Abstract
Objective
This study investigated if the incorporation of the bioadhesive polymers Carbopol 980, Carboxymethyl cellulose (CMC), and Aristoflex AVC in a fluoridated solution (NaF–900 ppm) would increase the solution’s protective effect against enamel erosion.
Methods
Enamel specimens were submitted to a 5-day de-remineralization cycling model, consisting of 2 min immersions in 0.3% citric acid (6x/day), 1 min treatments with the polymers (associated or not with fluoride), and 60 min storage in artificial saliva. Ultrapure water was used as the negative control and a 900 ppm fluoride solution as positive control. The initial Knoop microhardness (KHN1) was used to randomize the samples into groups. Another two microhardness assessments were performed after the first (KHN2) and second (KHN3) acid immersions, to determine initial erosion in the first day. The formula: %KHN alt = [(KHN3-KHN2)/KHN2]*100 was used to define the protective effect of the treatments. After the 5-day cycling, surface loss (SL, in μm) was evaluated with profilometry. Data were analyzed with 2-way ANOVA and Tukey’s tests (p < 0.05).
Results
For %KHN alt , the polymers alone did not reduce enamel demineralization when compared to the negative control, but Carbopol associated with NaF significantly improved its protective effect. The profilometric analysis showed that Carbopol, associated or not with NaF, exhibited the lowest SL, while CMC and Aristoflex did not exhibit a protective effect, nor were they able to improve the protection of NaF.
Conclusions
It is concluded that Carbopol enhanced NaF’s protection against initial erosion. Carbopol alone or associated with NaF was able to reduce SL after several erosive challenges.
Clinical significance
Carbopol by itself was able to reduce the erosive wear magnitude to the same extent as the sodium fluoride, therefore, is a promising agent to prevent or control enamel erosion.
1
Introduction
Dental erosion can be described as a loss of tooth minerals due to chemical dissolution, which occurs by intrinsic or extrinsic acids in the absence of microorganisms. Clinically, the initial sign of the erosive process in enamel is the appearance of a smooth and silk-glazed surface. Later, convex areas become flat, shallow concavities start to appear, the cusps become rounded, and restorations seem to rise above the adjacent tooth level . In more severe cases, the tooth can lose its entire morphology, resulting in unpleasant consequences, such as loss of vertical dimension and sensitivity .
The prevention of the erosive process and, consequently, the arrestment of the erosive lesions, can be influenced by several factors, including the exposure of the tooth to fluoride or other protective/remineralizing agents. The effectiveness of sodium fluoride (NaF) against dental erosion has been extensively investigated . Up until now, there was an indication of some anti-erosive effect , albeit in a limited way, possibly due to the high dissolution of calcium fluoride deposits under high acidic erosive conditions . In addition, fluoride does not present high substantivity in the oral cavity, especially when delivered in vehicles for daily application, such as mouth rinses and dentifrices .
In order to remain at therapeutic levels and effectively act in the oral environment, fluoride and any other active agent must withstand salivary clearance and the sheer forces associated with speaking, swallowing, and eating . To increase the bioavailability of drugs and actives in oral products, some bioadhesive polymers have been investigated. These polymers can potentially enhance the retention of actives within the oral cavity, improving their residence time . Nevertheless, studies investigating the addition of the bioadhesive polymers to fluoride-containing solutions are scarce. So far there is some evidence that these bioadhesive agents can influence the adsorption of fluoride, thereby enhancing its protective effect against erosion , but more studies are needed to confirm this fact.
In addition to their bioadhesive properties, some polymers also have the ability to form films over the dental surfaces, thus reducing the ionic exchange between the acid solution and the dental substrate . These polymers have already been tested as supplements of acidic beverages, in order to reduce their erosive potential , and as active ingredients in oral rinse solutions or toothpastes .
