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Current therapeutic options forendodontic biofilmsMARKUS
HAAPASALO & YA SHEN
Microbial biofilms in the infected root canal space are the
primary cause of apical periodontitis. Root canal
treatmenttherefore aims to either remove the biofilms from the root
canal or kill all of the microbes in the biofilms.Instrumentation
mechanically removes or disrupts biofilm organization and creates
sufficient space in the canal toallow effective irrigation and
disinfection to occur. While none of the mechanical or chemical
factors alone canpredictably eradicate the infective agents, their
combined action under optimal efforts is the key factor for
long-termsuccess of endodontic treatment and healing of the lesion.
In this article, the role and impact of various
mechanical(physical) and chemical means of attacking root canal
biofilms are discussed in light of relevant literature (Fig.
1).
Received 14 November 2011; accepted 30 June 2012.
Building blocks for success inendodontic treatment
Apical periodontitis (AP) is an inflammatory reactionof
periradicular tissues caused by a microbial infectionin the root
canal (1,2). Because the bacteria in thenecrotic root canal system
grow mostly in sessile bio-films, the success of endodontic
treatment depends oneffective elimination of such biofilms. The
necessaryelements in the control of endodontic infection arehost
defense, instrumentation and irrigation, locallyused intracanal
medicaments between appointments,root canal filling, and coronal
restoration (3,4).Chemomechanical preparation has been regarded
as
the key element of endodontic treatment (5,6). Impor-tantly,
mechanical canal preparation supports disinfec-tion by disturbing
or detaching biofilms that adhere tocanal surfaces and by removing
a layer of infecteddentin. Anatomical complexities often represent
physi-cal constraints that pose a serious challenge to adequateroot
canal disinfection. Even with the use of rotaryinstrumentation, the
nickeltitanium (NiTi) instru-ments currently available only act on
the central body ofthe canal, leaving canal fins, isthmi, and
cul-de-sacsuntouched after completion of the preparation
(710).These areas may harbor tissue debris and microbes andtheir
by-products (1113), which can prevent close
adaptation of the obturation material (14) and result
inpersistent periradicular inflammation (15,16).In addition to
mechanical preparation, irrigating
solutions with a strong antibacterial effect are neces-sary.
However, the available irrigants also face greatchallenges in
eliminating all of the biofilm from theroot canal. The protective
mechanisms underlyingbiofilm antimicrobial resistance are not fully
under-stood, although several mechanisms have been pro-posed
(17,18). These mechanisms include physical orchemical diffusion
barriers to antimicrobial penetra-tion into the biofilm, slow
growth of the biofilm dueto the limitation of nutrients, activation
of the generalstress response, and the emergence of a
biofilm-specificphenotype (19). Furthermore, recent studies
(2022)have given valuable information about the interactionof
endodontic disinfecting agents with dentin andother compounds
present in the necrotic root canal.As a result of such
interactions, the antimicrobial effec-tiveness of several key
disinfectants may be weakenedor even eliminated under certain
circumstances. It islikely that inactivation of the medicaments in
thechemical environment of the necrotic root canal is onereason for
the failure to completely eradicate themicrobes (23). Therefore,
resistant microbes cansurvive on the walls of the main root canal
after vig-orous chemomechanical treatment.
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Endodontic Topics 2012, 22, 7998All rights reserved
2012 John Wiley & Sons A/S
ENDODONTIC TOPICS 20121601-1538
79
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Given the inability of metal instruments to directlyplane the
walls of the complex internal surface geom-etry of teeth, the key
issue is the ability of antibacterialfluids to reach these spaces
and surfaces to effectivelyattack bacterial biofilm. Accessory
(lateral) canalsbranch from the main root canal, with
diametersranging from a maximum of 100 mm to a commonminimum of 10
mm in permanent molars (24). Suchnarrow orifices create a surface
tension barrier thatdoes not allow adequate mixing between the
irrigantand the liquid within the canal. The narrowing of theroot
canal apically (toward the root) poses a similarbarrier. Any fluid
flowing down the accessory canalsfrom the root canal will have a
laminar flow; turbulentflow will be not be achievable due to the
very lowReynolds numbers inherent at such small pipe diam-eters,
where edge effects and viscosity become themajor factors affecting
fluid dynamics (25). At thescale of the accessory canals, diffusion
of irrigant downthe concentration gradient will be the
dominantmechanism by which the agent moves along the
canal.Therefore, progress in the search for safe and moreeffective
irrigant delivery and agitation systems forroot canal irrigation is
necessary.In conclusion, the factors that remain a challenge in
cleaning and disinfecting the root canal space includebiofilm
resistance (17,18), poor penetration of themedicament/irrigant
(26), low concentration (27),short exposure time (28,29), small
overall volume
(30), and poor exchange of irrigants in the apical partsof the
root canal (24,25). In addition, inactivation ofthe medicament in
the root canal in the in vivo situa-tion may weaken the
effectiveness of endodontic treat-ment procedures and thus
contribute to the survival ofresistant bacteria and yeasts in the
root canal system.
Locations and characteristics ofendodontic biofilmsThe
localization of bacteria in the necrotic root canal isdependent
mainly on ecological factors such as avail-ability of nutrients in
the various parts of the canalsystem, redox potential (oxygen), and
composition ofthe infective microflora including positive and
negativebacterial interactions. In primary AP, the vast majorityof
the microbes are occupying the main root canal(s)and only a few
have invaded deeper into the dentin vialateral canals and dentinal
tubules (31,32). Apicalramifications, lateral canals, and isthmuses
connectingmain root canals have all been shown to harbor bac-terial
cells, which are also frequently organized inbiofilm-like
structures (13,33,34). In post-treatmentendodontic disease
(root-filled teeth with AP), thelocation of the microbes is
affected by several addi-tional factors such as the quality of the
root filling,main source of the nutrients (e.g. coronal versus
apicalleakage), and possible antibacterial effects of the
rootfilling materials (16). In addition, biofilms attached tothe
apical root surface (extraradicular biofilms) havebeen reported and
regarded as a possible cause ofpost-treatment apical periodontitis
(35). However, it isimportant to understand that in post-treatment
endo-dontic disease, the bacteria (or yeasts) must be able
tointeract with the host defense of the paradental tissuesto cause
the inflammatory response and tissue destruc-tion, which can then
be detected in the radiograph.A necrotic root canal also represents
a challenging
environment in which bacteria face toxic substancessuch as
bacteriocins and have to survive with limitedaccess to nutrients
and key elements such as iron. Thisis likely to result in various
survival strategies such asdecreasing metabolic activity and even
transforminginto the viable but non-culturable state (36). Chvezde
Paz et al. (37) also found that the low reactivity ofnon-growing
biofilm cells to the introduction of freshnutrients may be a
survival strategy employed bymicroorganisms in the oral cavity.
