Journal of Dental Research

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Incorporation of Casein Phosphopeptide-Amorphous Calcium Phosphate into a Glass-ionomer Cement

S.A. Mazzaoui, M.F. Burrow, M.J. Tyas, S.G. Dashper, D. Eakins and E.C. Reynolds
J DENT RES 2003 82: 914
DOI: 10.1177/154405910308201113

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 RESEARCH REPORTS
Biomaterials & Bioengineering
S.A. Mazzaoui1,2, M.F. Burrow1, M.J.
Tyas1, S.G. Dashper1, D. Eakins1, and
E.C. Reynolds1*
1School of Dental Science, The University of Melbourne,
711 Elizabeth Street, Melbourne, Victoria 3000, Australia;
2
and Dental Research Institute, The University of Zulia,
Maracaibo, Venezuela; *corresponding author,
e.reynolds@unimelb.edu.au
J Dent Res 82(11):914-918, 2003
ABSTRACT
Casein phosphopeptide-amorphous calcium
phosphate (CPP-ACP) nanocomplexes have been
shown to prevent demineralization and promote
remineralization of enamel subsurface lesions in
animal and in situ caries models. The aim of this
study was to determine the effect of incorporating
CPP-ACP into a self-cured glass-ionomer cement
(GIC). Incorporation of 1.56% w/w CPP-ACP into
the GIC significantly increased microtensile bond
strength (33%) and compressive strength (23%)
and significantly enhanced the release of calcium,
phosphate, and fluoride ions at neutral and acidic
pH. MALDI mass spectrometry also showed
casein phosphopeptides from the CPP-ACP
nanocomplexes to be released. The release of
CPP-ACP and fluoride from the CPP-ACP-
containing GIC was associated with enhanced
protection of the adjacent dentin during acid
challenge in vitro.
KEY WORDS: glass-ionomer cement, CPP-ACP,
calcium, inorganic phosphate and fluoride release,
microtensile bond strength.
Received July 30, 2001; Last revision July 23, 2003;
Accepted July 30, 2003
914
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International and American Associations for Dental Research
Incorporation of Casein
Phosphopeptide-Amorphous
Calcium Phosphate
into a Glass-ionomer Cement
INTRODUCTION
C asein phosphopeptide-amorphous calcium phosphate nanocomplexes
(CPP-ACP) have been shown to prevent enamel demineralization and
promote remineralization of enamel subsurface lesions in animal and human
in situ caries models (Reynolds et al., 1995, 1999, 2003). The anticariogenic
potential of CPP-ACP has been attributed to the ability of the CPP to
localize amorphous calcium phosphate at the tooth surface, thereby helping
to maintain a state of supersaturation with respect to tooth mineral
(Reynolds et al., 2003). Recently, the CPP-ACP have been shown to interact
with fluoride ions to produce an additive anticariogenic effect through the
formation of a stabilized amorphous calcium fluoride phosphate phase
(Reynolds et al., 1995; Reynolds, 1998).
Glass-ionomer cements (GICs) are water-based, tooth-colored, and
chemically adhesive materials used in dentistry as bases and restorations.
Microleakage around restorations remains a significant problem which can
lead to caries of the underlying tooth tissues (Pachuta and Meiers, 1995).
However, GICs are ion-releasing materials, and the incorporation into, and
slow release of fluoride ions from, the cement provides a significant
anticariogenic property (Forss, 1993).
In an approach to enhance the anticariogenic potential of a GIC, we
incorporated 1.56% w/w CPP-ACP into a selected commercial GIC and
investigated the cement's physical and chemical properties. Compressive
strength, net setting time, and microtensile bond strength to dentin, as well
as calcium, phosphate, and fluoride release at neutral and acidic pH values,
were determined.
MATERIALS & METHODS
Compressive Strength and Net Setting Time
Cylinders, 4 mm diam x 6 mm long, were made from the GICs for the
compressive strength test and discs, 10 mm diam x 5 mm thick, for the net
setting time test. The control GIC was prepared with a self-curing glass-ionomer
cement (Fuji IX GP, liquid batch No. 080561 and powder batch No. 061051, GC
International, Tokyo, Japan). The GIC containing CPP-ACP (RecaldentTM) was
prepared from the same batch, with 1.56% w/w CPP-ACP incorporated. The
CPP-ACP and GIC powder were manually mixed, and the powder:liquid ratio
used was as recommended by the manufacturer. Compressive strength and net
setting time tests were performed following ISO methods (ISO, 1991).
