Journal of Dental Research

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Chemical Interaction of Phosphoric Acid Ester with Hydroxyapatite

D. Fukegawa, S. Hayakawa, Y. Yoshida, K. Suzuki, A. Osaka and B. Van Meerbeek
J DENT RES 2006 85: 941
DOI: 10.1177/154405910608501014

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 RESEARCH REPORTS
Biomaterials & Bioengineering
D. Fukegawa1, S. Hayakawa3,
Y. Yoshida1*,2, K. Suzuki1,2,
A. Osaka2,3, and B. Van Meerbeek4
1Department
of Biomaterials, Okayama University Graduate
School of Medicine, Dentistry and Pharmaceutical Sciences,
2-5-1 Shikata-cho, Okayama, 700-8525, Japan; 2Research
Center for Biomedical Engineering, Okayama University, 3-
1-1, Tsushima, Okayama, 700-8530, Japan; 3Biomaterials
Laboratory, Graduate School of Natural Science and
Technology, Okayama University, 3-1-1, Tsushima,
Okayama, 700-8530, Japan; and 4 Leuven BIOMAT
Research Cluster, Department of Conservative Dentistry,
School of Dentistry, Oral Pathology and Maxillo-Facial
Surgery, Catholic University of Leuven, Kapucijnenvoer 7,
B-3000 Leuven, Belgium; *corresponding author,
yasuhiro@md.okayama-u.ac.jp
J Dent Res 85(10):941-944, 2006
ABSTRACT
Among functional monomers used in
contemporary dental adhesives, 10-
methacryloyloxydecyl dihydrogen phosphate
(MDP) has been found to interact chemically with
hydroxyapatite (HAp) most intensively and stably.
This effect was thought to be the basis of the
superior bonding effectiveness of MDP-based
self-etch adhesives to enamel/dentin. To elucidate
fully the chemical interaction and reactivity of
MDP with HAp, we used 31 P CP-MAS NMR
spectroscopy and powder x-ray diffraction. In an
aqueous ethanol solution, Ca ions were leached
from HAp to form, at short term, a MDP-calcium
salt (CaMHP 2 ) layered structure on the HAp
surface. When MDP was allowed to interact for
longer time (< 24 hrs), CaHPO4·2H2O precipitated
on top of this MDP-calcium salt layered structure.
In conclusion, the intense chemical interaction of
MDP with HAp must be ascribed to superficial
dissolution of HAp induced by the MDP
adsorption and subsequent deposition of MDP-
calcium salt with a solubility lower than that of
CaHPO4·2H2O.
KEY WORDS: acid monomer, NMR, XRD,
adsorption, demineralization.
Received January 7, 2006; Last revision May 15, 2006;
Accepted June 6, 2006
A supplemental appendix to this article is published
electronically only at http://www.dentalresearch.org.
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International and American Associations for Dental Research
Chemical Interaction of
Phosphoric Acid Ester
with Hydroxyapatite
INTRODUCTION
A mong contemporary dental adhesives, self-etch adhesives are clinically
most easy to use and therefore very attractive for routine use in a busy
daily practice. The so-called 'mild' self-etch adhesives demineralize dentin
only partially, leaving some hydroxyapatite (HAp) crystals around collagen
within a submicron hybrid layer (Van Meerbeek et al., 2003). Such residual
HAp may serve as a template for additional chemical interaction with the
adhesive's functional monomer, and is especially regarded as essential to the
long-term stability of the bond (Inoue et al., 2005). Among functional
monomers contained in contemporary self-etch adhesives, the monomer 10-
methacryloyloxydecyl dihydrogen phosphate (MDP) was found to adhere to
HAp most readily and intensively (Yoshida et al., 2004). On the basis of
Yoshida's adhesion-decalcification concept (AD-concept; Yoshida et al.,
2001), the bond between MDP and HAp should be stable, because the
MDP-calcium salt hardly dissolves in water (Yoshida et al., 2004).