These various phasesof microbial interaction with the surface
appear to
Fig. 1. Green symbols: factors that effect the develop-ment and
characteristics of endodontic biofilms. Redsymbols: factors that
are important in eradication ofbiofilms and biofilm bacteria.
Haapasalo & Shen
80
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require the production of extracellular polymers thatassist in
initial adhesion, maintenance of biofilm struc-ture, and detachment
from matrix-enclosed aggre-gates. Overall, the unique root canal
environmentalconditions are expected to influence biofilm
structureand function (16). Endodontic biofilm morphologydiffers
considerably from individual to individual, andthe reasons for that
deserve further investigation butmay conceivably be related to
different biofilm com-position, type and availability of nutrients,
and overallduration of the infection (16,26,38).
Effect of instrumentationon biofilmsThe purpose of root canal
preparation in the contextof endodontic therapy is to: (i) shape
the canals to anadequate geometry; (ii) clean the canal system by
pro-moting access for disinfection solutions (this strategyhas been
termed chemomechanical canal preparation);and (iii) make it
possible to place a high-quality rootfilling (4).
Microbiologically, the goal of instrumenta-tion and irrigation is
to remove or kill all of the micro-organisms in the root canal
system, and neutralize anyantigenic/biological potential of the
microbial com-ponents remaining in the canal. If this goal
couldpredictably be achieved at the first appointment,
mosttreatments could be completed in one visit, if only thetime
available would allow it. In cases where this (com-plete
eradication of root canal microorganisms) cannotbe achieved, the
goal of instrumentation and irrigationis to create optimal
conditions for the placement of anantibacterial inter-appointment
dressing in order tofurther enhance the disinfection of the
canal.Mechanical instrumentation is the core method for
bacterial reduction in the infected root canal. With thelaunch
of nickeltitanium (NiTi) rotary systems,perhaps too much credit was
given to these systems asbeing the sole solution to challenges in
root canaltreatment. Regarding the direct efficacy in the removalof
bacteria, it is important to notice that no differencewas found
between hand and rotary instruments (39).Dalton et al. (40)
compared the ability of stainless-steel K-type files and NiTi
rotary instruments toremove bacteria from infected root canals
using salineas the irrigating solution. The canals were sampled
formicrobes before, during, and after instrumentation. Inthis
study, only about one-third of the canals wererendered
bacteria-free, and no significant difference
was detected between canals instrumented with handfiles and
rotary instruments. An in vivo study thatapplied correlative light
and electron microscopictechniques to evaluate residual intracanal
infectionafter instrumentation with stainless-steel hand files
inmesio-buccal canals and NiTi instruments in mesio-lingual canals
of the same lower molars showed thatthere was no difference in
their respective ability toeliminate infection (33). In addition,
Carver et al. (41)evaluated the in vivo antibacterial efficacy of a
hand/rotary technique in mesial root canals of necrotic man-dibular
molars. Root canal cleaning and shaping withhand and rotary
instrumentation and irrigation with6.0% sodium hypochlorite showed
a significant reduc-tion in the log colony-forming unit (CFU)
counts.However, bacteria still remained in the canals.Moreover,
mechanical disinfection can also be
related to the removal of a layer of infected dentin, orat least
of incompletely mineralized predentin (6). Ithas been shown that
bacteria might penetrate dentinaltubules to depths of 200 mm or
more (42,43). Com-plete uniform enlargement of a root canal by 200
mmis not achieved with any contemporary instrument;this appears to
be an unattainable goal for anymechanical canal preparation
technique (44,45).It has been shown that the amount of
mechanically
prepared canal surface and, perhaps equally, theamount of
disturbed biofilm in the main root canaldepends on the canal type
(10,45). Rotary instru-ments perform comparably poorly in long oval
canalssuch as distal canals in lower molars, specificallybecause
they do not mechanically prepare 60% or moreof the canal surface
under these conditions (45,46). Inthe case of an infected root
canal, any bacterial biofilmon the instrumented canal surfaces is
likely to be dis-turbed or removed, although some of the
bacterialcells may become embedded within the smear of tissue(47).
The bacterial biofilm on the uninstrumentedsurfaces is likely to
remain mechanically undisturbed,except by the displacement of any
pulpal tissue ordentinal debris from the prepared part of the
canal. Itis possible that changes in the ecology of the root
canalsystem may have a significant influence on the survivaland
death of bacteria on the uninstrumented surface.Nevertheless, the
uninstrumented surfaces should stillbe regarded as contaminated.A
newly developed self-adjusting file (SAF)
(ReDent-Nova, Raanana, Israel) was designed toaddress the
shortcomings of traditional rotary files by
Current therapeutic options for endodontic biofilms
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adjusting itself to the cross-section of the canal (48).The SAF
system uses a hollow vibrating instrument,which allows for
continuous irrigation with NaOCl orethylenediaminetetraacetic acid
(EDTA) throughoutthe instrumentation process (Fig. 2). Irrigants
areexchanged and taken to the apical root canal as a resultof the
vibration and in-and-out motion of the SAF.The compressible NiTi
tube can adapt itself to theoval-shaped canal while its abrasive
blades are pressedagainst the walls to promote root canal
enlargement.When compared with NiTi instrumentation, it hasbeen
reported that the SAF leaves fewer unpreparedareas in anterior
teeth (49) and molar root canals(48,50). Siqueira et al. (51)
compared the capability ofSAF and rotary NiTi instrumentation to
eliminateEnterococcus faecalis populations from extractedhuman
teeth. Long oval canals from mandibular inci-sors and maxillary
second premolars were infectedwith E. faecalis for 30 days in order
to form biofilm-like structures. Preparation of long oval canals
withthe SAF was significantly more effective than rotaryNiTi
instrumentation in reducing intracanal E. faecaliscounts. Data
regarding the incidence of negativeand positive cultures revealed
that in the SAF group,80% of the samples were rendered free of
detectablelevels of E. faecalis, whereas instrumentation withrotary
NiTi instruments resulted in only 45% of thesamples being
culture-negative. The SAF system hasthe potential to be
particularly advantageous in pro-moting the disinfection of
oval-shaped canals.
However, it is presently unknown whether canalpreparation with
the SAF, and in particular its poten-tial to debride canal walls
better, will lead to improvedclinical outcomes.The reported
incidence of isthmuses in the mesial
root of mandibular molars ranges from 5489%,mostly in the middle
and apical thirds (52). In addi-tion to these structures being
inaccessible to instru-ments, instrumentation can in fact further
complicatethe cleaning of these areas. Paqu et al. (45) showedthat
dentin debris is formed and packed into theisthmus area during
rotary instrumentation withoutirrigation. Accumulated debris
certainly has a negativeimpact on the sealability of root canals,
but it may alsohamper disinfection in cases with apical
periodontitis.Endal et al. (53) found that even copious
irrigationduring and after instrumentation with solutions
dis-solving both organic and inorganic matter was not ableto
prevent or remove the debris packed into theisthmus area between
the main root canals (Fig. 3),where bacteria may be present in the
form of biofilms(33). Although mechanical instrumentation
togetherwith the use of irrigants in the canal is often
quiteeffective, complete cleanliness of these inaccessibleareas is
difficult to achieve. In an in vivo study, Burle-son et al. (54)
examined the efficiency of hand/rotarytechniques in removing
biofilm/necrotic tissue in themesial roots of necrotic human
mandibular molars.Following extraction, histological preparation,
andstaining, cross-sections from the 1- to 3-mm apicallevels were
evaluated for percentage of biofilm/necrotic debris removal.