Microtensile Bond Strength to Dentin
Non-carious human molars stored in saline solution containing thymol were
used within 2 mos following extraction. The use of human teeth for the study
was approved by The University of Melbourne Human Research Ethics
J Dent Res 82(11) 2003 CPP-ACP in a Glass-ionomer Cement 915

Committee. Bar-shaped Table 1. Physical Properties and Mode of Failure of CPP-ACP-containing GIC and Control
specimens, half GIC and half
dentin, were prepared with Group Compressive
GIC (Fuji IX GP, batch Strength (MPa)
No.9909021, GC International,
Japan) and the same GIC 1.56% w/w
containing 1.56% w/w CPP- CPP-ACP/GIC 170 + 29a,b
Control GIC 138 + 21
ACP. Microtensile bond
strength tests were performed a M + SD (n = 6-17).
as previously described b Significantly different from control value (p < 0.05).
(Phrukkanon et al., 1998; c Mode of failure:
Tanumiharja et al., 2000), and Type 1 adhesive failure between the restoration and the dentin.
the specimens were stressed in Type 2 partial adhesive failure between the GIC and the dentin and partial cohesive failure in the GIC.
Type 3 cohesive failure in the dentin.
tension at a cross-head speed Type 4 cohesive failure in the GIC.
of 1 mm/min until failure. d Mean frequency value.
Mean bond strength values
were calculated according to
the standard formula (ISO,
1991), and we observed the fractured specimens in a scanning
electron microscope (SEM 515; Phillips, Eindhoven, The
Netherlands) to assess the mode of failure (Phrukkanon et al.,
1998).
Ion Measurements and CPP Detection
We prepared discs, 6 mm diam x 2 mm thick, using the GIC
with and without 1.56% w/w CPP-ACP. The GIC was mixed, as
described above, injected into the molds, condensed, and
allowed to set at 37C and 100% RH for 1 hr. During setting,
the bottom and top of the filled molds were covered by mylar
strips and microscope slides under hand pressure. The discs
were removed from the molds, placed in individual sealed
plastic tubes, and incubated at 37C in either 2 mL of de-
ionized water (pH 6.9) (Milli-Q Water, Millipore Corporation,
Melbourne, Victoria, Australia), or 2 mL of 50 mM sodium
lactate buffer at pH 5.0. The solutions were changed every 24
hrs for 3 days, and the release of calcium, inorganic phosphate,
and fluoride ions was measured in each solution. Calcium
concentrations were determined by atomic absorption
spectrophotometry (Adamson and Reynolds, 1995), inorganic
phosphate colorimetrically (Itaya and Ui, 1966), and fluoride
ion with the use of an ion-selective electrode (Ion 85
Radiometer, Copenhagen, Denmark). The release of the ions
was expressed as mmol/mm2 surface area of the GIC exposed.
The presence of CPP in the solutions was determined by
Matrix Assisted Laser Desorption/Ionisation-Mass Spectrometry
(MALDI-MS) (Voyager-DE, Perseptive Biosystems; Framingham,
MA, USA) with a matrix of 2,5-dehydroxybenzoic acid in 66%
water, 33% CH3CN, and 1% formic acid.
Acid Demineralization Test
Box-shaped cavities (6 mm x 2 mm x 1.5 mm) along the
cemento-enamel junction of human molars were prepared. The
cavity margins were finished with a slow-speed cylindrical bur,
giving a cavo-surface angle as close as possible to 90. The
cavities were filled with the GIC with and without the
incorporated 1.56% w/w CPP-ACP. The teeth were then stored at
37C and 100% relative humidity for 24 hrs. The GIC surfaces
were finished and polished, and the integrity of the cavo-surface
margins was confirmed under a light microscope. Two coats of
an acid-resistant varnish were then applied to the tooth surface,
leaving a 1-mm window around the cavity margins. The teeth
were then placed in 25 mL of acid buffer containing 2.2 mM
Net Setting Bond Strength Mode of Failure Frequency
Time (sec) (MPa) Type 1c Type 2 Type 3 Type 4 Total
226 + 35a,b 10.6 + 4.0a,b 2d 11b 0 4b 17
186 + 25 8.0 + 2.7 2 5 0 10 17
CaCl2, 2.2 mM NaH2PO4, and 50 mM acetic acid (pH 5.0) (ten
Cate and Duijsters, 1982) for 4 days at 37C. The acid solution
was replaced every 24 hrs. After 4 days, longitudinal sections
(100 mm) were obtained through the center of each restoration as
described previously (Reynolds, 1997). The sections were
observed under polarized light with quinoline as the imbibing
medium (Wefel et al., 1985). A first-order red wavelength plate
was inserted between the crossed polarizers to highlight the
changes in the demineralized dentin, and a 10 3 - m m-length
graticle was used to measure the areas of the lesions. The area of
the body of the lesion was measured from the margin of the
restoration to 568 + 3 m from the restoration. Five
measurements were made of each lesion.
