Quantitative Analysis of the Adhesion of Cariogenic Streptococci to Orthodontic Metal Brackets

Transcription

1 Original Article Quantitative Analysis of the Adhesion of Cariogenic Streptococci to Orthodontic Metal Brackets Sug-Joon Ahn a ; Bum-Soon Lim b ; Hyeong-Cheol Yang c ; Young-Il Chang d Abstract: The aim of this study was to analyze the adhesion of cariogenic streptococci to orthodontic metal brackets in terms of the type of bacterial strains, the incubation time, and saliva coating. Two strains of Streptococcus mutans (S. mutans LM7 and S. mutans OMZ65) and two strains of S. sobrinus (S. sobrinus B13 and S. sobrinus 6715) were used. Twenty metal brackets were incubated with either unstimulated whole saliva or phosphate-buffered saline for two hours. The bacterial adhesion assays were then performed by incubating the tritium-labeled streptococci with saliva-coated or noncoated orthodontic brackets for three, six, or nine hours. The results showed a characteristic binding pattern according to the type of bacterial strains used. S. mutans OMZ65 showed the highest amount of adhesion, whereas S. sobrinus B13 showed the lowest amount of adhesion. Generally, an extended incubation time increased the adhesion of cariogenic streptococci, and the amount of adhesion was the highest after nine hours of incubation. The saliva coating did not significantly influence the adhesion of bacteria. However, this saliva-mediated adhesion differed according to incubation time. The saliva coating tended to gradually decrease the adhesion by the extended incubation time, compared with the noncoated controls. This study indicates that each strain of cariogenic streptococci has a characteristic adhesion pattern and the type of bacterial strain, the incubation time, and saliva influenced the adhesion. (Angle Orthod 2005;75: ) Key Words: Adhesion; Cariogenic streptococci; Orthodontic metal bracket INTRODUCTION Enamel demineralization or white spot lesions around orthodontic appliances is a common side effect of orthodontic treatment. The placement of orthodontic appliances creates a favorable environment for the aca Assistant professor, Dental Research Institute and Department of Orthodontics, College of Dentistry, Seoul National University, Seoul, South Korea. b Associate Professor, Department of Dental Biomaterials Science, College of Dentistry, Seoul National University, Seoul, Korea. c Assistant Professor, Dental Research Institute and Department of Dental Biomaterials Science, College of Dentistry, Seoul National University, Seoul, South Korea. d Professor, Dental Research Institute and Department of Orthodontics, College of Dentistry, Seoul National University, Seoul, South Korea. Corresponding author: Bum-Soon Lim, BS, MS, PhD, Seoul National University, College of Dentistry and IBEC Dental Biomaterials Science and Dental Research Institute, 28 Yeongundong, Chongro-ku, Seoul, South Korea ( nowick@snu.ac.kr) Accepted: November Submitted: October by The EH Angle Education and Research Foundation, Inc. cumulation of microorganisms, which cause demineralization or exacerbate the effects of any preexisting caries. The incidence of enamel demineralization after fixed orthodontic appliance can involve up to 50% of the patients. 1,2 Despite the recent advances in orthodontic materials and techniques, the development of demineralization around the brackets during orthodontic treatment has not been overcome. Preventing these lesions during treatment is an important concern for orthodontists because the lesions are unesthetic, unhealthy, and potentially irreversible. 3 The enamel demineralization is caused by organic acids, produced mainly by mutans streptococci (MS), 4 which are known to be the prime causative organisms of dental caries. 5,6 Cariogenic MS are currently classified into seven species: Streptococci cricetus, S. ratti, S. mutans, S. sobrinus, S. downei, S. ferus, and S. macacae. 7 Of these, S. mutans and S. sobrinus have been the most frequently isolated species from the human oral cavity and have been implicated as the main causative organisms of human dental caries. 8 Recently, several epidemiologic studies have shown that S. sobrinus is more closely associated with a high caries activity than S. mutans. 9,10 In addition, S. sob- 666