Cleanliness results at the 1-,2-, and 3-mm levels were 80%, 92%,
and 95% forcanals and 33%, 31%, and 45% for isthmuses,
respec-tively. Interestingly, a recent case report (55) showedthat
a complex, variable, multi-species biofilm waspresent along the
entire length of the isthmus in whichthe tooth had been initially
treated 10 years earlier andthen re-treated 2 years ago. Both
Gram-positive andGram-negative organisms were detected, surviving
inan extremely harsh and nutrient-deficient environmentthat had
existed for more than a decade after root canaltreatment.In
summary, instrumentation plays an important
role in helping to remove biofilm from those areaswhere the
instrument can gain direct contact with theroot canal wall. In
addition, shaping of the main canalfacilitates effective irrigation
by creating the necessaryspace for needle penetration and
sufficient irrigant
Fig. 2. Self-adjusting file system. The hollow structureof the
file and elasticity of NiTi allow the file to comeinto contact with
the root canal dentin walls in a largerarea than is possible with
traditional rotary files.
Haapasalo & Shen
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flow. Regardless, challenges remain in many areas dueto anatomy
and the resistance of biofilms.
Effect of various irrigatingsolutions on root canal
biofilmsAntimicrobial agents have often been developed andoptimized
for their activity against fast-growing, dis-persed populations of
a single species. However,microbial communities growing in biofilms
areremarkably more difficult to eradicate with antimicro-bial
agents, and microorganisms in mature biofilmscan be extremely
resistant for reasons that have yet tobe fully explained. Most of
the endodontic studies onbiofilms have been conducted with
monocultures byallowing cells to grow on membranes, glass, or
plasticand divide under a continuous or frequent supply offresh
nutrients from a few hours to a few days(27,28,5659). As the
influence of root canal dentinand other surfaces on the expression
of novel biofilmphenotypes has not yet been touched upon,
conclu-sions and decisions reached from studies of monocul-ture
biofilm in the laboratory must be taken with greatcaution. Such
models may not reflect the reality of theinfected root canal and
thus may give misleading inter-pretations, especially regarding the
effects of antimi-crobials on biofilm bacteria. Therefore, it is
importantto develop multi-species in vitro biofilm models with
aclose similarity to oral/endodontic in vivo biofilms(Fig. 4).
Given that the current instrumentation techniquesalone are
unable to render root canals bacteria-free, achemical irrigant is
regarded as necessary to assist inreducing the amount of bacteria
and their toxicby-products. In addition, an ideal irrigant
shouldremove organic and inorganic debris and have low orno tissue
toxicity (60). While none of the irrigatingsolutions/disinfecting
agents presently used in endo-dontic treatment are able to do all
of the requiredtasks, many of them can have an impact on
biofilms,either by dissolving the film, killing the microbes
resid-ing in the biofilm, or by helping to break down ordetach the
film from the surface.
Sodium hypochlorite
Sodium hypochlorite (NaOCl) is the most popularand important
irrigating solution (61). In water,NaOCl ionizes into the sodium
ion, Na+, and thehypochlorite ion, OCl-, establishing equilibrium
withhypochlorous acid (HOCl). Hypochlorous acid isresponsible for
the antibacterial activity; the OCl- isless effective than
undissolved HOCl.NaOCl is commonly used in concentrations
between
0.5% and 6%. It is the only irrigant in Endodontics thatcan
dissolve organic tissue, including the organic partof the smear
layer. It should be used throughout theinstrumentation phase.
Dunavant et al. (27) comparedthe efficacy of 1% or 6% NaOCl with
that of 2% chlor-hexidine (CHX), Smear Clear, and MTAD against
Fig. 3. Three-dimensional reconstruction micro-CT scans of the
mesial root canal system of the mandibular molarunder
investigation. (A) Three-dimensional micro-CT reconstruction after
instrumentation. Prepared canal areas areindicated in blue, and
untouched areas are indicated in red. (B) Superimposition of the
apparent accumulated hardtissue debris areas are indicated in
yellow. The canal space and empty space in the isthmus area after
instrumentationare indicated in silver. (C) Root filling material
is indicated in silver; the non-filled area in the ribbon-shaped
isthmusthat includes debris and void is shown in dark violet.
Current therapeutic options for endodontic biofilms
83
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E. faecalis biofilms in an in vitro model system. Theirmodel
consisted of biofilms grown in a flow cellsystem. Biofilms were
immersed in test irrigants for 1or 5 minutes. Results showed that
both concentrationsof NaOCl provided statistically significantly
betterbiofilm killing than any of the other agents tested. 6%NaOCl
also removed biofilm cells. In an ex vivobiofilm study, Clegg et
al. (62) demonstrated a differ-ence in the effectiveness of 6% and
3% NaOCl againstbiofilm bacteria, the higher concentration being
moreeffective. Bacterial cells in the root canal encounter aharsh
ecological milieu. Recently, one in vitro study(38) evaluated the
biofilm formation capability ofstarved E. faecalis cells on human
dentin and the sus-ceptibility of the biofilm to 5.25% NaOCl. The
find-ings showed that E. faecalis cells in the starvationphase
could develop biofilm on human dentin. Bio-films of starved cells
were more resistant to 5.25%NaOCl than those of stationary
cells.Despite some good in vitro results, the more limited
antimicrobial effectiveness of NaOCl in vivo is disap-pointing.
The poor in vivo performance compared tothe in vitro effect may be
caused by problems in pen-etration to the most peripheral parts of
the root canalsystem such as fins, anastomoses, apical canals,
lateralcanals, and dentin canals. Also, the presence of
inacti-vating substances such as exudate from the periapicalarea,
pulp tissue, dentin collagen, and microbialbiomass counteracts the
effectiveness of NaOCl (63).One of the unknown areas regarding the
effect of
NaOCl on biofilms is the role of EPS (extracellularpolymeric
substance) in this interaction. Althoughsome in vitro studies have
shown a complete disap-pearance of the biofilm with strong sodium
hypochlo-rite treatment, one cannot exclude the possibility
thatwhile some/most of the biofilm may have been dis-solved, the
effect can also be caused by detachment ofthe biofilm from its
substrate in the in vitro environ-ment. Nevertheless, as NaOCl is
the only solution inEndodontics that can at least to some extent
dissolvebiofilm in addition to direct killing of microbes insidethe
film, it should be regarded as the main disinfectingsolution during
chemomechanical preparation ofinfected root canals.
