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Endodontic Topics 2012, 22, 99–123 2012 © John Wiley & Sons A/S
All rights reserved ENDODONTIC TOPICS 2012
1601-1538

Advanced therapeutic options for

endodontic biofilms
ANIL KISHEN

Limitations in endodontic disinfection are collectively due to the biofilm mode of bacteria in root canal systems,
anatomical complexities, dentin structure/composition, and limitations associated with chemical disinfectants.
Consequently, advanced disinfection strategies are developed and tested in Endodontics. The primary aim of these
advanced anti-biofilm strategies is to eliminate biofilm bacteria from the uninstrumented portions and anatomical
complexities of the root canal system without inducing untoward effects on healthy tissues. This article outlines
various challenging factors in root canal disinfection, and describes in detail different advanced therapeutic
strategies for endodontic biofilms such as antibacterial nanoparticles, antimicrobial photodynamic therapy,
laser-assisted root canal disinfection, ozone, and herbal/enzyme alternatives.

Received 21 January 2012; accepted 3 March 2012.

Biofilm as a therapeutic target in to antimicrobials when they are in a biofilm mode of

root canal treatment growth (7).
The resistance mechanisms in a bacterial biofilm to
The infected root canal harbors a polymicrobial popu- antimicrobial agents may generally include the follow-
lation of aerobic, anaerobic, Gram-positive, and ing: (i) resistance associated with the extracellular poly-
Gram-negative bacteria in a biofilm mode of growth meric matrix; (ii) resistance associated with growth rate
(1–5). Gram-positive and Gram-negative bacteria have and nutrient availability; or (iii) resistance associated
profound differences in their three-dimensional cell with the adoption of a resistance phenotype. It is recog-
architecture. The membrane barrier of a bacterial cell nized that no single mechanism may account for the
limits the diffusion of antimicrobials into the cytosol. general resistance to antimicrobials. It is apparent that
The membrane barriers of a Gram-positive bacterium these mechanisms act in concert within the biofilm,
consist of a relatively thicker but porous cell wall made and amplify the effect of small variations in the suscep-
up of inter-connected peptidoglycan layers surround- tible phenotypes (8,9). Nevertheless, bacteria in a
ing a cytoplasmic membrane. The teichoic acid resi- biofilm are protected from antimicrobials by unique
dues of the cell wall contribute to the negative charge, mechanisms that are mostly due to certain peculiarities
which serves as binding sites for cationic molecules. of biofilm growth and structure. Schematic diagrams
Conversely, the cell envelope of a Gram-negative bac- of the hypothesized mechanisms of antimicrobial re-
terium is composed of an outer membrane, a thinner sistance in biofilm bacteria are shown in Figure 1.
peptidoglycan layer, and a cytoplasmic membrane. A mature bacterial biofilm is composed of multiple
Movement of molecules across a Gram-negative cell layers of bacteria embedded in a self-made matrix
wall is strictly regulated at the outer membrane, which formed of extracellular polymeric substance (EPS).
is rich in lipopolysaccharides (6). Thus, the suscepti- The EPS has the potential to modify the response of
bility of a bacterium to an antimicrobial will depend the resident bacteria to antimicrobials by acting as a
upon the type of cell wall it possesses. In addition to “diffusion shield” and “reaction neutralizer” against
the inherent resistance to antimicrobials, bacteria are the chemical effects of antimicrobials. The EPS, with
observed to demonstrate considerably high resistance its highly charged and interwoven structure, deters

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Fig. 1. Schematic diagram showing different methods by which bacteria in a biofilm gain resistance to antimicrobials
(AM).

penetration of antimicrobials (10–12). The barrier proliferate in the post-treatment phase (15). The bac-
effect of EPS is further enhanced by the extracellular teria in biofilms can up-regulate the expression of
substances and enzymes retained within the matrix. stress-response genes, shock proteins, and multi-drug
Certain constituents of the biofilm matrix may react pumps (efflux pumps), changing the biofilm bacteria
chemically and directly neutralize different antimicro- to a more resistant phenotype (17). Thus, the nature
bial agents such as iodine, iodine-polyvinylpyrollidone of the biofilm structure and physiological characteris-
complexes, chlorine, and peroxygens (13,14). There is tics of resident microorganisms offer the biofilm bac-
also a localized high density of bacterial cells in a teria an inherent resistance to antimicrobials (18,19).
biofilm structure. This spatial arrangement of cells will The current concepts emphasize endodontic disease
expose the cells in the deeper layers of the biofilm to as an example of a biofilm-mediated infection (20).
less nutrients and redox potential than the cells on the Ricucci & Siqueira (20) revealed a very high preva-
biofilm surface. Because the degree of nutrient and gas lence of bacterial biofilms in the apical root canals of
gradients increases with the thickness and maturity of both untreated and treated teeth with apical periodon-
a biofilm, the influence of growth rate and oxygen on titis. The arrangement pattern of the bacterial com-
the antimicrobial resistance is particularly marked in munity in the root canal is noted to be consistent with
aged biofilm. The resistance associated with biofilm the acceptable criteria to include apical periodontitis in
bacteria is also linked with the slow growth and star- the set of biofilm-mediated diseases. The authors also
vation of bacterial cells residing in a biofilm (14,15). suggested that the biofilm morphology/structure
Certain bacterial cells growing in a biofilm commu- varied from case to case, and no unique pattern for
nity, when exposed to unfavorable environmental endodontic infection was determined. Elimination or
stress or low-level antimicrobials, form survivor cells significant reduction of endodontic bacterial biofilms
called persister cells (16). The persister cells are non- and prevention of recontamination of the root canal
growing phenotypic variants of the general cell popu- after treatment are the essential elements for successful
lation. Following the purging of unfavorable stresses, outcomes of endodontic treatment. However, clinical
the persister cells grow rapidly in the presence of nutri- studies have shown that even after meticulous chemo-
ents. Biofilm populations are rich in persister cells; mechanical disinfection and obturation of the root
these cells would survive treatment procedures and canals, bacteria may still persist in the uninstrumented

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 Advanced therapeutic options for endodontic biofilms

canal lumen in many situations is found to communi-

cate with another root canal lumen via an isthmus.
These complexities will account for 30–50% of the root
canal wall left uninstrumented during routine root
canal instrumentation (22). The inability of the endo-
dontic irrigants/medicaments to penetrate the com-
plexities of the root canal system will cause bacterial
biofilms to persist in these niches after cleaning and
shaping procedures. Nair et al. showed that following
one-visit conventional endodontic treatment, the teeth
revealed microbial biofilm in the inaccessible recesses
and diverticula of instrumented main canals, the inter-
canal isthmus, and accessory canals (21).

Structure and composition of dentin

The bulk of the dentin structure is traversed by
Fig. 2. Different challenges in the disinfection of endo- S-shaped dentinal tubules. The tubular nature of den-
dontic biofilms. tinal tubules makes dentin a porous structure, and
bacteria have been shown to possess the ability to
invade dentinal tubules (23). The degree of bacterial
portions and anatomical complexities of the root canal
penetration varies between different areas of a tooth
(21). Therefore, it is vital to understand that the
and the number of patent dentinal tubules present
current limitations in endodontic disinfection strate-
(23). The inability of antimicrobials to penetrate the
gies are not only due to the biofilm mode of bacterial
infected dentinal tubules results in the survival of the
growth within the root canals, but also collectively due
bacterial population within the dentin (reservoir of
to the anatomical complexities of the root canal
infection). Berutti et al. showed that irrigating the
system, dentin structure/composition, and factors
canal with sodium hypochlorite (after removing the
associated with the chemical disinfectants (Fig. 2).
smear layer) rendered the dentinal tubules bacteria-
Consequently, advanced disinfection strategies are
free only to a depth of 130 mm from the canal lumen
developed and tested in Endodontics to circumvent
(24), beyond which surviving bacteria were detected.
these challenges. Ideally, these disinfection strategies
In addition, dentin is a biological composite that is
should eliminate biofilm bacteria from the uninstru-
made up of an inorganic phase (carbonated hydroxy-
mented portions and anatomical complexities of the
apatite), an organic phase (collagenous and non-
root canal system without inducing untoward effects
collagenous proteins), and a water phase. The
on dentin substrate and periradicular tissue.
chemicals used within the root canal can interact with
the organic and inorganic components of the dentin
Challenging factors in root matrix, which induces a buffering effect on their anti-
canal disinfection microbial effects. This will lead to the observed time-
dependent and depth effects of chemicals in the root
dentin (25). The buffering effect offered by the dentin
Root canal anatomy
will be more significant clinically since only small
The complexities of the root canal system, in addition volumes of irrigants are used in root canals.
to the structure and composition of the root dentin,
are decisive limiting factors in endodontic disinfection.
Irrigation dynamics in the root canal
The root canal system is a highly complex anatomy.
The accessory canals, lateral canals, apical ramifications, Endodontic irrigants are primarily liquid antimicro-
and transverse anastomoses all contribute to the com- bials used to combat microbial biofilms in the root
plexities in root canal anatomy (22). The main root canal system. The process of irrigant delivery within

