Biochemical characterisation of navicular hyaline cartilage, navicular fibrocartilage and the deep digital flexor tendon in horses with navicular disease


Viitanen, M.; Bird, J.; Smith, R.; Tulamo, R.M.; May, S.A.

Research in Veterinary Science 75(2): 113-120

2003


The study hypothesis was that navicular disease is a process analogous to degenerative joint disease, which leads to changes in navicular fibrocartilage and in deep digital flexor tendon (DDFT) matrix composition and that the process extends to the adjacent distal interphalangeal joint. The objectives were to compare the biochemical composition of the navicular articular and palmar cartilages from 18 horses with navicular disease with 49 horses with no history of front limb lameness, and to compare navicular fibrocartilage with medial meniscus of the stifle and collateral cartilage of the hoof. Cartilage oligomeric matrix protein (COMP), deoxyribonucleic acid (DNA), total glycosaminoglycan (GAG), metalloproteinases MMP-2 and MMP-9 and water content in tissues were measured. Hyaline cartilage had the highest content of COMP and COMP content in hyaline cartilage and tendon was higher in lame horses than in sound horses (p<0.05). The concentration of MMP-2 amount in hyaline cartilage was higher in lame horses than in sound horses. The MMP-2 amounts were significantly higher in tendons compared to other tissue types. Overall, 79% of the lame horses with lesions had MMP-9 in their tendons and the amount was higher than in sound horses (p<0.05). In horses with navicular disease there were matrix changes in navicular hyaline and fibrocartilage as well as the DDFT with potential implications for the pathogenesis and management of the condition.

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ELSEVIER
Research
in
Veterinary
Science
75
(2003)
113-120
www.elsevier.com/locate/rvsc
Biochemical
characterisation
of
navicular
hyaline
cartilage,
navicular
fibrocartilage
and
the
deep
digital
flexor
tendon
in
horses
with
navicular
disease
M.
Viitanen
*,
J.
Bird,
R.
Smith,
R.-M.
Tulamo,
S.A.
May
Royal
Veterinary
College,
FAEMS,
University
of
London, London,
UK
Faculty
of
Veterinary
Medicine,
Department
of
Clinical
Veterinary
Sciences,
University
of
Helsinki, Helsinki,
Finland
Accepted
6
March
2003
Abstract
The
study
hypothesis
was
that
navicular
disease
is
a
process
analogous
to
degenerative
joint
disease,
which
leads
to
changes
in
navicular
fibrocartilage
and
in
deep
digital
flexor
tendon
(DDFT)
matrix
composition
and
that
the
process
extends
to
the
adjacent
distal
interphalangeal
joint.
The
objectives
were
to
compare
the
biochemical
composition
of
the
navicular
articular
and
palmar
cartilages
from
18
horses
with
navicular
disease
with
49
horses
with
no
history
of
front
limb
lameness,
and
to
compare
navicular
fibrocartilage
with
medial
meniscus
of
the
stifle
and
collateral
cartilage
of
the
hoof.
Cartilage
oligomeric
matrix
protein
(COMP),
deoxyribonucleic
acid
(DNA),
total
glycosaminoglycan
(GAG),
metalloprotein-
ases
MMP-2
and
MMP-9
and
water
content
in
tissues
were
measured.
Hyaline
cartilage
had
the
highest
content
of
COMP
and
COMP
content
in
hyaline
cartilage
and
tendon
was
higher
in
lame
horses
than
in
sound
horses
(p
<
0.05).
The
concentration
of
MMP-2
amount
in
hyaline
cartilage
was
higher
in
lame
horses
than
in
sound
horses.
The
MMP-2
amounts
were
significantly
higher
in
tendons
compared
to
other
tissue
types.
Overall,
79%
of
the
lame
horses
with
lesions
had
MMP-9
in
their
tendons
and
the
amount
was
higher
than
in
sound
horses
(p
<
0.05).
In
horses
with
navicular
disease
there
were
matrix
changes
in
navicular
hyaline
and
fibrocartilage
as
well
as
the
DDFT
with
potential
implications
for
the
pathogenesis
and
management
of
the
condition.
©
2003
Elsevier
Science
Ltd.
All
rights
reserved.
Keywords.•
Navicular
disease;
Navicular
hyaline
cartilage;
Navicular
fibrocartilage
1.
Introduction
Navicular
disease
is
estimated
to
account
for
one-
third
of
all
chronic
forelimb
lamenesses
in
horses
(Col-
les,
1982).
Despite
the
high
incidence
of
this
disease,
and
continuing
research
into
the
nature
of
the
problem,
the
exact
cause
remains
unknown.
Currently
there
are
two
basic
theories
regarding
the
aetiology:
the
vascular
the-
ory
and
mechanical
theory.
Adams
(1969)
described
navicular
disease
as
a
condition,
which
begins
with
bursitis
of
the
navicular
bursa,
between
the
flexor
ten-
don
and
the
navicular
bone,
and
ultimately
leads
to
degenerative
and
erosive
lesions
of
fibrocartilage.
Corresponding
author.
Tel.:
+358-505664862;
fax:
+358-98136994.
E-mail
address.•
(M.
Viitanen).
It
is
generally
accepted
that
navicular
disease
is
a
de-
generative
disease
involving
the
distal
sesamoid
(navicu-
lar
bone),
navicular
bursa
and
deep
digital
flexor
tendon
(DDFT).
In
advanced
cases
of
the
disease,
pathological
findings
include
erosion
and
ulceration
of
the
fibrocarti-
lage
on
the
flexor
surface
of
the
bone,
and
tearing
of
the
DDFT
in
contact
with
the
bone
(Asquith
and
Kivipelto,
1994).
No
lesions
of
the
navicular
hyaline
cartilage
have
been
reported.
Diagnosis
is
based
on
clinical
examination,
nerve
blocks
and
radiographs.
