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The effect of polymerization mode on monomer

conversion, free radical entrapment, and
interaction with hydroxyapatite of commercial
self-adhesive cements

ARTICLE in JOURNAL OF THE MECHANICAL BEHAVIOR OF BIOMEDICAL MATERIALS JUNE 2015

Impact Factor: 3.42 DOI: 10.1016/j.jmbbm.2015.02.019

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Research Paper

The effect of polymerization mode on monomer

conversion, free radical entrapment, and interaction
with hydroxyapatite of commercial self-adhesive
cements

Paulo Henrique Perlatti DAlpinoa,n, Marlia Santos Silvaa,

Marcus Vincius Goncalves Vismarab, Vinicius Di Hipolitoa,
Alejandra Hortencia Miranda Gonzaleza, Carlos Frederico de Oliveira Graeffb
a
Biomaterials Research Group, School of Dentistry, UNIAN/SPUniversidade Anhanguera de So Paulo, So Paulo, SP,
Brazil
b
DF-FC, UNESPUniversidade Estadual Paulista, POSMATPrograma de Ps-Graduao em Cincia e Tecnologia de
Materiais, Bauru, SP, Brazil

art i cle i nfo ab st rac t

Article history: Objectives: This study evaluated the degree of conversion, the free radical entrapment, and
Received 19 November 2014 the chemical interaction of self-adhesive resin cements mixed with pure hydroxyapatite,
Received in revised form as a function of the polymerization activation mode among a variety of commercial self-
15 February 2015 adhesive cements.
Accepted 19 February 2015 Materials and methods: Four cements (Embrace WetBond, MaxCem Elite, Bix SE, and RelyX
Available online 26 February 2015 U200) were mixed, combined with hydroxyapatite, dispensed into molds, and distributed
Keywords: into three groups, according to polymerization protocols: IP (photoactivation for 40 s); DP
Self-adhesive resin cement (delayed photoactivation, 10 min self-curing plus 40 s light-activated); and CA (chemical
Hydroxyapatite activation, no light exposure). Infrared (IR) spectra were obtained and monomer conversion
Chemical interaction (%) was calculated by comparing the aliphatic-to-aromatic IR absorption peak ratio before
FT-IR and after polymerization (n10). The free radical entrapment values of the resin cements
EPR were characterized using Electron Paramagnetic Resonance (EPR) and the concentration of
X-ray diffraction spins (number of spins/mass) calculated (n3). Values were compared using two-way ANOVA
and Tukeys post-hoc test (5%). X-ray diffraction (XRD) characterized the crystallinity of
hydroxyapatite as a function of the chemical interactions with the resin cements.
Results: The tested parameters varied as a function of resin cement and polymerization
protocol. Embrace WetBond and RelyX U200 demonstrated dependence on photoactivation
(immediate or delayed), whereas MaxCem Elite exhibited dependence on the chemical
activation mode. Bix SE presented the best balance based on the parameters analyzed,
irrespective of the activation protocol.

n
Correspondence to: Universidade Anhanguera de So PauloUNIAN SP, Programa de Mestrado em Biomateriais em Odontologia,
Rua Maria Cndida, 1.813 6.1 andar, Bloco G, So Paulo CEP: 02071-013, SP, Brazil. Tel.: 55 11 29679058/55 11 29679077.
E-mail address:
paulodalpino@yahoo.com (P.H.P. D'Alpino).

