Fixed appliances are the most widely used devices in clinical orthodontics. They consist of bands, brackets, and archwires, providing broad indications with precise control of tooth movement (Fleming and Johal, 2010). These appliances are bonded directly to the enamel surface, which easily create plaque-retentive areas that hinder oral hygiene. This increases the risks of plaque accumulation, which can lead to enamel demineralization, dental caries, and gingival inflammation (Ren et al., 2014; Miller et al., 2016; Salerno et al., 2024). In contrast, clear aligners are removable appliances which were manufactured from thermoplastic materials using advanced computer-aided design and computer-aided manufacturing (CAD/CAM) technology with characteristic of esthetic appearance and comfort (Tartaglia et al., 2021; Bichu et al., 2023). Patients can easily remove clear aligners to clean their teeth. This helps reduce plaque retention and maintains periodontal health. However, effective treatment requires wearing aligners for more than 22 hours daily, which may compromise the natural cleansing effect of saliva and promote plaque accumulation on tooth surfaces and inside the aligners (Galan-Lopez et al., 2019). Therefore, a systematic comparison of these two orthodontic appliances and their impact on the oral microenvironment is essential for optimizing treatment strategies.

Recent studies have mainly compared the effects of fixed appliances and clear aligners on clinical periodontal parameters such as plaque index and bleeding on probing, with a prevailing consensus that clear aligners exert less adverse impact on periodontal health (Chhibber et al., 2018; Jiang et al., 2018; Llera-Romero et al., 2023). Advances in molecular biology have enabled the use of high-throughput sequencing, such as 16S rRNA gene sequencing, to analyze changes in the oral microbiota. These methods sequence conserved bacterial gene regions in environmental samples, providing a comprehensive profile of microbial composition, diversity, and community structure. They also offer high sensitivity, strong specificity, and rapid detection (Youssef et al., 2009; Overmyer et al., 2021). Despite these advances, the influence of different orthodontic appliances on oral microbiome homeostasis remains debated. Some studies have shown lower subgingival microbial abundance in clear aligners users compared with fixed appliance patients, suggesting a greater capacity for maintaining microbial balance (Levrini et al., 2015). Other reports have indicated that fixed appliances may promote colonization by cariogenic bacteria and shift microbial metabolism toward an acidogenic phenotype, whereas clear aligners appear to trigger only transient inflammatory responses (Gong et al., 2024). Conversely, Wang Q et al. have found through metagenomic analysis of saliva that both orthodontic methods exert microbial dysbiosis, and clear aligners show no significant advantage (Wang et al., 2019). These inconsistencies highlight the need for further studies integrating multidimensional indicators to clarify the underlying mechanisms.

Reactive oxygen species (ROS), a collective term for oxygen-derived reactive molecules such as hydroxyl radicals and superoxide anions, play extensive biological roles in living organisms (Dumitru et al., 2025). The sources of ROS can be classified into two major categories: endogenous and exogenous. Endogenous sources derive from cellular and mitochondrial metabolism, immune activity, and oral microbiome imbalance. While, exogenous sources originate from alcohol, smoking, xenobiotics, dental materials, diets, and radiation (Chen et al., 2019). Previous study showed that ROS play dual roles in maintaining oral microbial homeostasis (Baltacıoğlu et al., 2014). During dysbiosis, pathogenic bacteria, including Fusobacterium nucleatum and Porphyromonas gingivalis, stimulate host neutrophils to release excessive ROS, thereby inducing oxidative stress and driving chronic inflammation. On the other side, physiologically generated ROS from commensal bacteria can suppress pathogen colonization and support microbial balance (Tamaki et al., 2009; Kang et al., 2019; Sies and Jones, 2020). Substantial evidence has linked ROS to the pathogenesis of periodontitis and dental caries (Liu et al., 2017; Yu et al., 2023; Pouliou and Piperi, 2024). However, the dynamic fluctuations of ROS during orthodontic treatment and their underlying interactions with the oral microbiota remain poorly investigated.

Therefore, this study aims to integrates clinical periodontal assessment, 16S rDNA gene sequencing, and ROS detection. It systematically compared the effects of fixed appliances and clear aligners on periodontal indices, oral microbial community structure, and ROS levels. The study further investigates the correlations between ROS, oral microbiota, and clinical periodontal parameters, thereby providing a theoretical basis for the development of antioxidant and antimicrobial materials tailored for orthodontic patients.

