8840
J. Phys. Chem. B 2008, 112, 8840–8848
New Insights into Structural Alteration of Enamel Apatite Induced by Citric Acid and Sodium Fluoride Solutions Xiaojie Wang,† Arndt Klocke,‡ Boriana Mihailova,*,† Lubomira Tosheva,§ and Ulrich Bismayer† Institute of Mineralogy and Petrology, Department of Earth Sciences, UniVersity of Hamburg, Grindelallee 48, D-20146 Hamburg, Germany, Department of Orthodontics, College of Dentistry, UniVersity Hospital Eppendorf, D-20246 Hamburg, Germany, and DiVision of Chemistry and Materials, Manchester Metropolitan UniVersity, Chester Street, Manchester, M1 5GD, U.K. ReceiVed: March 21, 2008; ReVised Manuscript ReceiVed: May 5, 2008
Attenuated total reflectance infrared spectroscopy and complementary scanning electron microscopy were applied to analyze the surface structure of enamel apatite exposed to citric acid and to investigate the protective potential of fluorine-containing reagents against citric acid-induced erosion. Enamel and, for comparison, geological hydroxylapatite samples were treated with aqueous solutions of citric acid and sodium fluoride of different concentrations, ranging from 0.01 to 0.5 mol/L for citric acid solutions and from 0.5 to 2.0% for fluoride solutions. The two solutions were applied either simultaneously or consecutively. The citric acidinduced structural modification of apatite increases with the increase in the citric acid concentration and the number of treatments. The application of sodium fluoride alone does not suppress the atomic level changes in apatite exposed to acidic agents. The addition of sodium fluoride to citric acid solutions leads to formation of surface CaF2 and considerably reduces the changes in the apatite P-O-Ca framework. However, the CaF2 globules deposited on the enamel surface seem to be insufficient to prevent the alteration of the apatite structure upon further exposure to acidic agents. No evidence for fluorine-induced recovery of the apatite structure was found. Introduction Dental erosion is the destruction of dental hard tissues caused by acids without the involvement of microorganisms.1 The acid may be of endogenous (from within the body) or exogenous (from outside the body) origin. Compared to the process of caries development, enamel erosion involves a more widespread and rapid dissolution and leads to progressive loss of the enamel surface over a long period of time.2,3 It has been suggested that dental erosion is largely irreversible and cannot be recalcified because there is no suitable matrix for crystal growth.4 Enamel erosion caused by exogenic acids originating from acidic food or beverages has been receiving more and more attention in recent years because of the increased consumption of such drinks and food.5 The enamel erosion caused by citric acid has been of particular interest because citric acid is contained in a variety of fruits and juices.6 Citric acid is also widely used to add a sour taste to food and soft drinks. Citric acid is potentially hazardous to dental enamel for two reasons. First, citric acid is a tribasic carboxylic acid and may dissolve enamel by the following reaction with hydroxylapatite: Ca10(PO4)6(OH)2 + 2H+ T 10Ca2+ + 6PO43- + 2H2O.7 Second, the citrate ion (C6H5O7)3- (cit) is a calcium-chelating ligand and can form a soluble Ca-cit complex, promoting further dissolution.8,9 The erosive potential of citric acid related to dental hard tissues has been proven by methods such as surface profilometry, microhardness testing, and scanning * Corresponding author. Telephone: +4940 42838 2052. Fax: +4940 42838 2422. E-mail:
[email protected]. † University of Hamburg. ‡ University Hospital Eppendorf. § Manchester Metropolitan University.
