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British Journal of Oral and Maxillofacial Surgery xxx (2016) xxx–xxx

Experimental model of osteonecrosis of the jaw in rats

treated with zoledronic acid
M.E. Kolpakova a,b,∗ , A.A. Zubarevɑ a , T.D. Artamonova a , E.K. Lisovskaya a , S.G. Chefu a,b ,
O.D. Yagmurov a , A.I. Yaremenko a , T.D. Vlasov a,b
a First I.P. Pavlov State Medical University, 197022, Lev Tolstoy Str., 6/8, St. Petersburg, Russian Federation
b Almazov North-West Federal Medical Research Centre, Akkuratov Str., 2, St. Petersburg, 197341, Russian Federation

Accepted 9 October 2016

Abstract

We have examined the development of medication-related osteonecrosis of the jaws (MRONJ) in rats with no previous accumulation of
zoledronic acid in the mandible. Ten male Wistar rats (weight 350–400 g) were anaesthetised with chloral hydrate 450 mg/kg intraperitoneally
and the first and second mandibular molars on the left side were extracted. The five experimental rats were given six injections of zoledronic acid
0.18 mg/kg over the next four weeks (total dose 1.08 mg/kg). Two injections were given at once as an intravenous bolus injection (0.36 mg/kg).
Then rats were given 4 injections (0.18 mg/kg) with 1 week interval over the next four weeks, after which they observed for a further four
weeks. The five control rats were injected with saline. At the end of the eighth week, the animals were killed by asphyxiation in a carbon
dioxide chamber, and their bone structure was visualised using cone-beam computed tomography (CT) and Galaxis software. We then studied
the mandibles histopathologically to investigate the incidence of necrosis and infiltration of inflammatory cells. The cone-beam CT images
in the experimental group showed deficiencies in the bone structure in the extracted molar area of the lower alveolar ridge. The histological
findings in the mandibles of the group given zoledronic acid showed necrosis and infiltration of inflammatory cells, which were not present
in the control group. We conclude that the immediate effect of zoledronic acid on the bone tissue during regeneration is an important factor
in the development of MRONJ, in addition to the previously reported effects of the duration of treatment with zoledronic acid.
© 2016 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Keywords: jaw; tooth extraction; zoledronic acid; bisphosphonates; osteonecrosis; MRONJ

Introduction According to data from the American Association of Oral

Bisphosphonates (including zoledronic acid) have been used
in the treatment of various diseases for over 40 years.1–3
∗ Corresponding author at: First I.P. Pavlov State Medical University,
Almazov North-West Federal Medical Research Centre.
E-mail addresses: patho@yandex.ru (M.E. Kolpakova),
a.zubareva@bk.ru (A.A. Zubarevɑ), tany0207@rambler.ru
(T.D. Artamonova), katyrina08@mail.ru (E.K. Lisovskaya),
chefusveta@yandex.ru (S.G. Chefu), oraz.yagmurov@gmail.com
(O.D. Yagmurov), ayaremenko@me.com (A.I. Yaremenko),
tvlasov@yandex.ru (T.D. Vlasov).
and Maxillofacial Surgeons the most important use of zole-
dronic acid, a bisphosphonate, is as a therapeutic agent for
conditions associated with malignancies, including hypercal-
caemia in malignancy, and bony metastases of tumours such
as breast, prostate, and lung cancer. It is usually given as an
intravenous injection. However, owing to possible adverse
effects (such as osteonecrosis of the mandible) it must be
given with caution.4 Randomised studies have shown that
the duration of treatment with zoledronic acid is one of the
main risk factors for osteonecrosis, the risk being 0.5% over a
one-year treatment period, 1.0% over a two-year period, and

http://dx.doi.org/10.1016/j.bjoms.2016.10.006
0266-4356/© 2016 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Kolpakova ME, et al. Experimental model of osteonecrosis of the jaw in rats treated with zoledronic
acid. Br J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.bjoms.2016.10.006
YBJOM-5031; No. of Pages 4
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2 M.E. Kolpakova et al. / British Journal of Oral and Maxillofacial Surgery xxx (2016) xxx–xxx

