materials

Article
Cytotoxicity of Light-Cured Dental Materials
according to Different Sample Preparation Methods
Myung-Jin Lee 1,2 , Mi-Joo Kim 1 , Jae-Sung Kwon 1 , Sang-Bae Lee 3, * and Kwang-Mahn Kim 1,2, *
1 Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of
Dentistry, Seoul 03722, Korea; [email protected] (M.-J.L.); [email protected] (M.-J.K.);
[email protected] (J.-S.K.)
2 Brain Korea 21 PLUS Project, Yonsei University College of Dentistry, Seoul 03722, Korea
3 Dental Device Testing and Evaluation Center, Yonsei University College of Dentistry, Seoul 03722, Korea
* Correspondence: [email protected] (S.-B.L.); [email protected] (K.-M.K.);
Tel.: +82-2-2228-3089 (S.B.L.); +82-2-2228-3082 (K.M.K.); Fax: +82-2-364-9961 (K.M.K.)

Academic Editor: Mohan Jacob

Received: 16 January 2017; Accepted: 9 March 2017; Published: 14 March 2017

Abstract: Dental light-cured resins can undergo different degrees of polymerization when applied
in vivo. When polymerization is incomplete, toxic monomers may be released into the oral cavity.
The present study assessed the cytotoxicity of different materials, using sample preparation methods
that mirror clinical conditions. Composite and bonding resins were used and divided into four
groups according to sample preparation method: uncured; directly cured samples, which were cured
after being placed on solidified agar; post-cured samples were polymerized before being placed
on agar; and “removed unreacted layer” samples had their oxygen-inhibition layer removed after
polymerization. Cytotoxicity was evaluated using an agar diffusion test, MTT assay, and confocal
microscopy. Uncured samples were the most cytotoxic, while removed unreacted layer samples
were the least cytotoxic (p < 0.05). In the MTT assay, cell viability increased significantly in every
group as the concentration of the extracts decreased (p < 0.05). Extracts from post-cured and removed
unreacted layer samples of bonding resin were less toxic than post-cured and removed unreacted
layer samples of composite resin. Removal of the oxygen-inhibition layer resulted in the lowest
cytotoxicity. Clinicians should remove unreacted monomers on the resin surface immediately after
restoring teeth with light-curing resin to improve the restoration biocompatibility.

Keywords: biocompatibility; cytotoxicity; oxygen-inhibition layer; sample preparation; resin-based

materials

1. Introduction
Resin-based dental materials are widely used in restorative dentistry, owing to their many desirable
qualities, including excellent esthetic outcome, easy handling, favorable mechanical properties, and
improved bonding efficiency [1–3]. However, concerns remain over the biocompatibility of unreacted
resin monomers following incomplete polymerization of such light-cured materials [4–6]. Extracts from
resin composites have been reported to have genotoxic, mutagenic, and estrogenic effects [7–11].
Therefore, toxicity levels need to be experimentally determined to clarify the safety of resin-based
dental materials in clinical settings [7,8,12,13].
There have been contradictory reports concerning the biocompatibility of resin-based materials.
Ruey-Song Chen et al. [14] reported the cytotoxicity of three dentin bonding agents on human dental
pulp cells. In contrast, Alexander Franz et al. [15] reported no significant cytotoxic effects of dental
bonding substances on L929 cells. Another study tested the same resin-based materials and reported
very different results [16]. However, these studies differed in sample preparation methods, cell lines,

Materials 2017, 10, 288; doi:10.3390/ma10030288 www.mdpi.com/journal/materials

Materials 2017, 10, 288 2 of 12

application methods, and types of products. Perhaps most importantly, different products undergo
different degrees of conversion and, thus, produce different amounts of monomer extract, thereby
leading to large variations in cytotoxicity [10,17–19]. Therefore, standardized sample preparation and
curing procedures are necessary to assess the actual toxicity of dental resin-based materials.
The international standard ISO 7405, which addresses the biocompatibility of dental materials
and devices, states that biocompatibility tests should be performed on materials in an “as-used state.”
In the case of light-curing materials, the standards recommend that light curing be performed in the
presence of oxygen, reflecting the conditions that are experienced in clinical use [20]. According to the
recommendations, light-curable composites should be polymerized using a light-curing unit for 20 s
and then used in biocompatibility tests. However, complete curing is not always possible in clinical
practice because of the existence of saliva or anatomical problems [21,22]. The incomplete curing thus
leads to the release of cytotoxic leachable monomers. Hence, toxicity should be tested in vitro and
in vivo to elucidate the actual effects of dental materials.
Accordingly, in the present study, we evaluated the cytotoxicity of two types of light-curing resin
under four different conditions, including uncured, direct light-cured, post-cured, and post-cured
samples, with the removal of the oxygen-inhibition layer. We aimed to assess the cytotoxicity of
resin-based dental materials under conditions that closely mimic those encountered in clinical practice.

2. Materials and Methods

2.1. Test Cells

L929 mouse fibroblasts (Korean Cell Line Bank, Korea) were cultivated in RPMI 1640 (Sigma,
Irvine, CA, UK) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1%
penicillin/streptomycin (Invitrogen, Grand Island, NY, USA) in a humidified incubator with 5% CO2
at 37 ◦ C. Confluent cells were detached using 0.25% trypsin (Sigma, St. Louis, MO, USA), and aliquots
of separated cells were sub-cultured. Cells between the 7th and 14th passages were used for the
experimental procedures.