This study comprehensively evaluated the potential impact of clear aligners and fixed appliances on the oral micro environmental and periodontal health by analyzing saliva, supragingival plaque, and GCF samples collected at T0, T1, and T2. High-throughput 16S rDNA sequencing was integrated with clinical periodontal indices (PI, GI, BOP, PD) and ROS detection. The findings revealed differences between the two orthodontic modalities in maintaining of periodontal health, microbial community composition and functional potential, and oxidative stress levels.

The present study observed a progressive increase in PI, GI, PD and BOP in patients treated with fixed appliances as treatment duration extended. Conversely, patients in the CA group exhibited only minor fluctuations in PI and PD (Figure 2). These results demonstrated that clear aligners might provide potential advantage over fixed appliances in limiting plaque accumulation and reducing gingival inflammation. At T1 and T2, PI was significantly higher in FA group compared with CA group (Figure 2). These differences were likely attributable to the inherent design of the two orthodontic systems. The brackets, archwires, and bands of fixed appliances generate multiple plaque-retentive sites that were relatively difficult to clean thoroughly. On the contrary, the removability of clear aligners facilitated effective oral hygiene practices and might lowered the burden of plaque control (Kozak et al., 2021; Manphibool et al., 2023; Rouzi et al., 2023). As a sensitive indicator of gingival inflammation, BOP was used for early gingivitis detection and patient monitoring. Our results suggested that clear aligners might alleviate gingival inflammation. Moreover, our findings were also consistent with those reported by Chhibber et al, further supporting the potential positive role of clear aligners in maintaining periodontal health during orthodontic therapy (Chhibber et al., 2018).

To comprehensively evaluate changes in oral microecology, three representative sample types were selected: saliva, supragingival plaque, and GCF. Saliva offers a comprehensive representation of the diversity and abundance of oral microorganisms and has been widely recognized as a reliable tool for assessing the risk of caries and periodontal diseases (Haririan et al., 2014; Pedersen et al., 2018; Garcia et al., 2024). Supragingival plaque reflects the diversity and quantity of cariogenic bacteria, which are critical in the development of caries (Qudeimat et al., 2021), while GCF serves as an indicator of gingival and periodontal inflammatory status (Bibi et al., 2021; Yang et al., 2021). In this study, microbial communities of saliva, supragingival plaque, and GCF were profiled using 16S rDNA high-throughput sequencing for comprehensive and sensitive assessment (Shen et al., 2022).

Alpha diversity analysis reflects species richness and evenness within individual samples. Our study showed that, no significant differences were found in Chao1, ACE, Shannon, Simpson indices between CA and FA groups or among different time points in each group. These results were align with those of Liu et al (Liu et al., 2024), indicating that during the early treatment phase, neither appliance type significantly affected the overall richness and evenness of the oral microbiome. Beta diversity analysis assesses inter-sample community dissimilarities. Interestingly, our results demonstrated an obvious relatively separation of microbial community structures between CA and FA groups in saliva, supragingival plaque, and GCF (Figure 4). These findings suggested that while the “quantity” of microbial diversity remained largely stable, the “quality” of microbial community composition was to some extent influenced by different types of orthodontic appliances. The divergence might be attributed to two main factors. First, the surface characteristics of fixed appliances or clear aligners played an important role (Low et al., 2011; Kozak et al., 2021). The rough surfaces of fixed brackets and bands were prone to promote biofilm retention. Similarly, the long-term wear of clear aligners could lead to micro-abrasions and fissures on the surfaces of aligners which also favorable for the colonization of the plaque. Second, distinct impacts on the oral microenvironment, including variations in local oxygen tension and pH, contribute to this divergence. Notably, previous research had shown that as early as 48 hours after clear aligners placement, specific bacterial taxa (e.g., Streptococcus and Corynebacterium) could be detected colonizing on clear aligners’ surfaces (Low et al., 2011).

At the phylum level, Firmicutes, Actinobacteria, Proteobacteria, Fusobacteria, and Bacteroidetes were consistently identified as dominant taxa in both groups. Within Firmicutes, several genera were closely associated with oral diseases, including Streptococcus, Lactobacillus, and Veillonella. Among them, Streptococcus mutans which were highly acidogenic and aciduric, facilitated biofilm formation, and contributed to enamel demineralization (Shiotsu-Ogura et al., 2019). Whereas, Streptococcus sanguinis functioned as an early colonizer which might be potentially involved in plaque initiation. Actinobacteria also constituted a critical component of the oral microbiome, with Actinomyces species frequently linked to various oral pathologies. Fusobacteria were another key group, exemplified by Fusobacterium nucleatum, which played an important role in periodontal disease, caries, oral infections, and systemic disorders. Likewise, Bacteroidetes also had a huge impact on oral health and disease. For instance, genera such as Porphyromonas, Prevotella, and Tannerella were strongly implicated in periodontal disease, caries, and overall health condition (Yang et al., 2025).

