A comparative study on monomer elution and cytotoxicity of different adhesive restoration materials

TL;DR: Except for SR and QF, all other adhesive restoration materials showed different degrees of toxicity along with different monomer release kinetics, indicated that the cytotoxicity of the materials increased with the monomers release.

Abstract: This study evaluated monomer release and cytotoxicity of different adhesive restoration materials used for dental restorations. The extracts (1, 2, and 7 days) of three types of adhesive dental restoration materials, [Quixfill (QF), Silorane Restorative (SR), and Ketac N 100 Restorative (KR)], and the adhesive resins, [XP Bond (XP), Silorane Primer (SP), Ketac N 100 Primer (KP), and Silorane Bond (SB)] were analyzed using high performance liquid chromatography/mass spectrometry (HPLC-MS). The cytotoxicity levels were determined at different time points (24, 48, and 72 h) of cell culture using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. All adhesive resin materials showed monomer release at varying amounts with the highest release after 7 days. The lowest amount of release was observed in QF and the highest with KP. Bis-Phenol A (BPA) was not detected in SP and KR that contain bisphenol-A diglycidyl ether dimethacrylate (bis-GMA). Decamethylpenthasiloxane (D5) was ...

Summary

Introduction

• Resin-based composite materials and amalgam are both accepted as appropriate materials for the direct restoration of class I and II cavities in restorative dentistry.
• In an attempt to overcome the disadvantages of glass ionomers, different types of glass ionomers have been developed including resin-modified glass ionomers. [7].
• Several studies have shown the elution of different monomers such as bis-GMA, TEGDMA, and UDMA, from methacrylate-based resin restoration materials [12] but limited information is available on the monomer release from silorane-based restorative materials. [12–15].
• The in vitro cytotoxicity tests have the advantage of easy detection of the influence of a material on isolated cells growing in culture plates.[17].
• The null hypotheses tested were that adhesive restoration materials with different chemistry would not show significant difference in monomer elution kinetics and in cytotoxic effect on cell cultures.

Specimen preparation

• Materials were placed in polytetrafluoroethylene molds and processed according to the manufacturer`s instructions in a laminar flow chamber (Bioair, Siziano, Italy).
• The materials were then photopolymerized using light-emitting diode (LED, Elipar Free Light, 3 M ESPE, St. paul, USA; Light output: 1007 mW/cm2) according to the manufacturers’ instructions (40 s for QF, SR and KR; 20 s for XP, SP, SB, and KP).
• The power output of the unit was measured with a radiometer (Cure-Rite, Dentsply-Caulk) before the placement of each restoration.
• In total, 27 specimens of adhesive restoration materials and 54 specimens of primers and adhesives were prepared.

Preparation of extracts

• All specimens from material groups were divided into three subgroups according to the extraction time intervals (1, 2, 7 days).
• The disk-shaped specimens of all subgroups were immersed separately in 10 mL of culture medium (extract solution, DMEM, Sigma) immediately after photopolymerization.
• The cell suspension was stained with Trypan Blue (Sigma Chemical Co.), counted using a Neubauer chamber and seeded in 24-well plates (Nunc, Sigma–Aldrich, Copenhagen, Denmark) at a density of 1 × 104 cells/well.

HPLC analysis

• The analysis of extracts from adhesive materials and reference solutions of the monomers were performed using LC/MS System (Waters Alliance 2695 Separations Module HPLC System, Waters 2996 Photodiode Array Detector and Waters Micromass ZQ Mass Spectrometer, Meadows Instrumentation Inc., Bristol, USA).
• A diode array detector was used to analyze the extracts except for SR, and the mass detector was chosen for SR extracts.
• The injection volume was 20 μL and the elution time for the extracts was 15 min including bis-GMA, whereas for the remainder it was 6 min.
• Calculating and comparing the area of the monomer peaks at different extraction time intervals performed a semi-quantitative comparison of the eluted silorane monomers.

Cultivation of L929 fibroblasts

• The murine fibroblast cell line L929 in the third passage was obtained (HUKUK Cell Line Collection, no: 92123004 Foot and Mouth Disease Institute, Ankara, Turkey).
• The cells were grown as monolayer culture in 25 cm2 flasks in Dubelcco’s modified Eagle’s medium (DMEM, Sigma Chemical Co.) supplemented with 10% fetal bovine serum (FBS, Hyclone, Utah, USA) and 1% penicillin/streptomycin at 37 °C in an incubator (Heraeus, Germany) containing 5% CO2 at 95–98% humidity.
• Next, they were centrifuged and suspended in 24-well tissue culture plates of 1-mL aliquots, containing 5 × 104 cells/mL.

