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Initiation of degenerative joint damage by experimental bleeding combined with loading of the jointA possible mechanism of hemophilic arthropathy.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 6, June 2004, pp 2024–2031
DOI 10.1002/art.20284
© 2004, American College of Rheumatology
Initiation of Degenerative Joint Damage by
Experimental Bleeding Combined With Loading of the Joint
A Possible Mechanism of Hemophilic Arthropathy
Michel J. J. Hooiveld, Goris Roosendaal, Kim M. G. Jacobs, Marieke E. Vianen,
H. Marijke van den Berg, Johannes W. J. Bijlsma, and Floris P. J. G. Lafeber
altered, as shown by histologic damage. Histologic analysis also revealed signs of synovial inflammation. These
effects were not observed 10 weeks after the experimental bleedings in joints that did not undergo forced
loading.
Conclusion. Experimental joint bleedings when
combined with loading of the affected joint resulted in
features of progressive degenerative joint damage,
whereas similar joint hemorrhages without joint loading
did not. This might reflect a possible mechanism of joint
damage in hemophilia.
Objective. To investigate the effect of a limited
number of experimental joint bleedings, combined with
loading of the affected joint, on the development of
progressive degenerative joint damage.
Methods. The right knee of 8 mature beagle dogs
was injected with freshly collected autologous blood 3
times per week for 4 weeks, to mimic a limited number
of joint hemorrhages occurring over a short period. To
ensure loading of the experimental joint, the contralateral control knee of the animals was fixed to the trunk 4
hours per day, 3 days per week. Ten weeks after the last
injection, cartilage tissue and synovium were collected
from both knees to analyze features of joint degeneration. Cartilage was prepared for analysis of proteoglycan turnover (synthesis, retention, release, and content)
and histologic features. Synovium was prepared for
histologic analysis.
Results. The rate of proteoglycan synthesis was
significantly increased, characteristic of degenerative
cartilage damage as seen in osteoarthritis. Release of
newly formed proteoglycans (as a measure of retention)
and total loss of proteoglycans from the cartilage matrix
were increased. Cartilage matrix integrity was adversely
Blood-induced arthropathy is the most common
clinical phenomenon in patients with severe hemophilia
(⬍1% clotting factor VIII or IX) (1). Current opinion
regarding the pathogenesis of hemophilic arthropathy is
that recurrent intraarticular bleeding leads to production of catabolic factors by inflamed synovium. Although
synovectomy improves clinical status in these patients, it
does not end the progressive cartilage damage (2,3).
Moreover, hemophilic arthropathy has been described
to be more degenerative in nature, as seen in osteoarthritis, as opposed to inflammatory, as in rheumatoid
arthritis (4).
Previous studies have shown that synovitis is
indeed not primarily responsible for the cartilage damage after intraarticular bleeding, but rather that exposure of cartilage to blood induces direct adverse effects
(5,6). These cartilage alterations are primarily induced
by the combination of mononuclear cells and red blood
cells. This combination of blood cells has been found to
act on chondrocyte activity, leading to a long-lasting
inhibition of cartilage matrix synthesis and an increased
loss of matrix components in vitro (6). These in vitro
effects have been sustained for at least 10 weeks (7).
Supported by grants from Aventis Behring (Zaventem, Belgium) and the van Creveld Foundation (Utrecht, The Netherlands).
Michel J. J. Hooiveld, PhD, Goris Roosendaal, MD, PhD,
Kim M. G. Jacobs, BSc, Marieke E. Vianen, BSc, H. Marijke van den
Berg, MD, PhD, Johannes W. J. Bijlsma, MD, PhD, Floris P. J. G.
Lafeber, PhD: University Medical Center Utrecht, Utrecht, The
Netherlands.
Address correspondence and reprint requests to Floris
P. J. G. Lafeber, PhD, Department of Rheumatology & Clinical
Immunology, F02.127, University Medical Center Utrecht, PO Box
85500, 3508 GA, Utrecht, The Netherlands. E-mail:
F.Lafeber@azu.nl.
