Preventive Effect of Different Toothpastes on Enamel Erosion: AFM and SEM Studies

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1 SCANNING VOL. 36, (2014) Wiley Periodicals, Inc. Preventive Effect of Different Toothpastes on Enamel Erosion: AFM and SEM Studies MARCO LOMBARDINI, MATTEO CECI, MARCO COLOMBO, STEFANO BIANCHI, AND CLAUDIO POGGIO Section of Dentistry, Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, University of Pavia, Pavia, Italy Summary: The aim of the present in vitro study was the evaluation of new formulation toothpastes on preventing enamel erosion produced by a soft drink (Coca Cola), using atomic force microscopy (AFM) and scanning electron microscopy (SEM). Fifty enamel specimens were assigned to 10 groups of 5 specimens each. 1: intact enamel, 2: enamel þ soft drink, 3: intact enamel þ BioRepair Plus-Sensitive Teeth, 4: enamel þ soft drink þ BioRepair Plus-Sensitive Teeth, 5: intact enamel þ BioRepair Plus-Total Protection, 6: enamel þ soft drink þ BioRepair Plus-Total Protection, group 7: intact enamel þ Sensodyne Repair & Protect, 8: dentin þ soft drink þ Sensodyne Repair & Protect, 9: intact dentin þ Colgate Sensitive Pro Relief, 10: dentin þ soft drink þ Colgate Sensitive Pro Relief. The surface of each specimen was imaged by AFM and SEM. The root mean-square roughness (R rms ) was obtained from the AFM images and the differences in the averaged values among the groups were analyzed by ANOVA test. Comparing groups 4, 6, 8, 10 (soft drink þ toothpastes) Colgate Sensitive Pro Relief promoted enamel remineralization, while BioRepair Plus-Sensitive Teeth, Biorepair Plus-Total Protection and Sensodyne Repair & Protect provided lower effectiveness in protecting enamel against erosion. Significance: the use of new formulation toothpastes can prevent enamel demineralization. SCANNING 36: , Wiley Periodicals, Inc. Key words: AFM, dentistry, SEM Address for reprints: Claudio Poggio, Section of Dentistry, Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, Policlinico San Matteo, Piazzale Golgi 3, Pavia, Italy claudio.poggio@unipv.it Received 9 September 2013; Accepted with revision 18 November 2013 DOI: /sca Published online 11 December 2013 in Wiley Online Library (wileyonlinelibrary.com). Introduction Dental erosion has been defined commonly as the chemical dissolution of the hard tissues by acids of non-microbiological origin (Hemingway et al., 2006). Dental erosion is a common problem in modern societies, owing to the increased consumption of acid drinks such as soft drinks, sport drinks, fruit juices, which in turn have a high potential to provoke dental demineralization (Barbour et al., 2007). Previously erosion lesions in the cervical area were reported mostly in elderly people. However dietary changes and inadequate oral hygiene have led to erosion becoming more frequent among young people. The development of erosion involves a chemical process in which the inorganic phase of the tooth is demineralized, thereby reducing the hardness of the tooth substrates. Subsequent abrasive challenges through brushing increase the loss of the tooth substrates (Lippert et al., 2004b). The loss of substance by erosion is a dynamic process with periods of demineralization and remineralization. Thus, preventive measures against erosion are required. Dental enamel consists of 95% calcium hydroxyapatite, 4% water, and 1% organic mineral. Enamel is organized in prisms; aprismatic enamel with a thickness of up to 100 mm has been reported to be present at the enamel surface, which is generally more highly mineralized than subsurface. The early stages of dental erosion are characterized by a softening of the enamel surface to a depth of the order of 1 10 mm. Many studies have been carried out to understand the process of enamel demineralization at the early stages, but there are still little known about if these early stages are reversible (Finke et al., 2000). Biological and chemical factors in the oral environment influence the progress of dental erosion. Saliva provides protective effects by neutralizing and clearing the acids; it is also a source of inorganic ions necessary for the remineralization process (Lussi et al., 2006). Enamel has no spontaneous capability to repair when affected by specific dental pathologies such as caries, abrasions or fractures because it contains no cells (Oshiro

