Accepted Manuscript

Title: A comparison between effect of photodynamic therapy

by LED and calcium hydroxide therapy for root canal
disinfection against Enterococcus faecalis: A randomized
controlled trial

Author: <ce:author id="aut0005"

author-id="S1572100016300953-
680fa1207f5928d4e1fe08dda501e28a"> Mohammad
Asnaashari<ce:author id="aut0010"
author-id="S1572100016300953-
681300497778b7c22014d70afc0716c3"> Hengameh
Ashraf<ce:author id="aut0015"
author-id="S1572100016300953-
de1e0fe51cec5b6b6dd8f4f2c8d66f7b"> Afsaneh
Rahmati<ce:author id="aut0020"
author-id="S1572100016300953-
0074222affb1c784391a56d21413c200"> Neda
Amini

PII: S1572-1000(16)30095-3
DOI: http://dx.doi.org/doi:10.1016/j.pdpdt.2016.12.009
Reference: PDPDT 875

To appear in: Photodiagnosis and Photodynamic Therapy

Received date: 3-6-2016

Revised date: 8-12-2016
Accepted date: 24-12-2016

Please cite this article as: Asnaashari Mohammad, Ashraf Hengameh, Rahmati
Afsaneh, Amini Neda.A comparison between effect of photodynamic therapy by
LED and calcium hydroxide therapy for root canal disinfection against Enterococcus
faecalis: A randomized controlled trial.Photodiagnosis and Photodynamic Therapy
http://dx.doi.org/10.1016/j.pdpdt.2016.12.009

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A comparison between effect of photodynamic therapy by LED and calcium

hydroxide therapy for root canal disinfection against Enterococcus faecalis: A

randomized controlled trial

1
Mohammad Asnaashari, 2Hengameh Ashraf, 3Afsaneh Rahmati*(Corresponding Author), 4Neda Amini

1
Professor of Endodontics, Laser Application in Medical Sciences Research Center, Shahid Beheshti

University of Medical Sciences, Tehran, Iran

2
Professor of Endodontics, Endodontic Department, Dental School, Shahid Beheshti University of Medical

Science, Tehran, Iran

3
Assistance Professor, Endodontic Department, Dental School, Hamedan University of Medical Science,

Hamedan, Iran

4
Dentist, Professional Trainee, University of Texas Health Science Center, Houston

1
Highlights
-Antibacterial Photodynamic Therapy (aPDT) and calcium hydroxide therapy as auxiliary
adjunct methods for common endodontic retreatments displays disinfectant properties.

- Efficacy against E.faecalis is better in LED photodynamic therapy compared to calcium

hydroxide therapy.

- APDT is capable of disinfecting the canals in a single visit root canal treatment. This results in
a fewer number of visits and less chair time.

- Advantages of aPDT with LED over lasers include greater effectiveness due to a broader
spectrum, ease and safety of use, affordability and less heat production.

Abstract

Background: Insufficient root canal disinfection is one of the main reasons for persistent periapical
pathology. Photodynamic therapy (PDT) has been proven effective in disinfecting infected root canals.
The aim of this study was to evaluate the antimicrobial effect of photo activated disinfection (PAD)
when using toluidine blue as photosensitizer and a LED lamp after the conventional treatment, and
comparing it with calcium hydroxide therapy in vivo.

Methods: This clinical trial includes 20 patients with molars requiring endodontic retreatment. After the
conventional treatment, first microbiological samples were obtained using sterile rotary ProTaper F2 file
and 3 paper points and transferred to a microbiology laboratory. Group 1 (n=10) specimens underwent
PAD with photosensitizer (PS) solution (0.1 mg/ml TB) and irradiation with Fotosan light emitting diode
(LED) lamp (635nm, 200mW/cm2) for 60s. Creamy Ca(OH)2 paste was used in group 2 (n=10) for two
weeks. A second sample was then obtained. The samples were cultured and then bacterial colonies
were counted. Data included number of colony forming units (CFUs) before and after treatments,
analyzed by t-test and analysis of covariance (ANCOVA) using SPSS vs.18.

