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Immature articular cartilage is more susceptible to blood-induced damage than mature articular cartilageAn in vivo animal study.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 2, February 2003, pp 396–403
DOI 10.1002/art.10769
© 2003, American College of Rheumatology
Immature Articular Cartilage Is More Susceptible to
Blood-Induced Damage Than Mature Articular Cartilage
An In Vivo Animal Study
Michel J. J. Hooiveld, Goris Roosendaal, Marieke E. Vianen, H. Marijke van den Berg,
Johannes W. J. Bijlsma, and Floris P. J. G. Lafeber
showed a mild synovitis in animals of all ages, but
catabolic inflammatory activity was found only in immature animals.
Conclusion. Joints of skeletally immature dogs
appeared to be more susceptible than joints of mature
dogs to the adverse effects of a joint hemorrhage. These
data suggest that for humans, specifically young children are at risk for joint damage after a joint hemorrhage.
Objective. Cartilage of young but skeletally mature dogs is more susceptible to blood-induced damage
than that of old dogs. The aim of the present study was
to investigate whether cartilage of skeletally immature
individuals is even more adversely affected by exposure
to blood than that of mature individuals, as suggested
by clinical practice experience with humans.
Methods. Right knees of 3 groups of 6 beagle dogs
(skeletally immature, young mature, and old animals)
were injected with autologous blood on days 0 and 2. On
day 4, cartilage matrix proteoglycan turnover (content,
synthesis, and release), synovial inflammation, and
cartilage-destructive properties of the synovial tissue
were determined and compared with those of the left
uninjected control knees.
Results. Subsequent to intraarticular bleeding,
cartilage proteoglycan content decreased in an agedependent manner, with the largest decrease occurring
in cartilage of immature animals. Proteoglycan synthesis per cell also decreased in an age-dependent manner,
with the largest decrease occurring in the immature
animals. Cartilage proteoglycan release increased in all
3 groups, but the decrease was not age dependent.
Interestingly, immature animals showed a large increase in cartilage DNA content upon exposure to blood,
whereas mature animals did not. Histologic analysis
Joint damage due to recurrent joint hemorrhages
is a common feature in patients with hemophilia (1). The
mechanism of blood-induced joint damage is not fully
understood. In previous studies, we have shown that the
combination of mononuclear cells with red blood cells,
as present in whole blood, is responsible for the initial
adverse changes in cartilage matrix turnover (2). These
initial changes are at first independent of inflammatory
mediators such as tumor necrosis factor ␣ (TNF␣) or
interleukin-1␤ (IL-1␤). Therefore, synovitis, which often
accompanies degenerative cartilage damage, is not primarily involved (3). In addition, it was demonstrated in
in vivo and in vitro studies that only a transient exposure
of cartilage to blood is needed to induce long-lasting
changes to cartilage matrix turnover, independent of
synovitis, resembling degenerative cartilage damage
(4,5).
In addition, it appeared that young age predisposed toward an increased risk of cartilage degeneration
after joint hemorrhage (6). In an in vivo animal study,
cartilage in young but skeletally mature individuals
showed more severe adverse changes in cartilage matrix
turnover than did cartilage in older animals. Furthermore, the adverse changes in cartilage matrix turnover
were found to be long lasting in both groups, but were
Michel J. J. Hooiveld, MSc, Goris Roosendaal, MD, PhD,
Marieke E. Vianen, BA, 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 Michel J. J.
Hooiveld, MSc, University Medical Center Utrecht, Room F02.127,
PO Box 85500, 3508 GA Utrecht, The Netherlands. E-mail:
M.J.J.Hooiveld@AZU.NL.
Submitted for publication June 21, 2002; accepted in revised
form October 16, 2002.
396
BLOOD-INDUCED JOINT DAMAGE
more profound in the younger group (6). These data
support the idea that early treatment of joint hemorrhages, specifically in younger individuals, is to be advised. This corroborates current ideas on treatment of
patients with hemophilia. It has been found that hemophilia patients show little arthropathy in adulthood if
they receive immediate treatment of their first joint
hemorrhages, which usually occur early in life (7).
Moreover, prophylactic treatment with clotting factors
in hemophilia patients is preferable to on-demand treatment (8). In very young (i.e., skeletally immature)
patients, prophylactic treatment yielded a better clinical
outcome after 22 years of followup (9).
