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Author's personal copy journal of dentistry 38 (2010) 868–874
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Impact of two toothpastes on repairing enamel erosion produced by a soft drink: An AFM in vitro study Claudio Poggio a,*, Marco Lombardini a, Marco Colombo b, Stefano Bianchi b a b
Department of Operative Dentistry, University of Pavia, Policlinico ‘‘San Matteo’’, Piazzale Golgi 3, 27100 Pavia, Italy Department of Endodontics, University of Pavia, Italy
article info
abstract
Article history:
Objectives: The aim of the present in vitro study was the evaluation of two toothpastes
Received 2 May 2010
(Sensodyne Pronamel and Biorepair Plus on repairing enamel erosion produced by a soft
Received in revised form
drink (Coca Cola), using Atomic Force Microscopy (AFM).
13 July 2010
Methods: Fifty extracted human central incisors free of caries were selected and divided in a
Accepted 20 July 2010
treatment and a control half; they were kept in artificial saliva during whole experimentation. The treatment halves were divided into five groups; group 1: demineralization with soft drink; group 2: demineralization with soft drink + Pronamel; group 3: demineralization with
Keywords:
soft drink + Biorepair Plus; group 4: intact enamel + Pronamel; group 5: intact enamel + -
Enamel
Biorepair Plus. Specimen demineralization was carried on in 4 intervals of 2 min. In groups 2,
Surface properties
3, 4, and 5 the toothpastes were applied for 3 min at 0, 8, 24 and 36 h. The surface of each
Toothpastes
specimen was imaged by AFM and Rrms, root-mean-square roughness, and Maximum Depth
Atomic Force Microscopy (AFM)
of the cavities were registered. Results: Amongst treatment specimens of groups 1, 2, and 3 a statistically significant difference (P < 0.01) in Rrms and Maximum Depth values was registered: the toothpastes reduced enamel demineralization. No statistical differences in Rrms values were registered between the two toothpastes. Conclusions: The toothpastes tested (Pronamel and BioRepair Plus) offer a degree of protection from erosive drinks. # 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Surface demineralization and remineralization processes have an important role for mineralized tissues, such as bones and teeth. Dental enamel is the hardest substance of the body because of its high mineral content. The ultrastructure of enamel has been widely investigated, showing that the inorganic components are distributed in the form of rods or prisms,1 which are composed of hexagonal hydroxyapatite crystals. Both aprismatic and prismatic crystals can be found at enamel surface; aprismatic enamel is generally more mineralized and more resistant to acid challenge.2
In adults, enamel does not contain any cells and is therefore not capable of self-regeneration. Any damage is irreversible, as there is no biological process capable of repairing damaged enamel. For this reason, any reparative action must be provided by materials or substances that are extraneous to dental tissue metabolism. These substances are either synthetic or are precipitated from saliva.3 Tooth structure undergoes continuous demineralization and remineralization: if this balance is interrupted, demineralization will lead to a progressive deterioration of tooth.4,5 Erosive tooth wear in the oral environment has gained more importance from the dental practice since the decline in dental caries in many industrialized countries.6
* Corresponding author. Tel.: +39 0382 516257/39 3398124925; fax: +39 0382 516224. E-mail address:
[email protected] (C. Poggio). 0300-5712/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2010.07.010
Author's personal copy journal of dentistry 38 (2010) 868–874
Changes in dietary habits have led to excessive consumption of acidic food and beverages, which are the most widespread extrinsic factors that cause dental erosion.7 Typical acid sources come from the diet, medications, occupational exposure and lifestyle activities.8,9 The characteristics of erosion may be due to several factors, including the chemical properties of the erosive medium and the frequency and method of contact between acid and tooth. 