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Fig. 3. Schematic diagram showing (a) closed system and (b) open system set-ups. A micro-CT image of a shaped canal
from a closed system (c) following delivery of cesium chloride. A vapor lock with an air bubble on the top was evident
along the apical end of the canal space (open arrowheads). (d) Open system after the canal was filled with cesium
chloride. The solution in this case reached the apical 0–2 mm of the canal space (arrow). Reproduced with permission
from Tay et al. (29).

the root canal is called irrigation, and irrigation within the entire root canal system. The final efficiency
dynamics deal with how irrigants flow, penetrate, and of endodontic disinfection will depend upon both its
exchange within the root canal space and the forces chemical and physical effectiveness (26–28). It is
produced by them. Hence, in endodontic disinfection, important to realize that even the most powerful irri-
the process of delivery is as important as the antibac- gant will be of no use if it cannot penetrate the apical
terial characteristics of the irrigants. The overall objec- portion (up to the working length) of the root canal,
tives of root canal irrigation are as follows: (i) To interact with the root canal wall, and exchange fre-
inactivate bacterial biofilms, inactivate endotoxin, and quently within the root canal system (26).
dissolve tissue remnants/smear layer (chemical effects) In an in vivo situation, a tooth root is enclosed in a
from the infected root canals. The chemical effective- bone socket and thus a root canal is believed to behave
ness will depend upon the concentration of the anti- as a closed-end channel, which in turn causes gas
microbial irrigants and the duration of interaction entrapment at its closed end during irrigation (vapor-
between irrigants and infected material. (ii) To allow lock effect) (Fig. 3) (29). Recently there have been
the flow of irrigant throughout the root canal system several computational fluid dynamics (CFD) analyses
so as to detach the biofilm structures and loosen/flush carried out to study the nature and pattern of irrigant
out the debris from the root canals (physical effects). flow within the root canal space (30,31). These studies
The physical effectiveness will depend upon the ability have demonstrated that irrigants, when expressed into
of irrigation to generate optimum streaming forces the apical portion of the root canal, experience a

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 Advanced therapeutic options for endodontic biofilms

Fig. 4. Contour images obtained from computational fluid dynamic analysis showing (a) shear wall stress pattern and
(b) turbulence intensity pattern resulting from irrigation with end-vented needle.

turbulent flow near the exit (orifice) of the needle, in the physical detachment of biofilms, was signifi-
followed by a reflux flow, and finally a laminar flow cantly less on the walls of the root canal compared to
backwards toward the pulp chamber, allowing the irri- the center of the root canal lumen (30–32). In order
gant to exit the root lumen. The irrigant flow was to circumvent the above challenges, endodontic irri-
noted to be significant only to about 1–2 mm apically gation needs to be combined with strategies that apply
from the exit of the needle (32) (Fig. 4). Furthermore, pressure gradients to the irrigant, such as ultrasonic
the shear stress exerted by the irrigant flow, which aids agitation, sonic agitation, or apical negative pressure

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Fig. 5. Schematic diagram showing different anti-biofim strategies. AM: antimicrobial.

(33–36). The application of pressure gradients to irri- been modified with chemicals or surface preparations
gants can improve fluid dynamics within the root canal in order to hinder bacterial adherence and subsequent
and subsequently result in significant biofilm elimina- biofilm formation (37,38). Considering the nature of
tion. These aspects of irrigation dynamics will not be the challenges presented by the root canal environ-
covered here. This article will focus on advanced thera- ment and endodontic microbes, a reliable therapeutic
peutic strategies to disinfect endodontic biofilms. requirement of endodontic disinfection should be to
eliminate the biofilm structure and destroy the resi-
dent bacteria completely, even in locations untouched
Advanced therapeutic strategies for by root canal instrumentation procedures. During this
endodontic biofilms process, it is crucial that these therapeutic methods do
not cause any physical, mechanical, and/or chemical
Generally, therapeutic strategies against bacterial bio-
changes to the root dentin. In the following para-
films focus on (i) inactivating the resident bacteria in
graphs, different advanced therapeutic approaches for
the biofilm structure or (ii) disrupting the biofilm
endodontic biofilms will be reviewed.
structure and simultaneously killing the resident
microbes. Figure 5 shows a schematic representation
of different anti-biofilm strategies. The above objec-
Antibacterial nanoparticles
tives are achieved by different antimicrobials and/or
treatment strategies. They include the application of Nanoparticles are microscopic particles with one or
antimicrobials that (i) produce slow destruction of more dimensions in the range of 1–100 nm. Nanopar-
the biofilm structure; (ii) destroy persister cells or ticles are recognized to have properties that are very
quorum-sensing signals in a biofilm; (iii) diffuse into unique from their bulk or powder counterparts. Anti-
the biofilm structure and kill bacteria; (iv) are used in bacterial nanoparticles have been found to have a
combination with other strategies that enhance their broad spectrum of antimicrobial activity and a far
diffusion into the biofilm structure; and (v) destroy lower propensity to induce microbial resistance than
both the biofilm matrix and the resident bacteria in a antibiotics. It is documented that magnesium oxide
biofilm structure (12). Recently, anti-biofilm strategies (MgO) and calcium oxide (CaO) slurries acted upon
have also been directed toward preventing biofilm for- both Gram-positive and Gram-negative bacteria in a
mation. With this in mind, biomaterial surfaces have bactericidal manner (39), while a zinc oxide (ZnO)

