Inhibition effects of different toothpastes on demineralisation of incipient enamel lesions

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(1)Inhibition Effects of Different Toothpastes on Demineralisation of Incipient Enamel Lesions Halenur Altana / Zeynep Göztaşb / Kamil Kahramanc / Mahmut Kuşd / Gul Tosune. Purpose: To evaluate the inhibitory effects of different toothpastes on demineralisation of incipient enamel lesions using a toothbrush simulator. Materials and Methods: Fifty enamel specimens were prepared from extracted human molars. The specimens were randomly assigned to the following groups (n = 10/group): 1. no treatment (control); 2. toothpaste containing arginine (ProRelief, Colgate;); 3. fluoride toothpaste (Pronamel, Sensodyne GlaxoSmithKlein); 4. tooth mousse containing casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) (Recaldent, GC); 5. toothpaste (Restore, Dr. Collins) containing bioactive glass (NovaMin, GlaxoSmithKlein). All specimens were exposed to pH cycling. The remineralising agents were applied to the samples with a toothbrush simulator for 2 min twice a day for five days. The weight percentage of mineral changes for the elements calcium (Ca), phosphorus (P), sodium (Na) and silica (Si) were measured by SEM energy-dispersive x-ray spectroscopy (SEM-EDX). SEM revealed properties of treated enamel surfaces. The data were analysed using one-way ANOVA. Results: Statistically significantly higher levels of Ca and P were found in all groups compared to the control (p < 0.05). Conclusions: The toothpastes’ efficacy of inhibiting demineralisation depended on the active ingredients in the respective toothpaste. The demineralisation inhibition efficacy of the tested toothpastes depended on the active ingredients in the toothpaste. Key words: arginine, bioglass, brushing, enamel, remineralisation, simulator Oral Health Prev Dent 2019; 17: 179–185. doi: 10.3290/j.ohpd.a42202. D. ental caries is a chronic disease that can be stopped and even reversed, if it is treated at an early stage.33,38 This can be achieved by controlling changes that occur on the early enamel surface lesions.27 Under optimal conditions, ions such as calcium (Ca+2), phosphate (PO4-3), hydroxide (OH-), and fluoride (F) must be present in sufficient. a. Assistant Professor, Department of Paediatric Dentistry, Faculty of Dentistry, Gaziosmanpaşa University, Tokat, Turkey. Study hypothesis, experimental design, performed the experiments and statistical analysis, contributed to the discussion, wrote the manuscript. b. Paediatric Dentist in Private Practice, Ankara, Turkey. Experimental design, statistical analysis, wrote the manuscript, contributed to the discussion.. c. Paediatric Dentist in Private Practice, Istanbul, Turkey. Experimental design, performed the experiments.. d. Associate Professor, Selcuk University Chemical Engineering Department and Selcuk University Advanced Technology Research and Application Center, Konya, Turkey. Performed SEM and SEM-EDX.. e. Professor, Department of Paediatric Dentistry, Faculty of Dentistry, Selcuk University, Konya, Turkey. Study hypothesis.. Correspondence: Halenur Altan, Department of Paediatric Dentistry, Faculty of Dentistry, Gaziosmanpaşa University, Kaleardı Street, Ali Sevki Erek Campus,Tokat, Turkey 60100. Tel: +90-356-212-4222; e-mail: halenuronat@gmail.com . Vol 17, No 2, 2019. Submitted for publication: 22.08.17; accepted for publication: 17.11.17. quantities around the crystals to maintain the mineral balance between tooth enamel and oral fluid.34 Plaque bacteria create organic acids from carbonhydrates in a cariogenic acid attack, thereby releasing hydrogen (H+) ions. An increase in the H+ concentration in the plaque surrounding the teeth (low pH value) causes a decrease in the OH- concentration.37 Lastly, demineralisation begins by releasing calcium from the dental hard tissue, maintaining a neutral pH.37,38 To remineralise the demineralised enamel on the surface of teeth, the necessary ions must diffuse into these regions.32 However, remineralisation through saliva is slow.38 Saliva provides protection against acid attack by diluent action over the acids and has a buffering capacity which neutralises dietary acids. In addition, saliva is supersaturated with respect to tooth mineral content, and thus provides minerals, especially the calcium and phosphate necessary for remineralisation.5 Van der Veen and Mattausch40 examined the natural remineralisation phase and reported that the majority of lesions were stable even after two years, with no signs of regression. However, this remineralisation is not sufficient, and additional remineralisation is necessary.19,40 Fluoride remineralisation is the approach most frequently used to treat initial caries lesions.18 Fluoride improves the quality of mineralised tooth tissues by reducing the relative. 179.

