Characteristics of oral microbiota and oral health in the patients treated with clear aligners: a prospective study

doi:10.21203/rs.3.rs-3103267/v1

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Research Article

Characteristics of oral microbiota and oral health in the patients treated with clear aligners: a prospective study

https://doi.org/10.21203/rs.3.rs-3103267/v1

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Journal Publication

published 29 Sep, 2023

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Objectives

In this prospective clinical study, the effect of clear aligners on periodontal health and oral hygiene was examined. As the same time, microbial changes of the aligner tray and subgingival microbiota community were investigated.

Methods

The study recruited fifteen patients, and clinical parameters were recorded at three different time points: before the initiation of aligner treatment (T0), one month after treatment onset (T1), and three months after treatment onset (T3). Plaque samples were collected from the inner surface of aligners and subgingival sulcus at each of these time points. The microbial composition of the samples was analyzed using 16S rRNA gene sequencing, and changes were evaluated based on the abundance of amplicon sequence variants (ASVs).

Results

Reduction in plaque index and improvement in periodontal health were observed. In aligner tray plaque samples, the relative abundance of Streptococcus increased significantly, as well as the richness and diversity of microbiota decreased substantially as the duration of treatment time. In subgingival plaque samples, alpha and beta diversity of microbiota did not change significantly.

Conclusions

Clear aligner treatment have no significant impact on periodontal health and subgingival microbiota composition of patients. The structure of the aligner tray microbiota altered significantly at both phylum and genus levels and attracted a unique and less divers microbiota community.

Clinical Relevance:

Clear aligner treatment have no significant impact on periodontal health and subgingival microbiota composition of patients.

Plaque biofilm

16S rRNA gene sequencing

Clear aligner

Orthodontics

Dentistry

Adult patients seeking orthodontic treatment have increased recently[1]. They expressed a desire for more visually appealing and comfortable alternatives to conventional fixed equipment[2]. The popularity of the clear aligner is due to its transparency and low stiffness, which satisfy patients' needs. Clear aligner was introduced to orthodontic treatment in the 1990s, and began to spread to other nations in 2001[3]. Its indications have increased over the past 20 years, going from minor crowding or diastemas to complex cases[4]. According to studies, clear aligner treatment showed encouraging results in the aspects of improved periodontal health and decreased prevalence of white spot lesions when compared to fixed appliances[5, 6].

Clinical results and corresponding microbial communities are closely related. The host’s health and wellbeing are significantly influenced by the oral microbiota. If the local environment undergoes significant changes, it could alter the host-microbe equilibrium. This, in turn, could lead to an elevation in the likelihood of developing oral diseases. [7]. Clear aligners are made from transparent thermoformed plastic materials, which cover the teeth surfaces entirely and create a closed environment. As the inner surface of the aligner comes into direct contact with the tooth surface, the microbial composition of the aligner tray plaque can have a direct impact on the health of the enamel. In addition, observing how the structure of the subgingival flora changes can help us determine whether a patient is likely to develop periodontal disease during clear aligner treatment. Therefore, it’s noteworthy to determine the microbial characteristics of tray plaque microbiota and subgingival microbiota in patients treated with clear aligners. Due to their capacity to provide a more comprehensive and sensitive quantity of data with regard to microbial diversity (including unculturable bacteria), high-throughput methods based on 16S rRNA gene sequencing have been widely employed in research on bacterial phylogeny and taxonomy[8, 9].

However, there are limited prospective studies that investigated the microbiota characteristics of clear aligner patients by using next-generation sequencing technology[10, 11]. Traditional microbiological identification techniques, such as PCR and DNA-DNA hybridization, are applied to examine specific types of pathogenic bacteria[12, 13]. But these findings only offer a glimpse into the changes that occur within the microbial community and may not be representative of the dynamics of oral bacteria communities.

