Antibacterial Effect of Chlorhexidine- Containing Glass Ionomer Cement in vivo: A Pilot Study

Abstract

This in vivo pilot study was carried out to test the antibacterial effect of glass ionomer containing chlorhexidine (test group) in comparison to conventional glass ionomer (control group). Fifty 6- to 11-year-old children with one occlusal lesion in a molar were randomly allocated to test and control groups in a parallel-group design. The cavity walls and one half of the floor were cleaned and restored with one of the materials without dentine conditioning. The restorations were removed after 7 days. Dentine samples were taken from the cleaned (affected dentine) and noncleaned area (infected dentine) at baseline and at day 7. Samples were anaerobically and aerobically cultivated for mutans streptococci, lactobacilli and total viable bacterial count (TVC) following common laboratory procedures. ANCOVA was used to test for treatment effects. Seven days after treatment, a significant decrease in anaerobic and aerobic bacterial counts (p = 0.0001) was shown. Lower numbers of anaerobic lactobacilli (p = 0.02), TVC (p = 0.008) and aerobic lactobacilli and TVC (p = 0.03), but not of mutans streptococci, were indicated in the test group compared to the control group. A significant reduction in aerobic lactobacilli from infected dentine treated with the glass ionomer containing chlorhexidine (p = 0.05) was observed whereas in affected dentine, anaerobic mutans streptococci, lactobacilli and TVC and aerobic TVC and mutans streptococci were significantly lower in the test group 7 days after treatment (p = 0.01). We conclude that the present pilot study revealed lower microorganism counts in chlorhexidine-containing glass ionomers than in conventional glass ionomers for both affected and infected dentine over a 7-day period.

Full text

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Original Paper
Caries Res 2007;41:102–107
DOI: 10.1159/000098042
Antibacterial Effect of Chlorhexidine-
Containing Glass Ionomer Cement in vivo:
A Pilot Study
J.E. Frencken
a
S. Imazato
c
C. Toi
d
J. Mulder
b
S. Mickenautsch
e
Y. Takahashi
c
S. Ebisu
c
a
WHO Collaborating Centre for Oral Health Care Planning and Future Scenarios and
b
Department of
Preventive and Restorative Dentistry, University of Nijmegen, Nijmegen , The Netherlands;
c
Department of
Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, Osaka , Japan;
d
Dental Research Institute, Faculty of Health Sciences, and
e
Division of Public Oral Health, University of the
Witwatersrand, Johannesburg , South Africa
cilli and TVC (p = 0.03), but not of mutans streptococci, were
indicated in the test group compared to the control group.
A significant reduction in aerobic lactobacilli from infected
dentine treated with the glass ionomer containing chlorhex-
idine (p = 0.05) was observed whereas in affected dentine,
anaerobic mutans streptococci, lactobacilli and TVC and aer-
obic TVC and mutans streptococci were significantly lower
in the test group 7 days after treatment (p = 0.01). We con-
clude that the present pilot study revealed lower microor-
ganism counts in chlorhexidine-containing glass ionomers
than in conventional glass ionomers for both affected and
infected dentine over a 7-day period.
Copyright © 2007 S. Karger AG, Basel
Minimal intervention approaches for managing den-
tal caries have gained importance in the last decade. One
such minimal intervention approach is called the atrau-
matic restorative treatment (ART). This approach relies
on hand instruments for removing infected carious tooth
tissues and on adhesive restorative materials to fill the
cavity and adjacent pits and fissures [Frencken et al.,
1996]. ART has the potential to provide preventive and
largely pain-free restorative care to a large segment of the
Key Words
Glass ionomer, antibacterial  Chlorhexidine  Atraumatic
restorative treatment  Residual caries
Abstract
This in vivo pilot study was carried out to test the antibacte-
rial effect of glass ionomer containing chlorhexidine (test
group) in comparison to conventional glass ionomer (con-
trol group). Fifty 6- to 11-year-old children with one occlusal
lesion in a molar were randomly allocated to test and control
groups in a parallel-group design. The cavity walls and one
half of the floor were cleaned and restored with one of the
materials without dentine conditioning. The restorations
were removed after 7 days. Dentine samples were taken
from the cleaned (affected dentine) and noncleaned area
(infected dentine) at baseline and at day 7. Samples were an-
aerobically and aerobically cultivated for mutans strepto-
cocci, lactobacilli and total viable bacterial count (TVC) fol-
lowing common laboratory procedures. ANCOVA was used
to test for treatment effects. Seven days after treatment, a
significant decrease in anaerobic and aerobic bacterial
counts (p = 0.0001) was shown. Lower numbers of anaerobic
lactobacilli (p = 0.02), TVC (p = 0.008) and aerobic lactoba-
Received: December 20, 2005
Accepted after revision: July 19, 2006
Dr. J.E. Frencken
WHO Collaborating Centre for Oral Health Care Planning and Future Scenarios
PO Box 9101
NL–6500 HB Nijmegen (The Netherlands)
Tel. +31 24 361 4050, Fax +31 24 354 0265, E-Mail j.frencken@dent.umcn.nl
© 2007 S. Karger AG, Basel
0008–6568/07/0412–0102$23.50/0
Accessible online at:
www.karger.com/cre

Antibacterial Effect of Glass Ionomer
with Chlorhexidine
Caries Res 2007;41:102–107
103
world population at low cost [Yip et al., 2001; Micke-
nautsch et al., 2002; Schriks and van Amerongen,
2003].
