Biofilm removal by 6% sodium hypochlorite activated by different irrigation techniques

Abstract

To compare the removal of biofilm utilizing four irrigation techniques on a bovine root canal model.
Fifty dentine specimens (2x2 mm) were infected with biofilm. The samples were then adapted to previously created cavities in the bovine model. The root canals were irrigated twice with 2 mL of 6% sodium hypochlorite for 2 minutes (4 minutes total). Following initial irrigation, the different treatment modalities were introduced for 60 s (3 x 20 s intervals). The evaluated techniques were needle irrigation, endoactivator, passive ultrasonic irrigation and laser activated irrigation (photon induced photoacoustic streaming). The controls were irrigated with distilled water and conventional needle irrigation. Subsequently, the dentine samples were separated from the model and analyzed using a scanning electron microscope (SEM). Fifteen operative fields were scanned per block and SEM pictures were captured. Two calibrated evaluators examined the images and collected data using a 4-degree scale. Non-parametric tests were used to evaluate for statistical significance among the groups.
The group undergoing laser-activated irrigation using photon induced photoacoustic streaming exhibited the most favorable results in the removal of biofilm. Passive ultrasonic irrigation scores were significantly lower than both the endoactivator and needle irrigation scores. Sonic and needle irrigation were not significantly different. The least favourable results were found in the control group.
Laser activation of 6% sodium hypochlorite significantly improved the cleaning of biofilm infected dentine followed by passive ultrasonic irrigation. This article is protected by copyright. All rights reserved.

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Received Date : 19-Jul-2013
Revised Date : 17-Sep-2013
Accepted Date : 05-Oct-2013
Article type : Original Scientific Article
Biofilm removal by 6% sodium hypochlorite activated by different irrigation techniques
R. Ordinola-Zapata
1
, C. M. Bramante
1
, R. M. Aprecio
2
, R. Handysides
2
, D. E. Jaramillo
2
1
Department of Endodontics, Dental School of Bauru, University of São Paulo, Bauru, Brazil,
2
Department of
Endodontics, School of Dentistry, Loma Linda University, California, USA.
Running Head: Biofilm removal by different irrigation techniques
Keywords: laser activated irrigation, photoacoustic streaming, biofilms, irrigant solutions.
Corresponding author:
Ronald Ordinola-Zapata DDS, MSc
Faculdade de Odontologia de Bauru, University of São Paulo, Al. Octávio Pinheiro Brisolla, 9-75, CEP 17012-
901, Bauru, São Paulo, Brazil
e-mail: ronaldordinola@usp.br
Abstract
Aim To compare the removal of biofilm utilizing four irrigation techniques on a bovine root
canal model.
Methodology Fifty dentine specimens (2x2 mm) were infected with biofilm. The samples were then
adapted to previously created cavities in the bovine model. The root canals were irrigated twice with 2

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mL of 6% sodium hypochlorite for 2 minutes (4 minutes total). Following initial irrigation, the
different treatment modalities were introduced for 60 s (3 x 20 s intervals). The evaluated techniques
were needle irrigation, endoactivator, passive ultrasonic irrigation and laser activated irrigation
(photon induced photoacoustic streaming). The controls were irrigated with distilled water and
conventional needle irrigation. Subsequently, the dentine samples were separated from the model and
analyzed using a scanning electron microscope (SEM). Fifteen operative fields were scanned per
block and SEM pictures were captured. Two calibrated evaluators examined the images and collected
data using a 4-degree scale. Non-parametric tests were used to evaluate for statistical significance
among the groups.
Results The group undergoing laser-activated irrigation using photon induced photoacoustic
streaming exhibited the most favorable results in the removal of biofilm. Passive ultrasonic irrigation
scores were significantly lower than both the endoactivator and needle irrigation scores. Sonic and
needle irrigation were not significantly different. The least favourable results were found in the
control group.
Conclusions Laser activation of 6% sodium hypochlorite significantly improved the cleaning of
biofilm infected dentine followed by passive ultrasonic irrigation.
Introduction
The aim of irrigation in root canal treatment is to improve the cleaning and disinfection
process of the root canal system (Siqueira & Roças 2008). Irrigants play multiple roles in
endodontic therapy. They are necessary from an antimicrobial aspect since the mechanical
instrumentation process is insufficient on its own to remove the microbial load (Byström &
Sundqvist 1983). Sodium hypochlorite (NaOCl) is considered the main root canal irrigant
because of its tissue dissolution and antimicrobial properties. While some microscopic studies

