Basic Research—Technology

An Investigation on the Antibacterial and Antibiofilm

Efficacy of Cationic Nanoparticulates for Root Canal
Disinfection
Anil Kishen, BDS, MDS, PhD,* Zhilong Shi, PhD,† Annie Shrestha, BDS,* and
Koon Gee Neoh, PhD†

Abstract
This study aimed to investigate the antibacterial and
antibiofilm efficacy of cationic nanoparticulates for root
canal disinfection. Experiments were performed in two
M icroorganisms that persist after endodontic treatment would have entered the
root canal during treatment, survived disinfection procedures, and persisted after
root canal obturation (1). Otherwise, they would have entered the root canal after
stages. In stage 1, experiments were conducted to endodontic treatment is completed via interfacial gaps and subsequently survived the
examine the physical properties of three types of nano- harsh endodontic environment prevailing in the root-filled teeth (2). Endodontic seal-
particulates. The antibacterial properties of nanopar- ers are expected to possess antimicrobial property and provide optimum seal against
ticulates alone and nanoparticulates mixed with zinc fluid leakage (3–5). However, compromise in the physical properties of root canal
oxide– eugenol– based sealer were studied. In stage 2, sealers and the lack of adhesion between sealer and core filling material or between
the ability of nanoparticulates-treated dentin to prevent sealer and dentin may cause fluid leakage (6 –11). Studies have revealed Enterococcus
bacterial adherence was examined. Zinc oxide nano- faecalis as the most dominant and occasionally the only species to persist in endodon-
particulates, chitosan nanoparticulates, a mixture of tically treated teeth (12–14). This highlights the ability of E. faecalis to resist endodon-
zinc oxide and chitosan nanoparticulates, and zinc ox- tic disinfectants and survive nutrient-stringent conditions within root-filled teeth (12,
ide nanoparticulates with multilayered coating of chi- 13). E. faecalis has the capacity to adhere and form biofilm on dentin under different
tosan were tested. This study showed that the incor- environmental conditions (15–18). The presence of smear layer, conditioning with
poration of nanoparticulates did not alter the flow saliva, and starvation have been shown to increase the adherence of E. faecalis to
characteristics of sealer but improved the direct anti- dentin, whereas chlorhexidine treatment has the reverse effect (19, 20).
bacterial property and the ability to leach out antibac- Antibacterial nanoparticulates are found to have higher antibacterial activity than
terial components. There was a significant reduction in antibacterial powders. This is because of the higher surface area and charge density of
the adherence of Enterococcus faecalis to nanoparticu- nanoparticulates, which enable them to achieve a greater degree of interaction with the
lates-treated dentin (p ⬍ 0.05). These experiments negatively charged surface of bacterial cells (21–23). Chitosan (CS) is a nontoxic
highlighted the potential advantage of nanoparticu- biopolymer derived by the deacetylation of chitin. Chitin is a natural polymer occurring
lates in root canal disinfection. (J Endod 2008;34: in the exoskeleton of the crustaceans. It is a bioadhesive that readily binds to negatively
1515–1520) charged surfaces and has excellent antimicrobial and antifungal activities. One of the
proposed mechanisms of antibacterial property of CS is that the interaction between
Key Words positively charged CS and negatively charged bacterial cell would alter the bacterial cell
Bacterial adherence, chitosan, Enterococcus faecalis, permeability resulting in the leakage of intracellular components and cell death (24).
nanoparticles, zinc oxide The objectives of this study were twofold: (1) to investigate the physical and
antibacterial properties of different nanoparticulates and nanoparticulates-mixed zinc
oxide– eugenol– based endodontic sealer and (2) to examine the ability of different
nanoparticulates treated dentin to prevent adherence of E. faecalis. Zinc oxide nano-
From the *Department of Restorative Dentistry and †De- particulates (ZnO-NPs), CS nanoparticulates (CS-NPs), a mixture of zinc oxide and CS
partment of Chemical & Biomolecular Engineering, National
University of Singapore, Singapore. nanoparticulates (CS/ZnO-NPs), and zinc oxide nanoparticulates with multilayered
Supported by grant NUS ARF R-224-000-028-112. coating of CS (CS-layer-ZnO-NPs) were tested. The physical and antibacterial properties
Address requests for reprints to Dr Anil Kishen, National Univer- of the nanoparticulates were characterized, whereas direct-contact and membrane-
sity of Singapore, National University Hospital, 5 Lower Kent Ridge restricted antibacterial assays were conducted to evaluate the antibacterial properties of
Road, Singapore 119074. E-mail address: [email protected].
0099-2399/$0 - see front matter
the nanoparticulates-mixed endodontic sealer. The direct-contact assay shows the abil-
Copyright © 2008 American Association of Endodontists. ity of antibacterial components from the sealer to kill microbes adhering superficially to
doi:10.1016/j.joen.2008.08.035 the dentin wall, whereas the membrane-restricted assay tests the ability of antibacterial
components to kill microbes shielded in the depth of dentinal tubules (15). A fluores-
cence microscopy– based adherence assay was used to examine the effect of nanopar-
ticulate treatment on the adherence of E. faecalis to dentin.

