Journal of Equine Veterinary Science 35 (2015) 577583

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Journal of Equine Veterinary Science

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Original Research

Levels of Cytokines and Matrix Metalloproteinases 2 and 9

in the Synovial Fluid of Osteoarthritic Horses Treated With
Pamidronate
Emilio A. De Simone a, Gustavo Perrone b, Nicols Caggiano a, Yael Lastra a, Florencia Rubatino a,
Julieta Daz a, Araceli Ferretto a, Cristian Montes de Oca a, Emilio Roldn c,
Mara Angelina Chiappe Barbar a, *
a

Department of Animal Physiology, School of Veterinary Sciences, University of Buenos Aires, Autonomous City of Buenos Aires, Argentina
Department of Equine Production, School of Veterinary Sciences, University of Buenos Aires, Autonomous City of Buenos Aires, Argentina
c
Gador S.A, Autonomous City of Buenos Aires, Argentina
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 30 October 2014
Received in revised form 12 March 2015
Accepted 25 March 2015
Available online 1 April 2015

The aim of this study was to evaluate the effect of pamidronate on the clinical score and
the secretory prole of inammatory biomarkers (interleukin [IL]-6, tumor necrosis factor
alpha [TNF-a], matrix metalloproteinase [MMP]-2, and MMP-9) in the synovial uid in
clinically healthy horses and in horses with joint disease. Healthy horses and horses with
joint symptoms were examined and subjected to a standardized clinical evaluation of the
locomotor system. The clinical condition was evaluated by a global score. Matrix metalloproteinases 2 and 9 were measured by gel zymography. The concentration of cytokines
(IL-6 and TNF-a) in synovial uid was determined by enzyme linked immunosorbent assay
(ELISA). Pamidronate treatment signicantly improved the clinical condition of horses
with osteoarthritis (OA). Values of IL-6 (pg/mL) were similar (ns) in the healthy control
group (102.2
26.94) and at day 3 of treated (TD) group (113.9
18.33). Tumor necrosis
factor alpha level, at day 3 of treatment, was signicantly lower in TD groups than in
untreated osteoarthritis. Treated group registered a fast increase in MMP-9 activity but till
days 21 and 60 it was not detectable. No signicant differences were found in the MMP-2
activity between groups. We concluded that treatment with pamidronate has a benecial
effect on the clinical score of horses with OA and can reduce proinammatory cytokines
(IL-6 and TNF-a) and MMP-9 at different stages after treatment.
2015 Elsevier Inc. All rights reserved.

Keywords:
Horse
Pamidronate
Cytokine
Matrix metalloproteinase
Osteoarthritis

1. Introduction
Osteoarthritis (OA)
evolves to painful and
arthritis usually occurs
dietary imbalances,

is an inammatory disease that

degenerative joint damage. Osteoas a result of physical overtraining,
or growth disordersdincluding

* Corresponding author at: Mara Angelina Chiappe Barbar, Department of Animal Physiology, School of Veterinary Sciences, University of
Buenos Aires, Chorroarn 280, Ciudad Autnoma de Buenos Aires
C1427CWO, Argentina.
E-mail address: mach@fvet.uba.ar (M.A.C. Barbar).
0737-0806/$ see front matter 2015 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jevs.2015.03.194

osteochondrosisdduring development [1,2]. Osteoarthritis

is one of the major causes of economic loss in sport horses
because it results in either temporary or permanent, premature retirement from sports competitions and racing [3].
The aforementioned risk factors lead to the occurrence of
repeated and incompletely resolved articular microtrauma
that maintain and increase the severity of the inammatory
process. Furthermore, an abnormal maturation of the
cartilage might result in the formation of cartilage aps and
intraarticular bone fragments that can predispose to articular damage [2]. Osteoarthritis is characterized by joint
damage that affects the articular cartilage, the adjacent

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E.A. De Simone et al. / Journal of Equine Veterinary Science 35 (2015) 577583