Considering the potential of some bioadhesive polymers to increase the substantivity of fluoride in the oral cavity and to create dental protective films, the objectives of this study were as follows: 1. To evaluate whether the incorporation of bioadhesive polymers in a fluoridated solution would increase the interaction of fluoride with the enamel surface, and consequently, its protection against enamel erosion; and 2. To verify if the bioadhesive polymers tested have the ability to form acid-resistance films over the enamel surface, exerting an anti-erosive effect. The null hypothesis is that the polymers tested, associated or not with NaF, will not present a protective effect against enamel erosion.
2
Materials and methods
2.1
Experimental design
This study tested two experimental factors: the polymer treatment with four levels (control- ultrapure water, Carbopol 980, Carboxymethyl cellulose (CMC), and Aristoflex AVC) and the presence (or not) of sodium fluoride, both in an erosion-remineralization cycling model using bovine enamel specimens (n = 104). The response variables were surface microhardness (KHN) for initial erosion analysis, and surface loss (μm), the latter measured by stylus profilometry at the end of the cycling (after 5 days).
2.2
Specimen preparation
Fresh, non-damaged bovine incisors were collected for this study. The crowns were separated from the roots and stored in 0.1% thymol solution at 4 °C until required. One hundred four cylindrical enamel specimens (3 mm in diameter and 2.1 mm in height) were obtained from the labial surface using a custom-made diamond-coated trephine mill adapted to a circular cutting machine. The specimens were embedded in acrylic resin (ExtecFast Cure Acrylic, ExtecCorp, Enfield, CT, USA) using a silicone mold, which consisted of a cavity with 2 mm deep and 6 mm in diameter. Also, in the bottom of the mold there was a second level cavity with a 0.1 mm deep and 3 mm in diameter, in which the specimens were positioned with the enamel surface facing down . On the lower side of the mold there was a projection in the shape of a line, which produced a lateral notch in the specimen. This allowed the exact repositioning of the specimen at the time of the surface profile measurement (before and after testing).
After the resin cure, the specimens were attached to a metal holder, and the 0.1 mm high enamel surface was removed and ground flat using aluminum oxide abrasive papers in sequential grits of 1200, 2400, and 4000 (FEPA-P, Struers, Ballerup, Denmark), in a polishing device (DP-10, Panambra Industrial e Técnica SA, São Paulo, SP, Brazil) under water irrigation for 30, 60 and 120 s, respectively. After each paper grit change, specimens were kept in ultrasonic baths in distilled water for 10 min to remove waste and abrasive grains. The prepared specimens were examined under a stereomicroscope (Carl Zeiss—Stemi 2000 −20X) to ensure the absence of cracks or other surface defects. After preparation, the specimens were stored in ultrapure water at 4 °C to avoid dehydration.
The initial microhardness (KHN1) of all specimens was assessed using a Knoop Microhardness Tested (FM-700, Future-Tech, Tokyo, Japan) fitted with a 50 g load, for 10 s. Three indentations were performed, 100 μm apart from each other. The recorded values were averaged, and the baseline mean value obtained from all samples was 325.4 (±18.5). The samples presenting variation in 10% of the mean were replaced.
Specimens were randomly divided into eight groups (n = 13), according to the type of polymer used: Carbopol 980 (Noveon, Cleveland, OH, USA), Carboxymethyl Cellulose (CMC—Synth, Diadema, São Paulo, Brazil), and Aristoflex AVC (PharmaSpecial, Santana de Parnaiba, São Paulo, Brazil), associated or not with 900 ppm sodium fluoride (Synth, Diadema, Sao Paulo, Brazil). The control groups were ultrapure water (negative control) and a 900 ppm fluoride solution (positive control).
2.3
Preparation of the solutions
The concentration of the polymers used in this study was determined based on their effect in the viscosity of the solutions, which was intended to simulate a mouth rinse. Several preliminary studies were conducted to determine the highest amount of the polymers that could be added, without interfering with the solutions viscosity. Since Carbopol presented the ability to turn the solution into a gel at lower concentrations, the highest possible concentration determined for this polymer was used as standard for the other polymers, preventing that the concentration was a potential confusing factor.
