Abstract
Objectives
Human dental erosion caused by acids is a major factor for tooth decay. Adding polymers to acidic soft drinks is one important approach to reduce human dental erosion caused by acids. The aim of this study was to investigate the thickness and the structure of polymer layers adsorbed in vitro on human dental enamel from polymer modified citric acid solutions.
Methods
The polymers propylene glycol alginate (PGA), highly esterified pectin (HP) and gum arabic (GA) were used to prepare polymer modified citric acids solutions (PMCAS, pH 3.3). With these PMCAS, enamel samples were treated for 30, 60 and 120 s respectively to deposit polymer layers on the enamel surface. Profilometer scratches on the enamel surface were used to estimate the thickness of the polymer layers via atomic force microscopy (AFM). The composition of the deposited polymer layers was investigated with X-ray photoelectron spectroscopy (XPS). In addition the polymer–enamel interaction was investigated with zeta-potential measurements and scanning electron microscopy (SEM).
Results
It has been shown that the profilometer scratch depth on the enamel with deposited polymers was in the range of 10 nm (30 s treatment time) up to 25 nm (120 s treatment time). Compared to this, the unmodified CAS-treated surface showed a greater scratch depth: from nearly 30 nm (30 s treatment time) up to 60 nm (120 s treatment time). Based on XPS measurements, scanning electron microscopy (SEM) and zeta-potential measurements, a model was hypothesized which describes the layer deposited on the enamel surface as consisting of two opposing gradients of polymer molecules and hydroxyapatite (HA) particles.
Significance
In this study, the structure and composition of polymer layers deposited on in vitro dental enamel during treatment with polymer modified citric acid solutions were investigated. Observations are consistent with a layer consisting of two opposing gradients of hydroxyapatite particles and polymer molecules. This leads to reduced erosive effects of citric acid solutions on dental enamel surfaces.
1
Introduction
Dental enamel is one of the hardest materials of the human body . Yet, as is well known, enamel can be destroyed by acids present in dietary components, especially in soft drinks. This destructive process, occurring without the involvement of bacteria, is known as dental erosion . The process of dental erosion is influenced by a number of extrinsic and intrinsic factors including the formulation of the soft drinks, biological factors and the socio-economic status of the consumer . An important approach to reduce erosion is to modify the erosive agents and, thus, to reduce the erosive effects against human dental enamel.
The process of enamel dissolution caused by acids was investigated in several studies. Surface softening and enamel loss are most often caused by the consumption of acidic soft drinks containing citric and/or phosphoric acids . Enamel surfaces can be characterized by profilometry to measure the loss of dental enamel dependent on e.g. pH, acid concentration and time. Atomic force microscopy based nanoindentation is one relatively new advanced method to characterize the nano mechanical properties of enamel surfaces. Using this method, it is possible to determine the erosive effects of acids against human dental enamel after short time (early stage) erosion .
Some mechanisms are known that can modify and decrease the dissolution process of human dental enamel caused by the acids. The degree of saturation of the erosive solution can be changed with respect to hydroxyapatite (HA), the main component of the dental hard tissue. The addition of Ca 2+ and PO 4 3− ions to citric acid solutions (CASs) changes the chemical equilibrium of these ions between the enamel surface and the erosive agent and leads to a reduced enamel softening . Also, the addition of different compounds, such as citrate and fluoride to CAS has been examined in order to determine their ability to reduce dental enamel erosion.
Another complementary approach to reduce dental erosion is to modify the acidic soft drinks with food-approved polymers. To date, only few studies have investigated the erosion reducing effect of such polymers added to acidic solutions . In our previous study, we showed that the treatment of human dental enamel with polymer modified citric acid solutions (PMCASs) led to reduced softening of the enamel surfaces compared to samples treated with unmodified CAS . In addition, we presented qualitative evidence that the polymers interact with the hydroxyapatite of the human dental enamel and build up a protective layer on the enamel surface. The polymers propylene glycol alginate (PGA), highly esterified pectin (HP) and gum arabic (GA) were highly effective in protecting dental enamel against citric acid induced dental erosion. Nevertheless, until now the morphology and structure of such protective polymer layers are unknown. The aim of our study was, therefore, to use combined microscopic and spectroscopic approaches to characterize the morphology and structure of the polymer layers formed on the enamel surfaces. Atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used, amongst other techniques, to investigate the thickness, composition and structure of these protective layers. In addition, based on our results, a descriptive model of the protective layer was developed.
