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Effects of Fluoride and Aluminum from Ionomeric Materials on S. mutans Biofilm

¹ Faculty of Dentistry of Piracicaba, UNICAMP, Av. Limeira, 901, CEP 13414-903, Piracicaba, São Paulo, Brazil;
² Faculty of Dentistry of Bauru, USP, Bauru, São Paulo, Brazil; and
³ Eastman Department of Dentistry and Center for Oral Biology, University of Rochester, NY, USA;

*corresponding author, JCury{at}fop.unicamp.br

	ABSTRACT

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Ionomeric materials release different proportions of fluoride and aluminum. Their simultaneous effect on the acidogenicity and composition of S. mutans biofilm is unknown. Six cylindrical specimens of each material (Ketac-fil, Vitremer, Fuji-Ortho LC, F-2000, and Z-100) were incubated with S. mutans GS-5 in culture media containing 5% sucrose (w/v). The media were changed daily for seven days, during which the pH and concentrations of fluoride and aluminum were determined. Furthermore, the concentrations of these ions and insoluble polysaccharide were determined in the biofilm formed at the end of the experimental period. The results showed that all the materials tested released fluoride. However, Vitremer released the highest amount of aluminum and was the most effective in reducing the acidogenicity of S. mutans biofilms. It also significantly affected both biofilm formation and composition. Thus, this study suggests that aluminum released by ionomeric materials may enhance the biological effects of fluoride.

KEY WORDS: aluminum • biofilm • fluoride • glass-ionomer cements • S. mutans

	INTRODUCTION

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Ionomeric materials have shown anticariogenic and antimicrobial properties (Benelli et al., 1993) which have basically been attributed to their ability to release fluoride over extended periods of time (Wilson and McLean, 1988). Although the antibacterial activity of these materials has been extensively studied, data in the literature are not conclusive in establishing whether this biological activity is solely due to fluoride release (Seppä et al., 1992; Yap et al., 1999). Furthermore, there are other elements that are simultaneously released from ionomeric materials (Forss, 1993)—aluminum, for example, which is one of the major constituents, is also leached for long periods of time (Nakajima et al., 1997; Savarino et al., 2000).

The antibacterial activity of aluminum salt solutions against cariogenic micro-organisms has been previously reported (Oppermann and Rölla, 1980). In addition, the aciduric property of Streptococcus mutans depends on ATPase activity, and the inhibitory effect of fluoride on this enzyme is enhanced by aluminum (Sturr and Marquis, 1990). Thus, by interfering with bacterial growth and metabolism, the simultaneous release of aluminum and fluoride by dental materials may play an important role in preventing caries. The possibility of these anticariogenic properties of restorative or bonding dental materials has not been considered, and if evidence thereof were to be shown, it could be used to improve their quality.

Therefore, the aim of this study was to evaluate the effects of fluoride and aluminum released from different dental materials on the formation, composition, and acidogenicity of S. mutans biofilms.

	MATERIALS & METHODS

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Experimental Design
Six disc specimens (11.0 mm in diameter and 1.5 mm in thickness) were prepared from each of the following materials: (1) glass-ionomer cement, Ketac-fil (ESPE, Seefeld, Germany); (2) resin-modified glass ionomer, Vitremer (3M, Dental Products Division, St. Paul, MN, USA); (3) resin-modified glass ionomer, Fuji-Ortho LC (G.C. Corporation, Tokyo, Japan); (4) polyacid-modified resin, F-2000 (3M, Dental Products Division, St. Paul, MN, USA); and (5) composite resin, Z-100 (3M, Dental Products Division, St. Paul, MN, USA) as a negative control. The specimens were prepared according to the manufacturers’ specifications, at room temperature (23 ± 1.0 °C and 50 ± 5% relative humidity) according to ISSO specification #7489 and under aseptic conditions, in a sterile stainless steel mold. Additionally, a piece of cotton string was attached to each matrix before the cement set so that the disks could be individually suspended in test tubes in the inoculated culture media during the experiment. The media were changed daily during the experimental period of seven days, and the pH and the concentrations of fluoride and aluminum in the media were determined daily. The composition of the biofilm formed on the material was analyzed at the end of the experimental period, which was repeated six times.

Biofilm Formation
The micro-organism used in this experiment was Streptococcus mutans GS-5. The bacterial inoculum was prepared as detailed elsewhere (Koo et al., 2002). Briefly, individual colonies were isolated from 18- to 24-hour cultures of S. mutans GS-5 and suspended in sterile 0.89% NaCl. The cell suspension yielded 1-2 x 10⁷ colony-forming units per mL. A 50-µL aliquot of the S. mutans suspension was inoculated in 4.0 mL of a culture medium comprised of 15 g/L of casein peptone, 5 g/L of thiotone, 1 g/L of glucose, 50 g/L of sucrose, and 50 mmol/L potassium phosphate buffer, pH 7.2. Biofilms of S. mutans were formed on the surfaces of specimens suspended in batch cultures for 7 days at 37°C. The pH and the concentrations of fluoride and aluminum were determined in the media from each day. The composition of the biofilms formed on the specimens was analyzed on the 7th day.

