21. Shahad F.doc J Bagh College Dentistry Vol. 27(4), December 2015 The effect of Pedodontics, Orthodontics and Preventive Dentistry 132 The Effects of Nano-Hydroxyapatite and Casein Phosphopeptide-Amorphous Calcium Phosphate in Preventing Loss of Minerals from Teeth after Exposure to an Acidic Beverage (An In vitro Study) Shahad Zahed Al-Janabi, B.D.S. (1) Zeyneb A.A. Al-Dahan, B.D.S., M.Sc. (2) ABSTRACT Background: This study aimed to evaluate the effect of antierosive agents (10% Nano-Hydroxyapatite (NHA), 10% Casein Phophopeptide-Amorphous Calcium Phosphate (CPP-ACP), and combination of 10% NHA and 10% CPP- ACP) on loss of minerals from enamel surface of permanent teeth treated with antierosive agents when exposed to an acidic beverage and investigate the morphological changes of treated enamel surface after demineralization with cola based beverage under Scanning Electron Microscope (SEM). Materials and Methods: Sixty maxillary first premolars were randomly divided into four groups, 15 teeth for each group. Group I treated with 10% NHA, Group II treated with 10% CPP-ACP, Group III treated with 10% NHA and 10% CPP-ACP, and Group IV did not treat with any remineralizing agents. The teeth were immersed in the remineralizing solutions (10% NHA, 10% CPP-ACP, and combination of 10% NHA and 10% CPP-ACP) for 4 minutes twice daily for 28 days and then stored in the artificial saliva. The samples were immersed for 40 minutes in 20 ml Pepsi cola (PH=2.5). Atomic Absorption Spectrophotometer (AAS) was used to record the calcium and phosphorus concentrationsin Pepsi cola before and after demineralization with cola based beverage. SEM also usedto examine the morphological changes occurs in enamel surface of each group after demineralization with cola based beverage. Results: Statistically, there is a highly significant increase in calcium concentration in Pepsi cola (mg/dl) after demineralization with cola based beverage. Group I showed the lowest changes in calcium concentration values among the three studied groups. Group II was the next, which also showed lower changes in calcium concentration values, then group III while the highest changes were recorded in group IV. There is a highly significant reduction in phosphorus concentration in Pepsi cola (mg/dl) after demineralization with cola based beverage. Group I showed the lowest changes in phosphorus concentration values among the three studied groups. Group II was the next, which also showed lower changes in phosphorus concentration values, then group III while the highest changes were recorded in group IV. Statistically, a highly significant difference was showed in calcium and phosphorus concentrations between the four studied groups after demineralization with cola based beverage. Group IV has a highly significant difference in comparison to group I, group II, and group III. Conclusions: Both the remineralizing agents (NHA and CPP-ACP) were found to be effective in inhibiting the demineralization caused by cola based beverage.The combination of NHA and CPP-ACP had no synergistic effect on remineralization. Key words: Nano-Hydroxyapatite, Casein Phophopeptide-Amorphous Calcium Phosphate, Erosion, Scanning Electron Microscope. (J Bagh Coll Dentistry 2015; 27(4):132-137). INTRODUCTION Dental erosion is defined as the irreversible loss of tooth substance by a chemical process not involving bacteria. The main cause of dental erosion is acid exposure (1) ; this may be caused by a sudden increase in consumption of soft drinks, diet cokes and fruit juices, which seems to be more significant compared to the other etiologic factors (2). Considering the deteriorating effects of acidic beverages on the microhardness of dental enamel, it is essential to search for a material that induces remineralization of erosive lesions. The results of a large number of studies have shown that tooth pastes and mouth washes containing NHA have the potential to remineralize initial erosive lesions (3). (1) M.Sc. student, Department of Pedodontics and Preventive Dentistry, College of Dentistry, University of Baghdad. (2) Professor, Department of Pedodontics and Preventive Dentistry, College of Dentistry, University of Baghdad. Nanotechnology is defined as the construction of materials, parts and useful systems with nanometer dimensions in longitudinal scale and subsequent application of the novel characteristics of this minimal scale technology (4, 5). NHA is one of the byproduct of nanotechnology, with high tissue biocompatibility; therefore production of hydroxyapatite crystals in