Finally, the solutions were prepared dissolving 0.1 g of the polymer in 100 ml of distilled water, in a temperature-controlled environment (25 °C ± 1 °C) to avoid alterations in their kinematic viscosity. To achieve homogeneous solutions, they were placed inside a mixer (SpeedMixer—FlackTek, Landrun, SC, USA) programed at 1500 rpm for 10 min. In the groups containing NaF, 0.2 g/100 ml (900 ppm) was added to the solutions.
The pH of all solutions was adjusted to 4.5. This low pH was used to favor the deposition of CaF2-like globular structures on dental surface . In addition, this acidic pH helped the maintenance of the low viscosity of the solutions. The solutions using Carbopol presented pH values lower than 4.5, therefore 0.1 M KOH solution was used to increase their pH to 4.5. The Aristoflex and CMC solutions presented a natural pH between 6.1 and 6.7, so HCl was used to decrease their pH to 4.5.
For the viscosity measurements, a kinematic viscometer (TV2000AKV, Tamson Instruments, Bleiswijk, Netherlands), using a Cannon–Fenske glass tube number 100 was used, at 36 °C. Kinematic viscosity was measured by the time taken for the fluid to flow through a capillary tube under the gravity force at a given temperature and was converted directly to a kinematic viscosity using a simple calibration constant provided for each tube. Different sized capillaries were available to support fluids of different viscosities. The unit of kinematic viscosity is centistoke (cSt) ( Table 1 ).
| Solutions | Initial pH | Viscosity cSt (36 °C) |
|---|---|---|
| NaF (900 ppm F) | 6.3 | 0.79 |
| Aristoflex + NaF | 6.1 | 1.01 |
| Aristoflex | 5.7 | 1.89 |
| Carbopol + NaF | 4.4 | 1.28 |
| Carbopol | 4.2 | 4.92 |
| CMC + NaF | 6.4 | 2.49 |
| CMC | 6.7 | 1.83 |
2.4
Erosive challenge and remineralization
The erosive challenge was performed with a 0.3% citric acid solution, natural pH of approximately 2.6, which was obtained by adding 3 g/l of citric acid (Dinâmica, Diadema, São Paulo, Brazil) to 1000 ml of ultrapure water. The challenge consisted of immersing specimens in the citric acid for 2 min, six times a day. Polymer treatment with the test solutions was performed twice a day, after the first and last acid challenges, for 1 min. Between the acid exposures and 30 min before the polymer treatment, the specimens were stored in artificial saliva for 1 h. Artificial saliva was prepared using the following formula : 22.1 mmol/l NaHCO 3 ; 16.1 mmol/l KCl; 14.5 mmol/l NaCl; 2.6 mmol/l KH 2 PO 4 ; 0.8 mmol/l H 3 BO 3 ; 0.7 mmol/l CaCL 2 *2H 2 O; 0.4 mmol/l KSCN; and 0.2 mmol/l MgCl 2 *6H 2 O, adjusted to pH 7.0. The erosive challenge was repeated for 5 days. In the overnight period, the specimens were kept in relative humidity, at 4 °C. Fig. 1 shows a schematic chart illustrating the erosive cycle.
2.5
Microhardness analysis
The initial erosion was assessed by means of a microhardness test, with the same parameters previously described, and performed twice: after the first acid challenge (KHN2), and after the second acid challenge (KHN3).
After taking the microhardness measurements, the protective effect of the solutions against the acid challenge was determined by means of the Percentage of Microhardness Alteration (%KHN alt ), using the formula %KHN alt = [(KHN3-KHN2)/KHN2]*100.
