Basic Research—Technology

Residual Activity of Chelating Agents and their Combinationswith Cetrimide on Root Canals Infected with EnterococcusfaecalisCarmen Mar�ıa Ferrer-Luque, DDS, MD, PhD,* Amanda Conde-Ortiz, BDS,*Mar�ıa Teresa Arias-Moliz, DDS, PhD,† Mariano Jos�e Valderrama, PhD,‡

and Pilar Baca, DDS, MD, PhD§

Abstract

Introduction: The use of chelating agents inendodontic treatment is required to remove the smearlayer and to achieve better disinfection within the rootcanal system. The aim of this study was to evaluatethe residual antimicrobial activity of 17% ethylenedia-minetetraacetic acid (EDTA), 7% maleic acid (MA),and 10% lactic acid (LA) alone and combined with0.2% cetrimide (CTR) on infected root canals withEnterococcus faecalis. Methods: Biofilms of E.faecalis were grown in uniradicular roots for 4 weeks.After preparation the roots were randomly divided into 6groups (n = 15) according to the final irrigation regimenas follows: 5 mL of 17% EDTA; 5 mL of 17% EDTA +0.2% CTR; 5 mL of 7% MA; 5 mL of 7% MA + 0.2%CTR; 5 mL of 10% LA; and 5 mL of 10% LA + 0.2%CTR. Samples were collected for 60 days to denotethe presence of bacterial growth. Fisher and Pearsonc2 tests were applied to compare the percentages ofspecimens without E. faecalis regrowth at all thetime periods. Results: There were significant differ-ences in bacterial regrowth between the groups withand without CTR. There were not significant differencesover time among the 3 groups with CTR or among the 3without CTR. Conclusions: The residual activity againstE. faecalis of 7% MA + 0.2% CTR, 17% EDTA + 0.2%CTR, and 10% LA + 0.2% CTR was greater than the useof chelating agents alone. (J Endod 2012;38:826–828)

Key WordsBiofilms, cetrimide, EDTA, Enterococcus faecalis,lactic acid, maleic acid

From the *Department of Operative Dentistry, School of DentiInvestigation, School of Pharmacy, and §Department of Preventive D

Supported by the Research Group CTS-167 of the Junta de AndAddress requests for reprints to Dr Carmen Mar�ıa Ferrer-Luque,

18071, Granada, Spain. E-mail address: cferrer@ugr.es0099-2399/$ - see front matter

Copyright ª 2012 American Association of Endodontists.doi:10.1016/j.joen.2012.01.013

826 Ferrer-Luque et al.

The improvement of irrigating protocols is essential during and after root canalpreparation to achieve better disinfection and ensure greater residual antimicrobial

activity in the root canal system. Removal of debris and smear layer in root canalsenhances access of the irrigating solutions into the dentinal tubules as well as adhesionof sealers to the root canal walls (1–3).

Although solutions of ethylenediaminetetraacetic acid (EDTA), citric acid (CA),phosphoric acid, maleic acid (MA), and lactic acid (LA) have shown their ability toremove the smear layer from instrumented root canals (4–7), the antimicrobialeffectiveness of these agents against Enteroccocus faecalis can vary depending onthe chelating agent used (8, 9). EDTA, phosphoric acid, and CA have not provedeffective in eradicating E. faecalis biofilms (8), whereas MA alone or in combinationwith cetrimide (CTR) has demonstrated capacity to eradicate in vitro E. faecalisbiofilms at lower concentrations than those recommended for clinical use (9).In turn, LA, an organic acid widely used as preservatives in foods and more biologicallyacceptable than other chelating agents (10), has shown both the ability to removesmear layer (4) and higher bond strength of a resin adhesive sealer to human dentin(11) when used at concentrations of 10%–20%.

The addition of CTR, a cationic surfactant that possesses bactericidal activityplus the capacity to eradicate in vitro E. faecalis (12), reduces the surface tensionof chelating solutions (13, 14). CTR also increases the capacity of EDTA and CA toeradicate E. faecalis biofilms (9) without affecting their ability to remove calciumfrom instrumented root canals (15). However, the effect of the combined use of CTRwith EDTA, MA, or LA acids on the bacteria that persist after root canal treatmenthas not been studied yet. The aim of this study was, therefore, to evaluate the residualantimicrobial activity of 17% EDTA, 7%MA, and 10% LA alone and combined with 0.2%CTR in root canals infected with E. faecalis.