subchondral bone, and the synovial membrane. Pain is

another important factor associated with the inammatory
process leading to progressive loss of joint function and
turning into a performance-limiting factor.
During the onset of OA, the changes in the properties of
normal cartilage impact on subchondral bone, causing an
increase in the resorptive activity of osteoclasts and bone
turnover [4]. In addition, the production of anabolic factors
by chondrocytes decreases, together with an increase in the
release of proteases and other catabolic factors involved in
joint and bone damage [5]. Vascular changes and upregulation of inammatory cytokines and nitric oxide production in the subchondral region and synovial uid are also
observed in OA [6,7].
During the initial phase of OA, macrophages and neutrophils participate in the cascade of inammatory
response by secreting proinammatory cytokines, tumor
necrosis factor alpha (TNF-a), and interleukin (IL)-6 [811].
In turn, the inammatory cytokines induce the release of
matrix metalloproteinases (MMPs) [4,12], which are known
to be involved in articular cartilage degradation [13].
Tumor necrosis factor alpha is a key cytokine in the inammatory process and is known to increase vascular
permeability by inducing the endothelium to express
adhesion molecules and cellular migration factors that
promote leukocyte diapedesis [14]. Moreover, IL-6 is
involved in the degradation of proteoglycans in the articular cartilage [15].
Although MMPs are zinc-dependent endopeptidases
involved in tissue repair, they are also associated with the
development of arthritis in humans [16] and in horses [17].
Matrix metalloproteinases 2, 3, 9, and 13 are especially
involved in cartilage matrix degradation [18].
Many different pharmaceutical products have been
used to treat OA with the aim of reducing associated
inammation and pain; however, these drugs have
adverse effects on the extracellular matrix and bone
metabolism. For example, corticosteroids are widely used
in the management of equine OA for their highly effective
anti-inammatory properties but are associated with
enhancement of cartilage proteolysis, subchondral bone
resorption, microfractures, and direct inhibition of osteoblast function. The aim of this article was to evaluate the
effects of pamidronate in horses with OA. Although
tiludronate was the rst bisphosphonate licensed to treat
navicular disease in horses [19], pamidronate, an aminobisphosphonate, is widely used in human medicine for the
treatment of metabolic bone disease. Information on the
anti-inammatory effects of these drugs in equines is
scarce [20].
Pamidronate belongs to the family of aminobisphosphonates and is extensively used for the palliative
treatment of cancer metastasis due to its osteoclast inhibitory effect [2124]. Aminobisphosphonates have a more
potent antiresorptive effect compared with other
bisphosphonates. Pamidronate has been used in the treatment of osteolysis associated with Pagets disease [25] and
is known to have analgesic effects in patients suffering from
cancer and other related disorders. In addition,
bisphosphonates can inhibit apoptosis of osteocytes and
osteoblasts and induce the proliferation of osteoblasts

[20,26,27]. Another mechanism by which pamidronate inhibits bone resorption is by stimulating osteoblast inhibitory activity on osteoclasts [28]. Pamidronate acts by
inhibiting the mevalonate pathway [29]. Because of its
analgesic effect and its antiresorptive capacity, pamidronate could be an alternative treatment for OA [30].
Bisphosphonates might have anti-inammatory properties, particularly during the onset of arthritis [31]. The
aim of this study was to evaluate the effect of pamidronate
on the clinical score and the secretory prole of inammatory biomarkers (IL-6, TNF-a, MMP-2, and MMP-9) in
the synovial uid in clinically healthy horses and in horses
with joint disease.
2. Materials and Methods
2.1. Animals, Synovial Samples, and Experimental Design
At the beginning of the study, healthy horses and horses
with joint symptoms were examined and subjected to a
standardized clinical evaluation of the locomotor system. The
clinical condition was evaluated by a global score comprising
six items: (1) lameness degree (05), (2) tenderness (03),
(3) presence of pain under forced exion (03), (4) volume of
synovial uid extracted (03), (5) color of synovial uid (05),
and (6) synovial uid viscosity (04). The score was based
on the American Association of Equine Practitioners lameness score [32] with the addition of a wide evaluation
methodology focused in the tibiotarsal joint. This joint is an
important site of OA prevalence in jumping horses [33,34].
The following groups were considered: (1) control
group (n 8) clinically healthy young animals (24 years).
Young animals were chosen to be certain that since birth
they had no history of joint disease or disorders of bone
metabolism. The clinical score of this group was 4. (2)
Animals with joint disease that were treated (TD) (n 8,
with clinical score >5 at the beginning of treatment, aged
between 48 years). This group was evaluated and
analyzed at different stages: baseline or pretreatment
group (TD0), day 3 (TD3), day 10 (TD10), day 21 (TD21), and
day 60 (TD60). Animals included in TD group were animals
with clinical signs and clinical history of chronic recurrent
episodes of active OA with poor outcome to conventional
treatment modalities.
Radiographic analysis of both tibiotarsal joint was performed in all animals included in the experiment, control,
and TD groups. TD group had radiographics alterations of
articular cartilage in the joints and subchondral bone
resorption.
On days 0 and 9, horses with joint disease were treated
with 90 mg of IV disodium pamidronate (Aminomux
90 mg, Gador). There is no detailed information of
pamidronate dosage and posology for horses in the bibliography; however, divided doses was recommended. In
this article, we used low doses of pamidronate (0.40.8 mg/
kg intravenously) administered in two doses 9 days apart
[20,35]. Synovial uid samples were obtained on days 0, 3,
10, 21, and 60 by sterile aspiration from the tibiotarsal joint.
Samples were centrifuged at 2,000g for 10 minutes, and the
supernatant was kept at 70 C. The nal clinical score in
the pamidronate-treated group was considered on day 60.