Radiographic
changes,
previously
regarded
as
diagnostic
of
navicular
disease
(MacGregor,
1984),
can
also
occur
in
horses
without
lameness
(Ackerman
et
al.,
1977),
and
radiographic
signs
correlate
poorly
with
lameness
(Wright,
1993).
The
diagnosis
"navicular
disease"
is
not
always
re-
stricted
to
problems
of
the
navicular
area
alone.
The
0034-5288/$
-
see
front
matter
©
2003
Elsevier
Science
Ltd.
All
rights
reserved.
doi:10.1016/50034-5288(03)00072-9
114
M
Viitanen
et
aL
/
Research
in
Veterinary
Science
75
(2003)
113-120
disease
may
be
diagnosed
when
the
horse
has
a
front
limb
lameness
that
is
alleviated
with
a
palmar
digital
nerve
block
(Stashak,
1987;
Turner,
1986).
However,
a
palmar
digital
nerve
block
alleviates
pain
arising
from
all
structures
in
the
palmar
half
of
the
foot,
not
only
just
the
navicular
bone
(Dyson,
1986),
so
this
leads
to
con-
fusion
particularly
in
comparing
accounts
relating
to
the
success
rates
of
different
treatments.
Intra-articular
an-
algesia
of
the
distal
interphalangeal
joint
(DIP-joint)
or
the
navicular
bursa
is
often
used
to
add
further
infor-
mation
on
the
origin
of
the
pain.
These
techniques
are
not
specific
to
the
injected
space
only,
and
can
block
out
pain
from
the
sole
of
the
foot
(Dyson
and
Kidd,
1993;
Keegan
et
al.,
1996;
Schumacher
et
al.,
2000;
Schum-
acher
et
al.,
2001).
Synovial
fluid
studies
have
shown
that
the
results
obtained
from
the
navicular
bursa
and
the
DIP-joint
are
very
similar
in
horses
that
are
not
lame
(Viitanen
et
al.,
2000).
In
horses
with
navicular
disease,
the
relative
activity
of
metalloproteinases
was
increased,
and
glycosaminoglycan
(GAG)
levels
were
decreased
both
in
the
synovial
fluid
of
the
DIP-joint
and
in
bursal
fluids
(Viitanen
et
al.,
2001).
Understanding
the
molecular
composition
of
the
ex-
tracellular
matrix
will
enable
more
light
to
be
thrown
on
the
aetiopathogenesis
of
navicular
disease.
Cartilage
oligomeric
matrix
protein
(COMP)
is
synthesised
in
ligament,
tendon,
meniscus,
and
articular
cartilage
(Murray
et
al.,
2001).
Studies
in
human
have
demon-
strated
that
it
may
be
used
as
a
prognostic
marker
in
rheumatoid
arthritis
and
osteoarthritis.
Mechanical
loading
(Murray
et
al.,
2001)
may
influence
the
distri-
bution
of
COMP
in
articular
cartilage.
COMP
degra-
dation
in
synovial
fluids
from
advanced
joint
disease
may
be
due
to
metalloproteinases
(MMP),
gelatinolytic
activity
(Misumi
et
al.,
2001).
Areas
of
involvement
of
MMPs
in
pathological
pro-
cesses
include
tissue
destruction,
fibrotic
diseases
and
weakening
of
the
matrix
(Woessner,
1998).
Cartilage
destruction
in
osteoarthritis
(OA)
is
associated
with
in-
creased
levels
of
several
matrix
MMPs,
including
the
gelatinase
MMP-2
(Clegg
et
al.,
1997;
Thompson
et
al.,
2001).
Activation
of
MMPs
occurs
in
joint
disease,
and
in
vitro
stimulation
of
equine
articular
cells
and
tissues
causes
not
only
an
increase
in
MMP
production,
but
also
an
increase
in
amount
of
activated
enzyme
released
(Clegg
and
Carter,
1999).
Studies
of
synovial
fluid,
from
horses
with
navicular
disease,
collected
from
the
navicular
bursal
fluid
and
DIP-joint,
showed
significant
changes
in
bursal
metal-
loproteinase
activities,
GAG
and
hyaluronan
content
(Viitanen
et
al.,
2001).
Different
types
of
tissues
are
present
in
the
navicular
area
and
therefore
changes
in
levels
of
MMPs
or
GAGs
may
not
only
be derived
from
cartilage,
but
also
from
tendon
and
ligaments.
The
hypothesis
of
this
study
accepts
the
greater
complexity
of
tissue
types
present
in
the
navicular
area
in
comparison
to
the
synovial
joint,
proposes
that
navicular
disease
is
a
process
analogous
to
degenerative
joint
dis-
ease
(osteoarthritis)
which
leads
to
similar
changes
in
navicular
fibrocartilage
and
in
DDFT
matrix
composi-
tion.
The
subsidiary
hypothesis
was
that
this
process
extends,
simultaneously,
to
the
adjacent
synovial
cavity,
the
DIP-joint,
and
navicular
hyaline
cartilage.
2.
Materials
and
methods
2.1.
Tissue
source
A
total
of
67
horses
were
used
in
this
study.
Samples
were
collected,
after
euthanasia,
from
18
horses
diag-
nosed
clinically
with
navicular
disease
[having
a
mean
age
of
10.8
(0.5)].
Control
horses
without
a
history
of
front
limb
lameness
comprised
34
middle-aged
horses
[5-14
years,
mean
8.8
(0.6)],
8
old
horses
[>18
years,
mean
22
(0.8)]
and
7
young
horses
[<2
years,
mean
0.9
(0.3)].
No
differences
between
the
sexes
were
observed
in
these
experiments;
therefore
the
data
were
not
segre-
gated
on
the
basis
of
gender.
Horses
with
navicular
disease
came
from
the
Royal
Veterinary
College
and
an
abattoir
near
Bristol.
