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Orthodontically induced osteocyte apoptosis under different force magnitudes

in rats: an immunohistochemical study

Article  in  European Journal Of Oral Sciences · August 2017

DOI: 10.1111/eos.12366

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Eur J Oral Sci 2017; 1–10 Ó 2017 Eur J Oral Sci
DOI: 10.1111/eos.12366 European Journal of
Printed in Singapore. All rights reserved
Oral Sciences

Hassan E. Kassem1 , Iman M.

Orthodontically induced osteocyte Talaat2,3, Afaf El-Sawa4, Hanan
Ismail1, Abbas Zaher1
apoptosis under different force 1
Department of Orthodontics, Faculty of
Dentistry, Alexandria University, Alexandria,
Egypt; 2Department of Clinical Sciences,
magnitudes in rats: an College of Medicine, University of Sharjah,
Sharjah, United Arab Emirates; 3Department

immunohistochemical study of Pathology, Faculty of Medicine, Alexandria

University, Alexandria, Egypt; 4Department of
Oral Biology, Faculty of Dentistry, Alexandria
University, Alexandria, Egypt

Kassem HE, Talaat IM, El-Sawa A, Ismail H, Zaher A. Orthodontically induced

osteocyte apoptosis under different force magnitudes in rats: an immunohistochemical
study.
Eur J Oral Sci 2017;00: 1–10. © 2017 Eur J Oral Sci
We investigated the effect of different force magnitudes on osteocyte apoptosis in a
model of orthodontic tooth movement. Forty-nine male Sprague Dawley rats
(7–9 wk of age) were divided into light- and heavy-force groups (n = 21 each group)
and a control group (n = 7). A coil spring delivered pressure (either 10–15 g or
20–25 g) to the left maxillary first molar. The rats were sacrificed 1, 3, or 5 d after
Hassan Elsayed Kassem, Faculty of
placement of the appliance. Sections of the maxillary first molars were immunos- Dentistry, Alexandria University, Champilion
tained for caspase-3. Upon force application, the number of apoptotic osteocytes Street, Azarita, Alexandria, Egypt
significantly increased in the pressure side at 1 d and remained the same at 3 d and
E-mail: hassan.kassem@dent.alex.edu.eg
5 d. However, there was no significant difference in the number of apoptotic osteo-
cytes between the two force groups. We conclude that osteocyte apoptosis appears
to increase under orthodontic loading, reaching a plateau after 1 d. However, Key words: caspase-3; orthodontic tooth
osteocyte apoptosis seems to be independent of the magnitude of orthodontic forces movement; programmed cell death
tested. Accepted for publication June 2017

