Chubinskaya, S.

Hakimiyan, A.A.

Oegema, T.

Pascual Garrido, C.

Rappoport, L.

Wimmer, M.A.

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S78 Osteoarthritis and Cartilage Vol. 16 Supplement 4
synovial membrane nodules showed blood vessels containing red blood
cells, as well as, osteoclasts. Type I and type IIb loose bodies, however,
did not show them.
Conclusions: It is well known that loose bodies grow from proliferation
of cartilage without blood supply in the joint cavity, and that enchondral
ossiﬁcation is able to develop only under the condition of having a blood
supply. As synovial membrane nodules were also classiﬁed to the same
type as loose bodies and more than half of osteochondral and osseous
loose bodies contained blood vessels with red blood cells, the loose
bodies were thought to be caught in the synovial membrane and to be
modiﬁed as the result of a blood supply. Considering the results of this
study, various histologic characteristics of loose bodies in osteoarthrosis
resulted from modiﬁcations including cartilage proliferation in the joint
cavity and enchondral ossiﬁcation in the synovial membrane.
150 INHIBITION OF CELL DEATH PREVENTS CARTILAGE
DEGRADATION IN ACUTE TRAUMA MODEL
S. Chubinskaya, C. Pascual Garrido, A.A. Hakimiyan, L. Rappoport,
M.A. Wimmer, T. Oegema Jr.. Rush University Medical Center, Chicago,
IL, USA
Purpose: Post-traumatic arthritis is one of the most common causes of
secondary osteoarthritis (OA). The aim of our study was to investigate the
effect of anti-apoptotic agents (non-ionic surfactant P188 and inhibitors
of caspases 3 and 9) on cartilage degradation after a single impact to
human ankle cartilage.
Methods: Human normal tali (cartilage with bone attached) obtained
from organ donors were impacted with a nominal stress of 25−30 MPa.
Cartilage from both left and right ankles was used; one served as an
un-impacted control and a second was impacted with a 4mm indenter.
Full thickness 8mm cartilage plugs consisted of impacted (4mm core)
and immediately adjacent (4mm ring) areas were removed and either
collected at day 0 or cultured with or without P188 (8mg/ml), caspase 3
inhibitor (10 uM), or caspase 9 inhibitor (2 uM). The effect of treatments
was assessed at days 0, 1, 2, 7 and 14 post injury by live/dead cell assay,
apoptosis (Tunel Stain), histology with Safranin O staining, and proteo-
glycan content released into the media. At each time point histological
assessment with a modiﬁed Mankin score and the number of viable and
apoptotic cells were evaluated in the superﬁcial and middle-deep layers
separately.
Results: An impact to articular cartilage caused cell death by necrosis
primarily in the superﬁcial layer of the impacted core. By day 14 of culture,
the number of dead cells in this area was increased by more than 30%.
The impact also caused cell death by apoptosis which was observed in
both, impacted core and adjacent ring. Tunel-positive chondrocytes were
present in all cartilage layers. During culture, cell death by apoptosis
spread out to the areas that did not experience the impact and the number
of apoptotic cells was statistically increased by day 14 not only in the core,
but also in the ring. All three treatments improved cell survival by necrosis
at all time points. However, with regard to apoptosis, P188 appeared
to be the most effective among chosen treatments: already at day 2,
the number of Tunel-positive chondrocytes was signiﬁcantly reduced in
the superﬁcial layer of both core and ring (13% ±0.58 and 9%±4.8
respectively) in comparison to the non-treated group (33%±5.49 and
30%±0.53 respectively, p< 0.01). It also inhibited expansion of apoptosis
through the adjacent ring area. Within the ﬁrst 7 days, P188 was able
to maintain apoptosis in the ring at a steady level (about 20% of Tunel-
positive cells) vs 60% (p< 0.05) detected in the untreated control. Among
caspase inhibitors, anti-caspase-3 was more effective in reduction of
apoptosis than anti-caspase-9. P188 and caspase-3 inhibitor exhibited
a similar effect on cartilage integrity. Mankin score under both treatments
remained within a normal range (4±0.5), while in the untreated core
it was 7±0.5. Similar results were observed for the ring area. The
caspase 9 inhibitor did not improve histological appearance of cartilage
sections. Only the P188 treatment signiﬁcantly reduced the amount of
proteoglycans released into the media at all time points.
Conclusions: This study documented that P188 and caspase 3 inhibitor
were effective in promoting chondrocyte survival and thus protecting
cartilage from degeneration in acute trauma model. Combination of
these anti-catabolic treatments with anabolic agents may provide novel
therapeutic approaches to stimulate proper cartilage repair in order to
prevent post-traumatic OA.
151 SMALL HEAT-SHOCK PROTEINS, NOVEL IDENTIFIED
MEDIATORS OF MATRIX GENE EXPRESSION, ARE
DIFFERENTIALLY EXPRESSED IN OA CHONDROCYTES
