Biomedicine & Pharmacotherapy 79 (2016) 120–128

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Original article

Chondroprotective effects of the combination chondroitin

sulfate-glucosamine in a model of osteoarthritis induced by anterior
cruciate ligament transection in ovariectomised rats
María Carmen Terencioa , María Luisa Ferrándiza , María Carmen Carcellera , Ramón Ruhíb ,
Pere Dalmaub , Josep Vergésc, Eulàlia Montellc, Anna Torrentc , María José Alcaraza,*
a
Department of Pharmacology and IDM, University of Valencia, Av. Vicent Andrés Estellé s/n, 46100 Burjasot, Valencia, Spain
b
Technological Extraction Department, Bioiberica S.A., Pol. Ind. “Mas Puigvert” Crta. N-II, Km 680.6, 08389 Palafolls, Barcelona, Spain
c
Pre-Clinical R&D Department, PharmaScience Division, Bioiberica S.A., Francesc Macià 7, 08029 Barcelona, Spain

A R T I C L E I N F O A B S T R A C T

Article history: Context: The efficacy of the combination chondroitin sulfate-glucosamine (CS-GlcN) in the treatment of
Received 21 January 2016 knee osteoarthritis (OA) has been suggested in recent clinical studies. In vitro reports have also suggested
Accepted 8 February 2016 anti-inflammatory and anti-resorptive effects of this combination.
Objective: The aim of this study was to characterize the effects of CS-GlcN on joint degradation in vivo
Keywords: including the assessment of inflammation and bone metabolism in a model of OA.
Anterior cruciate ligament transection Materials and methods: We have used the OA model induced by anterior cruciate ligament transection
model
(ACLT) in ovariectomised rats. CS-GlcN was administered daily (oral gavage) from week 0 until week
Osteoarthritis
Chondroitin sulfate-glucosamine
12 after ovariectomy at the dose of 140 (CS) + 175 (GlcN)(HCl) mg/kg. Histochemical analyses were
Ovariectomised rats performed, the levels of biomarkers and inflammatory mediators were measured by luminex or ELISA
and bone microstructure was determined by mCT.
Results: CS-GlcN protected against cartilage degradation and reduced the levels of inflammatory
mediators such as interleukin-1b and tumor necrosis factor-a in the affected knee. In addition, serum
biomarkers of inflammation and cartilage and bone degradation including matrix metalloproteinase-3,
C-telopeptide of type II collagen and the ratio receptor activator of nuclear factor kB ligand/
osteoprotegerin were significantly decreased by CS-GlcN. This treatment also tended to improve some
bone microstructural parameters without reaching statistical significance.
Discussion and conclusions: These results demonstrate the chondroprotective effects of CS-GlcN in vivo, in
the experimental model of ACLT in ovariectomised rats, and suggest that this combination may be useful
to control the joint catabolic effects of inflammatory stress. These findings could have clinical relevance
related to the prevention of joint degradation by CS-GlcN and support the potential development of OA
treatments based on this combination.
ã 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction epigenetic mechanisms involved in the development of OA [3] as

Osteoarthritis (OA) is a complex multifactorial disease that may
progressively affect the structure of all joint tissues. Some
characteristic alterations include progressive hyaline articular
cartilage loss, low-grade synovitis and changes in subchondral
bone and periarticular tissues [1]. OA is the most prevalent joint
condition and a leading cause of disability in the world [2]. In the
last years, major efforts have been made to identify genetic and
* Correspondence author:
E-mail address: maria.j.alcaraz@uv.es (M.J. Alcaraz).
well as the role of ageing, obesity, joint injury, biomechanical
stress, inflammation, hormone levels, metabolic disease, etc. [4,5].
Recent investigations have also stressed the interest of OA
phenotypes identification to understand the variety of pathophys-
iological processes responsible for disease progression in order to
provide a more personalized clinical approach [6]. Osteoporosis is
the most common skeletal disease in humans [7]. This condition is
characterized by a low bone mineral density and deterioration of
bone microarchitecture, which results in bone loss and fragility
leading to fractures [8]. Age and weight are main risk factors for the
development of osteoporosis although a number of other factors
also play a role such as height, estrogen deficiency and chronic

