Key words:
Biocompatibility - dental materials - toxic substances
INTRODUCTION
With the evolution of esthetic dentistry, self-adhesive resin cements have become
indispensable in clinical practice. These materials are used in several procedures,
for example, cementation of indirect restorations, porcelain laminate veneers, and
fixed prostheses, especially because of their low solubility in water and the strength
of their bond to enamel and dentin.[1],[2] Because of these properties, the use of self-adhesive resin cements is associated
with a lower degree of infiltration and marginal staining, lower postoperative sensitivity,
in addition to reinforcement of the bond between the restoration and the tooth.[3],[4] The large number of resin cements available in the market and the introduction of
self-adhesive systems have increased their use in clinical practice, especially because
they simplify the cementation technique, eliminating the need for previous treatment
of the tooth substrate and decreasing sensitivity. In addition, these cements present
a strong bond to dentin, similar to that of conventional adhesive cements.[5]
[6]
[7]
Adequate conversion of monomers into polymers is essential to maximize the physical
properties and clinical performance of resin cements, as well as to reduce their cytotoxicity.[8] Polymerization may be influenced by several factors, for example, ceramic translucency,[9],[10] thickness,[9],[11],[12] curing time,[13] type of curing unit,[14],[15] battery level,[16] light intensity, wavelength, and type of initiator.[17] Different types of curing light units have been proposed and assessed for the photopolymerization
of restorative materials, always with the goal of enhancing physical properties and
clinical performance and consequently reducing cytotoxicity.[18]
[19]
[20]
Several attempts have been made over recent years to enhance polymerization rates,
and it is currently known that the molecular cross-linking density of methacrylate-based
resin materials can be improved with the use of high temperatures either before or
during polymerization.[21],[22] Several authors have demonstrated superior physical properties of resin materials
as a result of a higher degree of conversion of monomers into polymers obtained through
different light-curing methods employing heat.[23]
[24]
[25]
[26]
Cytotoxicity measurement based on cellular behavior and viability is the first step
in assessing the biocompatibility of dental materials for subsequent use in clinical
practice.[27] Cytotoxicity depends on the quality and amount of monomers and derivatives released,
which may irritate the pulp and oral soft tissues and eventually lead to a toxic reaction.[28],[29] Methacrylate-based dental materials are known to present a high level of cytotoxicity
and are therefore likely to penetrate the pulp and induce cytotoxic effects.[30]
Several protocols have been used to assess cellular behavior, viability, and cytotoxicity,
including the trypan blue exclusion assay, chromium release assay, DNA synthesis,
and cellular metabolism (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
[MTT]) assay. In particular, the MTT is considered a relatively simple assay, yet
as thorough and reliable as the others, and therefore it is widely used to determine
cytotoxicity of different materials in cell cultures.[31]
[32]
[33]
The objective of this study was to assess, in vitro, the influence on cytotoxicity of heat treatment applied before photopolymerization,
while mixing three self-adhesive resin cements, in an NIH/3T3 fibroblast cell culture,
based on cell viability measures.
Methods
Sample preparation
Three self-adhesive resin cements were used in this in vitro study: RelyX U200 (3M ESPE, Saint Paul, Minnesota, USA), Multilink N (Ivoclar Vivadent,
Schaan, Liechtenstein), and BisCem (Bisco Inc., Schaumburg, Illinois, USA) [Table 1]. Resin cement dispensers were sterilized with ethylene oxide (Esteriliplus, Porto
Alegre, Rio Grande do Sul, Brazil), and the necessary amounts of base and catalyst
paste to produce specimens (9 mm diameter x 1 mm thickness) were dispensed onto a
sterilized glass slide. Specimens were immediately prepared in three different forms:
(1) no heat treatment while mixing the pastes (control); (2) jet of warm air (37°C)
distant 10 cm from the slide for 10 s while mixing; and (3) jet of hot air (60°C)
distant 10 cm from the slide for 10 s while mixing. All specimens were subsequently
light cured for 20 s using a VALO Cordless light-emitting diode curing unit (Ultradent,
Salt Lake City, Utah, USA).
Table 1:
Self-adhesive resin cements used, composition, light.curing time, batch, and manufacturer
|
Cement
|
Composition
|
Light-curing time (s)
|
Batch
|
Manufacturer
|
|
HEMA: Hydroxyethylmethacrylate, Bis-GMA: Bisphenol A glycidyl methacrylate
|
|
RelyX U200
|
Silanated filler (glass powder), dimethacrylate monomers, 1-benzyl-5-phenylbarbituric
acid, calcium salt, 1,12-dodecanediol dimethacrylate, sodium p-toluenesulfonate, silanated
silica, calcium hydroxide, methacrylated aliphatic amine, titanium dioxide
|
20
|
622725
|
3M ESPE
|
|
Multilink N
|
Dimethacrylate, HEMA, barium glass, ytterbium trifluoride, spherical mixed oxides
|
20
|
U44037
|
Ivoclar
|
|
BisCem
|
Bis-GMA, dimethacrylate monomer, glass particles, and acid monomer
|
20
|
1500003825
|
Bisco
|
Cell culture
The cells used in this study were NIH/3T3 mouse fibroblasts (ATCC®-American Type Culture
Collection-TCC, Old Town, Maryland, USA) cultured in Dulbecco’s Modified Eagle Media
(DMEM; Invitrogen®, Carlsbad, California, USA). This medium was supplemented with
10% of fetal bovine serum, 100 U/mL of penicillin (Gibco), 100 U/mL of streptomycin
(Gibco), and 100 μg/mL of gentamycin (Gibco). Cells were kept in a humidified incubator
at a temperature of 37°C and 5% of CO2.