Thus, the current study intended to examine the effects of clear aligners on patients' periodontal health and oral hygiene, as well as phylogenetically define and compare the diversity of the microbiota in the aligner tray and subgingival regions before and after 3 months of treatment by using 16S rRNA gene sequencing. To our knowledge, this is the first study to simultaneously investigate the changes in both the structure of the aligner plaque biofilm and subgingival flora in patients using clear aligners.

Subject population

The study involved fifteen individuals (six male and nine female) who were treated with Invisalign clear aligners. These patients, aged between 19–35 years, were recruited from the orthodontic department at XXX Hospital of Stomatology, XXX University, and provided informed consent prior to enrollment. The research received formal ethical approval from the Institutional Review Board of XXX Hospital of Stomatology (WCSHIRB-D-2021-443).

The patients were enrolled based on the following criteria:

(1) No missing permanent teeth except for the third molars. No crowns or orthodontic history.

(2) No periodontal diseases, caries and white spot lesions.

(3) Undergone supra-and subgingival ultrasonic scaling one months before orthodontic treatment.

(4) Good oral hygiene, conformity, and no smoking.

(5) No antibiotics or hormones for two months prior to starting the study.

(6) No pregnancy or systemic diseases.

Clinical examination and sample collection

The study visits were scheduled at T0 (30 seconds after the application of first passive aligner), T1 (one month after treatment onset), and T3 (three month after treatment onset). At each time point, sample collection and clinical examinations were conducted. During the first three months, no periodontal therapy was performed. Before participating in the trial, patients were taught the standard Bass method and how to properly clean their aligners. They were instructed to clean their aligners at the same time as brushing their teeth. Patients were instructed to wear aligners minimum of 20 hours per day and switch to a new set every ten days. No cleaning their teeth and aligners or eating for two hours before each appointment.

The clinical parameters (plaque index (PI)[10], probing depth (PD)[11], and bleeding index (BI)[14]) and frequency of tooth brushing per day recorded by an experienced dentist at each time point. Tray plaque samples were scraped from the inner surface of aligners by using sterile cotton swabs. Subgingival plaque samples were obtained from anterior and posterior teeth (maxillary first molars, maxillary first central incisors, mandibular first molars and mandibular first central incisors) using the previously described procedures[15], and combined into one sample. Following collection, all plaque samples were sorted in a phosphate-buffered saline solution within a 1.5 ml Eppendorf tube at − 80°C until further analysis.

DNA extraction

The OMEGA Soil DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA, USA) was employed to extract total genomic DNA samples in accordance with the manufacturer's guidelines. The resultant samples were then stored at -20°C until further analysis. The quantity and quality of the extracted DNAs were assessed using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively.

16S rRNA Gene Amplicon Sequencing

For amplification of the bacterial 16S rRNA genes V3-V4 region, the forward primer 338F (5'-ACTCCTACGGGAGGCAGCA-3') and reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3') were employed. Additionally, sample-specific 7-bp barcodes were included into the primers for multiplex sequencing. PCR was performed using the following components: 5 µl of buffer (5×), 0.25 µl of Fast pfu DNA Polymerase (5U/µl), 2 µl (2.5 mM) of dNTPs, 1 µl (10 uM) of each Forward and Reverse primer, 1 µl of DNA Template, and 14.75 µl of ddH2O. The thermal cycling program consisted of an initial denaturation step at 98°C for 5 minutes, followed by 25 cycles comprising of denaturation at 98°C for 30 seconds, annealing at 53°C for 30 seconds, and extension at 72°C for 45 seconds, with a final extension at 72°C for 5 minutes. After amplification, PCR amplicons were purified using Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The amplicons were then pooled in equal amounts, and 480 bp sequencing was carried out on the Illumina NovaSeq PE250 platform at Shanghai Personal Biotechnology Co., Ltd (Shanghai, China).