http://dx.doi.org/10.1016/j.biopha.2016.02.005
0753-3322/ ã 2016 Elsevier Masson SAS. All rights reserved.
 M.C. Terencio et al. / Biomedicine & Pharmacotherapy 79 (2016) 120–128
diseases including hypertension, hyperlipidemia, diabetes melli-
tus, and OA [9]. In particular, estrogen deficiency is involved in both
the early and the late phases of bone loss in postmenopausal
women and it also contributes to bone loss in aging men [10]. Both
OA and osteoporosis are main health issues with an enormous
socioeconomic impact as the number of older people increases.
Although an inverse relationship between OA and osteoporosis
has been suggested (reviewed in [11]), different reports indicate
that osteopenia and osteoporosis occur in a significant proportion
of patients with severe knee or hip OA [12] and longitudinal bone
mineral density loss, at a site remote from the knee, is associated
with progressive loss of cartilage in OA knees [13]. Several studies
have also shown that changes in bone can be detected early in the
development of OA with osteoporosis subjacent to the subchondral
sclerosis area [14] and within the tibiae of patients with knee OA
[15]. Epidemiological studies have shown an increased prevalence
of OA in postmenopausal women suggesting a relationship
between estrogen deprivation and the development of OA
[16,17]. In fact, estrogen deficiency-related OA has been proposed
as a subset of OA [6].
Inflammation may be a factor influencing the development of
OA and osteoporosis [18]. Synovitis is associated with clinical
symptoms of OA as well as with damage of the adjacent cartilage
[19]. An increased production of pro-inflammatory mediators such
as interleukin-1b (IL-1b), tumor necrosis factor-a (TNFa) and
prostaglandin E2 (PGE2) can play a role in suppression of
extracellular matrix synthesis and expression of degradative
enzymes which results in focal loss of cartilage in knee joints
[20,21]. In addition, pro-inflammatory cytokines increase osteo-
clast activity and contribute to bone resorption [22].
Biochemical biomarkers are an important research area in OA
that will support new drug developments, preventive medicine,
and medical diagnostics for OA [23]. Early changes in joint and
bone during OA may be followed by determinations of biomarkers
[24]. Studies in OA patients have shown significant increases in
serum levels of markers of cartilage destruction (reviewed in [25]).
Within catabolic mediators, the levels of matrix metalloprotei-
nases (MMPs) in serum have been related with the progression of
OA and the effects of drugs [26]. In addition, markers of bone
metabolism are useful to investigate the physiopathology of bone
alterations and to monitor the efficacy of drug treatments in
osteoporosis (reviewed in [27]) and also in OA combined with
markers of cartilage turnover [28]. Some relevant examples are
osteocalcin (a marker of osteoblast differentiation), receptor
activator of nuclear factor kB ligand (RANKL) which is a key
inducer of osteoclastogenesis [29], and osteoprotegerin (OPG)
which acts as a competitive inhibitor of RANKL [30,31].
There are limited treatment options for OA and they are mainly
based on symptom management with non-pharmacological and
pharmacological approaches according to patient’s characteristics
Fig. 1. Experimental design and animal groups.
121
and the presence of risk factors [32]. There is a great unmet need
for effective and safe therapeutic interventions for OA able to
inhibit disease progression (reviewed in [33]). In recent years,
chondroitin sulfate (CS) and glucosamine (GlcN) have been widely
used in OA [34]. Recent clinical studies suggest the efficacy of the
combination CS-GlcN to improve function, control pain [35,36] and
reduce the loss of cartilage volume over 24 months in patients with
knee OA [37].
Animal models are relevant to study the influence of treatments
on the whole joint and to assess systemic effects. These models can
reproduce key aspects of human OA and are essential to
understand the early events occurring during disease progression
and to test the safety and efficacy of therapies. In experimental OA,
little is known of the possible effects of CS-GlcN. Although a
combination of CS with glucosamine sulfate reduced cartilage
degradation and pain in rat ACLT [38], CS-GlcN did not exert
significant effects on OA induced by partial medial meniscectomy
in rabbits [39]. The anterior cruciate ligament transection (ACLT)
model in rats exhibits a number of characteristics similar to human
OA [40]. This experimental model mimics posttraumatic OA and is
widely used for the assessment of new treatments that may control
the progression of OA. Ovariectomy (OVX) in rats has been
proposed as a model of postmenopausal OA although the changes
