Photodynamic Therapy for Endodontic Disinfection

Nikolaos S. Soukos, DDS, PhD,* Peter Shih-Yao Chen, DMD, MS,

Jason T. Morris, DMD, MS,

Karriann Ruggiero, BS,* Abraham D. Abernethy, BS,* Sovanda Som, BS, MS,*
Federico Foschi, DDS,* Stephanie Doucette, BS,* Lili Luschke Bammann, DMD, PhD,

Carla Raquel Fontana, DDS,* Apostolos G. Doukas, PhD,

and
Philip P. Stashenko, DMD, PhD

Abstract
The aims of this study were to investigate the effects of
photodynamic therapy (PDT) on endodontic pathogens
in planktonic phase as well as on Enterococcus faecalis
biofilms in experimentally infected root canals of ex-
tracted teeth. Strains of microorganisms were sensi-
tized with methylene blue (25 g/ml) for 5 minutes
followed by exposure to red light of 665 nm with an
energy fluence of 30 J/cm
2
. Methylene blue fully elim-
inated all bacterial species with the exception of E.
faecalis (53% killing). The same concentration of meth-
ylene blue in combination with red light (222 J/cm
2
)
was able to eliminate 97% of E. faecalis biofilm bac-
teria in root canals using an optical fiber with multiple
cylindrical diffusers that uniformly distributed light at
360 degrees. We conclude that PDT may be developed
as an adjunctive procedure to kill residual bacteria in
the root canal system after standard endodontic
treatment. (J Endod 2006;32:979984)
Key Words
Biofilms, endodontic bacteria, Enterococcus faecalis,
methylene blue, photodynamic therapy, root canals
T
he goal of endodontic treatment is to prevent and, when required, to cure endodon-
tic disease, apical periodontitis (1). This principle is supported by classic studies
that demonstrate significantly higher success rates, by approximately 10% in teeth that
are minimally infected at the time of treatment, compared to grossly infected teeth with
necrotic pulps (2). Similarly, teeth that give a negative culture for bacterial growth at the
time of root canal filling have a higher success rate than teeth that are culture positive
12-26% higher) (3). Although the bulk of the infecting microorganisms are removed
during endodontic instrumentation, residual bacteria are readily detectable in approx-
imately one-half of teeth at the time of placement of a filling material, despite extensive
irrigation with sodium hypochlorite (NaOCl) (4). Recently, Nair et al. (2005) detected
histologically biofilm bacteria in intercanal isthmi and accessory canals of the apical 3
mm in 14 of 16 teeth, which was surgically removed on completion of the root canal
treatment in a single visit (5). Scanning electron microscopic investigations have dem-
onstrated bacterial penetration up to 1000 m into dentinal tubules in a laboratory
model (6). The presence of a smear layer after instrumentation reduces the effective-
ness of irrigants and temporary dressings in disinfecting dentinal tubules (7). Addition-
ally, the complexity of the root canal system with its isthmuses, ramifications, and
dentinal tubules make complete debridement of bacteria with instrumentation and
irrigation alone almost impossible (8).
Microorganisms associated with endodontic failures can be detected in the ma-
jority of failing cases using cultural methods, and can be identified in virtually all cases
using PCR amplification of the microbial 16S rRNA genes (9, 10). The data indicate that
the bacteria present in treatment failures are distinct fromthose present in infected root
canals before endodontic treatment. Failing cases are associated with high proportions
of gram-positive aerobic and facultative organisms, versus the predominance of strict
anaerobes at presentation. This scenario has been substantiated in controlled studies
that examined changes in residual root canal bacteria after treatment; facultative bac-
teria were more resistant to treatment than anaerobes in a monkey model (11). En-
terococcus faecalis, which is rarely found in large proportions in untreated root
canals, is highly associated with failures (12). However, some studies have failed to
detect E. faecalis and have implicated other taxa including Pseudomonas, Staphylo-
cocci, and Streptococci as causative of failures (9, 13). 16S rRNA analyses have given
an even more diverse picture of bacteria associated with failure, with species including
Pseudoramibacter, Proprionibacterium, Dialister, and Filifactor in addition to En-
terococcus (10). Large numbers of isolates including Actinomyces, Streptococci,
Peptostreptococcus, and Prevotella in addition to Enterococci have also been re-
ported (14).