Removing infected dentine using hand excavators re-
sults in a steep decline of cultivable microorganisms in
the cavitated dentine lesion, sometimes to a level of no
detection [Massara et al., 2002; Toi et al., 2003]. This im-
plies, however, that not all cultivable microorganisms are
always removed [Smales et al., 1998; Nadanovsky et al.,
2001; Santiago et al., 2005]. Whether the residual infected
dentine will cause the caries process to progress further
is equivocal. Some researchers call for total removal of all
caries-affected tissues [Weerheijm et al., 1999] whereas
others have shown that the carious lesion does not
progress if residual caries is left behind under properly
sealed restorations [Mertz-Fairhurst et al., 1998; Massara
et al., 2002; Maltz et al., 2002; Kidd, 2004]. So far, re-
search has shown that few ART restorations fail because
of secondary caries development over a period of 6 years
[Mandari et al., 2003; Frencken et al., 2006; Lo et al., ac-
cepted].
Notwithstanding the low percentage of secondary car-
ies reported, the survival of ART restorations would most
probably increase and, consequently, the need for refill/
repair would most probably decrease if more microor-
ganisms could be killed in the process of cavity cleaning.
Recently, an in vitro study showed a slight caries-inhibit-
ing effect of a chlorhexidine-containing glass ionomer
without compromising its physical characteristics [Taka-
hashi et al., 2006].
The aim of the present pilot study was to test in vivo
the antibacterial effect of this chlorhexidine-containing
glass ionomer in comparison to a non-chlorhexidine-
containing glass ionomer.
Materials and Methods
Study Population and Treatment Allocation
The present study was carried out at the mobile dental ser-
vices of the Division of Public Oral Health of the Witwatersrand
University in Johannesburg, South Africa, in October 2004. The
ethics committee of the University approved the study proposal
under protocol No. 40442. The study inclusion criteria were:
(1) healthy people with at least 1 sizable cavity in occlusal sur-
faces in different quadrants of primary or permanent teeth and
(2) a signed written consent of the individual or guardian. People
having tooth cavities with (expected) pulpal involvement and
those medically compromised were excluded. As the present
study was of a pilot nature, the sample size was not determined
on the basis of a power calculation.
A parallel-group study design was chosen. Allocation of the
test material to the individual was done on an alternating basis.
The start of the allocation sequence was based on the outcome of
a flip of a coin. The selection of individuals took place at the out-
patient department of the dental school by one instructed staff
member. Eligible tooth cavities in included individuals were X-
rayed to confirm absence of pulpal involvement. A staff member
of the Public Oral Health Division (S.M.) performed the treat-
ment. Prior to this, the study individuals were provided with oral
health education messages and emergency care if needed. Staff
members of the Division of Public Oral Health provided addi-
tional oral care at the end of the experimental period.
Restorative Materials Used
The experimental glass ionomer was prepared by incorporat-
ing chlorhexidine diacetate (Sigma Aldrich, Steinheim, Germa-
ny) into the powder of the control glass ionomer at 1% (w/w). The
control material was a conventional restorative glass ionomer
(Fuji IX, GC, Tokyo, Japan).
Treatment Procedures
The treatment was carried out by one dentist (S.M.) in a sub-
urban area of Johannesburg in the morning. The treatment pro-
cedure followed those described by Massara et al. [2002]. The
teeth were isolated with cotton wool rolls. The cavity opening was
enlarged if needed with a sterile hatchet. The cavity walls were
then cleaned using sterile small-size excavators. In order to assess
the effect of the experimental material, the floor of the cavity was
arbitrarily divided into a mesial and distal part. Infected dentine
was removed from the distal part only with a small excavator.
