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Emdogain Stimulates Matrix Degradation by Osteoblasts
S. Goda, H. Inoue, Y. Kaneshita, Y. Nagano, Y. T. Ikeo, J. Iida and N. Domae
J DENT RES 2008 87: 782
DOI: 10.1177/154405910808700805
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International and American Associations for Dental Research
RESEARCH REPORTS
Biological
S. Goda1*,3, H. Inoue2,3, Y. Kaneshita3,
Y. Nagano3, Y. T. Ikeo1, J. Iida4,
and N. Domae3
Emdogain Stimulates Matrix
Degradation by Osteoblasts
Departments of 1Biochemistry, Orthodontics, 2Physiology,
and 3Internal Medicine, Osaka Dental University, 8-1 Kuzuha
Hanazono-cho, Hirakata-shi, Osaka, 573-1121, Japan; and
4Department of Laboratory Medicine and Pathology, Cancer
Center, University of Minnesota, Minneapolis, USA;
*corresponding author, [email protected]
J Dent Res 87(8):782-787, 2008
INTRODUCTION
ABSTRACT
Emdogain has been used clinically for periodontal
regeneration, although the underlying molecular
mechanisms are not clear at present. In this study,
we hypothesized that Emdogain stimulated
degradation of type I collagen via osteoblasts. We
showed that Emdogain enhanced cell-mediated
degradation of type I collagen in an MMPdependent manner. Although MG-63 cells
spontaneously produced a zymogen form of
MMP-1, treatment with Emdogain significantly
induced the generation of the active form of this
enzyme. We demonstrated that MMP-3 was
produced from MG63 cells in response to
Emdogain in a MEK1/2-dependent manner.
Concomitantly, blocking of MEK1/2 activation by
U0126 significantly inhibited the generation of the
active form of MMP-1 without affecting the total
production of this collagenase. These results
suggest that Emdogain facilitates tissue
regeneration through the activation of the
collagenase, MMP-1, that degrades matrix
proteins in bone tissue microenvironments.
KEY WORDS: Emdogain, matrix metallo proteinases, osteoblasts.
major objective of periodontal therapy is to stimulate regeneration of
A
periodontal ligament (PDL) and alveolar bone. When applied to
periodontal defects, Emdogain induces periodontal regeneration and
improves therapeutic efficacy in concert with periodontal surgery
(Gestrelius et al., 1997; Sculean et al., 2001; Cochran et al., 2003).
Although previous studies showed that Emdogain promotes regeneration of
PDL, root cementum, and alveolar bone in vivo (Hammarström et al., 1997;
Heijl et al., 1997; Boyan et al., 2000), the mechanisms of its action still
remain unclear.
Osteoblasts play a key role in remodeling bone by regulating matrix
turnover, such as type I collagen. Matrix metalloproteinases (MMPs) are
secreted by mesenchymal stromal lining cells and osteoblasts to generate the
initiation sites for osteoclastic bone resorption at the beginning of the
remodeling cycle (Sasaki et al., 2007). MMPs are endopeptidases that play a
primary role in the degradation of extracellular matrix (ECM) proteins (Vu
and Werb, 2000). Collagenases, including MMP-1, -8, -13, and -14,
hydrolyze native collagens to generate ¼ and ¾ fragments, which are
substrates for gelatinases and stromelysins (Visse and Nagase, 2003). Thus,
it is hypothesized that the catalytic activity of collagenases initiates matrix
turnovers in remodeling bone. Because Emdogain activated osteoblast cells
to enhance mRNA expression of type I collagen, osteopontin, bone
sialoprotein, and osteocalcin, it has potential therapeutic value as a material
for the enhancement of bone remodeling (Yoneda et al., 2003).
Previous studies have shown that Emdogain regulates the production of
MMP-1 and MMP-8 in gingival crevicular fluid (Okuda et al., 2001),
although the functions of these collagenases are not clear. The purpose of
this study was to characterize Emdogain for its ability to enhance matrix
turnover and the production of MMPs, with the osteoblast cell line, MG-63.
MATERIALS & METHODS
Cell Culture
MG-63 cells were purchased from the American Type Culture Collection
(Manassas, VA, USA) and maintained in DMEM supplemented with 10% heartinactivated FBS, 2 mM glutamine, and 100 units/mL of penicillin/streptomycin.
Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air.
Reagents and Antibodies
Received July 10, 2007; Last revision March 23, 2008;
Accepted May 19, 2008
782
Anti-phospho-p44/42 (Thr202/Tyr204) and anti-p44/42 antibodies were
purchased from Cell Signaling Technology (Danvers, MA, USA). Emdogain
was obtained from Seikagaku-kogyo Corporation (Tokyo, Japan). U0126,
MMP-1, and MMP-3 antibodies were purchased from Calbiochem (Darmstadt,
Germany). DQ-collagen I was purchased from Molecular Probes (Eugene, OR,
USA).
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International and American Associations for Dental Research
J Dent Res 87(8) 2008
MMPs Produced by Emdogain in Osteoblasts
783
DQ-Collagen I
Degradation Assay
Coverslips were coated with 25
␮ g/mL of quenched fluorescence
substrate DQ-collagen I. MG-63 cells
(5 x 10 4) were incubated with 100
␮ g/mL Emdogain for 20 hrs,
followed by incubation on DQcollagen I-coated plates for an
additional 4 hrs. Cells were fixed
with 2% paraformaldehyde and
examined with 488-nm (excitation)
and 533-nm (emission) fluorescence
by confocal microscopy (Olympus
LSM-GB200 confocal microscope,
Olympus, Tokyo, Japan) with an oil
immersion lens. Degradation of DQcollagen I was visualized in an
optical section as a green fluorescent
signal. Differential interference
contrast (DIC) was shown to
visualize cells cultured on the matrix.
Western Blot Analysis
MG-63 (1 x 106) cells were incubated
in serum-free medium with 50, 100,
and 200 ␮g/mL of Emdogain for 24
hrs. The conditioned media were
Figure 1. Emdogain enhanced MG-63 cells’ degradation of type I collagen. MG-63 cells were incubated in
the presence (d, e, f) or absence (a, b, c) of 100 ␮g/mL Emdogain for 20 hrs and then incubated on glass
concentrated in Amicon Centriprep
coated with 25 ␮g/mL of quenched fluorescence substrate DQ-collagen I for an additional 4 hrs. TIMP-2
concentrators (MW cut-off, 10 kDa)
(20 ␮M) was incubated with Emdogain and MG-63 cells as described above (g, h, i). Degradation of type
(Millipore Corporation, Bedford, MA,
I collagen (green fluorescence) was detected by confocal microscopy (samples: excitation, 488 nm;
USA) up to 10-fold to visualize
emission, 530 nm). Pictures were taken at 40x magnification (a-i). Differential interference contrast (DIC)
images are shown. These data are representative of more than 3 independent experiments.
proteins in Western blotting analysis.
For studying phosphorylation of
p44/42, we incubated MG-63 (1 x 106)
medium prepared from HT1080 cells stimulated with ConA was
cells in serum-free medium with 50, 100, or 200 ␮ g/mL of
used to localize the pro-, intermediate, and active forms of MMP-2.
Emdogain for the indicated periods described in the text. Samples
were separated on 8% or 10% SDS polyacrylamide gels (SDSPAGE) under reducing conditions. Proteins were electrophoretically
RESULTS
transferred to Immobilon-P membranes and were incubated for 1 hr
Emdogain Stimulated the Degradation
with primary antibodies in PBS containing 0.05% Tween-20 and
10% Blockace (Dainippon Pharm. Co., Tokyo, Japan). Peroxidaseof Type I Collagen by MG-63
conjugated secondary antibody (Amersham Biosciences,
Matrix metalloproteinases (MMPs) and tissue inhibitors of
Piscataway, NJ, USA) was used at a 1:1000 dilution, and
metalloproteinases (TIMPs) produced by osteoblasts play an
immunoreactive bands were visualized by means of Super Signal
essential role in bone remodeling and regeneration (Krane,
west pico chemiluminescent substrate (PIERCE Biotechnology Inc.,
1995; Opdenakker et al., 2001). Previous clinical applications of
Rockford, IL, USA). Signals on each membrane were analyzed with
Emdogain showed enhanced tissue regeneration, including bone
VersaDoc 5000 (BIO-RAD, Hercules, CA, USA).