in knee cartilage observed after OVX are relatively mild and
cartilage turnover may be transient [41]. Nevertheless, the
combination of ACLT and OVX in rats represents a model of OA
and bone loss that may be useful to test chondroprotective
interventions [42,43]. We have recently reported the suitability of
this model for the evaluation of drugs targeting cartilage and bone
alterations [44].
The aims of the present study were to determine whether CS-
GlcN could regulate cartilage and bone alterations associated to
inflammation in vivo. For this purpose, we have used the ACLT
model of OA in ovariectomised rats. In addition to histological
techniques, we have included biomarker determinations and mCT
imaging for a detailed analysis of CS-GlcN effects.
2. Materials and methods
2.1. OVX + ACLT
The protocols were approved by the institutional Animal Care
and Use Committee (University of Valencia, Spain). All studies
were performed in accordance with European Union regulations
for the handling and use of laboratory animals, and are reported in
accordance with the ARRIVE guidelines for reporting experiments
involving animals [45]. A total of 45 female Wistar rats (180–200 g)
from Janvier (Le Genest Saint Isle, France) were used in this study.
They were housed at the animal facility (School of Pharmacy,
University of Valencia, Spain), with controlled temperature
122 M.C. Terencio et al. / Biomedicine & Pharmacotherapy 79 (2016) 120–128
(21
2  C), 12 h dark-light cycle, and water and food ad libitum. Rats
were randomly divided into three groups (n = 15 per group) before
the experiment. All surgical procedures were carried out under
deep anaesthesia with isoflurane (1.5%, Esteve S.A., Barcelona,
Spain) which was followed by the subcutaneous injection of
butorphanol (2 mg/kg, Pfizer, Madrid, Spain), just after the surgery.
OVX was performed on 10-week old rats (180–200 g body weight,
n = 15 rats/group) (day 0) and 2 weeks after (week 2) ACLT was
performed via an incision on the medial aspect of the right knee
joint capsule. Another group of animals underwent sham
operations (Sham group) (Fig. 1). The combination CS-GlcN
(Bioibérica S.A., Barcelona, Spain) was administered daily (oral
gavage of a dispersion in water) from day 0 until week 12 after
ovariectomy (OVX-ACLT CS-GlcN group) at the dose of 140
(CS) + 175 (GlcN)(HCl) mg/kg, which correspond approximately
to the usual human dose of 1200 and 1500 mg/day, respectively.
GlcN was glucosamine hydrochloride pharmaceutical grade (code
F1590, batch 11/0013) and CS was bovine chondroitin sulfate
sodium 100% (code F0425, batch 10/0005) according to the
specifications outlined in the European Pharmacopoeia. The sham-
operated (Sham) and OVX + ACLT (OVX-ACLT Vehicle) groups,
received the same volume of vehicle (water). After week 12,
animals were anesthetized with isoflurane (1.5%), blood was
collected by cardiac puncture and finally rats were sacrificed by
isoflurane overdose. All determinations were performed at this
time point.
2.2. Histopathology
After dissection of skin, the whole knee joints were fixed in 4%
paraformaldehyde in phosphate-buffered saline solution. Samples
were decalcified in 10% EDTA, dehydrated in graded ethanol,
cleared in toluene and embedded in paraffin. The knees were
transected in the frontal plane. Serial sections were cut (7 mm)
using a Leica RM2255 microtome (Leica Biosystems, Nussloch,
Germany) and mounted on SuperFrost glass slides (Menzel-Gläser,
Braunschweig, Germany). Sections were deparaffinised in xylol,
Fig. 2. Histological analysis of the ACLT knees. Frontal sections were stained with haematoxylin and eosin. Representative images of sections from patella, femur, tibia and
synovial membrane are shown. Bars: 200 mm (patella, femur, tibia) and 100 mm (synovial membrane). Original magnification: 100 (patella, femur, tibia) and 40 (synovial
membrane).
rehydrated in graded ethanol and stained with haematoxylin and
eosin and safranin O. The sections were mounted on a DPX medium
(Panreac, Barcelona, Spain) and examined under a light microscope
(Nikon Eclipse E800, Izasa S.A., Valencia, Spain) and a Nikon Digital
Camera DXM1200 using Nikon ACT-1 software. Histopathological
grading of cartilage degradation was performed in accordance with
the Osteoarthritis Research Society International (OARSI) histopa-
thology grading and staging system [46]. In this system, score is a
combined value defined as assessment of OA grade x OA stage. Grade
is defined as OA depth progression into cartilage (0–6), and stage is
defined as the horizontal extent of cartilage involvement (0–4). In
addition, proteoglycan depletion was determined in sections stained
with safranin O and scored from 0 (no proteoglycan depletion) to 3
(maximal depletion). All evaluations were performed in a blinded
