Anna
Kovtun
a,
Diana
Kozlova
a,
Kathirvel
Ganesan
a,
Caroline
Biewald
b,
Nadine
Seipold
b,
Peter
Gaengler
b,
Wolfgang H.
Arnold
b and
Matthias
Epple
a
aInstitute of Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Universitaetsstrasse 5-7, 45117, Essen, Germany. E-mail: matthias.epple@uni-due.de
bFaculty of Health, School of Dentistry, University of Witten/Herdecke, Alfred-Herrhausen-Strasse 50, 58448, Witten, Germany
First published on 22nd November 2011
One of the main problems in dental medicine is the growth of bacterial biofilms on tooth surfaces which cause caries and periodontitis. We have developed a new system for oral hygiene and dental treatment that consists of either a paste or a rinsing solution containing calcium phosphate nanoparticles, functionalized with the antibacterial agent chlorhexidine. As calcium phosphate is the natural component of tooth mineral, it can lead to the remineralization of damaged enamel, while chlorhexidine prevents the colonization of the tooth surface by bacteria. In the form of a paste, a bifunctional system with both mineralizing and antibacterial properties is obtained. The nanoparticles may also stick to open dentin tubules at the root surface due to their coating with carboxymethyl cellulose. In vitro studies on teeth show that the paste sticks well to the root surface and closes dentin tubules.
Chlorhexidine (1) is very common in dental medicine as an antibacterial agent against gram-positive and gram-negative bacteria and fungi.19 It is used to treat periodontal diseases20 in the form of pastes, sprays or mouth rinses.21–23. However, chlorhexidine is toxic at high concentrations for eukaryotic cells because of its interaction with membrane proteins, e.g.Na+-K+-ATPase.24 Babich et al. showed a toxic effect of chlorhexidine at high concentrations on fibroblasts after 1, 24 and 72 h.25
Here, we have loaded calcium phosphate nanoparticles with chlorhexidine to combine mineralization ability and antibacterial effect. To achieve an optimal loading with chlorhexidine and to enhance the adhesion properties on the tooth surface, the nanoparticles must be modified by adhesive polymers to make them more sticky. For this, we used carboxymethyl cellulose (CMC). This leads to a good adhesion on dental surfaces, remineralization, and occlusion of dentin tubules and bactericidal effect. The nanoparticles can be applied either as dispersion (“mouth rinse”) or as paste.
The resulting dispersion was centrifuged at 2540 g (z300 instrument from Hermle Labortechnik GmbH, Wehingen, Germany) to remove the counter ions and a possible excess of organic additives, then redispersed in ultrapure water and centrifuged again. The obtained wet product had the consistency of a paste (i.e. a highly viscous, toothpaste-like material) and a content of solid of 22 ± 4 wt% as measured by thermogravimetry. The preparation of the nanoparticulate paste is schematically shown in Fig. 1.
Fig. 1 Schematic diagram of the preparation of a paste of CaP-CMC-CHX nanoparticles. Y1, Y2 and Y3 are tube connectors. The distances between two connections were: Y1 to Y2 = 10 cm; Y2 to Y3 = 100 cm. |
The release of chlorhexidine from the nanoparticles was studied by dissolution experiments. 1 g of the nanoparticulate paste (i.e. including 78 wt% of water) was redispersed in 200 mL of artificial saliva (83 mL H2O, 15 mL KCl (1 M), 1 mL CaCl2 (150 mM), 1 mL KH2PO4 (90 mM) in 100 mL solution, pH 7) and stirred for 0, 1, 2 or 3 days. Then the particles were collected by centrifugation (2540 g) and subjected to thermogravimetric and elemental analysis.
For chemical analysis, the paste was dried in air at room temperature for 24 h. The resulting particles had a spherical morphology, a diameter around 150–200 nm, and a negative zeta potential (−37 ± 6 mV) due to the outer shell of CMC, whereas unfunctionalized calcium phosphate particles had a zeta potential around zero.26Fig. 2 shows a representative scanning electron micrograph. The particles were stable as a colloidal dispersion in water for only about an hour before sedimentation occurred, a fact that points to ongoing agglomeration of the primary nanoparticles. However, for the projected application as tooth remineralisation material, an application as a paste is convenient, and therefore we prepared the particles in the form of a paste by partial removal of water by centrifugation (content of solid about 20–25 wt%). During the application, agglomerates will be redispersed during the mechanical action of a toothbrush or a polishing cup.
