ENDODONTICS

MTADA Review of a Promising Endodontic Irrigant

Zahed Mohammadi, D.M.D., M.S.D.

ABSTRACTThe essential role of microorganisms in the initiationand perpetuation of pulp and periapical pathosis hasbeen well documented. The major objective in endo-dontic therapy is to disinfect the entire root canal sys-tem. Considering the complex anatomy of the rootcanal system, complete elimination of bacteria bycleaning the root canal with instrumentation aloneis unlikely. Therefore, an appropriate antimicrobialirrigant is needed to decrease the microbial load, es-pecially in necrotic and retreatment cases. Anotherimportant issue in endodontics is the smear layer. Al-though there is no general consensus on removal ofthe smear layer, it seems that removing it enhancesthe sealing ability of the root canal filling. MTAD isan endodontic irrigant with both antibacterial andsmear layer removal abilities. The aim of this paper isto review these different aspects of MTAD.

Microorganisms and their byproducts play an essential role in thedevelopment and perpetuation of pulp and periapical diseases.̂ "^The elimination of microorganisms from infected root canal sys-

tems is a complicated task. Numerous measures, including the useof various instrumentation techniques, irrigation regimens andintra-canal medicaments, have been described to reduce the mi-crobial load inside the root canal system. There is no solid evidencethat demonstrates that mechanical instrumentation alone resultsin a bacteria-free root canal system. When the complex anatomy ofthe root canal system* is considered, this is not surprising.

In vitro and clinical evidence have shown that mechani-cal instrumentation leaves significant portions of the root canalwalls untouched,' and complete elimination of bacteria by in-strumentation alone is unlikely to occur.* It is assumed, but notdemonstrated, that any pulp tissue left in the root canals canserve as nutrient for any remaining bacteria. Furthermore, tis-sue remnants also impede the antimicrobial effects of root canalirrigants and medicaments. Therefore, some form of irrigationand disinfection is necessary to remove residual tissue and to killmicroorganisms.

Another issue of great importance during root canal treat-ment is the smear layer. The effect of the smear layer on the mi-croleakage of obturated root canals is still controversial. Someauthors suggest that keeping the smear layer may block the den-tinal tubules and limit bacterial or toxin penetration by alteringthe dentinal permeability.''"' On the other hand, some believethat the smear layer must be completely removed from the sur-face of the canal wall because it can harbor bacteria and can bedetrimental to the effective disinfection of dentinal tubules by

The New York state Dental Journal • AUGUST/SHPTEIVIBER 2O12 4 7

preventing irrigant and medicaments from penetrating into thedentinal tubules. It can also act as a barrier between obturatingmaterials and the canal wall and, thus, interfere with the forma-tion of an appropriate seal.''°"''̂

A systematic review of in vitro studies showed that smearlayer removal improves the fiuid-tight seal of the root canal sys-tem." Prior to the introduction of MTAD, there was no irrig-ant capable of removing the smear layer and disinfecting the rootcanal system simultaneously. MTAD consisted of a mixture of atetracycline isomer (doxycycline), an acid (citric acid) and a de-tergent (Tween-80).''''

Rationale for Local Application of AntimicrobialsWhile systemic antibiotics appear to be clinically effective as anadjunct in certain surgical and nonsurgical endodontic cases,their administration is not without the potential risk of adversesystemic effects, particularly possibilities of allergic reactions, tox-icity, side effects and development of resistant strains of microbes.Systemic administration of antibiotics relies upon circulation tobring the active drug to an infected site thatmay no longer possess a normal vascula-ture, including the necrotic pulpless toothand the periradicular tissues; therefore, thelocal application of antibiotics may be amore effective mode of delivery."

as effective as citric acid in removing the smear layer.̂ ^ Anotherstudy showed that teeth with retrograde IRM or amalgam fillingsplaced following doxycycline irrigation of the retro-cavities hadsignificantly less dye leakage.^"

