ONLINE ONLY

Effects of clodronate on early alveolar bone

remodeling and root resorption related to
orthodontic forces: A histomorphometric
analysis
Josefina Choi,a Seung-Hak Baek,b Jae-Il Lee,c and Young-Il Changd
Seoul, Korea

Introduction: The objective of this study was to evaluate the short-term effects of clodronate, a first-generation
bisphosphonate, on early alveolar bone remodeling and root resorption related to orthodontic tooth movement.
Methods: The samples consisted of 54 sex-matched Wistar rats (weight, 180-230 g) allocated to the 2.5 mmol/L
clodronate, 10 mmol/L clodronate, and control groups (n 5 18 for each group). After application of a nickel-
titanium closed-coil spring (force, 60 g) between the maxillary central incisor and first molar, 2.5 mmol/L of
clodronate, 10 mmol/L of clodronate, or saline solution was injected into the subperiosteum adjacent to the
maxillary first molar every third day. All animals received tetracycline, calcein, and alizarin red by
intraperitoneal injection at 1, 6, and 14 days, respectively. The amounts of tooth movement were measured at
3, 6, 9, 12, and 15 days. The animals were killed at 4, 7, and 17 days. Histomorphometric analyses of bone
mineral appositional rate, labeled surface, percentage of root resorption area, and number of root resorption
lacunae of the mesiobuccal root of the maxillary first molar at 4, 7, and 17 days were done. One-way analysis
of variance (ANOVA) with the post-hoc test were done for statistical analyses. Results: Rats in the 10 mmol/L
clodronate group had significant decreases of tooth movement (12 and 15 days, P \0.05) and percentages
of root resorption area and numbers of root resorption lacunae (7 day, P\0.05), and increases of labeled surface
and mineral appositional rates (17 day, P \0.05) over those of the 2.5 mmol/L clodronate and control groups.
Conclusions: Although clodronate might decrease root resorption related to orthodontic tooth movement, pa-
tients should be informed about a possible decrease in the amount of tooth movement and a prolonged period of
orthodontic treatment. (Am J Orthod Dentofacial Orthop 2010;138:548.e1-548.e8)

R
ecently, orthodontists have been treating aging
populations with malocclusion and providing
care for esthetic or periodontal problems. These
patients have various metabolic bone diseases associ-
ated with excessive bone resorption. Identifying the fac-
tors related to bone or root resorption after application
of orthodontic forces remains a challenge.
Clodronate (dicloromethylene-1,1 bisphosphonate) is
a first-generation bisphosphonate and synthetic nonhydro-
lyzable analog of pyrophosphate.1-3 Because it can
effectively control the formation and dissolution of
From the School of Dentistry, Seoul National University, Seoul, Korea.
a
Postgraduate student, Department of Orthodontics.
b
Associate professor, Department of Orthodontics, Dental Research Institute.
c
Professor, Department of Oral Pathology, Dental Research Institute.
d
Professor, Department of Orthodontics.
The authors report no commercial, proprietary, or financial interest in the prod-
ucts or companies described in this article.
Reprint requests to: Young-Il Chang, Department of Orthodontics, School of
Dentistry, Dental Research Institute, Seoul National University, Yeonkun-
dong #28, Jongro-ku, Seoul, 110-768, Republic of Korea; e-mail, drchang@
plaza.snu.ac.kr.
Submitted, October 2009; revised and accepted, January 2010.
0889-5406/$36.00
Copyright Ó 2010 by the American Association of Orthodontists.
doi:10.1016/j.ajodo.2010.01.031
calcium phosphate in vitro, as well as mineralization and
bone resorption in vivo, it has become a major
antiresorptive drug for the prevention and treatment of
postmenopausal osteoporosis and various metabolic bone
diseases associated with excessive bone resorption.4 Addi-
tionally, it is used to decrease the number and rate of frac-
tures in patients with breast cancer and myeloma-related
osteolysis and to relieve metastatic bone pain caused by
solid tumors.5-9
In issues related to orthodontic tooth movement,
bisphosphonates are known to inhibit root resorption in
rats in a dose-dependent manner.10-14 Liu et al15 reported
that the number of osteoclasts on the pressure side of the
periodontal ligament during orthodontic tooth movement
was decreased in animals injected with clodronate. They
suggested that the anti-inflammatory properties of clodr-
onate could be helpful in the treatment of periodontitis
and that this inhibitory effect was dose-dependent.15
Other studies demonstrated the inhibitory action of clodr-
onate on bone resorption by apoptosis of osteoclasts
through incorporation of clodronate into the cells.16,17
However, to our knowledge, the relationship be-
tween the dose of clodronate and early bone
548.e1
548.e2 Choi et al American Journal of Orthodontics and Dentofacial Orthopedics
November 2010

