ORIGINAL ARTICLE

Gene expression profile altered by

orthodontic tooth movement during
healing of surgical alveolar defect
Eun-Kyung Choi,a Jae-Hyung Lee,b Seung-Hak Baek,c and Su-Jung Kimd
Seoul, Korea

Introduction: We explored the gene expression profile altered by orthodontic tooth movement (OTM) during the
healing of surgical alveolar defects in beagles. Methods: An OTM-related healing model was established where
a maxillary second premolar was protracted into the critical-sized defect for 6 weeks (group DT6). As controls,
natural healing models without OTM were set at 2 weeks (group D2) and at 6 weeks (group D6) after surgery.
Total RNAs were extracted from dissected tissue blocks containing the regenerated defects and additionally
from sound alveolar bone as a baseline (group C). mRNA profiling was performed using microarray analysis.
Results: Functional annotations of gene clusters based on differentially expressed genes among groups
indicated that the gene expression profile of group DT6 had a stronger similarity to that of group D2 than to
group D6. The genes participating in high woven-bone fraction in group DT6 could be identified as TNFSF11,
MMP13, SPP1, and DMP1, which were verified by quantitative real-time polymerase chain reactions.
Conclusions: We investigated at the gene level that OTM can affect the healing state of surgical defects serving
as favorable matrices for OTM with defect regeneration. It would be a basis on selecting putative genes to be
therapeutically applied for tissue-friendly accelerated orthodontics in the future. (Am J Orthod Dentofacial
Orthop 2017;151:1107-15)