Mitochondrial activity

• Cell metabolic activity was evaluated through the mitochondrial activity analysis using MTT test that detects the presence of succinic dehydrogenase enzyme (SDH) active in viable cells [22].
• The succinic dehydrogenase activity has shown to be a reasonable representative of mitochondrial activity in the cells and reflects both cell number and activity.
• Subsequently, the absorbance at 570 nm was measured using spectrophotometer with a microplate reader (Asys UVM 340, Biochrom, Victoria, Australia) with a reference of 690 nm.
• The cell viability was calculated according to the Equation (1):.

Statistical analysis

• Data were analyzed using a statistical software package (SPSS Software V.20, Chicago, IL, USA).
• The differences in the release of each component from different adhesive materials were analyzed using one-way analysis of variance and repeated measures of variance analysis to determine the variability of the monomer elution between three different extraction time intervals.
• Bonferroni post hoc test was used to determine the differences (1) Cell viability (%) = (Optical densities of test groups∕Optical densities of control groups) × 100 between the toxicity of the materials.
• The relationship between the monomer release and toxicity was analyzed using the Spearman’s rho correlation test (−1 < r < 1).
• P values less than 0.05 were considered to be statistically significant in all tests.

Morphologic assessment

• L929 cells in the culture medium showed typical fibroblastic morphology .
• The cells incubated with QF extracts for 48 h showed a spindle-shaped morphology similar to the typical fibroblastic morphology of control cells.
• The cultures incubated with SR (1 and 2 days) extracts for 72 h were spindle-shaped in appearance similar to the control cells.
• The cells incubated with KP extracts for 24 h were round in shape and additionally led to the enlargement of the intercellular space.
• The cells treated with SP for 48 h and KR extracts for 72 h had morphologies similar to that of the cells incubated with KP extracts for 24 h .

Cell viability

• After 72 h, 1- and 2-days extracts of SR were biocompatible to the cells and similar to the results of 48 h .
• When the toxicities of the 1-day extracts of all tested materials after different incubation periods were compared, differences among the cell survival rates of KP, KR, SP, and XP were found to be statistically not significant (p > 0.05).
• The chromatogram of silorane restorative extract of 2 days.

Discussion

• This study was undertaken to measure the amount of monomers released from different types of adhesive restoration materials with different monomer types and to assess the cytotoxicity of these materials on L929 mouse fibroblast cultures at different time intervals.
• The monomers were identified based on the mass spectra of each peak in the chromatogram.
• The cytotoxic effect of adhesive materials on L929 cells was investigated using MTT assay where the reduction of MTT by viable cells is a good indicator of toxicity.[22].
• The cytotoxicity of the materials increased with the monomer release where the correlation was significant after 48 h.

Conclusion

• From this study, the following could be concluded: (1) All tested adhesive restorative materials showed monomer release kinetics at varying degrees at the time intervals tested.
• (2) The least amount of monomer release was detected for the hybrid posterior resin composite, QF, containing UDMA, and TEGDMA. (4) The most toxic material was KP after 1 day but the differences among the toxicities after 2 and 7 days were not statistically significant between materials tested.
• (5) After 48 h, 1- and 2-days extracts of SR and all extracts of QF were biocompatible with the cells.
• (6) The amount of monomer release and cytotoxicity of the materials indicated weak but significant correlation especially after 48 h.

Figures

Table 2. Monomers, abbreviations, chemical formulas, molecular weights, and chemical structures of the investigated adhesive restoration materials in this study.

Table 3.  calibration values obtained by linear regression analysis for monomers tested in this study using hPlc–Ms.

Table 4. The mean values and standard deviations of the amounts of monomers (μg/ml) released from each tested material.

Table 1. The brands, types, manufacturers, batch numbers, and chemical compositions of the adhesive restorative materials tested in this study.