Photodynamic therapy (PDT) has been used as a treatment for cancer and other
nonmalignant diseases (15). PDT is based on the concept that a certain, nontoxic,
photosensitizing agent known as 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 that are cytotoxic to cells of the target tissue
(16). Although visible light can also kill bacteria after their treatment with an appro-
priate PS (17), PDT has never been used to treat any specific bacterial infections in
humans. Gram-negative bacteria are less susceptible to photoinactivation compared
with gram-positive species (18); however, PS bearing a cationic charge can increase
their killing (19, 20). A wide range of oral bacteria could be killed by red light after
From the *Applied Molecular Photomedicine Laboratory,

Department of Cytokine Biology, The Forsyth Institute, Bos-

ton, Massachusetts;

Harvard School of Dental Medicine, Bos-
ton, Massachusetts; and the

Wellman Center for Photomedi-
cine, Massachusetts General Hospital, Boston, Massachusetts.
Address requests for reprints to Dr. Nikolaos Soukos,
Applied Molecular Photomedicine Laboratory, The Forsyth In-
stitute, 140 The Fenway, Boston, MA 02115-3799. E-mail
address: nsoukos@forsyth.org.
0099-2399/$0 - see front matter
Copyright 2006 by the American Association of
Endodontists.
doi:10.1016/j.joen.2006.04.007
Basic ResearchTechnology
JOE Volume 32, Number 10, October 2006 Photodynamic Therapy for Endodontic Disinfection 979
sensitization with the cationic PS toluidine blue and methylene blue
(2123). Partial inactivation of S. intermedius biofilms in root canals
of extracted teeth using toluidine blue Oand light that was applied at the
orifice of the access cavity has been reported recently (24).
In the present study, we have investigated the photodynamic effects
of methylene blue on two gram-positive and four gram-negative com-
mon endodontic pathogens using short exposure times to PS and light of
665 nm. In addition, we have explored the photodynamic effects of
methylene blue in killing E. faecalis biofilms in dentinal tubules of
single-rooted extracted human teeth using an optical fiber with multiple
cylindrical diffusers, which uniformly distributed light at 360 degrees.
Materials and Methods
Photosensitizer
Methylene blue (Sigma, St. Louis, MO) was dissolved in brain heart
infusion (BHI) broth and filter-sterilized immediately before use. The
concentration of 25 g/ml (67 M) was used. The ultraviolet-visible
absorption spectra of methylene blue in BHI broth were recorded from
200 to 800 nm using quartz cuvettes with 1-cm path length on a diode-
array spectrophotometer. The absorption spectra of methylene blue in
BHI broth were characterized by a Soret band maximumat 290 nmand
a long-wavelength maximum at 666 nm as shown previously (23).
Microorganisms
The following common endodontic pathogens were used in this
study: Porphyromonas gingivalis (ATCC 33277), P. intermedia (ATCC
25611), Fusobacterium nucleatum nucleatum (ATCC 25586), Pep-
tostreptocococcus micros (ATCC 33270), Porphyromonas endodon-
talis (ATCC 35406), and E. faecalis (ATCC 29212). Cultures of P.
gingivalis and P. endodontalis were maintained by weekly subculture
in plates comprised of trypticase soy agar (Becton, Dickinson, and Co.,
Sparks, MD), brain heart infusion agar (Becton, Dickinson, and Co.),
yeast extract (Fisher Biotech, Fair Lawn, NJ), 6 g/ml N-acetylmuramic
acid, 5 g/ml hemin, 0.3 g/ml vitamin K, and 5% sheep blood. Tryp-
ticase soy agar with 5% sheep blood (Northeast Laboratories, Water-
ville, ME) was the culture media for the remaining bacterial species. For
experimental purposes, organisms were grown in the presence of 80%
N
2
, 10% H
2
, 10% CO
2
at 35
o
c in an anaerobic chamber for 72 h;
harvested by centrifugation; and resuspended in BHI broth. Cells were
dispersed by vortexing and repeated passage through Pasteur pipettes.
Cell numbers were measured in a spectrophotometer (wavelength, 600
nm; 0.1 optical density unit equals approximately 10
8
cells/ml) in 1-ml
cuvettes.
Light Source
The irradiation source was a diode laser (BWTEK Inc., Newark,
DE) with an output power of 1 Watt and a central wavelength of 665 nm.
The system was coupled to a 1-mm optical fiber that delivered light into
a lens, which formed a uniform circular spot on the base of the 24-well
plate, 2 cm in diameter. The laser possessed a spectral stability of 2
nm with an output power stability of 10 mW. Power measurements
were quantified with a power meter (Ophir Optronics LTD, Danvers,
MA). Distance adjustments between the lens and the illuminated plates
created fields of irradiation with appropriate dimensions and power
densities.