Results: A significant difference between results of before and after treatment of both groups (calcium
hydroxide therapy p= 0.02 < 0.05, PAD p<0.0001) indicated the efficacy of both treatments. The mean
numbers for log 10 CFUs/mL before calcium hydroxide therapy and PAD with LED irradiation was
10.1968 and 11.3773. After treatment, the mean numbers were 9.4202 and 8.3772, respectively. The

2
difference in results after treatment between groups was significant (p= 0.01< 0.05) and indicate that
PAD was more effective.

Conclusion: PAD and calcium hydroxide therapy, as auxiliary methods adjunct to conventional root canal
therapy, are both effective in root canal disinfection. In comparison with calcium hydroxide therapy,
PAD leads to a greater reduction in enterococcus faecalis number in the infected root canals.

Introduction:

Successful root canal therapy relies on the removal of pathogenic microflora from the root canal.
Debridement of the root canal forms an important aspect of the endodontic triad.1Insufficient root
canal disinfection may be one of the main reasons for endodontic failure and persistent periapical
pathology.2

The results of endodontic re-treatments are highly affected by the existence of intracanal
microorganisms compared to those of primary endodontic treatments.3
During a conventional root canal treatment, mechanical instrumentation and chemical debridement
using antimicrobial irrigants such as sodium hypochlorite (NaOCl) and chlorhexidine are
performed. Subsequently, a routine intracanal antibacterial dressing (usually calcium hydroxide)
is used as an inter-appointment medicament. Next, the root canal is sealed with an inert material
to retain the integrity and health of the periradicular tissues. 4

Conventional disinfection techniques, however, do not ensure complete disinfection of the root
canals.5
Anatomical complexities such as bacterial growth as a biofilm and so on are the factors that impede
complete disinfection of the root canal system. 5, 9

Several cultural and molecular biology studies have shown that Enterococcus faecalis is the most
prevalent bacterial species detected in root canal-treated teeth.2,4 Its prevalence reaches up to 90%
of cases. E. faecalis is nine times more common in root canal-treated teeth than other bacteria
causing primary infections. Studies have shown a 94% success rate with a negative culture before

3
obturation, which decreased to 68% with a positive culture for E. faecalis.2

E. faecalis demonstrates resistance to irrigants used in chemomechanical preparation because of

its ability to penetrate dentinal tubules. Its ability to form biofilms in root canals plays an important
role in its resistance and persistence following intracanal antimicrobial procedures. E. faecalis is
also extremely resistant to chemicals, including calcium hydroxide, and is capable of adapting to
varying conditions.4, 7

Antimicrobial photodynamic therapy (aPDT) relies on the application of a photosensitizer (PS),

oxygen for bacterial damage and a light source. After treating the site of infection by PS, the light
source which coincides with the peak absorption of the PS, is illuminated to generate singlet
oxygen and free radicals. This causes bacterial cell damage8 and death of microorganisms.9
This technique is minimally invasive, non-resistant and simple to repeat. 8 So it can be used as an
adjuvant to conventional endodontic treatment to eliminate the bacterial load. 9 In vitro studies
using aPDT have revealed that it has an excellent bactericidal potential against E. faecalis.4,10,11

APDT has been proposed as an appropriate antimicrobial treatment to maximize root canal
disinfection11 and is an effective supplement in one-visit root canal disinfection.

It has been found that PSs, which possess a marked cationic charge, can bind to and penetrate
bacterial cells rapidly. As a result, PSs possess a high degree of selectivity for killing
microorganisms compared to the host mammalian cells.12

The single-appointment endodontic treatment of teeth with apical periodontitis versus a two-visit
treatment is an area of endodontics that has been largely debated.13, 14

Introducing treatment protocols that can predictably disinfect the root canals in one visit can help
to resolve this debate In this case, the idea of speeding up disinfection of the root canal while
preserving efficacy sounds interesting. In this regard, the usage of aPDT as an alternative is
increasingly common in endodontic treatments.1, 13, and 14

