SCANNING VOL. 9999, 1–8 (2015) © Wiley Periodicals, Inc.
Protective Effect of Casein Phosphopeptide-amorphous Calcium Phosphate on Enamel Erosion: Atomic Force Microscopy Studies MATTEO CECI,1 MARIA MIRANDO,1 RICCARDO BELTRAMI,2 MARCO CHIESA,1 1 AND CLAUDIO POGGIO 1 2
Department of Clinical, Surgical, Diagnostic and Pediatric Sciences - Section of Dentistry. University of Pavia, Italy Deptartment of Brain, Behavioral Sciences-Section of Statistics University of Pavia, Italy
Summary: The aim of this study was to investigate the in vitro effect of a casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste (GC Tooth Mousse- TM, GC Corporation, Tokyo, Japan) on preventing enamel erosion, by using Atomic Force Microscopy (AFM). 30 human incisors, were equally assigned to 6 groups: intact enamel, enamel þ soft drink, enamel þ TM, enamel þ TM þ soft drink, enamel þ soft drink þ TM, enamel þ soft drink þ TM þ soft drink. Specimens were observed through atomic force microscopy (AFM). The most common topographical parameters were determined, such as the surface roughness (Rrms). The use of soft drink on intact enamel has roughened the surface of the sample. The application of the CPP-ACP paste on non-treated enamel made the surface smoother. A significant decrease in roughness was seen after remineralization with CPP-ACP paste. Significant differences were recorded when comparing softened enamel with softened enamel remineralized with CPP-ACP paste. Comparing eroded enamel with demineralized/remineralized specimens, the application of a CPP-ACP paste leads to a significant reduction in roughness values. AFM images of enamel surface treated with CPP-ACP resulted in less morphological changes of the tooth substrate when compared with the only eroded enamel surface morphology; thus, indicating that CPP-ACP paste promoted remineralization. Specimens’ surface roughness remained similar regardless that the protective agent is used before or after exposure to coke or
Conflicts of interest: None Address for reprints: Claudio Poggio Department of Clinical, Surgical, Diagnostic and Pediatric Sciences - Section of Dentistry, Policlinico “San Matteo&rdquo Piazzale Golgi 3, 27100 Pavia, Italy. E-mail: [email protected] Received 3 February 2015; revised 15 March 2015; Accepted with revision 7 April 2015 DOI: 10.1002/sca.21219 Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com).
between two demineralizing cycles. The results confirmed the effectiveness of the CPP-ACP paste on preventing enamel erosion produced by soft drinks. SCANNING 9999:XX–XX, 2015. © 2015 Wiley Periodicals, Inc. Key words: AFM/other scanned probe microscopes, Dentistry, Surface analysis
Introduction Dental erosion is defined as the loss of tooth substance due to chemical processes not involving bacteria (Lussi et al., 2011). Although erosive lesions have a multifactorial etiology, the increasing consumption of acidic food, and soft drinks has become an important factor driving their development (Lussi et al., 2004). Many strategies have been developed for the prevention and treatment of erosion (Magalh~aes et al., 2009). Toothpastes were considered effective and affordable vehicles to improve enamel resistance to erosive attacks (Kato et al., 2010). The incorporation of protective agents in toothpastes has become increasingly common; also because dental sensitivity, a problem often related to acid erosion, is a common complaint among patients. Currently, conventional fluoride-based toothpastes do not seem to be able to effectively protect teeth against erosion (Moron et al., 2013). Recently, new toothpastes formulations have been introduced to contrast enamel and dentin erosion (Ganss et al., 2013). Among the large amount of commercially available products, several newly toothpastes were subject of our previous studies (Poggio et al., 2013; Lombardini et al., 2014). In recent years, different agents for inhibiting erosion have been widely investigated, such as casein phosphopeptides with amorphous calcium phosphate complex (CPP-ACP) (White and Gracia, 2011). It has been suggested that casein phosphopeptides have the ability to stabilize calcium phosphate in solution by binding
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amorphous calcium phosphate with their multiple phosphoserine residues, thereby allowing the formation of small CPP-ACP clusters (Ranjitkar et al., 2009). The probable remineralizating mechanism of CPP-ACP is its ability to localize CPP-ACP nanocomplexes at the tooth surface, which brings about buffering of calcium, and phosphate free ion activities, thereby helping to maintain a state of super saturation with respect to tooth enamel negating demineralization and enhancing remineralization (Hegde et al., 2007). CPP-ACP complex increase the level of calcium and inorganic phosphate ions at the tooth surface, thereby permitting immediate enamel surface remineralization (White and Gracia, 2011). The aim is to create a calcium and phosphate reservoir that can bind stably to plaque and dental surfaces (Manton et al., 2008). It has been demonstrated that the application of a CPP-ACP paste could prevent enamel demineralization produced by soft drink (Poggio et al., 2010; Grewal et al., 2013; Poggio et al., 2013). The application of a CPP-ACP paste either before or before and after inoffice bleaching protocols proved to be able to prevent negative changes of roughness and hardness to enamel (Cunha et al., 2012). Therefore recently casein phosphopeptide-amorphous calcium phosphate and fluoride (CPP-ACPF) due to its added fluoride content has shown improved ability to remineralize initial caries (Oliveira et al., 2014; Shetty et al., 2014). Changes in tooth structure due to extrinsic factors have been widely investigated. 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 (Bertassoni et al., 2010). 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 dentin (Parker et al., 2014). The aim of this study was to investigate the in vitro effect of CPP-ACP paste on preventing enamel erosion, by using Atomic Force Microscopy.