Submitted for publication May 20, 2003; accepted in revised
form February 16, 2004.
2024
JOINT BLEEDINGS AND LOADING IN HEMOPHILIC ARTHROPATHY
Moreover, in vitro experiments showed that chondrocyte
apoptosis is involved in these unfavorable events (8).
In vivo animal studies have shown that adverse
changes in cartilage matrix can be induced by a single
4-day experimental intraarticular bleeding. These
changes were still present after a 12-day recovery period
and appeared to be independent of primary synovial
inflammation (9). However, the in vivo effects were less
pronounced than those observed in vitro. Apparently,
factors in vivo that are not present in vitro are able to
partially counteract the unfavorable effects of blood on
cartilage.
In the clinical setting it has been shown that early
treatment of joint bleedings in patients with hemophilia
may protect against arthropathy in later years; starting
treatment with clotting factors after the third joint
hemorrhage during early childhood results in better
clinical outcome after 22 years of followup (10). However, this early prophylaxis cannot completely prevent
arthropathy. This corroborates our findings, from in
vitro and animal studies, that only a few bleeding
episodes may induce permanent adverse alterations in
cartilage matrix turnover and, consequently, cartilage
degeneration after a period of years.
In contrast with these data, however, are the
results of a previous in vivo study in which we found that
a limited number of experimental joint bleedings in dogs
did not lead to adverse changes in cartilage matrix
turnover, evaluated 10 weeks after the last injection (7).
This suggests that in vivo, factors in addition to blood
exposure are needed for induction of progressive degenerative features of joint damage. Therefore, in the
present study we combined a series of experimental joint
bleedings with loading of the affected joint to investigate
whether the latter is of importance in the development
of characteristics of degenerative joint damage after
joint bleeding.
MATERIALS AND METHODS
Animals. Eight skeletally mature beagle dogs (mean ⫾
SD age 1.8 ⫾ 0.1 years, mean ⫾ SD weight 9.9 ⫾ 1.2 kg) were
obtained from the animal facilities of Utrecht University. They
were housed in groups of 2 and were let outside daily for at
least 2 hours on a large patio. The animals were fed a standard
commercial diet with water ad libitum. Approval for this study
was obtained from the University Animal Experiments Ethical
Committee.
Experimental hemorrhages. Freshly collected autologous blood was injected into the right knee with the animal
under short-term anesthesia (Domitor/AntiSedan; Orion
Farmos, Turku, Finland). The intraarticular injection of blood
was confirmed by placing a second needle on the contralateral
2025
side of the joint, through which blood flowed after injection.
When this occurred, the second needle was removed and 3 ⫾
1 ml of blood (mean ⫾ SD) was injected. Thus, blood was
added to synovial fluid, closely mimicking conditions that occur
in hemophilia. Distension of the knee was moderate and was
visible in the first 24 hours and palpable until 48 hours after the
injection. No cumulative effect in this respect was observed.
Anticoagulants were not used. The contralateral control knee
was not injected at all, to prevent possible traumatic bleeding.
Blood was injected 3 times per week for 4 consecutive weeks.
We have found that up to 14 repeated injections with vehicle in
7 weeks (as a control for the experimental injections), using the
same protocol of 2 needles entering the joint, did not result in
changes to cartilage or synovial tissue (ref. 11, and Hooiveld
MJJ, et al: personal observation).
In addition to the blood injections, the animals underwent a forced loading protocol for the injected joints. The
contralateral control knee was fixed to the trunk for 4 hours
per day, 3 days per week. Thus, during 7% of the duration of
the experiment, the blood-injected knee was forced to be
loaded. Importantly, this not only results in loading during the
fixation procedure, but also facilitates normal usage of the
blood-injected joints during the time between the periods of
forced loading. The frequency of loading was based on the
findings of previous studies in which mechanically induced
cartilage damage combined with this loading profile led to
features of osteoarthritis (12). At the end of the experiment, 10
weeks after the last injection (week 14), the dogs were killed by
intravenous injection of 10 ml of Euthesate (200 mg/ml; Cefa
Sante Animal BV, Maassluis, The Netherlands).
