Evaluation to determine the caries remineralization potential of three dentifrices: An in vitro study

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1 Original Article Evaluation to determine the caries remineralization potential of three dentifrices: An in vitro study Arun Balakrishnan, Jonathan R, Benin P, Arvind Kuumar Department of Conservative Dentistry and Endodontics, Rajas Dental College, Tirunelveli, Tamil Nadu, India Abstract Aim: The aim of this study was to evaluate the remineralizing potential of three different remineralizing agents (GC tooth Mousse, Clinpro tooth crème and SHY-NM) on demineralized tooth surfaces using micro CT and microhardness. Materials and Methods: Forty five freshly extracted mandibular premolars were collected and enamel specimens were prepared. The samples were assigned to three groups with fifteen specimens in each group. The specimens were then demineralized using McInne s demineralizing solution in two cycles. After that, remineralization was carried out in two cycles for 30 days using Casein phosphopeptide - Amorphous calcium phosphate (CPP - ACP), 0.21% sodium fluoride - Tricalcium phosphate (f-tcp) and Calcium Sodium Phosphosilicate (CSP) containing tooth pastes for groups I, II, III respectively. The specimens were evaluated for Linear attenuation co-efficient using micro CT (Scanco TM ) and Vicker s Micro Hardness (Schimadzu TM ) testing at different time periods. The results were tabulated and statistically analysed. Results: It was observed that all the three remineralizing agents used in the study significantly increased the Linear Attenuation Co-efficient and Vicker s hardness number values of the enamel specimens following 15 days and 30 days application. Conclusion: CPP ACP showed the better remineralizing potential than the other two agents and there was no statistical significant between f-tcp and CSP groups. Keywords: Casein phosphopeptide amorphous calcium phosphate; calcium sodium phosphosilicate; micro computed tomography; vicker s microhardness number; 0.21% sodium fluoride - tricalcium phosphate INTRODUCTION Dental caries is a world-wide chronic disease, easily detectable, and reversible at an early stage, but irreversible and destructive after cavitation. Its dynamic nature of reversible and irreversible process makes its management time specific and critical. In the past, surgical approach for early surface lesions made the tooth crippled and posed irreparable damage. Recently, biological approaches focused on application of remineralization (RML) agents to early carious lesions, aimed at controlling demineralization (DML), and promoting RML. [1] DML is the process of mineral loss from the tooth structure while RML is the mineral gain in the form of hydroxyapatite to tooth structure. [2,3] RML agents creates a supersaturated environment around the early lesion; thus, preventing mineral loss and forces calcium and phosphate ions in the vacant areas. Universally, these agents contain calcium phosphate with or without fluoride. [3] Address for correspondence: Dr. Arun Balakrishnan, Pooja Vikas Nagar, 52 Pattathanam, Kollam , Kerala, India. Date of submission : Review completed : Date of acceptance : Casein Phosphopeptide Amorphous Calcium Phosphate (CPP ACP), (Tooth Mousse, GC India) was introduced as a RML agent in the year [4] It contains nanocomplexes of milk protein CPP with ACP. It has been claimed that it promotes RML of the carious lesions by maintaining a supersaturated state of essential minerals, at the same time it also hinders colonization of dental surfaces by cariogenic bacteria. [5] Clinpro tooth crème (3M ESPE) is a 0.21% w/w sodium fluoride (NaF) anti caries dentifrice that contains 950 ppm fluoride and a functionalized tricalcium phosphate (f-tcp) ingredient. One major advantage of this calcium phosphate system is that it is stable in aqueous environment and also does not affect the fluoride activity added in the dentifrices. Furthermore, it has been suggested that fluoride combination with f-tcp not only provides greater RML in terms of microhardness and fluoride uptake, but also decreases the dose of fluoride required to achieve the same degree of RML. [6] Access this article online Quick Response Code: Website: DOI: /