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 process10; this is the reason why patients with diminished salivary flow are more exposed to dental erosion and decay.11 Many studies have demonstrated that the early stages of erosion result in the softening of the enamel surface up to a depth of less than 1 mm.12–14 Surface softened enamel is not capable of complete rehardening by exposure to saliva although rehardening of surface softened enamel by the exposure to calcifying solutions in vitro and in vivo has been reported. Fluoride dentifrice had a protective effect on eroded enamel subjected to brushing abrasion.15 Currently, conventional fluoride-containing toothpastes do not appear to be able to protect efficiently against erosion.16 Recently, two toothpastes have been introduced: Pronamel and BioRepair Plus. Their main characteristic is the ability to contrast enamel erosion. Sensodyne Pronamel (GlaxoSmithKline, Brentford, Middlesex, UK) is a derivative of Sensodyne toothpaste; it is made of bioavailable sodium fluoride (1450 ppm fluoride) and also contains potassium nitrate to help prevention against enamel erosion and mitigate the effects of dentinal hypersensitivity.17 Its biocompatibility has been enhanced by removing sodium lauryl sulphate from the composition. BioRepair Plus (Coswell S.p.A., Bologna, Italy) is a fluoride free toothpaste made of hydroxyapatite nanocrystals, which have been introduced because of their excellent biological properties, lack of toxicity and inflammatory and immunological responses. The hydroxyapatite microparticles are completely identical to the mineral that forms dentine and enamel. In the case of the enamel, the microparticles action takes place via their ability to bond to natural tissues, thus filling microgaps in the enamel.18 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.19–21 In a previous study AFM have been used to investigate the protective effect of a mouthrinse on enamel softened by a demineralizing beverage.22 In the present in vitro study Atomic Force Microscopy was used to investigate the surface roughening during dental erosion caused by a soft drink. Our chief aim was to study the role played by two toothpastes (Pronamel and BioRepair Plus) on repairing enamel erosion.
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 h1 and stored in artificial saliva (pH 7.0, 14.4 mM NaCl; 16.1 mM KCl; 0.3 mM Cl26H20; 2.9 mM K2HPO4; 1.0 mM CaCl22H2O; 0.10 g/100 ml sodium carboxymethylcellulose) during the whole experimentation.5 The specimens were cut longitudinally, with a high-speed diamond rotary bur with a water-air spray; one half 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.7 Samples were placed into Teflon moulds measuring 10 mm 8 mm 2 mm, embedded in flowable composite resin and polymerized. 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.22 Measurements were performed in triplicate and average values calculated. Two toothpastes were used: Pronamel and BioRepair Plus. The samples were randomly assigned to 5 groups, each made of 10 teeth (Fig. 1): group group group group group
1: 2: 3: 4: 5:
soft drink, soft drink + Pronamel, soft drink + BioRepair Plus, intact enamel + Pronamel, intact enamel + BioRepair Plus.
The treatment specimens of groups 1, 2, and 3 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.6 The toothpastes were applied neat onto the surface of the specimens to cover the enamel surface without brushing in the treatment groups 2, 3, 4, and 5 and wiped off with distilled water washing; the matching control specimens received no treatment. The toothpaste was 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.
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-meansquare roughness, Rrms, was obtained from the AFM investigations by testing, 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 data were obtained by averaging on at least 20 selected lines of the image.23 Measurements were performed on the treatment specimens and on the matching controls.
2.2.
2.
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Statistical analysis
Materials and methods
Specimens were prepared from 50 extracted human incisors free of caries and defects. After the extraction, the teeth were
Differences in the averaged values amongst the groups were analyzed by ANOVA test. Statistical difference was set at P < 0.01.
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[(Fig._1)TD$IG]
Fig. 1 – Flow chart. Table 1 – Mean roughness values (Rrms) and Maximum Depth of the cavities obtained in the five groups on the exposed and the not-exposed specimens. Groups
Treatment
Rrms
Group 1: soft drink
Exposed Not-exposed Exposed Not-exposed Exposed Not-exposed Exposed Not-exposed Exposed Not-exposed
0.32 0.01 0.05 0.01 0.15 0.02 0.06 0.02 0.17 0.02 0.07 0.03 0.07 0.04 0.07 0.02 0.06 0.05 0.07 0.02
Group 2: soft drink + Pronamel Group 3: soft drink + BioRepair Plus Group 4: intact enamel + Pronamel Group 5: intact enamel + BioRepair Plus
3.
