth and sample quality. Subsequent data analyses were performed on the obtained ASVs.

Microbiome diversity and community structures

Alpha diversity was analyzed by calculating the Chao1 and Shannon indexes to evaluate the species richness and diversity of communities (Fig. 2). The Shannon index and Chao1 index of saliva and supragingival plaque microbiome revealed no statistically significant difference between the two groups and within group at different time points (p > 0.05). As for the indexes of the saliva and supragingival plaque samples, no statistically significant difference was demonstrated between the two samples within the same group at T₀ (p > 0.05). However, at T₁, the two indexes for saliva were significantly higher than those for supragingival plaque in both RA and CA groups (p < 0.05), indicating a potential impact of orthodontic appliances on the oral microbiota.

Fig. 2.

Shannon index and Chao1 index among groups. (a) Shannon Index. (b) Chao1 Index. CA-S, saliva microbiome of group CA; CA-P, supragingival plaque microbiome of group CA; RA-S, saliva microbiome of group RA; RA-P, supragingival plaque of group RA. T₀: before treatment; T₁: after 6-month follow-up. * p < 0.05, ** p < 0.03, statistically significant differences between groups

To better elucidate community diversity, beta diversity was analyzed by performing the principle coordinates analysis (PCoA) with Bray-Curtis distance, analysis of similarities (ANOSIM), and Adonis analysis (Fig. 3). The distance of each subgroup was measured based on the similarities in the microbial structure. A significant difference in the microbiome composition (ANOSIM: p = 0.012, R: 0.0838) was observed in saliva samples, but not in plaque samples (Fig. 3b, c).

Fig. 3.

Beta diversity based on Bray Curtis distance. (a) Principal coordinate analysis (PCoA) of all groups. (b) Analysis of similarities (ANOSIM) analysis of saliva samples. (c) ANOSIM analysis of plaque groups

Furthermore, the unweighted Unifrac distance of saliva and supragingival plaque within group CA between different time points showed statistically insignificant difference (p > 0.05, Adonis), whereas that within group RA showed significant difference (p < 0.05, Adonis), suggesting the existence of RA has brought about a transformation in the composition of oral microbial communities. In consistent with the results of ANOSIM, at T₁, significant difference (p < 0.05, Adonis) was only observed in the unweighted Unifrac distance of saliva, but not in plaque between CA and RA groups (p > 0.05, Adonis). In addition, analysis of beta diversity using the unweighted Unifrac distance of metric revealed a statistically significant difference in the composition of the salivary microbiome compared to the supragingival plaque microbiome within the same group across different time points (p < 0.05, Adonis) (Additional file 3: Table S2).

Comparison of ASVs abundance and microbial composition

To precisely delineate the specific alterations in the oral microbiome, taxonomic analysis of the sequences was conducted and sequences were classified into 170 genera. The results indicated that the top 10 genera accounted for over 60% of the total sequences (Fig. 4a), and the top 10 genera included Nesseria (9.93%), Streptococcus (9.10%), Corynebacterium (7.30%), Leptotrichia (6.55%), Actinomyces (6.34%), Prevotella (5.95%), Veillonella (4.81%), Fusobacterium (3.51%), and Capnocytophaga (3.50%).

Fig. 4.

Alterations in microbial composition at the genus level. (a) Percentage of the relative abundance of the top 10 genera. (b) Comparison of relative abundance in genus Lachnospiraceae [G-2], Enterobacter, Bacteroides, and Bifidobacterium between T₀ and T₁ in group RA. (c) Histogram of Lactobacillus among groups in different time points. The data are presented as the mean of the relative abundance with standard error of mean (SEM). ∗p < 0.05, q < 0.05; ∗∗p < 0.03, q < 0.05, statistically significant differences between groups

At the genus level, group RA exhibited significant increases in Lachnospiraceae [G-2], Enterobacter, Lactobacillus, Bacteroides, and Bifidobacterium in supragingival plaque samples, and Enterobacter, Bacteroides, and Lactobacillus in saliva samples from T₀ to T₁ (p < 0.05 and q < 0.05) (Fig. 4b), while no statistically significant alteration was found in saliva and supragingival plaque for group CA (p > 0.05 or q > 0.05). Furthermore, no statistically significant difference was observed in the bacteria at the genus level in saliva and supragingival plaque between the two groups at either T₀ or T₁ (p > 0.05, q > 0.05).

