Abstract
Accurate evaluation of programmed cell death, or apoptosis, in chondrocytes is essential to studying cartilage injury. We evaluated four methods of detecting chondrocyte-programmed cell death in formalin-fixed, paraffin-embedded cartilage after experimental osteochondral fracture. Human osteochondral explants were subjected to experimental fracture in a manner known to induce high levels of chondrocyte-programmed cell death. After 4 days in culture, specimens were fixed and analyzed for programmed cell death using: (1) terminal deoxynucleotidyl transferase end labeling; (2) DNA denaturation analysis using an antibody specific for single-stranded DNA; (3) immunohistochemistry using antisera specific for active caspase-3; and (4) in situ oligonucleotide ligation. Quantitative analysis of programmed cell death levels for each technique was performed comparing injured and uninjured areas of cartilage. We observed differences between injured and uninjured areas of cartilage using the four methods. Human cartilage fixed in zinc-formalin and embedded in paraffin is amenable to programmed cell death analysis using any of four independent methods, each of which ostensibly has some advantages in terms of assaying different steps along the apoptotic pathway. Using the protocols described in this article, investigators may have additional tools to identify and quantify chondrocytes undergoing programmed cell death after experimental cartilage injury.