2.2. Test Materials and Sample Preparations

Two types of dental light-curing materials, composite resin (C groups; 3M ESPE Filtek™ Z350XT,
St. Paul, MN, USA) and bonding resin (B groups; 3M ESPE Adper Scotchbond™, St. Paul, MN, USA),
were used in the present study (Table 1); the selection of test materials was based on the previous
studies [2,15,17]. The test specimens were prepared to a thickness of 2 mm and a diameter of 5 mm in
a Teflon mold. They were sterilized using ethylene oxide gas treatment. In consideration of clinical
process of using the materials, these were divided into four different conditions of sample preparation,
as follows (Table 2): group 1—uncured (CU, BU) samples were used without being polymerized; group
2—direct light cured (CD, BD) samples were placed on the solidified agar without being polymerized
and cured directly using a light curing unit (3M Elipar Free Light 2, St. Paul, MN, USA, 650 mW/cm2 )
for 20 s at a distance of 3 mm from the samples according to manufacturer’s instructions; group
3—post-cured (CP, BP) samples were used after being polymerized; group 4—“removed unreacted
layer” (CR, BR) samples were polymerized using a translucent polyethylene film to protect the resin
materials from oxygen exposure. Following sample preparation, the samples were polished using
#1500-grit silicon carbide paper for 15 s. A schematic illustration of the sample preparation methods is
shown in Figure 1.
Materials 2017, 10, 288 3 of 12
Materials 2017, 10, 288 3 of 12

Figure 1. A representative illustration of the different sample preparation methods. (a) Samples were
Figure 1. A representative illustration of the different sample preparation methods. (a) Samples were
used without any curing; (b) samples were cured on agar with a light curing unit for 20 s; (c) samples
used without any curing; (b) samples were cured on agar with a light curing unit for 20 s; (c) samples
were used
were usedafter
afterpolymerization;
polymerization; (d)(d) samples
samples were
were polymerized
polymerized andand the unreacted
the unreacted resin resin monomer
monomer layer
layer was removed afterward.
was removed afterward.

Table 1. Summary of commercially available materials used for the cytotoxicity evaluation.
Table 1. Summary of commercially available materials used for the cytotoxicity evaluation.
Product Manufacturer Type Formulation
Product Manufacturer Type Formulation
Bis-GMA, UDMA, Bis-EMA, PEGDMA, TEGDMA
3M ESPE, St. Paul, Composite Bis-GMA, UDMA, Bis-EMA, PEGDMA,
Filtek™ Z-350XT resins, combination of 20 nm silica filler, 4 to 11 nm
MN,
3M USA
ESPE, St. Paul, resin TEGDMA resins, combination of 20 nm
Filtek™ Z-350XT Composite resin zirconia filler, zirconia/silica cluster filler
MN, USA silica filler, 4 to 11 nm zirconia filler,
Adper 3M ESPE, St. Paul, Bonding Bis-GMA, UDMA, HEMA, glycerol
zirconia/silica dimethacrylate
cluster filler
Scotchbond™ MN, USA resin (GDMA), modified polyacrylic acid, ethanol, water
Bis-GMA, UDMA, HEMA, glycerol
Adper 3M ESPE, St. Paul,
Bonding resin dimethacrylate (GDMA), modified
Scotchbond™ MN, USA
polyacrylic acid, ethanol, water
Table 2. Experimental conditions.

Product Material Group Code Method of Sample Preparation Curing Condition

Table 2. Experimental conditions.
CU Uncured No polymerization
Filtek™
Composite CD Direct cured
Method of Sample Direct light curing
Product MaterialC Group Code Curing Condition
resin CP Preparation
Post-cured Indirect light curing
Z-350XT CURemoved unreacted
Uncuredlayer NoIndirect
polymerization
Filtek™ CR light curing
Composite CD Direct cured Direct light curing
C
BU Uncured No polymerization
resin CP Post-cured Indirect light curing
AdperZ-350XT CR Removed unreacted layer Indirect light curing
Bonding BD Direct cured Direct light curing
B BU Uncured NoIndirect
polymerization
Adper resin BP Post-cured light curing
Scotchbond™ Bonding resin
BD Direct cured Direct light curing
B
BR BPRemoved unreacted
Post-curedlayer Indirect
Indirect lightlight curing
curing
Scotchbond™
BR Removed unreacted layer Indirect light curing
2.3. Degree of Conversion (DC)
The measurements of the DC (n = 5) were evaluated by Fourier Transform Infrared Spectroscopy
(FTIR; Vertex 70, Bruker Optik, Ettlingen, Germany). The spectrometer was coupled to a horizontal
attenuated total reflectance (ATR) device consisting of a diamond crystal 2 mm in diameter
(Platinum ATR-QL, Bruker Optik, Baden-Württemberg, Germany). The diameter of the measured
Materials 2017, 10, 288 4 of 12

2.3. Degree of Conversion (DC)

The measurements of the DC (n = 5) were evaluated by Fourier Transform Infrared Spectroscopy
(FTIR; Vertex 70, Bruker Optik, Ettlingen, Germany). The spectrometer was coupled to a horizontal
attenuated total reflectance (ATR) device consisting of a diamond crystal 2 mm in diameter (Platinum
ATR-QL, Bruker Optik, Baden-Württemberg, Germany). The diameter of the measured surface was
800 µm, the wave number range of the spectrum was 2000–1400 cm−1 and the FTIR spectra were
recorded with two scans/s at a resolution of 4 cm−1 . To determine the percentage of the remained
unreacted double bonds, the DC was assessed as the variation of the absorbance intensities peak area
ratio of the methacrylate car-bon double bond (peak 1634 cm−1 ) and those of an internal standard
(aromatic carbon double bond; peak at 1608 cm−1 ) during polymerization, in relation to the uncured
material [23]:
!
1634 cm−1 /1608 cm−1 Peak height after curing


DCPeak height (%) = 1 − × 100 (1)
(1634 cm−1 /1608 cm−1 )Peak height before curing

2.4. Eluent Preparation

Preparation of extracts from the samples was performed in accordance with international
standards [24]. Because groups 2 and 3 had the same eluents, the extracts were divided as follows:
“Uncured”, “Post-cured”, and “Removed unreacted layer”. Despite the limitation of extraction for
hydrophobic material used in this study, choice of eluent was based on previous studies [2,8,18] and
consideration of cellular experiment. The appropriate amount of each sample was added to a culture
medium (RPMI 1640; Gibco, Grand Island, NY, USA) supplemented with 10% FBS inside a sterilized
glass bottle. The extraction ratio was 0.2 g/mL, according to ISO 10993-12. The samples and extraction
solution were incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 for 24 h. After incubation,
the culture medium containing material extracts was filtered through 0.22 µm cellulose acetate filters
(Millipore, Sigma, St. Louis, MO, USA) and the extracts were used for the cytotoxicity testing in the
MTT assay.

