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Original Paper

Caries Res 2006;40:172–177 DOI: 10.1159/000091120

Inhibition of Cultivable Bacteria by Chlorhexidine Treatment of Dentin Lesions Treated with the ART Technique

Nazan Kocatas Ersin

a Atac Uzel

b Arzu Aykut

a Umit Candan

a Cemal Eronat

a

Departments of a Pedodontics and b

Biology, Ege University, I zmir , Turkey

TVC, S. mutans and lactobacilli. After 6 months, applica-tion of chlorhexidine exhibited a greater signifi cant re-duction in TVC (p = 0.013), and a signifi cant reduction in S. mutans compared to the nondisinfected group (p ! 0.001). A signifi cant reduction in lactobacilli counts was observed in both groups after 6 months, but the differ-ence between the disinfected and nondisinfected groups was not signifi cant (p = 0.056). ART was found to be ef-fective in reducing the cultivable microfl ora and chlorhex-idine-gluconate-based cavity disinfectant might serve as a suitable additional agent in inhibiting the residual bac-teria in the dentine.

Copyright © 2006 S. Karger AG, Basel

Atraumatic restorative treatment (ART) is a minimal-ly invasive technique to remove soft and demineralized carious dental tissues using hand instruments followed by placement of glass ionomer cement (GIC) as the restor-ative material [Frencken and Holmgren, 1999]. It has been developed for the treatment of caries in parts of world with limited resources but is gaining acceptance in developed countries for the management of early child-hood caries, especially for treatment of very young chil-dren with rampant caries who cannot cooperate suffi -

Key Words Atraumatic restorative treatment � Glass ionomer cement � Chlorhexidine � Disinfectants � Total viable counts � Streptococcus mutans � Lactobacilli

Abstract The aim of this study was to examine the changes in the cultivable microfl ora of carious dentin before and after atraumatic restorative treatment (ART) and investigate the inhibitory effect of chlorhexidine-gluconate-based cavity disinfectant in the microfl ora. Using a split mouth design, 35 primary molar pairs with class II carious le-sions in 35 patients (mean age 7.31 8 0.47 years) were selected. The total viable counts (TVC), Streptococcus mutans and lactobacilli were fi rst measured in the center of the infected demineralized lesion and then from the hard dentine after caries removal by the ART technique. Chlorhexidine-gluconate (2%)-based cavity disinfectant was applied to one of the molar pairs and the other mo-lar received no disinfectant treatment. Thereafter, all of the teeth were restored with glass ionomer cement (GIC). Cavities were reassessed after 6 months and again den-tine samples were microbiologically investigated. Re-moval of carious dentine by ART signifi cantly reduced

Received: January 26, 2005 Accepted after revision: July 18, 2005

Nazan Kocatas ErsinDepartment of PedodonticsSchool of Dentistry, Ege UniversityBornova-Izmir, 35100 (Turkey)Tel. +90 232 388 64 31, Fax +90 232 388 03 25, E-Mail nazan@dent.ege.edu.tr

© 2006 S. Karger AG, Basel0008–6568/06/0402–0172$23.50/0

Accessible online at:www.karger.com/cre

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ciently to allow conventional restorative treatment [Smales and Gao, 2000; Carvalho and Bezerra, 2003]. However, critics of this technique argue that all carious dentine is not removed from the hand-prepared cavity as effectively as by rotary burs [Bjørndal et al.,1997; Weer-heijm and Groen, 1999] and cariogenic bacteria can sur-vive incarceration under GIC restorations [Foley and Blackwell, 2003].

Some fi lling materials have cariostatic properties sup-posedly ensuring reduction of remaining microorgan-isms. It is reported in many studies that GIC has antibac-terial properties due to the release of fl uoride [Benelli et al., 1993; Weerheijm and Groen, 1999]. Studies have re-ported the antibacterial potential of GICs in vitro [Ben-derli et al., 1997; Friedl et al., 1997; Herrera et al., 1999] and reduced plaque formation of Streptococcus mutans on GIC restorations has also been observed in vivo [Svan-berg et al., 1990]. However, only a few studies showed a direct correlation between fl uoride release and antibacte-rial properties of GICs [Palenik et al., 1992; Friedl et al., 1997]. Another study failed to confi rm such a correlation and found no antibacterial activity of GICs at all [Yap et al., 1999].

Various concepts have been used to reduce or elimi-nate microorganisms underneath the restorations. Differ-ent approaches have been described, among them the addition of chlorhexidine to GIC or the application of chlorhexidine-containing disinfectants following cavity preparation are just a few options [Ribeiro and Ericson, 1991; Meiers and Kresin, 1996; Botelho, 2003].

