European Journal of Orthodontics, 2019, 1–12

doi:10.1093/ejo/cjz035
Original article

Downloaded from https://academic.oup.com/ejo/advance-article-abstract/doi/10.1093/ejo/cjz035/5506479 by The Edward G Miner Library user on 21 July 2019
Original article

Effects of mesenchymal stem cell transfer

on orthodontically induced root resorption
and orthodontic tooth movement during
orthodontic arch expansion protocols: an
experimental study in rats
Nisa Gul Amuk1, , Gokmen Kurt2, Erol Karsli3, Servet Ozcan4,
Mustafa Burak Acar4, Mehmet Amuk5, Ayca Lekesizcan6 and
Cem Abdulkadir Gurgan7
1
Department of Orthodontics, Faculty of Dentistry, Erciyes University, Kayseri, 2Department of Orthodontics, Faculty
of Dentistry, Bezmialem Vakif University, Istanbul, 3Orthodontic Private Practice, Adana, 4Genome and Stem Cell Cen-
ter (GENKÖK), 5Department of Oral and Maxillofacial Radiology, Faculty of Dentistry, and 6Department of Histology
Embryology, Faculty of Medicine, Erciyes University, Kayseri, and 7Department of Periodontology, Faculty of Dentis-
try, Nuh Naci Yazgan University, Kayseri, Turkey

Correspondence to: Nisa Gul Amuk, Department of Orthodontics, Faculty of Dentistry, Erciyes University, 38039 Melikgazi,
Kayseri, Turkey. E-mail: nisa.gul86@hotmail.com