Genus-level analysis showed that Prevotella was significantly more abundant in saliva and supragingival plaque samples from FA group than CA group. Previous studies had identified Prevotella as a key periodontal pathogen that produced diverse virulence factors capable of directly damaging periodontal tissues or inducing host immune responses, thereby driving periodontitis (Jiang et al., 2014; Sharma et al., 2022). Among its species, Prevotella intermedia had been reported to co-aggregate with Porphyromonas gingivalis and Fusobacterium nucleatum to form subgingival biofilms, enhancing pathogenicity and accelerating disease progression (Das et al., 2023). The present findings suggested that fixed appliances might be associated with the colonization of Prevotella, thereby increasing the potential risk of periodontitis. These results were consistent with those of Zheng et al., who also noted elevated Prevotella abundance in patients treated with fixed appliances (Zheng et al., 2024). Furthermore, the positive correlation between Prevotella and PI was reported previously, which supported the higher PI in FA group (Xie et al., 2025). This indicated that fixed appliances might facilitate plaque accumulation, with Prevotella contributing to biofilm formation and heightened inflammation risk.

In saliva, the FA group also showed significantly greater abundance of Veillonella at both T1 and T2 compared with CA group. Elevated Veillonella levels had been reported in periodontitis patients, where their metabolic products could impair periodontal tissues and trigger inflammatory responses (Ortiz et al., 2022). The current findings indicated that fixed appliances might be linked to Veillonella proliferation, thereby increasing susceptibility to periodontal inflammation. This observation was consistent with the findings of Shokeen Bhumika et al. who reported that fixed appliances might increase the risk of periodontal inflammation (Shokeen et al., 2022).

The present study demonstrated that the relative abundance of Lactobacillus was significantly higher in saliva, supragingival plaque, and GCF samples from the FA group compared with the CA group. Previous studies had shown that Lactobacillus, characterized by its acidogenic and aciduric properties, acted as a major cariogenic bacterium involved in the initiation and progression of dental caries (Gondo et al., 2024). Elevated levels of Lactobacillus had consistently been observed in saliva and supragingival plaque of caries patients compared with healthy individuals, supporting the notion that fixed appliances might facilitate its proliferation and thereby increase the risk of caries.

Similarly, Corynebacterium was significantly enriched in saliva and GCF samples from the FA group relative to the CA group. Evidence indicated a strong association between Corynebacterium and the onset of dental caries, highlighting its potential role in disease initiation (Jiang et al., 2014). The present findings therefore suggested that fixed appliances were easier to create an oral environment conducive to the growth of cariogenic bacteria, ultimately increasing caries susceptibility. This interpretation was consistent with the results of Wang and Shokeen Bhumika et al., further reinforcing the conclusion that fixed appliances might pose more risks for caries development (Wang et al., 2019; Shokeen et al., 2022).

Moreover, at both T1 and T2, the relative abundance of Rothia and Lautropia in GCF was significantly higher in CA group compared with FA group. Previous studies had demonstrated that Rothia contributed to oral health through its immunomodulatory and metabolic functions, with its abundance closely associated with a healthy oral status (Liu et al., 2023; Lundtorp Olsen et al., 2023). Similarly, Lautropia was frequently detected in healthy individuals and is regarded as a beneficial genus linked to oral homeostasis (Liu et al., 2023). The present findings therefore indicated that clear aligners were associated with the colonization of health-related taxa such as Rothia and Lautropia. These results were consistent with those reported by Zheng and Shokeen et al., supporting the notion that clear aligners might offer some unique advantages in maintaining oral health during orthodontic treatment (Shokeen et al., 2022; Zheng et al., 2024).

