| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
RESEARCH REPORT |
1 Department of Orthodontics, College of Dentistry, Seoul National University, 28-22 Yunkeun-Dong, Chongro-Ku, Seoul 110-744, Korea (ROK); and
2 Department of Oral Medicine and Oral Diagnosis, College of Dentistry, Seoul National University;
*corresponding author, Titoo{at}chollian.net
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-amylase, secretory IgA, acidic proline-rich proteins, and cystatins adhered to all kinds of brackets, though the amino acid composition of pellicles differed between bracket types. Some of these proteins increased the binding of S. gordonii to saliva-coated brackets. However, salivary pellicles decreased the binding of S. mutans. Collectively, salivary pellicles were found to play a significant role in the initial adhesion of oral streptococci to orthodontic brackets.
KEY WORDS: salivary pellicle • Streptococci • orthodontic bracket
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The wearing of orthodontic appliances has been found to induce decreased pH, increased plaque accumulation, and elevated Streptococcus mutans colonization, which enhance susceptibilities to enamel white-spot formation (Balenseifen and Madonia, 1970; Mizrahi, 1982). Recently, salivary components were identified on various orthodontic materials (Lee et al., 2001). In view of the selective nature of protein adsorption, there might be differences in the compositions of pellicles and the adherent patterns of bacteria on different kinds of orthodontic brackets. This study was undertaken to identify the salivary components of pellicles formed on various orthodontic brackets, and to determine the effects of these pellicles on the adherence of Streptococcus gordonii and Streptococcus mutans.
|
MATERIALS & METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Preparation of Orthodontic Brackets
Four different kinds of orthodontic brackets were used: stainless-steel metal (Korean Smart, Dae-Seung, Seoul, Korea), monocrystalline sapphire (MCS) (Inspire, Ormco/A Company, Glendora, CA, USA), polycrystalline alumina (PCA) (Transcend 6000 series, 3M/Unitek, Monrovia, CA, USA), and polycarbonate plastic (Silkon m, American Orthodontics, Sheboygan, WI, USA). All were upper bicuspid brackets of Roth prescription with a 022 x 028 slot. Each type of bracket was incubated in 2 mL of UWS with agitation for 2 hrs at room temperature, and washed 3 times with Tris-buffered saline (TBS) (10 mmol/L of Tris-HCl, 154 mmol/L of NaCl; pH 7.5). Experimental bracket pellicle (EBP) was obtained by the addition of 0.5% sodium dodecyl sulfate (SDS) and lyophilized immediately.
Amino Acid Composition Analysis
The EBPs for amino acid composition analysis were obtained from 100 of each type of bracket by the addition of 60% formic acid and then lyophilized. Samples obtained were hydrolyzed with 6 N HCl at 110°C for 24 hrs in evacuated sealed tubes and dried. Hydrolysates were analyzed by means of a Hewlett-Packard model 1100 series amino acid analyzer. Cysteine was analyzed by oxidative hydrolysis, and tryptophan by hydrolyzation with 4 N methanesulfonic acid.
Characterization of Bracket Pellicles
EBPs from 50 of each type of bracket were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, gels were stained for 2 hrs with 0.25% Coomassie brilliant blue (Sigma Chemical, St. Louis, MO, USA), and the molecular weights of the salivary components were compared with standard low-molecular-weight markers (Amersham Pharmacia Biotek, Piscataway, NJ, USA).
Western blotting was performed on a polyvinylidene fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA, USA). Unbound sites on the membrane were blocked in TBS containing 2% bovine serum albumin (BSA) for 3 hrs and then incubated with rabbit antisera to a panel of salivary proteins for more than 5 hrs at room temperature. After being washed in TBS containing 0.01% BSA, each blot was incubated with a 1:2500 dilution of alkaline-phosphatase-conjugated affinity-purified goat anti-rabbit IgG (Bio-Rad Laboratories, Hercules, CA, USA) for 3 hrs. The blots were then washed and visualized with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate alkaline phosphatase color-developing reagents (Bio-Rad). For negative controls, the same experiments were performed with rabbit pre-immune sera as a primary antibody. All test samples were assayed at least three times.
Radioactive Labeling and Preparation of Bacteria
Streptococcus gordonii strain DL1 and Streptococcus mutans strain KPSK-2 (from the Department of Oral Microbiology, College of Dentistry, Seoul National University) were used. Radiolabeling was performed by incubation in 10 mL of brain heart infusion broth containing 50 µCi [3H] thymidine ([methyl-3H] thymidine, Amersham Pharmacia Biotech) for 16 hrs anaerobically at 37°C with a loopful of bacteria. The tritium-labeled bacteria were harvested by centrifugation at 3500 x g for 10 min and washed in Hanks' Balanced Salt Solution (Gibco, Grand Island, NY, USA) supplemented with 4 mmol NaHCO3, 1.3 mmol CaCl2, 0.8 mmol MgCl2, and 0.5% BSA (HBSS-BSA, pH 7.2). After being washed twice, pellets were re-suspended in HBSS-BSA and finally adjusted to a final concentration of 5 x 108 cells per mL at A660 with the use of a Petroff-Hauser cell counter (Hausser Scientific Partnership, Horsham, PA, USA).