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slurry acted in a bacteriostatic manner and exhibited quently the number of adhering bacteria is reduced.
stronger antibacterial activity against Gram-positive Irrigation with sodium hypochlorite and subsequently
than Gram-negative bacteria (40). The antibacterial with chlorhexidine (CHX) significantly reduced the
powders of MgO, CaO, and ZnO generated active adherence of E. faecalis to dentin (72% reduction)
oxygen species, such as hydrogen peroxide and super- (56,57), though the by-product of this interaction is a
oxide anion radical, which are responsible for their concern. These findings highlight the fact that chemi-
antibacterial effect. Nanoparticles, with their high cals which alter the physico-chemical properties of
surface area, charge density, and greater degree of dentin may influence the nature of bacterial adherence
interaction with cells, exhibited higher levels of anti- and the adhesion force to dentin. The quantum
bacterial activity (41). The electrostatic interaction size effect of nanoparticles permits them to exhibit
between positively charged nanoparticles and nega- superior interaction with bacteria and dentin substrate.
tively charged bacterial cells, and the accumulation of When cationic nanoparticles in an aqueous suspension
a large number of nanoparticles on the bacterial cell are allowed to settle onto the dentin surface (nega-
membrane, have been associated with the increase in tively charged), the cationic nanoparticles adhere to
membrane permeability and rapid loss of membrane the dentin surface via an electrostatic interaction.
function. Heavy metal ions are known to have differ- Although this interaction between nanoparticles and
ent effects on bacterial cell functions (42–44). Copper dentin is weak and easily disrupted, it can impede
ions induce oxidative stresses (45) and affect the redox bacterial re-colonization and biofilm formation (56).
cycling, resulting in cell membrane and DNA damage. Chitosan (CS) is a natural non-toxic biopolymer
Zinc ions above the essential threshold level inhibit derived from the deacetylation of chitin. It binds to
bacterial enzymes including dehydrogenase (46), negatively charged surfaces and has excellent anti-
which in turn impedes metabolic activity (47). Silver microbial and antifungal activities. The exact mechan-
ions inactivate proteins and inhibit the ability of DNA isms of the antibacterial action of CS and its derivatives
to replicate (48). Nanoparticles synthesized from have still not been elucidated. Nonetheless, even in the
powders of silver (Ag), copper oxide (CuO), and ZnO case of CS nanoparticles, the electrostatic interaction
are currently used for their antimicrobial activity (49). between the positively charged CS nanoparticles and
Adherence of microorganisms to a tissue or bio- the negatively charged bacterial cell membrane is
material surface is recognized to be an important and believed to alter bacterial cell permeability and loss of
the earliest step in the establishment of a biofilm- function (58). A recent study examined the antimicro-
mediated infection. Adherence of microorganisms to a bial properties of ZnO and resin-based root canal
substrate enables the microbes to evade the normal sealers loaded with CS and ZnO nanoparticles (59).
flushing action of saliva and allows the microbes to This study demonstrated that the addition of anti-
survive harsh growth conditions (50–53). Bacterial bacterial nanoparticles in root canal sealers improves
adherence experiments have demonstrated that endo- the direct (based on a direct antibacterial assay) and
dontic irrigants reduce the post-treatment adherence diffusible (based on a membrane-restricted anti-
of E. faecalis to root dentin. Nevertheless, different bacterial assay) antibacterial effects in root canal
chemicals produce dissimilar degrees of bacterial sealers. Studies have also shown that the application of
adherence to root dentin. Final irrigation with EDTA CS nanoparticles reduces the adherence of E. faecalis
following sodium hypochlorite (5.2%) produced a to root dentin. The treatment of root dentin with
minimal reduction (33%) in the bacterial adherence to ZnO nanoparticles, ZnO–CS mixed nanoparticles,
root dentin. A 5 min application of 17% EDTA (pH CS-layer-ZnO nanoparticles, or CS nanoparticles
7.3) produced a 20–30 mm zone of demineralization produces an 80–95% reduction in the adherence of E.
in dentin (54,55). Demineralization of dentin exposes faecalis to dentin. Root dentin treated with chlorhexi-
collagen, which forms an excellent substrate for dine and then with nanoparticulates shows the
binding many bacterial species including E. faecalis. maximum reduction (97%) in bacterial adherence
This could be the possible reason for the increased (59). Previous studies have highlighted good antibac-
adherence of E. faecalis to root dentin treated with terial substantivity after using 2% chlorhexidine gel for
EDTA. When sodium hypochlorite is used as the final 7 days in root canal treatment (60). But chlorhexidine
irrigant, the exposed collagen is removed; subse- was not able to entirely remove the bacteria from

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the dentinal tubules of teeth that were infected with of polyisoprene (PI) or polycaprolactone (PCL) and
E. faecalis (61). Studies have also shown that the nanometric bioactive glass 45S5 (BAG) was employed.
addition of nanoparticles did not deteriorate the flow Incorporation of BAG fillers into PI and PCL ren-
characteristics of the root canal sealer. Sealer loaded dered the resulting composite material bioactive and
with ZnO nanoparticles shows better antibacterial permitted improved mineralization (69). Although it
properties and the ability to diffuse antibacterial com- was concluded that a composite of PI, PCL, and BAG
ponents, which is of particular importance in a post- indicated a promising application as a “single” root
treatment root canal environment (59). Another study canal filling material, more rigorous investigations are
tested the efficacy of CS nanoparticles and ZnO nano- warranted in this area.
particles in eliminating bacterial biofilm and the effect Studies so far have shown that most tested nanopar-
of aging (conditioning with tissue fluids) on their anti- ticles possess high antibacterial properties when com-
bacterial properties. E. faecalis strains in planktonic pared with their powder counterparts. The high
and biofilm forms were tested in this study. It was reactivity resulting from the nanometric dimension
demonstrated that the rate of bacterial killing by nano- and their ability to resist aging for longer durations are
particles depended on the concentration and duration some of the advantages. Most cationic antibacterial
of interaction. Total elimination of planktonic bacteria particles show excellent interaction with biomaterials,
was observed in contrast to the biofilm bacteria, which bacteria, and biofilms. In root canal therapy, they may
survived even after 72 hours of interaction. Both CS be applied as a slurry or in combination with sealers.
nanoparticles and ZnO nanoparticles were found to Although they have the ability to diffuse antimicrobial
retain their antibacterial properties after aging for 90 components deep in dentin tissue, more research is
days (62). required in order to study their ability to inactivate
Bioactive glass (BAG) has received considerable bacterial biofilms in the anatomical complexities and
interest in root canal disinfection due to its antibacte- uninstrumented portions of the root canal system.
rial properties. BAG consists of SiO2, Na2O, CaO2, Their interaction with host tissues/immune cells also
and P2O5 at different concentrations. The antibacterial requires additional investigation. Furthermore, it is of
mechanism of BAG has been attributed to a combina- key importance to complement research on antibacte-
tion of several factors including: (i) a high pH; (ii) an rial nanoparticles with research on procedures to
increase in osmotic effects; and (iii) Ca/P precipitation deliver these nanoparticles within the root canal
(63). The feasibility of using BAG for root canal dis- system. The successful application of nanoparticles in
infection has been tested in vitro (64–66). However, Endodontics will depend on both the effectiveness of
when compared with calcium hydroxide, BAG showed antimicrobial nanoparticles and the delivery method
significantly less antibacterial effects (65). In addition, used to disperse these particles into the anatomical
BAG application did not effectively prevent recontami- complexities of the root canal system.
nation of instrumented root canals (66). It has been
suggested that an ideal preparation of 45S5 bioactive Antimicrobial photodynamic
glass suspension/slurry for root canal disinfection
therapy
should combine the ability to induce a high pH with
the capacity to continuously release alkaline species Antimicrobial photodynamic therapy (APDT) is a
(67). It was demonstrated that a BAG nanometric two-step procedure that involves the introduction of a
slurry had a 12-fold higher specific surface area than photosensitizer (Step 1: photosensitization of the
the micrometric counterpart. Nevertheless, the latter infected tissue) followed by light illumination (Step 2:
produced considerably higher alkalinity and antimicro- irradiation of the photosensitized tissue) of the sensi-
bial efficacy. This was in contrast to the previous report tized tissue, which would generate a toxic photo-
by the same group that showed higher antibacterial chemistry on the target cell, leading to cell lysis. The
efficacy with the shift from micron- to nano-sized wavelength of the light should be at the specific wave-
treatment materials (68). In another related applica- length that corresponds to the absorption wavelength
tion, BAG was used to promote mineral deposition in of the photosensitizer. The photosensitizer molecule
the root canal, which could ultimately replace the use in its ground state is a spectroscopic singlet (S0). After
of endodontic sealers. Toward this end, a combination absorption of the photon, it passes from the ground

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 Advanced therapeutic options for endodontic biofilms

