Effect of Adding Tricalcium Phosphate to Fluoride Mouthrinse on Microhardness of Demineralized Primary Human Tooth

Effects of Fluoride Plus TCP Mouthrinse on Microhardness of Primary Enamel

EFFECT OF ADDING TRICALCIUM PHOSPHATE TO FLUORIDE MOUTHRINSE ON MICROHARDNESS OF DEMINERALIZED PRIMARY HUMAN TOOTH

Praphasri Rirattanapong1, Kadkao Vongsavan1, Chavengkiat Saengsirinavin2 and Pimonchat Phuekcharoen3

1Department of Pediatric Dentistry, Faculty of Dentistry; 2Research Unit, Faculty of Dentistry, Mahidol University, Bangkok; 3Dental Department, Khao Khitchakut Hospital, Chantaburi, Thailand

Abstract. The purpose of the present study was to evaluate the effect of fluoride mouthrinse containing tricalcium phosphate on microhardness of demineralized primary enamel. Thirty-six sound primary incisors were immersed in a demineralizing solution (pH 4.4) for 96 hours at 37oC to create artificial caries-like lesions. After artificial caries formation, the specimens were randomly divided into 3 groups (with 12 specimens in each group): Group A: deionized water; Group B: 0.05% NaF plus 20 ppm tricalcium phosphate mouthrinse and Group C: 0.05% NaF mouthrinse. All the specimens were immersed for 1 minute at 37ºC three times per day for 7 days in the respective mouthrinse among pH cycling. The surface microhardness was examined using a Vickers hardness tester (100 grams for 15 seconds) at baseline, before and after the pH-cycling procedure. Data were analyzed using one-way ANOVA and Tukey’s multiple comparison tests with a significance level of 0.05. After treatment, Group A had a significantly lower surface microhardness value than the other two groups (p=0.000); however, there was no significant difference between Groups B and Cp ( =0.728). We concluded fluoride mouthrinse containing tricalcium phosphate and fluoride mouthrinse have similar remineralizing effects on microhardness of demineralized primary teeth. Keywords: fluoride mouthrinse, microhardness, primary enamel, remineralization, tricalcium phosphate

INTRODUCTION than treatment. Fluoride is an important popular agent for promoting reminer- Dental caries are common among alization and is available in a variety of children in Thailand (Narksawat et al, forms, such as toothpaste, mouthrinse, gel 2011). The prevention of dental caries in and solution (Marinho et al, 2003). children and adolescents is a priority for Many studies have confirmed the dental practices and is more cost-effective benefits of fluoride mouthrinses against Correspondence: Praphasri Rirattanapong, caries over the past 30 years, especially Department of Pediatric Dentistry, Faculty of among children (Marinho et al, 2003, 2004; Dentistry, Mahidol University, 6 Yothi Street, Divaris et al, 2012). School based programs Bangkok 10400, Thailand. for dental health have been conducted Tel: 66 (0) 2200 7821 ext 30 world-wide as a public health preventive E-mail: praphasri.rir @mahidol.ac.th measure (Reich et al, 2002). Fluoride is not

