Abstract
Objectives
The consumption of acidic soft drinks may lead to demineralization and softening of human dental enamel, known as dental erosion. The aims of this in vitro study were to determine: (i) if different acids with a similar sensorial acidic taste lead to different hardness loss of enamel and (ii) if the fruit acids tartaric, malic, lactic or ascorbic acid lead to less hardness loss of enamel than citric or phosphoric acid when their concentration in solution is based on an equivalent sensorial acidic taste.
Methods
Enamel samples of non-erupted human third molars were treated with acidic solutions of tartaric (TA), malic (MA), lactic (LA), ascorbic (AA), phosphoric (PA) and citric (CA) acids with a concentration that gave an equivalent sensorial acidic taste. The acidic solutions were characterized by pH value and titratable acidity. Atomic force microscopy (AFM) based nanoindentation was used to study the nano mechanical properties and scanning electron microscopy (SEM) was used to study the morphology of the treated enamel samples and the untreated control areas, respectively.
Results
The investigated acids fell into two groups. The nano hardnesses of MA, TA and CA treated enamel samples (group I) were statistically significantly greater ( p < 0.05) than the nano hardnesses of PA, AA and LA treated enamel samples (group II). Within each group the nano hardness was not statistically significantly different ( p > 0.05). The SEM micrographs showed different etch prism morphologies depending on the acid used.
Significance
In vitro, the acids investigated led to different erosion effects on human dental enamel, despite their equivalent sensorial acidic taste. This has not been reported previously.
1
Introduction
Dental health is influenced by various factors . One of them is dental erosion. This is defined as the dissolution of dental hard tissue without involvement of bacteria . Acids present in food and beverages are one major etiological factor responsible for the erosive lesions of dental enamel . Since the mid-1990s the effects of surface softening and enamel loss caused by the consumption of acidic soft drinks have been investigated. Soft drinks containing citric acid were investigated with respect to their erosive effects, specifically dissolution and softening of human enamel .
Different parameters determine the erosive effects of soft drinks on enamel. Important parameters to characterize the erosive potential of acidic solutions are the pH value and the titratable acidity . The primary dissolution of human enamel depends on the pH value and the corresponding pKa value of acids . With lower acid pH the erosive softening of human enamel increases dramatically . Hannig et al. tested different acids with the same pH value for their calcium and phosphate dissolution from bovine enamel . It was shown that ascorbic acid caused the highest erosion effect and malic acid the lowest. In addition, the titratable acidity parameter best described the erosive potential of acid solutions. The titratable acidity is the amount of sodium hydroxide solution (NaOH), which is required to reach a neutral pH . It was shown that the higher the value of titratable acidity the greater the erosion effects .
Different compositional aspects are important for the use of different acids in soft drinks. From the perspective of the manufacturer, the concentration of acid in soft drinks is limited and the acid used should be able to reach a specific pH, which is necessary for the application. A further aspect for consumers is that, besides a generally preferred acidic taste of the soft drink the pH-values, concentration or type of acids or titratable acidity are not important for their choice of soft drinks. Rather, for consumers, the sensorial taste of a beverage is important . There is a wide range of fruit acids that can be used in soft drinks or other food, e.g. malic acid, tartaric acid, ascorbic acid or lactic acid .
It is understood how acids interact with hydroxyapatite (HA) surfaces . It has been shown that mono-, di- and tricarboxylic acids chemically adsorb onto the enamel surface and dissolve Ca 2+ ions out of the HA surface . The carboxylic acids: MA, TA and LA showed increased dissolution of Ca 2+ and PO 4 3− ions out of a bovine HA surface compared to other acids tested .
A newer approach for using acids in soft drinks is to establish an adjusted concentration of the different acids that results in an equivalent sensorial acidic taste. From sensorial tests it is known that different acids can have an equivalent sensorial acidic taste by using the concentrations shown in Table 1 . The useful and sensitive sensory method can be used to adjust the acidic concentration so that it becomes sensorially equal for soft drinks. The previous sensorial tests were conducted by a trained panel. This panel consisted of various people who individually assessed the taste of the acid solutions. The panel identified the concentrations that give an equivalent acidic taste. The erosive effects of such sensorially adjusted acidic solutions on human dental enamel have not been investigated previously. Therefore, the aims of this in vitro study were to determine: (i) if different acids with a similar sensorial acidic taste lead to different hardness loss of enamel and (ii) if the fruit acids tartaric, malic, lactic or ascorbic acid lead to less hardness loss of enamel than citric or phosphoric acid when their concentration in solution is based on the equivalent sensorial acidic taste.
