International Journal of

Molecular Sciences
ArticleMicro-Osteoperforations Induce TNF-α Expression and
Accelerate Orthodontic Tooth Movement via TNF-α-Responsive
Stromal Cells
Ria Kinjo, Hideki Kitaura * , Saika Ogawa, Fumitoshi Ohori, Takahiro Noguchi, Aseel Marahleh
Yasuhiko Nara, Adya Pramusita, Jinghan Ma, Kayoko Kanou and Itaru Mizoguchi,

Division of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Tohoku University, 4-1,
Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan; ria.kinjou.p5@dc.tohoku.ac.jp (R.K.);
saika.ogawa.a4@tohoku.ac.jp (S.O.); fumitoshi.ohori.t3@dc.tohoku.ac.jp (F.O.);
takahiro.noguchi.r4@dc.tohoku.ac.jp (T.N.); marahleh.aseel.mahmoud.t6@dc.tohoku.ac.jp (A.M.);
yasuhiko.nara.q6@dc.tohoku.ac.jp (Y.N.); adya.pramusita.q6@dc.tohoku.ac.jp (A.P.);
ma.jinghan.s1@dc.tohoku.ac.jp (J.M.); kanou.kayoko.s7@dc.tohoku.ac.jp (K.K.); mizo@tohoku.ac.jp (I.M.)* Correspondence: hideki.kitaura.b4@tohoku.ac.jp; Tel.: +81-22-717-8374

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Citation: Kinjo, R.; Kitaura, H.;
Ogawa, S.; Ohori, F.; Noguchi, T.;
Marahleh, A.; Nara, Y.; Pramusita, A.;
Ma, J.; Kanou, K.; et al.
Micro-Osteoperforations Induce
TNF-α Expression and Accelerate

Abstract: Micro-osteoperforations (MOPs) have been reported to accelerate orthodontic tooth movement (OTM), and tumor necrosis factor (TNF)-α has been reported to play a crucial role in OTM.
In this report, the influence of MOPs during OTM was analyzed. We evaluated the expression of
TNF-α with and without MOPs by RT-PCR analysis. A Ni-Ti closed coil spring was fixed between
the maxillary left first molar and the incisors as an OTM mouse model to move the first molar in
the mesial direction. MOPs were prepared on the lingual side and mesial side of the upper firstmolars. Furthermore, to investigate the target cell of TNF-α for osteoclast formation during OTM
with MOPs in vivo, we created four types of chimeric mice in which bone marrow of wild-type (WT)or TNF receptor 1- and 2-deficient mice (KO) was transplanted into lethally irradiated WT or KO mice.
The results showed that MOPs increased TNF-α expression, the distance of tooth movement andosteoclast formation significantly. Furthermore, mice with TNF-α-responsive stromal cells showed asignificant increase in tooth movement and number of osteoclasts by MOPs. We conclude that MOPs
increase TNF-α expression, and tooth movement is dependent on TNF-α-responsive stromal cells.

Orthodontic Tooth Movement via
TNF-α-Responsive Stromal Cells. Int.

Keywords: micro-osteoperforations; TNF-α; orthodontic tooth movement; osteoclast

J. Mol. Sci. 2022, 23, 2968. https://doi.org/10.3390/ijms23062968
Academic Editor: Jung Eun Kim

1. Introduction
Received: 24 February 2022
Accepted: 8 March 2022
Published: 9 March 2022
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A key concern for orthodontics is the lengthy duration of orthodontic treatment.
Therefore, shortening the duration of orthodontic treatment is an active research area. Many
methods have been tested to accelerate tooth movement, including biological, mechanical,
physical and surgical approaches [1]. Surgical procedures are the most consistent andachieve the desired results. Surgical methods that accelerate orthodontic tooth movement
are based on the regional acceleratory phenomenon (RAP), which involves an enhanced
remodeling process in response to harmful stimuli [2].
Various techniques have been developed to accelerate orthodontic tooth movement(OTM) to coordinate these biological processes. In 1959, a method was introduced toaccelerate OTM by cutting the cortical bone between the teeth, leaving minor penetrationsin the medullary bone after exposing the buccal and lingual alveolar bone with a fullthickness flap. A subapical horizontal cut that connects the interdental cuts is an osteotomystyle approach that penetrates the entire thickness of the alveoli [3]. This cut was introduced
to corticotomy in 2008, with alveolar grafting and flap elevation, as a new technique foraccelerating orthodontic bone formation [4]. Additionally, a less invasive cortical incision
was also introduced. In these methods, a surgical blade was inserted into the alveolar bonehorizontal and interdentally without raising the flap [5]. In addition, minimal invasive

