Vol. 112 No. 5 November 2011

ENDODONTOLOGY Editor: Larz S.W. Spångberg

Cytotoxicity evaluation of Gutta Flow and Endo Sequence BCsealersKeivan Zoufan, DDS, MDS,a Jin Jiang, DDS, PhD,b Takashi Komabayashi, DDS, MDS, PhD,c

Yu-Hsiung Wang, DMD, PhD,d Kamran E. Safavi, DMD, Med,e andQiang Zhu, DDS, PhD,f Farmington, Connecticut; and Dallas, TexasUNIVERSITY OF CONNECTICUT AND BAYLOR COLLEGE OF DENTISTRY

Objective. This study evaluated the cytotoxicity of GuttaFlow and EndoSequence BC sealers and compared them withAH Plus and Tubli-Seal sealers.Study design. Samples (0.5 mg) of freshly mixed or set BC, GuttaFlow, AH Plus, and Tubli-Seal sealers were elutedwith 300, 600, and 1,000 �L cell culture medium for 24 and 72 hours. L929 cells were seeded into 96-well plates at3 � 104 cells/well and cultured with 100 �L eluate from each eluate group. Cells cultured only with culture mediumserved as control. After 24 hours’ incubation, the cytotoxicity was evaluated by MTT assay. Cell viability wascalculated as the percentage of the control group, and the results were analyzed with a one-way analysis of variance.Results. For the freshly mixed sealer, cell viability in the AH Plus group was less than in all of the other 3 sealergroups. The Tubli-Seal sealer group had less cell viability than the EndoSequence BC and GuttaFlow sealer groups. Forthe set sealer, the Tubli-Seal and AH Plus groups had less cell viability than the EndoSequence BC and GuttaFlowsealer groups. There was no cell viability difference between the EndoSequence BC and GuttaFlow sealer groups inthe either freshly mixed or set sealer group.Conclusions. The GuttaFlow and EndoSequence BC sealers have lower cytotoxicity than the AH Plus and Tubli-Seal

sealers. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112:657-661)

Endosequence BC sealer has recently been developedto improve the seal of root canal filling (Brasseler,Savannah, GA). According to the manufacturer, it iscomposed of zirconium oxide, calcium silicates, cal-

aEndodontic Resident, Division of Endodontology, School of DentalMedicine, University of Connecticut Health Center.bAssistant Professor, Division of Endodontology, School of DentalMedicine, University of Connecticut Health Center.cAssistant Professor, Department of Endodontics, Baylor College ofDentistry.dAssistant Professor, Division of Pediatric Dentistry, School of Den-tal Medicine, University of Connecticut Health Center.eProfessor and Chair, Division of Endodontology, School of DentalMedicine, University of Connecticut Health Center.fAssociate Professor, Division of Endodontology, School of DentalMedicine, University of Connecticut Health Center.Received for publication Jan. 7, 2011; returned for revision Mar. 17,2011; accepted for publication Mar. 28, 2011.1079-2104/$ - see front matter© 2011 Mosby, Inc. All rights reserved.

doi:10.1016/j.tripleo.2011.03.050

cium phosphate monobasic, calcium hydroxide, filler,and thickening agents. BC sealer is a premixed ready-to-use injectable bioceramic cement paste that requiresthe presence of water to set. It is based on the rootcanal repair material Bioaggregate.1,2 BC sealer hasthe strongest antimicrobial activity among the testedsealers, including Epiphany, AH Plus, Apexit Plus,Tubli-Seal, and Sealapex, owing to a high pH, hy-drophilicity, and active calcium hydroxide diffu-sion.1 BC sealer is equivalent to AH Plus in rootcanal sealing ability.3

GuttaFlow is a silicone-based sealer consisting of amixture of gutta-percha powder, poly-dimethylsiloxaneand nanosilver particles (Coltene/Whaledent, CuyahogaFalls, OH). It comes in a unidose capsule and is injectedafter mixing. GuttaFlow with primer showed better resis-tance to bacterial penetration than the sealers RC Sealer,Epiphany, EndoREZ, AcroSeal, Apexit, AH Plus, andRoekoSeal.4 Recent studies demonstrated that the sealing

ability of GuttaFlow was better or equal to AH Plus.5,6

657

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Root canal sealer should not only have a goodsealing ability, but also have good biocompatibility.There are few reports to evaluate the cytotoxicity ofGuttaFlow and BC sealers.7,8 The purpose of the pres-ent study was to examine the cytotoxicity of BC andGuttaFlow sealers and compare them with traditional rootcanal sealers AH Plus and Tubli-Seal.

