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