Adhesion of Streptococci to Orthodontic Brackets
Twenty of each type of bracket were incubated in 2 mL of UWS with agitation for 2 hrs at room temperature. For negative controls, the same procedure was performed with TBS instead of UWS. After being washed 3 times in TBS, pellicle-formed brackets were incubated in 2 mL of HBSS-BSA containing 1 x 109 tritium-labeled bacteria with agitation for either 3 or 6 hrs at 37°C. The brackets were then washed with HBSS-BSA and transferred to scintillation vials. The radiolabeled bacteria were solubilized from the brackets by incubation with 300 µL of 8 M urea, 1 M NaCl, and 1% SDS as previously described (Lee et al., 2001). Then, 3.5 mL of scintillation cocktail was added, and the number of adherent cells was determined with the use of a Beckman LS-5000TA liquid scintillation counter. The radioactive counts were divided by the total counts per minute of the bacterial suspension solution. Each assay was repeated 6 times. To analyze binding affinities and interaction effects with respect to the bracket types, incubation times, and saliva-coating, we used three-factor ANOVA. Multiple comparisons were done by the Bonferroni t test at a significance level of = 0.05.
Bacterial Overlay Assay
PVDF and nitrocellulose membranes (Sigma Chemical) were used in the solid-phase assay. After SDS-PAGE and Western transfer, membranes were incubated in TBS containing 2% BSA for 3 hrs, and then incubated overnight with 10 mL of HBSS-BSA containing 2.5 x 109 cells of each bacterium at 37°C. After being washed 3 times in HBSS-BSA, the membranes were air-dried and exposed overnight to a Fuji imaging plate (BAS-TR2040S; Fuji photo film, Tokyo, Japan). This image was then developed with the use of a BAS-1500 image reader (Fuji photo film). For negative controls, the same experiment was performed without bacterial overlay. All assays were repeated at least 3 times.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Immunodetection of the pellicle components with monospecific antisera identified the 13- to 20-kilodalton (kd) molecule as cystatins, the 30-kd molecule as acidic proline-rich proteins (PRPs), the 55-kd molecule as -chain of secretory IgA (sIgA), the 60-kd molecule as -amylase, and the 120-kd molecule as low-molecular-weight mucin (MG2). High-molecular-weight mucin (MG1) was not found in the bracket pellicles (Appendix Table, www.dentalresearch.org).
|
).
Amino Acid Composition Profiles
Amino acid composition profiles of the EBPs from 4 types of brackets differed each other and from UWS. The EBPs generally contained higher proportions of arginine and tryptophan, but less aspartic acid, glutamic acid, and tyrosine. A remarkable difference was found in the proportions of proline and histidine. The EBP on the plastic brackets contained twice as much proline, and the EBP on the PCA brackets contained two to four times as much histidine than those on the other brackets (Table 1).
|
presents binding affinities of streptococci with respect to bracket types, incubation times, and saliva-coating. The results of three-factor ANOVA indicate that main factors, such as bracket types, incubation times, and saliva-coating, have significant effects on the binding affinities of both species, though interaction effects were dependent upon species types.
|
).
Streptococcus mutans
Saliva-coating generally decreased the binding affinity of S. mutans, though binding affinity increased with extended incubation time (p < 0.001). The interaction effects between saliva-coating and incubation times, and between bracket types and incubation times, were statistically significant (p < 0.001). Binding affinities in the non-coated group were increased more than those in the saliva-coated group by extended incubation time. Binding affinity for MCS brackets was increased less than for other brackets by extended incubation time (Table 2).
Bacterial Overlay Assays
Overlay assays of S. gordonii showed that binding was mediated by several salivary proteins: acidic PRPs, a molecule of about 40 kd, -amylase, and MG2 (Fig. 2). In particular, its binding to MG2 was prominent. A 40-kd molecule was thought to be of the proline-rich protein family, which is consistent with this size.
|
). Binding of S. gordonii to acidic PRPs was detected only in the nitrocellulose membrane. In addition, its bindings to a 40-kd molecule and -amylase were more evident in the nitrocellulose membrane. These results can be explained by the physico-chemical surface properties of the membranes. However, none of the salivary proteins contributed to the binding of S. mutans in the solid-phase assays (data not shown). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-amylase, sIgA, and MG2. These salivary proteins are known to act as receptors for bacterial adhesion (Liljemark et al., 1979; Murray et al., 1992; Scannapieco et al., 1993). Therefore, EBPs may offer bacterial binding sites and present a potential risk of dental plaque formation and enamel demineralization.
