Journal of Dental Research

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In vivo Dentin Remineralization by Calcium-phosphate Cement

M.C. Peters, E. Bresciani, T.J.E. Barata, T.C. Fagundes, R.L. Navarro, M.F.L. Navarro and S.H. Dickens
J DENT RES 2010 89: 286 originally published online 5 February 2010
DOI: 10.1177/0022034509360155

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 RESEARCH REPORTS
Clinical

M.C. Peters1*, E. Bresciani2,

T.J.E. Barata3, T.C. Fagundes4,
In vivo Dentin Remineralization
R.L. Navarro5, M.F.L. Navarro4,
and S.H. Dickens6
by Calcium-phosphate Cement
1
University of Michigan, School of Dentistry, Department of
Cariology, Restorative Sciences and Endodontics, Room 2345,
1100 N. University, Ann Arbor, MI 48109, USA; 2University of
Michigan, School of Dentistry, Department of Orthodontics
and Pediatric Dentistry; 3University of North Parana, Londrina-
Brazil, Department of Operative Dentistry; 4University of São
Paulo-Brazil, Bauru School of Dentistry, Department of Dental
Materials, Endodontics and Operative Dentistry; 5University of
São Paulo-Brazil, Bauru School of Dentistry, Department of
Oral Maxillo-Facial Surgery; and 6American Dental Association
Foundation, Paffenbarger Research Center, National Institute
of Standards and Technology; *corresponding author, mcpete@
umich.edu

J Dent Res 89(3):286-291, 2010

Abstract
Minimally invasive caries-removal procedures
remove only caries-infected dentin and preserve
caries-affected dentin that becomes remineralized.
Dental cements containing calcium phosphate pro-
mote remineralization. This study evaluated the
in vivo remineralization capacity of resin-based
calcium-phosphate cement (Ca-P) used for indirect
pulp-capping. Carious and sound teeth indicated for
extraction were randomly restored with the Ca-P base
or without base (control), followed by adhesive res-
toration. Study teeth were extracted after three
months, followed by elemental analysis of the cavity
floor. Mineral content of affected or sound dentin at
the cavity floor was quantified by electron probe
micro-analysis to 100-µm depth. After three months,
caries-affected dentin underneath the Ca-P base
showed significantly increased calcium and phos-
phorus content to a depth of 30 µm. Mineral content
of treated caries-affected dentin was in the range of
healthy dentin, revealing the capacity of Ca-P base to
promote remineralization of caries-affected dentin.
KEY WORDS: caries, remineralization, Ca-P
cement, bioactive cement, RCT.
DOI: 10.1177/0022034509360155
Received December 23, 2008; Last revision June 25, 2009;
Accepted July 7, 2009
Introduction
M inimally invasive treatment concepts use adhesive materials to
stabilize lesions by halting the bacterial caries process and providing
caries-affected tissue an opportunity to heal (Peters and McLean, 2001a,b).
Contemporary tissue-saving treatments, such as ultraconservative car-
ies removal (Mertz-Fairhurst et al., 1998; Ribeiro et al., 1999; Maltz et al.,
2002), atraumatic restorative techniques (Frencken et al., 1994), and indirect
pulp-capping procedures (Bjørndal et al., 1997; Bjørndal and Larsen, 2000),
assume that caries can be halted and affected tissue can be remineralized.
Healing of remaining affected dentin may be encouraged by the use of bioac-
tive, ion-releasing base materials, e.g., glass-ionomer or Ca-P cements (Mukai
et al., 1998; Dickens et al., 2003, 2004; Exterkate et al., 2005). Recent in vitro
studies in an artificial caries model showed that calcium phosphate from a
resin-based calcium-phosphate cement (RCPC) was able to remineralize dis-
eased tooth tissue, showing the potential of promoting dentin repair (Dickens
et al., 2003, 2004; Dickens and Flaim, 2008). During remineralization experi-
ments, hydroxyapatite and/or other calcium phosphates precipitated in the
lesions, resulting in significantly increased mineral content (Dickens et al.,
2003; Dickens and Flaim, 2008). These in vitro experiments suggest that
where complete removal of carious tissue is contra-indicated, these cements
may induce remineralization of mineral-deficient dentin. The mineral phase
in RCPC is based on the resin-free calcium-phosphate cement, developed as
a bone-regenerating material (Chow et al., 2000). The incorporation of cal-
cium phosphates into dual-curing resins enhanced clinical handling and per-
formance, yielding command cure and increased strength and dentin adhesion
(Dickens et al., 2004).
This proof-of-principle study investigated the potential clinical benefit of
RCPC. Following standardized caries removal procedures, the bioactivity of
RCPC-base material on caries-affected and sound dentin was evaluated in
adhesively restored teeth after 3 mos of in vivo service. We tested the hypoth-
esis that placement of RCPC on caries-affected dentin would increase calcium
and phosphorus content of mineral-depleted dentin over time. In vitro elemen-
tal analysis of the cavity floor by electron probe micro-analysis (EPMA)
techniques complemented evaluation of clinical hardness at re-entry.