formation (Heijl, 1997), suggesting that osteoblasts function to
Gelatin Zymography
The conditioning medium prepared as described above was
subjected to gelatin zymography to visualize MMP-2 and MMP-9
as described previously (Goda et al., 2006). MG-63 (1 x 106) cells
were incubated in serum-free DMEM with 50, 100, or 200 ␮g/mL
of Emdogain for 24 hrs. The conditioning medium was resolved
under non-reducing conditions on 10% SDS-PAGE impregnated
with 1 mg/mL gelatin. Gels were rinsed once in 2.5% Triton X-100
for 30 min at room temperature and then incubated in a developing
solution [50 mM Tris-HCl (pH 7.6), 5 mM CaCl2, 1 ␮M ZnCl2]
for 8-16 hrs. Gels were stained with Coomassie blue and de-stained
according to a standard protocol. Areas of gelatinolytic activity
were detected as transparent bands. As a standard, a conditioning
facilitate the process. We therefore utilized human osteoblasts,
such as the MG-63 cell line, as a model system to evaluate
Emdogain in the process of matrix turnover. We previously
demonstrated that a quenched fluorescent derivative of type I
collagen (DQ-collagen I) served as a substrate for MMPs for
studying proteolysis by living cells (Goda et al., 2006). In this
study, we used this assay system to examine the ability of MG63 cells to degrade type I collagen. Degradation of DQ-collagen
I was visualized in an optical section as green fluorescent
signals. Although unstimulated MG-63 cells resulted in minimal
signals of the degradation of type I collagen (Fig.1a, 1c),
Emdogain (100 ␮g/mL) enhanced the degradation of type I
collagen visualized by the green fluorescence signals (Fig. 1d,
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International and American Associations for Dental Research
784
Goda et al.
J Dent Res 87(8) 2008
Figure 2. Expression of MMPs from Emdogain-stimulated MG-63 cells.
MG-63 cells (1 x 10 6 cells) were incubated in serum-free media
containing 50, 100, and 200 ␮g/mL of Emdogain for 24 hrs and in
the conditioned media as described in MATERIALS & METHODS. (A)
The concentrated media were separated on 8% SDS-PAGE, blotted with
anti-MMP-1 antibody, and visualized with a Super Signal west pico
chemiluminescent substrate. Molecular markers (kDa) are shown in the
left column. Quantification of MMP-1 (upper panel) was performed
densitometrically with NIH image software. The intensities of each band
are depicted as percent of maximum value (lower panel). (B) The same
conditioned media were subjected to gelatin-zymography as described
in MATERIALS & METHODS. As a standard, conditioned medium
prepared from HT1080 cells stimulated with ConA was used to localize
inactive (upper) and active (lower) forms of MMP-2 and are shown as
lines. These data are representative of more than 3 independent
experiments.
MMP-8, MMP-13, and MMP-14) from MG-63 cells stimulated
by Emdogain. MG-63 cells spontaneously produced MMP-1 in
1f). Given the fact that type I collagen is a substrate for MMPs,
the serum-free conditioned medium with 57 kDa (major) and
we tested whether the tissue inhibitor of metalloproteinases
47 kDa (minor) molecular masses, which correspond to latent
(TIMP-2) would inhibit the proteolysis of type I collagen.
and active forms of this enzyme, respectively. Quantification of
TIMP-2 almost completely abrogated the proteolysis by the
MMP-1 was performed densitometrically with NIH image
cells (Fig. 1g, 1i), indicating that MMPs plays a key role in the
software (Fig. 2A, upper). The intensities of each band are
proteolysis of type I collagen.
depicted as percent of maximum value. Importantly, when cells
Emdogain Enhanced the Generation
were cultured in the presence of Emdogain, the generation of
of an Active Form of MMP-1 in MG-63 Cells
the active form of MMP-1 was observed to occur in a dosedependent manner (Fig. 2A). We separated the same samples
We then evaluated the production of collagenases (MMP-1,
(as in Fig. 2A) via gelatin
zymography to test whether this
enzyme was activated, since
previous studies have shown that
MMP-2 could act as a collagenase
to some extent (Nagase and
Woessner, 1999). While un stimulated MG-63 cells spon taneously produced a latent form of
MMP-2, neither the total amount of
production nor the activation status
of this enzyme was altered by
stimulation with Emdogain (Fig.