For decades, the bone-trapped osteocyte was consid-
ered a passive ‘place holder’ or a ‘quiescent’ cell that
mainly served a structural, rather than a functional,
role (1). Despite the intricate and pervasive cell- and
fluid-filled network formed by osteocytes, clues to their
function were concealed for many years. The renais-
sance of interest in osteocytes started in the second half
of the 20th century when several reports started to shed
light on their dynamic nature (2).
Osteocyte apoptosis was first observed to increase in
bones with high modeling and remodeling rates, such
as in the calvarial bone or osteophytic bone of infants,
in comparison with sites of low remodeling, such as in
adult iliac crests or femoral heads (3). Henceforth, sev-
eral experimental models were established to study
osteocyte apoptosis in relation to mechanical loading.
Osteocyte apoptosis was found to increase in vivo in
instances of hyperphysiological loading (4), fatigue
damage (5–8), and bone unloading (9, 10), as well as
under compressive strain in cell cultures (11). On the
other hand, osteocyte survival was promoted in
response to physiological loading (4), fluid shear stress
(12, 13), under tension in cell cultures (11, 14), and
under tension in ex vivo bone specimens (15). These
findings suggest that osteocyte apoptosis plays a regula-
tory role in bone modeling and remodeling in response
to mechanical loading.
Moreover, osteocyte apoptosis was found to be
associated with osteoclast recruitment. The incidence of
osteocyte apoptosis prior to osteoclast recruitment and
subsequent bone resorption was reported in several stud-
ies (9, 10, 16). Pharmacological blocking of the apoptotic
pathway blocked both fatigue-induced osteocyte apopto-
sis and osteoclast bone resorption (7). These findings
support the hypothesis that dying osteocytes may be
directly or indirectly involved in the signaling pathway
for osteoclast recruitment and function (2). A suggested
mechanism was that apoptotic osteocytes localized to
areas of bone microdamage cued neighboring viable
osteocytes to release osteoclastogenic signals (8).
Orthodontic tooth movement can be considered a
process of mechanically induced bone modeling and the
consequent remodeling response; hence, the changing
perspectives of bone physiology are expected to impact
our understanding of the mechanism by which teeth are
orthodontically moved (17). Specifically ablating osteo-
cytes in a transgenic mouse model of orthodontic tooth
movement resulted in reduced osteoclast numbers at
compression sites in addition to smaller amounts of
tooth movement (18).
Several studies have shown that osteocyte apoptosis
is spatially and temporally related to osteoclast recruit-
ment and bone resorption during orthodontic tooth
movement. The percent of apoptotic osteocytes
2 Kassem et al.
increased at compression sites adjacent to hyalinized
periodontal ligament (PDL) (19). The peak of osteocyte
apoptosis preceded the onset of osteoclast recruitment
(19–21). These findings suggest that osteocyte apoptosis
may lie along the pathway starting from the application
of orthodontic force and culminating in osteoclast acti-
vation, bone resorption, and tooth movement.
Terminal deoxynucleotidyl transferase-mediated
deoxyuridine triphosphate (dUTP) nick-end labeling
(TUNEL) has previously been used as an assay for
osteocyte apoptosis in orthodontic tooth-movement
experiments (19–21). However, the TUNEL assay has
been criticized for being unable to distinguish osteocyte
apoptosis from necrosis (22) and for giving false-posi-
tive reactions as a result of DNA nicking during tissue
processing (2). Instead, use may be made of the fact
that caspase-3 is a member of a family of cysteine pro-
teases that play an important role in the apoptotic
pathway. Activation of caspase-3 is considered the
point of no return for the apoptotic pathway (23).
Immunostaining for caspase-3 in histological sections is
considered as being highly specific for the detection of
osteocyte apoptosis (2). However, MOIN et al. (21)
found a non-significant increase in the number of cas-
pase-3-positive osteocytes at pressure sites of orthodon-
tically loaded first molars in mice.
Even so, we hypothesized that if osteocyte apoptosis
plays a regulatory role of the biological reactions in
response to orthodontic loading, a direct relationship
would exist between osteocyte apoptosis and the magni-
tude of the orthodontic force. To the best of our
knowledge, no studies have reported the effect of differ-
ent force magnitudes to osteocyte apoptosis in a model
of orthodontic tooth movement.
Hence, the main objective of the present study was
to quantify osteocyte apoptosis in response to two dif-
ferent magnitudes of force in a rat model of orthodon-
tic tooth movement. The null hypothesis is that the
number of apoptotic osteocytes does not differ with a
change in magnitude of the orthodontic force.
Material and methods
This study was a single-center randomized controlled ani-
mal experiment. The experimental design was approved by
the Ethics Research Committee of the Faculty of Den-
tistry, Alexandria University (Alexandria, Egypt). The
experiment was conducted, and results reported, in accor-
dance with Animal Research: Reporting of In Vivo Exper-
iments (ARRIVE) guidelines (24) and the National
Institutes of Health guide for the care and use of labora-
tory animals (25).
Experimental animals
A total of 49 male Sprague Dawley rats, 7–9 wk of age
(average weight: 150  20 g), were used in this study. The
weight of the rats was checked at the time when the appli-
ance was placed (to determine eligibility for inclusion) and
at the time of the rat’s death. The rats were housed in the
vivarium of the Medical Research Institute, Alexandria
University, under normal laboratory conditions with a
12-h light/12-h dark cycle using natural lighting, ambient
temperature and humidity. The rats were fed powdered
rodent chow and water ad libitum. It was decided a priori
that any rat experiencing loss of ≥20% of their original
weight (i.e. at the time of placement of the appliance)
would be excluded from the experiment.
Sample size estimation
The primary outcome of the study was the percentage of
caspase-3-positive osteocytes as a proxy for the percentage
of apoptotic osteocytes. The sample size was calculated
using software PS Power and Sample Size Calculations
Version 3.0 (Vanderbilt University, Nashville, TN, USA)
(26). Based on previous findings of the percentage of
apoptotic osteocytes (19, 20), the sample size was calcu-
lated using an independent-samples t-test to detect a
between-group mean difference of 5% and an SD of 2%,
with a type I error probability of 0.05, a power of 80%,
and equal size of groups. The minimum number of rats to
be included in each group was calculated to be four. It
was decided to include seven rats in each group to account
for any losses during the experiment.
Randomization and masking
Simple randomization was performed without stratification
or blocking with equal allocation ratios. The random
sequence was generated by an Internet-based free access
random number generator (27). The table for the alloca-
tion of the rats was kept in a sealed envelope in the office
of the head of the animal house accessed by the attending
veterinarian. The operator was masked to the assignment
of the rat until after the rat was considered eligible for
inclusion in the experiment according to both age and
weight. Once a rat was deemed eligible, the operator con-
sulted the attending veterinarian to reveal the allocation of
the rat from the sealed envelope. Further masking of the
operator was not possible until the rat was killed owing to
the nature of the intervention.
Forty-nine rats were randomized into a total of seven
groups. Seven rats served as a control. The test rats
(n = 42) were divided according to the magnitude of the
orthodontic force and the duration of force application, as
follows:
(i) Light-force group (n = 21): divided according to day
of sacrifice (d 1, d 3, or d 5; n = 7 per group).
(ii) Heavy-force group (n = 21): divided according to day
of sacrifice (d 1, d 3, or d 5; n = 7 per group).
Orthodontic tooth movement
Before placement of the orthodontic appliance, the rats
were anesthetized with a combination of ketamine
hydrochloride (90 mg kg 1) and xylazine (5–8 mg kg 1),
injected intramuscularly according to the Canadian Coun-
cil on Animal Care guidelines. A nickel-titanium (NiTi)
closed-coil spring (NiTi Closed Coil Springs 0.008” 9
0.030” (0.20 9 0.76 mm) UltraLight; G&H Wire Com-
pany, Franklin, IN, USA), attached to the left maxillary
first molar and the maxillary incisors, was used to deliver
a mesial force to the molar. The coil was tied to the teeth