whole experimentation (Farina et al., ’99). High-speed diamond rotary bur with a water-air spray was used to cut the teeth at the enamel-dentin junction. The specimens were embedded in Orthodontic Resin (L.D. Caulk, Milford, DE) and placed into Teflon moulds measuring 10 8 2 mm. The labial surfaces were ground wet using 240, 400, and 600 grit silicon carbide paper (Buehler, Lake Bluff, IL) and polished with 1.0 and 0.05 mm alumina suspension (Buehler) using an Ecomet 3 Grinder-Polisher (Buehler) to expose a flat enamel, ca 3–6 mm (Tantbirojin et al., 2008). The baseline root mean-square roughness, Rrms, was measured for all the specimens before starting experimentation. No statistical difference in Rrms values (150 5) was recorded, suggesting that the specimens may be comparable (Poggio et al., 2013).
Demineralization and Remineralization
A soft drink (Coca Cola; Coca Cola Company, Milano, Italy) was chosen for the demineralization process. The pH at 20˚C, buffering capacity, and concentration of calcium and phosphate of the beverage were measured. The pH of coke was measured with a pH meter (Accumet AB15, Fisher Scientific, Pittsburgh, PA). Measurements were performed in triplicate and average values calculated (Poggio et al., 2013). For the remineralization treatment, a CPP-ACP paste (GC Tooth Mousse, GC Corporation, Tokyo, Japan) was evaluated. The samples were then assigned to 6 groups, each made of 5 teeth (Fig. 1):
Materials and Methods Specimens Preparation
Specimens were prepared from 30 human incisors, extracted for periodontal reasons. Soft tissue debris was eliminated and teeth were inspected for cracks, hypoplasia, and white spot lesions. After 60 min disinfection procedures in 5.25% sodium hypochlorite solution; teeth were stored in artificial saliva (pH 7.0, 14.4 mM NaCl; 16.1 mM KCl; 0.3 mM Cl2.6H20; 2.9 mM K2HPO4; 1.0 mM CaCl2.2H2O; 0.10 g/ 100 ml sodium carboxymethylcellulose) during the
Fig 1. Flow chart.
M. Ceci et al.: Effect of casein phosphopeptide-amorphous calcium phosphate on enamel erosion
Group 1: no material applied and used as control. Group 2: soft drink applied. Group 3: GC Tooth Mousse applied. Group 4: GC Tooth Mousse þ soft drink applied. Group 5: soft drink þ GC Tooth Mousse applied. Group 6: soft drink þ GC Tooth Mousse þ soft drink applied.
The control specimens (group 1) were taken on storage for the whole experimentation and they did not receive any treatment. The specimens of groups 2, 4, and 6 were immersed in 6 mL of the soft drink for 2 min at room temperature before rinsing with deionized water. Four consecutive intervals of the immersion procedure were carried out at 0, 8, 24 and 36 h for a total of 8 min (Tantbirojin et al., 2008). GC Tooth Mousse was applied neat onto the surface of the specimens of groups 3, 4, 5, and 6 without brushing for 3 min at 0, 8, 24, and 36 h and then wiped off with distilled water washing. In group 4 the paste was applied before demineralization with Coca Cola, while in group 5 after demineralization. Finally in group 6 the remineralization treatment was performed between two acid exposures.
3
Statistical Analysis
Differences in Rrms values among the groups were analyzed by ANOVA test. Post hoc Tuckey test was performed to assess the differences between the different groups. Statistical difference was set at P < 0.05.
Results Descriptive statistics of Rrms values are reported in Table I. ANOVA showed significant differences among the groups and post-hoc Tuckey test confirmed significant differences between pairs of groups (Table II). Group 2 showed the highest Rrms values, while group 3 showed the lowest Rrms values (P < 0.05). Significant differences were recorded between group 1 with groups 2 and 3 (P < 0.05). Tuckey test showed significant differences when comparing group 2 alternatively with groups 4, 5, and 6 (P < 0.05). Similar Rrms values were obtained for groups 4, 5, and 6 (P < 0.05).
