European Journal of Pharmacology 794 (2017) 8–14

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

The combined therapy with chondroitin sulfate plus glucosamine sulfate or

chondroitin sulfate plus glucosamine hydrochloride does not improve joint
crossmark
damage in an experimental model of knee osteoarthritis in rabbits
Jorge A. Roman-Blas, Aránzazu Mediero, Lidia Tardío, Sergio Portal-Nuñez, Paula Gratal,
⁎
Gabriel Herrero-Beaumont , Raquel Largo
Bone and Joint Research Unit. IIS-Fundación Jiménez Díaz UAM and Cooperative Research Thematic Network on Aging and Frailty (RETICEF), Madrid,
Spain

A R T I C L E I N F O A BS T RAC T

Keywords: Osteoarthritis is the most common chronic joint disorder especially during aging. Although with controversies,
Osteoarthritis glucosamine, both in its forms of sulfate and hydrochloride, and chondroitin sulfate are commonly employed to
Cartilage damage treat osteoarthritis. Due to the modest improve in the symptoms observed in patients treated with these drugs
Synovial inflammation alone, a formulation combining both agents has been considered. The discrepant results achieved for pain
Subchondral bone
control or structural improvement in osteoarthritis patients has been attributed to the quality of chemical
Chondroitin sulfate
Glucosamine sulfate
formulations or different bias in clinical studies. The current study has been designed to test the effects of two
different combined formulations with adequate pharmaceutical grade of these drugs in osteoarthritic joints, and
to explore the underlying mechanisms modulated by both formulations in different osteoarthritis target tissues.
Knee osteoarthritis was surgically induced in experimental rabbits. Some animals received the combined
therapy (CT)1, (chondroitin sulfate 1200 mg/day + glucosamine sulfate 1500 mg/day), or the CT2 ((chondroitin
sulfate 1200 mg/day + glucosamine hydrochloride 1500 mg/day). Neither CT1 nor CT2 significantly modified
the cartilage damage or the synovial inflammation observed in osteoarthritic animals. Treatments were also
unable to modify the presence of pro-inflammatory mediators, and the synthesis of metalloproteinases in the
cartilage or in the synovium of osteoarthritic animals. Combined therapies did not modify the decrease in the
subchondral bone mineral density observed in osteoarthritic rabbits. Therapies of chondroitin sulfate plus
glucosamine sulfate or chondroitin sulfate plus glucosamine hydrochloride failed to improve structural damage
or to ameliorate the inflammatory profile of joint tissues during experimental osteoarthritis.

1. Introduction membrane contributes to this process as a pro-inflammatory cytokine

Osteoarthritis is the most common cause of morbidity and disability
in advanced middle aged and elderly wordwide. It is a chronic articular
disorder characterized by progressive cartilage destruction, bone repair
response and relevant synovial changes.
Synovitis and subchondral sclerosis have gained attention in
osteoarthritis pathology (Goldring and Goldring, 2010; Samuels
et al., 2008; Sellam and Berenbaum, 2010). Synovitis occurs in over
90% of patients with osteoarthritis (Roemer et al., 2011) and is a major
risk factor associated with both progression of cartilage loss and signs
and symptoms of disease (Sellam and Berenbaum, 2010). Indeed,
synovitis is associated with pain, disease severity and progression,
being therefore a potential target for therapy in osteoarthritis. Synovial
reservoir, but the mechanism underneath is still unclear. In turn,
subchondral bone sclerosis is associated with significant articular
cartilage destruction in the late stages of osteoarthritis (Dieppe,
1999; Goldring and Goldring, 2010). Recently, we have demonstrated
that an increased remodeling in the subchondral bone also aggravates
cartilage damage during early stages of osteoarthritis (Bellido et al.,
2010b; Calvo et al., 2007; Castañeda et al., 2010).
The goal of treatment for osteoarthritis are to reduce symptoms and
slow disease progression. Chondroitin sulfate and glucosamine, in its
forms of sulfate or hydrochloride, are natural compounds considered as
Symptomatic Slow Acting Drugs for osteoarthritis (SYSADOAS).
Although the use of these compounds cause a great deal of controversy,
chondroitin sulfate, glucosamine sulfate and glucosamine hydrochlor-