Sequence analysis

QIIME2 2019.4 was applied for microbiota bioinformatics with some modification based on the official instructions (https://docs.qiime2.org/2019.4/tutorials/). Initially, the raw sequence data was demultiplexed using the demux plugin, followed by primer cutting using the cutadapt plugin. Subsequently, the DADA2 plug-in [16] was employed to filter, denoise, merge and remove chimeras from sequences at a sequencing depth of approximately 50,000 reads. The non-singleton amplicon sequence variants (ASVs) were then aligned using mafft. Finally, taxonomy was assigned to ASVs using the classify-sklearn naïve Bayes taxonomy classifier in the feature-classifier plugin against the HOMD (v15.2) database [17, 18].

Bioinformatics and statistical analysis

The analysis of sequence data was primarily conducted using QIIME2 and R packages (v3.2.0). Alpha diversity indices, such as the Chao1 richness estimator, Observed species, Shannon diversity index, and Simpson index, were calculated at the ASV level in QIIME2 and visualized as box plots to determine the level of diversity within the microbial communities. Furthermore, beta diversity analysis was performed utilizing weighted UniFrac distance metrics to investigate the structural variation of microbial communities across samples. The results were visualized using principal coordinates analysis (PCoA). The ANOSIM test was used in QIIME2 to evaluate the significance of differentiation of microbiota structure among groups. Normality and lognormality test were performed and posthoc multiple comparisons were analyzed by ANOVA or Friedman test to assess whether there were any statistically significant differences between each pair of groups at the phyla and genera level, as well as for for PI, PD, BI, and frequency of tooth brushing. Differences were considered statistically significant if they had a P value less than 0.05, 0.01 or 0.001. The statistical analyses were carried out using IBM SPSS Statistics version 25 (IBM Inc., Armonk, NY, USA).

Clinical results

The clinical parameters of the 15 individuals were presented in Table 1. Decreased PI, PD, and BI were presented during the aligner treatment. PI significantly decreased at T1 compared with T0 (P<0.05). PD was significantly lower at T1 and T3 compared to T0. The daily brushing frequency was increased significantly after treatment (P<0.05).

Table 1

Clinical parameters of the subjects
Time points	Plaque index (PI) (Mean ± S.D)	Probing depth (PD) (Mean ± S.D)	Bleeding index (BI) (Mean ± S.D)	Frequency of brushing (Mean ± S.D)
T0	2.15 ± 1.00	1.47 ± 0.27	0.90 ± 0.62	2.00 ± 0.38
T1	1.49 ± 0.69*	1.30 ± 0.27*	0.64 ± 0.62	2.87 ± 0.64**
T3	1.49 ± 0.71	1.26 ± 0.22**	0.61 ± 0.50	3.13 ± 0.64***
*Statistically significant difference: PI: T0 vs T1, P = 0.0151; PD: T0 vs T1, P = 0.0444, T0 vs T3, P = 0.0044; Frequency of brushing: T0 vs T1, P = 0.0013, T0 vs T3, P = 0.0001.

Sequence analysis

A total of 4,011,952 high-quality reads were obtained from 45 tray plaque samples, with an average of 89,154 ± 27,889 (ranging from 41,018 to 157,976) sequence per sample. Additionally, a total of 3,820,839 high-quality reads were acquired from 45 subgingival plaque samples, with an average of 84,908 ± 44,450 (ranging from 41,149 to 215,758) sequence per sample. To investigate changes within the tray plaque and subgingival plaque microbiota, the distribution and relative abundance of microbes at the phylum and genus level were examined.

Alpha diversity

Chao 1 and Observed species indexes were used to evaluate microbial richness, while Shannon and Simpson indices were utilized to assess the microbial diversity in plaque samples. In aligner tray plaque samples, richness and diversity of microbiota were decreased as the duration of treatment (Fig. 1, a). As compared to at T0, the richness of tray microbiota decreased significantly at T3, and the diversity of tray microbiota was significantly lower at T1 and T3 (P<0.05). In subgingival plaque samples, richness indexes decreased slightly, and diversity indexes stayed relatively stable during the treatment (Fig. 1, b).

