THE KOREAN JOURNAL of
ORTHODONTICS
Original Article
pISSN 2234-7518 • eISSN 2005-372X
http://dx.doi.org/10.4041/kjod.2012.42.3.138
Effect of fangchinoline on root resorption during rat
orthodontic tooth movement
Xingfu Bao
Min Hu
Yi Zhang
Ferdinand Machibya
Ying Zhang
Huan Jiang
Dongsheng Yu
Department of Orthodontics, School
of Stomatology, Jilin University,
Changchun, China
Objective: To evaluate the short-term effect of fangchinoline, an antiinflammatory drug widely used in Asia, on root resorption that is associated with
orthodontic tooth movement. Methods: Twenty-four Wistar rats were randomly
divided into 6 groups. Mesial forces of 0, 50, or 100 g were applied to the maxillary
first molar of the rats in each group for 14 days by activating nickel-titanium
closed-coil springs. One-half of the rats receiving each of these treatments also
received injections of 200 μL fangchinoline every 2 days. Finally, movement of
the maxillary first molars was measured using digitized radiographs. The molars
were extracted and the surfaces of the root resorption craters were recorded using
a scanning electron microscope. The distance the molars moved and resorptionarea ratio was measured, and results were analyzed using 2-way ANOVA tests.
Results: There were no statistical differences in the distances the first molars
moved under 50 or 100 g force, regardless of treatment with fangchinoline.
However, the resorption area ratios were significantly smaller in those rats that
were treated with both tension and fangchinoline than in those rats treated by
tension alone. Conclusions: Fangchinoline reduced the resorption area ratio in
rats and is therefore an important means of alleviating root resorption.
[Korean J Orthod 2012;42(3):138-143]
Key words: SEM, Root resorption, Tooth movement
Received January 9, 2012; Revised March 7, 2012; Accepted April 12, 2012.
Corresponding author: Min Hu.
Professor, Department of Orthodontics, School of Stomatology, Jilin University, 1500,
Qinghua Road, Changchun, Jilin 130021, China.
Tel +86-431-8879-6023 e-mail humin@jlu.edu.cn
This work was supported by Specialized Research Fund for the Doctoral Program of
Higher Education (SRFDP 20110061110072) and National Natural Science Foundation of
China (NSFC 81170999).
The authors report no commercial, proprietary, or financial interest in the products or companies
described in this article.
© 2012 The Korean Association of Orthodontists.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
(http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and
reproduction in any medium, provided the original work is properly cited.
138
Bao et al • Effect of fangchinoline on root resorption
INTRODUCTION
Orthodontically induced root resorption (OIRR) is a
sequela of orthodontic treatment that affects nearly 100%
of treated patients. 1 OIRR can induce cementum and
dentin damage, depending on its severity. Although the
normal root structure might later be partially repaired by
cementum, severe resorption can destroy it, leading to
the failure of orthodontic treatment. Because of its harm
to dental health, great efforts have been made to identify
mechanotherapies that might reduce or eliminate root
resorption.
OIRR is related to total apical displacement, treatment
duration, age, gender, malocclusion type, root shape, type
of appliance, treatment modality, and force regimen. 2
However, a hypofunctional periodontium may also
contribute to OIRR.3 The primary cells responsible for
this process are the odontoclasts. 4 A group of proinflam matory factors and enzymes are involved in the
diffe ren tiation, maturation, and activation of these
cells.5 Increased knowledge of this process has caused
investigators to focus on certain inhibitors as potential
treatments. Fangchinoline (C 37H40N2O6) is an alkaloid
that is extracted from bisbenzylisoquinoline and is widely
used in Asian countries as an anti-inflammatory drug.
Fangchinoline acts through more mechanisms, and is
safer, than other anti-inflammatory agents.