Results from this study were compared with the results
obtained in a study using an experimental procedure that was
identical with respect to animals, housing, feeding, blood
injections, and followup, except that in the earlier study (of 6
dogs) no forced loading was applied (7). Importantly, in that
study, in which the dogs did not undergo forced loading, they
were reluctant to load the experimental blood-injected joints
during and shortly after the period of injection, as indicated by
the fact that they walked on 3 limbs and did not load the bloodinjected joint.
After the dogs were killed at the end of the experiments, both hind limbs were amputated and synovium and
cartilage were collected and processed within 2 hours. Procedures were carried out under laminar flow conditions. Synovium from the suprapatellar pouch and cartilage from the
condyles and plateau of both knee joints were collected under
aseptic conditions and analyzed according to standard procedures as described below.
Cartilage preparation. Cartilage samples were obtained from weight-bearing areas and were scored according to
a slight modification of the Mankin criteria (13). The cartilage
was cut as thick as possible, taking care to exclude the
underlying bone, and was then cut into square pieces (mean ⫾
SD 6.2 ⫾ 3.1 mg). Within 3 hours after death of the animals,
the cartilage tissue samples were individually placed in culture
medium in 96-well round-bottomed microtiter plates, for a
1-hour equilibration period. For both femoral and tibial cartilage, the proteoglycan synthesis rate, DNA content, and levels
of proteoglycan retention, release, and content were determined. In addition, samples were prepared for histologic
analysis.
2026
Measurement of chondrocyte activity. Proteoglycan
synthesis rate. As a measure of chondrocyte matrix synthesis,
the synthesis of proteoglycan, one of the main matrix components, was determined by assessment of the rate of sulfate
incorporation (14). Briefly, after 1 hour of preincubation, 148
kBq of Na235SO4 (NEX-041-H, carrier free) (DuPont, Kortrijk, Belgium), in 10 ␮l Dulbecco’s modified Eagle’s medium
was added to 200 ␮l of culture medium. After a 4-hour labeling
period, samples were washed twice with ice-cold phosphate
buffered saline and digested with papain (Sigma, Zwijndrecht,
The Netherlands). Sulfated proteoglycans (glycosaminoglycans
[GAGs]) were precipitated by addition of cetylpyridinium
chloride (Sigma). The 35SO42⫺-labeled proteoglycans in the
precipitate were measured by liquid scintillation analysis. The
rate of sulfate incorporation was normalized to the specific
activity of the medium and expressed as nmoles of sulfate
incorporated per hour per gram wet weight of cartilage tissue.
Because the proteoglycan synthesis rate is dependent
on cell numbers, the DNA content of the samples was determined as a measure of cellularity. Part of the papain digest was
used for measurement of DNA, using the fluorescent dye
Hoechst 33258. Calf thymus DNA (D-4764; Sigma) was used
as a reference. The amount of sulfate incorporated was also
normalized to the DNA content of the samples and expressed
as nmoles per hour per milligram DNA.
Proteoglycan retention and release. As a measure of
retention of newly synthesized proteoglycans in the cartilage,
the release of 35SO42⫺-labeled proteoglycans in a 3-day culture
medium was determined, using experimental procedures that
have been described previously (14). Labeling with 35SO42⫺
was performed as described above. After this 4-hour labeling,
samples were rinsed thoroughly and subsequently incubated in
fresh medium for 3 days. GAGs were precipitated with Alcian
blue. The 35SO42⫺-labeled GAGs were measured by liquid
scintillation analysis, and release was normalized to specific
activity of the medium and the wet weight of the explants,
expressed as nmoles per milligram released in 3 days. Because
production of newly formed proteoglycans depends on the
proteoglycan synthesis rate, release of these newly formed
proteoglycans was also normalized to proteoglycan synthesis
and expressed as the percentage release of newly formed
proteoglycans.
In addition, the Alcian blue–precipitated GAGs were
quantified spectrophotometrically with chondroitin sulfate as a
reference. This revealed the total release of proteoglycans
(resident and newly formed), expressed as GAG released per
wet weight of the cartilage sample (milligrams per gram).