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Journal of Adhesion Science and Technology
ISSN: 0169-4243 (Print) 1568-5616 (Online) Journal homepage: https://www.tandfonline.com/loi/tast20
A comparative study on monomer elution and
cytotoxicity of different adhesive restoration
materials
Tuğba Toz, Arlin Kiremitçi, Anıl Sera Çakmak, Oya Ünsal Tan, Erhan Palaska,
Menemşe Gümüşderelioğlu & Mutlu Özcan
To cite this article: Tuğba Toz, Arlin Kiremitçi, Anıl Sera Çakmak, Oya Ünsal Tan, Erhan Palaska,
Menemşe Gümüşderelioğlu & Mutlu Özcan (2017) A comparative study on monomer elution
and cytotoxicity of different adhesive restoration materials, Journal of Adhesion Science and
Technology, 31:4, 414-429, DOI: 10.1080/01694243.2016.1215768
To link to this article: https://doi.org/10.1080/01694243.2016.1215768
Published online: 01 Aug 2016.
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JOURNAL OF ADHESION SCIENCE AND TECHNOLOGY, 2017
VOL. 31, NO. 4, 414429
http://dx.doi.org/10.1080/01694243.2016.1215768
A comparative study on monomer elution and cytotoxicity of
dierent adhesive restoration materials
Tuğba Toz
a
, Arlin Kiremitçi
b
 , Anıl Sera Çakmak
c,d
 , Oya Ünsal Tan
e
, Erhan Palaska
e
,
Menemşe Gümüşderelioğlu
c,d
and Mutlu Özcan
f
a
Faculty of Dentistry, Department of Restorative Dentistry, School of Dentistry, Istanbul Medipol University,
Istanbul, Turkey;
b
Faculty of Dentistry, Department of Restorative Dentistry, Hacettepe University, Ankara,
Turkey;
c
Faculty of Engineering, Department of Chemical Engineering, Hacettepe University, Ankara, Turkey;
d
Faculty of Engineering, Department of Bioengineering, Hacettepe University, Ankara, Turkey;
e
Faculty of
Pharmacology, Department of Pharmaceutical Chemistry, Hacettepe University, Ankara, Turkey;
f
Dental
Materials Unit, Center for Dental and Oral Medicine, Clinic for Fixed and Removable Prosthodontics and Dental
Materials Science, University of Zurich, Zurich, Switzerland
ABSTRACT
This study evaluated monomer release and cytotoxicity of dierent
adhesive restoration materials used for dental restorations. The
extracts (1, 2, and 7days) of three types of adhesive dental restoration
materials, [Quixll (QF), Silorane Restorative (SR), and Ketac N 100
Restorative (KR)], and the adhesive resins, [XP Bond (XP), Silorane Primer
(SP), Ketac N 100 Primer (KP), and Silorane Bond (SB)] were analyzed
using high performance liquid chromatography/mass spectrometry
(HPLC-MS). The cytotoxicity levels were determined at dierent
time points (24, 48, and 72h) of cell culture using 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. All
adhesive resin materials showed monomer release at varying amounts
with the highest release after 7days. The lowest amount of release
was observed in QF and the highest with KP. Bis-Phenol A (BPA) was
not detected in SP and KR that contain bisphenol-A diglycidyl ether
dimethacrylate (bis-GMA). Decamethylpenthasiloxane (D5) was not
eluted from SR. Except for SR and QF, all other adhesive restoration
materials showed dierent degrees of toxicity along with dierent
monomer release kinetics. The correlation between the monomer
release and cytotoxicity of the materials indicated that the cytotoxicity
of the materials increased with the monomer release (Spearman’s rho
correlation coecient – r). The correlation after 48h was statistically
signicant (r=−0.342, p=0.017).
Introduction
Resin-based composite materials and amalgam are both accepted as appropriate materials
for the direct restoration of class I and II cavities in restorative dentistry. However, according
to the current dental concepts, resin composites are considered as the most suitable direct
posterior lling materials since they also allow for minimal invasive restorations.[1]
KEYWORDS
Adhesion; cytotoxicity;
high-performance liquid
chromatography; L929 cell
culture; monomer release
ARTICLE HISTORY
Received 12 May 2016
Revised 7 July 2016
Accepted 12 July 2016
© 2016 Informa UK Limited, trading as Taylor & Francis Group
CONTACT Tuğba Toz ttoz@medipol.edu.tr

