UNIVERSIDADE ESTADUAL PAULISTA
“JÚLIO DE MESQUITA FILHO”
Instituto de Ciência e Tecnologia
Campus de São José dos Campos
ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2024.e3904
1
Braz Dent Sci 2024 Jan/Mar; 27 (1): e3904
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Antimicrobial activity of Er,Cr:YSGG and Ultrasonic on
E. faecalis
biofilm in the mesial root canal systems of lower molars
Atividade antimicrobiana de Er,Cr:YSGG e Ultrassônico no biofilme de
E. faecalis
nos sistemas de canais radiculares mesiais
de molares inferiores
Ghufran Ismail IBRAHIM1 , Hussein Ali JAWAD1
1 - University of Baghdad, Institute of Laser for Postgraduate Studies. Baghdad, Iraq.
How to cite: Ibrahim GI, Jawad HA. Antimicrobial activity of Er,Cr:YSGG and Ultrasonic on
E. faecalis
biolm in the mesial root canal
systems of lower molars. Braz Dent Sci. 2024;27(1):e3904. https://doi.org/10.4322/bds.2024.e3904
ABSTRACT
The internal topography of the root canal is complex, especially for the permanent molar’s mesial root. In response
to such issues, improved irrigation techniques have been created, which use laser pulses to agitate uids and
improve microbial deposit removal. Objective: To assess the effectiveness of the Er,Cr:YSGG laser with a wavelength
of 2,780 nm via photon-induced photoacoustic streaming (PIPS) protocol which agitated of 2% chlorohexidine
(CHX) in removing mature Enterococcus faecalis (
E. faecalis
) biolm in root canal systems of lower molars.
Material and Methods: The mesial roots of lower rst and second molars were separated and inoculated with
E. faecalis
bacterial suspension for 30 days. The roots were irrigated with CHX, some of them were agitated with a
passive ultrasonic device (PUI), while the other roots were agitated by an Er,Cr:YSGG laser in PIPS at 60 µs/pulse,
5 Hz, (0.25, 0.5, 0.75, and 1) W. An atomic force microscope (AFM) was used as a new method to get the results in
the isthmus area; the obtained results from each group were compared with each other. Results: Based on the AFM
and SEM analyses, laser and ultrasonic activation groups showed higher antimicrobial efcacy than the conventional
syringe irrigation group (P<0.05). Conclusion: Based on the investigation’s ndings, the activation of 2% CHX
solution by Er,Cr:YSGG laser in PIPS and PUI offers better mature bacterial biolm removal in the mesial root of
lower human molars than the same irrigant with the SI technique.
KEYWORDS
2% Chlorhexidine gluconate; Atomic force microscope; Enterococcus faecalis biolm; Er,Cr:YSGG laser;
Passive ultrasonic activation.
RESUMO
A topograa interna do canal radicular é complexa, especialmente para a raiz mesial do molar permanente. Em resposta
a esses problemas, foram criadas técnicas aprimoradas de irrigação, que utilizam pulsos de laser para agitar uidos
e melhorar a remoção de depósitos microbianos. Objetivo: Avaliar a ecácia do laser Er,Cr:YSGG com comprimento
de onda de 2.780 nm via protocolo de streaming fotoacústico induzido por fótons (PIPS) que agitou clorohexidina
a 2% (CHX) na remoção de Enterococcus faecalis maduro (
E. faecalis
) biolme em sistemas de canais radiculares
de molares inferiores. Material e Métodos: As raízes mesiais de 28 primeiros e segundos molares inferiores foram
separadas e inoculadas com suspensão bacteriana de
E. faecalis
por 30 dias. As raízes foram irrigadas com CHX,
sendo algumas delas agitadas com aparelho ultrassônico passivo (PUI), enquanto as demais raízes foram agitadas
com laser Er,Cr:YSGG em PIPS a 60 µs/pulso, 5 Hz (0,25, 0,5, 0,75 e 1) W. Um microscópio de força atômica (AFM)
foi utilizado como um novo método para obter os resultados na área do istmo; os resultados obtidos de cada grupo
foram comparados entre si. Resultados: Com base nas análises de AFM e SEM, os grupos de ativação por laser e
ultrassom apresentaram maior ecácia antimicrobiana do que o grupo de irrigação com seringa convencional (P<0.05).