Vol 46 No. 3 May 2015 539 Southeast Asian J Trop Med Public Health the only agent used for remineralization; The objectives of this in vitro study calcium and phosphate have also been were to evaluate and compare the effects used (Featherstone, 2008). Calcium and of fluoride mouthrinse with fluoride con- phosphate ions are the primary constitu- taining tricalcium phosphate mouthrinse ents of tooth enamel (Gurunathan et al, on the microhardness of demineralized 2012). Adequate quantities of these ions enamel in primary human teeth. must be present for remineralization to occur (Cury and Tenuta, 2009). Scheme- MATERIALS AND METHODS horn and colleagues (1999) evaluated the addition of calcium and phosphate Specimen preparation to fluoride toothpaste or mouthrinse can Thirty-six human primary incisors improve remineralization and increase were used for this study and stored in fluoride uptake. normal saline solution at room tempera- Tricalcium phosphate has been de- ture until use. The radicular part of each veloped by fusing beta-tricalcium phos- tooth was removed. The specimens were phate (β-TCP) with sodium lauryl sulfate embedded in self-cured acrylic resin, with or fumaric acid (Karlinsey and Pfarrer; the labial surface leveled on top and lying 2012). This combination is designed to flat and parallel to the horizontal plane. assist with the fluoride in remineraliza- Specimens were polished using silicon tion (Twetman et al, 2004; Shen et al, 2011). carbide sandpaper using progressively Tricalcium phosphate provides catalytic fines grit (400, 600, 1,200, 2,000 and 2,500) amounts of calcium to boost fluoride ef- to obtain a flat smooth surface and then ficacy and can be mixed with fluoride in stored in deionized water at room tem- mouthrinse or dentifrice, because it does perature until use. The baseline micro- not react before reaching the tooth surface hardness of the enamel was measured on (Karlinsey et al, 2010a). When the trical- the labial surface using a Vickers indenter cium phosphate comes into contact with (FM-700e Type D, Future-tech,Tokyo, the tooth surface and is moistened by sa- Japan) with 100 grams of force for 15 liva, it breaks down, making the calcium, seconds (Rirattanapong et al, 2012). Four phosphate and fluoride ions available for indentations per test were performed and the tooth. The fluoride and calcium then the microhardness value was an average react with the weakened enamel to pro- of the four readings. vide a seed for enhanced mineral growth This study was approved by The (Karlinsey et al, 2010b; Shen et al, 2011). Ethics Committee of Mahidol University. Many studies have evaluated reminer- (MU-DT/PY-1RB 2013/049.0509). alization of enamel lesions on permanent Demineralizing and remineralizing solu- tooth by various fluoride and calcium tions preparation phosphate containing mouthrinses (Kumar Demineralizing solution 1 (D1) con- et al, 2008; Puig-Silla et al, 2009; Moezizadeh sisted of 2.2 mM CaCl2, 2.2 mM NaH2PO4, and Alimi, 2014). However, there have 0.05 M acetic acid with the pH being been no studies comparing the efficacy adjusted to 4.4 using 1M KOH. Demin- of fluoride only mouthrinse with fluoride eralizing solution 2 (D2) contained the combined with tricalcium phosphate same components as D1, but the pH mouthrinse on microhardness of demin- was adjusted to 4.7 by the addition of eralized enamel in primary human teeth. 1M KOH. The remineralizing solution

540 Vol 46 No. 3 May 2015 Effects of Fluoride Plus TCP Mouthrinse on Microhardness of Primary Enamel

(R) was comprised of 1.5 mM CaCl2, 0.9 were immersed and agitated for 1 minute, mM NaH2PO4, and 0.15 M KCl with the three times a day in one of the treatment pH being adjusted using 1 M KOH to 7.0 rinses at room temperature (Moi et al, (Thaveesangpanich et al, 2005). The de- 2008). All of the specimens were placed mineralizing and remineralizing solutions in remineralizing solution overnight at were freshly prepared for each pH-cycling 37oC in a controlled environment incuba- period and the pH was checked each time tor shaker at 150 rpm (Series 25 incubator prior to use. shaker, Ramsey, MN). At the end of the Artificial caries lesion formation pH-cycling period, the microhardness for each tooth was measured by the Vickers Each specimen was immersed in 3 indenter test. ml of D1 and incubated at 37oC (Sheldon manufacturing, model 1545, Cornelius, Statistic analysis OR) for 4 days (Thaveesangpanich et al, The one-way analysis of variance 2005). The specimens were then immersed (ANOVA) and Tukey’s multiple com- in artificial saliva. The artificial saliva parison tests were used to compare mi- used consisted of 0.65 grams per liter KCl crohardness values at baseline, before and (British Pharmacopoeia, Norwrick, UK), after pH-cycling and compare the mean