JOURNAL OF ADHESION SCIENCE AND TECHNOLOGY 415
e polymerizable matrix of resin materials usually contains one or more base monomers
such as bisphenol-A diglycidyl ether dimethacrylate (bis-GMA) and/or urethane dimeth-
acrylate (UDMA), diluent co-monomers such as ethylene glycol dimethacrylate (EGDMA)
and/or triethylene glycol dimethacrylate (TEGDMA), and various additives such as pho-
toinitiators (e.g. camphoroquinone), co-initiators (e.g. dimethyl-aminobenzoic-acid- ester),
polymerization inhibitors and photostabilizers (e.g. benzophenone).[2] Despite the improve-
ments in adhesive technologies, drawbacks such as polymerization shrinkage within such
materials could not be eliminated.[3] In order to overcome the shortcomings of conventional
resin-based composites, dental siloranes have been introduced that consist of a new organic
matrix.[4] Siloranes present low polymerization shrinkage due to the ring-opening oxirane
monomer, increased hydrophobicity, and thereby increased biocompatibility compared to
the conventional resin-based composites.[5]
As an alternative to resin composites, glass ionomers are also commonly used for a num-
ber of applications such as base or liner materials, as luting cements for indirect restorations,
or as temporary or permanent restorative materials for direct restorations. Glass ionomers
present several advantages of biocompatibility, low coecient of thermal expansion, the
ability to adhere to the moist tooth structure without necessitating an intermediate agent
that decreases the chair-side of the treatment procedure and anticariogenic properties due
to uoride release compared to methacrylate-based resin composites. However, they have
some drawbacks such as poor polishability and less favorable mechanical properties, surface
wear, or decreased fracture resistance over time.[6] In an attempt to overcome the disad-
vantages of glass ionomers, dierent types of glass ionomers have been developed including
resin-modied glass ionomers.[7]
e application of adhesive restoration materials in dentistry has been extensively pro-
moted over the last decade.[8] However, concerns in the dentistry over the generation
of biodegradation products have also increased and possible eects of byproducts from
composite resin matrix degradation on the function of host cells and micro-organisms are
being questioned.[9] e release of unpolymerized components from the polymerizable
resin matrix as a result of incomplete polymerization of adhesive restoration materials may
inuence the biocompatibility of the restorations.[2] Consequently, the amount of mono-
mer elution plays an important role in biocompatibility of adhesive restorative materials.
Chromatographic techniques are helpful in the analysis of compounds released from
resin-based dental llings. e components extracted from resin-modied glass ionomers,
compomers, and resin composites could be determined using gas chromatography/mass
spectrometry (MS).[10] Unfortunately, base-monomer analysis by gas chromatography is
almost impossible due to their low volatility and decomposition at higher temperature in the
injection port of the gas chromatograph. One of the few available techniques suitable for the
analysis of high-molecular-mass compounds is high-performance liquid chromatography
(HPLC), mainly coupled with mass spectrophotometry or diode array detection.[11] Several
studies have shown the elution of dierent monomers such as bis-GMA, TEGDMA, and
UDMA, from methacrylate-based resin restoration materials [12] but limited information
is available on the monomer release from silorane-based restorative materials.[12–15]
By denition, cytotoxicity of an agent is the cascade of molecular events that interferes
with macromolecular synthesis, causing unequivocal functional and structural damage
in a cell culture. e interactions of the materials and their components with the cells
at a molecular level are responsible for tissue reactions such as inammation, necrosis,

416 T. TOZ ET AL.
immunological alterations, genotoxicity, and apoptosis.[16] e in vitro cytotoxicity tests
have the advantage of easy detection of the inuence of a material on isolated cells growing
in culture plates.[17] A number of methods have been developed such as lactate dehydro-
genase assay, bromodeoxiuridine assay [18], and uorescence microscopy [19] in order to
investigate the cytotoxicity of dental resin materials. However, 3-(4,5-dimethyl-2-thiazolyl)-
2,5- diphenyl-2H tetrazolium bromide (MTT) assay is considered to be more useful to
estimate cell densities in small culture volumes and has some advantages, such as simplicity,
accuracy, reliability, and eciency.[20]
e objective of this study, therefore, was to measure the amount of monomers released
from dierent types of adhesive restoration materials with dierent monomer types using
HPLC-MS and HPLC-Ultraviolet (UV) and to assess the cytotoxicity of these materials on
L929 mouse broblast cultures at dierent time intervals. e null hypotheses tested were
that adhesive restoration materials with dierent chemistry would not show signicant
dierence in monomer elution kinetics and in cytotoxic eect on cell cultures.
Materials and methods
Specimen preparation
Disk-shaped specimens from the tested restorative materials including etch-and-rinse adhe-
sive system ‘XP’ (XP Bond, Dentsply De Trey, Germany), posterior hybrid composite ‘QF’
(Quixll, Dentsply De Trey, Germany), silorane composite ‘SR’ used with its own self-etch
adhesive system including primer ‘SP’ and bond ‘SB’ (Silorane, 3M ESPE, St. Paul, USA),
and nano-ionomer ‘KR’ applied with its own primer ‘KP’ (Ketac N 100, GC, Tokyo Japan)
were prepared (Table 1). Materials were placed in polytetrauoroethylene (Teon) molds
and processed according to the manufacturer`s instructions in a laminar ow chamber
(Bioair, Siziano, Italy). e etch-and-rinse adhesive system (XP), primer (SP and KP), and
adhesive resin (SB) disks were 5mm in diameter and 1mm in thickness and adhesive res-
toration materials (QF, SR, and KR) were 5mm in diameter and 4mm in thickness. e
adhesive restorative materials were applied in two layers, while the primers and adhesives
were applied in one layer and the surfaces of all materials were covered with a transpar-
ent strip to prevent the formation of air-inhibited surface layer. e materials were then
photopolymerized using light-emitting diode (LED, Elipar Free Light, 3M ESPE, St. paul,
USA; Light output: 1007mW/cm
2
) according to the manufacturers’ instructions (40 s for
QF, SR and KR; 20 s for XP, SP, SB, and KP). e power output of the unit was measured
with a radiometer (Cure-Rite, Dentsply-Caulk) before the placement of each restoration.
In total, 27 specimens of adhesive restoration materials and 54 specimens of primers and
adhesives were prepared.
High-performance liquid chromatography
Preparation of extracts
All specimens from material groups were divided into three subgroups (n=9 for the adhe-
sive restoration materials; n=18 for primers and adhesives) according to the extraction
period (1, 2, 7days). e extraction period was adjusted accordingly for the methyltetra-
zolium (MTT) test procedure. Immediately aer photopolymerization, the specimens in

