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doi:10.1111/j.1365-2591.2011.01941.

A multiparametric assay to compare the


cytotoxicity of endodontic sealers with primary
human osteoblasts

M. Z. Scelza1, A. B. Linhares2, L. E. da Silva3, J. M. Granjeiro2,4,5 & G. G. Alves2,4


1
Laboratory of Experimental Cell Culture, Department of Endodontics, Fluminense Federal University (UFF), Niteroi; 2Clinical
Research Unit, Antônio Pedro Hospital, Fluminense Federal University (UFF), Niteroi; 3Department of Statistics, Fluminense
Federal University (UFF), Niterói; 4Molecular and Cell Biology Department, Institute of Biology, Fluminense Federal University
(UFF), Niteroi; and 5National Institute of Metrology, Standardization and Industrial Quality, Duque de Caxias, Brazil

Abstract dye exclusion test). Results from each test and exper-
imental time were compared by 2-way analysis of
Scelza MZ, Linhares AB, da Silva LE, Granjeiro JM,
variance (anova).
Alves GG. A multiparametric assay to compare the cytotoxicity
Results All endodontic sealers had strong cytotoxic-
of endodontic sealers with primary human osteoblasts. Inter-
ity 24 h after mixing, according to all parameters
national Endodontic Journal, 45, 12–18, 2012.
evaluated. At a longer setting period (7 days), viability
Aim To compare the cytotoxicity of four endodontic for Sealapex was significantly increased (P < 0.05) and
sealers (Sealapex, Pulp Canal Sealer EWT, Real Seal Pulp Canal Sealer achieved levels of cytocompatibility
and MTA Fillapex) either 1 or 7 days after mixing, similar to the control group. The anova indicated a
when assessed through a multiparametric analysis general correlation between the cytotoxicity of the
employing human primary cells closely related to materials and the time after mixing, with some level of
periapical tissues. dependence on the cell viability assay employed.
Methodology Extracts of each sealer were prepared Conclusions All materials had high cytotoxic levels
following 24-h exposure to culture media, at either for human primary cells, mostly on a time-dependent
24 h or 7 days after mixing. Primary human osteo- basis, as shown by three different cell viability tests.
blasts were exposed to extracts for 24 h, at 37 C with
Keywords: biocompatibility, multiparametric assay,
5% CO2, and cell viability was evaluated by a multi-
endodontic sealer, human primary osteoblasts.
parametric assay assessing sequentially, on the same
cells, mitochondrial activity (XTT), membrane integrity Received 14 April 2011; accepted 30 July 2011
(neutral red test) and total cell density (crystal violet

cements are placed in intimate contact with periapical


Introduction
tissues for prolonged times of exposure, there is always
Several compositions for root canal sealers have been the possibility of degradation products’ leaching
developed and employed with the purpose of achieving through the dentinal tubules, lateral and accessory
more desirable physicochemical properties, as well as canals or apical foramina, which may damage both the
improved biocompatibility with periapical tissues (Al- periodontal ligament and alveolar bone (Bernath &
Hiyasat et al. 2010). These materials may be organized Szabo 2003). Therefore, it is important to know the
according to their chemical composition, which is general biocompatibility of such materials, especially
usually based on calcium hydroxide, bioceramics, zinc with regard to the tissues and cell types most related to
oxide and eugenol, or epoxy resin. As endodontic the area of ultimate concern (Haustveit et al. 1984,
Huang & Chang 2002), in this case, the root canal
Correspondence: Gutemberg Alves, R. Outeiro de São João
system.
Batista, s/n - Centro Niterói - RJ - Brazil. 24020-150 (e-mail: The relevance of the biological evaluation of medical
gutemberg_alves@id.uff.br). and dental devices may be measured by the existence of

12 International Endodontic Journal, 45, 12–18, 2012 ª 2011 International Endodontic Journal
Scelza et al. Multiparametric cytotoxicity test of endodontic sealers