http://dx.doi.org/10.1016/j.jmbbm.2015.02.019
1751-6161/& 2015 Elsevier Ltd. All rights reserved.
84 journal of the mechanical behavior of biomedical materials 46 (2015) 83 92
Conclusions: Choice of polymerization protocol affects the degree of conversion, free radical
entrapment, and the chemical interaction between hydroxyapatite and self-adhesive resin
cement mixtures.
1. Introduction
Resin cements are a class of dental materials of choice for
bonding metal, ceramic and indirect composite restorations.
These resin-based materials are generally classied as a func-
tion of their activation reaction as self-cured (chemically
activated), light-cured (photoactivated), or dual-cured (a com-
bination of both activation reaction) (Burrow et al., 1996). Based
on interaction with tooth substrates, resin cements can be also
classied into three categories: etch-and-rinse, self-etch, and a
recently developed group of resin cements known as self-
adhesive systems (SLCs) (Carvalho et al., 2004; Pegoraro et al.,
2007). The main objective of the introduction of SLCs was to
overcome the drawbacks of other types of cements used to join
indirect restorations to tooth preparations (Radovic et al., 2008).
These materials require no technique-sensitive steps, such as
acid etching, priming, or bonding (De Munck et al., 2004). The
adhesion strategies employed with SLCs also allow the forma-
tion of secondary reactions between the self-adhesive resin
and hydroxyapatite, forming chemical bonds (De Munck et al.,
2004). This innovative bonding mechanism represents an
important characteristic when compared to other resin
cements, which are essentially micromechanical in nature
(Van Meerbeek et al., 1993).
Resin-based cements are also subjected to the oral environ-
ment, in spite of the thin layer that results from seating
indirect restorations. The polymerization process starts with
light exposure or through a self-curing mechanism, if no light
is present. Considering this clinical scenario, the polymeriza-
tion protocol of resin cements is of paramount importance in
the subsequent restoration performance (Svizero et al., 2013). It
is claimed that the best mechanical properties are achieved
when base and catalyst pastes are mixed and immediately
photoactivated (Pegoraro et al., 2007). On the other hand, the
chemical cure mechanism proceeds slowly and is expected to
ensure polymerization in those areas where light is unable to
reach (Pereira et al., 2010). As the polymerizing network
develops further, the rate of radical propagation eventually
becomes limited by diffusion, and the polymerization rate
decelerates, providing only a limited conversion, even in the
presence of unreacted monomer and free radicals (Halvorson
et al., 2002). Conversely, under a delayed photoactivation
condition, the cements is chemically activated at rst and the
polymerization reaction progresses slowly, especially in areas
where the curing light is unable to reach the material (Pereira
et al., 2010). In this way, a higher end conversion would be
expected. Increased degree of conversion has been strongly
correlated with improved mechanical properties of resin-based
materials (Ferracane and Greener, 1986). Previous studies
focused on the inuence of different activation protocols in
& 2015 Elsevier Ltd. All rights reserved.
conventional resin cements (Pereira et al., 2010; Svizero et al.,
2013). However, studies are necessary to understand the
inuence of these protocols for the SLCs that present distinc-
tive formulations, low initial pH, dual-cure polymerization, and
the ability to interact with hydroxyapatite.
Concerns have been expressed over the chemical com-
position of the self-adhesive resin cements, specically
regarding the need for balanced formulae due to the
polymerization reaction occurs in an acidic environment
(Di Hipolito et al., 2012; Vaz et al., 2012). This category of
resin cement presents methacrylate monomers that con-
tain phosphoric acid esters that simultaneously deminer-
alize and inltrate both the smear layer and the underlying
dentin, providing micromechanical bonding (Gerth et al.,
2006). At the same time, it is important that the pH be
neutralized in order to avoid impacting the end conversion,
considering the effect of both new methacrylate monomers
formulation and the technology to initiate polymerization
(Vaz et al., 2012). On the other hand, it has been claimed
that a glass ionomer concept was added to the formulation
in order to neutralize the initial low pH, which increases
from 1 to 6 (Radovic et al., 2008). Thus, the comprehension
of the dynamic process in which the demineralization/
monomer permeation process and the polymerization
kinetics coexist in this category of material is of paramount
importance.
Clinically, restorative materials must set in a reasonably short
time, in order to be practical, and immediate photoactivation
may optimize the initial cement properties necessary to with-
stand clinical stresses that affect indirect restorations cemented
to the remaining tooth structure (Pereira et al., 2010). It is claimed
that immediate photoactivation limits the time available for
polymer growth and thus inhibits the formation of a rigid
polymer network (Neves et al., 2005). On the other hand, the
chemical activation protocol is expected to provide the resin
cement optimal mechanical properties over time, in areas where
polymerization light energy is unable to reach, improving the
overall polymerization of the cement at that location (Camilotti
et al., 2008; Sideridou et al., 2003). However, different polymeriza-
tion protocols may affect the resin cements in different ways,
depending on a variety of parameters tested (Pereira et al., 2010;
Svizero et al., 2013). Some resin cements depend on immediate
photoactivation, whereas others seem more dependent on the
chemical activation mode. In this way, it is important to evaluate
this new category of dual-cure resin cements, considering among
others their particular acidic characteristics. Another concern is
related to the analysis of the biocompatibility of dental materials,
in that the nature and amount of components released during
their clinical application are most important. Monomer-release
studies are commonly investigated using different solvents such