Chlorhexidine digluconate and CHX-Plus
Chlorhexidine digluconate is widely used in disinfec-tion in
Dentistry because of its antimicrobial activity(6466). It has
gained considerable popularity inEndodontics as an irrigating
solution and as an intra-canal medicament. However, CHX has no
tissue-dissolving capability and therefore it cannot replacesodium
hypochlorite.CHX permeates the microbial cell wall or outer
membrane and attacks the bacterial cytoplasmic orinner membrane
or the yeast plasma membrane. Inhigh concentrations, CHX causes
coagulation of intra-cellular components (67). One of the reasons
for thepopularity of CHX is its substantivity (i.e. continued
Fig. 4. Scanning electron micrographs of biofilms with mixed
bacterial flora including numerous spirochetes.(A) Three-week-old
biofilm. (B) Six-week-old biofilm after 2% CHX treatment for 3
minutes shows tightly coiledspirochetes and a few damaged bacterial
cells.
Haapasalo & Shen
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antimicrobial effect) because CHX binds to hard tissueand
remains antimicrobial. However, similar to otherendodontic
disinfecting agents, the activity of CHXdepends on the pH and is
also greatly reduced in thepresence of organic matter (65). Several
studies havecompared the antibacterial effect of NaOCl and 2%CHX
against intracanal infections and have shownlittle or no difference
between their antimicrobialeffectiveness (6871). Clegg et al. (62)
evaluated theex vivo effectiveness of sodium hypochlorite, CHX,and
MTAD against biofilms grown on apical dentin.Six percent sodium
hypochlorite was the only solutioncapable of disrupting and
completely removing thebiofilm after 15 minutes of exposure. 2% CHX
killedthe biofilm bacteria but was not able to disrupt thebiofilm
structure (62). Although CHX may kill thebacteria, the biofilm and
other organic debris are notremoved by it. Residual organic tissue
may have anegative effect on the quality of the permanent
rootfilling seal, necessitating the use of NaOCl
duringinstrumentation.Surface-active agents have been added to
several dif-
ferent types of irrigants in order to lower their surfacetension
and to improve their penetration into the rootcanal. Recently, a
few studies have been published inwhich the antibacterial activity
of a chlorhexidineproduct with surface-active agents (CHX-Plus,
VistaDental Products, Racine, WI) has been compared toregular CHX,
both with a 2% chlorhexidine concen-tration. One study (29) showed
superior killing ofbiofilm bacteria by the combination product.
Anotherstudy (26) examined the susceptibility of multi-species
biofilm bacteria at different phases of biofilm growthto 2% CHX
and CHX-Plus. The multi-species biofilmswere grown from plaque
bacteria on collagen-coatedhydroxyapatite discs in brainheart
infusion broth fortime periods ranging from 2 days to several
months.Fresh nutrients were added weekly for the first 3weeks,
followed by a nutrient-deprivation phase, whenfresh medium was
added only once a month. Biofilmsof different ages were subjected
to a 1-, 3-, or10-minute exposure to 2% CHX or CHX-Plus.
Theproportion of killed bacteria in mature biofilms (3weeks or
older) was lower than in young biofilms (2days, 1 or 2 weeks) after
treatment with both CHXproducts, the reduction being much greater
with theregular 2% CHX (26). The resistance of mature bio-films
under the nutrient-limiting phase (612 weeks)to CHX remained stable
and was similar to 3-week-oldbiofilm (Fig. 5). CHX-Plus showed
higher levels ofbactericidal activity at all exposure times
compared to2% CHX, which may indicate that the surfactant
com-ponent in CHX-Plus facilitated penetration of thedisinfectant
into the biofilm. Overall, this study dem-onstrated that bacteria
in mature biofilms andnutrient-limited biofilms are more resistant
to CHXkilling than bacteria in young biofilms. The result
alsoemphasizes the importance of standardizing the age ofthe
biofilm cultures to allow comparisons betweenstudies. It is likely
that the biofilms in in vivo rootcanals are almost always older
than 3 weeks; thereforethe results of in vitro experiments with
biofilmsyounger than 3 weeks should be evaluated withcaution.
Fig. 5. The proportion of viable cells (volume) of biofilms of
different ages after treatment with CHX and CHX-Plus.There was a
significant difference in the proportion of killed bacteria
depending on the type of the disinfecting agent,exposure time, and
age and nutrient supply of the biofilm.
Current therapeutic options for endodontic biofilms
85
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MTAD
Bio Pure MTAD (Dentsply Tulsa Dental, Tulsa, OK)was introduced
to Endodontics in 2003. This mixtureof a tetracycline isomer
(doxycycline), citric acid, and adetergent has been shown to be
effective in smear layerremoval (72,73). Some in vitro experiments
indicatedthat MTAD has a strong antibacterial effect. It hasbeen
suggested to be more effective than NaOCl andEDTA against E.
faecalis (74) and mixed bacteria (75).However, some of these
results were later challengedin studies which found the
antibacterial effect ofMTAD to be inferior to 6% NaOCl and 2%
chlorhexi-dine (27). Furthermore, Dunavant et al. (27) reportedthat
1% NaOCl killed six times more E. faecalis inbiofilms (99.78%) than
MTAD did (16.08%). Pappenet al. (76) tested the efficacy of MTAD,
Tetraclean,and five experimental solutions against biofilm
bacte-ria. Tetraclean was more effective against two-week-old
polymicrobial biofilm than MTAD. A comparisonof biofilm killing by
MTAD and the experimental solu-tions indicated that the type of
detergent in the medic-ament mixture may have been of major
importance inthe effectiveness of the solutions against biofilms
(76).
QMiX
QMiX (Dentsply Tulsa Dental) is a new irrigatingsolution which
contains EDTA, chlorhexidine, and adetergent (surface-active
agent); its pH is slightlyabove neutral (Fig. 6) (77,78). A
surface-active agentdecreases the surface tension of solutions and
increasestheir wettability (79). Also, it enables better
penetra-tion of an irrigant into the root canal (80).The effect of
QMiX against E. faecalis and mixed
plaque biofilms was evaluated in a recent study
usingthree-week-old biofilms grown on collagen-coatedhydroxyapatite
discs under anaerobic conditions (80).The killing of bacteria
inside the biofilm was measuredby confocal laser scanning
microscopy (CLSM) andviability staining. The results demonstrated
that QMiXand 2% NaOCl were superior to 1% NaOCl, 2% CHX,or MTAD by
killing two to twelve times more biofilmbacteria in one to three
minutes. 2% NaOCl was moreeffective than QMiX at 1 minute against
plaquebiofilm bacteria, but at 3 minutes QMiX had killedmore
bacteria (65.3%) than any other solution tested(80). NaOCl
solutions stronger than 2% could not betested in the model because
of the strong bubbleformation, which made CLSM impossible.