Materials and Methods

All the chemicals used in this study were of analytical grade and were purchased
from Sigma Aldrich (St Louis, MO) unless otherwise stated. CS obtained from CarboMer
Inc (San Diego, CA) was refined twice by dissolving in dilute acetic acid solution. The

JOE — Volume 34, Number 12, December 2008 Antibacterial and Antibiofilm Efficacy of Nanoparticulates 1515
Basic Research—Technology
solution was filtered and the CS was precipitated with aqueous sodium mencement of mixing, the weight was removed and the diameter of the
hydroxide and then dried in a vacuum oven for 24 hours at 40°C. The sealer disc was measured. The experiments were repeated three times.
degree of deacetylation was 84% as determined by elemental analysis
using the Perkin-Elmer Model 2400 elemental analyzer (Waltham, MA). The Determination of Antibacterial Activity of
CS-NPs were synthesized according to the method previously reported Nanoparticulates with Sealer
(21). In brief, CS was dissolved in 1 v/v% HOAc solution at a concen- The direct-contact and membrane-restricted antibacterial assays
tration of 0.5 w/v%, and the pH was raised to 4.6 to 5 with 10 N NaOH. described by Kayaoglu et al. (15) was adopted in this study. E. faecalis
CS-NP was formed upon adding 5 mL 0.25% sodium tripolyphosphate in was incubated overnight at 37°C under agitation in AC medium. An
water to 15 mL CS solution under stirring at a speed of 1,000 rpm. The aliquot (2 mL) of the culture was added to the broth and incubated for
nanoparticles were separated by centrifugation at 20,000 rpm for 30 6 to 8 hours (37°C) until the exponential growth phase was reached.
minutes. The supernatant was discarded, and the CS-NP was extensively The bacterial concentration in the medium was then adjusted to an
rinsed with water to remove any NaOH and then freeze-dried before optical density of 0.1 (at 600 nm). Filter paper discs (Whatman no. 2,
further use. 4-mm diameter) were immersed in bacterial suspension for 10 min-
CS-layer-ZnO-NP was prepared by the electrostatic layer-by-layer utes. The discs were later removed from the suspension and drained of
technique as described previously (25). The layer-by-layer deposition the excess liquid. For direct-contact antibacterial assay, approximately
of polyelectrolytes was performed by dispersing the ZnO nanoparticles 15 ␮L of sealer, freshly mixed materials as described by the manufac-
(Sigma Aldrich) into anionic hyaluronan solution (1 mg/mL in 0.14 turer, was placed onto the discs with the bacteria in a 96-well micro-
mol/L aqueous NaCl) followed by the cationic CS solution (1.5 mg/mL in plate at 37°C. Four discs were used to test each sealer. The contact time
0.1 mol/L acetic acid containing 0.14 mol/L NaCl). In between disper- between sealers and paper discs was 30 minutes. Subsequently, the bulk
sion, the content was centrifuged and rinsed with 0.14 mol/L NaCl for 3 of the sealer was wiped off the discs, transferred to vials containing 2 mL
minutes at 10,000 rpm and washed thoroughly. A total of three bilayers of phosphate-buffered saline, and vortexed (30 seconds) followed by
of CS and hyaluronan were dispersed on the outer aspect of ZnO-NP. mild ultrasonication (5–7 minutes) (power: 100 W, frequency: 50 Hz).
One hundred microliters of the sample after serial dilutions was plated
Experiment 1: Characterization of Physical and Antibacterial and incubated aerobically at 37°C for 24 hours to determine the CFUs.
Properties The filter paper discs without any contact with sealer were used as the
Zeta potential is described as the overall charge of a particle in a control. In the membrane-restricted antibacterial assay, a filter mem-
specific medium. It is a measure of the magnitude of repulsion or brane (Whatman, 0.22-␮m pore size) was placed over the disc with
attraction between particles and provides insight into the electrostatic bacteria before the placement of the sealer. The membrane-restricted
interaction between surfaces. In this experiment, the zeta potential of antibacterial assay was then conducted as described earlier. The data
the nanoparticles was measured by a zeta potential analyzer (Zeta Plus, were evaluated for statistical significance using a two-sample t test. The