using 0.008” (0.20 mm) stainless-steel ligature wire. Light-
cured low-viscosity composite (Filtek Z350 XT; 3M ESPE,
St Paul, MN, USA) was used to secure the ligature wire
around the maxillary incisors (Fig. 1).
To deliver the desired force magnitude, the distance
between the maxillary first molar and the maxillary incisors
was measured to the nearest 0.5 mm using a conventional
Michigan-O periodontal probe with Williams markings
(Hu-Friedy Mfg. LLC., Chicago, IL, USA). A segment of
the NiTi closed-coil spring, measuring 3 mm less than the
molar–incisor distance, was cut to deliver 10–15 g of force
and another segment of the NiTi closed-coil spring, mea-
suring 6 mm less than the molar–incisor distance, was cut
to deliver 20–25 g of force, as verified by a tension gauge
(Correx; Haag-Streit, Koeniz, Switzerland) (28).
The entire procedure of placement of the orthodontic
appliance lasted 30–40 min. Following the procedures, the
rats were placed in separate cages and were monitored until
recovery. The control rats did not receive any intervention.
Test rats were killed at completion of the time course.
Specimen preparation
The left maxilla was dissected and cleaned carefully of soft
tissues. The harvested maxillae were immediately fixed in
10% neutral phosphate-buffered formalin (pH 7.4) over-
night at 4°C. The maxillae were subsequently washed with
phosphate-buffered saline (PBS) for 5 min. The specimens
were decalcified in 10% formic acid at 4°C (8, 29, 30).
End of calcification was checked using both manual test-
ing and X-ray examination. Samples were subsequently
dehydrated in a series of ethanol solutions of ascending
concentrations, cleared by xylene, and infiltrated with
paraffin wax at 60°C for 2–3 h. Paraffin blocks of the
specimens were subsequently prepared (31). Serial trans-
verse sections of the first molar region, of 5 lm thickness,
were cut using the microtome Leica RM 2125R7 (Leica
Microsystems, Nussloch, Germany), from the inter-radicu-
lar alveolar bone toward the root apex (32). Sections were
prepared with hematoxylin and eosin staining for
Fig. 1. The orthodontic appliance in situ showing a nickel-
titanium (NiTi) spring stretched between the left maxillary
first molar and the incisors. The custom-made wire gag is
shown in place.
Orthodontically induced osteocyte apoptosis 3
descriptive histology and with immunohistochemistry for
histomorphometry.
Immunostaining for caspase 3
Sections were deparaffinized in xylene and rehydrated in
graded concentrations of ethanol. Heat-mediated antigen
retrieval was carried out by boiling the sections in 10 mM
citrate buffer (pH 6.0) for 10–20 min followed by cooling
at room temperature for 20 min. Subsequently, sections
were washed with PBS and blocked with Hydrogen Perox-
ide Block (Thermo Fisher Scientific, Fremont, CA, USA)
for 5 min followed by UV Block (Thermo Fisher Scientific)
for 5 min at room temperature. Sections were incubated
with primary rabbit polyclonal caspase 3 antibody (CPP32)
Ab-4 (Thermo Fisher Scientific) at a concentration of 5–
10 lg ml 1 for 30 min at room temperature. To develop
the signal, sections were incubated with Quanto Amplifier
(Thermo Fisher) for 10 min, rinsed by PBS from wash bot-
tle followed by a PBS wash bath for 5 min, incubated in
Quanto Polymer (Thermo Fisher) for 10 min, rinsed again
in the same manner, and incubated in DAB Quanto
(Thermo Fisher) for 5 min. All reagents were as provided
in the UltraVision Quanto Detection System (HRP DAB;
Thermo Fisher). Sections were then counterstained with
hematoxylin. Negative controls were established by omit-
ting the primary antibody in the incubation step. Lympho-
cytes in tonsil sections were used as positive controls.
Immunostaining for tartrate-resistant acid
phosphatase
The same protocol as used for caspase 3 was used for
immunostaining for tartrate-resistant acid phosphatase
(TRAP) in order to identify osteoclasts. Sections were
incubated with mouse monoclonal primary antibody to
TRAP (Clone 26E5) (Thermo Fisher) at a concentration
of 1:20 for 30 min at room temperature overnight. Nega-
tive controls were established by omitting the incubation
step with primary antibody. Tonsil macrophages served as
positive controls.
Outcomes
The primary outcome of the investigation was the percent-
age of apoptotic osteocytes, determined as the percentage
of caspase-3-positive immunostained osteocytes relative to
the total number of osteocytes. The number of osteoclasts
identified as the number of TRAP-positive immunostained
multinucleated cells was considered a secondary outcome.
Area of observation
The cross-section from the midbuccal root of each left
maxillary first molar was used for observation and quan-
tification. For every specimen, three sections for each
assay were taken at 100 lm intervals (33, 34). Images were
taken using an Olympus BX51 (Olympus, Tokyo, Japan)
light microscope equipped with a Nikon DX100 digital