state to its first excited state (S1). From this state, the shown the selective killing of microbial cells over host
photosensitizer can return again to the ground state or cells, especially for the photosensitization periods and
it can pass into a triplet excited state (T1) via intersys- light fluence required for the antimicrobial effects.
tem crossing. The photosensitizer in the triplet state is Soukos et al. compared the effect of APDT using a
extremely reactive; it can then react further by one or combination of Toluidine blue O (TBO) and red light
both of the following pathways to destroy the cell. (i) against S. sanguis and human gingival keratinocytes
Type I reaction: the photosensitizer in the triplet state and fibroblasts. They reported no reduction in the
can react with a target other than oxygen by hydrogen human cell viability whereas the bacteria were effec-
or electron transfer, resulting in radical ions that can tively killed (78). Soncin et al. reported the selective
react with oxygen to yield cytotoxic species such as killing of S. aureus over human fibroblasts and kerati-
hydrogen peroxide, superoxide anion, hydroxyl, and nocytes (4–6-fold) when subjected to APDT using
lipid-derived radicals. (ii) Type II reaction: the photo- cationic pthalocyanine and relatively low light fluen-
sensitizer in the triplet state can transfer the excitation cies (79). Meanwhile, George & Kishen demonstrated
energy to ground-state molecular oxygen to produce a 97.7% success rate in killing Enterococcus faecalis
excited-state singlet oxygen (1O2) (70). compared to 30% human fibroblast dysfunction fol-
Singlet oxygen is a strong oxidizing agent and thus lowing Methylene blue-mediated APDT (80).
highly reactive; it has a lifetime of less than 0.04 ms in
Photosensitizers and light sources
a biological environment and a radius of action of less
than 0.02 mm (71). The reactions of singlet oxygen A photosensitizer is a chemical agent that, when acti-
with cellular targets lead to cell death. The two basic vated with light at a specific wavelength, reacts with
mechanisms that have been proposed to account for the surrounding molecular oxygen to produce highly
this lethal damage to bacterial cells are DNA damage reactive singlet oxygen. Toxicity may become an issue
and cytoplasmic membrane damage. For both Gram- if a high concentration/volume of photosensitizer is
positive and Gram-negative bacteria, it has been applied to a tissue in order to obtain a more significant
reported that APDT breaks single and double- treatment response. Lack of in vivo stability is another
stranded DNA and causes the disappearance of the issue associated with toxicity because the toxicity
plasmid super-coiled fraction (72,73). It has been sug- profile of the breakdown products may also need to be
gested that, during lethal photosensitization, singlet evaluated. Despite all of these impediments, there are
oxygen may interact with photo-oxidizable amino acid a large number of photosensitizers potentially useful in
residues such as His, Cys, Trp, and Tyr in one protein APDT, several of which are currently in various stages
molecule to produce reactive species, which may in of clinical trials for FDA approval. Over the last
turn interact with residues or free amino groups in decade, research has shown that compounds based on
another protein to form cross-links. In some cases it is phenothizinium chromophore are emerging as prom-
thought that free radicals may be involved (74). Pre- ising candidates for use as photosensitizers in APDT
vious studies have shown that the photo-oxidative (77). The phenothiazinium group of photosensitizers
effect caused by a phenothiazinium photosensitizer in such as Methylene blue and TBO are generally
bacteria could lead to damage in multiple targets such accepted photosensitizers for clinical application (81).
as DNA (73), membrane integrity (75), protease activ- Phenothiaziniums are usually cationic molecules with
ity, and lipopolysaccharides (LPS) (76). George & a core structure composed of a planar tricyclic aro-
Kishen reported functional impairment of the cell wall, matic ring system that functions as the chromophore
extensive damage to chromosomal DNA, and degra- (82). In addition to phenothiaziniums, cationic por-
dation of membrane proteins following Methylene phyrins (83), phthalocyanines (84), and chlorins (85)
blue-mediated APDT of E. faecalis (77). These find- have gained popularity as photosensitizers due to their
ings strongly support the hypothesis that APDT can ability to inactivate both Gram-positive and Gram-
represent a viable alternative because the mode of negative bacteria. Currently, photosensitizers such as
action on microbial cells is markedly different from Methylene blue, TBO, rose bengal, erythrosine,
that typical of most antimicrobial agents. chlorin (e6), and hematoporphyrin have been investi-
APDT has the potential to destroy microbial cells as gated for their antimicrobial potential against oral
well as mammalian cells. Yet several studies have pathogens.

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Fig. 6. Schematic diagram showing different methods of combining nanoparticles and photosensitizers.

Conjugating photosensitizers to various agents or efficiently inactivate both Gram-positive and Gram-
chemical moieties can result in improved photosensi- negative species (89). This was demonstrated by pre-
tizers for APDT. These modifications are commonly paring a conjugate of chlorin (e6) and a poly-l-lysine
aimed at improving their antibacterial or anti-biofilm chain (20 lysine residues), which after 1 min incuba-
efficacy and/or reducing their toxicity. Bezman et al. tion and illumination with red light, killed > 99% of
(86) covalently bound a photosensitizer (rose bengal) the Gram-positive Actinomyces viscosus and Gram-
to small polystyrene beads that were allowed to sensi- negative Porphyromonas gingivalis. (89). Polo et al.
tize bacterial cells. The modified photosensitizer was used conjugates between poly-l-lysine and porphycenes
expected to bind to the outer membrane of the bac- with significant phototoxic activity against Gram-
teria and, upon activation, generate reactive oxygen negative bacteria (90). Hamblin et al. demonstrated
species (ROS), which would then diffuse into the cells, the effectiveness of a poly-l-lysine-ce6 conjugate with a
resulting in cell death. Friedberg et al. (87) covalently chain length of lysines against both Gram-positive and
bound a photosensitizer to a monoclonal antibody Gram-negative species (85). Nanoparticles are ideal
(Mab) that binds to cell surface antigens expressed on carriers of photosensitizer molecules for APDT. The
P. aeruginosa and causes specific killing of the target combination of nanoparticles with photosensitizer
bacteria after light activation. Other researchers (88) molecules has emerged as a new interdisciplinary
have synthesized bacterio-chlorophyllide molecules research field. Some nanomaterials, such as TiO2,
(photosensitizers) conjugated to rabbit immunoglob- ZnO, and fullerenes as well as their derivatives, can
ulin G (IgG). The conjugated bacterio-chlorophyll generate singlet oxygen. On other occasions a photo-
(Bch1)–IgG with high specificity to protein A residues sensitizer molecule is combined with nanoparticles.
exposed on the cell wall of Staphylococcus aureus was Figure 6 shows different strategies that have been used
found to be 30 times more efficacious than other to combine nanoparticles with photosensitizers in
molecules that were tested. The higher efficacy of order to enhance the efficacy of APDT. They are (i)
Bchl–IgG was explained by its exclusive position on photosensitizers supplemented with nanoparticles; (ii)
the bacterial cell wall. Therefore, photo-generated oxi- photosensitizers encapsulated within nanoparticles;
dative species are confined to the cell wall and its (iii) photosensitizers bound or loaded to nanopar-
vicinity, which is a highly susceptible domain for pho- ticles; and (iv) nanoparticles themselves serving as
todynamic action. photosensitizers. Recently, the effect of APDT on E.
Soukos and co-workers formed a hypothesis that, by faecalis biofilm and human dental plaque bacteria was
covalently conjugating a suitable photosensitizer to a investigated in vitro using Methylene blue-loaded
poly-l-lysine chain, a bacteria-targeted photosensitizer poly(lactic-co-glycolic) (PLGA) nanoparticles (posi-
delivery vehicle could be constructed which would tively and negatively charged) that activated with red

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 Advanced therapeutic options for endodontic biofilms