way by two independent observers.
2.3. Determination of local inflammatory mediators
The knees were amputated, skin was dissected and the rest of
tissue was immediately frozen in liquid nitrogen and pulverized in
a Freezer/Mill 6750 (Spex SamplePrep, Metuchen, NJ, USA). The
powder was extracted with 2 ml of A buffer pH 7.4 [10 mM 4-
(hydroxyethyl)-1-piperazine-ethane-sulfonic acid (HEPES, pH 8),
1 mM EDTA, 1 mM ethylene glycol bis(b-aminoethylether)-N,N,N0 ,
N0 -tetraacetic acid (EGTA), 10 mM KCl, 1 mM dithiothreitol, 5 mM
NaF, 1 mM Na3VO4, 1 mg/ml leupeptin, 0.1 mg/ml aprotinin and
0.5 mM phenylmethylsulfonyl fluoride]. After centrifugation at
1200g (10 min at 4  C), supernatants were collected, centrifuged at
9000 g (15 min at 4  C) and used to measure cytokines by ELISA. IL-
1b and TNFa were measured by using the kits from R&D Systems
(Minneapolis, MN, USA) with a sensitivity of 5.0 pg/ml. PGE2 was
determined by radioimmunoassay [47].
2.4. Determination of serum biomarkers
Serum was used for the determination by ELISA of C-telopeptide
of type II collagen (CTX-II, serum pre-clinical Cartilaps, Nordic
 M.C. Terencio et al. / Biomedicine & Pharmacotherapy 79 (2016) 120–128
Biosciences, Herlev, Denmark, sensitivity of 6.3 pg/ml) and MMP-3
(R&D systems, sensitivity of 19.0 pg/ml).
OPG, RANKL and osteocalcin were determined in serum
samples by luminex, with sensitivity of 2.3, 3.3 and 7.0 pg/ml,
respectively (Millipore Corporation, Billerica, MA, USA).
2.5. X-ray micro-computed tomography (mCT)
The hind paws were kept wrapped in gauze soaked in 0.9% NaCl
at 20  C until analysis by mCT. Samples were analysed with a
SkyScan 1172 mCT unit (Bruker microCT NV, Kontich, Belgium).
Three-dimensional trabecular micro-architecture was analysed at
the metaphysis and epiphysis of the tibia. Samples were imaged
with an X-ray tube voltage of 50 kV, current of 200 mA, at a
scanning voxel size of 5.5 mm and with the use of an aluminium
filter (0.5 mm in thickness). The scanning total angular rotation
was 185 with an angular increment of 0.4 . Datasets were
reconstructed using a modified Feldkamp algorithm [48] and
segmented into binary images using adaptive local thresholding
prior to analysis. Bone volume fraction (BV/TV; %), bone surface
density (BS/TV; mm1), trabecular thickness (Tb.Th; mm), trabec-
ular number (Tb.N; mm1) and volumetric bone mineral density
(vBMD; mg/cm3) were measured.
2.6. Statistical analysis
The results are presented as mean
standard deviation (SD), n:
number of animals. The level of statistical significance was
determined by using one-way analysis of variance (ANOVA)
followed by Bonferroni’s test. Histological scoring was analyzed
by a non-parametric test (Kruskal-Wallis followed by Dunn’s post-
test). p values lower than 0.05 were considered significant.
Fig. 3. Histological analysis of the ACLT knees. Frontal sections were stained with safranin-O. Representative images of sections from patella, femur and tibia are shown. Bars:
200 mm (patella) and 500 mm (femur, tibia). Original magnification: 100 (patella) and 40 (femur, tibia).
123
3. Results
3.1. Histological assessment
Knee sections from patella, femur, tibia and synovial mem-
brane stained with hematoxylin and eosin are shown in Fig. 2.
Cartilage degradation was evident in the OVX-ACLT Vehicle group
compared with Sham. Focal damage, fibrillation and fissures in
cartilage surface accompanied by loss of cellularity and chondro-
cyte clustering were present. These alterations were more evident
in patella. Loss of safranin-O staining was observed in OVX-ACLT
Vehicle group (Fig. 3) as well as mild synovitis and focal bone loss.
To evaluate the severity of cartilage degradation, the OARSI
histological scoring system was used and proteoglycan loss was
also scored (Fig. 4). Significant increases in cartilage degradation
and proteoglycan depletion scores were observed in the OVX-
ACLT Vehicle group, compared with sham-operated animals.
Morphology of joint structures was better in animals treated with
the combination CS-GlcN in comparison with the OVX-ACLT
Vehicle group. Correspondingly, score values for cartilage
degradation and proteoglycan depletion were significantly lower
in the OVX-ACLT CS-GlcN group with reductions of approximately
90%.
3.2. Inflammatory mediators in the joint
In the affected knee, the levels of IL-1b, TNFa and PGE2 were
significantly enhanced in the OVX-ACLT Vehicle group compared
with Sham (Fig. 5). Treatment of rats with CS-GlcN significantly
reduced the levels of both pro-inflammatory cytokines by
approximately 46%. In addition, PGE2 levels were partly reduced
by CS-GlcN without reaching statistical significance (p = 0.38).
124 M.C. Terencio et al. / Biomedicine & Pharmacotherapy 79 (2016) 120–128