camera (Nikon, Tokyo, Japan) and stored in 24-bit true-
color JPEG format (Fig. 2).
The images at 9200 magnification were imported to
Adobe Photoshop CS (Adobe Systems, San Jose, CA,
USA) image processing software. An overlay was placed
over the image to designate the area used for
4 Kassem et al.
measurement. The area of measurement for caspase-3-
positive osteocytes was a rectangle of 200 9 100 lm. The
osteoclasts were counted in the PDL adjacent to the length
of the observation area (17). Counting was carried out by
the principal investigator on the computer image and the
results obtained were confirmed by simultaneously examin-
ing the sections under microscopy at higher magnification.
Empty osteocyte lacunae were counted as caspase-negative
osteocytes. Tartrate-resistant acid phosphatase-positive
multinucleated cells at the surface of the alveolar bone in
the designated areas were counted as osteoclasts. For
every specimen, the outcome was calculated as the average
of three sections.
To mask the outcome assessor, the tabs on the slides
holding the rat identification number were covered with
masking tape. Once the outcomes were assessed in every
slide, the tabs were unmasked and the outcomes were
recorded according to the rat identification number. How-
ever, the outcome assessor could not be blinded to the
obvious differences between the pressure and tension sides,
and between the teeth that had undergone orthodontic
tooth movement and the control teeth.
Intra- and inter-rater reliability
Twenty slides per assay were selected at random. The total
number of osteocytes, the number of caspase-positive
osteocytes, and the number of TRAP-positive osteoclasts
were re-counted by the principal investigator and the sec-
ond author. Based on a two-way random-effects model,
the intra-class correlation coefficients were calculated. Cor-
relation coefficients for intra-rater and inter-rater reliabil-
ity for the number of osteocyte lacunae were 0.891 and
0.875 respectively; for the number of caspase-positive
osteocytes the intraclass correlation coefficients were 0.984
and 0.987, respectively; and for the number of TRAP-posi-
tive osteoclasts the intraclass correlation coefficients were
0.914 and 0.947, respectively.
Statistical analysis
Statistical analysis was performed using the software SPSS
Statistical Package for Social Sciences (SPSS for Windows,
version 16; SPSS, Chicago, IL, USA). Outcome variables
were tested for normality using the Kolmogorov–Smirnov
Fig. 2. Region of interest for quantification. (A) Cross-sections were obtained from  100 lm of the inter-radicular bone to 
400 lm toward the apex. Three sections were taken at 100-lm intervals. (B) Overlay used to designate the area used for observa-
tion. The line ‘l’ is the mesiodistal line passing through the center of the root and parallel to the direction of tooth movement.
The points ‘m’ and ‘d’ are the intersection of the mesial and distal areas and the line l with the surface of mesial and distal alveo-
lar bones. Bo, bone; P, periodontal ligament; R, root.
normality test and Q-Q plots. All variables examined ex-
hibited a normal distribution across the groups except for
the osteoclast number especially at the tension side, which
consistently departed from normality. The paired t-test
and Wilcoxon signed-rank test were used to compare the
outcome at the pressure and the tension sides. One-way
and two-way ANOVAS were used for comparison of the per-
centage of apoptotic osteocytes across loading and day of
assessment, and the Kruskal–Wallis multiple comparisons
test was used for comparison of the osteoclast number
between the study groups. Corresponding post-hoc tests
were used where applicable. Statistical significance was
predefined at P ≤ 0.05.
Results
Welfare of the rats
The mean age and weight of the rats at the start of the
experiment were 8.00  0.53 wk and 184.40  3.14 g,
respectively, with no statistically significant difference
among the study groups. None of the rats had lost
more than 10% of their body weight by the end of the
experimental period. Three rats did not complete the
experiment because of damage of the orthodontic appli-
ance and one rat was excluded as a result of the devel-
opment of a skin lesion. Several specimens were
excluded because of error in tissue processing. Six rats
in every group were included in the analysis. A flow
chart of the experiment is shown in Fig. 3.
Descriptive histology and immunostaining
The mid-buccal root of the rat maxillary first molar
subjected to orthodontic forces showed the classical
changes of compression of the PDL fibers at the site of
compression on the mesial side of the root and the
stretching at the site of tension on the distal side.
Hyalinization of the PDL at the compression site was
seen in a few sections at 1 d in both the light- and
heavy-force groups. At 3 and 5 d, varying degrees of
hyalinization was almost always detected. Undermining

