Basic Research—Technology
Short-Term Cytotoxicity Assessment of Components of the
Epiphany Resin-Percha Obturating System by Indirect
and Direct Contact Millipore Filter Assays
Khalid Merdad, BDS, MSc, FRCD(C),* Alvaro Elizeu Pascon, DDS, MSD, PhD,*
Gajanan Kulkarni, BDS, LLB, MSc, DPedDent, PhD, FRCD(C),‡
Paul Santerre, BSc, MScE, PhD,† and Shimon Friedman, DMD*
Abstract
The Epiphany Resin-Percha Obturating System was as-
sessed for cytotoxicity, compared with gutta-percha
and AH-Plus sealer. Specimen disks (Resilon, gutta-
percha), filled glass rings (sealers), or imbibed cellulose
disks (primer, thinning resin) were placed over Millipore
filters in direct or indirect contact with HeLa cell mono-
layer, incubated for 2 hours, and stained with tetrazo-
lium blue. Cytotoxicity was rated by the surrounding
unstained zone: none (0 mm), mild (ⱕ7 mm), moderate
(7–12 mm), or marked (⬎12 mm). Data were analyzed
with one-way ANOVA and post hoc pairwise t tests.
Unstained zones indicating moderate cytotoxicity were
significantly larger (p ⬍ 0.05) for Epiphany primer than
for thinning resin and for freshly mixed AH-Plus than for
Epiphany sealer. Set sealers (24 and 48 hours), gutta-
percha, and Resilon elicited noncytotoxic responses. In
conclusion, cytotoxicity of set Epiphany sealer and Re-
silon was comparable with that of set AH-Plus and
gutta-percha. Cytotoxicity of freshly mixed Epiphany
sealer, primer, and thinning resin did not exceed that of
freshly mixed AH-Plus. (J Endod 2007;33:24 –27)
Key Words
AH-Plus sealer, cytotoxicity, Epiphany Resin-Percha Ob-
turating System, gutta-percha
From the *Department of Endodontics, †Department of
Biological and Diagnostic Sciences/Biomaterials, and ‡Depart-
ment of Pediatric and Preventive Dentistry, Faculty of Den-
tistry, University of Toronto, Ontario, Canada.
Address requests for reprints to Dr. Shimon Friedman,
Faculty of Dentistry, 124 Edward Street, Toronto, Ontario M5G
1G6, Canada. E-mail address: s.friedman@utoronto.ca.
0099-2399/$0 - see front matter
Copyright © 2007 by the American Association of
Endodontists.
doi:10.1016/j.joen.2006.10.003
24 Merdad et al.
R oot filling materials should be biocompatible (1), to avoid periradicular inflamma-
tion in reaction to leaching toxic products. Such response may delay healing and
adversely affect the outcome of treatment (2). In addition, root filling materials should
possess adequate sealing properties (1), to seal the root canal system against bacterial
ingress (3, 4).
Conventional root filling materials include a core material and a sealer. Gutta-
percha, the common core, was reported to be less toxic than other root filling materials
tested (5), and is the gold standard for assessing alternative core materials. However, it
has poor sealing properties (6). A frequently used type of sealer, the epoxy resin-based
AH 26 and AH-Plus (Dentsply DeTrey, Konstanz, Germany), is cytotoxic when freshly
mixed (7). AH-Plus, being free of formaldehyde, is less cytotoxic than AH 26 (7). Other
sealers are also cytotoxic to a varying degree (2, 8).
The resin-based Epiphany Resin-Percha Obturating System (Pentron Clinical
Technologies, Wallingford, CT) includes Resilon, a thermoplastic, synthetic polyester
polymer core material, containing fillers of bioactive glass, bismuth oxychloride, and
barium sulfate. The Epiphany sealer is a dual curable composite resin, with a matrix of
a BisGMA, ethoxylated BisGMA, UDMA, and hydrophilic difunctional methacrylates, and
fillers of calcium hydroxide, barium sulfate, barium glass, and silica.
In vitro studies of the Epiphany system have suggested an improved seal against
bacterial ingress over gutta-percha and AH 26 sealer (9, 10), and modest reinforcement
of teeth against vertical root fracture (11). Both findings have been attributed to bond-
ing of Epiphany sealer to root dentin and the Resilon core, resulting in a monoblock of
dentin-sealer-core (10, 11). In an animal study (12), coronally inoculated teeth filled
with the Epiphany system developed less periapical inflammation than teeth filled with
gutta-percha and AH 26 sealer. The short time allowed for bacterial ingress suggested
that the inflammation could be interpreted as a tissue response to the material rather
than to bacterial ingress (13–15). Thus, the Epiphany system may have displayed better
tissue compatibility than the AH 26 sealer control. All of the above (9, 10 –12) suggest
potential advantages for the clinical application of the Epiphany system.
The purpose of this study was to evaluate the cytotoxicity of the Epiphany system’s
components using the indirect contact Millipore filter assay, and in a modified, direct
contact assay.