(2) Altan et al. Fig 1. Toothbrushing simulator.. amounts of carbonated apatite. The reaction between hydroxyapatite and low concentrations of fluoride has been postulated to be an ionic exchange, in which fluoride replaces hydroxyl ions in the crystal lattice structure. The replacement of hydroxyl groups with the smaller fluoride ions should result in a more stable apatite structure. If an OH- ion in pure hydroxyapatite is completely replaced by a fluoride ion (F-), the resulting mineral is fluorapatite Ca10(PO4)6F2.10,18 In addition to the use of fluoride dentifrices, new agents such as Pro-Relief technology have been suggested as a means of stopping caries progression.3 Arginine is an amino acid naturally present in saliva. Arginine bicarbonate is comprised of an amino acid complex with calcium carbonate particles. It adheres to the surface of negatively-charged dentin and interacts with calcium carbonate; thus, it blocks the dentin tubules.4,12 Toothpaste containing a complex of arginine is used to control caries and increase the accuracy of treatment. Only a few studies have reported on the remineralisation potential in initial caries.3 Milk and milk derivatives have been proven to protect teeth.14,26,29,30 Casein phosphopeptide (CPP), which is a milk protein derived from cow’s milk using a selective deposition method, is obtained as a result of cleavage by trypsin. A nanocomplex of CPP-ACP consists of calcium, phosphate, and casein phosphopeptides. Studies have shown that the nanocomplex of CPP-ACP prevents enamel demineralisation and facilitates remineralisation of the enamel surface.14,26,30 The main function of casein phosphopeptides is to modulate the bioavailability of calcium phosphate by maintaining ionic phosphate and calcium supersaturation, which increases remineralisation.8,36 The role of ACP is also said to control the pre-. 180. cipitation of CPP with calcium and phosphate ions. The nanoclusters formed by CPP and ACP buffer the activities of free calcium and phosphate ions in the plaque fluid, helping to maintain supersaturation with respect to enamel mineral, and acting as a calcium-phosphate reservoir, thereby reducing enamel demineralisation and enhancing remineralisation.6,25 Bioactive glasses have been adopted as a potential bioactive material for bone healing.15,20 Based on this feature, studies have examined the effects of bioactive glass on remineralisation in dentistry. Bioactive glass is an inorganic compound consisting of silica calcium, sodium, and phosphate. When it comes in contact with saliva, it quickly releases calcium and phosphorus ions, which are necessary for remineralisation, into the saliva.1 NovaMin technology (GlaxoSmithKlein; Brentford, UK), which was first developed for the treatment of dentin hypersensitivity, treats the root surface of caries by preventing dentin demineralisation. When the active ingredient in NovaMin – inorganic chemical calcium sodium phosphorus silicate – comes in contact with saliva, water or any other bodily fluid, it binds to the tooth surface and initiates the remineralisation process on dental enamel.21,42 The present in vitro study aimed to assess the inhibition effect of pro-arginine technology on initial enamel lesions and compare its efficacy with fluoride toothpaste (Pronamel, Sensodyne GlaxoSmithKlein; Warren, NJ, USA), a tooth mousse containing CPP-ACP (Recaldent, GC; Tokyo, Japan), and a toothpaste (Restore, Dr Collins; Los Angeles, CA, USA) containing bioactive glass (NovaMin, GlaxoSmithKlein; Brentford, UK). The null hypothesis is that none of the tested toothpastes inhibit enamel demineralisation.. MATERIALS AND METHODS Enamel Specimen Preparation In this study, mandibular third molars were used. Malformed, hypoplastic and fractured teeth were excluded from the study. Tissue remnants were cleaned from 25 freshly extracted, caries-free wisdom teeth and stored in 0.2% thymol with distilled water at 4°C. The crowns of all teeth were removed 2 mm below the cementoenamel junction using a slow-speed diamond saw with water cooling (Isomet, Buehler; Lake Bluff, IL, USA). Crowns were sectioned off buccally and lingually, after which 3x3 mm (height x width) windows were prepared on the middle third of the enamel of the 50 samples obtained. Other tooth surfaces outside the windows were coated with nail varnish. Enamel surfaces were not abrasively polished. . Demineralisation Procedure All samples were incubated in demineralisation solution at 37°C for three days, and artificial carious lesions were produced. The demineralisation solution consisted of 2.0 mmol/l calcium, 2.0 mmol/l phosphate, and 75 mmol/l acetate at a pH of 4.3. All samples were rinsed with distilled water for 1 min and dried with air spray. Oral Health & Preventive Dentistry.