Int. J. Mol. Sci. 2022, 23, 2968. https://doi.org/10.3390/ijms23062968

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piezoelectric surgery has been introduced with a piezo-surgical knife rather than using
a surgical blade [6]. Recently, cortical incisions were performed using surgical lasers.
Piezocision and laser-assisted flapless corticotomy via the Er:YAG laser accelerated canine
retraction in a randomized controlled trial [7]. It has been reported that cortical incisions
with the Er-Cr:YSGG laser accelerated orthodontic tooth movement in a rabbit OTM
model [8]. In a recent study, piezocision and the Er:YAG laser incision were successfulin accelerating tooth movement in a rat OTM model [9]. These surgical methods foraccelerating OTM are based on local acceleration phenomena.
Perforations of the alveolar bone are a surgical method that is termed micro-osteoperforations(MOPs), and MOPs can be used to accelerate OTM [10]. Microscopic tissue reactions fromapplying this procedure in animal studies have been examined, and the results revealthat MOPs showed RAP [11]. Several studies introduced MOPs with clinical replicationin humans [10,12,13]. The reports suggest that MOPs accelerate OTM effectively. Perforations to the alveolar bone around the teeth have also been reported to accelerate OTM
in experimental animal models [11,14–17]. Perforation procedures of the alveolar bone
were found to induce expression of inflammatory cytokines because of enhanced osteoclast
formation [11]. Thus, this procedure enhances bone remodeling and tooth movement during OTM. However, despite its proven effectiveness, the onset mechanism that accelerates
OTM via MOP is poorly understood.
Osteoclasts derived from hematopoietic stem cells act a role in bone resorption andbone remodeling [18]. There are two essential factors for osteoclast formation: receptor
activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor(M-CSF) [19]. Moreover, tumor necrosis factor (TNF)-α, which is a pro-inflammatory
cytokine, may also induce osteoclast differentiation [20–23] and may induce osteoclasts inbone erosive diseases as well [24].
OTM is dependent on alveolar bone remodeling by compression-force-associatedosteoclasts and tension-force-associated osteoblasts, which results in bone resorption andbone formation, respectively [25,26]. The orthodontic force, induces cytokines, growth
factors and neurotransmitters, which, when added to the compressed side in the periodontal
ligament, induces osteoclasts, and the tooth moves as the osteoclasts resorb the bone.
Several reports showed that the compression force in OTM induces expression of
TNF-α [27–34]. In our previous study, we also found that TNF-α plays important roles in
an OTM mouse model by using both TNF receptor 1- and TNF receptor 2-deficient (KO)mice. Here, the distance the tooth moved was found to be shorter than that of the wild-type(WT) mice [35–37].
In our previous study, four kinds of chimeric mice were generated by a bone marrow transplantation method using WT and KO mice: (i) mice with stromal cells andmacrophages bearing TNF receptors; (ii) mice with only stromal cells bearing TNF receptors; (iii) mice with only macrophages bearing TNF receptors; and (iv) mice with stromalcells and macrophages that are deficient in TNF receptors. Osteoclast formation was evaluated after injection of TNF-α into the supracalvariae of these chimeric mice. The results
indicated that stromal cells contribute more to osteoclast formation than macrophages, although both macrophages and stromal cells were found to be direct targets of TNF-α [22,23].
Furthermore, the study showed that chimeric mice can be used to examine the type of cellthat TNF-α targets for osteoclast formation during OTM. The results of this study suggested
that the response of stromal cells to TNF-α is a crucial factor for osteoclast formation andbone resorption in OTM [37]. However, the effects of MOPs on TNF-α expression during
OTM remain unknown. In this study, we analyzed TNF-α expression during experimental
tooth movement with MOPs. We also investigated whether tooth movement is acceleratedby MOPs-induced TNF-α using mice that are deficient in the expression of TNF receptors.
Furthermore, chimeric mice were used to investigate the contribution of each TNF-α target
cell type in vivo for tooth movement and osteoclast formation in OTM with MOPs.

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2. Results
2.1. MOPs Increase Tooth Movement and Osteoclast Formation
After the mice were anesthetized, the mice were fixed in a supine position. Next,
the mouth was kept open by pulling the lower incisors caudally. The left corner of themouth was gently stretched laterally to access the upper left first molar. We made twopoints for MOPs. One MOP was placed about 1.0 mm mesial to the maxillary left firstmolar. The other MOP was placed about 1.0 mm palatal to the maxillary left first molar(Figure 1A). These MOPs were created by inserting a 0.5-mm diameter round steel bur inthe alveolar bone with a slow-speed handpiece. Perforations were approximately 0.5 mm
in diameter and 0.25 mm in depth. Hemostasis was achieved by using cotton pellets after
the MOPs procedure. To achieve OTM under anesthesia, a Ni-Ti closed coil spring (Tomy,
Fukushima, Japan) was fixed to the alveolar bone of the maxillary incisors and the left
maxillary first molar. The left maxillary first molar was mesially moved. The appliance
was tied to the upper first molar and incisor with a 0.1-mm diameter stainless steel wire.
Based on the manufacturer’s instructions, the appliance was adjusted to 10 g, as described
previously [35,37] (Figure 1B). Four mice were used in each group. The distance between
the upper first and second molars was measured by impressing the teeth with Examixfine
injection type material (GC Co., Tokyo, Japan) after OTM. The samples were immersed
in 4% paraformaldehyde for fixation after the impression process. The distance the teeth
moved was assessed using a stereoscopic microscope (VH-7000; Keyence, Osaka, Japan), as
described previously [35,37] (Figure 1B).