Conclusão: Com base nos achados da investigação, a ativação da solução de CHX a 2% pelo laser Er,Cr:YSGG em PIPS
a (60 µs/pulso, 5 Hz, 0,75 W) oferece melhor remoção de biolme bacteriano maduro na raiz mesial da raiz humana
inferior molares do que o mesmo irrigante com as técnicas SI e PUI.
PALAVRAS-CHAVE
2% Gluconato de clorexidina; Ativação ultrassônica passiva; Biolme de Enterococcus faecalis; Er,Cr: laser YSGG;
Microscópio de força atômica.
2
Braz Dent Sci 2024 Jan/Mar; 27 (1): e3904
Ibrahim GI et al.
Antimicrobial activity of Er , Cr: YSGG and Ultrasonic on E. faecalis biofilm in the mesial root canal systems of lower molars
Ibrahim GI et al. Antimicrobial activity of Er, Cr: YSGG and Ultrasonic on E. faecalis
biofilm in the mesial root canal systems of lower molars
INTRODUCTION
The primary pathogenic agents of the dental
pulp and periapical infections are bacterial and
other microbial pathogens in the root canal
system. Enterococcus faecalis (
E. faecalis
) is a
gram-positive, facultative and anaerobic bacteria
which is the most commonly identied after failed
root canal therapy and considered to be extremely
difcult to clear with conventional methods [1].
The most commonly used antibacterial irrigation
solution is chlorhexidine gluconate (CHX),
which is a broad-spectrum antibacterial fluid
that works to combat both gram-positive and
gram-negative bacteria as well as yeasts [2].
CHX has been utilized as a final irrigant or
intracanal medicament in endodontics. Also,
it is recommended as an alternative to NaOCl,
particularly in cases of open apex, root resorption,
and root perforation.
CHX’s antibacterial action is pH-dependent,
with an optimal range of 5.5 to 7, as well as
concentration-dependent, so irrigating with 2 percent
chlorhexidine is better than 0.12 percent [3].
It is critical to work on novel strategies to ensure
irrigation uid reaches inaccessible places, thereby
improving endodontic outcomes. Recently, the
laser photon-induced photoacoustic streaming
(PIPS) technology has demonstrated encouraging
results in the elimination of biofilm from the
root canal surface [4,5]. This method involves
activating the irrigation uid within the root canal
via complex photoacoustic and photomechanical
phenomena [6]. Each impulse generated by the
PIPS tip gets absorbed by the water particles,
resulting in the production of a powerful “shock
wave” that results in the creation of effective
uid streaming within the root canal system [7].
PIPS technique is used by an Erbium laser family
at sub-ablative settings. An erbium, chromium:
yttrium scandium gallium garnet (Er,Cr:YSGG) is
a type of water-absorbing laser with a wavelength
of 2,780 nm [8]. It has been suggested that the
hydrokinetic energy employing promotes dental
canal disinfection with no thermal harm to the
underlying tissues [9,10].
Atomic force microscopy (AFM) is a useful
technique for studying the shape and texture
of various surfaces. Surface texture includes
roughness, waviness, and aws [11]. This method
has been widely utilized to investigate the
mechanisms of antibacterial substance activity
on bacteria [12]. Many kinds of research using
AFM have been used in a variety of dental elds
of study [13,14].
The most previous work on irrigation activation
methods has been done about the improvement
of biofilm removal by the laser agitation of
chlorhexidine in an infected single root canal with
Enterococcus faecalis biolm obtained after a short
incubation period [15,16]. The goal of this study
was to examine whether mechanical agitation by
Er,Cr:YSGG laser improves the effectiveness of 2%
chlorhexidine against 30-day-old
E. faecalis
biolm
in human mandibular root canal systems, and
use of AFM to investigate the impact of different
irrigation protocols on the topography of the surface
by analyzing isthmus surface properties.