4. Discussion

Development of effective therapies remains a major challenge

in OA. Both CS and GlcN have shown symptomatic effects and
potential disease-modifying effects in OA although the results of
some clinical trials have been controversial (reviewed in [32,49]).
In vitro investigations have demonstrated that the combination CS-
GlcN may exert synergic effects on cartilage with stimulation of
chondrocyte metabolism and down-regulation of degradative
enzymes [50,51]. Recent clinical studies have suggested that
treatment with CS-GlcN delays the structural progression of knee

Fig. 4. Cartilage degradation and proteoglycan depletion were scored in frontal

sections of knee joints as indicated in materials and methods. Results are expressed
as mean
SD (n = 5); +p < 0.05 with respect to sham; *p < 0.05 compared to control
(Kruskal-Wallis followed by Dunn’s post-test).

3.3. Serum biomarkers

Serum levels of CTX-II, a biomarker of cartilage degradation,

were significantly increased in OVX-ACLT Vehicle animals in
comparison with sham-operated rats (Fig. 6). The group treated
with CS-GlcN showed a significant reduction in the serum levels of
this biomarker. In addition, the concentration of the proteolytic
enzyme MMP-3 was elevated in OVX-ACLT Vehicle group
compared with Sham whereas CS-GlcN treatment significantly
reduced MMP-3 levels. We also measured in serum the levels of
several proteins relevant to bone metabolism such as osteocalcin,
RANKL and OPG. As shown in Fig. 6, treatment with CS-GlcN
tended to increase osteocalcin levels and significantly reduced the
ratio RANKL/OPG by approximately 60%.