Because release depends on the content of proteoglycans, total
release was also normalized to proteoglycan content and
expressed as the percentage release of the total amount of
proteoglycans.
Determination of cartilage matrix characteristics. Proteoglycan content. The amount of GAG in cartilage was determined as a measure of the proteoglycan content of the
cartilage samples (14). In the papain digests, GAGs were precipitated and stained with Alcian blue, as described above. The GAG
content was expressed as GAG weight normalized to the wet
weight of the cartilage explants (milligrams per gram).
Cartilage histology. Four cartilage samples from the
femoral condyle and 4 samples from the tibial plateau in the
weight-bearing area of each knee were fixed in 4% phosphate
HOOIVELD ET AL
buffered formalin for histochemistry analysis. After fixation,
samples were dehydrated, placed in xylene, and subsequently
embedded in paraffin. Sections (4 ␮m) were deparaffinized
and stained with Safranin O–fast-green–iron hematoxylin and
scored according to a slight modification of the Mankin criteria
(13). With this modification, the maximum possible score is 11
instead of 14, with an average score of 5 for cartilage with
established osteoarthritis (4,13). At least 3 sections from each
of the 8 specimens from each knee were stained and scored
independently, in random order, by 2 observers.
Assessment of synovial inflammation. Three pieces of
synovial tissue were obtained from the suprapatellar pouch of
each knee. They were fixed in 4% phosphate buffered formalin
for histochemistry analysis. Deparaffinized sections were stained
with hematoxylin and eosin. To determine the degree of inflammation, at least 3 sections from each of the 3 specimens from each
knee were stained and scored. Synovial inflammation was defined
based on lymphocyte infiltration, hyperplasia of the synovial
lining cells, and hypertrophy of the synovial membrane, using a
slight modification of the scoring system of Goldenberg and
Cohen (15) as described previously (14). Two independent observers, unaware of the source of the tissue, scored the tissue
samples separately and in random order.
Statistical analysis. Power analysis was performed to
calculate the number of animals needed in each group to
achieve statistical power of ␣ ⫽ 0.05 and ␤ ⫽ 0.10, based on
the differences observed in our previous experiments (7,9). We
calculated that 6 animals per group would be sufficient to
achieve reliable statistical outcomes. Cartilage parameters
were determined in 6 cartilage explants from fixed locations
from both femoral condyles and tibial plateaus. Because, for all
parameters, no differences were found for values obtained
from the 6 samples from the condyles and the 6 samples from
the plateau, the means of the 12 samples for all parameters
were taken as representative of that particular knee. A retrospective comparison with previously published data (7) was
made. Mean ⫾ SEM values (n ⫽ 8 animals for the current
data, n ⫽ 6 for the retrospective data [the group with joint
loading and group without joint loading, respectively]) are
presented. To determine the statistical significance of differences between control and experimental knees within a group,
we used a nonparametric test (Wilcoxon’s 2-sample test).
Differences in the contralateral control joints between the 2
groups were tested with a nonparametric test for independent
samples (Mann-Whitney U test). For comparison of the effects
of blood injections with forced loading compared with blood
injections alone, the difference between blood-injected joints
and contralateral control joints was calculated and averaged
for each group. Percentage differences were calculated for
biochemical parameters and absolute differences for histologic
parameters. For comparison of the 2 groups in terms of these
parameters, the Mann-Whitney U test was again used. Statistical analyses were performed using SPSS, version 10.0. P
values less than or equal to 0.05 were considered significant.