2 ADHESION OF STREPTOCOCCI TO METAL BRACKET rinus has been associated with smooth-surface caries, 11 and an association of S. sobrinus with the development of caries in orthodontically treated children has been reported. 12 S. mutans is the predominant species and is often found alone, but S. sobrinus is usually detected in the teeth harboring S. mutans. 13 Among the many orthodontic appliances, orthodontic brackets may play a significant role in enamel demineralization because they are attached to the dentition continuously during almost all of the orthodontic treatment period and their complex design provides a unique environment, which impedes access to the tooth surfaces for cleaning. A previous report showed extensive patterns of plaque accumulation associated with bonded orthodontic brackets. 14 In particular, metallic brackets have been found to cause specific changes in the oral environment, such as a decrease in ph and an increase in plaque accumulation. 15 This indicates that orthodontic brackets impose a potential risk for enamel demineralization. Therefore, an analysis of the adhesion of S. mutans and S. sobrinus to orthodontic brackets is important for developing preventive methods on enamel demineralization during orthodontic treatment. Few studies, however, have investigated the adhesion of S. sobrinus and the role of saliva coating on the adhesion of cariogenic streptococci. The aim of this study was to quantitatively evaluate the adhesion of several cariogenic streptococci to orthodontic metal brackets with respect to incubation time, the saliva coating, and the type of cariogenic streptococci. MATERIALS AND METHODS Saliva collection Unstimulated whole saliva (UWS) was collected from a 33-year-old healthy volunteer using a spitting method. Saliva was collected into a chilled centrifuge tube in the icebox and centrifuged at 3500 g for five minutes, as described previously. 16 The resulting supernatants were immediately used for the pellicle formation and bacterial adhesion assays. Radioactive labeling and preparation of cariogenic streptococci S. mutans strains LM7 and OMZ65 and S. sobrinus strains 6715 and B13 (from the Department of Oral Microbiology and Immunology, College of Dentistry, Seoul National University) were used in this study. The bacteria were stored at 70 C in Trypticase (GIBCO, Grand Island, NY) with 3% yeast extract (TYE) broth containing 40% glycerol. Radiolabeling was performed by incubating a loop of bacteria in 10 ml of TYE broth 667 containing 50 Ci [ 3 H] thymidine ([methyl- 3 H] thymidine, Amersham Pharmacia Biotech, Piscataway, NJ) for 16 hours anaerobically at 37 C. The tritium-labeled bacteria were harvested by centrifugation at 3500 g for five minutes and washed in Hank s balanced salt solution (GIBCO) supplemented with 4.0 mmol NaHCO 3, 1.3 mmol CaCl 2, 0.8 mmol MgCl 2, and 0.5% bovine serum albumin (HBSS-BSA, ph 7.2). After washing twice, the pellets were resuspended in HBSS-BSA and adjusted to a final concentration of cells per ml at A 660 using a Petroff- Hauser cell counter (Hauser Scientific Partnership, Horsham, Penn). Adhesion of streptococci to orthodontic metal brackets Stainless-steel metal bracket (Korean smart, Dae- Seung, Seoul, Korea) was used in this study. These are upper bicuspid brackets of a Roth prescription with a slot. Twenty brackets were incubated in 2.0 ml of UWS with agitation for two hours at 37 C. For the negative control tests, the same procedure was performed with sterile phosphate-buffered saline (PBS, ph 7.2) instead of UWS. After washing three times in PBS, the brackets were incubated in 2.0 ml of HBSS-BSA containing tritium labeled bacteria with agitation for three, six, or nine hours at 37 C. The brackets were then washed three times with HBSS-BSA and transferred into scintillation vials. The radiolabeled bacteria were dislodged from the brackets by incubating them with 300 L of 8.0 M urea, 1.0 M NaCl, and 1% sodium dodecyl sulfate with agitation for one hour at 37 C, as described previously. 