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Correlation of bone resorption induced by orthodontic tooth
movement and expression of RANKL in rats
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Seifi M. DDS. MS., 2Jessri M. DDS. MS
1Associate Professor, Dept. of Orthodontics, Dental School, Shahid Beheshti University of Medical Sciences,
Tehran- Iran
2 Dentist
Abstract
Background and Aim: The product of RANKL gene expression has a fundamental role in bone
resorption and activates the chain of osteoclast differentiation from its predecessor cells. Reports
of this gene participation and role in physiologic and pathologic bone resorption led to
hypothesis that it may play a role in bone resorption induced by orthodontic tooth movement.
Materials & Methods: A closed coil spring (Dentaurum Ispringen, Germany) was fixed by two
pieces of ligature wires to mesial side of the maxillary right first molar and anteriorly to both
incisors of fifteen 7 week old Wistar male rats to tip the first molars mesially. The maxillary left
first molar of each animal used as an internal control group for the contralateral tooth. The
animals were sacrificed on the 21st day of the experiment and the tissue from the mesial side of
maxillary molars was used for PCR test. Wilcoxon Sign Rank test was used for densitometric
analysis of RANKL mRNA on the electrophoresis gel.
Results: Densitometric analysis showed a significant increase in expression of gene in the
experimental group compared to the control one (P<0.002). Genuineness of the PCR tests was
verified by household gene of GAPDH.
Conclusion: Based on the data obtained in controlled environment of the study, RANKL
expression increased significantly following bone resorption induced by orthodontic tooth
movement.
______________________________________________________________________________
Key words: Orthodontic tooth movement, RANKL, Root resorption.
Received on:October 2008
Final revision :November2008
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Accepted on :December2008
Introduction
During implementation of orthodontic forces, significant changes occur in periodontal
ligaments(1). Orthodontic forces remodel alveolar bone(2); this phenomenon induces tooth
movement. Osteoclasts play an important role in bone resorption(3,4), hence studying their
molecular characteristics assist in understanding their role in clinical phenomena. Receptor
Activator of Nuclear factor kappa β Ligand (RANKL) which is one of the Tumor Necrosis
Factors, is consisted of almost 300 amino acids and its major role in bone structure, has been
observed to be in osteoclasts differentiation and action(5).
A previous study demonstrated resorbing deciduous teeth pulp to have higher amounts of
RANKL when compared to a non-resorbing control group(6). This finding, considering active
bone and tooth resorption upon permanent teeth eruption, could suggest an important role for
RANKL in physiologic bone and root resorption. RANKL has been also shown to express in
fibroblasts and osteoclasts of in a resorbing PDL by histochemical analysis(7).
Previous studies suggest that RANKL expression in correlation with inflammatory bone
resorption due to lipopolysaccharide injection in rats’ gingival tissues increases in osteoclasts of
the region(8). This is clearly indicating that RANKL expression in endothelial, inflammatory and
periodontium cells increases during bone resorption. Also a close association between occlusal
trauma and RANKL expression in bone resorption site has been demonstrated8.
When RANKL function is restricted by OPG, periodontitis induced bone resorption is
significantly lower than the normal situation(9). This finding is also strongly suggesting a close
interaction between bone resorption and RANKL expression.
These findings demonstrate RANKL expression in bone resorption. Therefore, the aim of this study was to investigate the role of RANKL in bone resorption induced by orthodontic tooth
movement.
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Method and Materials:
Fifteen albino male Wistar rats aged seven weeks, with the mean weight of 200 gr were chosen
randomly. In each animal, the maxillary first molar, received orthodontic treatment and
considered control group, while the contralateral teeth which did not receive orthodontic
treatment, served as internal group. During the experiment period the manipulation and treatment
of the animals was performed according to the approved protocol of Institutional Animal Care
and Usage Committee and the approval of ethical committee of the dental School of Shahid
Beheshti University of Medical Sciences. To minimize the risk of trauma or discomfort to the
rats and displacement of the appliances, the animals were fed with ground laboratory chow and
drink water ad libitum. The animals were weighed before and after treatment to assess the whole
body effects of the intervention.
Orthodontic appliance placement: On the first day of the experiments the animals were
anesthesized with a Ketamine hydrochloride 50 mg/kgbw (Ketamine Hydrochloride, Alfasan,
Holland) injection containing 6mg/kgbw Xylazine HCL (Xylazine hydrochloride, Rompoun,
Bayer, Leverkusen, Germany).
Phosphoric acid gel 37.4% (Dentaurum® Group, Ispringen, Germany) was applied for 30
seconds to etch the mesial, palatal and buccal surfaces of the maxillary left first molars. The teeth
were then wiped with disposable brush (Dentaurum® Group, Ispringen, Germany) soaked in
distilled water and dried with air syringe. A closed Ni-Ti coil spring (0.002 × 0.008 inch 12 mm
Dentaurum ®- Ispringeu Germany) was used to drift the first molar mesially. By the means of a
force measuring gauge (Dentaurum® Group, Ispringen, Germany), it was proved that each
spring was capable of delivering 60gf. The springs were tightened to the cervical third of the first
maxillary right molars and then fixed anteriorly by using ligature-wire loops (Dentaurum® Steel
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ligature wire0.010 inch, Dentaurum® Group, Ispringen, Germany) around the cervical third of
the maxillary incisors. Anatomic position and structural development of both upper incisors,
suggested these teeth as appropriate anchorage units for protraction of first molars. In order to
prevent mucosal trauma the ligature wire ends were covered and secured with composite resin
(Self cure Degufill, Degussa AG, AG, Frankfurt, Germany).
