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Silica Nanoparticles to Polish Tooth Surfaces for Caries Prevention
1 Department of Physics, Clarkson University, 8 Clarkson Ave., Potsdam, NY 13699, USA; Correspondence: * corresponding author, isokolov{at}clarkson.edu
Although silica particles have been used for tooth polishing, polishing with nanosized particles has not been reported. Here we hypothesize that such polishing may protect tooth surfaces against the damage caused by cariogenic bacteria, because the bacteria can be easily removed from such polished surfaces. This was tested on human teeth ex vivo. The roughness of the polished surfaces was measured with atomic force microscopy (AFM). A considerably lower nanometer-scale roughness was obtained when silica nanoparticles were used to polish the tooth surfaces, as compared with conventional polishing pastes. Bacterial attachment to the dental surfaces was studied for Streptococcus mutans, the most abundant cariogenic bacteria. We demonstrated that it is easier to remove bacteria from areas polished with silica nanoparticles. The results demonstrate the advantage of using silica nanoparticles as abrasives for tooth polishing.
Key Words: silica nanoparticles • polishing • enamel • carious • Streptococcus mutans • atomic force microscopy
Tooth enamel is damaged gradually during the human lifetime, depending on a person’s dietary habits. Some cariogenic bacteria living in human mouths can survive on tooth surfaces and form colonies (Loesche, 1986). The acid produced by these bacteria causes enamel to demineralize, and, consequently, causes caries. The polishing of dental surfaces is a well-known practice in dentistry (Roulet and Roulet-Mehrens, 1982; Christensen, 1990; Gurgan et al., 1997; Berry et al., 1999; Banerjee et al., 2000). This is used to make it difficult for plaque and cariogenic bacteria to accumulate on tooth surfaces. Thus, polishing is a preventive procedure used as a primary defense against dental problems. Micron-sized silica particles are a typical component of conventional polishing pastes. In the first-order approximation, one could expect that the smaller the abrasive particles, the smoother the polished surface. An example of such an approach is known as the chemical-mechanical planarization process (Berdyyeva et al., 2003; Lu et al., 2005), used in the semiconductor industry. The chemical-mechanical planarization technique uses various nanometer-sized particles to polish surfaces of semi-conductor wafers to a sub-nanometer level. However, attaining such a smooth surface requires considerable research and optimization. Thus, it is debatable whether a noticeable improvement in the roughness of tooth enamel can be achieved with the use of nanosized abrasives. Second, what level is beneficial to decrease the roughness of tooth surfaces? Bacterial attachment is biochemical in nature. It might be simply insensitive to a change in roughness at the nanometer level. In the present study, the primary technique of choice was Atomic Force Microscopy (AFM). Previously, AFM has been successfully used for the study of dental surfaces (Schaad et al., 1993; El Feninat et al., 1998; Yoshida et al., 1999; Rodriguez et al., 2006; Gruverman et al., 2007) as well as bacteria and eukaryotic cells (Sokolov et al., 1996, 2001, 2007; Berdyyeva et al., 2005; Sokolov, 2006). Here we used it not only to investigate the roughness of tooth surfaces, but also to study the ease of bacterial removal from tooth enamel. Our first hypothesis, based on the approach used in chemical-mechanical planarization, was that the tooth surface could be made smooth if we polished it with nanosized abrasives. Silica particles were the material of choice because of their biocompatibility and low cost. Our second hypothesis was that the roughness we attained by polishing with nanoparticles would play a visible role in the removal of bacteria from the tooth surface. In general, the forces of interaction of an organic substance with flat surfaces are comparatively weaker than with rough corrugated surfaces, because of the increased area of the rough surface compared with the flat one. In the case of bacterial attachment, it may not necessarily be that simple. For example, bacteria could still attach strongly even to a rather smooth surface, so the difference in adhesion could be negligible when we consider the change of roughness at the nanoscale level.
Tooth Samples The tooth samples used for the research were deciduous exfoliated human teeth (the donors’ identities were kept anonymous by the institutional review board, and the study was approved for exemption). The surfaces were not treated with any chemicals before study. Each tooth was cut into 4 pieces by means of a cutter (Indigo, Inc., Waterloo, ON, Canada), and each piece was attached to a glass slide with water-insoluble glue (Loctite E-30CL Hysol® Epoxy Adhesive, Ellsworth Adhesives, Germantown, WI, USA) with the enamel exposed face-up for polishing. The glue was allowed to cure overnight.
Bacterial Culture To allow the bacterial films to grow on the tooth surfaces, the tooth samples were immersed overnight in the bacterial suspension, as described above, in a water bath (Model #406015, American Optical, Buffalo, NY, USA) at 37°C. The sample was rinsed from the sides with de-ionized water to remove excess bacterial colonies.
Atomic Force Microscopy (AFM)
In this work, we used AFM as both a microscope and a soft brush (the rastering AFM tip) to remove bacteria from the tooth surface while imaging the tooth surface in contact mode. This technique is analogous to the action of a single fiber of a soft cleaning brush. The AFM probe scans rather quickly over the tooth surface and moves with constant load force, which helps to monitor removal of the bacteria in a highly controlled manner.
