Antibacterial and Anti-biolm Activity of AH Plus

with Chlorhexidine and Cetrimide

M. Estela Bail on-S anchez, DDS,* Pilar Baca, DDS, MD, PhD,

Matilde Ruiz-Linares, DDS, PhD,

and Carmen Mara Ferrer-Luque, DDS, MD, PhD

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

C for 1 hour in a humid atmosphere, ensuring contact between

the bacterium and the material, we turned the plate to a horizontal
position; then 220 mL BHI was added to each well and gently mixed
with the pipette for 1 minute. Finally, 50 mL was taken from each
well, and serial dilutions were performed and plated for viable cell
counting. Each material was tested twice, and the procedure was
performed in duplicate (n = 4/group). Four wells with AH Plus alone
and noninfected were included as negative controls.
Biolm Formation on Material Tested
To study E. faecalis biolm formation, we used a methodology
based on the Calgary biolm device (commercially available as the
MBEChigh throughput [HTP]; Innovotech, Edmonton, Alberta,
Canada), originally described by Ceri et al (28) and adapted and
modied for E. faecalis (6) and the materials tested.
The top half of the MBEC-HTP is a lid with 96 identical pegs, in
this case used to make the silicon molds, which were sterilized for
45 minutes in hydrogen peroxide gas plasma (Sterrad-50; Advanced
Sterilization Products, Johnson & Johnson, Irvine, CA). Each material
was introduced in the molds to obtain identical cylinders. The
dimensions of each cylinder were 6 mm (height) by 3.90 mm
(maximum diameter). The amount of sealer in each cylinder was
approximately 0.065 mL. A sterile orthodontic wire 1 cm in length
was placed in each cylinder while the material set, always leaving
approximately 3 mm of it outside the material to facilitate manipulation
(Fig. 1).
The 96-well microtiter plate was inoculated with 180 mL/well of
the suspension of E. faecalis in BHI, after which the cylinders of
each material were introduced. The microtiter plate-cylinder set was
then placed on a rocking table (model Swing Sw 8 10000-00015;
OVAN, Badalona, Spain) and incubated at 37