Summary
Objectives: The aim was to evaluate the effects of mesenchymal stem cell (MSC) transfer to
periodontal ligament (PDL) on the inhibition and/or repair of orthodontically induced root resorption
(OIRR) during and after arch expansion and on the orthodontic tooth movement (OTM) rate of the
maxillary first molar teeth of rats.
Material and methods: Sixty Wistar rats were divided into three groups as the untreated group,
MSC and control injections during the expansion period group (EMSC-EC), and MSC and control
injections at the retention period group (RMSC-RC). Fifty grams of orthodontic force was applied to
the maxillary first molar teeth of the rats for 14 days in the vestibular direction, and then, 20 days
of retention was carried out. MSCs and control injections were performed every 3 days in the EC,
RC, EMSC, and RMSC groups. At the end of the experiment, samples were prepared for OTM
evaluation, mRNA expression analysis, micro-computed tomography measurements, cementum
thickness calculations, and structural examinations.
Results: The amount of OTM in EMSC group was significantly higher than in EC group (P < 0.001).
MSC transfer during the expansion and retention periods reduced the number of resorption
lacunae, volumetric and linear resorptive measurements, and cyclooxygenase-2 and receptor
activator of nuclear factor kappa B ligand (RANKL) mRNA expression levels, and increased the
osteoprotegerin (OPG) expression levels, OPG/RANKL ratio, and cementum thickness in the EMSC
and RMSC groups.
Conclusions: MSC transfer to PDL during expansion increased the amount of OTM. Injection of
MSC during the retention period was found to be slightly more effective in prevention and/or
repair of OIRR than MSC transfer during the expansion period.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Orthodontic Society.
1
All rights reserved. For permissions, please email: journals.permissions@oup.com
2 European Journal of Orthodontics, 2019
Introduction
Orthodontic arch expansion in the transversal direction may be
defined as the movement of the posterior teeth towards the ves-
tibular alveolar bone. Posterior arch expansion by biologically
friendly light forces has been stated as the philosophy of ortho-
dontic tooth movement (OTM) with fixed appliances (1, 2), along
with the other indications such as creating space in dental arches,
providing transversal intermaxillary harmony by correction of
tooth positions, and improvement of smiling by enlarging the
buccal corridors. In addition, it was reported that buccal crown
tipping is an expected and unavoidable effect of rapid maxillary
expansion (RME) that has been conducted especially with tooth-
borne and/or tooth-tissue-borne appliances (3–5), although only
skeletal expansion has been aimed rather than the movement of the
teeth through the alveolar bone.
The success of OTM is directly related to tissue vitality, cellular
and connective tissue response, periodontal health, and integrity
of the dental structures. Buccolingual tooth movement can occur
with or through the alveolar bone due to the characteristics of
orthodontic forces (6). Under the appropriate orthodontic forces,
the periodontal structures are expected to be reconstructed on
the molecular, cellular, and tissue levels (7), whereas uncontrolled
heavy forces induce indirect bone resorption that causes tissue
necrosis, extreme inflammatory results, delay in OTM, and ortho-
dontically induced inflammatory root resorption (OIIRR) (8–10).
Considering the biological and mechanical variables, buccal tip-
ping of posterior teeth may threaten the health of the dental and
periodontal structures in the case of movement through the enve-
lope of the alveolar process, which may be resulted with reduc-
tion of alveolar bone height, bone dehiscence defects, and OIIRR
(11, 12). Even though mechanical factors such as the duration,
direction, distribution, and degree of orthodontic forces may be
managed (13), biological factors are generally outside the control
of the orthodontist.
After the removal of orthodontic forces, the natural repair
process is activated for both soft and hard dental structures (14,
15). The repair of resorption lacunae is generally characterized
by new cementum formation, which is provided by maturation
and differentiation of progenitor reparative stem cells of the peri-
odontal ligament (PDL) (16, 17). Due to the immunomodulatory
function and potential of stem cells for proliferation and gener-
ation of a cementum/PDL-like complex (18, 19, 20), these mesen-
chymal-derived stem cells have been accepted as important actors
in periodontal homeostasis (17). Although many mechanical and
biological methods have been used to encourage appropriate pro-
genitor cell population of the resorption site, these approaches
typically aimed to stimulate cellular and mechanical activation in-
directly and were dependent on the duration, frequency, and mag-
nitude of mechanical stimulation forces. However, transfer of stem
cells into the PDL was found to be a successful method for direct
induction of cellular and molecular activation for repairing the
resorption process (21).
In addition to the reparative effects of stem cells on resorption
lacunae, these cells may also have important roles in the organiza-
tion of PDL (14, 22) and remodelling of the alveolar bone (23).
These factors that are directly related to tooth movement may also
be effective on the rate of OTM. Although there are some hypoth-
eses like the possible effects of stem cell transplantation on OTM
(17, 24), no study has been conducted on this issue. Moreover,
although the reparative effects of mesenchymal stem cells’ (MSCs)
transfer to PDL have been reported during the OTM in the mesial
direction, which generally occurs in the envelope of the alveolar
bone process and may be accepted as routinely used OTM in clini-
cal orthodontic practice (21), there has been no reports on the
effects of MSCs’ transfer to PDL during and after orthodontic
force application to the molar teeth towards the buccal vestibu-
lar bone, which is also generally observed during RME protocols
Downloaded from https://academic.oup.com/ejo/advance-article-abstract/doi/10.1093/ejo/cjz035/5506479 by The Edward G Miner Library user on 21 July 2019
in addition to fixed appliances. Therefore, the aims of this study
were to evaluate the effects of MSCs’ transfer to PDL on inhibition
and/or repair of orthodontically induced root resorption (OIRR)
during and after the buccal tooth movement of the maxillary first
molar teeth of rats and to determine the effects of MSCs’ transfer
to PDL on the OTM rate of the maxillary first molar teeth of rats
during arch expansion by digital model analysis, micro-computed
tomography (Micro-CT) analysis, evaluation of the transcrip-
tional alterations in osteoprotegerin (OPG), receptor activator of
nuclear factor kappa B ligand (RANKL), cyclooxygenase-2 (Cox-
2), histological analysis, and scanning electron microscopy (SEM)
evaluation.
Materials and methods
The study was supported by the Scientific and Technological
Research Council of Turkey (TUBITAK) with the project number
116S400. Animal selection, management, and experimental proto-
cols were approved by Erciyes University, Regional Animal Research
Ethics Committee (approval code: 14/146). The experiments were
carried out in the Hakan Çetinsaya Experimental and Clinical
Research Center (HÇRC) in Kayseri, Turkey.
Animals
Sixty 12-week-old male Wistar rats with a mean weight of
257.66 ± 43.10 g were produced and maintained in HÇRC and
randomly divided into 3 groups as the negative control (untreated
group, n = 20), injections during the expansion period (n = 20),
and injections at the retention period groups (n = 20) by the vet-
erinarian of the HÇRC who is not a researcher of this study. All
animals were housed in pre-numbered hygienic polycarbonate
cages to accommodate four animals per cage. They subjected to
a 12-hour light to dark cycle at the constant temperature of 22 ±
2°C, and the rats were fed with a standard pellet diet and tap
water ad libitum. The physiological requirements, care, and gen-
eral conditions of rats were controlled by the responsible veterin-
arian of HÇRC regularly.
Experimental design
A split-mouth experimental design was used in this study. Right
maxillary molar teeth received MSC injections whereas the contra-
lateral side served as the positive control with the Dulbecco’s phos-
phate-buffered saline (DPBS) solution injections for expansion MSC
injection and retention MSC injection groups. The experimental pro-
cesses were carried out on the groups as follows:
Expansion MSC injection (EMSC) and expansion-injection con-
trol group (EC)/20 rats: These animals received MSC injections and
DPBS solution injections during the active orthodontic expansion
period of 2 weeks. After the expansion period, a retention period of
20 days was carried out, and these animals were not subjected to any
injections during the retention period.
Retention MSC injection group (RMSC) and retention-injection
control group (RC)/20 rats: The animals in this group did not receive
any injections during the active expansion phase whereas the MSC
N. Gul Amuk et al.
injections and DPBS solution injections were performed during the
retention period of expansion.
The negative control group (NC)/20 rats: These animals, whose left
maxillary molar teeth were used for the analyses, did not receive any
experimental application or injection, and were killed at the end of
the 34-day experiment as in the other groups.
Orthodontic appliance and expansion protocol
The rats received orthodontic appliances and injections under gen-
eral anaesthesia by intraperitoneal injection of 1.0 mg/kg keta-
mine hydrochloride (Gedeon Richter Ltd, Budapest, Hungary) and
0.5 mg/kg xylazine (Rompun; Bayer, Leverkusen, Germany). A spe-
cial oral retractor was used for providing appropriate mouth open-
ing, head position, and soft-tissue retraction.
Orthodontic expansion was achieved by bonding of a helical
spring fabricated from 0.016 × 0.022-inch beta-titanium wire to
the retention regions constructed on the palatal surface of max-
illary first molars (Figure 1a). The retention areas consisted of
roughened enamel and a shallow notch that was created above
the papilla using a low-speed handpiece (below 15 000 rpm,
INTRAmatic L-motor 181 DBN, KaVo Dental Excellence, Santa
Katarina, Brazil) and a fine-needle diamond bur. The appliances
were fixed with resin (Transbond XT Light Cure Adhesive, 3M,
Ruschlikon, Switzerland) using a total-etch, total bond technique.
The upper incisors were also connected to each other using bond-
