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Contents lists available at ScienceDirect

Archives of Oral Biology

journal homepage: www.elsevier.com/locate/archoralbio

Sclerostin injection enhances orthodontic tooth movement in rats 7

a,1 a,b,1 c c a d a
Wenxin Lu , Xuan Zhang , Fiona Firth , Li Mei , Jianru Yi , Changyang Gong , Hanshi Li ,
⁎ ⁎
Wei Zhenge, , Yu Lia,
a
State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan
University, China
b
3E Dental Clinic, Chengdu, China
c
Discipline of Orthodontics, Department of Oral Sciences, Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, New Zealand
d
Department of Medical Oncology, Cancer Center, and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, China
e
State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Oral and Maxillofacial Surgery, West China Hospital of
Stomatology, Sichuan University, China

A R T I C LE I N FO A B S T R A C T

Keywords: Objective: It was aimed to investigate the in vivo effects of local injection of sclerostin protein on orthodontic
Sclerostin tooth movement.
Osteoclastogenesis Design: A total of 48 rats underwent orthodontic mesialization of the maxillary first molars on both sides. Local
Orthodontic tooth movement injection was given at the compression side in the alveolar bone on both maxillary sides, with sclerostin protein
Sost protein, Rat
carried by hydrogel on one side, and the same volume of normal saline carried by hydrogel on the other side
Osteoclast
Orthodontics
serving as the control. After two weeks, the tooth movement amount and effects on the periodontium were
assessed through micro-computed tomography (μCT) analysis, tartrate-resistant acid phosphatase (TRAP)
staining and immunohistochemistry (IHC) analysis.
Results: After two weeks of intervention, tooth movement was significantly greater in the 4 μg/kg and 20 μg/kg
sclerostin injection groups, compared to the control. Analysis of the furcation area of the maxillary first molar
showed that the 20 μg/kg group had significantly decreased BV/TV. At the compression side, the number of
TRAP-positive osteoclasts was significantly increased in 20 μg/kg group compared to the control. The expression
of RANKL was statistically higher in all the sclerostin groups, while the expression of OPG was statistically lower
in the 4 μg/kg and 20 μg/kg groups, compared to the control. At the tension side, the expression of RUNX2 and
COL-1 was statistically higher in the 20 μg/kg group compared to the control.
Conclusions: Local injection of sclerostin protein in the alveolar bone at the compression side accelerates OTM in
rats by promoting osteoclastogenesis.

1. Introduction Kennedy, 2014). The ablation of osteocytes in alveolar bone in trans-

Orthodontic tooth movement (OTM) is a process of coupling be-
tween bone resorption and bone formation under mechanical loading
(Wise & King, 2008). In addition to the key roles of osteoclasts and
osteoblasts (Proff & Römer, 2009), the function of osteocytes has also
been considered to be important in bone remodeling during OTM
(Bellido, 2014; Bonewald, 2010). For example, osteocytes have been
found to sense loading stimuli and regulate the differentiation of os-
teoclasts and osteoblasts during bone remodelling (Boyce et al., 2010;
Glass & Gerard, 2007; Matsumoto, Iimura, Ogura, Moriyama, &
Yamaguchi, 2013; Proff & Römer, 2009; Schaffler, Cheung, Majeska, &
genic mice was shown to significantly decrease the distance of OTM and
the number of osteoclasts at the compression site (Matsumoto et al.,
2013). The downregulation of osteocytes on bone formation was at-
tributed to the secretion of sclerostin (Schaffler et al., 2014).
Sclerostin, the SOST gene protein product, is nearly exclusively
expressed by osteocytes in mature bone (Epstein, Hamersma, &
Beighton, 1979; Poole et al., 2005). It has been found that sclerostin can
suppress the activity and viability of osteoblasts through antagonizing
Wnt/β-catenin signaling by integrating LRP4, 5 and 6 (Bezooijen et al.,
2004; Lei et al., 2015; Li et al., 2005; Semënov, Tamai, & He, 2005).
The SOST expression is known to be responsive to mechanical strain,