Using an agar diffusion test, Carson et al.̂ * evaluated theantimicrobial activities of 6% and 3% sodium hypochlorite(NaOCl), 2% and 0.12% chlorhexidine gluconate (CHX), and0.01% and 0.005% doxycycline (Doxy) on Peptostreptococcus mi-cros, Prevotella intermedia, Streptococcus sanguis and Lactobadllus

acidophilus. For P. micros, P. intermedia, S. sanguis, both concen-

trations of doxycycline were more effective than other agents.For L. acidophilus, the effectiveness of doxycycline was less thanother materials. Pinheiro et al." found that 85.7% of Enterococcusfaecalis isolates from canals of root-filled teeth with periapical le-sions was susceptible to tetracycline and doxycycline. Chai et al.̂ *showed that oxytetracycline was 100% effective in eliminating £.faecalis biofilm.

TetracyclinesTetracyclines (including tetracycline-HCl,minocycline, demeclocycline and doxycy-cline) are a group of broad-spectrum antibi-otics that are effective against a wide range ofmicro-organisms.'''' They are bacteriostatic innature.''"' This property may be advantageousbecause, in the absence of bacterial cell ly-sis, antigenic byproducts, such as endotoxin,are not released.''* Tetracyclines have manyother unique properties, such as the inhibition of mammalian col-lagenases, which prevents tissue breakdown,'"^'''' and the inhibitionof clastic cells'^"'" that results in anti-resorptive activity.^' Infiam-matory diseases such as periodontitis include a pathological excessof tissue collagenases that may be blocked by tetracyclines, leadingto enhanced formation of collagen and bone formation.^"

Tetracyclines have been used to remove the smear layer frominstrumented root canal walls,̂ '̂̂ ^ for irrigation of retrogradecavities during periapical surgical procedures^" and as an intra-canal medicament.^^ Barkhordar et al. '̂' showed that doxycyclineHCl eliminated the smear layer in a concentration-dependentmanner from the instrumented root canal walls. Results showedthat 100 mg/ml doxycycline HCl was more effective than lowerconcentrations in removing the smear layer. A scanning electronmicroscopy (SEM) study demonstrated that tetracycline HCl was

Another issue of great

importance during root

canal treatment is the

smear layer. The effect

of the smear layer on

the microleakage of

obturated root canals

is still controversial.

Substantivity of TetracyclinesTetracyclines readily attach to dentin andare subsequently released without losingtheir antibacterial activity.̂ "* This propertycreates a reservoir of active antibacterialagent, which is then released from the den-tin surface in a slow and sustained manner.In periodontics, several studies have beenconducted on the antibacterial substan-tivity of tetracyclines. A periodontal studyshowed that both concentrations of tetra-cycline demonstrated residual antibacterialactivity and the antibacterial substantivityof the three solutions in descending orderwas: 50 mg/ml tetracycline > 10 mg/mltetracycline > 0.12% CHX.-̂ In the field ofendodontics, Abbott et al.'* demonstratedthat tetracyclines form a strong reversible

bond with hard tissues and that they exhibit slov/ release over anextended period of time. Other studies have sho'OTi the substan-tivity of doxycycline for up to four weeks.^'''°

BioPure MTADAntibacterial activityBioPure (Dentspfy, Tulsa Dental, Usa, OK), otherwise known as MTAD,

is a root canal irrigant introduced by Torabinejad et al. in 2003.^*The solution is a mixture of 3% doxycycline, 4.25% citric acid anda detergent (0.5% Polysorbate 80)."

Several studies have evaluated the effectiveness of MTADfor disinfection of root canals. Torabinejad et al. have shown thatMTAD is able to remove the smear layer '̂' and is effective against£. faecalis.^^-^'^ Shabahang et al.̂ * demonstrated that the use ofMTAD was more effective than 5.25% NaOCl in disinfecting root

4 8 AUGUST/SEPTEZVIBER 2O12 • The New York State Dental Journal

canals. However, Tay et al.' ' found that when MTAD is appliedto 1.3% NaOCl-irrigated dentin, its antimicrobial substantivityis reduced. They attributed this phenomenon to the oxidation ofMTAD by NaOCl in a manner similar to the peroxidation of tet-racycline by reactive oxygen species.