2
Materials and methods
2.1
Sample preparation
For this in vitro study, non-erupted human third molars ( n = 12) were used and prepared as described previously . The teeth were extracted and disinfected, and the roots were carefully removed. Pieces (approx. 2 mm × 3 mm) were cut from the tooth crowns with a low-speed saw (Isomet, Buehler GmbH, Düsseldorf, Germany) using a water-cooled diamond blade (Buehler GmbH, Düsseldorf, Germany), and then 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 down from 6 to 1 μm) to obtain smooth surfaces. 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 (UT) control area which was protected against acid solution exposure.
2.2
Test solutions and layer building
The erosive solutions were prepared as described previously . Three different polymer solutions were prepared containing propylene glycol alginate (PGA), highly esterified pectin (HP) and gum arabic (GA) (1%, w/w), respectively. To obtain the polymer modified citric acid solutions (PMCASs) the pH of the polymer solutions was adjusted by addition of citric acid (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) to pH = 3.3 (Knick pH meter 765 Calimatic, Knick Elektronische Messgeräte GmbH & Co. KG, Berlin, Germany). As control, a non-polymer modified citric acid solution (CAS) with the same pH was used. For erosive treatment the enamel samples were placed in a glass beaker with approx. 50 mL of each acid solution under constant stirring. For each solution, 3 samples were immersed for 30, 60 and 120 s, respectively. The erosive treatment was stopped by removal and rinsing the enamel samples with deionized water for 20 s. Then the samples were dried with compressed air and the PVC-tape was removed carefully.
2.3
Profilometry and atomic force microscopy (AFM)
Directly after the PMCAS or CAS treatment of the enamel samples, a profilometer (Taylor Hobson FormTalysurf Series 2, Taylor Hobson, Leicester, England) was used to make scratches in the polymer layers on the enamel and subsequently the scratch depths were measured with AFM. The scratches (line scans) with a length of approximately 4 mm were created in the direction from the reference surface area to the eroded surface area. The cone shaped diamond tip had a tip radius of 2.00 μm. The force applied via the tip to the enamel surface was 0.87 mN .
Topographic images (image size 25 μm × 25 μm) of the profilometer scratches were recorded with an atomic force microscope (AFM, Digital Instruments Dimension 3100, Veeco Instruments, Santa Barbara, CA, USA) operating in tapping mode and using silicon cantilevers (OMCL AC160TS-W, Olympus, Tokyo, Japan). The spring constant of the cantilevers ranged between 20 M/m and 100 M/m and their length was 125 μm with a resonance frequency of 200–300 kHz. The AFM was used to measure the depth of the surface scratches caused by the profilometer . It was necessary to capture the AFM images in tapping mode to avoid damaging the softened enamel or the polymer layer. Raw AFM images were subjected to first order flattening prior to further analysis. Image processing was performed with Gwyddion 2.21 free SPM data analysis software ( http://gwyddion.net/ ). With the software (negative U-profile over the scratch) 9 line profiles with a line profile distance of 2.5 μm were used to determine the mean scratch depth.
2.4
X-ray photoelectron spectroscopy (XPS)
To investigate the atomic composition of the layer deposited on the enamel surfaces, the energy of the binding electrons was determined by angle dependent X-ray photoelectron spectroscopy with a Quantum 2000 (PHI Co., Chanhassen, MN, USA) and a focused monochromatic Al Kα source (1486.7 eV) for excitation. The pass energy was 23.5 eV and the reference was the C1s peak of the C C bond. The signal at 346.2 349.7 eV represents the Ca electrons of the 2p orbital. By increasing the angle of the focused X-ray beam from 0° (horizontal to the enamel sample surface) to 90° (perpendicular to the enamel sample surface) the penetration depth of the X-ray beam increased. This gave the opportunity to measure the atomic composition of the polymer layers depending on the penetration depth of the focused X-ray beam.