4
In a review of studies performed between 2000 and 2014 on the antimicrobial effect of aPDT and
its application to endodontic therapy, the authors noted that aPDT has been reported as a promising
antimicrobial therapy in numerous studies. Despite these results, it is still necessary to establish
different parameters so that aPDT can be used with maximum effectiveness in reducing pathogenic
endodontic bacteria.15The majority of studies on aPDT have considered using lasers as their light
source, but the high cost of lasers along with required safety measures have made them an
undesirable tool. Recently, a light-emitting diode (LED) lamp (Fotosan; CMS Dental,
Copenhagen, Denmark) emitting light in the red spectrum with a power peak of 628 nm has been
established for use in aPDT.9

In vitro studies have shown that this aPDT method exhibits an excellent bactericidal potential
against E. faecalis. The LED (630 nm) aPDT method and in vivo calcium hydroxide therapy have
never been reliably compared.10,11,16 Therefore, the purpose of this in vivo study was to compare
the effects of 2 weeks of calcium hydroxide therapy with aPDT by an LED 630-nm lamp and
toluidine blue (TB) for disinfection of human molar root canals.

Methods:

A total of 20 patients, receiving treatment from the endodontic department of Shahid Beheshti
Dental School, with periapical (PA) lesions and previously (more than two years ago)
endodontically treated molars, were randomly selected to participate in this randomized clinical
trial.

Following studies by Souza et al.14, we calculated the sample volume with the power and sample
size software VS.2.1.31 (Department of Biostatistics, Vanderbilt University). The calculation was
performed considering a significance level of <0.05 and a study power of 90% for detecting 0.6 ×
100 with a standard deviation of 0/4 × 100.

The protocol was approved by the Center of Application of Laser in Medical Science Ethics
Committee of Shahid Beheshti University of Medical Science.

5
The inclusion criteria were as follows: (1) patients with previously treated molar; (2) visible PA
lesion in radiography; (3) no clinical symptoms such as pain and swelling; and (4) no existing
systemic disease. (Figure 1)

Patient selection and treatments were performed by different practitioners. Microbiologic

sampling was performed by a third practitioner. All the 3 practitioners were blind about the study
groups.

Informed consent was obtained from all the participants. Each patient’s radiograph was checked
to confirm the presence of a PA lesion. The treatment group underwent root canal re-treatment in
several steps.

After rubber dam isolation, an access cavity was obtained. To minimize the bacterial load, access
cavities and the surrounding area were irrigated with 2% chlorhexidine solution.17
Gutta percha and sealer removal following the initial cleaning of the canals was performed using
chloroform solvent, hand files, and hedstrom files 15# (Maillefer Instruments SA, Ballaigues,
Switzerland) in a crown-down manner. The canals were irrigated with 5 mL of 2.5% NaOCl and
sterile saline solution.
Working length was verified using an apex locator (Raypex 5 VDW, Germany) and confirmed
with radiographic images, and the canals were cleaned and shaped using hand and rotary files up
to F2 ProTaper Universal (Dentsply Maillefer, Ballaigues, Switzerland) (300 rpm, 100 n/s) to the
working length. Irrigation was performed with 5 mL of 2.5 % NaOCl. To neutralize NaOCl, the
canals were irrigated with 5% sodium thiosulfate for 1 min at the end of the procedure, and the
final irrigation was completed with 5 ml of normal saline. F2 ProTaper file was used in the canals
with 18-22 mm of working length and an approximate diameter of F2 size. Bacterial biofilm
sample was collected by a F2 Protaper file for 10 s. Bacterial sampling was also performed using
3 sterile #30 paper points to the working length. Paper points were left inside the canal for 15 s.
This procedure is considered as the initial microbial sampling of the canals. The samples, the F2
file, and the paper points were then transferred to the laboratory in 1 ml brain-heart infusion (BHI)
broth medium and were incubated for 24 h at 37◦C.

6
Following the removal of gutta-percha, the participants were equally divided into two groups:
photodynamic therapy using light-emitting source and calcium hydroxide therapy. Selection was
performed randomly using a computer generated random number table.