⁎
Correspondence to: Bone and Joint Research Unit, IIS-Fundación Jiménez Díaz, Avda. Reyes Católicos, 2, 28040 Madrid, Spain.
E-mail addresses: jaromanblas@gmail.com (J.A. Roman-Blas), aranzazu.mediero@quironsalud.es (A. Mediero), ltardio@fjd.es (L. Tardío), sportal@fjd.es (S. Portal-Nuñez),
paula.gratal@fjd.es (P. Gratal), gherrero@fjd.es (G. Herrero-Beaumont), rlargo@fjd.es (R. Largo).

http://dx.doi.org/10.1016/j.ejphar.2016.11.015
Received 19 July 2016; Received in revised form 8 November 2016; Accepted 9 November 2016
Available online 12 November 2016
0014-2999/ © 2016 Published by Elsevier B.V.
J.A. Roman-Blas et al.
ide are commonly employed to treat osteoarthritis (Henrotin et al.,
2014). In fact, international guidelines for the treatment of sympto-
matic knee osteoarthritis recommend the use of these products
especially in mild-to moderate osteoarthritis or as a step 1 medication
(Bruyère et al., 2016; Henrotin et al., 2014). Furthermore, these
compounds have shown to have anti-inflammatory effects when
administered at high doses both in experimental models and in in
vitro studies (Herrero-Beaumont et al., 2008; Largo et al., 2010, 2009;
Martínez-Calatrava et al., 2010). Glucosamine sulfate and chondroitin
sulfate also attenuated the increased subchondral bone turnover,
structural damage and mineralization in early stages of experimental
osteoarthritis (Wang et al., 2007).
Due to the modest improve in the symptoms observed in patients
treated with glucosamine sulfate or chondroitin sulfate, a formulation
combining both agents has been also considered for osteoarthritis,
aiming to achieve a synergistic effect ameliorating the pain and
improving joint function. Different clinical trials studying these com-
binations have shown discrepant results (Henrotin et al., 2014;
Hochberg et al., 2016; Román-Blas et al., 2016). These discrepancies
have been attributed to formulation differences, uneven quality of
products tested or other sponsor-dependent bias (Eriksen et al., 2014;
Herrero-Beaumont et al., 2007; Hochberg et al., 2016; Vlad et al.,
2007). However, the potential different effects of these combinations
on the different osteoarthritis target tissues, such as cartilage and
synovial synthesis of pro-inflammatory and structural mediators, are
still unknown.
Thus, in the present study, our aims were to compare the structural
effects of two different combined formulations, chondroitin sulfate plus
glucosamine sulfate and chondroitin sulfate plus glucosamine hydro-
chloride of high quality pharmacological profile, in a rabbit model of
osteoarthritis, and to explore the underlying mechanisms modulated by
both formulations in the main target tissues of the disease.
2. Material and methods
2.1. Experimental model of osteoarthritis in rabbits
In this study, 20 male New Zealand white rabbits (3.0 ± 0.3 kg body
weight) (Granja San Bernardo, Navarra, Spain) were used. Rabbits
were allowed to adapt to the facilities for 2 week before osteoarthritis
induction. Animals were then randomly assigned to four different
groups (n =5 each): 1. healthy rabbits (control); 2. rabbits with
experimental osteoarthritis (group OA); 3. rabbits with experimental
osteoarthritis that received the combined therapy 1 with their food
(group OA+CT1), consisting in chondroitin sulfate 1200 mg/day plus
glucosamine sulfate 1500 mg/day (Tedec-Meiji Farma, S.A. Alcalá de
Henares, Madrid, Spain); and 4. rabbits with experimental osteoar-
thritis that received the combined therapy 2 with their food (group OA
+CT2), consisting in chondroitin sulfate 1200 mg/day plus glucosa-
mine hydrochloride 1500 mg/day (Droglican, Bioibérica, Barcelona,
Spain). The doses of the drugs employed and the administration via
were chosen based on previously published data where a significant
therapeutic effect was observed (Herrero-Beaumont et al., 2008; Largo
et al., 2010, 2009). Two weeks after starting the treatments, destabi-
lization surgery was performed in OA and OA+CT groups, by cruciate
ligament transection and partial medial meniscectomy in both knees of
each rabbit as previously described (Bellido et al., 2010b; Martínez-
Calatrava et al., 2012). Animals were euthanized 12 weeks after the
osteoarthritis surgery with an overdose of pentobarbital. At this
