an ex vivo study

EFFECTIVENESS OF DIFFERENT ENDODONTIC IRRIGANTS AGAINST ENTEROCOCCUS FAECALIS : AN EX VIVO STUDY.

Fabio Luis Miranda Pedro1, Lenieser Fajardo Nunes1, Thiago Machado Pereira1, Kellin Pivatto1, Cyntia Rodrigues de Araujo Estrela1, Octavio Amezcua2, Alvaro Henrique Borges1 1 Oral Sciences, University of Cuiaba, Brazil 2 Oral Sciences, University of Guadalajara, México

CORRESPONDING AUTHOR: [email protected]

ABSTRACT Aim: To evaluate the effectiveness of different endodontic irrigants against Enterococcus faecalis (ATCC 29212). Methods: Seventy bovine mandibular incisors were prepared, inoculated with a bacterial strain for 60 days and divided into the following groups: positive control; negative control; 2.5% NaOCl; 17% EDTA; 0.2% chitosan; 2.5% NaOCl + 0.2% chitosan; and 2.5% NaOCl + 17% EDTA. The irrigation protocol was performed using an experimental peristaltic pump device, with the irrigating solutions circulating within the apparatus at a constant flow for 10 min. Paper-point samples were then collected from the root canals and immersed in 7 mL of brain heart infusion broth, followed by incubation at 37°C for 48 h. Bacterial growth was assessed by turbidity of the culture medium. Results: E. faecalis was present in all samples after the use of different irrigants. Conclusion: The different irrigants tested were not effective in completely eliminating dentin bacterial contamination with E. faecalis. KEYWORDS: Chitosan. Enterococcus Faecalis. Ethylenediaminetetraacetic Acid. Sodium Hypochlorite. http://dx.doi.org/10.19177/jrd.v6e5201898-103

INTRODUCTION

microbial species, allowing attachment

faecalis is a Gram-positive, facultative

to the dentin surface and consequent

anaerobic microorganism with the

2

The ultimate goal of endodontic

formation of dense bacterial biofilms .

ability to form biofilms, acquire several

treatment is to eliminate bacteria from

Among the microorganisms present in

virulence

infected root canals by mechanical

the root canal microflora, Enterococcus

tubules, and grow in extreme conditions,

instrumentation combined with the use

faecalis is a major pathogen associated

such as at elevated pH4. Therefore, its

of irrigating solutions1. The root canal is

with treatment failure in cases of

present

an accessible environment for several

persistent endodontic infections3. E.

warrants further investigation.

factors,

degree

invade

of

dentinal

pathogenicity5

Pedro et al • Journal of Research in Dentistry 2018, 6(5):98-103

Sodium hypochlorite (NaOCl) is

Collection (ATCC 29212), was used as the

polypropylene Eppendorf tubes (Cral,

a commonly used antimicrobial irrigant

biological indicator. The bacterial strain

SP, Brazil) containing BHI broth and

in endodontics, mainly because of

was inoculated into 7 mL of brain heart

autoclaved for 30 min at 120°C.

properties such as the ability to dissolve

infusion

organic

matter,

instruments,

broth

(Difco

lubricate

the

Laboratories, MI, USA) and incubated at

neutralize

EXPERIMENTAL DESIGN

toxic

37°C for 24 h. The experimental

antimicrobial

suspensions were prepared by culturing

Five milliliters of BHI broth

activity and ability to remove the smear

the strain on the surface of BHI agar

were mixed with 5 mL of the bacterial

layer when used in combination with

(Difco Laboratories), following the same

inoculum.

incubation conditions. The bacterial cells

inoculated with E. faecalis for 60 days

were suspended in saline to give a final

using sterile syringes of sufficient

agents6,7.

and

(BHI)

Despite

its

8

chelating agents , NaOCl is associated with

6

cytotoxic

effects .

