Archives of Oral Biology 102 (2019) 83–92

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Archives of Oral Biology

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

Acetaminophen reduces apical root resorption during orthodontic tooth T

movement in rats
⁎
Masato Kaku , Taeko Yamamoto, Yuka Yashima, Jin Izumino, Haruka Kagawa, Kazutaka Ikeda,
Kotaro Tanimoto
Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima
734-8553, Japan

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

Keywords: Objective: The present study aimed to investigate the inhibitory effect of acetaminophen on apical root re-
Orthodontic tooth movement sorption during orthodontic tooth movement by controlling inflammation in the periodontal ligament and apical
Root resorption pulp tissue.
Inflammation Methods: Human periodontal ligament and pulp cells were subjected to 10 kPa of cyclic tensile force (CTF) in a
Acetaminophen
Flexcell Strain Unit for 48 h. Then, 10 and 100 μM acetaminophen were added to the culture medium, and the
expression of interleukin (IL)-1B, receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis
factor (TNF)α, and colony stimulating factor 1 (CSF1) were evaluated. In an animal experiment, the upper first
molars of 7-week-old rats were moved mesially by applying 10 g of orthodontic force. After 30 days of force
application, the effects of acetaminophen on apical root resorption were examined.
Results: In both the periodontal ligament and pulp cells, the expression levels of IL-1B, TNFα, RANKL, and CSF1
were significantly higher in the CTF-treated group than in the control group. However, the expression levels of
these factors were decreased by acetaminophen administration. High expression of IL-1B, TNFα, RANKL, and
CSF1 at the root apex were also detected immunohistochemically in rats after tooth movement, but were de-
creased by acetaminophen administration. In addition, the number of odontoclasts and the amount of apical root
resorption were significantly decreased in the acetaminophen group. Importantly, no significant difference in
tooth movement was observed between the acetaminophen and control groups.
Conclusions: These results suggest that acetaminophen can reduce severe root resorption in the apex area
without disturbing orthodontic tooth movement.

1. Introduction
In orthodontic patients, root resorption is crucial side effect.
Rakhshan and colleagues reported that the frequency of root resorption
after orthodontic tooth movement is around 91.8% (Rakhshan,
Nateghian, & Ordoubazari, 2012). Many factors lead to root resorption
during tooth movement, such as patient age (Nanekrungsan,
Patanaporn, Janhom, & Korwanich, 2012), the orthodontic force ap-
plied (Roscoe, Meira, & Cattaneo, 2015), the treatment period
(Rakhshan et al., 2012), and malocclusion type (Elhaddaoui, Benyahia,
Azeroual, Razine, & Bahije, 2016). It is thought that root resorption at
the root apex occurs due to inflammation of the apical root tissue, in-
cluding the periodontal ligament and apical dental pulp. Indeed, pre-
vious reports of human studies showed that root resorption is much
more severe in vital teeth than in pulpless teeth (Remington, Joondeph,
Artun, Riedel, & Chapko, 1989; Spurrier, Hall, Joondeph, Shapiro, &
Reidel, 1990). Therefore, it is thought that apical root resorption can be
reduced by the administration of non-steroidal anti-inflammatory
drugs. However, the effects of non-steroidal anti-inflammatory drugs on
root resorption are controversial. Kirschneck reported that meloxicam
reduced orthodontically induced root resorption as well as tooth
movement velocity in rats (Kirschneck, Meier, Bauer, Proff, &
Fanghänel, 2017). Gameiro showed that administration of celecoxib did
not reduce odontoclasts and root resorption during tooth movement
(Gameiro et al., 2008). However, Yamamoto demonstrated that lox-
oprofen administration significantly decreased the number of odonto-
clasts and apical root resorption without disturbing tooth movement
(Yamamoto et al., 2018). Therefore, further experiments are needed to
confirm the effects of non-steroidal anti-inflammatory drugs on root
resorption and the rate of tooth movement.