Although it appears that intraarticular bleedings
have the most significant impact in young patients, the
effects of a joint hemorrhage in skeletally immature
individuals are not known. It is therefore useful to
determine whether joints of immature individuals are
even more sensitive to the destructive effects of blood.
Knowledge in this respect could be meaningful in terms
of the underlying mechanisms of blood-induced joint
damage as well as in decision-making in clinical treatment of joint hemorrhages in very young patients.
MATERIALS AND METHODS
Animals. Eighteen female beagle dogs were obtained
from the Animal Facilities of Utrecht University. They were
housed in groups of 3 per pen and were fed a standard
commercial diet and water ad libitum. Animals were divided
into 3 groups: 6 skeletally immature animals (mean ⫾ SD age
0.7 ⫾ 0.02 years), 6 young but skeletally mature animals (2.4 ⫾
0.3 years), and 6 old animals (7.7 ⫾ 0.7 years). Immaturity was
confirmed by radiographs of the knee. The Utrecht University
Animal Experiments Ethical Committee had given ethical
approval for this study.
Experimental procedure. In all dogs, freshly collected
autologous blood obtained from the vena jugularis was injected into the right knee on day 0 and day 2, mimicking a
single intraarticular bleeding, thus guaranteeing the presence
of blood in the joint for 4 days. We chose not to use
anticoagulants. We know from previous studies that fibrincontaining plasma has no effect on the cartilage, and we know
that coagulated blood has effects similar to those of anticoagulated blood (2). We also know that blood does not (fully)
coagulate after autologous intraarticular injection; 2 days after
an injection, unclotted blood can be aspirated from the joint
(Hooiveld MJJ, Lafeber FPJG: unpublished observations).
Moreover, hemophilia patients are treated with coagulation
factor as soon as possible (within several hours) after a joint
bleed.
During the injection procedure, the animals were
briefly anesthetized using medatomidine/antesedan (Pfizer,
Capelle a/d IJssel, The Netherlands). The contralateral control
knee was not injected at all, in order to prevent possible
traumatic bleeding. Previous studies showed that control injec-
397
tions with saline did not have any effect on cartilage or
synovium (10). The intraarticular injection of blood was confirmed by placing a second needle on the contralateral side of
the joint in which blood appeared during injection. When this
occurred, the second needle was removed, and 4 ⫾ 2 ml of
blood (mean ⫾ SD) was injected intraarticularly. On day 4,
all 18 animals were killed using intravenous injection of 10 ml
of pentobarbital (200 mg/ml; CEVA, Naaldwijk, The Netherlands).
Synovium and cartilage were collected aseptically from
both knee joints within 1 hour postmortem and were analyzed
according to standard procedures as described below. In
addition, the cartilage of both hip joints from different young
mature animals was collected in order to test the catabolic
activity of culture supernatants of synovial tissue on healthy
mature homologous cartilage.
Cartilage tissue preparation and analysis. Cartilage
was cut as thick as possible, taking care to exclude the
underlying bone, from both femoral condyles and the tibial
plateau. Two samples from femoral condyles of each knee
were fixed in 4% phosphate buffered formalin for histochemistry. Deparaffined sections were stained with Safranin O–fast
green–iron hematoxylin and scored according to the modified
Mankin criteria (11). Due to the short duration of the experiment, only early signs of degeneration were expected, so the
emphases were on loss of Safranin O staining and articular
surface irregularities of the cartilage. At least 3 sections from
each of the 2 specimens of each knee were stained and scored
independently in random order by two observers.
Six explants from each femoral and tibial cartilage
were used for biochemical analyses of proteoglycan synthesis,
release, and content, as well as DNA content. Samples (mean
⫾ SD 5.4 ⫾ 2.8 mg) were cultured, each individually in 200 ␮l
culture medium, in 96-well tissue culture plates, according to
standard procedures as described previously (12). The culture
medium consisted of Dulbecco’s modified Eagle’s medium
(DMEM; Gibco, Breda, The Netherlands) supplemented with
ascorbic acid (85 ␮M; Sigma, Uithoorn, The Netherlands),
glutamine (2 mM; Gibco), penicillin (100 IU/ml; Yamanouchi
Pharma, Leiderdorp, The Netherlands), streptomycin sulfate
(100 ␮g/ml; Fisiopharma, Palomonte, Italy), and 10% heatinactivated pooled beagle serum. The mean of the outcome
values of the 6 samples, from each condyle and tibial plate, was
taken as representative of that knee.