Figure 1. Orthodontic tooth movement and region of MOPs in mice: (A) Schematic of the appliancefixed between incisors and the first molar in mice and MOPs. One MOP (black dot) was placed about
1.0 mm mesial to the maxillary left first molar. The other MOP (black dot) was placed about 1.0 mm
palatal to the maxillary left first molar. (B) Pictures of tooth movement and measurement of OTM in
mice. The red double arrow between the maxillary left first molar (M1) and maxillary left second
molar (M2) on the solid line connecting the central fossae of the two molars in silicone impressions
was evaluated by stereoscopic microscopy.

After six days of mechanical loading, no significant difference between no MOPs and
MOPs was observed for the WT mice (Figure 2A,B). On Day 12, tooth movement was greaterfor the WT mice with MOPs than that of the WT mice without MOPs (Figure 2A,B). Furthermore, tartrate-resistant acid phosphatase (TRAP) staining was performed on sections
from the mesial side of the distobuccal root of the left maxillary first molars. Osteoclasts
were detected along the alveolar bone on the pressure side of the tooth in the WT mice,

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4 of 17and a similar number of osteoclasts were detected in both of the WT mice groups on Day 6(Figure 2C,D). Significantly more osteoclasts were observed on the alveolar in the pressure
side of the tooth on Day 12 than on Day 6, and the number of osteoclasts detected in the
WT mice with MOPs on Day 12 was significantly greater than the WT mice without MOPs
(Figure 2C,D). To evaluate the effect of MOPs, we examined TRAP-stained transverse
sections of a tooth on the opposite side. There was no significant difference between themice groups with and without MOPs on both Day 6 and Day 12 (Figure 2E,F).

Figure 2. Effect of MOPs on orthodontic tooth movement and histology analysis of MOPs on
orthodontic tooth movement: (A) Images of teeth after 0, 6 and 12 days of experimental loading withand without MOPs in WT mice. (B) Distance of tooth movement with and without MOPs in WT
mice after 0, 6 and 12 days of experimental loading. (C) TRAP-stained histological sections of the
distobuccal root of the maxillary left first molar after 6 and 12 days of experimental loading with andwithout MOPs in WT mice. Arrows indicate the direction of OTM. (D) Evaluation of the number ofosteoclasts on the mesial side of the distobuccal upper-left first molar. (E) Histological examination
was performed to evaluate the area affected by MOPs. TRAP-stained sections of the opposite sideof WT mice with and without MOPs on Day 6 and Day 12. (F) The number of osteoclasts on the
opposite side of WT mice with and without MOPs on Day 6 and Day 12. The results are presented
as the mean ± standard deviation (n = 4). ** p < 0.01 indicate significant differences, which wereanalyzed by using the Scheffe test. Scale bars = 100 µm.

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5 of 172.2. MOPs Induce TNF-α Expression
We showed previously that in the absence of a TNF-α response, osteoclast formationand tooth movement in a mouse model decreased [35–37]. Therefore, we evaluated whether
MOPs induced TNF-α expression. To evaluate TNF-α mRNA expression around the left
maxillary first molar after MOPs, alveolar bones were excised from the maxilla around theleft maxillary first molar after 0, 6 and 12 days of MOPs. TNF-α mRNA expression was
evaluated. WT mice with MOPs showed a significant increase in TNF-α mRNA level whencompared with that of WT mice without MOPs on Day 0 (Figure 3).

Figure 3. Time course of TNF-α expression by WT mice with MOPs. The alveolar bones of the left
side maxillary first molar were excised after 0, 6 and 12 days with MOPs to evaluate the expressionof TNF-α mRNA around the left maxillary first molar after MOPs. TNF-α mRNA expression was
evaluated by real-time RT-PCR. The levels of TNF-α mRNA were normalized to the levels of GAPDH.
The results are presented as the mean ± standard deviation (n = 4). ** p < 0.01 indicated significant
differences, which were analyzed by using the Scheffe’s test.

2.3. MOPs Induce Tooth Movement and Osteoclast Formation Is Dependent on TNF-α
On Day 6, there was no significant difference between the WT and KO mice. Moreover,there was also no significant difference in tooth movement between the WT and KO micewith or without MOPs (Figure 4A,B). On Day 12, tooth movement was greater in the
WT mice than that of the KO mice. Furthermore, tooth movement in the WT mice with
MOPs was greater than that observed for the WT mice without MOPs. However, there
was no significant difference between the KO mice with or without MOPs after 12 days
(Figure 4A,B).
TRAP staining of sections from the mesial side of the distobuccal root of the maxillaryleft first molars was performed. Osteoclasts were observed along the alveolar bone on
the pressure side of the tooth in the WT mice, and a similar number of osteoclasts weredetected in the KO mice on Day 6 (Figure 4C,D). The number of osteoclasts in the WT
mice with MOPs was also similar to that observed in the KO mice with MOPs on Day 6.
No significant difference in the osteoclast numbers between the WT and KO mice with orwithout MOPs on Day 6 was observed (Figure 4C,D). In contrast, many osteoclasts wereobserved along the alveolar bone on the pressure side of the tooth in the WT mice, whereas
fewer osteoclasts were observed in the KO mice on Day 12. There were more osteoclasts
in the WT mice than in the KO mice on Day 12 in OTM (Figure 4C,D). The number ofosteoclasts in the WT mice with MOPs was much greater than observed in the WT mice
without MOPs on Day 12. (Figure 4C,D). However, the number of TRAP-positive cells in
the KO mice with MOPs was similar in number to the TRAP-positive cells in the KO miceon Day 12. There was no significant difference in osteoclast numbers between the KO andKO mice with MOPs on Day 12 (Figure 4C,D).