Thereafter, a baseline sample from the cleaned (i.e. affected) dis-
tal and the noncleaned (i.e. infected) mesial part were taken using
a sharp small-size sterile excavator. The extent and depth of the
cavity were recorded using an endodontic file. The cavity was re-
stored with either test or control material but without condition-
ing the tooth surfaces.
Patients were recalled after 7 days. Using the previously re-
corded cavity depth and extension as reference points, the resto-
ration was removed with the aid of a high-speed diamond bur
until the deepest part was reached. The remaining part of the res-
toration was removed with a sterile hand excavator. Thereafter, a
sample was taken from the distal (affected dentine) part and from
the mesial (infected dentine) part using a sterile sharp small-size
hand excavator. The cavities were completely cleaned by hand ex-
cavation and restored using the normal ART procedure. The col-
lected samples were placed in preweighed microcentrifuge tubes
(Elkay, Costelloe, Co. Galway) containing 1 ml of reduced trans-
port fluid (RTF) [Syed and Loesche, 1972], fortified with 20%
(v/v) fetal calf serum and 15% (v/v) glycerol. During collection, all
dentine samples were kept cool in a closed cooling box, contain-
ing frozen ice packs. The microbiology samples were transported
to the laboratory where tubes were placed in the freezer and pro-
cessed within 6 h of return. At the laboratory the tubes were re-
weighed and the final weight was calculated to estimate the mass
of the dentine sample in milligrams per milliliter of RTF.
Microbiological Procedures
Bacterial Cultivation
The dentine samples were homogenized with a vortex mixer
(MT19 Chiltern, London, UK) for 30 s and log diluted by 1.0 !

Frencken /Imazato /Toi /Mulder /
Mickenautsch
/Takahashi /Ebisu
Caries Res 2007;41:102–107
104
10
1
in phosphate-buffered saline. All samples were inoculated
with a spiral plater Model D (Spiral System, Cincinnati, Ohio,
USA) onto trypticase-yeast-cystine-bacitracin agar supplement-
ed with 15% sucrose and bacitracin (0.2 U/ml) [Van Palenstein
Helderman et al., 1983] for the selective isolation of mutans strep-
tococci, and Rogosa agar (Oxoid, Basingstoke, UK), a medium
selective for lactobacilli [Rogosa et al., 1951]. The total viable
count (TVC) of all predominant bacteria was determined on tryp-
tone soy agar (Oxoid) supplemented with 5% (v/v) horse blood.
Duplicate sets of plates were prepared for each sample of which
one was incubated aerobically and the other anaerobically in an
atmosphere containing 10% CO
2
(Carbon Dioxide Gas Generat-
ing Kit, Oxoid) at 37
° C for a total of 3 days. Plates selectively in-
oculated for mutans streptococci and lactobacilli were allowed to
develop at room temperature for 24 h to enhance colony develop-
ment. TVC were made for each bacterial species. Bacterial counts
were recorded as colony-forming units (CFU) per milliliter of
RTF. Counts below 10 CFU were beyond detectable limits and re-
corded as 0 = not detectable.
Identification of Bacterial Species
Colonies of mutans st reptococci were morphot yped according
to the criteria described by Emilson [1983] and van Palenstein
Helderman et al. [1983], and biochemical fermentation tests
[Shklair and Keene, 1974] were used to verify identification. On
Rogosa agar, lactobacillus species were recognized as fine, opaque
colonies, which appeared microscopically as Gram-positive rod-
shaped cells, with a negative catalase reaction.
Statistical Analyses
The bacterial counts were calculated to reflect the number of
CFU per milligram dentine and log transformed (log
10
– 1) to
normalize the data. The independent variables were treatment
group (test/control) and type of dentine (infected/affected). The
dependent variables were mutans streptococci, lactobacilli and
TVC that were cultured anaerobically and aerobically. An analy-
sis of covariance (ANCOVA) using the general linear model pro-
cedure (SAS version 9.1, SAS Institute, Cary, N.C., USA) was used
to compare the levels of bacterial counts by type of dentine at
baseline and the effect of the treatment on bacterial numbers 7
days after treatment. Statistical significance was set at  = 0.05.
R e s u l t s
Background Information
Fifty individuals, each with one eligible cavity equally
divided over the test and control group, participated in
the pilot study. The ages of the participants varied from
6 to 11 years. Both study groups consisted of 2 primary
and 23 permanent molars each containing a sizable cav-
ity in the occlusal surface.