DNA content. The DNA content of cartilage was
determined as a measure of its cellularity. Cartilage samples
were digested for 2 hours at 65°C with 3% papain (Sigma) as
described (13). Part of the papain digest was used for measurements of DNA using the fluorescent dye Hoechst 33258
(Sigma). Calf thymus DNA (Sigma) was used as a reference
standard.
Proteoglycan synthesis. As a measure of chondrocyte
matrix synthesis, the ex vivo synthesis of proteoglycans (one of
the main matrix components) was determined by assessment of
the rate of sulfate incorporation (12). Briefly, cartilage samples
were cultured for 4 hours in the presence of 14.8 MBq
Na235SO4 (carrier free; DuPont NEN, Hoofddorp, The Netherlands) added in 10 ␮l DMEM to the culture medium. After
this pulse-labeling period, samples were washed and digested
with 3% papain for 2 hours at 65°C. Glycosaminoglycans
(GAGs) were precipitated by addition of cetylpyridinium
398
chloride (Sigma). The 35SO42⫺-labeled GAGs in the precipitate were measured by liquid scintillation analysis. The rate of
sulfate incorporation, calculated with reference to the specific
activity of the medium, was normalized to the DNA content of
the explants and is expressed as nmoles of sulfate incorporated
per hour per ␮g DNA of the cartilage sample (nmoles/h*␮g
DNA).
Proteoglycan release. The release of proteoglycans was
determined as a measure of loss of matrix components (12).
Cartilage explants were precultured for 1 day, and then the
amount of GAGs released into refreshed culture medium
during 4 subsequent days of culture was assessed by precipitation with Alcian blue (Fluka, Zwijndrecht, The Netherlands).
The release of GAGs was measured photospectrometrically
and is expressed as mg GAG released per gram wet weight of
cartilage sample.
Proteoglycan content. The amount of cartilage GAG
was determined as a measure of the proteoglycan content of
the cartilage samples (12). In the papain digest, GAGs were
precipitated and stained with Alcian blue. The GAG content is
expressed in mg GAG normalized to the wet weight of the
cartilage explants.
Synovial tissue preparation and analysis. Five pieces
of synovial tissue from the suprapatellar pouch were taken
from each knee. Three were fixed in 4% phosphate buffered
formalin for histochemistry. Deparaffined sections were
stained with hematoxylin and eosin. To determine the degree
of inflammation, at least 3 sections from each of the 3
specimens of each knee were stained and scored. Synovial
inflammation was defined by lymphocyte infiltration, hyperplasia of the synovial lining cells, and hypertrophy of the synovial
membrane as described previously (12), a slight modification
of the scoring system described by Goldenberg and Cohen
(14). Two independent observers who were blinded to the
source of the tissue scored the tissue samples separately and in
random order.
Two synovial tissue samples (mean ⫾ SD 60 ⫾ 3 mg)
from each experimental and control knee were cultured to
assess cartilage-destructive activity. Each of the two samples
was cultured for 4 days in 5 ml culture medium. The supernatants of these cultures were harvested, rendered cell free by
centrifugation at 351g for 10 minutes, and tested in 50%
(volume/volume) concentration for catabolic activity on homologous, mature, healthy hip cartilage using procedures as
described above. Release and synthesis of proteoglycans by hip
cartilage were determined. Culture supernatants of each tissue
sample were tested on 8 cartilage samples per culture condition. The average result in the 16 samples was considered
representative of the synovial tissue of the knee from which the
two synovium samples were taken.