16 Then, 3.5 ml of the scintillation cocktail was added, and the number of adherent cells was determined using a Beckman LS-5000TA liquid scintillation counter (Beckman Instruments, Fullerton, Calif). The radioactive counts were divided by the total counts per minute of the bacterial suspension solution and the binding affinity of the cariogenic streptococci is defined as the percentage adhesion. All the test samples were counted in triplicate and each experiment was repeated six times. Three-way factorial analysis of variance (ANOVA) was used to analyze the binding affinities and the interaction effects with respect to the type of species, incubation times, and saliva coating, the Bonferroni t-tests at a significant level of 0.05 were used for the multiple comparisons between the different groups. RESULTS Tables 1 and 2 show the results of the three-way factorial ANOVA on the adhesion of the cariogenic streptococci with respect to the type of the strains, in-

3 668 AHN, LIM, YANG, CHANG TABLE 1. Means and SD of the Binding Affinities of the Cariogenic Streptococci With Respect to the Strains (Streptococcus mutans LM7 and OMZ65, and S. sobrinus B13 and 6715), the Incubation Times (three, six, and nine h), and the Saliva Coating (Saliva-Coated Group and Noncoated Control). The Binding Affinity of the Cariogenic Streptococci is Defined as the Percentage Adhesion Species Strain Saliva Three h Six h Incubation Time Nine h Total Streptococcus mutans LM7 Control 0.14 (0.04) 0.21 (0.08) 0.27 (0.09) 0.21 (0.09) Saliva coated 0.14 (0.04) 0.17 (0.09) 0.23 (0.11) 0.20 (0.11) Total 0.14 (0.04) 0.19 (0.08) 0.25 (0.10) 0.19 (0.09) OMZ65 Control 0.24 (0.08) 0.30 (0.06) 0.38 (0.12) 0.31 (0.11) Saliva coated 0.24 (0.06) 0.35 (0.08) 0.34 (0.08) 0.31 (0.09) Total 0.24 (0.07) 0.33 (0.07) 0.37 (0.10) 0.31 (0.10) Streptococcus sobrinus B13 Control 0.11 (0.04) 0.17 (0.06) 0.22 (0.09) 0.17 (0.08) Saliva coated 0.13 (0.06) 0.16 (0.06) 0.20 (0.07) 0.16 (0.07) Total 0.12 (0.06) 0.17 (0.05) 0.21 (0.08) 0.17 (0.07) 6715 Control 0.11 (0.05) 0.15 (0.06) 0.24 (0.13) 0.16 (0.10) Saliva coated 0.14 (0.07) 0.22 (0.14) 0.22 (0.09) 0.19 (0.10) Total 0.13 (0.06) 0.19 (0.11) 0.23 (0.11) 0.18 (0.10) Total Control 0.15 (0.08) 0.22 (0.09) 0.28 (0.13) 0.22 (0.11) Saliva coated 0.17 (0.07) 0.24 (0.12) 0.25 (0.11) 0.22 (0.11) Total 0.16 (0.07) 0.23 (0.10) 0.27 (0.12) 0.22 (0.11) TABLE 2. The Results of the Three-Way Factorial ANOVA of the Binding Affinities With Respect to the Strains (Streptococcus mutans LM7 and OMZ65, and S. sobrinus B13 and 6715), the Incubation Times (three, six, and nine h), and the Saliva Coating (Saliva-Coated Group and Noncoated Control) a Source DF SS MS F P Multiple Comparisons b Strains OMZ65 LM7, B13, 6715 Incubation times h Saliva coating Saliva coating incubation times Strains saliva coating Strains incubation times a DF indicates degree of freedom; SS, sum of squares; and MS, mean squares. b Bonferroni t-test was performed at the level of cubation times, and saliva coating. The results indicate that two main factors such as the type of strains and incubation times had significant effects on the adhesion of the cariogenic streptococci, whereas the saliva coating did not have a significant influence on the binding affinities. A difference in the interaction effects was statistically significant only between saliva coating and incubation times (P.05). There was significant difference in the adhesion according to the strains (Figure 1; Table 1). Multiple comparisons demonstrated that S. mutans OMZ65 showed significantly higher amount of adhesion than the other strains (P.000, Table 2). S. sobrinus B13 showed the lowest amount of adhesion, but this was not statistically significant compared with S. mutans LM7 and S. sobrinus 6715 (Tables 1 and 2). The order of the amount of adhesion was S. mutans OMZ65, S. mutans LM7, S. sobrinus 6715, and S. sobrinus B13 (Figure 1). This indicates that each strain of the cariogenic streptococci has a characteristic binding pattern. The extended incubation time increased the adhe- FIGURE 1. Adhesion of cariogenic streptococci (Streptococcus mutans LM7 and OMZ65 and S. sobrinus B13 and 6815) to the orthodontic metal brackets with respect to the incubation times (three, six, and nine hours).

