Identification of important interactions between subchondral bone ...

CHAPTER 7: PAPER IV
Detection of key proteases to degrade human OA cartilage ex vivo; evaluated by an aggrecan
degradation marker
In human metabolic inactivated OA cartilage incubated in MMP buffer, MMP-3 and -7, and
ADAMTS-4 and -5 all increased aggrecan degradation (AGNx-II) compared to non-stimulated
vehicle (fig. 1E). Addition of the MMP inhibitor, GM6001, to the proteases attenuated this
release of the aggrecan fragments (data not shown). We did not observe any changes in the
release of AGNx-II fragments by MMP-1, -9 and -13, compared to vehicle (fig. 1E).
The cathepsins did not affect the levels of AGNx-II significantly; however, we
observed a tendency towards a decrease in the levels of AGNx-II compared to vehicle (fig. 1F).
Collagen type II degradation in normal, cytokine-induced, and OA cartilage
In normal bovine cartilage, the endogenous proteinases activated by the MMP-buffer, degraded
the collagen type II in to fragments that were able to be detected by the biomarker, CTX-II. The
MMP inhibitor, GM6001, was able to attenuate CTX-II (Table 1). The bovine cartilage pre-
cultured with cytokines for 4 and 11 days before metabolic inactivated and incubated in MMP
buffer, increased the levels of CTX-II compared to normal bovine cartilage, which were also
attenuated in the presence of GM6001. However, the human OA cartilage did not generate CTX-
II fragments as in the bovine samples. The cysteine protease inhibitor, E64, did not affect the
CTX-II levels. The biomarker, NBC2, was not detectable in the MMP buffer (Table 1).
The proteinases activated by the cysteine buffer in both bovine and human
cartilage, generated fragments that could be detected by both biomarkers of collagen type II
degradation; CTX-II and NBC2 (Table 2). The MMP inhibitor, GM6001 showed a tendency to
decrease CTX-II levels (P=0.057) in normal bovine cartilage, whereas the same inhibitor showed
a tendency to increase CTX-II levels in pre-cultured bovine cartilage (11 days) and in human OA
cartilage (Table 2). Additionally, GM6001 increased NBC2 in human OA and bovine normal and
pre-cultured cartilage. The cysteine protease inhibitor, E64, decreased the levels of CTX-II in
human OA and bovine normal and pre-cultured (11 days) cartilage. E64 also decreased the levels
of NBC2 in normal bovine cartilage, whereas the cysteine protease inhibitor increased the levels
of NBC2 in bovine cartilage pre-cultured with cytokines for 4 days (Table 2). The bovine
cartilage pre-cultured with cytokines for 4 and 11 days before metabolic inactivated and
incubated in cysteine buffer, decreased the levels of CTX-II compared to normal bovine cartilage
(Table 2), which was the opposite effects from the one seen in MMP buffer (Table 1).
Furthermore, the human OA cartilage had CTX-II levels corresponding to the cartilage pre-
cultured with cytokines for 11 days (Table 2). Moreover, the levels of NBC2 were also lower for
the pre-cultured cartilage (11 days) compared with the normal cartilage. However, the human OA
cartilage had greatly higher levels of NBC2 compared to bovine cartilage in general (Table 2).
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