Torabinejad et al.'^ compared the antibacterial effects ofMTAD with those of NaOCl and EDTA by using standard in vitromicrobiological techniques and reported that MTAD was signifi-cantly more effective against E. faecalis. Kho and Baumgartner^*compared the antimicrobial efficacy against £ faecalis of 1.3%NaOCl/BioPure MTAD with that of the combined alternate useof 5.25% NaOCl and 15% EDTA for root canal irrigation. Theirfindings showed consistent disinfection of infected root canalswhen a combination of 5.25% NaOCl/15% EDTA was used.However, the combination of 1.3% NaOCl/BioPure MTAD leftnearly 50% of the canals contaminated with E. faecalis. In an-other study, the antimicrobial effect against E. faecalis of MTAD,two of its components (doxycycline and citric acid) and sodiumhypochlorite were assessed in two in vitro models using two dif-ferent methods. In the tooth model, NaOCl and doxycycline weremore effective than the control in killing E. faecalis at shallowbur depths into dentin; but at deeper bur depths, the NaOCl wassuperior. In the agar diffusion model, NaOCl produced less inhi-bition of bacteria than MTAD or doxycycline.'^

Ghoddusi et al.'* evaluated the effect of MTAD as a final irri-gant on bacterial leakage of the root canal and its interaction withtwo conventional root canal sealers (AH-Plus or Rickert). Accord-ing to their findings, it took longer for bacteria to penetrate wheneither EDTA or MTAD was used for smear layer removal. Fur-thermore, the root canals obturated with AH-Plus showed signifi-cantly longer duration of resistance to bacterial penetration thancanals obturated with Rickert.

Using the zone of inhibition test and Enterococcus faecalis asa test microorganism, Davis et al." demonstrated that BioPureMTAD showed significantly more zones of microbial inhibitionthan 5.25% NaOCl, 2% CHX, and Dermacyn (Oculus Innovative Sci-ences, Petaluma, CA). Newberry et al.''° determined in vitro the anti-microbial effect of MTAD as a final irrigant on eight strains ofE. faecalis and measured the minimum inhibitory concentration(MIC) and the minimum lethal concentration (MLC) of MTAD.After irrigating with 1.3% NaOCl, the root canal and the exter-nal surfaces were exposed to MTAD for five minutes. The resultsshowed that this treatment regimen was effective in completelyeliminating growth in seven of eight strains of E. faecalis. TheMIC/MLC tests showed that MTAD inhibited most strains of E.faecalis growth when diluted 1:8192 times and killed most strainsof E. faecalis when diluted 1:512 times.

Shabahang et al."*̂ evaluated the effect of the addition orsubstitution of chlorhexidine for doxycycline and compared thesethree formulations in their ability to disinfect extracted humanteeth infected with E. faecalis. The results showed that although

the addition of chlorhexidine did not negatively impact the ef-ficacy of MTAD, the substitution of this antimicrobial agent fordoxycycline significantly reduced the efficacy of the solution.

Substantivity of MTADAs stated earlier, tetracyclines (including doxycycline) readily at-tach to dentin and are subsequently released without losing theirantibacterial activity.̂ ^ The presence of doxycycline in MTAD sug-gests that MTAD may have some substantive antimicrobial ac-tion." In an in vitro study, Mohammadi and Shahriari*^ showedthat the substantivity of MTAD was significantly greater than thatof CHX and NaOCl. Another study demonstrated that the sub-stantivity of 100% MTAD was significantly greater than the twoother concentrations (10% and 1%) of MTAD.*'

MTAD and BiofilmsThe term biofilm was introduced to designate the thin-layeredcondensation of microbes that may occur on various surfacestructures in nature. Free-floating bacteria existing in an aqueousenvironment, or planktonic microorganisms, are a prerequisitefor biofilm formation.'* Such films may, thus, become establishedon any organic or inorganic surface substrate where planktonicmicroorganisms prevail in a water-based solution.