4 ADHESION OF STREPTOCOCCI TO METAL BRACKET FIGURE 2. Adhesion of cariogenic streptococci to the orthodontic metal brackets with respect to the saliva coating (saliva-coated group and noncoated control) and the incubation times (three, six, and nine hours). FIGURE 3. Adhesion of cariogenic streptococci (Streptococcus mutans LM7 and OMZ65 and S. sobrinus B13 and 6815) to the orthodontic metal brackets with respect to the saliva coating (saliva-coated group and noncoated control). sion of the cariogenic streptococci (P.000, Figures 1 and 2; Table 1). The level of the bacterial adhesion increased significantly as a result of the extended incubation time, and the amount of adhesion was the highest in the sample after nine hours of incubation (Table 2). S. mutans OMZ65 showed the highest adhesion after nine hours of incubation, whereas S. sobrinus B13 had the lowest adhesion after nine hours of incubation. The saliva coating did not significantly influence the adhesion of the cariogenic streptococci (P.815, Figure 3; Table 1). However, the adhesion as a result of the saliva coating was affected significantly by the incubation times. The saliva coating tended to gradually decrease the adhesion by the extended incubation time, compared with the noncoated control. This was proved by the statistically significant interaction effect 669 between the saliva coating and incubation time (Table 2). Therefore, the level of adhesion in the noncoated group increased more than the saliva-coated group, as a result of the extended incubation time (Figure 2). Although there was no statistically significant interaction effect between the saliva coating and bacterial strains (P.119), the saliva-mediated adhesion was somewhat different according to the type of bacterial strains. Adhesion of S. mutans LM7 and S. sobrinus B13 was decreased slightly by saliva coating, whereas that of S. sobrinus 6715 was increased by saliva coating (Figure 3; Table 1). This also reflects the characteristic binding pattern of the cariogenic streptococci. DISCUSSION A significant difference in the adhesion was observed among the cariogenic streptococci strains. S. mutans OMZ65 adhered to the bracket surfaces significantly more than the other type of strains irrespective of the incubation time and saliva coating. The other three streptococci strains also showed a different amount of adhesion, although there was no statistically significant difference (Figures 1 and 3; Tables 1 and 2). This indicates that different strains showed different amounts of adhesion, although they belong to the same species. The order based on adhesion amount was S. mutans OMZ65, S. mutans LM7, S. sobrinus 6715, and S. sobrinus B13. This suggests that each strain of cariogenic streptococci has a characteristic adhesion pattern irrespective of the type of species. This study highlighted the role of the saliva and incubation time in modulating adhesion of cariogenic streptococci. Adhesion in the noncoated control reflects the genuine relationship between the bracket surface and bacterial adhesion. The adhesion in the noncoated group was increased by the extended incubation time and was the highest after nine hours of incubation. However, the level of adhesion in the saliva-coated group was different from that in the noncoated sample. The change in adhesion as a result of saliva coating varied according to the incubation time and the bacterial strain. At three hours of incubation, the level of adhesion in the saliva-coated sample was greater in all bacterial strains compared with that in the noncoated control. However, the saliva coating tended to gradually decrease the level of adhesion as a result of the extended incubation time, compared with the noncoated control group (Figure 2). Therefore, the adhesion level in the noncoated control group was increased more than that in the saliva-coated group as a result of the extended incubation time, and the level of adhesion in