286
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J Dent Res 89(3) 2010 In vivo Dentin Remineralization   287

Materials & Methods

TOTAL
A prospective randomized controlled
clinical trial investigated clinical effi- 27 teeth
cacy of RCPC in permanent human
teeth. The study protocol was approved
by governing Institutional Review
Boards (UM #H03-00001883, FOB-
C S
Carious group Sound group
USP #126/2003, and CONEP-Brasil
(N = 14) (N = 13)
#9846). The clinical phase (August-
December, 2005) included 44 adult
patients of the Bauru School of
Dentistry, who signed informed con- CE and CU CN SE and SU SN
sent, presenting 87 periodontally Treated group Non-treated group Treated group Non-treated group
involved carious and healthy teeth, indi- (N = 10) (N = 4) (N = 10) (N = 3)
cated for extraction. Teeth were pre-
pared and randomly restored with/
without RCPC-base. Prior to applica-
tion of RCPC-base, the cavity floor was CE SE
Treated and etched Treated and etched
etched or left unetched (Fig. 1), fol- (N = 5) (N = 5)
lowed by an adhesive composite
resin restoration according to common
clinical protocol (etch-bond-restore).
Randomization according to random CU SU
number table was concealed in num- Treated (un-etched) Treated (un-etched)
bered envelopes. Patients were blinded (N = 4, 1 lost) (N = 4, 1 lost)
to treatment delivered, while clinicians
were not. Clinical, physical, radio- Figure 1. Sample distribution.
graphic, and microbial characteristics of
cavity walls were evaluated at baseline
and 3 mos later at re-entry after extraction. All patients presented equal subgroups (N = 5): CE, SE with pre-treatment of dentin
after 3 mos for extraction. Extracted teeth were prepared for exten- (Total Etch®, Ivoclar Vivadent AG, Schaan, Liechtenstein) and CU,
sive in vitro physical and microbiological testing of dentinal walls. SU without pre-treatment. Application and light-curing of RCPC
This report focuses on the mineral content of caries-affected, demin- were followed by the application of a thin film of vaseline to
eralized dentin at the cavity floor of 27 randomly selected carious facilitate restoration removal at re-entry. Subsequently, remaining
and sound teeth. cavity walls were etched and bonded (Excite®, Ivoclar Vivadent
AG, Schaan, Liechtenstein), and a microfilled composite resin
Standardization of Caries-affected Dentin (Epic®-TMPT Restorative, Parkell Inc., Edgewood, NY, USA)
restoration was placed. Restoration margins were re-etched and
After the cavity was opened and circumferential caries removed, re-bonded (same agents) to achieve optimal seal.
leaving a clean hard dentino-enamel junction, central carious dentin
was removed with a polymer bur (SmartPrep-System®, SSWhite
Specimen Preparation
Burs, Inc., Lakewood, NJ, USA). This self-limiting instrument
produced a standardized affected dentin surface of about 25 KHN Three mos later, restored teeth were extracted and restorations
Knoop hardness (Peters, unpublished observations). Cavity walls in removed, followed by careful partial removal of base material,
sound teeth were instrumented with the polymer bur according to exposing half of the cavity floor. Dentin at the cavity floor was
the same protocol. After standardized caries removal, hardness of clinically assessed for its hardness. All 27 teeth were fixed,
the cavity floor was clinically assessed by means of an explorer embedded, and longitudinally sectioned through the cavity (~ 3
(Kidd et al., 1993). sections/tooth, 200 µm thick) in preparation for elemental analy-
sis. Two teeth (1 per subgroup, CU and SU) provided only 1
Clinical Treatment Protocol section and were excluded from further analysis.

