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Influence of Resin Monomers on Growth of Oral Streptococci

¹ Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan; and
² Department of Oral Biology, The Dental School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4BW, UK;

^* corresponding author, takahasi{at}dent.osaka-u.ac.jp

	ABSTRACT

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Ethyleneglycol dimethacrylate monomers have been previously reported to stimulate the growth of certain caries-associated bacteria on the basis of turbidity measurements. To elucidate the detail of this effect, we examined the influence of resin monomers on the growth of Streptococcus sobrinus and Streptococcus sanguis by determination of bacterial numbers (colony-forming units), morphological observation, and chemical analysis. Although the absorbance values in the stationary phase of bacterial suspension were increased in the presence of ethyleneglycol monomers, no significant differences were observed for bacterial numbers throughout the incubation period. Scanning electron microscopy observation revealed the formation of sparse vesicular material surrounding bacterial cells when incubated with ethyleneglycol monomers, and these products were proved to be resin polymers. The results demonstrate that the apparent biomass increase during incubation with ethyleneglycol monomers is due not to promotion of bacterial multiplication, but to the polymerization of resin monomers to form vesicular structures attached to cells.

KEY WORDS: Streptococcus sobrinus • Streptococcus sanguis • resin monomer • bacterial growth

	INTRODUCTION

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Resin composites are cured by polymerization of methacrylate monomers, but complete polymerization is not possible due to the increase in rigidity and steric hindrance, so that the degree of conversion of resin composites is only 50 to 70% (Rueggeberg et al., 1990; Tarumi et al., 1999; Imazato et al., 2001). Residual unpolymerized monomers leach out from the cured materials in a wet environment (Tanaka et al., 1991; Spahl et al., 1998; Pelka et al., 1999) and are known to cause several problems, such as a decrease in mechanical properties (Asmussen, 1984; Lee et al., 1998) or toxic effects to pulpal cells (Stanley et al., 1975; Geurtsen et al., 1998; Gwinnett and Tay, 1998). In addition, it has been reported that ethyleneglycol monomers promoted the growth of Streptococcus sobrinus or Lactobacillus acidophilus (Kawai et al., 1988b; Hansel et al., 1998), and the possibility of the eluted monomers accelerating the growth of bacteria in the interfacial gap between teeth and restorative materials has been suggested. However, these previous studies demonstrated the growth-stimulating effects based simply on the results of absorbance measurements, and the detail of the biological influence of resin monomers on bacteria has not yet been fully clarified.

In this study, to examine the hypothesis that increase in the turbidity of bacterial suspension in the presence of resin monomers may not be caused by promotion of bacterial growth but by chemical reaction of monomers, we measured the numbers of two species of oral streptococci during incubation with various methacrylate monomers and investigated the biomass-increase phenomenon by scanning electron microscopy (SEM) observation and chemical analysis.

	MATERIALS & METHODS

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Bacteria and Resin Monomers
Streptococcus sobrinus B13 and Streptococcus sanguis ST3R were stored at -20°C in Brain Heart Infusion (BHI, Becton Dickinson, Sparks, MD, USA) broth containing 50% glycerol, and grown at 37°C in BHI broth. In the preliminary experiments, the suspensions of these species were confirmed to exhibit increase in turbidity when incubated with ethyleneglycol monomers.

Each of 4 resin monomers used for commercial composites was dissolved in 100% dimethyl sulfoxide (DMSO, Wako Pure Chemicals Industries, Osaka, Japan) and added to BHI broth to give the concentrations shown in the Table. The concentrations approximated the maximum solubility of each monomer in water (Kawai et al., 1988a). The final concentration of DMSO in each specimen was 2.0%, and BHI broth containing 2.0% DMSO without addition of any monomers served as control.

The statistical significance of difference between controls and experimental groups was analyzed by means of Mann-Whitney’s U test (p < 0.05).

SEM Observation
A portion of the suspension was taken every 2 hrs from the culture incubated in the same manner as in the absorbance measurements, and centrifuged at 3000 rpm for 10 min, then washed with 0.1 mol/L sodium cacodylate buffer at pH 7.4. The specimens were then fixed in 0.1 mol/L half-Karnovsky’s solution containing 2% paraformaldehyde and 2.5% glutaraldehyde for 1 hr, and dehydrated in ascending graded ethanol. After being freeze-dried and platinum-coated, the specimens were examined by SEM (JSM-5310LV, JEOL, Tokyo, Japan).

