Identification of important interactions between subchondral bone ...

1478 S.H. Madsen et al. / Steroids 76 (2011) 1474–1482
Fig. 2. The effects of prednisolone in anabolic-induced bone and cartilage from femoral heads. Femoral heads were cultured in the presence of 100 nM prednisolone (PRED)
and/or 100 ng/ml IGF-I for 2 weeks. Cell viability was measured with alamar blue after 2 weeks (A). Biomarkers were measured after 1 week (black bars) and 2 weeks (gray
bars) (B–F). The number of osteoclasts was measured with TRAP activity (B). The bone resorption and formation were measured by the collagen type I markers, CTX-I (C) and
PINP (D), respectively. Cartilage degradation and formation were measured by the collagen type II markers, CIIM (E) and PIINP (F), respectively. PRED [100 nM] + IGF-I
[100 ng/ml] treatment decreased bone resorption, but not bone formation. Furthermore, PRED [100 nM] + IGF-I [100 ng/ml] treatment increased cartilage formation after 2
weeks. Each treatment n P 4.
osteoblasts were cultured in the presence of DEX, cell viability was
reduced by 55% (p < 0.05), compared to the vehicle (Fig. 4A). The
same pattern was seen in the presence of both DEX and BMP-2,
where viability decreased by 35% (p < 0.05), compared to BMP-
2 stimulation alone (Fig. 4A). DEX also decreased the nodule formation
by 65% (p < 0.05), compared to vehicle (Fig. 4B). Nodule
formation was induced by addition of the positive control [34],
BMP-2, by 2.4-fold (p < 0.05) compared to vehicle (Fig. 4B). Furthermore,
addition of DEX to BMP-2-stimulated osteoblasts, led
to a further increase in nodule formation by 1.8-fold (p < 0.05)
(Fig. 4B). ALP production by the osteoblasts was reduced when cultured
with DEX, both in the presence (by 45%) or absence of BMP-
2 (by 85%) (p < 0.05) (Fig. 4C), correlating with the cell viability
measurements (Fig. 4A).
3.5. Dexamethasone decreases viability and function of both
mature osteoclasts and their precursors
When pre-osteoclasts were cultured either on bone or plastic,
DEX reduced the viability of the cells by more than 95%
(p < 0.01), as well as the osteoclast marker TRAP by more than
98% (p < 0.01) (Fig. 5A and B). Furthermore, the release of calcium
during resorption of bone, decreased by 85% (p < 0.01) when treated
with DEX (Fig. 5C). To investigate whether this effect was specific
to RANKL induction of osteoclastogenesis, an experiment
omitting RANKL was conducted [31]. In these cells, we again observed
a reduction in cell viability in the presence of DEX (data
not shown), thus showing that the reduction in viability is related
to the macrophages rather than specifically to the osteoclasts. In
mature osteoclasts, DEX decreased cell viability by 80%
72
(p < 0.01) on both bone and plastic (Fig. 5D). The number of osteoclasts
was decreased by 70% (p < 0.01) on bone and 75%
(p < 0.01) on plastic (Fig. 5E). Bone resorption decreased by 40%
(p < 0.05) (Fig. 5F). Interestingly, the overall level of suppression induced
by the DEX on mature osteoclasts was lower than the level
of suppression induced in the pre-osteoclasts.
4. Discussion
Intra-articular corticosteroid injections have been tested for
beneficial effects on inflammation and joint destruction, and appear
to have some applicability by reducing pain in OA patients
[2,40,41]. However, how GCs affect the individual cell types in
the joint is not known. Thus, this study focused on elucidating
whether GCs have any effects on cartilage turnover. Furthermore,
since subchondral bone has been shown to play an essential role
in the development of OA, and since the effects of GCs on bone cells
are still discussed [27,42], we extended the study by performing a
thorough characterization of the effect of GCs on the two major
bone cell types, namely osteoblasts and osteoclasts. This study
used four model systems to investigate how GCs affect the cells
of the joint both in the setting of intact tissues, as femoral heads
or cartilage explants (resting, catabolically active or anabolically
active), and as separate cell cultures of osteoblasts (resting or differentiating)
and osteoclasts (differentiating or mature and on
plastic or bone).
Several studies of GC-mediated effects on chondrocyte function
have been published; however no clear-cut conclusion has been
presented. These studies have indicated both beneficial and detrimental
effects of GCs on cartilage, [6,7] however, the studies also