MATERIALS AND METHODSWe adapted the experimental methods from previous

studies.9,10 L929 mouse fibroblasts were obtained fromAmerican Type Culture Collection (ATCC, Manassas,VA). The cells were grown in Eagle minimum essentialmedium (ATCC) supplemented with 10% fetal bovineserum (Hyclone Laboratories, Logan, UT) and 1% anti-biotic/antimycotic cocktail (300 U/mL penicillin, 300�g/mL streptomycin, and 5 �g/mL amphotericin B;Gibco BRL, Gaithersburg, MD) under standard cell cul-ture conditions (37°C, 100% humidity, 95% air, 5% CO2).

The root canal sealers tested in this study wereEndoSequence BC, GuttaFlow, AH Plus Jet (DentsplyDeTrey, Konstanz, Germany), and Tubli-Seal Xpress(SybronEndo, Orange, CA).

The cytotoxicity of the different sealers was tested in2 ways. In 1 set of experiments, set sealer was exam-ined. The sealers were mixed according to the manu-facturers’ instructions, placed into 24-well plates at 0.5g/well, and incubated for 72 hours in a cell cultureincubator to allow the sealer to set. In another set ofexperiments, freshly mixed sealers were placed into the24-well plates (0.5 g/well). The freshly mixed and setsealers were incubated with 3 different amounts of cellculture medium (300 �L, 600 �L, and 1,000 �L) for 24and 72 hours (1 day and 3 days). Each sealer had a totalof 12 eluate groups to be evaluated.

For the cell cytotoxicity assay, L929 cells wereseeded into 96-well plates at 3 � 104 cells/well andincubated for 24 hours to allow adhesion. Then 100 �Lsealer eluate from the different eluate groups was addedto the culture wells. Untreated cells served as a controlgroup. After a 24-hour incubation period, the cell via-bility was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay accord-ing to the manufacturer’s instructions (ATCC).

Cell viability was calculated as the percentage relativeto the control group, and the results were analyzed with aone-way analysis of variance. Post hoc tests were donewith Schaffer test. Each experiment was performed 3times.

RESULTSFresh sealer

When cells were cultured with the 1-day eluate of

fresh sealers, AH Plus had greater cytotoxicity than the

other 3 sealers, and Tubli-Seal was more cytotoxic thanthe BC and GuttaFlow sealers in the 300 and 600 �Leluate groups (Fig. 1). In the 1,000 �L eluate groups,AH Plus was more cytotoxic than all of the othersealers, and there was no difference in cytotoxicityamong BC, Tubli-Seal, and GuttaFlow sealers (Fig. 1).

When cells were cultured with the 3-day eluate offresh sealers, AH Plus exhibited more cytotoxicity thanthe other 3 sealers, and Tubli-Seal sealer was morecytotoxic than BC and GuttaFlow sealers in the 300,600, and 1,000 �L eluate groups (Fig. 2). There was nocytotoxicity difference between GuttaFlow and BCsealers in all of the eluate groups (Fig. 2).

Set sealerWhen the L929 cells were cultured with the 1-day

eluate of the set sealers, AH Plus and Tubli-Seal weremore cytotoxic than GuttaFlow and BC sealers, andTubli-Seal was more cytotoxic than AH Plus in the 300�L eluate groups (Fig. 3). In the 600 �L eluate groups,Tubli-Seal sealer was more cytotoxic than all of theother sealers, and no difference in cytotoxicity wasobserved among AH Plus, BC, and Tubli-Seal sealers(Fig. 3). In the 1,000 �L eluate group, Tubli-Seal sealerwas more cytotoxic than the AH Plus and BC sealers.No difference in cytotoxicity was observed between theTubli-Seal and GuttaFlow sealers (Fig. 3).

When the cells were cultured with the 3-day eluate ofthe set sealers, AH Plus and Tubli-Seal were more cyto-toxic than GuttaFlow and BC in the 300 �L and 600 �Leluate groups (Fig. 4). AH Plus sealer was more cytotoxicthan Tubli-Seal sealer in the 600 �L eluate groups (Fig.4). In the 1,000 �L eluate groups, Tubli-Seal sealer wasmore cytotoxic than AH Plus sealer. No difference was

Fig. 1. Cell viability of L929 cells after culture with 1-dayeluate of freshly mixed sealers.

observed among AH Plus, BC, and GuttaFlow sealers

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(Fig. 4). There was no difference between GuttaFlow andBC sealers in all of the eluate groups (Fig. 4).

Observation of sealer settingWe also observed that when the tested sealers were

left on the benchtop at room temperature, BC sealer didnot set, even after 2 months (Fig. 5). The other 3 sealershad set when they were checked after 24 hours (Fig. 5).No indentation or penetration could be made in thesealer pellets after setting.

