Accepted Manuscript

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

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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:n-chiniforush@farabi.tums.ac.ir,

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.

References:

1. Torabinezhad M, Handysides R, Khademi A, Bakland L. Clinical imlications of

the smear layer in endodontics: A review. OralSurg Oral Med Oral Pathol

2002;94:658-666.

2. Bahcall JK, Brass JT. Understanding and evaluating the endodontic file. Gen

Dent 2000;48:690-692.

3. Sedgley G.Root canal irrigation –a historical perspective. Jhist Dent 2004;52: 61-

67.

4. Bystrom A, Sunqvist G. Bacterical evaluation of the efect of 0.5 persent sodium

hypochloite in endodontic. Oral Surg Oral Pathol 1983;55:307-12.

5. Xu Y, Yong MJ, Battagiino RA, Morse LR, Fontana CR, Pagonis TC, et al.

Endodontic antimicrobial photodynamic therapy: safety assessment in

mammalian cell cultures. J Endod 2009 Nov;35(11):1567-72.

6. Bonsor SJ, Nichol R,Reid TM, Pearson GJ. Microbiological evaluation of

photoactivated disinfection in endodontics (an in vivo study). Br Dent J

2006;200:33741.

7. Sjogren U,Figdor D,Persson S,Sundqvist G. Influence of infection at the time of

root filling on the outcome of endodontic treatment of teeth with apical

periodontitis. Int Endo J 1997;30:297-306.

8. SundqvistG, Figdor D, Persson S, Sjogren U. Analysis of teeth with failed

endodontic treatment and outcome of conservative re-treatment. Oral Surg Oral

Med Oral Pathol 1998;85:86-93.

9. Berutti E, Marini R, Angeretti A. Penetration ability of different irrigant into

dentinal tubules. J Endod 1997;23:725-7.

10. Kouchi Y, Ninomiya J, Yasuda H, Fukui K, Moriyama T, Okamato. Location of

Strepptococcus Mutans in the dentinal tubules of open infected root canals. J Dent

Res 1980;59:2038-2046.

11. Gatot A, Arbelle J, Leiberman A, Yanai-Inbar I. Effects of sodium hypocholorite

on soft tissues after its inadvertent injection beyond the root apex. J Endod

1991;17:573-74.

12. Neaverth EJ, Swindle R. Aserious complication following the inadvertent

injection of sodium hypochlorite outside the root canal system. Compend

1990;11:474-81.

13. Taylor EL, Brown SB. The advantages of aminolevulinic acid photodynamic

therapy in dermatology. J Dermatolog Treat 2002;13 Suppl 1:S3-11.

14. Macmillan JD, Mxwell WA, Chichester CO. Lethal photosensitization of

microorganisms with light from a continuous-wave gas laser. Photochem Photobiol

1966 Jul;5(7):555-65.

15. Moan J,Berg K. The photodegrdation of porphyrins in cells can be used to

estimate the life time of singlet oxygen. Photochem Photobiol 1991;53:549-53.

16. Dougherty TJ,Gomer CJ, Henderson BW, et al. Photodynamic therapy. J Natl

Cancer Inst 1998;90:889-905.

17. Chan Y, Lai Ch. Bactericidal effects of different laser wavelengths on

periodontopathic gems in photodynamic therapy. Lasers Med Sci 2003;18:51-5.

18. Wilson M, Dobson J, Sarkar S, Sensitization of periodontophatogenic bacteria to

killing by light from a low-power laser light in the presence of a photosensitizer. J

Appl Bacteriol 1995;78:569-574.

19. Tuchin VV. Tissue Optics: Light scattering methods and instruments for medical

diagnosis. The international Society for optics and photonic (SPIE), Bellingham,

Wash, USA, 2007.

20. Pashley DH. Smear layer: Overview of structure and function. Proc Finn Dent

Soc 1992;88 (suppl 1):215-24.

21. Bystrom A, Sundqvist G. The antibacterial action of sodium hypochlorite and

EDTA in 60 cases of endodontic therapy. Int Endod J 1985;18:35-40.

22. Gettleman BH, Messer HH, Eldeeb ME. Adhesion of sealer cements to dentin

with and without the smear layer. J Endod 1991;17:15-20.

23. Economides N, Liolios E, Kolokuris I, Beltes P. Long-term evaluation of the

influence of smear layer removal on the sealing ability of different sealers. J Endod

1999;25:123-5.

24. Pallares A, Faus V, Glickman GN. The adaptation of mechanically softened

gutta-percha to the canal walls in the presence or absence of smear layer: a

scanning electron microscopic study. Int Endod J 1995;28:266-9.

25. Kouvas V, liolios E, Vassiliadis L, Parissis-Messimeris S, Butsiukis A. Influence

of smear layer depth of penetration of three endodontic sealers: an SEM study.

Enod Dent Traumatol 1998;14:191-5.

26. Vassiliadis L, Liolios E, Kouvas V, Economdes N. Effect of smear layer on

coronal microleakage. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996;

82:315-20.

27. Taylor JK, Jeansonne BG, Lemon RR. Coronal leakage: effects of smear layer,

obturation technique, and sealer. J Endod 1997;23:508-12.

28. Timpawat S, Vongsavan N, Messer NH. Effect of removal of smear layer on

apical microleakage. J Endod 2001;27:351-3.

29. Pashley DH, Livingston MJ. Effect of molecular size on permeability coefiicients

in human dentin. Arch Oral Biol 1978;25:391.

30. Whittaker DK, Kneale MJ. The dentin-predentin interface in human teeth. Br

dent J 1979;146:43-6

31. Carrigan PJ, Morse DR, Furs ML, Siani IH. A scanning electron microscopic

wvaluationof human dentinal tubules according to age and location. J Endod

1984;8:359-63.

32. Seltzer S. Pain control in dentistry-diagnosis and management. Philadelphia: JB

Lippincott; 1978:106-15.

33. Paque F, Luder HU, Sener B, Zehnder M. Tubular sclerosis rather than the

smear layer impedes dye penetration into the dentine of endodontically

instrumented root canals. Int Endod J 2006;39:18-25.

34. Michelich V, Schuster GS, Pashley DH. Bacterial penetration of human dentine,

invitro. J Dent Res 1980;59:1398.

35. Reeder OW, Walton RE, Livingston MJ, Pashley DH. Dentin permeability:

dentine determinants of hydraulic conductance. J Dent Res

1978;57:187.

36. Pashley DH, Livingston MJ, Greenhill JD. Regional resistances to

Fluid flow in human dentin, In vitro. Arch Oral Biol 1978;23:80

37. Foster KH, kulid JC, Weller RN. Effects of smear layer removal on the diffusion of

calcium hydroxide through radicular dentin. J Endod 1993;19:136-40.

38. Vasiliadis L, Darling AI, Levers BG.The amount and distribution of sclerotic

human root dentine. Archives of oral biology 1983;28:649-9.

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