Photodynamic Therapy of Bacterial Suspensions
Aliquots of bacterial suspensions (10
8
/ml) were placed in sterile
microcentrifuge tubes and were centrifuged (7000 rpmfor 4 minutes).
The supernatants were aspirated and then methylene blue (25 g/ml)
in BHI broth was added to a final volume of 1 ml. Cultures were resus-
pended with the PS and placed in the wells of 24-well plates for 5
minutes before they were exposed to light. All wells were irradiated with
red light from the diode laser for 5 minutes in the dark at room tem-
perature. The light exposure was from above with an irradiance of 100
mW/cm
2
and a fluence of 30 J/cm
2
. All plates were kept covered during
the illumination to maintain the sterility of the culture. After illumination
of the appropriate wells, serial dilutions of the contents of each well
were prepared in BHI broth, and 100-l aliquots were spread over the
surfaces of blood agar plates. Survival fractions in each well were cal-
culated by counting the colonies on the plates (LPS) and dividing
by the number of colonies from controls (LPS) that were kept at
room temperature for a period equal to the irradiation time. Other
experimental groups were: (a) bacteria treated only with the PS
(LPS) that were kept in plates at room temperature, and (b) bac-
teria exposed to light in the absence of the PS (LPS). The average
survival fractions of the three wells per group were determined in each
experiment, and summary data were obtained by calculating the mean
standard error average from 2 to 4 experiments for each microor-
ganism. Differences between means were analyzed for statistical signif-
icance by Students t test.
Tooth Specimens
Sixty freshly extracted single-rooted human teeth were stored in
0.5% NaOCl for 2 to 8 weeks. Root segments were prepared to a stan-
dard length of 12 mm by decoronation using a rotating diamond saw
(#911H, Brasseler USA, Savannah, GA) at 20,000 rpm under water-
cooling. Patency of apical foramina was standardized by inserting a size
15K-file (Dentsply Maillefer, Tulsa, OK). A file measurement was taken
at the point where the size 15 K-file was just visible and individual
working length was calculated 0.5 mm short of this measurement. The
instrumentation sequence was performed by an endodontist and con-
sisted of the following instruments: Gates Glidden Burs (Dentsply
Maillefer) sizes 4 and 2 for the coronal 4 mm, ProTaper S1 (Dentsply
Tulsa Dental, Tulsa,OK), followed by .06 taper Profile series 29
(Dentsply Tulsa Dental) files sizes 4 (.216 ISO equivalent) to 6 (.465
ISOequivalent) in a crown-down manner, to achieve a master apical file
size of .465 (ISO equivalent). An Aseptico Endo ITR (Dentsply Tulsa
Dental) electric motor was used with an 8:1 gear reduction mini-head
contra-angle. Final apical patency was achieved with a size 25 K-file
(Dentsply Maillefer) to allow for an adequate apical aperture for flush-
ing of microbial aggregates. Canals were irrigated using 6% NaOCl after
each change of instrument as described previously (25). After canal
preparation an aliquot of 1 ml of 17% EDTA solution was left in situ for
3 minutes for smear layer removal, and was replaced by 1 ml of 6%
NaOCl for 3 minutes. The tooth specimens were then placed in sterile
microcentrifuge tubes containing 1 ml of phosphate buffered saline
(PBS). Teeth were subsequently autoclaved at 121C for 20 minutes.
After autoclave sterilization, PBS was aspirated from the microcentri-
fuge tubes under sterile conditions. Six specimens were fractured and
processed for analysis with a scanning electron microscope, to confirm
smear layer removal as well as the patency of dentinal tubules as de-
scribed below.
E. faecalis Infection
Fifty-four root specimens were transferred into sterile microcen-
trifuge tubes under sterile conditions. One milliliter of BHI broth con-
taining 10
9
microorganisms of E. faecalis (ATCC 29212) was syringed
into the prepared root canal system using a Pro Rinse 30 gauge irriga-
tion needle (Dentsply TulsaDental). Cell numbers were measured in a
spectrophotometer (wavelength 600 nm; 1 optical density unit equals
approximately 10
9
cells /ml) in 1 ml cuvettes. After injection, the entire
specimen was fully covered by the medium. The tubes were kept in a
Basic ResearchTechnology
980 Soukos et al. JOE Volume 32, Number 10, October 2006
chamber with 80% N
2
, 10% H
2
, and 10% CO
2
at 35
o
C for 3 days. After
incubation, the medium was aspirated from the tubes under sterile
conditions. Six root specimens were fractured and processed for scan-
ning electron microscopy (SEM). The remaining 48 specimens were
used for PDT studies.