as water, saliva, and organic solvents (Sideridou et al., 2003).
 journal of the mechanical behavior of biomedical materials 46 (2015) 83 92
Considering that SLCs are frequently applied to newly exposed
dentin, especially in total crown preparations, and the fact that
the polymerization reaction in methacrylate-based resins is
radical-mediated, according to a study (Andrzejewska, 2004) to
evaluate entrapped free radicals in the resin cements and their
interaction with tooth substrates as a function of the activation
protocol, is of clinical relevance.
Drawing upon three hypotheses, this study attempts to
investigate the effects of activation protocols on the degree of
conversion, free radical entrapment, and the integrity of the
hydroxyapatite crystal structure, when evaluating different
SLCs. The following research hypotheses were tested: (1) the
degree of conversion will not be negatively inuenced by the
polymerization activation mode and brand of resin cement;
(2) the different activation mode will signicantly affect the
density of spins of the resin cements tested; and (3) the
integrity of the hydroxyapatite (HA) crystal (as monitored in
the 211 orientation) will be inuenced by the brand of SLCs
tested and by the polymerization activation mode used.
2. Materials and methods
2.1. Experimental design
In this in vitro study, the degree of conversion, free radical
entrapment, and the integrity of the hydroxyapatite crystal-
line structure were evaluated on resin cement mixtures
incorporating known amounts of hydroxyapatite, according
to the following factors: (1) SLCs at four levels: Embrace
WetBond, MaxCem Elite, Bix SE, and RelyX U200; and (2)
activation mode at three levels: immediate photoactivation
(IP), delayed photoactivation (DP), and chemical activation
(CA). The characteristics of the resin cements selected are
described in Table 1. Twelve groups were categorized and
treated according to the various polymerization protocols
(Table 2).
2.2. Degree of conversion
2.2.1. Specimen preparation
Specimens were prepared by mixing 0.035 g of dry hydroxyapatite
(HA) with 0.25 g of the SLCs. As a starting compound, a HA with a
high degree of crystallinity and purity (Ca10(PO4)6(OH)2) was used.
The SLCs were mixed according to the manufacturers instruc-
tions in terms of working and setting times, and then the HA was
gently incorporated using manual mixing. The unpolymerized
resin cement/HA mixtures were then placed into a Teon mold
(2 mm thick, 6 mm in diameter), positioned over a polyester strip.
After lling the mold to excess, the material surface was covered
with a Mylar strip and a glass slide and compressed to extrude
excess material. The glass slide was then removed, leaving the
Mylar strip. The distal end of the curing light guide was placed
within 0.1 mm of the top Mylar surface. Specimens of groups IP
and DP were photoactivated for 40 s on the top and bottom
surfaces. Photoactivation was performed after the working time.
After the photoactivation procedures (IP and DP groups), the
specimens were removed from the molds and then stored in
lightproof recipients for 24 h at room temperature (22 1C). The
light-curing procedure was performed using a poly-wave LED
85
light-curing unit (Bluephase, Ivoclar Vivadent, Schaan, Liechten-
stein) having an irradiance of 1200 mW/cm2 s. Prior to testing,
and throughout the experiment, the light output was monitored
using a handheld radiometer (Model 100, Demetron Research
Corp., Danbury, CT, USA). Specimens from the chemically acti-
vated group (CA group) were kept in the molds for 24 h at room
temperature in the dark prior to their removal. Ten replications
were made for each test condition (n10).
2.3. FT-IR analysis
The specimens were placed and vertical pressed against a
horizontal diamond element of an attenuated total reec-
tance (ATR) unit (MIRacle, PIKE Technologies, Madison, USA)
of a Fourier transform infrared spectrometer (IRPrestige-21,
Shimadzu, Tokyo, Japan) in order to obtain the infrared
(IR) spectrum. IR spectra were collected between 1680 and
1500 cm  1 using 16 scans at 2 cm  1 resolution. The speci-
mens were placed one at a time in the sample holder of the
device, and spectra were recorded. A small amount of
uncured resin cement from each material was also scanned
and its spectrum was used as unpolymerized reference. The
ratio between the peak absorbance value of the aliphatic
(CC) at 1636 cm  1 to the aromatic C C group absorption at
1608 cm  1 in the cured and uncured states was used to
calculate the degree of conversion of aliphatic (C C) into
(CC). The results were expressed as the percentage of
aliphatic C C remaining after light exposure to that available
in the unpolymerized condition (Rueggeberg et al., 1990).
Average conversion values were calculated, and a two-way
ANOVA was performed. Tukeys post-hoc test was used as a
multiple comparison test, at a pre-set alpha of 0.05.
2.4. Magnetic properties
2.4.1. Specimen preparation
Specimens were prepared for Electron Paramagnetic Reso-
nance (EPR) analysis in the same way as previously described,
except that the unpolymerized resin cement/HA mixtures were
placed into a rectangular Teon mold (8 mm  2 mm  2 mm). In
addition, three replicas were made for each test condition (n3).
2.5. EPR analysis
Spectra were acquired using an EPR instrument (Magnettech
Miniscope MS300, Berlin, Germany) operating at X-Band  9.43
GHz. The main EPR parameters were modulation amplitude
(from 0.2 to 0.4 mT) and microwave power (0.5012 mW). The
g-factors were determined by reference to the signal of the
diphenylpicrylhydrazyl (DPPH) with a known value of 2.0036.
The density of spins was determined by comparing the EPR
spectrum of the unknown specimens (cements) to that of a
sample with a known spin concentration (standard sample).
Each free radical has a spin different from zero, so by measuring
the spin density one is measuring the free radical density (Poole
and Charles, 1983). For that standard, an amorphous silicon
sample with a calibrated spin concentration of 1.13  1015 spins/
grams was used (DAlpino et al., 2014). The number of spins in
the resin cements of each experimental group was then
86 journal of the mechanical behavior of biomedical materials 46 (2015) 83 92