light (wavelength 665 nm). The cationic Methylene light. Soukos et al. conducted APDT experiments on a
blue-loaded nanoparticles exhibited greater bacterial range of endodontic pathogens using Methylene blue
phototoxicity in both planktonic and biofilm phases. as the photosensitizer and reported complete elimina-
The nanoparticles were found to concentrate mainly tion of all of the bacteria except for E. faecalis (53%)
on the bacterial cell walls at all tested time points. It (96). In yet another study, significant antibacterial
was concluded that cationic Methylene blue-loaded effects on suspensions of S. intermedius, P. micros,
PLGA nanoparticles have the potential to be used as P. intermedia, and F. nucleatum were reported by Wil-
carriers of Methylene blue for antimicrobial APDT in liams et al. following APDT with TBO and red light
endodontic treatment (91,92). (101). Some of the tissue-specific challenging factors in
It is crucial to note that most photosensitizers easily the application of APDT for endodontic disinfection
form aggregates in aqueous medium, which may lead are the penetration of the activating light energy into
to a self-quenching effect upon excitation, thus reduc- the infected tissue, penetration of the optimum photo-
ing the yield of singlet oxygen (1O2) formation (93). sensitizer concentration into the infected tissue,
Studies have shown that a relatively high proportion limited availability of environmental oxygen in the
of aggregated photosensitizers in water may not favor infected tissue, and the ability of excess photosensitizer
the formation of singlet oxygen. To increase the effi- to induce dentin discoloration.
cacy of APDT, it is preferable to prepare the photo- Light propagation through tissue involves processes
sensitizer in its monomeric form by formulating it of reflection, absorption, scattering, and transmission.
in suitable carriers. Most studies involving APDT of Generally about 4–6% of light tends to be reflected. In
microbial pathogens use deionized water (DI) or biological tissue, absorption is mainly due to the pres-
phosphate buffered saline (PBS) to dissolve the ence of free water molecules, proteins, pigments, and
photosensitizer. Some studies, in which a photo- other macromolecules. The absorption co-efficient
sensitizer was dissolved in Brain–Heart Infusion strongly depends upon the wavelength of the incom-
(BHI) broth, reported a reduced bactericidal effect ing light/laser irradiation. Scattering of light in tissue
with the tested photosensitizer (91,94). This reduc- has the utmost effect on light intensity and direction-
tion in antibacterial effect was attributed to the pres- ality. Scattering and refraction of light causes a widen-
ence of serum proteins in the BHI broth (94–96). ing of the light beam, resulting in the loss of fluence
Light sources used for APDT can be coherent (lasers) rate (power per unit area) and a change in the direc-
or non-coherent (lamps). The choice of the light tionality of the light beam (97). In an effort to
source is dictated by the location, required light dose, improve the antimicrobial efficacy of APDT in the root
and choice of photosensitizer. Laser provides mono- canal system (tissue-specific approach), George &
chromatic, coherent, and collimated light, offering a Kishen dissolved Methylene blue in different formula-
wide range of output powers. Laser light can easily be tions: water, 70% glycerol, 70% poly ethylene glycol
coupled into a fiber optic cable that can serve as a (PEG), or a mixture of glycerol:ethanol:water (MIX)
delivery system (probe) while irradiating complex in a ratio of 30:20:50, and analyzed the photophysical,
anatomy such as a root canal. Nd:YAG, KTP, HeNe, photochemical, and photobiological characteristics
GaAlAs and diode lasers, light emitting diodes (99). They showed that aggregation of Methylene
(LEDs), and xenon-arc lamps have been employed for blue molecules was significantly higher in water when
APDT. The superiority of one type of light source compared with the other formulations. The MIX-
over another has not been clearly demonstrated and based Methylene blue formulation had effective pene-
hence the use of lasers or lamps depends upon the tration into dentinal tubules and enhanced singlet
specific application (97). oxygen generation, which in turn improved bacteri-
cidal action. A significantly higher impairment of the
bacterial cell wall and extensive damage to chromo-
APDT in Endodontics
somal DNA were observed when Methylene blue in a
In the Endodontic literature, Meire et al. (98) and MIX-based formulation was used, as compared to
George & Kishen (99,100) showed that E. faecalis water (77). The same authors also showed that the
could effectively be killed by APDT with photosensi- incorporation of an oxidizer and an oxygen carrier
tizers such as Methylene blue and TBO along with red with a photosensitizer formulation in the form of an

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Kishen

emulsion produces significant photo-oxidation capa- Laser-assisted root canal

bility, which in turn facilitates a comprehensive disrup- disinfection
tion of the mature endodontic biofilm structure (100).
Prokaryotic and eukaryotic cells possess families of The nature of the laser–tissue interaction is influenced
membrane proteins termed efflux pumps. The efflux by (i) the properties of the laser such as wavelength,
pumps act to remove amphiphilic molecules from energy density, and pulse duration; and (ii) the optical
within the cell. Given that many drugs are amphiphilic characteristics of the tissue such as absorption, reflec-
in nature, efflux pumps can effectively remove these tion, transmission, and scattering. Different types of
molecules from both prokaryotic and eukaryotic cells. lasers may produce different effects on the same tissue,
Efflux is the process in which bacteria transport com- and the same laser can have varying effects on different
pounds that are potentially toxic (such as drugs or tissues. The nature of light absorption and transmis-
chemicals) outside the cell (102). Many of these efflux sion is wavelength dependent. It should be noted that
pump systems have broad substrate profiles that allow the intensity of light will not remain constant through-
structurally diverse drugs/chemicals/compounds to out a definite volume of tissue. Therefore, laser effects
be extruded. Efflux pump expression has been shown will change depending upon the depth of penetration.
to be enhanced in biofilm bacteria when compared Generally, the clinician controls the following param-
with their planktonic counterparts. Inhibiting bacterial eters while operating a laser system: (i) applied power
efflux with an efflux pump inhibitor (EPI) reverses (power density); (ii) total energy applied over a given
the resistance to antimicrobials generated by the area of tissue (energy density); (iii) rate and duration
efflux pump. Tegos & Hamblin (103) showed that of laser irradiation (pulse repetition); and (iv) mode of
phenothaizinium dyes, which are structurally charac- energy delivery (continuous/pulsed energy, direct/
terized as amphipathic cations, were substrates of indirect tissue contact) (113).
multi-drug efflux pumps (MEP). They showed that One of the major disadvantages of current endodon-
inhibitors of bacterial MEP, when used in combina- tic antimicrobial irrigants is that their bactericidal
tion with phenothaizinium dyes, potentiated APDT effect is mostly limited to the main root canal lumen
(103,104). Since efflux pumps are highly active in (24). Lasers are primarily used in root canal disinfec-
bacterial biofilms, they are considered to be effective tion to enhance the degree of microbial elimination
targets for anti-biofilm measures (105,106). Kishen subsequent to cleaning and shaping procedures. Laser-
et al. have demonstrated the enhanced ability of EPI in assisted root canal disinfection requires the root canal
combination with phenothiazinium photosensitizers to be prepared in a conventional approach because the
to disinfect biofilm bacteria (107). parameters of the laser used for disinfection do not
Different in vivo studies that examined the efficacy of produce ablative effects on dentin tissue (113,114).
APDT in root canal disinfection have been summa- Infrared lasers such as CO2, Nd:YAG, diode, and
rized in Table 1 (108–111). These studies concluded Erbium lasers have been used for endodontic disinfec-
that a combination of chemomechanical preparation tion. The bactericidal effect of lasers depends upon the
and APDT would bring about the maximum reduc- wavelength characteristics and laser energy used, and
tion in microbial loads. Current research is directed in most cases is due to their thermal effects. The
toward potentiating the anti-biofilm efficacy of APDT laser-induced thermal effect will produce an alteration
by combining the advantages of photodynamic effects in the bacterial cell wall leading to changes in the
with bioactive micro- (92) and nanoparticles (112). osmotic gradients, swelling, and cell death. However,
Currently, APDT is not considered as an alternative when applied to root dentin, the depth of penetration
but rather a possible supplement to the existing pro- of the laser would depend on parameters such as wave-
tocols in root canal disinfection. Further research is length and power density. Generally, the depth of pen-
mandatory in order to improve the anti-biofilm effi- etration decreases with an increase in the degree of
cacy of APDT in the presence of tissue inhibitors, absorption by the tissue. It is also noted that Gram-
optimize light delivery within the root canal, optimize negative bacteria show a higher resistance to laser irra-
new photosensitizers (and formulations) for applica- diation than Gram-positive bacteria. This higher
tion in the root canal, and achieve a well-defined pro- resistance of Gram-negative bacteria is attributed to
tocol for endodontic applications. their cell wall characteristics (113).

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 Advanced therapeutic options for endodontic biofilms

Table 1: Summary of relevant in vivo studies carried out using antimicrobial photodynamic therapy
Author/Date Objective and Materials Methodology Conclusion

Bonsor et al., Aimed to evaluate the antimicrobial Irrigation with 20% citric acid and Cleaning and shaping resulted in
2006 (108) efficacy of root canal disinfection 2.25% sodium hypochlorite complete bacterial killing in
by combining conventional PDT with TBO and diode laser 86.7% of samples
endodontic treatment with PDT (12.7 mg/L, 100 mW, 120 s) Combination of cleaning and
Clinical study on 32 root canals Samples collected by filing shaping + PDT resulted in
from 14 patients complete bacterial killing in
96.7% of samples

Bonsor et al., Aimed to compare the effect of a Procedure similar to previous study Combination of 20% citric acid and
2006 (109) combination of 20% citric acid PDT resulted in complete
and PDT with the use of 20% bacterial killing in 91% of
citric acid and 2.25% sodium samples
hypochlorite on bacterial load in 20% citric acid and 2.25% sodium
prepared root canals hypochlorite resulted in complete
64 patients were used bacterial killing in 82% of
samples

