Brazilian Dental Journal (2013) 24(3): 235-240 ISSN 0103-6440 http://dx.doi.org/10.1590/0103-6440201302000 Department of Pediatric Dentistry Effect of Sodium Trimetaphosphate and Public Health, Dental School, UNESP - Universidade Estadual on Hydroxyapatite Solubility: Paulista, Araçatuba, SP, Brazil An In Vitro Study Correspondence: Prof. Dr. Alberto Carlos Botazzo Delbem, Rua José Bonifácio, 1193, 16015-050 Araçatuba, SP, Brasil. Tel. +55-18- 3636-3314. e-mail: adelbem@foa. José Antonio Santos Souza, Jackeline Gallo do Amaral, João Carlos Silos unesp.br, [email protected] Moraes, Kikue Takebayashi Sassaki, Alberto Carlos Botazzo Delbem This study evaluated the effect of different concentrations of sodium trimetaphosphate (TMP) with and without fluoride (F) on the concentration of calcium (Ca), phosphorus (P) and F in hydroxyapatite (HA). Synthetic HA powder (0.15 g) was suspended (n=6) in solutions (75 mL) of TMP at 0%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 2.0%, 4.0%, 6.0%, 8.0% and 10% concentrations in the presence and absence of 100 ppm F and subjected to a pH-cycling process. The precipitates were filtrated, dried at 70° C for 24 h and ground onto a fine powder. The concentrations of F (KOH (CaF2) and HCl (FA) soluble), Ca (Arsenazo III), and P (molybdate method) in HA were determined. The Ca P, and Ca/P ratio data were subjected to Tukey’s test and the F data were subjected to Student-Newman-Keuls test (p<0.05). The addition of TMP to the samples reduced F deposition to 98% (p<0.001). The groups containing 100 ppm F and 0.4% or 0.6% TMP exhibited a higher Ca concentration than the group containing only 100 ppm F (p<0.05). Furthermore, the HA treated with 0.2% and 0.4% TMP and 100 ppm F showed a higher Ca/P ratio than the other groups Key Words: hydroxyapatite, (p<0.001). In conclusion, TMP at 0.2%, 0.4% and 0.6% concentrations combined with trimetaphosphate, F seemed to be able to precipitate HA with low solubility. However, especially at high demineralization, concentrations, TMP interferes with F deposition on HA. solubility, fluoride. Introduction was superior in inhibiting dental caries in vitro because it Fluoride (F) is the most important agent for preventing prevents acid diffusion into the enamel. It has also been dental caries (1); however, at the same time, its extensive observed a higher F content in the enamel after use of ingestion is related to dental fluorosis (2). Thus, F in toddlers this mouthrinse, although the type of F deposits on the must be used with caution. As a safety measure, oral health enamel has not been identified: loosely bound F (calcium promotion programs for toddlers (baby clinics, 0-3 years) fluoride - CaF2) or firmly bound F (fluorapatite - FA) (11). currently use topical solutions with low F concentrations in Other studies demonstrated that an adequate TMP/F ratio the dental office (0.02% NaF/day) instead of F-containing can improve the effectiveness of F-based products even at dentifrice (3,4). However, the 0.02% NaF solution shows low F concentrations (12-14). Nevertheless, the mechanism lower anticaries efficacy (1,5,6). Addition of inorganic of TMP action is not yet completely clear. Using the study phosphate can be performed to improve the effectiveness designs used so far, it has not been possible to define of mouthrinses containing 0.02% NaF (7,8). the mechanism of TMP action when associated with F at Polyphosphate has a strong affinity for the different concentrations. The analysis of the effect of TMP hydroxyapatite (HA) surface and resistance against with and without F on HA allows the evaluation of the successive acid attacks while reducing enamel dissolution changes that occur by combining these two additives, as (9). Sodium trimetaphosphate (TMP), a cyclic condensed well as shed light on the effects of TMP on the deposition phosphate, has lower capacity to bind to HA than of CaF2 and FA. polyphosphates, but it remains bound to enamel for a longer The aim of this study was to evaluate the effect of period (10). Studies have demonstrated the reduction of different sodium TMP concentrations (0%, 0.1%, 0.2%, enamel solubility, suggesting an affinity of TMP to enamel 0.4%, 0.6%, 0.8%, 1.0%, 2.0%, 4.0%, 6.0%, 8.0% and 10%) surface and/or HA (7,8,10). with and without F (100 ppm) on the concentration of Manarelli et al. (7) investigated whether addition of calcium (Ca), phosphorus (P) and F in HA when subjected TMP in a mouthrinse could improve its effectiveness against to demineralization and remineralization. dental