MATERIAL AND METHODS
Ethical approval (10-2022-488) was obtained
from the Research and Ethics Committee of
Institute of Laser for Postgraduate Studies,
University of Baghdad.
Specimen selection and preparation
A total of forty-two extracted mandibular rst
and second molars without root canal llings, root
caries, or restorations were obtained and cleaned
immediately after extraction. All samples were
placed in a glass container containing distilled
water with 0.1% thymol crystals (Lab Grade, Lab
Alley, Texas, USA) until the day of the experiment.
A diamond disc (OSA-E28, Osakadent group
ltd., Guangdong, China) was used for cutting off
the teeth crowns. The mesial roots of all molar
teeth were separated from the rest of the teeth
to supply roots with a length of 12 mm from
the apex. A stainless-steel K-type hand le #10
(Dentsply, Maillefer, Ballaigues, Switzerland)
was used to locate the site of the apical foramen.
After sighting the file tip through the apical
foramen, 1 mm was subtracted from the file
length that was measured, and this value was
used as the working length (WL).
All root canals were prepared to this WL up to
#25/.04 NiTi engine les (X3 Never Break Serious,
Easyinsmile, New Jersey, USA) at speed and
torque recommended by the manufacturer. During
instrumentation, 1 mL of 5.25% NaOCl (Cerkamed,
StalowaWola, Poland) was administered after
each le size, using a 30-gauge irrigation needle
with side vents (Endotop, Hang ZhouEndoTop
Medi-Tech Co. ltd., Zhejiang, China). 1 ml of 17%
3
Braz Dent Sci 2024 Jan/Mar; 27 (1): e3904
Ibrahim GI et al.
Antimicrobial activity of Er , Cr: YSGG and Ultrasonic on E. faecalis biofilm in the mesial root canal systems of lower molars
Ibrahim GI et al. Antimicrobial activity of Er, Cr: YSGG and Ultrasonic on E. faecalis
biofilm in the mesial root canal systems of lower molars
EDTA was used as the nal irrigant (Cerkamed,
StalowaWola, Poland). The uid was left inside
the canals for 3 minutes, within this period the
solution was activated with an ultrasonic device
(Guilin Woodpecker Medical Instrument Co. ltd,
Guangxi, China) for 30 seconds.
At the last stage of the process, all the root
samples were irrigated with 5 mL of distilled water
(Pioneer Company, Baghdad, Iraq) to eliminate
the remains irrigation solutions. The root canals
and the outer surfaces of the teeth were dried,
internally with paper points (Sure-endo, Sure
Dent Corporation, Gyeonggi-do, Korea) and
externally with paper towels. All roots were placed
individually in Eppendorf tubes in an upright
posture (Lab Serv, Thermo fisher Scientific,
Gurugram, India) and autoclaved for 20 minutes
at 121 °C and 15 psi pressure.
Bacterial inoculation
After sterilization, paper points were used to
dry the root canals then all canals were inoculated
with bacterial suspension of
E. faecalis
except for
three roots which act as a negative control.
E. faecalis
(ATCC 4083) taken from its frozen
stock was streaked onto agar plate (Himedia,
Mumbai, India) and cultured for 24 hours at 37 °C.
Multiple
E. faecalis
colonies were taken from the
agar plate and activated by being placed in brain
heart infusion (BHI) broth (Himedia, Mumbai,
India) a day before. Then 1 mL of bacterial infusion
was diluted by adding 8 mL of normal saline
to obtain a suspension equal to the McFarland
standard 1.5 × 10 8 colony forming units per
milliliter (CFU/ml). The suspension of bacteria
was injected into the cleaned root canals using
a disposable syringe and a 30-gauge irrigation
needle until they were lled completely. Each root
specimen was immersed in 1.5 mL of BHI broth
after introducing the bacteria into the canals. All
sample tubes were stored in a warm environment
at 37 °C under aerobic conditions for 30 days.
Re-inoculation was conducted every three days to
be sure of the presence of viable bacteria during
the period of incubation, and the BHI was replaced
daily with a new one. All procedures were carried
out in a sterile environment.
