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RESEARCH REPORT |
Department of Biomedical Engineering, University Medical Centre Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
* corresponding author, C.G.van.Hoogmoed{at}med.rug.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: enamel • pellicle • biosurfactant • microbial adhesion forces • atomic force microscopy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The aim of the present work was, first, to measure the interactive forces between enamel with and without a salivary pelicle and S. sobrinus HG 1025 and S. mutans ATCC 25175 and, second, to determine whether S. mitis biosurfactant affected the interactive forces between the two mutans streptococcal strains and the enamel.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The mutans streptococcal pre-cultures were used to inoculate second 10-mL cultures, which were grown for 16 hrs. Subsequently, these suspensions were centrifuged at 4000 g, washed twice with adhesion buffer (2 mmol/L potassium phosphate, 50 mmol/L potassium chloride, 1 mmol/L calcium chloride; pH 6.8), and re-suspended in adhesion buffer to a concentration of 105 cells per mL. We used the S. mitis BMS pre-culture to inoculate a 1400-mL culture. After 18 hrs of growth, cells were harvested by centrifugation at 4000 g, washed twice with adhesion buffer, and re-suspended in 200 mL demineralized water.
Crude biosurfactant was produced by gentle stirring of the S. mitis BMS suspension for 2 hrs at room temperature. Subsequently, the organisms and the biosurfactant released were separated by centrifugation at 10,000 g. To ensure complete removal of all bacteria, we centrifuged the supernatant twice at 10,000 g. The crude biosurfactant was re-suspended in demineralized water and purified by acid precipitation with concentrated HCl down to pH 2.0. After the supernatant was decanted, the precipitate was washed twice with acidic water (pH 2) and collected by centrifugation at 4000 g. After being redissolved in water, the acid precipitate was freeze-dried and stored at –20°C.
Preparation of Enamel Particles
Enamel particles were prepared from bovine dental incisors, as approved by the university’s medical ethical committee. First, the labial surface was cut from the tooth. The back of the enamel surface was ground with abrasive paper (1200 grit) until all dentin was removed. Subsequently, the enamel slabs were ground in a ball-mill to particles of 10–50 µm in diameter. After attachment to the AFM cantilever, enamel particles were dipped for 30 min in reconstituted human whole saliva to create a salivary pellicle, with care taken not to dip the cantilever in the saliva. Biosurfactant, from a 1.5 mg/mL solution in adhesion buffer, was adsorbed for 10 min onto both a bare and a pellicle-coated enamel particle, already attached to the AFM cantilever.
Saliva
Human whole saliva from 20 healthy volunteers of both sexes was collected into ice-chilled cups after salivary flow was stimulated by the chewing of Parafilm®. The medical ethical committee approved the collection of human saliva, and volunteers gave their informed consent. After the saliva was pooled and centrifuged at 12,000 g for 15 min at 4°C, phenylmethylsulfonylfluoride, as a protease inhibitor, was added to a final concentration of 1 mM. The solution was again centrifuged, dialyzed for 48 hrs at 4°C against demineralized water, and freeze-dried for storage. Finally, we prepared a lyophilized stock by mixing freeze-dried material originating from, in total, 2 L saliva. Reconstituted human whole saliva was prepared from the lyophilized stock by dissolution of 1.5 mg/mL in adhesion buffer.
Atomic Force Microscopy (AFM)
A 10-mL quantity of a mutans streptococcal suspension was filtered through an Isopore polycarbonate membrane (Millipore Corporation, Billerica, MA, USA) with a pore size of 0.8 µm. The pore size was chosen slightly smaller than the size of the streptococci, to immobilize the bacteria by mechanical trapping (Kasas and Ikai, 1995). After immobilization, the filter was fixed on a glass surface with double-sided sticky tape. An enamel particle was fixed to the silicon nitride V-shaped AFM cantilever with the use of a minute amount of Pattex Super Mix glue (Henkel, Düsseldorf, Germany) with the aid of a Micromanipulator Leica DMIL (Leica Microsystems, Wetzlar, Germany). When appropriate, an attached enamel particle was dipped in reconstituted human whole saliva or in a biosurfactant solution.
We used a Nanoscope III AFM (Digital Instruments, Santa Barbara, CA, USA), operating in the contact mode, to measure interactive forces. A silicon nitride V-shaped cantilever (Veeco Instruments, Inc., Woodbury, NY, USA), with a probe curvature of ~ 50 nm, was used for measurements at room temperature in Millipore water. We experimentally determined spring constants by measuring the resonance frequency of each tip used, from which the spring constant could be calculated according to
 | (1) |
where k is the spring constant, f the true resonance frequency, and a is a proportionality constant provided by Veeco. Spring constants measured were similar to those provided by the manufacturer (0.06 Nm–1) and were assumed not to be influenced by the attachment of an enamel particle. Force-distance curves were subsequently taken with a z-displacement of 2000 nm and a scan rate of 1.99 Hz. Integral and proportional gains of the feedback loop were about 2 and 3, respectively. The slopes of the retraction force curves in the region where probe and sample are in contact were used to convert the voltage into a cantilever deflection. The conversion of deflection into force was carried out as has been previously described (Dufrêne et al., 2001). The point of zero separation was defined as the onset of the constant-compliance regime for the retraction curve.
When the interacting surfaces approach one another, an electrosteric repulsive force F is always generated (Vadillo-Rodríguez et al., 2004); it decays exponentially with distance D according to
 | (2) |
where F0 is the repulsive force at zero separation distance, and 
is the decay length of the repulsive force. The retraction curves can show local maxima in adhesion forces, and since these did not occur in all curves, we recorded the percentage of curves displaying adhesion forces. Furthermore, the largest adhesion force (Fmax), as well as the maximum distance (Dmax) at which an adhesion force occurred, were registered.