In dental contexts, a well-known and extensively studied bio-film structure is established during the attachment of bacteria toteeth to form dental plaque. Here, bacteria free in saliva (planktonicorganisms) serve as the primary source for the organization of thisspecific biofilm.''* However, in endodontics, the biofilm concept hasthus far gained limited attention. It has been discussed mainly with-in the framework of bacterial appearances on root tips of teeth withnon-vital pulps.*' Such bacterial aggregations have been thought tobe the cause of therapy-resistant apical periodontitis.**'''' Bacterialcondensation (that is, biofilms) on the walls of infected root canalshave been observed, but have not been described in great detail.**

Anti-microbial agents have often been developed and opti-mized for their activity against fast-growing, dispersed popula-tions containing a single microorganism.*' However, microbialcommunities grown in biofilms are remarkably difficult to eradi-cate with anti-microbial agents, and microorganisms in maturebiofilms can be notoriously resistant for reasons that have yet tobe adequately explained.*' There are reports showing that micro-organisms grown in biofilms could be two- to 1000-fold moreresistant than the corresponding planktonic form.*' Clegg et al.'°evaluated the effectiveness of three concentrations of NaOCl(6%, 3% and 1%), 2% CHX and BioPure MTAD on apical dentinbiofilms in vitro. Their findings indicated that 6% NaOCl was theonly irrigant capable of both rendering bacteria nonviable andphysically removing the biofilm.

Dunavant et al.'^ evaluated the efficacy of 6% NaOCl, 1%NaOCl, Smear Clear, 2% CHX, REDTA and BioPure MTADagainst E. faecalis biofilms using a novel in vitro testing sys-

The New York State Dental Journal • AUGUST/SEPTEMBER 2O12 4 9

tem. Biofilms grown in a flow cell system were submerged intest irrigants for either one or five minutes. There was a sig-nificant relationship between the test agent and the percent-age kill of the biofilm bacteria. No significant relationshipbetween time and percentage kill was found. The percentagekill of the bioñlm bacteria was: 6% NaOCl (>99.99%), 1%NaOCl (99.78%), Smear Clear (78.06%), 2% CHX (60.49%),REDTA (26.99%) and BioPure MTAD (16.08%). There was asignificant difference between 1% and 6% NaOCl and all otheragents, including Smear Clear, 2% CHX, REDTA and BioPureMTAD. Therefore, both 1% NaOCl and 6% NaOCl were moreefficient in eliminating £. faecalis biofilm than the other solu-tions tested.

Giardino et al." evaluated the efficacy of 5.25% NaOCland MTAD against E. faecalis biofilm and found that only 5.25%NaOCl was able to disgrégate and remove the biofilm at everytested time period.

On the whole, it seems that NaOCl is the only endodonticirrigant that can disrupt and remove microbial bioñlm from theinfected root canals.

Smear Layer RemovalMany instrumentation techniques have been proposed to shaperoot canals to facilitate their complete obturation. Less attention

has been directed toward the ability of these techniques to com-pletely clean and disinfect the root canal system. Studies showthat currently used methods of instrumentation, especially rotaryinstrumentation techniques, produce a smear layer that coversroot canal walls and the openings to the dentinal tubules."'^*The smear layer consists of organic and inorganic substances,including fragments of odontoblastic processes, microorganismsand necrotic materials. The presence of this smear layer preventspenetration of intracanal medication into the irregularities ofthe root canal system and the dentinal tubules and also preventscomplete adaptation of obturation materials to the prepared rootcanal surfaces."

Various organic acids, ultrasonic instruments and lasershave been used to remove the smear layer. Based upon availableevidence, it seems that these agents and methods do not providecomplete disinfection of the root canal spaces in all cases whenused in one-visit root canal therapy. Because of the ineffectivenessof these techniques, many practitioners rely upon the placementof Ca(OH)2 in the root canals to assist in canal disinfection.^'-"As a result, root canal therapy has to be performed in more thanone appointment.

