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Volume 2017, Article ID 9650420, 7 pages
https://doi.org/10.1155/2017/9650420

Research Article
Cytotoxicity of Etch-and-Rinse, Self-Etch, and Universal Dental
Adhesive Systems in Fibroblast Cell Line 3T3

Yasmine Mendes Pupo,1 Cintia Fernanda de Freitas Bernardo,2

Francielly Fernanda de Freitas A. de Souza,2 Milton Domingos Michél,3
Camila Nunes de Morais Ribeiro,4 Sandro Germano,5 and Daniela Florencio Maluf4,5
1
Department of Restorative Dentistry, Federal University of Parana (UFPR), Curitiba, PR, Brazil
2
School of Dentistry, Tuiuti University of Parana (UTP), Curitiba, PR, Brazil
3
Department of Materials Engineering, State University of Ponta Grossa (UEPG), Ponta Grossa, PR, Brazil
4
Department of Biomedicine, Tuiuti University of Parana (UTP), Curitiba, PR, Brazil
5
Department of Pharmacy, Federal University of Parana (UFPR), Curitiba, PR, Brazil

Correspondence should be addressed to Yasmine Mendes Pupo; [email protected]

Received 13 July 2016; Accepted 17 October 2016; Published 10 January 2017

Academic Editor: Jessem Landoulsi

Copyright © 2017 Yasmine Mendes Pupo et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

The aim of this study was to evaluate in fibroblast cultures the direct cytotoxic effects of etch-and-rinse, self-etch, and universal
adhesive systems. The sterile glass cover slips (𝑛 = 3) were then immersed in culture medium to obtain the eluates for the
experimental groups: (1) Adper Single Bond 2; (2) Ambar; (3) Adper Scotchbond Multi-Purpose; (4) Scotchbond Universal;
(5) Ambar Universal; and (6) OptiBond All-In-One. As a negative control, sterile glass cover slips were immersed in culture medium
only. After 24 h, the eluate obtained was applied on fibroblast culture. Cell viability and cell morphology were evaluated by MTT
assay and SEM, respectively. Data were analyzed by Kruskal–Wallis and Mann–Whitney tests (𝛼 = 0.05). All adhesive systems
except universal reduced cell viability in 3T3 cells to between 26.04% and 56.57%, and Scotchbond Universal and Ambar Universal
reduced cell viability to 2.13% and 3.57%, respectively, when compared to the negative control. Cytoplasmic membrane shrinkage
and cell-free areas with residual membrane fragments from dead cells were observed. In conclusion, improvements in universal
adhesive system formulations and their mechanisms of action are not accompanied by increased toxicity compared with those in
other systems, warranting commitment to the use of these dentin-pulp complexes.

1. Introduction adhesive systems are recommended because they improve

the contact between resin-based restorative materials and
The evolution of technologies for clinical procedures in den- the walls of prepared cavities [2, 3]. However, previous
tistry has led to a wide variety of marketed materials. How- studies have reported that there are various substances with
ever, in addition to the esthetic care properties and durability biological effects that are potentially toxic following release
of these products, the evaluations of biocompatibility with from adhesive systems [4]. Numerous materials have been
dental structures are required to optimize compatibility of developed for dental applications. However, more tissue-
pulp-dentin tissue complexes [1]. Light composite materials friendly materials remain desired, and the biocompatibility of
are commonly used in restorative dentistry, and the field of dental materials is an increasingly important area of research.
adhesive dentistry encompasses the use of these materials Although the severity of adverse effects varies, the risks of
in conjunction with adhesive systems for the restoration or toxicity remain an important consideration for materials that
reanatomization of lost dental tissues and for shape changes. are placed in contact with oral tissues [3].
To improve the retention of restorative procedures and Adhesive systems commonly comprise bifunctional
to seal tooth-restoration interfaces from microorganisms, monomers and hydrophobic and hydrophilic monomers,
2 Scanning