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579

2.3. Determination of IL-6 and TNF-a Levels

The concentration of cytokines (IL-6 and TNF-a) in
synovial uid was determined by commercial enzyme
linked immunosorbent assay (ELISA) kits (OptEIA BD Biosciences, San Diego, CA) as per manufacturers instructions.
Cytokines were analyzed in synovial uid because it has
previously been demonstrated that this biological uid is
more suitable than serum [4]. Cytokine levels were derived
from a standard curve. Undiluted samples were analyzed.
Reactions were elicited by adding TMB substrate solution
(Biosciences, San Diego, CA) and interrupted by adding 1M
sulfuric acid. Plates were read at 450 nm in a Rayto 2100C
microplate reader (China).
2.4. Statistical Analysis

Fig. 1. Clinical score (mean
standard deviation) for control, pretreated (TD0),
and treated at different stages (TD3, TD10, TD21, and TD60). TD0, TD3, TD10,
and TD21 differ signicantly vs. control (**P < .01) and vs. TD60 (*P < .05).

Data were expressed as mean
standard deviation.

Comparisons were performed using a nonparametric
analysis of variance (KruskalWallis test) followed by
Dunns posttest. Signicant differences were indicated as *P
< .05, **P < .01, and ***P < .001.

2.2. Gel Zymography

The gelatinolytic activities of MMP-2 and MMP-9 were
measured by zymography as described by Gruber et al [36].
All samples were analyzed for protein concentration by the
Bradford method [37]. Aliquots of 100 mg of each sample
were briey mixed with a buffer and loaded on a 10%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
with 0.2% porcine skin gelatin (Sigma). After electrophoresis, gels were washed with 50 mM Tris-HCl pH 7.5 plus
2.5% Triton X-100 for 45 minutes and then with a solution
of 5 mM CaCl2, 1 mM ZnCl2, 50 mM TrisHCl pH7.5 plus 2.5%
Triton X-100 pH 7.5 for another 45 minutes. Finally, gels
were incubated with a solution containing 50 mM TrisHCl,
10 mM CaCl2, and 200 mM NaCl pH 7.5 at 37 C for 24 hours.
Gels were stained with 0.2% (wt/vol) Coomassie Brilliant
Blue R-250 for 2 hours and then bleached with decolorizing
solution (25% vol/vol isopropanol plus 10% vol/vol acetic
acid). Inhibition controls were performed by incubating
with 5 mmol/L EDTA. The densitometric study of the bands
generated by the gelatinolytic activity was done using the
Image J software (National Institutes of Health, MD).
Zymographic activity was expressed as a percentage of the
control group. Data for different gels were standardized
with the help of control samples. As MMP-9 is not always
present in synovial uid, we worked with MMP-9 data in
a qualitative scale (negative, lightly positive, and strong
positive).

3. Results
Pamidronate treatment signicantly improved the
clinical condition of horses with OA, as measured by the
clinical score. The comparative analysis showed that
treatment with pamidronate 60 days after bisphosphonates application signicantly lowered the clinical score in
animals with joint disease vs. TD0 animals (P < .05) and
that there was no signicant difference between the control group and the TD group at this time (Fig. 1; Table 1).
Cytokine levels were measured after synovial uid
extraction. Synovial values of IL-6 (pg/mL) were similar in the
healthy control group (102
26.94), the TD3 group (113.9

18.33), and TD60 group (78.87
26.17) (ns); however, TD0,
TD10, and TD21 groups had greater IL-6 values (P < .05 vs.
control; Fig. 2). Tumor necrosis factor alpha dropped
considerably by day 3 in the treated animals (TD3)
(6.44pg/ml
4.71) and was statistically signicant compared
with TD0 group (P < .05), but compared with the control
group (15.2 pg/mL
12.7), the difference was not statistically
signicant due to the wide scattering of data (Fig. 3).
Synovial MMP-2 levels showed no signicant differences between groups (Fig. 4). Matrix metalloproteinase 9
was scored as (1) absent, (2) slightly positive, and (3) strong

Table 1
Measured parameters (mean
SD) for control, pretreated (TD0), and treated at different stages (TD3, TD10, TD21, and TD60).
Parameters