3.4. Bone architecture

Representative images of mCT analysis are shown in Fig. 7. In

OVX-ACLT Vehicle group, strong bone loss and alterations in bone
microarchitecture were detected at the metaphysis (Table 1), with
reductions in bone volume fraction (BV/TV), bone surface density
(BS/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N)
and volumetric bone mineral density (vBMD) with respect to
Sham. Treatment with the combination CS-GlcN increased the
values of all these parameters, especially of volumetric bone
mineral density without reaching statistical significance (p = 0.09).
Fig. 5. Levels of pro-inflammatory mediators in homogenates from ACLT knees.
Minor changes at the epiphysis were present in OVX-ACLT Vehicle
IL-1b, and TNFa were measured by ELISA. PGE2 was measured by radioimmunoas-
rats compared with Sham animals and no modification was say. Results show mean
SD (n = 6); ++p < 0.01 with respect to sham;
observed after pharmacological treatment. *p < 0.05 compared to control (ANOVA followed by Bonferroni’s test).
 M.C. Terencio et al. / Biomedicine & Pharmacotherapy 79 (2016) 120–128
Fig. 6. Levels of serum biomarkers. CTX-II and MMP-3 were measured by ELISA
Serum levels of osteocalcin, OPG and RANKL were determined by luminex. Results
show mean
SD (n = 12–15); ++p < 0.01 with respect to sham; *p < 0.05 compared
to control (ANOVA followed by Bonferroni’s test).
125
OA in patients with less disease severity. This structural effect was
observed in the medial plateau in early OA and in the lateral
plateau in moderate OA [37]. Nevertheless, controversial results
have been reported [52]. The current study focused on the possible
effects of CS-GlcN in a model of OA in ovariectomised rats to study
its influence on cartilage and bone metabolism. We provide
evidence of in vivo protective effects of this combination at both
levels. Our results indicate that CS-GlcN significantly reduced
cartilage erosion and proteoglycan depletion. These effects on
cartilage were reflected by the reduction in serum levels of the
biomarker CTX-II. Our data thus confirm the anti-catabolic effects
of this combination reported by in vitro studies using bovine
cartilage explants or human OA chondrocytes [51,53–55].
ACLT leads to joint instability and posttraumatic OA [56]. In
addition to biomechanical factors, biochemical mediators may
negatively modify chondrocyte metabolism [57]. In particular,
inflammation may be a component of the early events of OA,
preceding cartilage changes [58] and it plays a role in the
progression of OA after ACLT in rats [59]. Cytokines play a central
role in the complex signaling network regulating inflammation
and joint destruction. Therefore, pro-inflammatory cytokines such
as IL-1b and TNFa inhibit the synthesis of extracellular matrix
molecules and upregulate a number of catabolic mediators
including MMPs and aggrecanases leading to extracellular matrix
degradation [60]. Our data indicate that CS-GlcN, can exert in vivo
anti-inflammatory effects which could be related to the down-
regulation of IL-1b and TNFa. These findings are in line with the
reported in vitro inhibition of inflammatory responses by CS-GlcN
[54,61]. Pro-inflammatory cytokines activate signaling pathways
such as mitogen-activated protein kinases and transcription
factors including nuclear factor-kB leading to the transcription
of pro-inflammatory and catabolic genes. In vitro studies have
demonstrated that the components of this combination inhibit the
nuclear translocation and activation of this transcription factor
(reviewed in [62]). Therefore, it is likely that this mechanism of
action contributes to the in vivo anti-inflammatory and anti-
catabolic effects of CS-GlcN.
The baseline MMP-3 level in serum is a significant predictor of
joint space narrowing in knee OA [63]. In addition, serum MMP-
3 levels in knee OA patients are significantly greater in generalized
OA compared with non generalized disease [64]. We have
demonstrated the inhibitory effects of CS-GlcN on serum levels
of this protease which plays a relevant role in joint destruction in
experimental OA [65]. Therefore, the down-regulation of MMP-
3 may contribute to the inhibitory effects of CS-GlcN on cartilage
damage in this experimental model.
Little is known of CS-GlcN influence on bone metabolism. In
vitro anti-resorptive activity of CS-GlcN has been shown in co-
cultures of human OA subchondral osteoblasts and pre-osteoclasts
which may be related to a reduction in the RANKL/OPG ratio [66],
an index of osteoclastic activation. Our results have confirmed in
vivo the modification of this ratio towards a reduced osteoclastic
activation and indicate that CS-GlcN may help to control the
dysbalance between osteoblastic and osteoclastic activity that
results in bone resorption. Pro-inflammatory cytokines are
produced at local sites of pathology and they influence bone
metabolism. It is known that pro-inflammatory cytokines increase
osteoclast activity leading to bone resorption [22]. Furthermore, IL-
1–induced bone loss is dependent upon RANKL which favors the
formation and survival of osteoclasts as well as the apoptosis of
osteoblasts [67]. Our data thus suggest that events elicited by the
inhibition of cytokines may result in protective effects on bone.
ACLT in ovariectomized rats induces significant trabecular bone
metaphyseal alterations of the tibia [44] and CS-GlcN administra-
tion tended to counteract all these changes although no statistical
126 M.C. Terencio et al. / Biomedicine & Pharmacotherapy 79 (2016) 120–128

Fig. 7. Representative images of epiphysis and metaphysis of tibia by mCT analysis. Quantification of trabecular bone parameters in both regions is shown in Table 1.

significance was reached. Further studies would be necessary to which could lead to the development of novel therapeutic
characterize the in vivo effects of CS-GlcN on bone metabolism. approaches for the prevention and management of OA.
There were some limitations to this study. The pathophysiology
of human OA is complex and no experimental model can reproduce
Acknowledgements
human disease. These data have been obtained in a rat model of
post-traumatic OA in the presence of osteoporosis. The results may
The authors thank Ms. Anna Blanco for her technical assistance.
vary in experimental models mimicking different OA subtypes, in
The present study was supported by grants from Bioibérica S.A. and
larger animals and human subjects. Despite these limitations, the
Spanish Ministerio de Economía y Competitividad, ISCIII, FEDER
results of this study using CS-GlcN by oral route at doses equivalent
(RETICEF RD12/0043/0013). The funding sources had no role in the
to those in humans support the potential of this combination to
conduct of the study; collection, management, analysis, interpre-
prevent joint degradation. In addition, our findings provide a
tation of the data; and preparation of the manuscript. A.T., P.D., R.R.,
foundation for a better understanding of CS-GlcN effects in OA as
E.M. and J.V. are employed by Bioibérica S.A.
well as for designing animal experiments and human studies

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