d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 1493–1501

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Functional monomer impurity affects adhesive

performance

Kumiko Yoshihara a , Noriyuki Nagaoka b , Takumi Okihara c ,

Manabu Kuroboshi c , Satoshi Hayakawa d , Yukinori Maruo e ,
Goro Nishigawa e , Jan De Munck f , Yasuhiro Yoshida g ,
Bart Van Meerbeek f,∗
a Center for Innovative Clinical Medicine, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama
700-8558, Japan
b Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, 2-5-1

Shikata-cho, Kita-ku, Okayama 700-8558, Japan

c Division of Chemical and Biological Technology, Graduate School of Natural Science and Technology, Okayama

University, 3-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan

d Biomaterials Laboratory Graduate School of Natural Science and Technology, Okayama University, 3-1-1

Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan

e Department of Occlusion and Removable Prosthodontics, Okayama University, 2-5-1 Shikata-cho, Kita-ku,

Okayama 700-8558, Japan

f BIOMAT, Department of Oral Health Research, KU Leuven (University of Leuven) & Dentistry, University Hospitals

Leuven, Kapucijnenvoer 7 blok a bus 7001, B-3000 Leuven, Belgium

g Department of Biomaterials and Bioengineering, Graduate School of Dental Medicine, Hokkaido University, Kita 13,

Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8586, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Objective. The functional monomer 10-MDP has been considered as one of the best perform-
Received 30 May 2015 ing functional monomers for dental adhesives. Different adhesives containing 10-MDP are
Received in revised form 9 July 2015 commercially available, among which many so-called ‘universal’ adhesives. We hypothesize
Accepted 25 September 2015 that the quality of the functional monomer 10-MDP in terms of purity may affect bonding
performance.
Methods. We therefore characterized three different 10-MDP versions (10-MDP KN provided
Keywords: by Kuraray Noritake; 10-MDP PCM provided by PCM; 10-MDP DMI provided by DMI) using
Functional monomer NMR, and analyzed their ability to form 10-MDP Ca salts on dentin using XRD. The ‘imme-
NMR diate’ and ‘aged’ micro-tensile bond strength (␮TBS) to dentin of three experimental 10-MDP
TEM primers was measured. The resultant interfacial adhesive-dentin ultra-structure was char-
Impurity acterized using TEM.
Adhesion Results. NMR disclosed impurities and the presence of 10-MDP dimer in 10-MDP PCM and 10-
Dentin MDP DMI. 10-MDP PCM and 10-MDP DMI appeared also sensitive to hydrolysis. 10-MDP KN,

∗
Corresponding author. Tel.: +32 16 33 75 87; fax: +32 16 33 27 52.
E-mail addresses: k-yoshi@md.okayama-u.ac.jp (K. Yoshihara), nagaoka@okayama-u.ac.jp (N. Nagaoka), okihara@cc.okayama-u.ac.jp
(T. Okihara), mkurohos@cc.okayama-u.ac.jp (M. Kuroboshi), satoshi@okayama-u.ac.jp (S. Hayakawa), ykmar@md.okayama-u.ac.jp
(Y. Maruo), goro@md.okayama-u.ac.jp (G. Nishigawa), jan.demunck@med.kuleuven.be (J. De Munck), yasuhiro@den.hokudai.ac.jp
(Y. Yoshida), bart.vanmeerbeek@med.kuleuven.be (B. Van Meerbeek).
http://dx.doi.org/10.1016/j.dental.2015.09.019
0109-5641/© 2015 Published by Elsevier Ltd on behalf of Academy of Dental Materials. All rights reserved.
1494 d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 1493–1501

on the contrary, contained less impurities and dimer, and did not undergo hydrolysis. XRD
revealed more intense 10-MDP Ca salt deposition on dentin induced by 10-MDP KN. The
adhesive based on the experimental 10-MDP KN primer resulted in a significantly higher
‘immediate’ bond strength that remained stable upon aging; the ␮TBS of the experimental
10-MDP PCM and 10-MDP DMI adhesives significantly dropped upon aging. TEM revealed
thicker hybridization and more intense nano-layering for 10-MDP KN.
Significance. It was concluded that primer impurities and the presence of 10-MDP dimer
affected not only hybridization, but also reduced the formation of 10-MDP Ca salts and
nano-layering. 10-MDP in a high purity grade is essential to achieve durable bonding.
© 2015 Published by Elsevier Ltd on behalf of Academy of Dental Materials. All rights
reserved.