For the first time, Torabinejad et al.'* showed that MTADwas an effective solution for the removal of the smear layer anddid not significantly change the structure of the dentinal tubules

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5O AUGUST/SEPTEIVIBER 2O12 • The New York State Dental Journal

when canals were irrigated with sodium h5^ochlorite and under-went a final rinse of MTAD. In another study, they showed thatalthough MTAD removed most of the smear layer when used asan intracanal irrigant, some remnants of the organic componentof the smear layer remained scattered on the surface of the rootcanal walls.'^

The effectiveness of MTAD to completely remove the smearlayer was enhanced when low concentrations of NaOCl wereused as an intracanal irrigant before the use of MTAD as a fi-nal rinse. This regimen did not seem to significantly change thestructure of the dentinal tubules.'* Tay et al." revealed that bothirrigants created a zone of demineralized collagen matrices ineroded dentin and around the dentinal tubules, with the mildlyacidic BioPure MTAD being more aggressive than EDTA. Thesedemineralized dentin zones create the opportunity for dentinhybridization by infiltration of hydrophilicadhesives/sealers. However, the potentialconsequences of compaction of hydropho-bic sealers against air-dried, collapsed col-lagen matrices and hydrolytic degradationof incompletely infiltrated matrices remainunresolved. In an ultrastructural study, Tayet al.'° showed that BioPure MTAD cre-ated a thicker demineralized dentin ma-trix (5-6 micrometers) than EDTA (1-2micrometers). De-Deus et al.*' found thatthe demineralization kinetics promptedby BioPure MTAD were significantly fasterthan those prompted by a 17% solution ofEDTA.

Bone apatite has

long been known to

be a major carbonate

reservoir, providing

buffering for all acid-

base disturbances and

maintaining the body's

acid-base balance.Effects of MTAD on DentinMachnick et al." evaluated the effect of MTAD on the flexuralstrength and modulus of elasticity of dentin. Dentin bars wererandomly assigned to eight groups treated with various concen-trations of NaOCl (5.25%, 2.65%, 1.31% and 0.66%), a 17% so-lution of EDTA, MTAD, saline, or, according to a clinical protocol,with 1.3% NaOCl and then five minutes of MTAD. Specimenswere subjected to ñexur.al strength and modulus of elasticity tests.Findings showed no statistically significant differences betweenvarious groups except for a reduction in fiexural strength for the2-h MTAD group and the EDTA group. A significant reduction ofmodulus of elasticity for the 2-h MTAD group, EDTA group and0.6% NaOCl also was noted. There was no significant differencein flexural strength and modulus of elasticity between the dentinbars exposed to saline or MTAD when applied according to theclinical protocol.

In summary, it seems that MTAD is suitable for clinicaluse as prescribed without affecting the physical properties ofthe dentin.

Buffering Effect of Dentin on MTADBone apatite has long been known to be a major carbonate res-ervoir, providing buffering for all acid-base disturbances andmaintaining the body's acid-base balance.'^ With a quite similarchemical composition, dentin can be expected to have a corre-sponding buffering effect on acids and bases.

Wang and Hume^* showed that dentin was a strong bufferagainst acids. Buffering against alkali (NaOH) was weaker but,nevertheless, considerable. Dentin chips weighing 250 mg wereable to keep the pH unchanged after the addition of 3 mmol of HClor 2 mmol of NaOH. Inorganic apatites are supposed to be mainlyresponsible for the buffering effect of dentin. However, the factthat whole dentin is a more effective buffer than hydroxyapatitesuggests that other inorganic and even organic components alsocontribute to the buffering. Camps and Pashley*' found that or-

ganic components of dentin alone accountedfor 1.5% of the total buffering capacity.

The root canal milieu is a complex mix-ture of a variety of organic and inorganiccompounds. Hydroxyapatite, the main com-ponent of dentin, is the major representa-tive of inorganic components present. Inaddition, inflammatory exúdate, enteringthe apical root canal in purulent infections,is rich in proteins such as albumin.**

The relative importance of the vari-ous organic and inorganic compounds inthe inactivation of root canal disinfectantshas been studied to a limited extent.** Dif-ficulties in designing experiments that willgive reliable and comparable data were oneof the great challenges for researchers for

many years. Ultimately, Haapasalo et al.*^ introduced a new den-tin powder model for studying the inhibitory effect of dentin onvarious root canal irrigants and medicaments. Portenier et al.**investigated the inhibitory effects of dentin and bovine serum al-bumin (BSA) on the antibacterial activity of chlorhexidine andMTAD and found that the presence of dentin or BSA caused amarked delay in the killing of bacteria by both medicaments.