(3) Altan et al. Table 1 pH cycling and treatment steps Treatment day. Time. Solutions. Treatment 1 08:00: rinsing with distilled water. Approximately 1 h needed to treat all specimens with brushing simulator in artificial saliva. 2 min. 4.1 mM KH2PO4, 4.0 mM Na2HPO4, 24.8 mM KHCO3, 16.5 mM NaCl, 0.25 mM CaCl2, pH 7.0. Demineralisation (9:00–15:00). 6h. 2 mM CaCl2, 2 mM PO4, 75 mM for acetate buffer, pH 4.3. Treatment 2 15:00: rinsing with distilled water. Approximately 1 h needed to treat all specimens with brushing simulator in artificial saliva. 2 min. 4.1 mM KH2PO4, 4.0 mM Na2HPO4, 24.8 mM KHCO3, 16.5 mM NaCl, 0.25 mM CaCl2. Remineralisation 016:00–08:00 (next day). 16 h (overnight). 1.5 mmol/l calcium, 0.9 mmol/l phosphate, 150 mmol/l, potassium chloride and 20 mmol/l, cacodylate buffer at pH 7.0. Rinse with distilled water. SEM-Energy Dispersive Spectroscopy (SEM-EDX) Analysis At the end of demineralisationprocedure, the mineral contents (weight percentage) of demineralised enamel were measured with SEM-EDX (LEO 440, K61n, Leike Zeiss; Cambridge, UK). The SEM was operated in the conventional highvacuum mode (20 Kv). Samples were not gold-sputter coated, so that wet sample measurements could be performed. SEM-EDX analysis was carried out at three different points on the samples, and %wt calcium (Ca), %wt phosphorus (P), %wt sodium (Na), and %wt silica (Si) were measured.. pH Cycling, Simulated Toothbrushing The specimens were randomly assigned to the following groups (n = 10/group): 1: no treatment (control); 2. toothpaste containing arginine (ProRelief, Colgate Palmolive; New York, NY, USA); 3. fluoride toothpaste (Pronamel, Sensodyne GlaxoSmithKlein; Warren, NJ, USA); 4. tooth mousse containing CPP-ACP (Recaldent, GC; Tokyo, Japan); 5. toothpaste (Restore, Dr. Collins) containing bioactive glass (NovaMin, Glaxosmithkline). Group 2 to 5 samples were treated for 2 min with the remineralising dentifrices. A custom-made toothbrushing machine was employed with circular (clockwise) motion (Fig 1). The head of a toothbrush with soft bristles was attached to the machine, along with a 125 g load to provide constant contact. During brushing, the brush head was positioned parallel to the demineralised enamel surface. Each sample received 16,000 brush strokes (800 cycles/ min), as described by Ozalp and Tulunoglu.23 Each sample was covered with 20 ml of toothpaste and brushed twice a day for five days.35 The toothpaste was renewed after 5000 and 10,000 brush strokes. Artificial saliva was used to dilute the toothpastes (5 ml dentifrice, 15 ml saliva) used for brushing. Artificial saliva was prepared at pH 7 (4.1 mM KH2PO4, 4.0 mM Na2HPO4, 24.8 mM KHCO3, 16.5 mM NaCl, 0.25 mM CaCl2). Vol 17, No 2, 2019. After the first brushing, each sample was rinsed with distilled water, then immersed in 20 ml of demineralisation solution (2 mM CaCl2, 2 mM PO4, 75 mM for acetate buffer, pH 4.3) for 6 h at 37°C. After the demineralisation challenge, the enamel samples were rinsed with distilled water for 1 min and then treated again with toothpastes for 2 min. Samples were subsequently rinsed with distilled water for 1 min. Then, each crown was immersed in 20 ml of remineralisation solution for 16 h. The remineralisation solution contained 1.5 mmol/l calcium, 0.9 mmol/l phosphate, 150 mmol/l, potassium chloride and 20 mmol/l, cacodylate buffer at pH 7.0 (Table 1). The samples were stored in de/re-mineralisation solutions in an incubator at 37°C. The de-/remineralisation solutions and artificial saliva were changed daily.9. Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDX) analysis After de-/remineralisation cycling, samples were completely dried and coated with gold film, then examined in SEM in conventional high-vacuum mode (20Kv). SEM images (LEO 440, Leike Zeiss) were obtained to evaluate the surfaces at 1000X and 2500X magnification. The mineral contents (%wt) of demineralised enamel were measured with SEMEDX (LEO 440, Leike Zeiss).. Statistical Analysis The results were analysed statistically using one-way ANOVA and Tukey’s honest significant differences (HSD) tests at p = 0.05. SPSS 21.0 (IBM; Armonk, NY, USA) was used for the statistical analysis of the results.. RESULTS SEM-EDX Analysis The weight percentages of mineral changes for calcium, phosphorus, sodium and silica are presented in Table 2.. 181.