⁎
Corresponding author.
E-mail address: mkaku@hiroshima-u.ac.jp (M. Kaku).

https://doi.org/10.1016/j.archoralbio.2019.04.002
Received 25 January 2019; Received in revised form 2 April 2019; Accepted 4 April 2019
0003-9969/ © 2019 Elsevier Ltd. All rights reserved.
M. Kaku, et al. Archives of Oral Biology 102 (2019) 83–92

A systematic review of randomized controlled trials revealed that

although non-steroidal anti-inflammatory drugs have greater efficiency,
they also have greater side effects (Zhang et al., 2010). Therefore, non-
steroidal anti-inflammatory drugs should be administered carefully,
especially in young children. In contrast, acetaminophen is widely used
for pain relief and as an antipyretic agent by all age groups, because of
its lesser side effects (Zhang, Jones, & Doherty, 2004). Although acet-
aminophen has little anti-inflammatory effects, recent studies have
shown that acetaminophen can suppress local inflammation by in-
hibiting cyclooxygenase-2 (Hinz, Cheremina, & Brune, 2008; Lee et al.,
2007).
Therefore, in this study, we observed the effects of acetaminophen
on apical root resorption and the expression of interleukin (IL)-1B, re-
ceptor activator of nuclear factor kappa-B ligand (RANKL), tumor ne-
crosis factor (TNF)α, and colony stimulating factor 1 (CSF1) in the root
apex area after experimental tooth movement as well as the rate of
tooth movement in rats. Moreover, because the root apex area includes Fig. 1. Schematic drawing of experimental tooth movement. The upper right
the periodontal ligament and apical pulp tissues, we examined the ef- molar were moved mesially by closed-coil spring with magnitude at 10 g for 30
fect of acetaminophen on the protein expression of IL-1B, TNFα, days.
RANKL, and CSF1 in human periodontal ligament and pulp cells after
cyclic tensile force (CTF) application.

2. Materials and methods

2.1. Cell culture

Human periodontal ligament cells were obtained from ScienCell

Research Laboratories (San Diego, CA, USA), and human pulp tissues
were obtained from three orthodontic patients who provided consent.
Ethical approval for this study was obtained from the Ethics Committee
of Hiroshima University. The periodontal ligament and pulp cells were
cultured in Dulbecco’s Modified Eagle’s Medium (Nissui
Pharmaceutical Co., Ltd., Tokyo, Japan), containing 10% fetal calf
serum (FCS; Biological Industries, Kibbutz Beit-Haemek, Israel), 32 U/
mL penicillin-G (Meiji Seika, Tokyo, Japan), 250 μg/mL amphotericin B
(Nacalai Tesque, Kyoto, Japan), and 60 μg/mL kanamycin (Meiji Seika).
The cells were cultured at 37 °C in the presence of 5% CO2, and the
medium was changed twice a week. After the cells reached confluence,
they were detached by treatment with 0.25% trypsin/ethylenediami-
netetraacetic acid for 5 min. Then, the cells were subcultured in a 100-
mm culture dish at 3 × 105 cells per well. For all experiments, cells at
the fourth and sixth passages were used.

2.2. Cyclic tensile force application and the addition of acetaminophen

A previous study showed that CTF mimics the force of orthodontic

tooth movement (Hu et al., 2015). Nokhbehsaim also used CTF to re-
produce orthodontic force (Nokhbehsaim et al., 2010). Therefore,
periodontal ligament and pulp cells (1 × 105) were cultured on a silicon
membrane, and CTF was applied with a strain unit (FX-2000; Flexcell
International Co., Hillsborough, NC, USA). The cells were loaded with
10 kPa (12% cell elongation) of tensile force at a frequency of 30 cy-
cles/minute. Then, the cells were cultured with or without acet-
aminophen (10 or 100 μM; Yoshida Pharmaceutical Co., Ltd., Tokyo,
Japan). The dose of acetaminophen was determined in reference to the
dosage of loxoprofen for in vitro study (Yamamoto et al., 2018), and the
effects of CTF on the expression levels of IL-1B, TNFα, RANKL, and
CSF1 were examined.
Fig. 2. Measurement of the amount of tooth movement by computed tomo-
Cells were treated in four groups as follows: Group 1 (control): no
graphy.
CTF and without acetaminophen, group 2: 10 kPa CTF for 48 h without
acetaminophen, group 3: 10 kPa CTF for 48 h with 10 μM acet-
aminophen, and group 4: 10 kPa CTF for 48 h with 100 μM acet-
aminophen.