electron microscopy (SEM), which revealed enamel softening and loss after treatment with aqueous solutions and beverages containing citric acid.10–13 The profound effect of fluoride on reducing the incidence of caries is well documented.14 It is suggested that when a low concentration of fluoride is applied to dental enamel, F- ions can replace OH- ions in hydroxylapatite crystals, and such hybrid crystals are sometimes referred to as fluorhydroxylapatite (Ca10(PO4)6(F,OH)2),15,16 which is more resistant to acidic challenge than hydroxylapatite.17 When a high concentration of fluoride is used, a CaF2-like layer is formed on the enamel surface that may serve as a reservoir of fluorine inducing the reprecipitation of mineral in the form of fluorapatite or fluorhydroxylapatite interfering with further demineralization.14 However, a consensus about the effectiveness of fluoride in reducing the formation of erosive lesion in enamel has not yet been reached.18–24 It has been stated that moderate fluoride concentrations such as those commonly used in toothpastes cannot prevent enamel erosion due to acidic challenges.20,21 On the other hand, topical solutions and gels with high fluoride concentration might have a protective effect, especially in combination with moderate acidic pH values and extended exposure time due to the enhancement of CaF2 formation on the enamel surface.22–24 Hence, it was suggested that acidulated fluoride agents may be more effective in preventing erosion as compared to gels of neutral pH because of the formation of a denser and thicker CaF2 layer.24 It is thought that moderate acidic pH values slightly accelerate the dissolution of enamel apatite, which increases the Ca concentration in the solution near the surface and facilitates the deposition of CaF2 globules:24,25
10.1021/jp802492d CCC: $40.75 2008 American Chemical Society Published on Web 06/28/2008
Enamel Apatite: Citric Acid and Fluoride Exposure
J. Phys. Chem. B, Vol. 112, No. 29, 2008 8841
Ca5(PO4)3OH + 7H+ f 5Ca2++3H2PO4- + H2O,
against acid-induced erosion. In addition, the impact of citric acid-based agents on enamel apatite was compared with that on single-crystal hydroxylapatite of geological origin.
Ca2+ + 2F- f CaF2 (1) In addition, artificially eroded enamel is believed to retain more fluoride as compared to sound enamel because of the increased surface area, which offers a higher number of possible retention sites for the fluoride;23,26 hence, the increased amount of adsorbed fluoride should enhance the formation of CaF2like material. While the effect of citric acid and fluorine-containing agents on dental enamel morphology and hardness has been described in detail, there is limited information on the underlying ultrastructural, atomic level changes. The technique of attenuated total reflectance infrared (ATR IR) spectroscopy has recently been demonstrated as a nondestructive and easy handling surface-sensitive method for studying biomaterials.27–30 A great advantage of ATR IR spectroscopy as an analytical method for dental material research is its ability to probe the structure of outermost layers of the tooth tissue within micrometer and submicrometer thickness. Hence, ATR IR spectroscopy permits repeated analyses of the sample surface at sequential stages of chemically induced transitions. Thus, it seems a very suitable method for determination of time-lapsed chemical changes in enamel surfaces exposed to agents that may induce demineralization/remineralization processes. A previous study that combined different analytical methods including ATR IR spectroscopy and Ca leaching analysis revealed that the position of the major ATR IR peak near 1010 cm-1 is indicative for acid-induced alteration of the atomic bonding in superficial enamel apatite.27 This ATR IR absorption peak arises from the antisymmetrical ν3(PO4) mode of apatite, and the peak shift to higher wavenumbers points to occurrence of violated P-O-Ca linkages in enamel apatite (i.e., to changes in the atomic level structure of the mineral component of enamel).27 The treatment with agents containing citric acid loosens and breaks a part of the Ca-O bonds in apatite, which leads to stiffening of the adjacent P-O bonds and thus to a shift of the major absorption peak to higher wavenumbers.27 The aim of the present study is to analyze the local structure and stability of Ca-O-P atomic linkages in enamel apatite subjected to citric acid by applying ATR IR spectroscopy and complementary SEM imaging as well as to investigate the role of fluorine-containing reagents in preserving the apatite structure