1.3% over a three-year period.5 Different types of trauma to
the oral cavity, including extraction of teeth,6,7 are also risk
factors for osteonecrosis in the context of a history of treat-
ment with bisphosphonates -for example, medication-related
osteonecrosis of the jaw (MRONJ).
We hypothesised that trauma to the mandible combined
with an intake of zoledronic acid is an important factor, so
we examined the likelihood of the development of MRONJ
in the mandible after an injury such as extraction of a tooth,
with no previous accumulation of zoledronic acid but with
simultaneous injection of the drug, in rats.
Material and methods
All animal experiments were approved by the ethics commit-
tee of the Almazov North-West Federal Medical Research
Centre in St. Petersburg, Russian Federation, and were done
in accordance with the UK Animals (Scientific Procedures)
Act, 1986, and the National Institutes of Health Guide for
the Care and Use of Laboratory Animals (NIH Publications
No. 8023, revised 1978). The animals were maintained on
a 12 hour light/dark cycle and were provided with food and
free access to water. Male Wistar rats (weight, 350–400 g)
were anaesthetised with chloral hydrate 450 mg/kg intraperi-
toneally and had the first and second left mandibular molars
removed with HULL Tooth Forceps DI030R#101 (Aesculap,
Inc., Central Valley, PA, USA).
A serial number was assigned to each animal. After the
extraction of mandibular molars, the rats in the experimental
group (n = 5) were given zoledronic acid (Rezorba, Pharm-
synthez, St. Petersburg, Russia) 0.36 mg/kg (1 ml) as an
intravenous bolus into the tail vein. Over the next four weeks,
the rats in this group were given six further injections of
zoledronic acid 0.18 mg/kg (total dose: 1.08 mg/kg). Two
injections were given at once as an intravenous bolus injection
(0.36 mg/kg). Then rats were given 4 injections (0.18 mg/kg)
with 1 week interval over the next four weeks, after which
they observed for a further four weeks. Rats in the control
group (n = 5) had a similar operation, but were subsequently
injected with saline solution. At the end of the eighth week,
the animals were killed by asphyxiation in a carbon diox-
ide chamber. Afterwards, their bone structure was visualised
using cone-beam computed tomography (CBCT; Galileos,
Sirona Dental, Salzburg, Germany) and Galaxis software
(Sirona Dental). The cone-beam technique involves a single
◦
360 scan in which the source of the beam and a recipro-
cating area detector move synchronously around the object.
We obtained high-contrast images of the bones, and evalu-
ated the measured volume of the injury based on three single
3-dimensional measurements. Cone-beam CT provides cor-
related axial, coronal, and sagittal perpendicular images for
evaluation of pathological conditions of the mandible. The
following variables were used for the scan: radiation exposure
setting 0.07 mSv; tube voltage 80 kV; current intensity 4 mA;
duration of scan 14 seconds; thickness of scan 0.15 mm.
We then studied the mandible histopathologically to assess
the amounts of necrosis, infiltration of inflammatory cells,
and tissue regeneration. After the animals had been killed,
fragments of the mandible 1 cm × 1 cm in size were placed
in a 4% paraformaldehyde solution for 24 hours at 4 ◦ C,
followed by decalcification with 10% ethylenediaminete-
traacetic acid disodium salt (Trilon B) for four weeks.
Tissue blocks were removed and embedded in paraffin at
a temperature of 56 ◦ C. Paraffin sections 5 ␮m thick were
cut longitudinally and stained with haematoxylin and eosin
(Sigma-Aldrich, St Louis, MO, USA). The stained sections
were examined under a light microscope with 20× objective
lenses, and images were obtained using a digital camera (DP-
2 BSW; Olympus, Tokyo, Japan).
Statistical analysis
Analyses were made with the aid of IBM SPSS software
(version 20, IBM Corp, Armonk, NY, USA). The significance
of differences between the groups was assessed using the
Mann-Whitney U test. Probabilities of < 0.01 were accepted
as significant.

Table 1
Volume of bony defect in the control and experimental groups.
Variable (mm) Control Mean (SD) value

Animal 1 Animal 2 Animal 3 Animal 4 Animal 5

Length 1.35 (0.34) 2.67 (0.03) 1.39 (0.24) 2.12 (0.07) 2.14 (0.06) 1.93 (0.56)
Width 0.92 (0.09) 1.67 (0.05) 1.06 (0.11) 1.57 (0.04) 0.84 (0.01) 1.21 (0.38)
Depth 1.42 (0.29) 1.41 (0.04) 1.77 (0.36) 1.54 (0.04) 0.80 (0.01) 1.39 (0.36)