A
his-
tory
was
obtained
either
at
the
time
of
the
clinical
ex-
amination
carried
out
at
the
Royal
Veterinary
College
or
via
a
questionnaire
sent
to
the
owner
of
the
horse.
Horses
were
diagnosed
as
having
navicular
disease
if
they
had
been
lame
and
had
shown
a
positive
response
to
a
palmar
digital
nerve
block.
All
diseased
horses
used
in
this
study
had
a
history
of
a
chronic
navicular
problem
(more
than
2
years).
Both
front
legs
were
ex-
amined
in
all
but
three
diseased
horses.
The
cartilages
and
tendon
of
the
control
groups
used
in
this
study
were
obtained
from
horses
destroyed
for
reasons
other
than
front
limb
lameness.
Only
one
leg
from
each
horse
was
used.
Full
thickness
cartilage
samples
were
harvested
from
the
whole
articular
surface
of
the
navicular
hyaline
cartilage
and
navicular
fibrocartilage.
DDFT
samples
were
collected
from
the
area
opposite
to
the
navicular
bone
(Fig.
1).
Collateral
cartilage
was
obtained
from
the
medial
side
of
the
hoof,
and
the
proximal
part
of
the
cartilage
was
collected.
The
medial
meniscus
of
the
stifle
was
also
used
for
this
study.
Collateral
cartilage
and
meniscus
samples
and
samples
analysed
from
old
and
young
horses
were
collected
from
sound
animals.
Col-
lateral
cartilage
and
meniscus
samples
only
came
from
middle-aged
horses
(5-15
years).
Samples
were
assessed
macroscopically,
and
stored
at
—20°C
until
analysed.
Tissue
wet-weights
were
taken
before
freezing.
Navicu-
lar
hyaline
cartilage,
navicular
fibrocartilage
and
tendon
samples
from
middle-aged
horses
were
divided
into
four
groups:
1,
sound
horses
with
no
macroscopic
lesions
in
the
navicular
fibrocartilage
or
tendon;
2,
sound
horses
M.
Viitanen
et
al.
/
Research
in
Veterinary
Science
75
(2003)
113-120
115
Distal
interphalangeal
join
Navicular
hyaline
cartilage
Navicular
bone
Navicular
fibrocartilage
Navicular
bursa
Deep
digital
flexor
tendon
Fig.
1.
Diagrammatic
representation
of
a
sagittal
section
of
the
foot.
with
macroscopic
lesions
in
the
navicular
area;
3,
lame
horses
with
no
macroscopic
lesions
in
the
navicular
area;
and
4,
lame
horses
with
macroscopic
lesions
in
the
na-
vicular
area.
Lesions
were
assessed
by
eye,
fraying
of
the
cartilage
and
tendon
surfaces
were
counted
as
a
lesion.
Slight
yellowish
discoloration
in
the
navicular
fibrocar-
tilage
or
DDFT
was
considered
to
be
a
normal
age-re-
lated
sign,
and
was
not
counted
as
a
lesion
(Colles,
1979).
All
the
horses
in
groups
with
lesions
had
changes
in
both
the
navicular
fibrocartilage
and
DDFT.
Throughout
the
text,
these
groups
are
referred
to
as
lame
and
sound
groups
with
or
without
lesions.
2.2.
Determination
of
DNA
content
The
cellularity
of
the
tissues
was
determined
by
as-
sessing
the
total
DNA
content
of
the
tissue
following
papain
digestion.
DNA
was
quantified
using
the
bis-
benzimidazole
fluorescent
dye
technique,
Hoechst
33258
(Kim
et
al.,
1988).
The
Hoechst
33258
dye
was
used
at
a
concentration
of
0.2
mg/ml,
in
0.01
mol/L
Tris,
1.0
mmol/L
EDTA,
and
0.1
mol/L
NaCl.
Fluorescence
of
aliquots
of
the
digests
was
evaluated
by
spectrofluori-
metry:
emission
in
the
range
400-550
nm
for
an
excita-
tion
wavelength
of
365
nm
was
determined.
Standard
curves
were
determined
using
solutions
of
highly
poly-
merised
calf
thymus
DNA
of
known
concentration.
2.3.
Extraction
of
proteoglycan
and
protein
Cartilage
and
tendon
was
extracted
twice
with
4.0
mol/L
guanidine
hydrochloride
(GnHCl)
in
0.05
mol/L
sodium
acetate
buffer,
pH
5.8,
containing
proteinase
inhibitors
[pepstatin
(1.0
µg/ml,
1,10-phenanthroline
(1.0
mmol/L),
iodoacetic
acid
(1.0
mmol/L),
phenylm-
ethylsulphonyl
fluoride
(1.0
mmol/L)]
at
4
°C
for
24
h
(Platt
and
Bayliss,
1994).
2.4.
Determination
of
COMP
GnHCl
extracts
were
precipitated
twice
with
95%
ethanol,
50
mM
sodium
acetate
(-20
°C
overnight),
centrifuged
at
8000g
for
30
min
and
freeze-dried.
Pellets
were
resuspended
with
sample
buffer
and
the
quantity
of
COMP
in
the
supernatant
was
determined
using
a
het-
erologous
inhibition
ELISA
(Smith
et
al.,
1997).
2.5.
Determination
of
GAG
Cartilage
and
tendon
samples
were
digested
with
papain
(25
mg/ml;
Sigma
type
III),
in
50
mmol/L
sodium
phosphate
buffer,
pH
6.5,
2.0
mmol/L
EDTA,
2.0
mmol/
L
N-acetylcysteine
for
a
minimum
of
6
h
at
60
°C.
The
concentration
of
total
glycosaminoglycan
in
the
digests
was
determined
by
the
dimethylene
blue
dye
assay
(Farndale
et
al.,
1986).
Standard
curves
for
the
assay
were
constructed
using
purified
chondroitin
sulphate
from
shark
cartilage.