2B), suggesting that the catalytic
activity of MMP-2 is not involved
in Emdogain-stimulated type I
collagen degradation. Thus, the
zymography shown in Fig. 2B
served as a loading control for the
Western blot illustrated in Fig. 2A.
MMP-14 is a membrane-associated
collagen ase that promotes
degradation of type I collagen and
activates pro-MMP-2 (Seiki,
Figure 3. Emdogain induced activation of ERK in MG-63 cells. (A) MG-63 cells were stimulated with 100
2003). However, MMP-14 was not
␮g/mL EMD for the indicated times at 37°C. Cells were harvested, and lysates were resolved in 10% SDSactivated in cell lysates in MG63
PAGE and then transferred to a PVDF membrane. The membrane was immunoblotted with anti-phosphocells cultured in the presence of
p44/42 antibody (upper panel) and then stripped and immunoblotted with anti-p44/42 antibody (middle
Emdogain at any concentration
panel). Molecular markers (kDa) are shown in the left column. (B) MG-63 cells were treated for 30 min with
(not shown), consistent with the
U0126 (5 and 10 ␮M) before stimulation with 100 ␮g/mL Emdogain for 5 min. Cells were harvested, and
lysates were resolved in 10% SDS-PAGE and then transferred to a PVDF membrane. The membrane was
lack of activation of pro-MMP-2
immunoblotted with anti-phospho-p44/42 antibody (upper panel), and then stripped and immunoblotted
(Fig. 2B). Emdogain did not affect
with anti-p44/42 antibody (middle panel). Molecular markers (kDa) are shown in the left column.
the production of MMP-8 or
Quantification of phosphorylation of p44/42 was performed densitometrically and corrected to the amount
MMP-13 (not shown). Given the
of total p44/42 protein. The intensities of each band are depicted as percent of maximum value (lower
panel). These data are representative of more than 3 independent experiments.
fact that MMP-1 is a potent
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International and American Associations for Dental Research
J Dent Res 87(8) 2008
MMPs Produced by Emdogain in Osteoblasts
785
collagenase (Ohuchi et al., 1997),
analysis of these data suggests that
the active MMP-1 plays a key role
in the proteolysis of type I collagen
by Emdogain-stimulated MG-63
cells.
Activation of MEK-ERK was
Involved in the Degradation of
Type I Collagen by Emdogainstimulated MG-63 Cells
Previous studies showed that
Emdogain stimulates MAP kinases
in monocytic and periodontal
ligament cells, inducing the
formation of osteoclasts and a
mitogenic response, respectively
(Matsuda et al., 2002; Itoh et al.,
2006). When MG-63 cells were
cultured in the presence of 100
␮g/mL Emdogain, ERK1/2 was
phosphorylated in a time-dependent
manner, with the maximum
phosphorylation at 5 min (Fig. 3A,
upper and lower panels). The total
amount of ERK protein was not
affected under the experimental
conditions (Fig. 3A, middle panel).
A synthetic specific inhibitor for
ERK1/2, U0126, abolished the
activation of this kinase in both
unstimulated and Emdogainstimulated MG-63 cells (Fig. 3B).
Previous studies have shown
that the activation of ERK1/2 plays
a key role in the induction of MMPs,
including MMP-1 (Goda et al.,
2006). We therefore tested whether
this signaling pathway was involved
in the proteolysis of type I collagen
by Emdogain-stimulated MG-63
cells. Consistent with our results
(Fig. 1), Emdogain enhanced
proteolysis of type I collagen by
Figure 4. MEK-ERK pathways are important for Emdogain-stimulated MG-63 cell-mediated degradation
MG-63 cells compared with
of type I collagen. (A) MG-63 cells were pre-treated for 30 min with U0126 (10 ␮M) and then incubated
unstimulated cells (Fig. 4A, d and f
in the presence or absence of 100 ␮g/mL Emdogain for 20 hrs. Cells were cultured on glass coated with
vs. a and c). Importantly, U0126
25 ␮g/mL of the quenched fluorescence substrate, DQ-collagen I. Degradation of type I collagen (green
completely inhibited the proteolysis
fluorescence) was detected by confocal microscopy (fluorescence: excitation, 488 nm; emission, 530 nm).