which contain carboxylic acid- or phosphoric acid-derived 2.1. Material Preparation. Aliquots (10 𝜇L) of the adhesive
radicals and/or added organic or mineral acid derivatives. systems (SB, AM, SBU, AMU, and OPT) were pipetted in
Moreover, these monomeric components are present in sextuplicate into sterile circular microscopy coverslips (G-
solvents, such as water, alcohol, and acetone, and also in 13C100) of 13 mm diameter and 0.13 mm thickness (Glasscyto,
aromatic amines and filler particles [5]. Time-dependent Bioslide Technology, Walnut, CA, USA). For Scotchbond
cytotoxicity of the monomers hydroxymethyl methacrylate Multi-Purpose (MP), 5 𝜇L primer and 5 𝜇L bond were
(HEMA), bisphenol A diglycidyl dimethacrylate (Bis-GMA), pipetted. The aliquots were light-cured with LED (Valo,
and urethane dimethacrylate (UDMA) has been shown in Ultradent Products Inc.; South Jordan, USA; irradiance:
deep cavities and direct contact with pulp tissue [6]. Conse- 1400 mW cm2 ) for 10 s. Coverslips containing adhesive sys-
quently, basic cellular functions, such as proliferation, tems were disposed into sterile 6-well plates where 3 mL per
enzyme activity, and mitochondrial respiration, are report- well of culture medium RPMI supplemented with 10% fetal
edly compromised, with concomitant changes in cell mor- bovine serum (FBS) and antibiotics (penicillin/streptomycin
phology and membrane integrity [6]. To provide broader 100 IU/100 𝜇g⋅mL−1 ) was added. The plates were incubated at
indications, other components have been added to universal 37∘ C for 24 hours. Subsequently, culture media containing
adhesive systems, and these may cause changes in the bio- leached components of the adhesive systems were collected
logical behaviors of dentin-pulp complexes. and sterilized by filtration through 0.22 𝜇m membrane filters
With the evolution of adhesive systems with various com- to obtain sterile eluates for cell application. The same proce-
ponents, prior removal of the smear layer using conven- dure was conducted with coverslips in the absence of adhesive
tional approaches has been made redundant by self-etching system to characterize the negative control.
approaches that maintain the smear layer as part of the
adhesion substrate [2]. Previous studies recommend selective 2.2. Cell Viability Analysis. Cell viability was determined
etching of enamel margins to accommodate self-etching according to mitochondrial activity in proliferation assays.
adhesives that do not allow conditioning of the enamel at In these assays, soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-
the same depth as with phosphoric acid before application in diphenyltetrazolium bromide (MTT) salt is converted to
two steps [7]. Accordingly, this approach has been established insoluble formazan crystals by mitochondrial succinate dehy-
by new universal adhesives, which have similar composition drogenase (SDH) in viable cells, dissolved in dimethyl sulfox-
to that of one-step self-etching primer adhesives. In these imine (DMSO), and formazan concentrations are measured
primer adhesives, the methacrylic monomers found in total- spectrophotometrically at 570 nm.
etch adhesives have been replaced by functional monomers, The cells were concentrated in 1 mL of RPMI containing
such as methacryloyloxydecyl dihydrogen phosphate (MDP), 10% SFB and antibiotics, and the numbers of viable cells were
leading to characteristic acidic functions and subsequent determined using the trypan blue method. Considering cell
chemical adhesion. viability of >90%, suspensions of 3 × 105 cells were seeded
Due to standardization and reproducibility of cytotoxicity into 96-well plates (100 𝜇L/well). Plates were incubated for
determinations in cell cultures, in vitro cytotoxicity assays 24 h at 37∘ C in 5% CO2 to allow cell adhesion. Culture
are suitable for assessments of biocompatibility of adhesive medium was then removed and cells were treated with the
systems containing MDP and can be considered prerequisite conditioned media from test materials in triplicate at 37∘ C
for understanding the biological risks of these materials overnight. Control cells were treated with nonconditioned
during initial setting. Accordingly, in vitro tests using cell medium. Subsequently, the cells were washed with 200 𝜇L of
cultures can be used to rapidly generate sensitive, inexpen- sterile PBS (37∘ C) in duplicate, and 100 𝜇L of MTT reagent
sive, convenient, and repeatable material classifications [4, 8]. solution (Sigma-Aldrich) in sterile PBS (0.5 mg/mL) was
Therefore, the aim of this study was to evaluate the cytotoxic added to each well and incubated at 37∘ C for 4 h. Culture
effects of etch-and-rinse, self-etch, and universal adhesive medium containing MTT was then replaced with 100 𝜇L
systems. The hypothesis was that universal dentin adhesive of pure DMSO to dissolve formazan crystals. Cell viability
containing MDP may be less cytotoxic on fibroblasts than was then evaluated spectrophotometrically at 570 nm using a
conventional and self-etch dentin adhesives systems. microplate reader (ELX 800, BioTek Instruments; Winooski,
VE, USA). The MTT assay was conducted three times to con-
2. Material and Methods firm reproducibility. Absorbance data were expressed relative
to the control group and percent viability was calculated. Data
Included adhesive systems are described in Table 1 and are presented as mean ± standard deviations.
included (1) two-step etch-and-rinse, Adper Single Bond 2
(SB, 3M ESPE; St. Paul, MN, USA); (2) two-step etch-and- 2.3. Cell Morphology Analysis Using Scanning Electron
rinse, Ambar (AM, FGM; Joinville, SC, Brazil); (3) three-step Microscopy (SEM). 3T3 fibroblast cells were seeded at 6 × 104
etch-and-rinse, Adper Scotchbond Multi-Purpose (MP, 3M cells/well in 6-well microplates containing coverslips (13 mm
ESPE; St. Paul, MN, USA); (4) two-step etch-and-rinse or diameter and 0.13 mm thickness) for sterile microscopy.
one-step self-etch, Scotchbond Universal (SBU, 3M ESPE; St. Cells were treated with conditioned media from adhesive
Paul, MN, USA); (5) two-step etch-and-rinse or one-step self- systems, which were light-cured for 10 s and stored for 24 h,
etch, Ambar Universal (AMU, FGM; Joinville, SC, Brazil); as described above. After treatment, cells that remained
and (6) One-step self-etch, OptiBond All-In-One (OPT, Kerr; attached to glass coverslips were fixed with 1 mL of 2.5
Orange, CA, USA). glutaraldehyde solution for 24 h and were then dehydrated
Scanning 3