Group 1: Photodynamic Therapy Using a Light-emitting Diode Source

Photodynamic therapy was performed by applying 0.5 mL of 0.1 mg/mL Toluidine blue according
to the manufacturer’s (MDD, CMS Dental Denmark, Korea) structure as a PS. The solution was
left in the canal for 5 min as pre-exposure time. APDT was performed using LED Fotosan 630
(MDD, CMS Dental Denmark, Korea), Endo tip (1 mm2 diameter), producing light with a
wavelength of 620-640 nm (85%) with peak of 630 nm, and intensity of 2-4 mW/cm2 for 60 s. The
energy density (fluence) was 1.2-4.4 mJ/ cm2.The Endo tip was positioned in the canal until the
maximum possible length and then radiation was performed. After irradiation Toluidine blue was
then removed by rinsing the root canals with 5 mL sterile saline solution. The final microbiological
sampling was performed similar to the first sampling method by shaving the dentin with a F2
rotary file to the working length. The samples were then transferred to the laboratory in 1 ml of
BHI solution.

Group2: Calcium Hydroxide Therapy

In the second experimental group, a creamy mix of calcium hydroxide was used as an intracanal
dressing, and the access cavity was temporized with >4-mm-thick Cavit (3M ESPE Dental-USA).
The final bacterial sampling was performed after 2 weeks with a sterile F2 rotary file to the
working length by shaving the dentin for 10 s. The files were transferred to the laboratory in BHI
solution. The canals were then obturated using cold lateral compaction of gutta-percha (Meta
Biomed, Chungbuk, Korea) and AH26 (Dentsply Maillefer) as a sealer. The tooth was temporized
with Cavit (3M ESPE Dental- USA), and the patient was referred for final restoration.

After 24 h incubation at 37◦C in BHI, the samples were cultured in bile aesculinazide agar, a
special culturing environment for E. faecalis to isolate the bacteria. The samples were cultured in
10 different dilutions of 1/10, 1/100, 1/1000, 1/10000 and more diluted. The countable sample was
then selected from the cultures.

7
Following incubation, the colonies were counted with a colony counter and grid (Diartajhiz,
Tehran, Iran). Only those samples obtained before treatments that were positive for E. faecalis
were retained, and all negative samples were excluded. Following the counting, the colony's
precise number was calculated by multiplying the result by the dilution ratio.

Data included the number of CFUs before and after treatments, analyzed by t-test and analysis of
covariance (ANCOVA) using SPSS v.18.

Results:

The number of colonies was decreased in both the after treatment groups compared to the pre-
treatment (control) groups. (Table 1 and Graphs 1 and 2)

The mean numbers for log 10 CFUs/mL before calcium hydroxide therapy and aPDT with LED
irradiation were 10.1968 and 11.3773, respectively. After treatment, they were 9.4202 and 8.3772,
respectively. (Table 1)

Decrease in the number of colonies was more evident in the aPDT group in which the difference
was significant (p = 0.01< 0.05), despite the lower number of colonies in the calcium hydroxide
group prior to the procedure.

Independent t-test analysis between the two pre-treatment groups did not show any significant
difference. The two groups were almost homogeneous.

Paired t-test indicated a significant difference between the results of before and after treatment of
both groups (calcium hydroxide therapy, p = 0.02 < 0.05; aPDT, p < 0.0001), which indicated the
efficacy of both treatments.

ANCOVA was used to evaluate the effect of the type of intervention on the post-treatment results
by controlling the bacterial load before treatments. It indicated that the type of intervention was
likely effective. (p= <0.0001)

Discussion

Treatment failure and persistent periapical pathology can result from microbial infections
because of inadequate disinfection of the root canals. 2

8
Bacteria may still be retained in 40% to 60% of the canals even after using chemomechanical
preparations with antimicrobial irrigants such as saline solution and NaOCl. The antimicrobial
irrigants are not sufficient for complete disinfection of the canals.2, 14, 18, 19 to inhibit bacterial
growth, the combination of an inter-appointment antibacterial medication with a calcium
hydroxide paste has been reported.1, 2

In a systematic review on antimicrobial activity of calcium hydroxide, Mohammadi et al. stated

that calcium hydroxide is more effective against common endodontic pathogens than against E.
faecalis and Candida albicans.20 Refractory endodontic infection cases have been frequently
detected with E. faecalis.2,7
Because of this reason, and the fact that gram-negative species are easily eliminated than gram-
positive species such as E. faecalis,21 the present study focuses on the effects of antimicrobial
treatments on this bacterial species.