HEMA [15]. 10-MDP’s performance may also degrade with time,

1. Introduction
Phosphate functional monomers are designed to chemi-
cally interact with dentinal hydroxyapatite, being thought
of importance for bond durability. Among many functional
monomers, 10-MDP was most shown to improve bonding
effectiveness in laboratory and clinical research [1–6]. In
our previous research, 10-MDP’s chemical interaction with
hydroxyapatite (HAp) of tooth tissue was proven using diverse
chemical analytical tools, such as XRD, XPS and NMR [7–9].
Furthermore, 10-MDP was documented to self-assemble into
so-called ‘nano-layering’, a process driven by the deposi-
tion of 10-MDP Ca salts with low solubility [8,10–12]. Each
nano-layering consists of two sublayers of parallel oriented 10-
MDP monomers in opposite direction. 10-MDP’s methacrylate
group is directed inwards, enabling mutual co-polymerization
between two opposed monomers. Its functional phosphate
group is directed outwards, capturing Ca released from dentin
thanks to the etching effect of 10-MDP and so coupling adja-
cent nano-layers. Further in-depth research into the process of
nano-layering is warranted to assess the relevance and actual
contribution of this compact 3D structure to the stability of
the adhesive interface.
The functional monomer 10-MDP was originally synthe-
sized by Kuraray (today Kuraray Noritake, Tokyo, Japan) and
has been introduced in 1981 (following ‘Clearfil SE Bond’ Tech-
nical Information from Kuraray Noritake). The actual 10-MDP
patent expired in 2011, and has led other companies to develop
and launch 10-MDP adhesives, many of them have been
referred to as so-called ‘universal’ adhesives, such as Adhese
Universal (Ivoclar Vivadent, Schaan, Liechtenstein), All-Bond
Universal (Bisco, Schaumburg, IL, USA), Clearfil Universal Bond
(Kuraray Noritake), Futurabond U (Voco, Cuxhaven, Germany;
although the MSDS document does not specify what the
‘acidic adhesive monomer’ is), G-Premio Bond (GC, Tokyo,
Japan) and Scotchbond Universal (3M ESPE, Seefeld, Germany).
Besides indicated for direct and indirect restorative proce-
dures, universal adhesives allow the dentist to opt for either
an etch-and-rinse (E&R) or self-etch (SE) application protocol.
Bonding effectiveness of the different commercial 10-MDP-
based adhesives was found to vary [13,14]. Self-evidently,
difference in composition is the most plausible reason. Bond
strength was shown to depend on the actual concentration of
10-MDP [12]. The interfacial interaction potential of 10-MDP
with HAp was found to be inhibited by other monomers like
as the monomer is sensitive to hydrolytic degradation [16].
To date, the effect of 10-MDP’s purity on bonding effective-
ness is unknown, although several chemical companies (PCM,
Krefeld, Germany; DMI, San Diego, CA) commercially offer the
functional monomer to be used for dental purposes. Different
purities are reported in the accompanied technical documen-
tation. Moreover, patent literature not clearly describes the
synthesis process and actual purification process [17,18]. We
therefore investigated in this study three different 10-MDP ver-
sions, provided by three different companies, on their purity
and their chemical interaction potential as well as bonding
effectiveness to dentin. The null hypothesis tested was that
the adhesive performance of experimental primers varying for
the 10-MDP version was not different when they were used as
part of a 2-step SE adhesive protocol.
2. Materials and methods
Three different 10-MDP monomers were selected, as they were
provided by DMI (further referred to as ‘10-MDP DMI’), by