Discussion Atomic Force Microscopy (AFM) Observations
After 24 hrs from the last procedure of demineralization and remineralization (Grewal et al., 2013), specimens were observed with an atomic force microscopy AutoProbe CP 100 (Themormicroscopes, Veeco, Plainview, NY), equipped with a piezoelectric scanner, which can cover an area of 100 100 mm2 with a range of 7 mm in the z-direction. The most common topographical parameters were determined, such as the surface roughness (Rrms). Rrms 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 30 30 mm2 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 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. TABLE I
Enamel outermost layer is often aprismatic. Because of its high mineral content, this prism-free enamel layer had a dense arrangement, less permeable, gradually worn off during mastication but still retained in protected areas (Poggio et al., 2009; Badr and Ibrahimet, 2010). In order to standardize the specimens, teeth surfaces were grounded flat, and polished, eliminating natural variations from enamel surface (Torres et al., 2010; Poggio et al., 2013) Specimens were kept in artificial saliva during the whole experimentation simulating the complexity of the oral environment as the artificial saliva enhances the action of CPP-ACP paste by interacting with hydrogen ions and forms calcium hydrogen phosphate lead to enamel mineralization (Gedalia et al., 2011). Different demineralizing solutions of various compositions are available. Majority of them are composed of calcium and phosphate with acetic acid or lactate. The concentration of each component influences the final pH and the sample exposure time (Lussi et al., 2004). For the demineralization process coke was selected in this study. The beverage was kept at a constant temperature of 20˚C. The
Descriptive statistics of Rrms values
Groups 1 2 3 4 5 6
Intact enamel Intact enamel þ soft drink Intact enamel þ GC Tooth Mousse Intact enamel þ GC Tooth Mousse þ soft drink Intact enamel þ soft drink þ GC Tooth Mousse Intact enamel þ soft drink þ GC Tooth Mousse þ soft drink
Mean
Std. Dev.
Min
Max
134907 238.21 61.8684 154222 163166 144514
34.7438 22.31211 22.82576 21.63184 31.04506 31.34432
80198 205.02 43066 126.21 135.96 106.51
166.96 264.31 89.95 182.18 209.32 182.92
SCANNING VOL. 9999, 9999 (2015)
4 TABLE II groups
Tukey multiple comparisons of means between pairs of
Groups
1
1 2 3 4 5 *
2
3
4
5
6
0.0000*
0.0000* 0.0000*
0.9589 0.0000 0.0000*
0.4042 0.0005* 0.0000* 0.8847
0.9930 0.0000* 0.0000* 0.9997 0.7497
Significant differences.
deminaralizing potential was stressed, replenishing the soft drink every 2 min to ensure that it was carbonated, and to reduce the buffering effect from ions dissolved from the enamel surface (Poggio et al., 2013). The pH at 20˚C, buffering capacity, and concentration of calcium and phosphate of the beverage were measured. Remineralization cycle was carried out at an interval of 0, 8, 24, and 36 h so as to simulate the application of any topically applied remineralizing agent (Tantbirojin et al., 2008; Poggio et al., 2013; Agrawal et al., 2014). AFM microscopy was used in the present study to verify the protective effect of casein phosphopeptideamorphous calcium phosphate (CPP-ACP) on eroded enamel surface with images of high contrast and resolution. According to our previous studies, AFM analyzes topographical aspects of the tooth surface qualities like when enamel was treated with CPP-ACP paste; a network with precipitated crystals was evident which was further confirmed by SEM (Poggio et al., 2013). Similarly we have showed that the CPP-ACP
paste significantly reduces the erosion cavities depth. This was assessed by AFM data analysis through scaling concept and continuum stochastic equations which revealed the remineralizing mechanism of calcium phosphate paste (Quartarone et al., 2008). One-way ANOVA shows that there is a significant difference between groups. Multiple group posthoc Tukey test showed significant differences between pairs of groups. The baseline Rrms of intact enamel (group 1) of the present study was almost similar according to our earlier study with a range of 50–120 nm (Quartarone et al., 2008). This variation in surface roughness accounts on anatomical topography of tooth varying with geographical location, age, oral environment (Fig. 2). Significant differences were recorded between intact enamel (group 1) with soft drink applied (groups 2) and CPP-ACP paste applied (group 3). The application of coke on intact enamel has roughened the surface of the sample (group 2). The process of erosion causes, in fact, an increase of Rrms values. The three dimensional AFM topographic image showed that enamel surface exposed to cola drinks produced an early pattern of demineralization with evidence of interprismatic mineral loss (Fig. 3). Contrariwise, the application of the CPP-ACP protective paste on the non-treated enamel made the surface of the sample smoother (group 3). In fact, a considerable and statistically significant decrease in Rrms was seen after remineralization with CPP-ACP paste. CCP-ACP paste results in formation of a superficial homogeneous layer (Fig. 4).
Fig 2. Three dimensional AFM topographic image of intact enamel surface (group 1).
M. Ceci et al.: Effect of casein phosphopeptide-amorphous calcium phosphate on enamel erosion
Fig 3.