Control

TD0

Clinical score
IL-6 (pg/mL)
TNF-a(pg/ml)
MMP-2 (% of control)

2.1
0.2
102.2
26.94
15.2
12.7
100
66

6.3
251.8
30.02
148

TD3

0.5a
61.98c
24.25d
45

8.2
113.9
6.44
107

TD10

0.5a
18.33
4.71
62

7.5
275
48.2
181

TD21
1.2a
20.73c
5.2e
116

Abbreviations: IL, interleukin; MMP, matrix metalloproteinase; SD, standard deviation; TNF, tumor necrosis factor.
No signicant differences were found in the MMP-2 activity between groups.
a
P < .01 vs. control.
b
P < .05 vs. TD0.
c
P < .05 vs. control, TD3, and TD60.
d
P < .05 vs. control and TD3.
e
P < .01 vs. control and TD3 group.

6.2
239.3
51.33
124

TD60

0.13a
71.25c
22.52e
6.6

3.8
78.87
24.43
120

0.3b
26.17
16.96d
42

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E.A. De Simone et al. / Journal of Equine Veterinary Science 35 (2015) 577583

Fig. 2. Interleukin 6 levels in synovial uid (mean
standard deviation)

from control, pretreated (TD0), and treated at different stages groups (TD3,
TD10, TD21, and TD60). Levels of TD0, TD10, and TD21 were signicantly
different vs. control, TD3, and TD60 group (*P < .05).

positive. The results in control group showed that only

three animals were slightly positive for MMP-9 (Fig. 5 and
Table 2). Only one animal of TD0 group had strong MMP-9
activity. Pamidronate seems to stimulate an acute increase
in synovial MMP-9 activity because six of eight animals of
TD3 group were positive (three slightly and three strong).
However, TD10 group only had a strong positive MMP-9
activity, and TD21 and TD60 had only negative MMP-9
activity.
4. Discussion
In this study, we observed that animals treated with
pamidronate showed a signicant improvement in the
clinical score compared with the TD0 group. The clinical
changes were characterized by a decrease in the signs of
inammation, less or no pain under forced exion, and
increased ability to work.

Fig. 3. Tumor necrosis factor alpha levels in synovial uid (mean
standard
deviation) for control, pretreated (TD0), and treated at different stages (TD3,
TD10, TD21 and TD60). TD10 and TD21 groups presented signicant differences from both the control and TD3 groups (**P < .01). TD0 and TD60
presented signicant differences vs. the control and TD3 groups (*P < .05).

Fig. 4. Matrix metalloproteinase 2 activities in joint synovial uid for control, pretreated (TD0), and treated at different stages (TD3, TD10, TD21, and
TD60). No signicant differences were found in the MMP-2 activity between
groups.

It has previously been observed that pamidronate can

reduce pain intensity and inammation in humans with
ankylosing spondylitis [31]. Although OA is generally
considered a degenerative gradual disorder, it can also be
considered an inammatory process.
In this study, no signicant differences were found in
MMP-2 activity between clinically healthy animals, TD0,
and TD animals. Pamidronate would seem to stimulate a
sharp increase in the activity of MMP-9, but at days 21 and
60, animals treated with pamidronate had an absence of
MMP-9 activity, which indicated that it could have a
benecial effect in the medium term. These ndings are in
line with previous results in which the treatment with
bisphosphonates inhibited MMP activity [38,39]. This
phenomenon will result in a lower degradation rate of the
extracellular matrix and the articular cartilage surface.
Furthermore, the low activity of MMPs in synovial uid
reduced leukocyte migration, inammation, and associated
pain. The latter observation is related to the therapeutic
prescription of pamidronate in humans, where this drug is
known to inhibit subchondral bone resorption, thus leading
to the amelioration of pain in cancer bone metastases [40].
Given that high osteoclast activity plays an important role
in the degradation of the adjacent subchondral bone and in
synovial inammation causing pain adjacent to subchondral bone [41], pamidronate-induced osteoclast
apoptosis will probably result in a decrease of bone turnover and the anti-inammatory-related effect.
Our results show that TNF-a levels in OA were signicantly decreased at day 3 of treatment and increased on
days 10, 21, and 60. Moreover, on day 3 in the TD group, IL-6
and TNF-a levels were similar to those in healthy animals.
Studies carried out in cultures of peripheral leukocytes
obtained from patients with osteoporosis treated with
pamidronate showed an increase in IL-6 and TNF-a production during the rst 24 hours after the rst dose [42].
Cancer patients treated with IV pamidronate showed an
increase in IL -6 levels that lasted 7 days [43]. These results
differ from other published studies performed in rheumatoid arthritis patients, whose levels of IL -6 and TNF-a were