Beta diversity

Alterations in microbial community composition were assessed at T0, T1 and T3 in tray plaque samples and subgingival plaque sample by using weighted UniFrac distance measurements. In the tray plaque samples, beta diversity was significantly different at T1 and T3 compared to T0 (P<0.05) ,and was similar at T1 and T3 (Fig. 2, a). However, in subgingival plaque samples, beta diversity remained consistent across time periods (Fig. 2, b). The principal coordinate analysis (PCoA) was employed to visualize the variations in tray and subgingival microbiota at different time points. At T1 and T3, the microbiota communities on the tray tended to form a cluster distinct from those at T0 (Fig. 3, a). Conversely, the subgingival microbial community structure remained similar over time (Fig. 3, a).

Microbial composition and relative abundances

A total of 14 and 10 phyla were identified in tray plaque and subgingival plaque samples respectively. Community structures of tray plaque and subgingival plaque at different time points at phylum level was showed in Fig. 4. Figure 5 displayed the relative abundances of major phyla (abundances at ≥ 1%). In tray plaque, the abundance of Proteobacteria and Firmicutes significantly increased, while the abundance of Actinobacteria decreased significantly at T3 compared to T0 (P<0.05) (Fig. 5, a). There were no significant changes observed in abundances of other phyla over different usage periods. In subgingival plaque, an increased abundance of Proteobacteria and a decreased abundance of Firmicutes were observed. Actinobacteria exhibited an increasing trend from T0 to T1 but returned to baseline level at T3 (Fig. 5, b).

Figure 6 presented the heatmap analysis of the relative abundance of tray and subgingival microbiota at the genus level. The top 80 genera were identified and compared at T0, T1 and T3. The microbial distribution of the core genera (abundances at ≥ 1%) of tray plaque and subgingival plaque at different time points is provided in Fig. 7. In aligner tray plaque, the abundances of Streptococcus and Haemophilus increased significantly at T1 and T3 compared to T0 (P<0.05) (Fig. 7, a). Whereas, compared to T0, the abundance of Actinomyces, Corynebacterium, Rothia, and Lautropia decreased considerably at T1 and T3 (P<0.05) (Fig. 7, a). In the subgingival plaque, Haemophilus increased steadily during treatment time and its relative abundance was considerably greater at T1 and T3 compared to T0 (P<0.05) (Fig. 7, b). The abundance of Lautropia increased significantly at T1 (P<0.05), then decreased to a similar level at T3 compared to T0 (Fig. 7, b). Compared to T3, the abundances of Veillonella, Pseudomonas, Streptococcus, Corynebacterium, Prevotella, Actinomyces, Capnocytophaga, and Selenomonas were higher at T0. Conversely, the abundances of Fusobacterium, Neisseria, Porphyromonas, Rothia, Peptidiphaga, and Aggregatibacter were lower at T0 compared to T3.

Clear aligners have increased in popularity in recent years as an alternative to traditional fixed appliances, particularly amongst adult patients[1]. Many studies investigating the impact of clear aligners and fixed appliances on oral health have concluded that clear aligner treatment is more advantageous for maintaining patients' periodontal health and plaque control compared to fixed orthodontics[6, 19, 20]. The primary reason for this is that clear aligners are removable and are more convenient for oral hygiene. The majority of studies examining the microbiological alterations in patients using clear aligners have focused on variations in the abundance of certain periodontopathogens and cariogenic bacteria[13, 15, 21, 22]. The changes in the oral microbial ecology have, however, not been extensively studied. In this three-month prospective study, we investigated the shift in the aligner tray plaque and subgingival plaque microbial community of clear aligner patients treated with Invisalign system by using 16S rRNA gene sequencing technique.