Variable (mm) Experimental Mean (SD)value

Animal 6 Animal 7 Animal 8 Animal 9 Animal 10

Length 9.59 (0.19)* 5.62 (0.25)* 8.69 (0.30)* 7.20 (0.05)* 7.88 (0.14)* 7.80 (1.50)*
Width 1.81 (0.12) 2.22 (0.05)* 1.79 (0.16) 2.20 (0.09)* 1.89 (0.11) 1.98 (0.21)
Depth 1.81 (0.12) 2.45 (0.11)* 3.66 (0.31)* 2.61 (0.06)* 3.76 (0.04)* 2.86 (0.83)*
Volume of bony defect (mm3 ) 31.42 30.57 56.93 41.34 55.99 43.25 (14.87)*
∗ p = 0.0076

Please cite this article in press as: Kolpakova ME, et al. Experimental model of osteonecrosis of the jaw in rats treated with zoledronic
acid. Br J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.bjoms.2016.10.006
YBJOM-5031; No. of Pages 4
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Fig. 1. Cone-beam computed tomographic scans of rats’ mandibles (control

group). The extracted molar area is denoted by an arrow (horizontal view).

Fig. 3. Histological sections showing the healing process in the mandible of

the control group (injected with saline solution). (Haematoxylin and eosin,
original magnification x 200).

Fig. 2. Cone-beam computed tomographic scans of rats’ mandibles (experi-

mental group). Extracted molar area shows osteonecrosis (arrow) (horizontal
view).

Results

There were no changes in the cone-beam CT images of the

bone structure of the lower alveolar ridge in the control group
(Fig. 1). The structure of the mandible was homogeneous,
and the extraction socket had smooth, well-delineated edges.
The lamina of the cortical bone had not been destroyed and Fig. 4. Histological sections of bone in the area of tooth extraction from the
the mean (SD) volume of the mandibular defects was 3.45 experimental group injected with zoledronic acid (total dose 1.08 mg/kg).
Note the presence of necrotic areas and infiltration of inflammatory cells.
(2.50) mm3 (Table 1). (Haematoxylin and eosin, original magnification x 200).
The images in the experimental group showed deficien-
cies in the bone structure in the extracted molar area of the
lower alveolar ridge (Fig. 2). The bone also had a heteroge-
neous structure and showed sequestration and destruction of
the cortical layer. The mean (SD) volume of the mandibular
defects in the experimental group was 43.25 (14.87) mm3 ,
and there were significant differences between the control
and experimental groups (p <0.01) (Table 1).

Effect of zoledronic acid on the mandible

The areas from which the teeth had been extracted showed
findings typical of local tissue necrosis. The histological find-
ings in the mandibles of the experimental group (which had
been given zoledronic acid) differed from those in the con-
trol group (Fig. 3). Lesions developed only in rats treated
with zoledronic acid, and these lesions were characterised by Fig. 5. Histological sections of bone in the area of tooth extraction from the
experimental group injected with zoledronic acid (total dose: 1.08 mg/kg).
necrosis and infiltration of inflammatory cells. Representa- Note the presence of necrotic areas and the fibrous structure of the osteons.
tive histological images from rats treated with zoledronic acid (Haematoxylin and eosin, original magnification x 200).
showed that the osteons had a fibrous structure (Figs. 4 and 5).