Separation
of
protein
fragments
was
achieved
using
sodium
dodecyl
sulphate—polyacrylamide
gel
electro-
phoresis
(SDS—PAGE).
Aliquots
of
GnHCl,
ethanol
precipitated
extracts
were
analysed
on
a
tissue
wet
weight-
equivalent
basis,
by
Western
blotting
onto
polyvinylidene
fluoridene
membrane.
Detection
of
`aggrecanase'
and
the
metalloproteinase
generated
G1
domain
of
aggrecan
was
achieved
using
polyclonal
antisera
that
specifically
recognise
the
neo-carboxy
terminal
regions
of
the
inter-
globular
domain
produced
by
their
proteolytic
activity
(Lark
et
al.,
1997).
The
antisera,
which
recognise
the
aggrecanase
and
metalloproteinase
cleavage
sites,
were
raised
against
NITEGE
and
FVDIPEN
sequences,
re-
spectively
(Lark
et
al.,
1997).
2.6.
Determination
of
matrix
metalloproteinases
Relative
amounts
of
MMP-2
and
-9
were
assayed
by
gelatin
zymography
(Sepper
et
al.,
1994).
Gelatin
was
polymerised
in
8%
polyacrylamide
gels.
Dry
samples
(3
mg)
or
wet
samples
(10
mg)
of
tissue
were
mixed
with
100
µI
sample
buffer
containing
0.471
M
Tris,
0.256
mol/
L
H
3
PO
4
,
20%
glycerol,
0.04%
bromophenol
blue
and
6%
sodium
dodecyl
sulphate.
Samples
were
heated
for
30
min
at
60
°C
and
10
µI
aliquots
of
each
sample
was
loaded
onto
the
gels.
Polymorphonuclear
neutrophils,
separated
from
equine
blood,
were
used
as
an
internal
standard
in
each
gel
(Raulo
and
Maisi,
1998).
The
in-
tensity
of
the
zymogen
bands
was
compared
to
internal
standard
and
assessed
using
computer
assisted
image
116
M
Viitanen
et
al.
/
Research
in
Veterinary
Science
75
(2003)
113-120
analysis
of
the
gels
(Raulo
and
Maisi,
1998).
For
each
sample,
the
intensity
of
the
enzyme
band
was
calculated
by
comparison
to
the
amount
in
the
standard
(neu-
trophils),
thereby
giving
relative
metalloproteinase
amount.
The
standard
was
applied
in
each
gel,
enabling
samples
to
be
compared
between
gels
(Clegg
et
al.,
1997).
2.7.
Determination
of
water
content
Portions
of
tissue
of
known
wet
weight
were
freeze
dried
for
24
h.
The
remaining
tissue
was
measured
and
the
water
content
calculated.
2.8.
Statistical
analysis
Comparison
of
means
between
different
groups
was
calculated
using
the
one-way
ANOVA
test.
3.
Results
All
results
are
presented
in
Tables
1-3.
3.1.
Disease-related
changes
in
navicular
tissues
Typically
tissues
with
lesions
contained
less
DNA
than
similar
tissues
without
lesions
but
these
differences
were
not
statistically
significant.
No
lesions
in
the
navicular
hyaline
cartilage
were
noted
in
this
study.
However,
hyaline
cartilage
from
lame
horses
with
lesions
in
the
fibrocartilage
had
the
highest
content
of
COMP,
and
this
difference
was
sta-
tistically
significant
when
compared
to
the
hyaline
car-
tilage
from
sound
horses
(p
<
0.05).
Tendons
from
lame
horses
with
lesions
had
also
significantly
higher
COMP
values
than
tendons
from
sound
horses
(p
<
0.05).
Total
GAG
was
calculated
as
a
proportion
of
the
wet
weight
of
tissue.
The
highest
GAG
values
were
identified
in
hyaline
cartilage
from
sound
horses
without
lesions
(p
<
0.05).
Hyaline
cartilage
from
both
sound
and
lame
horses
with
lesions
in
the
navicular
fibrocartilage
had
significantly
lower
GAG
values
than
the
hyaline
carti-
lage
from
horses
without
lesions
(p
<
0.05).
Similarly,
fibrocartilage
of
sound
horses
with
no
lesions
had
sig-
nificantly
higher
GAG
values
than
fibrocartilage
of
both
sound
and
lame
horses
with
lesions
(p
<
0.05).
The
presence
of
`aggrecanase'
and
the
metalloproteinase
generated
cleavage
sites
adjacent
to
the
G1
domain
of
aggrecan
were
identified
in
all
tissue
types
but
no
major
differences
in
content
were
found
between
any
of
the
groups.
Navicular
hyaline
cartilage
from
lame
horses
with
lesions
had
significantly
higher
MMP-2
values
than
that
of
sound
horses
(p
<
0.05).
Fibrocartilage
of
lame
horses
with
lesions
had
higher
MMP-2
amounts
than
that
of
sound
horses
but
this
difference
was
not
sta-
tistically
significant.
Tendons
of
both
lame
and
sound
horses
with
lesions
had
significantly
higher
relative
MMP-2
amounts
when
compared
with
the
tendons
of
animals
without
lesions
of
the
fibrocartilage
(p
<
0.05).
Overall,
79%
of
the
tendons
of
lame
horses
with
le-
sions
had
MMP-9
present.
