Effect of CPP ACP paste on mechanical properties of bovine enamel as determined by an ultrasonic device

Journal of Dentistry (2006) 34, 230 236 www.intl.elsevierhealth.com/journals/jden Effect of CPP ACP paste on mechanical properties of bovine enamel as determined by an ultrasonic device Kanako Yamaguchi a, Masashi Miyazaki a, *, Toshiki Takamizawa a, Hirohiko Inage a, B. Keith Moore b a Department of Operative Dentistry, Nihon University Graduate School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-Ku, Tokyo 101-8310, Japan b Department of Restorative Dentistry, Division of Dental Materials, Indiana University School of Dentistry, 1121 West Michigan Street, Indianapolis, IN 46202, USA Received 22 March 2005; received in revised form 7 June 2005; accepted 13 June 2005 KEYWORDS Enamel; CPP ACP; Ultrasonic device; Sonic velocity; Remineralization; Demineralization Summary Objective: The purpose of this study was to determine the effect of Casein Phosphopeptide Amorphous Calcium Phosphate (CPP ACP) paste on demineralization of bovine enamel by measuring changes in the ultrasound transmission velocity. Methods: The enamel specimens were prepared by cutting bovine teeth into blocks. The specimens were stored in 0.1 M lactic acid buffer solution (ph 4.75, Ca 0.75 mm, P 0.45 mm) for 10 min twice a day, and then stored in the artificial saliva (ph 7.0). Other specimens were stored in a 10-times diluted solution of CPP ACP paste and a placebo paste without CPP ACP for 10 min, followed by 10 min immersion into a demineralization solution twice a day before storage in the artificial saliva. The propagation time of longitudinal ultrasonic waves was measured by a Pulser-Receiver (Model 5900, Panametrics) with a transducer (V112, Panametrics). Six specimens were used for each condition, and one-way ANOVAs followed by the Tukey HSD tests (az0.05) were done. Results: The sonic velocity was found to decrease with time for specimens stored in the demineralization solution. On the other hand, a significant increase in sonic velocity was found for specimens stored in the CPP ACP solution. Conclusions: From the result of this study, it was suggested that the conditions of deand remineralization of the enamel structure could be measured non-destructively by using an ultrasonic pulse method. It could be concluded that the inorganic * Corresponding author. Tel.: C81 3 3219 8141; fax: C81 3 3219 8347. E-mail address: miyazaki-m@dent.nihon-u.ac.jp (M. Miyazaki). 0300-5712/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2005.06.005

Re- and demineralization of enamel substrate 231 components contained in high concentrations in CPP ACP acted to enhance remineralization of the enamel structure. Q 2005 Elsevier Ltd. All rights reserved. Introduction In the oral environment, tooth structure undergoes continuous demineralization and remineralization. 1,2 If this balance is disrupted, demineralization will progress leading to a deterioration of the tooth structure. Milk and milk products such as cheese have been shown to have anticariogenic properties in human and animal models. 3,4 It has been suggested that the mechanism of this action is due to a direct chemical effect from phosphoprotein casein and calcium phosphate components of the cheese. 5,6 Casein phosphopeptides (CPP) have the ability to stabilize calcium phosphate in solution through binding amorphous calcium phosphate (ACP) with their multiple phosphoserine residues. This allows the formation of small CPP ACP clusters. CPP ACP should have an anti-caries protective effect, by suppressing demineralization, enhancing remineralization, or possibly a combination of both. 7 Ultrasonic imaging is used in many fields as a noninvasive technique and offers considerable potential in diagnosis as well as serving as a tool for research. 8 10 The first attempt to apply an ultrasonic diagnostic method to tooth structure was done with opththalmographic equipment. 11 Since then, sonic properties of enamel and dentine have been reported by several researchers. 12 14 Ultrasonic devices are also used to detect carious lesions 15 17 and to measure the remaining dentine thickness between the tooth surface and pulp chamber. 18 Recently, these devices were used to evaluate adhesive defects of dentine bonding systems. 19 It has been indicated that the sonic velocity proportionally increases with the volumetric concentration of inorganic components. 