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Figure 4. MOPs-induced tooth movement and osteoclast formation is dependent on TNF-α:(A) Images of teeth after 6 and 12 days of OTM in WT and KO mice with and without MOPs.
(B) Measured distance of tooth movement in WT and KO mice with and without MOPs after 6 and12 days of experimental loading. (C) TRAP-stained histological sections of the distobuccal root of
the maxillary left first molar in WT and KO mice with and without MOPs after 6 and 12 days ofexperimental loading. (D) The TRAP-positive osteoclast cell number at the alveolar bone surface
around the distobuccal root of the left maxillary first molar after 6 and 12 days of OTM for WT and
KO mice with and without MOPs. The results are given as the mean ± standard deviation (n = 4).
** p < 0.01 and * p < 0.05 indicate significant differences, which were analyzed by using the Scheffe
test. Scale bars = 100 µm.

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7 of 172.4. MOPs Increased Osteoclast Formation and Tooth Movement In Vivo via TNF-α-Responsive
Stromal Cells
MOPs affected the OTM distance and osteoclast formation in OTM. Four kinds ofchimeric mice were generated to analyze which cell type is targeted by TNF-α in order to assess their contribution to MOP-assisted OTM. The chimeric mice prepared were:(i) bone marrow cells of WT transplanted into lethally gamma-irradiated WT mice (WT > WT);(ii) bone marrow cells of WT transplanted into lethally gamma-irradiated KO mice (WT > KO);(iii) bone marrow cells of KO transplanted into lethally gamma-irradiated WT mice (KO > WT);and (iv) bone marrow cells of KO transplanted into lethally gamma-irradiated KO mice(KO > KO). After 12 days of tooth movement with or without MOPs, the distance of OTM
was evaluated, and osteoclasts along the alveolar bone on the compression side werecounted as osteoclasts. The distance of OTM in the WT > KO and KO > KO chimeric mice
was significantly shorter than the distance of OTM in the WT > WT and KO > WT chimericmice (Figure 5A,B). No significant difference was observed between the distance of OTM
in the WT > KO and KO > KO chimeric mice or between the WT > WT and KO > WT
chimeric mice. Furthermore, the distances of OTM in the WT > WT and KO > WT mice with
MOPs were significantly longer than the distances of OTM in the WT > WT and KO > WT
mice without MOPs, respectively (Figure 5A,B). However, no significant difference wasobserved between the distance of OTM between the WT > KO with MOPs and WT > KOmice without MOPs or between the KO > KO with MOPs and KO > KO mice without
MOPs (Figure 5A,B).
The osteoclasts number along the alveolar bone on the compression side in the
WT > KO and KO > KO mice was also significantly lower than the number of TRAPpositive cells in the WT > WT and KO > WT mice (Figure 6C,D). Moreover, no significant
difference was observed in the number of osteoclasts along the alveolar bone on the compression side between the WT > KO and KO > KO mice or between the WT > WT and
KO > WT mice (Figure 5C,D). Furthermore, the number of osteoclasts along the alveolarbone in the compression side in the WT > WT and KO > WT mice with MOPs was significantly higher than the number of osteoclasts in the WT > WT and KO > WT mice without
MOPs, respectively (Figure 5C,D). However, no significant difference was observed in thenumber of osteoclasts on the alveolar bone in the compression side between the WT > KOwith MOPs and WT > KO mice without MOPs or between the KO > KO with MOPs andKO > KO mice without MOPs (Figure 5C,D).
2.5. TNF-α Induces RANKL Expression in Stromal Cells in a Dose-Dependent Manner
We evaluated the possibility that TNF-α-induced RANKL expression is dose dependent. Bone marrow stromal cells were cultured with several doses of TNF-α (0, 1, 10 and
100 ng/mL). Total RNA was obtained from adherent cells after 4 days of culturing. The
increasing RANKL expression in stromal cells was observed to be dependent on the TNF-α
dose (Figure 6A).
2.6. TNF-α Enhances Osteoclast Formation in a Chimeric Co-Culture System, Which Used KO
Osteoclast Precursors and WT Stromal Cells
To evaluate whether TNF-α-induced expression of RANKL in stromal cells and enhanced osteoclast formation are dependent on the dose of TNF-α in a co-culture, we used
KO osteoclast precursors to eliminate the direct effect of TNF-α on osteoclast precursors
in osteoclast formation. We cultured WT stromal cells and KO osteoclast precursors withseveral doses of TNF-α (0, 1, 10 and 100 ng/mL), 10−6 M prostaglandin E2 and 10−8 M
1,25(OH)2 D3 . We counted the osteoclast number in the co-culture. It showed that the osteoclast number increased significantly in a TNF-α dose-dependent manner (Figure 6B,C), andthe percentage of resorption pits also increased significantly in a TNF-α dose-dependent
manner (Figure 6D,E).