Three dimensional AFM topographic image of enamel exposed to coke (group 2).
Significant differences were recorded when comparing softened enamel (group 2) with softened enamel remineralized with CPP-ACP paste (group 4, 5, and 6). Comparing eroded enamel with demineralized/remineralized specimens, the application of a CPP-ACP paste
Fig 4.
5
leads to a significant reduction in roughness values. AFM images of enamel surface treated with CPP-ACP (before or after exposure to cola drinks) resulted in less morphological changes of the tooth substrate when compared with the only eroded enamel surface
Three dimensional AFM topographic image of intact enamel surface trated with CPP-ACP paste (group 3).
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Fig 5. Three dimensional AFM topographic image of enamel remineralized by CPP-ACP paste before exposure to soft drink (group 4).
morphology; thus, indicating that CPP-ACP paste promoted remineralization (Figs. 5 and 6). Similar Rrms values were obtained in groups 4, 5, and 6. According to these results, specimens’ surface roughness remained similar regardless that the protective agent is used before or after exposure to coke (group
4 and 5 respectively) or between two demineralizing cycles (group 6). These morphological aspects of enamel surface were confirmed by three dimensional AFM images (Fig. 7). Differently from our previous studies, the protective effect of the CPP-ACP paste on enamel erosion was
Fig 6. Three dimensional AFM topographic image of enamel remineralized by CPP-ACP paste after exposure to soft drink (group 5).
M. Ceci et al.: Effect of casein phosphopeptide-amorphous calcium phosphate on enamel erosion
7
Fig 7. Three dimensional AFM topographic image of enamel surface treated with CPP-ACP paste between two demineralizing cycles (group 6).
tested not only by applying the remineralizing agent only after exposure to coke. In this study CPP-ACP paste was applied even before attack with coke and between two acid exposures. Rrms values and AFM observations from the present in vitro study confirmed such a remineralizing property of the CPP-ACP paste regardless of when it is applied. Therefore the CPP-ACP protective effect was not modified by a following exposure to acids.
Conclusion Within the limitations of this in-vitro study model, the results confirmed the effectiveness of the CPP-ACP paste on preventing enamel erosion produced by soft drinks.
References Agrawal N, Shashikiran ND, Singla S, Ravi KS, et al. 2014. Atomic force microscopic comparison of remineralization with casein-phosphopeptide amorphous calcium phosphate paste, acidulated phosphate fluoride gel and iron supplement in primary and permanent teeth: an in-vitro study. Contemp Clin Dent 5:75–80. Badr SB, Ibrahim MA. 2010. Protective effect of three different fluoride pretreatments on artificially induced dental erosion in primary and permanent teeth. J Am Sci 6:442–451. Bertassoni LE, Habelitz S, Pugach M, et al. 2010. Evaluation of surface structural and mechanical changes following remineralization of dentin. Scanning 32:312–319.
Cunha AG, Vasconcelos AA, Borges BC, et al. 2012. Efficacy of in-office bleaching techniques combined with the application of a casein phosphopeptideamorphous calcium phosphate paste at different moments and its influence on enamel surface properties. Microsc Res Techniq 75:1019–1025. Farina M, Schemmel A, Weissmuller G, Cruz R, Kachar B, Bisch PM. 1999. Atomic force microscopy study of tooth sufaces. J Struct Biol 125:39–49. Ganss C, Shulze K, Schlueter N. 2013. Toothpaste and erosion. Monogr Oral Sci 23:88–99. Gedalia I, Dakuar A, Shapira L, Lewinstein I, Goultschin J, Rahamim E. 2011. Enamel softening with Coca-Cola and rehardening with milk or saliva. Am J Dent 4:120–122. Grewal N, Kudupudi V, Grewal S. 2013. Surface remineralization potential of casein phosphopeptide-amorphous calcium phosphate on enamel eroded by cola-drinks: An in-situ model study. Contemp Clin Dent 4:331–337. Hegde MN, Shetty S, Pardal D. 2007. Remineralization of enamel subsurface lesion using CPP-ACP - A quantitative energy dispersive X-ray analysis. J Conserv Dent 10:19–25. Kato MT, Lancia M, Sales-Peres SH, Buzalaf MA. 2010. Preventive effect of commercial desensitizing toothpastes on bovine enamel erosion in vitro. Caries Res 44:85–89. Lombardini M, Ceci M, Colombo M, Bianchi S, Poggio C. 2014. Preventive effect of different toothpaste on enamel erosion: AFM and SEM studies. Scanning 36:401–410. Lussi A, Jaeggi T, Zero D. 2004. The role of diet in the aetiology of dental erosion. Caries Res 38:34–44. Lussi A, Schlueter N, Rakhmatullina E, Ganss C. 2011. Dental Erosion - an overview with emphasis on chemical and histopathological aspects. Caries Res 45:2–12. Magalh~aes AC, Wiegand A, Rios D, Honorio HM, Buzalaf MA. 2009. Insights into preventive measures for dental erosion. J Appl Oral Sci 17:75–86. Manton DJ, Walker GD, Cai F, Cochrane NJ, Shen P, Reynolds EC. 2008. Remineralization of enamel subsurface