2.5. Agar Diffusion Test

This test was performed to evaluate the nonspecific cytotoxicity of test materials after diffusion
through agar according to ISO 10993-5 [24]. Cell suspensions (2.5 × 105 cells/mL) in a 10 mL volume
were seeded in 100 mm diameter cell culture dishes (SPL, Pocheon-Si, Gyeonggi-Do, Korea) and
incubated at 37 ◦ C in a humidified atmosphere with 5% CO2 . After 24 h, the medium was replaced
with 10 mL of freshly prepared agar medium containing 2× RPMI 1640 (Sigma, Irvine, Ayrshire,
UK). Following solidification of the liquid culture medium, 10 mL of neutral red solution (0.01% in
phosphate-buffered saline, Sigma, St. Louis, MO, USA) was added in the dark for 20 min. Excess
neutral red solution was aspirated, and the test specimens were placed on the agar surface along with
the positive (latex sheet) and negative controls (Teflon mold) in the same cell culture dish.
After 24 h of incubation, the decolorization index and lysis index were assessed using an optical
microscope according to ISO 7405 [20]. The decolorized zones were scored as follows: 0 = no
decolorization detectable; 1 = decolorization only under the specimen; 2 = decolorization in a zone not
greater than 5 mm from the specimen; 3 = decolorization in a zone not greater than 10 mm from the
specimen; 4 = decolorization in a zone greater than 10 mm from the specimen; 5 = the total culture is
decolorized. Cell lysis was defined as a loss of cell membrane integrity, visible under light microscopy.
Cell lysis was scored as follows: 0 = no cell lysis detectable; 1 = less than 20% cell lysis; 2 = 20%–40%
cell lysis; 3 = 40%–60% cell lysis; 4 = 60%–80% cell lysis; 5 = greater than 80% cell lysis. The test was
performed in quadruplicate.
Materials 2017, 10, 288 5 of 12

2.6. MTT Assay

The MTT assay is a colorimetric assay used to measure cell viability. Yellow water-soluble MTT
is metabolically reduced in viable cells to a blue-violet insoluble formazan. This test was performed
in accordance with ISO 10993-5 [24]. The L929 cells were seeded at a density of 1 × 105 cells/mL
into 96-well plates (SPL, Pocheon-Si, Gyeonggi-Do, Korea) and maintained in culture for 24 h to form
a semi-confluent monolayer. The culture medium was removed from the wells, and 100% extractions
from samples or serial dilutions of extractions using culture medium (50%, 25%, 12.5%, and 6.25%)
in a 100 µL volume were placed into each well. After 24 h, the culture medium was removed and
replaced with 50 µL of MTT solution in phosphate-buffered saline (PBS, 1 mg/mL). The MTT solution
was then discarded and 100 µL of isopropanol was added to each well. The plates were shaken until
all crystals were dissolved. The absorbance was spectrophotometrically measured using an ELISA
reader (Epoch, BioTek, Winooski, VT, USA) at 570 nm. The assay was conducted in triplicate.

2.7. Cytotoxicity Evaluation

Cytotoxicity was assessed by exposing 100% of each extraction type of composite resins and
bonding resins to the cells for 24 h, followed by staining with calcein AM and ethidium homodimer-1
(Molecular Probes, Eugene, OR, USA) for observation under a confocal laser microscope (LSM700,
Carl Zeiss, Thornwood, NY, USA). Live cells were stained to produce a green fluorescence, and bright
red fluorescence was observed from dead cells. Live and dead cells were quantified according to
normalized surface areas of staining intensity using ImageJ software.

2.8. Statistics
Statistical analysis was performed using one-way analysis of variance (ANOVA) and the
independent t-test (equal variance not assumed); p ≤ 0.05 were considered to be statistically significant.
SPSS PASW version 21.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.

3. Results

3.1. Degree of Conversion (DC)

Both materials showed significant differences in degree of conversion according to the method of
sample preparation, as indicated in Table 3.

Table 3. Results of the degree of conversion.

Test Materials Degree of Conversion (%)

CU 0
CD 74.52 ± 2.28
CP 87.59 ± 1.51
CR 95.96 ± 0.07
BU 0
BD 48.71 ± 2.09
BP 61.14 ± 3.48
BR 94.45 ± 0.22

3.2. Agar Diffusion Test

The decolorization zones of the experimental materials are presented in Figure 2.
A Teflon mold was used as a negative control, which caused no cellular lysis, whereas the latex
sheet, a positive control, resulted in a cytotoxicity score of 4. The cytotoxicity scores of the various
preparations are shown in Table 4. Uncured resins (CU, BU) had the highest cytotoxicity. Samples with
the oxygen-inhibition layer removed (CR, BR) showed lower cytotoxicity than post-cured samples (CP,
BP). There was no significant difference between the cytotoxicity of composite resin and bonding resin.
Materials 2017, 10, 288 6 of 12
Materials 2017, 10, 288 6 of 12

Figure 2.
Figure 2. Decolorization
Decolorization zones
zones of
of experimental
experimental materials
materials inin the agar diffusion
the agar diffusion test.
test. The
The empty
empty Teflon
Teflon
mold (negative control), the latex sheet from the latex glove (positive control), and
mold (negative control), the latex sheet from the latex glove (positive control), and experimental experimental
samples of
samples of (a)
(a) uncured
uncured composite
composite resin
resin (CU);
(CU); (b)
(b) directly
directly cured
cured composite
composite resin
resin (CD);
(CD); (c)
(c) post-cured
post-cured
composite resin (CP); (d) composite resin after removing the unreacted layer (CR); (e) uncured uncured
composite resin (CP); (d) composite resin after removing the unreacted layer (CR); (e) bonding
bonding
resin (BU);resin (BU); cured
(f) directly (f) directly
bondingcured
resinbonding
(BD); (g)resin (BD); bonding
post-cured (g) post-cured bonding
resin (BP); resin resin
(h) bonding (BP);
(h) bonding
after removing resin
theafter removing
unreacted the(BR)
layer unreacted layer (BR)
were located were located inpositions.
in predetermined predetermined positions.
Representative
Representative images are shown after experiments were
images are shown after experiments were performed in triplicate. performed in triplicate.