Materials and MethodsOne hundred four anterior human teeth stored in 0.1% thymol solution at 4�C

were decoronated to obtain roots 12 mm in length. We followed a protocol previouslyapproved by the Ethics Committee of the University of Granada. A customized model ofeach tooth was made (16). Each root and its corresponding model were autoclaved at121�C. After sterilization, the specimens were incubated in brain-heart infusion (BHI)broth for 24 hours at 37�C to ensure no bacterial contamination.

stry, †Department of Microbiology, School of Dentistry, ‡Department of Statistics and Operativeentistry, School of Dentistry, University of Granada, Granada, Spain.aluc�ıa, Spain.Department of Operative Dentistry, School of Dentistry, Campus de Cartuja, Colegio M�aximo s/n.E-

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Basic Research—Technology

From a subculture of E. faecalis (ATCC 29212), a standard

suspension was prepared in BHI to obtain 1� 107 colony-forming unitsper mL (CFU/mL). Afterwards, 1.2 mL of this suspension and thesterilized tooth were added to an Eppendorf tube and were incubatedfor 4 weeks under aerobic conditions at 37�C, with reinoculation per-formed every 7 days. The cultures were checked for purity by Gram stainand colony morphology, and 2 samples were processed to confirm thepresence of E. faecalis biofilm by scanning electronmicroscopy (SEM).

The working length was established for each tooth by introducinga #10 K-file (Dentsply Maillefer, Ballaigues, Switzerland) into the canaluntil it was observed at the apical foramen and then decreasing the filelength by 1 mm. The apexes of the teeth were sealed with cyanoacrylate.Each tooth was then inserted into its customized model, and the inter-face was sealed with cyanoacrylate. The root canals were instrumentedby using the ProTaper system (Dentsply Maillefer) to working length byusing an F3 master apical file (Dentsply Maillefer), following the manu-facturer’s instructions. During preparation, 1 mL of 5.25% NaOCl wasused after each file.

Once instrumented, the roots were randomly divided into 6 groups(n = 15) according to the final irrigating solution: group I, 5 mL of 17%EDTA; group II, 5 mL of 17% EDTA + 0.2% CTR; group III, 5 mL of 7%MA; group IV, 5 mL of 7% MA + 0.2% CTR; group V, 5 mL of 10% LA;and group VI, 5 mL of 10% LA + 0.2% CTR. In each group, 2 other spec-imens not contaminated with E. faecalis received the same treatmentand served as negative controls. All the irrigating solutions were deliv-ered through a 3-mL Luer-Loc syringe coupled to a 30-gauge needle tip.

After preparation, the root canals were dried with sterile paperpoints (Dentsply Maillefer) and filled with BHI broth. A #15 K-file(Dentsply Maillefer) was placed in the canal to within 1 mm of workinglength, and the canal was circumferentially filed for 10 seconds.Three consecutive sterile paper points were introduced into the canalto absorb the BHI broth. The paper points were transferred to anEppendorf tube containing 5 mL of BHI broth. After collection of thefirst bacterial sample, the canal was refilled with the same broth, andsamples were collected daily for 60 days. All collected samples wereincubated 24 hours at 37�C. Turbidity was recorded for each specimenas an indicator of bacterial growth. Once turbidity was present, bacteriawere identified to ensure that there was no contamination other thanE. faecalis.

Statistical AnalysisThe percentages of specimens without E. faecalis regrowth were

compared between each chelating agent and its corresponding associ-

TABLE 1. Specimens without E. faecalis Recolonization after Different Time Perio

5 10 15

n (%) n (%) n (%)

Group I 12 (80.0) 9 (60.0) 6 (40.0Group II 15 (100.0) 11 (73.3) 11 (73.3Comparison P value† .112 .350 .070Group III 15 (100.0) 9 (60.0) 8 (53.3Group IV 15 (100.0) 11 (73.3) 11 (73.3Comparison P value† 1.000 .350 .225Group V 13 (87.0) 9 (60.0) 3 (20.0Group VI 14 (93.3) 10 (66.6) 10 (66.6Comparison P value† .500 .500 .013

Comparison among the 3 irrigation groups without CTR (17% EDTA, 7% MA, and 10% LA) and with CTR (17

Pearson c2 test gave no statistically significant differences at any of the time periods evaluated.