RESULTS
Effects of blood injection when combined with
forced loading. Chondrocyte activity. The rate of proteoglycan synthesis, expressed per milligram wet weight of
JOINT BLEEDINGS AND LOADING IN HEMOPHILIC ARTHROPATHY
2027
Figure 1. Proteoglycan turnover, measured 10 weeks after the last intraarticular injection of blood into the right knee of the animals. Values are
the mean and SEM from 8 animals, for the blood-injected joints (solid bars) and the contralateral control joints (open bars). Asterisks indicate a
statistically significant difference versus control joints. A, Proteoglycan synthesis rate as measured by the rate of sulfate incorporation. Proteoglycan
synthesis per mg wet weight of tissue was significantly increased in the experimental joints (39 ⫾ 8%; P ⬍ 0.01). The proteoglycan synthesis rate per
cell was equally increased (30 ⫾ 10%; P ⬍ 0.01) (data not shown). B, Release of newly formed proteoglycans, as a measure of the retention of these
newly formed proteoglycans, during 3 days. An increased release of newly formed proteoglycans was observed in the experimental joints (30 ⫾ 14%;
P ⬍ 0.03). C, Total release of glycosaminoglycan (GAG) during 3 days of ex vivo culture. A 16 ⫾ 6% increase in the release of GAG was found
in the experimental joints (P ⬍ 0.03). When normalized to the GAG content, a similar increase was observed (14 ⫾ 4%; P ⬍ 0.02) (data not shown).
These results show that after blood exposure, GAGs (resident and newly formed) are lost from the cartilage matrix.
tissue, was significantly enhanced in the experimental
joints (blood-injected with forced loading) compared
with the contralateral control joints. The mean ⫾ SEM
increase was 39 ⫾ 8% (P ⬍ 0.01) (Figure 1A). This
increase in the synthesis rate was not due to a significant
change in chondrocyte number, since the rate of proteoglycan synthesis per DNA content (as a measure of
chondrocyte number) was also significantly higher in the
experimental joints (30 ⫾ 10%; P ⬍ 0.01). The difference in DNA content between control and experimental
joints was only 7 ⫾ 4% (P not significant). The release of
newly formed proteoglycans, as a measure of retention
of these newly formed proteoglycans, was significantly
increased in the blood-injected joints with forced loading compared with the contralateral control joints (30 ⫾
14%; P ⬍ 0.03) (Figure 1B). Even when normalized to
the proteoglycan synthesis rate, a 5% increase in release
of newly formed proteoglycans was observed. Release of
the total amount of proteoglycans (resident and newly
formed [measured as Alcian blue–precipitated GAG])
2028
HOOIVELD ET AL
Figure 2. Changes in cartilage matrix integrity after exposure to blood.
Values are the mean and SEM from 8 animals, for the blood-injected
joints (solid bars) and the contralateral control joints (open bars).
Asterisks indicate a statistically significant difference versus control joints.
A, Total content of glycosaminoglycan (GAG) in cartilage, as measured
by Alcian blue precipitation of digested cartilage samples. There was a
small, statistically nonsignificant, decrease in proteoglycan content in the
experimental joints. B, Cartilage integrity based on histologic findings.
Cartilage samples were stained with Safranin O, and cartilage degeneration was scored according to a modification of the Mankin score. There
was an increase in the histologic score in cartilage from the experimental
joints (0.9 ⫾ 0.2 units; P ⬍ 0.01).
was significantly increased in the experimental joints
(16 ⫾ 6%; P ⬍ 0.03) (Figure 1C). When total proteoglycan release was normalized to proteoglycan content
(percentage release), a similar increased loss of these
matrix molecules was found (14 ⫾ 4%; P ⬍ 0.02).
Cartilage integrity. As a measure of cartilage integrity, proteoglycan content was determined. There
appeared to be a small decrease (mean ⫾ SEM 2 ⫾ 3%)
in the amount of proteoglycans in the blood-injected
joints with forced loading (P not significant) (Figure
2A). However, when cartilage integrity was evaluated
histologically, a statistically significant increase in the
modified Mankin score in the blood-injected joints with
forced loading, compared with the contralateral control
joints, was found (change of 0.9 ⫾ 0.2 units; P ⬍ 0.01)
(Figure 2B). Changes consisted mainly of slight (1 point)
to moderate (2 points) losses of Safranin O staining, and
surface irregularities (1 point). Although the mean difference of 0.9 units might seem small, it should be kept in
mind that the average score of cartilage with established
osteoarthritis has been reported to be ⬃5 (4,13).