20 The degree of mineralization and the differences in the histological structures resulted in differences of the sonic velocities in each specimen. Therefore we focused on using an ultrasonic pulse method and to identify the conditions of de- and remineralization with time. The purpose of this study was to evaluate the effect of CPP ACP paste on demineralization by measuring changes in the speed of sound using an ultrasonic device, and by microscopic observation with field emission scanning electron microscopy (FE-SEM). The null hypothesis tested was that changes in de- and remineralization would not be detected by the ultrasound technique. Materials and methods Fabrication of the specimens Freshly extracted bovine incisors without cracks or erosions were cleaned and stored in physiological saline for up to 2 weeks. The teeth were sliced into 1 mm sections in different directions with a lowspeed diamond saw (Buehler Ltd, Lake Bluff, IL, USA). Each enamel slab was carefully shaped into a rectangular form (4 mm!4 mm!1 mm) with a superfine diamond point (ISO #021, Shofu, Inc., Kyoto, Japan) to make the specimen walls parallel to each other. Each surface of the specimen was ground on wet #600 to #2000-grit SiC paper successively. Thickness and size of the specimens were measured by means of a dial gage micrometer (CPM15-25DM, Mitutoyo, Tokyo, Japan). The composition of topical paste and artificial saliva used in this study was listed in Table 1. A CPP ACP paste used was Tooth Mousse (GC Corp., Tokyo, Japan), a water-based sugar-free soft wet mixture of Recaldent. The specimens were stored in the 0.1 M lactic acid buffer solution (ph 4.75, Ca 0.75 mm, P 0.45 mm) for 10 min and then placed in the artificial saliva (group De). Other specimens were stored in the 10-times diluted solution of CPP ACP paste (group TM), and Placebo Paste without CPP ACP (group PP) for 10 min prior to the storage of the specimens in the demineralizing solution. Table 1 Composition of topical paste and artificial saliva. Code Paste Topical paste TM 1% (w/v) CPP ACP containing paste PP CPP ACP-free placebo paste Component wt% Artificial saliva NaCl 0.08 KCl 0.12 MgCl 2 0.01 K 2 HPO 4 0.03 CaCl 2 0.01 CMC-Na 0.10 IEW 99.6 CMC-Na, sodium carboxymethyl cellulose; IEW, ion exchanged water; ph value was adjusted to 7.0 by CO 2.

232 Specimens were stored in the 0.1 M lactic acid buffer solution and then in the artificial saliva at 37 8C. The specimens were stored in the demineralizing solution twice a day during 4 weeks of test periods. An additional group was not treated but simply stored in artificial saliva for the same total length of time (group control). Measuring of the propagation time of ultrasonic waves The ultrasonic equipment used to measure sound velocity consisted of a Pulser-Receiver (Model 5900PR, Pamametrics, Waltham, MA, USA), a transducer for longitudinal waves (V112, Panametrics) and an oscilloscope (Wave Runner LT584, LeCroy Corp., Chestnut Ridge, NY, USA) as shown in Fig. 1. 21,22 Measurements were done before and 7, 14, 21, and 28 days after the start of the tests. The equipment was initially calibrated using a standard calibration procedure with the calibration blocks made of 304 Stainless Steel (2211M, Panametrics) 2.5, 5.0, 7.5, 10.0 and 12.5 mm thick. The transducer was oriented perpendicular to the contact surface of each specimen to get the echo signal. The ultrasonic waves propagate from the transducer to the tooth, then are reflected off the surface of the tooth or transmitted through the tooth. Each measurement was conducted at 23G1 8C and 50G5% relative humidity (RH) in the temperature-controlled room. The number of specimens used was six pieces for each condition. Statistical analysis The sonic velocity for each specimen was subjected to one-way ANOVA followed by Tukey HSD test for comparison among the different treatments (az0.05). The statistical analysis was carried out with the Sigma Stat w software system (SPS, Inc., Chicago, IL, USA). FE-SEM observation For the ultrastructure observation of enamel surfaces by FE-SEM, specimens stored in each condition were dehydrated in ascending concentrations of tert-butanol (50% for 20 min, 75% for 20 min, 95% for 20 min, and 100% for 2 h), and then transferred to a critical-point dryer for 30 min. The surfaces were coated in a vacuum evaporator, Quick Coater Type SC-701 (Sanyu Denshi, Inc., Tokyo, Japan), with a thin film of Au. The specimens were observed in FE-SEM (ERA 8800FE, Elionix Ltd, Tokyo, Japan). Results K. Yamaguchi et al. The results of the sonic velocities obtained from the specimens are summarized in Table 2 and Fig. 2. The average sonic velocity in bovine intact enamel (control) ranged 6060 6074 m/s, and no significant differences were found among the different storage periods. The sonic velocities of specimens immersed in demineralization solution (De group) were found to decrease with time (5671 5507 m/s) and significant differences were found when compared to the control (p!0.001). The same tendency was found for the specimens treated with the placebo paste (PP group). On the other hand, increase in the sonic velocities was found for specimens stored in the 10-times diluted solution of CPP ACP paste (TM group), and there was no significant difference compared to that of control until 21 days of storage. After 28 days, a significant increase in sonic velocity was found for Figure 1 Experimental set-up of the ultrasonic device for detection of tooth de- and remineralization.