Brookhaven Instruments, NY) with palladium electrodes at room tem- differences observed between substrates were considered significant at
perature, and the mean of six readings was calculated. The particle size p ⬍ 0.05.
of CS-NP and ZnO-NP was determined from field emission scanning
electron microscopy (JEOL 6700F; JEOL Ltd., Tokyo, Japan). Experiment 2: The Effect of Nanoparticulates on the
The antibacterial efficacies of nanoparticulates were tested on E. Adherence of Bacteria to Dentin
faecalis (American Type Culture Collection 29212). Cells grown over- One hundred noncarious single-rooted teeth maintained in phos-
night at 37°C under agitation in All Culture (AC) medium (Sigma Al- phate-buffered saline solution were used in this study. The crown (at the
drich) were washed twice and resuspended in deionized water to 107 cementoenamel junction) and the apical portion of the teeth were
cells/mL before use. One milliliter of inoculum and three concentra- ground to obtain a uniform root length of 8 mm. The teeth were verti-
tions (2, 5, and 10 mg) of CS-NP, ZnO-NP, CS-layer-ZnO-NP, and CS/ cally sectioned along the midsagittal plane into two halves. These dentin
ZnO-NP were added aseptically into each well of 24-well plates and sections were divided randomly into five equal groups and were exposed to
incubated at 37°C under agitation. One hundred microliters of the in- different chemical treatment. Specimens in group 1 were treated with 5.2%
oculums was removed from each well after 2, 4, and 8 hours; and NaOCl for 30 minutes. Group 2 specimens were treated with 5.2% NaOCl for
plated. Bacterial inoculum without nanoparticulates was taken as the 30 minutes and 17% EDTA (Merck KGaA, Darmstadt, Germany) for 5 min-
control group. Colony-forming units (CFUs) were determined after 24 utes. Group 3 specimens were treated with 17% EDTA for 5 minutes and
hours of incubation at 37°C and expressed as log CFU/mL. The exper- 5.2% NaOCl for 30 minutes. Specimens in group 4 were treated with chlo-
iment was performed in triplicates. rhexidine for 30 minutes, and specimens in group 5 were left untreated.
Grossman Type 801 cement containing zinc oxide powder (Roth After treatment with different irrigants, specimens from each group were
International Ltd, Chicago, IL) and eugenol liquid (Dentsply, Konstanz, further divided into five subgroups and were treated with 30 mg of nano-
Germany) was used in this experiment. The rheologic properties of the particulates mixed with approximately 1 mL of deionized water. Specimens
sealers before and after the addition of nanoparticulates were studied in subgroup 1 were treated with ZnO-NP, subgroup 2 was treated with
according to the International Standard Organization standard 6876: CS-NP, subgroup 3 was treated with CS/ZnO-NP, and subgroup 4 was treated
2001. The International Standard Organization specification for the flow with CS-layer-ZnO-NP for 24 hours. Control specimens without any treat-
of the sealer is based on the diameter of a film of sealer between two ment were maintained in all the groups. The concentration and duration of
glass plates at room temperature. CS-NP, ZnO-NP, or CS/ZnO-NP mix- irrigants used in this study were similar to the earlier experiments con-
ture (mass ratio 1:1) was mixed with the powder component of the ducted to investigate the influence of different endodontic irrigants on the
sealer at a ratio of 15:100. All cements were manually prepared by adherence and adhesion force of E. faecalis to root canal dentin (26).
mixing the powder with a liquid component in a ratio of 3 g/mL. Ce- A single colony of E. faecalis maintained in Tryptone Bile X-Glu-
ments were delivered (0.5 mL) via a 1-mL syringe onto the glass plate. curonide agar (Merck, Darmstadt, Germany) was transferred to 50 mL
Three minutes after the commencement of mixing, a second glass plate of AC medium. Bacterial cells were allowed to grow overnight (14
(20 g) was placed on top of the sealer, followed by a 100-g weight to hours) at 37°C in an orbital incubator at 120 rpm. Dentin blocks after
make a total mass of 120 g on the sealer. Ten minutes after the com- designated treatment with nanoparticulates were rinsed in distilled wa-