remodeling, as well as root resorption during orthodon-

tic tooth movement, has not been proven with histomor-
phometric studies. Therefore, the purpose of this study
was to evaluate the short-term effects of clodronate on
early alveolar bone remodeling and root resorption re-
lated to orthodontic tooth movement, measured by fluo-
rescent bone markers.

MATERIAL AND METHODS

The sample consisted of 54 Wistar rats (approximate
weight, 180-230 g; age, 8 weeks) allocated into the 2.5
mmol/L clodronate group, the 10 mmol/L clodronate
group, or the control group (n 5 18; 9 males and 9 females
in each group) to prevent a sex-related bias. This study was
conducted under an approved protocol and guidelines es-
tablished by the Institutional Animal Care and Use Com-
mittee of Seoul National University (SNU-090306-4).
After the animals were placed under general anes-
thesia with zylazine hydrochloride (40 mg/kg, Rompun,
Bayer Korea, Seoul, Korea) and zolazepam (5 mg/kg,
Virbac, Carros, France), the initial body weights and
distances between the tip of the maxillary central inci-
sors and the mesiobuccal cusp tip of the maxillary first
molars were measured with a digital caliper (700-126,
Mitutoyo, Kawasaki, Japan). Grooves were prepared
at the cervical line of the maxillary incisors by using Fig 1. Application of the orthodontic nickel-titanium
a low-speed hand piece (Vmax, Nakanishi, Tochigi, closed-coil spring.
Japan) to prevent the ligatures from dislodging.
Since several studies demonstrated that 40 to 60 g of
force stimulates substantial tooth movement of the mo- Two animals from each group were killed on the 4th,
lars in rats, a nickel-titanium closed-coil spring (0.10 3 7th, and 17th days after spring placement. The speci-
0.30-in; force, 60 g; Ormco, Orange, Calif) was con- mens of each half of the right and left sides with the
nected with a 0.010-in steel ligature wire between the adjacent bone tissue were fixed by using 70% ethanol,
maxillary first molar and central incisor (Fig 1).18-20 dehydrated, and embedded in Osteobed resin (Poly-
After the ligatures were tied, composite resin sciences, Warrington, Pa).21 The specimens were cut
(Transbond XT Light Cure Adhesive Paste, 3M Unitek, parallel to the mesiodistal long axis of the maxillary first
Monrovia, Calif) was cured over the ligature wire to molar and then prepared as 40 to 50 mm thickness spec-
prevent slippage. The mandibular incisors were reduced imens by using a cutting and grinding system (Exakt
to prevent breakage of the orthodontic appliance. Apparatebau, Nordstedt, Germany).22 As the observa-
Volumetrically equivalent dosages of 2.5 mmol/L or tion area, the mesiobuccal root of the maxillary first
10 mmol/L of clodronate solution (50 mL; dichloro- molar was chosen because it is the largest of the maxil-
methylene diphosphonic acid, Sigma, St Louis, Mo) lary first molar’s 5 roots and is in approximately the
were injected into the subperiosteum area adjacent to same plane with the applied force.18,23
the maxillary first molar every third day for the experi- During the grinding procedure, the specimen was
mental groups with a 31-gauge ultra-fine insulin syringe checked with a microscope (SZ 61, Olympus, Tokyo,
(BD, Franklin Lakes, NJ) during the experimental Japan) to prepare the most suitable slides for histomor-
period. An equal volume of saline solution was injected phometric analysis. An average of 6 slides for each
into the same area for the control group. specimen was obtained; this was sufficient to perform
Tetracycline (25 mg/kg, Sigma) on the 1st day, cal- the histomorphometric analysis.
cein (20 mg/kg, Sigma) on the 6th day, and alizarin red After examination of the fluorescent labeling by us-
(10 mg/kg, Sigma) on the 14th day were given to all ing fluorescence microscopy (BX-51, Olympus), the
animals by intraperitoneal injection. specimens were stained with hematoxylin and eosin.
American Journal of Orthodontics and Dentofacial Orthopedics Choi et al 548.e3
Volume 138, Number 5