Fangchinoline inhibits 90% of interleukin (IL)-1 and
tumor necrosis factor-alpha (TNF-α) production at a
dose of 10 µg/mL.6 Fangchinoline also partially inhibits
cyclooxygenase and human IL-6,7 is a nonspecific calcium
channel inhibitor,8,9 and has an antithrombosis function.10
At the same time, fangchinoline has effective antioxidant
and radical-scavenging activity.11 Our laboratory recently
found fangchinoline to be a potential cathepsin K (CK)
inhibitor. Fangchinoline is an excellent candidate for
inhibiting root resorption because it is safe and has multiple anti-inflammatory effects.
The aim of this study was to examine the effects of
fangchinoline on root resorption during tooth movement.
We hypothesized that fangchinoline would limit root
resorption during orthodontic tooth movement.
week old male Wistar rats, with an average weight of
200 g, were purchased from the Animal Center of Jilin
University. Rats were randomly allocated to 1 of 6 control
or treatment groups. There were 4 rats in each group.
Rats in the treatment groups had either 50 g or 100 g
of orthodontic force applied to their maxillary right
first molars, while control rats were not subjected to
orthodontic force (0 g). Treatment and control groups
were further divided into 2 subgroups, based on whether
they received fangchinoline injections. Rats in the 0 g(+),
50 g(+), and 100 g(+) groups received 40 μL of 5 μg/μL
fangchinoline every 2 days. Rats in the 0 g(-), 50 g(-), and
100 g(-) groups received no fangchinoline.
We based the dose of fangchinoline on a study by Onai
et al.6 and the unpublished results of our earlier study. All
of the rats were observed for 7 days prior to beginning the
experiment, to ensure that they were healthy. Rats were
fed a soft diet during the 14 days experiment.
Experimental protocol
Orthodontic appliances were installed on the rats while
under anesthesia with SuMianXin (Institute of Military
Medicine Science, Changchun, China) (0.2 mL/kg) IM.
The appliances consisted of a nickel-titanium coil spring
(ShengMaTe Co., Shanghai, China) tied between the
maxillary incisors and right first molar (Figure 1). The
force of the springs was measured using an ergometer
before installing them. We covered the end of the ligature
wire with a piece of resin in order to prevent appliance
loosening.
Fangchinoline injection
In our experiment, 200 μg fangchinoline was given
every 2 days. Twenty milligrams fangchinoline (YuanYe
Co., Shanghai, China) was added to 4 mL distilled water,
MATERIALS AND METHODS
Animals
All experimental procedures were approved by the
Institutional Animal Care and Use Committee of the local
district government and the Animal Care Commissioner
of Jilin University.
We used an online sample-size calculator for clinical
trials and scientific experiments (hedwig.mgh.harvard.
edu) to determine the appropriate sample size for a
study with a statistical power of 0.8. Twenty-four 8
www.e-kjo.org
http://dx.doi.org/10.4041/kjod.2012.42.3.138
Figure 1. Rat tooth movement model. A nickeltitanium
coil spring was activated after insertion between the first
molar and incisors in order to move the molar mesially.
139
Bao et al • Effect of fangchinoline on root resorption
forming an injectable suspension with a concentration
of 5 μg/μL. We injected 40 μL fangchinoline every 2 days
into the subperiosteum adjacent to the maxillary first
molar of the rats in the 0 g(+), 50 g(+), and 100 g(+)
groups. The suspension was stored at 4oC and stirred
before each injection.
then removed and carefully cleaned to reveal clear root
surfaces. All teeth were dried and observed under an
SEM (JSM-6390A; JEOL Ltd., Tokyo, Japan) at the same
distance and orientation. Images of the mesial surfaces of
both distal roots were saved as digital photographs.
Evaluation and statistical procedure
The distance between the contact points of the first and
second molar in each rat was measured using ImageJ
software (version 1.44; National Institutes of Health,
USA). The resorption area and area of the entire mesial
surface of the 2 distal roots were measured separately
using the same software. We obtained the resorption-area
ratio by dividing the crater area by the total surface area
(Figure 2). Each measurement was made 3 times by the
same examiner, and the mean of these values was used
in further analyses. We performed a 2-way ANOVA to
compare the paired treatment groups using SPSS version
17 (SPSS Inc., Chicago, IL, USA).