Kuraray Noritake (referred to as ‘10-MDP KN’), and by PCM
(referred to as ‘10-MDP PCM’). The purity grade of 10-MDP DMI
was reported by DMI to be 90%, versus 83% reported by PCM for
10-MDP PCM. The purity grade of 10-MDP KN was not released
by Kuraray Noritake, but was informed to be higher than that
of the other 10-MDP’s tested and to be the same as that of
10-MDP included as functional monomer in the commercial
adhesive Clearfil SE Bond (Kuraray Noritake).
Three respective experimental primers were prepared
to consist of a 15:45:40 wt% solution of 10-MDP/ethanol-
D6/water-D2 and were stored for 1 month at 37 ◦ C. The pH of
the experimental primers was 2.66 for 10-MDP DMI, 2.03 for
10-MDP KN, and 2.73 for 10-MDP PCM.
2.1. Nuclear magnetic resonance spectrocopy (NMR)
NMR is a research technique that exploits the magnetic prop-
erties of atomic nuclei and can provide detailed information
about the electronic structure of a molecule. In order to
investigate the molecular structure of 10-MDP and poten-
tial impurities, we analyzed the three 10-MDP experimental
primers using 1 H (with respect to hydrogen), 13 C (with respect
to carbon) and 31 P (with respect to phosphorus) NMR spec-
troscopy. The three experimental primers were disposed
 d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 1493–1501
in 5-mm diameter NMR glass tubes (528PP, Wilmad glass,
Vineland, NJ). An NMR spectrometer (JNM-ECS 400, JEOL,
Tokyo, Japan) was employed to acquire 1 H NMR spectra with a
30 s relaxation delay between pulses to allow complete relax-
ation, 31 P NMR and 13 C NMR spectra, both with a 2 s delay
between pulses. 1 H NMR and 13 C NMR spectra were referenced
externally to neat chloroform-d3 (ı = 7.27 ppm); 31 P NMR spec-
tra were referenced externally to neat 85% aq. phosphoric acid
(ı = 0.00 ppm).
2.2. X-ray diffraction analysis (XRD)
XRD is a tool used for identifying the atomic and
molecular structure of crystals or materials with a
highly ordered microstructure. Fifteen dentin specimens
(10 mm × 8 mm × 1 mm) were cut from fifteen bovine
mandibular front teeth (n = 5 for each experimental group),
after which the exposed surfaces were ground using SiC paper
(#600). The 15:45:40 wt% 10-MDP/ethanol/water monomer
solutions were applied on dentin as well as on glass plates
by lightly rubbing with a micro-brush (Centrix Benda Brush,
Centrix, Chelton, CT, USA). After 20 s, the samples were
strongly air-dried prior to being further chemically analyzed
using XRD.
The surface structures of the dentin specimens and glass
plates treated with the experimental monomer solutions were
examined by thin-film X-ray diffraction (TF-XRD) using an X-
ray diffractometer (Cu K␣1 1.5406 Å, RINT2500, Rigaku, Tokyo,
Japan), operated under 40 kV acceleration and 200 mA current,
and a scanning rate of 0.02◦ /s, with the angle of the incident
X-ray beam fixed at 1.0◦ .
2.3. Energy dispersive X-ray spectroscopy (EDS)
EDS is an analytical technique used for elemental analysis.
Since crystals precipitated upon mixing 10-MDP DMI with
ethanol, the chemical element composition of solely 10-
MDP DMI was determined using EDS (Noran Voyager III M3100,
NORAN Instruments, Middleton, Wisconsin, USA), connected
to a scanning electron microscope (SEM; DS-720, Topcon Corp.,
Tokyo, Japan), after having dissolved 10-MDP DMI in ethanol
and the precipitate separated.
2.4. Micro-tensile bond strength (TBS) to dentin
Three respective experimental primers were prepared to
consist of 15 wt% 10-MDP dissolved in 45 wt% ethanol and
40 wt% water, to which 1 wt% camphorquinone (CQ)/ethyl-4
dimethylaminobenzoate (amine) (Sigma–Aldrich, St. Louis,
MO) was added to achieve a final concentration of 14.7 wt%
