comparision of biocompatibility and cytotoxicity of two new root canal sealers

Acta histochemica 112 (2010) 567—575

0065-1281/$ - sdoi:10.1016/j.

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Comparision of biocompatibility and cytotoxicity oftwo new root canal sealers

Nimet Gencoglua, Goksel Senerb, Gulden Z. Omurtagc, Ayfer Tozanc,Bahar Uslud, Serap Arbake, Dilek Helvacioglua,�

aDepartment of Endodontics, Faculty of Dentistry, Marmara University, Buyuk C- iftlik sok. No: 6, Nis-antas-ı,Istanbul, TurkeybDepartment of Pharmacology, Faculty of Pharmacy, Marmara University, Haydarpasa, Istanbul, TurkeycDepartment of Pharmaceutical Toxicology, Faculty of Pharmacy, Marmara University, Haydarpasa, Istanbul, TurkeydDepartment of Histology and Embryology, Faculty of Medicine, Marmara University, Haydarpasa, Istanbul, TurkeyeAcibadem University, Department of Histology and Embryology, Gulsuyu Maltepe, Istanbul 34848, Turkey

Received 4 April 2013; received in revised form 9 June 2013; accepted 11 June 2013

KEYWORDSBiocompatibility;Cytotoxicity;EndoREZ;GuttaFlow;Kerr pulp canal sea-ler;Root canal;Rats

ee front matter & 2013acthis.2013.06.005

ing author. Fax: +90 212ess: dhelvacioglu@gma

SummaryThe aim of this study was to investigate the remote organ toxicity and connective tissue reaction of two new root canal sealers (‘‘GuttaFlows’’ and ‘‘EndoREZs’’) and to compare them with zinc oxide eugenol sealer using biochemical and histopatho-logical parameters. A total of 60 white albino Wistar rats were used in the study. 0.1 ml of GuttaFlows, EndoREZs or Kerr Pulp Canal Sealers were administered subcutaneously into the mid-dorsal thoracic region of rats (15 in each group). Control rats were given saline only. Rats were decapitated after 24 h, on day 7 and on day 30 of the experiment and tissue samples from lung, liver, kidney and skin were removed for the determination of malondialdehyde (MDA) and glutathione (GSH) levels. In parallel, tissues were also examined histologically. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels, and creatinine and blood urea nitrogen (BUN), concentrations (BUN) were measured to assess liver and kidney functions, respectively. Tumor necrosis factor (TNF) and lactate dehydro-genase (LDH) were also assayed in serum samples. No statistical differences were found among the control and EndoREZs, GuttaFlows and Kerr Pulp Canal sealers regarding tissue MDA, GSH levels or serum parameters (p40.05) at all time points examined. Both of the new root canal sealers showed good compatibility and acceptable tissue toxicity.& 2013 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights reserved.

246 52 47.il.com (D. Helvacioglu).

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N. Gencoglu et al.568

Introduction

The main objective of successful endodontictreatment is proper cleaning and shaping of theroot canal, as well as total obturation of the canalspace with an inert, dimensionally stable andbiologically compatible material. Currently, themost frequent obturation methods employ a solidcore such as gutta-percha cemented in the rootcanal with a sealer. Since these materials will be indirect contact with periapical tissues for a pro-longed period of time, their biocompatibility is ofprimary importance (Nguyen, 1984). Previous stu-dies have shown that almost all sealers have somedegree of toxicity in contact with living tissue(Browne and Friend, 1968; Friend and Browne,1968; Olsson and Wennberg, 1985; Ørstavik andMjor, 1988; Yes-ilsoy et al., 1988).

