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Effects of the addition of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP)

on mechanical properties of luting and lining glass ionomer cement

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2016 Mater. Res. Express 3 075405

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 Mater. Res. Express 3 (2016) 075405 doi:10.1088/2053-1591/3/7/075405

PAPER

Effects of the addition of casein phosphopeptide-amorphous

RECEIVED
23 April 2016
calcium phosphate (CPP-ACP) on mechanical properties of luting
REVISED
22 June 2016
and lining glass ionomer cement
ACCEPTED FOR PUBLICATION
28 June 2016
PUBLISHED
Farzin Heravi1, Hossein Bagheri2, Abdolrasoul Rangrazi3 and Seyed Mojtaba Zebarjad4
22 July 2016 1
Professor of Orthodontics, Dental Research Center, School of Dentistry, Mashhad University of Medical Sciences, Mashhad, Iran
2
Assistant Professor of Dental Materials, Dental Materials Research Center, School of Dentistry, Mashhad University of Medical Sciences,
Mashhad, Iran
3
Dental Research Center, School of Dentistry, Mashhad University of Medical Sciences, Mashhad, Iran
4
Professor of Materials Engineering, Faculty of Engineering, Shiraz University, Shiraz, Iran
E-mail: rangrazi.r@gmail.com

Keywords: CPP-ACP, glass ionomer, mechanical properties, luting

Abstract
Recently, the addition of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) into
glass ionomer cements (GICs) has attracted interest due to its remineralization of teeth and its
antibacterial effects. However, it should be investigated to ensure that the incorporation of CPP-ACP
does not havesignificant adverse effects on its mechanical properties. The purpose of this study was to
evaluate the effects of the addition of CPP-ACP on the mechanical properties of luting and lining GIC.
The first step was to synthesize the CPP-ACP. Then the CPP-ACP at concentrations of 1%, 1.56% and
2% of CPP-ACP was added into a luting and lining GIC. GIC without CPP-ACP was used as a control
group. The results revealed that the incorporation of CPP-ACP up to 1.56%(w/w) increased the
flexural strength (29%), diametral tensile strength (36%) and microhardness (18%), followed by a
reduction in these mechanical properties at 2%(w/w) CPP-ACP. The wear rate was significantly
decreased (23%) in 1.56%(w/w) concentration of CPP-ACP and it was increased in 2%(w/w).
Accordingly, the addition of 1.56%(w/w) CPP-ACP into luting and lining GIC had no adverse effect
on the mechanical properties of luting and lining GIC and could be used in clinical practice.

Introduction

In recent decades, glass ionomer cements (GICs) have been widely used as a luting agent, a cavity liner, and for
bases and restorative material. Some important characteristics of GICs include fluoride ion release, a low
thermal coefficient of expansion almost near the tooth, the bonding ability to the tooth and metallic surfaces and
biocompatibility [1]. Since the introduction of GIC to the dental profession by Kent Wilson in 1972 [2], its
mechanical and antibacterial features have undergone significant improvement. Many studies have examined
the effects of the addition of other materials such as hydroxyappatite (HA) [3], bioactive glass (BAG) [4] and
TiO2 [1] on GIC characterization. Recently, the incorporation of casein phosphopeptide-amorphous calcium
phosphate (CPP-ACP) into dental cements such as GICs was noted due to its effects such as remineralization of
the tooth and its antibacterial activity. The anticariogenic potential of CPP-ACP has been attributed with the
effect of reducing bacterial growth and the capability of CPP to localize amorphous calcium phosphate at the
tooth surface, thereby helping to maintain a state of saturation of calcium and phosphate ions and consequently
remineralizing the tooth [5]. The main advantage of CPP-ACP over fluoride is its non-toxicity in fluorosis [6].
Many researchers have evaluated the effect of different CPP-ACPs on tooth remineralization [7–9]. However, it
must be investigated to ensure that the incorporation of CPP-ACP into luting and lining GIC does not have
significant adverse effects on its mechanical properties. To the best of our knowledge, the effect of the addition of
CPP-ACP on the mechanical characteristics of conventional luting and lining GICs has not been evaluated to

© 2016 IOP Publishing Ltd

Mater. Res. Express 3 (2016) 075405 F Heravi et al

Figure 1. 3-point flexural strength test.

date. Therefore, the aim of this study was to evaluate the effects of the incorporation of CPP-ACP into a luting
and lining cement on the flexural strength, diametral tensile strength, microhardness and wear rate (WR). The
null hypothesis of this study was that the addition of CPP-ACP would not affect the mechanical behavior of
luting and lining GIC.