Re- and demineralization of enamel substrate 233 Table 2 Average sonic velocity in bovine enamel stored in various conditions. Group 0 7 14 21 28 Control 6073 (180) 6060 (180) 6068 (187) 6074 (186) 6068 (185) De 6012 (276) 5671 (151) 5570 (157) 5558 (122) 5507 (144) TM 6089 (308) 6240 (334) 6280 (255) 6296 (250) 6409 (286) PP 6005 (142) 5800 (105) 5725 (104) 5678 (141) 5618 (155) Unit, m/s; ( ), SD; NZ6. Values connected by vertical lines indicate no significant difference (po0.05). TM group compared to those of other three groups (p!0.001). Representative pictures of SEM observations of enamel specimens are shown in Fig. 3. SEM observations revealed different morphological features brought about by storage conditions. Demineralization of the enamel surfaces was more pronounced with the longer test period for the demineralization and negative control specimens (De and PP groups). On the other hand, enamel specimens treated with CPP ACP paste (TM group) revealed slight changes in their morphological features. Discussion Although there is a consensus that the use of human teeth is more relevant for conducting in vitro studies, bovine teeth were used in this study. The key experimental parameters that need to be considered are the characteristics of the subject panel, the physical design of the model, the type of hard tissue substrate and the method of assessing mineral status, and the study design. Each parameter must be carefully considered in relation to the objectives of the research. 23 The advantage of using bovine teeth instead of human teeth is that they are easy to obtain in large quantities in good Figure 2 Change in sonic velocity in bovine enamel stored in various conditions. condition, and have less composition variables than human enamel. 24 Bovine teeth have large flat surfaces and would not have had prior caries challenges that might affect the test results. Mineral distribution in the carious lesions in bovine teeth is reported similar to that found in human teeth, and structural changes in human and bovine teeth are similar. 24 Care should be taken when drawing conclusions from the data since there are many factors that affect the results obtained in vitro De- and remineralization process are difficult to detect at early stages of formation on enamel by visual inspection. There are many diagnostic techniques for detecting the demineralization which occurred as a result of the caries process. 25 29 Sonic velocity related to the mineral content of the body of the enamel lesion has been reported. 16 When the tooth surface suffers demineralization, the mineral volume concentration at the tooth surface as well as the specific sonic velocity should decrease. The sonic velocity proportionally increases with the volumetric concentration of mineral components and is an index of degree of mineralization; larger values of the sonic velocity indicate a higher degree of mineralization. As previously described, the orientation of the enamel prism may modify the sonic velocity. 30 According to the plane of section of the teeth, the enamel prism are at different angles to the direction of ultrasonic beam. To avoid this kind of effect on sonic velocity, the enamel specimens were obtained from labial surfaces of the bovine teeth. The role of CPP ACP has been described as localization of ACP at the tooth surface which buffers the free calcium and phosphate ion activities, helping to maintain a state of supersaturation with respect to enamel depressing demineralization and enhancing remineralization. 31 The presence of CPP ACP might permit a rapid return to resting calcium concentrations and allows more immediate remineralization of enamel substrate. It has demonstrated that CPP could still be detected on the tooth surface 3 h after consuming xylitol gum containing CPP ACP. Other

234 K. Yamaguchi et al. Figure 3 FE-SEM observation of enamel surfaces (original magnification;!10,000). studies have demonstrated that CPP ACP in a mouthwash significantly increased the level of calcium and inorganic phosphate ions in supragingival plaque with the CPP bound to salivary pellicle and to the surface of bacteria in the plaque biofilm. 