ever, Se addition reduced this expression. In contrast, exposure
to DON, NIV and T-2 toxin caused depressed TIMP-1 and TIMP-3
levels compared with the Control; this reduction was reversed
with Se addition. Se addition also reversed the expression. α2
macroglobulin that was lowered by the three Toxins.
Conclusions: This study indicates that three toxins (DON, NIV
and T-2 toxin) all depress type II collagen and aggrecan expres-
sion in tissue engineered cartilage grafts cultured in vitro. The
levels of MMP-1 and MMP-3 were increased but the levels of
TIMP-1, TIMP-3 and α2 macroglobulin were all decreased in the
presence of Toxins. Addition of Se reversed all of the expression
effects produced by these Toxins. These in vitro results provide
evidence for the potential biological mechanisms underlying carti-
lage degradation in the pathogenesis of KBD and how Selenium
addition reverses these pathological effects.
REGULATION OF MICROSOMAL PROSTAGLANDIN E2
SYNTHASE-1 AND 5-LIPOXYGENASE-ACTIVATING
PROTEIN/5-LIPOXYGENASE BY 4-HYDROXYNONENAL IN
S.-H. Chen, Q. Shi, H. Fahmi, M. Benderdour
Univ. of Montreal, Montreal, QC, Canada
cyclooxygenase-2 (COX-2) decreased dramatically after 24 hrs
of incubation with 4-hydroxynonenal (4-HNE), a product of lipid
peroxidation. This study aimed to investigate whether HNE is re-
sponsible for the shunt from COX-2 to 5-lipoxygenase-activating
protein (FLAP)/5-lipoxygenase (5-LOX) in human osteoarthritic
Methods: OA chondrocytes were treated with 10 μM of 4-HNE
at different times of incubation (0 to 72 hrs). The protein level of
mPGES-1 was evaluated by Western blot and that of prostaglandin
E2 (PGE2) and leukotriene 4 (LTB4) was determined by EIA using
commercial kits. The level of mPGES-1 and FLAP/5-LOX mRNA
was measured by real-time RT-PCR.
Results: HNE induced the production of both PGE2and LTB4 by
chondrocytes, but in opposite fashion. The level of PGE2increased
during the short period of stimulation (0-24 hrs), whereas that of
LTB4 increased after a long period of stimulation (48 and 72
hrs), where the level of PGE2decreased. The Western blot data
showed that protein expression of mPGES-1 increased gradually
in treated cells with 4-HNE, to reach a maximum level after 16
h of incubation. At mRNA level, 4-HNE induced mPGES-1 and
FLAP/5-LOX expression after 4 and 48 h of incubation respectively.
Conclusions: Our results showed the shunt from the COX-2 to
FLAP/5-LOX pathway in HNE-induced human OA chondrocytes.
The increase in LTB4 level may be due to the decrease in that
of PGE2. Further experiments are in progress in other to de-
termine the molecular mechanism underlying this switch in OA
EFFECT OF THREE DIFFERENT CHONDROITIN SULFATES
IN HUMAN OSTEOARTHRITIS CARTILAGE
S. Kwan Tat1, J.-P. Pelletier1, F. Mineau1, N. Duval2,
1Osteoarthritis Res. Ctr., Notre-Dame Hosp., Univ. of Montreal
Hosp. Res. Ctr. (CRCHUM), Montreal, QC, Canada;2Duval
Clinique Orthopédique, Laval, QC, Canada
Purpose: During the osteoarthritis (OA) process, the altered
metabolism of the chondrocytes is responsible for the extensive
breakdown of the cartilage in which pro-inflammatory cytokines
and matrix metalloproteases (MMPs) are highly implicated. Previ-
ous studies have demonstrated that chondroitin sulfate (CS) exerts
a protective effect on the cartilage. However, due to differences
in CS in terms of origin, purity and the production/purification
process, we compared the effects of three different types of CS
on human OA cartilage.
Methods: Three types of CS were tested: namely CS#1 (porcine,
purity: 90.4%), CS#2 (bovine, purity: 93.0%), and CS#3 (Bioibérica
S.A.; bovine, purity: 99.9%). Treatment with each CS at 200 and
1000 μg/ml were performed in human OA cartilage explants in the
presence/absence of IL-1β, and the protein modulations of factors,
including PGE2, IL-6, and MMP-1, were investigated by specific
ELISA. The CS effect on the expression of the pro-anabolic factor,
collagen type II, was also investigated on OA chondrocytes using
Results: In the presence of IL-1β, CS#3 at 200 μg/ml, but not
at 1000 μg/ml, reduced the level of MMP-1, PGE2 and IL-6.
CS#2 followed the same trend as CS#3, however, at a much
higher concentration, 1000 μg/ml. On the other hand, CS#1 was
much less efficient in reducing the same catabolic markers and
very surprisingly, in the absence of IL-1β, it increased the three
catabolic factors tested, PGE2, IL-6, and MMP-1. As expected
IL-1β inhibits the gene expression level of the collagen type II; only
CS#3 was able to limit this inhibition. CS#1, in the presence or
absence of IL-1β, further decreased collagen type II expression.
Conclusions: This study provides data on the effect of different
CS on the cartilage metabolism. In this context, CS prescribed
for alleviating OA symptoms should be taken with care as the
origin, purity and/or production/purification of the CS compound
could orientate the current OA disease process toward increased
PATHOLOGY OF THE OUTERBRIDGE IV LESION;
L.L. Johnson1, C. Verioti1, J. Gelber1, D. D’Lima1, M. Spector2
1Shiley Ctr. for Orthopedic Res. and Ed., La Jolla, CA;2Woman’s
Brigham Hosp., Harvard Univ., Boston, MA
Purpose: The purpose of this study is to examine the gross and
microscopic characteristics of the Outerbridge IV lesion that may
serve as the foundation for cartilage repair.
Methods: Human osteochondral specimens having Outerbridge
IV lesions were harvested following total knee surgery. They were
subject to visual examination before and after Safranin O staining.
Correlative histology was examined.
Results: The stained gross specimens showed cartilaginous ag-
gregates on the surface as well as multiple small depressions.
The microscopy showed cartilaginous aggregates on the surface