The aims of this study were (1) to examine the changes in the cultivable fl ora before and after ART; (2) to investigate the effectiveness of chlorhexidine-glu-conate-based cavity disinfectant on the microfl ora of den-tin excavated by ART after 6 months underneath a GIC restoration.

Materials and Methods

Study Population This study was conducted at a primary school which was lo-

cated in a rural area of Izmir. Thirty-fi ve healthy students of both sexes with no complicating medical history participated in the study. The children who had taken antibiotics within the last month were excluded. The mean age of the children was 7.31 8 0.47 years, the mean dfs score was 7.60 8 4.93 and the mean plaque score was 1.66 8 0.54 according to Silness and Löe [1964].

The study was approved by the Ethical Committee of Ege Uni-versity, state of Izmir, Turkey. The parents of the children were informed about the aim of this trial well in advance and written signed informed consent was obtained.

Treatment Procedures and Sample Collection The study was based on a split-mouth design. Primary molars

which were clinically asymptomatic and vital with class II carious lesions were selected. The lesions were smaller than one third of the entire tooth crown with the involvement of one approximal surface and were similar in size.

The restorations were performed in the classrooms by two den-tists trained in the ART technique. The other dentist acted as ‘blinded’ evaluator who inspected the completeness of caries re-moval after cavity preparation using the conventional optical and tactile criteria. The evaluator also determined randomly which tooth was assigned to the disinfected or nondisinfected group. Car-ies lesions were removed using only the ART technique according to the recommendations of Frencken and Holmgren [1999].

The tooth was isolated with sterile cotton rolls before sampling to avoid contamination with saliva. The fi rst sample was collected with a sterile ART excavator (Hu-Friedy 131/132, 14-9-8, USA) from the center of the demineralized lesion before starting the re-moval of caries (sample 1). Thereafter, all soft carious dentine was removed from the fl oor of the cavity with the ART instruments. A second sample was collected using a same size sterile excavator from the hard dentin, when cavity preparation was judged to be complete by the evaluator (sample 2). Twenty repeated samples were performed in order to ensure that similar amounts of mate-rial were obtained from the sampling sites (mean 8 SD, 0.30 8 0.04 mg for the demineralized dentin, 0.27 8 0.06 mg for the hard dentin). Teeth with pulpal involvement were excluded. For cavity depth measurements, a sterilized curved endodontic fi le No. 45 was used without its sharp end and with an adapted stop. The measure-ment was transferred to a sterilized endodontic ruler and recorded for all teeth. This procedure guided the later removal of restorative material and collection of the third sample as described by Massara et al. [2002]. The prepared cavity was then cleaned using a moist cotton pellet with distilled water followed by a dry pellet. The cleaned cavity and adjacent pits and fi ssures were conditioned with Ketac-Molar conditioner (3M ESPE Dental Products, St. Paul, Minn., USA) for 30 s. Then, the cavity was rinsed and dried with cotton pellets. Consepsis (Ultradent, South Jordan, Utah, USA), a 2% chlorhexidine-gluconate-based cavity disinfectant was applied to one of the molar pairs after conditioning the cavity. It was left in contact with the dentine for 1 min and air-dried for 10 s. Ketac-Molar (3M ESPE) powder and liquid were prepared following the manufacturer’s instructions and applied using the fi nger press tech-nique. Excess material was removed, occlusal adjustment was per-formed and the restoration was covered with Ketac-Molar glaze and light cured for 10 s. The other tooth was restored with the same restorative material without using the disinfectant.

After 6 months, the teeth were evaluated under clinical condi-tions according to the modifi ed criteria of Ryge [1980] at the Pedi-atric Clinic of Ege University. Rubber-dam was used and GIC was removed with the help of high-speed burs, until almost complete removal of the cement. The deepest layer was removed by cleaving the GIC with a sterile excavator having as reference the depth of the cavity previously determined. It was possible to avoid inadver-tent removal of the dentine adjacent to the GIC, which could change the results. The new samples were collected for microbial analysis taking care to remove them from the same place (sam-ple 3). After the collection of third samples, the teeth were again restored with Ketac-Molar.