Materials and methods

In this study, various weight percentages (1%, 1.56% and 2%) of CPP-ACP powder were incorporated into a
selected commercial luting and lining GIC (GC Corporation, Tokyo, Japan). Then, investigation was done to
determine the flexural strength, diametral tensile strength, Vickers microhardness and WR of the CPP-ACP-
containing GIC and control group (GIC without CPP-ACP).

CPP-ACP synthesis
CPP was dissolved at 10 g l-1 in deionized distilled water (DDW) at room temperature and stirred using a
magnetic stirrer until a homogeneous suspension was prepared. Anhydrous calcium chloride (Merck, Germany)
and disodium hydrogen phosphate (Merck, Germany) were added very slowly to the CPP suspension to reach
final concentrations of 100 and 60 mM, respectively. The pH level of the suspension was held at pH=9 by the
addition of NaOH (Merck, Germany). The solution was centrifuged and washed to extract the product from the
reaction. The precipitant was lyophilized to produce CPP-ACP powder. The amorphous state of the powder was
examined one day and one month after production by x-ray diffraction (XRD) analysis.

Flexural strength
To determine the flexural strength according to ISO9917-2 [10], 25 × 2 × 2 mm bar-shaped specimens were
prepared using a Teflon mold. Thirty samples were made for each weight percentage of CPP-ACP and the
control group. The specimens were stored in distilled water at 37 °C for 24 h. Then, the flexural strength was
measured by a universal testing machine (STM20, SANTAM, Tehran, Iran) with a crosshead speed of 1mm min-
1
and gauge length (distance between the supports ) of 20 mm (figure 1). The force at the fracture point was
recorded and the flexural strength ( F.S) was calculated using the following equation:

3Fl
F. S =
2wh2

where F is the flexural load at fracture, l the distance between the supports (20.0 mm), w the specimen width
(2 mm) and h the specimen height (2 mm).

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Mater. Res. Express 3 (2016) 075405 F Heravi et al

Figure 2. DTS test.

Figure 3. Vickers indent in the CPP-ACP-containing GIC sample.

Diametral tensile strength (DTS)

In this test, according to ADA Standard no.66 [11] a Teflon mold with holes (6 mm diameter and 3mm height)
was fabricated. Then, from this mold, thirty specimens were prepared and stored under the same conditions as
the flexural strength test. Each specimen was compressed diametrically at a crosshead speed of 0.5 mm min−1
until failure detected by a SANTAM device (figure 2). The DTS was calculated by the following formula.
P
DTS =
pdh
where P is the load at fracture, d is the diameter of the samples and h is the height of the samples.

Microhardness
A Teflon mold (6 mm diameter and thickness of 3 mm) was used to prepare disk-shaped samples for each group
(n=30). The mold was filled by the prepared cement, the excess was removed and a Mylar strip and glass slides
were placed on the filled hole. The specimens were kept until set as described above. The hardness of the surfaces
was measured for samples using the Vickers microhardness measuring device (Buhler Microhardness Tester,
Model No.1600-6125, Germany). A 200 gf load was applied through the indenter with a dwell time of 15 s. Three
indentations (figure 3) were made for each sample and the mean was recorded as Vickers hardness.

Wear rate (WR)

Thirty disk-shaped specimens 6 mm in diameter and 3 mm in height were made for each group as described for
the microhardness testing. The WR test was performed by a pin-on-disk tribometer machine that was fabricated
according to ASTM G99-95 standard [12]. Each sample was attached to the pin and the pin was attached to the

3
Mater. Res. Express 3 (2016) 075405 F Heravi et al

Figure 4. Pin-on-disk wear test.

Figure 5. XRD patterns of CPP-ACP during the different times of (a) one day and (b) one month after production.

device holder and the inferior surface of the sample was placed on a disk (figure 4). After 2000 cycles, each sample
was weighed and the mass reduction was calculated. The WR was determined by the following formula.

Dm
WR =
n (pd )

where Dm is the mass loss, d the diameter of wear track (3.5 cm) and n the number of cycles (2000).

Results

Characterization of CPP-ACP
The diffraction patterns of the CPP-ACP samples after different periods are demonstrated in figure 5. As shown,
no sharp peaks were identified in these spectra, confirming the amorphous state of the calcium phosphate phase.