erosion. The solution containing 100 ppm F and 0.4% TMP presented a greater protective effect under erosive Material and Methods conditions than a solution containing 225 ppm F. In another Synthesis of HA study (8), the same mouthrinse (100 ppm F and 0.4% TMP) HA was synthesized according to Qu and Wei (15): 1 Braz Dent J 24(3) 2013 -1 mol L (300 mL) calcium nitrate solution (Ca(NO3)2.H2O; 340; Biotek, St. Paul, MN, USA) with a wavelength of 650 Sigma-Aldrich Corp. St. Louis, MO, USA) and 0.3 mol L-1 (600 nm by adopting the Arsenazo III colorimetric method (17). mL) diammonium phosphate solution ((NH4)2HPO4; Sigma- Aliquots of 5 µL were taken from the samples (diluted 1:10 Aldrich Corp.) were prepared. The pH of each solution was and partly neutralized) and added to 50 µL of deionized raised to 10-12 by adding NH4OH (29.5%). The diammonium water and arsenazo. For calibration, standards containing phosphate solution was added slowly to the calcium 40 to 200 µg Ca/mL were used. Phosphorus was measured nitrate solution (2-5 mL/min) under constant agitation at by the molybdate method (colorimetric method) for an 37° C in order to precipitate the fully-crystallized HA. The aliquot of 20 µL from the samples, which were subsequently precipitates were aged for 7 days at 37° C while the pH added to a mixture of 50 µL molybdate solution and 20 µL was maintained around 10 for the growth and formation of reactive reducer, as described by Fiske and Subbarow of a single crystalline phase. (18), at a wavelength of 660 nm; standards containing 1.5 The precipitate was collected by filtration using a to 24 µg P/mL were used. Buchner funnel attached to a vacuum system (-600 mmHg), washed repeatedly with deionized water (250 Fluoride Analysis (CaF2 and FA) mL/0.15 g of HA), and dried at 70° C for 24 h. The For F analysis (CaF2 and FA), 1 mg of HA powder was precipitate was then ground onto a fine powder. The HA weighed into pre-weighed micro-centrifuge tubes, and -1 powder was heated at 1,000° C for 2 h to decrease the 1.0 mL of 1 mol L KOH was added for CaF2 extraction, 2- 2- amount of impurities such as CO2, CO3 , HPO4 and water according to Caslavska et al. (19). After 24 h under (16). Six samples of approximately 0.15 g were taken for continuous agitation (Shaker, SK-300; Lab. Companion), the F, Ca, and P analysis. samples were centrifuged for 20 min at 2,900 g. A 0.5 mL aliquot of the supernatant was neutralized with 0.5 mL of Demineralization and Treatment TISAB II (total ionic strength adjustment buffer) modified -1 Solutions (75 mL) of TMP (Na3P3O9, Sigma-Aldrich Corp.) with 1 mol L HCl (8.2 mL HCl/L). CaF2 was determined by were prepared at 0.0%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, using a specific electrode (Orion 9409; Orion Research, 1.0%, 2.0%, 4.0%, 6.0%, 8.0%, and 10% concentrations, Inc., Boston, MA) and a reference electrode (Orion 900100; associated with or without 100 ppm F (NaF, Merck, Orion Research, Inc.) connected to an ion analyzer (Orion Darmstadt, Germany). Synthetic HA powder (0.15 g) was 720plus; Orion Research, Inc.). J.A.S. Souza et al. suspended (n=6) in each of the prepared solutions. Samples For FA determination, the precipitate was washed of the HA powder (n=6) were suspended in deionized water thrice with deionized water and once with methanol. After and used as negative control. After 15 min of stirring at methanol evaporation (overnight at 60° C), 1 mL of 1 mol pH 7.0, the pH of the suspensions was slowly reduced L-1 HCl was added, and the samples were homogenized for -1 to 4.0 using 1 mol L nitric acid (HNO3, Merck) under 30 s by vortexing and agitated for 1 h at room temperature. agitation. After 30 min of equilibration, the pH of each Next, 0.5 mL aliquots of these samples were added to 0.5 solution was raised to 7.0 by the addition of 1 mol L-1 mL of TISAB II modified with 20 g NaOH/L. The FA samples sodium hydroxide (NaOH, Merck). This pH-cycling process were analyzed in a similar manner as CaF2. was repeated thrice, ending with pH 7.0 at 37° C (15). The precipitates were filtrated, dried for 24 h at 70° C and ground Statistical Analysis onto a fine powder. After completion of this process, the F (CaF2 and FA), Ca, and P (mg/g) concentrations were precipitates were separated by filtration using a Buchner subjected to a normality test (Shapiro-Wilk’s test) and a funnel attached to a vacuum system (-600 mmHg), washed homogeneity test (Cochran’s test) to determine the most five times with deionized water (250 mL/0.15 g of HA) to appropriate data analysis.