Synovial inflammation. Synovial inflammation
was assessed 10 weeks after the last experimental hemorrhage, a period with intermittent forced loading of the
blood-exposed joint. The score was slightly but statistically significantly increased in experimental versus contralateral joints (mean ⫾ SEM 1.5 ⫾ 0.3 versus 0.6 ⫾
0.1; P ⬍ 0.01).
Validity of use of the contralateral joint as a
control during forced loading. It could be questioned
whether the contralateral control joints of the animals
with intermittent forced loading were proper controls,
although forced loading was applied for only 7% of the
total experiment time. Fixing the contralateral joint to
the trunk could induce changes in cartilage that would
make these joints inappropriate to use as controls. For
this reason we retrospectively compared the findings in
contralateral control joints of the animals used in the
present study with the values from animals that underwent an identical protocol but without the intensified
loading (7). These data are presented in Table 1. There
were no significant differences observed for any of the
chondrocyte activity, cartilage integrity, or synovial in-
Table 1. Parameters measured in the contralateral control joints of the animals with experimental
hemorrhages and forced loading and of the animals with experimental hemorrhages but no forced
loading*
Chondrocyte activity
Proteoglycan synthesis, nmoles sulfate/hour/gm wet weight
Proteoglycan synthesis, nmoles sulfate/hour/mg DNA
Release of newly formed proteoglycans, nmoles/gm in 3 days
Release of newly formed proteoglycans, %
Total release of proteoglycans, mg GAG/gm wet weight
Total release of proteoglycans, %
Cartilage integrity
Proteoglycan content, mg GAG/gm wet weight
Histologic damage, modified Mankin score
Synovial inflammation, modified Goldenberg/Cohen score
Group with
joint loading
(n ⫽ 8)
Group without
joint loading
(n ⫽ 6)
6.9 ⫾ 0.3
45.7 ⫾ 2.0
0.8 ⫾ 0.1
26.3 ⫾ 1.0
3.0 ⫾ 0.2
10.7 ⫾ 0.5
6.6 ⫾ 1.1
36.7 ⫾ 6.5
NA
NA
3.0 ⫾ 0.3
9.9 ⫾ 0.9
26.1 ⫾ 1.0
0.9 ⫾ 0.1
0.6 ⫾ 0.1
26.6 ⫾ 0.7
1.0 ⫾ 0.2
0.6 ⫾ 0.2
* The group without joint loading consisted of animals from a previous study (7). Values are the mean ⫾
SEM. There were no statistically significant differences between the 2 groups for any of the parameters
investigated in the contralateral control joints. NA ⫽ not available; GAG ⫽ glycosaminoglycan.
JOINT BLEEDINGS AND LOADING IN HEMOPHILIC ARTHROPATHY
Figure 3. Effect of loading of the experimental joint on cartilage
matrix turnover. Values are the mean and SEM percentage change in
experimental versus contralateral control joints in the animals without
intensified joint loading (n ⫽ 6) (open bars) and the animals with
intensified joint loading (n ⫽ 8) (hatched bars). P values for differences between the groups with and without loading are indicated;
asterisks indicate statistically significant change within each group,
compared with the contralateral joints. A, Changes in proteoglycan
synthesis. Both groups showed a significant increase in proteoglycan
synthesis in the experimental knee compared with that in the contralateral knee, suggesting repair activity. The difference between the group
with and the group without loading was not significant. B, Changes in
total release of glycosaminoglycan (GAG). In experimental joints that
had not been loaded, release of GAG was decreased compared with
the contralateral joints, but the difference was not significant. However, if loading was applied, an increase in release of GAG, and
consequently a loss of cartilage matrix, was observed (P ⬍ 0.05).
flammation parameters measured. Thus, fixation of the
contralateral control joint does not alter cartilage matrix
turnover in this joint. This indicates that the use of the
contralateral joint as a control as was done in the present
study, even in combination with the forced loading
protocol, is appropriate.
Effect of forced loading versus no forced loading
after blood exposure. In a group of animals on which we
have reported previously (7), beagle dogs were studied
using a protocol identical to the one in the present study,
but without the forced loading. To evaluate the effects of
experimental bleeding with versus without forced loading, the results of that study were compared with the
present findings.