Zinc oxide eugenol sealers, which have been usedfor many years, release potentially cytotoxicconcentrations of eugenol (Hume, 1986; Schmalzet al., 2000). On the other hand, although resin-based sealers are popular, their cytotoxicity andmutagenic effects are debatable (Kleinsasser et al.,2004; Schweikl et al., 1998). Under clinical condi-tions, the polymerization processes of resin mate-rials are usually incomplete and almost all thecomponents can be detected in extracts of poly-merized materials. Previous studies have shownthat extractable monomers and/or other organicand inorganic ingredients may induce local andsystemic adverse effects (Spahl et al., 1998).Moreover, resin-based materials may elicit biologi-cal effects with underlying genetic mechanismsresulting in mutation in vitro. There are very littledata on the cytotoxic and genotoxic effects ofmethacrylate, which is a component of manysealers, on human cells (Kleinsasser et al., 2004).

Recently EndoREZs, a hydrophilic resin-basedroot canal sealer, was developed. It contains zincoxide, barium sulphate, resins and pigments withina matrix of urethane dimethacrylate (UDMA) resin.Although microleakage of EndoREZs has beenproperly investigated, the cytotoxic effects andbiocompatibility of the material are still not clear(Kardon et al., 2003; Kazemi et al., 2003).

More recently, silicon-based sealers have beendeveloped and their clinical and biocompatibilityhave been found promising. Another new recentlyintroduced material is GuttaFlows, which includesboth silicon and gutta-percha.

Reports in the literature on cytotoxicity havebeen based on in vivo or in vitro tests. However, incontrast with the in vitro studies, in vivo testsprovide information on acute reactions and long-term effects of implantation materials. Some

materials may affect distant organs such as kidney,liver, lung and change biochemical parameters(Economides et al., 2005; Omurtag et al., 2005).In some studies, the cytotoxic effects of endodonticsealers on macrophage activities have been inves-tigated. Activation of macrophages and cytokinesand the subsequent formation of reactive oxygenand nitrogen species are of central pathogenicimportance in the development of oxidative tissuedamage and organ dysfunction. Polymorphonuclearleukocyte (PMN) extravasation can lead to vasculardysfunction as well as parenchymal cell dysfunction(Neviere et al., 1999). Besides their direct dama-ging effects on tissues, these reactive speciesappear to trigger the accumulation of leukocytesin the tissues involved, and thus cause furtherinjury through activated neutrophils. It has beenshown that activated neutrophils secrete enzymes,such as myeloperoxidase, elastase, proteases andliberate oxygen radicals (Reiter et al., 2001). Lipidperoxidation mediated by oxygen free radicals isbelieved to be an important cause of destructionand damage to cell membranes.

Accordingly, this study was designed to investi-gate if GuttaFlows, EndoREZs or Kerr Pulp CanalSealers have any toxic effects by activating macro-phages and thus generating reactive species, whichultimately could lead to oxidative tissue damage inthe lungs, liver, kidneys and skin. Toxicity wasexamined using biochemical measurements, suchas tissue malondialdehyde (MDA) and glutathione(GSH) or serum lactate dehydrogenase (LDH) andTNF-a analysis, and parallel histopathological ap-proaches, while the functional impairments weremonitored by tests for hepatic and renal functions.

Materials and methods

Sixty albino Wistar rats (weighing 200–250 g)were used in this study. The study was performedon 4 groups of animals; Control, Kerr Pulp CanalSealers (Kerr, Romulus, MI, USA), GuttaFlows

(Roeko, Coltene Whaledent, Langenau, Germany)and EndoREZs (Ultradent Products Inc, SouthJordan, UT, USA) groups. Each group consisted of15 rats. Rats were administered a single dose of0.1ml dental materials subcutaneously into thedorsal thoracic mid-region and decapitated after24 h, 7 days and 30 days of the experiment. Allexperimental protocols were approved by theMarmara University Animal Care and Use Commit-tee (No: 09.28.2004-49). The authors have nocommercial interest in the products investigatedin this study.

Comparision of biocompatibility and cytotoxicity of two new root canal sealers 569

Histological analysis

For histological analysis, samples of the tissueswere fixed in 10% neutral buffered formalin andprepared for routine paraffin wax embedding. 6 mmthick section of lung, kidney and liver tissue werestained with hematoxylin and eosin and skinsections were stained with Masson’s trichromestain. All the slides were examined under a bright-field light microscope (Olympus-BH-2, OlympusCorporation, Tokyo, Japan). An experienced histol-ogist who was unaware of the treatment conditionsmade the histological assessments.