Scanning Electron Microscopy
A total of 12 human extracted teeth with a single canal (six for
demonstration of the smear layer removal and the patency of dentinal
tubules, six for demonstration of E. faecalis infection) were selected for
SEM. Longitudinal grooves were cut with a diamond bur using a high-
speed water-cooled handpiece both on palatal/lingual and buccal sur-
faces of each root to facilitate vertical splitting with a chisel after canal
instrumentation. The tooth specimens were prepared and infected as
described above. Each sample was split into two halves with a stainless
steel chisel. The section with the most visible part of the apex was fixed
in 3.7%glutaraldehyde in 0.2 Msodiumcacodylate buffered solution at
4C for 24 h. After dehydration in a graded concentration ethanol series,
samples were air dried and mounted on an SEM stub, gold sputtered
and observed with a JEOL JSM 6400 scanning electron microscope
(JEOL Corporation, Tokyo, Japan). SEM microphotographs were ob-
tained at a standard magnification of 1500 at each third (coronal,
middle and apical) and on the fracture surface. Bacterial penetration in
dentinal tubules was measured with image analyzer (ImageJ, National
Institutes of Health, USA).
Photodynamic Treatment of E. faecalis Biofilms
Forty-eight root specimens were prepared and infected as de-
scribed above, and were subjected to PDT using 25 g/ml of methylene
blue in BHI broth. The specimens were put in 1.5-ml microcentrifuge
tubes under sterile conditions and then the canals were filled to the level
of the access cavity with methylene blue solution using a Pro Rinse 30
gauge irrigation needle (Dentsply TulsaDental). After injection, the en-
tire specimen was fully covered by methylene blue for 5 minutes. After
incubation, the drug solution was aspirated and the root specimens
were removed from the tubes. Light was then applied for 5 minutes
using a plastic, flexible optical fiber with cylindrical diffusers (Cuda
Technologies Inc., Jacksonville, FL) that was connected to the same
diode laser described above (Fig. 1A,B). This optical fiber with a diam-
eter of 500 m was able to uniformly distribute light at 360 degrees to
a length of 10 cm. The total energy dose delivered to the fiber from the
diode laser was 140 J. However, part of this energy was lost before light
entered the root canal system. We were able to calculate the energy dose
delivered to the root canal system by sacrificing one fiber. Specifically,
we cut the part of the fiber that was inserted in each root specimen (1.2
cm) and measured the energy dose coming out of the tip of the remain-
ing fiber. The amount of energy delivered in the root canal system was
found 70 J. The fluence of light energy (J/cm
2
) distributed within each
root specimen was estimated as follows. The master apical file taken to
working length has an apical diameter of 0.465 mmwith a coronal taper
of 0.06 mm/mm. The diameter of the most coronal extent for a 12 mm
working length is: 0.465 mm12 0.06 mm1.185 mm. Assuming
the instrumented root canal is a truncated cone, whose surface is equal
to that of the canal master, then its surface area (neglecting the area of
the bases) is given by
Lateral Surface Area r R

R r
2
H
2
(1)
where R is the radius of the upper base (1.185/2 0.592 mm), r is the
radius of the lower base (0.465/2 0.232 mm), and His the height (12
mm). Therefore
Surface area 31.6 mm
2
0.316 cm
2
,
Energy fluence 70/0.316 J/cm
2
222 J/cm
2
, and
Power density Energy fluence/Time of irradiation 222/300
Jcm
-2
/second 0.74 W/cm
2
740 mW/cm
2
.
The fiber optic was wiped with ethanol after the completion of each
light exposure. Three separate experiments were carried out (16 root
specimens per experiment). Each experiment included the following
four groups: PDT group (four specimens), Only PS (four specimens),
Only light (four specimens), and No light/No PS (control group) (four
specimens).
After all treatments, the contents of root canals were sampled by
flushing the root canal systems with a coronal application of 1-ml BHI
broth with a Pro Rinse 30 gauge irrigation needle (Dentsply TulsaDen-
tal). The bacterial suspension was collected into an Eppendorf tube
from the apical foramen. After vortexing for 15 to 20 s, serial dilutions
were prepared and 100-l aliquots were spread over the surfaces of
blood agar plates that were kept anaerobically for 7 days. Survival frac-
tions in each root specimen were calculated by counting the colonies on
the plates and dividing by the number of colonies from controls (No
light/No PS) that were kept at room temperature for periods equal to
irradiation times. Differences between means were analyzed for statis-
tical significance by Students t test.