⁎
Corresponding authors at: State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology,
Sichuan University, 14 Renmin South Road Third Section, Chengdu, 610041, China.
E-mail addresses: zhengwei81101@163.com (W. Zheng), yuli@scu.edu.cn (Y. Li).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.archoralbio.2018.12.011
Received 30 August 2018; Received in revised form 12 December 2018; Accepted 26 December 2018
‹(OVHYLHU/WG$OOULJKWVUHVHUYHG
W. Lu et al.
with a reduction of SOST transcripts and sclerostin level under an in-
creased load, and an increase in SOST expression and sclerostin secre-
tion under unloading (E Michael, 2013; Robling et al., 2008). This also
explains the phenomenon that reduced mechanical stress in astronauts
and bedridden patients causes bone loss and disuse osteoporosis, and
SOST knock-out mice have appeared to be resistant to mechanical un-
loading-induced bone loss (Chuwen et al., 2009; Mcgeelawrence et al.,
2013).
The role of sclerostin in osteoblast-mediated bone formation has
been extensively studied. Most of the previous studies involving scler-
ostin used sost−/− animal models or in vivo injections of sclerostin
antibody (Scl-Ab) to stimulate bone regeneration and suppress bone
loss (Chen et al., 2015; Shu et al., 2017; Suen et al., 2015; Taut et al.,
2013). For example, Scl-Ab treatment can prevent bone loss, create
greater alveolar crest height and higher alveolar bone density in rats
with experimental periodontitis (Chen et al., 2015; Taut et al., 2013).
However, only a few studies have focused on the relationship between
sclerostin and osteoclasts to date. A study using the sost−/− animal
model found that there were fewer tartrate-resistant acid phosphatase
(TRAP)-positive cells and fewer osteoclasts in SOST knock-out mice
(Shu et al., 2017). Recombinant human sclerostin interference has been
found to enhance receptor activator of nuclear factor kappa B ligand
(RANKL) expression and the RANKL/osteoprotegerin (OPG) ratio in
MLP-Y4 osteocyte-like cells as well as the osteoclastic-inducible ability
of osteocytes (Shu et al., 2017). Periodontal ligament cells subjected to
a light compressive force showed an increased expression of SOST/
sclerostin, which is consistent with the expression of osteoclastic in-
ducible cytokines. It has been speculated that the enhanced expression
of SOST/sclerostin may be associated with hypoxia during OTM (Shu
et al., 2017; Suen et al., 2015). To date, it is still unclear whether the
direct injection of sclerostin affects the bone remodeling during OTM,
especially the process of osteoclastogenesis.
The aim of the study was to investigate the in vivo effects of local
injection of sclerostin protein on OTM in rats. The hypothesis was that
the in vivo local injection of sclerostin protein could promote osteo-
clastogenesis and OTM.
2. Materials and methods
2.1. Experimental animals
Fifty-two 6-week-old male Wistar rats (weight range 180–200 g)
were used for the experiments. Two rats died during the experimental
period, and another two failed to complete the experiment because of
failed force-applying devices. Therefore, 48 rats were used as valid
samples. The rats were housed in plastic cages under a 12-hour light/
dark photoperiod with access to standard rat food and plain water for
one-week to allow adaption to the experimental environment before the
interventions. Soft rat food was fed after the interventions. The study
was a self-control design. Each rat had experimental tooth movement
models applied to both sides of the maxilla - one side was subjected to
sclerostin and the other side was subjected to normal saline injection.
2.2. Orthodontic tooth movement model
This study used the classical experimental OTM model that has been
described previously (Fig. 1) (Taut et al., 2013). A 50 g force to induce
tooth movement was generated using nickel-titanium coil springs (3 M
Unitek, Monrovia, CA). Following anesthesia with intraperitoneal in-
jection of 10% chloral hydrate (3–4 mg/kg), the coil springs were li-
gated bilaterally between the central incisor and first molar with 0.010-
inch stainless steel ligatures and fixed by a light-cured glass ionomer
cement (3 M Unitek, Monrovia, CA). The incisors of rats erupt along
with tooth attrition. As we bonded resin on the maxillary incisors, the
teeth were supposed to have no attrition and eruption during our ex-
periment. Based on that, we didn’t trim the incisors. A micropore was