O
rthodontic tooth movement (OTM) is occasion-
ally attempted toward the periodontal defect
area induced by periodontitis, trauma, congen-
ital deformity, or alveolar surgery, but OTM might pro-
duce side effects such as uncontrolled or impaired
tooth movement, root resorption, and poor defect
healing.1,2 OTM into an alveolar defect becomes a
challenging issue, since there is no well-established
protocol allowing efficient tooth movement concomi-
tant with favorable defect regeneration. Although
a
Graduate School, Kyung Hee University, Seoul, Korea.
b
Department of Life and Nanopharmaceutical Sciences and Department of
Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University,
Seoul, Korea.
c
Department of Orthodontics, School of Dentistry, Dental Research Institute,
Seoul National University, Seoul, Korea.
d
Department of Orthodontics, School of Dentistry, Kyung Hee University, Seoul,
Korea.
All authors have completed and submitted the ICMJE Form for Disclosure of
Potential Conflicts of Interest, and none were reported.
Supported by National Research Foundation grant funded by the Korea govern-
ment (number 2014R1A1A3052072).
Address correspondence to: Su-Jung Kim, Department of Orthodontics, Oral
Biology Research Institute, Kyung Hee University School of Dentistry, 1 Hoegi-
Dong, Dongdaemoon-Ku, Seoul 130-701, Korea; e-mail, ksj113@khu.ac.kr.
Submitted, May 2016; revised and accepted, October 2016.
0889-5406/$36.00
Ó 2017 by the American Association of Orthodontists. All rights reserved.
http://dx.doi.org/10.1016/j.ajodo.2016.10.039
the biologic responses of periodontal tissues to ortho-
dontic forces have been systematically investigated for
when a tooth moves surrounded by healthy periodon-
tium, there is still a lack of understanding of the differ-
ential biologic mechanisms taking place in the
periodontal defect encompassing a moving tooth.3,4
When a surgical intervention such as corticotomy,
osteotomy, or bone grafting is planned to improve the
quality and the quantity of bone matrix for a subsequent
tooth movement,5 both mechanisms of surgically
induced wound healing and orthodontically induced
alveolar remodeling might work cooperatively. It is
well known that a surgical alveolar defect undergoes a
healing process involving the progressive states of coag-
ulum, granulation tissue, osteoid deposit, immature
woven bone, and mature lamellar bone.6,7 The
changing structural composition of the regenerated
tissue over time might affect the rate of tooth
movement. The regenerated tissue containing a high
fraction of woven bone relative to lamellar bone
and marrow would be the best matrix for tooth
movement because woven bone provides low tissue
resistance by inducing transient osteopenia, and
because highly recruited and activated bone cells
synergistically enhance alveolar remodeling activity on
the bone surrounding moving teeth.8,9 Conversely,
1107
1108
OTM-induced alveolar remodeling has been used to pro-
mote periodontal regeneration.10 OTM is known to
involve cytokine-mediated bone adaptive responses
similar to the wound healing process, generating an os-
teopenic state of woven bone and subsequent stimula-
tion of bone apposition and mineralization.11 It was
postulated that these complementary potentials of
OTM and defect healing are worthy of being therapeuti-
cally applied, based on the identification of woven-bone
capacity as a common denominator at the molecular
level.
Recent studies have demonstrated the differential
gene expression in woven bone compared with lamellar
bone in the mechanically loaded ulna,12 comparative
gene expression between the healing of an alveolar
defect and osseointegration,13 and enhanced expression
of an arbitrarily selected gene in the periodontium dur-
ing OTM.14-16 However, little is known about gene
expression profiles of multiple genes during the
healing of an alveolar defect with or without OTM
over time. Since clinical bone quality varies depending
on the place and the conditions that determine cellular
density and activity, bone density and architecture,
and the proportions of organic and inorganic matrix, a
specialized animal model would be required for such
studies.17 Furthermore, in pursuit of exploring specific
biologic modulators of woven bone to aid long-lasting
rapid tooth movement through the regenerated tissue,
more comprehensive screening of molecular events
needs to be conducted.
The aim of this study was to elucidate the gene
expression profile altered by OTM during the healing
process of surgical alveolar defects in beagles. Based
on this, we explored putative genes that might have
participated in differential gene expression between
OTM-related healing and natural healing groups using
bioinformatics of gene clustering in beagles.
MATERIAL AND METHODS
Four male beagles, 18 to 24 months of age, were
housed according to the guidelines of the institutional
animal care and use committee of Kyung Hee University
Medical Center (KHMC IACU 14-044). They were allo-
cated into 4 groups according to the healing condition
of the surgical defect: group DT6, 6 weeks of healing
with OTM; group D6, 6 weeks of natural healing without
OTM; group D2, 2 weeks of natural healing without
OTM; and group C, no intervention as a baseline control
(Fig 1). The observation time points were based on our
previous study,18 to determine intergroup similarity of
the healing state of group DT6 along with the woven-
bone state at 2 weeks or with the lamellar bone state
at 6 weeks of healing. Under general anesthesia by
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Choi et al
intramuscular injection of zoletil 50 (0.25 mg/kg; Virbac
Laboratories, Carros, France), a 4-wall, cuboidal-shaped,
critical-sized defect of 5 (mesiodistal) 3 5
(buccolingual) 3 7 (vertical) mm that included the
extraction socket of the maxillary first premolar was
created by surgical bur. The distal wall of the defect
was prepared 1.5 mm away from the mesial root of the
maxillary second premolar to prevent immediate fracture
of the interdental bony wall during OTM (Fig 1, A and
B). As postoperative care, anti-inflammatory analgesics
(Ketopro; Uni Biotech, Chungnam, Korea) and antibi-
otics (Gentamycin; Komipharm International, Siheung,
Korea) were injected for 3 days, and 0.12% chlorhexidine
gluconate dressing was used for 14 days. In group DT6,
orthodontic brackets were bonded on the buccal sur-
faces of the maxillary second premolar and canine, and
sectional stainless steel wire of 0.019 3 0.025 in was
passively inserted between the 2 teeth. An immediate
force of 100 g was applied for mesial traction of the sec-
ond premolar toward the defect and readjusted for
6 weeks (Fig 1, D). Time-course changes of defect heal-
ing status were observed by serial radiographic images
taken at 0, 2, 4, and 6 weeks after surgery (Rextar X; Pos-
kom, Goyang, Korea). After the animals were killed by
direct injection of zoletil 50 (50 mg/kg) into the heart
at each observation time, tissue blocks were harvested
including the regenerative tissue in the defect between
the roots.
Total RNA was extracted either from the regenerated
tissue in the defect and adjacent alveolar bone sur-
rounding the root of the maxillary second premolar
(groups D2, D6, and DT6) or from the sound alveolar
bone tissue around the maxillary premolar roots (group
C). The purity and integrity of the extracted RNA was
evaluated by spectrophotometry at an optical density
ratio of 260/280 with an Agilent 2100 Bioanalyzer (Agi-
lent Technologies, Palo Alto, Calif). The Affymetrix
Whole Transcript Expression Array was used to measure
multiplexed mRNA expression according to the manu-
facturer's protocol (GeneChip Whole Transcript plus
reagent kit; Affymetrix, Santa Clara, Calif). Signal inten-
sity values were computed using the Affymetrix Gene-
Chip Command Console software. The raw data were
normalized with the robust multiaverage method, and
the median value of probe intensities in the same
gene was used to represent the expression level of the
gene.
For bioinformatic cluster analysis, principal compo-
nents analysis has been performed using “prcomp”
function in R statistical language (version 3.1.2; www.
r-project.org). The hierarchical clustering heat map
was obtained using complete linkage and Euclidean dis-
tance as a measure of similarity. Differentially expressed
Choi et al 1109