X-ray and scanning electron microscope (SEM) observation
All rats were killed by decapitation at the end of the
experiment. After 14 days of force application, the amount
of tooth movement was measured on cephalometric
radiographs, as previously reported.12 The maxillary right
first molar and the adjacent alveolar bone was extracted
in toto for further examination. All tissues were soaked in
5% sodium hypochlorite solution for 12 hours, at which
time the alveolar bone was delicately removed, exposing
the 5 roots of the first molar. The periodontal ligament
residue of the distobuccal and distopalatal roots were
RESULTS
Tooth movement
None of the rats had any distance between the first
and second molar crown prior to the experiment. Tooth
movement occurred in all of the rats in the treatment
groups by the end of the experiment (Table 1). No force
was loaded on the rats in either control group and no
movement had occurred. Statistical analysis indicated
that the distance of tooth movement in the 50 g and 100 g
groups was significantly greater than that in the 0 g group
(p < 0.05). However, there was no significant difference in
the distance of tooth movement between the 50 g and 100
g groups, irrespective of fangchinoline injection (p > 0.05).
Root resorption-area ratio
Root resorption was not detected in the control group,
and smooth cementum could be seen on the mesial
surface of the distobuccal and distopalatal roots. However,
root resorption was observed among the treatment group
rats, mainly in the cervical and middle one-third of
the root (Figure 3). There was no significant difference
between the 50 g(-) group and the 100 g(-) group in
resorption area ratio. However, the resorption area ratio
was less severe in the fangchinoline treatment groups than
in the non-fangchinoline treatment groups, irrespective
Figure 2. Calculation of the resorption area ratio in
the molars of Wistar rats. A, Representative scanning
electron microscope micrograph of the mesial surface of
the distopalatal root. B, Diagram indicating the area of
the crater (black) as compared to the total root surface
area (gray). The resorption area ratio was calculated by
dividing the area of the crater by the total root surface
area (resorption area ratio = black area/gray area).
Table 1. Tooth movement (mm) after 14 days of treatment
Group
0 g force
50 g force
100 g force
F
p-value
With Fangchinoline
0
0.235 ± 0.136*
0.275 ± 0.07*
26.752
0.000 (force)
Without Fangchinoline
0
0.285 ± 0.138*
0.265 ± 0.05*
*p < 0.05 vs 0 g force.
140
http://dx.doi.org/10.4041/kjod.2012.42.3.138
www.e-kjo.org
Bao et al • Effect of fangchinoline on root resorption
of force magnitude (p < 0.05) (Table 2).
DISCUSSION
In this study, we used SEM and X-ray to quantify root
resorption and tooth movement. After 14 days, the
maxillary first molar moved no further in the 50 g(+)
and 100 g(+) groups than it did in the 50 g(-) and 100
g(-) groups. These results agree with those of Gonzales
et al.,12 indicating that there is no significant difference
in tooth movement after application of force at these
magnitudes. Rat tooth movement is divided into 3 phases:
an initial phase lasting 2 days, a lag phase, and a final
linear increment of tooth movement.13,14 In our study,
tooth movement over the 14 days was relatively slow
and was the result of the teeth shifting in the periodontal
ligament space. Because odontoclasts and osteoclasts are
similar, we expected that when the same amount of force
Figure 3. Representative scanning electron microscope micrographs of the distal root surface of molars from Wistar rats.
A, 50 g force and no fangchinoline; B, 50 g force with injection of 40 μL of 5 μg/μL fangchinoline every 2 days; C, 100
g force and no fangchinoline; D, 100 g force with injection of 40 μL of 5 μg/μL fangchinoline every 2 days. Different
sizes of resorption craters can be observed on the mesial surface of the root image from each group. Craters are found
primarily on the cervical and middle one third of each root (white arrow).