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prepared bone using a reamer on the alveolar bone at the mesial side of
the maxillary first molar root to allow the injection of either sclerostin
protein (experimental side) or normal saline (control side) (Fig. 1).
2.3. Application of intervention (sclerostin) and control (saline)
The rats were divided into three groups, receiving three different
concentrations of sclerostin injection, i.e. 0.8 μg/kg, 4 μg/kg or 20 μg/
kg (n = 16 in each group). Each rat received 0.1 ml local direct injec-
tion of sclerostin (R&D systems, MN, USA) in the alveolar bone at the
mesial side (compression side) of the maxillary first molar at the left or
right side randomly (which became the experimental side). The scler-
ostin was carried by a polyethylene glycol-polycaprolactone-poly-
ethylene glycol (PECE) hydrogel (a synthesized thermosensitive hy-
drogel that undergoes a sol-gel-sol transition as temperature increases)
(Gong et al., 2009). The control side of the maxilla received the same
volume of normal saline carried by the PECE hydrogel. The micropores,
prepared using a reamer and used for sclerostin injection, were on the
alveolar bone approximately 4 mm mesial from the maxillary first
molar on both the right and left sides of the maxilla.
After two weeks of force application, all rats were sacrificed by
overdose intraperitoneal injection of anesthesia. The maxillae were
dissected for micro-computed tomography (μCT) analysis, TRAP
staining and immunohistochemistry (IHC) analysis.
2.4. Distance of OTM
The magnitude of OTM was measured by the distance between the
distal of the maxillary first molar to the mesial of the maxillary second
molar at the cervical level using digital slide calipers on the micro-CT
images. The distance of OTM was measured in triplicate by an operator
who was blind to the grouping to calculate the mean values.
2.5. Micro computed tomography (μCT) analysis
The maxillary molars and the adjacent alveolar bone were fixed in
formalin and scanned using micro computed tomography (μCT80,
Scanco Medical, Bassersdorf, Switzerland). The region of interest was a
cuboid extracted from the furcation area of the first molar of
400 μm × 400 μm × 500 μm (length × width × thickness, in the sa-
gittal plane), which has been considered to have reproducible mor-
phological landmarks in the literature (Fig. 2) (Yamaki, Kataoka,
Ohtsuka, & Miyazaki, 2012). Three different regions were selected and
measured for the calculation of the mean values. Bone volume fraction
(BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and
trabecular separation (Tb.Sp) were analyzed with the innate software.
2.6. Tartrate-resistant acid phosphatase (TRAP) staining and
immunohistochemical (IHC) analysis
Following μCT analysis, all samples were decalcified and dehy-
drated using a graded series of ethanol, and embedded in paraffin.
Sections were cut to a thickness of 5 μm and used for TRAP staining and
IHC analysis. For the compression side, the periodontal ligament and
alveolar bone mesial to the upper one-third of the mesial buccal root of
maxillary first molar was chosen to be the region of interest. For the
tension side, the periodontal ligament and alveolar bone distal to the
upper one-third of the distal buccal root of maxillary first molar was
chosen to be the region of interest. Three sections were used randomly
for analysis of IHC staining and counting of TRAP-positive cells per
animal. TRAP staining was performed to examine osteoclast formation
using a commercial kit (Sigma, St Louis, MO, USA) following the
manufacturer's instructions. Multinucleated TRAP-positive cells (at
least 2 nuclei) adjacent to the alveolar bone at the compression side of
the first molar were counted as osteoclasts. Total numbers of the cells
were counted and calculated by the area to get the average number of
W. Lu et al.
Fig. 2. The region of interest of the maxillary first molar for μCT analysis.
The yellow square frame indicates the region of interest. The white arrow in-
dicates the direction of orthodontic tooth movement (OTM). (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article).
Fig. 3. The distance of orthodontic tooth movement (OTM) after two weeks of force application.
* and ** indicate P < 0.05 and P < 0.01, respectively.