*Recolonization was evaluated daily.†One-tailed Fisher test.

JOE — Volume 38, Number 6, June 2012

ation with CTR by a one-tailed Fisher test. A modified version of the c2

test was used to compare the 3 irrigating groups with and withoutCTR. The level of statistical significance was set at P <.05. All statisticalanalyses were performed by means of SPSS 15.0 software (SPSS Inc,Chicago, IL).

ResultsIn all the groups, negative cultures were obtained within 4 days of

final irrigation. Table 1 shows the specimens without regrowth ofE. faecalis after final irrigation during the 60 days of evaluation. Overall,the combined use of CTR with chelating agents reduced the regrowth indisinfected root canals. The pair-by-pair comparisons between thegroups with 17% EDTA, 7% MA, and 10% LA and their correspondinggroups with 0.2% CTR were statistically significant at certain timeperiods (Table 1). There were no statistically significant differencesamong the 3 groups with CTR or among those without CTR. All sterilitycontrols showed an absence of turbidity.

DiscussionThe biomechanical preparation in endodontic treatment includes

the use of chelating agents to remove the smear layer created duringinstrumentation of the root canals, thereby allowing antiseptics to pene-trate deeper into the dentin (12). In this study we described the residualantimicrobial activity of 6 chelating solutions on root canals of humanteeth cultured with E. faecalis by using a previously published method-ology (16). A 60-day follow-up period was selected because in this timeframe a previous study (17) showed residual antimicrobial activity ofCTR combined with chlorhexidine (CHX), thus allowing for compari-sons to be drawn.

Our results showed that combinations of 0.2% CTR with 17%EDTA, 7% MA, and 10% LA as final irrigating solutions inhibited theregrowth of bacteria within the root canals more effectively than finalirrigation with EDTA, MA, and LA acids alone. Irrigation protocols of2.5% NaOCl/17% EDTA and 2.5% NaOCl/7% MA followed by 0.2%CTR have shown 100% inhibition of E. faecalis biofilm formation indentin (18), whereas in infected root canals, final irrigation regimenswith EDTA and MA followed by the combination of 2% CHX + 0.2% CTRhave recently been seen to exert a much more prolonged residualactivity than EDTA or MA followed by 5.25% sodium hypochlorite (17).

When the chelating agents of study were not combined with CTR,the percentage of bacterial regrowth was higher in all the time periods,occurring more rapidly in the specimens treated with 10% LA, followedby 17% EDTA and 7% MA. The best results were obtained with 7% MA,

ds (initial sample, n = 15)

Evaluated time (days)*

20 25 30 60

n (%) n (%) n (%) n (%)

) 4 (26.6) 3 (20.0) 3 (20.0) 3 (20.0)) 10 (66.6) 10 (66.6) 10 (66.6) 10 (66.6)

.033 .013 .013 .013) 7 (46.6) 6 (40.0) 5 (33.3) 4 (26.6)) 10 (66.6) 10 (66.6) 10 (66.6) 10 (66.6)

.231 .136 .072 .033) 3 (20.0) 3 (20.0) 3 (20.0) 3 (20.0)) 9 (60.0) 9 (60.0) 9 (60.0) 9 (60.0)

.030 .030 .030 .030

% EDTA + 0.2% CTR, 7% MA + 0.2% CTR, and 10% LA + 0.2% CTR) by means of modified version of

Residual Activity of Chelating Agents on Infected Root Canals 827

Basic Research—Technology

which only showed significant differences at 60 days with respect to itscorresponding solution with 0.2% CTR. It is known that 7% MA hasgreater residual antimicrobial activity against E. faecalis biofilms(18) as well as a greater ability to remove smear layer from the apicalroot canal than 17% EDTA (7). The highly acidic pH of MA couldexplain its great residual and antimicrobial activities. On the otherhand, 10% LA, reportedly similar to 15% EDTA solution in both smearlayer removal (4) and the capacity to increase fracture resistance inendodontically treated teeth (19), showed the highest percentages ofbacterial regrowth over time in our study. This finding could be dueto its reduced capacity to eradicate enterococci biofilms when usedat concentrations below 20% (results not published).