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lesions in situ by the use of three commercially available sugar-free gums. Int J Paediatr Dent 18:284–290. Moron BM, Miyazaki SS, Ito N, Wiegand A, Vilhena F, Buzalaf MA, Magalh~aes AC. 2013. Impact of different fluoride concentrations and pH of dentifrices on tooth erosion/ abrasion in vitro. Aust Dent J 23:106–111. GM Oliveira, AV Ritter, HO Heymann, E Swift Jr, et al. Remineralization effect of CPP-ACP and fluoride for white spot lesions in vitro J Dent 42 2014; 1592–1602 AS Parker, AN Patel, R Al Brotos, ME Snowden, et al. Measurement efficacy of calcium silicate for the protection and repair of dental enamel J Dent 42 2014; 121–129 C Poggio, M Lombardini, M Colombo, S. Bianchi, Impact of two toothpastes on repairing enamel erosion produced by a soft drink: an AFM in vitro study J Dent 38 2010; 868–874 C Poggio, M Lombardini, A Dagna, M Chiesa, S. Bianchi, Protective effect on enamel demineralization of a CPP-ACP paste: An AFM in vitro study J Dent . 37 2009; 949–954 C. Poggio, M. Lombardini, P. Vigorelli, M. Ceci, Analysis of Dentin/Enamel Remineralization by a CPP-ACP Paste: AFM and SEM Study Scanning 35 2013; 366–374
E Quartarone, P Mustarelli, C Poggio, M. Lombardini, Surface kinetic roughening caused by dental erosion: An atomic force microscopy study J Appl Phys 103 2008; 104702– 104706 S Ranjitkar, JA Kaidonis, LC Richards, GC. Townsend, The effect of CPP-ACP on enamel wear under severe erosive conditions Arch Oral Biol 54 2009; 527–532 S Shetty, MN Hegde, TP. Bopanna, Enamel remineralization assessment after treatment with three different remineralizing agents using surface microhardness: an in vitro study J Conserv Dent 17 2014; 49–52 D Tantbirojin, A Huang, MD Ericson, S. Poolthong, Change in surface hardness of enamel by a cola drink and a CPP-ACP paste J Dent 36 2008; 74–79 CP Torres, MA Chinelatti, JM Gomes-Silva, FA Rizoli, MA Oliveira, RG Palma-Dibb, et al. Surface and subsurface erosion of primary enamel by acid beverages over time Braz Dent J 21 2010; 337–345 AJ White, LH Gracia, ME. Barbour, Inhibition of dental erosion by casein and casein-derived proteins Caries Res 4 2011; 13–20
Protective Effect of Casein Phosphopeptide-amorphous Calcium Phosphate on Enamel Erosion: Atomic Force Microscopy Studies MATTEO CECI,1 MARIA MIRANDO,1 RICCARDO BELTRAMI,2 MARCO CHIESA,1 1 AND CLAUDIO POGGIO 1 2
Department of Clinical, Surgical, Diagnostic and Pediatric Sciences - Section of Dentistry. University of Pavia, Italy Deptartment of Brain, Behavioral Sciences-Section of Statistics University of Pavia, Italy
Summary: The aim of this study was to investigate the in vitro effect of a casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste (GC Tooth Mousse- TM, GC Corporation, Tokyo, Japan) on preventing enamel erosion, by using Atomic Force Microscopy (AFM). 30 human incisors, were equally assigned to 6 groups: intact enamel, enamel þ soft drink, enamel þ TM, enamel þ TM þ soft drink, enamel þ soft drink þ TM, enamel þ soft drink þ TM þ soft drink. Specimens were observed through atomic force microscopy (AFM). The most common topographical parameters were determined, such as the surface roughness (Rrms). The use of soft drink on intact enamel has roughened the surface of the sample. The application of the CPP-ACP paste on non-treated enamel made the surface smoother. A significant decrease in roughness was seen after remineralization with CPP-ACP paste. Significant differences were recorded when comparing softened enamel with softened enamel remineralized with CPP-ACP paste. Comparing eroded enamel with demineralized/remineralized specimens, the application of a CPP-ACP paste leads to a significant reduction in roughness values. AFM images of enamel surface treated with CPP-ACP resulted in less morphological changes of the tooth substrate when compared with the only eroded enamel surface morphology; thus, indicating that CPP-ACP paste promoted remineralization. Specimens’ surface roughness remained similar regardless that the protective agent is used before or after exposure to coke or
Conflicts of interest: None Address for reprints: Claudio Poggio Department of Clinical, Surgical, Diagnostic and Pediatric Sciences - Section of Dentistry, Policlinico “San Matteo&rdquo Piazzale Golgi 3, 27100 Pavia, Italy. E-mail: [email protected] Received 3 February 2015; revised 15 March 2015; Accepted with revision 7 April 2015 DOI: 10.1002/sca.21219 Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com).