Table 1: Test materials, classifications, and compositions.

Adhesive system
Lot number Classification Composition
(manufacturer)
Bis-GMA, HEMA, polyacrylic acid,
Adper Single Bond 2 (3M
#N508311 Two-step etch-and-rinse poly(itaconic acid), water, ethanol,
ESPE)
dl-CQ, silica (10% wt).
UDMA, HEMA, 10-MDP, hydrophilic
methacrylated monomers, ethanol, silica
Ambar (FGM) #240215 Two-step etch-and-rinse
nanofiller, photoinitiators, coinitiators,
stabilizers.
Primer: HEMA, polyalkenoic acid
Adper Scotchbond Primer: #N560292
polymer, water.
Three-step etch-and-rinse
Bond: Bis-GMA, HEMA, tertiary amines,
Multi-Purpose (3M ESPE) Bond: #N551363
photoinitiator.
Bis-GMA, HEMA, ethanol, water, silane
treated silica, 2-propenoic acid 2-methyl-,
Scotchbond Universal (3M Two-step etch-and-rinse or reaction products with 1, 10-decanediol
#569736
ESPE) one-step self-etch and phosphorous oxide (P2 O5 ),
copolymer of acrylic and itaconic acid,
CQ, 4-dimethylaminobenzoate, toluene.
UDMA, HEMA, 10-MDP potentiated,
Two-step etch-and-rinse or hydrophilic methacrylated monomers,
Ambar Universal (FGM) #030815
one-step self-etch ethanol, silica nanofiller, photoinitiators,
coinitiators, stabilizers.
GPDM, HEMA, GDMA, Bis-GMA,
OptiBond All-In-One
#5125872 One-step self-etch water, 2.5–3 acetone, ethanol, CQ, silica
(Kerr)
filler, sodium hexafluorosilicate.
Bis-GMA, bisphenol A diglycidyl methacrylate; HEMA, 2-hydroxyethyl methacrylate; CQ, camphorquinone; UDMA, urethane dimethacrylate; 10-MDP, 10-
methacryloyloxydecyl dihydrogen phosphate; GPDM, glycerol phosphate dimethacrylate; GDMA, glycerol dimethacrylate.

using an ethanol series of 30, 50, 70, 95, and 100% in 120
∗
SDH activity (% of viable cells)

concentration. Cells were then maintained in colloidal silica 100

∗ ∗
for 24 h and were sputter-coated with gold/palladium in a
metallizer (Shimadzu, Kyoto, Japan). Cell morphology of 3T3 80 ∗∗
fibroblasts was then assessed using SEM (Shimadzu). ∗∗∗
60 ∗∗∗∗ ∗∗∗∗
2.4. Data Analysis. Data from MTT assays were expressed 40
as percent viability relative to the negative control (cul-
ture medium; 100%). Statistical analyses were performed 20
using SPSS version 21 and differences were identified using 0
Kruskal–Wallis and Mann–Whitney nonparametric tests and Control SB AM MP SBU AMU OPT
were considered significant when 𝑝 < 0.05. Figure 1: Percentage (%) of cell viability of 3T3 cells; columns
with the same quantity asterisks did not differ significantly (Mann–
3. Results Whitney, 𝑝 > 0.05).