1516 Kishen et al. JOE — Volume 34, Number 12, December 2008
 Basic Research—Technology
ter and inoculated with 200 ␮L of E. faecalis (106cells /mL) in 24-well Rheological Properties
plates at 37°C for 1 hour. The specimens were subsequently removed, The diameter of the original sealer disc was 24.2 mm (standard
washed gently to remove loose nonadherent cells, stained with Baclight deviation [SD] ⫽ 0.6), the ZnO-NP–incorporated sealer disc was 28.6
(Molecular Probes, Eugene, OR), and observed under fluorescence mm (SD 1.3), the CS-NP–incorporated sealer disc was 26.3 mm (SD
microscope (Eclipse 80i; Nikon, Tokyo, Japan) using a 520-nm filter 0.7), and the mixture of the CS/ZnO-NP–incorporated disc was 27.7
under oil immersion (100⫻). The average number of bacteria adhered (SD ⫽ 1.1). The flow property of the sealer increased significantly after
was obtained by counting at least nine randomly chosen microscopic the incorporation of nanoparticulates at a nanoparticulates/sealer pow-
fields. All experiments were performed in triplicates. The results were der ratio of 15%.
subjected to one-way analysis of variance with a post hoc Tukey test. The
difference between two groups was considered statistically significant if
p ⬍ 0.05. Antibacterial Assay with Sealer
The antibacterial assay showed that the original zinc oxide– euge-
nol sealer has strong antibacterial activity (Fig. 1B). With the addition of
Results ZnO-NP and CS-NP to the sealer, the number of viable bacterial cells on
Experiments 1: Characterization of Physical and Antibacterial the paper discs decreased further by 2 logs and 1 log, respectively,
Properties compared with the original sealer. It was observed that the addition of
Characterization of Nanoparticulates CS/ZnO-NP results in the strongest antibacterial activity, although it was
not significantly higher (p ⫽ 0.36) than ZnO-NP (Fig. 1B).
The FE-SEM analysis showed that the average diameter of the
The insertion of a filter membrane in membrane-restricted anti-
CS-NP was approximately 70 nm, whereas the diameter of ZnO-NP was
bacterial assay significantly reduced the antibacterial activity of all seal-
60 to 100 nm. The zeta potentials of CS-NP and ZnO-NP were 49 mV and
ers (Fig. 1C). The membrane-restricted antibacterial assay showed the
22 mV, respectively, and the zeta potential of the CS/ZnO mixture at a
ability of antibacterial components to diffuse out from the sealers. The
mass ratio of 1:1 was 40 mV.
original zinc oxide– eugenol sealer did not exhibit any antibacterial
activity. The ZnO-NP–loaded sealer exhibited 3 log reductions. The
Antibacterial Activity of Nanoparticulates CS/ZnO-NP–loaded sealer exhibited the highest antibacterial activity
All the nanoparticulates tested showed bacterial killing, and the because of the synergistic effects between CS-NP and ZnO-NP. This ex-
rate of killing depended on the time and concentration used (Fig. 1A). periment showed that the addition of nanoparticulates enhanced the
CS-NP showed complete killing of bacteria after 8 hours. The other ability of the zinc oxide– eugenol sealer to leach out antibacterial com-
nanoparticulates showed a 3 to 4 log reduction in CFU. ponents. Figure 1D confirms the presence of bacterial cells on the

Figure 1. (A) The antibacterial effect of NPs on E. faecalis. (B) The number of viable adherent bacterial cells on the paper discs after a direct-contact antibacterial
assay. (C) The number of viable adherent bacterial cells on the paper discs after a membrane-restricted antibacterial assay. (D) A scanning electron micrograph of
the filter paper disc after immersing in the medium containing E. faecalis for 10 minutes.