2 Novamin (calcium sodium phosphosilicate) containing SHY-NM (Group Pharmaceuticals) is a bioactive glass in the class of highly biocompatible materials that were originally developed as bone regenerative materials. [7] These materials are reactive when exposed to body fluids and deposit Hydroxycarbonate apatite, a mineral that is chemically similar to natural tooth minerals. [8] All the three RML agents mentioned above differ in their composition and mechanism of action, yet each one has a promising ability to RML the enamel. Thus, this present study was undertaken to determine and compare the microhardness values as well as the RML potential of CPP-ACP (GC Tooth Mousse), 0.21% NaF with f-tcp (Clinpro) and bioactive glass Novamin (SHY-NM) containing dentifrice. MATERIALS AND METHODS Sample selection Forty five freshly extracted human single rooted permanent mandibular premolar teeth, which were extracted for orthodontic reasons were taken for the study. The teeth selected were free from dental caries, restorations or developmental defects. The samples were cleaned of calculus and soft-tissues and stored in artificial saliva. Sample grouping and preparation Forty five teeth were equally divided into three groups, which were treated with GC Tooth mousse, Clinpro tooth crème and SHY-NM dentifrices respectively. The teeth were sectioned horizontally using a diamond disc (Axis dental, Texas) with a slow speed straight hand piece (NSK Japan) at 15,000 rpm at the level of Cemento Enamel Junction CEJ, separating the crown part of the tooth. The cusp and occlusal surface of the crown were then removed following the same technique. Next, the mesial, distal, and lingual sides of the tooth block were cut to obtain flat surfaces. Finally, the buccal side was flattened and polished using 200, 400, 800, 1000, 1200 grit abrasive paper to obtain cuboidal tooth blocks of 4 mm 4 mm 6 mm from each tooth. [9] All other tooth surfaces except the buccal surface was painted with acid resistant nail varnish (Lakme, India) and mounted in self-cure acrylic resin with buccal surface facing upward and exposed. The specimens were then stored in artificial saliva. The samples were then stabilized individually on the micro computed tomography (U CT) scanning machine and scanned to determine the mineral content of enamel specimens at baseline. Similarly the samples were placed on to the table of Vicker s Hardness testing machine, stabilized, and indented with the indenter to determine the Vicker s Hardness Number (VHN) value at baseline. After determining the baseline U CT and micro hardness values, the specimens were stored in artificial saliva. DML of samples DML of the samples were performed using freshly prepared McInne s DML solution. It was carried out in two cycles, (DML1 and DML2) with an application time of 5 min at 24 h interval. After DML, the samples were washed in running water, damped dry and subjected to U CT and micro hardness evaluation. [10] RML of samples First cycle of RML1 After DML, the specimens were divided into three groups (Group I, Group II and Group III) and subjected for RML treatment. The RML pastes were respectively applied for the specific group specimens with cotton applicator tips, 3 min twice daily for 15 consecutive days. The samples were then washed under running tap water, stored in artificial saliva for 15 days and then subjected to U CT and microhardness test for determining the mineral content and surface micro hardness respectively after the first RML cycle. [10] Second cycle of RML2 After first cycle of RML, RML pastes were applied for 15 more days, following the same protocols as mentioned for the first cycle of mineralization and stored in artificial saliva and at the end of 30 days the samples were again subjected for U CT and micro hardness testing [Figure 1] to determine the mineral content and surface micro hardness. [10] Statistical analysis The recorded values were subjected to statistical analysis using the SPSS software. Paired-t-test, one-way ANOVA, and Post-hoc Duncan s Multiple Range test were performed to evaluate the U CT and micro hardness values. For the entire evaluation, P < 0.05 was considered to be statistically significant. RESULTS When the DML enamel specimens were subjected to a RML cycle with CPP ACP for 15 days (First cycle of RML), the VHN values showed a marginal recovery in micro hardness of ± VHN from ± When the RML cycle was repeated for 15 more days, the VHN value increased to ± 36.13, which was statistically significant (P < 0.05) [Table 1]. Similarly when the DML enamel specimens were subjected to the first cycle of RML, the Linear attenuation Co efficient (LAC) values showed a marginal recovery within a range of ± mg/cm 3 [Table 1]. When the RML cycle was repeated for 15 more days, the LAC value increased to a range of ± mg/cm 3, which was statistically significant (P < 0.05) [Table 1]. The micro hardness of enamel specimens when treated with f-tcp + NaF showed a mean increase of ±