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2.3. Measurement of IL-1B, TNFα, RANKL, and CSF1 protein

concentrations

The medium from the human periodontal ligament and pulp cell
cultures after CTF treatment with or without acetaminophen was col-
lected for enzyme-linked immune sorbent assay and centrifuged at
2000 rpm for 5 min. The protein concentration in the culture medium
was measured by comparison to standard curves using the following
kits: the Quantikine Human IL-1B Immunoassay Kit and Quantikine
Human TNFα Immunoassay Kit (R&D Systems, Inc., Minneapolis, MN,
USA) for IL-1B and TNFα; the Human RANKL Mini TMB ELISA
Development Kit (Peprotech, Rocky Hill, NJ, USA) for RANKL; and the
Quantikine Human CSF1 Immunoassay Kit (R&D Systems) for CSF1.

2.4. Animal studies

All animal experimental procedures were approved by the Ethics

Fig. 3. Observation of alveolar bone and root resorption. The number of os-
teoclasts at the tension side of the alveolar bone and odontoclasts at the root
apex, and the amount of root resorption were examined at square area
(1200 × 1200 μm2).
Committee of the Hiroshima University Faculty of Dentistry (A-15-116).
Fifteen 7-week-old Wistar rats were divided into three groups. The first
group was a negative control group for immunohistochemistry with no
tooth movement that was orally administered phosphate buffered saline
with a feeding needle. The second group underwent first molar move-
ment for 30 days and was orally administered phosphate buffered
saline. The last group underwent first molar movement for 30 days and
was orally administered a 2% acetaminophen syrup at 10 mg/kg/day
(AYUMI Pharmaceutical Co., Tokyo, Japan). The dose of acet-
aminophen used in the experiment was based on the human dosage and
the low dose of acetaminophen administered to rats in a previous study
(Gonzales et al., 2009). An orthodontic cobalt-chrome closed-coil
spring (0.009 × 0.030 in.; JM Ortho Co., Tokyo, Japan) was set on the
upper right molar and incisor by orthodontic ligature wire (0.008 inch;
TOMY, Inc., Tokyo, Japan) and bonded with a bonding agent

Fig. 4. The effects of acetaminophen sodium on the expression of IL-1B (A), TNFα (B), RANKL (C) and CSF1 (D) protein concentrations in periodontal ligament cells
stimulated by a 10-kPa CTF for 48 h. (Values are shown as the mean ± SD of at least three independent repeats. *P < 0.05, **P < 0.01, N = 4).