0.058 g/l MgCl2 (British Pharmacopoeia), microhardness values among the groups. 0.165 g/l CaCl2 (British Pharmacopoeia), Significance was set atp < 0.05. K2(HPO4)2 (Pharmacopoeia, Rockville, MA), KH2(PO4)3 (British Pharmacopoeia), RESULTS 2 g/l NaCO CH cellulose (Pharmacopoe- 2 3 The mean microhardness values at ia, Rockville, MA) and deionized water baseline, before and after the pH-cycling was added to make 1 liter (modified from are shown in Table 1. At baseline, the Amaechi et al,1999). Microhardness was mean microhardness value (±SD) was measured by a Vickers indenter test. 328.453±26.566 Vicker Hardness Number Grouping (VHN). The mean baseline microhardness After artificial carious lesion forma- values were not significantly different tion, the specimens were pooled and among the groups (p=0.271). randomly assigned to one of three groups Before pH-cycling period, the micro- (n=12): Group A (control) - deionized hardness values were not significantly water, Group B - 0.05% sodium fluoride different among the groups (p=0.526) and and 20 ppm tricalcium phosphate mouth- were at significantly lower values than rinse, Group C - 0.05% sodium fluoride baseline (p=0.000) having a 61.19±9.8% mouthrinse. reduction. pH-cycling process After pH-cycling, groups B and C An experimental process intended to had a significant increase in mean micro- imitate the changes in pH in the oral en- hardness compared to before pH-cycling. vironment for seven days was employed Group A had a significantly lower micro- in the present study (Yimcharoen et al, hardness than Groups B and C (p=0.000). 2011). Each cycle involved placing in D2 There was no significant difference in for 3 hours twice daily and placing in R microhardness values between Groups B for 2 hours in between. All the specimens and C after pH-cycling (p=0.728).

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Table 1 Microhardness values at baseline, before and after 7 days of pH-cycling.

Group Treatment Condition (Mean±VHN)

Baseline Before pH-cycling After pH-cycling

A Deionized water 332.332±23.218a 136.171±31.750b 179.318±25.593b B 0.05%NaF plus TCP 318.320±27.499a 122.106±32.051b 231.848±18.729c C 0.05%NaF 334.705±27.913a 124.338±31.199b 225.101±19.986c

Same letters indicate no statistically significant differencep ( ≥ 0.05).

DISCUSSION have shown that fluoride mouthrinse can promote enamel remineralization (Marin- The mean baseline microhardness ho et al, 2003, 2004; Divaris et al, 2012). value was 328.45±26.57 Vicker Hardness Number (VHN) and ranged from 264.85- Our study confirmed the ability of 367.61 VHN. Our study required a flat fluoride mouthrinse to improve micro- area to measure surface microhardness, so hardness of primary enamel lesion. This we used an area that was not the original confirms the protective effect of fluoride surface after preparation; it could explain mouthrinse on primary enamel, similar the range in the baseline values. Our mean to previous studies (Divaris et al, 2012; baseline microhardness value was similar Rirattanapong et al, 2015). to those found by Rirattanapong et al Calcium and phosphate have also (2012) (342.1±21.9 VHN) and Maupomé been used for remineralization. A new et al (1999) (344.2±32.4 VHN), but higher prospective calcium phosphate system than that found by Vongsawan et al (2014) can be prepared by modifying calcium (295.75±15.79 VHN). This difference could phosphate to form a more functional cal- be due to differences in tooth preparation cium phosphate that better interacts with as described above. Prior to pH-cycling, demineralized enamel to boost reminer- the mean microhardness reduced by alization (Karlinsey et al, 2012). Previous 61.19% from baseline. This reduction is studies found a synergistic effect between similar to a study by González-Cabezas fluoride and calcium phosphate (Karlin- et al (2012), which had a 66.67% reduction sey et al, 2009; 2010c; 2012). Karlinsey and in microhardness. The small difference colleages (2009) found fluoride with tri- between our study and their studies could calcium phosphate provided significantly be due to different pH levels, solution better surface strengthening than fluoride compositions or timing of demineraliza- alone for bovine enamel. tion (Cuy et al, 2002). Our findings showed the addition of The American Academy of Pediatric tricalcium phosphate provided no addi- Dentistry (2012) guidelines on fluoride tional benefit over the fluoride only prepa- therapy recommended fluoride mouth- ration. This differs from previous studies rinses as an adjunct to fluoride toothpaste that showed a synergistic effect between for individuals who are at high risk of tricalcium phosphate and fluoride (Karlin- developing dental caries. Many studies sey et al, 2009; 2012). Differences in study

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