Fig 1. Photographs of experimental procedures (A-D) and diagrams of animal groups (E-H). A and B,
surgically prepared critical-sized defect on beagle maxilla; C, natural healing model without OTM; D,
OTM-related healing model where second premolar was protracted into the defect. Animals were allo-
cated into 4 groups according to the healing condition: E, group D2, 2 weeks of natural healing; F, group
D6, 6 weeks of natural healing; G, group DT6, 6 weeks of healing with OTM; H, group C, baseline con-
trol with no intervention.

genes (DEGs) were defined as those with changes of at
least 1.5 fold between a pair of samples, at a false dis-
covery rate of 5%. Identified DEGs were clustered by
the Mfuzz package in R19 using average normalized
signal intensity values of individual genes as input
values. The number of clusters was chosen as 8, and
the fuzzifier coefficient M was set to 1.5. Gene ontology
terms for the genes were obtained from Ensembl Bio-
mart (http://www.ensembl.org/biomart/martview), and
gene ontology term enrichment analysis was per-
formed.4
For the verification of DEGs among groups, a
quantitative real-time polymerase chain reaction was
performed for the putative genes which might
contribute to the different bone healing states among
the groups. The tested genes were selected from the
category of biologic function regarding “extracellular
binding,” “bone morphogenesis,” “osteoclastogenesis,”
and “bone mineralization” as a result of functional
annotation analysis. cDNA was produced using the Su-
perscript II real-time polymerase chain reaction system
(Invitrogen, Karlsruhe, Germany) according to the man-
ufacturer's recommendations for oligo (dT) primed
cDNA-synthesis. Polymerase chain reaction was per-
formed using a Quant Studio Sequence Detection Sys-
tem (Applied Biosystems, Foster City, Calif) in 384-well
microtiter plates with a final volume of 10 mL. Optimum
reaction conditions were obtained with 5 mL of Universal
Master Mix (Applied Biosystems) containing dNUTPs,
magnesium chloride, reaction buffer, Ampli Taq Gold,
90 nM of primers and 250 nM fluorescence-labeled
TaqMan probe (4352930E; Applied Biosystems). Finally,

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1110 Choi et al

Fig 2. A, Hierarchical clustering heat map where the grouping of samples was explored; B,
3-dimensional principal component analysis to show distributional proximity among samples.
Group DT6 was included in the similar clustering to group D2, whereas group D6 showed similarity
to group C.

