Metabolomic and metagenomic analyses reveal distinct microenvironmental signatures between clear aligner–associated and tooth surface dental plaque

Clear aligners (CAs) create unique oral microenvironments that may alter oral microbial metabolism, potentially increasing the risk of disease. However, metabolomic differences between CA-facing and tooth surface plaque remain unexplored. We compared the metabolites in dental plaque from 40 participants, including 20 undergoing CA treatment. Plaque samples were collected from both CA-facing and tooth surfaces under good plaque (GP) and poor plaque (PP) control conditions. Metabolomic profiling was performed using untargeted metabolomics, followed by sparse partial least squares discriminant analysis (sPLS-DA), network analysis, pathway enrichment, and MaAsLin3 analysis. Plaque metagenomic sequencing was conducted on 20 non-CA-treated participants to further elucidate the potential mechanisms responsible for the observed metabolite changes. Amino acids, peptides, and analogues were the most common types (20.72%) among the 2502 metabolites identified. sPLS-DA demonstrated clear separation between CA-facing and tooth surface plaque metabolomes, with significant differences validated by permutational multivariate analysis of variance, particularly under PP conditions. Tooth surface plaque showed higher metabolite abundance (GP: 386 upregulated vs. 28 downregulated; PP: 570 vs. 85). Network analysis indicated stronger interactions in PP groups, especially CA-facing plaque. The plaque index was primarily the main factor driving the tooth surface metabolomic variation. In contrast, age-dominated CA-facing profiles, with site-specific responses under varying control. Integrated metagenomic–metabolomic analyses associated amino acid metabolism with dental plaque formation. Metabolites, such as 4-aminovaleric acid betaine and raffinose, positively correlated with plaque index. PP control exhibited higher microbial diversity, enriched species, and elevated D-amino acid metabolism enzymes. CAs create unique metabolomic microenvironments characterized by reduced metabolite diversity and altered bacterial metabolic pathways. These findings provide insights into new oral health risks with aligners and highlight the need for better plaque control during orthodontic treatment.