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Fig. 1. Orthodontic tooth movement model of rats
used in the study.
(A) The coil springs were ligated bilaterally between
the maxillary central incisor and first molar with
0.010-in stainless steel ligatures, and fixed by a light-
cured glass ionomer cement to generate 50 g force to
induce tooth movement. (B) The micropore prepared
by a reamer at the mesial side of the maxillary first
molar root on both sides per rat for the injection of
sclerostin protein.
cells per 1 mm2 for comparisons. The number of TRAP-positive cells
were counted by two independent investigators. Expression of RANKL,
OPG, RUNX2, OCN and collagen I (COL-1) in the periodontium was
evaluated by immunohistochemical staining (Abcam, Cambridge, MA,
USA). Semiquantitative analysis of average optical density (AOD) was
done through Image-Pro Plus software.
2.7. Statistical analysis
SPSS 22.0 was used for the statistical analyses. Data were presented
as mean ± standard deviation (SD). The independent student’s t-test
and one-way ANOVA analysis followed by the SNK methods of analysis
of variance were used for statistical comparison. P < 0.05 was con-
sidered statistically significant.
3. Results
3.1. Injection of sclerostin enhanced OTM
After two weeks of intervention, the distance of OTM was
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Fig. 4. μCT scan and analysis of furcation area of the maxillary first molar.
(A) μCT scan of maxillae in the experimental and control groups. (B) 3D reconstruction of the maxilla in the 20 μg/kg group. (C) μCT analysis of BV/TV. (D) μCT
analysis of Tb.N. (E) μCT analysis of Tb.Th. (F) μCT analysis of Tb.Sp. * and ** indicate P < 0.05 and P < 0.01, respectively.


W. Lu et al.
Fig. 5. TRAP staining at the mesial periodontium of maxillary first molar.
AB (alveolar bone), PDL (periodontal ligament) and R (root). The red arrows indicate TRAP-positive osteoclasts. (A) TRAP staining of the maxillary first molar in the
0.8 μg/kg group. (B) TRAP staining of the maxillary first molar in the 4 μg/kg group. (C) TRAP staining of the maxillary first molar in the 20 μg/kg group. (D) The
number of TRAP-positive osteoclasts on the compression side. ** indicates P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article).
0.65 ± 0.06 mm and 0.72 ± 0.04 mm in the 4 μg/kg and 20 μg/kg
sclerostin injection groups respectively, both significantly greater than
that in the control. The distance of OTM in the 4 μg/kg and 20 μg/kg
groups was significantly greater than that in the 0.8 μg/kg group
(0.58 ± 0.07 mm) (Fig. 3).
3.2. Sclerostin decreased alveolar bone BV/TV
Analysis of the furcation area of the maxillary first molar showed
that sclerostin decreased the BV/TV compared to the control. BV/TV
was 0.59 ± 0.12 in the 20 μg/kg sclerostin group, significantly lower
than that (0.75 ± 0.08) in the control group (Fig. 4A–C). No significant
difference was found among all the groups for alveolar bone Tb.N,
Tb.Th and Tb.Sp (Fig. 4D, E and F).
3.3. Sclerostin increased osteoclast formation and RANKL expression at the
compression side
The number of TRAP-positive osteoclasts was significantly higher at
the mesial periodontium of the maxillary first molar in the 20 μg/kg
sclerostin group compared to the control (Fig. 5). RANKL expression
was significantly higher in all the sclerostin groups compared to the
control (Fig. 6). Inversely, OPG expression was significantly lower in
the 4 μg/kg and 20 μg/kg groups compared to the control (Fig. 6). No
significant difference was found among all the groups for sclerostin
expression at the compression side (Fig. 6).