Flexural strength
The ANOVA test demonstrated a significant difference among the groups (P<0.001). The results of the Tukey
test showed that the difference between the groups was statically significant (P<0.001). By increasing the
percentage of CPP-ACP from zero to 1.56%(w/w), the flexural strength improved (29%), and it reduced in 2%
(w/w)(figure 6).

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Mater. Res. Express 3 (2016) 075405 F Heravi et al

Figure 6. Mean flexural strength of the CPP-ACP-containing GIC and control (GIC without CPP-ACP) samples.

Figure 7. Mean DTS of the CPP-ACP-containing GIC and control (GIC without CPP-ACP) samples.

Diametral tensile strength

The results of the ANOVA test revealed a significant difference among the groups (P<0.001) and the results of
the Tukey test also showed a significant difference between the groups (P<0.001). Similar to flexural strength
behavior, increasing CPP-ACP up to 1.56%w/w increased the diametral tensile strength (36% improvement).
However, at a concentration of 2%w/w CPP-ACP, the tensile strength decreased (figure 7).

Microhardness
The significant difference between the groups was demonstrated by the ANOVA test (P<0.001). The results of
the Tukey test showed a significant difference among the groups (P<0.001). Figure 8 demonstrates that the
microhardness improved by 18% when the concentration of CPP-ACP was increased to 1.56%w/w and after
that it decreased (figure 8).

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Mater. Res. Express 3 (2016) 075405 F Heravi et al

Figure 8. Mean Vickers microhardness of the CPP-ACP-containing GIC and control (GIC without CPP-ACP) samples.

Figure 9. Mean WR of the CPP-ACP-containing GIC and control (GIC without CPP-ACP) samples.

Wear rate (WR)

The ANOVA test showed a significant difference among the groups (P<0.001). The Tukey test verified that the
difference between the groups was significant (P<0.001). Figure 9 demonstrates that, by increasing the
percentage of CPP-ACP from 0%–1.56%(w/w), the WR decreased by 23%, then it increased in 2%(w/w) CPP-
ACP (figure 9).

Discussion

In this study, the CPP-ACP powder was synthesized using a wet-chemical method by adding certain amounts of
calcium and phosphate sources to an aqueous solution of CPP powder. The XRD patterns showed no sharp
diffraction peaks and thereby proved the amorphous state of CPP-ACP during different production times,

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Mater. Res. Express 3 (2016) 075405 F Heravi et al

Figure 10. Structure of Glass ionomer.

which confirmed the stabilization of ACP by CPP as described before. Without CPP, the calcium phosphate will
transform within minutes into crystalline hydroxyapatite.
Based on the results of this study, the null hypothesis was rejected. Our data showed that the addition of
CPP-ACP up to 1.56%(w/w) increased the flexural strength, diametral tensile strength, microhardness, and
wear resistance, followed by a reduction in these mechanical properties at 2%(w/w) CPP-ACP. In other words,
the modification of GIC with 1.56%(w/w) of CPP-ACP had maximum mechanical strength. This is inconsistent
with the study of Mazzoaui et al [13] which reported that incorporation of 1.56%(w/w) CPP-ACP improved the
mechanical properties of a restorative GIC. They observed the greater porosity in the fractured surfaces of the
control GIC relative to the CPP-ACP-containing GIC. The CPP-ACP in the GIC may have improved the
mechanical strength by the incorporation of the CPP-ACP into the crosslinked matrix of the GIC [13].
Furthermore, the released calcium ions from the CPP-ACP additive could induce additional crosslinking to the
GIC matrix (figure 10) and strengthen the cement more. In setting the reaction of the GIC, calcium poly salts are
formed first and this is followed by a formation of aluminum poly salts. These poly salts cross link with poly
anion chain [14]. In a higher concentration (2% w/w), the aluminum reaction sites may be occupied by calcium
ions, and consequently prevent the maturation during the setting reaction of the GIC. This can decrease the
mechanical strength of the cement.
Flexural forces create bending stress in materials and dental cement should resist this kind of stress in
different clinical circumstances. The results of this study showed a 29% improvement in the flexural strength of
GIC at 1.56%(w/w) CPP-ACP. Many studies have been done to evaluate the flexural strength of GICs doped
with other materials. However, no studies had been done to examine the effect of CPP-ACP on the flexural
strength of GICs. Sayyedan et al [15] showed that the addition of 1% and 3% forsterite (Mg2SiO4) nanoparticles
to GIC increased the flexural strength of the cement and that the optimum concentration was 1%. Elsaka et al [1]
evaluated the effect of the incorporation of 3%, 5% and 7%(w/w)TiO2 to a conventional glass ionomer. They
found that the addition of 3% TiO2 increased the flexural strength and after that a decrease was observed at a
higher concentration of the additive. Osinaga et al [16] showed that the incorporation of 5%–10%(w/w) zinc
sulfate into a GIC had no significant effect on the flexural strength. Korkmaz et al [17] investigated the flexural
strength of conventional luting glass ionomer after the addition of 5% chlorhexidine diacetate/cetrimide (CHX
+CT). Their research showed that CHX+CT decreased the flexural strength.
Tensile stress is one of the important considerations for mechanical and clinical aspects of dental cements. A
pure tensile test for brittle materials is difficult, because failure occurs at local concentrations (two fixed ends of a
specimen). The fabrication of dumbbell-shaped specimens is one way to overcome this problem, but this
process is difficult for GIC. One alternative, which has been applied in our study, is to determine the DTS. In
agreement with the flexural strength, the results of our research showed that 1.56%(w/w) CPP-ACP had
maximum DTS with 36% improvement, compared to the control group. Previous studies have also shown that
the DTS of GIC increased after the incorporation of 5% hydroxyapatite (HA) and 5% fluoroapatite [3], 1%
forsterite [15], 5% hydroxyapatite/yttria-stabilized zirconia (HA/YSZ) [18] and cellulose nanocrystals (CNC)
[19] into GIC.
The hardness of dental cement may affect its abrasive wear. Similar to the trend of the DTS and flexural
strength results, our Vickers microhardness test showed that 1.56% concentration of CPP-ACP had an 18%
increase in hardness. Yli-Urpo et al [4] observed that mixing concentrations of 10% and 30% of BAG with GIC
caused a reduction in hardness. Prentice et al [20] evaluated the effects of the addition of 1%, 2%, 5%, 10%, 15%