The presence of bacteria in the dentinal tubules hasbeen
associated with persistent root canal infection(81). Studies have
shown that bacteria can penetrateinto dentinal tubules, and the
depth of penetrationvaries from 200 mm to 1,500 mm (42,43,82).
Bacteriawithin the dentinal tubules may be poorly accessible toroot
canal irrigants, medicaments, and sealers becausethey may have
limited penetrability into the dentinaltubules. An in vitro study
(77) using a novel type ofdentin infection model found that QMiX
was equallyeffective at killing E. faecalis bacteria in dentin as
6%NaOCl: over 40% and 60% of the bacteria were killedby both at 1
minute and 3 minutes, respectively(Fig. 7). Both solutions were
more effective againstthe bacteria inside dentin than 1% or 2%
NaOCl or 2%CHX.
Ethylenediaminetetraacetic acid (EDTA)
EDTA is a calcium binder (chelator) that aids in theremoval of
the smear layer. The smear layer is mainlycomposed of dentin
particles embedded in an amor-phous mass of organic material that
forms on the innerroot canal walls during the instrumentation
procedure.The smear layer counteracts disinfectants and mayblock or
slow down the penetration of medicamentsinto the dentinal tubules
(42). It also interferes withthe adhesion of some and penetration
of all root filling
Fig. 6. Two combination products: (A) CHX-Plus; and(B) QMiX.
Haapasalo & Shen
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materials. Therefore, by facilitating the cleaning andremoval of
infected tissue, EDTA contributes to theelimination of bacteria in
the root canal. It has alsobeen shown that the removal of the smear
layer byEDTA improves the antibacterial effect of locally
useddisinfecting agents in deeper layers of dentin
(42,83).Chemicals that alter the physicochemical properties
of dentin might influence the nature of the bacterialadherence
and the adhesion force to dentin, which arefactors in biofilm
formation. Kishen et al. (84) inves-tigated the effects of
endodontic irrigants on theadherence of E. faecalis to dentin. The
bacteria adher-ence assay was conducted by using
fluorescencemicroscopy, and the adhesion force was measured byusing
atomic force microscopy. There were significantincreases in
adherence and the adhesion force afterirrigation of dentin with
EDTA, whereas NaOClreduced them. With the use of CHX, the force
ofadhesion increased, but the adherence assay showed areduction in
the number of adhering bacteria.However, the sequence in which
NaOCl and EDTAare used for canal irrigation has an impact on the
levelof dentin erosion on the main root canal wall (85).Sodium
hypochlorite used as a final irrigant solutionafter
demineralization agents causes marked erosion of
root canal dentin. So far, it is not known whether sucherosion
is harmful to the root dentin and the tooth.However, chemical
removal, even by strong surfaceerosion, may facilitate the removal
of biofilms from theuninstrumented parts of the root canal.
Mechanical agitation by sonic andultrasonic appliancesThe
hydrodynamic behavior of the irrigating solutionsplays an important
role in the effectiveness of irrigation(15,25). It also depends on
the working mechanism ofthe irrigant as well as the mechanism of
action of theequipment used to introduce and agitate the irrigantin
the canal (86). Irrigant agitation can be done manu-ally with a
needle and syringe or by machine-drivenforces such as in sonic and
ultrasonic agitation.
Sonic agitation
In 1985, Tronstad et al. (87) were the first to reporton the use
of a sonic instrument in Endodontics. Sonicirrigation is different
from ultrasonic irrigation in thatit operates at a lower frequency
(110 kHz) and pro-duces smaller shear stresses (88). The sonic
energy alsogenerates significantly higher amplitude or
greaterback-and-forth tip movement.Sonic activation has been shown
to be an effective
method for disinfecting root canals (89). The Endo-Activator
(Advanced Endodontics, Santa Barbara, CA)uses sonic energy to
agitate the irrigants in the rootcanal system (Fig. 8). The action
of the EndoActivatortip often produces a cloud of debris
originating fromthe canal contents. Vibrating the tip, in
combinationwith moving the tip up and down in short
verticalstrokes, synergistically produces a powerful hydrody-namic
phenomenon (30). It has been suggested that10,000 cycles per minute
is needed to optimize thedebridement and promote disruption of the
smearlayer (90). The EndoActivator System has beenreported to be
able to clean debris from lateral canals,remove the smear layer,
and dislodge clumps of simu-lated biofilm within the curved canals
of molar teeth(90). However, not all studies have reported
similarresults. Brito et al. (91) found that sonic activation
ofEDTA and NaOCl with the EndoActivator deviceafter chemomechanical
procedures on a straight singlecanal did not result in improved
disinfection comparedto conventional needle irrigation. An in vivo
study
Fig. 7. Viability staining and confocal laser scanningmicroscopy
of E. faecalis infected dentinal tubulestreated with different
antibacterial solutions for 3minutes each: (A) sterile water; (B)
2% NaOCl; (C) 6%NaOCl; and (D) QMiX.
Current therapeutic options for endodontic biofilms
87
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(92) reported that the EndoActivator did not enhancethe ability
of standard needle irrigation to eliminatecultivable bacteria from
root canals.Shen et al. (93) investigated whether mechanical
agi-
tation (ultrasonic or sonic) improved the effectivenessof
chlorhexidine against biofilm bacteria in vitro. Formechanical
agitation, an ultrasonic tip or an EndoAc-tivator (sonic) tip was
placed 5 mm above the top ofthe multi-species biofilm, which was
immersed in irri-gant. This was the minimum distance between
theultrasonic or sonic tip and the biofilm surface at
whichmechanical agitations did not disrupt or disperse thebacteria.
After treatment, the amount of dead bacteriain biofilms was
analyzed by viability staining and con-focal laser scanning
microscopy. The low-intensityultrasonic or sonic agitation that
does not disrupt ordisperse the biofilm bacteria improves the
action ofdisinfectants against biofilm bacteria. The
precisemechanisms of the enhanced killing have not beenidentified
and may be different in different situations.When the EndoActivator
tip was placed 5 mm over thetop of the biofilm in this study, the
acoustic stream wasconnected to the rapid movement of the
irrigatingsolution in a vortex around the biofilm (93).
Thisenhanced transport may be partially responsible for
theincreased killing of biofilm bacteria exposed to com-binations
of the disinfectant and mechanical agitation.The visual observation
of more bubbles exiting alongthe EndoActivator file during
irrigation indicates thatbubbles do not exit in a perfect linear
stream but theirflow is turbulent and chaotic; thus, creating a
columnof bubbles instead of a line of bubbles produces abetter
result. The formation of micro-bubbles gradu-
ally increasing in diameter until they collapse provokesvery
effective small implosions, which produce anirregular agitation of
the irrigant. The results showedthat the combined use of ultrasonic
or sonic vibrationand chlorhexidine produced a better
antimicrobialeffect against biofilms than chlorhexidine alone.