Results
The pH of the soft drink at 20 8C was 2.44; the buffering capacity was 0.0056. Concentration of calcium was 20.83 mg/ml, concentration of phosphate 175.7 mg/ml. Table 1 reports mean Rrms values and Maximum Depth (MD) of the cavities obtained through the demineralization process. In groups 1–5 the not-exposed halves of the specimens provided similar Rrms values (no statistical difference), suggesting that the groups may be comparable. Comparing exposed specimens of groups 1 (softened enamel) with group 2 (softened enamel + Pronamel) and 3 (softened enamel + BioRepair Plus), a not statistically significant difference (P > 0.01) in Rrms values was registered. Comparing groups 2 and 3, Pronamel and BioRepair Plus gave no statistically significant Rrms values, thus demonstrating a comparable remineralizing ability. In groups 4 and 5, no statistically significant difference was registered between exposed and not-exposed halves of the specimens and between the two groups.
Maximum Depth (mm) 0.50 0.15 0.17 0.07 0.20 0.09 0 0
As for the Maximum Depth of the cavities, there was a statistical significant difference between group 1 (softened enamel) and groups 2, 3 (softened enamel repaired by toothpastes). Fig. 2(a) shows an untreated specimen surface. Fig. 2(b) shows the demineralized surface of a specimen (group 1). Fig. 3 displays a specimen surface demineralized and then exposed to the protective effect of Pronamel (a) and BioRepair Plus (b) toothpastes (groups 2, 3). Fig. 4 shows an intact enamel surface exposed to the protective effect of Pronamel (a) and BioRepair Plus (b) toothpastes (groups 4, 5). The surfaces of the untreated teeth appear quite smooth (Fig. 1). A remarkable increase of enamel surface alterations was observed after the exposure to the acid drink, which causes a loss of material from the surface (Fig. 2). The final enamel morphology recalls a typical prismatic structure, with prism size ranging between 3 and 5 mm: the etching process involves mainly the inner area of the prism creating a honeycomb-like structure.22–24 There is not a statistically significant difference in Rrms between the exposed surfaces of groups 2 and 3 and between the not-exposed halves of groups 2 and 3. Regarding the tooth
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[(Fig._2)TD$IG]
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[(Fig._3)TD$IG]Fig. 2 – Intact enamel, untreated specimen surface (a); demineralized surface of a specimen (b).
Fig. 3 – Specimen surface demineralized and exposed to Pronamel (a) and BioRepair Plus (b) toothpastes (groups 2, 3).
[(Fig._4)TD$IG]
Fig. 4 – Intact specimen surface exposed to the protective effect of Pronamel (a) and BioRepair Plus (b) toothpastes (groups 4, 5).
surfaces treated with the toothpastes after exposure to the soft drink (groups 2, 3), the interprismatic cavities appear largely filled in, with a consequent decrease of their depth. Zones of intact enamel within the test area were interspersed with superficial adherent irregularities, appearing as granular or globular deposits. Moreover, the observation and the comparison of the exposed halves of groups 2 and 3 show that the treatment with the two toothpastes causes a different remineralizing process. After treatment with Pronamel, bulk material loss was observed: both interprismatic and prismatic enamel structures still appear evident. BioRepair Plus, through its hydroxyapatite nanocrystals, showed a progressive microparticles deposition: the interprismatic and prismatic enamel
structures appear to be completely hidden by a thick homogeneous apatitic layer. Concerning the Maximum Depth of the cavities, the treatment with the two toothpastes tested resulted in a statistically significant depth reduction.
4.