Materials and Methods
The methodology of the indirect contact Millipore filter assay strictly followed the
ANSI/ADA (16) and FDI (17) guidelines, defining the sample size (n ⫽ 10), cell type
(HeLa), and test specimens weight (ⱕ3.5 g) and diameter (7 mm).
Cell Cultures
HeLa cells (CCL-2; ATCC, Manassas, VA) were grown in Eagle’s ␣-minimum es-
sential medium with 10% fetal bovine serum and antibiotics (penicillin G 200 ␮g/ml,
streptomycin 200 ␮g/ml). Cell cultures were incubated at 37°C, 5% CO2, and 100%
humidity, with the medium changed every other day.
JOE — Volume 33, Number 1, January 2007

Millipore Filters
Millipore filter disks (47 mm diameter, 0.45 ␮m pore size; Milli-
pore Filter Corporation, Bedford, MA), were placed in 50-mm tissue
culture dishes (Falcon, Becton Dickinson, Franklin Lakes, NJ), and
covered with 6 ml of cell suspension (1.5 ⫻ 105 cells/ml). Culture
dishes were incubated for 24 hours at 37°C, 100% humidity, 5% CO2.
Specimens
Test materials were prepared according to manufacturers’ direc-
tions, and specimens were formed as follows:
1. Core materials: Resilon and gutta-percha (Obtura Spartan, Fen-
ton, MO) were molded into flat discs.
2. Sealers: Epiphany sealer and AH-Plus were placed in glass rings
(5 mm height). They were tested freshly mixed, then incubated at
37°C, 90 ⫾ 10% humidity and tested after 24 and 48 hours.
Epiphany sealer specimens were covered on both sides with My-
lar sheets to prevent contact with air and light-cured from both
sides for 20 seconds before incubation.
3. Primer and thinner: Epiphany primer and thinning resin were
imbibed in cellulose disks using a constant volume of 0.1 ml.
4. Epiphany system components combined: The sealer and primer
were mixed and placed into glass rings, Resilon was inserted in
the center, and the specimens were processed the same as the
sealers.
Figure 1. Direct cytotoxicity test with the Millipore filter test. (1, 2) Agar placed around the glass rings filled with the test materials. (3) The Millipore filter with HeLa
cells placed cell-side down in the culture dish, over the agar medium and test materials. (4) A second layer of agar placed on top of the filter to provide nutrition to
the cells.
JOE — Volume 33, Number 1, January 2007
Basic Research—Technology
Indirect Assay
Millipore filters with HeLa cell monolayer were rinsed once in
phosphate-buffered saline (PBS) and placed cell-side down on a cul-
ture dish containing nutrient agar (Difco, Becton Dickinson, Sparks,
MD). Three or four specimens were placed on the filters, and the dishes
were incubated for 2 hours at 37°C, humidified atmosphere of 5% CO2.
After incubation, specimens were discarded; filters were removed,
rinsed in PBS, and incubated overnight in a nitro blue tetrazolium dye
solution (Aldrich Chemical, Milwaukee, WI). They were then rinsed in
distilled water, air-dried, and assessed for staining pattern.
Direct Assay
The reproducibility of the protocol was verified in preliminary
tests. Similar materials and procedures were used as in the indirect
assay; however, specimens were placed at the bottom of a culture dish
and nutrient agar was added flush with the top of the specimen. Milli-
pore filters with HeLa cell monolayer were placed cell-side down over
the agar and embedded specimens, and covered with a second agar
layer (Fig. 1).
Controls
Negative controls were comprised of filters with cells placed on
agar without material specimens, and filters without cells placed on
agar with specimens, to assess interactions between test materials
Cytotoxicity of the Epiphany Resin-Percha Obturating System 25
Basic Research—Technology
and filters. The positive control was comprised of filters with cells
placed on agar with zinc oxide– eugenol and formocresol speci-
mens (18).
Analysis
Filters were examined for unstained zones at the material contact
areas. Increased unstained zone diameter indicated increased cytotox-
icity: (a) no unstained zone, noncytotoxic response; (b) ⬍7 mm, mild
response; (c) 7 to 12 mm, moderate response; and (d) ⬎12 mm,
marked response.
Testing was repeated three times, and mean and standard devia-
tion values were calculated for each test material and control. Data were
analyzed with one-way ANOVA and post hoc pair-wise t tests (p ⬍ 0.05).
Results
Results of both assays are plotted in Fig. 2. The positive control
elicited a marked cytotoxic response, whereas both negative controls
showed a noncytotoxic response (one column in graph). Both freshly
mixed sealers elicited a moderate cytotoxic response, with significantly
larger (p ⬍ 0.05) unstained zones around AH-Plus specimens than
around Epiphany specimens. The 24- and 48-hour set sealers, Resilon,
and gutta-percha were all characterized by noncytotoxic response. The
combined Epiphany system elicited similar responses as the Epiphany
sealer alone.
Epiphany primer and thinning resin elicited a moderate cytotoxic
response, with significantly larger (p ⬍ 0.05) unstained zones around
the primer.