Chondrocyte activity. Figure 3A shows the percentage difference in proteoglycan synthesis compared
with the contralateral joints, in animals that underwent
repeated hemorrhages with joint loading and animals
that underwent these hemorrhages without loading. No
significant difference between the 2 groups was observed. In both groups, repair activity was observed.
When normalized to DNA content, similar results were
found; DNA content in both groups did not change
significantly as a result of the intraarticular blood injec-
2029
tions (mean ⫾ SEM 7 ⫾ 11%; P not significant).
Unfortunately, no data on retention were available for
the group that did not undergo joint loading. The change
in release of proteoglycans, however, was significantly
different in the animals that did not undergo joint
loading compared with the forced loading group. Release was decreased by a mean ⫾ SEM of 12 ⫾ 6% in the
group without loading and increased by 16 ⫾ 6% in the
group that underwent loading (P ⬍ 0.02) (Figure 3B).
The same differences between groups were observed
when the release was normalized to proteoglycan content (⫺11 ⫾ 6% and ⫹14 ⫾ 4% in the group without
and the group with loading, respectively; P ⬍ 0.01).
Cartilage integrity. The proteoglycan content of
the cartilage was increased in the previous study in which
the animals did not undergo forced loading of the
experimental joints (7). In the present study with forced
loading, a decrease in proteoglycan content was observed, although the difference from the value in the
group that did not undergo loading was not statistically
significant (Figure 4A). The findings in the different
groups were paralleled by the histologic results. In the
study without forced loading of the experimental joints,
no significant differences between the control and the
Figure 4. Effect of loading of the experimental joint on cartilage
matrix integrity. Values are the mean and SEM percentage changes in
experimental versus control joints in the animals without intensified
joint loading (n ⫽ 6) (open bars) and the animals with intensified joint
loading (n ⫽ 8) (solid bars). P values for differences between the
groups with and without loading are indicated; asterisks indicate
statistically significant change within each group, compared with the
contralateral joints. A, Changes in total proteoglycan content. There
was a nonsignificant increase in proteoglycan content in the group
without loading, and a nonsignificant decrease in the group with
loading. The difference between the group with and the group without
loading was not significant. B, Changes in cartilage histologic findings
(graded according to a modification of the Mankin score). In the group
without loading the change was not significant, whereas in the group
with loading there was a significant increase in the histologic score
(P ⬍ 0.01).
2030
HOOIVELD ET AL
experimental joints were found (mean ⫾ SEM score
1.0 ⫾ 0.2 versus 0.8 ⫾ 0.1, respectively), whereas in the
present study a slight increase in histologically determined cartilage damage was evident. The change in
score was ⫺0.2 ⫾ 0.3 in the group without loading versus
⫹0.9 ⫾ 0.2 in the group with loading (P ⬍ 0.05) (Figure
4B).
Synovial inflammation. Inflammation of synovial
tissue as scored histologically did not change significantly in the group that did not undergo loading. In
contrast, as described above, a statistically significant
increase was observed in the forced loading group.
DISCUSSION
The present data demonstrate that a limited
series of joint bleedings with loading of the affected joint
adversely influences cartilage matrix turnover and integrity. In addition, increased (although still very mild)
synovial inflammation was found in the experimental
joints. The changes in chondrocyte activity are characteristic of early osteoarthritic cartilage (12,16). The
enhanced synthesis of proteoglycans is ineffective in
protecting against osteoarthritis, because the additionally formed proteoglycans are lost from the matrix, i.e.,
retention of these newly formed proteoglycans is decreased. Also, the total release of proteoglycans, mainly
resident ones, is increased. As a consequence, not
unexpectedly, decreased cartilage integrity is observed.
Although the effects are small and may not appear
dramatic, such changes observed 10 weeks after the last
blood injection might be the first step in the development of osteoarthritis. Comparable changes in chondrocyte activity, matrix integrity, and synovial inflammation
have been reported to occur 10 weeks after induction of
experimental osteoarthritis by anterior cruciate ligament
transection in dogs (12,17) or by surgically applied
chondral damage accompanied by transient intensified
loading of the affected joint (12,16).