ing materials at the interproximal surfaces. The springs were acti-
vated to deliver a force of 50 g and were not reactivated during
the 14-day expansion period. After the active expansion period,
the springs were removed, and a transpalatal retaining wire fab-
ricated from 0.016 × 0.022-inch stainless-steel wire was placed
under general anaesthesia (Figure 1b). Retention of arch expan-
sion was maintained for 20 days. No loss or reinsertion need of
the appliances was observed during the experiment, and no mid-
line diastema that indicates midpalatal suture opening was formed
in animals during the experiment.
Rat MSC isolation and cell culture
Visceral adipose tissue was surgically isolated from 8-week-old
Wistar albino rats and collected into antibiotic-antimycotic-con-
taining pre-cooled culture media. After physical disintegration of
the tissue, Type II collagenase (2.5 mg/ml, C6885; Sigma) solu-
tion was added on and incubated until the tissue was completely
disassociated. At the end of the incubation process, 70-µm cell
strainers were used to discard non-disassociated tissue particles.
Cells were washed with DPBS at ×350g for 5 minutes and plated
into 75 cm2 cell culture flasks. We changed the culture media
every third day of culture and expanded the culture when cells
became 90% confluent. After liberalization, cells were washed
Figure 1. Orthodontic expansion appliance (a) and orthodontic retention
appliance (b).
3
twice with DPBS and plated into the flask that has enlarged cul-
ture areas.
MSC characterization
Characterization of isolated Rat-MSCs was performed with fluor-
Downloaded from https://academic.oup.com/ejo/advance-article-abstract/doi/10.1093/ejo/cjz035/5506479 by The Edward G Miner Library user on 21 July 2019
escein isothiocyanate-conjugated CD44, and allophycocyanin-con-
jugated CD90 antibodies have been used as positive markers and
V450-conjugated CD45 (BD Bioscience) has been used as a negative
marker (25, 26) (Figure 2). Flow cytometry analysis was performed
on BD FACSAriaIII and using BD FACSDiva 8.0.1. software.
Green fluorescent protein clone transfection and
hygromycin selection
Green fluorescent protein (GFP) cDNA clone (Cat. No.: AG13105-
G-H; Sino Biological Inc.) was transformed into Escherichia coli
DH5α host as in established protocols and transformants were
selected on ampicillin-containing LB agar (50 µg/ml). MaxiPrep
plasmid isolation procedure was completed by using Endotoxin-
free Plasmid isolation kit (QIAGEN) and confirmation of the
plasmid was checked on agarose gel electrophoresis. GFP and
hygromycin-resistance-gene-containing plasmid was used for
transfection of rat-MSC. In order to determine the minimum in-
hibitory concentration (MIC), a broad range of antibiotic concen-
tration was applied on the cells ranging from 10 μg/ml to 200 μg/
ml, and it has been decided that 50 μg/ml hygromycin was enough
to kill the MSCs that did not take the plasmids. After transfec-
tion, cells were cultured in the presence of 50 μg/ml hygromy-
cin in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen,
Paisley, UK) for 15 days under selective pressure. Those that sur-
vived under the pressure of hygromycin were healthy prolifer-
ated and also were expressing the GFP gene. Concomitantly, the
GFP expression of transfected cells was checked on the fluores-
cent microscope (Nicon Eclipse Ti, Figure 3). After the established
hygromycin resistance, cells were cultured in non-selective pres-
sure media and expanded for application to rats.
Transfer of MSCs to the rats
The transfer of MSCs to the PDL of maxillary first molar teeth
was achieved by the method described in previous studies (21,27).
The DPBS injections were also made by the same method as in
the teeth in the MSC group. Injections were made under general
anaesthesia at the 1st, 4th, 8th, and 12th days of the expansion
phase in the EMSC and EC groups. In the RMSC and RC groups,
MSC and control injections were carried out on the 1st, 4th, 8th,
and 12th and 16th days of the retention phase. For the EC and
RC teeth, 15 µl DPBS solution was injected, whereas for the MSCs
injection groups, 2.5 × 105 cells were prepared in 15 µl of DPBS
solution for each tooth at each time point. Each injection to the
buccal side of the maxillary first molars was performed as infil-
trative and intraligamentary anaesthesia injections from the mid-
buccal point. Attention was paid to not create trauma in the rats
during the injections. The solutions were injected smoothly and
unpressurized.
At the end of 34 days, the experiment and control group rats
were killed with an overdose of an anaesthetic (200 mg/kg sodium
pentobarbital pentol, Abbott, USA). Twenty samples in each group
were allocated as six samples for real-time polymerase chain re-
action (RT-PCR) analysis, six samples for Micro-CT analysis,
six samples for histomorphometric analysis, and two for SEM
observations.
4 European Journal of Orthodontics, 2019
Figure 2. Characterization of the mesenchymal stem cell. Representative flow cytometry results showing the principal mesenchymal stem cell markers; CD44
and CD90 are the positive markers for rat mesenchymal stem cell, whereas CD45 is the negative marker.
Figure 3. Green fluorescent protein (GFP) shining in fluorescent microscopy
images of mesenchymal stem cells transfected with GFP prior to injection
(magnification: ×200).
Measurement of tooth movement
To measure the amount of tooth movement in the buccal direc-
tion, silicone impressions (Zetaplus c-silicone impression material;
Zhermack, Rovigo, Italy) were taken from the upper jaws of the rats
in the EMSC and EC groups just before the placement of the expan-
sion appliance (T0) and at the end of the active expansion process
(T1-before the transpalatal retaining wire placement). Plaster mod-
els were produced by pouring the silicone impressions with plaster
maximum in 10 minutes after taken from rats’ jaw in order to avoid
any distortion or variation on impressions. When the plaster mod-
els of the maxillae were trimmed, they were scanned with 3Shape
Downloaded from https://academic.oup.com/ejo/advance-article-abstract/doi/10.1093/ejo/cjz035/5506479 by The Edward G Miner Library user on 21 July 2019
model scanner (3Shape R700 3D Scanner, 3Shape A/S, Copenhagen,
Denmark) and digital models of maxillae and maxillary teeth were
produced (Figure 4). Digital model analyses on digital models were
carried out using 3Shape 3D software (2010, Version 1.0; 3Shape
A/S, Copenhagen, Denmark). The distance between the top of the
mesiopalatal cusp of the maxillary first molar and the midpoint of
third palatal rugae was measured for the right and left maxillary
molar teeth, and the amount of OTM was calculated by subtracting
the distance values of T0 from T1.
Determining expression levels of OPG, RANKL,
and Cox-2
The extracted teeth containing PDL tissue on their roots were placed
in 500 µl mRNA fixation solution (RNAlater RNA Stabilization
Reagent, Qiagen—Cat. No.76104) and stored at −20°C until RNA
isolation. RNA was isolated from the samples by a QIAamp RNA
Blood Mini Kit (QIAGEN—Cat. No. 52304), according to the man-
ufacturer’s protocol. A First Strand cDNA Synthesis Kit (Thermo
Scientific—Cat. No. K1612) was used for cDNA synthesized from
the RNA isolates. The mRNA expression levels of OPG, RANKL,
and Cox-2 were determined by RT-PCR using the ACTB house-
keeping gene for the internal control. For this purpose, Taqman
probes were used (list of primer and probe sequences used can be
found in the Supplementary Table; the primers were optimized to
give specific band in agarose gel electrophoresis). SuperHotTaq
DNA polymerase (Bioron GmbH) was used in RT-PCR reaction
and Rotor-Gene Q 6-plex (Qiagen) was used as RT-PCR instrument.
Negative (no-template) controls were used in reactions to make sure
that there is no RT-PCR contamination. Expression normalization
was achieved with relative quantitation using ACTB and the follow-
ing equation: 2.012^[(Norm. Ct) − (GOI Ct)] [Norm. Ct: normalizer
Ct value; in this experiment the ACTB gene (housekeeping gene) was
N. Gul Amuk et al.
used as the normalizer. GOI Ct: gene of interest Ct value]. For evalu-
ation of the presence of the GFP-transfected MSCs in the related tis-
sues, GFP mRNA expression was also investigated by conventional
endpoint RT-PCR analysis.
Micro-CT instrumentation and image capture
All maxillary blocks including the roots and surrounding alveolar
bone were subjected to micro-CT image capture using an X-ray
source (SkyScan 1272, Bruker, Belgium) with the potential of 70 kV
and amperage of 142 mA, through 360 degrees with a rotation step
of 0.5 degrees. The conversion of the data that were obtained from
the samples and scanned by micro-CT was achieved by NRecon
(Ver. 1.7.0.4; SkyScan, Bruker, Belgium) and CTAn software (Ver.
1.18; SkyScan, Bruker, Belgium). Serial coronal sections (5 μm) were
obtained such that the maxillae would include all of the molar teeth
crowns and roots.
Figure 4. Digital model of rats’ maxillae. The distance between top of the
mesiopalatal cusp of maxillary first molar and the midpoint of third palatal
rugae was measured for calculation of orthodontic tooth movement.
Figure 5. A cross-sectional slice of mesiobuccal root of the retention-injection
control group teeth. The distance between the deepest point of lacunae
and the outer line of root surface was measured of the deepest resorption
lacunae (a); the width of the lacunae was measured on the largest lacunae of
mesiobuccal and buccal roots (b).
5
Linear root resorption measurements on Micro–
CT images
Linear measurements were taken (millimetres) as the maximum
depth, maximum width of the resorption lacunae, and lengths of
the buccal and mesiobuccal roots. Number of lacunae were counted,
Downloaded from https://academic.oup.com/ejo/advance-article-abstract/doi/10.1093/ejo/cjz035/5506479 by The Edward G Miner Library user on 21 July 2019
and the depth of the deepest resorption lacunae (Figure 5a) and the
width of largest resorption lacunae (Figure 5b) of the mesiobuccal
and buccal roots were measured using the cross-sectional sections on
the CTAn software.
Volumetric root resorption measurements of Micro–
CT images
The buccal and mesiobuccal roots of the maxillary first molar teeth
were selected as the 3D region of interest (ROI) on the CTAn soft-
ware (Figure 6). The upper and lower borders of the ROI frame were
arranged between the beginning of furcation and end of the root
length vertically. The outer and inner borders of the ROI were the
pulp chamber outline and outer root prominences horizontally. After
ROI selection, 3D reconstructed images were obtained (Figure 7),
and volumetric parameters were recorded. Total root volume