several international standards dedicated to the issue four materials, representing different groups of root
(ASTM International 2007, ISO 7405:2008, Interna- canal sealer namely Sealapex (Kerr, Romulus, MI
tional Organization for Standardization 2009). The USA), Pulp Canal Sealer EWT (SybronEndo, Orange,
International Organization for Standardization (ISO) CA, USA), Real Seal (SybronEndo) and MTA Fillapex
dictates that tissue compatibility evaluation, both (Angelus, Curitiba, Brazil) and (ii) to contrast the
in vitro and in vivo, when applicable, should be results with previous reports employing different
performed as critical steps prior to the clinical applica- models. Cytocompatibility was assessed by a multi-
tion of the material or device (ISO 7405:2008). In this parametric assay employing human osteoblast in
context, in vitro cytotoxicity tests allow the employment primary culture (hOB), a cell type.
of relevant cell types and lineages, using simple,
controlled and reproducible test conditions, and in
Materials and methods
conformity with the principles of bioethics (Interna-
tional Organization for Standardization 2009). For This work is part of a project which was approved by
decades, such evaluations have been performed on the Antonio Pedro Hospital/Fluminense Federal Uni-
individual materials or in comparative studies testing versity Committee of Research Ethics. No human
most commercially available sealers such as Sealapex, beings or animal subjects were involved.
Real Seal, Pulp Canal Sealer and others (Leonardo et al.
2000, Bouillaguet et al. 2004, Huang et al. 2004,
Sample preparation
Vajrabhaya et al. 2006, Gorduysus et al. 2007, Brack-
ett et al. 2008, 2010, Gomes-Filho et al. 2009a,b, Xu Each endodontic sealer (Sealapex, Pulp Canal Sealer
et al. 2010). However, because some of the results from EWT, Real Seal and MTA Fillapex) was prepared as
such works are highly variable or even in conflict described on the manufacturers’ instructions (Table 1).
(Geurtsen 2001, Kim et al. 2010), it remains unclear Test samples consisting of conditioned media were
whether several of these materials are biocompatible or obtained according to ISO 10993-12:2007 (Interna-
how much the setting time affects biocompatibility. tional Organization for Standardization 2007). Briefly,
The issue of conflicting results raises the hypothesis 0.1 g of each sealer was immersed in 1 mL of serum-
that opposing results concerning the biocompatibility free Alpha-MEM (Gibco, Cergy-Pontoise, France) and
of some of these materials may be related to the incubated for 24 h at 37 C in a humidified chamber. A
employment of differing cell lines/types, as well as to second experimental group was obtained by preparing
the cell viability parameter assayed. Furthermore, the extracts of each sealer 7 days after preparation.
simultaneous evaluation of different cell viability
parameters may more accurately identify any possible
Multiparametric in vitro assay
cytotoxic effects of endodontic sealers with primary
human cells intimately related to the in vivo tissue Cytotoxicity was assessed in vitro according to interna-
response to endodontic sealers. Therefore, the aims of tional standards for the evaluation of dental materials
this study were (i) to compare the cytocompatibility of (ISO 7405:2008), with a multiparametric assay kit (In-

Table 1 Constituents of endodontic sealers

Product and manufacturer Composition Preparation mode

Sealapex (Kerr, Romulus, Catalyst, isobutyl salicylate resin, silicon dioxide The components were combined
MI, USA) bismuth trioxide, titanium dioxide pigment, base by mixing of equal
N-ethyl toluene solfanamide resin, silicon dioxide, portions by length of base and
zinc oxide, calcium oxide catalyst paste
Real Seal SE (SybronEndo, Bis-GMA, ethoxylated Bis-GMA, UDMA, The components were combined
Orange, CA, USA) hydrophilic monomers, fillers: calcium hydroxide, through a self-mixing tip
barium sulphate, barium glass, silica attached to a syringe
Pulp Canal Sealer EWT Powder: zinc oxide, silver, resin, thymol iodide, The components were combined
(Sybron Endo, Orange, liquid, eugenol, Canada balsam by mixing the powder
CA, USA) into liquid
MTA Fillapex (Angelus, Salicylate resin, diluting resin, natural resin, The components were combined
Curitiba, Brazil) bismuth trioxide, nanoparticulated silica, MTA, through a self-mixing tip
pigments attached to a syringe

ª 2011 International Endodontic Journal International Endodontic Journal, 45, 12–18, 2012 13
Multiparametric cytotoxicity test of endodontic sealers Scelza et al.