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Fig. 5. The effects of acetaminophen sodium on the expression of IL-1B (A), TNFα (B), RANKL (C) and CSF1 (D) protein concentrations in pulp cells stimulated by a
10-kPa CTF for 48 h. (Values are shown as the mean ± SD of at least three independent repeats. *P < 0.05, **P < 0.01, N = 4).
(CLEARFIL Mega Bond; Kuraray Noritake Dental, Inc., Tokyo, Japan)
and dental composite resin (CLEARFIL APeX; Kuraray Noritake
Dental). The magnitude of the force was measured by a tension gauge
(Shinpo Corp., Tokyo, Japan), and an initial force of 10 g was applied.
The upper molar was moved in a mesial direction, away from the upper
incisor with the closed-coil spring for 30 days (Fig. 1), and then the
distance between the upper first and second molar was measured as the
amount of first molar movement by computed tomography (Sky Scan
1176; Bruker micro CT, Kontich, Belgium) and a data viewer (Bruker
micro CT) (Fig. 2).
The animals were sacrificed under general anesthesia with mede-
tomidine (1.0 mg/ml; Kyoritsu Seiyaku Corporation, Tokyo, Japan),
midazolam (5.0 mg/ml; Sandoz, Tokyo, Japan), and butorphanol
(5.0 mg/ml; Meiji Seika Pharma, Tokyo, Japan), and then perfused with
4% paraformaldehyde. The maxillary bones were decalcified and em-
bedded in paraffin. Then, the specimens were sliced into 5-μm thick
sections in the frontal direction. For immunohistochemistry, the tissue
sections were deparaffinized and incubated with 3% H2O2 in methanol
for 30 min to inhibit endogenous peroxidase activity. After washing in
phosphate buffered saline, the sections were incubated with primary
antibodies against human IL-1B (rabbit polyclonal, 1:50; Abcam,
Tokyo, Japan), human TNFα (goat polyclonal, 1:100; R&D Systems,
Minneapolis, MN, USA), human RANKL (mouse polyclonal, 1:100;
Abcam), and human CSF1 (goat polyclonal, 1:100; Santa Cruz
Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. Then, the im-
munocomplexes were detected using the Histofine Simple Stain MAX-
Po Rabbit kit (Nichirei, Tokyo, Japan) for IL-1B, the Histofine Simple
Stain MAX-Po Goat kit (Nichirei) for TNFα and CSF1, and the Histofine
Simple Stain MAX-Po Mouse kit (Nichirei) for RANKL. The sections
were rinsed with phosphate buffered saline and treated with the
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substrate reagent 3, 3′-diaminobenzidine tetra-hydrochloride. Then, all
sections were counterstained with hematoxylin. The number of positive
cells for each factor at the root apex was determined (measurement
field: 1200 × 1200 μm2, Fig. 2).
To investigate the osteoclasts present on the pressure side of the
alveolar bone and the odontoclasts at the root apex, a measurement
field (1200 × 1200 μm2, Fig. 3) was defined. Specimens were subjected
to tartrate-resistant acid phosphatase staining, and the number of po-
sitive cells were counted. In each specimen, five sections, selected at 35-
μm intervals, were examined based on the results of previous studies
(Kaku et al., 2014; Sumi et al., 2017; Yamamoto et al., 2018). The
amount of apical root resorption in the same area was also investigated.
Digitized photomicrographs of each sample were obtained with ima-
ging software (NIH Image, Bethesda, MD, USA), and the root resorption
area was measured.
2.5. Statistical methods
The protein expression in pulp cells were evaluated using analysis of
variance and Fisher’s multiple comparison test. Student’s t-test was used
to compare the quantification of the immunohistochemically positive
cells, the tooth movement, number of osteoclasts and odontoclasts, and
root resorption areas between the control and acetaminophen groups,
and p values less than 0.05 were considered as statistically significant.
M. Kaku, et al.
Fig. 6. Immunohistochemical staining for IL-1B. (A) non-tooth movement without drug (B) tooth movement without drug (C) tooth movement with acetaminophen
(N = 5, Bar denotes 100 μm).
3. Results
3.1. In vitro studies
3.1.1. Effects of acetaminophen on the protein expression of IL-1B, TNFα,
RANKL, and CSF1 in periodontal ligament cells after CTF application
Compared to control periodontal ligament cells, the protein levels of
IL-1B, TNFα, RANKL, and CSF1 were increased in periodontal ligament
cells in the CTF group by the application of CTF. Compared to the levels
in the CTF group, the expression levels of IL-1B, TNFα, RANKL, and
CSF1 were reduced in the acetaminophen groups by acetaminophen
administration as follows. IL-1B levels were decreased dose-depen-
dently in the 10 μM and 100 μM acetaminophen groups by 0.64- and
0.50-fold, respectively (Fig. 4A). The amount of TNFα was significantly
decreased by 0.41- and 0.31-fold in the 10 and 100 μM acetaminophen
groups, respectively (Fig. 4B). The amount of RANKL was also slightly