In
contrast,
approximately
Table
1
Results
of
assays,
mean
(SE)
of
cartilage
oligomeric
matrix
protein
(COMP),
glycosaminoglycan
(GAG),
matrix
metalloproteinase
2
(MMP-2),
matrix
metalloproteinase
9
(MMP-9)
and
water
content
in
navicular
hyaline
cartilage
Control,
no
lesions,
N
=
27
Control,
with
lesions,
N
=
7
Diseased,
no
lesions,
N
=
8(15)d
Diseased,
with
lesions,
N
=
10(18)
d
Young
horses,
N
=
7
Old
horses,
N
=
8
DNA
µg/mg
of
wet
tissue
0.23
(0.03)
0.11
(0.01)
0.26
(0.03)
0.18
(0.02)
0.2
(0.06)
0.26
(0.09)
COMP
µg/mg
of
wet
tissue
2.7
(0.3)
1
'
2.1
(0.7)
3.5
(1.3)
4.6
(1.0)
a
0.9
(1.2)
a
4.2
(1.2)
a
GAG
µg/mg
of
wet
tissue
52.2
(3.3)c
39.1
(2.9)a
44.6
(5.0)
40.5
(2.7)a
50.7
(3.9)
36.6
(3.1)
a
MMP-2e
relative
amount
3.1
(0.9)
1.8
(0.4)
3.6
(0.9)
9.3
(2.2)a
4.0
(2.1)
2.0
(0.9)
MMP-9e
relative
amount
3.1
(1.2)
2.3
(0.6)
1.0
(0.4)
6.9
(2.1)
1.4
(0.5)
Water%
72.5
(0.5)
70.9
(0.6)
71.9
(0.8)
70.3
(1.7)
68.3
(3.4)
72.8
(0.4)
Hyaline
cartilage
of
horses
with
navicular
disease
and
macroscopic
lesions
in
navicular
fibrocartilage,
had
significantly
more
COMP
and
MMP-2
and
less
GAG
than
hyaline
cartilage
of
sound
horses.
In
navicular
hyaline
cartilage,
COMP
content
increased
and
GAG
content
decreased
with
age.
p
<
0.05,
in
comparison
with
sound
horses
without
fibrocartilage
lesions.
b
COMP
values
were
significantly
higher
in
hyaline
cartilage
than
in
any
other
tissues
analysed
(p
<
0.05).
GAG
values
were
significantly
higher
in
hyaline
cartilage
of
sound
horses
than
rest
of
the
tissues
except
hyaline
and
fibrocartilage
of
young
horses
(p
<
0.05).
d
N
is
number
of
horses
and
number
in
brackets
describes
individual
samples
from
which
means
were
derived.
'The
relative
amount
of
metalloproteinases
was
compared
between
samples.
To
determine
the
amounts
of
metalloproteinase,
a
neutrophil
standard
was
applied
to
each
gel,
and
the
samples
compared
against
it,
and
each
other.
M.
Viitanen
et
al.
/
Research
in
Veterinary
Science
75
(2003)
113-120
117
Table
2
Results
of
assays,
mean
(SE)
of
cartilage
oligomeric
matrix
protein
(COMP),
glycosaminoglycan
(GAG),
matrix
metalloproteinase
2
(MMP-2),
matrix
metalloproteinase
9
(MMP-9)
and
water
content
in
navicular
fibrocartilage,
meniscus
and
collateral
cartilage
Fibrocartilage
Meniscus
N
=
10
Collateral
cartilage
N
=
12
Control,
no
lesions,
N
=
27
Control,
with
lesions,
N
=7
Diseased,
no
lesions,
N
=
8(15)C
Diseased,
with
lesions,
N=
10(18)C
Young
horses,
N
=7
Old
horses,
N
=
8
DNA
µg/mg
of
wet
tissue
0.3
(0.05)
0.29
(0.06)
0.29
(0.04)
0.25
(0.01)
0.47
(0.2)
0.33
(0.04)
0.46
(0.1)b
0.21
(0.02)
COMP
µg/mg
of
wet
tissue
1.5
(0.2)
0.8
(0.3)
1.4
(0.3)
2.0
(0.7)
0.6
(0.3)
2.0
(0.4)
2.5
(0.8)
1.5
(0.4)
GAG
µg/mg
of
wet
tissue
43.5
(3.1)
25.2
(3.4)a
38.1
(3.7)
33.8
(2.1)a
46.6
(6.4)c
35.8
(6.7)a
34.8
(5.5)
42.2
(4.0)
MMP-2
relative
amount'
.
5.1
(0.9)
3.2
(0.2)
3.7
(0.6)
8.5
(2.5)
5.6
(1.1)
4.2
(0.8)
1.8
(0.2)
1.5
(0.3)
MMP-9
relative
amount'
.
2.8
(1.1)
4.0
(1.7)
1.4
(0.5)
3.1
(1.2)
3.1
(1.9)
Water%
71.2
(1.2)
70.5
(0.9)
72.1
(0.8)
67.1
(1.9)
69.7
(3.5)
70.8
(1.9)
61.3
(1.2y
67
(1.9)
Middle
aged
horses
(5-15
years)
were
the
source
of
meniscus
and
collateral
cartilage.
GAG
content
decreased
both
in
sound
and
navicular
diseased
horses
with
fibrocartilage
lesions.
GAG
content
also
decreased
with
age.
DNA
content
was
highest
and
the
water
content
lowest
in
meniscus.
8
p
<
0.05,
in
comparison
with
sound
horses
without
fibrocartilage
lesions.
b
The
DNA
content
was
higher
in
meniscus
than
in
navicular
hyaline
or
fibrocartilage
or
in
collateral
cartilage
(p
<
0.05).
c
Navicular
fibrocartilage
of
young
horses
had
higher
GAG
content
than
the
navicular
fibrocartilage
of
old
horses
(p
<
0.05).
d
Water
content
in
meniscus
was
lower
than
in
navicular
hyaline
or
fibrocartilage
or
in
collateral
cartilage
p
<
0.05.
e
N
is
number
of
horses
and
number
in
brackets
describes
individual
samples
from
which
means
were
derived.
f
The
relative
amount
of
metalloproteinases
was
compared
between
samples.
To
determine
the
amounts
of
metalloproteinase,
a
neutrophil
standard
was
applied
to
each
gel,
and
the
samples
compared
against
it,
and
each
other.