2 mL of template cDNA was added to the reaction
mixture. Amplifications were performed starting with a
10-minute template denaturation step at 95 C, followed
by 40 cycles at 95 C for 15 seconds and 60 C for 1 min-
ute. All samples were amplified in triplicate, and data
were analyzed with Sequence Detector software (Applied
Biosystems).
RESULTS
Standard radiographic images confirmed the healing
state of the defect in each group. More homogeneous
bone density was seen in the regenerated tissue covered
with a more intact and clear bony bridge up to the
marginal level in group DT6 (Fig 1, G) than in group
D6 (Fig 1, F). Simultaneously, controlled tooth migra-
tion was obtained, with a mean rate of 0.52 mm per
week. In group D2, the defect showed irregular
radiolucency without an intact bony bridge, indicating
active bone remodeling in the woven-bone state (Fig
1, E), in contrast to the image of the control group
C (Fig 1, H).
The microarray data are accessible through Gene
Expression Omnibus Series accession number
GSE85459. As a result of the RNA quality check, all sam-
ples passed the criteria suggested by Macrogen Next
Generation Sequencing (Seoul, Korea) as RIN greater
than 7.0, rRNA ratio greater than 1.0, and purity greater
than 1.0. The hierarchical clustering map showed the re-
lationships among the groups based on the gene
expression profiles (Fig 2, A). Group D6 was close to
group C. On the contrary, group DT6 was similar to
group D2. This finding was also supported by principal
components analysis to exhibit distributional proximity
among samples in 3 dimensions (Fig 2, B).
Volcano plots clearly display the differentially ex-
pressed genes between any 2 groups (Fig 3). There are
many overexpressed genes in group D2 compared with
groups C (Fig 3, A) and D6 (Fig 3, D). On the other
hand, not many DEGs were identified in the comparison
between groups DT6 and D2 (Fig 3, E). Interestingly,
there were more overexpressed genes in group DT6
compared with groups C (Fig 3, C) and D6 (Fig 3, F).
In total, 804 DEGs were clustered based on their gene
expressions using the fuzzy c-means algorithm imple-
mented in Mfuzz package, and 8 different gene expres-
sion patterns (clusters) were obtained (Fig 4). In clusters
1 and 2, the gene expressions for the 3 tested groups
(D2, D6, and DT6) showed no significant difference,
but the gene expression in the control group was lower
(cluster 1) or higher (cluster 2) than the one in the
other 3 groups. On the other hand, in cluster 3,
where gene ontology terms such as “kinesin complex

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Choi et al 1111

Fig 3. Volcano plots displaying the DEGs using red and blue dots in 6 pairs of groups. The x-axis rep-
resents the magnitude of fold changes ( log2) between 2 groups, and the y-axis is adjusted P value
( log2) with the Benjamini-Hochberg correction. A-C, Group C was compared with groups D2, D6,
and DT6, respectively, to serve as a relative baseline level. D-F, the comparisons between the test
groups. Group DT6 showed a low level of DEGs compared with group D2 (E), whereas group DT6
showed more DEGs compared with group D6 (F).

activation,” “hematopoietic progenitor cell differentia-
tio” “proteinaceous extracellular matrix,” “positive regu-
lation of cytokinesis,” and “inflammatory response”
were statistically enriched in the cluster member genes,
the gene expression level in group D2 was higher than
the one in the other groups.
Cluster 4 included the genes highly expressed in
both groups D2 and DT6 in contrast to groups D6
and C; this was consistent with previous results of
intergroup similarity (Fig 2). Gene ontology terms of
the top 5 upregulated subgroups in cluster 4 belonged
to “cell signaling,” “cell differentiation,” and “osteo-
clastogenesis.” In contrast, cluster 5 contained the
gene downregulated in both groups D2 and DT6 rela-
tive to group D6; the member genes of this cluster
were associated with “RNA binding,” “desmosome,”
“extracellular matrix binding,” and “ossification and
bone mineralization.” Clusters 6 and 7 manifested
highly expressed genes in group DT6 similarly to group
D6 rather than to group D2, and the member genes of
this cluster are involved with “biologic functions of
collagen remodeling,” “extracellular matrix binding,”
and “bone morphogenesis and mineralization.” Finally,
cluster 8 included the downregulated genes in groups
D2 and D6, relative to the control group C, with no
significance.
Among various genes showing significant differences
in gene ontology terms between groups DT6 and D6,
4 putative genes that might contribute to different states
of regenerated bone tissues were tested by quantitative
real-time polymerase chain reaction for further
verification: (1) matrix metalloproteinase 13, MMP13
(gene ontology term: metalloendopeptidase activity,
collagen catabolic activity, collagen binding, bone
morphogenesis, bone mineralization); (2) tumor necrosis
factor ligand superfamily member 11, TNFSF11
(gene ontology term: osteoclast differentiation,
TNFSF11-mediated signaling pathway); (3) secreted