Fig 2. Hematoxylin and eosin staining showing the effects of the clodronate on osteoclasts (OC) for A,
the control and B, the 10 mmol/L clodronate groups on day 7 after spring application (200 times mag-
nification). Alv, Alveolar bone; PDL, periodontal ligament.

Fig 3. Fluorescence microscopy images showing bone

labels for calculating the bone mineral apposition rate
(200 times magnification). Green lines, tetracycline;
orange lines, calcein; red lines, alizarin red.

The amounts of tooth movement were measured on

the 3rd, 6th, 9th, 12th, and 15th days after spring place-
ment. Distances between the tip of the maxillary central
incisors and the mesiobuccal cusp tip of the maxillary
first molars were measured by using digital calipers.
Histologic observation was performed with the
Olympus BX-51 microscope (Figs 2 and 3). Bone
mineral appositional rate (MAR), labeled surface (LS),
percentage of root resorption area (PRRA), and
number of root resorption lacunae (NRRL) of the
Fig 4. Hematoxylin and eosin staining showing the tracing
of root, pulp, and root resorption lacunae for calculating
the PRRA and NRRL on day 17 after spring application.
mesiobuccal root of the maxillary first molar were
measured and calculated with image-analyzing software
(KAPPA, Opto-electronics, Gleichen, Germany).24
Bone MAR of all the active bone-forming surfaces
was determined by measuring the distance between
548.e4 Choi et al American Journal of Orthodontics and Dentofacial Orthopedics
November 2010

Table I. Comparison of the amount of tooth movement (mm) according to time

Day after surgery Control 2.5 mmol/L clodronate 10 mmol/L clodronate Significance Multiple comparison test

3 1.57 6 0.92 1.71 6 0.81 1.61 6 0.67 NS NS

6 2.00 6 0.76 2.09 6 0.91 1.96 6 0.56 NS NS
9 2.27 6 0.80 1.99 6 0.56 1.90 6 0.46 NS NS
12 2.81 6 0.82 2.06 6 0.50 1.93 6 0.13 * (2.5C, 10C) \C
15 2.81 6 0.82 1.91 6 0.50 1.79 6 0.13 * (2.5C, 10C) \C

One-way ANOVA and the Scheffé multiple comparison test were done. Each group consisted of 18 samples. The numbers of samples in each group
were 18 at day 2; 12 at day 6; and 6 at days 9, 12 and 15 because rats were killed on these days.
NS, Not significant; * P \0.05.
C, control; 2.5C, 2.5 mmol/L clodronate; 10C, 10 mmol/L clodronate.