Table 1 Materials used in this study.

Material Lot Composition Working Setting Exposure Fillercontent

#/expiration time time duration W (%) V (%)
date (min) (min) (s)

Embrace WetBond
Pulpdent 130711 Co-monomers (mono-, di-, and tri-functional 36.6
2.0 3.0 40
Corporation, 2015-07 methacrylate monomers, Barium, glass, 39.0
Watertown, ytterbium triuoride, inert minerals.
MA, USA Automix system
69.9
MaxCem 5011290 GPDM, co-monomers (mono-, di-, and tri- 1.5 4.0 10 - 20 59.0
Elite 2015-03 functional methacrylate monomers, water,
Kerr acetone, and ethanol. Inert minerals and
Corporation, ytterbium uoride. Automix system
Orange, CA,
USA

Bix SE 1322421 Bis-GMA, UDMA, Gly-DMA, phosphate 70.0

2.0 4.0 10 - 20
Voco GmbH, 2014-12 monomers, initiators, stabilizers. Glass. 45.0
Cuxhaven, Automix system
Germany
Base: Methacrylate monomers containing
phosphoric acid groups, methacrylate
monomers, initiators, stabilizers, rheological
RelyX U200 132950065 additives
3M ESPE, St. 9 2014-12 Catalyst: Methacrylate monomers, alkaline 72.0
2.0 6.0 20
Paul, MN, llers, silanated llers, initiator components, 43.0
USA stabilizers, pigments, rheological additives.
Zirconia/silica llers. Clicker delivery system

Abbreviations: Bis-GMA: bisphenol A diglycidyl ether dimethacrylate; UDMA: urethane dimethacrylate; Gly-DMA: glycerol dimethacrylate;
GPDM: glycero-phosphate dimethacrylate. All information supplied by manufacturers.

Table 2 Experimental groups and curing protocols descriptions.

Polymerization mode used Group abbreviation Curing protocol description

Immediate Photoactivation IP (control) Photoactivation only for 40 s

Delayed photoactivation DP 10 min delay for chemical curing, followed by photoactivation for 40 s
Chemical activation CA Chemical activation only

