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Title: The comparison of penetration depth of two differentphotosensitizers in root canals with and without smear layer:An in vitro study
Author: Emad Kosarieh Sahar Sattari Khavas Arash RahimiNasim Chiniforush Norbert Gutknecht
PII: S1572-1000(15)30048-XDOI: http://dx.doi.org/doi:10.1016/j.pdpdt.2015.11.005Reference: PDPDT 715
To appear in: Photodiagnosis and Photodynamic Therapy
Received date: 20-9-2015Revised date: 5-11-2015Accepted date: 17-11-2015
Please cite this article as: Kosarieh Emad, Khavas Sahar Sattari, RahimiArash, Chiniforush Nasim, Gutknecht Norbert.The comparison of penetrationdepth of two different photosensitizers in root canals with and withoutsmear layer: An in vitro study.Photodiagnosis and Photodynamic Therapyhttp://dx.doi.org/10.1016/j.pdpdt.2015.11.005
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The Comparison of Penetration Depth of Two Different Photosensitizers in
Root Canals with and without Smear Layer: An in vitro study
Emad Kosarieh 1, Sahar Sattari Khavas 2, Arash Rahimi 3, Nasim Chiniforush 4, Norbert
Gutknecht5
1 DDS, MSc, Department of periodontics, Zanjan faculty of dentistry, Zanjan, Iran
2 DDS, MSc, Department of endodontics, Zanjan faculty of dentistry, Zanjan, Iran
3 DDS, MSc, Private practice, Karaj, Iran.
4 DDs, PhD candidate of laser dentistry, Laser Research Center of Dentistry, Tehran
University of Medical Sciences, Tehran, Iran
5 DDS, PhD, Aachen Dental Laser Center, University Aachen, Germany
Corresponding author: Nasim Chiniforush, DDs, PhD candidate of laser dentistry, Laser
Research Center of Dentistry, Tehran University of Medical Sciences, Tehran, Iran
Email:[email protected],
Tel/fax: 00982188994824, address: Laser Research Center of Dentistry , Dentistry Research
Institute, Tehran University of Medical Sciences, Enghelab Ave, Tehran, Iran.
Highlights:
1) ICG can penetrate in deeper regions of the root canal wall.
2) The creation of new methods in root canal disinfection in order to improve the
success rate of our treatment is necessary.
3) The usage of EDTA improved the mean of lateral penetration depth of ICG.
Background: The main objective of this study is to evaluate the penetration depth of
suggested photosensitizers in the lateral wall of the human root canal.
Materials& Methods: Forty extracted single-rooted human teeth with straight canals
that extracted for periodontal reasons were collected and stored in the sterile saline
until employment in the experiment. Teeth were decoronated to a standard 12mm
root segment using diamond
disc. After instrumentation of specimens, the external root surface was sealed with
two layers of nail polish to avoid environmental contamination. The apical foramen
was subsequently closed with composite material. Teeth were divided randomly in
two major groups consist of indocyanine green solution (ICG) and tolonium chloride
solution (TCH) with and without EDTA in their subgroups. Specimens in all groups
grooved longitudinally with a diamond disc and split in two halves with a stainless
steel chisel. The measurements were done by the stereo microscope under 20X
magnification in three zones of each specimen and the penetration depth of dye was
measured.
Results: The results of this study showed that the mean of lateral penetration depth
of ICG (224.04μm) was significantly (P<0.05) higher than TCH (70.15μm).Regarding
to the influence of EDTA, in ICG group without consideration to the different regions,
the usage of EDTA improved the mean of lateral penetration depth of ICG, but this
improvement was not statistically significant (P>0.05).
Conclusion: Further to the findings of this study, it could be assumed that ICG could
penetrate in deeper regions of the root canal wall. Keywords: Indocyanine green
solution, Tolonium chloride solution, EDTA, Penetration depth.
Introduction:
The main purpose of current endodontic techniques is eliminating bacteria within the
root canal system by using the combination of mechanical instrumentation and
chemical irrigation. The removal of infected tissue, elimination of bacteria within the
dentinal tubules and root canals, and prevention of recontamination after treatment
are the main objectives of endodontic treatments(1). To achieve these objectives the
treatment procedures for treatment of infected root canals should be included:
mechanical cleaning and shaping (2), irrigation with antimicrobial agents, such as
Sodium hypochlorite (NaOCl) and chlorhexidine, antibacterial dressing application,
sealing of the root canals with a 3-dimensional obturation and placing a coronal
seal(1,3).