2.6
Surface loss assessment
In order to maintain the reference surfaces for lesion-depth determination (profilometry) and to allow exact replacement, two parallel grooves were marked on the sides of the acrylic resin surface to serve as guides. Before the beginning of the erosive challenge, profiles of each specimen were obtained from the enamel surfaces with a contact profilometer (MaxSurf XT 20, Mahr-Goettingen, Germany). The diamond stylus moved from the first reference area in the acrylic resin to the second one (4.2 mm long). Three profile measurements were performed for each specimen at intervals of 0.25 mm.
At the end of the cycling (5 days), the final profiles were obtained in the same place as the first ones. The specimens were placed in a custom-made setting device in order to allow the exact replacement of the samples after the experimental procedures. Enamel loss was calculated after matching the baseline and post-polymer treatment profiles, using the previously described grooves as guides. The depth of the treated area was calculated based on the subtraction of the two profiles using a dedicated software (MarSurf XCR 20 4.50-07 SP3, 2011).
2.7
KOH-soluble fluoride determination
After the profilometric analysis, the amounts of KOH-soluble fluoride from enamel surfaces were determined based on a method described elsewhere . Briefly summarized, the specimens were individually stored in plastic containers with 0.5 ml of 1.0 M KOH solution, under gentle agitation at room temperature for 24 h. After this period, the specimens were rinsed, and a sample of the solution (0.25 ml) was transferred to a plastic vial and neutralized with 0.25 ml of 1 M HClO 4 . Then, 0.5 ml of TISAB II buffer was added to the tube. The fluoride content was determined by comparison to a similarly prepared standard curve using an ion-selective electrode (Perfection, Mettler Toledo, Schwerzenbach, Switzerland).
2.8
Energy-dispersive spectroscopy (EDS) and scanning electron microscopy (SEM)
Additional specimens were prepared, polished, and immersed in 0.3% citric acid for 10 min. After rinsing, the specimens were treated with the experimental treatment solutions for 1 min and dehydrated. The specimens were then mounted on aluminum stubs, sputtered with gold (Emitech SC7620 Sputter Coater, Moorestown, NJ, EUA) for 2 min, and taken to the scanning electron microscope (Inspect S50, FEI, Hillsboro, OR, EUA). The images were obtained in secondary electron mode (15 kV, 100 s) at 5.000 x magnification. The energy-dispersive spectroscopy (EDS) analysis was performed using an EDS-detector coupled at the scanning electron microscope, with spot size at 3.0 and voltage at 10 kV. The detector was used to assess the presence of fluoride in each sample. The weight percentage of fluoride was analyzed stoichiometrically.
2.9
Statistical analysis
Assumptions of normal distribution (Kolmogorov-Smirnov tests) were checked for all the variables tested. Descriptive and inferential statistical analyses were performed using Minitab 16.1.0 (2010 Minitab, State College, PA, USA) and Graphpad Prism (Graphpad Prism Software, La Jolla, CA, USA). Two-way ANOVA was performed for both profilometry and microhardness analyses, followed by Tukey’s test with 5% significance.
2
Materials and methods
2.1
Experimental design
This study tested two experimental factors: the polymer treatment with four levels (control- ultrapure water, Carbopol 980, Carboxymethyl cellulose (CMC), and Aristoflex AVC) and the presence (or not) of sodium fluoride, both in an erosion-remineralization cycling model using bovine enamel specimens (n = 104). The response variables were surface microhardness (KHN) for initial erosion analysis, and surface loss (μm), the latter measured by stylus profilometry at the end of the cycling (after 5 days).
2.2
Specimen preparation
Fresh, non-damaged bovine incisors were collected for this study. The crowns were separated from the roots and stored in 0.1% thymol solution at 4 °C until required. One hundred four cylindrical enamel specimens (3 mm in diameter and 2.1 mm in height) were obtained from the labial surface using a custom-made diamond-coated trephine mill adapted to a circular cutting machine. The specimens were embedded in acrylic resin (ExtecFast Cure Acrylic, ExtecCorp, Enfield, CT, USA) using a silicone mold, which consisted of a cavity with 2 mm deep and 6 mm in diameter. Also, in the bottom of the mold there was a second level cavity with a 0.1 mm deep and 3 mm in diameter, in which the specimens were positioned with the enamel surface facing down . On the lower side of the mold there was a projection in the shape of a line, which produced a lateral notch in the specimen. This allowed the exact repositioning of the specimen at the time of the surface profile measurement (before and after testing).
After the resin cure, the specimens were attached to a metal holder, and the 0.1 mm high enamel surface was removed and ground flat using aluminum oxide abrasive papers in sequential grits of 1200, 2400, and 4000 (FEPA-P, Struers, Ballerup, Denmark), in a polishing device (DP-10, Panambra Industrial e Técnica SA, São Paulo, SP, Brazil) under water irrigation for 30, 60 and 120 s, respectively. After each paper grit change, specimens were kept in ultrasonic baths in distilled water for 10 min to remove waste and abrasive grains. The prepared specimens were examined under a stereomicroscope (Carl Zeiss—Stemi 2000 −20X) to ensure the absence of cracks or other surface defects. After preparation, the specimens were stored in ultrapure water at 4 °C to avoid dehydration.
The initial microhardness (KHN1) of all specimens was assessed using a Knoop Microhardness Tested (FM-700, Future-Tech, Tokyo, Japan) fitted with a 50 g load, for 10 s. Three indentations were performed, 100 μm apart from each other. The recorded values were averaged, and the baseline mean value obtained from all samples was 325.4 (±18.5). The samples presenting variation in 10% of the mean were replaced.
Specimens were randomly divided into eight groups (n = 13), according to the type of polymer used: Carbopol 980 (Noveon, Cleveland, OH, USA), Carboxymethyl Cellulose (CMC—Synth, Diadema, São Paulo, Brazil), and Aristoflex AVC (PharmaSpecial, Santana de Parnaiba, São Paulo, Brazil), associated or not with 900 ppm sodium fluoride (Synth, Diadema, Sao Paulo, Brazil). The control groups were ultrapure water (negative control) and a 900 ppm fluoride solution (positive control).
2.3
Preparation of the solutions
The concentration of the polymers used in this study was determined based on their effect in the viscosity of the solutions, which was intended to simulate a mouth rinse. Several preliminary studies were conducted to determine the highest amount of the polymers that could be added, without interfering with the solutions viscosity. Since Carbopol presented the ability to turn the solution into a gel at lower concentrations, the highest possible concentration determined for this polymer was used as standard for the other polymers, preventing that the concentration was a potential confusing factor.
Finally, the solutions were prepared dissolving 0.1 g of the polymer in 100 ml of distilled water, in a temperature-controlled environment (25 °C ± 1 °C) to avoid alterations in their kinematic viscosity. To achieve homogeneous solutions, they were placed inside a mixer (SpeedMixer—FlackTek, Landrun, SC, USA) programed at 1500 rpm for 10 min. In the groups containing NaF, 0.2 g/100 ml (900 ppm) was added to the solutions.
The pH of all solutions was adjusted to 4.5. This low pH was used to favor the deposition of CaF2-like globular structures on dental surface . In addition, this acidic pH helped the maintenance of the low viscosity of the solutions. The solutions using Carbopol presented pH values lower than 4.5, therefore 0.1 M KOH solution was used to increase their pH to 4.5. The Aristoflex and CMC solutions presented a natural pH between 6.1 and 6.7, so HCl was used to decrease their pH to 4.5.
For the viscosity measurements, a kinematic viscometer (TV2000AKV, Tamson Instruments, Bleiswijk, Netherlands), using a Cannon–Fenske glass tube number 100 was used, at 36 °C. Kinematic viscosity was measured by the time taken for the fluid to flow through a capillary tube under the gravity force at a given temperature and was converted directly to a kinematic viscosity using a simple calibration constant provided for each tube. Different sized capillaries were available to support fluids of different viscosities. The unit of kinematic viscosity is centistoke (cSt) ( Table 1 ).