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have shown that complete dissolution of biofilms by sodium hypochlorite is possible using the
direct contact test (Del Carpio-Perochena et al. 2011), incomplete dissolution and residual
biofilm appears to be common under clinical conditions following full strength NaOCl irrigation
(Vera et al. 2012). Residual biofilm may contain viable bacteria and may decrease the interfacial
adaptation of root filling materials (Vera et al. 2012).
Significant information regarding the physical effect of fluids in root canals has been previously
reported (Chow 1983, Ahmad et al. 1987, Jiang et al. 2010, De Gregorio et al. 2010). These
studies have shown that positive or negative apical pressure can affect the diffusion of the
irrigant solutions into the root canal system, improving the cleaning ability (Chow 1983, De
Gregorio et al. 2010). In addition, the use of ultrasonic irrigation has been shown to improve the
cleaning efficacy of irrigants showing in many cases superiority in comparison to common
positive apical pressure techniques (Burleson et al. 2007).
Lasers have been used to produce cavitation of liquids, thereby increasing the cleaning ability of
the liquid (Lauterborn & Ohl 1997, Blanken 2007, Peel et al. 2011). When laser pulses are
focused into a limited volume of fluid, plasma is generated. Plasma formation can lead to rapid
heating of the material followed by an explosive expansion and the emission of a shock wave.
This is possible by the high absorption of the Er:YAG wavelength in water (Di Vito et al. 2012).
These lasers have been evaluated for the elimination of the smear layer and dentinal debris
(George et al. 2008) with promising results (de Groot et al. 2009). These techniques, referred to
as laser activated irrigation (de Groot et al. 2009) have been evaluated for endodontic irrigation
applications basically by using Erbium YAG (Er:YAG) or Er,CrYSGG lasers, with energy levels

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that vary from 25-300mJ (Blanken 2007, George et al. 2008, Blanken et al. 2009, Peters et al.
2011).
In this work, a novel tapered and stripped tip of a laser activated irrigation technique called
photon induced photoacoustic streaming (PIPS) at energy levels below those previously cited in
the literature (20mJ) was used. It has been demonstrated that the transition of the laser light from
the tip to the fluid creates a photoacoustic pressure wave throughout the liquid with no thermal
effects on the dentine surface (Di Vito et al. 2012). The efficacy of laser-activated irrigation to
clean biofilm-infected dentine has not been fully evaluated. This study compared the cleaning
ability of passive ultrasonic irrigation, Endoactivator (Dentsply Tulsa Dental, Tulsa, OK, USA),
needle irrigation and laser activated irrigation in conjunction with 6% NaOCl to clean in situ
biofilm infected bovine dentin.
Materials and methods
Biofilm development
Fifty sterile bovine dentine sections (2x2mm) were used. The samples were treated with
17% EDTA for 3 minutes to eliminate the smear layer produced during the sectioning process.
To induce dentine infection an in situ model was selected using a Hawleys orthodontic device.
The dentine surface exposed to the oral cavity was fixed 1mm above the surface to allow the
accumulation of plaque. One volunteer used the device continuously for 72h, except during oral
hygiene procedures, to generate biofilm (Human committee and ethic research approval,
CEP134/2010). Daily food diet was maintained. After the intraoral contamination process, each
sample was incubated in 2mL of BHI at 37º for 48h in aerobic conditions. Then, each sample

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was rinsed with 1mL of distilled water to remove culture medium and non-adherent cells (See
Figure 1).
The 50 specimens were randomly divided into 5 groups according to the final irrigation protocol
used. G1: conventional needle irrigation, G2: Endoactivator (Dentsply Tulsa Dental, Tulsa, OK,
USA), G3: passive ultrasonic irrigation, G4 Laser activated irrigation (PIPS, Fotona, Ljubljana,
Slovenia) and G5: control (distilled water).
Root canal irrigation model
A root canal irrigation model was developed using decoronated bovine incisors. The root
canals of 10 roots, 12 mm in length, were prepared to an apical size of 1.30 mm by using Gates
Glidden burs (Dentsply Maillefer, Ballaigues, Switzerland). Thereafter, a perforation (2.5 x 2.5
mm) was made 3 mm from the apical foramen to adjust the infected dentine block to the
perforation (Figure 1).
The infected dentine sections were fixed into the perforation site with the infected side placed
facing the root canal. The apical foramen was sealed with silicone (SOURCE) to provide a
closed system. This device allows the adaption of the infected area of the intraorally infected
dentine block at the same level of the apical area of the root canal of a bovine incisor tooth. Ten
bovine roots were used during the experiments. Each root was used a maximum of 5 times. The
irrigation protocol was divided in 2 steps:
All canals were irrigated with 2mL of 6% NaOCl (Clorox, Oakland, Ca, USA) delivered by
positive apical pressure using a 10mL syringe and a double side-vented needle (SybronEndo,
Glendare, CA, USA) inserted until 2mm from the apex. A flow rate of 1mL/10 s was used and
the NaOCl solution was left in the canal space for 2 min. After aspiration of the solution this

