Abstract
Objectives
Consumption of acidic soft drinks may lead to the dissolution and softening of human enamel, known as erosion. The first aim of this in vitro study was to test the hypothesis that food-approved polymers added to citric acid solutions (CAS) decrease the erosion of human dental enamel compared to citric acid solutions without these polymers. The second aim was to test the hypothesis that these polymers added to CAS form a polymer layer directly on the eroded enamel surface.
Methods
Enamel samples were obtained by embedding pieces of non-erupted human third molars in resin, grinding, and polishing them. CAS with pH values (pH: 2.3, 3.3 and 4.0) of typical soft drinks were prepared and modified by adding one of the following food-approved polymers (1%, w/w): highly esterified pectin (HP), propylene glycol alginate (PGA) and gum arabic (GA). The enamel samples were exposed to these polymer-modified citric acid solutions (PMCAS) or CAS not containing polymers, respectively, for different time periods (30, 60 and 120 s). Atomic force microscopy (AFM)-based nanoindentation was used to analyze the nanomechanical properties of the treated enamel samples and the control samples. The enamel nanohardness and the reduced elastic modulus of the samples treated with PMCAS were statistically analyzed (ANOVA, t -test) and compared to the mechanical properties of the samples treated with unmodified CAS. Thus treated enamel samples were imaged by scanning electron microscopy (SEM) to investigate the surface morphology of the different enamel samples.
Results
Enamel samples treated with PMCAS containing GA or PGA showed statistically significantly higher nanohardness ( p < 0.05) compared to samples treated with CAS. PMCAS containing HP did not reduce the enamel nanohardness loss significantly compared to the CAS treated enamel samples. The enamel samples eroded with PMCAS show generally a smoother surface compared to the enamel surfaces of samples treated only with CAS as detected by SEM. Therefore, it is hypothesized that the polymers possibly adsorb on the eroded enamel surface.
Significance
The present in vitro erosion study shows that some of the polymers used in this study may possibly adsorb like a protective layer directly onto the human enamel surface. For GA and PGA this possibly formed polymer layer reduces the erosive effects of citric acid solutions as shown by nanoindentation measurements.
1
Introduction
Dental erosion has been defined as the dissolution of dental hard tissue without involvement of bacteria . It is well known that acid containing food and beverages may lead to erosive damage of dental enamel . The acids in soft drinks are the major aetiological factor of dental erosion . Moreover, a wide range of extrinsic and intrinsic factors are involved in the process of dental erosion. A combination of these factors, e.g. high soft drink consumption, or eating disorders with acid regurgitation, leads to greatly enhanced tooth mineral loss .
The investigation of dental erosion has been recently of increasing interest for the dental materials science community . Since the mid-1990s studies investigated the intensity of enamel loss and surface softening caused by the consumption of acidic soft drinks. Citric and phosphoric acid containing soft drinks were frequently investigated with respect to their erosive effect of dissolution and softening of human enamel . The early stages of enamel erosion were characterized by demineralization of the enamel surface leading to softening and nanostructural changes. The subsequent stages of enamel erosion were characterized by material loss and the structural collapse of dental enamel .
To reduce or avoid erosive effects caused by acids in soft drinks, different strategies have been investigated. One strategy is to change the degree of saturation of the erosive agent with respect to hydroxyapatite (HA). Adding Ca 2+ and PO 4 3− ions to the citric acid solution (CAS) changes the chemical equilibrium of these ions between the surface and the erosive agent. Therefore, a reduced enamel softening during the acid exposure was possible .
Furthermore, other compounds have been tested for their capability to reduce enamel erosion. Some beverage-modifying agents, such as citrate and fluoride , have been successfully added to acidic solutions to reduce the dissolution of human enamel. The effect of proteins in the saliva which bind to the enamel surface has been investigated with respect to the reduction of erosive effects . Moreover, the interaction of milk-proteins with human enamel has been tested . In both cases, a reduced erosive enamel damage has been demonstrated.
Only a few studies have shown that polymers can affect the dissolution of the HA surface . Polymers have been investigated with respect to their potential to prevent or reduce the dissolution of Ca 2+ and PO 4 3− ions from the HA prisms . Such polymers may be useful to reduce erosion or to study enamel remineralization mechanisms . Barbour et al. have shown on artificial HA discs that some food-approved polymers, such as xanthan gum and carboxymethylcellulose reduce the dissolution of Ca 2+ and PO 4 3− ions caused by CAS. Two studies have shown in situ that xanthan gum and the combination of xanthan gum with calcium or polyphosphate reduce the softening of the enamel .