(remaining root volume plus resorption lacunae volume), resorp-
tion lacunae volume, and the ratio between the resorption lacunae
volume and total root volume were calculated for the buccal and
mesiobuccal roots of each specimen.
Histological preparation and histomorphometric
analysis
The right and left maxillae were dissected from the rats and fixed by
a formaldehyde solution (104002; Merck, Germany) of 4% concen-
tration, decalcified with a nitric acid solution (438073; Sigma, USA)
Figure 6. Region of interest selection on cross-sectional slice of mesiobuccal
root of the retention-injection control group teeth.
6 European Journal of Orthodontics, 2019
and then embedded in paraffin blocks. Serial horizontal sections of
5 μm from paraffin blocks were taken on slides. The paraffin of the
prepared sections was removed by standard histological methods
via xylol (108684; Merck, Germany) and passed through a graded
alcohol series. The sections were examined by staining with H&E
(105174; Merck; 446602; Carlo Erba) and Picrosirius Red (365548;
Sigma, USA). To evaluate the cementum thickness, one of each five
sections were selected from the cementoenamel junction to the ap-
ical end of the buccal root and investigated at ×40 magnification
(Olympus DP71 microscope). Cementum thickness measurement
was performed using the ImageJ software in a circular order on the
buccal side of the selected sections, and the average values were cal-
culated for all specimens (Figure 8).
Figure 7. Three-dimensional reconstructed images: resorption on the buccal
side of a coronal slice of root (mesiobuccal root of the retention-injection
control group teeth) (a), and healthy root surfaces on the buccal and palatal
side of a coronal slice of root (mesiobuccal root of the negative control group
teeth) (b). B, buccal alveolar side; P, palatal alveolar side.
Figure 8. Picrosirius staining of specimens from maxillary first molar
teeth of rats and circular order during cementum thickness measurement
(magnification: ×40). P, pulp; D, dentin; C, cementum; PDL, periodontal
ligament.
Scanning electron microscopy
The extracted teeth were submerged in 1% sodium hypochlorite
and then centrifuged at 1000 rpm. The specimens were dried for
1 week at 40°C in an incubator, and then, they were placed on a
retainer, plated with gold and observed with an SEM device (Zeiss,
GeminiSEM 500-71-08 Electron Microscopy, Germany). The re-
Downloaded from https://academic.oup.com/ejo/advance-article-abstract/doi/10.1093/ejo/cjz035/5506479 by The Edward G Miner Library user on 21 July 2019
sorptive changes on the mesiobuccal and buccal root surfaces were
evaluated in a structural manner on the SEM images (Figure 9).
Method error
The same investigator performed the measurements, and Micro-CT
measurements and tooth movement measurements were repeated
after 2 weeks’ interval by the same investigator. To assess the re-
producibility and reliability of the measurements, the Pearson and
Spearman’s correlation coefficients and Cronbach’s reliability tests
were applied. Cronbach’s alpha coefficients for intraclass correlation
were found to be within a range of 0.911–0.939 for OTM meas-
urements, 0.912–0.986 for linear Micro-CT measurements, and
0.885–0.989 for volumetric Micro-CT measurements. Correlation
coefficients were found to yield sufficient reliability for tooth move-
ment measurements (0.899–0.937), linear Micro-CT measurements
(0.809–0.993), and volumetric Micro-CT measurements (0.808–
0.981). Paired t-tests and Wilcoxon signed rank tests were used to
detect any systematic differences between the initial and replicated
measurements and revealed that the differences between the first and
second measurements were insignificant.
Statistical analysis
The data are presented as means standard deviation and 95% con-
fidence interval (CI). The Shapiro–Wilk normality test and Levene’s
variance homogeneity test were applied to the data. The Kruskal–
Wallis analysis was used for the inter-group comparisons of the
RT-PCR, Micro-CT, and histological analysis data that were not
normally distributed, whereas body weight and OTM data that
were found normally distributed and to have homogeneity of vari-
ance among the groups were tested by paired t-tests. Mann–Whitney
U-test was preferred for the pairwise comparisons. A P value of
smaller than 0.05 was accepted to be statistically significant.
Results
The rats gained weight 16.10 ± 14.40 g (95% CI: 9.16–23.04) in
RMSC and RC animals, 19.94 ± 17.40 g (95% CI: 11.55–28.33) in
EMSC and EC animals, and 32.65 ± 16.76 g (95% CI: 25.10–40.79)
in NC group animals until the end of the experiment. Weight gain in
NC group was significantly greater than those in experiment groups
(P < 0.010) whereas there was no significant difference between ex-
periment group animals. Three animals died because of the possible
reaction to the frequent and sequential general anaesthesia proce-
dures, and new animals were added to the experiment in place of
the missing ones.
Tooth movement
Statistically significant tooth movement was observed in both the
EMSC (0.91 mm) and EC (0.77 mm) groups during the expansion
phase (Table 1, P < 0.001). In the EMSC group, the amount of OTM
was found to be higher than that in the EC group, and the difference
was significant (P < 0.001).
N. Gul Amuk et al.
RT-PCR analysis
The GFP mRNA expression results revealed that there was GFP ex-
pression in the specimens of the EMSC and RMSC groups, which
indicates the presence of GFP-transfected MSC in PDL and the suc-
cess of MSC transfer.
There was a statistically significant difference among the Cox-2
(P < 0.001), OPG (P < 0.010), and RANKL (P < 0.011) levels, and
OPG/RANKL ratios (P < 0.003) of the groups (Table 2). The high-
est Cox-2 values were detected in the EC and RC groups, whereas
the lowest was in the NC group. The EMSC group had significantly
higher Cox-2 levels than those in the NC and RMSC groups (Table 3,
P < 0.009, P < 0.028 respectively). The OPG levels of the EMSC, RMSC,
EC, and RC groups were close to each other, whereas the NC groups’
value was significantly lower than those in the experiment groups. In the
RANKL evaluation, the lowest value was found in the NC group, and
there was no significant difference among the other groups, although
the RANKL values of the RMSC group were lower than those in the
EMSC, RC, and EC groups. The OPG/RANKL ratio was found to be
similar in the EMSC and RMSC groups and in the EC and RC groups.
Micro-CT analysis
The buccal and mesiobuccal roots volume and MB root length
values did not significantly differ among the groups (Table 4). The
EMSC and RMSC groups’ volumetric resorptive measurements of
the buccal and mesiobuccal roots were found to be close each other,
significantly higher than that of the NC group, and significantly
lower than those of the EC and RC groups (Table 5, P < 0.001). The
EMSC and RMSC groups’ buccal and mesiobuccal roots’ lacunae
counts were similar, whereas they were higher than that of the NC
Figure 9. Scanning electron microscopy images: resorption lacunae on the
buccal surfaces of buccal and mesiobuccal roots, ×40 (a) and ×1000 (b). B,
buccal alveolar side; HC, healthy cement; MB, mesiobuccal; RC, resorption
lacunae.
Table 1. Comparison of molar-midline distance at T0 and T1 and tooth movement amount of EMSC and EC groups. EMSC, expansion
mesenchymal stem cell injection; EC, expansion-injection control; T0, before the expansion; T1, end of the expansion, Diff, mean difference
between molar-midline distance from T0 to T1 and difference between tooth movement amount of EMSC and EC groups; SD, standard de-
viation; CI, confidence interval as lower-upper bound
EMSC (mm)
Mean ± SD 95% CI for Mean
T0 2.70 ± 0.20 2.60–2.79
T1 3.61 ± 0.14 3.54–3.68
Diff 0.91 ± 0.18 0.82–1.00
P <0.001
There is statistically significant difference at P < 0.05.
7
group and lower than those in the EC and RC groups (Table 4),
although a statistically significant difference among the experiment
groups was found only in the mesiobuccal roots’ lacuna count values
(Table 5, P < 0.005 to P < 0.037). In the linear measurements, the
B root length of the NC group was significantly higher than those
in the EC and RC groups (Table 5, P < 0.009), whereas the values
Downloaded from https://academic.oup.com/ejo/advance-article-abstract/doi/10.1093/ejo/cjz035/5506479 by The Edward G Miner Library user on 21 July 2019
of the other groups were close to each other. Although the lacunae
width and depth measurements of the buccal roots were lower in
the RMSC group than those in the EMSC group (Table 4), a sig-
nificant difference was found only between the NC group and the
experiment groups (Table 5, P < 0.006 to P < 0.043). In the lacunae
width measurement of the mesiobuccal roots, a statistically signifi-
cant difference was found in the RMSC-EC and RMSC-RC pairwise
matches (Table 5, P < 0.016, P < 0.021 respectively). The lacunae
depth of the mesiobuccal root was found to be significantly lower
in the NC group in comparison to the experiment groups (Table 5,
P < 0.009, P < 0.044), whereas there was no significant difference
among the EMSC, RMSC, EC, and RC groups.
As a general observation, it may be stated that the resorptive
changes were greater in the EMSC group than those in the RMSC
group although the differences were not statistically significant for
all pairwise comparison results.
Histological measurements
The cementum thickness values of the EMSC and RMSC groups
were similar and significantly higher than those in the EC and RC
groups (Tables 4 and 5, P < 0.018 to P < 0.021).
SEM observations
The buccal surfaces of the NC group specimens exhibited healthy areas
covered by undamaged and smooth cementum, whereas the deepest
and extensive resorption lacunae were observed in the EC and RC
groups with views of dentin tubules through the craters. The repair
layer on the resorption lacunae was recognized with smooth cement
surface coverage onto the dentin tubule orifices in the crater or with
shallowing of resorption craters in comparison to near lacunae borders.
Discussion
Easy accessibility of sources such as the adipose tissue of oral cavity,
periodontal and pulp tissues of primary teeth, or extracted wisdom
teeth has made MSC available in orthodontics (24). Prevention and
repair of OIRR and acceleration of OTM maintain their clinical im-
portance for orthodontists for years. Our study aimed to present the
effects of MSCs’ transfer to the PDL tissues on OIRR and OTM rate
during arch expansion.
EC (mm)
Mean ± SD 95% CI for Mean P
2.65 ± 0.17 2.57–2.73 0.066
3.42 ± 0.20 3.32–3.52 <0.001
0.77 ± 0.18 0.68–0.85 <0.001
<0.001
8 European Journal of Orthodontics, 2019