Table II. Comparison of the MAR (mm/day) near the mesiobuccal root of the maxillary first molars on days 4, 7, and 17
after spring application
Day after surgery Control 2.5 mmol/L clodronate 10 mmol/L clodronate Significance Multiple comparison test

4 7.68 6 4.71 6.37 6 5.16 4.90 6 2.73 NS NS

7 1.53 6 0.68 4.07 6 2.28 5.05 6 4.30 NS NS
17 3.52 6 1.31 3.58 6 1.12 6.62 6 1.47 * (C, 2.5C) \10C

One-way ANOVA and the Scheffé multiple comparison test were done. Each group consisted of 18 samples. The number of samples in each group at
each specific time was 6.
NS, Not significant; * P \.05.
C, Control; 2.5C, 2.5 mmol/L clodronate; 10C, 10 mmol/L clodronate.

the centers of 2 fluorescent bands (labels) and dividing RESULTS

by the time between labeling (mm/day).21,25
To quantify the overall remodeling activity of the
bone at the root surface, LS measurements were made
by using spliced images obtained under 200-times mag-
nification. The cumulative lengths of clearly identifiable
fluorescent LS were measured between the alveolar
bone crest and the apex of the tooth.21
The surface area of the whole root (SAWR, mm2),
surface area of root resorption lacunae (SARRL,
mm2), and surface area of the radicular pulp (SARP,
mm2) were calculated by tracing the pulp area and
roots with the image analyzing software (Fig 4).
PRRA was obtained by using the formula, SARRL/
(SAWR-SARP)] 3 100.26 NRRL measurements
were taken from 4 sections at 30-mm intervals and
averaged for the mesiobuccal root of the first molar
(Fig 4).
One operator (J.C.) did the counting and measuring
twice with a 2-week interval. Since there was no signifi-
cant difference, the first set of counts and measurements
was used for analysis.
Statistical analysis
One-way analysis of variance (ANOVA) and
a subsequent post-hoc test were performed. P values
less than 0.05 were considered statistically signifi-
cant.
The amounts of tooth movement were compared ac-
cording to time (Table I). Both the 2.5 and 10 mmol/L
clodronate groups showed significantly less tooth move-
ment on days 12 and 15 than did the control group
(P \0.05, respectively).
MAR of the bone was compared near the mesiobuc-
cal root area of the maxillary first molars on days 4, 7,
and 17 after spring application (Table II). The MAR
did not show a significant difference among the 3 groups
on days 4 and 7. However, the MAR on day 17 was sig-
nificantly increased in the 10 mmol/L clodronate group
compared with the control and the 2.5 mmol/L clodro-
nate groups (P \0.05, respectively).
The LS (mm) of bone was compared near the mesiobuc-
cal root of the maxillary first molars on days 4, 7, and 17 after
spring application (Table III). There was no significant dif-
ference in LS among the 3 groups on days 4 and 7. However,
LS in the 10 mmol/L clodronate group was significantly
greater compared with that of the control and 2.5 mmol/L
clodronate groups on day 17 (P \0.05, respectively).
PRRA and NRRL of the mesiobuccal root of the max-
illary first molars were compared on days 4, 7, and 17 af-
ter spring application (Tables IV and V). Although there
were no significant differences in PRRA and NRRL
among the 3 groups on days 4 and 17, the 10 mmol/L
clodronate group showed significant decreases in PRRA
and NRRL compared with the control group on day 7
(P \0.05).
American Journal of Orthodontics and Dentofacial Orthopedics Choi et al 548.e5
Volume 138, Number 5

Table III. Comparison of the LS (mm) near the mesiobuccal root of the maxillary first molars at days 4, 7, and 17 after
spring application
Day after surgery Control 2.5 mmol/L clodronate 10 mmol/L clodronate Significance Multiple comparison test

4 639.86 6 74.97 776.73 6 267.54 845.44 6 526.88 NS NS

7 516.67 6 109.55 522.30 6 89.44 513.32 6 68.28 NS NS
17 581.87 6 80.53 647.85 6 179.50 978.50 6 331.75 * (C, 2.5C) \10C

One-way ANOVA and the Scheffé multiple comparison test were done. Each group consisted of 18 samples. The number of samples in each group at
each specific time was 6.
NS, Not significant; * P \0.05.
C, Control; 2.5C, 2.5 mmol/L clodronate; 10C, 10 mmol/L clodronate.

