Cyclic Loading of Hyaline Articular Cartilage as a Tool in Cartilage Research

Introduction Understanding how mechanical loading affects cartilage is vital for understanding how osteoarthritis develops and how tissue engineered cartilage-scaffold constructs behave under conditions simulating in vivo cyclic loading. We have developed an in vitro dynamic loading chamber. In this report, development of chondrocyte apoptosis in unloaded and loaded bovine cartilage under these artificial in vitro conditions was assessed. Materials and Methods Cyclic loading was applied to 7 mm cartilage disc explants in a CO2-incubator (1) using sinusoidal waveform of 0.5 Hz-frequency and a peak stress of 0.5 MPa for up to 6 days. Loading periods of five second were followed by 100 second rest. The culturing media were changed on the 3th day. Explants were loaded (n=5) perpendicular to their long axis in radially-unconfined compression (Figure 1). Unloaded cartilage discs (n=5) cultured in loading chambers served as controls. The samples were embedded in paraffin. Methods to detect apoptosis 1) Transmission electron microscope disclosed apoptotic cells. These were quantified using 2) TUNEL reaction (singleand double-stranded DNA breaks) 3) staining of activated caspase-3 (the main effector caspase of apoptosis) 4) staining of single-stranded DNA. Morphometric counting using point counting principle and Leitz microscope and Diaplan lens system (Wetzlar, Germany) was performed. Ten high power fields were counted and results are expressed as the percentage of apoptotic chondocytes of all chondocytes.