In vitro model for the study of necrosis and apoptosis in native cartilage

Abstract

Apoptosis plays a role in everything from early development to ageing and in a host of disease states. Studying this important process in the in vivo state is critical, to understand its varied role and to open further avenues of therapeutic intervention. The present paper presents an ex vivo bovine articular cartilage model to study apoptotic and necrotic processes following acute injury. Ex vivo bovine articular cartilage was assessed 1, 3 and 6 days following holmium : YAG laser treatment (780 mJ). Markers to visualize cell viability, caspase-3 activity, changes in mitochondrial membrane potential and the degree of DNA fragmentation (TUNEL assay) were used alone or in various combinations. Standard histology and transmission electron microscopy (TEM) were also performed for a more comprehensive assessment. A significant progression (p < 0.05) of ethidium/caspase-3-positive signal depth at day 3 preceded a significant increase (p < 0.05) in TUNEL signal depth by day 6. The mitochondrial matrix marker CMXRos was shown to provide an alternative to calcein-AM for assessing cell viability. The identification of chondrocyte apoptosis morphology by TEM was not conclusive. Nevertheless, TEM revealed that cells which were clearly necrotic also stained positively for TUNEL, thus indicating the risk of using TUNEL alone for the assessment of apoptosis. The model described here allows the rapid, spatial and temporal determination of cell viability and of apoptotic and necrotic processes in whole-tissue specimens after acute injury, and permits study of the balance between these events. The assessment of healthy and diseased cartilage and of the effects of surgical, pharmaceutical or in vitro intervention are immediate applications of these protocols. Moreover, this model may be useful for the study of key mechanisms involved in apoptosis or for the establishment of other markers of apoptosis.