Table IV.Comparison of PRRA of the mesiobuccal root of the maxillary first molars on days 4, 7, and 17 after spring
application
Control 2.5 mmol/L clodronate 10 mmol/L clodronate
Day after surgery (n 5 6) (n 5 6) (n 5 6) Significance Multiple comparison test

4 34.91 6 18.93 32.03 6 4.17 28.09 6 4.67 NS NS

7 24.12 6 6.37 22.13 6 10.09 16.40 6 6.63 * NS†
17 17.00 6 7.50 16.75 6 9.40 14.13 6 6.95 NS NS

One-way ANOVA and the Scheffé multiple comparison test were done. Each group consisted of 18 samples. The number of samples in each group at
each specific time was 6.
NS, Not significant.
*P \0.05; †Although the 1-way ANOVA and the Scheffé multiple comparison test did not show statistically significant differences, the Mann-
Whitney test showed a significant difference on day 7 between the control and 10 mmol/L clodronate groups (P \0.05).

DISCUSSION The LS values of bone near the mesiobuccal root of

The finding that both the 2.5 and 10 mmol/L clodr-
onate groups showed significantly less tooth movement
on days 12 and 15 than the control group (P \0.05, re-
spectively, Table I) agreed with the results of Liu et al.15
The results of this study confirmed that clodronate in-
hibits bone resorption induced by orthodontic force.
This means that the 10 mmol/L clodronate groups
showed less tooth movement than did the 2.5 mmol/L
clodronate and control groups.
The MAR of bone near the mesiobuccal root area of
the maxillary first molars was compared on days 4, 7,
and 17 after spring application. The MAR has been
used to describe how fast the mineralization front pro-
ceeds during alveolar bone remodeling.27 The finding
that the MAR on day 17 significantly increased in the
10 mmol/L clodronate group compared with the control
and 2.5 mmol/L clodronate groups (P \0.05, respec-
tively, Table II) indicates that, if 10 mmol/L of clodro-
nate was given, the MAR could be increased in the
short term. However, further studies with multiple doses
should be performed for longer periods to verify the
dose-dependent tendency. Horie et al11 concluded that
clodronate stimulates bone formation in rats because
of a stimulatory effect on the differentiation process of
osteoblasts.
the maxillary first molars were compared on days 4, 7,
and 17 after spring application. Although several studies
have analyzed the effects of local administration of bi-
sphosphonates such as clodronate and risedronate on or-
thodontic tooth movement, none used multiple
fluorescent bone markers for the determination of LS
values.14,15 Measurement of the labeling surface of the
alveolar surface in rats can allow us to understand the
kinetics of bone turnover and to quantify the changes
in bone structure according to time.24
Similar to the MAR, the LS in the 10 mmol/L clodr-
onate group was significantly greater compared with
that of the control and 2.5 mmol/L clodronate groups
on day 17 (P \0.05, Table III). In the 10 mmol/L clodr-
onate group, although the labeling lines between calcein
and alizarin red were evident and wide (Fig 5), the tet-
racycline labeling lines were absent in the upper third
of the root. These findings suggest that alveolar bone re-
modeling might be started by day 8 and remodeling ac-
tivity increased until day 17; these findings coincide
with the peak MAR observed between the calcein and
alizarin red administered on days 6 and 14, respectively.
Furthermore, new bone formation was also detected on
the tension side by gross histologic observation in sam-
ples from days 8 and 12, consistent with the results of
Pavlin et al.28
548.e6 Choi et al
Table V. Comparison of NRRL of the maxillary first molars at days 4, 7, and 17 after spring application
Day after surgery Control 2.5 mmol/L clodronate
4 4.00 6 1.00 3.33 6 1.52
7 2.50 6 0.97 2.00 6 0.57
17 2.33 6 1.15 2.00 6 1.00
One-way ANOVA and the Scheffé multiple comparison test were done. Each group consisted of 18 samples. The number of samples in each group at
each specific time was 6.
NS, Not significant.
*P \0.05; †Although 1-way ANOVA and the Scheffé multiple comparison test did not show statistically significant differences, the Mann-Whitney
test showed a significant difference on day 7 between the control and 10 mmol/L clodronate groups (P \0.05).