Garcez et al., Analyzed the antimicrobial effect of Irrigation with 2.5% sodium First session produced 98.5%
2008 (110) PDT in association with hypochlorite, 3% hydrogen bacterial reduction (1.83 log
endodontic treatment peroxide, and 17% EDTA reduction)
20 patients were selected PDT with polyethyleneimine (PEI) Second session produced 99.9%
First session cleaning and chlorin (e6 [ce6]) conjugate bacterial reduction (1.14 log
shaping + PDT (2 min, 9.6 J, 240 s) reduction)
At the end of first session, the root Paper point sampling Second session PDT was observed
canal was filled with Ca(OH)2, to be more effective than first
and after 1 week, a second session
session of PDT was performed

Garcez et al., Studied antimicrobial effect of PDT PDT used polyethylenimine chlorin Endodontic therapy alone
2010 (111) combined with endodontic (e6) as a photosensitizer and a produced a significant reduction
treatment in patients with diode laser (40 mW, 4 min, in numbers of microbial species
necrotic pulp infected by 9.6 J) (only 3 teeth were free of
microflora resistant to a previous bacteria)
antibiotic therapy The combination of endodontic
30 teeth from 21 patients with therapy with PDT eliminated all
periapical lesions that had been drug-resistant species and all
treated with conventional teeth were bacteria-free
endodontic treatment and
antibiotic therapy were selected

The delivery of laser energy throughout the root canal (115). Schoop et al. (116) showed that the
canal system and the absorption of laser energy by Nd:YAG laser provided a bacterial reduction of 85% at
dentin tissues are important issues to consider in laser- a depth of 1 mm into the dentin when compared with
assisted root canal disinfection. The above issues will diode laser (810 nm), which produced a 63% bacterial
influence the degree of structural alteration in dentin reduction at a depth of 750 mm into the dentin. The
and the elimination of bacterial biofilm from the root difference in laser penetration and bacterial killing is
canal system. In a study aimed at increasing the effect attributed to the difference in the degree of absorption
of disinfection of the infected root canal, black Indian of different wavelengths of light within the dentin
ink or 38% silver ammonium solution was placed in the (116). Bergmans et al. tried to define the role of the
root canal before irradiating with pulsed Nd:YAG laser laser as a disinfecting tool by using Nd:YAG laser
(1,064 nm). The authors reported disinfection rates of irradiation on certain endodontic pathogens in vitro.
80% to 90% with Nd:YAG laser in the primary root They concluded that Nd:YAG laser irradiation is not

111
Kishen

an alternative but a possible supplement to existing Er:YAG irradiation produced a significant reduction in
protocols for root canal disinfection (117). In addi- the number of viable cells in most of the biofilms
tion, it was suggested that endodontic pathogens tested. Nevertheless, complete elimination of the
which grow as biofilms are difficult targets to eradicate biofilm structure/bacteria was not possible. Er:YAG
even with direct laser exposure. The bactericidal effect laser irradiation at all of the tested energy densities
of Erbium laser in a root canal model was observed to could not disinfect L. casei biofilm cells. It was men-
be inferior to that of an Nd:YAG laser. The thermal tioned that the L. casei decalcified the HA discs at a
energy produced by the Erbium laser is absorbed pri- depth of about 200 mm and invaded the porous decal-
marily by the surface structure due to the high affinity cified layer. It was speculated that the laser could not
to water molecules. Thus their bactericidal activities reach the base of the decalcified layer inhabited by the
tend to be higher on the surface (118). L. casei cells (122). It is important to realize that such
Several limitations that may be associated with the surface degradation and microbial penetration occurs
intracanal use of high-powered lasers cannot be over- deep within the anatomical complexities and dentinal
looked. The emission of laser energy from the tip of tubules in an endodontically infected tooth. The anti-
the optical fiber or laser guide is directed along the biofilm actions of the Er:YAG laser are influenced by
root canal and not necessary laterally to the root canal the water content, components of the extracellular
walls. Thus it is almost impossible to obtain uniform matrix, cell density, and absorption properties. The
coverage of the canal surface using a laser (119,120). temperature increase during irradiation ranges from
Because thermal damage to the periapical tissues is 7.3°C at 20 mJ to 40.2°C at 80 mJ. In another study,
possible, the safety of such a procedure is another Yavari et al. (123) examined the ability of high-
limitation. Direct emission of laser irradiation from the powered settings of Er and Cr:YSGG laser irradiation
tip of the optical fiber in the vicinity of the apical (2 W and 3 W output powers, respectively, for 16 s) to
foramen may result in the transmission of irradiation eradicate in vitro mono-species biofilms of E. faecalis
beyond the apical foramen, which may adversely affect (48 hours). It was concluded that, although 2 or 3 W
the supporting periradicular tissues. This effect can be of Er and Cr:YSGG lasers showed antibacterial
hazardous with teeth in close proximity to the mental properties on E. faecalis in root canal models, their
foramen or to the mandibular nerve (120). A modified effects were less remarkable than those of NaOCl
beam delivery system has been tested for Er:YAG solutions (123).
lasers. This system consists of a hollow tube that allows Early studies investigated changes in the ultrastruc-
for the lateral emission of radiation (side firing) rather ture of radicular dentin as a concomitant/adverse
than direct emission through a single opening at its effect of root canal disinfection with different lasers. It
terminal end (120). This new endodontic side-firing has been noted that, when used in a dry root canal,
spiral tip was designed to fit the shape and volume of both the near- and mid-infrared lasers produce char-
root canals prepared by nickel–titanium rotary instru- acteristic thermal effects on dentin. Human dentin
mentation. It emits the Er:YAG laser irradiation later- presents low absorption co-efficients in the near-
ally to the walls of the root canal through a spiral slit infrared range. Nonetheless, Nd:YAG laser irradiation
located all along the tip. The tip is sealed at its far end, is still able to melt the dentin surface (124). Moriyama
preventing the transmission of irradiation to and et al. (125) showed that morphological changes in
through the apical foramen of the tooth (121). The dentin are induced by Nd:YAG laser irradiation due to
goal of this tip improvement is to enhance the ability laser-induced thermal processes. In this case, the smear
of the laser-based antimicrobial effect to penetrate and layer is only partially removed and the dentinal tubules
destroy microbes in the lateral walls of root canals and are primarily closed due to the melting of inorganic
dentinal tubules. dentin material, and cracks are formed. Longer
Noiri et al. (122) investigated the anti-biofilm effect pulses produce the more evident effects of deeper
of Er:YAG lasers on in vitro mono-species biofilms of re-solidified structures due to the larger volume of
A. naeslundii, E. faecalis, L. casei, P. acnes, F. nuclea- melted material. Thus, increasing the number of pulses
tum, P. gingivalis, and P. nigrescens grown on hydroxy- may result in a more regular surface. However, the
apatite (HA) discs for 21 days (aerobically for 7 days high number of thermal cycles may lead to cracks
and anaerobically for 14 days). It was reported that (125). During the photothermal interaction, the tissue

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 Advanced therapeutic options for endodontic biofilms

Table 2: Summary of relevant in vivo studies carried out using laser-assisted disinfection
Author/Date Objective and Materials Methodology Conclusion

Koba et al., Evaluated the post-operative Nd:YAG (1 W, 15 pps for 1 No significant differences were
1999 (131) symptoms and healing after root second) found between the groups with
canal treatment with pulsed 5% NaOCl and 3% H2O2 used to respect to periapical healing
Nd:YAG laser disinfect (control)
44 teeth from 38 patients
Radiological evaluation used to
assess the reduction of periapical
lesions at 3 and 6 months

Dostálová Studied the ability of Er:YAG laser 5.25% NaOCl used to disifect Conventional treatment was
et al., 2002 radiation with a movable (control) effective in 60% of the
(132) waveguide to disinfect root Er:YAG (100 mJ, 30 pulses, root canals
canals repetition rate 4 Hz) Application of calcium hydroxide
Root canal of 44 premolars and Before and after treatment, the was effective in 80% of the root
molars were treated treated using colony-forming units were canals
a step-back technique; 10 teeth counted to determine 21 Er:YAG laser irradiation via
were then treated with calcium different microorganisms movable waveguide was effective
hydroxide and 22 teeth were in 100% of the root canals
irradiated with the waveguide