S. Lambrecht, G. Verbruggen, D. Elewaut, D. Deforce. Ghent University,
Gent, BELGIUM
Purpose: aBcrystallin (HSPb5) and HSP27 (HSPb1) belong to the family
of small heat-shock proteins. This family of proteins is only poorly de-
scribed in chondrocytes. We aimed to investigate the expression levels
of these proteins in chondrocytes isolated from healthy and osteoarthritis
(OA)-affected patients. The functional role of aBcrystallin in chondrocyte
metabolism was further explored using RNA-interference.
Methods:Western blot and real-time RT-PCR analysis were performed to
determine the expression levels of aBcrystallin and HSP27 in healthy and
OA chondrocytes cultured in alginate beads. RNA-interference mediated
gene knock-down was used to explore the role of aBcrystallin in chondro-
cyte biology by transfecting low concentrations of siRNA directed against
the aBcrystallin sequence in cultured chondrocytes. To determine the
differentiation status dependent expression of small heat-shock proteins,
phenotypically stable chondrocytes were seeded in low-density mono-
layer cultures to allow expansion and partial dedifferentiation. Expression
levels of aBcrystallin and HSP27 were monitored at different time points.
Results: Based on previous proteome screening analyses, aBcrystallin
and HSP27 were selected as potential interesting proteins in the de-
velopment of OA. A decreased abundance of these proteins was ob-
served in OA chondrocytes by Western blot. Moreover, real-time RT-
PCR conﬁrmed the differential expression between chondrocytes isolated
from visually intact and visually damaged zones of OA cartilage from
the same patient. The pro-inﬂammatory cytokines Il-1b and TNF-a, both
down regulated aBcrystallin and HSP27 expression. Transfection of low
concentrations siRNA in cultured chondrocytes resulted in an efﬁcient
knock down of aBcrystallin gene expression. This was accompanied
by an altered expression of the chondrocyte speciﬁc genes BMP-2,
aggrecan and collagen type II. The observed reduced expression of Ag-
grecan, COL2A1 and BMP-2 may reﬂect that a decreased expression of
aBcrystallin is associated with a more dedifferentiated cellular phenotype.
This hypothesis is supported by our observation that aBcrystallin showed
an initial decreased expression in monolayer-cultured dedifferentiating
chondrocytes.
Conclusions: In conclusion, our study clearly demonstrates the differ-
ential expression of small heat-shock proteins on the mRNA as well as
on the protein level between healthy and OA-affected articular chondro-
cytes, suggesting a distorted chaperone response in OA. This differential
expression seems to be a consequence of a cytokine driven mechanism.
Additionally, our data disclose aBcrystallin as a novel identiﬁed regulator
of matrix gene expression. The association between reduced aBcrystallin
levels and reduced levels of aggrecan, collagen type II and BMP-2, further
adds to the evidence that this protein might be involved in phenotypic
changes of chondrocytes during the development of OA.
152 IN VITRO VERSUS IN VIVO EXPOSURE OF CARTILAGE TO
BLOOD
N.W. Jansen, G. Roosendaal, J.W. Bijlsma, F.P. Lafeber. University
Medical Center Utrecht, Utrecht, NETHERLANDS
Purpose: Joint bleeds lead to joint destruction. Knowledge about the
mechanism of this blood-induced arthropathy has originated from both
in vitro and in vivo studies. Our group has shown that in vitro exposure
of cartilage to 50% v/v blood for 4 days leads to severe (−98%) and
long-lasting (−78% after 16 days) inhibition in cartilage matrix synthesis.
Also after an experimentally in vivo induced haemorrhage in the dog
knee joint, direct harmful effects were observed, including inhibition of the
cartilage matrix synthesis (−22%). But while in the in vitro experiments
this inhibition was long lasting, in the in vivo experiments, effects were
less outspoken and long-lasting. One of the differences between the in
vitro and the in vivo situation is that in the in vivo situation, the cartilage
is exposed to blood at the articular surface only, whereas in the in vitro
explant culture system the cartilage is exposed to 5 additional cutting
edges. Whether this difference in exposure of cartilage to blood can
explain the difference between the in vitro and in vivo studies on blood-
induced cartilage damage was subject of this study.
Methods: Human articular cartilage explants were exposed to blood
either totally, by submerging explants in 50% v/v blood, or to the articular
surface alone. For this purpose a speciﬁc culture device was developed
and validated. The effects of this exposure on the proteoglycan synthesis
rate and -release were determined.


150 INHIBITION OF CELL DEATH PREVENTS CARTILAGE DEGRADATION IN ACUTE TRAUMA MODEL 

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150 INHIBITION OF CELL DEATH PREVENTS CARTILAGE DEGRADATION IN ACUTE TRAUMA MODEL

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