C for 24 hours at 5 rocks

per minute. Biolms forming onto the cylinders were rinsed twice by
placing the cylinders in wells with 200 mL 0.9% saline solution for
2 minutes (each time) and then transferring them to a 96-well
microtiter recovery plate with 200 mL BHI/well, which was sonicated
in a water-table ultrasonic bath (model 5510EMT; Branson, Danbury,
CT). Ten minutes of ultrasonication were sufcient time to recover
biolm (with no signicant differences versus 20 minutes) without
affecting the cylinders integrity (height/diameter), which was also
determined by the optical density of the broth after subjecting the
uninfected cylinders to ultrasonication for 10 minutes.
Each material was tested twice, and the procedure was performed
in duplicate (n = 4/group). Four cylinders of AH Plus alone and
non-infected were included as negative controls. The disrupted biolms
were diluted serially and plated for viable cell counting; data were
transformed to a logarithmic scale.
Results of antimicrobial activity of the tested materials and
E. faecalis biolm formation onto them were respectively expressed
TABLE 1. Viable E. faecalis Counts by AH Plus Combined with CHX and/or CTR
by Using the Modied DCT
CTR
CHX
None 1% 2%
None 6.28 (0.06)*
,a
5.30 (0.13)
b
5.23 (0.28)
b
0.1% 4.65 (0.03)
c
4.52 (0.06)
d,e
4.56 (0.04)
c,d
0.2% 4.51 (0.06)
d,e
4.49 (0.02)
d,e
4.45 (0.06)
d,e,f,g
0.3% 4.42 (0.08)
e,f,g
4.39 (0.05)
e,f,g
0
0.4% 4.35 (0.08)
f,g,h
4.26 (0.09)
h
0
0.5% 3.24 (0.26)
i
3.04 (0.88)
j
0
The same superscript letters show differences not statistically signicant by Duncan test.
*Control: AH Plus without CHX and CTR.
TABLE 2. Viable Counts of E. faecalis Biolm Formed onto AH Plus Combined
with CHX and/or CTR
CTR
CHX
None 1% 2%
None 4.78 (0.06)*
,a
4.55 (0.09)
b
3.18 (0.07)
c
0.1% 1.95 (0.05)
d
0 0
0.2% 0 0 0
0.3% 0 0 0
0.4% 0 0 0
0.5% 0 0 0
Log
10
CFU + 1/mL. Mean (standard deviation).
The same superscript letters show differences not statistically signicant by Duncan test.
*Control: AH Plus without CHX and CTR.
Basic ResearchTechnology
978 Bailon-Sanchez et al. JOE Volume 40, Number 7, July 2014
as log
10
CFU + 1/mL. Eradication and inhibition of biolm formation
were understood as no bacterial growth or a log
10
reduction = 5
with respect to the control (AHPlus alone). Data were analyzed by using
1-way analysis of variance followed by the Duncan post hoc test to
compare among groups.
Results
All negative controls performed throughout the experiment
showed no bacterial growth. The overall results of antimicrobial activity
and the inhibition of E. faecalis biolmformation are shown in Tables 1
and 2, respectively, which include the values of the controls (AH Plus
alone). Figure 2 displays the antimicrobial activity as the log
10
reduction
in viable counts with respect to the control of all materials tested.
Regarding antimicrobial activity by DCT, AH Plus mixed only
with 1% and 2% CHX showed lower antimicrobial activity (respective
log
10
viable counts of 5.30 and 5.23) than the control (log
10
6.28).
The combinations of AH Plus with CTR showed a concentration-
dependent antimicrobial activity, although none of the tested
concentrations achieved eradication (log reduction = 5). CTR in the
Figure 1. Silicon molds and cylinders of material to test inhibition of biolm formation.
Figure 2. Antimicrobial activity expressed as log
10
reduction viable counts, with respect to the control (log
10
CFU/mL = 6.28 0.06), of AH Plus mixed with CHX
(A), CTR (B), or both (C and D).
Basic ResearchTechnology
JOE Volume 40, Number 7, July 2014 Antibacterial Activity of AH Plus 979
highest concentration (0.5%) obtained a 2.92 log
10
reduction with
respect to the control of AH Plus alone.
The inhibition of biolmformation obtained by AH Plus combined
with CHX showed a direct relationship with the concentration used, but
no full inhibition. However, CTR inhibited E. faecalis biolm formation
at a concentration of 0.2%.
The results of the combinations of both antimicrobials mixed with
AHPlus indicate a synergic antimicrobial effect seen in the antimicrobial
activity as well as in the inhibition of biolm formation. Thus, 0.3% CTR
was necessary to eradicate E. faecalis when combined with 2% CHX,
and only 0.1% CTR attained inhibition of biolmformation when mixed
with any CHX concentration.
Discussion
Although chemomechanical preparation is essential for elimi-
nating bacteria from the root canal system (2), an antibacterial effect
of root canal lling materials is helpful, because if bacteria remain in
dentinal tubules, they may become reservoirs for reinfection (29).
Endodontic cements are essential to ensure proper sealing of root
canals, and among their other properties, it would be desirable that
they exerted antimicrobial activity on contact with microbes and
biolms, ideally maintaining this effect over time (2).
To assay antimicrobial activity, we used a modication of the DCT
(25), which provides for a smooth and standardized surface, giving
optimal exposure to the bacteria. To test the inhibition of biolm
formation in this study, a system based on the Calgary device (28)
was developed, but replacing the original polystyrene peg-lids with
identical cement peg-lids for testing. Silicon molds from the impression