Discussion
Ideally, in vitro testing of biomaterials should match cell popula-
tions to those of the typical interaction site (19). However, investigators
(20, 21) have reported comparable results using different cell lines to
assess cytotoxicity. Rather than developing a specific cell line for this
study, the well-established HeLa cell line was used. It is recommended
in ANSI/ADA (16) and FDI (17) guidelines and ensures uniform cell
behavior and reproducibility of results, facilitating comparisons across
studies and test materials.
The indirect Millipore filter assay evaluates the effect on cells of
substances leaching from test materials. However, because materials
may exert a different effect on contact, the direct assay was developed to
complement the cytotoxicity assessment. In this assay, the filter-sup-
ported cells were placed passively over the test materials, avoiding the
concern of specimen weight effects on the cells.
Figure 2. Results of direct and indirect cytotoxicity assays using the Millipore
filter test in vitro. The diameter of the unstained zone is the measure of cytotox-
icity. P Ctrl, positive control; N Ctrl, negative control; Epi, Epiphany sealer; Thin
res, thinning resin.
26 Merdad et al.
Gutta-percha has been the material of choice for root canal filling,
in part because of its acceptable biocompatibility; therefore, it was
selected as the standard for comparison with Resilon. The noncytotoxic
response to gutta-percha observed after 2 hours was in agreement with
a previous study (5), where gutta-percha was noncytotoxic at 4 hours of
cell–material contact. However, in the previous study, a marked cyto-
toxicity was observed at 24 hours, suggested to be caused by zinc oxide
leaching out of gutta-percha over time (5).
The noncytotoxic response to Resilon after 2 hours suggested that
it was not more cytotoxic than gutta-percha in the short term. Changes
in cytotoxicity over time, as those observed for gutta-percha (5), were
not addressed by the present study. Thus the long-term compatibility of
Resilon requires further investigation.
AH-Plus was selected as the control sealer because of its wide-
spread use despite documented cytotoxicity when freshly mixed (8, 22).
It showed moderate cytotoxic effect immediately after mixing, but none
at 24 hours after mixing. Similarly, in a previous study (8) the initial
cytotoxicity of freshly mixed AH-Plus was undetectable after 4 hours.
The short-term cytotoxicity of AH-Plus has been attributed to release of
formaldehyde (23) and, to a lesser extent, to amines added to acceler-
ate the polymerization reaction (23, 24). The release of formaldehyde
appears to subside after setting, usually within 8 hours at 37°C (24).
Freshly mixed Epiphany sealer showed a moderate cytotoxic effect,
less than that of AH-Plus and undetectable at 24 hours. The initial cyto-
toxicity could be related to the monomer constituents of the dentine-
bonding agents, Bis-GMA and urethane dimethacrylate (UDMA) (25),
as well as the precursor Bis-MA. In addition, it could be aggravated by
the triethyleneglycol dimethacrylate (TEGDMA) component (26).
Difficulties were encountered during this in vitro experiment in
setting of the Epiphany sealer. Epiphany sealer requires body tempera-
ture and total elimination of air contact to set. It sets in 30 minutes in an
anaerobic environment, but in the presence of air, setting takes a week
and an uncured layer remains on the surface (27). This unpolymerized
monomer oxygen inhibition layer can form on the surface of resins
during polymerization (28). It significantly influences the biological
properties of resins (29) and has been implicated in increased toxicity
(30, 31).
The Epiphany self-etch primer is used to condition the canal walls
for bonding to the Epiphany sealer. It had a moderate cytotoxic effect,
greater than that of the Epiphany sealer and consistent with that of other
self-etch primers (25, 32). The cytotoxic effect of a self-etch primer
might be irreversible (32). Nevertheless, in clinical applications the risk
of exposing the periapical tissues to the toxicity of the primer is very low,
because the primer is coated on the canal walls with a paper point that
does not exceed the terminus of the root canal.
The thinning resin is the base part of the Epiphany sealer without
fillers, mixed with the sealer to adjust viscosity. Indeed, the cytotoxic
response to the thinning resin was similar to that of the freshly mixed
Epiphany sealer.
Root canal sealers are inserted into the canal space freshly mixed.
Toxicity of the sealer, even if it is short-lived, may provoke a transient
periapical inflammatory response (33) that may delay the healing pro-
cess (2). Therefore, root filling materials must successfully pass a clin-
ical risk assessment before they are applied clinically (34). Tissue com-
patibility is a critical component of this assessment (34), and in vitro
cytotoxicity assays are helpful as an initial step. This in vitro study indi-
cated that freshly mixed Epiphany sealer was less cytotoxic than the
widely used AH-Plus sealer, although both showed moderate cytotoxic-
ity. Notably, both sealers were noncytotoxic after 24 hours. Neverthe-
less, these in vitro results cannot be extrapolated to in vivo situations, as
materials cannot be biologically characterized by a single in vitro assay.
The current work should be followed with in vivo sensitization, implan-
JOE — Volume 33, Number 1, January 2007

tation, and mucosal irritation tests, and a usage test (16, 17, 34), to
biologically characterize the novel Epiphany Resin-Percha Obturating
System.
Acknowledgments
This study was supported by grants from the Canadian Acad-
emy of Endodontics Endowment, and Pentron Clinical Technolo-
gies.
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