Discussion
This study compared the effect of two toothpastes on human enamel demineralized by a soft drink. Atomic Force Microscope has been used to image and comparatively analyze enamel surface. AFM provides high-resolution
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images and is an important tool as a source of new structural information.1 In a previous study, enamel surfaces treated with demineralizing solutions were imaged by tapping mode AFM, showing a net boundary between exposed and unexposed enamel areas. AFM was used to study the effects of bleaching agents on enamel, as it is a suitable microscopic method to analyze biological objects under natural conditions.25,26 The authors concluded that AFM imaged non-dehydrated enamel surfaces. AFM nanoindentation was used to investigate the demineralization and remineralization of surface softened enamel,27,28 which provided a very sensitive measurement. An AFM nanoindentation study has shown that the early stage of enamel dissolution in situ results in the softening of the enamel surface up to a depth of less than 1 mm.29 Other studies showed that the amount of mineral loss increased in the presence of a demineralizing solution with increasing exposure time. 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 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.30 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: nontreated halves gave very similar Rrms results, allowing us to compare the groups to each other. However, it should be noted that natural tooth surfaces erode more slowly than polished surfaces.31 Specimens were cleaned with 5.25% NaOCl for 1 h, which could not alter enamel surface.1 It was demonstrated that erosion is correlated to pH; moreover, there was a negative correlation between calcium concentration and erosion, but no clear relationship between phosphate concentration and erosion.10 In a previous study, Coca Cola was the beverage with the lowest pH and highest concentration of calcium, so it was chosen for its highest erosion potential. There is a clear correlation between erosion and temperature of the beverages.5 In the present study, the beverages were kept at a constant temperature of 20 8C, in order to stress their demineralizing potential. 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.32 Acquired pellicle is a protective layer that covers enamel surface and consists mainly of salivary proteins: it has the ability to slow down the overall remineralization process. We did not intend to simulate the acquired pellicle as this barrier for acid diffusion could masque the potential of a toothpaste to protect from erosion. In an in vivo model, Hara et al.33 concluded that the acquired pellicle reduced dental erosion, but that this effect was limited to the less severe erosive challenge on enamel surfaces. Previous research suggests that human saliva has the ability to reharden the enamel surface in the oral environment, moreover enamel rehardening has often been reported
in vitro studies by various calcifying solutions.34,35 These calcifying solutions often possess a higher remineralization potential than human saliva, as their calcium and phosphate concentrations are higher. Surface precipitation of calcium phosphates is believed to occur in the form of an amorphous precursor phase, which undergoes a rapid transformation to crystalline apatite.36 Devlin et al.37 demonstrated that Coca Cola reduced the mean indentation hardness of enamel in the teeth, but the hardness was partially restored with artificial saliva. Lippert et al.27 stated that enamel rehardening by saliva was not observed. The authors added that a complete recovery of the initial surface hardness was not observed so far. In the present study, artificial saliva was not used during the erosion phase; its use would buffer the acidity of the cola drink and limit the demineralization process.6 For the remineralization phase natural saliva and acquired pellicle might influence the results provided. Soft drink tested showed high erosive potential on human enamel. In the oral environment, many factors can enhance or prevent demineralization such as mineral concentration, pellicle and plaque formation, salivary factors (flow rate and buffering capacity). Another important factor that must be taken into consideration is toothbrushing. Hemingway et al.10 demonstrated by optical profilometry that enamel softened by erosion is readily susceptible to abrasion trough toothbrushing. Lippert et al.27 investigated the toothbrush abrasion of surface softened enamel using AFM. They concluded that the amount of enamel lost due to toothbrushing was independent of the demineralization time and was lower compared to the mineral loss caused by demineralization treatments. We decided to avoid toothbrushing in order to stress the demineralizing potential of the soft drink chosen. In the present study, a sodium fluoride/potassium nitrate toothpaste (Pronamel) and a hydroxyapatite nanocrystals toothpaste (Biorepair Plus) were compared by AFM. After exposure to a demineralizing soft drink, the initial Rrms values were not regained after exposure to the two toothpastes. Though they provided similar Rrms values before and after treatment, the images obtained by AFM showed a different remineralizing pattern for the two toothpastes. Lussi et al.16 compared 5 fluoride-containing toothpastes on the prevention of erosion by a microhardness test. They underlined that the range of free-fluoride availability was quite similar in the 5 groups, which provided similar values of microhardness. Using an in situ erosion remineralization model and a microhardness test, Zero et al.38 concluded that fluoride dentifrice containing potassium nitrate significantly enhanced the remineralization of enamel previously subjected to an erosion challenge. After treatment with the demineralizing solution followed by Pronamel, both interprismatic and prismatic enamel structures still appear evident. Pronamel favors mineral deposition, thus providing a demineralization reduction, but in case of further acid challenges, no protective action will be obtained. On the contrary, BioRepair Plus showed a progressive microparticles deposition: the interprismatic and prismatic enamel structures appear to be completely hidden by a thick homogeneous apatitic layer. According to Roveri et al.18 this
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coating formation corresponds to an apatite deposition inside the demineralized areas of enamel surface. The treatment of the teeth surface with the CPP-ACP paste causes the formation of a layer that fills the interprismatic cavities, and partially cover the prisms faces. It is well known that the localization of ACP at the tooth surface buffers the free calcium and phosphate ion activities, thus helping to maintain a state of super saturation which depresses demineralization and enhances remineralization of the enamel, thus providing a protective action against further acid attack.