2 402 SCANNING VOL. 36, 4 (2014) et al., 2007). Therefore, when enamel is exposed to oral environment the only possibility to be reconstructed depends on the application of alloplastic materials, which provides to a sort of prosthetic restoration. Toothpastes have been considered effective and accessible vehicles to improve enamel resistance to erosiveattacks (Katoet al., 2010). The incorporation of protecting agents in toothpastes has become increasingly more common, because sensitivity is a common complaint among patients. Many types of toothpaste recently introduced are claimed to prevent erosion. Fluoride dentifrices have some protective effect on enamel eroded to brushing abrasion when immersed in vitro in a cola drink (Ganss et al., 2013). Currently, conventional fluoride-containing toothpastes do not appear to be able to protect efficiently against erosion (Moron et al., 2013). Recently, new toothpastes formulations have been introduced that claim to contrast enamel and dentin erosion. BioRepair Plus- Sensitive Teeth and BioRepair Plus-Total Protection (Coswell S.p.A., Bologna, Italy) are based on Zinc Hydroxyapatite (MicroRepair 1 ). Sensodyne Repair & Protect (GlaxoSmithKline, Brentford, Middlesex, UK) is made of Calcium Sodium Phosphosilicate (Novamin 1 ). Colgate Sensitive Pro Relief (Colgate- Palmolive) is based on Arginine 8% (Pro-Argin 1 ). Changes in tooth structure due to extrinsic factors have been widely investigated through scanning electron microscopy (SEM). This method requires proper specimen preparation and examination conditions: these procedures change the natural condition and/or part of the specimen structure. 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 (Bertassoni et al., 2010). The kinetic roughening of a surface is the process that takes place when material is removed from or added to the surface. Theoretical and experimental studies showed that this process might be often interpreted in terms of self-affinity concepts and simple scaling laws. However, whereas many works have been devoted to the investigation of film growth by chemical or physical vapor deposition methods, less attention has been reserved to the inverse problem of surface etching, polishing, or erosion (Barbour and Rees, 2004). Atomic force microscopy (AFM) is capable of giving images with atomic resolution with minimal sample preparation. This technique has been widely used to characterize the erosion of enamel and dentine (De-Deus et al., 2000). More recently, also AFM nanoindentation has been applied to the study of enamel erosion (Lippert et al., 2004a). The aim of the present in vitro study was the evaluation of four new formulation toothpastes on preventing enamel erosion produced by a soft drink (Coca Cola), using AFM and SEM. Materials and Methods Specimens Preparation Specimens were prepared from 50 human incisors free of caries and defects, extracted for periodontal reasons. After the extraction, the teeth were cleansed of soft tissue debris and inspected for cracks, hypoplasia and white spot lesions; they were disinfected in 5.25% sodium hypochlorite solution for 1 h and stored in artificial saliva (ph 7.0, 14.4 mm NaCl; 16.1 mm KCl; 0.3 m mm Cl 2 6H 2 O 2.9 mm K 2 HPO 4 ; 1.0 mm CaCl 2 2H 2 O; 0.10 g/100 ml sodium carboxymethylcellulose) during the whole experimentation (Farina et al., 99). The specimens were cut at the enamel dentin junction, with a high-speed diamond rotary bur with a water air spray. The labial surfaces near the enamel dentin junction were ground using silicon carbide papers (grades 600 1,200) under water irrigation to produce flat enamel surfaces. Samples were placed into Teflon moulds measuring 10 mm 8mm 2 mm, embedded in flowable composite resin and polymerized. The baseline root mean-square roughness, R rms, was measured for all the specimens before starting experimentation. No statistical difference in R rms values (150 5) was recorded, suggesting that the specimens may be comparable. Demineralization and Remineralization A soft drink (Coca Cola, Milano, Italy) was chosen for the demineralization process. The ph at 20 C, buffering capacity, concentration of calcium and phosphate of the beverage were measured (Poggio et al., 2013). Measurements were performed in triplicate and average values calculated. Four toothpastes were used: BioRepair Plus-Sensitive Teeth, BioRepair Plus-Total Protection, Sensodyne Repair & Protect, and Colgate Sensitive Pro Relief. The samples were then assigned to 10 groups, each made of 5 teeth (Fig. 1). The specimens of groups 2, 4, 6, 8, and10wereimmersedin6mlofthesoftdrinkfor 2 min at room temperature before rinsing with deionized water. Four consecutive intervals of the immersion procedure were carried out for a total of 8 min (Tantbirojin et al., 2008). The toothpastes were applied neat onto the surface of the specimens to cover the enamel surface without brushing and then wiped off with distilled water washing; the control specimens (group 1) were taken on storage for the whole experimentation and they did not receive any treatment. The toothpastes were applied to the enamel surfaces for 3 min at 0, 8, 24, and 36 h; during these intervals the specimens were kept in artificial saliva.