Results
Dark Toxicity and Light Effects
A very strong toxicity was shown on endodontic pathogens (79-
100%) after their incubation with methylene blue (25 g/ml) in the
dark for 5 minutes (Table 1). P. endodontalis and P. intermedia were
fully eliminated whereas E. faecalis remained unaffected (Table 1).
There was no reduction in the viability of all microbial species after
exposure to light alone with an irradiance of 100 mW/cm
2
and a fluence
of 30 J/cm
2
(data not shown).
Photodynamic Treatment In Vitro
The phototoxic effect of methylene blue on representative end-
odontic species is shown in Table 1. P. micros, P. gingivalis and F. nuc.
nuc. were fully eliminated after sensitization with methylene blue and
Figure 1. Optical fiber with a diameter of 500 m demonstrating cylindrical
diffusers that distribute light at 360 degrees (A). Irradiation of the root canal
system with red light of 665 nm (B).
Basic ResearchTechnology
JOE Volume 32, Number 10, October 2006 Photodynamic Therapy for Endodontic Disinfection 981
subsequent exposure to light (Table 1). PDT reduced the viability of E.
faecalis by 53% (p 0.004) (Table 1). The photodynamic effects of
methylene blue on both P. endodontalis and P. intermedia could not
be evaluated, because of their complete eradication by the drug alone as
reported above.
Scanning Electron Microscopy
SEM demonstrated open dentinal tubules after the removal of the
smear layer with NaOCl and EDTA (Fig. 2A,B). SEM showed invasion of
E. faecalis biofilms into the dentinal tubules of specimens infected with
the microorganism for 3 days (Fig. 2C,D). Specimens revealed pene-
tration of bacteria to a 400 to 500 m depth in a few canals. A dense
infection from the canal side reached up to 250 to 350 m, whereas a
moderate infection was usually seen up to 400 to 500 m.
Photodynamic Therapy of Root Canals
Exposure of E. faecalis biofilms in root canals to 25 g/ml meth-
ylene blue and subsequent illumination with light resulted in 97% re-
duction in viable cells (mean of values obtained fromthree independent
experiments with four tooth specimens per experiment, p 0.005)
(Fig. 3). Similarly, specimens treated with methylene blue or light alone
showed 83.2%(p 0.009) and 56.6%(p 0.03) killing respectively
(Fig. 3).
Discussion
Methylene blue, an organic dye belonging to the phenothiazine
family, has well-established photosensitizing properties and has been
used in PDT (26). Quite a few microorganisms including gram-positive
and gram-negative oral bacteria are known to be photoinactivated by
methylene blue (26). Several studies have shown phenothiazine dyes to
exert phototoxicity to both DNA and outer membrane of target species
(26). The hydrophilicity of methylene blue (27), and its low molecular
TABLE 1. Survival of microorganisms after their incubation only with
methylene blue (25 g/ml) or methylene blue (25 g/ml) and red light (30
J/cm
2
)
Species Only methylene blue PDT
P. micros 21 10.6 0
E. faecalis 94.5 17.6 47 12.1
F. nuc. nuc. 5.5 0.4 0
P. intermedia 0 0
P. gingivalis 6.7 0.1 0
P. endodontalis 0 0
Surviving species were expressed as percentage-of-controls receiving neither PS nor light. Each value
is the mean of values obtained from2 to 4 experiments (data fromeach experiment were representative
of three wells). Values are expressed as means standard error of the mean.
Figure 2. Scanning electron microscopy. Appearance of dentinal tubules (A) and their pulpal openings (B) after removal of the smear layer. E. faecalis biofilms on
the pulp canal wall (C) and in dentinal tubules (D).
Figure 3. Phototoxicity of E. faecalis biofilms in the root canal system after
incubation with 25 g/ml methylene blue for 5 minutes followed by treatment
with red light of 665 nm (222 J/cm
2
) and colony-forming assay. Each value is
the mean of counts of colony forming units that were recovered from the root
canal system of 12 specimens (three different experiments were combined).
Error bars denote the standard error of the mean. LPS, control group
subjected to no treatment; LPS, control group subjected to light only (p
0.03); LPS, control group subjected to methylene blue only (p 0.009);
LPS, test group subjected to PDT (p 0.005).
