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Abstract
Introduction: The use of root canal lling materials
with antibacterial activity can be considered benecial
to reduce the remaining microorganisms in the root ca-
nal system, where Enterococcus faecalis is often
found, and prevent recurrent infection. The aim of this
study was to evaluate the antimicrobial activity and ca-
pacity for inhibiting E. faecalis biolm formation of AH
Plus, alone and mixed with chlorhexidine (CHX),
cetrimide (CTR), and combinations of the two.
Methods: AH Plus alone and mixed with 1% and 2%
CHX, 0.1%0.5% CTR, and combinations of both
were tested to assess antimicrobial activity by a
modied direct contact test and determine inhibition
of E. faecalis biolm formation at 24 hours. The results
were expressed as log
10
viable counts. Eradication and
inhibition of biolm formation were understood as no
bacterial growth or log
10
reduction = 5 with respect to
the control (AH Plus alone). Results: AH Plus + CHX
showed a low antimicrobial activity with respect to
the control (at 2%, log
10
reduction = 1.30). None of
the tested concentrations achieved eradication or
inhibition of biolm. AH Plus + CTR showed a
direct relationship of concentration-antimicrobial effect,
reaching a log
10
reduction of 2.92 at 0.5%and inhibition
of biolm formation at 0.2%. With the combination
CHX + CTR, lower concentrations were needed for the
same effect, and eradication and inhibition of biolm
were achieved. Conclusions: The addition of CHX,
CTR, or some combination of both to AH Plus confers
it with bactericidal and anti-biolm activity against
E. faecalis. (J Endod 2014;40:977981)
Key Words
AH Plus, biolm, cetrimide, chlorhexidine, direct contact
test, Enterococcus faecalis
T
he most important objectives of endodontic treatment are the elimination of
microorganisms from the root canal system and the prevention of subsequent
reinfection. However, in some cases the complete elimination of bacteria from
the root canal system is impossible (1). The use of root canal lling materials with
antibacterial activity can reduce the number of remaining microorganisms (2, 3),
prevent recurrent root canal infection, and aid the repair process of apical and
periapical tissues (4). Enterococcus faecalis is often found in asymptomatic
failing root canaltreated teeth, in primary necrotic cases, and in persistent apical
periodontitis (5, 6). When grown as a biolm in the root canal, these bacteria
penetrate into dentinal tubules and resist nutritional deprivation (5).
A great variety of endodontic sealers are available commercially; most of them are
based on zinc oxideeugenol, epoxy resin, calciumhydroxide, and glass ionomers (7).
AH Plus (Dentsply DeTrey, Konstanz, Germany) is an epoxy resinbased root canal
sealer with good physical properties, good adhesion to dentin, and excellent uidity
and stability in aqueous solution (8). Aside from being biocompatible (9, 10), it is
not genotoxic (11) and exhibits good tissue tolerance, sealing ability, and long-term
dimensional stability (12). Its antimicrobial properties have been shown to be active
against Staphylococcus aureus, Escherichia coli, Streptococcus mutans, and
Staphylococcus epidermidis (4).
Chlorhexidine (CHX) is a potent antimicrobial frequently used in endodontics
(13, 14). Vianna et al (15) report that it is able to inactivate many endodontic-
resistant organisms in as little as 15 seconds of contact time. Cetrimide (CTR) is a
cationic surfactant that has demonstrated its ability to eradicate E. faecalis biolm
in vitro (14) and ex vivo (16, 17).