mediated by Emdogain-stimulated
Pictures were taken at 40x magnification (a-f). (B) The concentrated conditioned media prepared from
unstimulated MG-63 cells (lane 1), U0126 (10 ␮M) (lane 2), Emdogain-stimulated MG-63 cells (lane 3),
MG-63 cells (Fig. 4A, g and i).
and Emdogain-stimulated MG-63 cells cultured in the presence of U0126 (10 ␮M) (lane 4) were
These results suggest that the
separated on 8% SDS-PAGE. The membranes were blotted with anti-MMP-1 antibody and visualized with
activation of the ERK1/2 pathway as
a Super Signal west pico chemiluminescent substrate. Molecular-weight markers (kDa) are shown in the
stimulated by Emdogain is involved
left column. The same concentrated conditioned media were separated and transferred onto a membrane.
(C) The membranes were blotted with anti-MMP-3 antibody and visualized with a Super Signal west pico
in the production and/or activation
chemiluminescent substrate. Molecular-weight markers (kDa) are shown in the left column. Quantification
of MMP-1.
of MMP-1 (B) or MMP-3 (C) (upper panel) was performed densitometrically with NIH image software. The
U0126 significantly inhibited
peak heights of each density are depicted as percent of maximum value (lower panel). These data are
the activation of pro-MMP-1 in
representative of more than 3 independent experiments.
Emdogain-stimulated MG-63 cells,
although the total amount of the
collagenases was not altered (Fig.
4B). As a part of the study of the activation of pro-MMP-1,
through cleaving the prodomain of pro-MMP-1 between Gln80
stromelysin-1 (MMP-3) has been reported as a direct activator
and Phe 81 (Nagase et al., 1992). Although MG-63 cells
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International and American Associations for Dental Research
786
Goda et al.
spontaneously produced MMP-3, Emdogain significantly
enhanced MMP-3 production (Fig. 4C). Importantly, the
enhanced production of MMP-3 reverted to the basal level in
the presence of U0126 (Fig. 4C), suggesting that the activation
of ERK1/2 is involved in the production of MMP-3 from
Emdogain-stimulated MG-63 cells.
Taken together, these results suggest that Emdogain
facilitates the proteolysis of type I collagen by MG-63 cells
through the induction of MMP-3, which acts as an activator for
pro-MMP-1.
DISCUSSION
Previous studies demonstrated the indispensable role of cellmediated “collagenolysis” in bone formation and growth of the
skeleton (Holmbeck et al., 1999; Zhou et al., 2000). The
involvement of matrix remodeling in bone has been studied by
the elucidation of specific functional roles of MMPs, such as
MMP-9 and MMP-14 (Vu et al., 1998; Holmbeck et al., 1999;
Zhou et al., 2000). In this study, we demonstrated that
Emdogain stimulated osteoblasts to degrade type I collagen in
vitro through the catalytic activity of MMPs. Our results
suggest that the activation of MMP-1 is one of the mechanisms
for facilitating the degradation of type I collagen by Emdogainstimulated MG-63 cells, which plays a key role in the processes
of bone regeneration.
We demonstrated that MMP-1 was spontaneously produced
from unstimulated MG-63 and was activated in the presence of
Emdogain. Both degradation of type I collagen and the
activation of pro-MMP-1 were positively correlated with the
production of MMP-3 from Emdogain-stimulated MG-63 cells.
