The enamel active white spot lesion is the first clinical sign of the caries disease. Because of the mineral loss, especially in its center, such lesions are highly porous, facilitating the permeation of fluids and bacterial bio-products. The most recommended treatment for these lesions is the application of fluoride. Although fluorides are capable of remineralizing white spot lesions, such process is incomplete, mainly in the body of the lesion. Conversely, it has been shown that a caries infiltrant (Icon, DMG, Hamburg, Germany) can create a protective barrier inside the lesion and not only on its surface. That would block the diffusion channels for bacterial acids and the lesion would be sealed and stabilized. However, in the composition of this caries infiltrant there are biologically toxic components such as chloric acid and triethylene glycol dimethacrylate (TEGDMA). Taking that information into consideration and the fact that the manufacturer indicates the application of Icon in lesions reaching the first outer third of dentin, the aim of this study will be to investigate if this material diffuses through enamel and dentin and reaches the pulp cells in concentrations able to interfere with the cellular metabolism. Initially, twenty interproximal radiographs will be selected to determine the thickness of enamel and dentin in the proximal surfaces of primary molars. Then, 60 bovine incisors will be collected and from each crown a cylinder-shaped specimen containing enamel and dentin (5.6 mm diameter) will be punched. The specimens will be sanded until reaching the mean enamel and dentin thickness determined in the x-rays. The enamel surface will be subjected to a biological method to produce white spot lesions using S. mutans. Then, each specimen will be positioned in an artificial pulp chamber (APC). In the first part of the study, odontoblast-like cells (MDPC-23) will be seeded on the dentin surface of the specimens already mounted in the APCs, mimicking the architecture of the pulp tissue. On the demineralized enamel surface, the following treatments will be performed (n=10): deionized water (negative control); 35% hydrogen peroxide (positive control); Etch component (from Icon); Infiltrant component; Etch+Infiltrant components; and complete application of Icon (Etch+Dry+Infiltrant). After 72h from the enamel treatment, the MDPC-23 cells will be evaluated regarding cell viability. In the second phase of the study, the extract (culture medium containing the diffused products from the material) in contact with the cells will be collected and placed in contact with MDPC-23 cells previously seeded in the plate. After 72 hours of contact with the extracts, it will be evaluated the cell viability, total protein synthesis and the activity of alkaline phosphatase. The factor of the study will be the experimental groups (in 6 levels), while the response variables will be the cellular viability, the production of total protein and the alkaline phosphatase activity. The number of replicates by group will be 10 and the methodologies will be performed in duplicate. The statistical tests will be defined after analyzing the distribution of the dataset for each response variable and the homoscedasticity among groups to be compared. The adopted level of significance will be 5%.
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