3 Lombardini et al.: Preventive effect of toothpastes on enamel erosion 403 Fig 1. Flow chart. Atomic Force Microscopy (AFM) Observations After 24 h (Bertassoni et al., 2010), specimens were observed with an AFM AutoProbe CP 100 (Themormicroscopes, Veeco, Plainview, NY, USA), equipped with a piezoelectric scanner, which can cover an area of mm 2 with a range of 7 mm in the z-direction. The most common topographical parameters were determined, such as the surface roughness (R rms ). R rms is given by the standard deviation of the heights, obtained from the AFM images by testing, for each sample, at least 10 different film areas of mm 2 with a resolution of pixels. From the analyses of the AFM height profiles, it was also possible to estimate the erosion cavities depth of the enamel surface. The data were obtained by averaging on at least 20 selected lines of the image. Measurements were performed on the treatment specimens and on the matching controls. Statistical Analysis Differences in the averaged values among the groups were analyzed by ANOVA test. Statistical difference was set at p < Post hoc Bonferroni test was performed to assess the differences between the different groups. SEM Observations The specimens were gently air dried, dehydrated with alcohol, sputter-coated with gold, and observed under SEM Serial SEM microphotographs of the surfaces of each specimen at 1,000 original magnification were obtained. Results The surface roughness (R rms ) is an index of the surface quality. The mean R rms values recorded before experimentation was 150, the mean R rms value of group 1 (intact enamel) after immersion in artificial saliva was 148, suggesting that the groups were comparable. Tables I and II report mean R rms values with related standard deviations obtained in the groups. Comparing untreated specimens (group 1) with demineralized specimens (group 2), a statistically significant difference (p < 0.01) in R rms values was registered, with an increase of surface

4 404 SCANNING VOL. 36, 4 (2014) TABLE I Mean roughness values (R rms ) standard deviation obtained in five groups Groups Group 1: Intact enamel Group 3: Intact enamel þ BioRepair Plus-Sensitive Teeth Group 5: Intact enamel þ BioRepair Plus-Total Protection Group 7: Intact enamel þ Sensodyne Repair & Protect Group 9: Intact enamel þ Colgate Sensitive Pro Relief roughness passing from intact enamel to enamel exposed to Coca Cola. Comparing the R rms values of control groups 3, 5, 7, 9 with intact enamel (group 1) a decrease of surface roughness values suggests a remineralizating effect of all the toothpaste investigated. However this decrease was statistically significant for Sensodyne Repair & Protect and Colgate Sensitive Pro Relief. Comparing the R rms values of treatment groups 4, 6, 8, 10 (soft drink þ toothpastes) with group 2 (enamel only demineralized) a statistical difference (p < 0.01) was registered, suggesting a protective effect against enamel demineralization of all the biomimetic pastes. This protective effect appeared more pronounced for Colgate Sensitive Pro Relief, conforming this with the morphological analysis of AFM and SEM images (Figs. 2 5). Discussion Mean roughness values (R rms ) a a a b 59 5 c Different superscript letters indicate no significant differences (p > 0.01) between groups with the same letter. Groups with different superscript letters indicate significant differences between groups in vertical row (p < 0.01). In the present in vitro study, AFM and SEM were used to verify the protective effect of four toothpastes on Mean roughness values (R rms ) standard deviation obtained in five groups TABLE II Groups Group 2: Enamel þ soft drink Group 4: Enamel þ soft drink þ BioRepair Plus-Sensitive Teeth Group 6: Enamel þ soft drink þ BioRepair Plus-Total Protection Group 8: Enamel þ soft drink þ Sensodyne Repair & Protect Group 10: Enamel þ soft drink þ Colgate Sensitive Pro Relief Mean roughness values (R rms ) d e e e 93 7 f Different superscript letters indicate no significant differences (p > 0.01) between groups with the same letter. Groups with different superscript letters indicate significant differences between groups in vertical row (p < 0.01). enamel exposed to erosive action of a soft-drink. AFM was used to study tooth surfaces in order to compare the pattern of particle distribution in the outermost layer of the tooth surfaces (Farina et al., 99). It was found that AFM gives high-contrast, high-resolution images and is an important tool as a source of new structural information: tapping mode AFM (TM-AFM) images treated with demineralizing solutions are able to show net differences between exposed and unexposed enamel areas (Finke et al., 2000). There is a clear relationship between erosion and temperature of the beverages (Barbour et al., 2007). In this study, the beverage was kept at a constant temperature of 20 C. Although erosion proceeds more slowly in vivo than in vitro owing to the protective effect of saliva and acquired pellicle, the effect of temperature can be expected to be significant (Ranjitkar et al., 2009). In order to stress their deminaralizing potential, the soft drink was replenished every 2 min to ensure that it was carbonated and to reduce the buffering effect from ions dissolved from the enamel surface (Tantbirojin et al., 2008). Enamel surface is often aprismatic and more highly mineralized than enamel subsurface; however, enamel surface is completely removed by the polishing process and the resulting flat surface is as such not present in the oral cavity. This prismless enamel arises at the end of amelogenesis. Although this layer of enamel is more frequent on the surface of deciduous teeth, it can also be found on the surface of permanent teeth. It is known that the prism-free enamel is gradually worn off during mastication but it is retained in protected areas. Flat and polished specimens were used in the present study in an attempt to standardize specimens and remove natural variations in surface enamel between teeth and between different tooth sites and types, which may result in different responses to acid dissolution (Hemingway et al., 2006). However, it should be noted that natural tooth surfaces erode more slowly than polished surfaces (Ranjitkar et al., 2009). The specimens were cleaned with 5% NaOCl for 1 h, which could not alter enamel surface. The erosive potential of orange juice modified by food additives in enamel and dentine was evaluated; it was concluded that only the combination of calcium lactate pentahydrate and sodium linear polyphosphate reduced erosion (Scaramucci et al., 2012). The efficacy of three commercially available sugar-free chewing gums was compared, concluding that the superior remineralization activity of the gum was attributed to the presence of casein phosphopeptide amorphous calcium phosphate nanocomplexes (CCP-ACP) (Manton et al., 2008). A CPP-ACP cream was effective in remineralizing early enamel lesions of the primary teeth (Zhang et al., 2011), A CCP-ACP paste was found to be effective in promoting enamel remineralization (Poggio et al., 2013). The effects of nano-hydroxyapatite toothpastes on remineralization of bovine enamel and dentine subsurface lesions in vitro was evaluated. It was