Extraction medium
Immediately after the light-curing process, specimens from the three groups were immersed
in the DMEM medium. The specimen surface area to medium volume ratio was 3 cm2/mL, according to ISO 10993-12. Surface area was calculated based on the total dimensions
of the specimen, disregarding porosity. Extracts were tested for cell viability after
remaining 24 h and 7 days in the incubator.
Cytotoxicity assay
The MTT method was used to assess cytotoxicity. This assay measures the ability of
live cells to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT, Sigma) to insoluble blue-to-purple formazan crystals. At each treatment time
(24 h and 7 days), the culture medium was removed and 10% of an MTT solution (5 mg/mL)
in phosphate-buffered solution was added to each well. Subsequently, cultures were
incubated at 37°C, protected from light, until the presence of blue-to-purple formazan
crystals was observed. For the solubilization of formazan crystals, 100 μL of dimethyl
sulfoxide was added to each well, and absorbance was measured at 570 nm wavelength
using a spectrophotometer and an ELISA microplate reader (Benchmark Microplate Reader,
Bio-Rad Inc., Hercules, California, USA). The percentage of viable cells was calculated
and compared to the results obtained with the negative control (cells cultured in
DMEM). The assay was validated using a positive toxicity control (cells treated with
2% sodium hypochlorite).
Statistical analysis
The cytotoxicity of light-cured self-adhesive resin cements without previous heat
treatment and with warm and hot air treatment was compared in terms of cell viability
rates in NIH/3T3 mouse fibroblast cultures using three-way ANOVA followed by post hoc Student-Newman-Keuls. Examiners were blinded to group allocation. Results were expressed
as mean and standard deviation. Significance was set at 5%.
RESULTS
[Table 2] shows the cell viability results obtained for the three self-adhesive resin cements.
Groups treated with both temperatures (37°C and 60°C) showed cytotoxicity, with a
reduced number of viable cells, regardless of the cement used. Resin cement cytotoxicity
increased with time, with the highest values observed at 7 days for all cements.
Table 2:
Cell viability rates obtained using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay at 24 h and 7 days in light-cured self-adhesive resin cements without
and with previous heat treatment
|
Cement
|
24 h
|
7 days
|
|
No heat
|
37°C
|
60°C
|
No heat
|
37°C
|
|
Data presented as mean±SD. Different letters indicate statistical differences (ANOVA
or Student-Newman-Keuls test): P<0.05. SD: Standard deviation, ANOVA: Analysis of variance
|
|
RelyX
|
14.93±1.77A
|
14.56±0.71AC
|
23.25±0.45B
|
5.41±0.68D
|
6.24±1.73D
|
|
Multilink N
|
13.93±1.35A
|
12.24±1.15C
|
24.05±0.40B
|
6.33±1.06F
|
8.43±0.61F
|
|
BisCem
|
15.52±1.37A
|
15.89±0.30A
|
23.88±1.05B
|
5.78±0.69D
|
6.77±0.77D
|
At the 24h cell viability analysis, no differences were detected among the samples
not subjected to heat treatment in terms of cell viability. In the samples treated
with warm air (37°C), 24 h cell viability results were similar to those obtained without
heat treatment in both RelyX and BisCem samples. Multilink N, in turn, showed a significantly
lower cell viability rate. Finally, in the samples treated with hot air (60°C), all
three cements showed higher cell viability rates when compared to either the warm
air group (37°C) or the group with no heat treatment. However, no differences were
observed among the three self-adhesive resin cements subjected to 60°C heat treatment.
After 7 days of incubation, in turn, cell viability rates were lower than those obtained
at 24 h in both the group treated with warm air (37°C) and in the one not subjected
to heating. Conversely, in the group treated with hot air (60°C), cell viability results
showed a marked increase when compared with the nonheated group, in all self-adhesive
resin cements.
Furthermore, samples treated with hot air (60°C) were able to maintain high rates
of cell viability, showing similar results to those found at 24 h in the group not
subjected to heat treatment. Again, the three groups treated with hot air showed similar
cell viability results among themselves.
DISCUSSION
Several studies have been conducted over the past few years to analyze the biocompatibility
of methacrylate-based resin materials, which are known to have severe cytotoxic effects
on pulp tissues. In the present study, we measured cell viability rates in the extraction
medium in contact with cells after 24 h and 7 days of incubation. Not only did we
confirm the presence of toxicity in resin cements but also we found that toxicity
increases significantly with time.