Treatment groups
At the point of termination of the incubation
period, the liquid medium was drawn out of the
tubes, and the thirty-nine samples were irrigated
with 5 mL of distilled water, then divided randomly
into four groups: (A) Positive control group, which
did not get any sort of therapy (n=3) (B) Samples
were irrigated with 2% CHX delivered by irrigation
needle (n=12). (C) Passive ultrasonic activation
of 2% CHX (n=12), and (D) Er,Cr:YSGG laser at
{60µs/pulse, 5 Hz, (0.25, 0.5, 0.75, 1) W using
MZ6-6 mm length laser tip in PIPS protocol}
agitate of 2% CHX (n=12).
Each root of the last three groups was subjected
to several procedures: all samples’ surfaces were
cleaned with sterilized cotton pads immersed in
5.25% NaOCl, then they were mounted in plastic
tubes filled with alginate impression material
(Kromalgin, Vanninidental, Grassina, Italy) for easy
handling of the samples.
For group B, a 30-gauge irrigation needle
was used to irrigate the samples with 2% CHX and
the uid left inside the canals for 2 minutes, then
washed with 5 mL of distilled water.
Group C samples were also irrigated with 2%
CHX and left the uid inside the canals for 2 min,
then activated by a passive ultrasonic tip for 60 sec,
then all canals washed with 5 mL of distilled water.
The device tip was placed 1 mm shorter than the
estimated WL.
Group D samples were irrigated with 2% CHX
and left the uid inside the canals for 2 min, within
this time, the fluid was activated by 2780 nm
Er,Cr:YSGG laser (Biolase, Waterlase, iPlus, CA,
USA) for 60 sec. Infrared laser safety glasses
(Innovative Optics, Hemlock Lane North Maple
Grove, USA) were worn before laser activation.
A newly designed water Lase iPlus /MD glass
tip was used (MZ6 Zip tip diameter = 600 µm,
length = 6 mm) and the laser unit’s water, as well
as air spray were both set to “off “. During laser
work, the tip was put just into the canal opening,
remained stationary, and didn’t move apically into
the root canal. Laser operation proceeded for thirty
seconds of “on” time, followed by thirty seconds of
“off” time, and this sequence was repeated twice
(for a total of 60 seconds of activation). After that,
the canal was irrigated with 5 mL of distilled water.
A properly fitting paper point cones were
placed in the root canals to prevent tooth fragments
from entering the endodontic canals and isthmus
area. Without entering the root canals, all roots
were grooved longitudinally on their outer surface
using a diamond disc, and a chisel was used
to cut specimens in half. A middle area of the
4
Braz Dent Sci 2024 Jan/Mar; 27 (1): e3904
Ibrahim GI et al.
Antimicrobial activity of Er , Cr: YSGG and Ultrasonic on E. faecalis biofilm in the mesial root canal systems of lower molars
Ibrahim GI et al. Antimicrobial activity of Er, Cr: YSGG and Ultrasonic on E. faecalis
biofilm in the mesial root canal systems of lower molars
isthmus was marked about 6 mm from the apex
for examination by the AFM (Nanosurf, Liestal,
Switzerland) and SEM (Inspect F-50, FEI Electron
Optics International B.V., Netherlands) tools.
Statistical analysis
The data has been interpreted using the
Statistical Package for Social Sciences (SPSS)
version 21 (IBM, Armonk, New York, USA).
It was provided as a mean, while categorical
data was displayed using the standard deviation.
Data were expressed as a mean±SD. Analysis
of variance (ANOVA) was utilized to compare
the means of the tests. The least significant
difference (LSD) test was used to compute the
signicant differences among the tested means.
Results of p>0.05 were considered statistically
non-signicant, while p≤0.05 was considered a
signicant value.
RESULTS
The efcacy of laser activation and other
methods in the eradication of bacterial biolm in
the isthmus was assessed by using an AFM tool
that analyzes surface roughness to determine the
presence of the biolm in this region.
The uncultivated isthmus area was illustrated
in the two and three-dimensional images
(Figure 1a and 1b), in addition to a histogram that
showed the dominance of small particles with a low
mean diameter value equal to 52.82 nm (Figure 1c).