Table 4.
Table 4. Results
Results of
of the
the agar
agar diffusion
diffusion test.
test.

Test Materials Decolorization Index Lysis Index Interpretation

Test Materials Decolorization Index Lysis Index Interpretation
Positive control 4 5 Severely cytotoxic
Positive control 4 5 Severely cytotoxic
Negative control 0 0 Non-cytotoxic
Negative control 0 0 Non-cytotoxic
CUCU 44 4 4 Severely cytotoxic
Severely cytotoxic
CDCD 44 5 5 Severely cytotoxic
Severely cytotoxic
CP CP 33 4 4 Moderately
Moderately cytotoxic
cytotoxic
CR 2 3 Moderately cytotoxic
CR 2 3 Moderately cytotoxic
BU 4 4 Severely cytotoxic
BDBU 44 4 5 Severely cytotoxic
Severely cytotoxic
BP BD 43 5 4 Moderately cytotoxic
Severely cytotoxic
BR 2 3 Moderately cytotoxic
BP 3 4 Moderately cytotoxic
BR 2 3 Moderately cytotoxic
3.3. MTT Assay
3.3. MTT Assay of the cell viability assay are shown in Figure 3. The uncured samples (CU, BU) had a
The results
significantly higher
The results cytotoxicity
of the thanassay
cell viability any others, regardless
are shown of sample
in Figure 3. The preparation methods.
uncured samples (CU,The
BU)mean
had
(a±significantly
SD) cell viabilities for undiluted CU, CP, and CR were, 0.89% ± 0.83%, 21.40% ± 2.39%,
higher cytotoxicity than any others, regardless of sample preparation methods. and 48.67%
±The2.96%,
meanrespectively. A similarfor
(±SD) cell viabilities trend was found
undiluted CU,inCP,BU,
andBP,CR
andwere, 0.89% ±
BR (1.01% 15.15% ±
0.08%, 21.40%
± 0.83%, 1.41%
± 2.39%,
and 48.67%±± 0.71%,
and 21.18% 2.96%,respectively).
respectively. There was no
A similar significant
trend difference
was found in BU,inBP,
the viabilities
and BR (1.01%by CP±and CR,
0.08%,
which were 12.5% and 6.25%, respectively (p > 0.05). The viability increased in the
15.15% ± 1.41% and 21.18% ± 0.71%, respectively). There was no significant difference in the order of CU < CP <
CR in the composite resin groups, and this was the same in the bonding resin groups, i.e.,
viabilities by CP and CR, which were 12.5% and 6.25%, respectively (p > 0.05). The viability increasedBU < BP < BR.
in the order of CU < CP < CR in the composite resin groups, and this was the same in the bonding
resin groups, i.e., BU < BP < BR.
Materials 2017, 10, 288 7 of 12
Materials 2017, 10, 288 7 of 12

Materials 2017, 10, 288 7 of 12

Figure 3.
Figure 3. Cell
Cell viability
viability following
following exposure
exposure toto the
the extracts
extracts from
from (a)
(a) composite
composite resin
resin and
and (b)
(b) bonding
bonding
resin
resin atatdifferent
Figure different dilutions.
3. Celldilutions.
viability B: bonding
following
B: bonding resin;resin;
exposureC:to C: extracts
the composite
composite from
resin; resin;
U: (a) U: uncured;
composite
uncured; resin P:
andpost-cured;
P: post-cured; (b) bondingR:
R: unreacted
unreacted
resin
layer atlayer
removed. removed.
different dilutions. B: bonding resin; C: composite resin; U: uncured; P: post-cured; R:
unreacted layer removed.
The cell viabilities according to composite and bonding resin in each dilution are shown in
TheThe
cell viabilities according to composite and
andbonding resin in
in each dilution are
are shown
showninin
Figure 4. Thecell viabilities
viability by BP according
and BRto composite
was significantly bonding resinthat
higher than each
by CPdilution
and CR, respectively
Figure 4. The viability by BP and BR was significantly higher than that by CP and CR, respectively
(p <Figure 4. The viability
0.05), except by BP and and
for the post-cured BR was significantly
“Removed higherlayer”
unreacted than that by CP
groups at and CR,concentration
a 100% respectively
(p <(p
0.05),
< except
0.05), forfor
except thethe
post-cured
post-cured and “Removed
and “Removed unreacted
unreacted layer”
layer” groups
groups at aa 100%
at 100% concentration
concentration
and the “Removed unreacted layer” group at 50% concentration.
andand
the the
“Removed
“Removed unreacted
unreacted layer” group
layer” groupatat
50%
50%concentration.
concentration.