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Caries Res 2006;40:172–177 174

Microbiological Sampling and Culture Methods Samples were placed into Eppendorf tubes containing 1 ml of

reduced transport fl uid (RTF) [Syed and Loesche, 1972] and were kept in ice for no longer than 6 h before they were processed in the laboratory. The samples were then vortex-mixed to disperse bacte-rial aggregates and 0.1 ml of the samples were inoculated onto MS agar (MSA, Difco, Detroit, Mich., USA) supplemented with 15% sucrose and bacitracin (0.2 U/ml) (Sigma Chemical Co., St. Louis, Mo., USA) [Gold et al., 1973] for the selective isolation of S. mu-tans and Rogosa Agar (Oxoid, Basingstoke, UK) [Rogosa et al., 1951], a medium selective for lactobacilli. Total viable counts (TVC) of all cultivable bacteria were done on blood agar (brain heart infusion agar, Difco, Detroit, Mich., USA, supplemented with 5% human blood). The plates were incubated in an atmo-sphere containing 5% CO 2 and 95% N 2 at 37 ° C. After 48 h, TVC were assessed on each plate. Two to three isolates representing co-lonial types of S. mutans were subcultured for each sample, and biochemically differentiated by colony morphology, the presence of catalase [Coykendall, 1974], fermentation of inulin, mannitol, melibiose, raffi nose, sorbitol, production of acetoin and dextran, hydrolysis of arginin and esculin [Perch et al., 1974]. S . mutans NCTC 10449 was used as a reference strain. Fine, opaque colonies on Rogosa agar that were gram-positive, catalase-negative, rod-shaped cells were considered to be lactobacilli [Rogosa et al., 1951]. Counts below 10 colony forming units (CFU) were beyond detect-able limits and recorded as 0 (not detectable).

Statistical Analysis All data were analyzed with SPSS version 10.0 (SPSS Inc.,

Chicago, Ill., USA). TVC, S. mutans and lactobacilli were re-corded as CFU/ml of sample. As the microbiological counts were not normally distributed according to the Kolmogorov-Smirnov test, the values were log transformed and a symmetrical distribu-tion was achieved. The numbers of CFU collected in the center of the lesion before starting the removal of caries, from the hard dentin after excavation and from the same place after 6 months were compared using repeated-measures ANOVA and the paired t test between groups; the � 2 test was used for analysis of the cat-egorical variables. The critical level for statistical signifi cance was set as p ! 0.05.

Results

All of the patients were available for recall after 6 months and could be included in the microbiological analysis. All of the restorations were clinically scored as alpha, according to the modifi ed criteria of Ryge [1980] following the 6-month treatment interval.

Table 1 shows the log count means of TVC, S. mutans and lactobacilli recovered before starting the removal of caries (sample 1) and from the hard cavity wall after ex-cavation (sample 2) and after 6 months at the same place (sample 3) in both groups. When the fi rst and second sam-ples were compared, it was observed that the manual re-moval of carious dentine with the ART technique sig-nifi cantly reduced the bacterial counts for TVC, S. mu-tans and lactobacilli in both groups .

The results indicated that cavities which had been treated with 2% chlorhexidine gluconate exhibited sig-nifi cantly lower TVC and S. mutans after 6 months than untreated cavities (sample 2). The reduction in S. mutans under the restorations without use of the disinfectant af-ter 6 months was not statistically signifi cant in compari-son with the counts of S. mutans of the excavated dentin (sample 2). The difference in S. mutans between the two groups was statistically signifi cant (p ! 0.001). The mean number of TVC in the dentine without use of the disin-fectant was signifi cantly reduced in the third sample com-pared with the second sample. However, 2% chlorhexi-dine cavity disinfectant produced a greater reduction in TVC (p = 0.013). The reductions in lactobacilli after 6 months in both groups were statistically signifi cant, but no signifi cant difference was found between the groups (p = 0.056).

After 6 months, TVC were not detected in 4 (11.4%), S. mutans in 7 (20%) and lactobacilli in 18 (51.4%) sam-

Table 1. TVC, S. mutans and lactobacilli recovered from the center of the lesion before starting the removal of caries (sample 1), from the central part of the hard cavity wall after excavation (sample 2) and after 6 months at the same place (sample 3) in the nondisinfected and the disinfected groups

Nondisinfected group Disinfected group

sample 1 sample 2 sample 3 sample 1 sample 2 sample 3

TVC 5.5981.48a 4.5281.87b 3.8781.80c 5.5381.40a 4.4881.95b 2.9281.95c

S. mutans 4.8181.05a 3.6481.52b 2.9881.87b 4.7981.68a 3.5881.64b 1.2781.63c

Lactobacilli 4.4681.75a 3.3781.99b 1.7181.96c 4.5281.99a 3.2881.95b 1.5981.51c

log10 (CFU/ml), means 8 SD. Within each row, means with the same superscript letter are not signifi cantly different.