JOE — Volume 34, Number 12, December 2008 Antibacterial and Antibiofilm Efficacy of Nanoparticulates 1517
Basic Research—Technology
(A) (B)

Figure 2. Typical fluorescent microscopic images (A) showing bacterial adherence in untreated root canal dentin and (B) a lack of bacterial adherence in NP treated
dentin.

surface of the filter paper discs (fixed with 3% glutaraldehyde, followed restricted antibacterial assay showed that ZnO-NP and CS/ZnO when
by serial dehydration using ethanol) before contact with the sealer. mixed with zinc oxide– eugenol– based sealer displayed the ability to
leach out antibacterial components to reach deep into the dentinal
Experiment 2: The Effect of Nanoparticulates on the tubules (15). It is important to note that the original endodontic sealer
Adherence of Bacteria to Dentin (zinc oxide– eugenol) did not show any ability to leach out antibacterial
The typical fluorescence microscopic images showing bacte- components. However, the relevance of this finding in an in vivo end-
rial adherence in root canal dentin before and after treatment with odontic milieu is not well established.
nanoparticulates are shown in Figure 2. Figure 3A shows the per- The bacterial adherence assay highlighted reduced adherence of
centage reduction of bacterial adherence to dentin after treatment E. faecalis to the dentin surface treated with different irrigants. Never-
with different nanoparticulates. Figure 3B and C shows the percent- theless, different irrigants and irrigation regimens produced a different
age reduction in bacterial adherence to dentin irrigated with differ- degree of bacterial adherence. Chlorhexidine treatment produced the
ent irrigants and subsequently treated with different nanoparticu- maximum reduction, whereas final treatment with EDTA (in the hypo-
lates. The number of bacterial cells adhering to untreated dentin chlorite-EDTA group) produced the minimum reduction in the adher-
was taken as 100% adherence. Among the irrigants used, chlorhexi- ence of E. faecalis to dentin. These findings were similar to the previous
dine treatment produced the maximum reduction in bacterial ad- experiments conducted to study the influence of different irrigation regi-
herence to dentin (72% reduction) (Fig. 3D), whereas EDTA as the mens on bacterial adherence and adhesion force between bacteria and
last irrigant (hypochlorite-EDTA) produced the minimum reduction dentin (26). The findings from this study showed that the subsequent treat-
in bacterial adherence to dentin (33% reduction) (Fig. 3B). The ment of dentin with nanoparticulates produced a significant reduction in the
treatment of dentin with EDTA before sodium hypochlorite pro- number of E. faecalis cell adhering to dentin. Root canal dentin irrigated
duced 51% reduction bacterial adherence (Fig. 3C). For all three with chlorhexidine and later treated with nanoparticulates displayed a con-
irrigants, subsequent treatment with nanoparticulates produced a sistent reduction (⬃97%) in bacterial adherence.
significant further reduction in the E. faecalis adherence to dentin The zeta potential or surface charge of nanoparticulates can
(p ⬍ 0.05). Similarly, untreated dentin when treated with ZnO-NP, greatly influence its stability in suspension through the electrostatic
ZnO/CS-NP, or CS-layer-ZnO-NP produced ⬃95% reduction in bac- repulsion between particles. When positively charged nanoparticu-
terial adherence, whereas CS-NP produced ⬃83% reduction in bac- lates in an aqueous suspension are allowed to settle onto a substrate
terial adherence (Fig. 3A). There was no statistically significant surface, the positive charge of the nanoparticles allows adherence to
difference between the nanoparticulates-treated groups. negatively charged dentin surface. The electrostatic attraction be-
tween the particles and the dentin, which is a charged substrate,
Discussion would lead to rapid adhesion of the sedimenting particles. Even
This study suggested that all tested cationic nanoparticulates had though the force of adhesion between nanoparticulates and dentin
antibacterial activity and the addition of nanoparticulates also improved may be weak, when microbes reenter the root canal, the nanopar-
the antibacterial property of zinc oxide– eugenol– based sealer. The ticulates can electrostatically interact with the negatively charged
addition of nanoparticulates did not deteriorate the flow characteristics microbial cell to destroy them readily. This study highlighted that
of the sealer but at the same time decreased viscosity, leading to en- root canal surface treated with cationic antibacterial nanoparticu-
hanced flow of the sealer. The increased flow of the sealer after the lates such as ZnO-NP, CS/ZnO-NP, or CS-layer-ZnO-NP significantly
incorporation of ZnO-NP can be because of the better compatibility of inhibited bacterial adherence to dentin, which, in turn, would pre-
ZnO-NP with zinc oxide powder of the sealer. Although CS-NP has a vent bacterial recolonization and biofilm formation. Further exper-
higher zeta potential and better antibacterial property when used di- iments are needed to understand whether the inhibition of bacterial
rectly on planktonic bacteria than ZnO-NP, their antibacterial activity adherence by cationic nanoparticulates is by the killing of bacteria
diminished considerably when mixed with sealer. The ZnO-NP showed in the vicinity or by the direct effect of nanoparticulates on the
lesser antibacterial property when used directly on planktonic bacteria, bacteria-substrate interaction. The endurance of nanoparticulates-induced
but their antibacterial property increased noticeably when mixed with antibacterial effect on root canal dentin also needs further investigations. In
sealer. This difference in antibacterial activity may be attributed to the brief, this experimental investigation highlighted the potential advantage of
decrease in the release of CS-NP from the sealer and the difference in using ZnO-NP and CS-NP to inhibit bacterial recolonization in root canals
bactericidal mechanisms between CS-NP and ZnO-NP. The membrane- and to improve the antibacterial capabilities of endodontic sealers. How-