8

-1

The

specimens

were

Ethylenediaminetetraacetic acid (EDTA)

concentration of about 3x10 cells/mL ,

volume to fill the root canal. This

was the first chelating agent to be used

adjusted to No. 1 McFarland turbidity

procedure was repeated every 72 h,

for the purpose of facilitating the

standard.

always using 24-h pure cultures prepared

8,9

instrumentation of atretic canals . Therefore,

a

novel,

accessible,

and adjusted to No. 1 McFarland SOLUTION PREPARATION

turbidity standard. The teeth were

biocompatible and low-cost irrigating

maintained in a humid environment at

solution that promotes disinfection has

The following solutions were

37°C. Ten uncontaminated specimens

been the subject of research in several

tested in this experiment: 2.5% NaOCl

were used as a negative control group,

(Longevitá, GO, Brazil); 17% EDTA

serving as an aseptic control. Ten

(Longevitá);

chitosan

contaminated specimens served as a

(Longevitá). For the preparation of

positive control. Positive and negative

. It has a chelating

chitosan, 0.2 g of chitosan were diluted

control groups were kept without

properties

as

in 100 mL of 1% acetic acid, and the

irrigation

biodegradability,

mixture was stirred for 2 h using a

contamination. After the contamination

magnetic

obtaining

period, all specimens were filled with

toxicity . In endodontics, chitosan is

crystalline homogeneous solutions with

sterile distilled water. Sterile paper

considered an alternative to EDTA for

3.2 pH.

points (Tanari, Tanariman Indústria

10-12

studies

.

Chitosan

polysaccharide

is

a

natural

obtained

by

deacetylation of chitin from crab and shrimp shells capacity

12-14

and

biocompatibility, bioadhesion,

and

such

absence

of

cell

14

14,15

root canal irrigation

until

and

used

to

confirm

Ltda., AM, Brazil) were introduced into

endodontic

formation,

stirrer

0.2%

.

Because of the difficulty in controlling

and

TOOTH PREPARATION

the root canal and maintained for 1 min.

biofilm

investigation

Three

substances,

incisors had their crowns removed with

immersed in 7 mL of Letheen Broth

effective against resistant pathogens

a diamond disc (American Burrs, SP,

(Difco

such as E. faecalis, is necessary. The

Brazil) at a 90° angle to the long axis of

containing or added with neutralizers

objective of this ex vivo study was to

the

[lecithin,

evaluate the effectiveness of different

standardized to 16 mm and the canals

thiosulfate

endodontic irrigants against E. faecalis.

were emptied with a #15 K-Flex file

Brazil)], followed by incubation at 37°C

(Dentsply

Ballaigues,

for 48 h. The presence or absence of

METHODS

tooth.

Switzerland)

BIOLOGICAL INDICATOR

bovine

were

collected from each root canal and

chemical

Seventy

samples

mandibular

biocompatible

of

paper-point

Root

lengths

Maillefer, combined

were

sodium

Laboratories,

SP,

of the culture medium. The specimens

2.5% NaOCl (Longevitá). Root canals

were randomly divided into 7 groups

were dried and filled with 17% EDTA

(n=10) as described in Table 1.

remove the smear layer. All specimens

from

were individually placed in 1.5-mL

Culture

and

bacteria was determined by the turbidity

anaerobic coccus E. faecalis, obtained Type

(Art

80,

medium

with

(Biodinâmica, PR, Brazil) for 5 min to

American

Tween

a

conventional irrigation using 3 mL of

The Gram-positive facultative the

Laboratories),

|99


EVALUATION ANTIMICROBIAL

OF

THE

process have been investigated15-19. In the

models2,7,10,14. The choice of bovine dentin

OF

present study, the evaluation of the

for infection and disinfection tests has

turbidity of the culture medium and the

been well accepted14,20. Bovine dentin

qualitative evaluation of root dentin

appears to be a suitable substitute for

surface

SEM

human dentin, and factors such as ease

showed that the different endodontic

of obtaining and handling and the use of

irrigants and substances tested were not

bovine dentin in similar tests involving

effective in completely eliminating

the removal of smear layer further

dentin bacterial contamination with E.