Cell characteristics and functions have been used to analyze and investigate cytotoxicity
of methacrylate-based resin materials. Cell adhesion, proliferation, and metabolism
in 3T3, L929, and W138 fibroblast and osteoblast cell lines are among the parameters
that have been investigated.[31],[33],[34] In the present study, we assessed the behavior of mouse fibroblasts according to
modified parameters of Stanford. Although these cells are more sensitive to cytotoxicity
than human cells, they are indicated for this type of study by the American National
Standard ISO 10993-5 due to their reproducible growth rates, easy handling, and easy
availability when compared with primary cells and in addition being an immortal cell
line.[31] Cell inviability as determined by the MTT test does not necessarily mean a higher
occurrence of apoptosis and tissue necrosis; rather, it means that, in addition to
these events, there may also be a higher number of cells showing reduced metabolic
activity.
In this study, resin dispensers were sterilized and placed onto sterilized glass slides.
Then, the base and catalyst pastes were mixed (9 mm diameter x 1 mm thickness), light
cured, and immediately immersed in the monomer extraction medium. It is important
that materials are tested immediately after photopolymerization, to avoid the loss
of toxic substances that may be released by the material after light curing. The longer
the time elapsed between photopolymerization and cell viability analysis, the less
faithful and consequently less reliable the results will be. Studies have demonstrated[28] the relevance of immediate versus late cytotoxicity analysis of methacrylate-based
resin materials and its effects on cell vitality,[28]
[28]
[30] as well as the importance of effective photopolymerization in an attempt to minimize
cytotoxicity.[8]
Dioguardi et al.
[35] tested five different resin cements for cytotoxicity and found differences between
the brands assessed. Still, all cements presented low cytotoxicity rates, which remained
low even after 1 week of contact with cells. We did not confirm these findings of
low cytotoxicity in the present study. On the contrary, all toxicity values were high,
in all cements analyzed, both at the 24 h and at the 7-day analyses. In addition,
in our sample, cell viability continued to reduce with time, as observed on the 7-day
analysis.
Residual uncured monomers released during the light-curing process are one of the
factors responsible for the cytotoxicity of resin materials. However, according to
Goldberg,[28] there are other mechanisms that contribute to cytotoxicity, for example, leachable
components created by erosion or degradation over time, ion release, and bacteria
located at the interface between the tooth and the adhesive.
Uncured resin cement debris, such as monomers, degradation products, initiators, activators,
or stabilizers, produces cellular cytotoxicity. These products can be reduced through
enhancement of the polymerization process, as better cross-linking will result in
a better polymer. Heat treatment applied during the mixing of base and catalyst pastes
before polymerization has enabled a good reduction rate, according to some studies,[21],[22] producing a resin with superior properties. In the present study, heat treatment
at 60°C probably resulted in a higher rate of polymer cross-linking (enhanced polymerization)
and thus fewer residual monomers.
Ergun et al.
[8] reports that effective polymerization is one of the most important factors when
dealing with methacrylate-based dental materials so as to improve their physical properties,
clinical performance, and biocompatibility. Klein-Junior et al.
[25] and Ferracane and Condon[23] also showed that heat treatment before resin polymerization had a significant influence
on material properties. In the present study, samples not treated with a jet of hot
air (60°C) showed high cytotoxicity levels, at both 24 h and 7 days. Conversely, in
the group treated with hot air (60°C) before polymerization, cytotoxicity results
decreased, again at both 24 h and 7 days.
Resin cement cytotoxicity is known to increase with time.[35] In the present study, we compared cytotoxicity results after 24 h and 7 days of
incubation. The results showed that heat treatment had a significant effect in preventing
the increase of cytotoxicity. Samples treated with a jet of hot air (60°C) showed
7-day cell viability rates similar to those observed in nonheated samples at 24 h.
This finding is extremely relevant, as it suggests that heat treatment helps maintain
better cell viability rates when compared with nonheated samples.
There is a great concern that cytotoxicity mechanisms may be related to the release
of residual monomers during the conversion of monomers into polymers, i.e., to the
early stages of polymerization.[28] In this sense, the present study obtained markedly satisfactory results during early
polymerization, as heat treatment probably allowed for a higher degree of polymer
cross-linking and consequently resulted in a lower number of residual monomers.
Heat treatment before photopolymerization significantly increases monomer conversion
rates to above the levels observed with traditional methods.[36] This can be explained by the lower viscosity and increased mobility of radicals
as a result of heating. Moreover, the frequency of collision of active groups and
nonreacted radicals increases when curing temperature is below the glass transition
temperature, resulting in additional polymerization and a higher rate of conversion.[37-40
According to the present results, small physical modifications to the environment
where cement mixing and homogenization take place, for example, using a jet of hot
air at 60°C to heat the cement and glass slide, can play major roles in reducing material
cytotoxicity. Further studies are warranted to evaluate how this cytotoxicity can
be further reduced and thus cause less damage to patients.
Conclusions
The results obtained in the present study showed that heat treatment at 60°C, before
photopolymerization, while mixing self-adhesive resin cements, should be considered
as a strategy to reduce cytotoxicity of self-adhesive resin cements, as evidenced
by the results observed both at 24 h and 7 days of analysis.
Financial support and sponsorship
Nil.