However, the actual chemical interaction of MDP with HAp has not
been elucidated in full. Recently, Fu et al. (2005) reported that the reaction
of phosphoric acid esters (PAEs) with HAp produced PAEs-HAp complexes
that do not result from simple acid-base reactions, like those occurring with
Ca(OD)2 (deuterated calcium hydroxide) in either liquid or solid. Based on
liquid-state 31P NMR (phosphorus-31 nuclear magnetic resonance), the
authors concluded that PAEs can decalcify and adhere to HAp
simultaneously. Regrettably, detailed NMR spectroscopic analysis of the
reaction product was difficult, because mono- and di-esters reacted with
HAp in suspension. Consequently, many intermediate solid products are
expected to have been formed around the HAp particles; however, they are
barely detectable by conventional liquid NMR spectroscopy. Peak
assignment is rather difficult, due to severely overlapping NMR peaks. In
addition, many reaction products must have been formed, because PAEs
react with HAp via substitution of PO 4 3- - and OH - -groups and/or by
decalcification of HAp, resulting in the formation of diverse solid products.
For analysis of the latter solid materials, magic angle spinning multi-nuclear
magnetic resonance (MAS NMR) spectroscopy would be most appropriate,
since it can provide more definite evidence on the interaction patterns of
PAEs with HAp.
To clarify further the actual chemical interaction and reactivity of MDP
with HAp, we used 31P CP(cross-polarization)-MAS spectroscopy and
powder x-ray diffraction to characterize MDP-HAp samples.
MATERIALS & METHODS
HAp particles (surface area of 49 m2g-1, mean powder diameter of 19.45 ± 0.49
␮m) were obtained from Pentax (Tokyo, Japan). CaHPO4·2H2O particles were
purchased from Nacalai Tesque (Kyoto, Japan). MDP was provided by Kuraray
(Tokyo, Japan). MDP-coated HAp particles (referred to as MDP-HAp) were
prepared at room temperature as follows: Two gm of HAp particles were
941
942
Figure 1. Powder XRD patterns of MDP-HAp samples, CaMHP2, and
HAp. For comparison, the XRD pattern of CaHPO4·2H2O is also shown.
(a) Wide angle; (b) low angle.
dispersed in 10 gm of a mixed solution of MDP, absolute ethanol,
and distilled de-ionized water in a MDP:EtOH:H2O composition of
15:45:40 wt% while being stirred. The MDP-coated HAp particles
were separated from the mixed solution by centrifugation and
decantation, respectively, after 5 min, 1 hr, and 24 hrs (referred to
as MDP-HAp-5min, MDP-HAp-1h, and MDP-HAp-24h). The
separated HAp particles were then washed 3X with absolute
ethanol, and dried at room temperature in an ambient atmosphere.
In addition, MDP-calcium salt [calcium bis(10-methacryloyloxy-
decyl hydrogen phosphate) or CaMHP2 with a Ca/P molar ratio of
0.5] was also prepared through precipitation of MDP and CaCl2 in
water. Although, in an aqueous solution, the HAp surface is
expected to be demineralized due to the adsorption of MDP, in
absolute ethanol, HAp demineralization is expected to be
suppressed, because HAp barely dissolves in ethanol. Therefore, 2
gm of HAp particles were additionally dispersed in 10 gm of a
mixed MDP-ethanol solution in a MDP:EtOH composition of
25:75 wt%, while being stirred, so that we could examine the
adsorption of MDP by HAp. The MDP-HAp particles were
separated from the ethanol solution by centrifugation and
decantation after 24 hrs. The separated HAp particles were washed
3X with absolute ethanol, and then dried at room temperature in an
ambient atmosphere (referred to as MDP-HAp-24h-ethanol).
X-ray Diffraction (XRD)
The crystal phases of samples were identified by means of an x-ray
powder diffractometer (CuK ␣; 1.545 Å, RINT 2500, Rigaku,
Osaka, Japan), operated under 40 kV acceleration and 200 mA
current, and a scanning rate of 0.02° per sec. HAp and
CaHPO4·2H2O were also analyzed for comparison.
