In situ detection of superoxide anions within porcine articular cartilage

Cartilage was isolated from pig articular joints, and the production of reactive oxygen species (ROS) by chondrocytes embedded within the cartilage was assessed by two methods: the reduction of nitro blue tetrazolium and by the use of diaminobenzidine in the presence of manganese ions. Little constitutive generation of ROS was seen, but it could be detected after the addition of the calcium ionophore ionomycin. Further, the response seen was extremely heterogeneous; some cells showed a far greater release of ROS than others. Cells arranged in the columnar arrays of the deep zone were the most active, while those furthest from the cartilage/bone interface (i.e. nearer to the outer face of the cartilage) were unresponsive. Chondrocytes cultured in alginate beads also showed a similar heterogeneity in their response, suggesting that the isolation of these cells and the measurement of ROS production in a population is not representative of the true situation.