Identification of important interactions between subchondral bone ...

1480 S.H. Madsen et al. / Steroids 76 (2011) 1474–1482
Fig. 5. The effect of dexamethasone in pre-osteoclasts and mature osteoclasts. Osteoclasts were cultured in on either bone (black bars) or plastic (gray bars) in the presence or
absence of 100 nM dexamethasone (DEX). Pre-osteoclasts were cultured for 21 days (A–C) and mature osteoclasts for 14 days (D–F). Cell viability was measured with alamar
blue after 14 or 21 days (A and D). The number of osteoclasts was measured with TRAP activity (B and E) and bone resorption was measured by calcium release from bone (C
and F). DEX [100 nM] treatment decreased cell viability, osteoclast number and bone resorption in both pre-osteoclasts and mature osteoclasts. However, the effects of DEX
[100 nM] on pre-osteoclasts were higher than the effects on mature osteoclasts. Each treatment n P 5.
we used the bovine articular cartilage explants model that is presently
the preferred model for studying cartilage turnover ex vivo
[30,33]. The strong indications that OSM + TNF-a reduced the
ability to induce cartilage degradation in the presence of DEX or
PRED, evaluated by biomarkers and histology, suggest a chondroprotective
effect by GCs independent of bone cells. Furthermore,
histology showed that GC treatment had a positive effect on proteoglycans
under anabolic conditions, which is in line with a study
by Van der Kraan et al. that showed that PRED stimulates proteoglycan
synthesis in patellar cartilage, validated by [ 35 S] incorporation
[43]. However, the catabolic stimulated cartilage explants in
the presence or absence of DEX, were both close to be depleted
for proteoglycans, measured by the intensity of color (sGAG),
which was not as convincing as the 70% reduction of MMP-mediated
aggrecan degradation shown with the biomarker, 342-G2.
However, depletion of color may results from an early loss of aggrecanase-mediated
highly-sGAG-saturated aggrecan degradation,
leaving low-sGAG-saturated aggrecan to be degraded by MMPs at
day 19 [44], as we did not see any MMP-mediated degradation at
days 5 and 12. This suggests that the anti-catabolic effects of GC
on cartilage may be limited to the degradation mediated by MMPs.
Interestingly, GC treatment in resting conditions resulted in a small
loss of proteoglycans from the articular cartilage explants that we
were unable to detect using the MMP-mediated aggrecan degradation
marker, 342-G2. Thus, the two opposite effects of GCs treatment
(protecting in activated cartilage and degrading in resting
cartilage) indicate that the activation stage of the chondrocytes is
important in relation to the GC response. If the GC-mediated protective
effect results from an alteration of the chondrocyte phenotype
(preventing the induction/release of proteases) or that the
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GCs inhibit the cytokine pathway, still remains unclear. Yet, our
data indicate that a reduction of MMP activity is involved, as the
MMP-dependent biomarkers, 342-G2 and CIIM, are reduced. This
is in line with a recent study by Garvican et al. suggesting that
GCs reduce MMP-mediated cartilage degradation [9], although
other enzymatic pathways, such as the u-PA/plasminogen/plasmin-pathway,
also have been indicated [45]. Thus, in the search
for new disease modifying osteoarthritic drugs (DMOADs), we suggest
that the inhibition of protease pathways mediated by GCs
could be of interest.
We did not see any protective effects of GCs on the non-stimulated
bovine cartilage explants, as opposed to the results with cartilage
from the femoral head explants. The bovine cartilage
explants represent ‘‘healthy’’ cartilage, whereas the femoral heads
consist of cartilage in development, which contains more proliferating,
pre-hypertrophic and hypertrophic chondrocytes (which are
observed in OA cartilage) than the bovine cartilage explants [29].
This could explain why we have to induce a catabolic milieu with
cytokines in the bovine cartilage explants, before we see a protective
effect of GCs, and suggest that GCs mainly affect chondrocytes
which are activated, whereas resting chondrocytes appear to respond
less. We do acknowledge that we deal with cross-species
comparisons, which may cause the different effects observed in
this study. However, the anabolic and catabolic controls have the
same effects on the different species, suggesting the effects of
GC, or lack of same, are due to action from the GC and not a species-dependent
effect.
The present study clearly shows that GCs have demonstrably
large effects on cells of bone. While it for long has been known that
GCs induce apoptosis of osteoblasts and osteocytes in vivo, there is