Calcium hydroxide is known as a potentially valuable anti-endotoxin agent. Nevertheless, its effect
on microbial biofilms is subject to controversy. 20

In our study, both the biofilm (with rotary files) and planktonic bacteria (with paper points)
were collected as in vivo samples. The application of calcium hydroxide and aPDT decreased
the bacterial load of E. faecalis after the procedure; however, aPDT was more effective than
calcium hydroxide

To increase the validity of our study results, we prevented bias as much as possible. Patient
selection was performed randomly using a computer generated random number table to minimize
operation bias. Selection, treatment, and sampling were performed by different practitioners who
were blinded about the groups to control operative bias. Each canal was considered as both control
and case (before and after the procedure, respectively), which is the best method for controlling
measurement bias when comparing two single interventions.

The single-appointment endodontic treatment of teeth with apical periodontitis versus a two-visit
treatment is an area of endodontics that has been largely debated.5, 14
Introducing treatment
protocols that can predictably disinfect the root canals in one visit can resolve this controversy. In

9
this case, the idea of increasing the speed of disinfection of the root canal while preserving the
efficacy sounds interesting. In this regard, aPDT is being increasingly used as an alternative in
endodontic therapy.1, 13, 14

A systematic review assessed the bactericidal efficacy of aPDT against E. faecalis in infected
root canals. In 70% percent of the patients (12 of 17), aPDT was more effective in removing
E. faecalis than conventional endodontic therapy. Our results are also in concordance with this
review. However, considering the limitations of this review (inconsistency in methodology and
laser parameters used in the study cases), the authors pointed that aPDT application in reducing
E. faecalis is questionable. Therefore, further well-designed studies for investigating the role
of aPDT as a bactericidal agent in infected canals were required.11 This is the aim of our study.

In a literature review on the effects of photodynamic therapy on root canal disinfection, all reported
studies demonstrate a reduced number of root canal bacterial load after photodynamic therapy.
The efficacy of aPDT, especially for resistant bacterial species in the root canal, makes this
treatment a desirable alternative. Further studies such as randomized clinical trials are required to
accumulate level of evidence (LOE) for routine clinical applications of aPDT.22

To apply this method with increasing LOE in the clinic, safety trials and in vitro assessment were
first carried out, and in vivo assessment and RCT were performed later. On the basis of the results,
the clinical application of aPDT was recommended.

Xu et al. studied the effect of aPDT on the mitochondrial activity, cell maintenance, and apoptosis
in mammal cells. They concluded that aPDT can inhibit endodontic pathogens without altering the
host cell system.23

Nasim Kashef et al have evaluated the cytotoxicity of photodynamic therapy on human primary
fibroblasts with methylene blue and toluidine blue as sensitizers. They stated that FDP with these
materials has no significant effect on the cultured cells. 24

Rios et al. assessed the effect of the photodynamic therapy and LED bulb emission on E.
faecalis species in human extracted teeth. The survival rate of E. faecalis in the

10
NaOCl/toluidine blue treatment was significantly less than that in the other methods such as
sodium hypochlorite 6%/30 s, Toluidine blue 30 s, light emission light bulb 2-3 mm shorter
than the working length, and Toluidine blue and light/NaOCl.9

In a clinical trial, Garcez studied the antibacterial effect of aPDT with laser diode light source
in addition to the common chemical and mechanical cleaning methods in patients having two
visit sessions with calcium hydroxide as inter-session medicament.

The present study results showed that bacterial load decreases after the first session, which is
more evident and significant with aPDT. Thus, combining aPDT with endodontic treatments
can eliminate more bacterial load and can be effective in treating oral infections.17

In another study to investigate aPDT antibacterial effects, Garcez et al. demonstrated that only 3
of 21 canals in their samples were devoid of bacteria, while the canals treated with aPDT were
completely disinfected.25 For collecting the samples in their study, they used three paper points
that enabled the sampling of planktonic bacteria. However, in our present study, in addition to
three paper points, we collected a sample from the root canal walls using a similar procedure with
a rotary file and thus sampled the biofilm form of bacterial growth as well. This could explain the
negative culture results in the study of Garcez et al..