The sequential use of chelating agents and NaOCl has beenrecommended in final irrigation protocols (20–22). In the presentstudy, NaOCl was not used as a final irrigating solution because ithas shown no residual activity (17, 23). According to our results, thecombination of MA with CTR was more efficient than when usedalone. A further advantage of MA with respect to EDTA is that it hasno adverse interactions with CHX (24) and might even enhance itsantimicrobial potential (18).

In our study, the combination of chelating agents with CTRexhibited antimicrobial properties when used as final irrigatingsolutions, and their use might be beneficial in alternating irrigationregimen. Although the interaction between chelating agents and NaOClis known to decrease the antimicrobial activity of NaOCl (24–27), anirrigation regimen based on the alternating use of NaOCl and EDTAduring the preparation of the root canals recently led to a consistent,long-term elimination of E. faecalis biofilms as opposed to a moreconventional irrigation regimen (28). In this overall context, furtherstudies are needed to determine the effectiveness of chelating solutionscombined with CTR in an irrigation regimen involving alternation withNaOCl, and the combination of chelating agents with CHX as finalirrigating protocols in the preparation of infected root canals.

In conclusion, the residual antimicrobial activity againstE. faecalis of 7% MA + 0.2% CTR, 17% EDTA + 0.2% CTR, and 10%LA + 0.2% CTR was greater than the use of the chelating agents alone.

AcknowledgmentsThe authors thank Francisca Castillo P�erez for her technical

assistance.The authors deny any conflicts of interest related to this study.

References1. Cruz-Filho AM, Sousa-Neto MD, Savioli RN, Silva RG, Vansan LP, P�ecora JD. Effect of

chelating solutions on the microhardness of root canal lumen dentin. J Endod 2011;37:358–62.

2. Tagger M, Tagger E, Tjan AH, Bakland LK. Measurement of adhesion of endodonticsealers to dentin. J Endod 2002;28:351–4.

3. Saleh IM, Ruyter IE, Haapasalo MP, Ørstavik D. Adhesion of endodontic sealers:scanning electron microscopy and energy dispersive spectroscopy. J Endod2003;29:595–601.

4. Ayad MF. Lactic acid root canal irrigation for dowel and core treatment: a pilotstudy. J Prosthet Dent 2004;92:540–5.

5. Zehnder M. Root canal irrigants. J Endod 2006;32:389–98.

828 Ferrer-Luque et al.

6. P�erez-Heredia M, Ferrer-Luque CM, Gonz�alez-Rodr�ıguez MP. The effectiveness ofdifferent acid irrigating solutions in root canal cleaning after hand and rotary instru-mentation. J Endod 2006;32:993–7.

7. Ballal NV, Kandian S, Mala K, Bhat KS, Acharya S. Comparison of the efficacy ofmaleic acid and ethylenediaminetetraacetic acid in smear layer removal from instru-mented human root canal: a scanning electron microscopic study. J Endod 2009;35:1573–6.

8. Arias-Moliz MT, Ferrer-Luque CM, Espigares-Garc�ıa M, Baca P. Enterococcus fae-calis biofilms eradication by root canal irrigants. J Endod 2009;35:711–4.

9. Ferrer-Luque CM, Arias-Moliz MT, Gonz�alez-Rodr�ıguez MP, Baca P. Antimicrobialactivity of maleic acid and combinations of cetrimide with chelating agents againstEnterococcus faecalis biofilm. J Endod 2010;36:1673–5.

10. Spangberg L, Engstr€om B, Langeland K. Biologic effects of dental materials:3—toxicity and antimicrobial effect of endodontic antiseptics in vitro. Oral SurgOral Med Oral Pathol Oral Radiol Endod 1973;36:856–71.