Functional prediction using PICRUSt2 (KEGG database) revealed distinct microbial metabolic potentials between the FA and CA groups (Figure 8). The FA group exhibited significant enrichment in carbohydrate metabolism–related pathways, including glycan biosynthesis and metabolism, glycosaminoglycan degradation, and glycosyltransferases. Carbohydrate metabolism was a central energy acquisition pathway for microbial growth, and its heightened activity is typically associated with enhanced microbial proliferation (Guo et al., 2025). Moreover, the FA group showed significant enrichment in the Type II diabetes mellitus pathway and in proteins involved in lipopolysaccharide (LPS) biosynthesis. LPS, the major structural component of Gram-negative bacterial cell walls, was a potent endotoxin and pro-inflammatory mediator (Hicks and Jia, 2018). The FA group showed increased abundance of Gram-negative pathogens such as Prevotella, along with higher periodontal inflammation indices and elevated ROS levels. These observations were associated with enrichment in carbohydrate metabolism-related pathways dependent microbes, including Gram-negative pathogens, and LPS biosynthesis proteins. The FA group exhibited enrichment in pathways potentially linked to local inflammatory responses and oxidative stress, which was consistent with the compromised periodontal status. It is important to note that PICRUSt2 inferences represent potential functions, not measured activities. The predicted enrichment of the Type II diabetes mellitus pathway based on metagenomic functional prediction merely reflected the differences in the potential function of the microbial community rather than direct changes in host systemic glucose metabolism. The clinical significance of this finding required further validation and investigation through integration with host-derived metabolic indicators.

In contrast, the CA group showed significant enrichment of lipid metabolism-related pathways, including lipid metabolism, adipocytokine signaling, and fatty acid metabolism. Lipids and their metabolites played essential roles in maintaining cell membrane stability, mediating signal transduction (including inflammatory signaling), and regulating energy storage (Parsons and Rock, 2013; VanHook, 2020). Previous studies suggested that specific lipid metabolic activities could modulate inflammatory responses and alleviate oxidative stress (Andersen, 2022). In this context, clear aligners influenced the microbiota by enhancing lipid metabolic activity, which showed enrichments in pathways associated with lipid metabolism. These functional features might be associated with the relatively stable periodontal condition and reduced ROS levels observed in the CA group.

ROS is a kind of oxygen-containing molecules or free radicals with high reactivity. Most ROS exist in the organism for a very short time, and it will quickly react with other molecules or be scavenged by the antioxidant system (Dumitru et al., 2025). Previous studies have identified that the stable metabolites of ROS mainly include lipid peroxidation products (Malondialdehyde, 4-Hydroxynonenal), DNA oxidative damage markers (8-OHdG) and oxidized glutathione. Among these, 8-OHdG is the most stable product of oxidative DNA damage caused by ROS. Thus, in our study, 8-OHdG was detected to indirectly reflect ROS levels with ELISA kit based on highly sensitive double-antibody sandwich method. Two specific antibodies bind to different epitopes of the target antigen, forming an “antibody-antigen-antibody” sandwich complex. This enables highly sensitive and specific quantitative or qualitative detection (Lu et al., 2025). Given the short half-life and low concentrations of ROS in the oral environment, this technique offers a reliable and effective approach for accurate measurement.

Recent studies showed that long-term psychological stress can disrupt the redox balance, leading to excessive accumulation of ROS, which exceeds the antioxidant defense capacity of cells, triggering oxidative stress and subsequently damaging biological macromolecules such as DNA, RNA, proteins and lipids (Yu et al., 2024). Thus, in order to minimize the influence of psychological stress on ROS, we used the PSS-10 to control the confounding factors. Our results showed that there were no significant differences between FA group and CA group in T0, T1, and T2. Therefore, we considered that the ROS levels measured in this experiment were reliable. Our study demonstrated the temporal changes of ROS levels in saliva and GCF during the orthodontic treatment (Figure 9). In saliva, there were no significant differences of ROS levels in either FA group or CA group during treatment duration (T0, T1 and T3). Similarly, no significant differences of ROS levels in either FA group or CA group in GCF samples during treatment duration (T0, T1 and T3). Although there were no significant temporal changes of ROS levels in either saliva or GCF, the ROS levels in FA group were obviously higher than those in CA group at T2 with statistical significance (P < 0.001). As GCF originated directly from gingival tissues, it could be viewed as a sensitive marker of local periodontal inflammation and oxidative stress (Bibi et al., 2021). The pronounced elevation of ROS in GCF at T2 indicated that fixed appliances might exert greater oxidative stress on periodontal tissues compared to clear aligners in orthodontic treatment process. Specifically, several mechanisms might explain this effect. First, degradation of metallic components such as brackets and archwires in the FA group might release nickel and chromium ions into the oral cavity, catalyzing ROS production through Fenton-type reactions (Abedini et al., 2009; Mahmud et al., 2018). Second, increased plaque accumulation and gingival inflammation associated with fixed appliances, reflected by higher GI values, might activate neutrophils and other immune cells, leading to respiratory bursts which could release large amounts of ROS (Kanzaki et al., 2017). Third, alterations in microbial community structure, including enrichment of pathogenic genera (Prevotella and Veillonella), as well as enhanced metabolic activities such as elevated LPS production and carbohydrate metabolism, might also directly or indirectly stimulate ROS generation (Kang et al., 2019).