Leonardo Evaluated the antimicrobial effect Group I: cleaning and shaping only Er:YAG laser applied after cleaning
et al., 2005 of Er:YAG laser applied after Group II: cleaning and shaping and and shaping did not reduce
(133) cleaning and shaping of root Er:YAG laser application (63 mJ, microorganisms in the root canal
canals of dog teeth with apical output 15 Hz) system
periodontitis After coronal sealing, the root
40 root canals of dog premolars canals were left empty for 7 days
with periapical lesions were used before microbiological analysis

molecules absorb photons, resulting in the generation cal studies that have examined the antimicrobial effi-
of heat that is dissipated into the tissue. Since the tissue cacy of high-powered lasers in Endodontics (131–
needs some time to propagate the heat, longer pulses 133). There is no strong evidence currently available
will result in higher temperatures in deeper regions of to support the application of high-powered lasers in
the tissue, whereas for shorter pulses using the same endodontic disinfection.
average energy, higher temperatures will be observed Understanding the liquid irrigant–laser interaction
at the surface (126). The thermal damage in the tissue within the root canal is a new area of research interest.
is a temperature/time-dependent process. The result- This concept forms the basis for laser-activated irriga-
ant confinement of thermal stress would depend tion (LAI) and photon-initiated photoacoustic stream-
upon the laser pulse duration and the tissue absorp- ing (PIPS) in root canal disinfection (134–136). The
tion co-efficient (ma). The use of longer pulses will mechanism of interaction of Er,Cr:YSGG lasers with
lead to longer interaction times, resulting in more liquid irrigant in the root canal has been attributed to
evident thermal effects (127). The presence of water/ the efficient absorption of mid-infrared wavelength
irrigation solutions limits the thermal interaction light by water. This leads to vaporization of the irri-
of the laser beam on dentinal walls. The irradiation of gant and formation of vapor bubbles, which expand
root dentin with a diode laser (2.5 W, 15 Hz) and and implode with secondary cavitation effects. This
Nd:YAG laser (1.5 W, 100 mJ, 15 Hz) after irrigation process induces high-speed fluid motion into and out
with an irrigating solution produces a better dentin of the canal. The thermal component during this inter-
morphology (128,129). It has also been shown that action is moderate. The creation of bubbles is identical
the presence of water in the root canal space prevents in both water and sodium hypochlorite solutions. If
undesirable effects on dentin during the application of the liquid does not absorb the radiation, there is no
Erbium laser (130). Table 2 summarizes relevant clini- bubble, cavitation, pressure build-up, or fluid motion.

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Kishen

Fig. 7. Cross-sections of the root canal lumen at the 1-mm level with variable amounts of bacteria/biofilm after
(a) irrigation with photon-initiated photoacoustic streaming (PIPS)-activated NaOCl and (b) conventional NaOCl
irrigation. Original magnification 100 ¥. Reproduced with permission from Peters et al. (137).

At the beginning of the laser pulse, the laser energy is terial biofilm formed on root canal walls. This study
absorbed into a 2-mm-thick layer, which is instantly demonstrated that activated disinfection did not com-
super-heated to the boiling temperature at high pres- pletely remove bacterial biofilms from the apical
sure and and converted into vapor. This vapor at high third of the root canal and infected dentinal tubules.
pressure expands at high speed and provides an However, the finding that laser activation generated
opening in front of the fiber for the laser light. As the more negative bacterial samples and left less apical
laser continues to emit energy, the light passes through bacteria/biofilm than ultrasonic activation did war-
the bubble and evaporates the water surface in front rants further investigation (Fig. 7) (137). The current
of the bubble. Thus it drives a channel through the evidence for whether laser therapy can be recom-
liquid until the pulse ends (134,135). However, mended as an adjunct to chemomechanical disinfec-
further research is needed to examine the superiority tion of infected root canals is insufficient. This does
of lasers in eliminating root canal biofilms from the not necessarily imply that lasers should not be used as
anatomical complexities and the uninstrumented an adjunct to conventional chemomechanical root
portions of the root canal. canal treatment, but instead emphasizes the need for
PIPS is based on the direct shock wave generated by future high-quality studies in this field.
an Er:YAG laser in the liquid irrigant. The laser system
is equipped with a novel 400-mm-diameter radial and
Ozone
stripped tip, and subablative parameters (average
power 0.3 W, 20 mJ at 15 Hz) are used to produce Ozone (O3) is an energized, unstable gaseous form of
photomechanical effects when light energy is pulsed oxygen that readily dissociates back into oxygen (O2),
into the liquid. When activated in a limited volume of liberating a reactive form of oxygen, the singlet oxygen
fluid, the high absorption of the Er:YAG wavelength in (O1). The singlet oxygen is capable of oxidizing cells.
water, combined with the high peak power derived It has been suggested that ozone accomplishes its
from the short pulse duration that was used (50 msec), antimicrobial efficacy without developing drug resist-
results in a photomechanical phenomenon (136). ance (138,139). Ozone gas (HealOzone; KaVo,
Earlier it was demonstrated that mid-infrared laser Biberach, Germany) is currently used clinically for
energy, when delivered with conical modified fibers, endodontic treatment. However, results of studies on
influenced the configuration of shock waves and sub- its efficacy against endodontic pathogens have been
sequently produced improved photomechanical effects inconsistent. This inconsistency is attributed to the
in the root canal (121). Peters et al. studied the effi- lack of information about the optimum duration of
cacy of laser-activated and ultrasonically activated root application and concentration that should be used
canal disinfection with conventional irrigation, specifi- (140–142). In order to achieve a concentration that is
cally their ability to remove 3-week-old in vitro bac- relatively non-toxic toward periapical and oral mucosal

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 Advanced therapeutic options for endodontic biofilms

Table 3: Summary of relevant antimicrobial studies performed using ozone

Author/Date Objective/Methodology Conclusion

Estrela et al., 2007 To determine the antimicrobial efficacy of ozonated The irrigation of infected human root canals with
(143) water, gaseous ozone, 2.5% sodium hypochlorite, ozonated water, 2.5% NaOCl, 2% chlorhexi-
and 2% chlorhexidine in human root canals dine, and the application of gaseous ozone for
infected with E. faecalis for 60 days 20 min was not sufficient to inactivate E. faecalis

Hems et al., 2005 To evaluate the potential of ozone as an NaOCl was found to be superior to ozonated
(142) antibacterial agent using E. faecalis. The water in killing E. faecalis in broth culture and
antibacterial efficacy of ozone was tested against in biofilms
both broth and biofilm cultures. Ozone was
sparged for 30, 60, 120 and 240 s.

Nagayoshi et al., 2004 Effect of ozonated water against E. faecalis and S. Ozonated water application might be useful for
(140) mutans-infected dentin of bovine incisors root canal irrigation

Kuştarci et al., 2009 Evaluated the antimicrobial activity of potassium 2.5% NaOCl was superior in its antimicrobial
(148) titanyl phosphate (KTP) laser and gaseous ozone abilities compared with KTP laser and gaseous
in experimentally infected root canals (E. faecalis ozone
for 24 hours)