moment, femoral condyles were removed and fixed in 4% buffered
paraformaldehyde, decalcified for 4 weeks in a solution made up of
10% formic acid plus 5% paraformaldehyde, and embedded in paraffin.
The cartilage from the medial zone of the tibias was carefully dissected
and immediately frozen for protein studies. Furthermore, synovial
membranes were removed and divided in two pieces, to be processed
for paraffin embedding or for molecular biology studies. All experi-
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European Journal of Pharmacology 794 (2017) 8–14
ments were performed in accordance with Spanish regulations and the
Guidelines for the Care and use of Laboratory Animals, as drawn up by
the National Institutes of Health (USA). The protocol was approved by
the Institutional Ethics Committee.
2.2. Histopathological damage in the experimental model
Tissue damage was evaluated by two independent, blinded obser-
vers in paraffin sections of the cartilage and the synovial membrane of
the rabbits. Femoral cartilage sections of 4 µm were stained with
hematoxylin-eosin (H-E) and Alcian blue, and the histopathological
alterations were evaluated using the Mankin's grading system (Mankin
et al., 1971) as previously published (Alvarez-Soria et al., 2008;
Martínez-Calatrava et al., 2012). The histopathological study for each
rabbit was carried out in the most damaged zone of the cartilage. A
partial score for each category of the Mankin scale (structure abnorm-
alities, cellularity, matrix staining and tidemark integrity) was attrib-
uted, and these scores were combined for each section. Synovial
histopathology was evaluated in H-E stained sections according to
the Krenn scale (Krenn et al., 2002), as previously described (Alvarez-
Soria et al., 2006; Martínez-Calatrava et al., 2012). Briefly, lining
hyperplasia, fibrovascular alterations at the interstitium, and tissue cell
infiltration were independently evaluated using 0–3-point subscales,
where 0 indicates absence, 1 mild, 2 intermediate and 3 strong. The
total score was obtained from the sum of partial grades with a
maximum total score of 9.
2.3. Subchondral bone mineral density (sBMD)
After death, tibias were carefully dissected and analysed in a
densitometer PIXImusTM (software v1.4; GE Lunar, Madison, WI,
USA), as previously described (M Bellido et al., 2010a). For this
purpose, tibias were positioned similarly in the corresponding tray,
maintaining tibial plateau perpendicularly to the tray surface during
the scanning. Subchondral BMD was measured in the corresponding
ROI (region of interest, cm2) positioned in parallel with tibial plateau,
in the subchondral bone of each tibia.
2.4. Western blot studies
Tissues were homogenized in liquid nitrogen, and total proteins
were extracted as previously described (Martínez-Calatrava et al.,
2012). Protein concentration was determined and subsequently,
20 μg of total protein from each tissue was resolved on 10% acryla-
mide-SDS gels. After transfer to polyvinylidene difluoride (PVDF)
membranes (Millipore, Molsheim, France), membranes were blocked
in 5% skimmed milk in PBS-Tween 20 for 1 h at room temperature and
incubated overnight at 4 °C with anti-interleukin (IL)-1β (1:500, Santa
Cruz Biotechnology, Dallas, TX, USA), cyclooxygenase (COX)-2 (1:500,
Santa Cruz Biotechnology), inducible nitric oxide synthase (iNOS)
(1:500, Affinity Bioreagents, Golden, CO, USA), metalloproteinase
(MMP)-1 (1:500, Chemicon Int, Temecula CA, USA), MMP-3
(1:2000, Chemicon Int) and MMP-13 (1:200, Calbiochem,
Darmstadt, Germany). Antibody binding was detected by enhanced
chemoluminescence using peroxidase-labelled secondary antibodies,
and the results were expressed as arbitrary densitometric units (AU).
Loading control was performed on 15% acrylamide- SDS gels by
employing EZBlue gel staining reagent (Sigma-Aldrich, Madrid,
Spain) (Prieto-Potín et al., 2013).
2.5. Statistical analysis
Statistical analyses were performed using SPSS version 19.0 soft-
ware for Windows (SPSS, Chicago, IL, USA), and results were
expressed as the mean ± standard error of mean (S.E.M).
Comparisons between multiple groups used Kruskal-Wallis tests with
J.A. Roman-Blas et al. European Journal of Pharmacology 794 (2017) 8–14