Cytotox, Xenometrix, Germany), which evaluates three


Statistical analysis
different cell viability parameters sequentially on the
same cell culture (De-Deus et al. 2009). Analysis of Variance (ANOVA) was employed to test
Human osteoblasts on second passage (hOB) from the interactions of three sources of variation (material,
the HUAP Cell Culture Bank were subcultured for 24 h test method and time of extraction) with the propor-
at 37 C on 96-well culture plates at an initial cell tions of viable cells for each endodontic sealer, as
density of 10 000 cells per well and subsequently compared to the control group. Differences were
exposed to the conditioned media (as described in the considered statistically significant at the alpha = 0.05
previous section) for 24 h. One group (negative control level. The Shapiro-Wilk test was employed to determine
for cytotoxicity) was exposed only to culture medium if the results and different combinations of sources of
(Alpha-MEM). Each condition was tested on three variation were normally distributed. The Mann-Whit-
replicates and three different assays. ney U test with Bonferroni correction was performed to
compare results from different experimental times (1
and 7 days). All statistical analyses were performed
Mitochondrial dehydrogenase activity
with SPSS 10 (SPSS, Inc., Chicago, IL, USA).
After the exposure of hOB to each conditioned medium,
mitochondrial dehydrogenase activity was measured
Results
by the XTT assay. This test is based on the ability of
mitochondrial enzymes from metabolically active cells Figure 1 shows cell viability as evaluated by three
to reduce 2,3-bis(2-methoxy-4 -nitro-5-sulphophenyl)- different assays, after exposure to 24-h and 7-day
2H-tetrazolium-5-carboxanilide (XTT) molecules to a extracts of the tested sealers, and expressed as a
soluble salt of formazan, detectable by its absorbance at percentage of the control (cells exposed to uncondi-
480 nm, as measured by a UV–Vis microplate reader tioned medium). As seen in panels A, B and C, all
(Synergy II; Biotek Inst., Winooski, VT, USA). sealers had strong cytotoxic effects during the first 24 h
after mixing, as measured by all three methods
employed, because all extracts induced survival rates
Membrane integrity
of <20% of the control group. No significant difference
The same cells submitted to the XTT test were washed was found between the materials (P > 0.05).
and assayed with the neutral red uptake test (NR), When cells were exposed to extracts of the endodon-
which determines the levels of viable cell through tic sealers prepared 7 days after mixing, however, the
their membrane integrity. The vital dye NR is incor- pattern of cytotoxicity changed for several of the
porated through endocytosis and accumulates prefer- materials. Sealapex increased cell survival with time
entially on the lysosomes of membrane intact viable (P < 0.05), except for XTT reduction, whilst Pulp Canal
cells. After 3 h of exposition to the dye, cells were fixed Sealer EWT completely reversed its cytotoxic effects,
and the NR extracted be extracted and measured by presenting levels of cell survival and integrity equiva-
the optical density (O.D.) of the supernatant at lent to the control group and consistently higher than
540 nm, which directly relates to the proportion of all other groups, on all parameters evaluated. Real Seal
viable cells. had higher survival rates at 7 days than MTA Fillapex
and Sealapex for XTT reduction, but not the others
methods. There was no influence of time for MTA
Cell density
Fillapex and Real Seal.
After the NR test, fixed cells were washed and evalu- Table 2 summarizes the statistical analysis of the
ated for the total density of cells adhered, as estimated sources of variation and of their interactions by means
by the crystal violet dye exclusion test (CVDE). Cells of analysis of variance (anova) to three factors, for all
were treated with concentrated crystal violet dye (CV), variables of this study. A highly significant statistical
and after exhaustive washing, the dye was extracted difference (P < 0.01) based on the main effects
with a solution containing acetic acid and ethanol. As (endodontic sealer, method of analysis and period of
CV preferentially binds to DNA, the O.D. at 540 nm of time) and on the interactions of material with time
the dye extracted after washing was directly related to and of the method with the time (P < 0.01) was
the total amount of cells on each well. noted.

14 International Endodontic Journal, 45, 12–18, 2012 ª 2011 International Endodontic Journal
Scelza et al. Multiparametric cytotoxicity test of endodontic sealers

(a)

(b)

(c)

Figure 1 Cytotoxicity assay. Cytotoxic


effects of endodontic sealers on hOB by
XTT (a), NR (b) and crystal violet tests
(c), expressed as a percentage of control
(cells exposed to culture medium). Bars
indicate mean ± SD. (*) indicates signif-
icant difference to the control group
(P < 0.05). (a) indicates statistically sig-
nificant differences (P < 0.05) between
period of time with the same endodontic
sealer.

performance of a multiparametric assay, with three


Discussion
different cell viability tests, and (ii) the use of a human
In the present study, the cytotoxicity of four commer- primary cell culture more related to the periapical
cially available endodontic sealers was tested, employ- system. The importance of the use of an adequate cell
ing an in vitro methodological strategy that differs from model, employing human primary cells of a relevant
most previous works on these materials by: (i) the type on the study of endodontic materials, has been

ª 2011 International Endodontic Journal International Endodontic Journal, 45, 12–18, 2012 15
Multiparametric cytotoxicity test of endodontic sealers Scelza et al.