Calculations and statistical analysis. The mean values
of cartilage from condyles and plateaus did not differ significantly; therefore, values for condyles and tibia of each knee
were averaged (n ⫽ 12 samples per dog). The statistical
significance of the differences between control joints and
blood-injected joints was evaluated with a nonparametric test
for correlated data (Mann-Whitney U test) using the absolute
averaged values of each knee. The mean ⫾ SD values are
provided for each group of 6 dogs. Data are graphically
presented as the percentage change in experimental knees
versus contralateral control knees. Age dependency was ana-
HOOIVELD ET AL
Figure 1. Percentage change in total glycosaminoglycan (GAG) content of cartilage from blood-injected knees compared with that from
control knees. GAG content is expressed in mg GAG normalized to
the wet weight of the cartilage explants (mg/gm). Bars show the mean
and SD. Changes are shown for knee cartilage from immature animals
(mean ⫾ SD age 0.7 ⫾ 0.02 years), young mature animals (2.4 ⫾ 0.3
years), and old animals (7.7 ⫾ 0.7 years). The absolute values (mean ⫾
SD) of the GAG content of the control knees for these 3 groups were
37.0 ⫾ 2.7 mg/gm, 53.4 ⫾ 1.8 mg/gm, and 48.3 ⫾ 1.6 mg/gm,
respectively. In all groups, the total GAG content of the blood-injected
knees was decreased compared with that of the control knees (all P ⬍
0.05). Immature cartilage showed the largest decrease in this respect
(up to 19% loss compared with controls; P ⬍ 0.05). The change in
GAG content correlated linearly with age (r ⫽ 0.46, P ⬍ 0.05; n ⫽ 18).
lyzed by Spearman’s correlation analysis of these differences.
We used the percentage change because of the age-dependent
physiologic differences in some of the parameters under
control conditions (15,16). Two-tailed P values less than or
equal to 0.05 were considered significant.
RESULTS
Experimental intraarticular blood injections. After 48 hours, just prior to the second intraarticular
injection, swelling of the knee due to the first blood
injection was no longer palpable, presumably due to
absorption of the intraarticular blood. The joint swelling
was also not palpable on day 4, just before the animal
was killed. The dogs used the joints almost normally
within 2 hours after the injections and did not show signs
of pain or inconvenience.
Biochemical changes in cartilage ex vivo. The
total GAG content was statistically significantly diminished in all 3 groups, with the immature cartilage having
BLOOD-INDUCED JOINT DAMAGE
Figure 2. Percentage change in sulfate incorporation rate as a measure of proteoglycan (PG) synthesis of cartilage from blood-injected
knees compared with that from control knees. The sulfate incorporation rate is expressed as nmoles of sulfate incorporated per hour per
␮g DNA of the cartilage sample (nmoles/h*␮g DNA). Bars show the
mean and SD. Changes are shown for knee cartilage from immature,
young mature, and old animals. The absolute values (mean ⫾ SD) of
PG synthesis of the control knees for these 3 groups were 102.8 ⫾ 22.0
nmoles/h*␮g DNA, 35.9 ⫾ 6.3 nmoles/h*␮g DNA, and 24.9 ⫾ 4.6
nmoles/h*␮g DNA, respectively. In all groups, PG synthesis was
decreased after exposure to blood in vivo. The 36% decrease in PG
synthesis in immature cartilage (ⴱ ⫽ P ⬍ 0.05 versus control knees)
differed significantly from the decreases in the other groups (both P ⬍
0.05). The degree of decrease in PG synthesis correlated with age (r ⫽
0.45, P ⬍ 0.05; n ⫽ 18).
the largest decrease (19% compared with controls)
(Figure 1). There was a significant age-dependent loss of
GAG content after exposure to blood, as calculated by
correlation analysis (r ⫽ 0.46, P ⬍ 0.05; n ⫽ 18).
Proteoglycan synthesis, calculated per cell (i.e.,
per ␮g DNA), was statistically significantly decreased in
all 3 groups, with the immature group showing the
largest decrease (36% compared with controls; P ⬍
0.05) (Figure 2). The decrease in the immature group
differed significantly from those in the other 2 groups
(both P ⬍ 0.05). In addition, the decreases in proteoglycan synthesis upon exposure to blood changed linearly
with age (r ⫽ 0.45, P ⬍ 0.05; n ⫽ 18).
The total DNA content in both mature groups
did not change significantly after blood injections. However, it increased slightly in young mature cartilage,
whereas it decreased in old cartilage. In immature
cartilage, the DNA content increased significantly upon
exposure to blood (29% compared with controls; P ⬍
399
0.01) (Figure 3). This increase differed significantly from
those in the other 2 groups (both P ⬍ 0.05).