Fig. 1. Histopathology of the articular cartilage and the synovial membrane. A-D, alcian-blue stained sections of articular cartilage from OA+CT1 (A and B) and OA+CT2 (C and D). F-I,
Hematoxylin-eosin stained sections of synovial membranes from OA+CT1 (F and G) and OA+CT2 (H and I). E, Analysis of the total Mankin score in the groups studied. J, Analysis of the
global synovitis score employing the Krenn scale in the groups studied. The bars show the mean ± S.E.M. *P < 0.05 vs. control. N=9–10 samples per group. Control, osteoarthritic (OA),
osteoarthritic treated with combined therapy 1 (OA+CT1) and osteoarthritic treated with combined therapy 2 (OA+CT2) rabbits. The corresponding scale bar has been included in each
microphotograph.

Bonferroni correction of post hoc Mann–Whitney U tests. P < 0.05 was
considered significant.
3. Results
3.1. Histopathological assessment of articular cartilage and synovial
membrane
In order to assess whether these combined treatments were able to
modify the histopathological damage in the cartilage and synovial
membranes of the osteoarthritic rabbits, sections of internal femoral
condyles from right and left knees were stained and Makin score was
evaluated (Fig. 1A-E). The predominant histopathological findings in
untreated osteoarthritic rabbits were similar to those described in
patients with advanced osteoarthritis, such as cartilage clefts that
reached the inner zone, hypocellular cartilage together with frequent
cellular clones, moderate to severe reduction of the matrix staining
score and crossing of the tidemark by blood vessels. Cartilage lesions
observed in the OA+CT1 and OA+CT2 groups were similar to those
observed in OA group. As expected, the Mankin score was significantly
increased in OA group in comparison to control one (Fig. 1E). Neither
the combined therapy CT1 nor the combined therapy CT2 groups
showed significant differences in the Mankin score when compared to
OA animals (Fig. 1A-E).
Histopathologic analysis of the synovial membranes revealed
characteristic osteoarthritic lesions in untreated OA rabbits, including
hypercellularity, synovial stromal hyperplasia, increase angiogenesis
and extracellular matrix and increase on infiltrating cells in the
synovial stroma (Fig. 1F-I). No significant changes were observed in
any of the combined therapies applied in comparison with untreated
rabbits (Fig. 1F-J).
3.2. Pro-inflammatory cytokine presence in the cartilage and in the
synovial membrane
We studied if any of the combined therapies were able to modify the