When the tested sealers were placed in a cell cultureincubator for 24 hours, they all set (Fig. 6). BC releaseda clear liquid (Fig. 6).

DISCUSSIONThis study compared the cytotoxicity of BC and

Fig. 2. Cell viability of L929 cells after culture with 3-dayeluate of freshly mixed sealers.

Fig. 3. Cell viability of L929 cells after culture with 1-dayeluate of set sealers.

GuttaFlow sealers with the conventional root canal

sealers AH Plus and Tubli-Seal. In the freshly mixedsealer, AH Plus showed severe cytotoxicity in all of theeluates. The cytotoxicity decreased when it set, show-ing no cytotoxicity in the 1,000 �L eluate. A recentstudy also showed that AH plus was severely cytotoxicduring the first 3 days and became noncytotoxic after 3weeks.11 These findings agree with earlier studies thatthe cytotoxicity gradually decreased with time.12,13 Thehigher cytotoxicity when freshly mixed is due to theinitial release of formaldehyde or the epoxy resin com-ponent.14-16 Tubli-Seal was shown to be cytotoxicwhen freshly mixed and then set, even though thecytotoxicity decreased with the increase of eluate me-dium. Eugenol has been identified as one of the majoringredients responsible for the material’s cytotoxic-

Fig. 4. Cell viability of L929 cells after culture with 3-dayeluate of set sealers.

Fig. 5. Sealers were left on the laboratory bench top for 2months. BC sealer had not set.

ity.17,18 Several chemical additives, one of which is

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resins used for greater dentin adhesion, also may havepartially contributed to the tubliseal cytotoxicity.19

For BC sealer and GuttaFlow, the percentages of cellviability were �90%-100% in all the different eluateswith both freshly mixed and set sealer. There was nodifference for the cytotoxicity between the freshly mixedand the set sealer. They demonstrate biocompatibility atboth conditions. BC sealer is described by the manufac-turer as a bioceramic material, which is a ceramic productor component with osteoinductive properties used in med-ical and dental applications, mainly as implants and re-placements.20 A recent study also shows that BC sealer isbiocompatible.7 GuttaFlow is the newest silicone-basedsealer, manufactured by adding gutta-percha powder tothe silicone matrix. GuttaFlow demonstrated compara-tively low cytotoxicity compared with EndoREZ,AcroSeal, and Apexit.8 It also exhibited a comparativelylower cytotoxicity than the resin-based systems or AHPlus.21 Research regarding the organ toxicity and connec-tive tissue reaction of GuttaFlow has found that it hasgood compatibility and acceptable tissue toxicity.22 In arecent in vitro study, no genotoxicity of GuttaFlow wasobserved.23 Our results with GuttaFlow are consistentwith those studies in finding that GuttaFlow sealer hassimilar biocompatibility.

Being hydrophilic, BC sealer uses moisture withinthe root canal to complete the setting reaction. Moisturefacilitates the hydration reactions of calcium silicates toproduce calcium silicate hydrogel and calcium hydrox-ide, which partially reacts with the phosphate to formhydroxyapatite and water.1 Our observation that BCsealer only sets in a cell culture incubator shows that itrequires moisture to complete the hydration reaction.The clear fluid released from setting sealer is undoubt-

Fig. 6. Sealers were placed in cell culture incubator for 24hours. All sealers had set.

edly water resulting from the hydration reaction.1 A

recent study also shows that the EndoSequence rootrepair materials, which is similar to the BC sealer,become set only in a chamber of 100% humidity.24

The present study found that GuttaFlow and BC sealershave lower cytotoxicity than AH Plus and Tubli-Sealsealers. Other properties of GuttaFlow and BC sealers,such as solubility and dimensional stability, and the setting ofBC sealer in root canals, need to be further investigated.

The authors thank Ms. Jeanne Santa Cruz (Texas A&MHealth Science Center Baylor College of Dentistry) forcritical editing.

REFERENCES1. Zhang H, Shen Y, Ruse ND, Haapasalo M. Antibacterial activity

of endodontic sealers by modified direct contact test againstEnterococcus faecalis. J Endod 2009;35:1051-5.

2. Zhang H, Pappen FG, Haapasalo M. Dentin enhances the anti-bacterial effect of mineral trioxide aggregate and bioaggregate. JEndod 2009;35:221-4.

3. Zhang W, Li Z, Peng B. Assessment of a new root canal sealer‘sapical sealing ability. Oral Surg Oral Med Oral Pathol OralRadiol Endod 2009;107:e79-82.