Fig. 3. Flow diagram of the experiment.
Fig. 4. Photomicrograph of a cross section of the midbuccal
root in the rat maxillary first molar subjected to a heavy
orthodontic force for 3 d showing hyalinization (H) of the
periodontal ligament at the mesial side and several osteoclasts
starting undermining resorption at the edges of the hyalinized
area (arrows). B, bone; D, distal; H, hyalinization M, mesial;
P, periodontal ligament; R, root. Hematoxylin and eosin
(H&E) staining.
resorption became consistently evident from 3 d. Mult-
inucleated osteoclast cells were demonstrated in resorp-
tion lacunae adjacent to areas of hyalinization (Fig. 4).
In immunostained sections, caspase-3-positive osteo-
cytes showed both cytoplasmic and nuclear staining, in
addition to occasional cardinal signs of apoptosis, such
as nuclear shrinking (Fig. 5A). Osteoclasts in TRAP-
stained sections showed a positive reaction localized to
Orthodontically induced osteocyte apoptosis 5
the ruffled border (Fig. 5B). Representative photomi-
crographs of osteocyte apoptosis in the light- and
heavy-force groups at the different study time points
are shown in Fig. 6.
Apoptotic osteocytes number
Table 1 shows the percentage of apoptotic osteocytes
at the pressure and tension sides in each experimental
group. In the light-force group, there was a statistically
significant difference between the pressure side and the
tension side of 26.0% [standard error (SE) = 1.97]
(P = 0.001), 28.8% (SE = 1.95) (P = 0.001), and 24.3%
(SE = 2.88) (P = 0.001), at 1, 3, and 5 d, respectively.
Similarly, in the heavy-force group, the mean differ-
ences between the pressure side and the tension side
were statistically significant at 31.4% (SE = 2.23) at 1 d
(P = 0.002), 24.5% (SE = 4.33) at 3 d (P = 0.001), and
22.7% (SE = 3.52) at 5 d (P = 0.001). The mean differ-
ence in percentage of apoptotic osteocytes between the
pressure side and tension side in the control group was
2% (SE = 1.99), which was not statistically significant
(P = 0.360).
At the pressure side, there was a statistically signifi-
cant increase in the percentage of apoptotic osteocytes
between the light-force groups and the control group,
with mean differences of 25.8% (SE = 2.78) at 1 d
(P = 0.001), 29.3% (SE = 2.77) at 3 d (P = 0.001), and
22.9% (SE = 2.70) at 5 d (P = 0.001). Similarly, the
heavy-force groups showed a statistically significant dif-
ference compared with the control group at all days of
6 Kassem et al.