Analysis of the Culture Media
The pH of the media was measured daily by means of a pH electrode (± 0.01) calibrated with standard buffers, pH 4.0 and 7.0. After this determination, the media were centrifuged (1500 g/10 min) and the supernatant separated for further analysis. For fluoride analysis, duplicate aliquots of the supernatant were mixed with TISAB III at a ratio of 1:0.1. This was made by means of an ion-selective electrode, Orion 96-09 (Orion Research Inc., Boston, MA, USA) and a digital ion-analyzer, Orion EA-940, previously calibrated with various standard solutions (0.065, 0.125, 0.250, 0.500, and 1.000 µg of fluoride/mL). Aluminum was determined in duplicate by atomic absorption spectrometry with nitrous oxide, acetylene flame, and a hollow cathode lamp at 309.3 nm. The spectrophotometer (VARIAN-AA-50) was calibrated with five standard solutions ranging from 0.5 to 25.0 µg of aluminum/mL, and all samples were analyzed with no pre-treatment procedure. The sensitivity limit of this analysis was considered 0.1 µg of aluminum/mL. The results of fluoride and aluminum released were expressed in mmol/L. Means and standard deviations of fluoride and aluminum release were calculated for each analyzed day.

Biofilm Analysis
At the end of the experimental period, the biofilm formed around the discs was dip-washed twice in distilled/de-ionized water to remove loosely bound material. The disc surfaces were gently scraped by means of sterile curettes to remove the biofilm formed. The biofilm from each disk was transferred to a pre-weighed micro-centrifuge tube, and dried in vacuum desiccators over phosphorus pentoxide. The dry weight of each sample was determined to ± 0.10 µg. The chemical analysis for carbohydrate and inorganic composition of the biofilm was performed according to Cury et al. (2000), and the results were expressed in µg/mg of biofilm dry weight.

Statistical Analysis
The results of pH, fluoride, and aluminum in the media were analyzed by a split-plot model analysis of variance (MANOVA) followed by Tukey’s test (p < 0.05). For the biofilm composition, the ANOVA and Tukey test (p < 0.05) were used. Pearson’s correlation analysis was made for fluoride, aluminum in the media, and the biofilm composition.

	RESULTS

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Fig. 1 shows the pH changes of the culture media due to each of the materials tested, throughout the duration of the S. mutans biofilm formation. All the test materials significantly inhibited the pH drop compared with the negative control (Z-100) (p < 0.05). Statistically significant differences were observed on the first and second days among all materials (p < 0.05). On the third, fourth, sixth, and seventh days, the difference between the materials Fuji Ortho and Ketac-fil was not statistically significant (p > 0.05). These materials also did not differ from F 2000 in the last two days. However, Vitremer was more effective than all the other materials in inhibiting the pH drop throughout the entire experimental period (p < 0.05).

View larger version (16K): [in this window] [in a new window]   Figure 1. pH changes (Avg; SD; n = 6) in the culture media containing each material over time (days) of S. mutans biofilm formation.

View larger version (17K): [in this window] [in a new window]   Figure 2. Fluoride concentrations (Avg; SD; n = 6) in the culture media containing each material over time (days) of S. mutans biofilm formation.

View larger version (17K): [in this window] [in a new window]   Figure 3. Aluminum concentration (Avg; SD; n = 6) in the culture media containing each material over time (days) of S. mutans biofilm formation.

	DISCUSSION

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The antimicrobial properties of ionomeric materials have been demonstrated primarily by the agar diffusion method (Yap et al., 1999; Herrera et al., 2000). However, relatively little work has been done to evaluate the extent to which these materials can affect the formation and composition of biofilms.

All the test materials significantly affected the acid production of S. mutans biofilms, since the pH values of the culture media containing these materials were higher than those of the control Z-100 throughout most of the seven-day experimental period. This is consistent with reports from other studies (Seppä et al., 1992, 1995), in which fluoride-releasing materials significantly inhibited bacterial metabolism, resulting in lower pH drop. It is well-known that fluoride affects bacterial growth and metabolism (Hamilton, 1990), both directly (e.g., inhibition of enolase and ATPase) and indirectly (e.g., intracellular acidification). Among the test materials, Ketac-fil released significantly higher amounts of fluoride than others (Fig. 2). However, the higher release of fluoride by this material was not reflected as a greater inhibition of pH drop, suggesting that other additional factors may be involved. In fact, the material Vitremer was more efficient than Ketac-fil in reducing the acid production by S. mutans biofilms. Clearly, Vitremer released significantly higher amounts of aluminum (Fig. 3) and also inhibited the pH drop to a significantly greater extent during the entire seven-day experimental period than did the other test materials. However, this ionomeric material did not show the highest amount of fluoride release (Fig. 2), suggesting a synergistic effect. The findings are supported by the positive correlation observed between aluminum x pH, fluoride x pH, and aluminum x fluoride. A statistically significant correlation between aluminum and fluoride released in the culture media during sugar fermentation has also been found when ionomeric materials were immersed in water (Nakajima et al., 1997; Savarino et al., 2000).