2D & 3D images of not treated isthmus surface,
about 6 mm from the apex were shown in
Figure 2, and a mean diameter histogram
displayed dominant small particles with a
high mean diameter value of about 76.24 nm.
In Figure 3, the isthmus surface after being
treated with 2% CHX by irrigation syringe method
without any agitation was shown in AFM images.
Figure 1 - AFM imaging of not inoculated isthmus surface about 6 mm from the apex (a) Two and, (b) Three dimensions (c) Histogram
represents the number of particles for different particle’s mean diameter, red columns for large particle, orange columns for medium particles,
and green columns for small particles.
Figure 1
5
Braz Dent Sci 2024 Jan/Mar; 27 (1): e3904
Ibrahim GI et al.
Antimicrobial activity of Er , Cr: YSGG and Ultrasonic on E. faecalis biofilm in the mesial root canal systems of lower molars
Ibrahim GI et al. Antimicrobial activity of Er, Cr: YSGG and Ultrasonic on E. faecalis
biofilm in the mesial root canal systems of lower molars
In addition, the mean diameter of surface small
particles of about 57.42 nm was illustrated in
Figure 3c.
The isthmus of the treated root with
chlorhexidine gluconate activated with PUI was
shown in Figure 4. In Figure 5, the images of the
isthmus surface were exhibited after being treated
with 2% CHX that agitated with Er,Cr:YSGG laser
in PIPS at (60 µs, 5 Hz, 0.75 W), in addition
to a histogram that showed low value of small
particles mean diameter about 33.21 nm. Values
of the root mean square of positive control and
other test groups were statistically analysis
as illustrated in a statistical columns chart
(Figure 6). The lowest root mean square value
was presented in the 2% CHX group that agitated
by laser PIPS at (60 µs, 5 Hz, 0.75 W) (G D) and
the highest root mean square value was presented
in the positive control group (G A).To conrm the
results obtained from an AFM test, the isthmus
area was also evaluated by FE-SEM taken at 6
mm from the apex at (13000 and 50000 x) for
more detailed view of the biolm and bacterial
cocci. After bacterial incubation for 30 days, a
dense and heavy layer of bacterial biolm formed
on the isthmus surface occluding the dentinal
tubules, as shown in Figure 7a and Figure 8a. The
samples treated with the laser PIPS at (60 µs, 5
Hz, 0.75 W) and 2% CHX showed a clean surface
with open tubules and few smashed bacterial
biolms and debris remained on the surface, as
shown in Figures 7b and 8b.
Figure 2 - AFM imaging of inoculated not treated isthmus surface about 6 mm from the apex (a) Two and, (b) Three dimensions (c) Histogram
represents the number of particles for different particle’s mean diameter, red columns for large particle, orange columns for medium particles,
and green columns for small particles.
Figure2
6
Braz Dent Sci 2024 Jan/Mar; 27 (1): e3904
Ibrahim GI et al.
Antimicrobial activity of Er , Cr: YSGG and Ultrasonic on E. faecalis biofilm in the mesial root canal systems of lower molars
Ibrahim GI et al. Antimicrobial activity of Er, Cr: YSGG and Ultrasonic on E. faecalis
biofilm in the mesial root canal systems of lower molars
Figure 3 - AFM imaging of the isthmus surface after treatment with 2% CHX delivered by syringe irrigation (SI) (a) Two, and (b) Three
dimensions (c) Histogram represents the number of particles for different particle’s mean diameter, red columns for large particle, orange
columns for medium particles, and green columns for small particles.
DISCUSSION
Endodontic therapy’s main goal is to
completely clean the root canal, although this is
challenging due to the complicated structure of the
canal system. The microbial community of teeth
with persisting apical periodontitis is dominated by
simple gram-positive microorganisms.
E. faecalis
was selected for this investigation because
it is thought to be the most highly resistant
microbe detected in infected root canals, having
particular mechanisms for constructing a biolm,
signicant virulence-related factors, adherence
capacity to dentin collagen, survivability in harsh
Figure 3
circumstances, capability to resist root canal
treatment, and is easy to grow in vitro [17,18].