between two demineralizing cycles. The results confirmed the effectiveness of the CPP-ACP paste on preventing enamel erosion produced by soft drinks. SCANNING 9999:XX–XX, 2015. © 2015 Wiley Periodicals, Inc. Key words: AFM/other scanned probe microscopes, Dentistry, Surface analysis
Introduction Dental erosion is defined as the loss of tooth substance due to chemical processes not involving bacteria (Lussi et al., 2011). Although erosive lesions have a multifactorial etiology, the increasing consumption of acidic food, and soft drinks has become an important factor driving their development (Lussi et al., 2004). Many strategies have been developed for the prevention and treatment of erosion (Magalh~aes et al., 2009). Toothpastes were considered effective and affordable vehicles to improve enamel resistance to erosive attacks (Kato et al., 2010). The incorporation of protective agents in toothpastes has become increasingly common; also because dental sensitivity, a problem often related to acid erosion, is a common complaint among patients. Currently, conventional fluoride-based toothpastes do not seem to be able to effectively protect teeth against erosion (Moron et al., 2013). Recently, new toothpastes formulations have been introduced to contrast enamel and dentin erosion (Ganss et al., 2013). Among the large amount of commercially available products, several newly toothpastes were subject of our previous studies (Poggio et al., 2013; Lombardini et al., 2014). In recent years, different agents for inhibiting erosion have been widely investigated, such as casein phosphopeptides with amorphous calcium phosphate complex (CPP-ACP) (White and Gracia, 2011). It has been suggested that casein phosphopeptides have the ability to stabilize calcium phosphate in solution by binding
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SCANNING VOL. 9999, 9999 (2015)
amorphous calcium phosphate with their multiple phosphoserine residues, thereby allowing the formation of small CPP-ACP clusters (Ranjitkar et al., 2009). The probable remineralizating mechanism of CPP-ACP is its ability to localize CPP-ACP nanocomplexes at the tooth surface, which brings about buffering of calcium, and phosphate free ion activities, thereby helping to maintain a state of super saturation with respect to tooth enamel negating demineralization and enhancing remineralization (Hegde et al., 2007). CPP-ACP complex increase the level of calcium and inorganic phosphate ions at the tooth surface, thereby permitting immediate enamel surface remineralization (White and Gracia, 2011). The aim is to create a calcium and phosphate reservoir that can bind stably to plaque and dental surfaces (Manton et al., 2008). It has been demonstrated that the application of a CPP-ACP paste could prevent enamel demineralization produced by soft drink (Poggio et al., 2010; Grewal et al., 2013; Poggio et al., 2013). The application of a CPP-ACP paste either before or before and after inoffice bleaching protocols proved to be able to prevent negative changes of roughness and hardness to enamel (Cunha et al., 2012). Therefore recently casein phosphopeptide-amorphous calcium phosphate and fluoride (CPP-ACPF) due to its added fluoride content has shown improved ability to remineralize initial caries (Oliveira et al., 2014; Shetty et al., 2014). Changes in tooth structure due to extrinsic factors have been widely investigated. 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 (Bertassoni et al., 2010). 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 dentin (Parker et al., 2014). The aim of this study was to investigate the in vitro effect of CPP-ACP paste on preventing enamel erosion, by using Atomic Force Microscopy.
whole experimentation (Farina et al., ’99). High-speed diamond rotary bur with a water-air spray was used to cut the teeth at the enamel-dentin junction. The specimens were embedded in Orthodontic Resin (L.D. Caulk, Milford, DE) and placed into Teflon moulds measuring 10 8 2 mm. The labial surfaces were ground wet using 240, 400, and 600 grit silicon carbide paper (Buehler, Lake Bluff, IL) and polished with 1.0 and 0.05 mm alumina suspension (Buehler) using an Ecomet 3 Grinder-Polisher (Buehler) to expose a flat enamel, ca 3–6 mm (Tantbirojin et al., 2008). The baseline root mean-square roughness, Rrms, was measured for all the specimens before starting experimentation. No statistical difference in Rrms values (150 5) was recorded, suggesting that the specimens may be comparable (Poggio et al., 2013).