Nanoparticle Slurry for Polishing
Polishing Procedure
Roughness of the Polished Surfaces First, we estimated the degree of polishing by regular optical microscopy. Optical images of 4 dental surfaces—virgin, polished with CrestTM, NuProTM, and nanosilica slurry—are shown in the APPENDIX (Fig. 2  ). AFM measurements were performed on the areas identified as polished with optical microscopy. Representative 15 x 15 µm2 AFM images of the 4 corresponding surfaces (virgin, polished with CrestTM, NuProTM, and nanosilica slurry) are shown (Fig. 2).
Quantitative analysis of roughness was undertaken for the areas of 5 x 5 µm2. This was justified by the size of S. mutans bacteria. We assumed that the roughness should be analyzed over the area comparable with the bacterium size to contribute bacterial adhesion. This is plausible because it is unlikely that a bacterium would develop adhesion to an area much larger than its size. Statistical distributions for the roughness obtained are as shown (Fig. 3 ). The average roughness and standard deviation (with respect to the normal distribution) of the distributions are as follows: 21 ± 6 nm (virgin tooth), 22 ± 4 nm (CrestTM-polished), 25 ± 5 nm (NuProTM-polished), and 2.4 ± 1.4 nm (polished with the nanoparticle slurry). While the difference among the first 3 roughnesses is statistically insignificant, one can see a decrease by an order of magnitude for the surfaces polished with the nanoparticle slurry.
Bacterial Removal from the Surfaces with Different Roughnesses: AFM Study To correlate the removal of S. mutans bacteria from the tooth surface and the surface roughness, we used the raster AFM scanning method. To get a sufficient variation in roughnesses, we studied the bacterial removal on a tooth surface that was just slightly polished with nanoparticles (some areas having a roughness corresponding to that of the completely polished surface, as described previously). A series of AFM images of the slurry-polished tooth surface, with adherent S. mutans, was captured after a certain number of raster scans (Fig. 4 ). After the first scans, weakly attached bacteria were removed by the lateral force of the AFM probe (Fig. 4a). After several scans, more tightly attached bacteria were also removed (Fig. 4b). Finally, the bacteria with strongest adhesion were removed (Fig. 4c). This mimics the action of a single fiber of a soft cleaning brush. The total scanning time to collect those images was about 2 min, completing approximately a dozen full scans.
Calculating the roughness on the tooth surface, we found that the roughness of the areas in which the bacteria were removed after the first scans (Fig. 4c  , asterisks) ranged within 1.8 to 2.4 nm. In contrast, the areas in which bacteria were removed only after the complete series of scans (Fig. 4c, squares) had a roughness of 5.0 to 5.5 nm. In other words, bacteria were initially removed from smoother areas (roughness, 1.8–2.4 nm; Fig. 4a) and remained longer on the rougher ones (roughness, 5.0–5.5 nm; Figs. 4b, 4c). We obtained the same correlation in macroscale using soft brushes to clean away bacterial films (APPENDIX Fig. 3).
We compared tooth roughnesses for virgin, toothpaste-polished (regular CrestTM and NuProTM), and nanoparticleslurry- polished teeth. CrestTM toothpaste is not intended for polishing, so we expected to see no noticeable change in roughness after polishing with CrestTM toothpaste. NuProTM is a professional prophylactic toothpaste used for light polishing. The abrasives used in the NuProTM paste range from 1 to 180 µm (as estimated by optical microscopy). During polishing, however, these abrasives can be broken into smaller particles. Nevertheless, our roughness calculations showed no statistical difference in roughness (calculated for the micron scale area) after polishing with either of the pastes. Comparing this result with the macroscopic images of the polished surfaces, one can conclude that polishing with NuProTM does not improve surface smoothness at the micron scale, while resulting in noticeable removal of enamel at the macro (multi-micron) scale. Polishing with CrestTM toothpaste results in almost no enamel removal, at either the micro or macro scale. These results are not surprising, since neither toothpaste is intended to provide polishing at the micron scale. We showed that the use of silica nanoparticles for polishing of tooth surfaces allowed us to attain single-nanometer values of roughness, an order of magnitude better than the roughness of the surfaces polished with the professional fine polishing paste NuProTM. Analyzing the results in terms of bacterial removal, one can clearly see that S. mutans bacteria can be removed more easily from the smoother surfaces polished with silica nanoparticles than from the rougher ones. This can be helpful in protecting teeth against the damage caused by cariogenic bacteria. These results can be used in the formulation of new dental polishing pastes. The results presented here demonstrate an advantage of using silica nanosized particles as abrasives for the polishing of dental surfaces. There are obviously numerous unanswered questions, first among which is how long the effect of polishing will last, and whether the polished surface will inhibit mineralization and plaque formation. These and other questions will be addressed in future research.
Support from NanoBio Laboratory (NABLAB), Clarkson University, is acknowledged by IS.
A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/10/980/DC1. Received for publication January 22, 2008. Revision received June 18, 2008. Accepted for publication June 27, 2008.
Journal of Dental Research, Vol. 87, No. 10,
980-983 (2008)
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ABSTRACT
INTRODUCTION
