, Volume 467, Issue 7, pp 1877-1884,
Open Access This content is freely available online to anyone, anywhere at any time.
Date: 11 Apr 2009

Chondrocyte Apoptosis after Simulated Intraarticular Fracture: A Comparison of Histologic Detection Methods

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.

One or more of the authors (HK) have received funding from UCSF School of Medicine, UCSF Department of Orthopaedic Surgery, and San Francisco VA Medical Center.
Each author certifies that his or her institution has approved the human protocol for this investigation, that all investigations were conducted in conformity with ethical principles of research, and that informed consent for participation in the study was obtained.