Author's personal copy Protective effect on enamel demineralization of a CPP–ACP paste: an AFM in vitro study

Objectives: The aim of the present in vitro study was the evaluation of a CPP–ACP paste (Tooth Mousse) on preventing dental erosion produced by a soft drink (Coca Cola), using Atomic Force Microscopy (AFM). Methods: Thirty extracted human central incisors free of caries were selected and divided in a treatment and a control half. The treatment halves were divided in three groups—group 1: demineralization with soft drink (4 intervals of 2 min); group 2: demineralization with soft drink (4 intervals of 2 min) plus ToothMousse; group 3: intact enamel plus ToothMousse. In groups 2 and 3 Tooth Mousse was applied for 3 min at 0, 8, 24 and 36 h. The surface of each specimen was imaged by AFM and Rrms values were registered. Results: Among treatment specimens of groups 1 and 2, a statistically significant difference (P < 0.01) in Rrms values was registered: treatment of the specimens with CPP–ACP paste had a protective effect on enamel demineralization. In group 3 no statistically significant difference was registered between exposed and not exposed halves of the specimens. Conclusions: The use of a CPP–ACP paste had a protective effect on enamel demineralization in an in vitro model. # 2009 Elsevier Ltd. All rights reserved. * Corresponding author at: Department of Operative Dentistry, Policlinico ‘‘San Matteo’’, Piazzale Golgi 3, 27100 Pavia, Italy. Tel.: +39 0382 516257/3398 124925; fax: +39 0382 516224. E-mail address: [email protected] (C. Poggio). avai lab le at www.sc iencedi rect .com journal homepage: www.intl.elsevierhealth.com/journals/jden 0300-5712/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2009.07.011 Author's personal copy ability to stabilize calcium phosphate (ACP) in metastable solution. Through themultiple phosphoseryl residues, the CPP binds to forming nanoclusters of ACP, preventing their growth to the critical size required for nucleation and phase transformation. CPP–ACP has been demonstrated to have anticariogenic activity in laboratory, animal andhuman in situ experiments. TheCPP–ACP solutions have also been shown to significantly remineralize enamel subsurface lesions in vitro. CPP–ACP has been successfully incorporated into oral health products such as mouthrinses, sugar-free chewinggums, and a sports drink to reduce enamel erosion. Changes in tooth structure due to extrinsic factors have been widely investigated through SEM. This method requires proper specimen preparation and examination conditions: these procedures change the natural condition and/or part of the specimen structure. In contrast, Atomic Force Microscopy gives images with atomic resolution with minimal sample preparation. This technique has been widely used to characterize the erosion of enamel and dentin. More recently, also AFM nanoindentation has been applied to the study of enamel erosion. Whereas the material losses (absolute erosions) have been carefully characterized, only minor attention has been devoted to the investigation of tooth surface change during erosion and demineralization. The aimof the present in vitro studywas the evaluation of a CPP–ACP paste on preventing dental erosion produced by a soft drink, using Atomic Force Microscopy (AFM). 2. Materials and methods The overall experimental design is shown in Fig. 1. Specimens were prepared from 30 extracted human incisors free of caries and defects. After the extraction, the teeth were cleansed of soft tissue debris and inspected for cracks, hypoplasia and white spot lesions; they were disinfected in 5% sodium hypochlorite for 1 h and stored in artificial saliva (pH 7.0) during the whole experimentation. The specimens were cut longitudinally, with a high-speed diamond rotary bur with a water–air spray; onehalf served as a control, and the other half as treatment. The labial surfaces were ground using silicon carbide papers (grades 600–1200) under water irrigation to remove 50–100 mm and produce flat surfaces. Samples were placed into Teflonmouldsmeasuring 10 mm ! 8 mm ! 2 mm, embedded in flowable composite resin and polymerized. The CPP–ACP paste used was Tooth Mousse (GC Corp., Tokyo, Japan), a water based, sugar-free topical crème. A soft drink (Coca Cola, Italy) was chosen for the demineralization process. The pH at 20 8C, buffering capacity, concentration of calcium and phosphate of the beverage were measured. Allmeasurementswere performed in triplicate and average values calculated. The samples were randomly assigned to 3 groups, each made of 10 teeth: Group 1: Soft drink. Group 2: Soft drink plus Tooth Mousse. Group 3: Intact enamel plus Tooth Mousse. The treatment specimens of groups 1 and 2were immersed in 6 ml of the soft drink for 2 min at room temperature before rinsingwith deionizedwater. Four consecutive intervals of the immersion procedure were carried out. The remineralizing agents were applied onto the surface of the specimens to just cover the enamel surface without brushing in the treatment groups 2 and 3 and wiped off with distilled water washing; the matching control specimens received no treatment. The remineralizing paste was applied to the enamel surfaces for 3 min at 0, 8, 24 and 36 h. After that, the remineralizing agents were removed by rinsing the enamel surface with distilled water and the samples were immersed in artificial saliva again. 2.1. Atomic Force Microscopy (AFM) observations AFM images were collected with an Atomic Force Microscopy AutoProbe CP 100 (Themormicroscopes, Veeco), equipped with a piezoelectric scanner which can cover an area of 100 mm ! 100 mm with a range of 7 mm in the z-direction. The root-mean-square roughness, Rrms, was obtained from the AFM investigations by analysing, for each sample, at least 10 different film areas of 30 mm ! 30 mm with a resolution of 256 ! 256 pixels. From the analyses of the AFM height profiles, it was also possible to estimate the erosion cavities depth of the enamel surface. The datawere obtained by averaging on at least 20 selected lines of the image. Themeasurements were performed on the treatment specimens and on the matching controls. 2.1.1. Statistical analysis Differences in the averaged values among the groups were analyzed by ANOVA test. Statistical difference was set at P < 0.01.