Basic ResearchTechnology
982 Soukos et al. JOE Volume 32, Number 10, October 2006
weight and the positive charge allows passage across the porin-protein
channels in the outer membrane of gram-negative bacteria (28). Meth-
ylene blue predominantly interacts with the anionic macromolecule
lipopolysaccharide resulting in the generation of methylene blue dimers
(28), which participate in the photosensitization process (28, 29).
The results of our study revealed that oral endodontic pathogens
exposed only to methylene blue (25 g/ml) for 5 minutes in planktonic
phase demonstrated high cytotoxicity (Table 1). This toxicity, which led
to 79% to 100% reduction in cell numbers (Table 1), is similar to a
previous report (30). The addition of red light of 665 nmwith a fluence
of 30 J/cm
2
resulted in complete eradication of those species that were
incompletely eliminated by methylene blue (Table 1). Only E. faecalis
showed resistance to methylene blue in the absence of light (5.5%
killing), whereas its exposure to both methylene blue and light led to
53% killing (Table 1). Methylene blue has been used at concentrations
ranging from42 M(15.7 g/ml) to 100 M(37.4 g/ml) to achieve
photodestruction of E. faecalis invitro (27, 30). Recently, it was shown
that methylene blue in concentration of 100 g/ml did not exhibit any
dark toxicity on P. gingivalis and P. intermedia after incubation with
the dye for 1 minute (31). Complete elimination of both species was not
possible after incubation with methylene blue for 1 minute and subse-
quent exposure to red light of 650 nm with energy fluence of 60 J/cm
2
(31).
Methylene blue alone exhibited 83.2% reduction of E. faecalis
biofilm species in the root canal system of extracted human teeth. The
combined effect of methylene blue and red light did not lead to complete
eradication of E. faecalis biofilms (97% killing). The incomplete ste-
rility of the root canal system by PDT cannot be attributed to failure of
methylene blue and/or light to penetrate dentinal tubules. Infiltration of
dentinal tubules by methylene blue has been shown by light microscopy
as well as by confocal scanning laser microscopy (32), whereas there is
strong evidence that light propagates in dentin (33) with dentinal tu-
bules being the main scatterers (34). Therefore, the generation and
diffusion of the reactive free radicals responsible for the photodynamic
effect will be able to fully penetrate dentinal tubules, including the
conventionally unreachable areas, and eliminate residual microorgan-
isms. A possible explanation for the incomplete bacterial elimination is
the high power density (740 mW/cm
2
) used that may have caused a
rapid consumption of molecular oxygen in the low oxygenated dentinal
tubule microenvironments. As a result, during photoirradiation, bacte-
ria resided at oxygen tensions that were low enough to preclude or
minimize
1
O
2
-mediated damage (35). In this case, the oxygen depletion
could be partially overcome by reducing or fractionating the light de-
livery (36). Recent results obtained in our laboratory (unpublished
data) showed that incubation with a lower concentration of methylene
blue (6.25 g/ml) followed by exposure of the root canal systemto red
light with a power density of 100 mW/cm
2
for 5 minutes led to 60%
killing of E. faecalis, whereas methylene blue achieved 20% killing in
the absence of light. These results demonstrate the importance of opti-
mization of light dosimetry for bacterial photodestruction. Our hypoth-
esis is that the above dye and light parameters combined with longer
times of light exposure (up to 10 minutes) will lead to complete erad-
ication of microorganisms.
The results regarding the killing effects of light on E. faecalis root
canal biofilms in the absence of methylene blue were surprising (Fig.
3). Thermal profile tests in our study using a multimeter with a thermo-
couple showed an increase of temperature up to 45C in the root canal
during PDT, whereas the temperature rise on the outer surface of the
specimen did not exceed body temperature (data not shown). These
results show that there is heat development in the root canal during
irradiation. However, this temperature rise cannot eliminate E. faecalis
because this taxa can survive at 60C for 30 minutes (37). The other
possibility is that light activated bacterial endogenous chromophores. E.
faecalis cells cannot synthesize porphyrins (38), but they express a
cytochrome of the bd type with absorption peaks at 561 nm (cyto-
chrome b) and 626 nm (cytochrome d) (39). Red light may have
targeted cytochrome d leading to cell damage. Finally, the possibility of
a significantly higher heat development within the dentinal tubules com-
pared with that in root canals should not be excluded.