E.A. De Simone et al. / Journal of Equine Veterinary Science 35 (2015) 577583

581

Fig. 5. Gelatin zymography for detection of MMP-9 activity in a standard sample (line A), negative sample (B and E), slightly positive (D and F), and strong
positive (C). MMP, matrix metalloproteinase.

signicantly increased for 30 days after the treatment with

pamidronate [44] consistent with our own results at day 21.
Other authors suggest an anti-inammatory effect of
pamidronate evidenced by decreased IL-6 levels [45]. We
have also observed decreased levels of IL-6 after pamidronate treatment but only on days 3 and 60. In this study, the
increased TNF-a levels observed on days 10 and 21 after
treatment might be related to an exacerbation of the inammatory process, which could be benecial for the
resolution of chronic arthritis [46]. Tumor necrosis factor
alpha is a critical factor for NFkappa B expression which, in
turn, is responsible for MMP-9 expression [47,48]. In this
study, it could be assumed that MMP-9 activities in synovial uid did not increase after day 21 as a consequence of
the increased TNF-a levels on days 10 and 21; however,
further studies are required to conrm this hypothesis.
Many studies have focused on the role of certain MMPs
(MMP-1, MMP-3, and MMP-13) with collagenase activity,
whereas in this study, we have focused on the gelatinases
(MMP-2 and MMP-9), which play a major role in the
degradation of articular cartilage. Clodronate is a rst generation bisphosphonate, which acts as an inhibitor of MMP1 puried from human broblasts [49]. In vitro studies have
shown that alendronate could also inhibit leukocyte
migration by reducing MMP-2 activity [50,51]. Moreover,
the administration of alendronate can lead to the inhibition
of MMP-13 in patients with rheumatoid arthritis [52].
Osteoclasts, macrophage linage cells, are a major source
of MMP-9 in subchondral bone in OA joints. The increased
number and activity of osteoclasts in OA induce the
appearance of subchondral bone resorption areas and are
the reason for increased plasma activity of MMP-9 [53,54].
Pamidronate inhibits the MMP-9 activity through the induction of osteoclast apoptosis. The activity of MMP-9 has
been associated with rheumatoid arthritis and some other
types of OA in humans [55]. In vitro studies have shown an
inhibitory effect of alendronate on MMP-9 activity due to
its chelating effect [56]. In this article, we registered a fast
increase in MMP-9 activity, but it was not detectable until
days 21 and 60. The latter phenomenon could be important
because the inhibition of MMP-9 is associated with the
prevention of articular lesions.
Table 2
Number of animals with negative, slightly positive, and strong positive
gelatinolytic MMP-9 activity in synovial uid in control, TD0, TD3, TD10,
TD21, and TD60 groups.
MMP-9

Control

TD0

TD3

TD10

TD21

TD60

Negative
Slightly positive
Strong positive

5/8
3/8
d

7/8
d
1/8

2/8
3/8
3/8

7/8
d
1/8

8/8
d
d

8/8
d
d

Abbreviation: MMP, matrix metalloproteinase.

Potent MMP inhibitors such as doxycycline delay the

progression of OA lesions [57]. Furthermore, it has been
observed that glucosamine combined with chondroitin
sulfate inhibits MMP-9 activity and the joint damage
associated with adjuvant-induced arthritis in rats [58].
According to Clegg [59], MMP-9 activity is increased in
joint pathologic processes. In this article, the absence in the
activity of MMP-9 after 21 and 60 days of treatment suggests that bisphosphonates may be a safe alternative to
corticoids and other anti-inammatory therapies for the
treatment of OA.
5. Conclusions
We concluded that pamidronate treatment has positive
effect in the clinical score of horses with OA and reduces
proinammatory cytokines (IL-6 and TNF-a) and MMP-9
after treatment. Recent advances in the understanding of
molecular events in OA allow us to use novel drugs in this
disease. However, the real benet of these treatments in
horses should be further studied.
Acknowledgments
The authors thank UBACYT 20020120100092BA for
nancial support. The authors declared a conict of interest
(such as dened by JEVS policy). E.R. is employed in Gador
SA, the company that provides pamidronate (Aminomux).
All other authors have stated that they have no conict of
interest. M.A.C.B and E.A.D.S. designed research, performed
research, analyzed data, and wrote article. M.A.C.B performed the statistical analysis. E.R. participated in designed
research. G.P. and C.M.D.O performed eld research and
acquisition of clinical data. N.C., Y.L., F.R., J.D., and A.F. performed laboratory activity.
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