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ART and Chlorhexidine Caries Res 2006;40:172–177 175

ples and 3 samples (8.6%) had TVC more than 10 6 CFU/ml in the group without usage of the disinfectant. In the disinfected group, TVC were not observed in 9 (25.8%), S. mutans in 20 (57.1%) and lactobacilli in 22 (62.9%) samples and no microorganisms had values more than 10 6 CFU/ml. The results are shown for both groups in fi gure 1 a–c.

Discussion

Previous ART studies showed that the numbers of bac-teria were signifi cantly reduced to proportions that were considered to be of low clinical signifi cance (10 1 –10 3 CFU) [Bonecker et al., 2003; Toi et al., 2003] and com-parable to mechanical cavity preparation, which also does not completely eradicate all microorganisms [Kidd, 1991]. In the present study, it was also shown that ART was effective in reducing TVC, and the numbers of S. mutans and lactobacilli under fi eld conditions after care-ful standardization of methods.

It is reported that GIC has antibacterial properties due to the release of fl uoride which also potentiates reminer-alization, promoting a hardening of the layer of deminer-alized dentin left after lesion curettage [Massara et al., 2002]. However, most of these studies were carried out in vitro [Tam et al., 1997; Francci et al., 1999] or in situ [ten Cate and van Duinen, 1995] and studies carried out in vivo used teeth without dentin lesions [Mukai et al., 1993]. Although the publications about the fl uoride re-lease of GIC were important, they can hardly stimulate the conditions found in biological systems. Furthermore, it was shown in many reports that secondary caries was the most common reason for failure of GICs [Mjör, 1996, 1997; Wilson et al., 1997]. From the systematic review by Randall and Wilson [1999], no conclusive evidence for or against a treatment effect of inhibition of secondary caries by GIC was obtained. These results raised ques-tions about the infl uence of GIC fl uoride on dentin re-mineralization and its role in the antimicrobial properties of GIC.

The present study confi rmed that placement of GIC reduced bacteria in the cavity. Both TVC as well as lac-tobacilli were signifi cantly reduced after 6 months in the cavities in which no antimicrobial treatment was carried out and which were only restored with GIC.

Antibacterial agents are becoming more important in the suppression of the growth of bacteria under restora-tions to minimize the risk of recurrent caries [Lynch, 1996; Wicht et al., 2004]. One of the approaches is the

addition of antibacterial agents to GICs. Some authors [Ribeiro and Ericson, 1991; Sanders et al., 2002; Botelho, 2003] added chlorhexidine and showed the antimicro-bial effect of the GIC and also that chlorhexidine was ef-fective especially against Streptococcus species compared

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Fig. 1. Distribution of bacteria [log 10 (CFU/ml)] among samples after 6 months for the nondisinfected and disinfected group. ) = Nondisinfected; $ = disinfected. a Total viable count. b S. mutans. c Lactobacilli.

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Caries Res 2006;40:172–177 176

with other oral microorganisms as previously reported [Kidd, 1991; Järvinen et al., 1995]. However, it was re-ported that the incorporation of chlorhexidine into GIC resulted in minimal deterioration in the hardness, diam-etral strength and erosion of the materials, not seriously compromising their physical properties [Ribeiro and Er-icson, 1991; Sanders et al., 2002].

Another approach in reducing the potential of residu-al caries is the use of chlorhexidine-containing products as cavity disinfectants after tooth preparation [Meiers and Kresin, 1996]. In the present study, chlorhexidine-gluconate-based cavity disinfectant showed a signifi cant-ly larger inhibitory effect on TVC and a signifi cant reduc-tion in S. mutans after 6 months compared with the non-disinfected group. Complete elimination of TVC and S. mutans was observed in 25.7 and 57.1% of the disinfect-ed samples, respectively, and 68.6% had TVC and 97.1% had S. mutans below the clinically important level in this group after 6 months. It could be suggested that chlorhex-idine could penetrate deep into the tubules, sealing them and presumably enabling long-term release resulting in a larger reduction of microorganisms [Arends et al., 1997]. The reductions of lactobacilli were found in most of the samples of both groups. Since lactobacilli require an acid-ic environment, the low numbers found in both groups showed that the dentine sample environment after ART preparation during 6-month period was unfavorable for caries supported by other authors [Bonecker et al., 2003; Toi et al., 2003]. However, a signifi cant superior effect of

chlorhexidine on lactobacillus counts could not be dem-onstrated. This result was in contrast to Wicht et al. [2004] who found signifi cantly reduced levels of Lactobacillus species, whereas the total viable counts were not signifi -cantly affected after the application of chlorhexidine var-nish as a cavity disinfectant onto the carious dentine. A possible explanation for this might be the different treat-ment procedures and materials. Furthermore, they sug-gested that using a larger sample and a prolonged obser-vation period, chlorhexidine would have a signifi cant in-fl uence on TVC.