To the authors’ best knowledge, a potential effect of food-approved polymers on reducing the erosion of human enamel measured by nanoindentation has not been reported so far. Highly esterified pectin (HP), propylene glycol alginate (PGA) and gum arabic (GA), the polymers used in this current in vitro study, are normally used in sweets, jams, bakery products, ice cream and soft drinks. In these food systems, the polymers form gels to stabilize the food.
Therefore, the first aim of this in vitro study was to test the hypothesis that the above mentioned food-approved polymers, added to citric acid solutions (CAS), decrease the erosion of human dental enamel compared to CAS without such polymers, as measured by nanoindentation. The second aim was to test the hypothesis that these polymers, added to CAS, form a polymer layer on the enamel surface.
2
Materials and methods
2.1
Sample preparation
For this in vitro study non-erupted human third molars ( n = 87) were used and prepared as described previously . In brief, teeth were extracted and disinfected, and the roots were carefully removed. Pieces (approximately 2 mm × 3 mm) were cut from all over 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, Westerlo, Belgium). The samples were then ground with SiC paper (grit 1200–4000; Buehler GmbH, Düsseldorf, Germany) and polished with diamond powder dispersions (particle size ranging from 6 to 1 μm) to obtain smooth surfaces. Samples were stored in deionized water at room temperature until use. 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 an untreated area (UT) which was protected against solution exposure.
2.2
Test solutions
The solutions were prepared by dissolving the following polymers (1%, w/w) in deionized water: highly esterified pectin (HP), propylene glycol alginate (PGA) and gum arabic (GA). Polysaccharides were supplied by WILD GmbH & Co. KG (Eppelheim, Germany). All specifications known are given in Table 1 and their use or purity degree is regulated by the European Union directive (200/84/EG). To obtain the polymer-modified citric acid solutions (PMCAS), 50% (w/w) citric acid was added to adjust the pH to three different values (2.30 ± 0.01, 3.30 ± 0.01 and 4.00 ± 0.01) and controlled with a pH meter (Knick pH meter 765 Calimatic, Knick Elektronische Messgeräte GmbH & Co. KG, Berlin, Germany). This addition of citric acid may lead to minute changes in the polymer concentration. We estimate the deviation to be small because of adding only few droplets of citric acid (i.e. final polymer concentration 1%, w/w, ±0.01%). No other substances that could influence the erosive effect of enamel were added to the solutions. Control CAS was prepared similar but without addition of polymers.
| Highly esterified pectin (HP) | Propylene glycol alginate (PGA) | Gum arabic (GA) | |
|---|---|---|---|
| Source | Apple pectin | – | Acacia senegal |
| E-number | E 440 | E 405 | E 414 |
| Molecular weight | Not known | 10,000–600,000 g/mol | 350,000 g/mol |
| Level of esterification | 70–76% | ≥75% | – |
| Viscosity | 55 ± 5 mPa s, 2.5% solution | 50–175 mPa s, 2% solution | ≥60 mPa s, 25% solution |
| Others | Protein < 1% | – | – |
2.3
Erosive treatment of the enamel samples
The enamel samples were treated at room temperature with PMCAS or CAS for 30, 60, and 120 s, respectively, to simulate the normal intake times of soft drinks. For the erosion investigations three samples ( n = 3) were used for every PMCAS or CAS. For every sample 30 ml of PMCAS or CAS was used and the solution was stirred with a magnetic stirrer. After treatment, the samples were immediately rinsed with deionized water for 30 s and dried with compressed air. The PVC tape or residues of glue from the tape were removed carefully from the untreated areas of the enamel samples with ethanol soaked cotton swab. Samples were stored in air before investigation by nanoindentation.
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 nanohardness and the reduced elastic modulus of the enamel samples . For the indentations, the standard Berkovich diamond indenter with equilateral pyramidal area was calibrated with fused silica, and a standard tip area function was used. The indentations were made in air at room temperature in three steps: linear loading up to 5 mN from 0 to 15 s, holding this load for 5 s, and linear unloading to 0 mN within 15 s. An image of the surface was recorded before every indent to ensure that it was flat, clean, and free of damages. Indentations were made five times in both the treated area and the untreated area on every sample. The distance between the indents was kept to at least 20 μm to avoid interferences between them. Nanohardness and reduced elastic modulus were calculated with the TriboScope ® software (version 3.5).
2.5
SEM
Scanning electron microscopy (SEM) was performed with a LEO 440i SEM Scanning Electron Microscope (LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) operated at 15 kV. For SEM all samples were gold sputter-coated (approx. 10 nm) in vacuum.