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1112 Choi et al

Fig 4. Functional annotation of gene clusters upregulated or downregulated by OTM in the beagle
model with surgical alveolar defects. DEGs between OTM-related healing and natural healing models
at 6 weeks were significantly identified by clusters 4 and 5, including TNFSF11, MMP13, SPP1, and
DMP1 as bone-regulating representative genes.

phosphoprotein 1, SPP1 (gene ontology term: extracel-
lular matrix binding, ossification); and (4) dentin matrix
protein 1, DMP1 (gene ontology term: extracellular ma-
trix binding, nucleation of bone matrix). Significantly
upregulated mRNA expression was defined by a fold
change above 1.5 fold and downregulated expression
by a fold change below 0.67 (Fig 5). All tested genes
showed significant increases of fold changes in the
DT6-D6 group pairing comparison, consistent with the
result of the functional annotation analysis. Moreover,
the MMP13 mRNA level was upregulated in group
DT6 in all tested pairs of DT6-C, DT6-D2, and DT6-D6
(Fig 5, A). TNFSF11 mRNA expression was significantly
increased in the groups DT6 and D2, in comparison with
group D6 (Fig 5, B). SPP1 mRNA expression was
upregulated in group DT6 relative to the levels in groups
D2 and D6 (Fig 5, C). DMP1 mRNA expression was
exclusively increased only in the DT6-D6 group pairing
(Fig 5, D).
DISCUSSION
This study suggests the presence of biologically crit-
ical genetic pathways influenced by OTM during the
healing of surgical alveolar defects. As we noted for
the natural healing models (groups D2 and D6) and
the OTM-related healing model (group DT6), 3 signifi-
cant features of gene expression patterns were disclosed
(Fig 4). First, the genes differently expressed over time in
the natural healing model were displayed by clusters 3
through 6 (D2 vs D6). The genes that were increased in

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Choi et al 1113

Fig 5. Relative mRNA expression verified by quantitative real-time RT-PCR. The mRNA levels of A,
TNFSF11; B, MMP13; C, SPP1; and D, DMP1 showed significantly upregulated fold changes in group
DT6 compared with groups D6 and D2.

the woven bone at 2 weeks and then decreased at
6 weeks (KIF, IL-18, SLC, ASPN, AK, BGN, TNFSF11,
FOS, and COL11A1) were well contrasted with those
upregulated later in the matured bone at 6 weeks
(DSP, MMP13, DMP1, SPP1, and PHEX). Second,
DEGs between OTM-related healing and the natural
healing model at the same observation time point of
6 weeks were identified by clusters 4 and 5 (DT6 vs
D6). Group DT6 represented upregulated genes (AK,
TNFSF11, FOS, and COL11A1) and downregulated
genes (PHEX and MMP13) relative to the level of group
D6, showing similar fold changes to group D2. Third, in
contrast, the genes highly expressed in both groups DT6
and D6, representing a difference from group D2
(DMP1, SPP1, and MMP13) were reflected by cluster
6 (DT6 vs D2). Overall, the results showed that the
gene expression profile of group DT6 exhibited strong
similarity to that of group D2 in terms of functions
such as “immune response,” “acute inflammation,” “os-
teoclastogenesis,” and “extracellular matrix remodel-
ing,” while retaining the active remodeling potential of
group D6 regarding “bone morphogenesis” and “miner-
alization.”
As contributing factors for extending the period of
the woven-bone state by perpetuating matrix formation
and delaying lamellar maturation when the defect was
affected by OTM, the roles of periodontal ligament cells
and bone extracellular matrix proteins can be consid-
ered. It was reported that progenitor cells from peri-
odontal ligaments activated by OTM were responsible
for enhanced bone formation in the adjacent alveolar
defect, although the defect did not contain the root
initially.20,21 Nonetheless, it is not feasible to
separately identify the specific cytokines or signaling
transducers that participate in the regulatory
mechanisms of immune response, acute inflammation,
and bone regeneration.22,23 Focusing on controlling
the woven-bone fraction in the total bone matrix, there-
fore, we explored DEGs contributing particularly to bone
metabolic functions such as bone cell activation, bone
matrix stabilization, bone resorption, morphogenesis,
and mineralization. Because woven-bone formation
in a critical-sized defect depends on de novo formation,
mediated by a nucleation process, in addition to
appositional formation from the existing bone surfaces
by an osteoclast-osteoblast coupling process,24,25