Fig 5. Fluorescent microscopy images with bone labels
for A, the control and B, the 10 mmol/L clodronate
groups on day 17 after spring application.
The PRRA and NRRL of the mesiobuccal root of the
maxillary first molars were compared on days 4, 7, and
17 after spring application. There are controversies re-
garding the effect of bisphosphonates on root resorption.
Igarashi et al13,14 showed that systemic and topical
administration of bisphophonate could decrease or
prevent root resorption during orthodontic tooth
movement in animal models. Rogers et al16 suggested
that clodronate could inhibit recruitment of osteoclasts,
promote osteoclast apoptosis, or both.
Keles et al29 reported that the influx of osteoclasts
on the compression side of the molar roots was maximal
American Journal of Orthodontics and Dentofacial Orthopedics
November 2010
10 mmol/L clodronate Significance Multiple comparison test
3.00 6 1.00 NS NS
1.57 6 1.13 * NS†
1.66 6 0.57 NS NS
on day 4 and persisted until at least day 12 after force
application. King et al30 mentioned that clearance of os-
teoclasts from compression sites occurred between days
5 and 7 after appliance activation in rats. Pavlin et al28
found that the peak osteoclast concentration was on
day 6 after a light force application of 10 to 12 g. These
findings coincide with the results of this study that
showed that PRRA and NRRL in the 10 mmol/L clodr-
onate group were significantly decreased on day 7 com-
pared with the control group (P \0.05, Tables IVand V).
These results were also supported by histologic
examination in the 2.5 mmol/L clodronate group;
this demonstrated that tetracyclin labeling was
absent (Fig 5), confirming that the resorption occurred
before day 6. After that, intermittent calcein labeling
lines, indicating new bone formation, became more
evident in the lower third of the root (Fig 5).
Osteoclast apoptosis induced by bisphosphonate can
be considered a major mechanism for inhibition of bone
or root resorption.16,31 Rogers et al16 and Frith et al17
demonstrated that the simple bisphosphonates that
closely resemble PPi (such as clodronate, etidronate,
and tiludronate) can be metabolically incorporated
into nonhydrolyzable analogs of ATP, adenosine
50 -(ß,a)-dichloromethyl triphosphate, that accumulate
intracellularly in osteoclasts, resulting in induction of
osteoclast apoptosis. In addition, Liu et al32 suggested
that clodronate could impair the ability of the periodon-
tal ligament cells to support osteoclast formation, by de-
creasing RANKL expression and eventual promotion of
the osteoclast apoptosis. Therefore, clodronate’s meta-
bolic effects could result in decreased tooth movement
and root resorption.
Graham33 suggested that patients using bisphospho-
nate medication should not have invasive laser thera-
pies, miniscrew anchorage, and extractions. In
addition, the pharmacologic effect and half-life of bi-
sphosphonates, which can preferentially bind to actively
resorbing bone, inhibit orthodontic tooth movement and
the bone remodeling process, and can induce osteonec-
rosis in the maxilla and the mandible by impaired bone
American Journal of Orthodontics and Dentofacial Orthopedics
Volume 138, Number 5
healing, should be checked to warn patients about the
undesirable effects on orthodontic treatment.34,35
Several human studies demonstrated beneficial ef-
fects of clodronate (doses from 400 mg orally daily to
200 mg intravenously every 3 weeks) when adminis-
tered in the long term.7,9,36 In short-term studies,
women with postmenopausal osteoporosis obtained sig-
nificant increases in lumbar and femoral bone mineral
density.37 Higher doses of bisphosphonates are used
for oncologic indications than those used for osteoporo-
sis.38,39 Therefore, further long-term clinical and histo-
logic studies about orthodontic tooth movement are
needed.
CONCLUSIONS
Although clodronate might decrease root resorption
related to orthodontic tooth movement, patients should
be informed about the possible decrease in the amount
of tooth movement and the longer period of orthodontic
treatment.
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