Table 2. Root resorption area ratio after 14 days of treatment
Group
0 g force
50 g force
100 g force
F
p-value
With Fangchinoline
0
0.037 ± 0.010*
0.050 ± 0.022*
16.259
0.004 (force)
Without Fangchinoline
0
0.071 ± 0.021*
0.121 ± 0.042*
31.938
0.007 (group)
*p < 0.05 vs 0 g force.
www.e-kjo.org
http://dx.doi.org/10.4041/kjod.2012.42.3.138
141
Bao et al • Effect of fangchinoline on root resorption
was applied, there would be more tooth movement in the
fangchinoline group than in the non-fangchinoline group.
However, we found no difference between the tooth
move ment of groups that received fangchinoline and
those that did not. This is may be partially due to a lag in
tooth movement caused by hyalinization. Additionally,
there is still no consensus regarding the relationship
between root resorption and bone resorption; therefore,
further research is necessary in this regard.15 Experience
with human patients indicates that molars have a tendency to move toward open spaces along the arch. We
assumed this would also apply to rats: the second molar
would move forward after the movement of the first
molar. Because this process takes time to occur, we did
not expect any movement of the second molar within the
14 day experimental period. However, this effect should
be taken into account in studies with longer treatment
periods.
Several researchers have attempted to investigate the
precise mechanisms of root resorption. Orthodontic
movement is dependent on bone resorption on the pressure side and odontoclasts are now considered to be
responsible for root resorption. Odontoclasts are thought
to originate from circulating progenitor cells and have
characteristics that are similar to osteo clasts, such as
expression of CK, cathepsin D, matrix metalloproteinase-9,
H+-ATPase, and others. The activation of odontoclasts is
regulated by the nuclear factor kappa B/nuclear factor
kappa B ligand/osteoprotegerin (RANK/RANKL/OPG)
pathways.16,17
In the present study, there was no significant difference
in the resorption area ratio of 50 g(-) rats and 100 g(-)
rats. This result parallels those of Zhuang et al.,18 indicating that force level is not the sole contributor to root
resorption. Resorption areas in the 50 g(+) and 100 g(+)
groups were smaller than in the 50 g(-) and 100 g(-)
groups, respectively, supporting the hypothesis that
fangchinoline inhibits root resorption. CK is a cysteine
protease that is responsible for the degradation of collagen
and matrix proteins.19 CK inhibitor has been investigated
as a potential target for osteoporosis treatment.20 Our past
research indicated that fangchinoline is a CK inhibitor;
therefore, we assumed that this inhibitory effect would
also apply to resorption. Moreover, fangchinoline partly
inhibits IL-1 and TNF-α, which may also explain its
inhibitory effect on root resorption. A study by Jäger
et al. 21 indicated that systemic application of soluble
receptors to IL-1 and TNF-α reduces the number of
odontoclasts, lending further support to our hypothesis
that fangchinoline would limit root resorption during
orthodontic tooth movement.
Because root resorption craters are usually 3-dimensional, further investigation is needed to determine the
effect of fangchinoline on crater depth, as well as its
142
influence on the various phases of tooth movement.
CONCLUSION
Fangchinoline reduced the resorption-area ratio in rats
and is a valuable candidate for limiting root resorption.
Fangchinoline has no effect on the amount of tooth
movement of rat maxillary first molars under either 50 g
or 100 g of force.
REFERENCES
1. Brudvik P, Rygh P. Non-clast cells start orthodontic
root resorption in the periphery of hyalinized zones.
Eur J Orthod 1993;15:467-80.
2. Segal GR, Schiffman PH, Tuncay OC. Meta analysis
of the treatment-related factors of external apical root
resorption. Orthod Craniofac Res 2004;7:71-8.
3. Sringkarnboriboon S, Matsumoto Y, Soma K. Root
resorption related to hypofunctional periodontium in
experimental tooth movement. J Dent Res 2003;82:
486-90.
4. Harokopakis-Hajishengallis E. Physiologic root
resorption in primary teeth: molecular and histological events. J Oral Sci 2007;49:1-12.
5. Meikle MC. The tissue, cellular, and molecular regulation of orthodontic tooth movement: 100 years after
Carl Sandstedt. Eur J Orthod 2006;28:221-40.