calculated using the following formula (Poole and Charles, 1983;
Warren and Fitzgerald, 1977):
CT
N p 1
AM  2
PA
where N is the spin concentration (in spins); C is the standard
sample constant (1.13  1015); T is the temperature (in Kelvin); A
is the area under the resonance absorption curve; AM is the
modulation amplitude; and P is the incident microwave power.
For all measurements, the microwave power and modulation
amplitude were kept constant to avoid signal saturation and
distortion.
The identication of the free radicals was made with the
assistance of software (Easyspin 4.5.1). The ESR parameters
were obtained by tting the experimental data. The spin
concentration as a function of mass according to the experi-
mental groups was calculated dividing the number of spins by
the sample mass (in grams). The radicals were then calculated
as a function of the resin content. The concentration in the
resin portion of each product (by mass) was obtained by
thermal analysis (data not shown). Statistical analysis was
performed using a two-way analysis of variance (ANOVA)
followed by Tukeys post-hoc test at a pre-set alpha of 0.05.
2.6. Chemical interaction with hydroxyapatite
2.6.1. Preparation of hydroxyapatite (HA)/SLC samples
Samples were prepared for X-ray diffraction analysis (XRD) by
mixing 0.070 g of dry HA powder (Ca10(PO4)6(OH)2) with 0.5 g
of the freshly mixed but unset SLCs. The materials were
manipulated according to the manufacturers instructions
and were then gently mixed with HA on a glass plate until
a paste-like consistency was obtained. Then, the paste was
transferred and carefully spread onto an XRD specimen
holder. A homogeneous layer in which the HA was uniformly
distributed throughout the organic matrix of the resin
cements was obtained in all cases. The specimens were then
polymerized according to the activation protocols. The dry
HA power was also characterized for comparative analyses. A
different ratio was used here in order to ll out the specimen
holder to obtain the XRD patterns.
 journal of the mechanical behavior of biomedical materials 46 (2015) 83 92
2.7. XRD analysis
XRD patterns were collected using an X-ray diffractometer
(DMAX Ultima Rigaku International Corporation, Tokyo,
Japan) with CuK radiation, operating at 40 kV and 20 mA.
Scans were performed from 51 to 801 (2) at a step size of 0.021
and a scan speed of 21/min. Qualitative phase analysis was
carried out by using the Joint Committee on Powder Diffrac-
tionInternational Center for Diffraction Data (JCPDSICDD)
databases by comparing the peak intensity of pure HA with
that of the test specimens. In addition, a quantitative phase
analysis was also performed by comparing the peak intensity
of HA attributed to the 211 diffraction (highest peak of
crystallinity of pure HA) before and after mixing HA with
the SLCs. The results were expressed in peak intensity of the
mixture resin cement/HA (in cps) and also in the percent
decrease in crystallinity of HA using the strongest diffraction
orientation (2 1 1) as a guide (1730 cps), in comparison to that
of pure HA.
3. Results
3.1. FT-IR analysis
Experimental results are displayed in Table 3. The degree of
conversion ranged between 16.5% for RelyX U200 to 59.6% for
MaxCem Elite, when both cements were chemically activated.
When the cements were compared, signicantly lower conver-
sion averages were observed for Bix SE when immediately
photoactivated, and for RelyX U200 when chemically activated
or photoactivated after 10 min (po0.05). On the contrary, Max-
Cem Elite presented signicantly higher degree of conversion
averages, irrespective of the activation protocol (po0.05). In
terms of conversion, both Embrace WetBond and RelyX U200
demonstrated dependence on the photoactivation (immediate
or delayed), whereas MaxCem Elite exhibited signicantly higher
conversion averages when chemically activated. On the other
hand, Bix SE presented signicantly higher conversion averages
when chemically activated and for delayed photoactivation over
those seen in immediate photoactivation.
3.2. Free radical entrapment
The panels in Fig. 1 display characteristic electron paramagnetic
resonance spectra of the SLCs. The EPR spectra are characterized
by 9 lines. For the propagating radical g 2.00370.001 and isotropic
hyperne constant a is 21.570.1 G for CH3 and 11.770.1 G for CH2.
Table 3 Percent monomer conversion (%) (sd) of products tested using various polymerization protocols.
PRODUCT Immediate photoactivation (IP)
Embrace WetBond 32.3 (1.8) a,B
MaxCem Elite 52.3 (1,5) b,A
Bix SE 27.3 (1.7) b,C
RelyX U200 28.3 (3.9) a,B
(n 10).
Groups identied using similar letters (upper case for columns and lower case for rows) are not signicant (p40.05).
87
For the allylic radical g 2.00370.001 and a is 21.570.1 G related to
CH2. The calculated number of spins of the experimental groups,
normalized by weight (resin content), is presented in Table 4. The
spin concentration was found to be between 3.6  1016 spins/g for
MaxCem Elite (CA), and 1,560  1018 spins/g for RelyX U200 (IP).
Embrace WetBond and RelyX U200 presented signicantly higher
spin concentration averages, when photoactivated immediately or
after 10 min (po0.05). MaxCem Elite presented signicantly higher
averages of spin concentration when photoactivated after 10 min.
The same was observed for Bix SE. All of the materials tested
presented signicantly lower spin concentrations when the che-
mical activation was performed (po0.05).
3.3. XRD analysis
The panels in Fig. 2 displays the X-ray diffraction patterns of
SLCs tested. In each diffractogram, the crystalline peaks of pure
HA, as well as those of the resin cements/HA mixtures, are
displayed. The peaks and associated planes of HA diffracto-
grams match the crystallographic data sheet (74-0565) of
calcium hydroxide phosphate, Ca10(PO4)6(OH)2, obtained from
the PCPDFWIN database. In accordance with this database, the
crystalline pattern has a hexagonal cell with the following
parameters: a9.424 and c 6.879 . With respect to the HA/
resin cement interactions, X-ray diffraction patterns indicate
the presence of a hexagonal HA phase and an organic,
amorphous phase (seen as a broad hump in the baseline) at
2 between 101 and 301, associated with Embrace WetBond
(Fig. 2A). Similar ndings were observed in the diffractograms
of the mixture Bix SE/HA (Fig. 2C), irrespective of the activa-
tion protocol. The diffractograms indicating the interaction
between HA and RelyX U200 (Fig. 2D) show the presence of
peaks related to both Ca10(PO4)6(OH)2 and an unidentied
crystalline component at 2 17.91. In the diffractograms of
the mixture of MaxCem Elite and HA, two crystalline fractions
are present: one related to HA and another related to ytterbium
uoride (YbF3). These additional peaks in the diffractograms of
the MaxCem Elite/HA mixture were attributed to the presence
of the orthorhombic crystalline phase of YbF3. The peaks and
the crystalline planes associated match the crystallographic
data sheet of ytterbium uoride (711161).