3.1. Cytotoxic Effects of Adhesives. The treatment with the

adhesives resulted in cell death average of 2.13% to 56.57%, Following exposure to Adper Single Bond 2 (Figures 2(c)
demonstrating a moderate cytotoxic effect of the tested and 2(d)), large numbers of cells with regular morphology
systems (Figure 1). were observed, and near confluent cells showed abundant
cytoplasm with numerous elongated, fine cytoplasmic projec-
3.2. SEM Analyses of Cell Morphology. SEM micrographs tions adhering to glass surfaces. Similar characteristics were
of 3T3 fibroblasts after treatment with adhesive eluates are observed following exposure to Scotchbond Universal and
presented in Figures 2(a)–2(h) and 3(a)–3(f). These analyses Ambar Universal (Figures 3(a), 3(b), 3(c), and 3(d)). How-
demonstrated fusiform morphology of adherent positive ever, severe cytotoxic effects of the conditioned media from
control 3T3 cells (exposed only to culture medium), with some experimental materials were observed in fibroblast 3T3
large numbers of elongated cells on the coverslip surface cells (Figures 2(e), 2(f), 2(g), 2(h), 3(e), and 3(f)). Accord-
showing spindle shapes and cytoplasmic membrane pro- ingly, fewer cells were attached to the substrate. The analysis
cesses (Figures 2(a) and 2(b)). of cell morphology by SEM revealed the occurrence of death
4 Scanning

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Figure 2: Cell morphology in scanning electron microscopy (SEM) of 3T3 fibroblast cells. (a) Positive control group, large numbers of
3T3 fibroblasts with fusiform morphology, ×1000; (b) positive control group, spindle-shaped appearance with some cytoplasmic processes
originating from membranes covering the glass substrate surface, ×500; (c) SB, large numbers of fibroblasts with normal morphology, ×1000;
(d) SB, fusiform cells, uninucleation, and long and thin cytoplasmic membrane processes, ×500; (e) AM, altered morphology, rounded shapes,
and small sizes, ×1000; (f) AM, cytoplasmic membranes and debris from dead cells, ×500; (g) MP, severe cytotoxic effects, ×1000; (h) MP,
reduced numbers of cells, membrane rupture, and apoptosis, ×500.
Scanning 5

(a) (b)

(c) (d)

(e) (f)

Figure 3: Micrographs from SEM analyses of 3T3 fibroblasts. (a) SBU, no changes in morphology compared with those in the control group,
×1000; (b) SBU, abundant cytoplasm with numerous elongated thin cytoplasmic projections adhering to the glass surface, ×500; (c) AMU,
similar morphology to the control group, ×1000; (d) AMU, long, thin cytoplasmic membrane derived prolongations, ×500; (e) OPT, altered
morphology with ill-defined cellular limits, suggesting cell necrosis, ×1000; (f) OPT, morphological changes and ruptured membranes in 3T3
cells, ×500.

with consequent detachment from the glass substrate of than in vivo tests. Dental adhesives generally comprise
some cells. Furthermore, apoptosis was characterized by con- complex mixtures of crosslinked and functional hydrophilic
densation of the nucleus and cytoplasm, followed by mem- and hydrophobic monomers in solvents, such as acetone,
brane-bound fragmentation of the cell [5, 9, 10]. ethanol, and/or water. Although these adhesives contain low
concentrations of initiators and inhibitors of polymerization
4. Discussion reactions, previous studies have shown that monomers can
interfere with adaptive responses and can aggressively deplete
Efficacy and biocompatibility assessments are critical for vital cellular functions by generating oxidative stress and
the clinical validation of dental materials. Thus, several in exhausting antioxidant defense mechanisms [12].
vitro studies have investigated the cytotoxicity of dental Conventional or total-etch adhesive systems require dem-
adhesive systems and their components [4], and according ineralization of dental enamel and dentin substrates using
to Williams [11] these are more reproducible and convenient phosphoric acid conditioning before application [13]. The
6 Scanning