Assays

Serum levels of blood urea nitrogen, AST, ALT,creatinine, LDH and TNF-a

After decapitation, trunk blood was collected,the serum was separated to measure the aspartateaminotransferase (AST), alanine aminotransferase(ALT) levels and creatinine and blood urea nitrogen(BUN) as indicators of liver and kidney functions,respectively. Lactate dehydrogenase and TNF-awere also assayed in serum samples for theevaluation of generalized tissue damage.

BUN, AST, ALT and creatinine concentrations andLDH levels were determined spectrophotometri-cally using an automated analyzer (Olympus AU600, Diamond Diagnostic, Holliston, MA, USA)(Martinek, 1972). TNF-a was evaluated by anRIA-IRMA (radioimmunoassay-immunoradiometricassay) method. All samples were assayed induplicates using a commercial kit (BiosourceEurope S.A., Nivelles, Belgium). The activity ofradioactive assays was measured by a gammacounter (LKB Wallac 1270 Rack Gamma Counter,Turku, Finland). TNF-a in the serum samples wasexpressed as ng/ml.

Tissue levels of MDA, GSH and collagenIn order to evaluate the presence of tissue injury

in the skin and distant organs, tissue samples fromthe lung, liver, kidney and skin samples were storedat �80 1C for the determination of levels ofmalondialdehyde (an end-product of lipid perox-idation), glutathione (a key antioxidant), glu-tathione levels and collagen content (a marker offibrosis).

Tissue samples were homogenized with ice-cold150mM KCl for the determination of malondialde-hyde and glutathione levels. The MDA levels wereassayed for products of lipid peroxidation bymonitoring thiobarbituric acid reactive substance.Lipid peroxidation was expressed in terms of MDA

equivalents using an extinction coefficient of1.56� 105M–1 cm–1 and results are expressed as nmolMDA/g tissue. GSH measurements were performedusing a modification of the Ellman procedure(Beutler, 1975). Briefly, after centrifugation at3000 rev/min for 10min, 0.5ml of supernatant wasadded to 2ml of 0.3mol/l Na2HPO4 � 2H2O solution. A0.2ml solution of dithiobisnitrobenzoate (0.4mg/ml1% sodium citrate) was added and the absorbance at412nm was measured immediately after mixing. GSHlevels were calculated using an extinction coefficientof 13600M–1 cm–1. Results are expressed in mmolGSH/g tissue.

Tissue collagen was measured as a free radical-induced marker of fibrosis. Fresh tissue sampleswere cut with a razor blade, immediately fixed in10% formalin in 0.1M phosphate buffer (pH, 7.2)and processed for embedding in paraffin wax.Sections, approximately 15 mm thick, were ob-tained. Evaluation of collagen content was investi-gated according to the method of Lopez de Leonand Rojkind (1985), which is based on selectivebinding of the dyes Sirius red and fast green FCF tocollagen and non-collagenous components, respec-tively. Both dyes were eluted simultaneously byusing 0.1 N NaOH–methanol (1:1 v/v). Finally, theabsorbances at 540 and 605 nm were used todetermine the amount of collagen and protein,respectively.

Statistics

Statistical analysis was carried out using Graph-Pad Prism 3.0 (GraphPad Software, San Diego, CA,USA). All data were expressed as mean7SEM.Groups of data were compared with an analysis ofvariance (ANOVA) followed by Tukey’s multiplecomparison tests. Values of po0.05 were regardedas significant.