A B

Fig. 5. (A) Caspase-3-immunostained section showing positively stained osteocytes (arrows). (B) Tartrate-resistant acid phos-
phatase (TRAP)-immunostained section showing a positively stained osteoclast in a resorption lacuna.

A D

B E

C F

Fig. 6. Caspase-3-immunostained sections showing apoptotic osteocytes (arrows) in the light-force group at (A) 1 d, (B) 3 d, and
(C) 5 d, and in the heavy-force group at (D) 1 d, (E) 3 d, and (F) 5 d.

evaluation (P = 0.001). The mean differences between (SE = 3.20) at 3 d (P = 0.001), and 23.9% (SE = 3.20)
the control group and the heavy-force groups were at 5 d (P = 0.001). At the tension side, there was no
30.0% (SE = 3.21) at 1 d (P = 0.001), 25.5% statistically significant different between the light-force
 Orthodontically induced osteocyte apoptosis 7

Table 1
Prevalence of apoptotic osteocytes at the pressure and tension sides in each group

Pressure side*,† Tension side*,†

Study group Mean SD Min. Max. Mean SD Min. Max.

Control (n = 6) 22.4a 4.21 16.7 26.7 20.4a 2.70 16.7 23.5

Light force
1 d (n = 6) 48.2b 6.28 40.0 55.6 22.2a 2.67 18.8 26.3
3 d (n = 6) 51.7b 4.88 42.9 56.3 22.9a 6.32 14.3 29.4
5 d (n = 6) 45.4b 3.39 41.2 50.0 21.0a 5.75 15.0 29.4
Heavy force
1 d (n = 6) 52.4b 3.45 47.1 56.3 21.0a 4.68 15.8 27.8
3 d (n = 6) 47.9b 7.11 35.3 55.0 23.4a 5.68 16.7 31.3
5 d (n = 6) 46.3b 6.58 36.8 55.0 23.6a 7.70 15.0 33.3

Values are given as percentage (%) of apoptotic osteocytes. Max., maximum; Min., minimum.
*Different superscript letters denote a statistically significant difference within a row (paired t-test; significance level at P ≤ 0.05).
†
Different superscript letters denote a statistically significant difference within a column [post-hoc comparison based on Tukey’s honest sig-
nificant difference (HSD) test; equal variance assumed; significance level at P ≤ 0.05].

groups and the control group (P = 0.797), or between
the heavy-force groups and the control group
(P = 0.688).
Two-way ANOVA comparing the light- and heavy-
force groups showed a non-significant main effect of
both the force and the time point on the percentage of
apoptotic osteocytes at the pressure and tension sides.
There was no interaction between the force and time
point at the pressure and tension sides. This demon-
strates that the percentage of apoptotic osteocytes did
not vary differently in either the light- or heavy-force
groups along the study time points, either at the pres-
sure side or the tension side.
between 1 and 5 d in both the light-force group
(P = 0.003 for both) and the heavy-force group
(P = 0.003 for both). On the other hand, no significant
difference was found in the osteoclast number between
3 and 5 d in either group (P = 0.058, P = 0.064). The
tension side showed no statistically significant difference
between the time points in either force group.
When comparing the time points between the light-
and heavy-force groups, the osteoclast number showed
no statistically significant difference at the pressure or
the tension sides at 1 d (P = 0.575, P = 0.999), 3 d
(P = 0.38, P = 0.9992), or 5 d (P = 0.495, P = 0.999).