5 concluded that with the in vitro conditions chosen, toothpastes containing nanohydroxyapatite revealed higher remineralizing effects compared to amine fluoride toothpastes with bovine dentine (Tschoppe Lombardini et al.: Preventive effect of toothpastes on enamel erosion 405 et al., 2007). In an in vitro study it was stated that the application of 0.2% and 2% Chlorhexidine seemed to be effective in promoting the remineralization of demineralized dentine (Kim et al., 2011). Diamanti et al. Fig 2. 2D and 3D AFM images: (A) intact enamel (group 1) and (B) enamel treated with Biorepair Plus-Sensitive Teeth (group 3), (C) Biorepair Plus-Total Protection (group 5), (D) Sensodyne Repair & Protect (group 7), and (E) Colgate Sensitive Pro Relief (group 9).

6 406 SCANNING VOL. 36, 4 (2014) Fig 3. 2D and 3D AFM images: (A) enamel exposed to soft drink (group 2) and enamel demineralized and then remineralized with (B) Biorepair Plus-Sensitive Teeth (group 4), (C) Biorepair Plus-Total Protection (group 6), (D) Sensodyne Repair & Protect (group 8), and (E) Colgate Sensitive Pro Relief (group 10).

7 Lombardini et al.: Preventive effect of toothpastes on enamel erosion 407 Fig 4. SEM observations: (A) intact enamel (group 1) and enamel remineralized with (B) Biorepair Plus-Sensitive Teeth (group 3), (C) Biorepair Plus-Total Protection (group 5), (D) Sensodyne Repair & Protect (group 7), and (E) Colgate Sensitive Pro Relief (group 9). (2011) stated that the high fluoride toothpastes promoted remineralization and inhibited demineralization more effectively, than the calcium sodium phosphosilicate toothpastes. Calcium Sodium Phosphosilicate (Novamin) contained in toothpaste formulations adhered to exposed dentin surfaces. The layer formed was resistant to acid and mechanical challenges. Characterization of this layer indicated it was hydroxyapatite-like in nature (Wang et al., 2011). Que et al. (2010) study demonstrated that the new Pro-Argin formula toothpaste provided a significant reduction in dentin hypersensitivity when used over a period of 8 weeks in a clinical study. The AFM could analyze the topographical aspects of enamel samples and the effects of the demineralization/ remineralization processes on the enamel morphology,