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1114
osteoclastogenesis-related TNFSF11, collagenous
extracellular matrix protein-related MMP13, and non-
collagenous extracellular matrix protein-related SPP1
and DMP1 genes were specifically sorted out in our
study for further verification and future clinical applica-
tion (Fig 4).
TNFSF11, also known as osteoclast differentiation
factor, osteoprotegerin ligand, and receptor activator
of NF-kB ligand, encodes a member of the tumor necro-
sis factor cytokine family and functions as a key factor
for osteoclast differentiation and activation.26 TNFSF11
was upregulated in both groups DT6 and D2 to a similar
degree, but relatively downregulated in group D6, indi-
cating that TNFSF11 might play a critical role in delay-
ing full mineralization and maturation of the
regenerated tissue as enhanced by OTM, maintaining
the bone composition of group DT6 similar to that of
group D2. On the other hand, MMP13 was upregulated
in all experimental groups relative to the control, and the
MMP13 mRNA level was highest in group DT6 (Fig 5, B).
This implies that MMP13 participates in both resorption
and formation of bone matrix during defect healing, and
is overexpressed by OTM to modulate the bone remod-
eling activity, in accordance with previous reports that
MMP13 increased during connective tissue degrada-
tion, matrix formation, and bone remodeling as a colla-
genase subfamily member.27
As a well-known factor for the nucleation process,
especially in de novo bone formation,28,29 the small
integrin-binding ligand N-linked glycoprotein family
was interestingly detected. In particular, SPP1 and
DMP1 were noted by highly expressed fold changes in
both groups DT6 and D6, relative to group D2; that is,
SPP1 and DMP1 were more specifically upregulated
when OTM accompanied the defect, as verified by real-
time polymerase chain reaction. SPP1, also known as
osteopontin, was originally identified as the bridge be-
tween cells and hydroxyapatite in bone extracellular ma-
trix,30 but it has been recently documented to have an
inhibitory role on extracellular matrix mineralization
by way of posttranslational modification.31 SPP1 was
also demonstrated to be an important factor for trig-
gering an osteopenic state caused by a mechanical
load such as tooth movement.32 It was upregulated by
osteocytes first on the compression side and gradually
by osteoblasts on the tension side of the moving tooth.33
It might be that OTM increased the expression of SPP1,
affecting the defect healing process to prolong the
woven-bone state by counteracting other genes func-
tioning in full maturation. DMP1, as a transcriptional
component for activation of osteoblast-specific genes,34
is known to respond rapidly to mechanical stimulation
by promoting an early nucleation process in matrix
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Choi et al
mineralization.35 DMP1 expression was exclusively
increased in group DT6, even when matched against
group D6, suggesting that the regenerated tissue with
OTM could maintain the woven-bone state while the
initial nucleation process was still ongoing, potentially
to cope with the activated catabolic functions of genes
such as TNFSF11 and MMP13, and thus to engender
the equilibrium of woven-bone metabolism.
Our findings from beagle gene profiling contain
fundamental limitations: (1) our partial description
of whole mechanisms, deriving from the lack of
accuracy and specificity in microarray analyses36;
(2) possibly skewed interpretations on the specific
function of single genes, passing over their multifunc-
tional property when orchestrated with other genes37;
and (3) individual heterogeneity and the incomplete
gene ontology bank of beagles.38 Nevertheless, the
beagle model was preferred to resemble the clinical
situation that we intended to address in terms of tis-
sue dimension and biologic cycle of healing and re-
modeling.39
CONCLUSIONS
This study highlights in the gene level that OTM
could sustain a high fraction of woven bone in the
regenerative tissue of surgical alveolar defects that
serves as a favorable matrix for OTM and defect regen-
eration. This provides comprehensive understanding of
differentially expressed genes upregulated or downregu-
lated by OTM during healing of a surgical alveolar defect
and a basis for sorting out putative genes functioning as
biologic modulators of bone remodeling that could be
therapeutically applied aiming at tissue-friendly acceler-
ated orthodontics in the future.
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June 2017  Vol 151  Issue 6