Results

Histological analysis

LiverIn all groups, the liver tissue sections were

investigated to determine signs of hepatic degen-eration, sinusoidal dilatation, vasocongestion andincreased activity of Kupffer cells. Among the threeroot canal sealers, EndoREZs was the most dama-ging sealer on the liver tissue. Especially on day 7and day 30, hepatocyte degeneration, sinusoidaldilalation and vasocongestion were prominent. Novasocongestion was noticed on day 1 and day 7. The


activities of Kupffer cells were generally normal atall times. There was no inflammatory tissuereaction in liver. We conclude that all of the threesealers caused only a minor degree of liver injury(Figure 1).

KidneyIn all groups, the kidney tissue sections were

investigated to determine signs of tubular degen-eration, vasocongestion, glomerular degenerationand leukocyte infiltration. Of the three sealers,GuttaFlows proved to be the least toxic withregard to cellular injury. Vasocongestion, glomeru-lar degeneration and leukocyte infiltration werenearly absent in all kidney tissue sections for alldays with GuttaFlows. In Kerr Pulp Canal Sealerand EndoREZs groups, a slight tissue injury in termsof tubular cellular degeneration was revealed(Figure 2).

LungIn all groups, lung tissue sections were evaluated

for signs of edema, intra-alveolar hemorrhage,leukocytic infiltration, vasocongestion and dis-rupted alveolar architecture. Among thosecriteria, intra-alveolar hemorrhage was not noticedin any of the experimental groups. All of the agentspresented a slight degree of lung tissue damageincluding edema, leukocytic infiltration, vasocon-

Figure 1. Micrographs of liver from (a) EndoREZs group show( ), (b) Kerr Pulp Canal Sealer and (c) GuttaFlows groups onhepatocytic degeneration ( ). (d) Control group. Stained he

gestion and disrupted alveolar architecture(Figure 3).

Skin and connective tissueSkin tissue was investigated and scored to

determine the degree of tissue degeneration atthe levels of epidermis, dermis, hair follicles andsubcutaneous tissue. Very few signs of epidermaldegeneration were seen in the experimentalgroups. The total of other scoring criteria wasnearly equal in all groups revealing only slight skintissue damage (Figure 4).

AssaysSerum AST and ALT levels were determined as a

measure of hepatic function, while BUN and serumcreatinine levels were used for the assessment ofrenal function. The experimental root canal sealersdid not change the hepatic and renal functions(Table 1). Serum lactate dehydrogenase, which wasanalyzed to evaluate generalized tissue injury, wasfound not to be different from that of the controlgroup. TNF-a levels increased significantly(po0.05) in all groups.

There were no differences in MDA levels deter-mined in the lung, liver, kidney and skin tissuesbetween the control and the experimental groups(Table 2). Similarly, GSH levels in all the studiedtissues were not affected by the root canal sealers(Table 3).

ing vacuolated hepatocytes ( ) and sinusoidal dilatationday 30 after sealer treatment showing a slight degree of

matoxylin and eosin. Bars: 10 mm.

Figure 3. Micrographs of lung showing a low degree of interalveolar fusion (-), vasocongestion ( ) and slight edemain (a) EndoREZs, (b) Kerr Pulp Canal Sealer and (c) Gutta treated groups on day 30 after sealer treatment. (d) Controlgroup. Stained hematoxylin and eosin. Bars: 10 mm.

Figure 2. Micrographs of kidney showing slightly damaged tubular cells (-) in (a) EndoREZs, (b) Kerr Pulp Canal Sealerand (c) GuttaFlows groups on day 30 after sealer treatment indicating a low degree of kidney parenchymal damage. (d)Control group. Stained hematoxylin and eosin. Bars: 10 mm.


As an indicator of the level of fibrotic activity intissues, the collagen content was analyzed andwhen compared with the control, no differenceswere seen within experimental groups (Table 4).

Discussion

Many different studies have evaluated cytotoxi-city of root canal sealers using in vitro cell culture

Figure 4. Micrographs of skin of (a) EndoREZs, (b) Kerr Pulp Canal Sealer and (c) GuttaFlows groups on day 30 aftersealer treatment showing a thinning of epithelium (-). (d) Control group. Masson’s trichrome stain. Bars: 10 mm.