Notably, from T₀ to T₁, Lactobacillus was the only genus among the top 20 genera to exhibit a significant increase in both saliva and supragingival plaque in group RA, and it also demonstrated an increasing trend in group CA although without statistical significance (p > 0.05). Intergroup comparisons at T₀ and T₁ revealed no statistically significant differences in Lactobacillus levels between the two groups (Fig. 4c).

As shown in Fig. 5a, microbial distribution heatmap revealed significant differences at the species level between saliva and supragingival plaque. There were no significant differences in the top 30 species between the two groups or within groups between T₀ and T₁ (p > 0.05 or q > 0.05). At T₁, the detection of Streptococcus lactarius in saliva differed significantly between group RA and group CA (p < 0.05 and q < 0.05) with group RA exhibiting a significantly higher abundance (Fig. 5b). Bacteroides zoogleoformans was significantly up-regulated only in saliva of group RA (p < 0.05 and q < 0.05) (Fig. 5c). Lactobacillus strains, including L. pentosus, L. gasseri, L. salivarius, L. buchneri, L. panis, and L. reuteri genosp. 2, recognized as cariogenic bacteria, exhibited statistically insignificant increase in saliva and supragingival plaque in both groups (p > 0.05 or p > 0.05) (Fig. 5d-i). Interestingly, we found 73 species exhibiting significant different relative abundances between saliva and supragingival plaque in group CA at T₀, and the number increased to 111 at T₁ (p < 0.05 and q < 0.05). A similar trend was also seen in group RA, with the number increasing from 85 to 90 between T₀ and T₁ (p < 0.05 and q < 0.05), indicating notable differences in the relative abundance of dominant species between saliva and plaque within each group.

Fig. 5.

Alterations in microbial composition at the species level. (a) Heatmap of top 30 species that showed high relative abundance among groups. (b) Histogram of Streptococcus lactarius at the species level. (c) Histogram of Bacteroides zoogleoformans at the species level. (d-i) Histograms of Lactobacillus strains at the species level which showed an upward trend in saliva or plaque in the same group (p > 0.05 or q > 0.05). The data are presented as the mean value of the relative abundance with standard error of mean (SEM). ∗p < 0.05, q < 0.05

In order to identify the dominant bacterial species in group CA and group RA at T₁, LEfSe analysis was conducted (Fig. 6). Lactobacillus salivarius emerged as the predominant species in the supragingival plaque of group CA, while Lactobacillus gasseri, Cadiobacterium hominis, TM7 G 1 sp. oral taxon 347, and Bradyrhizobium elkanii were prominent species in the supragingival plaque of group RA (Fig. 6a). In saliva, Prevotella nanceiensis and Lactobacillus gasseri were prominent species in group CA, while Streptococcus tigurinus, Neisseria sicca, Lautropia mirabilis, Streptococcus sanguinis, Streptococcus vestibularis, and Actinomyces sp. oral taxon 448 were prominent in group RA (Fig. 6b).

Fig. 6.

Differential bacterial abundance of saliva and supragingival plaque at T₁ between groups. (a) Supragingival plaque bacterial abundance differences between groups at T₁ (p < 0.05 and LDA > 3.0). (b) Salivary bacterial abundance differences between groups at T₁ (p < 0.05 and LDA > 3.5). LDA, linear discriminant analysis

Discussion

To investigate the effect of clear aligners and traditional removable appliances on the saliva and supragingival plaque microbiome of mixed dentition patients, we recruited 14 patients treated with clear aligners and 11 patients treated with traditional removable appliances. This study demonstrated that both clear aligners and traditional removable appliances resulted in the increase of cariogenic bacteria Lactobacillus in both saliva and supragingival plaque. Specifically, though, the increase of Lactobacillus in saliva and supragingival plaque was only statistically significant for group RA, and the relative abundance of Lactobacillus showed no significant differences between CA and RA groups after a 6-month treatment. The results indicated that, at the microbial level, the potential cariogenic risk associated with application of clear aligners and traditional removable appliances remained similar between the two groups over the 6-month treatment period.