Experimental Section Sample Preparation. Noncarious human molars were collected and stored in distilled water at room temperature. The soft tissue of teeth was cleaned, and the root was discarded with a water-cooled high-speed hand piece. Slabs of size ∼3 × 3 × 1 mm3 were prepared from the middle one-third of the enamel surface using a diamond wire saw. The slabs were near parallel to the enamel-dentin junction function (i.e., approximately perpendicular to the sixfold symmetrical axis of hydroxylapatite). The outer part of the enamel slabs was further polished with silicon carbide papers of 1200-grit size and then with a 0.1-µm-sized colloidal silica suspension under continuous water cooling. Each slab was finally divided into several segments to be treated with the same type of agent but under various conditions (exposure time, concentration, etc.). Such a sample design avoids possible data misinterpretation coming from the tooth-to-tooth variability. The enamel samples were stored in distilled water before chemical treatment. One-sided polished samples of geological hydroxylapatite were cut from a monolithic single crystal. The cuts were also perpendicular to the sixfold axis of symmetry and sized ∼5 × 5 × 2 mm3. We used an original mineral specimen from Snarum, Buskerud, Norway, which is a traditional location for hydroxylapatite. The crystal structure and chemistry were verified by X-ray diffraction, Raman scattering, and infrared transmittance spectroscopy. Sample Treatment. Four different series of enamel apatite were considered: (i) treated with aqueous solutions of citric acid of different concentrations, (ii) treated first with a 0.1 mol/L citric acid solution and then with aqueous solutions of sodium fluoride of different concentrations, (iii) treated first with aqueous solutions of sodium fluoride of different concentrations and then with 0.1 mol/L citric acid solution, and (iv) treated with aqueous solutions of both citric acid and NaF. The exact concentrations and the pH values of the corresponding solutions are given in Table 1. Aqueous solutions of citric acid were prepared by dissolving anhydrous citric acid (Sigma-Aldrich) in distilled water. Aqueous solutions of sodium fluoride were prepared by mixing powdered NaF (Merck) and distilled water.
TABLE 1: Characteristics of the Aqueous Solutions Used for Enamel Treatment citric acid concentration (mol/L)
sodium fluoride
pH
concentration (%) pH
mixture of citric acid and sodium fluoride concentration of citric acid (mol/L)
concentration of NaF (%)
0.1 0.1 0.1
0.5 1.0 2.0
pH
Series 1 0.01 0.02 0.05 0.1 0.5
2.82 2.54 2.43 2.23 1.89
0.1 0.1 0.1
2.23 2.23 2.23
Series 2 and 3a 0.5 1.0 2.0
9.53 10.05 10.30
Series 4
a
4.15 4.88 5.24
Series 2 and 3 differ from each other in the order of treatment with different reagents. Series 2: first citric acid-containing solution and then NaF-containing solution. Series 3: first NaF-containing solution and then citric acid-containing solution.
8842 J. Phys. Chem. B, Vol. 112, No. 29, 2008 Each citric acid-NaF admixture solution was prepared by mixing 0.1 mol/L citric acid solution and NaF solutions of corresponding concentration. To analyze the sole effect of citric acid, the samples of series 1 were exposed to 10 mL of the corresponding citric acid solution for 10 min and then rinsed with distilled water. The chemical treatment was repeated many times, and ATR IR spectra were measured after 1, 2, 4, 6, 8, 10, 14, and 20 times of treatment. To check if fluorine alone leads to structural recovery of enamel exposed to citric acid, samples of series 2 were subjected to a multiple treatment consisting of repeated cycles of immersion of the sample into 10 mL of solution of citric acid for 10 min and subsequent rinsing with distilled water, and then immersion of the sample into 10 mL of NaF solution for 10 min and subsequent rinsing with distilled water. The concentration of citric acid was chosen to be 0.1 mol/L because the study of series 1 showed that such a concentration heavily affects the enamel surface even after a single treatment. ATR IR spectra were measured after the treatment with citric acid and after the treatment with NaF. After the last step of citric acid treatment, the immersion into NaF solution was prolonged to 48 h to ensure the detection of any structural recovery processes if such occur. To explore the ability of fluorine itself to