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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 Correspondence: * corresponding author, takahasi{at}dent.osaka-u.ac.jp
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
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.
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
Measurement of Absorbance and Bacterial Numbers Each species was inoculated at 1.0 x 106 CFU/mL to BHI broth containing each monomer, and incubated at 37°C. Every 2 hrs, a portion of the suspension was collected and the absorbance at OD550 measured by spectrophotometer (UV-1600, Shimadzu, Kyoto, Japan) during a 48-hour incubation period. At intervals, samples of the cultures were sonicated to disrupt the chain of bacterial cells, and spread on BHI agar plates after serial ten-fold dilution with BHI broth. The plates were incubated anaerobically for 48 hrs at 37°C, and the numbers of colony-forming units (CFU) were counted. The measurements of CFU numbers were conducted until 24 hrs had elapsed. Regarding TEGDMA, the absorbance and CFU numbers were also measured when added at 0.5, 0.1, or 0.01 mg/mL. All experiments were performed in triplicate for each condition. 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
Chemical Analysis
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.
When S. sobrinus B13 was cultured with lower concentrations of TEGDMA, significantly greater absorbance values in the stationary phase were also observed by the addition of 0.5 mg/mL of TEGDMA (p < 0.05), while the presence of 0.1 or 0.01 mg/mL of TEGDMA exhibited no influence on the absorbance value. Regarding CFU numbers, there were no significant differences between control and TEGDMA-containing cultures at any of the concentrations tested (Figs. 1E  , 1F).
SEM Observation
Chemical Analysis The FTIR spectrum obtained from the material formed around S. sobrinus B13 and S. sanguis ST3R (Figs. 3A , 3B) showed the same pattern. The TEGDMA-polymer and -monomer exhibited the similar spectrum as these experimental specimens (Figs. 3C, 3D), and the relative peak heights at 1640 cm–1 (C=C) for experimental specimens and TEGDMA-polymer were smaller compared with that for TEGDMA-monomer.
The materials formed around bacteria and TEGDMA-polymer exhibited the single peak at the retention time of 20 min for GC analysis. MS analytical profile for the experimental specimens demonstrated the identical pattern with TEGDMA-polymer and monomer (Figs. 3E –3H).
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.
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 for publication February 12, 2003. Revision received September 4, 2003. Accepted for publication February 4, 2004.
Journal of Dental Research, Vol. 83, No. 4,
302-306 (2004)
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