7
Mater. Res. Express 3 (2016) 075405 F Heravi et al

and 25% ytterbium fluoride (YbF3) and barium sulfate (BaSO4) on hardness. Their results showed that at
concentrations of 1% and 2%, hardness increased and after that the reduction of hardness occurred. Contreras
et al [21] found that the incorporation of 3% and 5% TiO2 nanopowder into GIC resulted in increased hardness.
Wear resistance is another important aspect in the mechanical surface property of GIC. There are several
methods for in vitro wear investigation of dental materials. Although many wear simulators are commercially
accessible, these devises may be expensive and are not promptly available at most materials laboratories. Thus,
there are widely used simple alternatives for wear examination such as pin-on-disk, ball-crater, reciprocating,
abrasive disk and taber abraser [22, 23]. In this research, the pin-on-disk method was used. The advantage of this
device was simple and reliable operation for the comparative wear study. In this research, the pin-on-disk
method was used. The results of our study showed that the addition of 1.56%(w/w) CPP-ACP into GIC caused a
23% reduction in the WR. In other words, the maximum wear resistance was achieved at GIC incorporated with
1.56%(w/w) of CPP-ACP, results which show consistency with other mechanical properties. In this study, the
effect of the addition of CPP-ACP on the WR of GIC was investigated for the first time. Few studies have been
done to evaluate the effect of the incorporation of materials into GICs on its WR. McKinney et al [24] showed
that the addition of silver to GIC caused a reduction in the WR.

Conclusions

In this study, CPP-ACP was synthesized and incorporated into a luting and lining GIC. The results proved that
CPP-ACP had an amorphous state during different production times. The mechanical behavior of the modified
cements by different CPP-ACPs was evaluated. The results of this study reject the null hypothesis, since the
addition of CPP-ACP into luting and lining cement affected the mechanical properties and it demonstrated that
the incorporation of CPP-ACP up to 1.56%(w/w) increased the flexural strength, diametral tensile strength,
microhardness, and wear resistance, followed by a reduction in these physical properties at 2%(w/w) CPP-ACP.
This study showed that maximum mechanical strength was achieved by modification of luting and lining GIC
with 1.56%(w/w) CPP-ACP and it is promising that this concentration could be applicable in dental practice.

Acknowledgments

The authors would like to thank the Vice-Chancellor for Research of the Mashhad University of Medical
Sciences for the financial support of this project (grant no. 930266). The results presented in this paper were
taken from a PhD student thesis in the Mashhad University of Medical Sciences.

Conflict of interests

The authors declare that there is no conflict of interest regarding the publication of this paper.

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