FigureCell
4. Cell viability
viability following
following exposure
exposure totothe
theextracts
extractsfrom
fromthe
thecomposite
composite resin
resin and bonding resin
Figure
Figure 4.
4. resin and bonding resin
resin
at different dilutions: (a) 100% concentration; (b) 50% concentration; (c) 25% concentration; (d) 12.5%
at different dilutions:
at different dilutions: (a) 100% concentration; (b) 50% concentration; (c) 25% concentration; (d) 12.5%
concentration; (e) 6.25% concentration (*: Statistically significant at p < 0.05).
concentration;
concentration; (e)
(e) 6.25%
6.25% concentration
concentration (*:
(*: Statistically
Statistically significant at pp << 0.05).
significant at
3.4. Live/Dead Image Assay®
3.4. Live/Dead Image Assay®®
3.4.
The cytotoxicity results were also confirmed by staining cells with calcein AM and ethidium
cytotoxicity
The cytotoxicity
homodimer-1 results were
(Molecular alsoEugene,
also
Probes, confirmed
confirmed by
OR, by staining
staining
USA) cells with
cells with calcein
for observation calcein AM
under AM and ethidium
and
a confocalethidium
laser
homodimer-1
homodimer-1 (Molecular
microscope(Molecular Probes,
(LSM700M,Probes, Eugene,
Eugene,
Carl Zeiss, OR,
OR, USA)
Thornwood, USA)
forUSA).
NY, for observation
observation
Intenseunder under
green afluorescencea confocal
confocal laser laser
wasmicroscope
observed
microscope
(LSM700M, (LSM700M,
from liveCarl
cellsZeiss,
and redCarl Zeiss, NY,
Thornwood, Thornwood,
fluorescence USA).
was NY, from
Intense
observed USA). Intense
greendead cellsgreen
fluorescence fluorescence
was
(Figure observed
5). wasfrom
from
The results observed
live cells
the
from
and live
red cells and
fluorescence red
was fluorescence
observed was
from observed
dead cells from
(Figure dead
5). Thecells (Figure
results from 5). The
the results
Live/Dead from
Assay ®
the
Live/Dead Assay image assay were in an agreement with the results of the agar diffusion test
® and
Live/Dead
imageMTT assay Assay
were®inimage
assay. assay were
an agreement within the
an agreement with
results of the thediffusion
agar results of the
test andagar diffusion
MTT assay. test and
MTT assay.
Materials 2017, 10, 288 8 of 12
Materials 2017, 10, 288 8 of 12

Figure5. 5.
Figure Confocal
Confocal laser
laser microscopy
microscopy images
images following
following calcein
calcein AM and AMethidium
and ethidium homodimer-1
homodimer-1 staining
staining of L929 cells. Cells were exposed to extracts from (a) uncured composite resin;
of L929 cells. Cells were exposed to extracts from (a) uncured composite resin; (b) directly (b) directly
cured
cured composite
composite resin; (c) post-cured
resin; (c) post-cured composite
composite resin; resin; (d)resin
(d) composite composite resin after
after removing removing layer;
the unreacted the
unreacted layer; (e) Live/Dead Assay® quantified the live and dead cells in equivalent surface areas
(e) Live/Dead Assay®quantified the live and dead cells in equivalent surface areas of composite resin;
of composite resin; (f) uncured bonding resin; (g) directly cured bonding resin; (h) post-cured
(f) uncured bonding resin; (g) directly cured bonding resin; (h) post-cured bonding resin; (i) bonding
bonding resin; (i) bonding resin after removing the unreacted layer; (j) Live/Dead Assay® quantified
resin after removing the unreacted layer; (j) Live/Dead Assay® quantified the live and dead cells in
the live and dead cells in equivalent surface areas of bonding resin. Live cells are stained green and
equivalent surface areas of bonding resin. Live cells are stained green and dead cells are stained red for
dead cells are stained red for confocal laser microscope images.
confocal laser microscope images.

4. Discussion
4. Discussion
A standardized protocol for biocompatibility evaluation is essential to assess the safety of
A standardized
dental materials. Severalprotocol for biocompatibility
studies have investigated evaluation is essential
the cytotoxicity oftoresin-based
assess the safety of dental
materials and
materials. Several studies have investigated the cytotoxicity
found that unreacted dental resin monomers are toxic to human gingival fibroblasts andof resin-based materials and found that
unreacted dental resin monomers are toxic to human gingival fibroblasts
keratinocytes. These monomers can be released from the final product, which can also be cytotoxic and keratinocytes. These
monomers can be released
itself, according to in vitro from the final
studies product,
[6,25]. which
Geurtsen et can
al. [9]also be cytotoxic
reported itself,
that the according to in vitro
bisphenol-A-glycidyl
studies [6,25]. Geurtsen et al. [9] reported that the bisphenol-A-glycidyl
methacrylate (Bis-GMA) monomer has the strongest cytotoxicity, followed sequentially by methacrylate (Bis-GMA)
UDMA,
monomer
TEGDMA, hasand theHEMA.
strongest In cytotoxicity,
addition, all resinfollowed sequentially
monomers exhibitedby UDMA, TEGDMA,genotoxicity
a dose-dependent and HEMA.
In[7,14,26–28].
addition, allFurthermore,
resin monomers it hasexhibited a dose-dependent
been reported that unreacted genotoxicity
resin-based[7,14,26–28].
materials Furthermore,
may release
it substances
has been reported that unreacted resin-based materials may release
into an aqueous environment for extended periods, possibly causing cell damage substances into an aqueousand
environment for extended
pulpal inflammation periods,
[8,18]. Thesepossibly
findingscausing cell damage
suggest and pulpal inflammation
that a cytotoxicity test is suitable[8,18].
forThese
the
findings suggest
evaluation thatbiocompatibility
of basic a cytotoxicity test is suitable for the evaluation of basic biocompatibility [24].
[24].
There
Therearearenumerous
numeroustechniques
techniques available
available for cytotoxicity evaluation,such
cytotoxicity evaluation, suchasasthe
theMTTMTTassay,
assay,
agardiffusion
agar diffusiontest,test,filter
filterdiffusion
diffusion test,
test, and
and pulp
pulp andand dentine
dentine usage test [20,24,29,30].
[20,24,29,30]. Two Twotest test
methodswere
methods wereusedused in in this study:
study: the theagaragardiffusion
diffusion testtest
andand MTT MTTassay. In the
assay. Inagar
the diffusion test,
agar diffusion
samples
test, sampleswerewereseparated from from
separated the cells
theby an agar
cells by an layer
agar mimicking the mucosal
layer mimicking the membrane, whereas
mucosal membrane,
in the MTT
whereas in theassay,
MTTextracts of the samples
assay, extracts of the were used,
samples mimicking
were constituents
used, mimicking leaching into
constituents the saliva.
leaching into
theThe endpoint
saliva. of the agar
The endpoint of thediffusion test istest
agar diffusion membrane
is membrane integrity, and and
integrity, thatthat
of the MTT
of the MTT assay
assayisis
mitochondrial
mitochondrial activity.
activity. TheThe
methodsmethods
address address different
different aspectsaspects of cytotoxicity,
of cytotoxicity, and the
and the results results
collectively
provide an overview of the cytotoxic potential of the samples [31]. Previous in vitro studiesvitro
collectively provide an overview of the cytotoxic potential of the samples [31]. Previous in have
studiescontradictory
yielded have yielded contradictory
findings regardingfindings regarding
the cytotoxicity the cytotoxicity
of resin-based of resin-based
dental materials dental
[6,16,27,31,32].
materials
These [6,16,27,31,32].
differences likely ariseThese differences
because in vitrolikely arise because
cytotoxicity tests doinnot vitro cytotoxicity
accurately tests
reflect thedo not
clinical
accurately reflect the clinical situation. In the clinic, resin-based materials
situation. In the clinic, resin-based materials are inserted into the oral cavity in a freshly mixed, are inserted into the oral
cavity in a polymerized
incompletely freshly mixed, incompletely
stage; local responses polymerized
are provoked stage;bylocal responses
unreacted or onlyarepartially
provoked by
reacted
unreacted or only partially reacted components. After polymerization,
components. After polymerization, the surface is usually polished to remove the oxygen inhibition the surface is usually
polished to remove the oxygen inhibition layer, which may cause the release of toxic constituents
layer, which may cause the release of toxic constituents from the material [8]. To optimize cytotoxicity
from the material [8]. To optimize cytotoxicity evaluation, suitable sample preparation methods are
evaluation, suitable sample preparation methods are important. In this study, the effect of these
important. In this study, the effect of these different conditions on the sample preparation of resin
different conditions on the sample preparation of resin based materials was investigated. We applied
based materials was investigated. We applied the various conditions shown in Table 2. The results
showed similar trends in both the agar diffusion and MTT tests; however, there was a significant
Materials 2017, 10, 288 9 of 12