formation of hybrid layers during total conditioning with and can also activate apoptosis [3]. Similarly, Bis-GMA alters
adhesive systems relies on superficial dentin demineraliza- cell cycle progression, elevates oxidative stress, and induces
tion by inorganic acids, which exposes collagen fibrils to apoptosis in a concentration-dependent manner. However,
infiltration by hydrophilic monomers [14]. However, dentin hydrophobic monomers of Bis-GMA have a high molecular
humidity continues to hamper the use of conventional weight and chemical features, such as high viscosity, low
adhesive systems [15] by preventing complete infiltration volatility, and low polymerization shrinkage, which result in
throughout the collagen matrix mesh, resulting in outbreaks stricter resins with lower susceptibility to hydrolytic media
for degradation of the bonding interface and higher rates [5, 22]. UDMA is also a viscous hydrophobic monomer, and
of postoperative sensitivity [13]. To address the detriments its high molecular weight leads to relative resistance of com-
of dentin humidity, self-etching adhesive systems in which posite resins. However, UDMA induces cellular changes at
monomer acids demineralize and infiltrate substrates simul- low concentrations, leading to pathological phenotypes and
taneously have been developed, precluding the use of acid cell death [23].
in a separate step to produce porosity of the substrate Adhesive systems also include other components that
[16]. Previous studies have shown lower cytotoxicity of self- alter their properties, and the present universal adhesive
etch adhesive systems and better responses in histological system contains functional monomers, such as MDP, Vitre-
tissues than that following use of total-etch adhesive systems bond Copolymer, and silane. MDP is a long carbon chain
[4]. The provision of a bond that is effective for various monomer that contributes hydrophobicity and hydrolytic
dental substrates is the main challenge of current dental stability [12]. Moreover, the presence of silane in this adhesive
adhesives [14]. In particular, one-step self-etch adhesive has allows direct application to the crowns of grafts [5]. Thus,
been introduced and classified as “universal” or “multimode” cell viability in the presence of universal adhesive systems
[17]. This multiapproach capability enables the clinician to likely reflects the presence of multiple components. A pre-
apply the adhesive with the so-called selective enamel etching vious study also demonstrated influences of monomer types
technique that combines the advantages of the etch-and-rinse and interactions on cytotoxic effects [1], reflecting differ-
technique on enamel, with the simplified self-etch approach ences in cytotoxicity concentrations, types and interactions
on dentine with additional chemical bonding on remnant car- of monomers, types of solvents, and molecular weights.
bonated apatite crystallites in those bonding substrates [18]. However, further studies are required to specifically define
The present study shows that adhesive systems can have relationships among adhesive components and interactions
metabolic effects in 3T3 fibroblasts, which were chosen to and cytotoxicity.
assess the toxicity of monomer materials due to the ease In the present SEM analyses, large numbers of cells
and speed of cell growth, and they are in line with ISO with normal morphology and cytoplasmic projections on
recommendations for evaluations of biological responses to coverslip surfaces were observed in the presence of SB, SBU,
dental materials [19]. The present data show that whereas and AMU adhesives. In contrast, the presence of adhesives
the Ambar (47.03%) and Adper Scotchbond Multi-Purpose AM, MP, and OPT led to reduced numbers of attached cells,
(43.43%) had severe cytotoxic effects, Adper Single Bond rounded and small morphology, and rupture of cytoplasmic
2 (73.96%) and OptiBond All-In-One (62.94%) were less membranes, reflecting widespread cell death [5, 9, 10].
cytotoxic. However, the Scotchbond Universal (97.44%) and One of the limitations of this study is that it is an in
Ambar Universal (96.05%) adhesive systems were better tol- vitro experiment; thus it may not directly reflect the clinical
erated by 3T3 fibroblasts, offering greater security to dentin- situation. Therefore, the use of more clinically relevant cells
pulp complex. Adhesive toxicity varied among the present is important [24]. The universal dental adhesives have few
adhesive systems and was likely related to the presence of studies reporting their clinical and biological performances
residual monomers in eluates. [8, 25]. In this study, universal dental adhesive showed the
Conventional self-etching adhesives have greater quan- highest fibroblast viabilities according to the MTT assay.
tities of hydrophobic monomers than universal adhesives, Hence, this universal adhesive offers a good alternative for
likely leading to greater toxicity. Accordingly, the present specific cases and has technical simplicity. However, further
MTT experiments reflected the presence of residual mono- longitudinal studies are required to investigate the effects
mers in eluates from total-etch adhesives. Residual mono- of other components following application using various
mers in the present adhesives included HEMA, Bis-GMA, techniques. These observations encourage studies in isolated
UDMA, and MDP. These compounds are known to change dental pulp stem cells.
cellular microenvironments by inducing the formation of
reactive oxygen species (ROS) and depleting antioxidants, 5. Conclusions
such as glutathione. Moreover, increased ROS levels are
directly related to the control of cell death by antioxidant Under the limitations of this study, improvements of formu-
genes and proteins [20]. HEMA is a monomer that improves lations and the mechanisms of action of universal adhesive
dentin bonding strength. Despite being less toxic, HEMA systems did not produce greater toxicity when compared with
has a low molecular weight and carries a hydroxyl group other systems.
with hydrogen binding affinity and is easily released in
aqueous solution [5, 6, 21, 22]. This monomer suppresses Competing Interests
the growth of many cells types, induces delays in cell cycle
progression of primary fibroblasts by increasing ROS levels, The authors declare that they have no competing interests.
Scanning 7

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Research International
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