Thevolume of killed cells was significantly correlated withthe time
of exposure, the type of medicament, and thetreatment group (sonic,
ultrasonic, or no mechanicalagitation) (93).
Ultrasonic agitation
Ultrasonics in Endodontics was introduced byRichman in 1956 for
the preparation of the accesscavity and for preparation as well as
obturation of thecanals (94). Twenty years later, Martin (95)
describedthe in vitro disinfectant action of ultrasonics,
demon-strating that the combined use of ultrasonics andsodium
hypochlorite could be more effective thaneither one on its own.The
ultrasonics used in Endodontics are acoustic
vibrations with frequencies around 25,000 cycles/second. From
the energy source, the ultrasonic wavesare transferred via a
transducer to a liquid, wherebywell-known physical phenomena occur.
One of these isan acoustic stream and is connected to the
rapidmovement of fluid particles in a vortex around theobject that
vibrates (88). Another phenomenoncaused by the ultrasonic vibration
is cavitation, whichis the formation of micro-bubbles that
graduallyincrease in diameter until they collapse, provokingvery
effective small implosions that produce an irregu-
Fig. 8. (A) The EndoActivator system has different-sized tips
and one handpiece. (B) EndoActivator with a smallplastic tip.
Haapasalo & Shen
88
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lar agitation of the liquid. Both of these effects areindicated
(88,95) as the principal reason why thedebris are removed from the
dentinal walls. It shouldalso be remembered that ultrasonics raise
the tem-perature of the liquid that surrounds the vibratingobject
(96).Numerous investigations have demonstrated that
the use of passive ultrasonic irrigation (PUI) after handor
rotary instrumentation resulted in a significantreduction in the
number of bacteria (96100) orachieved significantly better results
than syringe needleirrigation (99101). Carver et al. (41) found in
vivothat the use of ultrasonic irrigation following hand/rotary
instrumentation produced a significantlygreater reduction in CFU
counts in infected necrotichuman molars. Additionally, a
significantly higher per-centage of canals showed no microbial
growth insamples taken from the canals following the additionof
ultrasonic irrigation (80%) than following hand/rotary
instrumentation alone (27%). Histologicalspecimens from an in vivo
study by Burleson et al. (54)confirmed that one-minute use of
ultrasonically acti-vated irrigation following hand/rotary root
canalcleaning and shaping improved canal and isthmuscleanliness
because less necrotic debris/biofilm wasleft behind.The most
effective mechanism behind the effect of
ultrasound is the formation of cavitation bubbles(102104). These
bubbles are in a non-equilibriumstate and will oscillate and
collapse. The bubbledynamics involved is often complex because of
theproximity of the nearby tissues (105). The forcefulbubble
collapse with high-speed jetting could be har-nessed beneficially
(as in the removal of biofilm) orcould cause undesirable collateral
damage (106).High-intensity focused ultrasound (HIFU) is
appliedclinically to generate collapsing cavitation bubbles
influids and tissues, which collapse with high-speed jetsthat can
be used for drug delivery. However, itshould be remembered that the
distance from theultrasonic tip where cavitation can occur is
veryshort, in the range of 10100 mm (107). Recently,Shrestha et al.
(108) found that the collapsing cavi-tation bubbles used in HIFU
treatment resulted insignificant penetration (up to 1,000 mm) of
antibac-terial nanoparticles into the dentinal tubules. Thefindings
demonstrated the potential application ofHIFU-generated collapsing
cavitation bubbles todeliver antibacterial nanoparticles into the
dentinal
tubules and subsequently improve disinfection inEndodontics.
Photo-activated disinfectionPhoto-activated disinfection (PAD)
involves the use ofa photo-active dye (photosensitizer) that is
activatedby exposure to light of a specific wavelength in
thepresence of oxygen. The transfer of energy from theactivated
photosensitizer to available oxygen results inthe formation of
toxic oxygen species, such as singletoxygen and free radicals.
These very reactive chemicalspecies can damage proteins, lipids,
nucleic acids, andother cellular components (109111). PAD has
beenintroduced to Dentistry as a host-friendly way ofattacking
microorganisms in periodontal and endo-dontic infections. While
most other substances ormethods used in root canal disinfection are
directly orpotentially harmful to the host, PAD is claimed
tospecifically target microorganisms with no collateraldamage. It
involves the use of a photosensitizer (PS)that is activated by
light in the presence of oxygen.There are several factors
influencing photodamage
including the type, dose, incubation time, and local-ization of
the photosensitizer; the availability ofoxygen; the wavelength of
light; the light powerdensity; and the light energy fluence. An
importantcharacteristic of photodynamic therapy is its inherentdual
selectivity; first, by achieving an increased con-centration of the
photosensitizer by specific binding totarget tissues, and second,
by constraining the irradia-tion to a specified volume. In
antibacterial photody-namic therapy, photodestruction is mainly
caused bydamage to the cytoplasmic membrane and DNA(112,113).The
efficiency of PAD may depend on environmen-
tal and microbiological factors at the site of the infec-tion. A
fundamental difference in susceptibility to PADbetween
Gram-positive and Gram-negative bacteriahas been reported
(114,115). In general, neutral,anionic, or cationic PS molecules
can efficiently killGram-positive bacteria, whereas only cationic
PS mol-ecules or strategies that permeabilize the Gram-negative
permeability barrier in combination withnon-cationic PS molecules
are able to kill multiple logsof Gram-negative species. This
difference in suscepti-bility between species in the two bacterial
classifica-tions was explained by their physiology, as
theGram-positive species have a cytoplasmic membrane
Current therapeutic options for endodontic biofilms
89
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surrounded by a relatively porous cell wall composedof
peptidoglycan and lipoteichoic acid that allows PSmolecules to
cross. The cell envelope of Gram-negative species is composed of an
outer membrane, athin peptidoglycan layer, and a cytoplasmic
membraneas the innermost cell wall structure. The movementof
molecules across the Gram-negative cell wall isstrictly regulated
at the outer membrane that is richin lipopolysaccharides (LPS)
(116,117). Negativelycharged LPS molecules have a strong affinity
forcations such as calcium (Ca2+) and magnesium (Mg2+),the binding
of which is required for the thermody-namic stability of the outer
membrane. Antimicrobialphotosensitizers such as porphyrins,
phthalocyanines,and phenothiazines (e.g. Toluidine blue O and
Meth-ylene blue), which bear a positive charge, can directlytarget
both Gram-negative and Gram-positive bacteria(118,119). Toluidine
blue O and Methylene blue arecommonly used for oral antimicrobial
photodynamictherapy. The functioning of self-promoted
up-takepathways and protein transporters is modulated bycharged
entities such as cations. Therefore, the successof PAD in
eliminating bacteria from anatomical sitessuch as root canals could
be influenced by the cation-rich microenvironment persisting at
these sites.PAD conducted on endodontic biofilms has often
failed to achieve effective microbial killing, promptingmany
researchers to combine PAD with conventionalantimicrobial
strategies for superior performance(112,120123). Methylene blue has
been used as thephotosensitizer for targeting endodontic
micro-organisms in several studies (122,124126). The pho-todynamic
effects of Methylene blue were investigatedon multi-species root
canal biofilms comprised of fourspecies of microorganisms in
experimentally infectedroot canals of extracted human teeth (122).