32 The present study showed that the twice-daily use of the 10-times diluted CPP ACP paste resulted in an increase in sonic velocity, indicating CPP ACP paste enhances remineralization of enamel. Placebo paste without CPP ACP showed no protection of enamel against demineralization. It has been shown that CPPs are an excellent delivery vehicle for the co-localization of calcium and phosphate at the tooth surface, leading to anti-caries efficacy. CPP ACP is provided in the form of a mouthrinse, a tablet and sugar-free chewing gum. CPP ACP containing mouthrinse significantly increased plaque calcium and inorganic phosphate levels, and CPP ACP was incorporated into supragingival dental plaque by binding onto the surface of bacterial cells as well as to components of the intercellular plaque matrix. 32 Sugar-free chewing gum has an anticariogenic effect through the stimulation of saliva. 33 The chewing gum containing CPP ACP showed the highest level of enamel subsurface lesion remineralization independent of chewing frequency or duration. 34,35 Oral hygiene products such as toothpaste with anti-plaque chemicals or tooth brushes with functional design have been shown to provide benefits in terms of plaque removal. 36 On the other hand, the process of abrasion resulting from toothbrush and toothpaste is not fully understood. 37 In this study, a paste type formulation of CPP ACP was used. Patients can use this kind of oral hygiene paste just like tooth paste with toothbrushes and also apply the paste with cotton slabs. When a patient suffers from tooth sensitivity during home whitening procedure, the CPP ACP paste can be applied on the whitening tray to reduce sensitivity by remineralization. Paste type CPP ACP paste can be used with many application methods to prevent tooth erosion. The inclusions of active ingredients in oral care products to help prevent dental disease have been shown to contribute significantly to improvement and maintenance of oral health. 36 The results of this study reject the null hypothesis that the ultrasonic equipment does not detect

Re- and demineralization of enamel substrate 235 the de- and remineralization of enamel substrates. The ultrasonic method showed the ability to determine changes in ultrasonic velocity in specimens as a function of their demineralization. The data showed that CPP ACP paste has an ability to prevent demineralization and enhance remineralization of enamel substrate. Conclusions Within the limitations of this in vitro study, the following conclusions may be drawn. 1. The condition of demineralization of the tooth structure could be measured non-destructively by using an ultrasonic pulse method. 2. CPP ACP paste was effective in preventing demineralization of bovine enamel. CPP ACP paste enhanced remineralization of enamel substrate more effectively than placebo paste (CPP ACP-free). Acknowledgements This work was supported, in part, by grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to promote multi-disciplinary research project, and Grant-in Aid for Encouragement of Young Scientists (B) 17791370, and Grantin-Aid for Scientific Research (C) 17592004 from the Japan Society for the Promotion of Science. References 1. Featherstone JD. The science and practice of caries prevention. Journal of American Dental Association 2000; 131:887 99. 2. Featherstone JD. The continuum of dental caries evidence for a dynamic disease process. Journal of Dental Research 2004;83:C39 C42 [Spec Iss C]. 3. Reynolds EC, Johnson IH. Effect of milk on caries incidence and bacterial composition of dental plaque in the rat. Archives of Oral Biology 1981;26:445 51. 4. Rosen S, Min DB, Harper DS, Harper WJ, Beck EX, Beck FM. Effect of cheese, with and without sucrose, on dental caries and recovery of Streptococcus mutans in rats. Journal of Dental Research 1984;63:894 6. 5. Krobicka A, Bowen WH, Pearson S, Young DA. The effects of cheese snacks on caries in desalivated rats. Journal of Dental Research 1987;66:1116 9. 6. Harper DS, Osborn JC, Hefferren JJ, Clayton R. Cariostatic evaluation of cheeses with diverse physical and compositional characteristics. Caries Research 1986;20:123 30. 7. Reynolds EC. Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. Journal of Dental Research 1997;76:1587 95. 8. Louwerse C, Kjaeldgaard M, Huysmans MCDNJM. The reproducibility of ultrasonic enamel thickness measurements: an in vitro study. Journal of Dentistry 2004;32:83 9. 9. John C. Directing ultrasound at the cemento-enamel junction (CEJ) of human teeth: I. Asymmetry of ultrasonic path lengths. Ultrasonics 2005;43:467 79. 10. Arslantunali Tagtekin D, Öztürk F, Lagerweij M, Hayran O, Stookey GK, Çaliskan Yanikoglu F. Thickness measurement of worn molar cusps by ultrasound. Caries Research 2005;39: 139 43. 11. Baum G, Greenwood I, Slawski S, Smirnow R. Observation of internal structures of teeth by ultrasonography. Science 1963;139:495 6. 