Fig. 2 Scanning electron micrograph of the primary CaP-CMC-CHX nanoparticles. |
The IR spectra of the nanoparticles showed vibration bands of both CHX and CMC (Fig. 3). The adsorbed water molecules gave a weak absorption band around 1645 cm−1 (bending) and a broad absorption band around 3400 cm−1 (stretching vibration). The weak C–O bending vibration of carbonate group was observed at 1385 cm−1. The characteristic vibration bands of hydroxyapatite (1038, 604, and 565 cm−1) were clearly observed for the functionalized nanoparticles.
Fig. 3 IR data of pure CHX, pure CMC, and air-dried CaP-CMC-CHX nanoparticles (CaP). |
X-Ray powder diffraction data of calcium phosphate-CMC-CHX nanoparticles are shown in Fig. 4. The broad diffraction peaks show that the calcium phosphate nanoparticles consist of poorly crystalline hydroxyapatite in accordance to literature spectra.27
Fig. 4 X-Ray powder diffractogram of air-dried CaP-CMC-CHX nanoparticles. |
The composition of the nanoparticles was determined by thermogravimetric (Fig. 5) and elemental analysis. The first decomposition step of 5.7 wt% corresponds to the loss of residual water. The second and third steps (13.4 wt% and 12.9 wt%, respectively) correspond to the combustion of organic components (CMC and CHX). The fourth step (1.3 wt%) corresponds to the loss of CO2 from carbonated apatite. Note that the given numbers for the mass loss are not exact as the steps are overlapping. They were selected from visual inspection of the TG curve, taking into account the inflection points of the TG curve.
Fig. 5 Thermogravimetric analysis of the air-dried CaP-CMC-CHX nanoparticles. |
Table 1 shows the elemental analysis data of CaP-CMC-CHX nanoparticles. The nanoparticles contained about 48.6 ± 4.1 wt% of calcium phosphate (sum of calcium and phosphate; note that the exact stoichiometry of the calcium phosphate phase is unknown). The content of chlorhexidine was calculated from the nitrogen content to 13.1 ± 2.2 wt% according to eqn 1 with M(CHX) = 505.46 g mol−1 (C22H30Cl2N10):
(1) |
C/wt% | H/wt% | N/wt% | Ca/wt% | PO43−/wt% | Chlorhexidine/wt% (calculated) | Calcium phosphate/wt% (calculated) | Ca/PO43− molar ratio |
---|---|---|---|---|---|---|---|
18.5 ± 4.4 | 3.3 ± 0.8 | 3.4 ± 0.4 | 19.9 ± 2.9 | 28.7 ± 2.9 | 13.1 ± 2.2 | 48.6 ± 4.1 | 1.56 |
The content of chlorhexidine of 13.1 wt% in the dried particles corresponds to about 3 wt% in the wet paste which contains 78 ± 4 wt% water. This loading is high enough to achieve the desired bactericidal effect. According to Mohammadi et al., a bacteriostatic effect is obtained at a chlorhexidine concentration of 0.2% and a bacteriotoxic effect occurs at 2% of chlorhexidine.28 However, whereas in mouth rinses chlorhexidine is used at low concentrations of 0.1–0.2%, in tooth varnishes much higher concentrations are used, up to 40% without being toxic for humans.29
We determined the release kinetics of chlorhexidine and the dissolution of the particles under physiological conditions by dispersion of the paste in artificial saliva for 3 days and then subjected it to thermogravimetry and elemental analysis (Fig. 6 and Table 2). The particles released the chlorhexidine almost completely during the first day. The nanoparticles still contained carbon which indicates the presence of CMC; however, the amount of calcium phosphate increased from 48.6 to 72.0 wt% in two days. That leads to the conclusion that the nanoparticles will release the chlorhexidine during the first day in a clinical application, but will still attach to the tooth surface due to the presence of the sticky CMC. This will lead to the occlusion of dentin tubules and the remineralization of eroded teeth.30
Fig. 6 Thermogravimetry of CaP-CMC-CHX nanoparticles after dispersion for several days. |
Time/days | C/wt% | H/wt% | N/wt% | Ca/wt% | Chlorhexidine/wt% (calculated) | Calcium phosphate/wt% (calculated) |
---|---|---|---|---|---|---|
0 | 18.5 | 3.3 | 3.4 | 19.9 | 13.1 | 48.6 |
1 | 7.2 | 1.6 | 0.7 | 26.9 | 2.4 | 67.0 |
2 | 5.1 | 1.2 | 0.0 | 28.9 | 0.0 | 72.0 |
3 | 5.7 | 1.5 | 0.0 | 28.9 | 0.0 | 72.0 |
To follow the nanoparticles after application in the mouth, we additionally loaded them with fluorescein (40.97 mg L−1) which was added during the synthesis to the CMC solution. The addition of fluorescein had no significant influence on the morphology or size of the particles. The fluorescence spectrum of the fluorescein-loaded particles is shown in Fig. 7. At the excitation wavelength of 306 nm, the isolated dried powder showed a strong fluorescence at 551 nm (yellow-green colour).