6. Onai N, Tsunokawa Y, Suda M, Watanabe N, Nakamura K, Sugimoto Y, et al. Inhibitory effects of bisbenzylisoquinoline alkaloids on induction of proinflammatory cytokines, interleukin-1 and tumor
necrosis factor-alpha. Planta Med 1995;61:497-501.
7. Choi HS, Kim HS, Min KR, Kim Y, Lim HK, Chang
YK, et al. Anti-inflammatory effects of fangchinoline
and tetrandrine. J Ethnopharmacol 2000;69:173-9.
8. Kim HS, Zhang YH, Oh KW, Ahn HY. Vasodilating
and hypotensive effects of fangchinoline and tetrandrine on the rat aorta and the stroke-prone spontaneously hypertensive rat. J Ethnopharmacol 1997;
58:117-23.
9. Zhang YH, Fang LH, Ku BS. Fangchinoline inhibits rat
aortic vascular smooth muscle cell proliferation and
cell cycle progression through inhibition of ERK1/2
activation and c-fos expression. Biochem Pharmacol
2003;66:1853-60.
10. Kim HS, Zhang YH, Yun YP. Effects of tetrandrine
and fangchinoline on experimental thrombosis in
mice and human platelet aggregation. Planta Med
1999;65:135-8.
11. Gülçin I, Elias R, Gepdiremen A, Chea A, Topal
F. Antioxidant activity of bisbenzylisoquinoline
alkaloids from Stephania rotunda: cepharanthine and
fangchinoline. J Enzyme Inhib Med Chem 2010;25:44-
http://dx.doi.org/10.4041/kjod.2012.42.3.138
www.e-kjo.org
Bao et al • Effect of fangchinoline on root resorption
53.
12. Gonzales C, Hotokezaka H, Yoshimatsu M, Yozgatian
JH, Darendeliler MA, Yoshida N. Force magnitude
and duration effects on amount of tooth movement
and root resorption in the rat molar. Angle Orthod
2008;78:502-9.
13. Reitan K, Kvam E. Comparative behavior of human
and animal tissue during experimental tooth movement. Angle Orthod 1971;41:1-14.
14. Storey E. The nature of tooth movement. Am J Orthod
1973;63:292-314.
15. Wang Z, McCauley LK. Osteoclasts and odontoclasts:
signaling pathways to development and disease. Oral
Dis 2011;17:129-42.
16. Oshiro T, Shibasaki Y, Martin TJ, Sasaki T. Immunolocalization of vacuolar-type H+-ATPase, cathepsin K,
matrix metalloproteinase-9, and receptor activator of
NFkappaB ligand in odontoclasts during physiological
root resorption of human deciduous teeth. Anat Rec
2001;264:305-11.
17. Tsuji Y, Yamaza T, Kido MA, Goto T, Nakata S, Akamine A, et al. Expression of cathepsin K mRNA and
www.e-kjo.org
http://dx.doi.org/10.4041/kjod.2012.42.3.138
protein in odontoclasts after experimental tooth
move ment in the mouse maxilla by in situ hybridization and immunoelectron microscopy. Cell Tissue
Res 2001;303:359-69.
18. Zhuang L, Meng X, Li P, Bai Y. A pilot study on three
dimensional morphology of root by Micro-CT during
orthodontic root resorption. J Modern Stomatol 2010;
24:35-8.
19. Inaoka T, Bilbe G, Ishibashi O, Tezuka K, Kumegawa
M, Kokubo T. Molecular cloning of human cDNA for
cathepsin K: novel cysteine proteinase predominantly
expressed in bone. Biochem Biophys Res Commun
1995;206:89-96.
20. Henriksen K, Tanko LB, Qvist P, Delmas PD, Christiansen C, Karsdal MA. Assessment of osteoclast
number and function: application in the development
of new and improved treatment modalities for bone
diseases. Osteoporos Int 2007;18:681-5.
21. Jäger A, Zhang D, Kawarizadeh A, Tolba R, Braumann
B, Lossdörfer S, et al. Soluble cytokine receptor treatment in experimental orthodontic tooth movement in
the rat. Eur J Orthod 2005;27:1-11.
143