the various conditions shown in Table 2. The results showed similar trends in both the agar diffusion
and MTT tests; however, there was a significant difference in cell viability according to different sample
preparation methods for the same materials. This was also confirmed in the images obtained using
confocal laser microscopy. Uncured samples had the highest cytotoxicity, while the samples retaining
the oxygen-inhibition layer had a higher degree of toxicity than those that underwent the polishing
process. In this respect, the removal of the inhibition layer was found to be a crucial factor for increased
cell viability.
In this study, the degree of conversion was assessed using FTIR spectroscopy (Table 3). The degree
of conversion would indicate the proportion of polymerized products from their original monomer
state through free radical polymerization reactions. In other words, the lower the degree of conversion,
greater the proportion of unpolymerized monomers available to cells during the cytotoxicity tests.
The results indicated that the value for the degree of conversion was approximately 75% and 49%
for direct-cured composite resin and adhesive resin, respectively. The values then increased as
post-cured samples were analyzed (approximately 88% and 61% for composite resin and adhesive resin,
respectively). These values were, in fact, in agreement with previous studies that used either the same
or similar products [33,34]. However, the degree of conversion further increased to approximately 95%
for both products after removal of the oxygen inhibition layer, which is an extremely high value—such
a result has not been previously reported in the literature. It was clear that the degree of conversion of
the two materials, in descending order, was samples with unreacted layer removed > post-cured >
directly cured > uncured.
The results were then compared with the cytotoxicity tests. The agar diffusion test indicated that
both directly cured and uncured samples were more cytotoxic than both post-cured and those with
the unreacted layer removed. Additionally, Figures 3 and 4 indicated that the order of cell viability
following MTT assay was unreacted layer removed > post-cured > directly cured > uncured, for both
bonding resin and composite resin—an order equivalent to the degree of conversion. This finding was
further confirmed by fluorescence imaging in the Live/Dead Assay® . It is well known that oxygen
inhibits free radical polymerization and yields polymers with uncured surfaces [35]. Hence, it may
be the reason for the extremely high degree of conversion value for samples with the unreacted layer
removed. Removal of such a layer would have increased the degree of conversion and, consequently,
the adequate polymerization would have influenced the biocompatibility of the restoration [36].
As shown in Figures 3 and 4, there was a difference in cytotoxicity between composite and bonding
resins. Generally, composite resin has a higher filler content than bonding resin, which contains water,
alcohol, or acetone, although the contents differ depending on the product [19]. These hydrophilic
components may affect the solubility of bonding resin, i.e., cells may be more easily affected by
toxic materials from bonding resins. However, as the dilutions increase, the effects of hydrophilic
resin monomers, such as Bis-GMA, UDMA, TEGDMA or camphorquinone, have a critical impact on
total cell viability in the MTT assay. It has also been reported that filler contents appear to influence
polymerization [37]. The results shown in Figures 3 and 4 can be explained by these differences in
released components and filler contents.
Although the MTT assay revealed differences between dilutions and resin types, these differences
were not observed in the agar diffusion test. Because an agar overlay test only quantitatively
demonstrates the decolorization zone and lysis index, cytotoxicity is usually confirmed by other
qualitative methods such as an MTT assay.
This study showed that different methods of sample preparation led to different cytotoxicity
levels of dental resin. Because the present study was conducted on mouse fibroblast cells, as
recommended by international standards, conclusions regarding the possible toxicity in vivo are
limited [24]. A major concern regarding in vitro test data is the relatively poor correlation among
these tests. The International Standards Organization (ISO) recommends the use of established cell
lines, such as L929 mouse fibroblasts, for cytotoxicity tests. Because L929 cells are easy to prepare and
culture, they are commonly used for cell culture-based standardization of cytotoxicity studies [20,24].
Materials 2017, 10, 288 10 of 12