Table 1. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine,lactate dehydrogenase (LDH) and TNF-a levels in all groups (n ¼ 5 per group).

ALT (mg/dl) AST (mg/dl) BUN (mg/dl) Creatinine (mg/dl) LDH (U/L) TNF-a (pg/ml)

Control 78.078.6 172716.5 36.074.5 0.5670.02 19907165 4.070.2GR24 h 76.078.5 185714.2 41.074.6 0.570.04 18907361 8.670.3�

GR7d 69.0712.3 201720.1 39.073.5 0.670.02 24217420 6.971.1�

GR30d 68.079.9 176721.5 36.072.5 0.670.02 21107165 6.970.5�

ER24h 72.0710.2 196714.2 38.073.6 0.570.06 19347125 8.771.9�

ER7d 70.0712.3 198719.5 41.273.8 0.570.01 24317156 7.571.9�

ER30d 74.078.1 168724.3 42.074.0 0.670.08 19907189 8.171.2�

KR24 h 66710.2 16879.8 34.077.5 0.570.03 22107230 6.070.4�

KR7d 61.079.8 211712.3 35.073.2 0.670.01 25007320 5.070.9KR30d 72.0712.6 198712.8 38.072.2 0.670.01 26807256 8.270.2�

GR: GuttaFlow, ER: EndoREZ, KR: Kerr Pulp Canal Sealer.�po0.05: compared with control group.


tests, or in vivo tests such as implantation intoconnective tissue, osseous implantation and peri-radicular tissue response. In vivo tests are based onclinical or histological appraisal of tissue responseand probably correlate better than the in vitro test(Langeland, 1978).

In the present study, GuttaFlows, EndoREZs andKerr Pulp Canal sealers showed slight cytotoxicityin some organs such as liver, kidney and lung. Thesephenomena occurred while the subcutaneouslyinjected sealer was in direct contact with theblood circulation in the connective tissue of thetest animals. Apparently some of the sealer

ingredients (such as UDMA, eugenol or silicon)passed into the blood stream and were carried intotissues and affected distant organs (Langeland,1978). In the present study, tissue toxicity wasfound acceptable at the end of 30 days for allgroups. Blood serum parameters were not foundstatistically different from that of the controlgroup. Moreover, the root canal sealers weresubcutaneously injected in order to avoid thetrauma due to the surgical incision for theimplantation.

Studies reported in the literature have shown thatmost dental materials pose significant cytotoxic

Table 3. Tissue glutathione (GSH; mmol/g tissue) levelsin all groups (n ¼ 5).

Lung Liver Kidney Skin

Control 2.170.2 2.670.4 2.370.3 2.570.2GR24 h 1.970.3 1.870.8 2.070.4 2.070.4GR7d 2.070.4 1.970.3 2.370.2 1.870.4GR30d 2.470.2 2.370.5 2.270.2 1.970.1ER24h 2.170.3 2.470.1 1.970.1 2.170.1ER7d 2.570.4 2.070.2 2.170.2 1.970.1ER30d 2.570.1 1.770.1 2.270.2 2.370.2KR24 h 2.470.01 2.070.2 1.870.2 2.370.2KR7d 2.370.2 2.370.2 2.270.2 2.270.3KR30d 1.970.5 2.270.3 2.170.1 2.370.1

GR: GuttaFlow, ER: EndoREZ, KR: Kerr Pulp Canal Sealer. Valuesare represented as mean7SEM. The differences are notsignificant.

Table 2. Tissue malondialdehyde (MDA; nmol/g tissue)levels in all groups (n ¼ 5 per group).

