C o p y r i g h t b y N o t f o r Q u i n t e s s e n c e Not for Publication O ver the past few decades, a significant decline in caries prevalence has been observed in developed countries. 1 However, this decline has not been uniform for all tooth sur- faces. Epidemiological data show that pit-and-fissure sur- faces carry more than 85% of the total caries burden in US schoolchildren. 21 This finding is highly interesting in that these sites constitute only 12.5% of total tooth surfaces. 39 It has been widely accepted that pit-and-fissure sealants are the most effective means of reducing caries risk that arises from these sites. 37 Since its introduction by Buonocore 3 in 1955, acid etching has been an indispensable step of fissure sealant applica- tion. The rationale for the etching procedure is to create mi- croporosities into which the flow of resin-based materials is enhanced through increased wettability of enamel. The ap- plied resin hardens in taglike projections that attach the ma- terial to the surface. The effectiveness of the sealant is as- sessed by its retention and the ability to resist microleakage on the tooth surface. When lacking, the condition may lead to the formation or progression of caries on occlusal sur- faces. 36 In cases where the sealant is applied without rubber-dam isolation, changing of cotton rolls after rinsing off the etchant is a critical step in which salivary contamination of- ten occurs. Saliva contamination of the etched surface for as short as 1 second results in the formation of surface coat- Quantitative Microleakage EvaluatIon of Fissure Sealants Applied With or Without a Bonding Agent: Results After Four-year Water Storage In Vitro Zafer C. Cehreli a /H. Cem Gungor a Purpose: To evaluate the effect of long-term water storage on the microleakage of a fissure sealant applied with or with- out different bonding agents. Materials and Methods: Extracted human third molars were randomly assigned into 8 groups (n = 24/each). The oc- clusal surfaces were sealed with a fluoride fissure sealant material (Helioseal F) after one of the following pretreatments: 1. phosphoric acid etching only; 2. phosphoric acid etching + Single Bond; 3. phosphoric acid etching + Prime & Bond NT; 4. Clearfil SE Bond; 5. FL Bond; 6. One Up Bond F; 7. Prompt L-Pop; 8. Mac Bond II. All specimens were thermocy- cled (1000X), after which half of them (n = 96) were stored in distilled water for 48 h (group A), and the remaining half (n = 96) for 48 months (group B). The specimens were immersed in 0.5% basic fuchsin solution, sectioned, and digitally photographed. Microleakage was evaluated quantitatively using an open-source image analysis toolkit (ImageJ), and the data were analyzed statistically. Results: Four-year water storage significantly increased the amount of leakage in all test groups (p < 0.001). In both the absence and presence of water aging, the etch-and-rinse adhesives yielded the lowest microleakage scores (p < 0.001). In the 48-h group, the following ranking was achieved in terms microleakage values: phosphoric acid + Prime & Bond NT = phosphoric acid + Single Bond < Prompt L-Pop = phosphoric acid etching only < FL Bond < Clearfil SE Bond < Mac Bond II = One Up Bond F. In the 48-month group, the ranking changed as follows: phosphoric acid + Prime & Bond NT = phosphoric acid + Single Bond < Prompt L-Pop = FL Bond < Clearfil SE Bond = Mac Bond II = One Up Bond F < phos- phoric acid etching only. Conclusion: Long-term water storage significantly increased the microleakage of Helioseal F applied alone and with a bonding agent. Regardless of the storage term, the use of etch-and-rinse adhesives resulted in significantly less mi- croleakage compared to that achieved with self-etching adhesives or acid etching alone. The sealants placed without a prior bonding agent showed the greatest amount of leakage after four years. Keywords: microleakage, quantitative, etch-and-rinse adhesives, self-etching adhesives, fissure sealants. J Adhes Dent 2008; 10: 379-384. Submitted for publication: 27.07.07; accepted for publication: 19.09.07. Vol 10, No 5, 2008 379 a Associate Professor, Department of Pediatric Dentistry, Faculty of Dentistry, Hacettepe University, Ankara, Turkey. Correspondence: Dr. H. Cem Gungor, Department of Pediatric Dentistry, Fac- ulty of Dentistry, Hacettepe University, Sihhiye 06100, Ankara, Turkey. Fax: +90-312-324-3190. e-mail: hgungor@hacettepe.edu.tr