10
J.A. Roman-Blas et al.
Fig. 2. COX-2, IL-1β, iNOS, MMP-1, MMP-3 and MMP-13 presence in the articular cartilage. A-F: Densitometric analysis of protein presence in the articular cartilage, assessed by
western-blot. G, representative western-blot images for protein detection in the articular cartilage. EZ Blue stained gel used as protein loading control is also shown. The bars show the
mean ± S.E.M. *P < 0.05 vs. control. N=9–10 samples per group. Control (CTR), osteoarthritic (OA), osteoarthritic treated with combined therapy 1 (OA+CT1) and osteoarthritic treated
with combined therapy 2 (OA+CT2) rabbits.
expression of pro-inflammatory mediators in the articular cartilage and
in the synovial membrane of the osteoarthritic joints. The significant
increase in the IL-1β presence observed in the articular cartilage of
osteoarthritic rabbits was not modified by any of the combined
therapies (Fig. 2A, G). COX2 synthesis was also increased in articular
cartilage of the osteoarthritis group (Fig. 2B, G) and none of the
combined therapies was able to significantly decrease COX2 levels
(Fig. 2B, G). Similar results were observed for iNOS in the articular
cartilage (Fig. 2C, G). The combined therapies were also unable to
modify IL-1β (Fig. 3A, F) and COX2 (Fig. 3C, B) synthesis in the
synovial membrane of osteoarthritic rabbits.
3.3. MMP presence in the cartilage and in the synovial membrane
We also investigated whether any of the chondroitin sulfate/
Glucosamine combinations modified the synthesis of different MMPs
which have been demonstrated to be responsible for joint destruction
during osteoarthritis, such as MMP-1, MMP-3 and MMP-13 in the
articular cartilage and the synovial membrane. In the case of MMP-1,
we found that both the pro-enzyme and the active form were increased
in the articular cartilage of osteoarthritic (Fig. 2G) as well as in the
synovial membrane (Fig. 3F). When we analysed the expression of
MMP-3, we observed similar changes as those described for MMP-1, in
both articular cartilage (Fig. 2E, G), and synovial membrane (Fig. 3D
and F). Next we analysed the expression of MMP-13 in the same
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European Journal of Pharmacology 794 (2017) 8–14
tissues, and we found analogous results (Figs. 2F, G and 3E, F). None
of the combined therapies was able to significantly modify the presence
of these MMPs in the cartilage or in the synovial membrane of the
treated rabbits (Figs. 2 and 3).
3.4. Subchondral bone mineral density, OPG and RANKL synthesis
Since the beneficial effect of chondroitin sulfate or glucosamine
sulfate in osteoarthritis has been previously attributed to a putative
decrease in the resorptive activity of the subchondral bone cells (Tat
et al., 2007), we studied whether the combined therapies were able to
modify sBMD in experimental osteoarthritis. DXA analysis on tibias
demonstrated that osteoarthritic rabbits showed a clear decrease in
sBMD at the end of our study in comparison to control animals (0.48 ±
0.03 for OA vs 0.60 ± 0.03 g/cm2 for control, P < 0.05). None of the
combined therapies were able to significantly modify the sBMD (0.48 ±
0.03 for OA+CT1 and 0.51 ± 0.04 gr/cm2 for OA+CT2, both P > 0.05
vs. control).
We also studied the protein presence of both RANKL and OPG in
the subchondral bone of these rabbits. These factors are the main
regulators of bone homeostasis, and they have been found to be
dysregulated in the subchondral bone of osteoarthritic rabbits (Lv
et al., 2014). Although we did not observe significant changes in the
presence of OPG in the subchondral bone of OA rabbits in comparison
to control animals, RANKL was induced in this tissue (2.6 ± 0.4 for OA
J.A. Roman-Blas et al. European Journal of Pharmacology 794 (2017) 8–14

Fig. 3. COX-2, IL-1β, MMP-1, MMP-3 and MMP-13 presence in the synovial membrane. A-E: Densitometric analysis of protein presence in the synovial membrane, assessed by
western-blot. F, representative western-blot images for protein detection in the synovial membrane. EZ Blue stained gel used as protein loading control is also shown. The bars show the
mean ± S.E.M. *P < 0.05 vs. control. N=9–10 samples per group. Control (CTR), osteoarthritic (OA), osteoarthritic treated with combined therapy 1 (OA+CT1) and osteoarthritic treated
with combined therapy 2 (OA+CT2) rabbits.

vs. 1.03 ± 0.41.0 A.U. for control, P < 0.05). None of the combined for osteoarthritis is controversial. Preclinical and clinical studies
therapies were able to significantly modify RANKL presence (2.0 ± suggest that chondroitin sulfate, glucosamine sulfate and combinations
0.3 AU for OA+CT1 and 2.0 ± 0.5 AU for OA+CT2; P > 0.05 vs OA), nor could modify the disease progression but, at the same time, discre-
the ratio OPG/RANKL in the subchondral bone. pancies on the efficacy in modifying the course of the disease have been
also observed (Henrotin et al., 2014). In contrast to the negative results
found in the present study, in vitro research has demonstrated that
4. Discussion
pretreatment with GH reduces glycosaminoglycan release, production
of pathological interglobular domain aggrecan catabolites, mRNA
In this work, we have studied the effect of chondroitin sulfate plus
levels of ADAMTS-4 and -5 and ADAMTS-4 activity in bovine cartilage
glucosamine sulfate and chondroitin sulfate plus glucosamine hydro-
explants treated with IL-1α (Bascoul-Colombo et al., 2016).
chloride in combined therapy at structural level, as well as on the
Glucosamine hydrochloride also reduced the release of inflammatory
inflammatory and matrix degradative mediators in our destabilization
markers, prostaglandin E2 and nitric oxide induced by IL-1α.
model of osteoarthritis. High doses of chondroitin sulfate plus gluco-
Meanwhile, chondroitin sulfate alone did not have a significant effect
samine sulfate and chondroitin sulfate plus glucosamine hydrochloride
on IL-1α-induced cartilage degradation and inflammation.
did not prevent the histopathological changes observed in the non-
Furthermore, the combination of both compounds did not further
treated osteoarthritis group. Furthermore, no effects were observed in
inhibit IL-1α-induced activity (Bascoul-Colombo et al., 2016). Previous
the dysregulation of different osteoarthritis mediators at the articular
work carried out by our group also described positive effects for
cartilage and at the synovial membrane. Interestingly, the chondroitin
chondroitin sulfate in different experimental models of chronic arthri-
sulfate plus glucosamine sulfate and chondroitin sulfate plus glucosa-
tis. In this regard, we observed that intraperitoneal injection of
mine hydrochloride combinations were administered previously to
chondroitin sulfate was able to reduce the inflammatory response of
osteoarthritis induction, thus throwing into question the effect of
the synovial membrane, as well as to decrease the synovial histopatho-
SYSADOAS when used preventively at early stages of the disease.
logical lesions in chronic antigen-induced arthritis (Largo et al., 2010).
The use of chondroitin sulfate plus glucosamine sulfate and
Clinical data also reflect the controversy on chondroitin sulfate plus
chondroitin sulfate plus glucosamine hydrochloride in combination