Osteoclast number Discussion

The median (range) number of osteoclasts in each
group and at each time point is presented in Table 2.
There was no statistically significant difference between
the pressure and tension sides in the control group
(P = 0.157) and at 1 d in both the light-force
(P = 0.157) and the heavy-force groups (P = 0.655).
However, a statistically significant increase in the differ-
ence in osteoclast number between the pressure and
tension sides was found in the light-force group at 3 d
(P = 0.026) and at 5 d (P = 0.03) and in the heavy-
force group at 3 d (P = 0.020) and at 5 d (P = 0.026).
There was no statistically significant difference in
osteoclast number between the control group on one
side and either the light-force group (P = 0.269) or the
heavy-force group (P = 0.575) at 1 d. However, at 3
and 5 d, a statistically significant difference was found
between the control group and the light-force group
(P = 0.003 for both days) and between the control
group and the heavy-force group at 3 d (P = 0.003)
and at 5 d (P = 0.003). At the tension side, the osteo-
clast number did not differ significantly between the
control group and the light-force groups (P = 0.578) or
between the control group and the heavy-force groups
(P = 0.578).
There was a statistically significant difference in the
osteoclast number between 1 and 3 d, as well as
This study used a rat model of orthodontic tooth
movement to investigate the responsiveness of osteo-
cytes to mechanical stress of different magnitudes
through the apoptotic pathway. It was speculated that
if a proportional relationship existed between osteocyte
apoptosis and the magnitude of the applied force, then
a direct relationship between osteocyte apoptosis and
mechanical stress can be assumed, suggesting that
osteocyte apoptosis may act as a determinant of the
biological response to the application of orthodontic
force. This would offer future chances to modulate the
rates of orthodontically induced bone modeling and
remodeling. However, this proposition was not sup-
ported by the results of the study.
It is worth mentioning that this study is not attempt-
ing to define the rather ‘elusive’ optimal force (35). The
use of different force magnitudes intended to investigate
the alterability of the extent of osteocyte apoptosis with
varying force levels. Orthodontic tooth movement
serves as an excellent model for testing mechanical
bone loading. Hence, these investigations can be
extrapolated to other bone-loading models (36).
There were three incidences of appliance breakage in
the entire sample. This may be considered a reasonable
indication of the longevity of the appliance design used.
The short duration of the experiment does not allow us
8 Kassem et al.
Table 2
Prevalence of osteoclasts at the pressure and tension sides in each group
Pressure side*,†
Study groups Median Mode Min.
Control (n = 6) 0a 0 0
Light force
1 d (n = 6) 1a 1 0
3 d (n = 6) 3b,c 3 2
5 d (n = 6) 4c 4 3
Heavy force
1d(n = 6) 0.5a 0;1 0 1
3 d (n = 6) 3.5b,c 4 2
5 d (n = 6) 4.5c 5 3
Values are given as number of osteoclasts. Max., maximum; Min., minimum.
*Different superscript letters denote statistically significant difference within a row (Wilcoxon signed-rank test; significance level at
P ≤ 0.05).
†
Different superscript letters denote statistically significant difference within a column (post-hoc comparison based on Mann–Whitney
U-test; equal variance assumed; significance level at P ≤ 0.05).
to draw further conclusions on the stability of the
design in experiments of longer duration. However, in
other studies, this appliance design was proven stable
for up to 4 wk (37). The appliance breakage occurred
at the point of the attachment of the coil spring to the
stainless-steel ligature surrounding the maxillary inci-
sors, which may be attributed as interference with the
mandibular incisors. It is recommended to trim the
mandibular incisors to reduce such incidences of appli-
ance failure (38).
Osteocyte apoptosis was studied at the initial stage
of tooth movement and at study time points days 1, 3
and 5 thereafter. To enable comparison between the
two force groups and along the time points, it was nec-
essary to take histomorphometric measures were taken
before active bone resorption (39). Hence, the last time
point used was 5 d before the peak of osteoclast activa-
tion (8, 21).
In the present study, there was a significant increase
in the percentage of apoptotic osteocytes following the
application of orthodontic force. Similar results were
reported by HAMAYA et al. (19) using histological signs
of apoptosis in hematoxylin and eosin (H&E)-stained
sections, as well as by SAKAI et al. (20) using the
TUNEL assay. MOIN et al. (21) found a significant
increase in osteocyte apoptosis using the TUNEL
assay, whereas caspase-3-positive osteocytes showed
only a slight increase, which was not statistically signifi-
cantly different compared with the control values. This
may be attributed to the use of the unloaded contralat-
eral side as control. A distant effect of the application
of orthodontic tooth movement may be presumed
because the same study found an increase in osteoclast
number in the contralateral non-compression side (21).
The present study showed no statistically significant
difference in the percentage of apoptotic osteocytes
between 1, 3, and 5 d at the loading side. Similarly,
MOIN et al. (21) found a slight increase in the number
of caspase-3-positive osteocytes at 1 d and at 3 d and
to a lesser extent at 7 d; however, there were no
Tension side*,†
Max. Median Mode Min. Max.
1 1a 1 0 1
1 0a 0 0 1
4 0a 0 0 1
5 0a 0 0 1
0a 0 0 1
4 0a 0 0 1
5 0a 0 0 1
statistically significant differences between the time
points. On the other hand, using the TUNEL assay,
SAKAI et al. (20) and MOIN et al. (21) found a peak
increase of TUNEL-positive osteocytes at 12 and 24 h,
respectively. It is plausible that the current study
missed the peak of osteocyte apoptosis at 12 h, as
demonstrated by SAKAI et al. (20). The inclusion of
additional time points in the first few hours following
the application of orthodontic force could have
revealed a peak in osteocyte apoptosis. However,
TUNEL assay results have been criticized for failing to
differentiate osteocyte apoptosis and osteocyte necrosis
(22) in addition to the false positive-reaction from
DNA nicking during tissue processing (2).
The novel investigation in this study was the compar-
ison of the effect of force magnitude on osteocyte apop-
tosis. The results showed no significant difference
between the light-force group and the heavy-force
group at any time point studied. It can be argued that
the range of force magnitude used was not sufficient to
demonstrate such an effect despite the heavier force
applied being approximately double the magnitude of
the lighter force. It can be postulated that osteocyte
apoptosis occurs in response to mechanical stress at
light-force magnitudes; however, this effect is localized
and the increase in force magnitude does not result in
extension of the biological process to wider areas. This
attenuation of the biological effect of the mechanical
stress as the distance from its source increases was
demonstrated in a bone fatigue model in which the
number of caspase-3-positive osteocytes was highest at
the micro-crack site and decrased to control levels
within several hundred micrometers (8).
In the present study, the number of osteoclasts was
investigated as a surrogate for the progression of the
changes in the periodontal ligament associated with
orthodontic tooth movement. There was a significant
increase in the number of osteoclasts at the pressure
side starting at 3 d and to a greater extent at 5 d in
both force groups compared with the control group.