5 670 AHN, LIM, YANG, CHANG the saliva-coated sample was lower than that in the noncoated control after nine hours of incubation (Figure 2; Table 1). This can be explained by the difference in adhesion of the cariogenic streptococci to the salivary pellicle formed on the metal brackets. The orthodontic brackets are covered instantly by the salivary pellicle in the oral cavity. Therefore, the adhesion of oral microorganisms to the bracket surfaces is governed to a large extent by the properties of the adsorbed salivary protein layer. 17 In the noncoated samples, only the surface characteristics of the brackets affect the amount of adhesion, which is governed by thermodynamic rules. 18,19 Accordingly, a material with a high surface-free energy will attract more bacteria to its surface than a material with a low surface-free energy. 18,19 It has been suggested that metal brackets increase the level of bacterial adhesion because of their high surface energy compared with plastic and ceramic brackets. 20 A previous study showed that S. mutans adhered preferentially to metal brackets compared with plastic and ceramic brackets in the noncoated sample. 16 In contrast to the noncoated samples, the amount of adhesion in the saliva-coated samples was largely influenced by the salivary pellicle formed on the underlying materials. The salivary pellicle as a binding receptor can not only promote the adhesion 16,21 but can also prevent the adhesion by decreasing the surface-free energy of the underlying materials. 17,22 If a specific receptor is present in the salivary pellicle formed on the underlying material, the amount of adhesion will increase significantly because of saliva coating. In contrast, saliva coating would decrease the amount of adhesion unless the bacterial adhesion was mediated by the specific salivary proteins. Gibbons et al 23 reported that the adhesion of S. mutans is promoted mainly by high molecular weight mucin and slightly by acidic praline-rich proteins (acidic- PRPs) from saliva. The high molecular weight mucin was not detected, whereas the acidic-prps were detected from the salivary pellicle formed on the metal brackets. 16,21 It is possible that the adhesion of cariogenic streptococci may be mediated by the acidic- PRPs formed on the metal brackets at the initial stage. As the adhesion mediated by acidic-prps is gradually saturated by the increase in the incubation time, the influence of the salivary receptor on the adhesion of the cariogenic streptococci would be reduced and the level of adhesion will depend primarily on the surfacefree energy instead of the specific binding with the salivary receptor. Therefore, the saliva-mediated adhesion would be reduced by the extended incubation time because the saliva coating decreases the surface-free energy of the underlying brackets. The adhesion pattern would be changed significantly if high molecular weight mucin were present on the salivary pellicle formed on the bracket. This is consistent with other studies, which reported that the binding of S. mutans KPSK-2 decreased as a result of the saliva coating and that of S. gordonii DL1, which was mediated mainly by the low molecular weight mucin and acidic- PRPs of the salivary pellicle, increased by the salivary coating, as the incubation time was increased. 16,21 The decrease in adhesion amount may be also explained partly by the fact that the salivary pellicle can inhibit adhesion of the cariogenic bacteria to the saliva-coated brackets. It has been shown that lysozyme and lactoperioxidase components of the saliva are responsible for inhibiting the adhesion of S. mutans to the saliva-treated hydroxyapatite in vitro. 24 It was shown that the cariogenic streptococci has a very low binding affinity. Approximately % of the cells adhered to the metal brackets during the nine hours of incubation time. This may be primarily because of the inherent low binding affinity of cariogenic streptococci. This is consistent with a previous study, 25 which showed that the proportion of S. mutans was smaller than the other streptococci and comprised only 0.5% of the dental plaque after 24 hours. Despite the low binding affinity, the adhesion of cariogenic streptococci themselves on the bracket surfaces may be an important factor in the development of a cariogenic plaque in patients with poor oral hygiene or caries-active individuals. This is because the microbial mass increases within the first day primarily because of cell division. 26 Knowledge of the adhesion of cariogenic streptococci to orthodontic materials will highlight a better way of preventing enamel demineralization and white spot formation. This study will provide a primary step in identifying a means to interfere with the process of adhesion of pathogenic bacteria to the pellicle or plaque on the orthodontic appliances. CONCLUSIONS Each strain of cariogenic streptococci has a characteristic adhesion pattern to metal brackets. Generally, an extended incubation time increased the level of bacterial adhesion irrespective of the bacterial strains, whereas saliva coating tended to decrease gradually the adhesion because of extended incubation time. This study suggests that the adhesion amount of the cariogenic streptococci to the metal brackets is influenced by the strain, the incubation time, and the saliva coating. ACKNOWLEDGMENTS This work was supported by the Korean Science and Engineering Foundation (KOSEF) through the Intellectual Biointer-