| Acid | c acid g/L (mmol/L) | pH | pKa | Degree of dissociation (%) | Titratable acidity c NaOH (mmol/L) | Hardness loss (%) | |
|---|---|---|---|---|---|---|---|
| Group I | Malic acid (MA) | 1.12 (8.35) | 2.64 | pKa 1 = 3.46 | 15.14 | 0.80 | 48.6 |
| Tartaric acid (TA) | 1.00 (6.66) | 2.56 | pKa 1 = 2.98 | 38.02 | 0.42 | 47.5 | |
| Citric acid (CA) | 1.22 (6.35) | 2.62 | pKa 1 = 3.13 | 30.90 | 0.72 | 48.7 | |
| Group II | Lactic acid (LA) | 1.36 (15.1) | 2.63 | pKa 1 = 3.90 | 5.37 | 0.68 | 66.4 |
| Ascorbic acid (AA) | 3.00 (17.03) | 2.87 | pKa 1 = 4.20 | 4.68 | 0.67 | 59.6 | |
| Phosphoric acid (PA) | 0.85 (8.67) | 2.15 | pKa 1 = 2.16 | 97.72 | 0.53 | 64.0 | |
2
Materials and methods
2.1
Sample preparation
For this in vitro study non-erupted human third molars ( n = 53) were used and prepared as described previously . In brief, teeth were disinfected and the roots were carefully removed. Enamel pieces (approximately 2 mm × 3 mm) were cut from the tooth with a low speed saw (Isomet; Buehler GmbH, Düsseldorf, Germany) using a water cooled diamond blade (Buehler GmbH, Düsseldorf, Germany) and embedded in a resin (Stycast 1266; Emerson & Cuming, ICI, Wasterlo, Belgium). To obtain smooth surfaces, the samples were finally ground with SiC paper (grit 1200–4000; Buehler GmbH, Düsseldorf, Germany) and polished with aluminum oxide powder dispersions (particle size ranging from 6 μm to 1 μm). Prior to use, the samples were stored in deionized water at room temperature. Immediately before treatment with the test solutions, one half of the surface of each enamel sample was covered with PVC tape (Tesa AG, Hamburg, Germany) to obtain a reference area that was protected against acid solution exposure.
2.2
Acid solutions
The acid solutions were prepared in deionized water according to the acid concentrations given in Table 1 . The acids: tartaric acid (TA), malic acid (MA), lactic acid (LA), ascorbic acid (AA), citric acid (CA) and phosphoric acid (PA) were supplied by Rudolf Wild GmbH & Co. KG (Eppelheim, Germany). To quantify the erosive potential of the different acid solutions the pH value was measured with a pH-meter (Knick pH-meter 765 Calimatic, Berlin, Germany). In addition, the titratable acidity was calculated by measuring the amount of NaOH (0.1 mol/L) which was necessary to reach a neutral pH of 7.00.
2.3
Erosive treatment of the enamel samples
Four samples chosen at random ( n = 4) were used to investigate the erosive effect of each of the six acid solutions (overall n = 24). Each enamel sample was placed in a beaker with the acid solutions (approx. 40 mL) for 60 s under constant stirring. This treatment time relates to the neutralization properties of saliva and short term pellicle building time of saliva . Immediately after treatment, the samples were rinsed with deionized water for 30 s and dried with compressed air. The PVC tapes were removed from the untreated areas (UT) of the enamel samples. Residues of glue from the tape were removed carefully with ethanol soaked cotton swabs.
2.4
Nanoindentation
An atomic force microscope (Digital Instrument Dimension 3100; Veeco Instruments, Santa Barbara, CA, USA) equipped with a Hysitron TriboScope ® nanoindenter (Hysitron Inc., Minneapolis, MN, USA) was used to measure the nano hardness and the reduced elastic modulus of the enamel samples. For the indentations, a standard Berkovich diamond indenter with an equilateral pyramidal area was calibrated with fused silica and a standard tip area function was used. The enamel indentations were made in air at room temperature in three steps: linear loading up to 5 mN from 0 s to 15 s, holding this load for 5 s and linear unloading to 0 mN within 15 s. Before every indentation an image of the surface was recorded with the Berkovich tip to ensure that it was flat, clean, and free of damage. Five indentations were made in both the reference area and the treated surface area on every sample. The distance between the indents was kept to at least 20 μm to avoid interferences between them. Nano hardness and reduced elastic modulus were calculated with the TriboScope ® software (version 3.5).