2.5
Zeta-potential measurements
To characterize the surface charge (zeta-potential) of the polymer molecules and the HA particles (Merck KGaK, Darmstadt, Germany) a Zetasizer Nano-ZS (Malvern Instruments GmbH, Herrenberg, Germany) was used. The zeta-potential was measured over a pH range from 2 to 11.
2.6
Scanning electron microscopy (SEM)
SEM was performed with a LEO 440i SEM Scanning Electron Microscope (LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) operated at 15 kV, working distance 6–8 mm. For these investigations, the polymer solutions were prepared with 1% (wt/wt) polymer and 1% (wt/wt) HA particles (Merck KGaK, Darmstadt, Germany; also characterized by SEM measurements) in deionized water. The pH was adjusted with citric acid to pH 3.3 and the solutions were freeze dried for 48 h. For SEM imaging, samples were gold sputter-coated to approximately 10 nm thickness under vacuum (Edwards High Vacuum International, Crawley, West Sussex, UK).
2
Materials and methods
2.1
Sample preparation
For this in vitro study, non-erupted human third molars ( n = 12) were used and prepared as described previously . The teeth were extracted and disinfected, and the roots were carefully removed. Pieces (approx. 2 mm × 3 mm) were cut from the tooth crowns with a low-speed saw (Isomet, Buehler GmbH, Düsseldorf, Germany) using a water-cooled diamond blade (Buehler GmbH, Düsseldorf, Germany), and then 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 down from 6 to 1 μm) to obtain smooth surfaces. 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 (UT) control area which was protected against acid solution exposure.
2.2
Test solutions and layer building
The erosive solutions were prepared as described previously . Three different polymer solutions were prepared containing propylene glycol alginate (PGA), highly esterified pectin (HP) and gum arabic (GA) (1%, w/w), respectively. To obtain the polymer modified citric acid solutions (PMCASs) the pH of the polymer solutions was adjusted by addition of citric acid (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) to pH = 3.3 (Knick pH meter 765 Calimatic, Knick Elektronische Messgeräte GmbH & Co. KG, Berlin, Germany). As control, a non-polymer modified citric acid solution (CAS) with the same pH was used. For erosive treatment the enamel samples were placed in a glass beaker with approx. 50 mL of each acid solution under constant stirring. For each solution, 3 samples were immersed for 30, 60 and 120 s, respectively. The erosive treatment was stopped by removal and rinsing the enamel samples with deionized water for 20 s. Then the samples were dried with compressed air and the PVC-tape was removed carefully.
2.3
Profilometry and atomic force microscopy (AFM)
Directly after the PMCAS or CAS treatment of the enamel samples, a profilometer (Taylor Hobson FormTalysurf Series 2, Taylor Hobson, Leicester, England) was used to make scratches in the polymer layers on the enamel and subsequently the scratch depths were measured with AFM. The scratches (line scans) with a length of approximately 4 mm were created in the direction from the reference surface area to the eroded surface area. The cone shaped diamond tip had a tip radius of 2.00 μm. The force applied via the tip to the enamel surface was 0.87 mN .
Topographic images (image size 25 μm × 25 μm) of the profilometer scratches were recorded with an atomic force microscope (AFM, Digital Instruments Dimension 3100, Veeco Instruments, Santa Barbara, CA, USA) operating in tapping mode and using silicon cantilevers (OMCL AC160TS-W, Olympus, Tokyo, Japan). The spring constant of the cantilevers ranged between 20 M/m and 100 M/m and their length was 125 μm with a resonance frequency of 200–300 kHz. The AFM was used to measure the depth of the surface scratches caused by the profilometer . It was necessary to capture the AFM images in tapping mode to avoid damaging the softened enamel or the polymer layer. Raw AFM images were subjected to first order flattening prior to further analysis. Image processing was performed with Gwyddion 2.21 free SPM data analysis software ( http://gwyddion.net/ ). With the software (negative U-profile over the scratch) 9 line profiles with a line profile distance of 2.5 μm were used to determine the mean scratch depth.