Quantity of Dentine Sampled
The mean weight of dentine in milligrams sampled
from carious teeth is shown in table 1 . The quantity of
dentine collected ranged from a minimum of 0.1 mg to
134 mg per cavity sampled. A comparison using 2-way
ANOVA showed no significant difference (p = 0.37) be-
tween the weights of affected and infected dentine col-
lected at baseline and day 7, or between the two test ma-
terials.
Microbiology Outcomes at Baseline and 7 Days after
Treatment
The mean number and standard deviation of anaero-
bically and aerobically cultured TVC, mutans strepto-
cocci and lactobacilli by type of dentine, treatment group
and time are presented in table 2 . A comparison between
infected and affected dentine at baseline showed that the
numbers of anaerobic and aerobic TVC (p = 0.0001), mu-
tans streptococci (p = 0.0001) and lactobacilli (p = 0.004)
were significantly higher in infected compared with af-
fected dentine in both treatment groups. Only anaerobic
lactobacilli (p = 0.04) and aerobic TVC (p = 0.02) were
higher in infected than affected dentine on day 7.
Seven days after treatment, a significant decrease in
anaerobic and aerobic bacterial counts (p = 0.0001) was
shown. Lower numbers of anaerobic lactobacilli (p =
Table 1. The mean weight (mg), standard deviation (SD) and range of dentine by type of dentine, treatment
group and time
Type of dentine Treatment group n Day 0 Day 7
mean 8 SD
range
mean 8 SD
range
Infected GIC 25
26.0830.8
1.0–117.0
22.4830.1
2.0–134.0
GIC/CHX 25
30.7835.4
0.1–131.0
18.0822.4
0.1–88.0
Affected GIC 25
25.7824.3
0.1–85.0
25.6826.8
1.0–113.0
GIC/CHX 25
25.0825.7
0.1–94.0
14.5826.1
0.1–96.0
GIC = Glass ionomer; GIC/CHX = glass ionomer containing chlorhexidine.

Antibacterial Effect of Glass Ionomer
with Chlorhexidine
Caries Res 2007;41:102–107
105
0.02), TVC (p = 0.008) and aerobic lactobacilli and TVC
(p = 0.03), but not of mutans streptococci, were indicated
in the test material compared to the control group.
Microbiology Outcomes over the 7-Day Test Period
A statistically significant association between baseline
and 7 days after treatment, and type of dentine was evi-
dent in the numbers of anaerobic mutans streptococci
(p = 0.0001), TVC (p = 0.005) and in aerobically cultured
mutans streptococci (p = 0.004). In addition, TVC from
aerobic culture differed significantly between baseline
and 7 days after treatment, and between the control and
test material (p = 0.04).
Contrasts made between the type of dentine and test
material showed a significant reduction in aerobic lacto-
bacilli from infected dentine treated with the glass iono-
mer containing chlorhexidine (p = 0.05), whereas in af-
fected dentine, anaerobic mutans streptococci, lactoba-
cilli and TVC and aerobic TVC and mutans streptococci
were significantly lower in the test group 7 days after
treatment (p = 0.01; table 3 ).
Table 2. The mean number and standard deviation of log-transformed anaerobic and aerobic mutans streptococci, lactobacilli and
TVC (CFU/mg) by type of dentine, treatment group and time at baseline (0) and on day 7
Type of
dentine
Treatment
group
n TVC Mutans streptococci Lactobacilli
day 0 day 7 day 0 day 7 day 0 day 7
Anaerobic
Infected GIC 25
4.6881.16 2.5781.35 3.3480.99 1.4280.80 3.2781.38 1.9881.17
GIC/CHX 25
4.2681.30 1.9281.46 3.4081.59 1.0980.98 2.8381.89 1.3681.28
Affected GIC 25
2.9881.77 2.3381.37 1.8981.37 1.4880.80 2.0581.71 1.5980.94
GIC/CHX 25
2.9281.57 1.3581.14 2.1181.38 0.7880.73 2.0581.23 0.8880.75
Aerobic
Infected GIC 25
4.2581.35 2.5781.21 2.0281.06 1.1780.60 2.7581.43 1.8281.01
GIC/CHX 25
3.9881.61 1.7681.48 1.9581.24 0.9580.79 2.6981.74 1.2381.05
Affected GIC 25
2.6981.82 2.0481.26 1.2280.76 1.2780.64 1.9281.61 1.5980.85
GIC/CHX 25
2.8981.46 1.1381.04 1.3080.77 0.8080.75 1.7381.16 0.8780.74
GIC = Glass ionomer; GIC/CHX = glass ionomer containing chlorhexidine.