Teeth were divided into 2 main groups: C = carious (N = 14) and S

= sound (N = 13) (Fig. 1). Seven non-restored teeth, serving as
control [caries-affected CN (N = 4) and sound SN (N = 3)], were
extracted immediately after cavity preparation and standardized
caries removal. Components of RCPC, a two-paste system, and
their function are shown in Table 1. Prior to the application of
RCPC-base, both C and S teeth (N = 10) were randomized into 2
Quantitative Elemental Analysis (EPMA-WDS)
Using electron probe micro-analysis (EPMA) based on wavelength-
dispersive spectroscopy (WDS), we determined elemental composi-
tion of the cavity floor (Ngo et al., 1997). Quantitative elemental
analysis was carried out by EPMA spot analysis (Cameca SX-100
Microprobe Analyzer, Cameca Science & Metrology Solutions,

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288  Peters et al. J Dent Res 89(3) 2010

Table 1. Composition of the Resin-Based Calcium-phosphate Cement

(RCPC)
Composition and Function of Components (mass fraction % of each
component related to final composition)
Pyromellitic glycerol dimethacrylate PMGDM 15.6 Paste 1
Water 4.4
Dicalcium phosphate anhydrous DCPA 29.1
Sodium hexafluorosilicate Na2F6SiO4 0.1
Benzoyl peroxide BPO 0.8
BHT (stabilizer) BHT 0.0 Paste 2
Ethoxylated bisphenol A dimethacrylate EBPADMA 13.1
Calcium-enriched tetracalcium phosphate TTCP 36.8
N,N-Dimethylaminophenethanol DMAPE 0.2
Camphorquinone CQ 0.0

Gennevilliers, France) to measure weight-% of calcium (Ca) and

phosphorus (P) in each pre-determined point. Ca and P levels in each
tooth section (N = 75) were measured along 3 lines, starting at the
cavity floor toward the pulp (0-100 µm depth, 11 datapoints, 10 µm
apart). As internal control, a fourth line was measured distant from
the lesion in sound dentin. In total, 2409 datapoints were obtained,
1287 in caries-affected and 1122 in sound dentin, respectively.