Nanosizes enables higher compatibility with enamel crystals (6). NHA was used as a source of calcium and phosphorus in a sports drink. It has some advantages of high biocompatibility and chemical homogeneity with sound tooth enamel. Sports drink with NHA may prevent dental erosion (7). CPP-ACP nanocomplex had been shown to localize at the tooth surface and prevent enamel demineralization in laboratory, animal and human in situ trials (8). The CPP-ACP has also been shown to remineralize enamel subsurface lesions in situ when delivered in oral care products. The J Bagh College Dentistry Vol. 27(4), December 2015 The effect of Pedodontics, Orthodontics and Preventive Dentistry 133 proposed anticariogenic mechanism for CPP-ACP is the localization of amorphous calcium phosphate (ACP) at the tooth surface which buffers the free calcium and phosphate ion activities, thereby helping to maintain a state of supersaturation with respect to tooth enamel, preventing demineralizationand enhancing remineralization(9). In the present study, the effect of remineralizing agents was evaluated on preventing the loss of minerals from treated enamel surface of permanent teeth after exposure to an acidic beverage. As far as it is known there is no previous Iraqi study regarding the effect of NHA in preventing the loss of minerals from the enamel surface after exposure to an acidic beverage, for this reason this study was conducted. MATERIALS AND METHODS The Sample: Sixty sound human upper first permanent premolars freshly extracted for orthodontic purposes were used in this study with age 12 years old. Sample Preparation: The extracted teeth were cleaned using conventional hand piece and rubber cup with non- fluoridated pumice and stored in deionized water with thymol crystals at room temperature. The teeth were examined by visible light cure machine, any tooth had a visible fracture or a crack was discarded (10). A circle of 6mm in diameter were prepared on the buccal surface of each tooth by using adhesive tape. Then an acid resistant nail varnish was used to paint each tooth, the adhesive tape was removed leaving a circular window of 6 mm in diameter on the buccal surface of each tooth (11). Preparation of Remineralizing Solutions: Nano-Hydroxyapatite used in this study was hydroxyapatite Nanopowder Ca10 (PO4) 6 (OH) 2 from mknano, Mississauga, Canada. 10% NHA suspension solution, using 2 M HCl, PH adjusted to 7 (6, 12). 10% CPP-ACP is produced by hydrolysis of casein to form casein peptide, which is then complexed with amorphous calcium phosphate to form a casein phosphopeptide - amorphous calcium phosphate complex. The casein (10% w/v), trypsin (0.05% w/v), calcium chloride (100 mmol/L), disodium hydrogen phosphate (60 mmol/L) (13). Preparation of Artificial Saliva: To prepare one liter of artificial saliva, different materials in different concentrations were dissolved in (985.5) ml deionized water. These materials are: Sodium carboxymethyl cellulose (10 gm), Sodium chloride (1 gm), Sodium fluoride (0.0002 gm), Calcium chloride (0.05 gm), Potassium thiocyanate (0.01gm), Sorbitol (1 gm), Potassium chloride (1gm), Magnesium chloride (0.05 gm), Potassium phosphate (0.04 gm). Sodium carboxymethyl cellulose dissolved in 100 ml boiling water and after cooling, each other material was dissolved in deionized water and added to sodium carboxymethyl cellulose solution, pH was equal to 7 (14). Sample Grouping: Sixty teeth were divided into four groups according to the type of treatment. Group I: 15 teeth treated with 10% NHA solution. Group II: 15 teeth treated with 10% CPP-ACP solution. Group III: 15 teeth treated with [10% NHA and 10% CPP-ACP] solution. Group IV: 15 teeth left without treatment placed in artificial saliva. The teeth were treated with remineralizing solutions for 4 minutes twice daily, once in the morning and once at night for 28 days and then stored in artificial saliva between exposure to remineralizing solutions and for the remaining 12 hours overnight. Each tooth was rinsed and dried with a piece of cotton before and after each immersion for 2 minutes and then stored in artificial saliva. Incubator was used to maintain the temperature at 37ºC. After the samples were remineralized for 28 days, they were immersed in 20 ml Pepsi cola (pH=2.5) for 40 minutes (15). Biochemical Analysis of Pepsi Cola: Calcium of Pepsi cola was analysed by using flame atomic absorption spectrophotometer following standardized procedure. Inorganic phosphorus of Pepsi cola measured by using a ready-made kit of (Biomagrhreb, Tunisia) and