In addition, L929 cells are highly sensitive to the lytic action of cytotoxins, and exhibit a greater
decrease in cell viability than other cell lines [31,38]. This enables greater sensitivity in assessing the
degree of cytotoxicity.
Dental materials, such as resin composites and bonding agents, can harm teeth and the
surrounding soft tissues, and lead to hypersensitivity or other symptoms when applied clinically.
Therefore, tests methods that mimic in vivo conditions, such as a dentin barrier test will, no doubt,
be more clinically relevant. Although our protocol does not reflect clinical conditions as well as an
extended dentin barrier test or a long-term in vivo study, it is economical and easily available. Further
studies comparing and correlating cytotoxicity results with a dentin barrier test or in vivo test will
produce more clinically relevant results. Despite these limitations, the present study indicated that
the selection of an improper method may lead to false-negative cytotoxicity results. Hence, careful
consideration in selecting the sample preparation is required.
Furthermore, our findings may have implications for the selection of sample preparation method.
More specifically, clinicians should remove unreacted monomers on the resin surface immediately
after restoring teeth with light-curing resin to limit cytotoxic effects.

5. Conclusions
The cytotoxicity of resin-based dental materials depends on the sample preparation method.
Uncured materials were the most cytotoxic, followed by light-cured materials and those with
the oxygen-inhibition layer removed. Therefore, clinicians should ensure that the remaining
oxygen-inhibition layer is removed to improve the immediate cytocompatibility of restorations.

Acknowledgments: This research was supported by a grant (14172MFDS334) from the Ministry of Food and
Drug Safety.
Author Contributions: All authors conceived the experiments; Myung-Jin Lee performed the overall experiment
and writing of the manuscript; Mi-Joo Kim and Jae-Sung Kwon contributed to the data analysis and interpretation;
Sang-Bae Lee designed the experiment; and Kwang-Mahn Kim supervised the whole work.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Xia, Y.; Zhang, F.; Xie, H.; Gu, N. Nanoparticle-reinforced resin-based dental composites. J. Dent. 2008, 36,
450–455. [CrossRef] [PubMed]
2. Jandt, K.D.; Sigusch, B.W. Future perspectives of resin-based dental materials. Dent. Mater. 2009, 25,
1001–1006. [CrossRef] [PubMed]
3. Chai, Z.; Li, F.; Fang, M.; Wang, Y.; Ma, S.; Xiao, Y.; Huang, L.; Chen, J. The bonding property and cytotoxicity
of a dental adhesive incorporating a new antibacterial monomer. J. Oral Rehabil. 2011, 38, 849–856. [CrossRef]
[PubMed]
4. Sigusch, B.W.; Pflaum, T.; Volpel, A.; Schinkel, M.; Jandt, K.D. The influence of various light curing units on
the cytotoxicity of dental adhesives. Dent. Mater. 2009, 25, 1446–1452. [CrossRef] [PubMed]
5. Sigusch, B.W.; Volpel, A.; Braun, I.; Uhl, A.; Jandt, K.D. Influence of different light curing units on the
cytotoxicity of various dental composites. Dent. Mater. 2007, 23, 1342–1348. [CrossRef] [PubMed]
6. Moharamzadeh, K.; Van Noort, R.; Brook, I.M.; Scutt, A.M. Cytotoxicity of resin monomers on human
gingival fibroblasts and HaCaT keratinocytes. Dent. Mater. 2007, 23, 40–44. [CrossRef] [PubMed]
7. Ferracane, J.L. Elution of leachable components from composites. J. Oral Rehabil. 1994, 21, 441–452. [CrossRef]
[PubMed]
8. Huang, F.M.; Chang, Y.C. Cytotoxicity of resin-based restorative materials on human pulp cell cultures.
Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2002, 94, 361–365. [CrossRef] [PubMed]
9. Geurtsen, W.; Lehmann, F.; Spahl, W.; Leyhausen, G. Cytotoxicity of 35 dental resin composite
monomers/additives in permanent 3T3 and three human primary fibroblast cultures. J. Biomed. Mater. Res.
1998, 41, 474–480. [CrossRef]
Materials 2017, 10, 288 11 of 12

