current therapeutic options for endodontic biofilms

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

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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.

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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

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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.

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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.


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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.

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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.


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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.

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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.


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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.

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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


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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

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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.


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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-

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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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