DMFT scores, Plaque Index, and Gorelick Index were recorded before treatment and at 6-month intervals. None of the indicators underwent significant increases in the two groups, suggesting that both clear aligners and traditional removable appliances were effective in maintaining excellent oral hygiene. The lack of significant changes in these clinical parameters may be attributed to the increased frequency of teeth-brushing and reduced frequency of food intake among the study participants. The findings aligned with previous studies investigating the oral hygiene maintenance effects of clear aligners among teenagers and adults alike as no significant difference was found in plaque index compared to the fixed appliance [23, 27, 36, 37].

In the present study, we focused on patients in the mixed dentition stage and used 16S rDNA gene sequencing to investigate the different effects on oral microbiome among mixed dentition patients undergoing interceptive treatment using traditional removable appliances or clear aligners. As the results suggested, the alpha diversity of saliva and supragingival plaque microbiome in both groups was similar between T₀ and T₁. Previous researches have pointed out that mature biofilms are characterized by lower alpha diversity compared to the newly formed ones [17, 38–40]. Therefore, it could be speculated that there was no significant difference between two types of appliances in their performance in promoting biofilm maturation. However, the alpha diversity of saliva microbiome was significantly higher than that of supragingival plaque within both groups at T₁. This finding contrasted with previous researches reporting that a lower degree of evenness and diversity of the microbial community within saliva microbiome compared to supragingival plaque in children, both with and without caries [38, 39]. The observed differences may be attributed to the existence of various orthodontics appliances, which could alter the oral environment and microbial community structure [37]. Noteworthily, the correlation between oral microbial diversity and dental caries remains inconsistent. Qudeimat et al. [41] and Yang et al. [38] have found no statistically significant different microbial diversity in saliva and supragingival plaque microbiome between caries-affected and caries-free children. Conversely, it was suggested that significant differences existed in both taxonomic composition and co-occurrence patterns within the oral microbiome of caries-affected and caries-free children, demonstrating that the microbial alterations preceding the development of caries could potentially serve as a biomarker for early diagnosis and prediction of caries [42, 43].

Furthermore, the Bray-Curtis distance was calculated based on ASVs abundance information to compare the presence or absence of species between groups. The microbial species composition in saliva and supragingival plaque of group CA altered at a statistically insignificant level as the course of treatment increased, in agreement with a recent study by Zhao et al. [44].The distances showed only significant differences in saliva between the two groups at T₁, suggesting that different orthodontic appliances caused significant variations in the microbial species composition shift in saliva. Interestingly, other studies comparing the oral microbiome of patients treated with fixed orthodontic appliances and clear aligners, have also revealed significant differences in beta diversity between the two treatment modalities [23, 45–47]. Among them, Shokeen et al. [23] reported a significant difference between clear aligners and fixed appliances in the plaque communities, but not in the saliva suggesting that the wearable and easy-to-clean properties of both traditional removable appliances and clear aligners may minimize the unfavorable impact of fixed orthodontic appliances on microbiome of supragingival plaque.