Demineralization and Remineralization
A soft drink (Coca Cola; Coca Cola Company, Milano, Italy) was chosen for the demineralization process. The pH at 20˚C, buffering capacity, and concentration of calcium and phosphate of the beverage were measured. The pH of coke was measured with a pH meter (Accumet AB15, Fisher Scientific, Pittsburgh, PA). Measurements were performed in triplicate and average values calculated (Poggio et al., 2013). For the remineralization treatment, a CPP-ACP paste (GC Tooth Mousse, GC Corporation, Tokyo, Japan) was evaluated. The samples were then assigned to 6 groups, each made of 5 teeth (Fig. 1):
Materials and Methods Specimens Preparation
Specimens were prepared from 30 human incisors, extracted for periodontal reasons. Soft tissue debris was eliminated and teeth were inspected for cracks, hypoplasia, and white spot lesions. After 60 min disinfection procedures in 5.25% sodium hypochlorite solution; teeth were stored in artificial saliva (pH 7.0, 14.4 mM NaCl; 16.1 mM KCl; 0.3 mM Cl2.6H20; 2.9 mM K2HPO4; 1.0 mM CaCl2.2H2O; 0.10 g/ 100 ml sodium carboxymethylcellulose) during the
Fig 1. Flow chart.
M. Ceci et al.: Effect of casein phosphopeptide-amorphous calcium phosphate on enamel erosion
Group 1: no material applied and used as control. Group 2: soft drink applied. Group 3: GC Tooth Mousse applied. Group 4: GC Tooth Mousse þ soft drink applied. Group 5: soft drink þ GC Tooth Mousse applied. Group 6: soft drink þ GC Tooth Mousse þ soft drink applied.
The control specimens (group 1) were taken on storage for the whole experimentation and they did not receive any treatment. The specimens of groups 2, 4, and 6 were immersed in 6 mL of the soft drink for 2 min at room temperature before rinsing with deionized water. Four consecutive intervals of the immersion procedure were carried out at 0, 8, 24 and 36 h for a total of 8 min (Tantbirojin et al., 2008). GC Tooth Mousse was applied neat onto the surface of the specimens of groups 3, 4, 5, and 6 without brushing for 3 min at 0, 8, 24, and 36 h and then wiped off with distilled water washing. In group 4 the paste was applied before demineralization with Coca Cola, while in group 5 after demineralization. Finally in group 6 the remineralization treatment was performed between two acid exposures.
3
Statistical Analysis
Differences in Rrms values among the groups were analyzed by ANOVA test. Post hoc Tuckey test was performed to assess the differences between the different groups. Statistical difference was set at P < 0.05.
Results Descriptive statistics of Rrms values are reported in Table I. ANOVA showed significant differences among the groups and post-hoc Tuckey test confirmed significant differences between pairs of groups (Table II). Group 2 showed the highest Rrms values, while group 3 showed the lowest Rrms values (P < 0.05). Significant differences were recorded between group 1 with groups 2 and 3 (P < 0.05). Tuckey test showed significant differences when comparing group 2 alternatively with groups 4, 5, and 6 (P < 0.05). Similar Rrms values were obtained for groups 4, 5, and 6 (P < 0.05).
Discussion Atomic Force Microscopy (AFM) Observations
After 24 hrs from the last procedure of demineralization and remineralization (Grewal et al., 2013), specimens were observed with an atomic force microscopy AutoProbe CP 100 (Themormicroscopes, Veeco, Plainview, NY), equipped with a piezoelectric scanner, which can cover an area of 100 100 mm2 with a range of 7 mm in the z-direction. The most common topographical parameters were determined, such as the surface roughness (Rrms). Rrms 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 30 30 mm2 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 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. TABLE I
Enamel outermost layer is often aprismatic. Because of its high mineral content, this prism-free enamel layer had a dense arrangement, less permeable, gradually worn off during mastication but still retained in protected areas (Poggio et al., 2009; Badr and Ibrahimet, 2010). In order to standardize the specimens, teeth surfaces were grounded flat, and polished, eliminating natural variations from enamel surface (Torres et al., 2010; Poggio et al., 2013) Specimens were kept in artificial saliva during the whole experimentation simulating the complexity of the oral environment as the artificial saliva enhances the action of CPP-ACP paste by interacting with hydrogen ions and forms calcium hydrogen phosphate lead to enamel mineralization (Gedalia et al., 2011). Different demineralizing solutions of various compositions are available. Majority of them are composed of calcium and phosphate with acetic acid or lactate. The concentration of each component influences the final pH and the sample exposure time (Lussi et al., 2004). For the demineralization process coke was selected in this study. The beverage was kept at a constant temperature of 20˚C. The
Descriptive statistics of Rrms values
Groups 1 2 3 4 5 6
Intact enamel Intact enamel þ soft drink Intact enamel þ GC Tooth Mousse Intact enamel þ GC Tooth Mousse þ soft drink Intact enamel þ soft drink þ GC Tooth Mousse Intact enamel þ soft drink þ GC Tooth Mousse þ soft drink
Mean
Std. Dev.