The release of GAGs from the cartilage was
statistically significantly increased in all 3 groups (Figure
4). In contrast to findings for the other parameters, the
most profound increase was observed in the young
mature group (22%), significantly greater than those in
the immature (5%) and old (10%) groups (both P ⬍
0.05). Thus, the increase in GAG release upon exposure
to blood did not change linearly with age.
Histologic analyses of cartilage. All biochemical
analyses demonstrated an adverse effect of blood on
cartilage proteoglycan turnover; release increased, synthesis diminished, and content decreased as a consequence. This loss of proteoglycans from the cartilage
could be confirmed by the loss of Safranin O staining in
all blood-exposed condylar cartilage samples. Scoring of
loss of cartilage integrity showed that the immature
cartilage was graded (mean ⫾ SD) 2.0 ⫾ 1.4, versus
2.4 ⫾ 1.2 for both the young mature and old cartilage
groups (Table 1). These histologic differences between
the 3 groups were not statistically significant, although
Figure 3. Percentage change in total DNA content of cartilage from
blood-injected knees compared with that from control knees. Bars
show the mean and SD. The absolute values (mean ⫾ SD) of DNA
content for control knees were 0.18 ⫾ 0.03 mg/gm wet weight, 0.19 ⫾
0.02 mg/gm wet weight, and 0.17 ⫾ 0.02 mg/gm wet weight for
immature, young mature, and old cartilage, respectively. Both the
young mature and the old groups showed no statistically significant
change in DNA content after exposure to blood in vivo. The 29%
increase in DNA content of immature cartilage (ⴱ ⫽ P ⬍ 0.01 versus
control knees) differed significantly from changes in DNA content in
the other groups (both P ⬍ 0.05).
400
HOOIVELD ET AL
Figure 4. Percentage change in release of glycosaminoglycans
(GAGs) in cartilage from experimental knees compared with that from
control knees. GAG release is expressed as mg GAG released per
gram wet weight of cartilage sample. Bars show the mean and SD. The
absolute values (mean ⫾ SD) of GAG release for control knees were
4.6 ⫾ 0.4 mg/gm, 3.8 ⫾ 0.5 mg/gm, and 4.3 ⫾ 0.5 mg/gm for immature,
young mature, and old cartilage, respectively. GAG release was
enhanced in all groups after intraarticular blood injections (all P ⬍
0.05). The largest release of GAGs (22%) was found in young mature
cartilage. ⴱ ⫽ P ⬍ 0.05 versus each of the other groups.
exposed to synovial culture supernatants from control
and blood-injected knees of all 3 groups were measured
to determine catabolic activity of the synovium. No
statistically significant differences between the groups
were found in the catabolic activity of the synovia of the
control knees, either for release (Figure 5) or for
synthesis (Figure 6). Basal catabolic activity, measured
by the induced proteoglycan release, was not changed
significantly by blood injection in the young mature and
old animals. However, a statistically significant increase
in GAG release was induced by the experimental synovial culture supernatant from the immature animals
(38% compared with control; P ⬍ 0.05) (Figure 5).
Proteoglycan synthesis remained unchanged in the synovial culture supernatants from the 3 groups (Figure 6).
Histologic analyses of synovium. Histologically, a
very mild but statistically significant synovial inflammation was found in blood-injected versus control knees in
all 3 groups. The degree of inflammation did not differ
significantly between the 3 groups (Table 2), although
the highest score was found in the immature group. With
respect to the change in inflammatory activity compared
the immature and young mature cartilage showed the
most severe changes. A correlation of the effect with age
did not reach statistical significance, unlike the correlations with age of some of the biochemical parameters.
Catabolic activity of synovium. Proteoglycan release and synthesis in homologous mature hip cartilage
Table 1. Modified scores of cartilage damage*
Cartilage (age)
Immature (⬍6 months)
Young mature
(2–3 years)
Old (7–9 years)
Control
knee
Bloodinjected
knee
Change
(blood-injected
⫺ control)
0.1 ⫾ 0.3
0.4 ⫾ 0.5
2.0 ⫾ 1.4†
2.4 ⫾ 1.2†
1.9 ⫾ 1.0
2.0 ⫾ 0.4
1.3 ⫾ 1.0
2.4 ⫾ 1.2†
1.1 ⫾ 0.9
* Values are the mean Mankin score (11) ⫾ SD. After short-term
exposure of cartilage to blood in vivo, a slight increase in cartilage
degeneration was found. Due to the short duration of the experiment,
scoring was based on the loss of Safranin O stain from the cartilage and
on articular surface irregularities. Between the 3 groups, no significant
differences in cartilage degeneration were found. Within the 3 groups
(i.e., between control and experimental cartilage), the differences were
significant.