11. Ayad MF, Farag AM, Garcia-Godoy F. Effect of lactic acid irrigant on shear bondstrength of Epiphany adhesive sealer to human dentin surface. Oral Surg OralMed Oral Pathol Oral Radiol Endod 2010;109:e100–6.

12. Arias-Moliz MT, Ferrer-Luque CM, Gonz�alez-Rodr�ıguez MP, Valderrama MJ, Baca P.Eradication of Enterococcus faecalis biofilms by cetrimide and chlorhexidine.J Endod 2010;36:87–90.

13. H€ulsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment:mode of action and indications for their use. Int Endod J 2003;36:810–30.

14. Yilmaz Z, Basbag B, Buzoglu HD, G€um€usderelioglu M. Effect of low-surface-tensionEDTA solutions on the wettability of root canal dentin. Oral Surg Oral Med OralPathol Oral Radiol Endod 2011;111:109–14.

15. Zehnder M, Schicht O, Sener B, Schmidlin P. Reducing surface tension inendodontic chelator solutions has no effect on their ability to remove calciumfrom instrumented root canals. J Endod 2005;31:590–2.

16. Baumgartner JC, Johal S, Marshall JG. Comparison of the antimicrobial efficacy of1.3% NaOCl/BioPure MTAD to 5.25% NaOCl/15% EDTA for root canal irrigation.J Endod 2007;33:48–51.

17. Baca P, Mendoza-Llamas ML, Arias-Moliz MT, Gonz�alez-Rodr�ıguez MP,Ferrer-Luque CM. Residual effectiveness of final irrigation regimens on Enteroccusfaecalis-infected root canals. J Endod 2011;37:1121–3.

18. Baca P, Junco P, Arias-Moliz MT, Gonz�alez-Rodr�ıguez MP, Ferrer-Luque CM.Residual and antimicrobial activity of final irrigation protocols on Enterococcusfaecalis biofilm in dentin. J Endod 2011;37:363–6.

19. Ayad MF, Bahannan SA, Rosenstiel SF. Influence of irrigant, dowel type, and root-reinforcing material on fracture resistance of thin-walled endodontically treatedteeth. J Prosthodont 2011;20:180–9.

20. Yamada RS, Armas A, Goldman M, Lin PS. A scanning electron microscopic com-parison of a high volume final flush with several irrigating solutions: part 3.J Endod 1983;9:137–42.

21. Bystrom A, Sundqvist G. The antibacterial action of sodium hypochlorite and EDTAin 60 cases of endodontic therapy. Int Endod J 1985;18:35–40.

22. Johal S, Baumgartner JC, Marshall JG. Comparison of the antimicrobial efficacy of1.3% NaOCl/BioPure MTAD to 5.25% NaOCl/15% EDTA for root canal irrigation.J Endod 2007;33:48–51.

23. Weber CD, McClanahan SB, Miller GA, Diener-West M, Johnson JD. The effectof passive ultrasonic activation of 2% chlorhexidine or 5.25% sodium hypochloriteirrigant on residual antimicrobial activity in root canals. J Endod 2003;29:562–4.

24. Ballal NV, Moorkoth S, Mala K, Bhat KS, Hussen SS, Pathak S. Evaluation of chemicalinteractions of maleic acid with sodium hypochlorite and chlorhexidine gluconate.J Endod 2011;37:1402–5.

25. Grawehr M, Sener B, Waltimo T, Zehnder M. Interactions of ethylenediaminetetraacetic acid with sodium hypochlorite in aqueous solutions. Int Endod J2003;36:411–5.

26. Kuroiwa K, Nakayama H, Kuwahara T, et al. Augmenting effect of acetic acid for acid-ification on bactericidal activity of hypochlorite solution. Lett Appl Microbiol 2003;36:46–9.

27. Zehnder M, Schmidlin P, Sener B, Waltimo T. Chelation in root canal therapy recon-sidered. J Endod 2005;31:817–20.

28. Soares JA, Roque de Carvalho MA, Cunha Santos SM, et al. Effectiveness of chemo-mechanical preparation with alternating use of sodium hypochlorite and EDTA ineliminating intracanal Enterococcus faecalis biofilm. J Endod 2010;36:894–8.

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