Water plays antagonistic roles in dentin bonding. It is initially important to keep the interfibrillar spaces available to the infiltration of the adhesive system, but at the same time it impairs the infiltration of hydrophobic monomers, favors phase separation and enables the activity of dentin proteases. Proanthocyanidins are natural cross-linking reagents capable of increasing collagen stiffness and inactivating dentin endopeptidases. These phenolic compounds are abundantly found in grape seed extracts (GSE). Therefore, the aim of this study is to evaluate the quality of the dentin sealing promoted by the application of a two-step etch-and-rinse adhesive system using the cross-linked dry dentin technique, under constant simulated pulp pressure. The roots will be cut off and the crowns of 32 sound human molars will be adapted to a system that simulates a pressure equivalent to a 15 cm-high water column. After 24 hours, class I cavities involving half of the dentin thickness will be prepared in all teeth. Enamel and dentin will be conditioned with phosphoric acid, followed by abundant rinsing and dry blotting to keep dentin moist. Then, 20 µL of the following solutions will be applied for 60 seconds: (1) water (control solution), 5% GSE in 5% ethanol, or 5% GSE in 5% acetone. After rinsing the solutions, in half of the teeth from each group the dentin will be kept moist by removing the excess water with an absorbent paper (bonding technique control). In the other half, the dentin will be dried with air blasts for 60 seconds, uninterruptedly. Before application, a small amount of tetramethylrhodamine B isothiocyanate will be added to the Optibond S adhesive, and then it will be applied on enamel and dentin according to the manufacturer's instructions. The cavities will be restored with a composite resin and the teeth will be stored at 37oC for 24 hours in an incubator. For each group, fifty percent of the teeth will be immediate prepared for analysis and the remaining teeth will be stored in water, under pressure, for 6 months. The deionized water used in the pressure system will be replaced by a 0.1% sodium fluorescein solution, kept under pressure for additional 4 hours. Then, the teeth will be cut into 1 mm-thick slabs which will be prepared for analysis under a confocal laser scanning microscope (CLSM). The images will be evaluated as to the morphology of the interface and the quality of the dentin sealing provided by the different treatments and the results will be descriptively reported.
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