10-MDP, 44.1 wt% ethanol, 39.2 wt% water, 1 wt% CQ and
1 wt% amine. Mid-coronal dentin of fifteen extracted human
third molars (n = 5 for each experimental group; the teeth
were gathered following informed consent approved by the
Commission for Medical Ethics of KU Leuven under the file
number S57622) was exposed using a diamond saw (Buehler,
Illinois, USA) and next polished with #600 SiC paper. The
experimental primer was applied for 20 s followed by gently
air-drying, after which the bonding agent of Clearfil SE Bond
(Kuraray Noritake) was applied, and subsequently air-thinned
1495
and light-cured (BluePhase G2, Ivoclar Vivadent). Finally, a
micro-hybrid composite (Clearfil AP-X, Kuraray Noritake) was
applied to a thickness of 2 mm, after which the specimens
were stored for 24 h in water at 37 ◦ C (‘immediate’). The teeth
were cut into 1-mm2 stick-shaped micro-specimens with the
aid of a 0.3 mm diamond cut-off wheel (Struers, Ballerup,
Denmark) mounted in an Accutom-50 cutting machine
(Struers, Ballerup, Denmark). Half of the specimens were
additionally thermocycled (30 s immersion, alternatively, in
a 5 and 55 ◦ C water bath) during 100,000 cycles before being
subjected to ␮TBS (‘aged’). The micro-specimens were fixed
to a BIOMAT jig [19] with the aid of cyanoacrylate-based glue
(Model Repair II Blue, Dentsply-Sankin, Ohtawara, Japan), and
stressed at a crosshead speed of 1 mm/min until failure using
a universal testing device (LRX, Lloyd Instruments, Hamp-
shire, UK) equipped with a load cell of 100 N. The ␮TBS was
expressed in MPa, as derived from dividing the imposed force
(N) at the time of fracture by the bond area of the individual
specimen (mm2 ). The occurrence of failure prior to actual
testing was included in the calculation of the mean ␮TBS
as 0 MPa, with an explicit note of the number of pre-testing
failures (ptf). The data were statistically evaluated by two-way
ANOVA and Tukey HSD test (˛ = 0.05). The statistical analysis
was performed using SPSS (IBM, Armonk, NY, USA).
2.5. Interfacial characterization using transmission
electron microscopy (TEM)
Extracted non-carious human third molars (gathered fol-
lowing informed consent approved by the Commission for
Medical Ethics of KU Leuven under the file number S57622)
were used within 1 month of extraction (stored in 0.5% chlo-
ramine/water, 4 ◦ C). After removal of the occlusal crown third
using an Isomet diamond saw (Isomet 1000, Buehler, Lake
Bluff, IL, USA), the exposed dentin was wet-sanded (60 s, #600
SiC-paper) to produce a standard smear layer, after which
it was treated for 20 s with one of the three experimen-
tal 15:45:40 wt% 10-MDP/ethanol/water monomer solutions to
which 1 wt% CQ/amine was added. On top of the adhesive,
the flowable composite (Clearfil Protect Liner F, Kuraray Nori-
take) was applied and light-cured for 10 s using an Optilux
500 (Demetron/Kerr, Danbury, CT, USA) light-curing unit. After
bonding, the resin-bonded dentin specimens were stored for
1 day in distilled water at 37 ◦ C and further processed for TEM
following a protocol previously described in detail before [20].
Non-demineralized sections were cut (Ultracut UCT, Leica,
Vienna, Austria) to be imaged by TEM (80 kV JEM-1200 EX II
TEM, Jeol, Tokyo, Japan).
3. Results
3.1. NMR
31 PNMR of all 10-MDP samples showed a strong singlet peak
at 0.4 ppm, which must be assigned to the actual 10-MDP
monomer (Fig. 1a). In addition to this peak, a small peak
was detected at −14.5 ppm for 10-MDP PCM and 10-MDP DMI,
which was assigned to 10-MDP dimer (Fig. 1a).
1496 d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 1493–1501