reduced, by 0.83- and 0.74-fold, in the 10 and 100 μM acetaminophen
groups, respectively (Fig. 4C). The CSF1 protein concentration was
significantly reduced by 0.70-fold in the 100 μM acetaminophen group
(Fig. 4D).
3.1.2. Effects of acetaminophen on the protein expression of IL-1B, TNFα,
RANKL, and CSF1 in pulp cells after CTF application
Similar to what was observed in periodontal ligament cells, the
application of CTF increased the protein concentrations of IL-1B, TNFα,
RANKL, and CSF1. The expression levels of IL-1B, TNFα, and RANKL in
the 100 μM acetaminophen group were significantly decreased by
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Archives of Oral Biology 102 (2019) 83–92
0.55-, 0.67-, and 0.51-fold, respectively, compared to the levels in the
CTF group (Figs. 5A-C). The amount of CSF1 was decreased by 0.84-
fold in the 100 μM acetaminophen group (Fig. 5D).
3.2. Animal studies
3.2.1. Immunohistochemical staining
IL-1B, TNFα, RANKL, and CSF1 expression was not observed in rats
in the non-tooth movement control group (Figs. 6A, 7 A, 8 A, and 9 A).
After 30 days of tooth movement without acetaminophen administra-
tion, many positively stained cells for these factors were observed in the
periodontal ligament and pulp tissues (Figs. 6B, 7 B, 8 B, and 9 B). In
contrast, the immunoreactivity to these factors was weak in the peri-
odontal ligament and pulp tissues of the acetaminophen-administered
groups (Figs. 6C, 7 C, 8 C, and 9 C). Quantitative evaluation showed
that the numbers of IL-1B-, TNFα-, RANKL-, and CSF1-positive cells
were significantly reduced in the acetaminophen group compared to
the numbers in the tooth movement without acetaminophen group
(Table 1).
3.2.2. Tooth movement
The amount of tooth movement in the control and acetaminophen
groups was measured after 30 days. There was no significant difference
in the amount of tooth movement between the two groups (Fig. 10).
M. Kaku, et al.
Fig. 7. Immunohistochemical staining for TNFα. (A) non-tooth movement without drug (B) tooth movement without drug (C) tooth movement with acetaminophen
(N = 5, Bar denotes 100 μm).
3.2.3. The numbers of osteoclasts and odontoclasts and the amount of root
resorption
In both the control and acetaminophen groups, many osteoclasts
were observed in the alveolar bone on the pressure side (Fig. 11B and
D), and there was no statistically significant difference in the number of
osteoclasts in the alveolar bone on the pressure side between the two
groups (Fig. 12). Many odontoclasts appeared at the apical root area in
the control group (Fig. 11A), although there was a lesser number of
odontoclasts in the acetaminophen group (Fig. 11C), the difference
being statistically significant (Fig. 13). In the acetaminophen group, the
root resorption volume in the apical root area was significantly less
than that in the control group (Fig. 14).
4. Discussion
The root apex is a unique region that includes the periodontal li-
gament and apical pulp. Previous human study revealed that root re-
sorption during orthodontic tooth movement is much more severe in
vital teeth than in pulpless teeth (Remington et al., 1989; Spurrier et al.,
1990). Animal studies also showed that after orthodontic tooth move-
ment, the amount of inflammatory apical root resorption was sig-
nificantly less in pulpless teeth than in vital teeth (Kaku et al., 2014).
The expression of IL-1B, TNFα, RANKL, and CSF1 and odontoclasts
were clearly detected in the pulp tissue of vital teeth after tooth
movement. However, these factors were not observed in the control
group without tooth movement (Sumi et al., 2017). From these find-
ings, apical root resorption during tooth movement tends to become
severe than in the different parts of the roots, because apical root tissue
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include both periodontal ligament and apical dental pulp. Therefore,
these two tissues should be evaluated when investigating apical root
resorption during orthodontic tooth movement. Recently, it was re-
ported that human pulp cells treated with CTF expressed higher gene
and protein levels of IL-1B, TNFα, RANKL, and CSF1 than untreated
control cells, and that these upregulated gene and protein levels were
suppressed by the administration of loxoprofen. Furthermore, an in vivo
study showed that the expression of IL-1B, TNFα, RANKL, and CSF1
after orthodontic tooth movement was also decreased by loxoprofen
administration. Fewer odontoclasts and a smaller apical root resorption
area were observed in the loxoprofen group than in the control group
(Yamamoto et al., 2018). These results indicated that apical pulp in-
flammation can be counteracted by non-steroidal anti-inflammatory
drugs to reduce apical root resorption during orthodontic tooth move-
ment. However, because inflammation of the periodontal ligament