Statistical Analysis
Data from compressive strength, net setting time, areas of
demineralized lesions, and microtensile bond strength were
compared by Student's t test. Chi-squared distribution was used
to compare the mode of failure of fractured specimens. Data
from the ion release analyses were compared by a one-way
classification analysis of variance, with the least significant
difference test.
RESULTS
Physical Properties
Incorporation of 1.56% w/w CPP-ACP into the GIC resulted in a
23% increase in compressive strength, a 33% increase in
microtensile bond strength, and a 40-second increase in mean
setting time (Table 1). The increase in setting time did not exceed
the acceptable standard for a self-cured GIC (ISO, 1991). The
distribution in the mode of failure was analyzed by SEM. Type 2
fracture (partial adhesive and partial cohesive failure) was more
frequent with the CPP-ACP-containing GIC, whereas Type 4
(cohesive failure) was more frequent with the control (Table 1).
The microstructures of the 2 cements examined by SEM at a 360X
magnification appeared similar, although a more porous and
roughened fracture surface was observed with the control GIG
when compared with the CPP-ACP-containing cement (Fig. 1).
Ion and CPP Release
Mean values for the release of fluoride, calcium, and phosphate
ions in water (pH 6.9) and sodium lactate buffer (pH 5.0) from
the GIC containing 1.56% w/w CPP-ACP and the control GIC

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916
Figure 1. Representative scanning electron micrographs of fractured
cement surfaces of the GIC containing 1.56% w/w CPP-ACP (a) and the
control GIC (b).
are shown in Table 2. The pattern of fluoride release in water was
similar between samples and controls, with the highest release
occurring during the first 24 hrs and a slower but continued
release during the next two 24-hour periods. Fluoride release was
significantly higher in the pH 5.0 sodium lactate buffer than in
water for both materials. Significantly higher fluoride release was
found with the CPP-ACP-containing GIC when compared with
the control at both pH values. No calcium release was detected in
water from either material. However, calcium ion release was
detected with the CPP-ACP-containing GIC in the pH 5.0 sodium
lactate buffer. The release was low relative to fluoride but
continuous during the three 24-hour periods. At both pH values,
inorganic phosphate ion release was significantly higher from the
CPP-ACP-containing GIC than from the control material during
the first 24-hour period. The release of inorganic phosphate was
significantly higher in sodium lactate buffer at pH 5.0 than in
water for both materials. By MALDI-MS analysis, CPP could be
detected in the water and pH 5.0 buffer after 24 hrs' incubation
with the GIC containing CPP-ACP. The mass spectrum obtained
was the same as that for standard CPP-ACP.
Incorporation of the CPP-ACP into the GIC significantly
enhanced protection against acid demineralization of the
adjacent dentin (Fig. 2). The mean area of demineralized lesion
adjacent to the control GIC was 40,060 + 4870 mm2 (n = 12),
which was significantly larger (p < 0.05) than the area of the
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International and American Associations for Dental Research
Mazzaoui et al. J Dent Res 82(11) 2003
Figure 2. Representative photomicrographs of demineralized lesions in
dentin adjacent to the control GIC (a) and the GIC containing 1.5%
w/w CPP-ACP (b).
lesion (22,686 + 8075 m m 2 , n = 12) adjacent to the GIC
containing 1.56% w/w CPP-ACP.
DISCUSSION
Incorporation of 1.56% w/w CPP-ACP into a GIC was shown
to increase compressive strength and microtensile bond
strength, enhance the release of calcium, phosphate, and
fluoride ions, and enhance protection of the adjacent dentin to
acid demineralization.
A major reason for the use of GICs in a variety of clinical
applications is their capacity to bond chemically to different
surfaces such as enamel, dentin, and resin composite
(Akinmade and Nicholson, 1993). GICs are used routinely in
conjunction with resin composites (Li et al., 1996), in
Atraumatic Restorative Treatment (Frencken et al., 1996), in
tunnel restorations (Svanberg, 1992), and in the restoration of
primary teeth (Frankenberger et al., 1997). Bond strength,
therefore, is an important property of the GIC. The mean
microtensile bond strength of the CPP-ACP-containing GIC
was 33% higher than that of the control GIC. The testing
method used has been successfully used on specimens with
different dentin thicknesses and dentinal tubule orientations and
with disease-affected dentin specimens (Phrukkanon et al.,
1998). Therefore, factors such as the quality, depth, and
moisture of the dentin substrate (Burrow et al., 1994) were
believed not to affect the results of this study.
The most common mode of failure in the adhesion between
a GIC and the dentin during microtensile bond strength tests is
cohesive failure within the GIC (Tanumiharja et al., 2000).