The synergistic effect on the bacterial acidogenicity of aluminum and fluoride released from some dental materials (Figs. 1, 2, 3) is supported by the fact that aluminum can enhance the inhibitory activity of fluoride on ATPase isolated from S. mutans (Sturr and Marquis, 1990). The possible mechanism of the combined action of fluoride and aluminum to inhibit ATPase may involve the formation of ADP-Al-F₃ complex in the catalytic site of this enzyme (Lunardi et al., 1988). ATPase plays an important role in the maintenance of the intracellular pH by pumping out protons; inhibition of this enzyme disrupts the bacterial metabolism and the aciduric capability of S. mutans. In the present study, the pH drop was induced by sugar fermentation of biofilms, therefore simulating a cariogenic challenge in dental plaque. These conditions mimicking the caries process are relevant for the evaluation of fluoride release from dental materials (Carvalho and Cury, 1999), and the same approach should be considered for evaluating the anticariogenic effect of aluminum.

The analysis of the composition of biofilms (Table) also showed that the presence of higher concentrations of fluoride and aluminum during biofilm formation resulted in significant changes in their inorganic and insoluble polysaccharide concentrations. The high concentrations of aluminum released from Vitremer (almost twice those of other materials during the first 2 days of the experiment) may explain the presence of greater amounts of fluoride in the biofilm. Fluoride can bind to calcium, forming a calcium fluoride reservoir (Seppä et al., 1993), and possibly to aluminum, forming AlF₃ complexes. The concentration of aluminum was higher in Vitremer biofilms, although it did not reach statistical differences compared with those from Ketac-fil and F-2000. On the other hand, Vitremer was the only material that significantly reduced the concentration of extracellular insoluble polysaccharide in the biofilm formed, compared with the negative control (Z-100) used. Extracellular polysaccharides are synthesized by glucosyltransferase enzymes (GTFs) produced by S. mutans. GTFs are responsible for the synthesis of soluble and insoluble glucans, which not only contribute to the bulk of the biofilm but also serve as binding sites for oral bacteria, including S. mutans (Schilling and Bowen, 1992). Metal cations have been shown to inhibit the activity of several GTFs (Wunder and Bowen, 1999). Whether aluminum or AlF₃ soluble complexes interfere with the activity of these important plaque-building enzymes needs to be further elucidated. Insoluble polysaccharide concentration in dental plaque is associated with dental caries (Nobre dos Santos et al., 2002), and its reduction may have had a major impact on biofilm formation and accumulation. Additionally, the negative correlation between the concentrations of insoluble polysaccharide and fluoride in the S. mutans biofilm formed is consistent with reports from previous in situ studies (Cury et al., 1997, 2000).

Analysis of our data supports the idea that factors other than fluoride release may be involved in the antibacterial activity of ionomeric materials. Several studies have shown inconclusive results regarding correlation between fluoride release and antimicrobial activity of ionomeric materials (Seppä et al., 1995; Yap et al., 1999). Therefore, additional antibacterial mechanisms have been suggested, e.g., the low initial pH of the material during setting (Barkhordar et al., 1989), polyalkenoic acids, zinc oxide (Scherer et al., 1989), and HEMA (Benderli et al., 1997). Although the ionomeric materials release other substances in addition to fluoride and aluminum, such as Sr, Si, Ca, and Na (Forss, 1993), none of them is considered to have an antibacterial effect. Nevertheless, a possible effect of trace chemical substances, not yet identified in these materials, should not be ignored.

In conclusion, this study strongly suggests that aluminum released from dental materials plays an important role in inhibiting bacterial metabolism and growth by enhancing the biological activities of fluoride. However, considering the experimental design used, the findings should be confirmed experimentally with fluoride and aluminum solutions used at different concentrations.

	ACKNOWLEDGMENTS

 
We thank FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for the scholarship (Proc. No. 98/00496-4) and financial support (Proc. No. 98/05837-4) for this research during the graduate course of the first author at the Faculty of Dentistry of Piracicaba-UNICAMP, Brazil. The authors are grateful to Professor Glaucia Maria Bovi Ambrozano, Faculty of Dentistry of Piracicaba, UNICAMP, for assistance in statistical analysis. The manufacturers of the products used did not give financial support for this study. This work was based on a thesis submitted by the first author to the Faculty of Dentistry of Piracicaba, University of Campinas, SP, Brazil, in partial fulfillment of the requirements for the Master’s Degree in Dentistry (Cariology Area), and a preliminary report was presented at the 78th General Session of the IADR (Washington, DC, USA).

Received June 18, 2002; Last revision November 4, 2002; Accepted January 9, 2003

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