PADachieved a reduction in bacterial viability of up to 80%.The
results of this study suggested the potential ofPAD to be used as
an adjunctive antimicrobial proce-dure after standard endodontic
chemomechanicaldebridement, but also demonstrated the importanceof
further optimization of light dosimetry for
bacterialphotodestruction in root canals. Also, modified
PSformulations with improved photochemical and pho-tobiological
properties have shown that the nature ofthe PS solvent used for PAD
influences its bactericidalpotential (123,126,127). Methylene blue
dissolved ina mixture of glycerol, ethanol, and water (123,125),
aswell as a Methylene blue formulation containing an
emulsion of oxidizer and oxygen carrier (125),enhanced the
photodynamic effects of Methylene bluein vitro. Findings from a
recent study showed theefficacy of photodynamic therapy mediated
byMethylene blue dissolved in a mixture of glycerol,ethanol, and
water in the presence of an irradiationmedium
(perfluorodecahydro-napthalene) to eradicateE. faecalis biofilms in
the root canal system of experi-mentally infected human teeth
(128). The use ofMethylene blue mediated PAD with modified
PSformulations was found to enhance the efficacy ofPAD in
destroying Gram-positive E. faecalis biofilmand Gram-negative
Pseudomonas aeruginosa biofilm(115).PAD as an adjunctive technique
to standard endo-
dontic treatment may have potential in the clinicalsetting by
providing a large therapeutic windowwhereby residual root canal
bacteria can be killedwithout harming cells in the periapical
region. Futureexperimental studies should explore the use of
noveltechnologies for increased delivery of Methylene blueor
Toluidine blue O in dentinal tubules and the appli-cation of
supplemental hyperoxygenation in the rootcanal system to enhance
the photodynamic therapyeffect.
Local intracanal medicamentsIn the treatment of teeth with a
vital pulp, there is noneed for intracanal antibacterial
medication. However,in the treatment of apical periodontitis,
intracanalmedication has been recommended by many in orderto
eradicate the microbes that survive instrumentationand irrigation.
A variety of medicaments have beenused for this purpose. These
include calcium hydrox-ide [Ca(OH)2]; phenol compounds such as
eugenol,camphorated parachlorophenol (CMCP), and formo-cresol;
iodine potassium iodide (IPI); glutaraldehyde;and pastes containing
a mixture of antibiotics with orwithout corticosteroids.As an
inter-appointment dressing, a substance
should be selected that is not easily replaced by tissuefluid
and that can remain physically intact over weeksor months. A water
slurry of Ca(OH)2 combinesseveral attractive features (129,130) of
a good intra-canal dressing. It is strongly alkaline (pH 12.5)
anddissociates into calcium and hydroxide ions in aqueoussolution;
the latter provide antimicrobial effects (131)and tissue-dissolving
capacity (132). With its fairly low
Haapasalo & Shen
90
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solubility and mere physical presence, it may be used asan
intracanal dressing over long periods of time. Itsmost essential
function is then to obstruct bacterialregrowth. The antimicrobial
activity of calciumhydroxide seems dependent upon direct contact
withbacteria (130). Direct contact experiments in vitro(59) showed
that Ca(OH)2 was 100% effective ineliminating 2-day-old E. faecalis
biofilm in a mem-brane filter model. This could be attributable to
thefact that the pH of Ca(OH)2 remained high in
themembrane.However, owing to its poor solubility and
diffusibil-
ity, Ca(OH)2 is a rather inefficient antimicrobialagainst
microorganisms lodged in pulpal remnants,crevices of the canal, and
dentinal tubules (83,133). Aswell, the buffering ability of tissues
impacts pH levelchanges. Using scanning electron microscopy
andscanning confocal laser microscopy, Distel et al. (134)reported
that despite intracanal dressing withCa(OH)2, E. faecalis formed
biofilms in root canals.Both enterococci and yeasts sustain a high
alkalineenvironment and are able to survive in root canalsmedicated
with Ca(OH)2 (135,136). These results areamongst the reasons why
controversy has emergedover its usefulness as an antimicrobial
agent in rootcanal treatment. Although several clinical trials
haveobserved that root canals are rendered free of culti-vable
bacteria following its application for a week ormore (131,137),
others have found that micro-organisms can still be recovered from
a substantialnumber of medicated root canals (138140). Differ-ences
in findings may relate to the type of teethincluded in the studies
and the associated effectivenessof the biomechanical preparation,
sampling technique,and the extent to which Ca(OH)2 was eliminated
fromthe root canals prior to the sampling procedure.The clearly
poorer results in vivo in the root canal
indicate the presence of interfering factors that nega-tively
affect the outcome of the disinfection. Haapasaloet al. (63) and
Portenier et al. (20,21) studied theeffect of dentin and other
substances present in theroot canal milieu on the antibacterial
effect of com-monly used intracanal medicaments such as
calciumhydroxide, chlorhexidine, and IPI against E. faecalis.These
studies showed that all three disinfectants werenegatively affected
by the various substances tested,calcium hydroxide being
particularly sensitive to theinhibitory effect of a variety of
substances present inthe root canal.