Our findings suggest that PDT has the potential to be used as an
adjunctive anti-microbial procedure in endodontic treatment. However,
various light and drug parameters must be further explored to define the
appropriate dosimetry for eliminating root canal microorganisms. This
noninvasive technique would offer the following advantages in the hy-
pothetical case of its invivo application: rapid application of the drug in
the root canal and rapid bacterial killing after a short treatment time;
full penetration of biofilms and dentinal tubules by the PS; limited pen-
etration and cytotoxicity of PS and light into the periodontal ligament
and adjacent bone; and absence of thermal side effects in the tissues
surrounding the roots.
Acknowledgments
We thank Drs. Phil Stewart, The Center for BiofilmEngineering,
Montana State University-Bozeman, and Tom Pagonis, Department
of Endodontics, Harvard School of Dental Medicine, for critically
reading the manuscript. Carla Raquel Fontana is a fellow of CAPES
Foundation (Foundation for the Coordination of Higher Education
and Graduate Training), Brazilian government (process BEX 0547/
05-0). This work was supported by NIDCRgrants RO1-DE-14360 and
RO1-DE-16922.
References
1. rstavik D, Pitt Ford TR. Apical periodontitis. Microbial infection and host responses.
In: rstavik D, Pitt Ford TR, eds. Essential endodontology: prevention and treat-
ment of apical periodontitis. Oxford: Blackwell Science, 1998.
2. Sjgren U, Hagglund B, Sundqvist G, Wing K. Factors affecting the long-term results
of endodontic treatment. J Endod 1990;16:498504.
3. Sjgren 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
Endod J 1997;30:297306.
4. Bystrm A, Sundqvist G. Bacteriologic evaluation of the effect of 0.5 percent sodium
hypochlorite in endodontic therapy. Oral Surg Oral Med Oral Pathol
1983;55:30712.
5. Nair PNR, Henry S, Cano V, Vera J. Microbial status of apical root canal system of
human mandibular first molars with primary apical periodontitis after one-visit
endodontic treatment. Oral Surg Oral Med Oral Radiol Endod 2005;99:23152.
6. Haapasalo M, rstavik D. In vitro infection and disinfection of dentinal tubules. J Dent
Res 1987;66:13759.
7. Berutti E, Marine R, Angeretti A. Penetration ability of different irrigants into dentinal
tubules. J Endod 1997;23:7257.
8. Siqueira JF Jr, Arajo MCP, Garcia PF, Fraga RC, Dantas CJS. Histological evaluation
of the effectiveness of five instrumentation techniques for cleaning the apical third of
root canals. J Endod 1997;23:499502.
9. Rolph HJ, Lennon A, Riggio MP, et al. Molecular identification of microorganisms
from endodontic infections. J Clin Microbiol 2001;39:32829.
10. Siqueira JF, Rocas IN. Nested PCR detection of Centipeda periodontii in primary
endodontic infections. J Endod 2004;30:1357.
11. Mller AJ, Fabricius L, Dahlen G, Sundqvist G, Happonen RP. Apical periodontitis
development and bacterial response to endodontic treatment. Experimental root
canal infections in monkeys with selected bacterial strains. Eur J Oral Sci
2004;112:20715.
12. Peciuliene V, Balciuniene I, Eriksen HM, Haapasalo M. Isolation of Enterococcus
faecalis in previously root-filled canals in a Lithuanian population. J Endod
2000;26:5935.
13. Cheung GS, Ho MW. Microbial flora of root canal-treated teeth associated with
asymptomatic periapical radiolucent lesions. Oral Microbiol Immunol
2001;16:3327.
14. Hancock HH 3rd, Sigurdsson A, Trope M, Moiseiwitsch J. Bacteria isolated after
unsuccessful endodontic treatment in a North Am population. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod 2001;91:57986.
Basic ResearchTechnology
JOE Volume 32, Number 10, October 2006 Photodynamic Therapy for Endodontic Disinfection 983
15. Huang Z. A review of progress in clinical photodynamic therapy. Technol Cancer Res
Treat 2005;4:28393.
16. Dougherty TJ, Gomer CJ, Henderson BW, et al. Photodynamic therapy. J Natl Cancer
Inst 1998;90:889905.
17. Bertoloni G, Salvato B, DallAcqua M, Vazzoler M, Jori G. Hematoporphyrin-sensi-
tized photoinactivation of Streptococcus faecalis. Photochem Photobiol
1984;39:81116.
18. Malik Z, Ladan H, Nitzan Y. Photodynamic inactivation of Gram-negative bacteria:
problems and possible solutions. J Photochem Photobiol B 1992;14:2626.