MAS NMR Measurement
31 PMAS NMR spectra were recorded at 7.05 T (tesla) on a
VarianUNITY INOVA300 FT-NMR (Fourier transformed NMR)
spectrometer (Palo Alto, CA, USA), equipped with a CP-MAS
probe. The samples were placed in a silicon-nitride sample tube
with a diameter of 7 mm. The sample spinning speed at the magic
angle to the external magnetic field was 5 kHz. 31P CP-MAS NMR
spectra were taken at 121.4 MHz with 7.0-␮s pulse length (pulse
angle, ␲/2), 10-second recycle delays, and 1100-␮s contact time,
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International and American Associations for Dental Research
Fukegawa et al. J Dent Res 85(10) 2006
Figure 2. Solid-state 31P CP-MAS NMR spectra of MDP-HAp and
reference samples. (a,b) Solid-state 31P CP-MAS NMR spectra of MDP-
HAp samples, CaMHP2, and HAp, together with a liquid NMR spectrum
of MDP. For comparison, the 31 P CP-MAS NMR spectrum of
CaHPO4·2H2O is also shown. (c) 31P CP-MAS NMR spectra of MDP-
HAp in ethanol and of HAp.
where the signals from about 100-200 pulses were accumulated.
31P NMR chemical shifts are reported in ppm, with 85% H PO as
3 4
the external reference (0 ppm). For comparison, 31P CP-MAS
NMR spectra of HAp, CaHPO4·2H2O, and CaMHP2 (Ca/P molar
ratio = 0.5) were also measured. All NMR spectra were recorded in
a room for exclusive use of NMR, where the room temperature
was kept at 300°K by means of an air-conditioner.
RESULTS
MDP-HAp-24h-ethanol did not show any differences with HAp
(Figs. 1a, 1b). In contrast to HAp, the MDP-HAp samples and
CaMHP2 showed several strong peaks in the range of 2␪ =
2.24° to 6.86° (Fig. 1b). CaMHP2 revealed 3 characteristic
peaks in the range of 2␪ = 2.2° (d = 3.94 nm) to 6.9° (d = 1.29
nm), including 2␪ = 2.24° (d = 3.94 nm), 4.56° (d = 1.94 nm),
and 6.86° (d = 1.29 nm) (Fig. 1a), and 1 broad peak at 2␪ = 20°
that should be attributed to an amorphous phase (Fig. 1b). In
addition to the peaks representing HAp (range of 2␪ = 10.7° to
66.3°) (Fig. 1a), MDP-HAp-24h showed 3 characteristic peaks
in the range of 2␪ = 2.3° (d = 3.84 nm) to 6.8° (d = 1.31 nm),
including 2␪ = 2.3° (d = 3.84 nm), 4.52° (d = 1.95 nm), and
6.8° (d = 1.31 nm) (Fig. 1b), and 1 broad peak at 2␪ = 20° (Fig.
1a), due to the amorphous phase. Although relatively less
intense, MDP-HAp-1h and MDP-HAp-5min also showed the
latter 3 characteristic peaks (Fig. 1b), and the 1 broad peak
(Fig. 1a). These results suggest the presence of a crystalline
phase that consisted of a layered structure with d-spacing of
3.84, 1.95, and 1.31 nm. The XRD peak intensities assigned to
the layered structure obviously increased from 5 min to 24 hrs.
The strong peak at 2␪ = 11.8° (d = 0.75 nm) for MDP-HAp-
24h (Fig. 1b), accompanied by several peaks at 2␪ = 21.0° (d =
0.42 nm), 23.0° (d = 0.39 nm), and 29.4° (d = 0.30 nm), must
be ascribed to CaHPO4·2H2O (Fig. 1a). Thus, the very weak
peak at 2␪ = 11.8° for MDP-HAp-1h (Fig. 1a) must also be
assigned to CaHPO4·2H2O. The detection of the peak at 11.8°
(d = 0.75 nm) (Fig. 1a) became much stronger with increasing
reaction time from 1 to 24 hrs and is indicative of the formation
of CaHPO4·2H2O as a reaction product.
