Chondrocyte Apoptosis after Simulated Intraarticular Fracture: A Comparison of Histologic Detection Methods
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.
- Aigner T, Hemmel M, Neureiter D, Gebhard PM, Zeiler G, Kirchner T, McKenna L. Apoptotic cell death is not a widespread phenomenon in normal aging and osteoarthritis human articular knee cartilage: a study of proliferation, programmed cell death (apoptosis), and viability of chondrocytes in normal and osteoarthritic human knee cartilage. Arthritis Rheum. 2001;44:1304–1312. CrossRef
- Arnold MM, Srivastava S, Fredenburgh J, Stockard CR, Myers RB, Grizzle WE. Effects of fixation and tissue processing on immunohistochemical demonstration of specific antigens. Biotech Histochem. 1996;71:224–230. CrossRef
- Blanco FJ, Guitian R, Vazquez-Martul E, de Toro FJ, Galdo F. Osteoarthritis chondrocytes die by apoptosis: a possible pathway for osteoarthritis pathology. Arthritis Rheum. 1998;41:284–289. CrossRef
- Dapson RW. Fixation for the 1990’s: a review of needs and accomplishments. Biotech Histochem. 1993;68:75–82. CrossRef
- Didenko VV, Hornsby PJ. Presence of double-strand breaks with single-base 3’ overhangs in cells undergoing apoptosis but not necrosis. J Cell Biol. 1996;135:1369–1376. CrossRef
- Frankfurt OS, Krishan A. Identification of apoptotic cells by formamide-induced dna denaturation in condensed chromatin. J Histochem Cytochem. 2001;49:369–378.
- Frankfurt OS, Robb JA, Sugarbaker EV, Villa L. Monoclonal antibody to single-stranded DNA is a specific and sensitive cellular marker of apoptosis. Exp Cell Res. 1996;226:387–397. CrossRef
- Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K, Bursch W, Schulte-Hermann R. In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology. 1995;21:1465–1468.
- Hornsby PJ, Didenko VV. In situ DNA ligation as a method for labeling apoptotic cells in tissue sections: an overview. Methods Mol Biol. 2002;203:133–141.
- Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239–257.
- Kim HA, Lee YJ, Seong SC, Choe KW, Song YW. Apoptotic chondrocyte death in human osteoarthritis. J Rheumatol. 2000;27:455–462.
- Kim HT, Teng MS, Dang AC. Chondrocyte apoptosis: implications for osteochondral allograft transplantation. Clin Orthop Relat Res. 2008;466:1819–1825. CrossRef
- Marshman E, Ottewell PD, Potten CS, Watson AJ. Caspase activation during spontaneous and radiation-induced apoptosis in the murine intestine. J Pathol. 2001;195:285–292. CrossRef
- Matsuo M, Nishida K, Yoshida A, Murakami T, Inoue H. Expression of caspase-3 and -9 relevant to cartilage destruction and chondrocyte apoptosis in human osteoarthritic cartilage. Acta Med Okayama. 2001;55:333–340.
- Nuttall ME, Nadeau DP, Fisher PW, Wang F, Keller PM, DeWolf WE, Jr., Goldring MB, Badger AM, Lee D, Levy MA, Gowen M, Lark MW. Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro. J Orthop Res. 2000;18:356–363. CrossRef
- Perez HE, Luna MJ, Rojas ML, Kouri JB. Chondroptosis: an immunohistochemical study of apoptosis and Golgi complex in chondrocytes from human osteoarthritic cartilage. Apoptosis. 2005;10:1105–1110. CrossRef
- Roach HI, Aigner T, Kouri JB. Chondroptosis: a variant of apoptotic cell death in chondrocytes? Apoptosis. 2004;9:265–277. CrossRef
- Roach HI, Clarke NM. Physiological cell death of chondrocytes in vivo is not confined to apoptosis: new observations on the mammalian growth plate. J Bone Joint Surg Br. 2000;82:601–613. CrossRef
- Tbakhi A, Totos G, Hauser-Kronberger C, Pettay J, Baunoch D, Hacker GW, Tubbs RR. Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma. Am J Pathol. 1998;152:35–41.
- Willingham MC. Cytochemical methods for the detection of apoptosis. J Histochem Cytochem. 1999;47:1101–1110.
- Zamzami N, Kroemer, G. Apoptosis: condensed matter in cell death. Nature. 1999;401:127–128.
- Zhu C, Wang X, Hagberg H, Blomgren K. Correlation between caspase-3 activation and three different markers of DNA damage in neonatal cerebral hypoxia-ischemia. J Neurochem. 2000;75:819–829. CrossRef
- Zunino SJ, Singh MK, Bass J, Picker LJ. Immunodetection of histone epitopes correlates with early stages of apoptosis in activated human peripheral T lymphocytes. Am J Pathol. 1996;149:653–663.
- Chondrocyte Apoptosis after Simulated Intraarticular Fracture: A Comparison of Histologic Detection Methods
- Open Access
- Available under Open Access This content is freely available online to anyone, anywhere at any time.
Clinical Orthopaedics and Related Research®
Volume 467, Issue 7 , pp 1877-1884
- Cover Date
- Print ISSN
- Online ISSN
- Additional Links
- Industry Sectors