Purpose: To evaluate the effect of different enamel conditioning protocols and their re-application on the microleakage of fissure sealants placed following saliva contamination. Study design: The study included 156 human third molars in 16 subgroups (2×4×2) under two main groups (sealant type): Group A- hydrophobic resin sealant, 3M Clinpro Sealant; Group B- hydrophilic resin sealant, Ultraseal XT Hydro. Each group was then divided according to the type of surface conditioning; 1- Er,Cr:YSSG laser etching, 2- acid-etching, 3- acid-etching+etch-and-rinse adhesive (Prime&Bond® One Select) and 4- self-etching adhesive (Clearfil SE Bond). After contaminating the conditioned occlusal enamel surfaces with artificial saliva, fissure sealant was applied in half of the specimens (a), whereas in the other half, (b) the respective surface conditioning was repeated and then fissure sealant was placed. Following thermocycling, the samples were immersed in basic fuchsin, sectioned, and dye penetration was quantitatively assessed with ImageJ. Two-way ANOVA and Bonferroni post-hoc tests were used for statistical analyses (p<0.05). Results: The least microleakage was observed in A3b and A3a, whereas B4b and B4a were the subgroups with the highest microleakage. Following saliva contamination, when surface conditioning was not re-applied, the effects of fissure sealant types and surface conditioning were significant (p=0.005 and p<0.001, respectively). However, their interaction was insignificant (p=0.173). When surface conditioning was re-applied after saliva contamination, the effects of type of fissure sealant and surface conditioning (p=0.000, for both) and their interaction (p=0.004) were significant. Conclusions: 3M Clinpro Sealant was superior to Ultraseal XT Hydro. Re-application of Er,Cr:YSSG laser and the self-etching adhesive did not affect the microleakage of both fissure sealants. Without re-application, acid-etching+etch-and-rinse adhesive was superior to acid-etching only. However, both of them were similarly successful when they were re-applied following saliva contamination.

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