Since it was difficult to obtain reasonably intense peaks
assignable to MDP-adsorbed-to-HAp using 31P MAS-NMR, we
J Dent Res 85(10) 2006 Interaction of Phosphoric Acid Ester with HAp
Figure 3. Chemical interaction models of 10-methacryloyloxydecyl
dihydrogen phosphate (MDP) with hydroxyapatite (HAp). (I,II) The
ionic binding models of MDP interacting electrostatically with the
Ca 2+ ions of HAp, where either one of the P-OH groups of MDP
dissociated H+ to form one P-O- group, or two of the P-OH groups
of MDP dissociated H + to form two P-O - groups during chemical
interaction with HAp, respectively. (III) The covalent binding model
o f M D P c o n d e n s a t i o n w i t h t h e P O 43- i o n s o f H A p t o f o r m
pyrophosphate groups.
used 31P CP-MAS NMR with high-power proton decoupling
(Pines et al., 1972). The 31P NMR chemical shifts of the
various samples are summarized in the Appendix Table. The
liquid NMR spectrum of MDP (Figs. 2a, 2b) shows 2 sharp
peaks at 0.4 ppm and -13.5 ppm, which must be assigned to the
MDP monomer and the MDP dimer in phosphorus molar
percentages of 90.6 ± 2.5% and 9.4 ± 2.5%, respectively. In the
short term (MDP-HAp-5min), a broad peak at -0.2 to -0.7 ppm
appeared (Fig. 2b), and must be assigned to the adsorbed MDP
monomer [Figs. 3(I), 3(II)]. The peak around -0.7 ppm became
clearly more intense and shifted to -1.4 ppm after 24 hrs (Fig.
2b), and, when compared with the peaks at -1.4 and -2.2 ppm in
the CaMHP2 spectrum (Fig. 2b), must be assigned to CaMHP2,
which is in agreement with the former XRD results. Note that
in the 31P CP-MAS NMR spectra of the MDP-HAp samples,
the intensity of the peak at 1.5 ppm assignable to HPO42- of
CaHPO4·2H2O increased with reaction time (Fig. 2b). This
result is also in agreement with the XRD results (Fig. 1a) and
confirms the formation of CaHPO4·2H2O as a reaction product.
The very weak peaks at -6.3 ppm and -14.5 ppm in the 31P CP-
MAS NMR spectra of the MDP-HAp samples did not become
more intense with reaction time, as compared with the other
peaks (Fig. 2b). The -6.3 ppm peak (Fig. 2b) indicates that very
small amounts of pyrophosphate groups were formed on the
HAp surface [Fig. 3(III)], and the -14.5 ppm peak (Fig. 2b)
indicates that very small amounts of MDP dimer were also
adsorbed onto the HAp surface. In contrast, the 31P CP-MAS
NMR spectrum of MDP-HAp-24h-ethanol (Fig. 2c) showed the
broad and small peak at -0.2-0.7 ppm, which should be
assigned to the adsorbed MDP monomer [Figs. 3(I), 3(II)], the
small peak at -6.3 ppm, which should be assigned to
pyrophosphate groups [Fig. 3(III)], and the small peak at -14.5
ppm, which should be assigned to MDP dimer (regardless of
long-term reaction time). In addition, the peak at 1.5 ppm
assigned to HPO 4 2- of CaHPO 4 ·2H 2 O was not observed,
indicating that MDP was adsorbed onto HAp, while
demineralization of HAp did not occur in the ethanol solution.
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943
Figure 4. Proposed mechanism of MDP adsorption onto HAp and
precipitation of CaMHP2 and CaHPO4·2H2O (modified from Tanaka et
al., 1997).
