Cartilage Explant Culture with ADAMTS-4 and MMP-13

Recombinant human ADAMTS-4 (R&D Systems, Minneapolis, MN, USA) and MMP-13 (R&D Systems, Minneapolis, MN, USA) were diluted to 50 ng/ml in the tissue explant culture medium. Cartilage explant tissues were incubated in the control culture medium and 50 ng/ml ADAMTS-4 at 37 °C and 5% CO₂ for 7 days; or incubated in the control culture medium and 50 ng/ml MMP-13 at 37 °C and 5% CO₂ for 1, 4 or 7 days respectively. The plugs were then sliced perpendicular to the articular surface in 30 μm-thick sections by a vibratome (VT-1000S, Leica Microsystems Inc., Germany) in physiological buffer to preserve cell viability. Cartilage sections were washed thoroughly with PBS and pre-stained with Calcein AM and DAPI (Life Technologies, Grand Island, NY, USA) to identify live cells, and then affixed to a coverslip by applying a small drop of cyanoacrylate (Loctite, Westlake, OH, USA) to each end of the tissue sections but not the AFM testing regions as previously described^{23 (link)}.