1518 Kishen et al. JOE — Volume 34, Number 12, December 2008
 Basic Research—Technology
94
94 93
97 98 93 100

100 81

% reduction in bacterial adherence

% reduction in bacterial adherence

81
80
90

60
80
33

40
70

20
60

50 0
C S NP ZnO NP C S /ZnO NP C S -la ye r-ZnO Na OC l & C S NP ZnO NP C S /ZnO NP C S -la ye r-
NP EDTA ZnO NP

Untre a te d de ntin tre a te d with NP De ntin tre a te d with Na OC l & EDTA s ubs e que ntly tre a te d with NP

(A) (B)
96 98
93 94 97 96 97
100 83 100
72
% reduction in bacterial adherence

% reduction in bacterial adherence

80
51 80

60
60

40
40

20
20

0
EDTA & C S NP ZnO NP C S /ZnO NP C S -la ye r- 0
Na OC l ZnO NP C hlo rhe xidine C S NP ZnO NP C S /ZnO NP C S -la ye r-ZnO
NP

De ntin tre a te d with EDTA & Na OC l s ubs e que ntly tre a te d with NP De ntin tre a te d with c hlo rhe xidine s ubs e que ntly tre a te d with NP

(C) (D)
Figure 3. (A) The percentage reductions in the adherence of E. faecalis cells to root canal dentin (untreated with endodontic irrigants) treated with NPs. (B) The percentage
reduction in the adherence of E. faecalis cells to root canal dentin (previously treated with NaOCl and EDTA) before and after treating with NPs. (C) The percentage reduction
in the adherence of E. faecalis cells to root canal dentin (previously treated with EDTA and NaOCl) before and after treating with NPs. (D) The percentage reduction in the
adherence of E. faecalis cells to root canal dentin (previously treated with chlorhexidine) before and after treating with NPs.