(4) Altan et al. Table 2 Weight percentage of mineral changes (Δ) and standard deviations Groups. ΔCa. ΔP. ΔNa. ΔSi. 1: control. -1.69 ± 1.02a. -0.56 ± 0.08a. -0.04 ± 0.03a. -0.01 ± 0.01a. 2: arginine toothpaste. 1.13 ± 0.85b. 1.11 ± 0.13b. 0.21 ± 0.09b. 0.08 ± 0.04a. 3: fluoride toothpaste. 1.34 ± 0.04b. 1.19 ± 0.23b. 0.24 ± 0.05b. 0.11 ± 0.05a. 4: CPP-ACP toothpaste. 2.46 ± 0.48c. 3.04 ± 0.14c. 0.04 ± 0.23c. -0.01 ± 0.0a. 5: bioglass toothpaste. 0.63 ± 0.39b. 0.6 ± 0.18b. 0.26 ± 0.18b. 0.54 ± 0.21c. Different superscript letters in each column indicate statistically significant differences within the tested materials (p < 0.05).. In Group 4 (CPP-ACP containing toothpaste), the enamel surface treated with CPP-ACP toothpaste had a inhomogeneous appearance, with some pits not filled with mineral deposits. In contrast, globules of deposits were observed on the enamel surface (Fig 5).41 In Group 5 (toothpaste containing bioglass), mineral deposition took place selectively on enamel prisms. Mineral deposition was homogeneous and all pits and voids were covered. Small toothpaste particles were observed on the enamel surface (Fig 6).. DISCUSSION. Fig 2. Demineralised enamel surface (2500X magnification).. Levels of Ca and P increased statistically significantly in all groups compared to the control group (p < 0.05). The levels of Ca and P in group 4 (CPP-ACP) increased statistically significantly compared with groups 1, 2, 3, and 5. In group 5 (NovaMin), there was also a statistically significant increase in Na and Si.. SEM Analysis In group 1 (control group), interprismatic structures and porosities created by the different treatments are visible on enamel surfaces. Ordered and disordered distributions of hydroxyapatite crystallites are apparent in the incipient lesion area on the enamel surface (Fig 2). In group 2 (toothpaste containing arginine), partial coverage was observed on the enamel surface. The treated enamel surfaces retained calcified deposits but holes were evident on the enamel (Fig 3). Group 3 (fluoride toothpaste) showed enamel surface changes after pH cycling and brushing with toothpaste. The enamel surface was filled with mineral deposits, and appeared smooth and uniformly flat (Fig 4).. 182. In contemporary dentistry, priority is given to non-invasively managing initial molecular changes on the enamel surface through remineralisation intervention to prevent the caries process.7 Caries progression involves a continual imbalance between pathological and protective factors that result in the dissolution of apatite crystals and loss of calcium, phosphate, and other ions from the enamel surface.33,38 In present study, calcium, phosphate, sodium and silica ions were analyzed with SEM-EDX to evaluate the in vitro efficacy of four different remineralising agents on incipient enamel lesions. It was noticed that all of the tested toothpastes used in this study inhibited demineralisation on enamel, so that the null hypothesis was rejected. Fluoride is the cornerstone of the non-invasive management of incipient enamel lesions.27 Fluoride is effective in promoting mineral deposition and inhibiting mineral dissolution.39 In particular, fluoride ions remineralise the demineralised enamel by penetrating the surface of the reactive crystals and increasing the diffuseability of calcium and phosphate. Fluoride ions promote the formation of fluorapatite in enamel in the presence of calcium and phosphate ions produced during enamel demineralisation by plaque bacterial organic acids. Fluoride ions can also drive remineralisation of previously demineralised enamel if enough salivary or plaque calcium and phosphate ions are available when the fluoride is applied.31 In the fluoride toothpaste group, SEM-EDX showed positive mineral changes (calcium, phosphate, sodium and silica) in the newly covered enamel layer. The demineralised enamel prisms (control group) conOral Health & Preventive Dentistry.