19. Soukos NS, Ximenex-Fyvie LA, Hamblin MR, Socransky SS, Hasan T. Targeted anti-
microbial photochemotherapy. Antimicrob Agents Chemother 1998;42:25952601.
20. Soukos NS, Mulholland SE, Socransky SS, Doukas AG. Photodestruction of human
dental plaque bacteria: enhancement of the photodynamic effect by photomechanical
waves in an oral biofilm model. Las Surg Med 2003;33:1618.
21. Sarkar S, Wilson M. Lethal photosensitization of bacteria in subgingival plaque from
patients with chronic periodontitis. J Periodont Res 1993;28:20410.
22. Soukos NS, Wilson M, Burns T, Speight PM. Photodynamic effects of toluidine blue on
human oral keratinocytes and fibroblasts and Streptococcus sanguis evaluated in
vitro. Las Surg Med 1996;18:2539.
23. Soukos NS, Socransky SS, Mulholland SE, Lee S, Doukas AG. Photomechanical drug
delivery into bacterial biofilms. Pharm Res 2000;17:4059.
24. Seal GJ, Ng Y-L, Spratt D, Bhatti M, Gulabivala K. An in vitro comparison of the
bactericidal efficacy of lethal photosensitization or sodium hyphochlorite irrigation
on Streptococcus intermedius biofilms in root canals. Int Endod J
2002;35:26874.
25. Foschi F, Nucci C, Montebugnoli L, et al. SEM evaluation of canal wall dentine fol-
lowing use of Mtwo and ProTaper NiTi rotary instruments. Int Endod J
2004;37:8329.
26. Harris F, Chatfield LK, Phoenix DA. Phenothiazinium based photosensitizers: photo-
dynamic agents with a multiplicity of cellular targets and clinical applications. Curr
Drug Targets 2005;6:61527.
27. Wainwright M, Phoenix DA, Marland J, Wareing DRA, Bolton FJ. A study of photo-
bacterial activity in the phenothiazinium series. FEMS Immunol Med Microbiol
1997;19:7580.
28. Usacheva MN, Teichert MC, Biel MA. The interaction of lipopolysaccharides with
phenothiazine dyes. Las Surg Med 2003;33:3119.
29. Bartlett JA, Indig GL. Effect of self-association and protein binding on the photochem-
ical reactivity of triarylmethans. Implication of noncovalent interactions on the com-
petition between photosensitization mechanisms Type I and Type II. Photochem
Photobiol 1999;70:4908.
30. Usacheva MN, Teichert MC, Biel MA. Comparison of the methylene blue and toluidine
blue photobactericidal efficacy against gram-positive and gram-negative microor-
ganisms. Las Surg Med 2001;29:16573.
31. Krespi YP, Slatkine M, Marchenko M, Protic J. Lethal photosensitization of oral
pathogens via red-filtered halogen lamp. Oral Dis 2005;11:925.
32. Absi EG, Addy M, Adams D. Dentin hypersensitivity: the development and evaluation
of a replica technique to study sensitive and non-sensitive cervical dentin. J Clin
Periodontol 1989;16:1905.
33. Kienle A, Forster FK, Diebolder R, Hibst R. Light propagation in dentin: influence of
microstructure on anisotropy. Phys Med Biol 2003;48:N7N14.
34. Zijp JR, ten Bosch JJ. Theoretical model for the scattering of light by dentin and
comparison with measurements. Appl Opt 1993;32:4115.
35. Foster TH, Murant RS, Bryant RG, Knox RS, Gibson SL, Hilf R. Oxygen consumption
and diffusion effects in photodynamic therapy. Rad Res 1991;126:2963.
36. Muller S, Walt H, Dobler-Girdziunaite D, Fiedler D, Haller U. Enhanced photody-
namic effects using fractionated laser light. J Photochem Photobiol B
1998;42:6770.
37. Sherman JM. The streptococci. Bacteriol Rev 1937;1:397.
38. Frankenberg L, Brugna M, Hederstedt L. Enterococcus faecalis heme-dependent
catalase. J Bacteriol 2002;184:63516.
39. Winstedt L, Frankenberg L, Hederstedt L, von Wachenfeldt C. Enterococcus faecalis
V583 contains a cytochrome bd-type respiratory oxidase. J Bacteriol
2000;182:38636.
Basic ResearchTechnology
984 Soukos et al. JOE Volume 32, Number 10, October 2006