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3.4. Local sclerostin injection at the compression side did not suppress
RUNX2, OCN expression and COL-I formation at the tension side
At the distal periodontium of the maxillary first molar, the expres-
sion of sclerostin had no significant difference between the sclerostin
and control groups. As a sign of osteogenesis, the expression of RUNX2
was significantly higher in the 4 μg/kg and 20 μg/kg groups compared
to the control. Besides, the expression of COL-1 was significantly higher
in the 20 μg/kg group compared to the control (Fig. 7).
4. Discussion
Sclerostin, encoded by SOST, is a secreted cystine-knot protein ex-
pressed in bone and periodontium, specifically by osteocytes. It is a
negative regulator of osteoblast differentiation and function, and a well-
demonstrated inhibitor of bone formation (Bezooijen et al., 2004; Li
et al., 2005; Poole et al., 2005; Semënov et al., 2005). On the other
hand, sclerostin has also been showed to have a catabolic effect through
promotion of osteoclast activity (Xiaolin et al., 2015). In addition,
sclerostin induces expression of cathepsin K, TRAP, and carbonic an-
hydrase-2 in osteocytes, suggesting that it may have a role in osteolysis
of the extracellular matrix surrounding osteocytes to release mineral
from bone (Masakazu et al., 2013). Recently, osteoclasts derived from
aged mouse bone marrow have been found to express and secrete
sclerostin, leading to decreased osteoclast-stimulated osteoblast mi-
neralization. This mechanism may contribute to the decreased bone
mass that is exhibited with age (Weivoda & Merry Jo, 2014).
Expression of sclerostin during OTM has been reported as well. In a
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Fig. 6. IHC staining of RANKL, OPG and sclerostin at the compression side.
AB (alveolar bone), PDL (periodontal ligament) and R (root). The red arrows indicate strongly positive cells (deeper brown staining indicates greater expression). (A)
The IHC staining of RANKL, OPG and sclerostin at the compression side. (B) The average optical density of IHC staining of RANKL, OPG and sclerostin at compression
side. * indicates P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).


W. Lu et al.
Fig. 7. IHC staining of RUNX2, OCN, COL-1 and sclerostin at tension side.
AB (alveolar bone), PDL (periodontal ligament) and R (root). The red arrows
indicate strongly positive cells (deeper brown dye indicates greater expression).
The blue arrows indicate predentin. The green arrows indicate odontoblasts in
the odontoblast layer. (A) The IHC staining of RUNX2, OCN, COL-1 and scler-
ostin at tension side. (B) The average optical density of IHC staining of RUNX2,
OCN, COL-1 and sclerostin at tension side. * indicates P < 0.05. (For inter-
pretation of the references to colour in this figure legend, the reader is referred
to the web version of this article).
rodent study, the level of sclerostin increased on the compression side
after one week's orthodontic force application, which was in accordance
with expression of osteoclastogenic factors leading to tooth movement
(Shu et al., 2017). In another study, expression of sclerostin in the al-
veolar bone increased on the compression side and peaked on day 5;
whilst expression of sclerostin on the tension side significantly de-
creased on day 1 (Odagaki et al., 2018).
Thus, in the present study, we injected sclerostin at the mesial
(compression) side of the maxillary first molar of a rat orthodontic

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tooth movement model, with the hypothesis that sclerostin may en-
hance OTM through promoting osteoclastogenesis. Male rats were used
so as to exclude potential influences from oestrogen. The experimental
groups of rats received the complete intervention from 8 to 10 weeks of
age, which was roughly equivalent to the adolescent and early adult
period in mankind. Due to lack of related studies regarding local in-
jection of sclerostin protein, three concentrations of sclerostin were
selected. The PECE hydrogel used in this study has been evaluated and
reported by the previous research (Gong et al., 2009). The hydrogel
undergoes sol–gel–sol transition as the temperature increases, and it is
aqueous at room temperature and becomes gel at 37 ° centigrade, which
can sustain for at least 14 days.
This study found that with orthodontic force application, injection
of sclerostin protein at the compression side accelerated OTM. In ad-
dition, local injection of sclerostin protein seemed not to affect osteo-
genesis on the tension side. Micro-computed tomography analysis
showed lower BV/TV in the sclerostin groups, which were in ac-
cordance with increased TRAP-positive osteoclasts, as well as higher
RANKL expression whilst lower OPG expression. The changes above
were revealed in a sclerostin dosage-dependant manner, with the
20 μg/kg group showing the greatest osteoclastogenic effects. This is
consistent with a previous finding that sclerostin could increase the
ratio of RANKL/OPG expression in osteocytes in a dosage-dependent
manner (Wijenayaka et al., 2011).
A number of studies have identified the osteoclastogenesis promo-
tion function of sclerostin (Delgado-Calle, Sato, & Bellido, 2017;
Matsumoto et al., 2013; Ota et al., 2013; Wijenayaka et al., 2011);
however, the mechanisms of which remain elusive. It is well known that
sclerostin could down-regulate OPG expression through the Wnt sig-
naling pathway by interacting with LRP4, 5 and 6 (Bezooijen et al.,
2004; Epstein et al., 1979; Lei et al., 2015; Li et al., 2005; Poole et al.,
2005; Semënov et al., 2005). As a decoy receptor for RANKL, repression
of OPG may lead to enhanced osteoclastogenesis. On the other hand,
sclerostin could directly up-regulate the expression of RANKL to a great
extent in both osteocyte-like human primary osteoblasts and MLO-Y4
cells, resulting in elevated bone resorption (Ota et al., 2013;
Wijenayaka et al., 2011) However, the mechanisms underlying the
regulation of RANKL expression by sclerostin in osteocytes remain to be
clarified (Delgado-Calle et al., 2017). Additionally, some studies have
claimed that sclerostin could promote apoptosis of osteoblasts and os-
teocytes, which may recruit osteoclast precursors and enhance their
differentiation (Bellido, 2014; Matsumoto et al., 2013).
Interestingly, although local injection of sclerostin increased os-
teoclastogenesis during OTM at the compression side in the present
study, the osteoblastic activity was not reduced on the tension side. It
seems that local injection of sclerostin at the compression side of al-
veolar bone may not affect the tension side.
In addition, the nickel-titanium coil springs were stretched 2 mm
(50 g force) at the beginning of the study. The largest tooth movement
of rats was 0.76 mm in the present study. Thus, based on the elasticity
modulus of the nickel-titanium coil springs, the elongation of the
springs could still provide a force of no less than 35 g at the endpoint of
the study, which was still an optimal force for OTM of rats (Ibrahim,
Gudhimella, Pandruvada, & Huja, 2017).
One limitation of the present study was that only one time point,
two weeks, had been selected. OTM is a complex biological process, and
that different changes may be found at different stages (Wise & King,
2008). Therefore, future studies are suggested to increase time points to
observe the prolonged effects of local injection of sclerostin on OTM.
5. Conclusions
Local injection of sclerostin protein in the alveolar bone at the
compression side enhances tooth movement and osteoclastogenesis in
rats.
W. Lu et al.
Ethical approval
This in vivo animal study was approved by the Animal Care and
Ethics Committee of West China School of Stomatology, Sichuan
University (NO: SKLODLL2012 A025).
Competing interest
All authors report no potential conflict of interest.
Declarations of interest
None.
Acknowledgment
This work was supported by the National Natural Science
Foundation of China [NO. 11372202] and [NO. 81801018].
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