Another concern is the principal aspects of the accu-racy of the sampling method. Kidd et al. [1993] empha-sized that excellence in sampling performance was re-quired to make valid comparisons between samples. For this reason, training was carried out followed by repeated weight measurements on the samples obtained. Our re-sults confi rmed that it was possible to compare the sam-ples and there was no statistical difference in the weight measurements of the demineralized and hard dentin sam-ples.

The microbiological and clinical results of this work showed that ART was effective in reducing the cultivable microfl ora even under fi eld conditions. Furthermore, the use of chlorhexidine-gluconate-based cavity disinfectant might serve as a suitable additional agent in reducing the residual microfl ora. However, further clinical trials are necessary to determine its antibacterial and clinical ef-fects in the long term.

References

Arends J, Duschner H, Ruben JL: Penetration of varnishes into demineralized root dentin in vi-tro. Caries Res 1997; 31: 201–205.

Benderli Y, Ulukapi H, Balkanli O, Kulekci G: In vitro plaque formation on some dental fi lling materials. J Oral Rehabil 1997; 24: 80–83.

Benelli EM, Serra MC, Rodrigues AL, Cury JA: In situ anticariogenic potential of glass ionomer cement. Caries Res 1993; 27: 280–284.

Bjørndal L, Larsen T, Thylstrup A: A clinical and microbiological study of deep carious lesions during stepwise excavation using long treat-ment intervals. Caries Res 1997; 31: 411–417.

Bonecker M, Toi C, Cleaton-Jones P: Mutans streptococci and lactobacilli in carious dentine before and after atraumatic restorative treat-ment. J Dent 2003; 31: 423–428.

Botelho MG: Inhibitory effects on selected oral bacteria of antibacterial agents incorporated in a glass ionomer cement. Caries Res 2003; 37:

108–114.

Carvalho CK, Bezerra AC: Microbiological assess-ment of saliva from children subsequent to atraumatic restorative treatment (ART). Int J Paediatr Dent 2003; 13: 186–192.

Coykendall AL: Four types of Streptococcus mu-tans based on their genetic, antigenic and bio-chemical characteristics. J Gen Microbiol 1974; 83: 327–338.

Foley J, Blackwell A: In vivo cariostatic effect of black copper cement on carious dentine. Caries Res 2003; 37: 254–260.

Francci C, Deaton TG, Arnold RR, Swift EJ, Per-digao J, Bawden JW: Fluoride release from restorative materials and its effects on dentin demineralization. J Dent Res 1999; 78: 1647–1654.

Frencken JE, Holmgren CJ: Atraumatic restor-ative treatment (ART) for dental caries. Nij-megen: STI Book, 1999.

Friedl KH, Schmalz G, Hiller KA, Sham M: Resin-modifi ed glass ionomer cements: fl uoride re-lease and infl uence on Streptococcus mutans growth. Eur J Oral Sci 1997; 105: 81–85.

Gold OG, Jordan HV, van Houte JA: Selective me-dium for Streptococcus mutans. Arch Oral Biol 1973; 18: 1357–1364.

Herrera M, Castillo A, Baca P, Carrion P: Antibac-terial activity of glass-ionomer restorative ce-ments exposed to cavity-producing microor-ganisms. Oper Dent 1999; 24: 286–291.

Järvinen H, Pienihakkinen K, Huovinen P, Teno-vuo J: Susceptibility of Streptococcus mutans and Streptococcus sobrinus to antimicrobial agents after short-term oral chlorhexidine treatments. Eur J Oral Sci 1995; 103: 32–35.

Kidd EA, Joyston-Bechal S, Beighton D: Micro-biological validation of assessments of caries activity during cavity preparation. Caries Res 1993; 27: 402–408.

Dow

nloa

ded

by:

Uni

v. o

f Mic

higa

n, T

aubm

an M

ed.L

ib.

141.

213.

236.

110

- 9/

4/20

13 1

0:34

:53

PM

ART and Chlorhexidine Caries Res 2006;40:172–177 177

Kidd EA: Role of chlorhexidine in the manage-ment of dental caries. Int Dent J 1991; 41: 279–286.

