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Objective: Previous studies reported that bleaching agents are capable of altering the outer enamel or exposed dentine surfaces. In this study, transmission electron microscopy (TEM) characterisation in combination with energy dispersive x-ray spectroscopy (EDS) elemental mapping was performed to investigate the effect of hydrogen peroxide (HP) on the sub-surface of tooth structures. Materials and Methods: Bovine incisors (n=6) were assigned to three groups for treatments: 30% H 2 O 2 pH 3, 30% H 2 O 2 pH 7 and 1M sodium hydroxide (NaOH) for 16 hours. Samples were exposed to direct treatment with agents to allow easy access of the agents and uniform treatment across the entire sample sub-surfaces. Specimens were immersed in Karnovsky's fixative for three days, embedded in epoxy and polished. Using focussed ion beam (FIB), samples were milled ~50-100 µm below the treated polished surface to feature the ultra-fine structures for TEM analysis. Results: Enamel rods, inter-rods and crystalline structures within rods were discerned for sound and H 2 O 2 treated enamel. No structural difference in the mineral dense peritubular region or fibrous protein rich inter-tubular dentine was observed after acidic or neutral H 2 O 2 treatment. A complete loss in the structural integrity of enamel rod was observed after NaOH treatment for 16 hours without having any impact on dentine structure. Conclusions: TEM analysis produced high quality sub-surface images of tooth structures and revealed no deleterious effect on the structural integrity of the sub-surface enamel or dentine after direct bleaching with hydrogen peroxide.

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