tissue at the root apex is also crucial for apical root resorption, we
studied periodontal ligament and pulp cells both in vitro and in vivo in
this study.
Acetaminophen is frequently administered for pain management
and as an antipyretic. Although acetaminophen shows a weaker an-
algesic effect than non-steroidal anti-inflammatory drugs, it is used for
the treatment of all age groups as an alternative to non-steroidal anti-
inflammatory drugs because of its lower side effects (Zhang et al.,
2004). Its mechanism of action is thought to be similar to that of non-
steroidal anti-inflammatory drugs, and many reports have suggested
that acetaminophen acts as an anti-inflammatory agent through selec-
tive blockage of cyclooxygenase-2. Lee et al. examined the effect of
acetaminophen on prostaglandin release and gene expression related to
M. Kaku, et al.
Fig. 8. Immunohistochemical staining for RANKL. (A) non-tooth movement without drug (B) tooth movement without drug (C) tooth movement with acetaminophen
(N = 5, Bar denotes 100 μm).
prostaglandin production following the extraction of mandibular-im-
pacted wisdom teeth, which showed that acetaminophen is a selective
inhibitor of cyclooxygenase-2 (Lee et al., 2007). Hinz et al. investigated
cyclooxygenase inhibition in vitro and ex vivo following administration
of a 1000 mg dose of acetaminophen. The in vitro results showed that
acetaminophen induced a 4.4-fold selective inhibition of cycloox-
ygenase-2 when compared to cyclooxygenase-1 (Hinz et al., 2008).
Thus, it is now widely accepted that acetaminophen is a selective cy-
clooxygenase-2 inhibitor.
Graham et al. reported that acetaminophen does not inhibit severe
inflammation, such as that in rheumatoid arthritis and acute gout, but
can suppress lesser inflammation, such as that in tooth extraction
(Graham, Davies, Day, Mohamudally, & Scott, 2013). In this study,
periodontal ligament and pulp cells exposed to CTF expressed higher
protein levels of IL-1B, TNFα, RANKL, and CSF1 than the unexposed
control, and the upregulated expression of these factors was suppressed
by acetaminophen. Although there were no statistically significant
differences in RANKL expression in periodontal ligament cells and CSF1
expression in pulp cells between the control and acetaminophen groups,
it was assumed that these two factors would be decreased by additional
acetaminophen because acetaminophen induced a dose-dependent re-
duction in the effects of CTF. Moreover, after experimental tooth
movement in rats, high expression levels of IL-1B, TNFα, RANKL, and
CSF1 were detected in the periodontal ligament and pulp tissue of the
apical root area by immunohistochemistry, which were decreased by
the administration of acetaminophen. This suggested that acet-
aminophen can inhibit inflammation in periodontal ligament and pulp
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Archives of Oral Biology 102 (2019) 83–92
tissues following orthodontic tooth movement and dental trauma.
Furthermore, our present data showed that both the number of odon-
toclasts and the amount of apical root resorption were decreased in the
acetaminophen group. However, no significant difference in the
amount of tooth movement was observed between the acetaminophen
and control groups.
Meanwhile, the effects of acetaminophen on root resorption and the
rate of tooth movement are controversial. Kehoe et al. reported that the
control and acetaminophen groups exhibited similar degrees of tooth
movement for pig incisors (Kehoe, Cohen, Zarrinnia, & Cowan, 1996).
In addition, Gonzales et al. showed that there was no significant dif-
ference in the rate of tooth movement and the amount of root resorp-
tion between the control and acetaminophen groups (Gonzales et al.,
2009). However, in Gonzales’s study, supraphysiological levels of force
(50 g for rat molars) and a limited experimental period (2 weeks) were
used, and the drug was administered by placing it in drinking water. In
the present study, administration of acetaminophen by oral gavage
using a feeding needle ensured reliable intake of the full dosage.
Moreover, the previous study revealed that the amount of tooth
movement was significantly larger with less root resorption following
light orthodontic force application (10 g for rat molars) compared to
that following heavy force application (Gonzales et al., 2008). There-
fore, a difference in the rate of tooth movement and the amount of root
resorption may be observed between the previous study and ours. The
dose of acetaminophen is also important for the rate of tooth move-
ment. Hammad reported that the rate of tooth movement was inhibited
in rats that received 150 mg/kg acetaminophen (Hammad, EI-Hawary,
M. Kaku, et al. Archives of Oral Biology 102 (2019) 83–92