ever, additional studies need to be completed before recommending the use 4. Dagher FB, Yared GM. Influence of operator proficiency on the sealing ability of the
of antibacterial nanoparticulates within the root canals in vivo to prevent vertical condensation. J Endod 1995;21:335– 6.
5. De Almeida WA, Leonardo MR, Tanomaru Filho M, Silva LA. Evaluation of apical
biofilm formation. sealing of three endodontic sealers. Int Endod J 2000;33:25–7.
6. Bergenholtz G, Horsted-Bindslev P, Reit C. Textbook of Endodontology. ed 1. Oxford:
Acknowledgments Blackwell Publishing Limited; 2003.
The authors wish to thank Ms Shibi Mathew for the help during 7. Wu MK, De Gee AJ, Wesselink PR. Leakage of four root canal sealers at different
the adherence assay. thickness. Int Endod J 1994;27:304 – 8.
8. Zmener O, Spielberg C, Lamberghini F, Rucci M. Sealing properties of a new epoxy
resin-based root-canal sealer. Int Endod J 1997;30:332– 4.
References 9. Kopper PM, Figueiredo JA, Della Bona A, et al. Comparative in vivo analysis of the
1. Nair PN. Pathogenesis of apical periodontitis and the causes of endodontic failures. sealing ability of three endodontic sealers in post-prepared root canals. Int Endod J
Crit Rev Oral Biol Med 2004;15:348 – 81. 2003;36:857– 63.
2. Sundqvist G, Figdor D, Persson S, Sjogren U. Microbiologic analysis of teeth with 10. Negm MM. The effect of human blood on the sealing ability of root canal sealers: an
failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg in vitro study. Oral Surg Oral Med Oral Pathol 1989;67:449 –52.
Oral Med Oral Pathol Oral Radiol Endod 1998;85:86 –93. 11. Baumgartner G, Zehnder M, Paque F. Enterococcus faecalis type strain leakage
3. De Gee AJ, Wu MK, Wesselink PR. Sealing properties of Ketac-Endo glass ionomer through root canals filled with Gutta-Percha/AH plus or Resilon/Epiphany. J Endod
cement and AH26 root canal sealers. Int Endod J 1994;27:239 – 44. 2007;33:45–7.

JOE — Volume 34, Number 12, December 2008 Antibacterial and Antibiofilm Efficacy of Nanoparticulates 1519
Basic Research—Technology
12. Molander A, Reit C, Dahlen G. The antimicrobial effect of calcium hydroxide in root 19. George S, Kishen A. Effect of tissue fluids on hydrophobicity and adherence of En-
canals pretreated with 5% iodine potassium iodide. Endod Dent Traumatol terococcus faecalis to dentin. J Endod 2007;33:1421–5.
1999;15:205–9. 20. Yang SE, Cha JH, Kim ES, et al. Effect of smear layer and chlorhexidine treatment
13. Portenier I, Haapasalo H, Orstavik D, Yamauchi M, Haapasalo M. Inactivation of the on the adhesion of Enterococcus faecalis to bovine dentin. J Endod
antibacterial activity of iodine potassium iodide and chlorhexidine digluconate 2006;32:663–7.
against Enterococcus faecalis by dentin, dentin matrix, type-I collagen, and heat- 21. Shi Z, Neoh KG, Kang ET, Wang W. Antibacterial and mechanical properties of
killed microbial whole cells. J Endod 2002;28:634 –7. bone cement impregnated with chitosan nanoparticles. Biomaterials 2006;
14. Dahlen G, Samuelsson W, Molander A, Reit C. Identification and antimicrobial sus- 27:2440 –9.
ceptibility of enterococci isolated from the root canal. Oral Microbiol Immunol 22. Sawai J, Kawada E, Kanou F, et al. Detection of active oxygen generated from ceramic
2000;15:309 –12. powders having antibacterial activity. J Chem Eng Japan 1996;29:627–33.
15. Kayaoglu G, Erten H, Alacam T, Ørstavik D. Short-term antibacterial activity of root 23. Yamamoto O. Influence of particle size on the antibacterial activity of zinc oxide. Int
canal sealers towards Enterococcus faecalis. Int Endod J 2005;38:483– 8. J Inorg Mater 2001;3:643– 6.
16. Distel JW, Hatton JF, Gillespie MJ. Biofilm formation in medicated root canals. J 24. Rabea EI, Badawy ME, Stevens CV, Smagghe G, Steurbaut W. Chitosan as antimi-
Endod 2002;28:689 –93. crobial agent: applications and mode of action. Biomacromolecules 2003;
17. George S, Kishen A, Song KP. The role of environmental changes on monospecies 4:1457– 65.
biofilm formation on root canal wall by Enterococcus faecalis. J Endod 25. Tse CW, Leung YH, Tam KH, Chan WK, Djurisic AB. Tailoring and modifications of a
2005;31:867–72. ZnO nanostructure surface by the layer-by-layer deposition technique. Nanotechnol-
18. Kishen A, George S, Kumar R. Enterococcus faecalis-mediated biomineralized ogy 2006;17:3563– 8.
biofilm formation on root canal dentine in vitro. J Biomed Mater Res A 26. Kishen A, Sum CP, Mathew S, Lim CT. Influence of irrigation regimens on the adher-
2006;77:406 –15. ence of Enterococcus faecalis to root canal dentin. J Endod 2008;34:850 – 4.

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