JOURNAL OF ADHESION SCIENCE AND TECHNOLOGY 417
each subgroup were weighed (Mettler Toledo, Colombus, USA) and, except for SR, spec-
imens in all groups were placed separately into 10mL of 70% methanol/water solution
(Sigma–Aldrich, St Louis, MO USA). e extracting medium for the SR groups was pure
methanol solution as it is not soluble in methanol/water solution. e specimens were
incubated for 1, 2, and 7days at 37°C in dark. Aer the incubation period, the specimens
were removed and the extracts were analyzed using HPLC.
Table 1.The brands, types, manufacturers, batch numbers, and chemical compositions of the adhesive
restorative materials tested in this study.
Adhesive restor-
ative system
Type of the
material Manufacturer Batch number Chemical composition
Adhesive restora-
tive system
Type of the
material
Manufacturer Batch number Chemical composition
XP bond Two-step etch
and rinse
adhesive
system
Dentsply, De
Trey, Konstanz
Germany
0,707,000,223 Carboxylic acid-modified dimeth-
acrylate,phosphoric acid-modified
acrylate resin,urethane dimeth-
acrylate, TEGDMA,HEMA, butylated
benzenediol,ethyl-4-dimethylam-
inobenzoate,camphorquinone,
functionalizedamorphous silica,
t-butanol
Quixfill Methacyr-
late-based
hybrid pos-
terior resin
composite
07,030,000,799 Resin: urethane dimethacrylate
(UDMA);triethyleneglycol di-
methacrylate (TEGDMA); di- and
trimethacrylate resins; carboxylic
acid-modified dimethacrylate resin;
butylated hydroxy tolüene (BHT); UV
stabilizer, camphorquinone; ethyl-4-
dimethylaminobenzoateFillers: sila-
nated strontium aluminumsodium
fluoride phosphate silicate glass
Silorane primer Primer 3M ESPE,St. Paul,
USA
20,090,220 2-Hydroxyethyl methacrylate (HEMA)
bisphenol A diglycidyl ether
dimethacrylate (bis-GMA), water,
ethanol, phosphoric acid-meth-
acryloxy-hexylester mixture,
silane treated silica 1,6-Hexanediol
dimethacrylate,copolymer of acrylic
and itaconic acid,(Dimethylamino)
ethyl methacrylate,dl-Camphorqui-
none,phosphine oxide
Silorane bond Adhesive resin 20,090,220 Substituted dimethacrylate Silane
treated silica, triethylene glycol di-
methacrylate (TEGDMA), phosphoric
acid methacryloxy-hexylesters-
dl-Camphorquinone 1,6-Hexanediol
dimethacrylate
Silorane Silorane
restorative
20,090,220 3,4 epoxycyclohexylethylcyclopolyme-
thylsiloxane,bis-3,4-epoxycyclohex-
ylethylphenylmethylsilane, silanized
glass,yittrium floride, camphorqui-
none
Ketac N 100
Primer
Nano-ionomer
primer
3M ESPE 20,070,917 Water, HEMA, acrylic/itaconic acid
copolymer, photo-initiators
Ketac N 100 Photopolym-
erizedna-
no-ionomer
restorative
20,070,917 Paste A: silane-treated glass, si-
lane-treated ZrO2silica, silane-treat-
ed silica, PEGDMA, HEMA, bis-GMA,
TEGDMAPaste B: silane-treated
ceramic, silane-treated silica, water,
HEMA, acrylic/itaconic acidcopol-
ymer

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This study evaluated monomer release and cytotoxicity of different adhesive restoration materials used for dental restorations.