Gel and amino acid composition profiles showed qualitative and quantitative pellicle differences according to the bracket types (Fig. 1 and Table 1). Additionally, bracket pellicles were found to differ qualitatively from pellicles reported on enamel or other dental biomaterials (Appendix-Table, www.dentalresearch.org). These results reflect the selective nature of salivary protein adsorption to various substrate surfaces.
Interestingly, a difference was found between the protein adsorption patterns of the metal bracket and the bracket metal. MG1 was not present in the EBP formed on metal brackets, but was present in the experimental pellicle formed on bracket metal (raw material) (Lee et al., 2001). This may have been the result of a surface property change caused by bracket fabrication. This result is supported by the finding that the physical and chemical nature of materials significantly affected their physico-chemical surface properties and configuration of the pellicle coating (Baier and Glantz, 1978; Ruan et al., 1986).
Extremely large differences were observed between the binding affinities of the 2 streptococci (Table 2). S. gordonii adhered to the orthodontic brackets by a factor of 10 more than S. mutans. These results suggest that there is an ecological significance of S. gordonii associated with initial colonization on the orthodontic brackets. Despite its low binding affinity, the binding of S. mutans to the bracket surfaces may be an important factor in the development of cariogenic plaque in caries-active individuals or patients with poor oral hygiene, since microbial mass increases primarily as a result of cell division (Brecx et al., 1983).
In non-coated samples, only bracket surface characteristics affect binding affinity. According to thermodynamic rules, bacteria with high surface-free energy prefer high surface-free energy materials (Busscher et al., 1984; Van Dijk et al., 1987). It has been suggested that metal brackets increase bacterial adhesion because of their high surface energy compared with that of plastic and ceramic brackets (Eliades et al., 1995). Therefore, it might be expected that streptococci adhere preferentially to metal brackets, which have higher surface-free energy, because S. gordonii and S. mutans strains have high surface-free energy (Weerkamp et al., 1985; Kilian et al., 1989). The binding affinity of S. mutans for metal brackets was higher than its affinity for other brackets in the non-coated samples. In the case of S. gordonii, however, the binding affinity for non-coated metal brackets was second only to that for the PCA brackets (Table 2). This result may be associated with the surface characteristics of the PCA brackets. Basic amino acids were detected in higher proportions, whereas acidic amino acids were detected in lower proportions in the EBP of PCA brackets (Table 1). S. gordonii may interact specifically with surface charge on the PCA brackets.
Species differences of binding pattern were also detected in the solid-phase assays. S. gordonii DL1 interacted in a specific manner with several salivary proteins, namely, PRPs, -amylase, and MG2 (Fig. 2). The major binding region was associated with MG2 and the minor ones with PRPs and -amylase. These results are consistent with those of previous studies, which showed that PRPs, -amylase (Scannapieco et al., 1995), and MG2 (Murray et al., 1992) were binding receptors for S. gordonii. These specific interactions may explain the increased binding of S. gordonii to saliva-coated brackets. In particular, saliva-coating increased the binding of S. gordonii to plastic brackets more so than to other brackets (Table 2). This could be explained by the higher proportion of acidic PRPs in the gel profiles, the known receptors for S. gordonii, and higher proportions of proline in the amino acid composition profiles of EBPs from the plastic brackets (Fig. 1 and Table 1).
However, the adhesion of S. mutans KPSK-2 was not mediated by any salivary proteins. In fact, saliva-coating tended to decrease its binding affinity (Table 2). This may be explained by the fact that saliva-coating reduces the surface-free energy of the underlying materials (Weerkamp et al., 1985; Quirynen and Bollen, 1995) and partly by the fact that there is no adsorption of MG1 to the orthodontic brackets, which was known to promote the adhesion of S. mutans (Gibbons et al., 1986).
The present study indicates that several salivary proteins adhere to various orthodontic brackets, and some of these proteins play a significant role in the binding of oral bacteria. Information about the relationships between salivary pellicles and oral bacteria will allow us to achieve a better understanding of the adhesion of pathogenic micro-organisms to orthodontic appliances, and help find a means of interfering with the adherence of pathogenic bacteria to the pellicle on orthodontic appliances.
|
ACKNOWLEDGMENTS |
|---|
This article was based on a thesis submitted to the graduate faculty, Seoul National University, in partial fulfillment of the requirements for the PhD degree.
|
FOOTNOTES |
|---|
Received July 30, 2001; Last revision February 25, 2002; Accepted April 4, 2002
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Baier RE, Glantz POJ (1978). Characterization of oral in vivo films formed on different types of solid surfaces. Acta Odontol Scand 36:289–301.[Medline]
Balenseifen JW, Madonia JV (1970). Study of dental plaque in orthodontic patients. J Dent Res 49:320–324.