3 from ± VHN after 15 days [Table 2] and to ± VHN after 30 days, which was statistically significant (P < 0.05) [Table 2]. Similarly, the LAC value also increased to ± from ± after 15 days and to ± after 30 days treatment, which was statistically significant (P 0.05) [Table 2]. The micro hardness of enamel specimens when treated with Calcium sodium Phosphosilicate (CSP) toothpaste showed a mean increase of ± VHN from ± after 15 days [Table 2] and to ± after 30 days treatment, which was statistically significant [Table 2]. LAC values also increased to ± from ± after 15 days and to ± after 30 days, which was statistically significant [Table 1]. DISCUSSION Non-invasive intervention can transform a lesion from an active to an inactive state. The clinician needs to monitor the outcome of non-invasive measures and in cases where there is evidence of lesion progression to make a timely decision to intervene, using minimally invasive techniques and restore damaged tooth structure without weakening the tooth. Complementing traditional diagnostic methods with advanced, more sensitive methods will improve caries diagnostic efficiency and hence the dental care and treatment of patients. [4] The basic mechanism of RML involves the diffusion of calcium and phosphate ions from saliva and other topical sources aided by fluoride to build a hypermineralized, acid-resistant, fluorapatite like layer on the existing crystal remnants, which act as RML nuclei. [11,12] In this study, a comparative evaluation of the RML potential of three different commercially available dentifrices on DML tooth surfaces was evaluated using U CT and micro hardness testing. The buccal side of the sample was flattened and polished to obtain a comparatively flat surface to avoid any operational bias during Vicker s Micro hardness measurement. [10,13] DML with McInne s bleaching solution was performed in two cycles as it has been proved in the previous studies that only after completing the second cycle of DML after 24 h, there was a significant reduction in VHN. [13,14] Artificial saliva was used for storing the specimens in between the bleaching cycles, because it is believed that artificial saliva contributed to a slight increase in the micro hardness, after demineralization. [13] The RML treatment regimen of 3 min twice daily application was employed as per the manufacturer s recommendations and it has also been proved in the previous studies that the longer the duration of the RML agent in contact with the teeth, the better was the RML. [10,13] U CT is a microscopic version of CT that allows three-dimensional non-destructive visualization of Figure 1: Micro hardness indentation images on enamel specimens of group I, II, III 377

4 Table 1: Comparison of micro CT (mg/cm 3 ) between demineralization 2 cycle 1 cycle and demineralization 2 cycle 2 cycle in three groups Groups Demineralization 2 cycle 1 cycle P value Demineralization 2 cycle 2 cycle GP 1 DML ± c <0.05 DML ± b <0.05 GP 1 RML ± c <0.05 RML ± b <0.05 GP 2 DML ± b <0.05 DML ± a <0.05 GP 2 RML ± b <0.05 RML ± a <0.05 GP 3 DML ± a <0.05 DML ± a <0.05 GP 3 RML ± a <0.05 RML ± a <0.05 a,b,c with same superscript do not differ each other (Duncan s multiple range test), RML: Remineralization, DML: Demineralization, GP: Groups, CT: Compterised tomography Table 2: Comparison of Vicker s hardness number between demineralization 2 cycle 1 cycle and demineralization 2 cycle 2 cycle in three groups Groups Demineralization 2 cycle 1 cycle P value Demineralization 2 cycle 2 cycle GP 1 DML ± b <0.05 DML ± b <0.01 GP 1 RML ± b <0.05 RML ± b <0.01 GP 2 DML ± ab <0.001 DML ± a <0.001 GP 2 RML ± ab <0.001 RML ± a <0.001 GP 3 DML ± a <0.001 DML ± a <0.001 GP 3 RML ± a <0.001 RML ± a <0.001 a,b with same superscript do not differ each other (Duncan s multiple range test), RML: Remineralization, DML: Demineralization, GP: Groups P value P value the morphological characteristics of teeth and the determination of the mineral content in teeth and bones apart from providing both qualitative and quantitative data. Because of its advantages over the conventional equipments, U CT has been used as one of the evaluation criteria. In the present study, VHN was chosen over Knoop s Hardness Number because a square shape of indent obtained in VHN was easy and accurate to measure. CPP ACP can be used to prevent DML and promote RML of early enamel lesions and it has a short term RML effect in clinical in situ trials and long-term caries-preventing effect in the in vivo randomized control trial. [15,16] The proposed anticariogenic mechanism for CPP ACP is by the localization of ACP on the tooth surface, which buffers the free calcium and phosphate ion activities, thereby helping to maintain a state of supersaturation with respect to the tooth enamel and thus preventing DML and enhancing RML. Fluoride ions helps in RML by the formation of fluorapatite in enamel in the presence of calcium and phosphate ions produced during enamel DML. [16] Thus, the f-tcp and 950 ppm NaF containing dentifrice have an added advantage of fluoride in addition to novel f-tcp thus aiding in enhanced RML. The f-tcp contains 2% sodium lauryl sulfate, which prevents calcium phosphate reaction with fluoride and promotes formation of calcium fluoride. There are many studies in literature to support Novamin as a successful desensitizing agent. However, only a few studies are available to support the RML action of Novamin on enamel. [17,18] The present study compared the change in micro hardness and mineral content following treatment with CPP ACP, f-tcp + NaF and Novamin containing dentifrice over 30 days. It was observed that after 30 days no significant was seen between the CPP ACP and Novamin groups, but mean increase in micro hardness and mineral content was more in CPP ACP group compared to Novamin group. Hegde et al., [19] evaluated the RML of enamel by CPP ACP using Energy dispersive X-ray analysis and concluded that CPP ACP was successful in RML sub surface enamel and the extent of RML achieved was dose dependent and increased with increase the time of exposure and duration of the study. Thus, in the present study a greater increase in the micro hardness was seen since the specimens were subjected to 30 days cycle compared to the 5 days. [20] In another study conducted by Karlinsey, [21] he compared the effect of f-tcp ppm of fluoride combination with three other RML agents and measured changes in surface micro hardness of the enamel. At the end of 10 days of treatment, the micro hardness of enamel increased by ± 7.4 VHN. Karlinsey et al., compared 5000 ppm containing dentifrice with clinpro 5000 (5000 ppm fluoride + ftcp). The results showed that Clinpro 5000 showed a mean increase of ± 5.6 VHN, in micro hardness of enamel after 10 days. In the present study, the micro hardness of enamel treated with f-tcp + NaF showed a mean increase of ± from ± after 30 days, which was statistically significant (P < 0.05). The in the values could be attributed to the number of days the RML was carried out and also the RML agents used in both of the studies. 378

5 Rehder Neto et al., [22] assessed whether pastes containing CPP ACP and CSP control artificial caries lesion progression. The results showed that CSP containing paste had 7.1% increases in mineral content when compared to CPP ACP containing paste, which showed only 3.2%. The authors concluded that CSP containing paste, CPP ACP + F, were superior to CPP ACP paste. In 2011, Rehder Neto et al., [22] compared the RML potential of CPP ACP and CSP containing paste on acid softened enamel. They compared 4 products with control, (i) CPP ACP (MI paste, GC America); (ii) CPP ACP + Fluoride (MI Paste plus, GC America); (iii) CSP (Tooth revitalizing paste, Oravive); (iv) Fluoridated dentifrice (FD Sensodyne cool gel GSK); (v) Control (CO, unexposed to any product). The results showed that, the increase in Surface Micro Hardness (SMH) in CPP ACP Casein Phospho Peptide Amorphous Calcium Phosphate and CSP group did not differ significantly and was higher than control group. The present study also showed similar results, where no significant in micro hardness values was observed between the CPP ACP and Novamin group. The results of the present study showed that CPP ACP has the better RML potential among the three groups evaluated and it can be attributed due to the peculiar nature of CPP. CPP by stabilizing calcium phosphate in a metastable solution, facilitate high concentrations of calcium