It has been shown that residual bacteria are readily detectable in approximately one-
half of teeth just before obturation (4). Our inability to eliminate bacteria from the
infected root canals, leads to the requirement for retreatment and/or periradicular
surgery in order to perform a successful treatment against persistent infections (5).
There are some factors responsible for our inability to complete elimination of
bacteria from the canals such as: complexities of the root canal system (4,6,7) ,
inadequate instrumentation and missed canals (8).
Canal irrigation is most commonly done by NaOCl. Its penetration into dentinal
tubules is approximately 130 μm (9), whereas, scanning electron microscopy (SEM)
studies described bacterial penetration up to 1100 μm into dentinal tubules (10).
Meanwhile, it has cytotoxic and neurotoxic effects in extrusion into periapical
area(11,12). Therefore, the creation of new methods in root canal disinfection in
order to improve the success rate of our treatment is necessary. We need to develop
non-invasive and non-toxic novel antimicrobial strategies that are more efficiently
and faster than available antimicrobial agents and at the same time do not permit
pathogens to easily develop resistance (13). One available alternative to current
antimicrobial agents is lethal photosensitization (LP). The LP application to treat a
disease is known as photodynamic therapy (PDT) (14).
PDT is based on the concept that a nontoxic photosensitizer (PS) can be
preferentially localized in certain tissues and subsequently activated by light of the
appropriate wavelength to generate singlet oxygen and free radicals, which are
cytotoxic to cells of the target tissue (15) (Fig. 1). In biological systems, the lifetime of
singlet oxygen and its radius of action are very short (<0.04s & 0.02μm respectively)
(16), In the other words localization of the photosensitizer will define the site of initial
cell damage resulting from PDT. Thus the reaction will be placed in a very limited
space (localized response) and making it ideal for localized applications without any
effect on distant cells or molecules(16,17). It means that the penetration depth of the
photosensitizer in dentinal tubules and lateral canals will determine the killing effect
of PDT on microorganisms. It has been shown that methylene blue and toluidine
blue O are really effective photosensitizing agents for the inactivation of both gram-
positive and gram-negative periodontopathic bacteria (17,18).
ICG is a fluorescent dye that is used mainly in medical diagnostics(19). Nowadays,
its usage in dentistry as a photosensitizer is growing up because of its phototoxic
effects in combination with the use of lasers.
Smear layer (SL) contains inorganic and organic substances that contain
microorganisms, necrotic materials and odontoblastic processes fragments (20). It
has been shown that effectiveness of irrigants and intracanal medicaments in
disinfecting of dentinal tubules is diminished in the presence of “smear layer “ (21). It
has been shown that after removal of smear layer, adhesion of obturation materials
to the canal wall will be stronger (22,23). Other investigators showed that the
penetration of sealers to the dentinal tubules was 10 to 80 μm after removal of the
smear layer, whereas in cases with the intact SL, there was no
penetration(24,25).Regarding the influence of smear layer on microleakage of root
canal fillings several investigators have shown less dye leakage after removal of the
smear layer(26,27) whereas, others have reported no significant effect of SL removal
on the microleakage of root canals(28).
Up to now there is no study investigating the penetration depth of photosensitizers in
the dentinal tubules or lateral canals. The present study is conducted to investigate
the penetration depth of two kinds of photosensitizers and the influence of smear
layer on that.
Materials and Methods:
Teeth collection
Forty extracted single-rooted human teeth (upper central incisors and upper canines)
with straight canals that extracted for periodontal reasons were collected and stored
in the sterile saline until employment in the experiment. All patients who their tooth
was gathered for using in this study signed an informed consent that permits to use
their teeth in this study.
Preparation of specimens
Teeth were decoronated to a standard 12mm root segment using diamond disc
(Brasseler USA, Savana, GA). File measurement was taken at the point where the
tip of a size # 15 kerr files (Maillefer Instruments SA, Switzerland) become visible at
the apical foramen and 0.5mm will be subtracted to set the working length. Teeth
were instrumented in a crown-down manner by a set of M2 rotary files (VDW GmbH,
Germany) to achieve a master apical file size of M2# 40, 6% tapered at the working
length. The cleaning was done with 10 ml of 2.5% NaOCl throughout the
instrumentation sequence. The external root surface was sealed with two layers of
nail polish to avoid environmental contamination. The apical foramen was
subsequently closed with composite material.