2.4
X-ray photoelectron spectroscopy (XPS)
To investigate the atomic composition of the layer deposited on the enamel surfaces, the energy of the binding electrons was determined by angle dependent X-ray photoelectron spectroscopy with a Quantum 2000 (PHI Co., Chanhassen, MN, USA) and a focused monochromatic Al Kα source (1486.7 eV) for excitation. The pass energy was 23.5 eV and the reference was the C1s peak of the C C bond. The signal at 346.2 349.7 eV represents the Ca electrons of the 2p orbital. By increasing the angle of the focused X-ray beam from 0° (horizontal to the enamel sample surface) to 90° (perpendicular to the enamel sample surface) the penetration depth of the X-ray beam increased. This gave the opportunity to measure the atomic composition of the polymer layers depending on the penetration depth of the focused X-ray beam.
2.5
Zeta-potential measurements
To characterize the surface charge (zeta-potential) of the polymer molecules and the HA particles (Merck KGaK, Darmstadt, Germany) a Zetasizer Nano-ZS (Malvern Instruments GmbH, Herrenberg, Germany) was used. The zeta-potential was measured over a pH range from 2 to 11.
2.6
Scanning electron microscopy (SEM)
SEM was performed with a LEO 440i SEM Scanning Electron Microscope (LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) operated at 15 kV, working distance 6–8 mm. For these investigations, the polymer solutions were prepared with 1% (wt/wt) polymer and 1% (wt/wt) HA particles (Merck KGaK, Darmstadt, Germany; also characterized by SEM measurements) in deionized water. The pH was adjusted with citric acid to pH 3.3 and the solutions were freeze dried for 48 h. For SEM imaging, samples were gold sputter-coated to approximately 10 nm thickness under vacuum (Edwards High Vacuum International, Crawley, West Sussex, UK).
3
Results
The roughnesses (Ra and Rms) of the surfaces were measured by the AFM probe and are given in Table 1 . For CAS treated enamel samples the roughness values increased with the treatment time from 6.6 to 68.4. In comparison, the roughness values of the PMCAS treated enamel surfaces increases not or only slightly with increasing treatment time. It is known that profilometer measurements can cause scratches on eroded enamel surfaces . A typical light microscopy micrograph of a profilometer-caused scratch on an eroded polymer-coated dental enamel surface, obtained in our study, is shown in Fig. 1 . All samples showed scratches caused by the profilometer. The scratches were investigated with AFM ( Fig. 2 ) to determine the scratch depth. Enamel samples treated with PMCAS showed smooth and less eroded surfaces ( Fig. 2 ) compared to samples treated with pure CAS without polymer addition where strong erosion effects were recorded with AFM ( Fig. 2 ).
| Treatment time | CAS | PGA | HP | GA |
|---|---|---|---|---|
| Ra (nm) | ||||
| 30 s | 6.6 ± 0.5 | 1.9 ± 0.1 | 2.1 ± 0.0 | 1.7 ± 0.1 |
| 60 s | 7.6 ± 0.8 | 1.4 ± 0.1 | 1.9 ± 0.0 | 1.9 ± 0.1 |
| 120 s | 52.1 ± 4.2 | 1.9 ± 0.1 | 2.0 ± 0.3 | 2.3 ± 0.2 |
| Rms (nm) | ||||
| 30 s | 9.0 ± 0.8 | 2.8 ± 0.2 | 3.5 ± 0.0 | 2.8 ± 0.6 |
| 60 s | 11.4 ± 0.8 | 2.1 ± 0.1 | 3.7 ± 0.1 | 2.6 ± 0.2 |
| 120 s | 68.4 ± 6.1 | 3.2 ± 0.8 | 2.7 ± 0.4 | 3.1 ± 0.2 |
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