Groups Lung Liver Kidney Skin

Control 38.171.2 43.772.7 39.472.3 42.372.6GR24 h 41.372.1 47.773.6 44.774.5 40.674.3GR7d 40.372.0 44.774.3 43.272.4 47.674.2GR30d 37.071.8 48.575.0 35.676.7 46.274.1ER24h 37.973.6 47.775.6 38.072.6 41.273.7ER7d 40.471.6 47.773.2 43.175.6 46.075.1ER30d 38.972.1 50.273.9 41.275.5 44.573.4KR24 h 43. 172.3 42.572.6 41.273.4 42.972.3KR7d 39.373.1 47.075.4 42.674.4 47.674.5KR30d 41.972.5 44.273.9 39.57 6.5 45.575.3


Table 4. Tissue collagen content (mg/mg protein) of allgroups (n ¼ 5).

Lung Liver Kidney Skin

Control 19.570.4 19.670.97 9.570.3 27.071.1GR24 h 17.870.8 21.470.6 9.170.3 25.870.5GR7d 18.670.2 18.670.2 9.370.1 25.570.5GR30d 20.670.2 21.670.4 9.670.1 25.870.2ER24h 18.570.6 19.970.4 8.870.3 23.270.9ER7d 19.170.3 20.370.7 9.270.2 29.270.3ER30d 19.770.2 21.170.5 9.770.3 24.770.3KR24 h 17.970.4 20.670.4 8.770.2 25.970.7KR7d 20.870.6 21.770.7 9.470.2 28.770.5KR30d 20.970.2 20.370.4 10.570.2 27.970.4



risks, particularly in the freshly mixed condition, ina progressively decreasing time-related manner(Ørstavik and Mjor, 1988). Kardon et al. (2003),however, found in their study that EndoREZs didnot consistently set even after 7 days. In thepresent study, EndoREZs was found the mostdamaging on liver tissue at 24 h, but this harmfuleffect was alleviated with time. In the literature,there are only limited studies describing thecytotoxic effects of EndoREZs sealer.

Becce and Pameijer (2003) investigated thebiocompatibility of EndoREZs by in vitro cellculture using L929 cell lines and found a mildirritation with 60% rat survival. The result of invitro cytotoxicity tests may not highly correlatewith in vivo data. However, a limited number of invivo studies reported in the literature indicate thecytotoxicity of EndoREZs sealer. Louw et al. (2001)obturated teeth of primates with gutta-percha andEndoREZs or AH Plus (De Trey Dentsply, Konstanz,

Germany) with either short, flush or extruded rootcanal fillings and found inflammatory reaction in 90days only in overextended root fillings. Althoughlimited studies were found in literature aboutcytotoxicty of EndoREZs sealer, several studieshave been performed on urethane dimethacrylate,which is a component of EndoREZs.

Ratanasathien et al. (1995) found bisphenol-Adiglycidyl dimethacrylate (Bis-GMA) and urethanedimethacrylate to be more toxic than triethylene-glycol dimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA) in a period of 24 and 72 hafter exposing mouse fibroblasts and performing anMTT assay, which evaluates the mitochondrialenzyme activity. Kleinsasser et al. (2004) investi-gated the cytotoxicty of HEMA, TEGDMA, UDMA,Bis-GMA using a single cell microgel electrophoresis(comet) assay and found a relevant cytotoxicityeffect for all materials, but vitality levels were onlyat a critical level for Bis-GMA and TEGDMA.Regarding this information, it seems that extrac-table monomers or other ingredients in resin-basedEndoREZs sealer may produce some cytotoxiceffects at the outset, but these effects subsidewith time.

Recently introduced GuttaFlows sealer includesthe silicon-based sealer Roekoseal (RSA, Roeko,Coltene/Whaledent Inc, Langenau, Germany) com-bined with gutta-percha. Polydimethyl siloxane hasbeen used widely in prosthodontics because of itslow dimensional change (about 0.6–0.15%) and lowwater absorption. Silicon soft liners have beenproposed for use in patients with irritation of thedenture-bearing mucosa. Vulcanized or room tem-perature silicon is popular as maxillofacial materi-als for correcting facial defects because of its easeof processing and good physical properties. Alsosilicon-low temperature isotropic carbon (LTI) alloy