Teeth were divided randomly in four groups. In two groups, shaped canals were
irrigated with 17% EDTA for 2 minutes followed by irrigation with normal saline to
remove the smear layer and in other two groups; irrigation was done only by normal
saline. Then specimens were sterilized by autoclaving for 15 minutes at 121 °C.
In two groups, (EDTA group and non-EDTA group) TCH solution (PACT, Cumdente
GmbH, Germany) and in other two groups, ICG solution (EmunDo, A.R.C. laser
GmbH, Germany) were used. In all groups before filling the root canals with
photosensitizers, they dried again with paper cone, afterwards filled with suspected
photosensitizer and allowed to incubate for 10 minutes. After that the root canals
dried
again with paper cone. Therefore, our groups were as follows:
Group A: TCH solution in root canals without the smear layer.
Group B: TCH solution in root canals with the smear layer.
Group C: ICG solution in root canals without the smear layer.
Group D: ICG solution in root canals with the smear layer.
Stereoscopic microscopy
In this study, we used Nikon SMZ1500 stereo microscope (Nikon, Japan) for
measurement of penetration depth of suggested photosensitizers in the lateral wall
of root canals. Specimens in all groups grooved longitudinally with a diamond disc
(Brasseler USA,) and split in two halves with a stainless steel chisel and one of
them, which contained more root canal borders was chosen for measuring the
penetration depth of dye in the lateral wall of the canal. All measurements were done
by one of my colleagues who was blinded to this study using the stereo microscope.
The measurements were done under X20 magnification in three zones of each
specimen:
- Coronal zone: 4mm coronal part.
- Middle zone: 4mm middle part.
- Apical zone: 4mm apical part.
In each zone four measurements were done and the mean of them was considered
as penetration depth value at that site. The microscope was equipped by its own
software and all measurements were done by that.
Statistics:
Achieved data was evaluated by descriptive statistic methods via statistic software
SPSS 16. For evaluation of the influence of different variables include: group (ICG &
TCH), region(Apical, middle & coronal) and EDTA (with or without) as independent
variables and lateral penetration depth of photosensitizer as dependent variables,
the multi variable of ANOVA test was used. In this study, the P value<0.05 was
considered as significant.
Normalized distribution of collected data was assessed by Kolmogorov-Smirnov test.
Results:
Achieved data (Table.1) showed that the mean of lateral penetration depth without
consideration to the different regions and presence or absence of EDTA, in ICG
group was 224.04 μm whereas, in TCH group was 70.15μm (diagram. 1,2) and the
difference between them was statistically significant (P<0.001).The means of lateral
penetration depth of ICG in all regions were higher than TCH (diagram.3) without
consideration to the presence or absence of EDTA and this difference was
statistically significant (P<0.001).
The pictures of specimen in TCH and ICG groups with stereomicroscope (X20
magnification) were shown in Fig.1 and 2.
In both groups without consideration to the presence or absence of EDTA, the mean
of lateral penetration depth in coronal part was higher than middle, and in middle part
was greater than the apical part but in ICG group, the differences between coronal
and middle parts and middle and apical parts were not statistically significant
(P>0.05) whereas, the
difference between coronal and apical parts was statistically significant (diagram. 3).
In TCH group the differences between coronal and middle and coronal and apical
parts were statistically significant whereas between middle and apical was not
(diagram. 3).
Regarding to the influence of EDTA, in ICG group without consideration to the
different regions, the usage of EDTA improved the mean of lateral penetration depth
of ICG, but this improvement was not statistically significant(P>0.005). But in the
TCH group, the mean of lateral penetration depth of TCH into the lateral wall of the
canal was significantly improved by EDTA usage (P=0.004). With consideration to
the different regions, in ICG group in all regions using of EDTA improved the mean
of penetration depth of dye, but the differences were not statistically significant. In
TCH group, usage of EDTA improved the mean of penetration depth significantly
(P<0.001) in coronal part whereas, in the middle and apical parts only improved the
dye penetration, and differences were not statistically significant.
Discussion:
There are some factors responsible for the permeability of dentin to the small and
large molecules, solutions and bacteria such as: number and type of bacteria,
exposure time, and presence or absence of smear layer(1), surface tension,
molecular size, osmotic and hydrostatic pressure61. According to the Pashley and
Livingston(29), increasing in molecular size led to decrease in the permeability
coefficient in human root canal dentin. In their study dentin permeability was
decreased 100 fold after 19 fold increasing in molecular radius. Although regarding
fluoride and chlorhexidine, their permeability were much lower than expected
according to their molecular weight or size, suggesting their bond to the dentin. Like
that study in our study regardless of lower molecular weight of TCH in comparison to
the ICG, the mean penetration depth of ICG was significantly higher than TCH in all
regions (P<0.001) suggesting the bonding of TCH to the dentin. In this study
neither permeability coefficient, osmolality nor the surface tension of suggested
solutions were not assessed.