and phosphate ions, including CaHPO 4, which can diffuse into the enamel subsurface lesion. The RML efficacy of f-tcp group is due to the presence of fluoride compatible functionalized calcium phosphate ingredient that imparts superior RML at both the enamel surface and within the subsurface lesions, thereby boosting the enamel surface strength. CONCLUSION All the three RML agents used have significantly increased the LAC and VHN values of the enamel specimens after a period of 15 days. The LAC and VHN values are enhanced after additional 15 days treatment with RML agents. CPP ACP group has shown a statistically significant increase in the mineral content and micro hardness when compared to other two groups. Further, long-term clinical trials should be conducted to prove the superiority of these materials in the vital teeth. REFERENCES 1. Rao A, Malhotra N. The role of remineralizing agents in dentistry: A review. Compend Contin Educ Dent 2011;32: 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 Res 1975;9: Scott DB, Simmelink JW, Nygaard V. Structural aspects of dental caries. J Dent Res 1974;53: Reynolds EC. Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. J Dent Res 1997;76: Reynolds EC. Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: A review. Spec Care Dentist 1998;18: Karlinsey RL, Mackey AC, Stookey GK, Pfarrer AM. In vitro assessments of experimental NaF dentifrices containing a prospective calcium phosphate technology. Am J Dent 2009;22: Hench LL, Andersson O. Bioactive glasses. In: Hench LL, Wilson J, editors. Introduction to bioceramics. Singapore: World Scientific; p Andersson OH, Kangasniemi I. Calcium phosphate formation at the surface of bioactive glass in vitro. J Biomed Mater Res 1991;25: Lo EC, Zhi QH, Itthagarun A. Comparing two quantitative methods for studying remineralization of artificial caries. J Dent 2010;38: Hora BS, Kumar A, Bansal R, Bansal M, Khosla T, Garg A. Influence of McInnes bleaching agent on hardness of enamel and the effect of remineralizing gel GC tooth mousse on bleached enamel: An in vitro study. Int J Dent Res 2012:2: Featherstone JD. Dental caries: A dynamic disease process. Aust Dent J 2008;53: Featherstone JD, Duncan JF, Cutress TW. A mechanism for dental caries based on chemical processes and diffusion phenomena during in-vitro caries simulation on human tooth enamel. Arch Oral Biol 1979;24: Darshan HE, Shashikiran ND. The effect of McInnes solution on enamel and the effect of tooth mousse on bleached enamel: An in vitro study. J Conserv Dent 2008;11: Attin T, Müller T, Patyk A, Lennon AM. Influence of different bleaching systems on fracture toughness and hardness of enamel. Oper Dent 2004;29: Ferrazzano GF, Cantile T, Ingenito A, Chianese L, Quarto M. New strategies in dental caries prevention: Experimental study on casein phosphopeptides. Eur J Paediatr Dent 2007;8: Ten Cate JM. Current concepts on the theories of the mechanism of action of fluoride. Acta Odontol Scand 1999;57: Diamanti I, Koletsi-Kounari H, Mamai-Homata E, Vougiouklakis G. Effect of fluoride and of calcium sodium phosphosilicate toothpastes on pre-softened dentin demineralization in vitro. J Dent 2010;38: Vahid Golpayegani M, Sohrabi A, Biria M, Ansari G. Remineralization effect of topical novamin versus sodium fluoride (1.1%) on Caries-Like Lesions in Permanent Teeth. J Dent (Tehran) 2012;9: Hegde MN, Shetty S, Pardal D. Remineralization of enamel sub-surface lesions using casein phosphopeptide amorphous calcium phosphate (CPP ACP) quantative energy dispersive X-ray analysis (EDAX). J Conserv Dent 2007;10; Lata S, Varghese NO, Varughese JM. Remineralization potential of fluoride and amorphous calcium phosphate-casein phospho peptide on enamel lesions: An in vitro comparative evaluation. J Conserv Dent 2010;13: Karlinsey RL, Mackey AC, Walker TJ, Frederick KE, Blanken DD, Flaig SM, et al. In vitro remineralization of human and bovine white-spot enamel lesions by NaF dentifrices: A pilot study. J Dent Oral Hyg 2011;3: Rehder Neto FC, Maeda FA, Turssi CP, Serra MC. Potential agents to control enamel caries-like lesions. J Dent 2009; 37: How to cite this article: Balakrishnan A, Jonathan R, Benin P, Kuumar A. Evaluation to determine the caries remineralization potential of three dentifrices: An in vitro study. J Conserv Dent 2013;16: Source of Support: Nil, Conflict of Interest: None declared. 379

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