Table 2 Analysis of variance (anova) of the percentage of viable cells in relation to control

Variation Source Square sum Degrees of freedom Mean square Fobs P-value

Material 157 637.2 4 39 409.29 605.21 <0.0001


Method 1251.0 2 625.48 9.61 0.0002
Time 13 506.3 1 13 506.26 207.42 <0.0001
Material · Method 817.2 8 102.15 1.57 0.1454
Material · Method 47 792.8 4 11 948.19 183.49 <0.0001
Material · Method 546.4 2 273.19 4.20 0.0181
Material · Method · Time 610.7 8 76.34 1.17 0.3246
Residual 5860.5 90 65.12
Total 228 022 119

Material, endodontic sealers; Method, cell viability assay (XTT, NR or CVDE); Time, time after mixing (1 or 7 days).

pointed out previously (Huang & Chang 2002), in the Bryan et al. (2010) reported that the toxicity exhib-
light of several expected differences in the responses ited by Pulp Canal Sealer from 24 h to 5 weeks, using
from murine, non-tissue specific or immortalized cells. MC3T3-E1 murine preosteoblasts and the MTT assay,
For this, hOB cells have been considered closest to the was coherent with findings from other works (Geurtsen
ideal cells for cytocompatibility assays because the 2001, Brackett et al. 2008, 2010). Although previous
direct interaction of these cells with repair materials studies have shown that freshly mixed sealers rapidly
could play a critical role in the clinical setting (Zhu cause cell death (Bouillaguet et al. 2004, 2006), the
et al. 2000). It is possible that such a choice may clinical use of those materials may remain safe, as they
account for the rather singular results presented in this are introduced into canals in the unset state. In this
work, possibly due to a higher sensitivity of these cells. study, the cytotoxicity of Pulp Canal Sealer EWT was
In any case, the results point to high levels of high mostly in the first 24 h, decreasing after 1 week
cytotoxicity to human cells with all endodontic sealers for all parameters tested. According to the manufac-
tested. turer, setting of Pulp Canal Sealer EWT occurs between
Regarding Sealapex, the results disagree with previ- 60 and 120 min. Such a short setting time may explain
ous studies that showed this material to be noncyto- the higher biocompatibility when the extraction was
toxic at 24 h after mixing (Gomes-Filho et al. 2009b). performed at longer periods (1 week), when possi-
This might be one case where divergent results may bly there are no unrelated or degradation products
arise from the diverse experimental conditions, because released into the surrounding medium.
Gomes-Filho et al. (2009b) employed L929 mouse Several studies on the biocompatibility of Real Seal
fibroblasts and MTT assay, in contrast to the conditions produced highly variable results (Kim et al. 2010), with
described on the present work. There are reports of reports of either a severe toxicity (Bouillaguet et al.
divergent responses and sensibilities for the XTT and 2006) or a noncytotoxicity even at longer expositions
MTT assays on a cell-type-dependent basis (Scudiero (Scotti et al. 2008). According to the cell viability
et al. 1988). It was also reported that these methods, in parameters studied, this material is highly cytotoxic to
reality, do not evaluate the same enzyme systems, human osteoblasts at both 1 and 7 days. One expla-
because evidence was found that MTT reduction is nation for the Real Seal cytotoxicity may be related to
associated not only with mitochondria but also with its high resin content and possible incomplete poly-
the cytoplasm and with nonmitochondrial membranes merization of methacrylate, releasing toxic monomers
including the endosome/lysosome compartment and (Bouillaguet et al. 2006).
the plasma membrane (Berridge et al. 2005). However, MTA Fillapex was created in an attempt to combine
the results presented by XTT in this work are corrob- the physicochemical properties of a root canal sealer
orated by two other cell viability parameters, one of with the biological properties of MTA. The MTA-based
those (NR) recognized as more sensitive than MTT sealer, Endo-CPM-Sealer (COM Sealer; EGEO S.R.L.,
(Husoy et al. 1993, Putnam et al. 2002). A possible Buenos Aires, Argentina), according to the manufac-
explanation for the cytotoxicity of Sealapex comes from turer, exhibits similar chemical composition and so has
the calcium hydroxide, which produces high pH similar clinical indications as MTA. Endo-CPM Sealer
(Leonardo et al. 2000, Huang et al. 2004, Eldeniz et al. contains, besides CaCl2, calcium carbonate as a pH-
2007). reducing element, restricting the surface necrosis in