This demonstrates that the onset of osteoclast forma-
tion lags behind the peak of osteocyte apoptosis. Simi-
lar results were reported by other authors (19, 21). This
corroborates the temporal association between osteo-
cyte apoptosis and osteoclast recruitment. Although it
is not sufficient to associate osteocyte apoptosis directly
to osteoclastogenesis according to time precedence, it is
safe to say that osteocyte apoptosis appears to lie along
the pathway beginning at the application of the
mechanical force to osteoclast activation and bone
resorption.
In the present study, no significant difference in the
number of osteoclasts could be demonstrated between
the light- and heavy-force groups. Similar results were
reported in Beagle dogs (40) and in Wistar rats (41). TAD-
DEI et al. (42) found a significant increase in osteoclast
number with increasing force magnitude at 6 d, which
was not investigated in the current study. Conversely,
TOMIZUKA et al. (43), using repelling magnets to deliver
orthodontic force, found that the osteoclast number was
higher in the lighter force group at 1 and 3 d with no sig-
nificant difference between the two force groups at 7 d.
It is worth mentioning that the osteoclast activity may be
reflected in the count of TRAP-positive osteoclasts,
unlike caspase-3 immunostaining for osteocyte apopto-
sis, as cell apoptosis is an all-or-none event.
This rat model of orthodontic tooth movement
allowed us to study osteocyte apoptosis in vivo and to
correlate this event to different magnitudes of force in
an attempt to further our understanding of the impor-
tance of the apoptotic process along the pathway start-
ing from the application of mechanical stress to bone
resorption and orthodontic tooth movement. The
increase in number of apoptotic osteocytes corroborates
the results of previous studies of the incidence of osteo-
cyte apoptosis following mechanical stress and preced-
ing osteoclast recruitment. Inability to demonstrate an
effect of different force magnitude to osteocyte apopto-
sis might suggest that osteocyte apoptosis is not a
direct determinant of the biological reaction to mechan-
ical stress. This model of mechanical loading can be
extrapolated judiciously to mechanical loading models
other than orthodontic tooth movement. A caspase-3
knockout rat model could clarify the doubts cast by
other studies questioning the role of caspase-3 in osteo-
cyte apoptosis during orthodontic tooth movement and
could further test a direct effect of mechanical loading
on osteocyte apoptosis.
Possible limitations of this study include the classical
problem in rat studies that a normal physiologic tooth
position is transformed into an abnormal one, whereas
clinically the orthodontist strives to do the opposite
(44). Hence, despite offering better control of force
magnitude, time, and duration, the experimental model
is still different from the clinical situation. In addition,
histological sections are still two-dimensional represen-
tations of a three-dimensional tissue reaction. Although
efforts are made to take multiple sections to obtain a
realistic estimate of the quantity studied, it falls short
of a volumetric representation of the tissue reaction.
Optical projection tomography is promising in this
Orthodontically induced osteocyte apoptosis 9
regard (45). Moreover, it may be recommended to
employ further procedures to reduce the incidence of
appliance breakage encountered in the study (38).
According to the findings of the current study, osteo-
cyte apoptosis has been demonstrated to increase under
orthodontic loading as detected by caspase-3. However,
this study provided no support for the claim that
orthodontic force magnitude had an effect on osteocyte
apoptosis, at least in the range of force magnitudes
tested.
Conflicts of interest – The author and co-authors have no conflicts
of interest to declare.
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