In the last decade, antimicrobial agents have been incorporated into dental
materials to lend them antimicrobial activity (18, 19). The incorporation of CHX
and/or CTR into glass ionomer cement (GIC) (2022) is known to confer it with
benecial antibacterial properties. It was recently shown that the incorporation of
benzalkonium chloride and cetylpyridinium chloride into endodontic cements
improves their antibacterial effect on S. mutans, Lactobacillus casei, and
Actinomyces viscosus (23). In previous research, the authors of the present study
showed that the addition of 1% and 2% CHX, 0.1% up to 0.5% CTR, or combinations
of the two does not signicantly alter some important physical properties of AH Plus
(24). To evaluate the antimicrobial effects of endodontic sealers and pastes, the agar
diffusion test is widely used (4, 22, 23). Because this technique presents several
limitations, Weiss et al (25) described a direct contact test (DCT) assay designed to
overcome them. The DCT quantitatively assesses the effect of direct contact between
organisms and materials (26). Because bacteria in the root canal grow as biolm
From the *University of Granada, Campus de Cartuja, Colegio Maximo s/n;
Department of Preventive Dentistry, School of Dentistry, University of Granada, Campus
de Cartuja, Colegio Maximo s/n;
Department of Paediatric Dentistry, School of Dentistry, University of Granada, Campus de Cartuja, Colegio Maximo s/n; and
Department of Dental Pathology and Therapeutics, School of Dentistry, University of Granada, Campus de Cartuja, Colegio Maximo s/n, Granada, Spain.
Address requests for reprints to Dr Matilde Ruiz-Linares, Department of Stomatology, School of Dentistry, Campus de Cartuja, Colegio Maximo s/n, E-18071 Granada,
Spain. E-mail address: matr@ugr.es
0099-2399/$ - see front matter
Copyright 2014 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2013.11.020
Basic ResearchTechnology
JOE Volume 40, Number 7, July 2014 Antibacterial Activity of AH Plus 977
(27), particularly E. faecalis, it is necessary to determine the antimicro-
bial activity of materials that are able to inhibit its formation. Therefore,
the aim of this study was to evaluate the antimicrobial activity and
capacity of inhibiting E. faecalis biolm formation of the AH Plus sealer
mixed with either CHX or CTR and combined with both.
Materials and Methods
In both tests, antimicrobial activity and biolm formation, AH Plus
was used alone as the control group and combined with CHX
digluconate (liquid, 1% and 2%) (Guinama, Alboraya, Spain), CTR
(powder, 0.1%0.5%) (Sigma-Aldrich, Steinheim, Germany), and
both (concentrations of combinations are shown in Tables 1 and 2).
Concentrations of 10% and 20% CHX digluconate were combined
with AH Plus in a ratio 1/10 (v/v). CTR powder was added to AH Plus
in ratios ranging from 1/1001/20 (w/w) to obtain concentrations
from 0.1%0.5%. Combinations of both antimicrobials were prepared
with the different concentrations of CHX and CTR (v/w) and were added
in the same ratios to AH Plus as described above. All these procedures
were performed under sterile conditions in a laminar ow chamber.
In both tests the materials were left to set for 48 hours (24) in sterile
conditions.
The bacterial suspensions were prepared following the same
methodology for all experiments. From a subculture of E. faecalis
ATCC 29212, colonies were suspended in brain-heart infusion (BHI)
broth to match the optical density of the 1.0 McFarland standard
(3 10
8
colony-forming units [CFU] per mL), determined by using
a calibrated turbidimeter. This suspension was diluted 30-fold in
broth to obtain an initial bacterial suspension of approximately
1 10
7
CFU/mL to be used for DCT and biolm determination. The
experiments were performed under sterile conditions in a laminar
ow chamber (model Bio-II-B; Telstar S.A., Terrasa, Spain).
Antimicrobial Activity Test
To test the antimicrobial activity of materials, we used a method-
ology based on the DCT (25) adapted by Zhang et al (2). A 96-well
microtiter plate was used. To standardize the area of material tested,
a customized silicon mold was made to t inside each well in the plate,
leaving a small area on one side to allow us to introduce the material
always in equal amounts. The materials were introduced by using a
sterile syringe-needle system (BD DISCARDIT II; Becton Dickinson
S.A., Fraga, Huesca, Spain) in the space between the well and
customized silicon mold, and the material was left to set. Thereafter,
the molds were removed from each well, and the 96-well microtiter
plate was held vertically so that 10 mL of the bacterial suspension of
E. faecalis could be carefully placed over the materials. After incubation
at 37