For limitation of the inter-animal variation in response to

MSC injection; RC, retention injection control; NC, negative control; SD, standard deviation; CI, confidence interval as lower-upper bound; OPG, osteoprotegerin; RANKL, receptor activator of

0.001
0.010
Table 2. Intergroup comparisons of Cox-2, OPG, RANKL, and OPG/RANKL ratio values. EMSC, expansion mesenchymal stem cell injection; EC, expansion-injection control; RMSC, retention

0.011
0.003
P
metabolic activity, a split-mouth design was used in this study.
Adipose-derived MSCs were preferred due to their high potential

0.0014–0.0019
0.0002–0.0005
0.0000–0.0000
of differentiation to osteogenic and PDL differentiation (20,28).

4.72–7.19
Isolated MSCs were characterized using mesenchymal markers

for Mean
95% CI
CD44 and CD90. In order to prove the cells were not originated

Downloaded from https://academic.oup.com/ejo/advance-article-abstract/doi/10.1093/ejo/cjz035/5506479 by The Edward G Miner Library user on 21 July 2019
from blood vessels of adipose tissue, hematopoietic marker CD45
was investigated in flow cytometry analysis. The transfer of MSCs

0.0017 ± 0.0002
0.0004 ± 0.0001
0.0001 ± 0.0000
to the PDL of maxillary first molar teeth was performed accord-

5.96 ± 0.99
ing to the injection method described in previous studies (21, 27).
Mean ± SD
However, the amount of solution for injections reduced to 15 µl
with same amount of MSCs stated, because 2-point injection as
NC

midbuccal side infiltrative and midbuccal intraligamentary was

preferred for this study instead of the 4-point injection as infiltra-
0.0036–0.0096
0.0001–0.0018
0.0001–0.0013 tive and intraligamentary injections from the mesiovestibuler and
0.71–2.26 mesiopalatal corners. Because of the intraligamentary pressure,
for Mean
95% CI

some solution material including MSCs could be lost. So more so-

lution material was prepared than necessary. No bleeding or in-
flammation signs were observed after injections. The frequency of
0.0067 ± 0.0028
0.0010 ± 0.0006
0.0007 ± 0.0005

MSCs’ transfer injections were determined according to the reports

1.53 ± 0.58

of Hayashi et al. (29) and Robey et al. (30), which indicate the
Mean ± SD

short life span of transplanted stem cells about 3 days in host tis-
sue. Robey et al. (30) also stated that because most of the cells die
RC

in the first few days after transplantation into the tissue, thera-
peutic effects occurred in 3 days would be likely to persist long
0.0019–0.0031
0.0008–0.0017
0.0003–0.0004

term. For these reasons, all injections were performed every 3 days
2.07–5.32

in injection groups. At the end of the experiment, the presence of

for Mean
95% CI

GFP-transfected cells in PDL tissue was checked by the detection

of the GFP at mRNA level, which is an indication of cells’ stability
maintaining plasmid and is an evidence for reaching of the MSC to
0.0013 ± 0.0004
0.0004 ± 0.0000
0.0025 ± 0.005

the PDL tissue for regeneration. For the internal control of RT-PCR
3.70 ± 1.31

analysis, ACTB housekeeping gene, which is one of the most com-

Mean ± SD

monly used reference gene, (31) was selected. Although Kirschneck

RMSC

et al. (32) reported that PPIB and YWHAZ are more stable refer-
ence genes, Svingen et al. (31) stated that a gene does not have to
rank on top of the list to be suitable for normalization purposes as
0.0048–0.0090
0.0004–0.0015
0.0002–0.0012
0.58–2.87

long as they perform better than the exclusion criteria because of

for Mean

the discrepancy in stability rankings and contradiction in outcomes

95% CI

between different analytical methods.

The arch expansion was achieved by the movement of the max-
illary first molar teeth of rats towards the vestibular alveolar bone.
0.0070 ± 0.0016
0.0010 ± 0.0005
0.0007 ± 0.0003

The EC group showed 0.77 mm of tooth movement, which was

1.73 ± 0.92
Mean ± SD

slightly higher than the findings of Gonzales et al. (13) who deter-
mined 0.40–0.64 mm of OTM with applications of 50 g of force.
nuclear factor kappa B ligand; Cox-2, cyclooxygenase-2

This difference may be attributed to the different OTM measure-

EC

ment methods and different direction of OTM. With the transfer

There is statistically significant difference at P < 0.05.
0.0028–0.0046
0.0003–0.0015
0.0000–0.0014

of MSCs to the PDL tissues, the amount of OTM was increased to

0.25–3.98

0.91 mm, and the difference was statistically significant. Although

for Mean

hypotheses and estimations have been developed on the effects of

95% CI

MSCs on OTM, the main effects have not been reported previ-
ously. Adipose-derived MSCs were known to have high potential
of differentiation to osteogenic and PDL differentiation (20, 28),
0.0038 ± 0.0007
0.0009 ± 0.0005
0.0007 ± 0.0005
2.12 ± 0.1.50

and stem cells in PDL (PDLSC), which have MSC characteristics

Mean ± SD

(18), are known to have important roles in periodontal and osse-

ous remodelling during OTM (17). Zhang et al. (33) found that
EMSC

the track markers of PDLSC increased on both of the compression

and tension sides after 3 days of orthodontic force application. In
OPG/RANKL

addition, inflammatory markers such as interleukin (IL)-11 and

Cox-2 produced by stem cell induction in PDL regulate the pro-
RANKL

liferation and differentiation of osteoclasts and osteoblasts as well

Cox-2
OPG

as bone remodelling (34, 35, 36). In the light of this information,

N. Gul Amuk et al.
Table 3. Pairwise comparisons of Cox-2, OPG, RANKL level, and OPG/RANKL ratio values among groups. EMSC, expansion mesenchymal
stem cell injection; EC, expansion-injection control; RMSC, retention MSC injection; RC, retention injection control; NC, negative control;
OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor kappa B ligand; Cox-2, cyclooxygenase-2
Pairwise comparisons (P values)
EMSC/EC EMSC/RMSC EMSC/RC
Cox-2 0.009 0.028 0.082
OPG 0.754 0.175 0.931
RANKL 0.917 0.754 0.662
OPG/RANKL 0.917 0.117 0.662
Bold value indicates statistically significant difference at P < 0.05.
the possible stimulation of PDLSC by the transferred MSCs (37)
or direct MSCs activity in PDL tissues may exhibit an accelerat-
ing effect on OTM by accelerating the bone remodelling process
through the osteoblast–osteoclast turnover. In addition, PDLSC
cells reduce collagen I expression with OTM and recover the col-
lagen synthesis after force removal (14), and this mechanism was
previously interpreted as the possible OTM accelerator ability of
increased MSCs. (24). Furthermore, transplantation of stem cells
into the pressure site was hypothetically estimated to provide ac-
celeration of OTM, which was dependent on the idea of the delay
period prevention caused by hyalinized necrotic tissue removal
(24). Similar to these predictions, the comments of Huang et al.
(17) as ‘Due to their proliferation and differentiation potential,
stem cells in PDL could potentially be used for accelerating the
procedure of orthodontic treatment’ also supported our results
regarding OTM acceleration with MSCs’ transfer to PDL.
Resorption lacunae formation was evaluated on the buccal and
mesiobuccal roots, which were selected because of the midbuccal
location of the buccal root and the largest size of the mesiobuccal
root of the rats. Volumetric and linear resorption measurements
were performed on Micro-CT imaging whereas structural exam-
ination was also achieved by SEM images. Micro-CT evaluation
has been accepted as a precise and useful tool for both 2D and 3D
structural analysis of dental tissues by presenting serial sections in
any plane and 3D reconstructions (38). The similar root volume
values of the mesiobuccal roots in all groups were compatible with
the linear root length measurement results. As observed in the SEM
images, the main resorption lacunae were observed on the buccal
surfaces of the buccal and mesiobuccal roots rather than apical
root resorption, which is characterized by shortening of the root.
However, the buccal root length was found to be shorter in the EC
and RC groups than that in the NC group. This situation was inter-
preted with the small size and vulnerable structure of the buccal
root where the resorption process affected both the buccal surfaces
and length of the root. Nevertheless, the buccal root lengths of the
EMSC and RMSC groups were similar to each other and with the
NC group.
With MSCs’ transfer, the lacunae count values significantly
decreased in both the EMSC and RMSC groups. In compliance
with this finding, the resorption volume and resorption percentage
measurements were also decreased with MSCs application for the
expansion and retention periods. These results made think that
MSCs’ transfer probably provided an increase in the activation
and/or differentiation of periodontal MSCs, which differentiated
into reparative cells as osteoblasts or cementoblasts (37, 39) in
addition to direct differentiation transferred MSCs to reparative
cells. MSCs’ transfer during the expansion period presented both
9
EMSC/NC EC/RMSC EC/RC EC/NC RMSC/RC RMSC/NC RC/NC
Downloaded from https://academic.oup.com/ejo/advance-article-abstract/doi/10.1093/ejo/cjz035/5506479 by The Edward G Miner Library user on 21 July 2019
0.009 0.009 0.998 0.009 0.045 0.016 0.004
0.008 0.251 0.715 0.015 0.068 0.008 0.009
0.009 0.117 0.855 0.009 0.170 0.009 0.004
0.009 0.047 0.998 0.009 0.011 0.028 0.004
OTM acceleration and reparative effects for root resorption. The
metabolic request may lead MSCs to accelerate bone remodelling,
and tooth movement may be achieved with minimal or no necrotic
hyalinized tissue, which causes tooth movement delay and root re-
sorption during OTM. Moreover, IL-11 activated by Cox-2 with
force application is known to stimulate osteoblastic markers and
cementoblast-specific markers and increase the expression of bone
sialoprotein, which indicates that force-induced IL-11 stimulates
MSCs into differentiating to osteoblasts or cementoblasts during
the tooth movement (39). In addition, the cementum thickness val-
ues that were higher in the EMSC group than those in the EC and
RC groups supported the prevention effects during the expansion
period. So, it may be suggested that resorption lacunae formation
might be prevented in the expansion period by MSCs’ transfer.
However, the possible mechanisms of the results regarding acceler-
ated OTM and reduction of OIRR should be examined and clari-
fied in detail in future studies.
The RMSC group’s volumetric and linear resorption values were
slightly lower than those in the EMSC group, although the difference
was not significant. The retention period is known as the repair phase
of resorption lacunae by the mechanism of deposition of cementum
(16), which is activated after force removal as reattachment of PDL
fibres and collagen within the mineral layer (14, 22). MSCs’ transfer
may stimulate and enhance this healing process. In a supporting way
of this prediction, the OPG level was found higher, and the RANKL
level was found lower in the RMSC group than those in the EMSC
group, and these markers are well-known determinants of remod-
elling of hard tissues (40). In the light of these results, the slightly
lower resorptive changes in the RMSC group than those in the EMSC
group may be due to MSCs’ transfer time in the RMSC group that
probably created an enhancing effect on the natural healing period.
When evaluating all these findings, the possible undesired side-
effects of MSCs’ transfer to the related tissues should be kept in
mind. In the case of the distribution of locally transferred MSCs into
the systemic blood flow, uncontrolled migration and differentiation
potentials and some possible immortalization/transformation prop-
erties make it necessary to the implementation of relevant controls of
MSCs transplantation. Although no significant adverse events have
been reported at the 80 trials registered since 1995 (41), all risks
should be investigated in details at the further experimental animal
studies before using in humans.
In addition, due to limitations in study design such as add-
itional control groups like expansion/retention without injections
and injections of non-MSC groups could not be included because
of the aim of limited use of animals, and due to the limited general-
izability of the animal models to the biologic processes in humans,
the clinical applicability of this data is uncertain and further studies
 10

Table 4. Intergroup comparisons of volumetric (mm3) and linear (mm) micro-computed tomography measurements and histological cementum thickness (mm) values. EMSC, expansion
mesenchymal stem cell injection; EC, expansion-injection control; RMSC, retention MSC injection; RC, retention-injection control; NC, negative control; B, buccal; MB, mesiobuccal; resorp.,
resorption; vol., volume; SD, standard deviation; CI, confidence interval as lower-upper bound

EMSC EC RMSC RC NC

95% CI 95% CI 95% CI 95% CI 95% CI

Mean ± SD for Mean Mean ± SD for Mean Mean ± SD for Mean Mean ± SD for Mean Mean ± SD for Mean P

B-root vol. 0.127 ± 0.049 0.066–0.188 0.127 ± 0.039 0.079–0.175 0.121 ± 0.008 0.111–0.131 0.126 ± 0.025 0.095–0.157 0.149 ± 0.059 0.076–0.223 0.824
B-resorp. vol. 0.003 ± 0.001 0.001–0.004 0.016 ± 0.004 0.010–0.021 0.002 ± 0.001 0.001–0.003 0.023 ± 0.001 0.020–0.026 0.001 ± 0.000 0.000–0.002 <0.001
B-resorp. Per cent 0.023 ± 0.013 0.006–0.039 0.135 ± 0.044 0.079–0.190 0.020 ± 0.005 0.012–0.027 0.157 ± 0.125 0.001–0.313 0.007 ± 0.003 0.002–0.012 0.001
MB-root vol. 1.108 ± 0.025 1.077–1.139 1.149 ± .087 1.040–1.258 1.207 ± 0.032 1.167–1.247 1.246 ± 0.154 1.054–1.438 1.097 ± 0.164 0.893–1.301 0.188
MB-resorp. vol. 0.047 ± 0.021 0.021–0.073 0.094 ± 0.013 0.078–0.110 0.037 ± 0.007 0.027–0.046 0.100 ± 0.018 0.078–0.122 0.017 ± 0.005 0.010–0.023 <0.001
MB-resorp. per cent 0.042 ± 0.018 0.019–0.065 0.082 ± 0.015 0.063–0.102 0.031 ± 0.006 0.022–0.038 0.081 ± 0.013 0.064–0.097 0.016 ± 0.006 0.007–0.024 <0.001
B-lacunae count 1.80 ± 0.83 0.76–2.83 2.20 ± 1.30 0.58–3.81 1.20 ± 0.83 0.16–2.23 2.20 ± 0.84 1.16–3.23 0.40 ± 0.04 0.32–0.47 0.035
B-Root length 1.983 ± 0.113 1.842–2.125 1.792 ± 0.204 1.537–2.046 1.859 ± 0.142 1.683–2.036 1.186 ± 0.098 1.693–1.938 2.055 ± 0.063 1.976–2.134 0.022
B-lacunae width 0.146 ± 0.067 0.062–0.229 0.218 ± 0.092 0.103–0.332 0.088 ± 0.060 0.012–0.163 0.168 ± 0.046 .110-.225 0.002 ± 0.001 0.000–0.004 <0.001
B-lacunae depth 0.054 ± 0.021 0.026–0.081 0.080 ± 0.033 0.037–0.122 0.036 ± 0.025 0.004–0.067 0.072 ± 0.038 0.0243–0.119 0.004 ± 0.001 0.002–0.006 0.002
MB-lacunae count 2.60 ± 0.89 1.48–3.71 4.00 ± 0.70 3.12–4.87 2.20 ± 0.45 1.64–2.75 4.40 ± 0.89 3.28–5.51 0.80 ± 0.45 0.24–1.35 <0.001
MB-root length 2.470 ± 0.060 2.388–2.552 2.378 ± 0.092 2.263–2.493 2.519 ± 0.128 2.36–2.67 2.480 ± 0.046 2.421–2.538 2.639 ± 0.146 2.457–2.821 0.135
MB-lacunae width 0.262 ± 0.113 0.120–0.403 0.412 ± 0.170 0.200–0.623 0.186 ± 0.051 0.122–0.249 0.396 ± 0.174 0.179–0.612 0.170 ± 0.297 0.199–0.539 0.011
MB-lacunae depth 0.108 ± 0.063 0.028–0.187 0.112 ± 0.056 0.042–0.181 0.102 ± 0.049 0.040–0.163 0.170 ± 0.065 0.088–0.251 0.030 ± 0.018 0.006–0.053 <0.001
Cementum thickness (mm) 0.007 ± 0.002 0.005–0.009 0.003 ± 0.001 0.002–0.004 0.006 ± 0.001 0.004–0.007 0.004 ± 0.001 0.002–0.005 0.008 ± 0.000 0.007–0.008 0.006

There is statistically significant difference at P < 0.05.

European Journal of Orthodontics, 2019

Downloaded from https://academic.oup.com/ejo/advance-article-abstract/doi/10.1093/ejo/cjz035/5506479 by The Edward G Miner Library user on 21 July 2019
N. Gul Amuk et al. 11

Table 5. Pairwise comparisons of volumetric (mm3) and linear (mm) micro-computed tomography measurements and histological cemen-
tum thickness (mm) values among groups. EMSC, expansion mesenchymal stem cell injection; EC, expansion-injection control; RMSC,
retention MSC injection; RC, retention injection control; NC, negative control; B, buccal; MB, mesiobuccal; resorp., resorption; vol., volume;
SD, standard deviation

Pairwise comparisons (P values)

Downloaded from https://academic.oup.com/ejo/advance-article-abstract/doi/10.1093/ejo/cjz035/5506479 by The Edward G Miner Library user on 21 July 2019
EMSC/EC EMSC/RMSC EMSC/RC EMSC/NC EC/RMSC EC/RC EC/NC RMSC/RC RMSC/NC RC/NC

B-resorp. vol. 0.009 0.917 0.009 0.047 0.009 0.602 0.009 0.009 0.028 0.009
B-resorp. per cent 0.009 0.754 0.028 0.016 0.009 0.754 0.009 0.009 0.009 0.009
MB-resorp. vol. 0.009 0.602 0.009 0.016 0.009 0.917 0.009 0.009 0.016 0.009
MB-resorp. per cent 0.009 0.251 0.009 0.016 0.009 0.917 0.009 0.009 0.016 0.009
B-lacunae count 0.661 0.316 0.439 0.037 0.230 0.914 0.030 0.151 0.140 0.021
B-root length 0.073 0.295 0.059 0.251 0.834 0.674 0.009 0.754 0.076 0.009
B-lacunae width 0.141 0.203 0.462 0.007 0.036 0.346 0.007 0.028 0.034 0.007
B-lacunae depth 0.070 0.435 0.219 0.006 0.014 0.192 0.007 0.101 0.043 0.007
MB-lacunae count 0.037 0.439 0.024 0.006 0.009 0.371 0.006 0.009 0.005 0.006
MB-lacunae width 0.251 0.347 0.175 0.117 0.016 0.917 0.117 0.021 0.117 0.117
MB-lacunae depth 0.988 0.833 0.171 0.009 0.917 0.172 0.012 0.141 0.044 0.009
Cementum thickness 0.021 0.076 0.021 0.593 0.018 0.191 0.034 0.018 0.028 0.034

Bold value indicates statistically significant difference at P < 0.05.

should be conducted for obtaining knowledge regarding possible Funding

clinical effects of the advanced approaches introduced in this study.
In the case that these therapeutic applications could be improved
for clinical practice, MSC injections during the retention period
may be preferred in the repair of OIRR that occurs after RME pro-
tocols where an acceleration in OTM would not be desired during
the active expansion phase. On the other hand, if an acceleration
in OTM and simultaneous prevention and/or repair of OIRR are
required, MSC transfer can be performed during expansive ortho-
dontic force application.
Conclusions
Within the limitations of the current study, it can be concluded that
1. OIRR was observed on the buccal surfaces of the buccal and
mesiobuccal roots of the maxillary first molar teeth as a result of
50 g force application towards the vestibular alveolar bone dur-
ing arch expansion.
2. MSCs’ transfer to the PDL of rats’ maxillary first molar teeth was
achieved by the previously described injection method.
3. MSCs’ transfer to PDL during expansive orthodontic force appli-
cation towards the vestibular alveolar bone increased the amount
of OTM.
4. At the evaluation of the teeth that received MSCs during the
expansion and retention periods, the resorption lacunae count,
volumetric-linear resorptive measurements, Cox-2, and RANKL
mRNA expression levels reduced, and OPG level, OPG/RANKL
ratio, and cementum thickness in comparison to the EC and RC
groups increased.
5. The prevention and/or repair of OIRR was found slightly more
in teeth that received MSCs during the retention period than in
the teeth that received MSCs during the expansion period.
Supplementary material
Supplementary material is available at European Journal of
Orthodontics online.
The Scientific and Technological Research Council of Turkey (TUBITAK) with
the project number 116S400.
Conflict of Interest
None declared.
References
1. Pandis, N., Polychronopoulou, A. and Eliades, T. (2007) Self-ligating vs
conventional brackets in the treatment of mandibular crowding: a pro-
spective clinical trial of treatment duration and dental effects. American
Journal of Orthodontics and Dentofacial Orthopedics, 132, 208–215.
2. Damon, D.H. (1998) The Damon low-friction bracket: a biologically
compatible straight-wire system. Journal of Clinical Orthodontics, 32,
670–680.
3. Rungcharassaeng, K., Caruso, J.M., Kan, J.Y., Kim, J. and Taylor, G.
(2007) Factors affecting buccal bone changes of maxillary posterior teeth
after rapid maxillary expansion. American Journal of Orthodontics and
Dentofacial Orthopedics, 132, 428.e1–428.e8.
4. Garib, D.G., Henriques, J.F., Janson, G., de Freitas, M.R. and Fer-
nandes, A.Y. (2006) Periodontal effects of rapid maxillary expansion with
tooth-tissue-borne and tooth-borne expanders: a computed tomography
evaluation. American Journal of Orthodontics and Dentofacial Ortho-
pedics, 129, 749–758.
5. Garib, D.G., Henriques, J.F., Janson, G., Freitas, M.R. and Coelho, R.A.
(2005) Rapid maxillary expansion–tooth tissue-borne versus tooth-borne
expanders: a computed tomography evaluation of dentoskeletal effects.
The Angle Orthodontist, 75, 548–557.
6. Melsen, B. (1999) Biological reaction of alveolar bone to orthodontic
tooth movement. The Angle Orthodontist, 69, 151–158.
7. Krishnan, V. and Davidovitch, Z. (2006) Cellular, molecular, and tissue-
level reactions to orthodontic force. American Journal of Orthodontics
and Dentofacial Orthopedics, 129, 469.e1–469.32.
8. Vardimon, A.D., Graber, T.M., Voss, L.R. and Lenke, J. (1991) Determi-
nants controlling iatrogenic external root resorption and repair during
and after palatal expansion. The Angle Orthodontist, 61, 113–22; discus-
sion 123.
9. Odenrick, L., Karlander, E.L., Pierce, A. and Kretschmar, U. (1991) Sur-
face resorption following two forms of rapid maxillary expansion. Euro-
pean Journal of Orthodontics, 13, 264–270.
12
10. Brezniak, N. and Wasserstein, A. (2002) Orthodontically induced in-
flammatory root resorption. Part I: The basic science aspects. The Angle
Orthodontist, 72, 175–179.
11. Thilander, B., Nyman, S., Karring, T. and Magnusson, I. (1983) Bone
regeneration in alveolar bone dehiscences related to orthodontic tooth
movements. European Journal of Orthodontics, 5, 105–114.
12. Steiner, G.G., Pearson, J.K. and Ainamo, J. (1981) Changes of the marginal
periodontium as a result of labial tooth movement in monkeys. Journal of
Periodontology, 52, 314–320.
13. Gonzales, C., Hotokezaka, H., Yoshimatsu, M., Yozgatian, J.H., Darende-
liler, M.A. and Yoshida, N. (2008) Force magnitude and duration effects
on amount of tooth movement and root resorption in the rat molar. The
Angle Orthodontist, 78, 502–509.
14. Feng, L., Yang, R., Liu, D., Wang, X., Song, Y., Cao, H., He, D., Gan, Y.,
Kou, X. and Zhou, Y. (2016) PDL progenitor-mediated PDL recovery con-
tributes to orthodontic relapse. Journal of Dental Research, 95, 1049–1056.
15. Owman-Moll, P. and Kurol, J. (1998) The early reparative process of
orthodontically induced root resorption in adolescents–location and type
of tissue. European Journal of Orthodontics, 20, 727–732.
16. Barber, A.F. and Sims, M.R. (1981) Rapid maxillary expansion and ex-
ternal root resorption in man: a scanning electron microscope study.
American Journal of Orthodontics, 79, 630–652.
17. Huang, H., Yang, R. and Zhou, Y. (2018) Mechanobiology of periodontal
ligament stem cells in orthodontic tooth movement. Stem Cells Inter-
national, 2018, 1–7.
18. Seo, B.M., Miura, M., Gronthos, S., Bartold, P.M., Batouli, S., Brahim, J.,
Young, M., Robey, P.G., Wang, C.Y. and Shi, S. (2004) Investigation of
multipotent postnatal stem cells from human periodontal ligament. Lancet
(London, England), 364, 149–155.
19. Wada, N., Menicanin, D., Shi, S., Bartold, P.M. and Gronthos, S. (2009)
Immunomodulatory properties of human periodontal ligament stem cells.
Journal of Cellular Physiology, 219, 667–676.
20. Venkataiah, V.S., et al. (2019) Periodontal regeneration by allogeneic
transplantation of adipose tissue derived multi-lineage progenitor stem
cells in vivo. Scientific Reports, 9, 921.
21. Amuk, N.G., Kurt, G., Baran, Y., Seyrantepe, V., Yandim, M.K., Adan, A.,
Demir, S.A., Kiraz, Y. and Sonmez, M.F. (2017) Effects of cell-mediated
osteoprotegerin gene transfer and mesenchymal stem cell applications on
orthodontically induced root resorption of rat teeth. European Journal of
Orthodontics, 39, 235–242.
22. Franzen, T.J., Brudvik, P. and Vandevska-Radunovic, V. (2013) Peri-
odontal tissue reaction during orthodontic relapse in rat molars. European
Journal of Orthodontics, 35, 152–159.
23. Liu, H.C., E, L.L., Wang, D.S., Su, F., Wu, X., Shi, Z.P., Lv, Y. and
Wang, J.Z. (2011) Reconstruction of alveolar bone defects using bone
morphogenetic protein 2 mediated rabbit dental pulp stem cells seeded on
nano-hydroxyapatite/collagen/poly(L-lactide). Tissue Engineering. Part A,
17, 2417–2433.
24. Safari, S., Mahdian, A. and Motamedian, S.R. (2018) Applications of stem
cells in orthodontics and dentofacial orthopedics: current trends and fu-
ture perspectives. World Journal of Stem Cells, 10, 66–77.
25. Huang, S., Xu, L., Sun, Y., Lin, S., Gu, W., Liu, Y., Zhang, J., Chen, L.
and Li, G. (2016) Systemic administration of allogeneic mesenchymal stem
cells does not halt osteoporotic bone loss in ovariectomized rats. PLoS
One, 11, 1–15.
European Journal of Orthodontics, 2019
26. Tong, H., Wang, C., Huang, Y., Shi, Q., Fernandes, J.C., Dai, K., Tang, G.
and Zhang, X. (2013) Polyethylenimine600-β-cyclodextrin: a promising
nanopolymer for nonviral gene delivery of primary mesenchymal stem
cells. International Journal of Nanomedicine, 8, 1935–1946.
27. Gül Amuk, N., Kurt, G., Kartal Yandim, M., Adan, A. and Baran, Y.
(2018) A minimally invasive transfer method of mesenchymal stem cells to
Downloaded from https://academic.oup.com/ejo/advance-article-abstract/doi/10.1093/ejo/cjz035/5506479 by The Edward G Miner Library user on 21 July 2019
the intact periodontal ligament of rat teeth: a preliminary study. Turkish
Journal of Biology, 42, 382–391.
28. Tobita, M., Uysal, A.C., Ogawa, R., Hyakusoku, H. and Mizuno, H.
(2008) Periodontal tissue regeneration with adipose-derived stem cells.
Tissue Engineering. Part A, 14, 945–953.
29. Hayashi, M., Li, T.S., Ito, H., Mikamo, A. and Hamano, K. (2004) Com-
parison of intramyocardial and intravenous routes of delivering bone
marrow cells for the treatment of ischemic heart disease: an experimental
study. Cell Transplantation, 13, 639–647.
30. Robey, T.E., Saiget, M.K., Reinecke, H. and Murry, C.E. (2008) Systems
approaches to preventing transplanted cell death in cardiac repair. Journal
of Molecular and Cellular Cardiology, 45, 567–581.
31. Svingen, T., Letting, H., Hadrup, N., Hass, U. and Vinggaard, A.M. (2015)
Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis
of rat tissues under physiological and toxicological conditions. PeerJ, 3,
e855.
32. Kirschneck, C., Proff, P., Fanghänel, J., Wolf, M., Roldán, J.C. and
Römer, P. (2016) Reference genes for valid gene expression studies on
rat dental, periodontal and alveolar bone tissue by means of RT-qPCR
with a focus on orthodontic tooth movement and periodontitis. Annals of
Anatomy, 204, 93–105.
33. Zhang, L., Liu, W., Zhao, J., Ma, X., Shen, L., Zhang, Y., Jin, F. and
Jin, Y. (2016) Mechanical stress regulates osteogenic differentiation and
RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/β-
catenin pathway. Biochimica et Biophysica Acta, 1860, 2211–2219.
34. Liu, J., Li, Q., Liu, S., Gao, J., Qin, W., Song, Y. and Jin, Z. (2017) Peri-
odontal ligament stem cells in the periodontitis microenvironment are sen-
sitive to static mechanical strain. Stem Cells International, 2017, 1–13.
35. Yamaguchi, M., Shimizu, N., Goseki, T., Shibata, Y., Takiguchi, H., Iwa-
sawa, T. and Abiko, Y. (1994) Effect of different magnitudes of tension
force on prostaglandin E2 production by human periodontal ligament
cells. Archives of Oral Biology, 39, 877–884.
36. Matsumura, H., Nakayama, Y., Takai, H. and Ogata, Y. (2015) Effects of
interleukin-11 on the expression of human bone sialoprotein gene. Journal
of Bone and Mineral Metabolism, 33, 142–153.
37. Eggenhofer, E., Luk, F., Dahlke, M.H. and Hoogduijn, M.J. (2014) The life
and fate of mesenchymal stem cells. Frontiers in Immunology, 5, 148.
38. Parks, E.T. (2000) Computed tomography applications for dentistry.
Dental Clinics of North America, 44, 371–394.
39. Monnouchi, S., Maeda, H., Yuda, A., Hamano, S., Wada, N., Tomok-
iyo, A., Koori, K., Sugii, H., Serita, S. and Akamine, A. (2015) Mechanical
induction of interleukin-11 regulates osteoblastic/cementoblastic differen-
tiation of human periodontal ligament stem/progenitor cells. Journal of
Periodontal Research, 50, 231–239.
40. Abass, S.K. and Hartsfield, J.K. Jr. (2007) Orthodontics and external ap-
ical root resorption. Seminars in Orthodontics, 13, 246–256.
41. Sensebé, L., Krampera, M., Schrezenmeier, H., Bourin, P. and Giordano, R.
(2010) Mesenchymal stem cells for clinical application. Vox Sanguinis, 98,
93–107.