5.
12.
13.
14. 15.
Conclusions 16.
This in vitro study demonstrated that both toothpastes tested (Pronamel and BioRepair Plus) offer a degree of protection from erosive drinks, without statistical difference. The morphological interpretation of AFM images suggested that this protective effect is considered more evident for Biorepair Plus.
references
17.
18.
19.
20. 1. Farina M, Schemmel A, Weissmuller G, Cruz R, Kachar B, Bisch PM. Atomic force microscopy study of tooth surfaces. Journal of Structural Biology 1999;125:39–49. 2. Adebayo OA, Burrow MF, Tyas MJ. An SEM evaluation of conditioned and bonded enamel following carbamide peroxide bleaching and casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) treatment. Journal of Dentistry 2009;37:297–306. 3. Eisenburger M, Addy M, Hughes JA, Shellis RP. Effect of time on the remineralisation of enamel by synthetic saliva after citric acid erosion. Caries Research 2001;35:211–5. 4. Barbour ME, Finke M, Parker DM, Hughes JA, Allen GC, Addy M. The relationship between enamel softening and erosion caused by soft drinks at a range of temperatures. Journal of Dentistry 2006;34:207–13. 5. Yamaguchi K, Miyazaki M, Takamizawa T, Inage H, Moore BK. Effect of CPP-ACP paste on mechanical properties of bovine enamel as determined by an ultrasonic device. Journal of Dentistry 2006;34:230–6. 6. Tantbirojin D, Huang A, Ericson MD, Poolthong S. Change in surface hardness of enamel by a cola drink and a CPP-ACP paste. Journal of Dentistry 2008;36:74–9. 7. Ramalingam L, Messer LB, Reynolds EC. Adding casein phosphopeptide-amorphous calcium phosphate to sports drinks to eliminate in vitro erosion. Pediatric Dentistry 2005;27:61–7. 8. Lussi A, Jaeggi T, Zero D. The role of diet in aetiology of dental erosion. Caries Research 2004;38:34–44. 9. Barbour ME, Parker DM, Allen GC, Jandt KD. Human enamel erosion in constant composition citric acid solutions as a function of degree of saturation with respect to hydroxyapatite. Journal of Oral Rehabilitation 2005;32:16–21. 10. Hemingway CA, Parker DM, Addy M, Barbour ME. Erosion of enamel by non-carbonated soft drinks with and without toothbrushing abrasion. British Dental Journal 2006;201:447– 50. 11. Hay KD, Thomson WM. A clinical trial of the anticaries efficacy of casein derivatives complexed with calcium phosphate in patients with salivary gland dysfunction. Oral
21.
22.
23.
24.
25.
26.
27.
28.
29.
30. 31.
873
Surgery Oral Medicine Oral Patholology Oral Radiology and Endodontics 2002;93:271–5. Ten Cate JM, Arends J. Remineralization of artificial enamel lesions in vitro. III. A study of the deposition mechanism. Caries Research 1980;14:351–8. Lussi A, Kohler N, Zero D, Schaffner M, Megert B. A comparison of the erosive potential of different beverages in primary and permanent teeth using an in vitro model. European Journal of Oral Science 2000;108:110–4. Watari F. In situ Analysis of etching process of teeth by AFM. Journal of Electronic Microscopy 2005;54:299–308. Magalha˜es AC, Rios D, Delbem ACB, Buzalaf MAR, Machado MAAM. Influence of fluoride dentifrice on brushing abrasion of eroded human enamel: an in situ/ex vivo study. Caries Research 2007;41:77–9. Lussi A, Hellwig E, Zero D, Jaeggi T. Erosive tooth wear: diagnosis, risk factors and prevention. American Journal of Dentistry 2006;19:319–25. Rees J, Loyn T, Chadwick B. Pronamel and tooth mousse: an initial assessment of erosion prevention in vitro. Journal of Dentistry 2007;35:355–7. Roveri N, Battistella E, Bianchi CL, Foltran I, Foresti E, Iafisco M, et al. Surface enamel remineralization: biomimetic apatite nanocrystals and fluoride ions different effects. Journal of Nanomaterials 2009. article ID 746383. Finke