In saliva, ROS levels in FA group were significantly positive correlated with the relative abundance of Prevotella (r = 0.61, P < 0.05). Previous studies (Ibrahim et al., 2017) have shown that Prevotella and its metabolic products could stimulate immune cells to produce ROS through their pro-inflammatory effects. Prevotella abundance was strongly negatively correlated with Rothia abundance (r = -0.90, P < 0.001). As a potential beneficial genus, Rothia likely limited the proliferation of pathogenic bacteria such as Prevotella through competitive interactions or inhibitory substances (Lundtorp Olsen et al., 2023). These associations suggested that the salivary microenvironment in FA group might shift towards a pro-inflammatory and pro-oxidative state.

In GCF, ROS levels in CA group showed a significantly negative correlation with the relative abundance of Rothia (r = -0.65, P < 0.05). Considering the enrichment of Rothia observed in CA group, this finding indicated that Rothia or the microenvironment mediated by Rothia might exert antioxidant effects or mitigate oxidative stress, thereby providing a potential lead for developing microbiota-based antioxidant strategies.

A significant positive correlation was identified between GI and ROS levels in the FA group (Figure 11B). This observation aligned with the classical model in which plaque biofilms, reflected by increased GI, stimulated gingival tissues and elicited inflammatory responses. Activated neutrophils infiltrated the tissues and released large amounts of ROS. Excessive ROS subsequently caused periodontal damage such as protein and DNA oxidation and fibroblast apoptosis, thereby perpetuating a vicious cycle (Tamaki et al., 2009; Kanzaki et al., 2017). These findings further integrated the microbial alterations observed in FA group (increased pathogenic taxa) with the deterioration of periodontal parameters (elevated GI) and the enhancement of oxidative stress (increased ROS).

Several limitations should be noted. Notably, predicted functions from PICRUSt2 represent potential rather than actual microbial metabolic activity. First, the relatively small sample size (n = 12 per group; statistical power > 0.8; PASS 2021 software) largely dictated by practical and economic constraints might have reduced statistical power and limited the detection of subtle differences. Second, microbial functional analysis was based on 16S rDNA sequencing-derived predictions (PICRUSt2). These predictions represent potential functional capabilities of the microbial communities rather than direct measurements of microbial activity. While this method provides insights into functional potential, its accuracy is relatively lower than that of shotgun metagenomic or metatranscriptomic sequencing. Third, the associations observed between specific taxa and ROS levels were primarily statistical and lacked direct in vitro or in vivo validation to establish causality or clarify mechanistic pathways.

To overcome these limitations, future research should address these issues by increasing sample size and conducting multicenter studies to enhance generalizability. Advanced techniques such as metagenomic or metatranscriptomic sequencing could offer higher-resolution insights into microbial functional activities. Direct experimental validation in biofilm or animal models would help clarify the causal roles and molecular mechanisms of key taxa, such as Prevotella and Rothia, in ROS generation, inflammation, and periodontal tissue damage. Moreover, investigating microbiome-targeted interventions (e.g., probiotics or prebiotics) or antioxidant strategies may provide novel approaches to alleviate orthodontic treatment-related periodontal complications, particularly in patients with fixed appliances. Such studies will deepen our understanding of how different orthodontic modalities influence oral health and will provide stronger evidence to optimize periodontal management during orthodontic treatment.

This study examined the impact of clear aligners and fixed appliances on the oral microbiota of orthodontic patients. It also explored the relationship between microbial composition and ROS levels. Fixed appliances were associated with a higher relative abundance of periodontal and cariogenic pathogens, indicating a potential increased risk of periodontitis and caries. In contrast, clear aligners were linked to greater enrichment of beneficial taxa and observed alongside lower ROS levels in GCF, reflected by more favorable ROS indices. These findings suggest that clear aligners may provide potential advantages in preserving microbial homeostasis and reducing oxidative stress-related damage during orthodontic treatment. Additionally, correlation analysis identified Prevotella (a periodontal pathogen) and Rothia (a potentially beneficial genus) as key taxa likely involved in ROS modulation based on correlation analysis. These insights highlight promising research for monitoring oral health in orthodontic patients, preventing periodontal and caries-related complications, and guiding the development of novel oral materials with dual antimicrobial and antioxidant functions.