Table
3
Results
of
assays,
mean
(SE)
of
cartilage
oligomeric
matrix
protein
(COMP),
glycosaminoglycan
(GAG),
matrix
metalloproteinase
2
(MMP-2),
matrix
metalloproteinase
9
(MMP-9)
and
water
content
in
deep
digital
flexor
tendon
(DDFT)
Control,
no
lesions,
N
=
27
Control,
with
lesions,
N
=7
Diseased,
no
lesions,
N
=
8(15)
f
Diseased,
with
lesions,
N
=10(18)
f
Young
horses,
N
=7
Old
horses,
N
=
8
DNA
µg/mg
of
wet
tissue
0.33
(0.05)b
0.33
(0.08)
0.35
(0.03)
0.33
(0.04)
0.46
(0.1)
0.41
(0.06)
COMP
µg/mg
of
wet
tissue
1.7
(0.4)
0.6
(0.1)a
2.0
(0.5)
3.1
(0.7)
0.3
(0.05)a
3.2
(0.7)
GAG
µg/mg
of
wet
tissue
26.3
(2)c
26.4
(6.3)
24.9
(2.8)
28.0
(5.0)
30.7
(3.4)
26.6
(4)
MMP-2
relative
amountg
21.7
(5.2)
d
37.5
(7.5)a
16.9
(3)
8
34.7
(5.1)a
4.3
(1.7)
8
3.8
(1.4)a
MMP-9
relative
amountg
4.1
(1.1)
9.4
(2.7)
15.9
(5.3)a
25.6
(12.5)a
6.9
(2.1)
Water%
61.1
(1.6)e
60.9
(0.9)
65.2
(1.0)
63.9
(0.9)
61.1
(0.9)
63.6
(1.6)
Relative
amount
of
MMP-9
was
increased
in
horses
with
navicular
disease.
Both
sound
horses
and
navicular
diseased
horses
with
lesions
had
higher
MMP-2
amount
than
horses
without
lesions.
Relative
amount
of
MMP-2
was
highest
in
the
DDFT
than
other
tissue
types
analysed.
8
p
<
0.05,
in
comparison
with
sound
horses
without
lesions.
b
The
DNA
content
per
unit
weight
of
tissue
was
higher
in
the
DDFT
when
compared
to
navicular
hyaline
cartilage
and
collateral
cartilage
p
<
0.05.
c
DDFT
had
less
GAG
than
collateral
cartilage
and
fibrocartilage
(p
<
0.05).
d
DDFT
had
higher
MMP-2
amounts
than
other
tissue
types
analysed
p
<
0.05.
'Water
content
was
lower
in
DDFT
than
in
navicular
hyaline
or
fibrocartilage
or
collateral
cartilage.
f
N
is
number
of
horses
and
number
in
brackets
describes
individual
samples
from
which
means
were
derived.
g
The
relative
amount
of
metalloproteinases
was
compared
between
samples.
To
determine
the
amounts
of
metalloproteinase,
a
neutrophil
standard
was
applied
to
each
gel,
and
the
samples
compared
against
it,
and
each
other.
40%
of
all
tissues
from
sound
horses
had
MMP-9
present.
Approximately
50%
of
hyaline
and
fibrocarti-
lage
from
lame
horses
had
MMP-9
present.
The
MMP-9
amounts
in
tendons
of
lame
horses
with
lesions
were
significantly
higher
than
in
tendons
of
sound
horses
without
lesions
(p
<
0.05).
118
M
Viitanen
et
aL
/
Research
in
Veterinary
Science
75
(2003)
113-120
The
water
content
in
navicular
hyaline
and
fibrocar-
tilage
was
approximately
70%
and
did
not
significantly
vary
between
sound
and
lame
horses.
3.2.
Age-related
biochemical
changes
The
navicular
fibrocartilage
of
young
horses
had
a
higher
cellularity
when
compared
with
any
hyaline
car-
tilage
(p
<
0.05).
The
GAG
content
decreased
with
age
in
both
na-
vicular
hyaline
and
fibrocartilage
(p
<
0.05).
Interest-
ingly,
the
only
statistically
significant
age-related
change
in
MMP-2
was
a
peak
in
tendons
from
middle-aged
horses
(p
<
0.05).
No
measurable
MMP-9
was
present
in
young
horses,
whereas
approximately
50%
of
old
animals
had
MMP-9
in
the
DDFT.
3.3.
Comparison
of
fibrocartilages
The
DNA
content
per
unit
weight
of
cartilage,
a
measure
of
the
tissue
cellularity,
was
higher
in
meniscus
and
tendon
when
compared
with
navicular
hyaline
cartilage
and
collateral
cartilage
(p
<
0.05).
Navicular
fibrocartilages
had
more
DNA
than
hyaline
cartilages
but
these
differences
were
not
significant.
When
comparing
different
types
of
fibrocartilage,
the
meniscus
had
a
higher
COMP
content
than
collateral
cartilage
and
navicular
fibrocartilage
but
this
difference
was
not
statistically
significant.
COMP
values
were
significantly
higher
in
navicular
hyaline
cartilage
than
in
other
tissues
(p
<
0.05).
GAG
content
of
the
fibrocartilages
was
the
highest
in
collateral
cartilage
and
lowest
in
tendon.
Navicular
fi-
brocartilage
and
the
meniscus
had
similar
GAG
con-
tents.
Differences
were
statistically
significant
when
comparing
collateral
cartilage
and
fibrocartilage
to
the
tendons
(p
<
0.05).
When
comparing
tendons,
fibrocartilage,
collateral
cartilage
and
meniscus,
it
was
noted
that
the
relative
MMP-2
amount
was
highest
in
tendons
and
that
na-
vicular
fibrocartilage
had
higher
activity
levels
than
meniscus
and
collateral
cartilage.
These
differences
were
statistically
significant
only
for
tendons
(p
<
0.05).
There
was
no
MMP-9
detected
in
adult
collateral
car-
tilage
or
the
meniscus.