† P ⬍ 0.05 versus control cartilage.
Figure 5. Total glycosaminoglycan (GAG) release from mature homologous hip cartilage after exposure to control or experimental
synovial culture supernatants, as a measure of catabolic activity of the
synovium. Bars show the mean and SD. Between the 3 groups, no
statistically significant changes were found after exposure of cartilage
to culture supernatant of synovium from control knees. Compared
with control synovium supernatants, experimental culture supernatants
from immature synovium were able to induce increased GAG release
from cartilage (38%). Synovial culture supernatants from young
mature and old animals were unable to change GAG release. ⴱ ⫽ P ⬍
0.05 versus control synovium supernatants.
BLOOD-INDUCED JOINT DAMAGE
401
Figure 6. Sulfate incorporation as a measure of proteoglycan synthesis in homologous mature hip cartilage after exposure to control or
experimental synovial culture supernatants. The sulfate incorporation
rate is expressed as nmoles of sulfate incorporated per hour per ␮g
DNA of the cartilage sample (nmol/h*g). Bars show the mean and SD.
Between the 3 groups, no statistically significant changes were found
after exposure of cartilage to culture supernatant of synovium from
control knees. In addition, the experimental synovium culture supernatants did not alter the proteoglycan synthesis of healthy cartilage.
with the contralateral control, there was a tendency
toward an age-dependent effect of exposure to blood.
DISCUSSION
In recent years, we have shown in in vivo and in
vitro studies that a relatively short-term exposure of
cartilage to blood leads to lasting adverse changes in
chondrocyte activity and cartilage matrix integrity (2,3).
Table 2. Modified scores of synovial inflammation*
Synovium (age)
Immature (⬍6 months)
Young mature
(2–3 years)
Old (7–9 years)
Control
knee
Bloodinjected
knee
Change
(blood-injected
⫺ control)
0.8 ⫾ 0.4
0.5 ⫾ 0.5
2.8 ⫾ 0.8†
2.3 ⫾ 0.6†
2.0 ⫾ 0.9
1.8 ⫾ 0.3
0.8 ⫾ 0.7
2.1 ⫾ 0.9†
1.3 ⫾ 0.8
* Values are the mean inflammation score (modified from Goldenberg
and Cohen [14]) ⫾ SD. Short-term experimental intraarticular bleeding caused a slight increase in synovial inflammation. Between the 3
groups, no significant differences in synovitis were found. Within the 3
groups (i.e., between control and experimental joints), the differences
were significant.
† P ⬍ 0.05 versus control joints.
Results from the present in vivo study show that knee
cartilage of (skeletally) immature dogs after 4 days is
more susceptible to blood damage than that of young
mature and old dogs. The impact of the experimental
joint bleed appeared to be age related for several of the
parameters measured. This finding suggests that in case
of a hemorrhage, cartilage of immature individuals is at
greatest risk. This may be true not only in the case of
hemophilia, but also in the case of a single or a limited
number of traumatic bleedings.
The age-related decrease in proteoglycan content
was striking in this respect. The age-related inhibition of
synthesis of proteoglycans is in accordance with the
observations on proteoglycan content; the youngest animals show the most profound loss of proteoglycan
content. This was found when synthesis was normalized
to the amount of cells (i.e., the DNA content). However,
the DNA content showed an age-dependent increase.
The DNA content significantly increased in the immature animals, whereas no statistically significant change
was observed in young mature animals, and a slight
decrease in DNA content was seen in cartilage of old
animals. However, although proteoglycan synthesis is
decreased in the immature cartilage as a result of blood
injections, the cartilage from immature animals still
synthesizes larger quantities of proteoglycans per ␮g
DNA than cartilage from mature animals. This is because under control conditions, there is an agedependent decrease in proteoglycan synthesis (15), and,
specifically in immature cartilage, proteoglycan synthesis
is relatively high (17).