support its indication20. In addition, its

faecalis.

the

topography is an important factor in the

methodology used in this study should

survival of biofilms, since irregular

be considered when interpreting the

surfaces increase bacterial adhesion and

results, since statistical analyses were

retention14. Our methodology included

not possible. Although some authors

the use of a peristaltic pump device

have reported limited efficacy of NaOCl

connected to the entrance of the root

combined

and

canals in order to simulate the root canal

, there is still no report of a

irrigation process2,10. Subsequently, the

fully effective action of any of these

evaluation of the turbidity of the culture

solutions

medium of microorganisms collected

EFFECT

IRRIGATING SOLUTIONS To evaluate the antimicrobial efficacy of the irrigating solutions, a sterile urethane hose was connected to a polypropylene Eppendorf tube attached to the teeth and to the entrance of a peristaltic pump (Sarlo 90, SP, Brazil). The entrance of this apparatus was the urethane hose connected to the tube and its exit corresponded to the apical portion of the root canals. The irrigants circulated within the apparatus at a constant flow of 50 mL/min-1 for 10 min, as previously described by Estrela et al.10. After the 10-min period, each tooth was removed from the apparatus under aseptic conditions and irrigation was performed with 5 mL of sterile distilled water.

Paper-point

collection

was

performed as described earlier, followed by incubation at 37°C for 48 h. After evaluation of the culture medium, a 0.1mL inoculum was transferred to 7 mL of BHI broth and subsequently incubated at 37°C for 48 h. After 48 h, bacterial growth was examined by visual analysis of the turbidity of the culture medium. RESULTS The antibacterial efficacy of the chemicals studied is described in Table 2. None of the endodontic irrigating solutions and experimental substances tested effectively eliminated dentin bacterial contamination with E. faecalis.

endodontic

control

treatment

chitosan

during

remains

The

using

limitations

with

both

of

EDTA

16-19

DISCUSSION Infection

contamination

a

challenge, and new auxiliary chemical substances to help in the disinfection

against

E.

faecalis

in

endodontics. Several

from the root canals allowed us to studies

have

determine the microorganism growth

investigated the antimicrobial efficacy of

rate.

Studies

investigating

irrigants using different experimental

antimicrobial efficacy

of

the

irrigating

|100


solutions have used different methods, 15

such as the agar well diffusion method , 21

fluorescence 17

analysis .

assays ,

However,

and

SEM

methodological

differences may lead to results that

may be a viable alternative to the

research on the effectiveness of irrigants

irrigating solutions currently used in

in this scenario.

endodontics, given its effective action against

microorganisms

and

less

CONCLUSION

cytotoxic effects.

cannot be extrapolated to the clinical

E. faecalis was chosen as a

Based on the methodology and

setting or at least should be done with

biological marker because of its possible

irrigation protocol used in this study, it

caution.

microbial role in previously root-filled

can be concluded that the different

15

Active chlorine generated by the

teeth with persistent periapical lesions .

irrigants and substances tested were not

dissociation of NaOCl in aqueous

Owing to characteristics such as being

effective in eliminating dentin bacterial

medium is a powerful oxidizing agent

found more frequently in asymptomatic

contamination with E. faecalis.

that exerts an antimicrobial effect by

secondary endodontic infections and

1

irreversible oxidation of bacterial cells .

being able to invade dentinal tubules and

Although lower concentration solutions

compete with other microorganisms,

have

this Gram-positive, facultative anaerobic

1. Estrela C, Holland R, Estrela CR,

microorganism has gained an important

Alencar AH, Sousa-Neto MD, Pécora JD.

demonstrated

efficacy,

higher

antimicrobial

concentrations

of

1,2

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