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procedure was repeated one more time. The total time in this step (without taking into
consideration the 20 s of NaOCl application) was 4 minutes and 4mL of 6% NaOCl were used
for all the experimental groups (See Figure 1F).
Experimental procedures
Sodium hypochlorite solution was applied at a rate of 1mL/10 s and the irrigation
techniques test were performed for 20 s. Each procedure was repeated two more times. In all the
experimental groups the final amount of NaOCl used was 3mL in the last minute (See Figure
1F). The evaluated irrigation techniques were:
Group 1: Conventional needle irrigation using double side vented needles. In this technique the
needle was inserted until 2mm from the apex. Then, 1mL of NaOCl was applied using a flow
rate of 1mL/10 s and was left in root canal for 20 s, this procedure was repeated two more times
for a total period of 1 min of treatment.
Group 2: Endoactivator: 1mL of NaOCl was applied at the apical third followed by the sonic
activation of the irrigant by using a yellow endoactivator (15.02) tip for 20 s. This procedure
(irrigation/sonic activation) was repeated two more times for a total period of 1 min of sonic
treatment. The Endoactivator tip was inserted until 2mm from the apex.
Group 3: Passive ultrasonic irrigation (PUI): In this technique, a similar procedure was applied in
the same manner described for the endoactivator group, but an Irrisafe file 20.00 (Satelec,
Acteongroup, Merignac, France)
was used in conjunction with a Satelec P5 suprasson ultrasonic
unit (Suprasson P5; Satelec Acteongroup, Merignac, France) at a power setting of 4.
Group 4: Laser activated irrigation (LAI). An Er:YAG laser with a wavelength of 2940 nm
(Fidelis; Fotona, Ljubljana, Slovenia) was used to irradiate the root canals by using a 12 mm

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400-μm quartz tip. The laser operating parameters were: 20 mJ per pulse, 0.30W, 15 Hz, and 50
μs pulse duration. An endodontic fibre tip (PIPS, Fotona, Ljubljana, Slovenia) was placed into
the coronal access opening of the access cavity. One millilitre of NaOCl was applied and
activated for 20 s. This procedure was repeated 2 more times.
Group 5: Control, the initial irrigation procedures were similar to group 1, except that distilled
water was used for the initial and final irrigation procedures. In this technique, 4 mL of distilled
water were initially used for 4 minutes. For the final irrigation purposes, 1 mL of distilled water
was applied using a flow rate of 1mL/10 s and was left in the root canal for 20 s. This procedure
was repeated two more times for a total period of 1 min of treatment.
Following irrigation, the dentine blocks were detached from the root, treated for 1 min with 1mL
of 5% sodium thiosulfate and then fixed in formalin for 24 h. The samples were dehydrated with
alcohol, mounted on stubs sputter coated with platinum and observed using a scanning electron
microscope (XL30 FEG, Phillips, Eindhorn, Netherlands). Fifteen images from random areas
were obtained from each sample at 2400X magnification. One hundred fifty SEM pictures were
evaluated for each group. For quantification purposes a modified 4- score scale system was used
based on Bhuva et al. (2010).
Score 1: Clean dentine or residual isolated microbial cells that covers less than 5% of the
dentine. Absence of residual biofilm layers
Score 2: Residual isolated microbial cells covers 5% - 33% of the dentine. There is absence of
residual biofilm layers.
Score 3: Biofilm structures and microbial cells can be identified covering 34% - 66% of the
dentine.

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Score 4: Biofilm structures and microbial cells can be identified covering 67% - 100% of the
dentine.
Two evaluators with SEM experience evaluated the pictures in a blinded manner. The
evaluations were performed in two occasions with interval of 4-weeks. In cases of disagreement
between the evaluators the higher score was selected.
Statistical analysis was performed using the non-parametric Kruskal-Wallis and Dunn tests (p <
0.05). Kappa test was used to measure intra and inter rater agreement. Prisma 5.0 (GraphPad
Software Inc, La Jolla, CA, USA) was used as the analytical tool.
Results
Control specimens (distilled water irrigation) were characterized by the presence of a
thick biofilm layer covering the dentine structure. The presence of several morphotypes as cocci
and rods could be identified. From the 50-dentine blocks, 750 SEM images were examined (15
images for each sample).
The variability between examiners as measured by kappa coefficient was 0.78 (strong). The
intraobserver agreement was 0.82 and 0.85 for the first and second evaluator respectively. The
mean and median scores of the different groups are shown in Table 1. Distilled water irrigation
score was classified as 4 in all the SEM pictures evaluated. Kruskal Wallis - Dunn test showed
significant differences among the groups. Laser activated irrigation (LAI) had the lowest scores
compared to PUI, Endoactivator and needle irrigation (P < 0.05). PUI scores were lower than
both endoactivator and needle irrigation scores (P < 0.05). There was no difference between
endoactivator and needle irrigation (P > 0.05). The worst result was found in control group that