analyzed by UV visible recording spectrophotometry (Cecil CE 7200 UK) machine. SEM Examination: Representative specimens from all groups were randomly selected for SEM sample preparation. These were then examined by using Scanning electron microscope SEM (Tescan- Vega, USA) (6,12,15). Statistical Analysis Data were computerized and analyzed using SPSS 19.0 software. Student’s paired t-test was used to compare the calcium and phosphorus concentrations before and after demineralization with cola based beverage in the four studied groupsand ANOVA was used to compare the calcium and phosphorus concentrations among groups after demineralization with cola based J Bagh College Dentistry Vol. 27(4), December 2015 The effect of Pedodontics, Orthodontics and Preventive Dentistry 134 beverage in the four studied groups, followed by LSD test. RESULTS Statistically, there is a highly significant increase in calcium concentration in Pepsi cola (mg/dl) after demineralization with cola based beverage. Group I showed the lowest changes in calcium concentration values among the three studied groups (1.72 ± 0.08). Group II was the next, which also showed lower changes in calcium concentration values (1.96 ± 0.12), then group III (2.31 ± 0.08) while the highest changes were recorded in group IV (3.72 ± 0.12) as demonstrated in Table (1). There is a highly significant reduction in phosphorus concentration in Pepsi cola (mg/dl) after demineralization with cola based beverage. Group I showed the lowest changes in phosphorus concentration values among the three studied groups (9.56 ± 0.24). Group II was the next, which also showed lower changes in phosphorus concentration values (8.71 ± 0.18), then group III (7.68 ± 0.19) while the highest changes were recorded in group IV (5.59 ± 0.19) as shown in table (2). Statistically, a highly significant difference was showed in calcium and phosphorus concentrations between the four studied groups after demineralization with cola based beverage. Group IV has a highly significant difference in comparison to group I, group II, and group III (Table 3 and 4). As shown in figure (1): (a) a smooth and intact surface was obtained in the normal anatomical enamel surface before demineralization with cola based beverage. (b) Specimen treated with 10% NHA and demineralized with cola based beverage show that acicular crystals of NHA sedimented on the enamel surface. This sedimented new layer will resist the demineralization. (c) Specimen treated with CPP-ACP and demineralized with cola based beverage, microscopic irregularities on the enamel surface were observed as adherent granules or globules. The deposited minerals on the enamel surface following mobilization of calcium and phosphate from the CPP-ACP will resist the demineralization. (d) specimen treated with 10% NHA and 10% CPP-ACP and demineralized with cola based beverage show cubes like crystals formed from CPP-ACP, were disorderly distributed on the enamel surface and filled with NHA crystals. The new layer which sedimented on the enamel surface of the tooth didn’t resist the demineralization, so a honeycomb structure appeared in some regions on the surface of erosive lesions. (e) Specimen that not treated with any remineralizing agents and demineralized with cola based beverage show many micro pores and honeycomb structure were apparent on the enamel surface. This specimen can’t resist the demineralization. Table (1): Descriptive Statistics and State Difference of Calcium Concentration (mg/dl) in Pepsi Cola for the Four Studied Groups before and after Demineralization with Cola Based Beverage. Groups State Descriptive Statistics States' comparison (d.f.=14) Mean S.D. Min. Max. t-test P-value Sig. NHA Before 1.50 0.00 1.50 1.50 -11.03 0.000 HS After 1.72 0.08 1.59 1.87 CPP-ACP Before 1.50 0.00 1.50 1.50 -14.45 0.000 HS After 1.96 0.12 1.81 2.13 NHA+ CPP-ACP Before 1.50 0.00 1.50 1.50 -38.22 0.000 HS After 2.31 0.08 2.16 2.46 No treatment Before 1.50 0.00 1.50 1.50 -71.82 0.000 HS After 3.72 0.12 3.51 3.89 J Bagh College Dentistry Vol. 27(4), December 2015 The effect of Pedodontics, Orthodontics and Preventive Dentistry 135 Table (2): Descriptive Statistics and State Difference of Phosphorus Concentration (mg/dl) in Pepsi Cola for the Four Studied Groups before and after Demineralization with Cola Based Beverage. Groups State Descriptive Statistics States' comparison (d.f.