Statistical Analysis
Data were analyzed by ‘random coefficient models’, accounting for
the inherent variability of dentin (inter- and intra-tooth biologic
variation, lesion depth). We used a linear mixed model to fit the
amount of both calcium and phosphorus at each depth for each sec-
tion within a tooth. The slope was measured (i.e., change in phos-
phorus or calcium content with depth). A sample size of 12 sections
for CN and 36 sections for SE_SU_SN allowed for detection of a
0.006 slope difference with 90% power, at an alpha level of 0.05
(Diggle et al., 2002). This assumes that residual variance around the
regression line is 0.616 (based on actual results for phosphorus),
and 10 measurements were made for each section (at depths of 0,
10, 20, . . ., and 100 µm). Correlation between values was very high
for calcium, resulting in even more power for calcium.
Effects of treatment on EPMA data (means) were estimated
separately for sound and carious teeth by Random Coefficient
Models (Verbeke and Molenberghs, 2000). Models included
fixed effects of treatment, depth, and treatment-by-depth inter-
actions. Effect of depth was modeled by both a linear and a
quadratic trend to allow for a curved relationship as depth
increased. Linear and quadratic effects of depth were allowed to
differ by treatment. The model for EPMA data included random
intercepts and slopes for each tooth, allowing characteristic Ca-
and P-levels and fitted curves to vary randomly between teeth.
Although this proof-of-principle study was essentially explor-
atory, we used Bonferroni correction for multiple comparisons
within each depth. All statistical analyses were carried out with
the Proc Mixed procedure in SAS (SAS Institute, 2004).
Results
Clinical Evaluation (Baseline and 3-month Recall)
No adverse events related to RCPC material were reported. Clinical
characterization of affected (C) dentin at the cavity floor pre- and
post-caries removal (CR) showed the distribution of ‘soft/medium/
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Figure 2. Mineral content of the cavity floor (p-values in Table 2).
Connection of datapoints per group by a line provides the regression
lines for each group from cavity floor up to 100 µm into dentin towards
the pulp. (A) Calcium content (wt%) in caries-affected dentin from the
cavity floor up to 100 µm into dentin; CN (N=12 sections with N=36
datapoints at each depth): non-treated carious dentin ranged from 24
wt% (interface) up to 27 wt% (30 µm), and remained at 28-29 wt%
up to 100 µm; CE_CU (pooled; N=27 sections with N=81 datapoints
at each depth): treated carious dentin ranged from 29 wt% (0-40 µm)
to 30 wt% (up to 100 µm); SE_SU_SN (pooled; N=34 sections with
N=102 datapoints at each depth): sound specimens, whether treated
or not, showed consistent Ca-levels of about 31 wt% (range 30.1 –
31.8 wt%) at each depth (0-100 µm). Compared to CN, Ca-levels for
CE_CU were about 20 % higher at the interface, 11 % higher at 10 µm
and 5 % higher at 20 µm depth. (B) Phosphorus content (wt%) in caries-
affected dentin from the cavity floor up to 100 µm into dentin; sample
size per group is the same as above for calcium content determination.
CN: non-treated carious dentin ranged from 11.43 wt% (interface) up
to 12.72 wt% (30 µm), and remained at 13 wt% up to 100 µm; CE_CU
(pooled): treated carious dentin ranged from 13.09 wt% (interface)
increasing to 13.82 wt% in deeper areas; SE_SU_SN (pooled): sound
specimens, whether treated or not, showed very similar P-levels rang-
ing from 13.67 wt% to 14.39 wt%.
hard’ tissue to be 69/31/0 % (Pre-CR), 8/92/0 % (Post-CR), and
0/0/100 %, respectively, at re-entry after 3 mos (re-entry data only
for treated caries groups CE and CU). Sound dentin was ‘hard’ at
both timepoints: baseline and re-entry.
Quantification of Calcium Levels
Predicted Ca levels (wt%) are shown at intercept (interface, 0 µm
depth) and different depths for affected (CN), affected treated/based
(CE_CU, pooled), and sound (SE_SU_SN, pooled) dentin (Fig.
2A). The regression line for CN showed a pronounced quadratic
effect (p = -0.001), demonstrating increasing Ca levels from
the mineral-depleted cavity floor into deeper, less demineralized,

© 2010 International & American Associations for Dental Research

J Dent Res 89(3) 2010 In vivo Dentin Remineralization   289

Table 2. Mineral Content: p-values* for Comparisons of Groups

Calcium Content Phosphorus Content
Comparison  p-values p-values
Depth 0 µm 10 µm 20 µm 30 µm 0 µm 10 µm 20 µm 30 µm
Caries-affected CE vs. CN 0.0275 0.0622 0.9653 0.0091a 0.0240 0.0560 0.1137
  CU vs. CN 0.0126a 0.0465 0.1276 0.0114a 0.0429 0.1292 0.3007
  CE vs. CU 0.6200 0.7945 0.1353 0.9924 0.8357 0.6899 0.5778
Sound SE vs. SN 0.7529 0.2689
  SU vs. SN 0.9533 0.4556
  SE vs. SU 0.7820 0.7156
Pooled groups CN vs. SE_SU_SN < 0.0001a < 0.0001a 0.0002a 0.0024a < 0.0001a < 0.0001a < 0.0001a 0.0007a
  CE_CU vs. CN 0.0001a 0.0019a 0.0160a 0.0769 < 0.0001a 0.0006a 0.0079a 0.0525
  CE_CU vs. SE_SU_SN 0.0102a 0.0193 0.0370 0.0663 0.0525 0.0366 0.0312 0.0311
a
Significant: p ≤ 0.0166 with Bonferroni correction within caries-affected teeth and pooled comparisons.
* The p-values for comparisons of calcium and phosphorus levels (wt%) in caries-affected (CE, CU, CN) and sound (SE, SU, SN) dentin groups,
as well as between pooled caries-affected with base (CE_CU) and pooled sound (SE_SU_SN) dentin groups. After Bonferroni correction
within carious-affected teeth and pooled comparisons, no statistically significant difference in mineral content was observed between CE and
CU, or among sound subgroups (SE, SU, or SN) at the cavity floor or over deeper areas. At all depths, a significant difference in Ca- and
P-content was shown between untreated caries (CN) and pooled sound (SE_SU_SN) teeth. Calcium content: Within caries-affected groups,
CU values were significantly different from CN at the cavity floor. Pooled data were significantly different from CN up to 20-µm depth, while
these data was significantly different from SE_SU_SN only at the cavity floor. Phosphorus content: At the cavity floor, p-levels in both CE
and CU were significantly higher than in CN. Pooled groups were significantly different from CN up to 20-µm depth.