The observed damage corroborates our previous
in vitro and short-term in vivo data showing that blood
has direct harmful effects on cartilage (5,6,9). In addition, long-term studies in which animals were injected
with blood have shown that cartilage matrix is adversely
affected (18,19). Moreover, these data corroborate clinical findings that a limited number of joint hemorrhages
in childhood, before the initiation of prophylactic treatment, may result in joint damage more than a decade
later (10). Whether the changes observed in the present
study will indeed lead over a period of years to fullblown joint damage remains to be investigated.
Interestingly, in a recent study with identical
conditions except for the loading protocol, no significant
changes that would lead to progressive degenerative
joint damage were found 10 weeks after the last injection
(7). This in contrast to the results of previous studies
which have shown that even a less intensive injection
protocol (only 2 injections) was associated with adverse
changes in cartilage matrix turnover that were observed
shortly after the last injection and were sustained for 2
weeks (9). Apparently, such changes can be reversed
completely over a successive period of 8 weeks. Because,
after several experimental bleedings, dogs can easily
walk on 3 limbs (moving the affected fourth limb without
loading it), it is possible that the absence of loading
might have been protective against the development of
joint damage after a series of experimental joint hemorrhages in our recent study. This may have provided the
necessary “rest” to the joint to allow normalization of
the adversely altered parameters and prevention of
progressive degenerative changes. In the present study
the animals were deprived of this rest. Although still a
matter of debate, it has been suggested that articular
cartilage can easily recover from repeated injuries (20).
The direct effect of intensified loading of the
affected joint in this study was not investigated. A
delicate balance determines the equilibrium between
growth and breakdown of cartilage. It is known that
mechanical signals can lead to cartilage degeneration
(21,22). However, in vitro and in vivo experiments have
shown that mildly to moderately increased cartilage
loading leads to stimulation of cartilage matrix synthesis
rather than to damage (21–23). In addition, the amount
of time that the animals were exposed to intensified
loading in the present study, being less than 7% of the
total time, is not expected to lead on its own to adverse
changes in cartilage matrix turnover (12). Priming of the
cartilage, by diminishing repair capacity as a result of the
experimental joint bleedings, appears to be necessary for
the development of the progressive joint damage as
observed (16). This adverse priming of cartilage by blood
has also been demonstrated in the case of anterior
cruciate ligament transection, where joint bleeding during surgery has been reported to facilitate the development of experimental osteoarthritis (24).
The slight increase in synovial inflammation
might be important in relation to the slow but steady
progression of joint destruction 10 weeks after joint
bleeding. In contrast to the present study with forced
loading, in the study without forced loading no inflammation was observed 10 weeks after injection, and no
joint damage was observed (7). Although the initial
JOINT BLEEDINGS AND LOADING IN HEMOPHILIC ARTHROPATHY
cartilage degeneration is caused directly by blood, synovitis is initiated and might be essential in establishing the
progressive character of joint destruction. In contrast, it
could be possible that inflammation proceeds because of
progressive cartilage damage.
The data from the present study could have
clinical implications. From general practice in treatment
of hemophilia, it is known that a limited number of joint
bleedings has much more impact in childhood than
during adulthood. It is possible that due to differences in
pain perception between children and adults, loading of
the affected joint after a joint bleed may occur earlier
and with more intensity in children than in adults. The
fact that loading has an important part in blood-induced
joint damage, as described herein, might be explanatory
in this respect. Moreover, the notion that joint loading
plays a role in the process of hemophilic arthropathy is
corroborated by the centrifugal, progressive character of
cartilage damage in these patients (4,25). The peripheral, generally much less loaded, cartilage usually
appears normal in patients with hemophilia, whereas the
central cartilage in weight-bearing areas becomes diseased. These findings suggest that avoiding or minimizing joint loading (use, but no weight bearing) might be
helpful in protecting against hemophilic arthropathy.
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