Please cite this article in press as: Kolpakova ME, et al. Experimental model of osteonecrosis of the jaw in rats treated with zoledronic
acid. Br J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.bjoms.2016.10.006
YBJOM-5031; No. of Pages 4
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Discussion
In 2003 Marx et al,8 and in 2004 Ruggiero et al,9 published
studies on osteonecrosis that developed during treatment of
cancer with bisphosphonates, but even after more than 10
years, this topic is still not well understood. Do bisphos-
phonates affect the mineral metabolism of bones, and is the
accumulation of zoledronic acid in the bone a risk factor for
the development of osteonecrosis?
Experimental models of osteonecrosis usually mimic the
clinical picture associated with the continuous intravenous
use of bisphosphonates followed by extraction of teeth. In
previous experimental studies that involved histological eval-
uation of osteonecrosis, the period over which zoledronic
acid was given before extraction of the teeth varied from
three weeks10 to four weeks,11 and was even as high as eight
weeks.12 We did not give bisphosphonates before extraction
of the teeth, but obtained results similar to those obtained by
the other authors.13 We found that giving zoledronic acid at
the same time as extracting the teeth led to the development
of osteonecrosis.
Despite the small sample, our results are unidirectional
and show consistent development of MRONJ within the
study group. Because the rat’s mandible is characterised by
faster metabolism of bone than that of humans, which is
directly related to the high functional loads on this bone,
our study cannot be applied to clinical practice at this time.
Nevertheless, the fact that zoledronic acid has an immediate
and obvious influence on the development of osteonecrosis
deserves further study.
We conclude that this result at least proves that prolonged
exposure to bisphosphonates is not the only risk factor for
osteonecrosis, and the immediate effect of zoledronic acid on
the regeneration of bone is an important factor for the devel-
opment of MRONJ, together with the previously reported
effects of the duration of treatment with zoledronic acid.
Conflict of interest
We have no conflict of interest.
Ethics statement
All animal experiments were approved by the Ethics Com-
mittee at the Medical Research Centre and were carried out
in accordance with the UK Animals (Scientific Procedures)
Act, 1986 and the associated the National Institute of Health
Guide for the Care and Use of Laboratory Animals (NIH
Publications No. 8023, revised 1978).
Please cite this article in press as: Kolpakova ME, et al. Experimental model of osteonecrosis of the jaw in rats treated with zoledronic
acid. Br J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.bjoms.2016.10.006
Acknowledgements
The authors are grateful to Professor M. Galagudza for his
helpful suggestions during the preparation of the manuscript.
References
1. Ali-Erdem M, Burak-Cankaya A, Cemil-Isler S, et al. Extraction
socket healing in rats treated with bisphosphonate: animal model for
bisphosphonate-related osteonecrosis of jaws in multiple myeloma
patients. Med Oral Patol Oral Cir Bucal 2011;16, e879-83.
2. Jee JH, Lee W, Lee BD. The influence of alendronate on the healing
of extraction sockets of ovariectomized rats assessed by in vivo micro-
computed tomography. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 2010;110, e47-53.
3. Kobayashi Y, Hiraga T, Ueda A, et al. Zoledronic acid delays wound heal-
ing of the tooth extraction socket, inhibits oral epithelial cell migration,
and promotes proliferation of hydroxyapatite of oral bacteria, with-
out causing osteonecrosis of the jaw in mice. J Bone Miner Metab
2010;28:165–75.
4. Major P, Lortholary A, Hon J, et al. Zoledronic acid is superior to
pamidronate in the treatment of hypercalcemia of malignancy: a pooled
analysis of two randomized, controlled clinical trials. J Clin Oncol
2001;19:558–67.
5. Henry DH, Costa L, Goldwasser F, et al. Randomized, double-blind study
of denosumab versus zoledronic acid in the treatment of bone metastases
in patients with advanced cancer (excluding breast and prostate cancer)
or multiple myeloma. J Clin Oncol 2011;29:1125–32.
6. Barba-Recreo P, Del Castill Pardo de Vera JL, García-Arranz M, et al.
Zoledronic acid-related osteonecrosis of the jaws. Experimental model
with dental extractions in rats. J Craniomaxillofac Surg 2014;42:744–50.
7. Jabbour Z, El-Hakim M, Henderson JE, et al. Bisphosphonates inhibit
bone remodeling in the jaw bones of rats and delay healing following
tooth extractions. Oral Oncol 2014;50:485–90.
8. Marx RE. Pamidronate (Aredia) and zoledronate (Zometa) induced avas-
cular necrosis of the jaws: a growing epidemic. J Oral Maxillofac Surg
2003;61:1115–7.
9. Ruggiero SL, Mehrotra B, Rosenberg T, et al. Osteonecrosis of the jaws
associated with the use of bisphosphonates: a review of 63 cases. J Oral
Maxillofac Surg 2004;62:527–34.
10. Aghaloo TL, Kang B, Sung EC, et al. Periodontal disease and bisphos-
phonates induce osteonecrosis of the jaws in the rat. J Bone Miner Res
2011;26:1871–82.
11. Silva PG, Ferreira Junior AE, Teofilo CR, et al. Effect of different doses of
zoledronic acid in establishing of bisphosphonate-related osteonecrosis.
Arch Oral Biol 2015;60:1237–45.
12. Sakaguchi O, Kokuryo S, Tsurushima H, et al. Lipopolysaccharide aggra-
vates bisphosphonate-induced osteonecrosis in rats. Int J Oral Maxillofac
Surg 2015;44:528–34.
13. Dayisoylu EH, Üngör C, Tosun E, et al. Does an alkaline environment
prevent the development of bisphosphonate-related osteonecrosis of the
jaw? An experimental study in rats. Oral Surg Oral Med Oral Pathol
Oral Radiol 2014;117:329–34.