Since tooth movement is directly dependant upon bone resorption, bone resorption was
considered equal to bone resorption. Mesial side of alveolar bone was scratched by fine burs and
discs and sent for PCR examinations.
The obtained materials were then Snap-Frozen in liquid nitrogen and stored at -20ºC for later
extraction of mRNA. Specimens were kept in 5% EDTA solution for 24 hours and afterwards
they were sonicated for 15 minutes. After primary denaturation, to complete the strands, a final
fixation was performed at 72º C for 5 minutes.
By using reverse transcriptase enzyme (Super-Script II; Bibco, Wickliffe, OH, USA) and 2 μlit
of hexamer primers, cDNA was synthesized (at 42ºC for 1 h). Afterwards cDNA was amplified
using taq DNA polymerase (Amplitaq® DNA polymerase applied biosystems,CA, USA), PCR
buffer (Applied biosystems,CA,USA), specific primers for RANKL and GAPDH (Sigma
Aldrich, India), MgCl2 and according to Table 1 condition.
Table 1 - cDNA extension stages
Stage
Denaturation
Annealing
Extension
Temperature
94 °C
55°C
72°C
Duration
30 sec
30 sec
30 sec
In order to visualize DNA strands in UV transilluminator, PCR product was electrophoresed on
2% agarose gel and stained by ethidium bromide. PCR product length for RANKL measured 355
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base pairs and GAPDH was used as control gene. PCR expression was measured defining units
as 0,1,2,3 respectively representing none, low, medium, high and performed by a single
practitioner and assistant observer. The obtained results were analyzed by Wilcoxon signed-rank
test in the SPSS version 11.5 statistical package.
Results:
The animals had an 8-11% of weight gain during the experiment and this indicates that the
orthodontic appliances have not interfered with animals' nourishment. Statistical analysis of
tooth movement shows a significant change in control group. (Table 2)
Table 2- Orthodontic tooth movement(OTM) in study groups
Amount of OTM
Mean
0.8942mm
SD
0.063mm
Minimum
0.85mm
Maximum
0.98mm
According to the RT-PCR findings, RANKL mRNA was detectable in all experimental groups
and only 3 cases of control group. Statistical analysis i.e. Wilcoxon signed-rank of agar gel after
electrophoresis for detection of RANKL mRNA showed significant difference (p=0.002)
(Table3).
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Table 3-Densitometric findings of mRNA in control & experimental groups
0
1
2
3
RANKL (Control)
-
3
7
5
RANKL (Experiment)
12
1
2
-
RANKL (Control)
-
-
9
6
RANKL (Experiment)
-
-
8
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Discussion:
The results of comparison between inter-molars distance before and after the treatment signifies
the orthodontic treatment, which was a modification of Kobayashi (2000)(10) and Ashizawa
(1998)(11) method, to be effective in inducing tooth movement. This finding is in accordance with
Low(12) and Seifi (2003)(13) results.
Considering the fact that this is the first study investigating the association of RANKL expression
with orthodontic bone resorption, we do not have any peer study to compare our findings with.
However the results of other investigations which focused on this protein’s role on bone
resorption is in accordance with ours.
Although Ashizawa(1998)(11) designed his model to induce tooth movement, there was no reports
of the tooth movements which he made by his model and only reported the amount of bone
appositioned in alveolar sockets’ walls; which he had not observed a significant difference
between case and control groups. One may conclude that in Ashizawa’s model there was no
significant difference between case and control tooth movement. In his model, Ashizawa used a
metal plate over the occlusal surface of the teeth in order to attach the spring to them; this metal
plate could induce occlusal trauma and in the same time, the duration of his study, 6 days, in
comparison to ours, 21 days, seems insufficient to induce significant tooth movement.
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The method used to extract RANL mRNA was verified using GAPDH as control gene. RANKL
mRNA showed a significant increase in case groups which is consistent with Silva’s
(2008)findings(14) who has detected RANKL in periodontium of 65 patients suffering fro active
and aggressive periodontitis.
Garlet et al(2007)(15), studied the periodontium cytokines, they found out an increase in RANKL
expression in tension sides comparing to control group. Nishijima (2006) studied RANKL in
gingival sulcular fluid of 10 adloescents receiving orthodontic treatment(16). He found out RANKL
expression increases with force magnitude and is also dependant upon time.
RANKL was also observed to be of importance in differentiation and maturation of osteoclasts (17).
Since RANKL is essential in bone resorption process this finding is in accordance with previously
mentioned studies.
The mentioned studies results are consistent with the present study; however further studies are
needed for valid approval of the present study's findings.
Conclusion:
According to the results of the present study, the applied method is effective in inducing bone
resorption and tooth movement and could be used as a valid method to evaluate orthodontic tooth
movement and associated bone resorption.
On the other hand, PCR examination results suggested RANKL to be significantly increased on
pressure side in case group. This could be concluded that RANKL gene in controlled laboratory
environment is associated with orthodontic bone resorption.
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