8 408 SCANNING VOL. 36, 4 (2014) Fig 5. SEM observations: (A) eroded enamel (group 2) and eroded enamel treated with (B) Biorepair Plus-Sensitive Teeth (group 4), (C) Biorepair Plus-Total Protection (group 6), (D) Sensodyne Repair & Protect (group 8), and (E) Colgate Sensitive Pro Relief (group 10). in the presence of the studied toothpastes. The most common topographical parameter was therefore determined, such as the surface roughness (R rms ), to quantitatively evaluate the surface aspect. After demineralization with an acidic substance such as Coca Cola, the surface should appear much rougher. The process of erosion causes, in fact, an increase of R rms. The demineralizing process was investigated by comparing the fraction of intact enamel with the one treated with the soft drink. After the demineralization process,

9 cavities of diameter between 4 and 7 mm were formed. This is reflected in the values of R rms, which increased from 148 to 261 nm. As regards the effect of the toothpastes in the process of enamel remineralization, several considerations can be made. In control samples (groups 3, 5, 7, 9), enamel shows the typical aprismatic surface layer, on which it is possible to identify the precipitated crystals of the remineralizating agents. Instead, comparing the R rms values of control groups 3, 5, 7, 9 with intact enamel (group 1) a decrease in the values of R rms, which was statistically significant for Sensodyne Repair & Protect and Colgate Sensitive Pro Relief, suggests a remineralizating ability of all the products (Fig. 2). Comparing the R rms values of treatment groups 4, 6, 8, 10 (soft drink þ toothpastes) with group 2 (eroded enamel) a statistical difference (p < 0.01) was registered, suggesting a protective effect against enamel demineralization of all the toothpastes evaluated. This effect was numerically more evident for Colgate Sensitive Pro Relief (group 10) and this was confirmed by the morphological AFM images analysis (Fig. 3); in this case, in fact, the prismatic structure of hydroxyapatite, which typically becomes evident as a result of erosion, was not observed. This product seems to able to regenerate a homogeneous, very compact, thick and uniform surface layer. As for Biorepair Plus- Sensitive Teeth (group 4), Biorepair Plus-Total Protection (group 6) and Sensodyne Repair & Protect (group 8), enamel surface was not completely covered by the apatitic layer, but there were still areas of erosion (Fig. 3). The topographical and numerical AFM analysis was also confirmed by SEM images (Figs. 4 and 5). The demineralizing process was investigated by comparing the fraction of intact enamel with the one treated with the soft drink. It may be noted that in case of acid exposure, enamel is eroded and shows a high degree of surface porosity (Fig. 5). In control samples (groups 3, 5, 7, 9), enamel is formed by the typical aprismatic surface layer, on which it is possible to identify the mineral crystals, precipitated and not removed by washing (Fig. 4). As for the samples demineralized and then remineralized by means of Colgate Sensitive Pro Relief, the prismatic structure of hydroxyapatite, which typically becomes evident as a result of erosion, was not observed after the application of the toothpaste. In general, this remineralizing agent could regenerate a uniform, thick, homogeneous, and compact surface layer (Fig. 5). On the contrary, in the specimens treated with Biorepair Plus-Sensitive Teeth, Biorepair Plus- Total Protection and Sensodyne Repair & Protect, the enamel prismatic and interprismatic structure was still evident, so that it was not completely covered by the apatitic layer, with the crystals of the mineral agent randomly distributed along the enamel surface. In particular specimens demineralized and then remineralized with Biorepair Plus-Total Protection showed important signs of acid erosion (Fig. 5). It can be Lombardini et al.: Preventive effect of toothpastes on enamel erosion 409 concluded that all the analyzed toothpastes tend in different extent to remineralize the enamel surface. Among these, however, Colgate Sensitive Pro Relief appeared to be more effective in associating to the prismatic and interprismatic structure of demineralized enamel, which is important with regard to the reconstruction of the tissue and the recovery of its mechanical properties. Under the limitations of the present in vitro study, the application of the tested toothpastes is effective on preventing enamel erosion produced by a soft drink. References Barbour ME, Rees JS The laboratory assessment of enamel erosion: a review. 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