This was the predominant mode of failure of the control GIC
found in this study. The predominant mode of failure for the
CPP-ACP-containing GIC was partial cohesive failure in the
GIC and partial adhesive failure between the GIC and the
dentin. It has not been clearly established whether the mode of
failure for a particular material is associated with its bond
strength or elastic modulus. It is known that the pores in a solid
body act as stress-concentration points where fracture can
initiate (Griffith, 1920), and it has been speculated that this
explains the frequency of cohesive failure within a GIC
(Tanumiharja et al., 2000). SEM analyses of the fractured
surfaces of the different GICs did suggest a greater porosity of
the control GIC relative to the CPP-ACP containing GIC.
However, whether this was responsible for the difference in
bond strength is unknown. The CPP-ACP in the GIC may have
also directly increased microtensile bond strength by the
incorporation of the CPP-ACP nanoparticles into the cross-
linked matrix of the GIC.
J Dent Res 82(11) 2003 CPP-ACP in a Glass-ionomer Cement 917

With respect to the release of ions from the Table 2. Fluoride, Calcium, and Inorganic Phosphate Release from CPP-ACP-
GICs, it was shown that the fluoride release in containing GIC and Control at Neutral and Acid pH
sodium lactate buffer (pH 5.0) was significantly
higher than that in water (pH 6.9). This finding has Ion Release (mmol/mm2)
been previously reported for normal GICs (Forss, Water (pH 6.9) Sodium Lactate (pH 5.0)
1993). However, in the current study, fluoride Period CPP-ACP GIC Control GIC CPP-ACP GIC Control GIC
release was significantly higher from the CPP-ACP-
containing GIC than from the control GIC at both Fluoride release
pH values. It is possible that the CPP-ACP 1st 24 hrs 16.7 + 2.4a,b,c 10.1 + 1.2d 34.5 + 8.2b,c 24.8 + 0.5d
promoted the release of fluoride ions from the GIC 2nd 24 hrs 5.2 + 0.8b,c 3.2 + 0.3d 19.1 + 4.3b,c 14.5 + 1.4d
by forming casein phosphopeptide-amorphous 3rd 24 hrs 3.4 + 0.1b,c 2.4 + 0.1d 18.6 + 3.3b,c 15.3 + 0.9d
calcium fluoride phosphate (CPP-ACFP) Total 25.3 + 4.0b,c 15.7 + 1.6d 72.2 + 9.9b,c 54.6 + 1.4d
nanocomplexes (Reynolds, 1998), which were
released from the cement matrix. Calcium release
Significantly more inorganic phosphate was also 1st 24 hrs -e - 0.4 + 1b,c -
released from the CPP-ACP-containing GIC at both 2nd 24 hrs - - 0.3 + 1b,c -
pH values (5.0 and 6.9) than from the control GIC, 3rd 24 hrs - - 0.3 + 1b,c -
consistent with the addition of the stabilized Total - - 1.0 + 1b,c -
amorphous calcium phosphate. Calcium ions were
not detected at neutral pH with either GIC, and were Inorganic phosphate release
detected with the CPP-ACP-containing GIC only at 1st 24 hrs 0.4 + 0.1b,c - 0.9 + 0.2b,c 0.5 + 0.1d
acid pH. It is likely that a low level of calcium ion 2nd 24 hrs 0.1 + 0.1b,c - 0.5 + 0.1c 0.5 + 0.1d
release at neutral pH from the CPP-ACP-containing 3rd 24 hrs 0.1 + 0.1b,c - 0.5 + 0.1c 0.4 + 0.1d
GIC did occur, since CPP, Pi, and F release at this Total 0.6 + 0.1b,c - 1.9 + 0.1b,c 1.4 + 0.1d
pH was detected. However, this level of calcium
release was below the detection limit of the a M + SD (n = 6-12).
b Significantly different (p < 0.05) from control value at the same pH.
analytical method. Fuji IX is a non-calcium- c Significantly different (p < 0.05) from CPP-ACP GIC value at different pH.
containing GIC in which calcium ions have been d Significantly different (p < 0.05) from control value at different pH.
replaced by strontium ions (Wilson and McLean, e Not detected.
1988). Calcium is an ion not easily leachable from
GIC once the cement has set (Matsuya et al., 1984),
due to its rapid binding, in an insoluble form, to the
polyacrylic acid matrix (Crisp et al., 1976). Elution
of calcium, when it does occur, has been attributed to acid advice regarding the chemistry and composition of Fuji IX GP.
erosion of the cement matrix (Matsuya et al., 1984; Forss, The assistance of Joseph Palamara and Peter Riley is gratefully
1993). The CPP-ACP nanoparticles may have been physically acknowledged.
encapsulated into the set GIC, as has been found with
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