During orthodontic treatment, several variables effect periodontal health, including plaque accumulation, host immunity, hormone levels, the microbiological background of patients, and tooth mobility[23]. Among the various risk factors, plaque buildup is considered the most significant contributor to periodontal diseases. In our research, we observed a gradual decline in PI as well as improvements in patients’ periodontal health. When compared to T0, PI and PD was significantly decreased at T1 and T3, and BI showed decreasing trend. In addition, patients increased their tooth brush frequency during the orthodontic therapy (Table 1). The increased tooth brush frequency resulted in less plaque accumulation that contributed to maintaining good periodontal health during clear aligner treatment.

In aligner tray plaque, alpha diversity indices were significantly lower at T3 than at T0 (Fig. 1, a). This indicated that composition of tray plaque microbiota changed and aligners assembled a less diverse microbiota population. Dental plaque is a complicated biofilm that forms on the enamel of the teeth. Bacterial colonization first occurs through initial attachment of colonizers to the salivary pellicle on enamel, which is then followed by secondary colonization processes through interbacterial adhesion[24]. However, no study has been conducted on the biofilm formation process on the inner surface of aligners. Because of the various physicochemical qualities of aligner material and enamel, bacteria may have distinct affinities for teeth and aligners. Furthermore, clear aligners cover the whole tooth surface, resulting in a closed environment. This may inhibit the growth of some kinds of microorganisms. We hypothesize that these characteristics could potentially result in a reduction of the richness and diversity of plaque communities on the inner surface of aligners.

Gingivitis is caused by biofilm persistence at the gingival margin and in the gingival sulcus, and it may proceed to periodontitis, which results in the permanent loss of tooth-supporting components, in susceptible patients[25]. It is possible that a noticeable change in the microbial community, rather than the variations of particular periodontopathogens, is to blame for periodontal disorders[10]. The findings of this study showed that the richness and diversity indices of subgingival microbiota were comparable during orthodontic treatment (Fig. 1, b). This indicated that the subgingival microbial community structure was relatively stable over the course of clear aligner treatment. The following two considerations help explain this phenomenon. On the one hand, patients raised the frequency of their regular tooth brushing from 2.00 ± 0.38 to 3.13 ± 0.64 per day. Plaque biofilm was periodically removed from gingival margin, which helped avoiding gingival inflammation. On the other hand, the margin of Invisalign aligners is designed as scalloped, which does not make contact with gingiva and does not bring the bacteria on the aligner into the gingival sulcus.

Zhao et al. employed 16S rRNA gene sequencing to define and compare the salivary microbiota diversity of aligner patients before and six months after the treatment[11]. Their study results indicated that microbial composition of saliva did not alter significantly during the treatment. However, in tray plaque samples, significant differences can be seen between the microbiota composition at T0 and T1and T3 according to the beta analysis in our study. The major phyla identified in both studies were the same, but they were scattered in different abundances. Furthermore, we discovered bigger discrepancies in the outcomes of the two investigations at the genus level. This may be explained by two factors. On the one hand, saliva seeps into the spaces between the teeth and the aligners and contributes to the formation of tray plaque biofilm. On the other hand, the environment created by the aligner and the specific physicochemical properties of the aligner material accumulates unique microbiota that differ from the salivary microbiota. Dong et al. conducted a study to examine alterations in the microbiota of the inner surface of clear aligners after 24 hours of usage[27]. They discovered significant changes in bacterial distribution after a full day of wearing the aligner. At phylum level, the relative abundance of Firmicutes was increased, while Actinobacteria and Proteobacteria were decreased. At genus level, the abundance of Streptococcus, Haemophilus, and Porphyromonas increased, whereas that of Rothia, Lautropia, and Actinomyces decreased. Interestingly, we discovered similar changings in our research. This suggests that the structure of the plaque biofilm on the aligners is likely to remain stable in the short-term or long-term.