dentin. Regression lines for CE_CU (carious teeth that received
RCPC-base) were much flatter, indicating a more similar mineral
level at the cavity floor and in deeper, sound dentin. For sound
teeth, Ca levels appeared to be similar across all depths and all treat-
ments, and regression lines were flat.
Since no difference in Ca levels (Table 2) was observed
among sound subgroups (SE, SU, and SN) and between CE and
CU (base after etch or no-etch), data from these groups were
pooled, allowing for comparison among sound, and base and
no-base, caries-affected groups (Fig. 2A).
Quantification of Phosphorus Levels
Predicted P levels (wt%) are presented for affected (CN),
affected RCPC-treated (CE_CU, pooled), and sound (SE_SU_
SN, pooled) dentin at various depths (Fig. 2B). Linear and
quadratic effects were highly significant for CN. Linear effects
were significant for both CE and CU, while quadratic effects
were not (data not shown). For sound teeth, only SE showed a
statistically significant linear and quadratic effects in phospho-
rus levels (data not shown). The p-values (Table 2) for phosphorus-
level comparisons showed patterns similar to those of calcium
levels, resulting in similar pooling of data.
Discussion
A base material that promotes remineralization of affected dentin
and enhances tissue repair would be clinically beneficial and a use-
ful clinical treatment strategy. In vivo strontium and fluorine ion
penetration from GIC into dentin showed a penetration pattern
consistent with a remineralization process (Ngo et al., 2006).
In vivo remineralization data concerning Ca and P are lacking.
Studies supporting stepwise excavation have reported the repair
potential of carious dentin: Clinical assessment of affected dentin at
re-entry showed hard, dry, and dark dentin, characteristic of arrested
caries (Bjørndal et al., 1997; Bjørndal and Larsen, 2000). From a
caries-preventive point of view, a more mineralized (‘harder’) dentin
will not only be more resistant to mechanical forces, but will also
delay bacterial accumulation and penetration, halting a potentially
recurring caries-active process (ten Cate, 2001).
This study focused on the remineralization-enhancing ability of
resin-based Ca-P cement (RCPC). Clinical efficacy of this ion-
releasing base material to encourage tissue repair was explored after
3 mos of intra-oral service. At re-entry, qualitative clinical assess-
ment of residual affected dentin suggested lesion reversal, since the
dentin floor was hard, apparently remineralized after 3 mos.
The amount of caries to be removed is a point of continual
discussion among clinicians. We obtained a standardized end-
point by using a self-limiting polymer bur, leaving remaining
affected dentin of 25 KHN Knoop hardness (Peters, unpublished
observations; Boston, 2003). Since hardness is the only vali-
dated clinical measure (Kidd et al., 1993), this approach pro-
vided optimal standardization of the caries-removing process.
Remineralization was quantified by elemental analysis with
EPMA. Resulting weight-% (wt%) data for Ca- and P-content in
sound dentin were in the same range as those reported previ-
ously (Ngo et al., 1997; Hossain et al., 2003a,b), with Ca-levels
ranging from 27-32 wt% and P-content from 13-15 wt%.
Reports on artificially demineralized dental tissues showed a
decrease in mineral content for Ca- and P-levels, with the Ca:P
ratio close to 2.2 (Ngo et al., 1997). These data were corrobo-
rated by outcomes of this in vivo study, where similar levels and
ratios were detected in carious teeth.
In non-treated carious teeth, low Ca- and P-content at the
cavity floor indicated the amount of demineralization in the
outer lesion area. Mineralization levels gradually increased
toward the inner lesion area. This pattern followed the charac-
teristic hardness curve throughout a caries lesion, as described
previously (Ogawa et al., 1983). Mineral content peaked at
60-70 µm. The slight decrease of mineral toward the pulp (at