Min
Max
134907 238.21 61.8684 154222 163166 144514
34.7438 22.31211 22.82576 21.63184 31.04506 31.34432
80198 205.02 43066 126.21 135.96 106.51
166.96 264.31 89.95 182.18 209.32 182.92
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4 TABLE II groups
Tukey multiple comparisons of means between pairs of
Groups
1
1 2 3 4 5 *
2
3
4
5
6
0.0000*
0.0000* 0.0000*
0.9589 0.0000 0.0000*
0.4042 0.0005* 0.0000* 0.8847
0.9930 0.0000* 0.0000* 0.9997 0.7497
Significant differences.
deminaralizing potential was stressed, replenishing the soft drink every 2 min to ensure that it was carbonated, and to reduce the buffering effect from ions dissolved from the enamel surface (Poggio et al., 2013). The pH at 20˚C, buffering capacity, and concentration of calcium and phosphate of the beverage were measured. Remineralization cycle was carried out at an interval of 0, 8, 24, and 36 h so as to simulate the application of any topically applied remineralizing agent (Tantbirojin et al., 2008; Poggio et al., 2013; Agrawal et al., 2014). AFM microscopy was used in the present study to verify the protective effect of casein phosphopeptideamorphous calcium phosphate (CPP-ACP) on eroded enamel surface with images of high contrast and resolution. According to our previous studies, AFM analyzes topographical aspects of the tooth surface qualities like when enamel was treated with CPP-ACP paste; a network with precipitated crystals was evident which was further confirmed by SEM (Poggio et al., 2013). Similarly we have showed that the CPP-ACP
paste significantly reduces the erosion cavities depth. This was assessed by AFM data analysis through scaling concept and continuum stochastic equations which revealed the remineralizing mechanism of calcium phosphate paste (Quartarone et al., 2008). One-way ANOVA shows that there is a significant difference between groups. Multiple group posthoc Tukey test showed significant differences between pairs of groups. The baseline Rrms of intact enamel (group 1) of the present study was almost similar according to our earlier study with a range of 50–120 nm (Quartarone et al., 2008). This variation in surface roughness accounts on anatomical topography of tooth varying with geographical location, age, oral environment (Fig. 2). Significant differences were recorded between intact enamel (group 1) with soft drink applied (groups 2) and CPP-ACP paste applied (group 3). The application of coke on intact enamel has roughened the surface of the sample (group 2). The process of erosion causes, in fact, an increase of Rrms values. The three dimensional AFM topographic image showed that enamel surface exposed to cola drinks produced an early pattern of demineralization with evidence of interprismatic mineral loss (Fig. 3). Contrariwise, the application of the CPP-ACP protective paste on the non-treated enamel made the surface of the sample smoother (group 3). In fact, a considerable and statistically significant decrease in Rrms was seen after remineralization with CPP-ACP paste. CCP-ACP paste results in formation of a superficial homogeneous layer (Fig. 4).
Fig 2. Three dimensional AFM topographic image of intact enamel surface (group 1).
M. Ceci et al.: Effect of casein phosphopeptide-amorphous calcium phosphate on enamel erosion
Fig 3.
Three dimensional AFM topographic image of enamel exposed to coke (group 2).
Significant differences were recorded when comparing softened enamel (group 2) with softened enamel remineralized with CPP-ACP paste (group 4, 5, and 6). Comparing eroded enamel with demineralized/remineralized specimens, the application of a CPP-ACP paste
Fig 4.
5
leads to a significant reduction in roughness values. AFM images of enamel surface treated with CPP-ACP (before or after exposure to cola drinks) resulted in less morphological changes of the tooth substrate when compared with the only eroded enamel surface
Three dimensional AFM topographic image of intact enamel surface trated with CPP-ACP paste (group 3).
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Fig 5. Three dimensional AFM topographic image of enamel remineralized by CPP-ACP paste before exposure to soft drink (group 4).
morphology; thus, indicating that CPP-ACP paste promoted remineralization (Figs. 5 and 6). Similar Rrms values were obtained in groups 4, 5, and 6. According to these results, specimens’ surface roughness remained similar regardless that the protective agent is used before or after exposure to coke (group
4 and 5 respectively) or between two demineralizing cycles (group 6). These morphological aspects of enamel surface were confirmed by three dimensional AFM images (Fig. 7). Differently from our previous studies, the protective effect of the CPP-ACP paste on enamel erosion was
Fig 6. Three dimensional AFM topographic image of enamel remineralized by CPP-ACP paste after exposure to soft drink (group 5).
M. Ceci et al.: Effect of casein phosphopeptide-amorphous calcium phosphate on enamel erosion
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Fig 7. Three dimensional AFM topographic image of enamel surface treated with CPP-ACP paste between two demineralizing cycles (group 6).
tested not only by applying the remineralizing agent only after exposure to coke. In this study CPP-ACP paste was applied even before attack with coke and between two acid exposures. Rrms values and AFM observations from the present in vitro study confirmed such a remineralizing property of the CPP-ACP paste regardless of when it is applied. Therefore the CPP-ACP protective effect was not modified by a following exposure to acids.
Conclusion Within the limitations of this in-vitro study model, the results confirmed the effectiveness of the CPP-ACP paste on preventing enamel erosion produced by soft drinks.
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