Table 3. The percentage reduction and reduction in mean number and standard deviation (SD) of log-trans-
formed mutans streptococci, lactobacilli and TVC (CFU/mg) by type of dentine, treatment group and method
of incubation over the 7-day test period
Type of
dentine
Treatment
group
n TVC Mutans streptococci Lactobacilli
mean 8 SD
%
mean 8 SD
%
mean 8 SD
%
Anaerobic
Infected GIC 25
2.1181.26
45
1.9280.90
57
1.2981.28
39
GIC/CHX 25
2.3481.38
55
2.3181.29
68
1.4781.59
52
Affected GIC 25
0.6581.57
22
0.4181.06
22
0.4681.33
22
GIC/CHX 25
1.5781.36
54
1.3381.06
63
1.1780.99
57
Aerobic
Infected GIC 25
1.6881.28
40
0.8580.83
42
0.9381.22
34
GIC/CHX 25
2.2281.55
57
1.0081.01
51
1.4681.46
54
Affected GIC 25
0.6581.54
24
–0.0580.70
–4
0.3381.23
17
GIC/CHX 25
1.7681.25
61
0.580.76
38
0.8680.95
50
GIC = Glass ionomer; GIC/CHX = glass ionomer containing chlorhexidine.

Frencken /Imazato /Toi /Mulder /
Mickenautsch
/Takahashi /Ebisu
Caries Res 2007;41:102–107
106
Discussion
The present pilot study was carried out to investigate
whether a chlorhexidine-containing glass ionomer in-
hibited the growth of microorganisms left in infected
dentine under a restoration more than a conventional
glass ionomer. The mean values for the microbiological
variables were lower from affected than from infected
dentine at baseline. This provides evidence that the den-
tine samples were indeed taken from two different parts
within the dentine lesion and that infected dentine was
left behind. The operator had no difficulties in imple-
menting the clinical and microbiological protocol.
The amounts of infected and affected dentine collect-
ed at baseline and on day 7 were not significantly differ-
ent. This suggests that a similar mass of dentine was sam-
pled at each collection.
The two treatment groups did not differ significantly
at baseline for the microbiological variables under study.
Although baseline levels of anaerobic and aerobic mutans
streptococci, lactobacilli and TVC were similarly higher
in infected dentine compared with affected dentine, the
significant reduction in bacterial counts was more evi-
dent in affected dentine treated with the test material af-
ter 7 days. The mean numbers of mutans streptococci,
lactobacilli and TVC were clearly associated with the type
of dentine sampled, the test material and the number of
days after treatment. In addition, the response of the bac-
terial species investigated to the test materials suggests a
variance in sensitivity to the restorative materials. It
seems that a change in environment by restoring the
tooth cavity leads to a change in survival of microorgan-
isms that remain. Results of absolute microbiology values
in this study could not be related to other investigations
since the sampling design is unique in that data are pre-
sented from both affected and infected cav ity sites. Under
the current study setup with a low number of subjects,
chlorhexidine-containing glass ionomers have a superior
effect in inhibiting growth of microorganisms left under
restorations compared to conventional glass ionomers.
The finding in the present study is in line with those
reported by others, using anaerobic incubation. A marked
reduction in microorganisms, many to an ‘undetectable’
level, in carious dentine has been reported under tempo-
rary [Bjørndal et al., 1997; Maltz et al., 2002], glass iono-
mer [Weerheijm et al., 1993; Massara et al., 2002], resin
composite [Weerheijm et al., 1993] and resin-modified
glass ionomers and amalgam restorations [Kreulen et al.,
1997]. Considering the presence of a significant reduc-
tion in microorganisms over time, it is legitimate to ques-
tion the necessity for complete removal of infected den-
tine. Although evidence points towards a stop to the car-
ies process in incompletely cleaned and restored cavities,
many dental operators have difficulties in accepting this
evidence and would like to know the fate of the remain-
ing bacteria as questioned by Kidd [2004].
We conclude that the present pilot study showed a sta-
tistically significant difference in reduction of microor-
ganisms between chlorhexidine-containing glass iono-
mers and conventional glass ionomers for both affected
and infected dentine over a 7-day period. The difference
of the relative reductions in microorganisms between the
two types of glass ionomer warrants a follow-up study.
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