Various materials have been used in prior
experiments to produce bacterial biolm, such as
nitrocellulose membrane lters [19], hydroxyapatite
discs, or teeth previously extracted [20].
In this study, anatomically complicated extracted
human teeth were inoculated with
E. faecalis
in a
laboratory environment.
The time required for the development of
biolm differs between studies (15 minutes to
60 days) [21,22]. However, a prolonged period of
incubation results in more mature biolms. In the
current work, infected teeth underwent incubation
7
Braz Dent Sci 2024 Jan/Mar; 27 (1): e3904
Ibrahim GI et al.
Antimicrobial activity of Er , Cr: YSGG and Ultrasonic on E. faecalis biofilm in the mesial root canal systems of lower molars
Ibrahim GI et al. Antimicrobial activity of Er, Cr: YSGG and Ultrasonic on E. faecalis
biofilm in the mesial root canal systems of lower molars
Figure 4 - AFM imaging of middle isthmus surface after treatment with 2% CHX + passive ultrasonic activation (a)Two, and (b) Three dimensions
(c) Histogram represents the number of particles for different particle’s mean diameter, red columns for large particle, orange columns for
medium particles, and green columns for small particles.
Figure 4
at 37 °C for 30 days to allow mature
E. faecalis
biolm to grow. Chlorhexidine gluconate (CHX)
was chosen in this investigation because it has
broad-spectrum antibacterial activity and destroys
Enterococcus faecalis bacteria in the tubules of
the dentin, as well as its biocompatibility [23].
Also, it has proven a bacteriostatic effect at
low concentrations and a bactericidal effect at
high concentrations owing to potassium and
phosphorous leaking out and the coagulation of the
cytoplasm [24]. As reported by Ercan et al. [25],
both CHX and NaOCl were considerably effective
in minimizing microorganisms in teeth with dead
pulp, periapical disease, or both.
The effectiveness of irrigation is inuenced by
irrigation quantity, closeness to the apex, solution
temperature, and the dynamics of uids produced
by activation methods [26]. The utilization of
laser and ultrasonic devices has been proposed
as complementary techniques for improving
aqueous irrigation uid dispersion and activation.
In past experiments, both culture procedures and
molecular methods have been used to determine
the number of viable bacteria in the canals and
dentine tubules [27]. A confocal laser microscope
(CLSM) can also detect the viability of bacteria
colonizing the root canal walls as well as the
lateral canals and isthmus [28]. This study used
an atomic force microscopy (AFM) tool to analyze
the samples, which is easy to apply, precise,
available, and more economical than CLSM. AFM
is used as a supplementary tool for investigating
8
Braz Dent Sci 2024 Jan/Mar; 27 (1): e3904
Ibrahim GI et al.
Antimicrobial activity of Er , Cr: YSGG and Ultrasonic on E. faecalis biofilm in the mesial root canal systems of lower molars
Ibrahim GI et al. Antimicrobial activity of Er, Cr: YSGG and Ultrasonic on E. faecalis
biofilm in the mesial root canal systems of lower molars
antibacterial mechanisms by exposing the change
in the roughness of the surface inoculated with
E. faecalis
biolm. The topography of the surface
of the isthmus was studied, and measurements of
surface roughness, particle size, and particle analysis
were taken. Surface roughness was determined by
computing the parameters of the surface prole:
root mean square (Sq.), average roughness (Sa), and
maximum height (Sz.). The roughness parameters
are determined by analyzing topographical scans
of the sample’s isthmus surface [29]. In this study,
(sq.) was dependent, that is the root square of the
surface height distribution and is thought to be more
responsive to signicant deviations from the mean
line than average roughness [11].
Figure 5
Figure 5 - AFM imaging of middle isthmus surface after treatment with 2% CHX agitated by Er, Cr: YSGG laser at (60 µs, 5 Hz, 0.75 W) in PIPS
protocol (a)Two, and (b) Three dimensions (c) Histogram represents the number of particles for different particle’s mean diameter, red columns
for large particle, orange columns for medium particles, and green columns for small particles.
Figure 6 - Columns chart showing the root mean square roughness
values of positive control and other test groups, the red border’s
column represents the best result.