10. Wataha, J.C.; Lockwood, P.E.; Bouillaguet, S.; Noda, M. In vitro biological response to core and flowable
dental restorative materials. Dent. Mater. 2003, 19, 25–31. [CrossRef]
11. Schmalz, G. The biocompatibility of non-amalgam dental filling materials. Eur. J. Oral Sci. 1998, 106, 696–706.
[CrossRef] [PubMed]
12. Hume, W.R.; Gerzina, T.M. Bioavailability of components of resin-based materials which are applied to teeth.
Crit. Rev. Oral Biol. Med. 1996, 7, 172–179. [CrossRef] [PubMed]
13. Santerre, J.P.; Shajii, L.; Leung, B.W. Relation of dental composite formulations to their degradation and
the release of hydrolyzed polymeric-resin-derived products. Crit. Rev. Oral Biol. Med. 2001, 12, 136–151.
[CrossRef] [PubMed]
14. Chen, R.S.; Liu, C.C.; Tseng, W.Y.; Jeng, J.H.; Lin, C.P. Cytotoxicity of three dentin bonding agents on human
dental pulp cells. J. Dent. 2003, 31, 223–229. [CrossRef]
15. Franz, A.; Konig, F.; Lucas, T.; Watts, D.C.; Schedle, A. Cytotoxic effects of dental bonding substances as
a function of degree of conversion. Dent. Mater. 2009, 25, 232–239. [CrossRef] [PubMed]
16. Mohsen, N.M.; Craig, R.G.; Hanks, C.T. Cytotoxicity of urethane dimethacrylate composites before and after
aging and leaching. J. Biomed. Mater. Res. 1998, 39, 252–260. [CrossRef]
17. Ergun, G.; Egilmez, F.; Cekic-Nagas, I. The cytotoxicity of resin composites cured with three light curing
units at different curing distances. Med. Oral Patol. Oral Cir. Bucal. 2011, 16, e252–e259. [CrossRef] [PubMed]
18. Yalcin, M.; Ulker, M.; Ulker, E.; Sengun, A. Evaluation of cytotoxicity of six different flowable composites
with the methyl tetrazolium test method. Eur. J. Gen. Dent. 2013, 2, 292. [CrossRef]
19. Schedle, A.; Franz, A.; Rausch-Fan, X.; Spittler, A.; Lucas, T.; Samorapoompichit, P.; Sperr, W.;
Boltz-Nitulescu, G. Cytotoxic effects of dental composites, adhesive substances, compomers and cements.
Dent. Mater. 1998, 14, 429–440. [CrossRef]
20. Dentistry-Evaluation of Biocompatibility of Medical Devices Used in Dentistry; ISO 7405; International Standard:
Geneva, Switzerland, 2013.
21. Prasad, M.; Mohamed, S.; Nayak, K.; Shetty, S.K.; Talapaneni, A.K. Effect of moisture, saliva, and blood
contamination on the shear bond strength of brackets bonded with a conventional bonding system and
self-etched bonding system. J. Nat. Sci. Biol. Med. 2014, 5, 123–129. [CrossRef] [PubMed]
22. Kim, J.; Hong, S.; Choi, Y.; Park, S. The effect of saliva decontamination procedures on dentin bond strength
after universal adhesive curing. Restor. Dent. Endod. 2015, 40, 299–305. [CrossRef] [PubMed]
23. Durner, J.; Obermaier, J.; Draenert, M.; Ilie, N. Correlation of the degree of conversion with the amount
of elutable substances in nano-hybrid dental composites. Dent. Mater. 2012, 28, 1146–1153. [CrossRef]
[PubMed]
24. Evaluation of Medical Devices-Part 5: Tests for in Vitro Cytotoxicity; ISO 10993-5; International Standard: Geneva,
Switzerland, 2009.
25. Goon, A.T.; Isaksson, M.; Zimerson, E.; Goh, C.L.; Bruze, M. Contact allergy to (meth)acrylates in the
dental series in southern Sweden: Simultaneous positive patch test reaction patterns and possible screening
allergens. Contact Derm. 2006, 55, 219–226. [CrossRef] [PubMed]
26. Terakado, M.; Yamazaki, M.; Tsujimoto, Y.; Kawashima, T.; Nagashima, K.; Ogawa, J.; Fujita, Y.; Sugiya, H.;
Sakai, T.; Furuyama, S. Lipid peroxidation as a possible cause of benzoyl peroxide toxicity in rabbit dental
pulp—A microsomal lipid peroxidation in vitro. J. Dent. Res. 1984, 63, 901–905. [CrossRef] [PubMed]
27. Costa, C.A.; Vaerten, M.A.; Edwards, C.A.; Hanks, C.T. Cytotoxic effects of current dental adhesive systems
on immortalized odontoblast cell line MDPC-23. Dent. Mater. 1999, 15, 434–441. [CrossRef]
28. Anand, V.S.; Balasubramanian, V. Effect of resin chemistry on depth of cure and cytotoxicity of dental resin
composites. Mater. Sci. Eng. B 2014, 181, 33–38. [CrossRef]
29. Kwon, J.S.; Lee, S.B.; Kim, C.K.; Kim, K.N. Modified cytotoxicity evaluation of elastomeric impression
materials while polymerizing with reduced exposure time. Acta Odontol. Scand. 2012, 70, 597–602. [CrossRef]
[PubMed]
30. Schmalz, G.; Hiller, K.A.; Dörter-Aslan, F. New developments in the filter test system for cytotoxicity testing.
J. Mater. Sci. Mater. Med. 1994, 5, 43–51. [CrossRef]
31. Chen, F.; Wu, T.; Cheng, X. Cytotoxic effects of denture adhesives on primary human oral keratinocytes,
fibroblasts and permanent L929 cell lines. Gerodontology 2014, 31, 4–10. [CrossRef] [PubMed]
Materials 2017, 10, 288 12 of 12

32. Schweikl, H.; Hiller, K.A.; Bolay, C.; Kreissl, M.; Kreismann, W.; Nusser, A.; Steinhauser, S.; Wieczorek, J.;
Vasold, R.; Schmalz, G. Cytotoxic and mutagenic effects of dental composite materials. Biomaterials 2005, 26,
1713–1719. [CrossRef] [PubMed]
33. Galvão, M.; Costa, S.; Victorino, K.; Ribeiro, A.; Menezes, F.; Rastelli, A.N.D.S.; Bagnato, V.S.; Andrade, M.
Influence of light guide tip used in the photo-activation on degree of conversion and hardness of one
nanofilled dental composite. Laser Phys. 2010, 20, 2050–2055. [CrossRef]
34. Boaro, L.C.; Goncalves, F.; Guimaraes, T.C.; Ferracane, J.L.; Pfeifer, C.S.; Braga, R.R. Sorption, solubility,
shrinkage and mechanical properties of “low-shrinkage” commercial resin composites. Dent. Mater. 2013, 29,
398–404. [CrossRef] [PubMed]
35. Gauthier, M.A.; Stangel, I.; Ellis, T.H.; Zhu, X.X. Oxygen inhibition in dental resins. J. Dent. Res. 2005, 84,
725–729. [CrossRef] [PubMed]
36. Tezvergil-Mutluay, A.; Lassila, L.V.; Vallittu, P.K. Degree of conversion of dual-cure luting resins
light-polymerized through various materials. Acta Odontol. Scand. 2007, 65, 201–205. [CrossRef] [PubMed]
37. Ikeda, I.; Otsuki, M.; Sadr, A.; Nomura, T.; Kishikawa, R.; Tagami, J. Effect of filler content of flowable
composites on resin-cavity interface. Dent. Mater. J. 2009, 28, 679–685. [CrossRef] [PubMed]
38. Denizot, F.; Lang, R. Rapid colorimetric assay for cell growth and survival: Modifications to the tetrazolium
dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 1986, 89, 271–277. [CrossRef]

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