Table 5 displays the results of peak intensity of the resin
cement/HA mixture (in cps) and also in terms of percentage
reduction in comparison to that of pure HA at (2 1 1) diffraction
(1730 cps). The greatest reductions in peak intensity were
obtained when Bix SE was mixed to HA, irrespective of the
activation protocol. The percentages varied from 61.8 (DP group)
to 69.2 (CA group). On the other hand, the lowest reductions were
Delayed photoactivation (DP) Chemical activation (CA)
31.7 (0.8) a,B 27.5 (3.0) b,C
52.8 (3.1) b,A 59.6 (1.6) a,A
39.9 (1.6) a,B 40.8 (1.8) a,B
25.4 (1.0) a,C 16.5 (1.5) b,D
88 journal of the mechanical behavior of biomedical materials 46 (2015) 83 92
Fig. 1 Characteristic X-band electron paramagnetic resonance
spectra after 24 h of the self-adhesive resin cements according
to polymerization activation protocol.
obtained for RelyX U200 when photoactivated immediately
(IP group) or after 10 min (DP group), in which the percentages
were 42.7% and 49.7%, respectively. For the CA group, MaxCem
Elite demonstrated the lowest reduction of the peak intensity
(46.5%).
4. Discussion
The results of the present study indicate that, regarding the
degree of conversion, that both Embrace WetBond and RelyX
U200 cements depend mostly on the photoactivation (immediate
or delayed), whereas MaxCem Elite exhibits a higher dependence
of the chemical activation mode. In addition, Bix SE exhibited
dependence on chemical activation and the time for photoactiva-
tion (delay of 10 min). Thus, the rst research hypothesis, which
anticipated that the degree of conversion of a given SLC product
among the different polymerization modes would not be nega-
tively inuenced, was partially upheld by the experimental data.
It can be argued that the degree of conversion signicantly
varied among the experimental groups and that, for some of
them, the percentages were rather low. As described in the
FTIR methodology, the degree of conversion of aliphatic CC
into CC for each specimen was calculated. The results were
expressed as the percentage of aliphatic CC remaining after
polymerization to that available in the unpolymerized con-
dition. In this way, it would be expected that the materials
were equally compared using this methodology if the resin
cements presented a chemistry solely based in Bis-GMA. In
analyzing the material composition (Table 1), it can be noted
that the manufacturer provides no specic information. The
description includes mono-, di-, and tri-functional methacry-
late monomers, phosphate monomers, and others. In addi-
tion, the ller content in this type of restorative material
comprises an extremely important characteristic, determin-
ing the relative viscosity of the luting cement and thus
affecting polymer chain mobility and the ability of a growing
species to propagate. Concerns have been previously raised
as to whether the SLCs can be successfully used in clinical
applications because of the low percentages of conversion in
self-curing mode in the case of light attenuation (Vrochari
et al., 2009). On the other hand, it is important to mention
that there may be an additional glass ionomer-type reaction
occurring in the RelyX product at the same time as the free
radical polymerization (Nakamura et al., 2010). Thus, even if
low conversion values are seen, the testing performed does
not monitor other types of setting reactions that might also
contribute to the overall properties, and thus the potential
clinical success, of these products.
The weight content of ller particles varies between 60 and
75% in SLCs (Belli et al., 2009). It is suggested that a practical limit
to the amount of ller particles in resin cement formulations
must be far less than the densest pack limit possible (Darwell,
2009). Considering the same resin cement volume, very high ller
packing densities can be achieved with an appropriate selection
of the particle size and proportions. In this way, the ller packing
in resin cements differs from that in the dental composite resins,
where lower ller volumes are normally used. In the present
 journal of the mechanical behavior of biomedical materials 46 (2015) 83 92
Table 4 Mean entrapped free radical concentrations normalized by weight (sd)*, with respect to brand of self-adhesive
resin cements and polymerization activation mode.
PRODUCT Immediate Photoactivation (IP)
Embrace WetBond 231 (2.4) a,C
MaxCem Elite 21 (0.2) b,D
Bix SE 494 (2.9) b,B
RelyX U200 1560 (15.4) a,A
(n 3).
Values denoted using similar letters (upper case for columns and lower case for rows) are not signicantly different (p40.05).
n
(  1016 spins/g).
study, the volumetric ller content varied from 39.0 to 59.0%,
whereas the variation in weight content was between 39.9 and
72.0%. During polymerization, portion of the methacrylate groups
involved in the formation of the cross-linked matrix remains
unreacted, especially in the case of higher molecular weight
monomers (DAlpino et al., 2007). This fact, associated with high
ller levels decreases polymer radical mobility, and as a conse-
quence, there is a decrease in its reactivity: it tends to become
entrapped within the viscous, cured polymer network and is
incapable of causing additional chain lengthening or cross-
linking. The products with higher ller loading (such as RelyX
U200) react more slowly, and this fact may explain why this resin
cement exhibited a signicantly lower degree of monomer
conversion and a dependence on the delay time prior to photo-
activation (Pereira et al., 2010).
Previous studies recommended immediate photoactiva-
tion for SLCs, solely based on the degree of conversion
parameter (Aguiar et al., 2010; Arrais et al., 2014). In dual-
cured cements, both light and chemical activation procedures
generate reactive free radicals by cleavage of the initiator
molecules (Rueggeberg, 1999). In the propagation reaction,
these radicals are able to add to the double bonds of
methacrylate groups, thereby creating new radicals. This free
radical polymerization reaction occurs very rapidly, until no
additional monomer is able to react or the propagation
reaction is stopped by termination (Trufer-Boutry et al.,
2003a). The mobility of the developing polymer chains
becomes progressively more restricted, due to the increase
in the viscosity, the reduction in free volume, formation of
microgels, and chain entanglement (Neves et al., 2005). In this
way, the polymer network becomes rigid enough, its chains
become essentially immobile, and the propagation of free
radical reactions becomes diffusion limited, decreasing the
overall conversion value (Andrzejewska, 2004). This change
causes a signicant percentage of the methacrylate groups
(between 25 and 60%) to remain unreacted (Cook et al., 1999).
In resin-based restorative materials, mostly bifunctional
monomers are used and are able to react with four other
monomer molecules leading to the formation of polymer
chains and cross-linking. Although the network of a com-
posite includes not only the remaining double bonds but
also free radicals, which are located at functional groups