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do not show any significant effect against biofilm (P < 0.05). Representative SEM pictures and
distribution of the scores in the evaluated groups are shown in Figure 2.
Discussion
This study revealed that the disruption of biofilm by 6% NaOCl can be enhanced by
using LAI and PUI techniques. Most of the research available about cleaning ability of these
techniques has compared the efficacy to eliminate dentine debris (de Groot et al. 2009, Jiang et
al. 2010). However, there is a lack of evidence comparing the ability of PUI and LAI to improve
the cleaning of biofilm infected dentine (de Moor et al. 2009, Peters et al. 2011)
Several models of biofilms are used in endodontic research and the efficacy of NaOCl depends
on variables such as the method of biofilm growth (Bhuva et al. 2010), NaOCl concentration
(Ordinola-Zapata et al. 2013) and exposure time (del Carpio-Perochena et al. 2011). It could also
be considered that oral mixed biofilms can be more resistant and have a greater adhesion to
dentine in comparison to biofilms developed under laboratory conditions (Stojicic et al. 2012).
This detail can possibly explain why a previous study that used in vitro monospecies biofilms
found no difference between conventional and PUI irrigation (Bhuva et al. 2010). The authors
found that a Enterococcus faecalis biofilm can be completely dissolved by using 6mL of 1%
NaOCl for 2 minutes (Bhuva et al. 2010). In another direct contact test, Stojicic et al (2012)
found that 1–2% NaOCl destroyed Enterococcus faecalis biofilms in 3 min.
In the present study, similar to previous studies (Barthel et al. 2002, Peters et al. 2011)
intraorally developed dental plaque was used. The results showed the difficulty of conventional
needle irrigation in combination with 6% sodium hypochlorite to completely dissolve the dental

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plaque biofilm. This result is similar to studies performed in vivo (Vera et al. 2012). Even though
this research was not performed in a complex anatomy, the method used allows comparisons
between different NaOCl irrigation protocols using a standardized area of infected dentine at the
apical level. Despite the results obtained in the present study, one limitation to take into account
is the lack of actual anaerobic conditions such as those that actually appear in the root canal
environment, so the amount of residual biofilm may vary under those conditions.
Scanning electron microscopy is commonly used as an evaluative tool to observe infected dentin
(George et al. 2005). Although this technique allows only bidimensional and semi-quantitative
analysis, it provides the advantage of higher resolution and details of the dentine surface in
comparison to confocal microscopy or stereomicroscopy used in previous studies (de Groot et al.
2009, del Carpio-Perochena et al. 2011). To minimize bias, randomization and a considerable
numbers of images were taken of a small-predefined infected area placed at the apical third.
Similar to previous reports, the cleaning efficacy of the Endoactivator or sonic devices was
similar to needle irrigation (Brito et al. 2009, Uroz-Torres et al. 2010, Johnson et al. 2012, Seet
et al. 2012). This observation has been made by studies using scanning electron microscope
(Uroz-Torres et al. 2010, Seet et al. 2012), microbiological culture (Brito et al. 2009) or by
histological methods (Johnson et al. 2012). In general, it is accepted that ultrasonic irrigation
provides higher frequency and this improves the acoustic microstreaming of NaOCl in
comparison to the Endoactivator device (Jiang et al. 2010). According to Jiang et al. (2010) the
endoactivator device did not improve canal cleanliness regardless of frequency used or tip size.
These authors found that the amplitude of the endoactivator tip was 1 mm, which implies a high

