Identification of important interactions between subchondral bone ...

after 1 week (Fig. 2F). The addition of PRED to IGF-I stimulation
was however not different from the vehicle. On the contrary, after
2 weeks, PRED in combination with IGF-I showed a tendency to increase
cartilage formation compared to the anabolic control
(Fig. 2F).
3.3. Cytokine-induced cartilage turnover is decreased
by glucocorticoid treatment
The effect of PRED was tested on bovine cartilage explants.
However, since the effects of PRED were weak, we also tested the
more potent dexamethasone (DEX) [8] in a separate experiment.
The effects of PRED and DEX were measured at day 21.
After 21 days of culture, the viability of the bovine cartilage explants
increased with OSM + TNF-a stimulation by 1.6-fold
(p < 0.05) (Fig. 3A) and 1.7-fold (p < 0.01) (Fig. 3E), compared to
respective vehicles (Fig. 3A and E). However, this increase seemed
inhibited by DEX (p < 0.05) (Fig. 3E). Aggrecan degradation was induced
by OSM + TNF-a by more than 150-fold (p < 0.001) for both
experiments, and reduced by addition of DEX by 70% (p < 0.01);
however not to the level of the vehicle or DEX alone (Fig. 3B and
F). The same pattern was observed for collagen type II degradation,
where OSM + TNF-a stimulation increased the degradation by
more than 40-fold (p < 0.01), which was reduced by addition of
PRED and significantly by addition of DEX by 75% (p < 0.01)
(Fig. 3C and G). Aggrecan and collagen degradation were also measured
at days 5 and 12, but did not show any differences between
the vehicle and activated explants (data not shown). OSM + TNF-a
stimulation increased cartilage formation by more than 4-fold
(p < 0.01) in both experiments (Fig. 3D and H). Cartilage formation
S.H. Madsen et al. / Steroids 76 (2011) 1474–1482 1477
Fig. 1. The effects of prednisolone in resting and catabolic-induced bone and cartilage from femoral heads. Femoral heads were cultured in the presence of 100nM
prednisolone (PRED) and/or cytokines (O + T) [10 ng/ml] + [20 ng/ml] 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] treatment decreased viability of the cells in the femoral heads, osteoclast number, bone resorption, bone formation and cartilage degradation.
Each treatment n P 4.
71
was also induced by IGF-I stimulation by more than 8.5-fold
(p < 0.01) in both experiments (Fig. 3D and H). DEX inhibited formation
induced by OSM + TNF-a by 50% (p < 0.05) (Fig. 3H). PRED
or DEX by themselves could not induce cartilage formation
although there was a tendency towards increased formation when
combined with IGF-I (Fig. 3D and H). Generally, we saw the same
pattern for both GCs. However, PRED was less potent than DEX
as observed in the literature [8].
The biomarker data for aggrecan degradation were partly supported
by histology of the explants, using toluidine blue to detect
sulfated glycosaminoglycans (sGAGs) (Fig. 3I–T). The controls
worked; as shown in previous studies [30], stimulation with
OSM + TNF-a depleted the cartilage explants of sGAGs (Fig. 3J and
P), whereas IGF-I stimulation increased the content of sGAGs
(Fig. 3K and Q), compared to respective vehicles (Fig. 3I and O).
The cartilage explants treated with either PRED or DEX seemed to
have less sGAGs retained in the matrixes (Fig. 3L and R) than the
vehicle explants (Fig. 3I and O). On the other hand, when adding
DEX to OSM + TNF-a-stimulated cartilage explant, a small portion
of the sGAGs seemed to be retain in the matrix (Fig. 3S) compared
to the OSM + TNF-a-stimulated explant (Fig. 3P), although the visible
difference is almost lost on the digital images. Furthermore,
addition of PRED or DEX to the IGF-I-stimulated cartilage explants
seemed to increase the content of sGAGs (Fig. 3N and T).
3.4. The effect of dexamethasone depends on the activation
stage of osteoblasts
To investigate the effects of GCs on bone cells, the more potent
DEX [8] was used for further experiments/studies (Fig. 3). When