4. Eldeniz AU, Ørstavik D. A laboratory assessment of coronalbacterial leakage in root canals filled with new and conventionalsealers. Int Endod J 2009;42:303-12.

5. Bouillaguet S, Shaw L, Barthelemy J, Krejci I, Wataha JC.Long-term sealing ability of pulp canal sealer, AH-Plus, Gutta-Flow and Epiphany. Int Endod J 2008;41:219-26.

6. Vasiliadis L, Kodonas K, Economides N, Gogos C, Stavrianos C.Short- and long-term sealing ability of Gutta-Flow and AH-Plususing an ex vivo fluid transport model. Int Endod J 2010;43:377-81.

7. Zhang W, Li Z, Peng B. Ex vivo cytotoxicity of a new calciumsilicate-based canal filling material. Int Endod J 2010;43:769-74.

8. Eldeniz AU, Mustafa K, Ørstavik D, Dahl JE. Cytotoxicity ofnew resin-, calcium hydroxide– and silicone-based root canalsealers on fibroblasts derived from human gingiva and L929 celllines. Int Endod J 2007;40:329-37.

9. Alanezi AZ, Jiang J, Safavi KE, Spangberg LS, Zhu Q.Cytotoxicity evaluation of endosequence root repair material.Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:e122-5.

10. Donadio M, Jiang J, He J, Wang YH, Safavi KE, Zhu Q.Cytotoxicity evaluation of Activ GP and Resilon sealers in vitro.Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:e74-8.

11. Pinna L, Brackett MG, Lockwood PE, Huffman BP, Mai S, CottiE, et al. In vitro cytotoxicity evaluation of a self-adhesive,methacrylate resin-based root canal sealer. J Endod 2008;34:1085-8.

12. Lodiene G, Morisbak E, Bruzell E, Orstavik D. Toxicityevaluation of root canal sealers in vitro. Int Endod J2008;41:72-7.

13. Merdad K, Pascon AE, Kulkarni G, Santerre P, Friedman S.Short-term cytotoxicity assessment of components of the epiph-any resin-percha obturating system by indirect and direct contactMillipore filter assays. J Endod 2007;33:24-7.

14. Spangberg LS, Barbosa SV, Lavigne GD. AH 26 releases form-

aldehyde. J Endod 1993;19:596-8.

OOOOEVolume 112, Number 5 Zoufan et al. 661

15. Leonardo MR, da Silva LAB, Filho MT, da Silva RS. Release offormaldehyde by 4 endodontic sealers. Oral Surg Oral Med OralPathol Oral Radiol Endod 1999;88:221-5.

16. Huang TH, Ding SJ, Hsu TZ, Lee ZD, Kao CT. Root canalsealers induce cytotoxicity and necrosis. J Mater Sci Mater Med2004;15:767-71.

17. Serene TP, Vesely J, Boackle RJ. Complement activation asapossible in vitro indication of the inflammatory potential ofendodontic materials. Oral Surg Oral Med Oral Pathol OralRadiol Endod 1988;65:354-7.

18. Guigand M, Pellen-Mussi P, le Goff A, Vulcain JM, Bonnaure-Mallet M. Evaluation of the cytocompatibility of three endodon-tic materials. J Endod 1999;25:419-23.

19. Hauman CH, Love RM. Biocompatibility of dental materialsused in contemporary endodontic therapy: a review. part 2. Rootcanal filling materials. Int Endod J 2003;36:147-60.

20. Ribeiro DA, Matsumoto MA, Duarte MA, Marques ME, Salva-dori DM. Ex vivo biocompatibility tests of regular and whiteforms of mineral trioxide aggregate. Int Endod J 2006;39:26-30.

21. Bouillaguet S, Wataha JC, Tay FR, Brackett MG, Lockwood PE.

Initial in vitro biological response to contemporary endodonticsealers. J Endod 2006;32:989-92.

22. Gencoglu N, Sener G, Omurtag GZ, Tozan A, Uslu B, Arbak S,et al. Comparison of biocompatibility and cytotoxicity of twonew root canal sealers. Acta Histochem 2010;112:567-75.

23. Brzovic V, Miletic I, Zeljezic D, Mladinic M, Kasuba V, RamicS, et al. In vitro genotoxicity of root canal sealers. Int Endod J2009;42:253-63.

24. Damas BA, Wheater MA, Bringas JS, Hoen MM. Cytotoxicitycomparison of mineral trioxide aggregates and endosequencebioceramic root repair materials. J Endod 2011;37:372-5.

Reprint requests:

Dr. Qiang ZhuAssociate ProfessorDivision of Endodontology, MC 1715University of Connecticut Health CenterFarmington, CT 06030-1715

qzhu@nso2.uchc.edu