In all groups, the penetration depth of photosensitizers decreased from coronal part
of root segments to the apex without consideration to the presence or absence of
EDTA.
This finding could be explained by the greater number of dentinal tubules in coronal
parts in comparison to the apex region which are supported by previous studies that
showed that the number of dentinal tubules decreased from coronal to the apical
parts (30,31).
Carriganetal.(31) in 1984 showed that the mean number of tubules in cervical and
mid-root dentin were 254300 & 234060 respectively whereas in the apex area was
49140. They suggested that the greater number of tubules in coronal parts could be
responsible for rapidly increasing of bacteria through the coronal dentin (32).
Meanwhile, these findings are in accordance with the findings of another study that
was done by Paque and his coworkers(33) which showed statistically significant
decreasing of dye penetration from coronal to the apical parts of root canals.
Previous studies regarding permeability specifications of dentin to radioactive
albumin and tritiated water confirmed the restriction effect of SL on surface area
available to different size (small & large) molecules (in comparison to the amounts
achieved after its removal) and the bacterial penetration into the pulp (34,35).
Meanwhile, it has been shown that the huge increase in the amount of filtration
between unetched dentin and dentin acid-etched for 5 seconds was related to the
removal of SL100. Pashley et al. (36)in an in vitro study showed that SL was
responsible for 86% of resistance to movement of fluid through the dentin. Fogel and
Pashley in 1990 showed 50% reduction of root dentin conductivity in the presence of
smear layer(33).In accordance with the mentioned studies in this study in TCH group
removing the SL led to increase the penetration depth significantly (P=0.004). In ICG
group removing of SL resulted in increasing the penetration depth but the difference
was not statistically significant (P=0.7). This finding is comparable to the findings of
Foster et al. in 1993(37) in which EDTA and NaOCl were used for removing of the
smear layer and dressing of root canals was done by calcium hydroxide. They
showed the minimal effect of smear layer removing on the distribution of hydroxide
ions from root canals. On the other hands Paque F.(33) and his co-workers in 2006
showed that the dye penetration into the dentin in root canals that instrumented
endodontically was independent of smear layer presence or absence but, was
related to the function of tubular sclerosis. Tubular sclerosis is a physiologic
phenomenon starting from the third decade of life in the apical part of the root and
progresses coronally(38). In our study, the age of patients whose teeth were
gathered was unknown but as the teeth came from the patients suffering from
periodontal diseases, it could be assumed that most patients were over 30-years-old.
Therefore, the tubular sclerosis was a factor that may inhibit the influence of EDTA in
penetration depth of ICG.
Conclusion:
These findings showed us a higher penetration depth of ICG in comparison to the
TCH. Meanwhile, this amount was higher than that had been reported in previous
studies regarding the penetration depth of NaOCl. It could be assumed that ICG can
access to the microorganisms in deeper parts of the root canal wall. Therefore, it
could be a good alternative for TCH for using in PDT.
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Fig. 1: The specimen in TCH group under the stereo microscope with X20 magnification.
Fig. 2: The specimen in ICG group under the stereo microscope with X20 magnification.
Diagram1: Histogram related to the lateral penetration depth of ICG (in
microns) without consideration to the regions or presence or absence of
EDTA.
Diagram2: Histogram related to the lateral penetration depth of TCH (in
microns) without consideration to the regions or presence or absence of
EDTA.
Diagram3: Error bar related to the comparison between the mean of lateral penetration depth
(in microns) of ICG and TCH in different regions. The lateral penetration depth in all regions
was significantly higher in ICG group in comparison to the same region in TCH group.
Table1: The mean of penetration depth of ICG & TCH in different regions in
presence or absence of EDTA in microns.
Group Region EDTA Mean Std. Error of
Mean
ICG Apical Yes 142.94 24.43
No 136.85 40.02
Middle Yes 208.81 27.68
No 207.89 41.35
Coronal Yes 370.51 76.99
No 277.21 36.28
TCH Apical Yes 39.60 10.61
No 19.69 6.17
Middle Yes 76.56 13.64
No 42.32 5.66
Coronal Yes 163.20 23.17
No 79.51 13.88