The
main
result
from
measuring
the
water
content
was
that
the
mean
water
content
of
tendons
was
61.8%
(1.6)
and
of
the
meniscus
61.2%
(1.3)
and
both
were
significantly
lower
than
in
other
tissues
(p
<
0.05).
4.
Discussion
Navicular
disease
has
been
diagnosed
even
without
radiographic
evidence
of
lesions
in
the
navicular
fibro-
cartilage
(Ackerman
et
al.,
1977).
Eight
horses
in
this
study
were
diagnosed
clinically
as
having
pain
in
the
navicular
area,
but
there
were
no
gross
pathological
le-
sions
on
post-mortem
examination.
In
addition,
seven
horses
had
lesions
of
the
navicular
fibrocartilage,
with
no
history
of
lameness
or
other
signs
of
pain
in
the
area.
Slight
yellowish
discoloration
of
the
distal
and
proximal
ridges
of
the
palmar
surface
of
the
navicular
bone
is
thought
to
be
a
normal
ageing
change
(Cones,
1979).
However,
gross
pathological
changes,
including
ulcera-
tion
of
the
fibrocartilage
and
adhesions
between
the
DDFT
and
the
navicular
fibrocartilage,
are
usually
as-
sociated
with
clinical
signs
of
navicular
disease
(Wright
et
al.,
1998).
We
have
identified
for
the
first
time
bio-
chemical
differences
in
the
extracellular
matrix
compo-
sition
of
navicular
hyaline,
fibrocartilage
and
tendon
between
healthy
horses,
animals
which
are
lame
but
without
lesions,
animals
which
are
sound
with
lesions
and
lame
animals
with
lesions
of
the
fibrocartilage.
It
is
possible
that
sound
horses
with
lesions
might
have
de-
veloped
navicular-type
lameness
if
they
had
lived
longer,
and
equally
horses
with
pain
in
the
navicular
area
might
have
developed
lesions
later
on
in
their
life.
In
this
study,
erosion
of
navicular
fibrocartilage
was
reflected
in
biochemical
changes
in
the
extracellular
matrix
of
hyaline
cartilage.
The
COMP
content
of
hy-
aline
cartilage
and
tendon
in
horses
with
navicular
dis-
ease
was
significantly
higher
than
in
that
of
sound
horses.
Interestingly,
this
response
was
greater
in
hyaline
cartilage
than
in
fibrocartilage.
The
increase
in
COMP
production
may
be
an
attempt
to
repair the
tissue.
COMP
levels
were
increased
in
fibrocartilage
of
lame
horses
as
well
when
compared
with
fibrocartilage
of
sound
horses,
but
this
difference
was
not
statistically
significant.
It
has
been
hypothesised
that
increased
levels
of
COMP
are
produced
in
response
to
loading
(Smith
et
al.,
1997).
To
date,
there
are
no
reports
in
the
litera-
ture
of
the
navicular
hyaline
cartilage
showing
any
changes
in
navicular
disease,
and
direct
communication
between
the
navicular
bursa
and
the
DIP-joint
is
rare
(Bowker
et
al.,
1993).
However,
in
navicular
disease,
changes
observed
in
fluid
from
the
navicular
bursa
are
also
seen
in
the
synovial
fluid
of
the
DIP-joint
(Viitanen
et
al.,
2001).
One
possible
explanation
for
the
bio-
chemical
changes
observed
in
the
hyaline
cartilage
is
that
any
abnormal
pressure
exerted
on
the
bone,
from
a
palmar
direction,
will
be
transmitted
to
the
dorsal
surface,
and
the
hyaline
cartilage
could
therefore
be
subjected
to
the
same
forces
leading
to
the
similar
bio-
chemical
changes
as
those
seen
in
navicular
fibrocarti-
lage.
It
is
also
possible
that
some
smaller
molecules,
such
as
the
pro-inflammatory
cytokines
arising
from
erosion
sites,
may
pass
through
the
navicular
membrane
and
thus
cause
changes
in
the
DIP
joint
as
well.
Both
hyaline
cartilage
and
fibrocartilage
of
sound
and
lame
horses
with
fibrocartilage
lesions
had
a
sig-
nificantly
lower
GAG
content
than
comparable
cartilage
M.
Viitanen
et
al.
/
Research
in
Veterinary
Science
75
(2003)
113-120
119
collected
from
sound
and
lame
horses
without
lesions.
Loss
of
proteoglycan
from
cartilage
is
associated
with
osteoarthritis.
These
results
indicate
that
loss
of
GAG
is
associated
with
loss
of
extracellular
matrix,
and
not
pain
or
lameness,
which
are
the
main
clinical
reasons
for
di-
agnosing
navicular
disease.
Interestingly,
GAG
content
was
significantly
lower
in
navicular
hyaline
cartilage
in
horses
with
lesions
in
their
navicular
fibrocartilage
and
DDFT,
even
though
no
gross
pathological
lesions
were
found
in
the
hyaline
cartilage
itself.
This
indicates
that
the
disease,
which
affects
the
fibrocartilage,
may
also
induce
biochemical
changes
in
associated
tissues.
Analysis
of
the
`aggrecanase'
and
metalloproteinase-
generated
cleavage
fragments
of
aggrecan,
the
main
proteoglycan
in
cartilaginous
tissues,
did
not
indicate
any
variations
associated
with
disease.
The
most
abun-
dant
proteoglycan
in
both
hyaline
and
fibrocartilage
is
aggrecan.
This
is
lost
from
tissues
by
cleavage
at
two
principal
sites
near
to
the
G1
domain
by
activities
of
both
the
`aggrecanase'
and
metalloproteinase
families
of
enzymes.
Although
this
immunological
technique
is
semi-quantitative,
it
can
reveal
major
changes
in
the
expression
of
these
fragments
(Bird
et
al.,
2000).
Dis-
ease-related
changes
in
the
expression
of
these
epitopes
in
arthritic
cartilage
have
been
previously
reported
(Bonassar
et
al.,
1997).