Fig. 1 – Chemical characterization of 15 wt% 10-MDP/ethanol solution containing one of the three 10-MDP versions using
31 P NMR in (a), 13 C NMR after 24 h water storage in (b) and after 1 Mo storage in (c), and using 1 H NMR in (d). (a) 31 P NMR of

all 10-MDP samples showing a strong peak at 0.4 ppm, which must be assigned to the 10-MDP monomer. In addition to this
peak, a small peak was detected at −14.5 ppm for 10-MDP DMI and 10-MDP PCM, which was assigned to 10-MDP dimer. (b)
13 C NMR of 10-MDP PCM revealed a strong (4) and a weak (d) peak around 170 ppm. (c) After 1 Mo water storage,

10-MDP DMI also revealed the weaker ‘d’ peak, representing methacrylic acid and indicating that some hydrolysis of the
10-MDP monomer had occurred. (d) Using 1 H NMR, 10-MDP PCM showed additional peaks around 3.2 ppm. Two pairs of
distinct peaks were detected in the range from 3.9 ppm to 4.2 ppm for 10-MDP KN, which were assigned to the CH2 spacer
chain of 10-MDP. These peaks were much sharper, finer and intenser than those recorded for the other two 10-MDP’s. The
peaks revealed for 10-MDP DMI were also distorted.

13 C NMR analysis after 24 h of 10-MDP PCM revealed two
peaks around 170 ppm (Fig. 1b). The small peak refers to the
carboxyl group (‘d’) of methacrylic acid produced by hydrolysis
of the 10-MDP monomer. This ‘d’ peak was also detected after 1
Mo for 10-MDP PCM, but also for 10-MDP DMI (Fig. 1c); the peak
was not detected for 10-MDP KN after 24 h (Fig. 1b), nor after
1 Mo (Fig. 1c). In particular for 10-MDP PCM, several other, yet
unidentified peaks were detected at 8.4 and 46.6 ppm, which
were not detected for 10-MDP DMI and 10-MDP KN (Fig. 1b and
c).
Using 1 H NMR, the peak shapes from 3.8 to 4.1 ppm
that must be assigned to the CH2 groups of the 10-MDP
molecule, were sharper and more intense for 10-MDP KN
as compared to those detected for the two other 10-MDP’s
(Fig. 1d). 10-MDP DMI showed distorted peak shapes from
3.8 to 4.1 ppm; 10-MDP PCM showed additional peaks around
3.2 ppm (Fig. 1d).
3.2. XRD
Application of the three 10-MDP’s on dentin (D) resulted
in three distinct peaks at 2 = 2.52◦ (d = 3.51 nm), 2 = 4.84◦
(d = 1.82 nm) and 2 = 7.16◦ (d = 1.23 nm); they should be
assigned to 10-MDP Ca salts (Fig. 2a). The peak intensities
of these three 10-MDP characteristic peak differed in the
order of 10-MDP KN D > 10-MDP PCM D = 10-MDP DMI D. 10-
MDP KN showed an additional peak at 2 = 11.8◦ (d = 0.75 nm),
which should be attributed to DCPD (Fig. 2a).
When applied on a glass plate, 10-MDP KN GP and 10-
MDP PCM GP did not show any peaks (Fig. 2b). 10-MDP DMI GP
 d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 1493–1501 1497

Fig. 2 – XRD patterns of 10-MDP applied on dentin (D) in (a) and on a glass plate (GP) in (b), and EDS analysis of the salts of
10-MDP DMI in (c). (a) When the three 10-MDP’s were applied on dentin, three characteristic peaks were detected at
2 = 2.52◦ (d = 3.51 nm), 2 = 4.84◦ (d = 1.82 nm) and 2 = 7.16◦ (d = 1.23 nm); they were assigned to 10-MDP Ca salts. The peak
intensity differed in the order of 10-MDP KN D > 10-MDP PCM D = 10-MDP DMI D. 10-MDP KN revealed an additional peak at
2 = 11.8◦ (d = 0.75 nm), which should be attributed to DCPD. (b) When the three 10-MDP’s were applied on a glass plate, only
10-MDP DMI GP revealed the abovementioned three Ca-salt characteristic peaks. (c) EDS analysis of 10-MDP DMI revealed
four distinct peaks, representing respectively the elements of carbon, oxygen, sodium, and phosphate and thus indicating
that the 10-MDP DMI solution contained 10-MDP sodium salts.

however showed three peaks at 2 = 2.52◦ (d = 3.51 nm),
2 = 4.84◦ (d = 1.82 nm) and 2 = 7.16◦ (d = 1.23 nm).
3.3. EDS
When the three 10-MDP’s were dissolved in ethanol, only 10-
MDP DMI formed salts. EDS analysis of the 10-MDP DMI salts
revealed that it consisted of carbon, oxide, sodium and phos-
phate (Fig. 2c).
experimental 10-MDP KN primer, an about 1.0-␮m thick and
HAp-rich hybrid layer was formed (Fig. 4a). High magnifica-
tion of the adhesive layer immediately above the hybrid layer
clearly revealed intense nano-layering (Fig. 4b and c). The
hybrid layer produced by the 10-MDP PCM and 10-MDP DMI
versions was about 0.5-␮m thick and also rich in HAp (Fig. 4d
and g). High magnification of the adhesive layer again revealed
nano-layering (Fig. 4e, f, h, and i), although less intense than
that observed for 10-MDP KN.

3.4. TBS
The 10-MDP version as well as the aging imposed by long-
term thermo-cycling was found to have affected the ␮TBS
(two-way ANOVA, both p < 0.0001). Not all 10-MDP did however
degrade in a similar way as the interaction between the two
variables (10-MDP and aging) was found to be significant as
well (p = 0.0155). The ‘immediate’ ␮TBS to dentin of the 2-step
SE adhesive, including the experimental 10-MDP KN primer,
was significantly higher than that of the 10-MDP PCM and
10-MDP DMI versions. The ␮TBS of 10-MDP KN did not signif-
icantly decrease after 100,000 thermocycles. On the contrary,
the ‘aged’ ␮TBS of 10-MDP PCM and 10-MDP DMI significantly
decreased after long-term thermo-cycling (Fig. 3). Also, while
no pre-testing failures (ptf’s) were recorded for 10-MDP KN,
respectively 5 ptf’s (out of 22 micro-specimens) and 7 ptf’s
(out of 21 micro-specimens) were recorded for the ‘aged’ 10-
MDP PCM and 10-MDP DMI specimens.
3.5. TEM
TEM releaved tight interfaces at dentin for all experimental
10-MDP adhesives. For the 2-step SE adhesive, including the
4. Discussion
Functional monomers as part of self-etch adhesives are
designed to interact with HAp using the functional group at
one end, a phosphate group in case of 10-MDP, and at the same
time to co-polymerize through the methacrylate group at the
other end with the other resin monomers [21]. The spacer
of 10-MDP keeps the two functional groups separated by a
relatively long hydrophobic chain. The functional monomer
in ‘mild’ SE adhesives (self-)etches HAp of dentin, releasing
Ca, in an equilibrium with ionic interaction with Ca of HAp,
thereby forming 10-MDP Ca bonds. Upon sufficient release of
Ca from dentin, the 10-MDP monomers self-assemble into
nano-layers [11]. Each nano-layer consists of two rows of par-
allel oriented 10-MDP monomers, with the monomers in each
row directed in opposite direction. The methacrylate ends of
two opposed 10-MDP monomers can co-polymerize, while the
released Ca can bridge/link two adjacent nano-layers by Ca–P
salt formation. The obtained compact structure is thought to
contribute to the bond stability, while this assumption needs
to be confirmed and potential measures to intensify nano-
layering should be explored.
1498 d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 1493–1501

Fig. 3 – Graph presenting the micro-tensile bond strength (␮TBS) of the experimental adhesives with the SE primers based
on the three respective 10-MDP functional monomers, this after 24 h water storage (‘immediate’ ␮TBS) and after long-term
100,000 times thermo-cycling (‘aged’ ␮TBS). The numbers above each box express the mean ␮TBS (in MPa) with the
corresponding standard deviation (mean ± SD). Underneath between parenthesis, the number of pre-testing failures (ptf’s)
per total number of micro-specimens tested is mentioned. Different small letters underneath refer to statistically significant
differences between the experimental groups. 10-MDP KN revealed a significantly higher bond strength than 10-MDP PCM
and 10-MDP DMI. The ␮TBS of 10-MDP KN did not decrease upon aging, while the ␮TBS measured for 10-MDP PCM and
10-MDP DMI significantly decreased after long-term thermo-cycling. The boxes represent the spreading of the data between
the first and third quartile. The closed dot and the horizontal line in each box represent the mean and median, respectively.
The whiskers denote the range of variance; outliers are indicated by the open dot.

In this study, we investigated the effect of the quality or
more specifically the purity of 10-MDP as one of the best per-
forming functional monomers for a SE adhesive routine. Two
commercially available 10-MDP’s (10-MDP PCM, 10-MDP DMI)
and one 10-MDP version (10-MDP KN) provided by Kuraray
Noritake and used in the different Clearfil adhesive genera-
tions were tested. The experimental adhesive containing the
latter 10-MDP KN monomer revealed the significantly highest
‘immediate’ and ‘aged’ ␮TBS. Moreover, aging using long-
term thermo-cycling did not significantly reduce its ␮TBS, as
opposed to the significant decrease in ␮TBS upon aging for 10-
MDP PCM and 10-MDP DMI. Hence, the null hypothesis that
the three 10-MDP versions did not differ in bonding perfor-
mance was rejected.
In order to identify the reasons for this difference in
‘immediate’ and ‘aged’ bond strength, the three 10-MDP ver-
sions were chemically characterized using NMR. 31 P NMR
revealed that both 10-MDP PCM and 10-MDP DMI contain 10-
MDP dimer. Steric hindrance of the two (remaining) OH
groups of the dimer can be expected to result in a lower flexi-
bility and thus lower chemical reactivity of the dimer with HAp
than the single 10-MDP monomer. This is supported by TEM
that revealed a thicker hybrid layer with abundant HAp for
the experimental 10-MDP KN adhesive than for the other two
10-MDP versions. This higher etching efficacy of 10-MDP KN
may also explain its higher immediate bond strength as com-
pared to that of the other 10-MDP versions. The difference in
etching capacity also appeared from the pH of the prepared
experimental 10-MDP primers, being most acidic with a pH of
2.03 for 10-MDP KN, a pH of 2.66 for 10-MDP DMI and 2.73 for
10-MDP PCM.
As mentioned above, previous research demonstrated
that upon interaction of 10-MDP with Ca of HAp, 10-
MDP ionically bonded to HAp and even self-assembled in
nano-layering. XRD confirmed 10-MDP Ca salt formation and
nano-layering for all three 10-MDP versions upon 20-s inter-
action with dentin. Although in principle XRD cannot be
considered quantitative in terms of peak intensity, our previ-
ous studies revealed a clear correlation between the intensity
of the three nano-layering characteristic XRD peaks and
amount of nano-layering [11]. Much more intense nano-
layering was produced by 10-MDP KN than by the other
10-MDP monomers. High-magnification TEM corroborated
the XRD data. Ultra-structurally, more intense nano-layering
was observed with 10-MDP KN, clearly extending from
the hybrid layer into the adhesive resin. Although nano-
layering was also detected using the experimental adhesives
containing 10-MDP PCM and 10-MDP DMI, the amount of
nano-layering was clearly much smaller. Also this differ-
ence in nano-layering can be explained by the existence
of dimers, sterically hindering the formation of nano-
layering.
Remarkable was the detection by XRD of the three
nano-layering characteristic peaks when the experimental
10-MDP DMI primer was applied on a (Ca-free) glass plate.
EDS confirmed the presence of 10-MDP Na salts. When 10-
MDP DMI was dissolved in ethanol, the 10-MDP Na salts
remained. In water, the salts must have dissolved. These
sodium ions might represent impurities that consume 10-
MDP, by which it is less able to interact with Ca of HAp. This
sodium in 10-MDP DMI may have contributed to the lower
‘immediate’ bond strength.
 d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 1493–1501 1499

Fig. 4 – TEM photomicrographs illustrating the adhesive–dentin interface produced by the experimental 10-MDP KN primer
in (a–c), the experimental 10-MDP PCM primer in (d–f) and the 10-MDP DMI primer in (g–i), when they were applied on
dentin for 20 s and followed by the application of a low-viscosity resin. All experimental adhesives revealed tight interfaces
with dentin (no de-bonding). TEM of 10-MDP KN revealed a 1.0-␮m thick and HAp-rich hybrid layer (a). High magnification
of the adhesive layer clearly revealed intense nano-layering (b and c). TEM of 10-MDP PCM and 10-MDP DMI revealed a
0.5-␮m thick and HAp-rich hybrid layer (d and g). High magnification of the adhesive layer clearly revealed nano-layering (e,
f, h, and i) but in a lower degree than that observed for 10-MDP KN. PL: primer layer; D: dentin; HL: hybrid layer; LVR:
low-viscosity (composite) resin; NL: nano-layering.

1H NMR revealed distinct, sharp and fine peaks at around
4.0 ppm for 10-MDP KN, which should be assigned to the 10
CH2 spacer chain. The peak sharpness and intensity are
a clear indication of high purity. On the contrary, broader
and less intense peaks at around 4.0 ppm were detected
for 10-MDP PCM; even distorted peaks at around 4.0 ppm
were detected for 10-MDP DMI. 10-MDP PCM presented with
several peaks at 1.3, 1.7 and around 3.2 ppm. The latter peaks
might also represent the CH2 chain, which may have become
shorter by hydrolysis or may represent monomers with a
shorter CH2 chain that remained from the synthesis process.
All these additional peaks indicate that both 10-MDP DMI and
10-MDP PCM contain impurities. In addition, the peaks at 10
and 45 ppm disclosed by 31 C NMR must also be assigned to
1500 d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 1493–1501
impurities. As mentioned before, the purity grade of 10-MDP
released by the manufacturer was 83% for 10-MDP PCM and
90% for 10-MDP DMI. The purity of 10-MDP KN was not
disclosed, but thought to be higher than that of the other
two 10-MDP’s (confirmed by personal communication with
Kuraray Noritake). Most impurities could not be identified;
they may represent residual solvents and/or by-products. The
bond strength must have been affected by the impurities,
because the existence of impurities reduced the concentra-
tion of pure 10-MDP and some impurities may even have
inhibited bonding directly.
31 C NMR revealed a peak at 170 ppm for 10-MDP PCM. This
peak was assigned to methacrylic acid, indicating that some
10-MDP may have been hydrolysed [22]. 10-MDP DMI also
showed this peak after 1 Mo storage. When 10-MDP undergoes
hydrolysis, methacrylic acid and 10-hydroxydecyl dihydro-
gen phosphate are formed [23]. 10-Hydroxydecyl dihydrogen
phosphate can still react with HAp, but cannot polymerize
and thus prevents other 10-MDP monomers from interaction,
thereby not contributing to the bond strength. Aida et al.
[22] reported degradation of a HEMA and 10-MDP contain-
ing self-etch primer, as revealed by 13 C NMR. HEMA appeared
more sensitive for hydrolysis than 10-MDP; HEMA’s degra-
dation strongly affected bond durability [22]. On the other
hand, in our study, no signs of hydrolysis were observed
for 10-MDP KN. The sensitivity of the monomer for hydrol-
ysis depends on the monomer structure and the solvent.
10-MDP PCM and 10-MDP DMI may contain residual solvent
and/or by-products that may promote hydrolysis. Very likely,
the degradation of 10-MDP by hydrolysis recorded in this
study for 10-MDP PCM and 10-MDP DMI must have resulted
in the significantly decreased bond strength upon long-term
thermo-cycling.
It is concluded that the three 10-MDP’s studied in this
study clearly revealed a different purity. Differences in the
ultrastructure of the resultant hybrid layers were observed
for the three 10-MDP versions. Both the impurities and the
presence of dimers affected the etching efficacy of HAp, the
intensity of nano-layering and the ‘immediate’ bond strength.
Furthermore, these impurities may have promoted monomer
hydrolysis and so have affected bond durability as well. Hence,
the purity of 10-MDP present in commercial dental primers,
adhesives and cements can be expected to influence bonding
performance. Studies investigating the purity of monomers in
commercial dental adhesive materials are needed.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The current research was supported by a Grant-in-Aid for
Research Activity Start-up (26893156). We thank the respective
manufacturers, Kuraray Noritake (Tokyo, Japan), PCM (Krefeld,
Germany) and Designer Molecule (DMI, San Diego, CA) for pro-
viding the different 10-MDP monomers.
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