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probability of contact between the tip and the root canal wall decreasing its efficacy in
comparison to the ultrasonic movements that is in the range of 75 micrometres (Jiang et al.
2010).
In the present study minimal or negative canal wall contact of the Endoactivator and ultrasonic
device was expected due to the diameter of the root canal (1.3mm). Enlarging the canal also
allowed the Endoactivator and ultrasonic file tips to be placed at the same level of the infected
dentine in order to maximize their effectiveness. Conversely, the tip of the laser technique was
located in the access chamber and activated several millimetres coronal or distant from the target
point.
The use of shockwaves has gained the attention of some medical areas to treat biofilm related
diseases. Local deposition of energy as heat or light is necessary to induce cavitation (Lauterborn
& Ohl 1997) and photoacoustic streaming appears to be the mechanism of cleaning at the
liquid/dentine interface (Blanken, 2007, de Groot et al. 2009, Blanken et al. 2009). A previous
study has shown that this technique is effective in disrupting Pseudomona aureginosas and
plaque derived biofilm in the absence of antimicrobials (Krespi et al. 2008, Krespi et al. 2011,
Muller et al. 2011). Biofilm disruption can change the bacteria to their planktonic form, making
them more susceptible to antimicrobial agents (Kizhner et al. 2011).
One associated effect of the application of acoustic or photoacoustic waves on chemicals systems
is sonochemistry. Previous studies in the industrial area have shown that ultrasonics can enhance
the effectiveness of NaOCl disinfection (Duckhouse et al. 2004, Zifu et al. 2012). A previous
study showed that ultrasonic and laser activation increase significantly the reactivity of NaOCl
(Macedo et al. 2010). Temperature is also a variable that can influence the effectiveness of
NaOCl (Al- Jadaa et al. 2009). Two previous reports reported a rise of the root canal temperature

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after the use of passive ultrasonic activation (Cameron 1988, Al-Jadaa et al. 2009), which could
increase the ability of sodium hypochlorite to remove biofilm.
Parameters used in the laser
induced irrigation includes subablative power settings (20mJ) the use of the PIPS tip at the
coronal level avoids the undesired effects of the thermal energy on the dentinal walls (Di Vito et
al. 2012), thus, the cleaning ability of the laser could not be necessarily associated to a rise in the
temperature of the irrigant solution. The significant difference with this laser induced irrigation
technique (PIPS) in comparison to PUI may be attributed to the high peak powers created with
minimal energy (20mJ or less) with low pulse durations (50 μs) leading to pressure waves that
move irrigants in three dimensions distant to the tip position. The better cleaning ability of laser-
activated irrigation is in agreement with a previous study (Peters et al. 2011). Because the
cleaning effect of NaOCl is a time dependent phenomenon (del Carpio-Perochena et al. 2011)
the results can also reflect that there is acceleration in the dissolution and cleaning effect of
NaOCl when laser activated irrigation is used.
Due to the limited comparisons between acoustic and photoacoustic induced shockwaves future
studies are necessary for the understanding of laser-activated irrigation, including the effect of
activation time, the ability to avoid the accumulation of hard tissue debris and the cleaning
ability in presence of pulp tissue in complex anatomies.
Conclusions
Under the conditions of the current study, laser-activated irrigation using the photon-induced
photoacoustic streaming technique of 6% sodium hypochlorite significantly improved the cleaning of
biofilm-infected dentine compared to passive ultrasonic, sonic, or mere needle irrigation.

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Acknowledgements
Supported by FAPESP grants 2010/16002-4 and 2011/22283-9
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Figure Legends
Figure 1 A removable orthodontic device was used to induce the contamination of dentine (A).
Then, the blocks were incubated for 48 h in BHI (B). Image and schematic representation of
roots modified for the experiment (C, E). The pulp chamber walls were reconstructed by using
composite resin (c). A perforation was made at the apical portion in order to adapt the infected
dentine (b). The infected dentine sections were fixed into the perforation site facing the root
canal (arrow). The dentin block was set with fluid silicone (s). Representative SEM of a biofilm
infected dentin (D). The steps of the irrigation procedure are represented in (F). In the first step,
NaOCl was applied for 4 min. Then, the tested irrigation techniques were performed for 20
seconds and repeated two more times.
Figure 2 Representative images of the evaluated groups: Control (distilled water) showing
extensive biofilm colonization (A), needle irrigation (B), showing biofilm residual layers (*).
Dentine treated with the Endoactivator (C) and passive ultrasonic irrigation (D) showing residual
biofilms and bacteria. Clean dentine and open dentinal tubules can be seen in the laser activated
irrigation technique (E). Distribution of scores after the SEM evaluation (F).

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Table 1. Score distribution in the evaluated groups. Mean and Median are also presented.
Score 1 Score 2 Score 3 Score 4 Mean Median
*
Total
Control 0 0 0 150 4 4
a
150
Needle 23 40 56 31 2.63 3
b
150
Endoactivator 31 35 52 32 2.56 3
b
150
PUI 72 33 25 20 1.95 2
c
150
LAI 107 21 10 12 1.52 1
d
150
* Letters shows statistically significant differences between groups (Kruskal Wallis-Dunn’s test).

Accepted Article
This article is protected by copyright. All rights reserved.