Fig. 9. Immunohistochemical staining for CSF1. (A) non-tooth movement without drug (B) tooth movement without drug (C) tooth movement with acetaminophen
(N = 5, Bar denotes 100 μm).

Table 1
The number of positive cells after orthodontic tooth movement.
Factors without acetaminophen with acetaminophen

IL-1B 151.7 ± 22.5** 24.3 ± 9.5

TNFα 224.3 ± 4.0** 19.7 ± 4.5
RANKL 503.3 ± 55.1** 0
CSF1 124.7 ± 25.0** 26.7 ± 25.2

(**P < 0.01, N = 5).

& EI-Hawary, 2012). It was thought that this high dose of acet-
aminophen interfered with tooth movement, although the physiological
dose used in our study did not reduce tooth movement. Because the
dentin and cementum are mainly composed of matrix-dominant tissues
without blood vessels, it is more difficult to resorb than alveolar bone
(Goldie & King, 1984; Midgett, Shaye, & Fruge, 1981). Kurihara re- Fig. 10. Changes in the amount of tooth movement.
There was no significant difference in the amount of tooth movement between
ported that the root is more difficult to resorb than alveolar bone during
the control and the acetaminophen groups. Values are shown as the
orthodontic tooth movement because the cementum of the root is
mean ± SD of at least three independent repeats. (N = 5)
protected by cementoblasts (Kurihara, 2004). Moreover, in this study, a
number of osteoclasts and alveolar bone resorption were detected after
orthodontic tooth movement, while a small amount of root resorption Based on these findings, root resorption at the root apex after or-
was observed in the acetaminophen group. It is thought that following thodontic treatment can be decreased by administration of the proper
orthodontic tooth movement, alveolar bone remodeling occurs to pro- dosage of acetaminophen without delaying tooth movement. This
tect against external apical root resorption. That is why there was no suggests that acetaminophen is useful for reducing root resorption after
significant difference in the rate of tooth movement between the control orthodontic tooth movement due to its anti-inflammatory action and
and acetaminophen groups, which both showed small amounts of root better gastrointestinal safety compared to non-steroidal anti-in-
resorption. flammatory drugs.

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Fig. 11. Histological examination after orthodontic tooth movement. A and B :control group (tooth movement without drug), C and D: acetaminophen group (tooth
movement with acetaminophen). Many osteoclasts were found in the pressure side of alveolar bone after tooth movement both in the control and acetaminophen
groups (B and D). Many odontoclasts appeared at the apical root area in the control group (A), although there was less number of odontoclasts in the acetaminophen
group (C).

Fig. 13. The number of odontoclasts appeared in the apical root area.
Fig. 12. The number of osteoclasts appeared in the pressure side of the alveolar
The number of odontoclasts in the control group (tooth movement without
bone.
drug) was significantly higher than in the acetaminophen group (tooth move-
There was no significant difference between the control group (tooth movement
ment with acetaminophen) (Values are shown as the mean ± SD of at least
without drug) and the acetaminophen group (tooth movement with acet-
three independent repeats. **P < 0.01, N = 5).
aminophen) (Values are shown as the mean ± SD of at least three independent
repeats. (N = 5)
acetaminophen administration. The number of odontoclasts and the
amount of apical root resorption after orthodontic tooth movement
5. Conclusions
were decreased in the acetaminophen group. There was no significant
difference in the amount of tooth movement between the control and
Upregulation of the gene and protein expression of IL-1B, TNFα,
acetaminophen groups. These observations suggest that acetaminophen
RANKL, and CSF1 after the application of CTF was decreased by
has the potential to be effective for the reduction of severe apical root

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Fig. 14. The amount of root resorption in the apical root area.
The amount of root resorption was significantly greater in the control group
(tooth movement without drug) than in the acetaminophen group (tooth
movement with acetaminophen) (Values are shown as the mean ± SD of at
least three independent repeats. **P < 0.01, N = 5).
resorption without delaying tooth movement. However, further clinical
experiments are needed to confirm our findings.
Funding
This study was supported by JSPS KAKENHI from the Ministry of
Education, Culture, Sports, Science and Technology of Japan (Grant
Number 25862019).
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
We are grateful to prof. Kurihara, Shiba, Kato and Shukunami in
Hiroshima University Institute of Biomedical & Health Sciences.
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