12. Ng SY, Payne PA, Cartledge NA, Ferguson MWJ. Determination of ultrasonic velocity in human enamel and dentinee. Archives of Oral Biology 1989;34:341 5. 13. Löst C, Irion KM, John C, Nüssle W. Two-dimensional distribution of sound velocity in ground sections of dentine. Endodntics and Dental Traumatology 1992;8:215 8. 14. Mezawa S, Kawato T, Yoshida K, Nozaki H, Saito T, Tamura K, Onozawa M. Evaluation of human tooth structure with the ultrasonic imaging technique. Journal of Oral Science 1999; 41:191 7. 15. Peck SD, Briggs GAD. A scanning sonic microscope study of the small caries lesion in human enamel. Caries Research 1986;20:356 60. 16. Ng SY, Ferguson MWJ. Ultrasonic studies of unblemished and artificially demineralized enamel in extracted human teeth: a new method for detecting early caries. Journal of Dentistry 1988;16:201 9. 17. Yanıoğlu FÇ, Öztürk F, Hayran O, Analoui M, Stookey GK. Detection of natural white spot caries lesions by an ultrasonic system. Caries Research 2000;34:225 32. 18. Huysmans MCDNJM, Thijssen JM. Ultrasonic measurement of enamel thickness: a tool for monitoring dental erosion? Journal of Dentistry 2000;28:187 91. 19. Hamano N, Hanaoka K, Ebihara K, Toyoda M, Teranaka T. Evaluation of adhesive defects using an ultrasonic pulsereflection technique. Dental Materials Journal 2003;22: 66 79. 20. Lees S. Ultra-sonics in hard tissues. International Dental Journal 1971;21:403 17. 21. Miyazaki M, Inage H, Onose H. Use of an ultrasonic device for the determination of elastic modulus of dentine. Journal of Oral Science 2002;44:19 26. 22. Watanabe T, Miyazaki M, Inage H, Kurokawa H. Determination of elastic modulus of the components at dentine-resin interface using the ultrasonic device. Dental Materials Journal 2004;23:361 7. 23. Ogaard B, Rolla G. Intra-oral models: comparison of in situ substrates. Journal of Dental Research 1992;71:920 3. 24. Edmunds DH, Whittaker DK, Green RM. Suitability of human, bovine, equine, and ovine tooth enamel for studies of artificial bacterial carious lesions. Caries Research 1988;22: 327 36. 25. Huysmans MCDNJM, Longbottom C, Christie AM, Bruce PG, Shellis RP. Temperature dependence of the electrical resistance of sound and carious teeth. Journal of Dental Research 2000;79:1464 8. 26. Ando M, van Der Veen MH, Schemehorn BR, Stookey GK. Comparative study to quantify demineralized enamel in deciduous and permanent teeth using laser- and lightinduced fluorescence techniques. Caries Research 2001;35: 464 70.

236 27. Lussi A, Francescut P. Performance of conventional and new methods for the detection of occlusal caries in deciduous teeth. Caries Research 2003;37:2 7. 28. Reis A, Zach Jr VL, de Lima AC, de Lima Navarro MF, Grande RH. Occlusal caries detection: a comparison of DIAGNOdent and two conventional diagnostic methods. Journal of Clinical Dentistry 2004;15:76 82. 29. Higham SM, Pretty IA, Edgar WM, Smith PW. The use of in situ models and QLF for the study of coronal caries. Journal of Dentistry 2005;33:235 41. 30. Peck SD, Rowe JM, Briggs GA. Studies on sound and carious enamel with the quantitative sonic microscope. Journal of Dental Research 1989;68:107 12. 31. Reynolds EC. Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: a review. Special Care in Dentistry 1998;18:8 16. 32. 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 sugarfree chewing gum. Journal of Dental Research 2003;82: 206 11. K. Yamaguchi et al. 33. Beiswanger BB, Boneta AE, Mau MS, Katz BP, Proskin HM, Stookey GK. The effect of chewing sugar-free gum after meals on clinical caries incidence. Journal of American Dental Association 1998;129:1623 6. 34. Shen P, Cai F, Nowicki A, Vincent J, Reynolds EC. Remineralization of enamel subsurface lesions by sugarfree chewing gum containing casein phosphopeptide amorphous calcium phosphate. Journal of Dental Research 2001;80:2066 70. 35. Iijima Y, Cai F, Shen P, Walker G, Reynolds C, Reynolds EC. Acid resistance of enamel subsurface lesions remineralized by a sugar-free chewing gum containing casein phosphopeptide amorphous calcium phosphate. Caries Research 2004; 38:551 6. 36. Schafer F, Nicholson JA, Gerritsen N, Wright RL, Gillam DG, Hall C. The effect of oral care feed-back devices on plaque removal and attitudes towards oral care. International Dental Journal 2003;53(6 Suppl 1):404 8. 37. Imfeld T. Prevention of progression of dental erosion by professional and individual prophylactic measures. European Journal of Oral Science 1996;104:215 20.