implants, which are usually placed on a metalgraphite substrate in the form of either a subper-iosteal implant or an endosteal blade, are found tobe very biocompatible (Philiphs, 1981). However,there are still not many reported studies on thecytotoxicity of GuttaFlows. Gerosa et al. (2003)compared the cytotoxicity of GuttaFlow with PulpCanal Sealer and resin-based Acroseal (Septodont,Saint-Maur-des-Fosses, Cedex, France) and foundGuttaFlows less cytotoxic than Pulp Canal Sealerand Acroseal. Miletic et al. (2005) found nocytotoxic effects of the silicon-based sealer Roe-koseals on HeLa cells and mouse skin fibroblasts(L929). Gencoglu et al. (2003) investigated theconnective tissue response of RSA (Roekoseals) andfound new granulation tissue with fibrous tissueadjacent to RSA on day 30 after treatment. In thepresent study, GuttaFlows showed slightly cyto-toxic effects in all the investigated organs within aperiod of 24 h and this effect decreased in time andwas found acceptable. However, a lesser degree oftissue damage was found in kidney tissue of theGuttaFlows-applied group compared to the othergroups.

Although zinc oxide eugenol (ZOE) containingsealers are the most widely used sealers inendodontics, non-specific biocompatibility testsshowed eugenol elicited a pronounced tissueirritation (Langeland, 1978). Araki et al. (1993)demonstrated that the increased cytotoxicity ofZOE sealer was caused by its eugenol content.However, Hume (1986) indicated that variations inthe powder/liquid ratio of the ZOE mixture hadonly slight effects on eugenol availability. Hedemonstrated that when ZOE was in contact withthe soft tissue, a release of eugenol in sufficentconcentrations (10�3 M) to cause local cell deathwas likely to have occurred. If the contact area ofZOE at the root apex is small, then the area of celldeath is relatively confined and the rate of eugenolrelease declines sufficiently within 1–2 weeks toallow healing. A local effect at the root apex thatmight be therapeutic would be sensory nerveinhibition or death of local nerve endings (Hume,1986).

Kolokouris et al. (1998) also demonstrated thateugenol liberation from ZOE containing compoundswas initially high immediately after mixing, butdecreased over time with a progressive decrease inconnective tissue reaction. In the present study,Kerr Pulp Canal Sealer presented a slight cytotoxi-city compared to other two sealers, but this effectsubsided and was found acceptable in living organsat the end of 30 days. The effects of sealerscomponents on living organs have also beeninvestigated by other authors. Kolokouris et al.

(1998) investigated the effects of ZOE sealer on Zn,Ca and Cu concentrations in some living organs ofrats. They found that subcutaneous injection of aZOE sealer (Roth 811) changed the concentrationsof Zn, Ca and Cu in some organs on days 4 and 5after treatment. Economides et al. (2005) foundCalciobiotic root canal sealer (CRCS, Coltene/Whaledent Inc., Cuyahoga Falls, OH, USA) and Roth811 (Roth International, Chicago, IL, USA) to induceredistribution of zinc, whereas AH26 (De TreyDentsply, Konstanz, Germany) induced changes incalcium content in some organs of rats. Theseresults indicate that ZOE sealers might releaseconsiderable amounts of those compounds, whichwere then deposited in vital organs.

It appears that all sealers may cause someirritation reactions in organs distant from the sitesof injection or application. However, after finalsetting this reaction decreases. This phenomenonwas also confirmed in the present study. Althoughthese results were conducted in experimentalanimals, they are significant as such experimentalstudies cannot easily be conducted in humans.Nevertheless further clinical studies are needed toconfirm these results and to evaluate their rele-vance to the outcome of the treatment.

Acknowledgements

We would like to honor the memory of Dr. NursalGedik, our dear friend, who passed away in 2007.Without her contribution, it would not be possibleto accomplish the current study.

This research was presented in part at the 12thBiennial Congress of the European Society ofEndodontology (September, 2005), in Dublin, Ire-land.

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