Interestingly, this implies that in cartilage of
immature animals, chondrocytes are triggered and are
capable of proliferating upon receipt of a harmful signal.
Previous in vitro studies have shown that chondrocyte
apoptosis is induced by exposure of cartilage to blood
(18). This corroborates the loss of DNA in old individuals. It could be that the loss of healthy chondrocytes is
compensated for in immature cartilage, because cells are
still able to proliferate in this cartilage (19). Another
possibility is that chondrocytes in immature cartilage
change from a proteoglycan-synthesizing to a proliferative phenotype, as has been suggested before (20).
However, this is difficult to study, since detailed electron
microscopy analysis is needed. A simple increase in the
number of cells (light microscopy analysis) is not to be
expected in 4 days; most cells will still be in a mitotic
phase with a doubled amount of DNA, but they will still
be recognized as single cells.
Cell counting revealed a 6.5% increase in cartilage from blood-injected joints compared with that from
402
control joints (mean ⫾ SD 132 ⫾ 10 chondrocytes/mm2
versus 124 ⫾ 8 chondrocytes/mm2); although an increase
was found in 5 of the 6 animals, the mean increase was
not statistically significant. Nevertheless, in time this
could mean that specifically the immature cartilage
could recover from the harmful effects of exposure to
blood. Whether this is indeed the case, the outcome
after a longer recovery period has to be investigated.
Notwithstanding, the present data show that in the short
term, despite an increase in DNA, the new chondrocytes
in the immature cartilage cannot maintain matrix integrity shortly after exposure to blood, making the cartilage
vulnerable to further damage, which is in accordance
with observations from clinical practice.
It appeared that the cartilage from the young
mature animals, and not that from the immature animals, had the greatest increase in GAG release when
exposed to blood in vivo. This finding does not explain
the fact that GAG content was most profoundly diminished in the immature cartilage. One possible explanation could be that the immature cartilage lost its GAG
content at an earlier stage (i.e., during the exposure in
vivo), whereas this process of release was still present ex
vivo in the young mature cartilage, although with lower
intensity. This would be consistent with the abovementioned hypothesis, that although the cartilage of the
immature animals is most vulnerable to a bleeding, it
may have the better chance of recuperating.
On the one hand, the profound release of proteoglycans upon exposure to blood in the young mature
animals did not depend on synovial changes. On the
other hand, the synovium of immature animals was
significantly more triggered by exposure to blood than
was the synovium of the other 2 groups, as indicated by
its significantly greater potential to release proteoglycans from homologous explants. Proteoglycan synthesis
remained unchanged after addition of synovial culture
supernatants of all 3 groups. This suggests that exposure
to blood in the immature animals specifically triggers
activity of proteinases such as matrix metalloproteinases,
known to be involved in cartilage breakdown (21,22),
and, to a lesser extent, catabolic activity of cytokines
such as IL-1 and TNF␣, which are known to have
inhibitory effects on cartilage matrix synthesis (23,24).
Our results indicate that at a very early stage in immature dogs, the affected synovium adds to the harmful
effects of a joint bleed. This finding is in contrast to
previous observations in mature animals, in which synovitis occurs only at later stages (4).
These data suggest that in order to prevent a
more rapid degeneration of cartilage in immature indi-
HOOIVELD ET AL
viduals, it may be necessary to start prophylactic treatment of severe hemophilia at a very early age. Fischer et
al have reported that prophylaxis with clotting factors
after the first two hemorrhages in life, in most cases
occurring before adulthood, is more beneficial in the
long term than starting prophylaxis after more than
three hemorrhages (7). Although it remains arguable
whether only one joint hemorrhage is sufficient to
induce permanent joint damage, our data suggest that
one short-term exposure of cartilage to blood is sufficient to induce a temporary catabolic state in the joint.
Immature joints are more susceptible in this respect.
In summary, age has not previously been considered as a predictor for susceptibility to blood-induced
joint damage. We conclude from the present study that
joints of skeletally immature individuals are more susceptible than joints of mature individuals to the adverse
effects of a joint hemorrhage. These data suggest that
specifically in immature individuals, the treatment of
joint hemorrhages needs further attention, not only in
patients with hemophilia, but also after joint trauma
involving joint hemorrhages.
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