of peg-lids allowed us to obtain specimens of the same size, shape, and
surface smoothness. Such standardization of the techniques used is
considered a critical step for any biolm growth in an in vitro or
in vivo model system (30) involving comparisons.
AH Plus, an epoxy resinbased root canal sealer, was selected
because several in vitro (2, 4, 31), in vivo, and clinical studies
(32) indicate its suitability for endodontic therapy. CHX and CTR are
effective antimicrobial agents that have been incorporated successfully
in GICs (20, 22, 33). Furthermore, it was proven that the incorporation
of 2 antimicrobials belonging to the quaternary ammoniumgroup, such
as CTR, increase the antimicrobial effect of several endodontic sealers
(23), although these authors assayed cariogenic bacteria by the agar
diffusion test and recommended to determine their physical properties.
In this sense, it has been recently demonstrated that adding CHX, CTR,
or both to AH Plus at any of the combinations tested in this study does
not signicantly alter setting time, ow, solubility, and radiopacity (24).
The results remained consistently within the American National
Standards Institute/American Dental Association requirement for end-
odontic sealers (34). However, further characterization experiments
concerning the dimensional stability, adhesion to dentin/gutta-
percha, and leakage of the sealer/material set should be undertaken
before these combinations can be considered for clinical application.
Overall, these results are consistent with previous studies (2, 3,
35) and show that AH Plus alone was not capable of killing bacteria
or inhibiting E. faecalis biolm formation. However, the addition of
CHX, CTR, or CHX + CTR conferred antimicrobial properties to this
sealer cement, as observed in both tests; besides a direct relationship
between dose and effect, we conrmed that the combinations of CHX
and CTR ensured better results than their single use (17).
AH Plus-CHX at 1% and 2% showed a low antimicrobial activity
with respect to the control, with respective values of log
10
5.30 and
5.23 and signicant differences with respect to AH Plus alone
(log
10
6.28). From a microbiological point of view this would be
irrelevant, because log
10
reductions of 0.9 and 1.03 are very far from
the reduction of 5 considered eradication. The low antimicrobial
activity, similar for both concentrations, could be attributed to the
fact that CHX digluconate acts as a cross-linking agent (36), which
would be related to its low antimicrobial activity. Regarding the
inhibition of biolm formation, the results point to a limited effect.
AH Plus + 0.5% CTR achieved a log
10
reduction of 2.92; however,
a concentration of 0.2% CTR sufced to inhibit biolm formation. This
result is logical, because a greater concentration of antimicrobial agent
is needed for a bactericidal effect than for inhibiting properties such as
biolm formation (37).
In the past decade, antimicrobial agents have been added to dentin
adhesives and dental materials to enhance antimicrobial activity (8, 19).
The mixture of AH Plus with CHX + CTR was seen to improve the
antimicrobial effect. By using a DCT, 2% CHX combined with 0.3%
CTR achieved E. faecalis eradication with a log
10
reduction >5 with
respect to control, and 1% CHX + 0.1% CTR was able to inhibit
biolm formation completely. This enhanced capacity has been
demonstrated previously in irrigating solutions (14, 17). Moreover,
lower concentrations of the antimicrobial agents are sufcient to attain
the same results. Previous studies of GICs (21, 22) with incorporation
of CHX and CTR have shown that the antimicrobial properties are
accompanied by a deterioration of physical properties. This potential
is dose-dependent, however; 1% CTR does not exert this effect, as
opposed to the concentration of 2.5% (22). In this regard, it is essential
to ensure that the concentrations of antiseptics added to AH Plus are
effective as antimicrobials but do not alter their physical properties.
Conclusion
The DCT and biolm analysis method used here show that the
addition of CHX, CTR, or both to AH Plus proves benecial, increasing
the bactericidal and anti-biolm capacity of this material. However,
these are preliminary tests, and further ex vivo experiments in root
canals are needed to conrm these results in vitro.
Acknowledgments
The authors thank Francisca Castillo Perez and Gertrudis
Gomez Villaescusa for their technical assistance. This study was
supported by the Research Group CTS-167 of the Junta de
Andaluca, Spain.
The authors deny any conicts of interest related to this study.
References
1. Torabinejad M, Handysides R, Khademi AA, Bakland LK. Clinical implications of the
smear layer in endodontics: a review. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 2002;94:65866.
2. Zhang H, Shen Y, Ruse ND, Haapasalo M. Antibacterial activity of endodontic sealers
by modied direct contact test against Enterococcus faecalis. J Endod 2009;35:
10515.
3. Baer J, Maki JS. In vitro evaluation of the antimicrobial effect of three endodontic
sealers mixed with amoxicillin. J Endod 2010;36:11703.
4. Leonardo MR, da Silva LA, Tanomaru Filho M, et al. In vitro evaluation of antimi-
crobial activity of sealers and pastes used in endodontics. J Endod 2000;26:3914.
5. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: its role in root
canal treatment failure and current concepts in retreatment. J Endod 2006;32:938.
6. Arias-Moliz MT, Ferrer-Luque CM, Espigares-Garca M, Baca P. Enterococcus
faecalis biolm eradication by root canal irrigants. J Endod 2009;35:7114.
7. De Almeida WA, Leonardo MR, Tanomaru Filho M, Silva LA. Evaluation of apical
sealing of three endodontic sealers. Int Endod J 2000;33:257.
8. Siqueira JF Jr, Favieri A, Gahyva SM, et al. Antimicrobial activity and ow rate of
newer and established root canal sealers. J Endod 2000;26:2747.
9. Yamaguchi K, Matsunaga T, Hayashi Y. Gross extrusion of endodontic obturation
materials into maxillary sinus: a case report. Oral Surg Oral Med Oral Pathol
Oral Radiol Endod 2007;104:1314.
Basic ResearchTechnology
980 Bailon-Sanchez et al. JOE Volume 40, Number 7, July 2014
10. Neff T, Layman D, Jeansonne BG. In vitro cytotoxicity evaluation of endodontic
sealers exposed to heat before assay. J Endod 2002;28:8114.
11. Leyhausen G, Heil J, Reifferscheid G, et al. Genotoxicity and cytotoxicity of the epoxy
resin-based root canal sealer AH Plus. J Endod 1999;25:10913.
12. Leonardo MR, da Silva LA, Almeida WA, Utrilla LS. Tissue response to an epoxy
resin-based root canal sealer. Endod Dent Traumatol 1999;15:2832.
13. Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in
endodontics. Int Endod J 2009;42:288302.
14. Arias-Moliz MT, Ferrer-Luque CM, Gonzalez-Rodrguez MP, et al. Eradication of
Enterococcus faecalis biolms by cetrimide and chlorhexidine. J Endod 2010;
36:8790.
15. Vianna ME, Gomes BP, Berber VB, et al. In vitro evaluation of the antimicrobial
activity of chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol
Oral Radiol Endod 2004;97:7984.
16. Baca P, Junco P, Arias-Moliz MT, et al. Residual and antimicrobial activity of nal
irrigation protocols on Enterococcus faecalis biolm in dentin. J Endod 2011;
37:3636.
17. Baca P, Mendoza-Llamas ML, Arias-Moliz MT, et al. Residual effectiveness of nal
irrigation regimens on Enteroccus faecalis-infected root canals. J Endod 2011;
37:11213.
18. Imazato S. Antibacterial properties of resin composites and dentin bonding systems.
Dent Mater 2003;19:44957.
19. Pereira-Cenci T, Cenci MS, Fedorowicz Z, Marchesan MA. Antibacterial agents in
composite restorations for the prevention of dental caries. Cochrane Database
Syst Rev 2009;8:CD007819.
20. Takahashi Y, Imazato S, Kaneshiro AV, et al. Antibacterial effects and physical
properties of glass-ionomer cements containing chlorhexidine for the ART
approach. Dent Mater 2006;22:64752.
21. Tuzuner T, Kusgoz A, Er K, et al. Antibacterial activity and physical properties of
conventional glass-ionomer cements containing chlorhexidine diacetate/cetrimide
mixtures. J Esthet Restor Dent 2011;23:4655.
22. Turkun LS, Turkun M, Ertugrul F, et al. Long-term antibacterial effects and physical
properties of a chlorhexidine-containing glass ionomer cement. J Esthet Restor Dent
2008;20:2944.
23. Gjorgievska E, Apostolska S, Dimkov A, et al. Incorporation of antimicrobial agents
can be used to enhance the antibacterial effect of endodontic sealers. Dent Mater
2013;29:e2934.
24. Ruiz-Linares M, Bailon-Sanchez ME, Baca P, et al. Physical properties of AH Plus
with chlorhexidine and cetrimide. J Endod 2013;39:16114.
25. Weiss E, Shalhav M, Fuss Z. Assessment of antibacterial activity of endodontic sealers
by a direct contact test. Endod Dent Traumatol 1996;12:17984.
26. Vaidyanathan M, Sheehy EC, Gilbert SC, Beighton D. Antimicrobial properties of
dentine bonding agents determined using in vitro and ex vivo methods. J Dent
2009;37:51421.
27. Costerton JW, Stewart PS, Greenberg EP. Bacterial biolms: a common cause of
persistent infections. Science 1999;284:131822.
28. Ceri H, Olson ME, Stremick C, et al. The Calgary Biolm Device: new technology for
rapid determination of antibiotic susceptibilities of bacterial biolms. J Clin
Microbiol 1999;37:17716.
29. Oguntebi BR. Dentine tubule infection and endodontic therapy implications. Int
Endod J 1994;27:21822.
30. Coenye T, Nelis HJ. In vitro and in vivo model systems to study microbial biolm
formation. J Microbiol Methods 2010;83:89105.
31. Koulaouzidou EA, Papazisis KT, Beltes P, et al. Cytotoxicity of three resin-based root
canal sealers: an in vitro evaluation. Endod Dent Traumatol 1998;14:1825.
32. rstavik D. Materials used for root canal obturation: technical, biological and
clinical testing. Endod Topics 2005;12:2538.
33. Palmer G, Jones FH, Billington RW, Pearson GJ. Chlorhexidine release from an
experimental glass ionomer cement. Biomaterials 2004;25:542331.
34. ANSI/ADA. Specication No. 57 Endodontic Sealing Material. Chicago: ANSI/ADA;
2000.
35. Slutzky-Goldberg I, Slutzky H, Solomonov M, et al. Antibacterial properties of four
endodontic sealers. J Endod 2008;34:7358.
36. Plaut BS, Meakin BJ, Davies DJ, Richardson NE. The mechanism of interaction bet-
ween chlorhexidine digluconate and poly(2-hydroxyethylmethacrylate). J Pharm
Pharmacol 1981;33:828.
37. Maillard JY. Bacterial target sites for biocide action. J Appl Microbiol 2002;
92(Suppl):16S27S.
Basic ResearchTechnology
JOE Volume 40, Number 7, July 2014 Antibacterial Activity of AH Plus 981