Chemical Analysis
Each bacterial species was incubated for 24 hrs with TEGDMA at 1.0 mg/mL and washed with 0.1 mol/L sodium cacodylate buffer. Then, the biomass produced was stained with 0.025% Safranin (Merck, Darmstadt, Germany) for 10 min, and centrifuged at 500 rpm for 5 min to separate the materials formed around bacteria from the cells. After being dried at room temperature for 24 hrs, the unstained powdery material was collected and analyzed by Fourier transform infrared spectrophotometry (FTIR) and pyrolytic gas chromatography mass spectroscopy (GC-MS). For FTIR, 2 mg of the specimen was mixed with 200 mg of potassium bromide powder, and the absorbance peaks were obtained in the diffuse reflection mode (8200PC, Shimadzu). For GC-MS, a Curie point pyrolyzer (JHP-2, Japan Analytical Industry, Tokyo, Japan) was used for the pyrolysis unit for vaporization, and the system was mounted on GC (MS-GCG06, JEOL). A 100-µg quantity of the specimen was used, and pyrolysis was performed at 900°C for 5 sec with the chamber temperature at 180°C. A 30 m x 0.25 mm x 1.0 µm DB-1 column was used, and mass spectrometric detection was conducted with a mass selective detector (JMS-HX100/JMA-DA5000, JEOL). Experimental specimen peaks obtained by FTIR and GC-MS were compared with those of TEGDMA-polymer powder and TEGDMA-monomer.

	RESULTS

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Measurement of Absorbance and Bacterial Numbers
When 1.0 mg/mL of TEGDMA or DEGDMA was added to broth cultures of S. sobrinus or S. sanguis, the absorbance values in the stationary phase were significantly greater (p < 0.05) than those for controls (Figs. 1A, 1B). On the contrary, the CFU numbers in the presence of TEDGMA or DEGDMA were not significantly different from or less than those of the controls throughout the 24-hour incubation period (Figs. 1C, 1D). The addition of Bis-GMA or UDMA showed no influence on the absorbance or CFU for either species.

View larger version (41K): [in this window] [in a new window]   Figure 1. The absorbance values and CFU numbers (CFU/mL) of bacterial culture when incubated with various monomers. (A–D) In the presence of TEGDMA or DEGDMA, the absorbance values were significantly greater (p < 0.05) than those of controls in the stationary phase (A, S. sobrinus B13; B, S. sanguis ST3R). However, bacterial numbers were not significantly different from controls or less than controls throughout the incubation period (C, S. sobrinus B13; D, S. sanguis ST3R). (E,F) The absorbance values and bacterial numbers of S. sobrinus B13 incubated with the addition of 1.0, 0.5, 0.1, or 0.01 mg/mL of TEGDMA. Significantly greater absorbance values than in controls were obtained at the concentration of 0.5 mg/mL and over, whereas CFU numbers indicated no significant differences between control and all TEGDMA-added groups. The bar represents the standard deviation of the mean of three replicates.

SEM Observation
SEM observation demonstrated that sparse vesicular material was formed surrounding the bacterial cells at 12–14 hrs of incubation and thereafter with TEGDMA or DEGDMA for both species of bacteria. The amount of this vesicular material increased with the increase in the absorbance of the suspension, and bacterial cells were almost completely encapsulated in the stationary phase (Fig. 2). The production of vesicular materials was not observed at any incubation period in cultures containing Bis-GMA or UDMA.

View larger version (85K): [in this window] [in a new window]   Figure 2. SEM photographs of S. sobrinus B13 or S. sanguis ST3R after 18 hrs of incubation with 1.0 mg/mL of TEGDMA or without any monomers. (A) S. sobrinus B13 incubated without monomers. (B) S. sobrinus B13 incubated with TEGDMA. (C) S. sanguis ST3R incubated without monomers. (D) S. sanguis ST3R incubated with TEDGMA. Sparse vesicular material was formed around bacterial cells when incubated with TEGDMA (x7500).

View larger version (22K): [in this window] [in a new window]   Figure 3. FTIR and GC-MS analysis results of the materials formed around bacteria and TEGDMA. (A-D) FTIR spectrum for the material formed around S. sobrinus B13 (A), S. sanguis ST3R (B), TEGDMA-polymer (C), and TEGDMA-monomer (D). (E–H) GC-MS analysis results of the materials formed (E,F), TEGDMA-polymer (G), and TEGDMA-monomer (H).