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© 2010 International & American Associations for Dental Research

290  Peters et al. J Dent Res 89(3) 2010
depths of 90-100 µm; not significantly different) might be
explained by closer proximity to the pulp, where sound deep
dentin was less mineralized (Ogawa et al., 1983). Although
randomly chosen, this group may have included deeper lesions
compared with other groups not showing this phenomenon.
Sound subgroups presented no difference between treated and
non-treated teeth. Once mineral content was within normal limits,
equilibrium was maintained, and no additional mineral was depos-
ited. When compared with carious teeth, sound teeth showed higher
Ca- and P-levels over the entire depth measured, possibly caused by
differences in cavity depth. Cavity preparations in sound, vital teeth
were, for ethical reasons (post-operative sensitivity), limited to
outer dentin, resulting in a higher grade of mineralization than inner
dentin (Ogawa et al., 1983).
Acid-etching opens dentin tubules and facilitates adhesive pro-
cedures (Brännström and Johnson, 1974). Etching of cavity floor
prior to base placement did not result in increased mineral levels in
treated carious teeth after 3 mos compared with unetched teeth.
Despite patent tubules in etched dentin, remineralization occurred
to the same degree as in unetched dentin. This agrees with the con-
cept that lesion repair occurs only by precipitation onto residual
crystals and not by new nucleation of mineral on organic matrix
(Levine and Rowles, 1973; Daculsi et al., 1979; Tveit and Selvig,
1981; Klont and ten Cate, 1991). A definitive statement, however,
cannot be made, since the study was not powered to address the
acid-etch stratification. This question needs further study. Even
without enhanced remineralization after 3 mos, etching of dentin
may still be indicated to achieve optimal adhesion of base material
(Dickens et al., 2004).
After 5 mos of in vitro remineralization, artificial root caries
lesions (500 µm deep) showed lesion repair corresponding to
50-85% mineral gain and 40% reduction in lesion depth (Mukai
and ten Cate, 2002). Depth of mineral deposition in this in vivo
study was 30 µm. This is in accordance with data from in vitro
studies (range, 20-30 µm depth) (Mukai et al., 1998; Massara
et al., 2002; Kitasako et al., 2003; Exterkate et al., 2005). In our
in vivo study, after a period of 3 mos, mineral levels were similar
at interface and 100 µm into dentin. This supports the clinical
use of the Ca- and P-releasing base as a remineralization tool to
increase Ca- and P-levels in affected dentin.
The actual depth of demineralization and re-entry time may
have influenced the maximum depth at which remineralization
occurred. Further investigations focusing on mineral uptake from
RCPC base over deeper areas of mineral-depleted dentin (reminer-
alization distance) and at different timepoints (initial remineraliza-
tion mechanism, longer-term remineralization extent) are warranted
to reveal a complete picture of the in vivo potential of RCPC to
replenish Ca- and P-ions in caries-affected dentin.
ACKNOWLEDGMENTS
The authors thank Dr. R.B. Rutherford (University of Washington,
Seattle) for contributing to the study design and the manuscript, Carl
Henderson (UM-EMAL) for EPMA assistance, Kathy Welch
(UM-CSCAR) for invaluable statistical assistance, and S.S. White
Burs for providing SmartPrep-Systems®. This study was supported
by Dentigenix/Ivoclar-Vivadent AG (Schaan, Liechtenstein), by the
University of Michigan, and by CAPES #BEX3404-8 (Brazil).
Co-author SHD developed the cement at the Paffenbarger Research
Center, American Dental Association Foundation. She was not
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© 2010 International & American Associations for Dental Research
involved in sample collection or the analysis of data, but contributed
to manuscript writing. Preliminary data were presented at the 2006
Annual Meetings of the Academy of Operative Dentistry (Chicago),
the American Association for Dental Research (Abstr# 481,
Orlando), and the Academy of Dental Materials (São Paulo, Brazil).
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