that have reacted with only one methacrylate group, it is
expected that cross-linked polymers present lower free
radical densities. When the degree of conversion is the
only parameter assessed, these radical-mediated reaction
issues are not taken into consideration, because only the
89
Delayed Photoactivation (DP) Chemical Activation (CA)
281 (3.1) a,C 63 (2.2) b,D
66 (0.3) a,D 3.6 (0.1) c,C
594 (1.2) a,B 318 (0.9) c,A
1400 (14.8) a,A 159 (3.2) b,B
percentage of the monomers that convert into polymers is
calculated.
EPR spectroscopy is a very useful technique for studying
the structure of free radicals and for following the radical
molecule through various physically or chemically modied
pathways (Poole and Charles, 1983). In addition to qualitative
measurements, EPR is also quantitative. The absorption of
microwave energy in a sample is directly proportional to the
number of spins present. The area under the EPR absorption
spectrum is proportional to the number of free radicals
(spins) in the sample, and the number of free radicals is
proportional to the radiation energy absorbed in the sample.
Thus, the radiation dose may be determined by measuring
the relative concentration of radiation-generated free radicals
in a given material using EPR spectroscopy (Sagstuen and
Hole, 2009).
Free radicals are created during the photoactivation of resin-
based materials (Leprince et al., 2009). Some radicals terminate
by encountering other radical species and others become
trapped by vitrication of the system (Trufer-Boutry et al.,
2003a). EPR spectroscopy is an analytical tool which applies to
molecular systems having either an odd number of electrons
(free radicals) or more unpaired electrons. Two types of trapped
free radicals are detectable by EPR in polymerized composites:
propagating and allylic radical types (Trufer-Boutry et al.,
2003b). The former gives rise to an EPR spectrum with nine
weak and broad peaks, and the latter (allylic) to ve strong and
sharp peaks. Both spectra superimpose, resulting in a nine-line
spectrum with peaks of alternating intensities (Leprince et al.,
2009). As free radicals can be trapped in a solid matrix, they are
unable to recombine and the nine-line EPR spectrum persists
during a period of variable length, from a couple of days to
many months, depending on the storage conditions (Leprince
et al., 2009).
It has been previously established that the degree of
conversion is related to the number of unpolymerized term-
inal groups in which double bonds are present (Rueggeberg,
1999). On the other hand, the conversion reaction proceeds
through the formation of radicals under irradiant light, which
corresponds to the EPR spectrum. In this way, an increase in
the EPR signal intensity as a function of irradiation time
correlates to the propagation of the conversion reaction
(Leprince et al., 2009). In this way, recombination of radicals
can occur, either by bimolecular termination or due to
reactive diffusion, resulting in an increase of the degree of
conversion was observed 24 h after the photoactivation
procedure (2%) (Leprince et al., 2009). The various SLCs differ
90 journal of the mechanical behavior of biomedical materials 46 (2015) 83 92
Fig. 2 24-h X-ray diffraction patterns of the self-adhesive
resin cements: (a) Immediate photoactivation; (b) delayed
photoactivation; (c) Chemical activation. n crystalline peaks
of ytterbium uoride. means unidentied crystalline peak
at 2 17.91.
from each other in the speed of propagation of the conversion
reaction, probably because of differences in the composition
of the organic matrix, which is also known to inuence in the
degree of conversion (Rueggeberg, 1999).
Tooth preparation for indirect bonded restorations (i.e.,
composite/ceramic inlays, onlays, and veneers) can generate
signicant dentin exposure (Di Hiplito et al., 2014). Consid-
ering that self-adhesive luting cements are used to cement
indirect restorations in freshly cut dentin surfaces and that
no adhesive system is required, the presence of trapped free
radicals may cause pulpal damage, as these compounds can
be released by the action of substances in the oral environ-
ment such as water or saliva (Sideridou et al., 2003). In the
present study, the spectra of the resin cements follow what is
found in the literature: lower-intensity EPR signals were
observed, so there were signicantly fewer entrapped free
radicals when the chemical activation protocol was used,
irrespective of the material tested. In addition, the spin
concentrations varied among the materials tested. RelyX
U200 presented signicantly higher free radical entrapment
when photoactivated immediately or after 10 min. Thus, the
second hypothesis, in which it was speculated that different
activation protocols would affect the density of spins or free
radicals of the self-adhesive resin cements, was proven valid.
The present study also evaluated the inuence of SLCs on
the integrity of the crystalline structure of pure hydroxyapatite,
with which these materials were mixed. For all SLCs, it is
presumed that free radicals created in an acidic environment
demineralize the pure hydroxyapatite powder, which was some-
how altered when mixed with the different resin cements
tested. This effect was demonstrated by changes in the peak
intensities in the XRD diffractograms. In this case, the integrity
of the hydroxyapatite crystalline structure was evaluated. In the
present study it was advocated that if the 211 value decreases,
then there is a concomitant physical alteration of the HA
crystal. This does not infer a chemical bonding to HA, only
that the atomic arrangements of the atoms making up a
hydroxyapatite unit cell have been changed. It is important
to highlight that the 211 orientation represents the peak
with the highest value, so changes in this orientation were
more evident. The highest reductions were observed for
Bix SE/HA, irrespective of the activation protocol. On the
other hand, the lowest reductions were observed in RelyX
U200 when immediately photoactivated (IP group) as well
as after 10 min (DP group), in which the percent reductions
were 42.7% and 49.7%, respectively. For the CA group,
MaxCem Elite has the lowest reduction in the 211 peak
intensity (46.5%). Thus, the third hypothesis, which stated
that the integrity of the hydroxyapatite crystal would be
reduced by the brand of SLCs tested and by the polymer-
ization activation mode used, was accepted. The effect of
SLC in the modication of HA crystallinity differed depend-
ing on photoactivation protocol as well as on the resin
cement tested.
These changes in HA crystallinity are representative of the
power of SLCs to demineralize remaining tooth tissues and to
permeate these tissues. The acidic monomers incorporated in
the SLCs are not strong enough to etch through smear layers to
form a traditional hybrid layer along the resin-tooth inter-
face, with the absence of deeply penetrating resin tags into the
 journal of the mechanical behavior of biomedical materials 46 (2015) 83 92 91