Our primary study with the selective medium of bile aesculin revealed that there were no viable
cells in the treated sample. We used an enrichment medium to recover any possible viable E.
faecalis cells that could not grow on bile aesculin medium. APDT or calcium hydroxide may cause
some changes in E. faecalis cells, which result in no growth of the bacterial cells in the presence
of bile salts and other selective ingredients of BS medium. By using this method, we could recover
any possible target bacterial cells, and the count of viable cells could be reflected after CFU
determination.

aPDT and high-power lasers are considered as new methods of root canal disinfection.7 The use
of high-power lasers as light source can be harmful. 16 APDT uses a specific wavelength light for

11
the activation of the nontoxic photoactive dye (photosensitizer). The source of the light in aPDT
can be an LED lamp, a low-level laser, or a diode laser.11

In this study, in comparison with calcium hydroxide, aPDT lead to a greater reduction in E.
faecalis cell numbers in the root canal, which is consistent with the results reported by
Graces,17,25 Bonsor,27 and Foschi29.

Only one in vitro study has shown different results from the present study. Souza et al.14 studied
the antibacterial effects of aPDT with methylene blue and TB. Their study concluded that
aPDT was not effective against root canal bacteria. This can be attributed to the very low
concentration of the photosensitizer (concentration of 0.15 µg/ml) in their study. We used 0.1
mg/ml TB in our study and observed that it was effective in inhibiting bacterial growth.

In an in vitro investigation prior to this study, we observed that the effect of aPDT of LED
630-nm bulbs on root canal disinfection against E. faecalis was more prominent than 810-nm
laser. This may be due to the wider emitted light spectrum of LED.4

Other advantages of LED over lasers are that they are safer, more cost-effective, and easier to
handle. LED lamps have a lower thermal productivity and are less harmful to the tissues .28
They consume less energy and are easy to use.4 Therefore, our light source was LED.

On the basis of the results, we conclude that LED bulbs play an effective role in inhibiting the
growth of different species of endodontic bacteria. This is supported by the fact that gram-
negative species are destroyed more easily than gram-positive species such as E. faecalis,21
and aPDT causes elimination of various underlying pathogenic species of endodontic
diseases.29

Disinfection of root canals in clinical settings is of prime importance; hence, it is recommended

to evaluate the effectiveness of aPDT with different methods against polymicrobial infection
of root canals. 30 Moreover, additional investigations for changes in antibacterial properties of

12
aPDT with different concentrations of PS and exposure and pre -exposure time are
recommended.31

Conclusion
Photodynamic and calcium hydroxide therapies used as auxiliary adjunct methods for common
endodontic re-treatments exhibit disinfectant properties. LED photodynamic therapy is a better
disinfectant than calcium hydroxide therapy. It can be concluded that aPDT is capable of
disinfecting the canals in a single-visit root canal treatment. This results in a fewer number of visits
and less chair time. Moreover, the advantages of aPDT with LED over lasers include greater
effectiveness because of a broader spectrum, ease and safety of use, affordability, and less heat
production.

13
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285

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Figure 1: Stages of the clinical trial based on CONSORT recommendations

17
Graph 1: Log CFU before treatment

Graph1

18
 Graph 2: Log CFU after treatment

Graph2

19
 Table 1: Results of colony forming units count before and after treatment

Groups Counted Mean SD Min

Max
Calcium hydroxide before CFUs 6.9 ×1011 1.74×10 12 1.5×107
treatment Log 10 CFUs 10.1968 1.54734 5.3×1012
Calcium hydroxide after CFUs 4.5× 109 1.15×1012 3.4 ×106
treatment Log 10 CFUs 9.4202 1.40916 6.8 ×1011
aPDT before treatment CFUs 7.8× 1011 2.23× 1011 8.3×109
Log 10 CFUs 11.3773 0.89215 9.3×10 12
aPDT after treatment CFUs 7.95× 107 2.15 × 109 5.6×106
Log 10 CFUs 8.3772 1.08018 7.5×109

20