Lactobacillus is intimately associated with caries progression due to its acidogenicity and aciduricity. Saliva and supragingival plaque of caries patients have been detected with higher abundance of Lactobacillus than that of healthy individuals [17, 48–50]. In our study, although Lactobacillus increased in saliva and supragingival of both groups, only the increase in saliva and supragingival plaque of group RA showed statistically significant differences. This result was consistent with preceding researches [51–53] concerning the effect of removable orthodontic appliances on oral microbiome. Notably, the relative abundance was similar between group CA and group RA at T₁, indicating that both clear aligners and traditional removable appliances elevated caries risks, and the magnitude of increased caries risk between the two groups was similar at microbial level. A significant presence of Lactobacillus species, including L. gasseri, L. salivarius, L. plantarum, L. acidophilus, and L. panis has been consistently detected in saliva and caries lesions of patients with dental caries [20, 22, 36, 49]. In the present study, we found that L. salivarius was the prominent Lactobacillus species in supragingival plaque from group CA, while L. gasseri was the most abundant Lactobacillus species in supragingival plaque of group RA and saliva of group CA, as indicated by LEfSe analysis. This suggested that clear aligners and traditional removable appliances may lead to different caries-related Lactobacillus species attachment.

Interestingly, Scardovia wiggsiae and Streptococcus mutans, the well-recognized biomarkers for pediatric dental caries progression, were detected only in few samples in the present study. This observation warranted a redirection of focus towards a more diverse spectrum of bacterial species, since Streptococcus mutans is not the exclusive bacterium implicated in caries development [54]. Species related to caries activity, such as Saccharibacteria (formerly designated as TM7) [17] and various Streptococcus strains [55], were prominent in saliva and plaque of group RA at T₁, indicating a greater diversity of dominant cariogenic bacteria within this group. Moreover, the bacterial species identified as dominant through LEfSe analysis, including Saccharibacteria, Neisseria, Prevotella, and Streptococcus strains, have demonstrated multifaceted effects on periodontitis, another common oral cavity disease resulting from oral microbiome dysbiosis [56–67]. These findings indicated that orthodontic appliances exert a significant influence on the composition of oral microbiome, which consequently impact the entire oral microecosystem, potentially leading to a cascade of effects that influence the overall oral health status.

Another crucial observation was that microbial diversity between saliva and supragingival plaque within the same group became increasingly distinct after the 6-month orthodontic treatment. This finding indicated the potential of both clear aligners and traditional removable appliances to cause significant variation in the oral microbiome. Although the dominant genera and species were similar in the saliva and supragingival plaque from the two groups, microbial distributions at genus and species level between saliva and supragingival plaque within the same group exhibited significant difference. This was in accordance with Kahharova’s finding [68] of saliva and supragingival plaque among young children. These results indicated that relying solely on microbiome of saliva for caries risk prediction may be insufficient to yield accurate results, and that microbiome of supragingival plaque possesses the potential to foretell the development of dental caries.

Altogether, both clear aligners and traditional removable appliances resulted in increased Lactobacillus, subsequently elevating caries risk at the microbial level, but the magnitude of this elevated risk between groups was of no statistical significance. Considering that dental caries is a progressive disease that typically evolves over several months or years before manifesting as cavities, and that Lactobacilli species are more probable to be detected in associated carious lesions rather than in saliva and plaque, the observed increase in Lactobacillus levels in both groups indicated an elevated cariogenic risk following the use of orthodontic appliances for a duration of six months or more [69–71]. Furthermore, this finding stressed the importance of timely intervention upon detection of clues of caries, and it is also essential to conduct diligent monitoring and assessment of carious lesions, not only during the active phase of orthodontic treatment but also in the post-treatment period. This extended surveillance is vital to ensure that any cariogenic risks are promptly addressed and managed, thereby preventing the exacerbation of dental decay that could arise from the microbiome shifts associated with orthodontic interventions.