	DISCUSSION

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Although turbidity measurement of bacterial suspension has frequently been used for investigating the growth of bacteria, the results of this test do not directly indicate the bacterial numbers. Therefore, in this study, the net bacterial numbers (CFU) were counted in addition to simultaneous measurement of the absorbance. The results of the absorbance measurement coincided well with previous findings that the addition of ethyleneglycol monomers caused an increase in absorbance values in the stationary phase (Kawai et al., 1988b; Hansel et al., 1998). However, the number of bacterial cells was not greater than that in the control, even when TEGDMA or DEGDMA was added to the broth. It is of interest that SEM observation revealed that vesicular material was formed surrounding the bacterial cells specifically in the presence of ethyleneglycol monomers. In addition, the amount of this material was increased as absorbance of the suspension increased. Accordingly, it can be concluded that the relatively greater absorbance observed in the presence of ethyleneglycol monomers was due not to the increase in bacterial numbers but to the increase in particle size caused by formation of the vesicular materials surrounding the cells.

Since the vesicular materials produced around the bacteria were not stained by Safranin, Alcian blue, or Ruthenium red, these materials are not carbohydrates. We therefore carried out chemical analysis with FTIR and GC-MS to elucidate the constituents and found that the materials formed have a composition identical to that of TEGDMA-polymer. It was also confirmed that the materials were not aggregates of the monomer, since they were insoluble in organic solvents such as acetone or tetrahydrofurane. Consequently, it was proved that the ethyleneglycol monomer added to the broth was polymerized around the bacteria during their multiplication, and the cells were surrounded by this sparsely structured polymer. The apparent biomass-increase phenomenon, which has so far been reported to be due to promotion of bacterial growth, is possibly a misunderstanding, due to the investigators’ failure to take account of increased turbidity by the production of polymer around the bacteria.

Bis-GMA and UDMA have dimethacrylate groups as the polymerizable part in their molecules, but their polymerization mechanism is the same as that of ethlyleneglycol dimethacrylate monomers. However, the relative increase in turbidity of the bacterial cultures was observed only in the presence of ethyleneglycol monomers. It should be noted that, since Bis-GMA and UDMA have low solubility in water (Bowen, 1981), concentrations less than 0.1 mg/mL were used in the present study as in the previous reports (Kawai et al., 1988b; Hansel et al., 1998). The monomer TEGDMA also failed to cause an increase in absorbance at 0.1 mg/mL or lower concentrations. There may thus be a threshold in monomer concentrations for the occurrence of polymerization around bacterial cells, and it is not clear if the phenomenon is specific for ethyleneglycol monomers.

Several studies have described the release of considerable amounts of unpolymerized monomers from the cured composites (Tanaka et al., 1991; Spahl et al., 1998; Pelka et al., 1999), although the concentrations varied according to the size of the specimens and the amount of the elution medium. For instance, it has been reported that 1085.2 nmol/mL (0.31 mg/mL) of TEGDMA was eluted from the commercial composites after immersion in the medium for 24 hrs (Pelka et al., 1999). Condensation of monomers can take place when they leach into small spaces, such as tooth-restorative interfaces, so the amount of unpolymerized monomers eluted from restoratives appears to be in the range to cause polymerization around bacterial cells. The fact that more plaque accumulates on resin composites compared with other materials (Dummer and Harrison, 1982; Skjörland and Sönju, 1982) may be partly explained by this phenomenon, since sparse vesicular-structured polymer can be a scaffold for establishment of the bacterial community. Furthermore, the formation of surrounding resin polymer may act as a barrier to protect the bacterial cells, making bacteria more tolerant to chemical or physical attack.

Polymerization of methacrylate monomers is initiated by radical formation, which subsequently gives a chain reaction (McCabe, 1990). Therefore, production of resin polymer around bacterial cells is considered to have occurred by free radicals produced during multiplication or metabolism of S. sobrinus and S. sanguis, though the detailed mechanism is unclear. It is possible that the phenomenon is associated with production of hydrogen peroxide, since the species known to initiate polymerization (S. sobrinus, S. sanguis, and L. acidophilus) all produce peroxide, whereas Streptococcus mutans does not produce peroxide and does not initiate polymerization (Rupf et al., 2001). The explanation of why the phenomenon is observed only with certain bacterial species, as well as elucidation of the clinical relevance, remains to be determined.

	ACKNOWLEDGMENTS

 
This work was supported in part by a Grant-in-Aid for Scientific Research (13470402, 15209066) from the Japan Society for the Promotion of Science. This work was a part of the 21st Century COE entitled, "Origination of Frontier BioDentistry" at Osaka University Graduate School of Dentistry, supported by the Ministry of Education, Culture, Sports, Science, and Technology

Received February 12, 2003; Last revision September 4, 2003; Accepted February 4, 2004

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