DISCUSSION
There are many possible adsorption and reaction mechanisms
of MDP with HAp [Ca10(PO4)6(OH)2] via adsorption onto Ca2+
sites, reaction with PO43- groups of HAp, and/or by substitution
of OH - or PO 43- groups of the HAp lattice (Subirade and
Lebugle, 1991, 1993; Lebugle et al., 1994; Yoshida et al.,
2001, 2004; Yoshioka et al., 2002). Subirade and Lebugle
(1991, 1993) have grafted phosphorylated organic polymers
onto HAp surfaces by co-precipitation, and found that the
hydroxyl groups of HAp were substituted by PAE to form
inorganic-organic material connections. They reported that the
number of hydroxyl substitutions in HAp was dependent on the
ratio of PAEs to apatite. However, in the 1 H MAS NMR
experiment (not shown here), the number of hydroxyl groups of
HAp did not substantially change among the five-minute, one-
hour, and 24-hour interactions of MDP with HAp. Thus, the
hydroxyl substitutions in HAp were too few to be observed by
solid-state NMR, and were therefore considered negligible as
compared with other adsorption and decalcification processes.
Lebugle et al. (1994) reported that the modification of HAp
particles with n-dodecyl dihydrogen phosphate (C12H25PO4H2)
improved their dispersion in polymethylmethacrylate (PMMA),
suggesting that C 12 H 25 groups are grafted to the particle
surface. Ishikawa et al. (1995) also modified the surfaces of
HAp particles by treating them with water-acetone solutions of
ethyl dihydrogen phosphate (C2H5PO4H2). They also revealed
the formation of a layered structure on the particle surface.
Analysis of the abovementioned literature data and the
results obtained in this study indicates that the functional
monomer MDP initially (short-term or within 5 min) interacts
with the HAp surface and adheres to HAp. The 31P CP-MAS
NMR spectra of the MDP-HAp samples indicated that 1 or 2 of
the P-OH groups of MDP dissociated H+ to form P-O- groups
during chemical interaction with HAp. The broad line width of
the 31P CP-MAS NMR peak of MDP-HAp samples (Figs. 2a,
2b) must be attributed to the spreading of the chemical shift
distribution resulting from the different conformations of the
methacryloyloxydecyl group of MHP, and from the influence
of steric hindrance around the terminal methacryloyloxy groups
of MHP (Fig. 4). The conformation of the 10-methacryloyloxy-
decyl group of MHP will be clarified by 13C (carbon-13) MAS
NMR spectroscopy in our future work.
944
Also, the powder XRD analysis revealed that a layered
structure was already formed when MDP interacted with HAp
in a water-ethanol solution for only 5 min. This indicates
that MDP reacted with calcium ions leached from HAp by
decalcification in an aqueous solution. The molecular chain
length of MDP (ca. 1.95 nm) was in good agreement with
the d-spacing of 1.94 nm, as measured by XRD for
CaMHP2. The d-spacing of 3.94 nm must then be explained
by the formation of a MHP-layered structure (Fig. 4), as
modified from Tanaka's model (Ishikawa et al., 1995;
Tanaka et al., 1997). In addition, the detected formation of
CaHPO 4 ·2H 2 O indicates that not only Ca ions, but also
phosphate (HPO 4 2- ) ions, were dissolved from HAp by
demineralization in the aqueous solution. Due to the
electrophilic nature of the 10-methacryloyloxydecyl groups,
MDP is strongly acidic in a water-ethanol solution, by which
HAp is superficially dissolved through MDP adsorption, and
MDP-calcium salts with solubility lower than that of
CaHPO4·2H2O are deposited onto the modified HAp surface.
The 31P CP-MAS NMR results indicated that, in the short
term (within 5 min), the MDP monomer and dimer were
adsorbed onto HAp and at the same time superficially
decalcified HAp. The mechanism of MDP adsorption and HAp
decalcification, and subsequent deposition of calcium salts,
may then occur as follows:
• decalcification reaction induced by the MDP adsorption
(RPO4H2 with R = 10-methacryloyloxydecyl group) in the
aqueous solution
Ca10(PO4)6(OH)2 ➾ 10Ca2+ + 6PO43- + 2OH-
PO43- + RPO4H2 ➾ HPO42- + RHPO4-
• co-precipitation of calcium salts with low solubility
2Ca2+ + HPO42- + 2RHPO4- + 2H2O ➾
CaHPO4·2H2O + Ca(RHPO4)2
The amounts of CaHPO4·2H2O and Ca(RHPO4)2 (CaMHP2)
deposited onto HAp should then depend on the equilibrium of
the chemical reaction formula (1-3) and on the solubility of the
deposited salts in the water-ethanol environment.
