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Figure Legends
Figure 1. Orthodontic tooth movement and region of MOPs in mice: (A) Schematic of
the appliance fixed 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.
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 with and 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 and without MOPs in
WT mice. Arrows indicate the direction of OTM. (D) Evaluation of the number of
osteoclasts 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 side of 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 were analyzed by using the Scheffe
test. Scale bars = 100 μm.
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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 expression of 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.
Figure 4. MOPs-induced tooth movement and osteoclast formation is dependent on TNFα: (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 and 12 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 of experimental 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.
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
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experimental loading. (C) TRAP-stained histological sections 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 alveolar bone surface
around the distobuccal root of the left maxillary first molar after 12 days of OTM for
chimeric 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.
Figure 6. TNF-α induces RANKL expression in WT stromal cells and induced osteoclast
formation in a co-culture using KO osteoclast precursors to eliminate the effect of TNFα to osteoclast formation and WT stromal cells in a dose-dependent manner. (A) Realtime 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 number of 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)2D3 for 4 days. The results are
presented as the mean ± standard deviation (n = 4). ** p < 0.01 indicate significant
differences, which were analyzed by using the Scheffe’s test.
Figure 7. Schema of the mechanism of MOPs-enhanced osteoclast formation and MOPsaccelerated OTM.
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Figure S1. Here is a picture on orthodontic tooth movement. The maxillary central incisor
of the mouse is shown on the left side. In addition, the device was placed on the maxillary
left first molar in this study. The device was drilled just below the maxillary central incisor
in the anterior region and a wire was ligated. For the molars, a wire was passed through
the interdental space between the maxillary left first molar and second molar, and the
device was ligated.
Figure S2. TNFR1 and TNFR2 expressed on PDL cells in WT mice but not TNFRs KO
mice. Harvested maxillae from WT and TNFRs KO mice were fixed overnight in 4%
paraformaldehyde (diluted in PBS). Samples of maxillae were decalcified in 14%
ethylenediaminetetraacetic acid (EDTA) at room temperature for 1 month. The EDTA
solution was changed every 2 days. Decalcified maxillae were put in histological cassettes
and place them in a bag. Immerse the samples in 1000 mL of 30% ethanol, 70% ethanol,
80% ethanol, and 90%ethanol for 1 h each, 1000 mL of 95% ethanol for 3 hours, 1000
mL of 100% ethanol twice for 7 h and 12 h each, 1000 mL of xylene three times for 0.5
h, 1 h and 1.5 h each for dehydration, and then 1000 mL of liquid paraffin (56 °C) twice
for 7 h and 12 h each in an automatic tissue processor connected to a chemical hood to
allow the xylene to evaporate. Horizontal sections of the samples were cut at a thickness
of 4 µm. Sections of maxillae were taken at approximately 150 µm from the root branch
of the upperleft first molar. For immunohistochemistry, maxillae paraffin sections were
deparaffinized, rehydrated, and treated with 0.3%H₂O₂ in PBS for 15 minutes. Sections
were then blocked with 5% skim milk for 30 minutes at 37°C and treated with antiTNFR1 polyclonal antibody (rabbit polyclonal, 21574–1-AP, Proteintech, Rosemont, IL)
and anti-TNFR2 polyclonal antibody (rabbit polyclonal, 19272-1-AP, Proteintech,
- 31 -
Rosemont, IL) diluted to 1:50 in Can Get Immunostain solution B (Toyobo, Osaka, Japan),
overnight at 4°C. Sections were rinsed, then processed with VECTASTAIN Elite ABC
Kit PK 6101 (Vector, Burlingame, CA, USA) and treated with 3,3’-diaminobenzidine.
Hematoxylin was used for counterstaining. Immunohistochemical analysis showed that
there were TNFR1-positive cells and TNFR2-positive cells in the periodontal membrane
in WT mice but not TNFRs KO mice.
Figure S3. TNFR1 and TNFR2 were selectively expressed on osteoclast precursors in
chimeric mice. Bone marrow cells were cultured with M-CSF for 3day. The adherent cells
were incubated in NaN3 (0.1%) plus FBS-PBS (FBS, 1%) for 1 hours with FITCconjugated anti-TNFR1 mAb (Abcam, Cambridge, UK). They were then washed and
diluted with NaN3 plus FBS. Furthermore, the cells were also incubated for 1 hour with
PE-conjugated anti-TNFR2 mAb (BD Biosciences, San Jose, USA). TNFR1 and TNFR2
expression were analyzed by FACS. Osteoclast precursors of WT>WT and WT>KO
expressed TNFR1 and TNFR2, but KO>WT and KO>KO did not express TNFR1 and
TNFR2.
Figure S4. H-E staining of tissues from the OTM group with orthodontic tooth movement
for 12 days and from the MOPs group was performed. Alveolar bone resorption was
observed on the compression side, and slight new bone formation was observed on the
traction side.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Figure 7
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Figure S1
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Figure S2
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Figure S3
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Figure S4
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Acknowledgment
I would like to express my sincere gratitude to my supervisor, Prof. I. Mizoguchi of
Division of Orthodontics and Dentofacial Orthopedics, Tohoku University Graduate
School of Dentistry, for him direction and review throughout the whole process of
research. Additionally, I would like to express my deepest appreciation to Dr. H. Kitaura,
Dr. A. Kishikawa, Dr. S. Ogawa, Dr. F. Ohori, Dr. T. Noguchi, Dr. A. Marahleh, Dr. Y.
Nara, Dr. A. Pramusita for their elaborated guidance, considerable encouragement and
invaluable discussion that make my research of great achievement.
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