Lynch E: Antimicrobial management of primary root carious lesions: a review. Gerodontology 1996; 13: 118–129.

Massara ML, Alves JB, Brandao PR: Atraumatic restorative treatment: clinical, ultrastructural and chemical analysis. Caries Res 2002; 36:

430–436. Meiers JC, Kresin JC: Cavity disinfectants and

dentine bonding. Oper Dent 1996; 21: 153–159.

Mjör IA: Glass-ionomer cement restorations and secondary caries: a preliminary report. Quin-tessence Int 1996; 27: 171–174.

Mjör IA: The reasons for replacement and the age of failed restorations in general dental practice. Acta Odontol Scand 1997; 55: 58–63.

Mukai M, Ikeda M, Yanagihara T, Hara G, Kato K, Nakagaki H, Robinson C: Fluoride uptake in human dentine from glass-ionomer cement in vivo. Arch Oral Biol 1993; 38: 1093–1098.

Palenik CJ, Behnen MJ, Setcos JC, Miller CH: In-hibition of microbial adherence and growth by various glass ionomers in vitro. Dent Mater 1992; 8: 16–20.

Perch B, Kjems E, Ravn T: Biochemical and sero-logical properties of Streptococcus mutans in plaque samples from various human and ani-mal sources. APMIS 1974; 82: 357–370.

Randall RC, Wilson NHF: Glass-ionomer restor-atives: A systematic review of a secondary car-ies treatment effect. J Dent Res 1999; 78: 628–637.

Ribeiro J, Ericson D: In vitro antibacterial effect of chlorhexidine added to glass-ionomer ce-ments. Scand J Dent Res1991; 99: 533–540.

Rogosa M, Mitchell JA, Wiseman RF: A selective medium for the isolation and enumeration of oral lactobacilli. J Dent Res 1951; 30: 682–689.

Ryge G: Clinical criteria. Int Dent J 1980; 30: 347–358.

Sanders BJ, Gregory RL, Moore K, Avery DR: An-tibacterial and physical properties of resin modifi ed glass-ionomers combined with chlor-hexidine. J Oral Rehabil 2002; 29: 553–558.

Silness J, Löe H: Periodontal disease in pregnancy. II. Correlation between oral hygiene and peri-odontal condition. Acta Odontol Scand 1964;

22: 121–135. Smales RJ, Gao W: In vitro caries inhibition at the

enamel margins of glass ionomer restoratives developed for the ART approach. J Dent 2000;

28: 249–256. Svanberg M, Krasse B, Ornerfeldt HO: Mutans

streptococci in interproximal plaque from amalgam and glass ionomer restorations. Car-ies Res 1990; 24: 133–136.

Syed SA, Loesche WJ: Survival of human dental plaque fl ora in various transport media. Appl Microbiol 1972; 24: 638–644.

Tam LE, Chan GP, Yim D: In vitro caries inhibi-tion effects by conventional and resin-modi-fi ed glass-ionomer restorations. Oper Dent 1997; 22: 4–14.

ten Cate JM, van Duinen RN: Hypermineraliza-tion of dentinal lesions adjacent to glass-iono-mer cement restorations. J Dent Res 1995; 74:

1266–1271. Toi CS, Bönecker M, Cleaton-Jones PE: Mutans

streptococci strains prevalence before and after cavity preparation during atraumatic restor-ative treatment. Oral Microbiol Immunol 2003; 18: 160–164.

Weerheijm KL, Groen HJ: The residual caries di-lemma. Community Dent Oral Epidemiol 1999; 27: 436–441.

Wicht MJ, Haak R, Schutt-Gerowitt H, Kneist S, Noack MJ: Suppression of caries-related mi-croorganisms in dentine lesions after short-term chlorhexidine or antibiotic treatment. Caries Res 2004; 38: 436–441.

Wilson NH, Burke FJ, Mjör IA: Reasons for place-ment and replacement of restorations of direct restorative materials by a selected group of practitioners in the United Kingdom. Quintes-sence Int 1997; 28: 245–248.

Yap AU, Khor E, Foo SH: Fluoride release and antibacterial properties of new-generation tooth-colored restoratives. Oper Dent 1999;

24: 297–305.

Dow

nloa

ded

by:

Uni

v. o

f Mic

higa

n, T

aubm

an M

ed.L

ib.

141.

213.

236.

110

- 9/

4/20

13 1

0:34

:53

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