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Figure 5. Tooth movement in chimeric WT and KO mice with or without MOPs after 12 days of
OTM: (A) Images of tooth movements after 12 days of orthodontic force loading in chimeric WT and
KO mice with and without MOPs. (B) Measured tooth movement distance in chimeric WT and KO
mice with and without MOPs after 12 days of experimental loading. (C) TRAP-stained histologicalsections of the distobuccal root of the maxillary left first molar in chimeric WT and KO mice after
12 days of experimental loading. (D) The TRAP-positive osteoclast cell number along the alveolarbone surface around the distobuccal root of the left maxillary first molar after 12 days of OTM forchimeric WT and KO mice with and without MOPs. The results are given as the mean ± standard
deviation (n = 4). ** p < 0.01 indicate significant differences, which were analyzed by using the Scheffe
test. Scale bars = 100 µm.

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Figure 6. TNF-α induces RANKL expression in WT stromal cells and induced osteoclast formation ina co-culture using KO osteoclast precursors to eliminate the effect of TNF-α on osteoclast formationand WT stromal cells in a dose-dependent manner: (A) Real-time RT-PCR analysis of expression
levels of RANKL mRNA in WT stromal cells. Total RNA was obtained from WT stromal cells cultured
with TNF-α (0, 1, 10 and 100 ng/mL) for 3 days. (B) Images of TRAP-positive cells and (C) the numberof TRAP-positive cells. (D) Images of the resorption pits and (E) the percentage of resorption pits
in co-cultures of WT stromal cells and KO osteoclast precursors cultured with TNF-α (0, 1, 10 and
100 ng/mL) in the present of prostaglandin E2 and 1,25(OH)2 D3 for 4 days. The results are presented
as the mean ± standard deviation (n = 4). ** p < 0.01 indicate significant differences, which wereanalyzed by using the Scheffe test.

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3. Discussion
MOPs have been reported to accelerate OTM [10,12,13], and TNF-α has been reported
to play a significant role in OTM [35–37]. However, the effect of MOPs on TNF-α expression
during OTM remains unknown. In this study, the effect of MOPs on TNF-α expression
and OTM was investigated. A Ni-Ti closed coil spring was placed between the maxillaryleft first molar and the maxillary incisors as an OTM mouse model to move the first molar
in the mesial direction of C57BL/6J mice for a maximum of 12 days. MOPs were madeon the palatal side and mesial side of the upper first molars by using a round bur. Tooth
movement and histological effects with and without MOPs were assessed. Furthermore,
to investigate the target cell of TNF-α for osteoclast formation during OTM with MOPs
in vivo, we created WT > WT, KO > WT, WT > KO and KO > KO chimeric mice. MOPs
increased TNF-α expression and the distance of tooth movement significantly. Furthermore,
the WT > WT and KO > WT chimeric mice groups with MOPs significantly increased the
distance of tooth movement and number of osteoclasts when compared with the results
without MOPs. In the WT > KO and KO > KO mice, MOPS did not cause an increase in
tooth movement and the number of osteoclasts. The results suggest that MOPs accelerate
tooth movement, and this movement is dependent on the response of stromal cells to
TNF-α. This is the first report describing the effect of MOPs using KO mice and specificchimeric mice.
MOPs have been reported to induce inflammatory cytokines, TNF-α, IL-1, IL-3, IL-6,
IL-11 and IL-18, in rat periodontal ligament cells [11]. Moreover, it has been reported that
MOPs also increase OTM and enhance expression of inflammatory markers, including
TNF-α, in human gingival crevicular fluid [10]. These results suggest that these inflammatory cytokines induced by MOPs enhance osteoclast formation and bone resorption,
which accelerates OTM. However, these results do not explain how MOP-induced inflammatory cytokines enhance OTM. Several previous reports showed that TNF-α is induced
by orthodontic force, and TNF-α plays a significant role in osteoclast formation and bone
resorption in OTM [27–34]. Our previous studies also showed that TNF-α plays an important role in a mouse OTM model with an shorter average tooth movement distanceobserved for KO mice than that of WT mice [35,36]. An immunostaining study reported
the expression of TNF-α on the compression side of the periodontal ligament in a rat OTM
model which was enhanced by MOPs [16]. In this study, expression of TNF-α mRNA inmice was increased by MOPs. The results suggest that MOPs-induced TNF-α expression
may be linked with MOPs-induced acceleration of OTM. In our previous study, the distanceof tooth movement and osteoclast formation in WT and KO mice during an experimental
period were evaluated. A large amount of tooth movement occurred on Day 2 in bothgroups. From Days 4 to 8, large tooth movement was not observed in either group. On
Day 10, the distance of tooth movement increased in the WT mice, but not in the KO mice.
On Day 12, tooth movement increased in both groups, but the distance in the KO mice wassignificantly lower than that in the WT mice. The number of osteoclasts increased gradually
in both of mice. On Day 6, there was no significant difference in the number of osteoclasts inboth groups. However, the KO mice had significantly fewer osteoclast number than the WT
mice on Day 12 [35]. Therefore, in the present study, we evaluated both Day 6 and Day 12:
Day 6 was evaluated as a day when there is no difference, and Day 12 as the day when there
is a difference between the WT and KO groups. In the present study, on Day 6, there wasno significant difference of tooth movement and osteoclast formation with MOPs between
the WT and KO mice; however on Day 12, tooth movement and osteoclast formation weregreater in the WT mice with MOPs than the WT mice without MOPs. Conversely, there isno significant difference between the KO mice with MOPs and without MOPs. We foundthat the early stage of OTM is not affect by MOPs-induced TNF-α expression. We evaluatedhistological sections from the side opposite to the MOPs to evaluate the effect of MOPs on
the surrounding area’s osteoclast formation. Here, no difference in osteoclast formation
for the mice with and without MOPs after Day 6 and Day 12 was observed. These results
suggest that MOPs affected only the local area near the site of MOPs.

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A previous study investigated the contribution of macrophages and stromal cells toosteoclast formation induced by TNF-α in vivo [22,23]. These reports showed that stromalcells contributed more to osteoclast formation induced by TNF-α than macrophages. The
KO > WT chimeric mice showed an increase in osteoclast formation when compared with
the WT > KO chimeric mice [22,23]. Thus, these studies concluded that stromal cells inducedby TNF-α contributed more to osteoclast formation in vivo than that of macrophages. In
our previous study, four chimeric mice were used to determine the in vivo contributionof each cell type to OTM. The number of osteoclasts and distance of OTM were greaterin the WT > WT and KO > WT mice than those in the WT > KO and KO > KO mice. In
addition, the OTM distance and osteoclast numbers for the WT > KO mice were foundto not be significantly different to those observed for the KO > KO mice. The increase in
OTM distance and osteoclast numbers for the KO > WT mice was similar to those values
for the WT > WT mice. Moreover, the WT > KO mice showed significantly shorter OTM
distances and a lower osteoclast number than the WT > WT mice [37]. The findings showedthat the response of stromal cells to TNF-α is a crucial factor for osteoclast formation andbone resorption in OTM. In the present study, the amount of OTM in the WT > WT and
KO > WT mice with MOPs was higher than the amount in similar chimeric mice without
MOPs. However, no significant difference was observed between the WT > KO mice with
MOPs and the WT > KO mice without MOPs or between the KO > KO mice with MOPs
and the KO > KO mice without MOPs. The results indicate that MOPs induce TNF-α
expression, and the response of stromal cells to TNF-α is important for MOPs-enhanced
OTM and osteoclast formation.
Several studies showed that PDL cells expressed TNFRs [38–41]. Furthermore, it
has been reported that the expression levels of TNFRs in CD146 positive PDL cells werehigher than those in CD146 negative PDL cells, and TNF-α was found to have higher
level of biological effect in CD146 positive PDL cells than in CD146 negative PDL cells [38].
However, there is no report of the immunohistological examination for expression of TNFRs
in periodontal membrane. It is still unclear whether PDL cells express TNFRs. Therefore,
we examined whether TNFRs express in PDL cells by immunohistochemistry. TNFR1 andTNFR2 expressed in PDL cells in the WT mice but no TNFRs expressed in the KO mice
(Figure S1). The results indicated that TNFRs expressed as a protein level in PDL cells.
Furthermore, we investigated TNFR expression on bone marrow cells in each chimericmouse by fluorescent-activated cell sorting (FACS). Bone marrow macrophages of WT > WT
and WT > KO expressed TNFR1 and TNFR2 but not KO > WT and KO > KO (Figure S2).
The results suggest that at least the osteoclast precursors derived from hematopoietic cells
expressed TNFRs in PDL cells in the WT > WT and WT > KO mice. However, it is still
not clear exactly which kind of PDL cells expressed. Further studies are need to elucidate
these points.
Several reports have demonstrated that TNF-α enhances RANKL expression in stromalcells [22,23,42–45]. In the present study, we confirmed that TNF-α enhanced the RANKL
expression in stromal cells in a dose-dependent manner. Furthermore, we also evaluatedwhether TNF-α enhanced the osteoclast formation in a co-culture system consisting of TNFα-activated stromal cells and TNFRs and whether KO osteoclast precursors might eliminate
the direct TNF-α effect on osteoclast precursors as well as the TNF-α effect on stromalcells RANKL expression. The results demonstrated that TNF-α enhanced the RANKL
expression in stromal cells. Furthermore, TNF-α enhanced osteoclastogenesis and bone
resorption in the co-culture using KO osteoclast precursors in a dose-dependent manner.
The results indicate that increasing the amount of TNF-α enhances RANKL expression instromal cells, therefore enhancing osteoclast formation and bone resorption.
In a similar experiment, OTM in experimentally induced periodontitis in rats showed
an increased rate of distance of OTM compared with OTM without periodontitis. Thus,
periodontitis-induced bone loss was enhanced by OTM. It was concluded that OTM should
be initiated after successful periodontal therapy [46]. Conversely, it has been reported that
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rats [47]. It is unclear whether periodontal inflammation enhances OTM or not. Therefore,
further study is needed to elucidate this point.
In the present study, MOPs enhanced tooth movement and osteoclast formation via
TNF-α-responsive stromal cells. Furthermore, MOPs enhanced TNF-α expression in OTM.
This is probably because TNF-α acts on stromal cells to increase RANKL expression. MOPsenhanced TNF-α expression may increase the RANKL expression in stromal cells, andRANKL then enhances the osteoclast formation and tooth movement in OTM.
4. Materials and Methods
4.1. Mice
Nine- to ten-week-old C57BL6/J male mice were purchased from CLEA Japan (Tokyo,
Japan), and KO mice (Tnfrsf1atm1lmx Tnfrsf1btm1lmx ) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). The mice were housed under standard conditions (21–24 ◦ C,
12/12-h light/dark cycle). A granular diet (Oriental Yeast, Tokyo, Japan) was used as
feed for the mice to avoid excessive chewing power [35,37]. The protocols for all animal
procedures were approved and reviewed by the Tohoku University of Science Animal Careand Use Committee.
4.2. RNA Preparation and Real-Time PCR Analysis
To evaluate TNF-α expression around the maxillary left first molar after MOPs, alveolar bones of the maxilla around the maxillary left first molar were extracted at 0, 3, 6 and12 days after MOPs. To extract RNA, alveolar bone around the area of MOPs was dissected
and frozen in liquid nitrogen after mice were sacrificed, and then frozen alveolar bonesof left maxillae around the maxillary left first molar were crushed and homogenized byusing a Micro Smash MS-100R (Tomy Seiko Co. Ltd., Tokyo, Japan). These samples weresuspended in TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), and then total RNA was
isolated and purified from samples using RNeasy Mini Kit (Qiagen, Hilden, Germany),
as described previously [48]. The amount of total RNA was evaluated with 260 nm absorbance. For cDNA synthesizing by SuperScript IV Reverse Transcriptase (Invitrogen),cDNA synthesized from two µg of total RNA was used. TB Green Premix Ex Taq II (Takara,
Shiga, Japan) with the Thermal Cycler Dice Real Time System (Takara) were used forreal-time PCR. PCR was performed as follows: initial denaturation stage (30 s at 95 ◦ C),
amplification stage (50 amplification cycles with each cycle composed of a denaturation
step of 5 s at 95 ◦ C and an annealing step of 30 s at 60 ◦ C) and final dissociation stage (15s at 95 ◦ C, 30 s at 60 ◦ C and 15 s at 95 ◦ C). Glyceraldehyde 3-phosphate dehydrogenase(GAPDH) mRNA was used for normalizing expression levels. The following primers
were used as described previously [49]: GAPDH, 50 -GGTGGAGCCAAAAGGGTCA-30
and 50 -GGGGGCTAAGCAGTTGGT-30 ; TNF-α,50 - AGGCGGTGCTTGTTCCTCA-30 and
50 -AGGCGAGAAGATGATCTGACTGCC-30 ; and RANKL, 50 -CCTGAGGCCAGCCATTT-30
and 50 -CTTGGCCCAGCCTCGAT-30 .
4.3. Procedure for Bone Marrow Transplantation
In our previous studies, we established a method for WT and KO mice bone marrowtransplantation [22,23,37]. Mice were exposed to a lethal dose of gamma-irradiation (10 Gy).
Subsequently, bone marrow cells (1 × 106 cells in 100 µL PBS) from WT or KO mice weretransplanted via the tail vein of the WT or KO mice. We generated four kinds of chimericmice for investigating the contribution of TNF-α target cell types in vivo to MOPs-enhanced
OTM and osteoclast formation. The chimeric mice: (i) WT > WT, in which WT bone marrowcells from WT were transplanted into a lethal dose of gamma-irradiated WT; (ii) WT > KO,
in which WT bone marrow cells were transplanted into a lethal dose of gamma-irradiatedKO; (iii) KO > WT, in which the bone marrow cells of KO were transplanted into a lethal
dose of gamma-irradiated WT; and (iv) KO > KO, in which the bone marrow cells of KO
were transplanted into a lethal dose of gamma-irradiated KO.

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13 of 174.4. Sample Preparation for Histological Examination
After fixing, the samples were decalcified in 14% ethylenediaminetetraacetic acid
(EDTA) for 30 days. The solution was changed twice a week. After decalcification, thesamples were dehydrated by using graded ethanol and embedded in paraffin. Then, they
were sectioned in the horizontal plane at 4 µm. Sections at 100, 140, 180, 220 and 260 µm
apical to the maxillary left first molar bifurcation area were stained for TRAP activity toevaluate osteoclasts in every section. Fifty millimolar sodium tartrate, Fast Red Violet LB
Salt (Sigma-Aldrich, St. Louis, MO, USA) and Naphthol-ASMX-phosphate (Sigma-Aldrich)were used for TRAP staining. Osteoclasts were defined as multinucleated TRAP-positive
cells on the bone surface. They were counted by using light microscopy, as described
previously [35,37]. The area of counting was the mesial side of the distobuccal root of themaxillary left first molar, and every section was counted. The mean values were calculated
in all five sections, as described previously [35,37].
4.5. Procedure for Bone Marrow Stromal Cells
Bone marrow cells were obtained from the bone marrow cavity of the femora andtibia by flushing with PBS. After washing with PBS, the obtained bone marrow cells were
cultured in Dulbecco’s Modified Eagle Medium (Sigma-Aldrich) with 10% FBS, 100 µg/mL
streptomycin and 100 IU/mL penicillin G (Meiji Seika, Tokyo, Japan) for 2 weeks. After
culturing, the suspended cells were washed thoroughly with PBS. The remaining adherent
cells were detached by a trypsin-EDTA (Sigma-Aldrich). We used the cells as bone marrowstromal cells, as described previously [50].
4.6. Preparation of Osteoclast Precursors and Co-Culturing of Osteoclast Precursors from KO Miceand Stromal Cells from WTThe bone marrow cells of KO mice were obtained by flushing and cultured in αMEM that included 10% FBS, 100 µg/mL streptomycin and 100 IU/mL penicillin G with
100 ng/mL M-CSF for 4 days. After PBS washing, the adherent cells were obtained byusing trypsin-EDTA. The cells were used as osteoclast precursors. KO osteoclast precursors
and WT bone marrow stromal cells were co-cultured with several doses of TNF-α (0, 1,
10 and 100 ng/mL), 10–6 M prostaglandin and 10–8 M 1,25(OH)2 D3 (Sigma-Aldrich) in
a 96-well plate and in Osteo Assay Plate 96 Wells (Corning life Sciences, Corning, NY,
USA). The co-culture was maintained for 4 days and then terminated by fixation with 4%paraformaldehyde and stained with TRAP staining consisting of acetate buffer (pH 5.0),
naphthol AS-MX phosphate and 50 mM sodium tartrate Fast Red Violet LB salt. Cells
that were TRAP-positive and had two or more nuclei were considered to be osteoclasts.
Formation of pits was evaluated after 5 days of culturing.
4.7. Statistical Analysis
The Scheffe F test was performed to evaluate significance, and p values less than 0.05
were considered to be statistically significant. All quantitative results are presented as themean ± standard deviation.
5. Conclusions
MOPs increased OTM distance and TNF-α expression. The responsiveness of stromalcells to TNF-α is a crucial factor for osteoclast formation, and MOPs amplify this response,
leading to increased movement distance during OTM (Figure 7). This work provides atheoretical base for applications in humans. However, many variables come into play if
MOPs are to be practiced in humans, such as the number, location, and timing of MOPs.
These factors should be taken into consideration for MOPs-induced TNF-α to act effectively
on stromal cells in future experiments studying this application in humans.

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Figure 7. Schema of the mechanism of MOPs-enhanced osteoclast formation and MOPs-accelerated OTM.
Supplementary Materials: The following supporting information can be downloaded at:https://www.mdpi.com/article/10.3390/ijms23062968/s1.
Author Contributions: Conceptualization, R.K. and H.K.; methodology, H.K.; validation, R.K. and
H.K.; formal analysis, R.K., S.O. and T.N.; investigation, R.K., H.K., A.M., F.O., T.N., Y.N., A.P., J.M.
and K.K.; resources, H.K.; data curation, R.K.; writing—original draft preparation, R.K.; writing—review and editing, H.K.; supervision, I.M.; project administration, H.K.; funding acquisition, H.K.
and I.M. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported in part by JSPS KAKENHI grants from the Japan Society for the
Promotion of Science (No. 19K10397 to HK and No. 18K09862 to IM).
Institutional Review Board Statement: The protocols for all animal procedures were approved andreviewed by the Tohoku University of Science Animal Care and Use Committee.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available from the corresponding authors upon reasonable request.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
EDTA
GAPDH
IL
KO
M-CSF
MOPs
Ni-Ti
OTM
RANKL
RAP
TNFTRAP
WT

ethylenediaminetetraacetic acidglyceraldehyde 3-phosphate dehydrogenase
interleukin
TNF receptor 1- and TNF receptor 2-deficientmacrophage colony-stimulating factor
micro-osteoperforations
nickel-titanium
orthodontic tooth movement
receptor activator of nuclear factor κB ligand
regional acceleratory phenomenon
tumor necrosis factortartrate-resistant acid phosphatasewild-type

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