6 ADHESION OF STREPTOCOCCI TO METAL BRACKET face Engineering Center at the Seoul National University. We thank the Dae-Seung for kindly supplying brackets. REFERENCES 1. Gorelick L, Geiger AM, Gwinnett AJ. Incidence of white spot formation after bonding and banding. Am J Orthod. 1982; 81: Årtun J, Brobakken B. Prevalence of caries and white spots after orthodontic treatment with multibonded appliances. Eur J Orthod. 1986;8: Ogaard B. Prevalence of white spot lesions in 19-year-olds: a study on untreated and orthodontically treated persons 5 years after treatment. Am J Orthod Dentofacial Orthop. 1989;96: Featherstone JD. The science and practice of caries prevention. J Am Dent Assoc. 2000;131: Wisth PJ, Nord A. Caries experience in orthodontically treated individuals. Angle Orthod. 1977;47: Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev. 1986;50: Ulukapi H, Koray F, Efes B. Monitoring the caries risk of orthodontic patients. Quintessence Int. 1997;28: Babaahmady KG, Challacombe SJ, Marsh PD, Newman HN. Ecological study of Streptococcus mutans, Streptococcus sobrinus and Lactobacillus spp at sub-sites from approximal dental plaque from children. Caries Res. 1998;32: Beighton D, Rippon HR, Thomas HEC. The distribution of Streptococcus mutans serotypes and dental caries in a group of 5- to 8-year old Hamphire schoolchildren. Br Dent J. 1987;162: de Soet JJ, van Loveren C, Lammens AJ, Pavicic MJ, Homburg CH, ten Cate JM, de Graaff J. Differences in cariogenicity between fresh isolates of Streptococcus sobrinus and Streptococcus mutans. Caries Res. 1991;25: Madison KM, Bowen WH, Pearson SK, Falany JL. Enhancing the virulence of Streptococcus sobrinus in rats. J Dent Res. 1991;70: Fujiwara T, Sasada E, Mima N, Ooshima T. Caries prevalence and salivary mutans streptococci in 0 2 year- old children of Japan. Community Dent Oral Epidemiol. 1991;19: Rosenbloom RG, Tinanoff N. Salivary Streptococcus mutans levels in patients before, during, and after orthodontic treatment. Am J Orthod Dentofacial Orthop. 1991;100: Gwinnett AJ, Ceen RF. Plaque distribution on bonded brackets: a scanning microscope study. Am J Orthod. 1979; 75: Mitchell L. Decalcification during orthodontic treatment with fixed appliance an overview. Br J Orthod. 1992;19: Ahn SJ, Kho HS, Lee SW, Nahm DS. Roles of salivary proteins in the adherence of oral streptococci to various orthodontic brackets. J Dent Res. 2002;82: Weerkamp AH, van der Mei HC, Busscher HJ. The surface free energy of oral streptococci after being coated with saliva and its relation to adhesion in the mouth. J Dent Res. 1985;64: van Dijik J, Herkstroter F, Busscher H, Weerkamp A, Jansen H, Arends J. Surface-free energy and bacterial adhesion. An in vivo study in beagle dogs. J Clin Periodontol. 1987;14: Busscher HJ, Weerkamp AH, Van der Mei HC, Van Pelt AW, de Jong HP. Measurement of the surface free energy of bacterial cell surfaces and its relevance for adhesion. Appl Environ Microbiol. 1984;48: Eliades T, Eliades G, Brantley W. Microbial attachment on orthodontic appliances: I. Wettability and early pellicle formation on bracket materials. Am J Orthod Dentofacial Orthop. 1995;108: Ahn SJ, Kho HS, Kim KK, Nahm DS. Adhesion of oral streptococci to experimental bracket pellicles from glandular saliva. Am J Orthod Dentofacial Orthop. 2003;124: Quirynen M, Bollen CML. The influence of surface roughness and surface-free energy on supra- and subgingival plaque formation in man. A review of the literature. J Clin Periodontol. 1995;22: Gibbons RJ, Cohen L, and Hay DI. Strains of Streptococcus mutans and Streptococcus sobrinus attach to different pellicle receptors. Infect Immun. 1986;52: Roger V, Tenovuo J, Lenander-Lumikari M, Soderling E, Vilja P. Lysozyme and lactoperoxidase inhibit the adherence of Streptococcus mutans NCTC (serotype c) to saliva-treated hydroxyapatite in vitro. Caries Res. 1994;28: Nyvad B, Kilian M. Comparison of the initial streptococcal microflora on dental enamel in caries-active and in cariesinactive individuals. Caries Res. 1990;24: Brecx M, Theilade J, Attstrom R. An ultrastructural quantitative study of the significance of microbial multiplication during early dental plaque growth. J Periodont Res. 1983; 8: