d e n t a l m a t e r i a l s 2 9 ( 2 0 1 3 ) 888–897

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journal homepage: www.intl.elsevierhealth.com/journals/dema

Adhesive interfacial interaction affected by different

carbon-chain monomers

Kumiko Yoshihara a , Yasuhiro Yoshida b , Noriyuki Nagaoka c , Satoshi Hayakawa d ,

Takumi Okihara e , Jan De Munck a , Yukinori Maruo f , Goro Nishigawa f , Shogo Minagi f ,
Akiyoshi Osaka d , Bart Van Meerbeek a,∗
a KU Leuven BIOMAT, Department of Oral Health Research, KU Leuven (University of Leuven) & Dentistry, University Hospitals Leuven,
Belgium
b Department of Biomaterials, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
c Laboratory for Electron Microscopy, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,

Okayama, Japan
d Biomaterials Laboratory Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan
e Division of Chemical and Biological Technology, Graduate School of Natural Science and Technology, Okayama University, Okayama,

Japan
f Department of Occlusal and Oral Functional Rehabilitation, Okayama University Graduate School of Medicine, Dentistry and

Pharmaceutical Sciences, Okayama, Japan

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

Article history: Objectives. The functional monomer 10-methacryloxydecyl dihydrogen phosphate (10-MDP),
Received 20 February 2013 recently used in more self-etch adhesives, chemically bonds to hydroxyapatite (HAp) and
Received in revised form thus tooth tissue. Although the interfacial interaction of the phosphoric-acid functional
11 May 2013 group of 10-MDP with HAp-based substrates has well been documented, the effect of the
Accepted 14 May 2013 long carbon-chain spacer of 10-MDP on the bonding effectiveness is far from understood.
Methods. We investigated three phosphoric-acid monomers, 2-methacryloyloxyethyl dihy-
drogen phosphate (2-MEP), 6-methacryloyloxyhexyl dihydrogen phosphate (6-MHP) and
Keywords: 10-MDP, that only differed for the length of the carbon chain, on their chemical interaction
Dentin potential with HAp and dentin, this correlatively using X-ray diffraction (XRD) and trans-
Functional monomer mission electron microscopy (TEM). Commercial 6-MHP and 10-MDP containing adhesives
Adhesive were investigated as well.
X-ray diffraction Results. XRD revealed that on HAp only 10-MDP produced monomer-calcium salts in the
TEM form of ‘nano-layering’, while on dentin all monomers produced ‘nano-layering’, but with
a varying intensity in the order of 10-MDP > 6-MHP > 2-MEP. TEM confirmed that 10-MDP
formed the thickest hybrid and adhesive layer. XRD and TEM revealed ‘nano-layering’ for all
commercial adhesives on dentin, though less intensively for the 6-MHP containing adhesive
than for the 10-MDP ones.
Significance. It is concluded that not only the phosphoric-acid group but also the spacer group,
and its length, affect the chemical interaction potential with HAp and dentin. In addition,

∗
Corresponding author at: KU Leuven 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. Tel.: +32 16 33 75 87; fax: +32 16 33 27 52.
E-mail addresses: bart.vanmeerbeek@med.kuleuven.be, bart.vanmeerbeek@uzleuven.be (B. Van Meerbeek).
0109-5641/$ – see front matter © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dental.2013.05.006
 d e n t a l m a t e r i a l s 2 9 ( 2 0 1 3 ) 888–897
the relatively strong ‘etching’ effect of 10-MDP forms more stable monomer-Ca salts, or
‘nano-layering’, than the two shorter carbon-chain monomers tested, thereby explaining,
at least in part, the better bond durability documented with 10-MDP containing adhesives.
© 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Of all the ingredients of a dental adhesive, the functional
monomer is considered most important; it regulates the
interfacial interaction at tooth enamel and dentin [1,2]. Com-
monly, functional monomers in self-etch primers/adhesives
are acidic esters, originating from the reaction of a bivalent
alcohol with methacrylic acid and phosphoric/carboxylic acid
derivatives [3]. The diverse commercially available self-etch
adhesives contain each one or occasionally two specific func-
tional monomers. The wide variance in effectiveness data,
typically recorded for the different adhesives in the world-
wide conducted laboratory and clinical studies, should at least
in part be attributed to the diversity in kind of functional
monomers included in the particular adhesive formulations
[1–10].
Of the functional monomers contained in self-etch
adhesives, 10-methacryloyloxydecyl dihydrogen phosphate
(10-MDP) was found to adhere to hydroxyapatite (HAp) and
tooth tissue most readily and intensively [1,2,11]. According
to the adhesion–decalcification (AD) concept, the less solu-
ble the calcium salt of the acidic monomer, the more intense
and stable is the molecular adhesion to the HAp-based sub-
strate [1,12]. Indeed, adhesives containing 10-MDP revealed
rather consistently a favorable adhesive performance in many
laboratory and clinical studies, in particular also regarding
long-term bond durability [13–17]. Consequently, more man-
ufacturers recently marketed adhesives based on 10-MDP
monomer technology, like All-Bond Universal (Bisco, Schaum-
burg, IL, USA), Clearfil 3S Bond Plus (Kuraray Noritake Dental,
Tokyo, Japan), G-aenial Bond (GC, Tokyo, Japan), and Scotch-
bond Universal (3M ESPE, Seefeld, Germany).
The monomer 10-MDP possesses a phosphoric-acid func-
tional group as main adhesion promoter to interact with HAp,
a methacrylate polymerizable group for curing, of which the
importance with regard to bond strength and durability should
not be underestimated [18], and finally a 10-carbon chain or
decyl group as spacer to separate both other active groups.
The latter carbon spacer is known to influence monomer
characteristics such as flexibility, solubility, wetting, and the
hydrophobicity-hydrophilicity balance [1,3]. Actually, only a
few studies investigated the effect of the spacer of 10-MDP on
adhesive performance [9,19,20]. There is to our knowledge no
paper that searched for the underlying mechanisms by inves-
tigating the chemical interaction of different carbon-chain
monomers with HAp.
The objective of this study was therefore to investigate
the influence of the carbon-chain length of a phosphoric-
acid ester monomer on its chemical interaction potential
with HAp. This study consisted of three project parts: in
Project Part 1, the interaction with HAp of three functional
monomers, namely 2-methacryloyloxyethyl dihydrogen
phosphate (2-MEP), 6-methacryloyloxyhexyl dihydrogen
889
phosphate (6-MHP) or 10-MDP, all differing only for the
length of the carbon-chain spacer, was studied using X-ray
Diffraction (XRD); in Project Part 2, XRD was used to assess
potential nano-layering [21] at dentin, while transmission
electron microscopy (TEM) was used to ultra-morphologically
characterize the adhesive interface produced at dentin and so
to potentially confirm nano-layering morphologically when
detected by XRD; in Project Part 3, finally, the interfacial
interaction of one 6-MHP-based and three 10-MDP-based
commercial adhesives with dentin was evaluated chemically
using XRD and ultra-morphologically using TEM. The overall
null-hypothesis tested was that the carbon-chain length of a
phosphoric-acid ester functional monomer has no effect on
chemical interaction with HAp and dentin.
2. Materials and methods
The different project parts are schematically explained in
Fig. 1.
2.1. Project PART 1 (PP1): chemical interaction of
different carbon-chain monomers with HAp analyzed
using XRD
2.1.1. Preparation of monomer-coated HAp
2-MEP and 6-MHP-coated HAp particles (referred to as ‘2-
MEP HAp’ and ‘6-MHP HAp’, respectively) were prepared
at room temperature by dispersion of 0.2 g of HAp parti-
cles (surface area of 49 m2 g−1 , mean powder diameter of
19.45 ± 0.49 ␮m; Pentax, Tokyo, Japan) in 0.1 g of a mixed
solution of, respectively, 2-MEP and 6-MHP (Kuraray Noritake
Dental), in absolute ethanol and distilled de-ionized water
in a 2-MEP or 6-MHP:EtOH:H2 O composition of 15:45:40 wt%
under stirring. The 2-MEP or 6-MHP-coated HAp particles
were separated from the mixed solution by centrifuga-
tion and decantation, respectively, after 5 min (referred to
as ‘2-MEP or 6-MHP HAp 5min’), 1 h (referred to as ‘2-MEP
or 6-MHP HAp 1h’), and 24 h (referred to as ‘2-MEP or 6-
MHP HAp 24h’). The separated 2-MEP/6-MHP-HAp particles
were then washed three times with absolute ethanol, and
dried at room temperature in ambient atmosphere. Identical
compounds of 10-MDP were prepared similarly in our previous
study [22]. Kuraray Noritake Dental confirmed to have pro-
vided 10-MDP and 6-MHP in a high purity, while 2-MEP mainly
consists of pure monomer and some di-ester monomer. A
comparison will be made with commercial adhesives (see
below). The level of purity of the three functional monomers in
this experiment is thought to be of the same level of that of the
monomers contained in the respective commercial adhesives.
2.1.2. XRD
The crystal phases of powder samples were identified by
means of an X-ray powder diffractometer (CuK␣1 1.5406 Å,
890 d e n t a l m a t e r i a l s 2 9 ( 2 0 1 3 ) 888–897
Fig. 1 – Schematic explaining the whole study set-up consisting of three Project Parts and two analyses, XRD and TEM.
RINT2500, Rigaku, Tokyo, Japan), operated under 40 kV accel-
eration and 200 mA current, and a scanning rate of 0.02◦ per
second for 2Â/Â scan. The XRD data obtained for 2-MEP and
6-MHP were compared to the respective data obtained before
for 10-MDP [22].
2.2. Project PART 2 (PP2): interaction of different
carbon-chain monomers with dentin analyzed using XRD
and TEM
2.2.1. Preparation of dentin specimens treated with
monomer for XRD (PP2a)
Dentin specimens (10 × 8 × 1 mm) were cut from bovine
mandibular front teeth, after which the exposed surfaces
were ground using SiC-paper (#600). The same 15:45:40 wt% 2-
MEP or 6-MHP/ethanol/water monomer solutions as described
above were applied on dentin 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 further chem-
ical analysis using XRD. Identical compounds of 10-MDP were
prepared similarly in our previous study [22].
The surface structure of the dentin specimens treated with
the experimental monomer solutions were examined by thin-
film X-ray diffraction (TF-XRD) using an X-ray diffractometer
(RINT2500, Rigaku; same parameters as mentioned above),
with the angle of the incident X-ray beam fixed at 1.0◦ .
2.2.2. TEM of interfaces produced by experimental
adhesive formulations at dentin (PP2b)
Extracted non-carious human third molars (gathered fol-
lowing informed consent approved by the Commission for
Medical Ethics of KU Leuven) were used within 1 month
of extraction (stored in 0.5% chloramine/water, 4 ◦ C). After
removal of the occlusal crown third using an Isomet dia-
mond 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
with one of the three experimental 15:45:40 wt% 2-MEP or
6-MHP/ethanol/water monomer solutions. After 20 s, the sam-
ples were strongly air-dried and then light-cured using an
Optilux 500 (Demetron/Kerr, Danbury, CT, USA) light-curing
unit (40 s). On top of the adhesive, the flowable composite
(Clearfil Protect Liner F, Kuraray Noritake Dental) was applied
and again light-cured. 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 [23]. 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).
2.3. Project PART 3 (PP3): chemical interaction of
commercial adhesives with dentin analyzed using XRD
and TEM
2.3.1. Preparation of dentin specimens treated with a
commercial adhesive for XRD (PP3a)
Bovine dentin specimens (10 × 8 × 1 mm) were prepared for
XRD like described in PP2. The 6-MHP-containing adhesive,
‘Adper Easy Bond’ (3M ESPE), and the 10-MDP-containing
adhesives, ‘All-Bond Universal’ (Bisco), ‘Clearfil S3 Bond
(Kuraray Noritake Dental) and ‘ScotchBond Universal’ (3M
ESPE), were lightly rubbed on dentin with a micro-brush
(Centrix Benda Brush, Centrix). After 20 s, the samples were
strongly air-dried, after which XRD analysis was performed,
like it was described in PP2.
2.3.2. TEM of interfaces produced by the commercial
adhesives at dentin (PP3b)
Another set of extracted non-carious human third molars
was prepared in the same manner as described in PP2. The
 d e n t a l m a t e r i a l s 2 9 ( 2 0 1 3 ) 888–897 891

Fig. 2 – XRD of the three functional monomers, 2-MEP, 6-MHP and 10-MDP, after having interacted with synthetic
hydroxyapatite (HAp) powder for 5 min, 1 and 24 h, respectively. The XRD patterns of (untreated) HAp powder, CaHPO4 ·H2 O
(DCPD) and 10-MDP Ca salt served as references. (a) Powder XRD patterns of 2-MEP HAp. In addition to the peaks
representing HAp (2 = 10.7◦ –39.74◦ ), for ‘2-MEP HAp 24h’ a strong peak appeared at 2 = 11.8◦ (d = 0.75 nm), which is
accompanied by several peaks at 2 = 21.0◦ (d = 0.42 nm), 2 = 23.0◦ (d = 0.39 nm) and 2 = 29.4◦ (d = 0.30 nm). They must be
ascribed to DCPD. (b) Powder XRD patterns of 6-MHP HAp. Like for ‘2-MEP HAp 24h’, in addition to the peaks representing
HAp (2 = 10.7◦ –39.74◦ ), a peak was detected at 2 = 11.8◦ (d = 0.75 nm), though it was significantly less strong and not
accompanied by other peaks than in case of ‘2-MEP HAp 24h’. Consequently, only a relatively low amount of DCPD must
have been formed at 24 h. (c) Powder XRD patterns of ‘10-MDP HAp’. Already after 5 min, 10-MDP Ca salts were formed, as
evidenced by the three characteristic peaks at 2 = 2.24 (d = 3.94 nm), 2 = 4.56 (d = 1.94 nm), and 2 = 6.86 (d = 1.29 nm). The
intensity of these peaks increased with the longer the interaction time became. The additional peak of ‘10-MDP HAp 1h/24h’
at 2 = 11.8◦ (d = 0.75 nm), relatively weak at 1 h, but significantly more intense at 24 h, must be assigned to DCPD.

6-MHP-containing adhesive, ‘Adper Easy Bond’ (3M ESPE), and
10-MDP-containing adhesives, ‘All-Bond Universal’ (Bisco),
Clearfil S3 Bond (Kuraray Noritake Dental), and ‘ScotchBond
Universal’ (3M ESPE), were applied strictly following the
manufacturer’s instructions by lightly rubbing dentin with a
micro-brush (Centrix Benda Brush, Centrix). After 20 s, the
samples were strongly air-dried and then light-cured using an
Optilux 500 (Demetron/Kerr, Danbury, CT, USA) light-curing
unit. On top of the adhesive, the flowable composite (Clearfil
Protect Liner F, Kuraray Noritake Dental) was applied and again
light-cured. After bonding, the resin-bonded dentin speci-
mens were stored for 1 day in distilled water at 37 ◦ C and
further processed for TEM (JEM-1200 EX II TEM, and 300 kV
TEM JEM-3010, both Jeol) in the same manner as described in
PP2.
3. Results
3.1. PP1: chemical interaction of different carbon-chain
monomers with HAp analyzed using XRD
All samples showed diffraction peaks representing HAp (range
of 2Â = 10.7◦ –39.74◦ ) (Fig. 2). Powder-XRD of 2-MEP- and 6-MHP-
coated HAp disclosed the formation of dicalcium phoshate
dihydrate or DCPD (CaHPO4 ·2H2 O), but only after 24 h interac-
tion and very intense for 2-MEP, but not for 6-MHP (Fig. 2a and
b). The strong peak at 2Â = 11.8◦ (d = 0.75 nm) was accompanied
by several peaks at 2Â = 21.0◦ (d = 0.42 nm), 23.0◦ (d = 0.39 nm)
and 29.4◦ (d = 0.30 nm), all to be ascribed to DCPD. Besides
peaks representing DCPD, no other peaks that could have
represented monomer-Ca salts, were detected in the XRD pat-
terns of 2-MEP- and 6-MHP-coated HAp.
On the contrary, 10-MDP-coated HAp revealed three charac-
teristic peaks at 2Â = 2.24◦ (d = 3.94 nm), 4.56◦ (d = 1.94 nm) and
6.86◦ (d = 1.29 nm), which should be ascribed to the calcium
salt of MDP (‘CaMHP2 ) and thus the so-called ‘nano-layering’
(Fig. 2c); the peaks could already be detected at the 5-min XRD
pattern and became more intense after 1-h and 24-h expo-
sure. The DCPD-characteristic peak at 2Â = 11.8◦ (d = 0.75 nm)
could only clearly be detected after the 24-h exposure
(Fig. 2c).
3.2. PP2a: interaction of different carbon-chain
monomers with dentin analyzed using XRD
The TF-XRD pattern of ‘2-MEP D’ showed one small peak at
2Â = 4.68◦ (d = 1.89 nm), which was not detected for untreated
dentin (Fig. 3). The TF-XRD pattern of ‘6-MHP D’ showed
three characteristic peaks at 2Â = 3.19◦ (d = 2.77 nm), 2Â = 6.09◦
(d = 1.45 nm) and 2Â = 9.01◦ (d = 0.98 nm) (Fig. 3). Also three char-
acteristic peaks, though at slightly different positions, namely
2Â = 2.52◦ (d = 3.51 nm), 2Â = 4.84◦ (d = 1.82 nm) and 2Â = 7.16◦
(d = 1.23 nm), were detected for ‘10-MDP D’ (Fig. 3). An addi-
tional peak at 2Â = 11.8◦ was detected for ‘6-MHP D’ and
‘10-MDP D’, the latter most intense, should be attributed to
DCPD.
892 d e n t a l m a t e r i a l s 2 9 ( 2 0 1 3 ) 888–897
Fig. 3 – TF-XRD patterns of untreated dentin and of dentin,
on which the experimental 2-MEP (‘2-MEP D’), 6-MHP
(‘6-MHP D’) and 10-MDP (‘10-MDP D’) primers were rubbed
for 20 s. The TF-XRD pattern of ‘2-MEP D’ revealed one
small characteristic peak at 2 = 4.68◦ (d = 1.89 nm), which
could not be detected for untreated dentin. The TF-XRD
pattern of ‘6-MHP D’ showed three characteristic peaks at
2 = 3.19◦ (d = 2.77 nm), 2 = 6.09◦ (d = 1.45 nm) and 2 = 9.01◦
(d = 0.98 nm), though the latter two peaks could only weakly
be detected. The TF-XRD pattern of ‘10-MDP D’ also
revealed three characteristic peaks at 2 = 2.52◦ (d = 3.51 nm),
2 = 4.84◦ (d = 1.82 nm), and 2 = 7.16◦ (d = 1.23 nm), all being
very strong. DCPD was not detected on dentin.
3.3. PP2b: interaction of different carbon-chain
monomers with dentin analyzed using TEM
TEM of ‘2MEP D’ was not possible, because the interface
consistently separated during sample preparation. TEM of ‘6-
MHP D’ disclosed a 0.5-␮m thick and HAp-rich hybrid layer
(Fig. 4a and b). High magnification revealed 2.7-nm ‘nano-
layering’ (Fig. 4c). TEM of ‘10-MDP D’ revealed a 1.0-␮m thick
and HAp-rich hybrid layer (Fig. 4d and e). High magnification
of the adhesive layer clearly revealed intense ‘nano-layering’,
spread throughout the whole adhesive layer (Fig. 4f).
3.4. PP3a: chemical interaction of commercial
adhesives with dentin analyzed using XRD
The TF-XRD pattern of ‘Adper Easy Bond D’, with Adper Easy
Bond (3M ESPE) being a 6-MHP-based adhesive, did not reveal
any peaks (Fig. 5a). After 24-h interaction, the ‘Adper Easy
Bond 24h’ TF-XRD pattern revealed three small but charac-
teristic peaks at 2Â = 3.34◦ (d = 2.65 nm), 2Â = 6.46◦ (d = 1.37 nm)
and 2Â = 9.48◦ (d = 0.93 nm) (Fig. 5a), which should be assigned
to slight 6-MHP-Ca salt formation or ‘nano-layering’.
The TF-XRD pattern of ‘All-Bond Universal D’, with All-
Bond Universal (Bisco) being a 10-MDP-based adhesive,
revealed three characteristic peak at 2Â = 2.70◦ (d = 3.27 nm),
2Â = 5.12◦ (d = 1.73 nm) and 2Â = 7.35◦ (d = 1.20 nm) (Fig. 5b).
The TF-XRD pattern of ‘Clearfil S3 Bond D’, with Clearfil
S3 Bond (Kuraray Noritake Dental) being a 10-MDP-based
adhesive, also showed three characteristic peaks, namely
at 2Â = 2.57◦ (d = 3.44 nm), 2Â = 4.99◦ (d = 1.77 nm) and 2Â = 7.35◦
(d = 1.20 nm) (Fig. 5b). Likewise, the TF-XRD pattern of ‘Scotch-
bond Universal D’, with Scotchbond Universal (3M ESPE) being
a 10-MDP-based adhesive, also revealed three characteristic
peaks, namely at 2Â = 2.56◦ (d = 3.45 nm), 2Â = 5.04◦ (d = 1.75 nm)
and 2Â = 7.44◦ (d = 1.19 nm) (Fig. 5b). For all the three 10-
MDP-based adhesives; the characteristic three peaks must be
assigned to 10-MDP-Ca salt formation or ‘nano-layering’.
3.5. PP3b: chemical interaction of commercial
adhesives with dentin analyzed using TEM
TEM of ‘Adper Easy Bond D’ revealed that the 6-MHP-based
adhesive Adper Easy Bond (3M ESPE) did remove the smear
layer and formed a few hundreds nano-meter thin (Fig. 6a-1,2)
and HAp-containing hybrid layer (Fig. 6a-3). High magnifica-
tion revealed some nano-layering, in particular near the tubule
orifices where the adhesive infiltrated residual smear (Fig. 6a-
3,4). The width of nano-layering was about 2.7 nm.
TEM of ‘All-Bond Universal D’ revealed that the 10-MDP-
based adhesive All-Bond Universal (Bisco) did not completely
remove surface smear, while a 0.5-␮m thick hybrid layer was
formed (Fig. 6b-1,2,3). The width of the adhesive layer of All-
Bond Universal (Bisco) was 3 ␮m, and totally clear because
the adhesive does not contain filler (Fig. 6b-1,2). Dentin was
only partially demineralized, as HAp crystals were detected
to be scattered throughout the 500-nm thick hybrid layer
(Fig. 6b-2,3). High magnification confirmed nano-layering with
a periodicity of about 3.5 nm (Fig. 6b-4).
TEM of ‘Clearfil S3 Bond D’ revealed that the 10-MDP-based
adhesive Clearfil S3 Bond (Kuraray Noritake Dental) removed
surface smear, and formed a HAp-containing hybrid layer with
a thickness of 0.5–0.7 ␮m (Fig. 6c-1,2,3). Nano-layering with a
periodicity of about 3.5 nm was detected near the hybrid layer
(Fig. 6c-4).
Finally, TEM of ‘Scotchbond Universal D’ revealed that
the 10-MDP-based adhesive Scotchbond Universal (3M ESPE)
removed surface smear and formed a hybrid layer with a thick-
ness of 0.2–0.5 ␮m (Fig. 6d-1,2). Some nano-layering with a
periodicity of about 3.5 nm was discovered in the adhesive
layer (Fig. 6d-3,4).
4. Discussion
In search for improved bond durability to tooth tissue, we
investigated in this exploratory study the effect of the length
of the carbon-chain backbone of a phosphoric-acid ester
monomer on its interaction potential with HAp and dentin.
We correlatively used XRD and TEM, respectively, to chem-
ically characterize the interfacial interaction between the
functional monomer and HAp and to image the interfacial
ultra-structure produced at dentin. We selected the functional
 d e n t a l m a t e r i a l s 2 9 ( 2 0 1 3 ) 888–897 893

Fig. 4 – TEM photomicrographs illustrating the experimental 6-MHP primer in (a)–(c) and the experimental 10-MDP primer
in (d)–(f), when they were rubbed on dentin for 20 s and followed by the application of a low-viscosity resin. Noteworthy is
that TEM sections of ‘2MEP D’ could not be prepared because the interface consistently separated during specimen
preparation. TEM revealed that 6-MHP produced a 0.5-␮m thick hybrid layer (a) and (b). High-magnification TEM did not
reveal nano-layering (c). The use of the 10-MDP-based primer resulted in 1.0-␮m thick hybrid layer and an adhesive layer
with a thickness of about 2 ␮m (d) and (e). High-magnification TEM showed abundant nano-layering throughout the whole
adhesive layer (f). AR: adhesive resin; D: dentin; HL: hybrid layer; LVR: low-viscosity (composite) resin.

monomer 10-MDP, well documented for its intensive chemical
interaction potential with HAp, this documented to be to the
direct benefit of bond durability [13]. This monomer contains
of a polymerizable methylmethacrylate group, needed for cur-
ing, and a phosphoric-acid group, enabling ionic interaction
with Ca of HAp. Both groups are kept separate by a 10-fold
carbon chain. In this study, we compared the interfacial inter-
action of 10-MDP with that of two monomers that only differed
for the length of the carbon chain, namely consisting of only
2 methylene groups or 2-MEP, and 6 methylene groups or 6-
MHP. Both XRD and TEM revealed clearly different interfacial
interaction at both HAp and dentin for the three phosphoric-
acid ester monomers, by which the null-hypothesis should be
rejected.
Powder XRD revealed before [11] that 10-MDP ionically
bonded to HAp, already after 5 min, and more intense after
1- and 24-h exposure (Fig. 2c). The three detected peaks were
ascribed to a crystalline phase constituted of a layered struc-
ture of the 10-MDP Ca salts, previously also being referred to as
‘nano-layering’ [21]. The calcium salt DCPD was deposited at a
much later stage and only intensively detected in the 24-h XRD
pattern (Fig. 2c) [22]. DCPD was also detected when 2-MEP and
6-MHP interacted with synthetic HAp powder for 24 h. How-
ever, no other peaks, except those assigned to HAp and DCPD,
could be detected. In contrast to 10-MDP, 2-MEP and 6-MHP
did not produce monomer-Ca salts upon interaction with HAp
powder. Furthermore, unlike 10-MDP Ca salt, calcium salts of
2-MEP and 6-MHP could not be synthesized. A plausible rea-
son is that 2-MEP, although having the same methacryloxy and
phosphoric-acid ester group as 10-MDP, is completely soluble
in water in contrast to 10-MDP [20]. Hence, the calcium salts
of 2-MEP and 6-MHP might have been too soluble in water and
ethanol, unlike that of 10-MDP.
Applied on dentin, XRD of the 6-MHP primer revealed
the three characteristic peaks of nano-layering, as was also
detected for 10-MDP and for the latter much more inten-
sively (Fig. 3). These peaks must be assigned to 6-MHP Ca
salts. Also 2-MEP revealed one weak, but characteristic peak,
that might represent some 2-MEP Ca salt formation. These
nano-layering peaks were detected when the primers were
rubbed on dentin for only 20 s (PP2), while they were only
detected for 10-MDP upon interaction with HAp powder, but
not for 6-MHP and 2-MEP upon interaction with HAp powder
(PP1). Also the 10-MDP primer resulted in much more intense
nano-layering at dentin (PP2) than upon interaction with HAp
powder (PP1). This difference in interaction intensity between
HAp powder (PP1) and dentin (PP2), as also shown in our pre-
vious study, could be due to the small crystal size of HAp
in dentin and better reachable Ca in contrast of that of the
synthetic and more dense HAp powder [21]. Another reason
could be the difference in specimen-preparation protocol. In
PP1, the monomer-coated HAp powder was centrifuged and
thoroughly washed before being dried and analyzed using
XRD. On the contrary, in PP2 the monomer primers were
rubbed on dentin, which must have intensified the interaction
with dentinal HAp, and were subsequently also air-dried so
that monomer-calcium salts might have been deposited. Air-
drying the primed dentin reduced the amount of solvent, by
894 d e n t a l m a t e r i a l s 2 9 ( 2 0 1 3 ) 888–897

Fig. 5 – TF-XRD patterns of untreated dentin, and of dentin on which 4 commercial one-step self-etch adhesives, Adper Easy
Bond (3M ESPE) in (a), and All-Bond Universal (Bisco), Clearfil 3S Bond (Kuraray Noritake Dental) and Scotchbond Universal
(3M ESPE) in (b), were rubbed for 20 s. Adper Easy Bond (3M ESPE) was additionally allowed to interact with dentin for 24 h
(Adper Easy Bond D 24 h). The TF-XRD patterns of ‘6-MHP P’ and ‘10-MDP D’ from Fig. 3 served as reference in (a) and (b),
respectively. (a) The TF-XRD pattern of the 6-MHP-based Adper Easy Bond (3M ESPE) (‘Adper Easy Bond D’; 20-s rubbing) did
not reveal any peaks. The TF-XRD pattern of ‘Adper Easy Bond D 24h’ revealed three weak characteristic peaks at 2 = 3.34◦
(d = 2.65 nm), 2 = 6.46◦ (d = 1.37 nm) and 2 = 9.48◦ (d = 0.93 nm), which must be assigned to ‘nano-layering’ or 6-MHP Ca-salt
formation. (b) The TF-XRD pattern of All-Bond Universal (Bisco) rubbed on dentin for 20 s (‘All-Bond Universal D’) revealed
three intense characteristic peaks at 2 = 2.70◦ (d = 3.27 nm), 2 = 5.12◦ (d = 1.73 nm) and 2 = 7.35◦ (d = 1.20 nm). The TF-XRD
pattern of Clearfil S3 Bond (Kuraray Noritake Dental) rubbed on dentin for 20 s (‘Clearfil S3 Bond D’) revealed also three, but
less strong characteristic peaks at 2 = 2.57◦ (d = 3.44 nm), 2 = 4.99◦ (d = 1.77 nm) and 2 = 7.35◦ (d = 1.20 nm). The TF-XRD
pattern of Scotchbond Universal (3M ESPE) rubbed on dentin for 20 s (‘Scotchbond Universal D’) also showed three peaks at
2 = 2.56◦ (d = 3.45 nm), 2 = 5.04◦ (d = 1.75 nm) and 2 = 7.44◦ (d = 1.19 nm). For all the three 10-MDP-based adhesives, the three
peaks must be assigned to ‘nano-layering’ or 10-MDP Ca-salt formation.

which monomer-Ca salts might have been deposited earlier.
In general, the XRD-peak intensity is a measure of the quan-
tity of a given compound. Fukegawa et al. [11] confirmed by 31 P
NMR analysis that the XRD peaks assigned to 10-MDP Ca salts
were closely related to the amount of 10-MDP Ca salts formed
on HAp particles. Obviously, the difference in peak intensity
of the three characteristic peaks was in the same order of
10-MDP > 6-MHP > 2-MEP for both the HAp-powder and dentin
samples in respectively PP1 and PP2.
According to the AD concept [11,22], the amount of
monomer-Ca salts relative to DCPD depends on the intensity
of decalcification induced by the acidic monomer. However,
when the solubility of the monomer-Ca salts is higher than
that of DCPD, DCPD might have been deposited earlier than the
monomer-Ca salts. This means that the deposition of not only
DCPD but also of the monomer-Ca salts depends on the decal-
cification intensity. The peak intensity of DCPD in the 24-h
monomer-HAp samples (Fig. 2; PP1) was in order of 2-MEP > 10-
MDP > 6-MHP. The amount of DCPD formation is an indication
of the degree of dissolution of HAp. On the other hand, DCPD
is less stable than HAp, and thus cannot protect collagen as
well as the original apatite crystal [22]. Clearly more DCPD was
formed by 2-MEP than by 10-MDP, versus remarkably hardly
any for 6-MHP (Fig. 2; PP1). The latter lower DCPD formation of
the ‘6-MHP HAp 24h’ sample in PP1 might indicate that 6-MHP
also produced 6-MHP Ca salts; they were however washed
away using ethanol following the sample-preparation proto-
col for powder HAp in PP1 (Fig. 1) (unpublished data). Indeed,
6-MHP Ca salts were clearly formed when 6-MHP was rubbed
on dentin for 20 s. Hence, the small and slow deposition of
DCPC and monomer-Ca salts induced by 2-MEP and 6-MHP
may explain the lower decalcification ability of 2-MEP and 6-
MHP versus that of 10-MDP.
TEM in PP2 confirmed the XRD findings. Upon interaction
of the 10-MDP primer with dentin, nano-layering, the mor-
phologic manifestation of 10-MDP Ca salt formation, could be
observed throughout the whole adhesive layer (Fig. 4f). TEM
of ‘10-MDP D’ also revealed a dentin surface that appeared
to have been etched deeper (Fig. 4d,e) than when dentin was
rubbed with 6-MHP (‘6-MHP D’; Fig. 4a,b). Also, a thicker hybrid
layer was formed by the 10-MDP primer (Fig. 4d and e) than
by the 6-MHP primer (Fig. 4a and b). The morphologically
observed deeper etching and thicker hybrid layer confirmed
the higher etching ability of 10-MDP versus that of 6-MHP. XRD
revealed that 6-MHP did not form as much nano-layering as
10-MDP; any nano-layering was detected by TEM for 6-MHP, to
a certain extent also because of the narrower width of 6-MHP
nano-layering.
In PP3, when the commercial one-step self-etch and 6-
MHP-based adhesive Adper Easy Bond (3M ESPE) was rubbed
 d e n t a l m a t e r i a l s 2 9 ( 2 0 1 3 ) 888–897 895

Fig. 6 – (a) TEM photomicrographs of Adper Easy Bond (3M ESPE) applied on dentin. The photomicrographs were taken
using a 80 kV TEM (JEOL JEM-1200 EX II TEM, Jeol) in (a-1,2) and a 300 kV TEM (JEM-3010, JEOL) in (a-3,4). (a-1,2) Adper Easy
Bond (3M ESPE) appeared to have effectively dissolved the smear layer, while a hybrid layer of a few hundreds nano-meters
was formed. (a-3) The hybrid layer still contained residual HAp. (a-3,4) High-magnification TEM disclosed little
nano-layering, particularly near the tubule orifices where the adhesive had infiltrated residual smear. The periodicity of
nano-layering was about 2.7 nm. (b) TEM photomicrographs of All-Bond Universal (Bisco) applied on dentin. The
photomicrographs were taken using a 80 kV TEM (JEOL JEM-1200 EX II TEM, Jeol) in (b-1,2) and a 300 kV TEM (JEM-3010,
JEOL) in (b-3,4). (b-1,2) All-Bond Universal (Bisco) did not completely dissolve the smear layer, while a 0.5-␮m thick hybrid
layer was formed. The adhesive layer of All-Bond Universal (Bisco) was about 3 ␮m thick, and totally clear because the
self-etch adhesive does not contain any filler. (b-2,3) Dentin was only partially demineralized, with HAp crystals scattered
throughout the whole hybrid layer. (b-4) High-magnification TEM disclosed nano-layering with a periodicity of about
3.5 nm. (c) TEM photomicrographs of Clearfil S3 Bond (Kuraray Noritake Dental) applied on dentin. The photomicrographs
were taken using a 80 kV TEM (JEOL JEM-1200 EX II TEM, Jeol) in (c-1,2) and a 300 kV TEM (JEM-3010, JEOL) in (c-3,4). (c-1,2)
Clearfil S3 Bond (Kuraray Noritake Dental) appeared to have effectively dissolved the smear layer. A 0.5-␮m thick hybrid
layer was formed, while the dentin surface was also partially demineralized. (c-1,2) The hybrid layer contains much HAp.
(c-4) High-magnification TEM disclosed abundant nano-layering near the hybrid layer with a periodicity of about 3.5 nm. (d)
TEM photomicrographs of Scotchbond Universal (3M ESPE) applied on dentin. The photomicrographs were taken using a
80 kV TEM (JEOL JEM-1200 EX II TEM, Jeol) in (d-1,2) and a 300 kV TEM (JEM-3010, JEOL) in (d-3,4). (d-1,2) Scotchbond
Universal (3M ESPE) appeared to have effectively dissolved the smear layer. The interaction zone with dentin showed to be
partially demineralized, having left HAp-crystals dispersed in a 0.2–0.5 ␮m thick hybrid layer. (d-3,4) High-magnification
TEM disclosed little nano-layering in the adhesive layer with a periodicity of about 3.5 nm. AR: adhesive resin; D: dentin; HL:
hybrid layer; LVR: low-viscosity (composite) resin; NL: nano-layering.
896 d e n t a l m a t e r i a l s 2 9 ( 2 0 1 3 ) 888–897
Table 1 – List of commercial dental adhesive investigated.
Adhesive Manufacturer
Adper Easy Bond 3M ESPE, Seefeld, Germany
All-Bond Universal Bisco, Schaumburg, IL, USA
Clearfil S3 Bond Kuraray Noritake Dental, Tokyo, Japan
Scotchbond Universal 3M ESPE
a
Data provided by the respective manufacturers.
on dentin for 20 s, as per manufacturer’s instructions, XRD did
not disclose any peak (Fig. 5a), except those to be attributed
to HAp (not shown). Nevertheless, ‘Adper Easy Bond D 24h’,
representing the interaction of the adhesive with dentin for
24 h, revealed the three characteristic ‘nano-layering’ peaks,
though very weakly (Fig. 5a). TEM of the interface of Adper
Easy Bond (3M ESPE) at dentin disclosed nano-layering near
the dentin tubule. We hypothesize that unlike the adhesive
was cured, the presence of water within the dentin tubules
prevented some acidic monomers to polymerize adequately,
as they were dissolved in water, and thus were capable to con-
tinue to etch the surrounding dentin and thus to eventually
produce nano-layering at this particular location near a dentin
tubule [24].
In contrast to Adper Easy Bond (3M ESPE), all three 10-
MDP-based commercial adhesives, All-Bond Universal (Bisco),
Clearfil 3S Bond (Kuraray Noritake Dental) and Scotch-
bond Universal (3M ESPE) revealed the three characteristic
nano-layering peaks already after 20-s interaction (Fig. 5b).
Especially All-Bond Universal (Bisco) showed clear and intense
peaks. This adhesive does not contain filler (Table 1; Fig. 6b-
1,2). An experimental filler-free version of the one-step
self-etch and 10-MDP-based Clearfil 3S Bond (Kuraray Nori-
take Dental) also revealed stronger nano-layering peaks than
the commercial filler-containing adhesive (unpublished data).
The pH of all commercial adhesives is around 2.5 or higher,
by which they can be categorized as ‘ultra-mild’ self-etch
adhesives [25]. In this study, the hybrid layers produced
by the different commercial adhesives did not show that
much difference in ultra-structure, as was shown for the
6-MHP versus 10-MDP experimental primers investigated
by TEM in PP2. Because of differences in solvent and its
concentration, the etching ability can have been different [26].
In addition, all adhesives investigated in this study contain
HEMA. In our previous study [27], HEMA was shown using
quartz crystal microbalance (QCM) not only to decrease nano-
layering but also the etching ability. Compared to 6-MHP and
new phosphonic acid functional monomers synthesized by
Ivoclar-vivadent in another previous study [10], the functional
monomer 10-MDP clearly revealed the strongest 10-MDP Ca-
salt formation. 10-MDP has a relatively strong decalcification
ability, making Ca readily available to form 10-MDP Ca salts.
The functional monomer 10-MDP was used for the first
time for Clearfil Liner Bond 2V (Kuraray Noritake Dental); its
Compositiona pHa
HEMA, Bis-GMA, methacrylated phosphoric esters 2.5
(6-MHP), 1,6-hexanediol dimethacrylate,
methacrylate functionalized polyalkenoic acid
(VitrebondTM Copolymer), silica filler, ethanol,
water, initiators based on camphorquinone and
stabilizers
10-MDP, Bis-GMA, HEMA, water, ethanol, 3.2
photoinitiator system (CQ + amine)
10-MDP, Bis-GMA, HEMA, photo-initiators, 2.7
ethanol, water, silanated colloidal silica
10-MDP, dimethacrylate resins, HEMA, Vitrebond 2.7
copolymer, filler, ethanol, water, initiators, silane
bonding strategy involved the use of a self-etching primer,
based on the application of acidic polymerizable monomers
on dentin (and enamel) without a rinsing step. Such a self-
etching primer does not etch dentin (and enamel) as strongly
as phosphoric acid does. The self-etch procedure releases Ca
and phosphate ions from HAp, which remain at the interface
as the surface is not rinsed off. The relatively strong decalci-
fication ability of 10-MDP enables to form stable monomer-Ca
salts, manifested as ‘nano-layering’. This nano-layering is
thought to produce a better water-stable interface and thus to
contribute to bond durability. In this study, nano-layering was
also produced by 6-MHP, though less intensively; although the
structure of 6-MHP is similar to that of 10-MDP, its Ca-salt (it
could not be synthesized) is less stable than that of 10-MDP.
Acknowledgements
The current research was supported by the G.0496.10 research
grant of the Research Foundation – Flanders and by a Grant-
in-Aid for Challenging Exploratory Research (24659873). We
thank the respective manufacturers for providing the com-
mercial adhesives, and Kuraray Noritake Dental in particular
for providing the three phosphoric-acid ester monomers.
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