Fig. 7 Fluorescence emission spectrum of solid fluorescein-labelled air-dried CaP-CMC-CHX nanoparticles. |
The application of the fluorescein-labelled paste to teeth demonstrated that the nanoparticles stuck well onto the tooth surface, especially at the cervical and proximal areas (Fig. 8).
Fig. 8 Photograph of the fluorescein-labelled nanoparticulate paste on the tooth surface, 5 and 30 min after application. The green fluorescence indicates adsorbed nanoparticles. The paste was predominantly located at the cervical and proximal tooth surface. After 30 min it was still visible. |
We tested the antimicrobial activity of the nanoparticles at different concentrations of CaP-CMC-CHX paste against bacteria (Fig. 9). For this test, the paste was diluted to 2 wt% to obtain a dispersion by ultrasonication for 10 min. After further dilution by factors 10, 102, 103, 104 times, respectively, in ultrapure water, these dispersions were tested on the gram-negative strain Escherichia coli and the gram-positive strain Lactobacillus casei. The results showed an efficient inhibition of bacterial growth even after a 1:100 dilution, in good agreement with the literature data.28 However, more extensive studies are required to prove the activity against bacterial colonies (biofilms) occurring in the mouth.31
Fig. 9 Effect of 10 μL of CaP-CMC-CHX nanoparticles at different concentrations against Escherichia coli and Lactobacillus casei. The original dispersion of 2 wt% nanoparticles present in dispersion was diluted by factors of 10 (1), 102 (2), 103 (3), 104 (4). (5) shows a control experiment without chlorhexidine. |
In vitro experiments on human teeth showed the adsorption of the particles on the dental surfaces (Fig. 10 to 12). All control windows on enamel and root dentin surfaces showed the typical erosion patterns, i.e. prismatic demineralization of enamel, and open, empty dentin tubules.32,33 After gentle polishing; the treated windows showed well-adsorbed layers of CaP-CMC-CHX nanoparticles which were completely covering erosive niches, the prismatic demineralization of enamel as well as dentin tubule entrances. These features of surface SEM were confirmed by SEM of fractured samples (Fig. 13).
Fig. 10 Scanning electron micrograph of nanoparticle-treated enamel. Left: Overview of the surface. The nanoparticulate paste completely covers the surface. Right: Fractured sample with a dense cover of nanoparticles on the enamel surface. |
Fig. 11 Scanning electron micrograph of the eroded enamel surface of a control sample (left) and of an equivalent sample in which the surface was completely covered by the nanoparticulate paste after the application (right). |
Fig. 12 Scanning electron micrograph showing the eroded root surface of a control sample with open dentin tubules (left) and the root surface of an equivalent sample that was completely covered by the nanoparticles after application (right). |
Fig. 13 Scanning electron micrograph of the fractured window of a control sample and experimental sample. In the control window, the demineralized surface can be seen as a dark surface layer. In the experimental sample, the demineralized surface layer is covered by the nanoparticles. Small tags can be seen at the openings of the dentin tubules. |
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