(5) Altan et al Fig 3 SEM images of toothpaste containing arginine (left: 1000X; right: 2500X).. Fig 4 SEM images of fluoride toothpaste (left: 1000X; right: 2500X).. Fig 5 SEM images of toothpaste containing CPP-ACP (left: 1000X; right: 2500X).. Fig 6 SEM images of toothpaste containing bioglass (left: 1000X; right: 2500X).. tained numerous voids, whereas the voids following remineralisation with fluoride toothpaste showed substantial occlusion. SEM images of the enamel surface revealed that homogeneous, thin, and completely closed interprismatic spaces were formed without including mineral precipitation. In this study, two main reasons may explain the deficiency of mineral precipitation on the enamel surface. The first reason is the solubility of the compound containing bioavailVol 17, No 2, 2019. able fluoride. 15 ml of artificial saliva was used to dilute the toothpastes used for brushing; this amount may be decreased to become more effective. The second reason is the adhesion of fluoride compound to the enamel surface.22 This low remineralisation potential may be due to the fact that the low solubility of fluoride toothpaste could lead to precipitation of calcium and phosphate ions, which makes it more difficult for these ions reach the tooth surface.. 183.

(6) Altan et al. Arginine toothpaste (Pro Relief system) contains arginine amino acids and calcium carbonate.9 Its mechanism involves binding to the negatively charged dentin-enamel surface, creating a protective layer that covers surface defects and occludes the exposed dentinal tubules, consequently reducing sensitivity.9 Poggio et al24 and Lombardini et al17 reported that remineralisation of eroded enamel is supported by toothpaste containing arginine. When the tested groups were compared, fluoride toothpaste (group 3) was found to have an inhibition demineralisation potential similar to that of arginine toothpaste (group 2) according to SEM-EDX analysis; however, SEM images were different. Arginine toothpaste is a combination of arginine and an insoluble calcium compound. Despite similar fluoride ratios in groups 2 (1450 ppm F-) and 3 (1450 ppm F-), differences in SEM images and less increase in calcium and phosphate concentration compared to the fluoride group may be due to inhibitory effects between arginine protein and fluoride ions in toothpaste (group 3). CPP-ACP can infiltrate into the porosities of enamel lesions and diffuse down concentration gradients into the body of the lesion.7 The mechanism of action of the CPPACP nanocomplex effectively increases the level of bioavailable calcium and phosphate ions.22 In the present study, the highest mineral changes were found in the CPP-ACP group. SEM images clearly showed accumulation of CPPACP, a salient feature of remineralisation. The CPP-ACP mousse can be used alone in the treatment of initial enamel caries; however, in our opinion, it can also be combined with other toothpastes that have a low fluoride percentage. Doing so increases the remineralisation effect of the agents and protects from adverse systemic side effects by decreasing the amount of fluoride that is used. Bioglass rapidly exchanges with hydrogen cations (H3O+), bringing about the release of calcium and phosphate ions from the glass.13 When bioglass particles are attached to the surface of teeth, the number of sodium ions increases, and the pH of the local environment also increases. The increase in the number of silica ions on the enamel surface plays a key role in the collapse of hydroxyapatite in the early stages of caries and the creation of nucleation sites.11,16 When the mineral changes of surface enamel were examined in this study, the highest increase in the amount of sodium and silica ions was observed in the toothpaste containing bioactive glass. In the present study, it was found that toothpaste containing bioactive glass has a relatively high remineralisation potential compared to the other agents used. A limitation of this study is that enamel itself is not conductive, which is a problem when using EDX baseline assessment in high vacuum conditions without any coating.. 184. CONCLUSION The application of the tested toothpastes showed a preventive effect against enamel demineralisation to varying degrees. Among the materials tested, the toothpaste containing bioactive glass appears to be more effective in incorporating into prismatic and interprismatic structure of demineralised enamel.. REFERENCES 1. 2. 3. 4.. 5. 6. 7.. 8. 9. 10.. 11.. 12.. 13.. 14.. 15.. 16.. Allan I, Newman H, Wilson M. Antibacterial activity of particulate bioglass against supra- and subgingival bacteria. Biomaterials 2001;22:1683–1687. Amaechi BT, van Loveren C. Fluorides and non-fluoride remineralisation systems. Monogr Oral Sci 2013;23:15–26. Amaechi BT. Remineralisation therapies for initial caries lesions. Current Oral Health Reports 2.2 2015;2:95–101. 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