Identification of important interactions between subchondral bone ...

Methods
Sources of proteases, inhibitors and cytokines
CHAPTER 7: PAPER IV
Purified human matrix metalloproteinase-1 (MMP-1) and recombinant human MMP-7 were
purchased from Enzo life science, DK. Recombinant human Cathepsin-L and purified human
Cathepsin-S and Cathepsin-B were from BioVision, US. Recombinant human aggrecanases;
ADAMTS-4 and ADAMTS-5, were from Chemicon international, DK. Purified human MMP-9
and recombinant human MMP-3, MMP-13 and Cathepsin-K, were purchased from Biocol, DE.
The matrix metalloprotease inhibitor (R)-N4-Hydroxy-N1-[(S)-2-(1H-indol-3-yl)-1-
methylcarbamoyl-ethyl]-2-isobutyl-succinamide (GM6001) and the cysteine protease inhibitor
trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane, L-trans-3-Carboxyoxiran-2-carbonyl-L-
leucylagmatine, N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E64) were both
purchased from Sigma, DK. The cytokines, oncostatin M (OSM) was from Sigma, DK, and the
Tumor Necrosis Factor-α (TNF-α) was from R&D systems, UK.
Buffers
To evaluate endogenous activity of metalloproteinases (MMPs and ADAMTS’), cartilage explants
were incubated in an MMP buffer [50 mM Tris-HCL, 200 mM NaCl, 5 mM CaCl 2, 1 µM ZnCl 2,
0.05% NaN 3, 0.05% Brij-35, pH 7]. Activation of latent MMPs was performed by adding 1 mM
freshly made p-Aminophenylmercurie (APMA). To evaluate endogenous activity of cysteine
proteinases, cartilage explants were incubated in a cysteine buffer [100 mM Na-Acetate, 5 mM
EDTA, 0.05% NaN 3, pH 5]. Activation of latent cysteine proteinases was performed by adding
20 mM cysteine just before use. Both buffers were prepared with inspiration from Tabassi et al. 13 .
Ex vivo models
Human articular cartilage experiments
The human OA cartilage was obtained from female patients between the ages of 65-77 years
undergoing total knee arthroplasty. The patients were informed by both oral and written
communication at the hospital and gave their written consent before entering study, which was
approved by the Capital Region of Denmark, DK-3400, approval number HD-2007-0084. The
cartilage was metabolically inactivated (MI) by freeze-thaw cycles and cut into homologous
explants. Half of the MI explants were incubated in MMP buffer for 3, 6, and 9 h in the presence
or absence of the inhibitors, GM6001 [10 µM] and E64 [40uM], and the proteases [100 ng/ml];
MMP-1, -3, -7, -9, -13 and ADAMTS-4, -5. The other half were incubated in cysteine buffer for
3, 6, and 9 h in the presence or absence of the inhibitors, GM6001 [10 µM] and E64 [40 µM], and
the proteases [100 ng/ml]; Cathepsin-K, -B, -L, -S. At the end of each incubation time (3, 6, 9h),
conditioned buffer (about 300 µl) was extracted and stored frozen at -20°C until assayed for
specific biomarkers.
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