16 International Endodontic Journal, 45, 12–18, 2012 ª 2011 International Endodontic Journal
Scelza et al. Multiparametric cytotoxicity test of endodontic sealers

contact with the material and allows for the action of Berridge MV, Herst PM, Tan AS (2005) Tetrazolium dyes as
alkaline phosphatase and consequently the deposition tools in cell biology: new insights into their cellular
of mineralized tissue (Gomes-Filho et al. 2009a). Endo- reduction. Biotechnology Annual Reviews 11, 127–52.
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Krejci I (2004) Cytotoxicity and sealing properties of four
that can be explained by the similarity in its compo-
classes of endodontic sealers evaluated by succinic dehy-
sition and MTA based on Portland cement (Gomes-
drogenase activity and confocal laser scanning microscopy.
Filho et al. 2009b), which was already shown to be European Journal of Oral Sciences 112, 182–7.
biocompatible in culture of fibroblasts (Vajrabhaya Bouillaguet S, Wataha JC, Tay FR, Brackett MG, Lockwood PE
et al. 2006, Gorduysus et al. 2007). According to the (2006) Initial in vitro biological response to contemporary
present results, MTA Fillapex strongly affected cell endodontic sealers. Journal of Endodontics 32, 989–92.
viability in all assays employed (XTT, NT and CVDE) Brackett MG, Marshall A, Lockwood PE et al. (2008) Cytotox-
and for both experimental times. However, it remains icity of endodontic materials over 6-weeks ex vivo. Interna-
to be verified how Endo-CPM Sealer would perform tional Endodontic Journal 41, 1072–8.
with primary human cells in the multiparametric Brackett MG, Messer RL, Lockwood PE et al. (2010) Cytotoxic
model used in this study. response of three cell lines exposed in vitro to dental
endodontic sealers. Journal of Biomedical Materials Research
Part B Applied Biomaterials 95, 380–6.
Conclusion Bryan TE, Khechen K, Brackett MG et al. (2010) In vitro
osteogenic potential of an experimental calcium silicate-
High levels of cytotoxicity were detected for represen- based root canal sealer. Journal of Endodontics 36, 1163–
tatives of the major groups of endodontic sealers when 9.
evaluated by more than one cell viability parameter De-Deus G, Canabarro A, Alves G, Linhares A, Senne MI,
and employing primary human cells. The results imply Granjeiro JM (2009) Optimal cytocompatibility of a bioce-
to the need for either (i) more efforts on the search for ramic nanoparticulate cement in primary human mesen-
the ideal biocompatible endodontic sealer or (ii) a chymal cells. Journal of Endodontics 35, 1387–90.
profound reconsideration on the current in vitro cyto- Eldeniz AU, Erdemir A, Kurtoglu F, Esener T (2007) Evalua-
toxicity-testing approaches. As much of the variability tion of pH and calcium ion release of Acroseal sealer in
comparison with Apexit and Sealapex sealers. Oral Surgery
on the findings concerning endodontic sealers biocom-
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patibility might come from the experimental model
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chosen, it would be useful for researchers to prefer Geurtsen W (2001) Biocompatibility of root canal filling
those models employing human primary cells and materials. Australian Endodontic Journal 27, 12–21.
multiparametric evaluations. Gomes-Filho JE, Watanabe S, Bernabe PF, de Moraes Costa MT
(2009a) A mineral trioxide aggregate sealer stimulated
mineralization. Journal of Endodontics 35, 256–60.
Acknowledgements
Gomes-Filho JE, Watanabe S, Gomes AC, Faria MD, Lodi CS,
This research was financially supported by the Brazilian Penha Oliveira SH (2009b) Evaluation of the effects of
governmental entities FAPERJ (under the process no. endodontic materials on fibroblast viability and cytokine
E-26/110.909/2009), CNPq, FINEP, MCT and Decit- production. Journal of Endodontics 35, 1577–9.
Gorduysus M, Avcu N, Gorduysus O et al. (2007) Cytotoxic
MS.
effects of four different endodontic materials in human
periodontal ligament fibroblasts. Journal of Endodontics 33,
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