tissues, the ozone gas concentration currently used in models of Pseudomonas fluorescens. It was observed
Endodontics is 4 g/m3. This concentration has been that even low concentrations of ozone (0.1–0.3 ppm)
shown to be slightly less cytotoxic than NaOCl (2.5%). were able to completely kill bacteria after 15 or 30 min
Aqueous ozone (up to 20 mg/mL) showed essentially of contact time. However, the disinfectant action of
no toxicity to oral cells in vitro (143–145). ozone on biofilm models was less effective when com-
Hems et al. evaluated the potential of ozone as an pared with that on planktonic bacteria. In the biofilm
antibacterial agent using E. faecalis as the test microbe, models, only a decrease of two orders of magnitude
in both planktonic and biofilm cultures (48-hour-old was achieved. No increase in the anti-biofilm efficacy
biofilm grown on a cellulose nitrate membrane filter). was observed with increases in contact time (147).
Different interaction times ranging from 30 sec to Kuştarci et al. evaluated the antimicrobial activity of a
240 sec were applied to both cultures. It was con- potassium titanyl phosphate (KTP) laser and gaseous
cluded that ozone had an antibacterial effect on plank- ozone in experimentally infected root canals. It was
tonic E. faecalis cells and those suspended in fluid, but found that both the KTP laser and gaseous ozone have
little effect on cells embedded in a biofilm structure. a significant antibacterial effect on infected root canals,
The antibacterial efficacy of ozone was not comparable with the gaseous ozone being more effective than the
with that of sodium hypochlorite under the conditions KTP laser. However, 2.5% NaOCl was superior in its
tested in this study (142,143). Huth et al. assessed the antimicrobial abilities compared with the KTP laser
antimicrobial efficacy of aqueous (1.25–20 mg/mL) and gaseous ozone (148). Table 3 summarizes the
and gaseous ozone (1–53 g/m3) as an alternative relevant studies carried out to examine the antimicro-
antiseptic against endodontic pathogens in suspension bial efficacy of ozone.
and in a biofilm model. E. faecalis, Candida albicans, The reduced effectiveness of ozone against sessile
Peptostreptococcus micros, and Pseudomonas aeruginosa bacteria when compared with planktonic bacteria can
were grown in planktonic culture or in mono-species be attributed to different causes (147). (i) The EPS
biofilms in root canals for 3 weeks. It was concluded layer of the biofilm structure may form a physical/
that highly concentrated gaseous and aqueous ozone chemical barrier, preventing deeper penetration of the
was dose-, strain-, and time-dependently effective dissolved ozone into the biofilm structure (149). (ii)
against the tested microorganisms in suspension and in Biofilm is formed out of microbial colonies sur-
the biofilm test model (146). rounded by water channels through which the liquid
In another study, the antimicrobial efficacy of dis- movement is controlled by convective flow (149–
solved ozone was tested against planktonic and biofilm 150). Blockage of these channels in the biofilm by the

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Kishen

oxidation products of ozone may impede the further by 2% POV-I (87%), propolis (71%), MCJ (69%), and
penetration of ozone into the inner layers of the calcium hydroxide (55%) (162). Another study com-
biofilm structure (151). (iii) Phenotypically altered pared the in vitro effectiveness of MCJ with sodium
microbial communities possess enhanced resistance to hypochlorite and chlorhexidine gluconate to remove
antimicrobials in the deeper aspects of the biofilm. A the smear layer from the canal walls of endodontically
recent study has claimed that ozone dissolved in oil instrumented teeth. This study demonstrated that the
can be used as an intracanal medicament (152). efficacy of MJC was similar to NaOCl when used in
However, questions regarding the effect of surface conjunction with EDTA as an intracanal irrigant
tension on the flow characteristics of ozonized oil, (163).
chemical stability of ozonized oil, and the interaction Turmeric (Curcuma longa) is extensively used as a
of ozonized oil with root dentin and obturating food preservative in Southeast Asia. It has been used in
material must be answered before it can be applied traditional medicine for the treatment of numerous
in Endodontics (153,154). A systematic review by diseases. Curcumin (diferuloylmethane), the main bio-
Azarpazhooh & Limeback highlighted good evidence active component of turmeric, has been shown to have
of ozone biocompatibility with human oral epithelial a wide spectrum of biological actions including anti-
cells, gingival fibroblast, and periodontal cells. microbial, anti-inflammatory, and antioxidant activi-
However, conflicting evidence of the antimicrobial ties (164). A recent report suggested that curcumin
efficacy of ozone in Endodontics was emphasized in aqueous preparations exhibits phototoxic effects
(155). against Gram-positive and Gram-negative bacteria
(165). Triphala consists of the dried and powdered
fruits from three medicinal plants: Terminalia bel-
Herbal and enzyme alternatives
lerica, Terminalia chebula, and Emblica officinalis.
Some recent trends in anti-biofilm research are Triphala achieved 100% killing of E. faecalis in 6 min.
directed toward the application of natural extracts This may be attributed to its formulation, which con-
from plants to treat biofilm-mediated infection. tains three different medicinal plants in equal propor-
Natural polyphenols are present in a variety of plants tions; in such formulations, different compounds may
(156,157). They are characterized by the presence of help to enhance the potency of the active compounds,
more than one phenol unit per molecule (156). producing an additive or synergistic effect (166).
Polyphenols are well known for their antimicrobial Green tea polyphenols is prepared from the young
activity and have been used for food preservation. For shoots of the tea plant Camellia sinensis and showed
example, anacardic acid (found in the liquid extract of statistically significant antibacterial activity against E.
cashew nut shells) has been shown to exhibit anti- faecalis biofilms formed on tooth substrates (166).
microbial activity against Streptococcus mutans and It has been reported that phenol and natural phe-
Staphylococcus aureus (158,159). Many factors such as nolic compounds (except for ethyl linoleate and
bacterial species/strains, type of polyphenol, concen- tocopherol) significantly reduce the formation of bio-
tration of polyphenol, microbial cell density, synergis- films by P. aeruginosa. Experimental results indicate
tic effects of phenols with other antimicrobials, and that, for the tested P. aeruginosa strain, some of the
temperature can influence the antimicrobial properties tested phenolic and natural phenolic compounds were
of polyphenols. effective in reducing biofilm formation. At the concen-
Morinda citrifolia (MCJ) is an herb that has a broad tration level used in the experiments, not all of the
range of antibacterial, antiviral, antifungal, analgesic, tested compounds killed the bacterium, but some
anti-inflammatory, and immune-enhancing effects showed a significant effect in reducing the formation
(160,161). MCJ contains the antibacterial compounds of biofilms by P. aeruginosa. However, the exact
L-asperuloside and alizarin. An in vitro study investi- mechanism of the anti-adherence property was not
gated the antimicrobial activity of 2% chlorhexidine investigated (167). Table 4 summarizes the major
gel, propolis, MCJ, 2% povidone iodine (POV-I), and classes of antimicrobial compounds extracted from
calcium hydroxide on E. faecalis-infected root dentin. plants (164). The major advantages of using these
It was observed that chlorhexidine gluconate pro- herbal alternatives are easy availability, cost-
duced better antimicrobial efficacy (100%), followed effectiveness, increased shelf life, low toxicity, and lack

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Table 4: Summary of the major classes of antimicrobial compounds from plants (164)
Class Example(s) Mechanism

Phenolics Catechol Membrane disruption, binds to adhesins, complexes

with cell wall, inactivate enzymes
Hypericin Binds to adhesins
Warfarin Interaction with eukaryotic DNA (antiviral activity)

Terpenoids, essential oils Capsaicin Membrane disruption

Alkaloids Berberine Intercalate into cell wall and/or DNA

Piperine

Lectins and polypeptides Mannose-specific agglutinin Block viral fusion or adsorption

of microbial resistance. The combination of natural different antimicrobials (ranging from antimicrobial
polyphenols with nanoparticles and photodynamic irrigants to advanced antimicrobial methods such as
therapy should open new vistas in bacteria-specific lasers, photoactivated disinfection, and nanoparticles)
killing (targeted bacterial killing) without undue are employed in the management of infected root
effects on healthy tissues and mammalian cells. canal systems. Many of these advanced antimicrobial
It has also been suggested that various enzymes strategies show tremendous inhibitory effects on most
remove biofilm structures, especially disrupting the types of microbial biofilm in vitro. However, they
EPS from inanimate surfaces such as orthopedic should be subjected to animal and human studies in
implants (168). The two carbohydrate-containing order to determine their effectiveness in vivo, includ-
moieties of staphylococcal biofilms, a linear poly-b-(1- ing their side-effects on dentin and periradicular
6)-N-acetyl-D-glucosamine (PNAG) and teichoic tissues. Disruption of biofilm bacteria and prevention
acid, have been targeted using enzymes such as dis- of re-colonization are some examples of anti-biofilm
persin B and proteinase K (168–170). These studies efficacies that are presently not commonly examined.
have shown that rinsing an implant surface with It is also important to combine potent anti-biofilm
enzymes can prevent the formation of staphylococcal methods with good delivery systems in order to
biofilms. However, applying enzymes in an in vivo achieve essential goals in the root canal system. Atten-
situation that involves a multi-species pathogenic bac- tion to these issues could usher in a much-needed
terial biofilm may pose limitations due to specificity. new era of chemotherapeutic treatment of root canal
The effect of these enzymes on a biological substrate biofilms in the anatomical complexities and uninstru-
and their mechanisms of action need to be well under- mented potions of the root canal system.
stood before implementing such treatment strategies
in vivo. Further experiments are required to evaluate
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