Table 5 Quantitative analysis of intensity peak of HA attributed to the (2 1 1) diffraction after mixing HA with the self-
adhesive resin cements. (One specimen tested per group).

PRODUCT Immediate photoactivation (IP) Delayed photoactivation (DP) Chemical activation (CA)

Peak height (AU) % Reduction Peak height (AU) % Reduction Peak height (AU) % Reduction

Embrace WetBond 667 61.4 798 53.9 812 53.1

MaxCem Elite 790 54.3 843 51.3 926 46.5
Bix SE 546 68.4 660 61.8 532 69.2
RelyX U200 992 42.7 870 49.7 792 54.2

Reference: intensity peak of pure hydroxyapatite 1730 cps.

dentinal tubules (Vaz et al., 2012). In addition, it was also considered. For the development of future SLCs, studies
proven that this adhesion strategy relies not only on micro- are necessary to evaluate the longevity of indirect restora-
mechanical retention, but also on chemical interactions tions cemented with this category of resin cement over
between monomer acidic groups and hydroxyapatite (Gerth longer evaluation times.
et al., 2006). According to the results of the present study, it can
be speculated that the dynamic process, in which the acidity of
5. Conclusions
the cements is progressively neutralized, as well as the poly-
merization process, is inuenced by the SLCs and by the
Within the limitations of this study, the following conclu-
activation protocol. These events start immediately after mix-
sions can be made:
ing the base and catalytic pastes, and due to their rheological
properties, the cements are able to ow according each
1. Choice of polymerization protocols affects the degree of
material. Therefore, it is of paramount importance to compare
conversion of SLCs;
the results in the light of the parameters analyzed. In general,
2. Entrapped, unreacted free radical density of SLCs varies as
the resin cement Bix SE presented higher dependence on the
a function of the resin cement and of the activation;
chemical activation, in which higher conversion averages,
3. Brand of SLC and its polymerization activation mode can
lower free radical density, and higher reduction in HA crystal-
alter its interaction with the crystalline structure of
linity are observed. Although the cement MaxCem Elite also
hydroxyapatite; and
demonstrated a chemical dependence in terms of conversion
4. Based on the parameters evaluated, one of the tested
efciency and lower free radical density, this product inter-
products provided more optimal characteristics than did
acted less with the HA when chemically activated. In this
the others: improved degree of conversion, lower density
case, MaxCem Elite was more effective when immediately
of free radicals, and higher interaction with hydroxyapa-
photoactivated or after a delay of 10 min. On the other
tite, regardless of the activation mode.
hand, the cement RelyX U200, as previously discussed,
showed a higher dependence on delay time prior to
photoactivation in terms of conversion efciency. Conver-
sely, the EPR and XRD analyzes showed a higher depen- Acknowledgements
dence on chemical activation, considering the signicantly
lower spin (free radical) density and higher interaction of This study was partially supported by grants from CNPq nos.
479744/2010-6 and 163102/2011-2 (P.I. Paulo H. P. DAlpino)
the resin with HA. Although a higher dependence on the
This study was developed as partial fulllment of the
chemical activation in terms of free radical concentration
was observed, the cement Embrace WetBond showed requirements of Dr. Silvas Master degree (UNIANSP). The
higher dependence on the photoactivation, in terms of authors are grateful to Prof. Dayse Iara dos Santos (UNESP
degree of conversion and higher interaction with HA. Thus, Bauru) for the technical support in the XRD analysis.
the cement Bix SE presented the best balanced formula-
tion based on the parameters analyzed, demonstrating r e f e r e nc e s
higher effectiveness to demineralize hydroxyapatite, a
higher degree of conversion, and lower density of free
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