Bradway SD, Bergey EJ, Jones PC, Levine MJ (1989). Oral mucosal pellicle. Adsorption and transpeptidation of salivary components to buccal epithelial cells. Biochem J 261:887–896.[Medline]
Brecx M, Theilade J, Attström R (1983). An ultrastructural quantitative study of the significance of microbial multiplication during early dental plaque growth. J Periodontal Res 18:177–186.[Medline]
Busscher JH, Weerkamp AH, van der Mei HC, van Pelt AWJ, de Jong HP, Arends J (1984). Measurement of the surface free energy of bacterial surfaces and its relevance for adhesion. Appl Environ Microbiol 48:980–983.
Edgerton M, Levine MJ (1992). Characterization of acquired denture pellicle from healthy and stomatitis patients. J Prosthet Dent 68:683–691.[Medline]
Edgerton M, Lo SE, Scannapieco FA (1996). Experimental salivary pellicles formed on titanium surfaces mediate adhesion of streptococci. Int J Oral Maxillofac Implants 11:443–449.[Medline]
Eliades T, Eliades G, Brantley W (1995). Microbial attachment on orthodontic appliances: I. Wettability and early pellicle formation on bracket materials. Am J Orthod Dentofac Orthop 108:351–360.[Medline]
Gibbons RJ (1989). Bacterial adhesion to oral tissues: a model for infectious diseases. J Dent Res 68:750–760.
Gibbons RJ, Cohen L, Hay DI (1986). Strains of Streptococcus mutans and Streptococcus sobrinus attach to different pellicle receptors. Infect Immun 52:555–561.
Kilian M, Mikkelsen L, Henrichsen J (1989). A taxonomic study of viridans streptococci: description of Streptococcus gordonii sp. nov. and emended descriptions of Streptococcus sanguis (White and Niven 1946), Streptococccus oralis (Bridge and Sneath 1982), and Streptococcus mitis (Andrews and Horder 1906). Int J Syst Bacteriol 39:471–484.
Lee SJ, Kho HS, Lee SW, Yang WS (2001). Experimental salivary pellicles formed on surfaces of orthodontic materials. Am J Orthod Dentofac Orthop 119:59–66.[Medline]
Lee SY, Kim KK, Choe SJ (2001). Binding of oral streptococci to human fibrinogen. Oral Microbiol Immunol 16:88–93.[Medline]
Liljemark WF, Bloomquist CG, Ofstenhage JC (1979). Aggregation and adherence of Streptococcus sanguis: role of human salivary immunoglobulin A. Infect Immun 26:1104–1110.
Mizrahi E (1982). Enamel demineralization following orthodontic treatment. Am J Orthod 82:62–67.[Medline]
Murray PA, Prakobphol A, Lee T, Hoover CI, Fisher SJ (1992). Adherence of oral streptococci to salivary glycoproteins. Infect Immun 60:31–38.
Quirynen M, Bollen CML (1995). 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 22:1–14.[Medline]
Ruan MS, Di Paola C, Mandel ID (1986). Quantitative immunochemistry of salivary proteins adsorbed in vitro to enamel and cementum from caries-resistant and caries-susceptible human adults. Arch Oral Biol 31:597–601.[Medline]
Scannapieco FA, Torres G, Levine MJ (1993). Salivary -amylase: role in dental plaque and caries formation. Crit Rev Oral Biol Med 4:301–307.
Scannapieco FA, Torres GI, Levine MJ (1995). Salivary amylase promotes adhesion of oral streptococci to hydroxyapatite. J Dent Res 74:1360–1366.
Scheie AA (1994). Mechanisms of dental plaque formation. Adv Dent Res 8:246–253.[Abstract]
Shahal Y, Steinberg D, Hirschfeld Z, Bronshteyn M (1998). In vitro bacterial adherence onto pellicle-coated aesthetic restorative materials. J Oral Rehabil 25:52–58.[Medline]
van Dijk J, Herkstroter J, Busscher H, Weerkamp AH, Jensen H, Arends J (1987). Surface free energy and bacterial adhesion. An in vivo study in beagle dogs. J Clin Periodontol 14:300–304.[Medline]
Weerkamp AH, van der Mei HC, Busscher HJ (1985). The surface free energy of oral streptococci after being coated with saliva and its relation to adhesion in the mouth. J Dent Res 64:1204–1210.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| IADR Journals | Advances in Dental Research ® |
| Journal of Dental Research ® | Critical Reviews (1990-2004) |