12
J.A. Roman-Blas et al.
glucosamine sulfate efficacy as therapeutic option for osteoarthritis. A
late study using data from the Osteoarthritis Initiative (Raynauld et al.,
2016) has reported a long-term (6-year) protective structure-modifying
effect of combined chondroitin sulfate plus glucosamine sulfate. This
combined therapy significantly reduced cartilage volume loss as
assessed by automated quantitative MRI. However, this protective
effect was not associated with symptom improvement measured by
WOMAC (Raynauld et al., 2016). Chondroitin sulfate plus glucosamine
hydrochloride has been shown to possess a comparable efficacy to
celecoxib in reducing pain, stiffness, functional limitation and joint
swelling/effusion after 6 months in patients with painful knee osteoar-
thritis (Hochberg et al., 2016). Conversely, another just released study
has failed to demonstrate any beneficial effect of chondroitin sulfate
plus glucosamine sulfate in osteoarthritis (Román-Blas et al., 2016).
Our phase III, randomized, double-blinded, placebo-controlled, paral-
lel-group clinical trial comparing the combination of chondroitin
sulfate plus glucosamine sulfate versus placebo, showed no significant
differences between both arms on WOMAC total score, and WOMAC
pain and function subscales, the proportion of OMERACT-OA respon-
ders, and the consumption of rescue medication (Román-Blas et al.,
2016). Likewise, the use of chondroitin sulfate plus glucosamine sulfate
did not relieve the symptoms as assessed by WOMAC score total and
subscales or modify disease progression as estimated by joint narrow-
ing pace among patients with radiographic knee osteoarthritis (Yang
et al., 2015).
Taken together, all these contradictory results would indicate that
although chondroitin sulfate and glucosamine sulfate alone and in
different compositions may have a modest positive effect on improving
or preventing osteoarthritis progression in animals, the combination of
both compounds is not that good as previously described. Both drugs
have been previously administrated separately to rabbits in similar
doses, and they have demonstrated to be active after oral treatment
(Herrero-Beaumont et al., 2008; Iovu et al., 2012; Largo et al., 2009),
thus validating the drugs and the via of administration. Furthermore,
the lack of effect cannot be attributable to the pharmacological quality
of the compounds used in these experiments, since both formulations
were of high pharmacology quality. However, the oral administration of
both compounds at the same time could modify the bioavailability and
pharmacokinetic of these compounds when administered alone
(Jackson et al., 2010).
In conclusion, combined therapy of chondroitin sulfate plus
glucosamine sulfate and chondroitin sulfate plus glucosamine hydro-
chloride failed to prevent the structural damage and to decrease the
expression of pro-inflammatory and matrix degradative mediators at
joint tissues in an extensive experimental model of osteoarthritis.
Taken together with previous preclinical and clinical research, these
studies cast serious doubt on the suitability of these combinations as
treatments for knee osteoarthritis.
Funding
This work was partially supported by research grants from the
Instituto de Salud Carlos III (PI12/00144; PI13/00570; PI15/00340
and RETICEF RD12/0043/0008), co-funded by Fondo Europeo de
Desarrollo Regional (FEDER).
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