M, Jandt KM, Parker DM. The early stages of native enamel dissolution studied with atomic force microscopy. Journal of Colloid Interface Science 2000;232:156–64. Marshall GW, Chang YJ, Saeky K, Gansky SA, Marshall SJ. Citric acid etching of cervical sclerotic dentin lesions: an AFM study. Journal of Biomedical Material Research 2000;49:338–44. De-Deus G, Paciornik S, Pinho Mauricio MH, Prioli R. Realtime atomic force microscopy of root dentin during demineralization when subjected to chelating agents. International Endodontic Journal 2006;39:683–92. Poggio C, Lombardini M, Dagna A, Chiesa M, Bianchi S. Protective effect on enamel demineralization of a CPP-ACP paste: an AFM in vitro study. Journal of Dentistry 2009;37:949– 54. Quartarone E, Mustarelli P, Poggio C, Lombardini M. Surface kinetic roughening caused by dental erosion: an atomic force microscopy study. Journal of Applied Physics 2008;103:104702. Silverstone LM, Saxton CA, Dogon IL, Fejerskov O. Variation in the pattern of acid etching of human dental enamel examined by scanning electron microscopy. Caries Research 1975;9:373–87. Oshiro M, Yamaguchi K, Takamizawa T, Inage H, Watanabe T, Irokawa A, et al. Effect of CPP-ACP paste on tooth mineralization: an FE-SEM study. Journal of Oral Science 2007;49:115–20. Hegedus C, Bistey T, Flora-Nagy E, Keszthelyi G, Jenei A. An atomic force microscopy study on the effect of bleaching agents on enamel surface. Journal of Dentistry 1999;27:509–15. Lippert F, Parker DM, Jandt KD. In vitro demineralization/ remineralization cycles at human tooth enamel surfaces investigated by AFM and nanoindentation. Journal of Colloid Interface Science 2004;280:442–8. Lippert F, Parker DM, Jandt KD. Toothbrush abrasion of surface softened enamel studied with tapping mode AFM and AFM nanoindentaion. Caries Research 2004;38:464–72. Lippert F, Parker DM, Jandt KD. In situ remineralisation of surface softened human enamel studied with AFM nanoindentation. Surface Science 2004;553:105–14. Barbour ME, Rees JS. The laboratory assessment of enamel erosion: a review. Journal of Dentistry 2004;32:591–602. Ranjitkar S, Rodriguez JM, Kaidonis JA, Richards LC, Townsend GC, Bartlett DW. The effect of casein
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journal of dentistry 38 (2010) 868–874
phosphopeptide-amorphous calcium phosphate on erosive enamel and dentin wear by toothbrush abrasion. Journal of Dentistry 2009;37:250–4. 32. Yamaguchi J, Miyazaki M, Takamizawa T, Inage H, Kurokawa H. Ultrasonic determination of the effect of casein phosphopeptide-amorphous calcium phosphate paste on the demineralization of bovine dentin. Caries Research 2007;41:204–7. 33. Hara AT, Ando M, Gonza´lez-Cabezas C, Cury JA, Serra MC, Zero DT. Protective effect of the dental pellicle against erosive challenges in situ. Journal of Dental Research 2006;85:612–6. 34. Reynolds EC. Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. Journal of Dental Research 1997;76:1587–95.
35. Reynolds EC, Cai F, Shen P, Walker GD. Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum. Journal of Dental Research 2003;82:206–11. 36. Shen P, Cai F, Nowicki A, Vincent J, Reynolds EC. Remineralization of enamel subsurface lesions by sugarfree chewing gum containing casein phosphopeptideamorphous calcium phosphate. Journal of Dental Research 2001;80:2066–70. 37. Devlin H, Bassiouny MA, Boston D. Hardness of enamel exposed to Coca-Cola and artificial saliva. Journal of Oral Rehabilitation 2006;33:26–30. 38. Zero DT, Hara AT, Kelly SA, Gonza´lez-Cabezas C, Eckert GJ, Barlow AP, et al. Evaluation of a desensitizing test dentifrice using an in situ erosion remineralization model. Journal of Clinical Dentistry 2006;17:112–6.