Previous researches have suggested that elevated levels of Streptococcus and Actinomyces are linked with caries and white spot lesions, while an increase in Corynebacterium is associated with good oral health [28–30]. Jiang et al. found no notable variance in the relative abundance of Haemophilus among patients with and without caries[31]. In our study, we observed that the abundance of Streptococcus and Haemophilus significantly increased, and the abundance of Actinomyces and Corynebacterium decreased significantly in the tray plaque microbiota. Streptococcus is made up of several opportunistic pathogens such as Streptococcus salivarius, Streptococcus constellatus, Streptococcus sobrinus, Streptococcus parasanguinis, and Streptococcus mutans, which are proficient in lactic acid production and can increase the risk of caries development[32–34]. Dong et al. observed a decline in the pH of the aligner liquid as the duration of usage increased[27]. Clear aligner creates a closed environment and impedes the self-cleansing and buffering effect of saliva. Hence, we conclude that the increased Streptococcus level and the closed environment may account for the elevated acidity of the aligner liquid contents. This also implies that aligners should be cleaned on a regular basis in order to avoiding of developing white spot lesions or/and caries.

There is a strong relationship between the subgingival plaque composition and periodontal health [35, 36]. The general structure of the microbial community may be indicative of the periodontal condition. In our research, although subgingival microbial composition and structure were rather consistent before and after the treatment, substantial changes in abundance were seen in some bacteria across time periods. At genus level, we discovered that Haemophilus significantly increased at T3 compared to T0, while Lautropia showed a significantly increased abundance at T1 compared to T0 then declined to baseline level at T3 (Fig. 7, b). Previous studies indicated that Proteobacteria and Lautropia were more abundant in healthy periodontal pockets than diseased periodontal pockets[35, 36].

In this study, we investigated the effects of clear aligners on patients' periodontal health and changes of microbiota composition in the clear aligner tray plaque and subgingival plaque during the first three-month of orthodontic therapy. The results showed that clear aligners have no significant impact on periodontal health and subgingival microbiota composition of patients. The structure of the aligner tray microbiota altered significantly at both phylum and genus levels and attracted a unique and less divers microbiota community. In order to protect the integrity of enamel and periodontal health, patients are recommended to clean their teeth and aligners on a regular basis during clear aligner treatment. The primary limitations of our study were the limited number of participants and short observation period. To gain a more comprehensive understanding of the influence of clear aligners on oral health and oral microbiota, future research with a larger sample size and extended observation duration is required.

Author Contribution

Maierdanjiang Rouzi performed the sample collection, analyzed the data, and wrote and revised the manuscript. Qingsong Jiang, Haoxin zhang carried out clinical data collection and data analyses. Xiaolong Li helped perform the analysis with constructive discussions. Hu Long guided the analyses of oral microbiome. Wenli Lai contributed to the conception of the study.

Conflict of interest

The authors declare no competing interests

Funding

This work was supported by the National Nature Science Foundation of China (NSFC, NO. 82071147, NO. 82171000) and 2022 EMEA and APAC Align Research Award.

Ethical Approval

The research received formal ethical approval from the Institutional Review Board of XXX Hospital of Stomatology (WCSHIRB-D-2021-443), where the study was initiated.

Consent to Participate

All participants gave written consent.

Data availability statement

Derived data supporting the findings of this study are available from the corresponding author on request.

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No competing interests reported.

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published 29 Sep, 2023

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Editorial decision: Major revision
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Characteristics of oral microbiota and oral health in the patients treated with clear aligners: a prospective study

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Journal Publication

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Abstract

Objectives

Methods

Results

Conclusions

Clinical Relevance:

Figures

Introduction

Material and methods

Subject population

Clinical examination and sample collection

DNA extraction

16S rRNA Gene Amplicon Sequencing

Sequence analysis

Bioinformatics and statistical analysis

Results

Clinical results

Sequence analysis

Alpha diversity

Beta diversity

Microbial composition and relative abundances

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1