2.5
SEM measurements
Scanning electron microscopy (SEM) was performed with a LEO 440i SEM Scanning Electron Microscope (LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) operated at 15 kV and with a working distance of 6 mm. For SEM images all samples were gold sputter coated (approx. 10 nm) with an Edwards sputter coater S150B (Edwards High Vacuum International, Crawley, West Sussex, UK).
2.6
Statistical analysis
ANOVA analysis and a Scheffe t -test (95% confidence interval, p < 0.05) were performed with StatGraphics Centurion XV (StatPoint Inc., Warrenton, USA) to test the statistical significance of the nano hardness loss and the reduced elastic modulus of the human enamel samples. The factor investigated for significance was the acid type.
2
Materials and methods
2.1
Sample preparation
For this in vitro study non-erupted human third molars ( n = 53) were used and prepared as described previously . In brief, teeth were disinfected and the roots were carefully removed. Enamel pieces (approximately 2 mm × 3 mm) were cut from the tooth with a low speed saw (Isomet; Buehler GmbH, Düsseldorf, Germany) using a water cooled diamond blade (Buehler GmbH, Düsseldorf, Germany) and embedded in a resin (Stycast 1266; Emerson & Cuming, ICI, Wasterlo, Belgium). To obtain smooth surfaces, the samples were finally ground with SiC paper (grit 1200–4000; Buehler GmbH, Düsseldorf, Germany) and polished with aluminum oxide powder dispersions (particle size ranging from 6 μm to 1 μm). Prior to use, the samples were stored in deionized water at room temperature. Immediately before treatment with the test solutions, one half of the surface of each enamel sample was covered with PVC tape (Tesa AG, Hamburg, Germany) to obtain a reference area that was protected against acid solution exposure.
2.2
Acid solutions
The acid solutions were prepared in deionized water according to the acid concentrations given in Table 1 . The acids: tartaric acid (TA), malic acid (MA), lactic acid (LA), ascorbic acid (AA), citric acid (CA) and phosphoric acid (PA) were supplied by Rudolf Wild GmbH & Co. KG (Eppelheim, Germany). To quantify the erosive potential of the different acid solutions the pH value was measured with a pH-meter (Knick pH-meter 765 Calimatic, Berlin, Germany). In addition, the titratable acidity was calculated by measuring the amount of NaOH (0.1 mol/L) which was necessary to reach a neutral pH of 7.00.
2.3
Erosive treatment of the enamel samples
Four samples chosen at random ( n = 4) were used to investigate the erosive effect of each of the six acid solutions (overall n = 24). Each enamel sample was placed in a beaker with the acid solutions (approx. 40 mL) for 60 s under constant stirring. This treatment time relates to the neutralization properties of saliva and short term pellicle building time of saliva . Immediately after treatment, the samples were rinsed with deionized water for 30 s and dried with compressed air. The PVC tapes were removed from the untreated areas (UT) of the enamel samples. Residues of glue from the tape were removed carefully with ethanol soaked cotton swabs.
2.4
Nanoindentation
An atomic force microscope (Digital Instrument Dimension 3100; Veeco Instruments, Santa Barbara, CA, USA) equipped with a Hysitron TriboScope ® nanoindenter (Hysitron Inc., Minneapolis, MN, USA) was used to measure the nano hardness and the reduced elastic modulus of the enamel samples. For the indentations, a standard Berkovich diamond indenter with an equilateral pyramidal area was calibrated with fused silica and a standard tip area function was used. The enamel indentations were made in air at room temperature in three steps: linear loading up to 5 mN from 0 s to 15 s, holding this load for 5 s and linear unloading to 0 mN within 15 s. Before every indentation an image of the surface was recorded with the Berkovich tip to ensure that it was flat, clean, and free of damage. Five indentations were made in both the reference area and the treated surface area on every sample. The distance between the indents was kept to at least 20 μm to avoid interferences between them. Nano hardness and reduced elastic modulus were calculated with the TriboScope ® software (version 3.5).
2.5
SEM measurements
Scanning electron microscopy (SEM) was performed with a LEO 440i SEM Scanning Electron Microscope (LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) operated at 15 kV and with a working distance of 6 mm. For SEM images all samples were gold sputter coated (approx. 10 nm) with an Edwards sputter coater S150B (Edwards High Vacuum International, Crawley, West Sussex, UK).
2.6
Statistical analysis
ANOVA analysis and a Scheffe t -test (95% confidence interval, p < 0.05) were performed with StatGraphics Centurion XV (StatPoint Inc., Warrenton, USA) to test the statistical significance of the nano hardness loss and the reduced elastic modulus of the human enamel samples. The factor investigated for significance was the acid type.
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