protect the enamel from erosion induced by citric acid, samples of series 3 were prepared similarly to the samples in series 2, but the order of treatments was NaF and then citric acid. ATR IR spectra were measured after the treatment with NaF and after the treatment with citric acid. Additionally, to ensure the detection of any protective effects due to fluorine alone, enamel samples were immersed first in NaF solutions for 60 h and then treated with 0.1 mol/L citric acid solution. Finally, to investigate the effect of reagents containing both fluorine and citric acid, samples of series 4 were exposed to a multiple treatment for 10 min in 10 mL of admixture solutions of citric acid and NaF and then rinsed with distilled water. The pH values of the used admixture solutions correspond to mild acidic conditions (pH ≈ 4.2-5.2), which according to previous studies31,32 are favorable for the formation of CaF2 globules on the enamel surface. ATR IR spectra were measured after 1, 2, 4, 6, 8, 10, and 14 steps of treatment. In addition, to check the ability of the deposited CaF2 globules to protect the structure of enamel apatite from further acidic impact, we exposed the sample treated for 14 × 10 min with 0.1 mol/L citric acid + 0.5% NaF solution to a solution of 0.1 mol/L citric acid for 10 min. All the immersion processes were performed at room temperature in static liquids. For all samples, the time between two steps of acidic treatment was approximately 12 h. The samples were kept in distilled water between the treatments. The samples of the same series were taken from the same tooth; however, four different teeth were used to prepare the corresponding samples from series 1, 2, 3, and 4. Thus, samples within the same series are fully comparable, but weak spectral changes from one series to another might also be due to variation from one tooth to another. Analogous treatments with citric acid- and NaF-containing solutions were carried out on a series of samples of geological hydroxylapatite. Sample Characterization. IR Spectroscopy. The structure of the sample surface was examined by ATR IR spectroscopy. The ATR IR spectra were measured with a Bruker Equinox 55 FTIR spectrometer, using a Pike MIRacle ATR accessory with and a contact sample area of 1.8 mm in diameter. The spectra
Wang et al.
Figure 1. ATR IR spectra of enamel apatite single step treated with 0.1 and 0.5 mol/L citric acid solutions for 10 min.
were recorded with an instrumental resolution of 4 cm-1, averaging over 512 scans. The error in determining the peak positions was (2 cm-1. We used a Ge ATR crystal, and consequently, the characteristic penetration depth of our ATR IR experiments was approximately 700 nm. Preliminary experiments with a ZnSe ATR crystal, which has a smaller refractive index than Ge, and, hence, a longer penetration depth, revealed that the use of Ge as a refractive element ensures the collection of ATR spectra with negligible contribution of specular reflection. Thus, the ATR IR spectra presented here possess no heavily modified peaks due to the dispersion of the refractive index across an absorption band and/or due to a strong dependence of the effective thickness. The spectra were measured in the spectral range 570-4000 cm-1 and subsequently normalized to a constant penetration depth. SEM Imaging. The morphology of the treated samples as well as the formation of CaF2 globules was checked by SEM imaging. The micrographs were recorded with a JEOL 5600LV scanning electron microscope operating at 12 kV and using different magnifications up to 20 000×. Before SEM measurements, the surfaces of the examined samples were coated with gold. Results We focused our spectroscopic analysis on the phonon modes of the apatite P-O-Ca framework and in particular on the AIR IR peak near 1010 cm-1, whose shift to higher wavenumbers is indicative for occurrence of affected P-O-Ca atomic linkages.27 Effect of Citric Acid on Enamel. For aqueous solutions of citric acid with concentrations equal to or higher than 0.1 mol/ L, a peak shift and, therefore, alteration of atomic bonding in superficial enamel apatite are observed even after a single treatment for 10 min (Figure 1). The repetition of the citric acid treatment did not lead to further spectral changes, which indicates that the concentration of acidic molecules was high enough to affect the apatite atomic bonding in the whole available surface during the single treatment of 10 min. The use of lower citric acid concentrations (