Results were presented as an average of 50 force-distance curves, taken over 5 different organisms on 10 randomly selected locations per organism, and including 4 different enamel particles.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Note that the enamel surface for interaction is large enough to be classified as macroscopic in comparison with streptococci.
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). For bare enamel, the repulsive force at contact (Fig. 2A; Table) was significantly (p < 0.01, Student’s t test) larger for S. sobrinus (3.9 nN) than for S. mutans (0.9 nN). For both bacteria, this repulsive force decayed rapidly within 30–53 nm. After biosurfactant-coating of bare enamel particles, the range and magnitude of this repulsive force for S. sobrinus increased significantly, with p < 0.01 (Fig. 2B; Table). A biosurfactant coating on bare enamel also significantly (p < 0.01, Student’s t test) increased the range of the repulsive force for the S. mutans, although the magnitude of the repulsive force at contact was not affected (Table). The repulsive force at contact for S. sobrinus (4.6 nN) and S. mutans (5.5 nN) with pellicle-coated enamel was similar (Fig. 2C; Table), although ranging over significantly (p < 0.01) longer distances for S. mutans than for S. sobrinus. Upon adsorption of biosurfactant to pellicle-coated enamel, the range of the repulsive forces increased significantly (p < 0.01) for both strains, in line with the observations for bare enamel (Fig. 2D; Table).
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), it can be seen that adhesive forces were found in about 75% of the force-distance curves measured upon retraction of bare enamel away from S. sobrinus and S. mutans surfaces. The maximum adhesive force averaged 0.8–0.9 nN, occurring at a distance of 39–80 nm between the interacting surfaces. For bare enamel, the percentage of the force-distance curves showing adhesion with S. mutans decreased to 10% upon biosurfactant adsorption, while with S. sobrinus no adhesive forces were observed upon retraction. Also, in the presence of a salivary pellicle, only a small percentage of the force-distance curves with S. sobrinus showed adhesion, while no adhesive forces were observed upon retraction for S. mutans. A biosurfactant coating on the salivary pellicle had only a minor effect on the characteristics of the retraction curves. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The approach curves have been described to be most pertinent to bacterial adhesion (Vadillo-Rodríguez et al., 2004), and adhesion of micro-organisms is more difficult if the repulsive energy between two interacting surfaces increases. Since the range of this repulsive force far exceeded that of the electrostatic forces distinguished in the DLVO (Derjaguin, Landau, Verwey, and Overbeek) theory (Rutter and Vincent, 1980) for the interaction of colloidal particles with a surface, the nature of this repulsion has been suggested to be electrosteric (Camesano and Logan, 2000). The most important effect of a salivary pellicle on the force-distance curves measured is an increase in the range of this electrosteric repulsion. In line with this increased range after pellicle-coating of an enamel surface, both strains of mutans streptococci involved in this study adhered less to a salivary pellicle than to bare enamel, as established in a parallel-plate flow chamber (Van Hoogmoed et al., 2004). Moreover, S. mutans ATCC 25175 adhered in fewer numbers to a salivary pellicle than did S. sobrinus HG 1025, which, again, is in agreement with the range of the electrosteric repulsion for S. mutans. Pellicle compositions vary intra- and inter-individually, owing to, respectively, site-specific differences in composition and secretion of saliva (Carlén et al., 1998) and differences in salivary flow rates and genetic polymorphism among proline-rich proteins (PRPs) and amylase. When saliva donated by a group of volunteers was pooled, these differences were averaged out. As a consequence of centrifugation, the reconstituted saliva might be deficient in high-molecular-weight mucins, which is probably of minor relevance, since these proteins are hardly present in 30-minute pellicles as applied here, though valuable for Streptococcus mutans adhesion (Carlén and Olsson, 1995).
Biosurfactants are, in the majority of cases, released by micro-organisms. Well-known actions of biosurfactants are the solubilization of hydrocarbons for nutrient uptake and metabolism, and their antibiotic activities on several micro-organisms. In the last decade, biosurfactants have also been increasingly recognized as substances that alter surface properties, thereby preventing the adhesion of other harmful micro-organisms to that surface (Neu, 1996). In an earlier study (Van Hoogmoed et al., 2004), it was demonstrated that S. mitis biosurfactant inhibited adhesion of S. mutans ATCC 25175 and S. sobrinus HG 1025 to bare enamel. S. mitis biosurfactant could inhibit adhesion of S. sobrinus HG 1025 only to salivary pellicles, but less so the adhesion of S. mutans ATCC 25175. Here, these reductions can be attributed to an increased range of the electrosteric repulsion upon approach between the bacteria and the biosurfactant-coated pellicles.
In conclusion, this paper presents direct AFM measurements of interactive forces between enamel with and enamel without a salivary pellicle and two strains of mutans streptococci. In addition, we demonstrated that the major effects of applying an S. mitis BMS biosurfactant coating to the enamel was an increased range of the electrosteric repulsion upon approach of the interacting surfaces, explaining the reductions observed in adhesion of these mutans streptococci brought about by S. mitis biosurfactant coatings.
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ACKNOWLEDGMENTS |
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Received July 14, 2004; Last revision August 19, 2005; Accepted September 11, 2005
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REFERENCES |
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Camesano TA, Logan BE (2000). Probing bacterial electrosteric interactions using atomic force microscopy. Environ Sci Technol 34:3354–3362.
Carlén A, Olsson J (1995). Monoclonal antibodies against a high-molecular-weight agglutinin block adherence to experimental pellicles on hydroxyapatite and aggregation of Streptococcus mutans. J Dent Res 74:1040–1047.
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