Finally, the proposed mechanism of adhesion of MDP to
HAp and the simultaneous decalcification of HAp induced by
MDP adsorption are in good agreement with the results of Fu et
al. (2005). The combination of solid-state NMR spectroscopy
with XRD analysis clarified in detail the several different
chemical states of MDP adhering to HAp, and of the
precipitation/deposition mechanism of CaMHP 2 and/or
CaHPO4·2H2O onto HAp.
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Fukegawa et al. J Dent Res 85(10) 2006
ACKNOWLEDGMENTS
The authors used the solid-state MAS NMR facility of the
Venture Business Laboratory at the Graduate School of Natural
Science and Technology, Okayama University, Okayama,
Japan. The authors are grateful to Dr. E. Fujii and Dr. K.
Kawabata of the Industrial Technology Center of Okayama
Prefecture for their helpful support with particle size
measurement. This study was supported by a Grant-in-Aid for
Scientific Research from the Japan Society for the Promotion
of Science and by funds from Medical-Techno-Okayama.
REFERENCES
Fu B, Sun X, Qian W, Shen Y, Chen R, Hannig M (2005). Evidence of
chemical bonding to hydroxyapatite by phosphoric acid esters.
Biomaterials 26:5104-5110.
Inoue S, Koshiro K, Yoshida Y, De Munck J, Nagakane K, Suzuki K,
et al. (2005). Hydrolytic stability of self-etch adhesives bonded to
dentin. J Dent Res 84:1160-1164.
Ishikawa T, Tanaka H, Yasukawa A, Kandori K (1995). Modification
of calcium hydroxyapatite using ethyl phosphonates. J Mater
Chem 5:1963-1967.
Lebugle A, Subirade M, Delpech V (1994). Grafting of phosphorylated
molecules on apatite surface. In: Hydroxyapatite and related
materials. Brown PW, Constantz B, editors. Boca Raton, FL: CRC
Press, pp. 231-236.
Pines A, Gibby MG, Waugh JS (1972). Proton-enhanced nuclear
induction spectroscopy. A method for high resolution NMR of
dilute spins in solids. J Chem Phys 56:1776-1777.
(1) Subirade M, Lebugle A (1991). Solubility of an organic phosphate in
(2) an apatitic calcium phosphate. Ann Chim Fr 16:41-53.
Subirade M, Lebugle A (1993). Study of the mineral-organic interface
between a calcium phosphate and a phosphorylated monomer. Ann
Chim Fr 18:183-193.
Tanaka H, Yasukawa A, Kandori K, Ishikawa T (1997). Modification
(3) of calcium hydroxyapatite using alkyl phosphates. Langmuir
13:821-826.
Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P,
et al. (2003). Buonocore memorial lecture. Adhesion to enamel and
dentin: current status and future challenges. Oper Dent 28:215-235.
Yoshida Y, Van Meerbeek B, Nakayama Y, Yoshioka M, Snauwaert J,
Abe Y, et al. (2001). Adhesion to and decalcification of
hydroxyapatite by carboxylic acids. J Dent Res 80:1565-1569.
Yoshida Y, Nagakane K, Fukuda R, Nakayama Y, Okazaki M,
Shintani H, et al. (2004). Comparative study on adhesive
performance of functional monomers. J Dent Res 83:454-458.
Yoshioka M, Yoshida Y, Inoue S, Lambrechts P, Vanherle G, Nomura
Y, et al. (2002). Adhesion/decalcification mechanisms of acid
interactions with human hard tissues. J Biomed Mater Res 59:56-62.