The
lack
of
change
in
expression
with
the
onset
of
disease
is
thought
to
be
because
these
changes,
which
occur
principally
at
the
lesion
site,
are
too
subtle
to
be
detected
by
this
type
of
analysis.
Cartilage
destruction
in
osteoarthritis
(OA)
is
associ-
ated
with
increased
levels
of
several
matrix
metallopro-
teinases
(MMPs),
including
the
gelatinases
MMP-2
and
MMP-9.
While
increases
in
some
MMPs
may
be
de-
structive,
up-regulation
of
others
may
result
from
in-
creases
in
normal
tissue
turnover
(Thompson
et
al.,
2001).
MMP-2
amount
was
increased
in
navicular
hyaline
car-
tilage,
in
fibrocartilage
and
in
DDFT
of
lame
horses
with
lesions
even
though
the
difference
was
not
significant
in
the
case
of
fibrocartilage.
The
MMP-2
amount
was
sig-
nificantly
higher
in
all
tendon
tissues
when
compared
to
the
other
tissue
types.
This
could
indicate
higher
turnover
in
tendon
tissues
due
to
higher
forces
in
the
deep
digital
flexor
tendon
in
the
navicular
area.
High
relative
activity
of
MMP-9
is
observed
in
os-
teoarthritis
and
rheumatoid
arthritis.
As
much
as
79%
of
tendons
of
lame
horses
had
MMP-9
present
and
the
relative
amounts
were
higher
than
in
tendons
of
sound
horses.
This
may
possibly
explain
some
of
the
destruc-
tive
processes
in
tissues.
Increased
pressure
of
the
ten-
don
on
the
bone
may
have
a
role
in pathogenesis
of
navicular
disease
(Pool
et
al.,
1989).
Some
clinical
cases,
which
have
DDFT
lesions
in
the
absence
of
radio-
graphically
discernible
bone
pathology
have
been
de-
scribed
(Wright
et
al.,
1998).
Cartilage
analysed
in
this
study
was
obtained
from
the
entire
palmar
and
dorsal
surface
of
the
navicular
bone.
Regional
variation
in
the
biochemistry
of
equine
articular
cartilage
is
well
known
(Brama
et
al.,
2000).
Most
lesions
in
navicular
disease
occur
near
the
sagittal
ridge
of
the
navicular
bone
(Turner,
1986).
It
is
possible
that
if
the
samples
had
been
collected
only
from
the
affected
areas,
the
differences
between
the
groups
would
have
been
more
obvious.
However,
this
amount
of
tis-
sue
is
insufficient
to
complete
all
the
analyses
in
this
study.
In
this
study,
the
cellularity
of
fibrocartilage
de-
creased
with
age
suggesting
that
this
is
a
common
re-
sponse
in
cartilagenous
tissues
(Platt
et
al.,
1998).
A
decrease
in
GAG
content
with
age
was
also
observed
in
both
fibrocartilage
and
hyaline
cartilage.
However,
Platt
et
al.
(1998)
found
no
change
in
GAG
content
of
hyaline
cartilage
relative
to
tissue
dry
weight.
The
reasons
for
the
difference
between
these
findings
are
unclear,
but
may
be
attributable
to
the
number
of
adolescent
animals
used
in
the
studies
and
the
statistical
analyses used.
Age
also
has
an
influence
on
MMP-9
expression
in
these
tissues.
This
enzyme
was
identified
in
all
tissues
in
40%
of
adult
horses,
but
was
not
found
in
any
tissues
from
adolescent
horses.
The
reasons
for
this
increase
with
age
are
unclear,
but
are
consistent
with
a
proposed
role
in
tissue
maintenance
(Thompson
et
al.,
2001).
The
biochemistry
of
navicular
fibrocartilage
was
compared
to
other
fibrocartilages
since
these
are
thought
to
vary
depending
on
anatomical
locations
and
may
consequently
respond
differently
to
disease
pro-
cesses.
The
COMP
content
of
the
meniscus
was
higher
than
in
collateral
cartilage
and
navicular
fibrocartilage
suggesting
that
meniscus
is
adapted
to
resist
higher
loads
than
the
other
two
fibrocartilages.
GAG
levels
were
lowest
in
collateral
cartilage,
which
was
the
only
non-compressional
fibrocartilage
analysed.
Although
MMP-9
was
identified
in
40%
of
adult
healthy
fi-
brocartilage,
none
was
found
in
collateral
cartilage
or
meniscus.
In
this
study
we
have
identified
subgroups
within
animals
present
with
navicular
disease.
We
have
also
shown
that
biochemical
changes
occur
in
tissues
asso-
ciated
with
fibrocartilage
from
diseased
joints.
Horses
with
navicular
disease
have
increased
amounts
of
COMP
in
their
navicular
hyaline
cartilage.
MMP-2
amounts
are
increased
in
the
DDFT
and
navicular
hy-
aline
cartilage,
and
GAG
content
decreased
in
navicular
hyaline
and
fibrocartilage
in
diseased
joints.
GAG
content
also
decreased
in
sound
horses,
which
had
le-
sions
in
their
navicular
fibrocartilage.
This
study
has
associated
biochemical
changes
in
the
articular
cartilage
of
the
DIP-joint
with
changes
in
the
palmar
aspect
of
the
navicular
bone
and
the
bursa
in
navicular
disease.
In
this
disease
there
are
matrix
changes
in
navicular
hyaline
and
fibrocartilage
and
the
DDFT
with
potential
impli-
cations
for
the
clinical
significance
and
management
of
the
condition.
120
M
Viitanen
et
aL
/
Research
in
Veterinary
Science
75
(2003)
113-120
Acknowledgements
We
are
grateful
to
Finnish
Foundation
of
Veterinary
Sciences,
Research
and
Science
Foundation
of
Farmos
and
Home
of
Rest
for
Horses
for
financial
support.
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