MTAD and Dentin Bonding (Anticollagenolytic Activity)During the last two decades, chemical and technical advances havecontributed to increases in resin-dentin bond strength. However,the premature loss of bond strength is one of the problems thatstill affects adhesive restorations*' and markedly reduces their du-rability.'""''^ The loss of bond strength has been attributed mainlyto the degradation of the hybrid layer at the dentin-adhesive in-terface. Numerous publications have demonstrated this lack ofbond stability."-'*

The notion that deterioration of dentin collagen fibrils con-tributes to the mechanism responsible for bond degradation has

The New York state Dental Journal • AUGUST/SEPTEIVIBER 2O12

been reported.'''^' In this context, it has been speculated that adecreasing concentration gradient of resin monomer diffusionwithin the acid-etched dentin, and a subsequent resin elutionfrom hydrolj^ically unstable pol3mieric hydrogels within the hy-brid layers^^ leave the collagen fibrils unprotected and vulnerableto degradation by endogenous metalloproteinases (MMPs). TheMMPs are a group of 23 mammalian enzymes capable of degrad-ing all extracellular matrix components. Human dentin containsat least collagenase (MMP-8), gelatinases MMP-2 and -9, andenamelysin MMP-20.'''"*^ Dentin collagenolytic and gelatinolyticactivities'^ can be suppressed by protease inhibitors,''* indicatingthat MMP inhibition could be beneficial in the preservation ofhybrid layers.

Tetracyclines have also been shown to inhibit mammaliancollagenases. Inflammatory diseases such as periodontitis includea pathological excess of tissue collagenases that may be blockedby tetracyclines, leading to enhanced formation of collagen andbone formation. Doxycycline, a hydroxy derivative of tetracycline,is the most potent anticollagenase antibiotic among commerciallyavailable tetracyclines^" and is also relatively more potent againstmost periodontal pathogens.^°"^^

Machnick et al." compared the effect of MTAD and phos-phoric acid on the bond strength to enamel and dentin using aconventional OptiBond Solo Plus dentin adhesive system and re-ported that teeth endodontically treated vnth the MTAD protocolfor clinical use (20 min 1.3% NaOCl/5 min MTAD) might notneed any additional dentin conditioning before the application ofthe dental adhesive. Garcia-Godoy et al.̂ '* evaluated the structureof the hybrid layer formed after the use of EDTA or MTAD solu-tions when used as a final fiush. Findings showed that the BioPureMTAD hybrid layer was thicker than the 17% EDTA hybrid layer.Both the BioPure MTAD and EDTA caused a collapse of the den-tin matrix structure, which impeded sealer infiltration and theformation of high-quality hybrid layer bonding. The hybrid layerscreated in smear layer-covered dentin exhibited less potential fornanoleakage than the MTAD or EDTA hybrid layers. Wachlaro-wicz et al.*' reported that neither EDTA nor MTAD significantlyimproved Epiphany-dentin bond strengths when compared withNaOCl used alone.

On the whole, due to its broad-spectrum MMP-inhibitoryeffect, MTAD can significantly improve the resin-dentin bondstability.

Toxicity of MTADThere is only one study regarding the toxicity of MTAD. Zhang etal.*' examined the cytotoxicity of MTAD compared with that ofcommonly used irrigants and medications. L929 fibroblasts weregrown on cell culture plates and were placed in contact with vari-ous concentrations of test irrigants and medications. The cytotox-icity of these materials was evaluated 24 hours after incubationusing MTT assay. Results showed that MTAD was less cytotoxic

than eugenol, 3% H2O2, Ca(OH)2 paste, 5.25% NaOCl, Peridexand EDTA and was more cytotoxic than 2.63%, 1.31% and 0.66%NaOCl. //.

Queries about this article can be sent to Dr. lÁohavnmadi at zahed.moham-madi®smail.corr\.

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