Adherence to dentin and inter-species interactionsin a biofilm
appear to differentially affect the sensitiv-ity of microbial
species to calcium hydroxide. Brandleet al. (57) investigated the
effects of growth condi-tions (planktonic, mono- and multi-species
biofilms)on the susceptibility of E. faecalis, Streptococcus
sobri-nus, Candida albicans, Actinomyces naeslundii,
andFusobacterium nucleatum to alkaline stress. Findingsshowed that
planktonic microorganisms were mostsusceptible; only E. faecalis
and C. albicans survivedin saturated solution for 10 min (the
latter also for100 min). Dentin adhesion was the major factor
inimproving the resistance of E. faecalis and A. naeslun-dii to
calcium hydroxide, whereas the resistance todisinfecting agents by
S. sobrinus was dependent on amulti-species biofilm. In contrast,
the C. albicansresponse to calcium hydroxide was not influenced
bygrowth conditions. Tolerance to alkali, and possiblyother agents,
is likely to be connected to the expres-sion of phenotypes
resistant to these agents withinthe biofilm communities. There is
very little dataavailable about the effect of calcium hydroxide
onbiofilm bacteria. Chavez et al. (141) reported thatbacteria
isolated from infected root canals resistedalkaline stress better
in biofilms than in planktoniccultures.Endodontic infections are
polymicrobial and no
medicament is effective against all of the bacteriafound in
infected root canals. The combination oftwo medicaments may produce
additive or synergisticeffects. Evidence suggests that the
association ofcalcium hydroxide with CMCP has a broader
antibac-terial spectrum, a higher radius of antibacterial
action,and kills bacteria faster than mixtures of calciumhydroxide
with inert vehicles (142). Therefore,CMCP cannot be considered a
vehicle for calciumhydroxide, it is an additional medicament. While
invivo studies have indicated calcium hydroxide to be themost
effective all-purpose intracanal medicament, IPIand CHX may be able
to kill calcium hydroxide re-sistant bacteria. Supplementing the
antibacterial activ-ity of Ca(OH)2 with IPI or CHX preparations
wasstudied in bovine dentin blocks (143). While Ca(OH)2was unable
to kill E. faecalis in the dentin, Ca(OH)2combined with IPI or CHX
effectively disinfected thedentin. The addition of CHX or IPI did
not affect thealkalinity of the calcium hydroxide suspensions. It
maybe assumed that combinations also have the potentialto be used
as long-term medication.
Current therapeutic options for endodontic biofilms
91
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Can sealers and cements killbiofilm bacteria?
An ideal endodontic sealer should be biocompatibleand
dimensionally stable; it should seal well and have astrong,
long-lasting antimicrobial effect (144146).The antibacterial
activity of sealers may help to elimi-nate residual microorganisms
that have survivedchemomechanical preparation and thereby
improvethe success rate of endodontic treatment. It is expectedthat
the antibacterial activity of the root canal sealer, inits unset
stage (147), kills the organisms or theybecome deprived of
nutritional supply and space forregrowth if pathways to and from
the periapical tissueare effectively blocked.It should be
recognized that sealers with high anti-
microbial activity, especially formaldehyde-releasingZnOE (zinc
oxideeugenol) sealers such as N2, arealso toxic to cells and
tissues. Furthermore, sealers thatrelease antimicrobial substances
may also disintegrateto some extent during this stage. Most sealers
are onlyantimicrobially active during the setting period (147).For
a short time of a few hours or a few days, residualbacteria may be
killed. However, this may be enoughto control the residual
infection.One of the challenges in endodontic research has
been the lack of standardized in vitro and in vivoprotocols for
testing the antimicrobial effect of sealers.The agar diffusion test
(ADT) used to be the mostcommonly applied method to assess the
antimicrobialactivity of endodontic sealers (148150). However,the
limitations of this method are now well recog-nized. The results
obtained are not likely to reflect thetrue antimicrobial potential
of the various sealers ordisinfecting agents; therefore, ADT is no
longer rec-ommended to be used for this purpose in
endodonticresearch (151). A direct contact test (DCT),
whichcircumvents many of the problems of ADT, was firstintroduced
by Weiss et al. (152) for the evaluation ofthe antimicrobial effect
of endodontic sealers and root-end filling materials. The test is a
quantitative andreproducible assay that allows for the testing
ofinsoluble materials and can be used in standardizedsettings. One
study (147) used a modified DCT assayto evaluate the antibacterial
activity of seven differentendodontic sealers against E. faecalis
20 minutes aftermixing (fresh samples) and 1, 3, and 7 days
aftermixing (set samples). The findings showed that freshiRoot SP,
AH Plus, Sealapex, and EndoRez killed
E. faecalis effectively. iRoot SP, Sealapex, andEndoRez
continued to be effective for 3 days aftermixing. Sealapex and
EndoRez were the only oneswith continuing antimicrobial activity
even at 7 daysafter mixing. However, the direct contact test is
notdirectly applicable to studying the effect of the sealeron
biofilm bacteria and thus further development ofthe experimental
model is warranted.Antibacterial nanoparticulates are found to
have
higher antibacterial activity than antibacterial powders.This is
due to the greater surface area and chargedensity of
nanoparticulates, which enable them toachieve a higher degree of
interaction with the nega-tively charged surface of bacterial
cells. Chitosan (CS)is a non-toxic biopolymer derived from the
deacetyla-tion of chitin. It is a bioadhesive that readily bindsto
negatively charged surfaces and has excellentantimicrobial and
antifungal activities. Recently,Kishen et al. (153) examined the
ability of differentnanoparticulate-treated dentin to prevent the
adher-ence of E. faecalis. Results showed that the incorpora-tion
of nanoparticulates did not alter the flowcharacteristics of the
ZnOE sealer but improved thedirect antibacterial property and the
ability to leachout antibacterial components.
Eradication of root surface andother extraradicular
biofilmsBacteria (or yeasts) that have succeeded in establishinga
colony/biofilm on the root surface or in the periapi-cal tissue are
beyond the direct reach of conservative(non-surgical) treatment
methods. In addition to tra-ditional microbial micro- and
macro-colonies, a specialvariant of root surface biofilm is
calcified root surfacebiofilm. In vitro, the precipitation of
minerals inE. faecalis biofilm on dentin has been described
(154),and there are some reports where calculus-like depos-its on
the apical external root apex were responsible forroot canal
treatment failure (155).In a recent study, Ricucci & Siqueira
(16) evaluated
the prevalence of bacterial biofilms in untreated andtreated
root canals of teeth with apical periodontitis.Extraradicular
bacterial biofilms were observed in sixout of 100 specimens (6%),
four from teeth withuntreated canals and two from teeth with
treatedcanals. All of the cases showing an extraradicularbiofilm
exhibited clinical symptoms, and three of themwere associated with
sinus tracts. These findings indi-
Haapasalo & Shen
92
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cate that extraradicular infections in the form of bio-films or
planktonic bacteria are not common.Periapical biofilm colonies,
similar to biofilms in
general, are assumed to be resistant to systemic anti-biotic
treatment (156,157). Irrespective of thepathway of infection, when
actinomycosis-like coloniesin the tissue or root surface biofilm
have developed,surgical treatment including apicoectomy and
removalof the infected hard and soft tissues has been shown tobe
effective with an excellent long-term prognosis(158160).
ConclusionsThe complex anatomy of teeth and root canals
createsan environment that is a challenge to instrument andclean.
In addition, the complex chemical environmentof the root canal
prevents antimicrobial irrigating solu-tions and medicaments from
exerting their full poten-tial against the microorganisms found in
endodonticinfections. While our knowledge of persistent
infec-tions, disinfecting agents, and the chemical milieu ofthe
necrotic root canal has greatly increased, there is nodoubt that
more innovative basic and clinical researchis needed to improve and
optimize the use of existingmethods and materials, and to find new
techniques andmaterials (or combinations of materials) in order
toachieve the goal of predictable, complete disinfectionof the root
canal system in apical periodontitis.
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