=14) Mean S.D. Min. Max. T-test P-value Sig. NHA Before 11.50 0.00 11.5 11.5 30.92 0.000 HS After 9.56 0.24 9.24 9.96 CPP-ACP Before 11.50 0.00 11.5 11.5 59.62 0.000 HS After 8.71 0.18 8.28 8.97 NHA+ CPP-ACP Before 11.50 0.00 11.5 11.5 76.36 0.000 HS After 7.68 0.19 7.34 7.98 No treatment Before 11.50 0.00 11.5 11.5 117.86 0.000 HS After 5.59 0.19 5.25 5.84 Table (3): Descriptive Statistics and Group Difference for Calcium Concentration (mg/dl) in Pepsi Cola after Demineralization with Cola Based Beverage. Groups Descriptive Statistics Groups' comparison (d.f.=59) Mean S.D. Min. Max. F-test p-value Sig. NHA 1.72 0.08 1.59 1.87 1148.77 0.000 HS CPP-ACP 1.96 0.12 1.81 2.13 NHA+CPP-ACP 2.31 0.08 2.16 2.46 No treatment 3.72 0.12 3.51 3.89 Table (4): Descriptive Statistics and Group Difference for Phosphorus Concentration (mg/dl) in Pepsi Cola after Demineralization with Cola Based Beverage. Groups Descriptive Statistics Groups' comparison (d.f.=59) Mean S.D. Min. Max. F-test p-value Sig. NHA 9.56 0.24 9.24 9.96 1053.09 0.000 HS CPP-ACP 8.71 0.18 8.28 8.97 NHA+CPP-ACP 7.68 0.19 7.34 7.98 No treatment 5.59 0.19 5.25 5.84 (a) (b) (c) (d) (e) Figure (1): SEM Images of a Representative Specimen in Different Groups after Demineralization with Cola Based Beverage: (a) Sound Enamel Surface, (b) Group I (Treated with NHA, (c) Group II (Treated with CPP-ACP), (d) Group III (Treated with NHA and CPP- ACP), (e) Group IV (No Treatment). J Bagh College Dentistry Vol. 27(4), December 2015 The effect of Pedodontics, Orthodontics and Preventive Dentistry 136 DISCUSSION Dental erosion was effectively prevented by the increase in PH and the degree of saturation with respect to dental enamel (DSEn) which was caused by abundant release of calcium, phosphate and hydroxide ions from remineralizing agents, and the incapability of phosphoric acid to chelate with enamel calcium. Most calcium compounds are known to have low solubility and it makes them less manageable. However, in the present study the use of nanotechnology allowed the release of ions from hydroxyapatite in great quantities. The solubility was reported to dramatically increase per unit time due to the higher specific surface area and low crystallinity of the nano-sized crystals. Therefore more active constituents can be released (16-18). Due to low solubility of pure hydroxyapatite, no enough calcium and phosphorus were available to increase the stability of hydroxyapatite in the enamel and to prevent dissolution of the dental enamel. Since the surface area and proportion of atomicity increase with decreasing particle size, NHA has bioactive and biocompatible properties to be used in this study (19). Pepsi cola was used in this study to induce artificial erosive effect as in other studies (20,21). Pepsi cola is the most popular and most widely drinks used in the country. When the changes in calcium concentrations in Pepsi cola after demineralization with cola based beverage were measured, group I showed the lowest changes in calcium concentrations values among the three studied groups. Group II came the next, which also showed lower changes in calcium concentrations values, then group III while the highest changes were recorded in group IV. There is a highly significant increase in calcium concentrations before and after demineralization of all groups. This increase in calcium concentrations in Pepsi cola means that there was some loss of calcium from the enamel surface of the teeth after demineralization with cola based beverage. When also the changes in phosphorus concentrations in Pepsi cola after demineralization with cola based beverage were measured, group I showed the lowest changes in phosphorus concentrations values among the three studied groups. Group II came the next, which also showed lower changes in phosphorus concentrations values, then group III while the highest changes were recorded in group IV. There is a highly significant reduction in phosphorus concentration before and after demineralization of all groups. This reduction in phosphorus concentration in Pepsi cola means that the acidic nature of phosphoric acid had the ability to chelate calcium in saliva and on tooth surface, so their potential to cause erosion is strongly dependent on this effect (22). A highly significant difference in calcium and phosphorus concentration between the four studied groups after demineralization with cola based beverage. Group IV has a highly significant difference in comparison to group I, group II, and group III. The direction of calcium and phosphorous concentrations were inversely increased. So when the calcium concentration increased, the phosphorous concentration was decreased. 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