Given the high risk of dental caries in mixed dentition patients owing to poor dietary habits and oral hygiene that provide the retentive niche allowing bacteria to accumulate, it is crucial for patients undergoing interceptive orthodontic treatment and their guardians to prioritize effective plaque control measures, such as frequent and thorough tooth-brushing and tongue-cleaning [72]. The utilization of plaque-disclosing agents and other visual aids is recommended to inspire and educate pediatric patients, particularly those who may not strictly adhere to oral hygiene protocols. Fluoride-containing oral hygiene products including mouthwash and toothpaste, can serve as a beneficial supplement to daily oral hygiene routines [73–75]. Clinicians are advised to conduct an appropriate caries-risk assessment (CRA) during follow-up visits [69, 76]. Based on the CRA, personalized preventive and therapeutic strategies should be developed and recommended to patients to mitigate the risk of dental caries. These strategies may include dietary modifications, fluoride therapies, and behavioral interventions aimed at improving oral hygiene practices. Notably, close monitoring of white spot lesions upon each visit is essential, as they are indicative of early tooth demineralization and serve as a precursor to more advanced carious lesions [75, 77]. Regular assessment of these lesions is crucial for early intervention and prevention of further dental decay. Besides intraoral examinations, the cleanliness of the orthodontic appliances, especially the inner surfaces, should not be overlooked. In terms of dietary habits, it is recommended to reduce the intake of fermentable carbohydrates and substituted them with low cariogenic sweeteners. Emerging evidence supports the potential of natural substances like probiotics, paraprobiotics, postbiotics, and ozonized substances, in mitigating microbial dysbiosis and facilitating personalized prevention and treatment of dental caries during orthodontic treatment [72, 78–81].

Remarkably, the present study demonstrated a variation in the dominant cariogenic species in plaque biofilm between patients treated with clear aligners and traditional removable appliances, indicating a need for tailored anti-bacterial material optimization targeting different dominant cariogenic species associated with each appliance type, while the specific strategy warrants further investigation.

To be noted, despite that the clear aligners and traditional removable appliances showed a tendency to cause dental caries as suggested by the upregulated the pathogenic bacteria within a 6-month period, no significant variations in clinical parameters were observed. This was probably due to the relatively small sample size and short observation period. To enhance the clinical relevance of our findings, we emphasize the need for further research that includes a larger sample size, different age groups and a longer observation period covering the entire orthodontic treatment cycle, preferably encompassing the “phase II” orthodontic treatment after interceptive treatment.

Conclusions

Both clear aligners and traditional removable appliances, although showing no significant increase in caries-related indices, elevated the cariogenic risk at the microbial level in mixed dentition, with differences in the dominant cariogenic species in plaque biofilm. This underscores the critical importance of oral hygiene practices, including proactive prevention and treatment, as they appear to mitigate the potential for caries development despite the inherent risks posed by these orthodontic appliances.

Electronic supplementary material

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Abbreviations

CA

Clear aligners

RA

Traditional removable appliances

DMFT

Decayed, missing (due to caries), or filled tooth

PI

Plaque Index

GI

Gorelick Index

TB

Twin-block appliance

ECC

Early childhood caries

LEfSe

Linear discriminant analysis Effect Size

ASV

Amplicon Sequence Variant

OTUs

Operational taxonomic units

ANOSIM

Analysis of similarities

LDA

Linear discriminant analysis

CRA

Caries-risk assessment

Author contributions

CWX participated in the design of the study, data collection, analyses and interpretation, organized figures, and drafted the manuscript. CJJ participated in data collection, analyses and interpreted. BD offered critical advice to improve the study design and the registration procedure. WPQ contributed to data analyses, figure draft, manuscript revision and interpretation. SR led the study design, provided funding, and contributed to manuscript revision. All authors read and approved the final manuscript.

Funding

This work was supported by China Oral Health Foundation (No. A2021-122), the National Natural Science Foundation of China (Grant No. 82071146, 82371002, 81870804), the Sichuan Science and Technology Program (Grant No. 2021YJ0014), the Nature Science Foundation of Sichuan Province (Grant No. 2024NSFSC0540), as well as Align Technology Specialized Scientific Research Fund (21H0922).

Data availability

All raw sequences were deposited in the NCBI Sequence Read Archive under accession number PRJNA1129082. The SRA records are accessible with the following link: https://www.ncbi.nlm.nih.gov/sra/PRJNA1129082.

Declarations

Ethics approval and consent to participate

This research was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of West China Hospital of Stomatology, Sichuan University (WCHSIRB-D-2020-236) and written informed consents were obtained from the parents of all participating patients included in this paper.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.