A preliminary study on the relationship between iron and black extrinsic tooth stain in children

Black extrinsic tooth stain, which has long troubled many people, is common among children and influences the aesthetics of teeth. The pigment was proposed to be a black insoluble ferric compound, but this is controversial. To determine whether iron exists in black stain, we collected 10 samples of black stain and 10 samples of plaque separately from children with and without black stain using sterile titanium implant curettes, and analysed the samples by inductively coupled plasma-mass spectrometry. Iron was present in both black stain and plaque, with concentrations ranging from 76·12 to 1116·88 μg g⁻¹. The contents of iron in black stain were significantly higher than in plaque. Because bacteria may be involved in the aetiology of black stain, we assessed the functional genes of bacteria in black stain based on 16S rRNA gene sequencing results obtained using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States. Of 253 Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways tested, 56 differed in abundance between samples from children with and without black stain. Genera altered in black stain were related to many of the pathways. Some KEGG Orthology groups showed differences between black stain and plaque of control group were found to be related to iron. Significance and Impact of the Study: In this study, we have confirmed the existence of iron in black extrinsic tooth stain by ICP-MS. It was the first time the functional genes of bacteria in black stain were accessed and the genes associated with iron were found. These findings provided clues on the research of aetiology of black stain, which troubled millions of children. It also revealed the association between metabolic pathway of microbiota and oral phenomenon.