Previous studies demonstrated that MMP-3 results in direct
proteolysis of the Glu80-Phe81 bond in the prodomain of
MMP-1, thus activating it (Nagase et al., 1992). Furthermore,
MMP-1-mediated tumor invasion and matrix degradation
required MMP-3 as an activator (Benbow et al., 1996). Thus,
these results suggest that MMP-3 produced from Emdogainstimulated MG-63 cells would act as an activator for proMMP-1. Previous studies showed that Emdogain decreased the
production of MMP-1 in gingival crevicular fluid (Okuda et al.,
2001). Although the in vivo function of Emdogain needs to be
evaluated, it is possible that Emdogain may facilitate tissue and
bone regeneration by regulating MMP-1 expression from
distinct cell populations (i.e., neutrophils for tissue regeneration
and osteoblasts for bone remodeling). Previous studies showed
that Emdogain contains TGF-␤ and BMP-like activity (Suzuki
et al., 2005). Furthermore, BMP-2 enhanced regeneration of
bone and the production of MMPs (Wang et al., 1990;
Takiguchi et al., 1998; Fujisaki et al., 2006). Although the
details of the mechanisms of Emdogain for stimulating MMP-3
production from osteoblasts require further study, these results
implicate soluble factors such as BMP-2 and TGF-␤ in
Emdogain as potential activators for osteoblasts.
We have shown that the activation of ERK1/2 plays a key
role in the induction of MMP-3 from Emdogain-stimulated MG63 cells. Previous studies showed that activation of ERK1/2
plays a key role in the production of MMP-3 with various cell
types and extracellular stimuli (Tanimura et al., 2003; Hoberg et
al., 2007; Kajanne et al., 2007). Despite the fact that U0126
completely inhibited basal activation of ERK1/2 in MG-63
cells, the production of MMP-3 was approximately comparable
with that in the non-treated control cells. These results suggest
J Dent Res 87(8) 2008
that spontaneous production of MMP-3 from MG-63 cells was
independent of the activation of ERK1/2. Similarly, the
production of MMP-1 from Emdogain-stimulated MG-63 cells
was independent of the activation of ERK1/2. Furthermore,
these results suggest that the production of MMPs is regulated
via multiple signaling pathways stimulated by Emdogain in
MG-63 cells. Although the characterization of the mechanisms
of the production of MMP-3 from Emdogain-stimulated MG-63
cells is not clear at present, our results suggest that the induction
of MMP-3 is one of the key steps for facilitating matrix turnover
in bone environments.
In summary, our results suggest a model in which MMP-3
plays a role in bone regeneration by degrading matrix proteins
and/or by activating a potent collagenase, MMP-1 in
osteoblasts. In addition to ECM proteins, recent studies suggest
that MMPs degrade various “non-classic” substrates in vivo
(McCawley and Matrisian, 2001). For example, inflammatory
chemokines such as monocyte chemoattractant proteins (MCPs)
are a substrate for MMP-1 and MMP-3 and are inactivated by
the cleavage at specific sites within the molecules (McQuibban
et al., 2002). Thus, further studies will be needed to elucidate
fully the mechanisms of Emdogain-stimulated tissue and bone
formation by determining the substrates for MMPs, including
MMP-1 and MMP-3, in tissues.
ACKNOWLEDGMENTS
This work was supported by the High-Tech Research Center
Project for Private University, a matching fund subsidy from
MEXT, 2002-2006, and an Oral Implant Research Grant,
Osaka Dental University (07-02).
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International and American Associations for Dental Research
ERRATA
The name of one of the authors of the article entitled “Emdogain Stimulates Matrix Degradation by Osteoblasts”,
published in J Dent Res 87:782-787, 2008, was accidentally
misspelled. Y.T. Ikeo should be T. Ikeo. The authors regret
this error.
In the article by P. Radhakrishnan and J.J. Mao (“Nanomechanical
Properties of Facial Sutures and Sutural Mineralization Front”, J
Dent Res 83:470-475, 2004), the average elastic modulus of each sample was calculated from individual force volume images according
to the Hertz model as shown in the following equation:
3F (1 - n 2)
E = __________
4 = Rd3/2
Due to an error by a vendor managed by HighWire Press,
the JDR’s online hosting service, Dr. Sessle’s name was misspelled in the online publication, J Dent Res 87:797, 2008. The
error was corrected online on September 2, 2008. HighWire
Press regrets the error.
where E is the Young’s modulus, F is the applied nanomechanical
load, n is the Poisson’s ratio, R is the radius of curvature of the AFM
tip , and d is the amount of nano-indentation.
There was a typographical error in this equation, which should read:
3F (1 - n )
E = __________
4 = Rd3/2
The authors regret this error. 984
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International and American Associations for Dental Research