2.6
Statistical analysis
A multiway ANOVA and the Scheffe t -test (95% confidence interval, p < 0.05) analysis were performed with StatGraphics Centurion XV (StatPoint Technologies Inc.; Warrenton, VA, USA) to test the statistical significance of the nanohardness loss and the reduced elastic modulus of the human enamel samples. The factors investigated for significance were erosion time ( t = 30, 60, 120 s), polymer substance (HP, PGA, GA or CAS), and pH value (pH 2.3, 3.3, 4.0). For the measurements the samples were randomly divided to the different test groups.
2
Materials and methods
2.1
Sample preparation
For this in vitro study non-erupted human third molars ( n = 87) were used and prepared as described previously . In brief, teeth were extracted and disinfected, and the roots were carefully removed. Pieces (approximately 2 mm × 3 mm) were cut from all over 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, Westerlo, Belgium). The samples were then ground with SiC paper (grit 1200–4000; Buehler GmbH, Düsseldorf, Germany) and polished with diamond powder dispersions (particle size ranging from 6 to 1 μm) to obtain smooth surfaces. Samples were stored in deionized water at room temperature until use. 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 an untreated area (UT) which was protected against solution exposure.
2.2
Test solutions
The solutions were prepared by dissolving the following polymers (1%, w/w) in deionized water: highly esterified pectin (HP), propylene glycol alginate (PGA) and gum arabic (GA). Polysaccharides were supplied by WILD GmbH & Co. KG (Eppelheim, Germany). All specifications known are given in Table 1 and their use or purity degree is regulated by the European Union directive (200/84/EG). To obtain the polymer-modified citric acid solutions (PMCAS), 50% (w/w) citric acid was added to adjust the pH to three different values (2.30 ± 0.01, 3.30 ± 0.01 and 4.00 ± 0.01) and controlled with a pH meter (Knick pH meter 765 Calimatic, Knick Elektronische Messgeräte GmbH & Co. KG, Berlin, Germany). This addition of citric acid may lead to minute changes in the polymer concentration. We estimate the deviation to be small because of adding only few droplets of citric acid (i.e. final polymer concentration 1%, w/w, ±0.01%). No other substances that could influence the erosive effect of enamel were added to the solutions. Control CAS was prepared similar but without addition of polymers.
| Highly esterified pectin (HP) | Propylene glycol alginate (PGA) | Gum arabic (GA) | |
|---|---|---|---|
| Source | Apple pectin | – | Acacia senegal |
| E-number | E 440 | E 405 | E 414 |
| Molecular weight | Not known | 10,000–600,000 g/mol | 350,000 g/mol |
| Level of esterification | 70–76% | ≥75% | – |
| Viscosity | 55 ± 5 mPa s, 2.5% solution | 50–175 mPa s, 2% solution | ≥60 mPa s, 25% solution |
| Others | Protein < 1% | – | – |
2.3
Erosive treatment of the enamel samples
The enamel samples were treated at room temperature with PMCAS or CAS for 30, 60, and 120 s, respectively, to simulate the normal intake times of soft drinks. For the erosion investigations three samples ( n = 3) were used for every PMCAS or CAS. For every sample 30 ml of PMCAS or CAS was used and the solution was stirred with a magnetic stirrer. After treatment, the samples were immediately rinsed with deionized water for 30 s and dried with compressed air. The PVC tape or residues of glue from the tape were removed carefully from the untreated areas of the enamel samples with ethanol soaked cotton swab. Samples were stored in air before investigation by nanoindentation.
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 nanohardness and the reduced elastic modulus of the enamel samples . For the indentations, the standard Berkovich diamond indenter with equilateral pyramidal area was calibrated with fused silica, and a standard tip area function was used. The indentations were made in air at room temperature in three steps: linear loading up to 5 mN from 0 to 15 s, holding this load for 5 s, and linear unloading to 0 mN within 15 s. An image of the surface was recorded before every indent to ensure that it was flat, clean, and free of damages. Indentations were made five times in both the treated area and the untreated area on every sample. The distance between the indents was kept to at least 20 μm to avoid interferences between them. Nanohardness and reduced elastic modulus were calculated with the TriboScope ® software (version 3.5).
2.5
SEM
Scanning electron microscopy (SEM) was performed with a LEO 440i SEM Scanning Electron Microscope (LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) operated at 15 kV. For SEM all samples were gold sputter-coated (approx. 10 nm) in vacuum.
2.6
Statistical analysis
A multiway ANOVA and the Scheffe t -test (95% confidence interval, p < 0.05) analysis were performed with StatGraphics Centurion XV (StatPoint Technologies Inc.; Warrenton, VA, USA) to test the statistical significance of the nanohardness loss and the reduced elastic modulus of the human enamel samples. The factors investigated for significance were erosion time ( t = 30, 60, 120 s), polymer substance (HP, PGA, GA or CAS), and pH value (pH 2.3, 3.3, 4.0). For the measurements the samples were randomly divided to the different test groups.
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