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Chondrocyte apoptosis is not essential for cartilage calcification: Evidence from an in vitro avian model
Author(s) -
Pourmand Eric P.,
Binderman Itzhak,
Doty Stephen B.,
Kudryashov Valery,
Boskey Adele L.
Publication year - 2006
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.20977
Subject(s) - endochondral ossification , chondrocyte , calcification , cartilage , apoptosis , tunel assay , terminal deoxynucleotidyl transferase , biology , microbiology and biotechnology , chemistry , pathology , anatomy , medicine , biochemistry
The calcification of cartilage is an essential step in the process of normal bone growth through endochondral ossification. Chondrocyte apoptosis is generally observed prior to the transition of calcified cartilage to bone. There are, however, contradictory reports in the literature as to whether chondrocyte apoptosis is a precursor to cartilage calcification, a co‐event, or occurs after calcification. The purpose of this study was to test the hypothesis that chondrocyte apoptosis is not a requirement for initial calcification using a cell culture system that mimics endochondral ossification. Mesenchymal stem cells harvested from Stages 21–23 chick limb buds were plated as micro‐mass cultures in the presence of 4 mM inorganic phosphate (mineralizing conditions). The cultures were treated with either an apoptosis inhibitor or stimulator and compared to un‐treated controls before the start of calcification on day 7. Inhibition of apoptosis with the caspase inhibitor Z‐Val‐Ala‐Asp (O‐Me)‐fluoromethylketone (Z‐VAD‐fmk) caused no decreases in calcification as indicated by radioactive calcium uptake or Fourier transform infrared (FT‐IR) analysis of mineral properties. When apoptosis was inhibited, the cultures showed more robust histological features (including more intense staining for proteoglycans, and more intact cells within the nodules as well as along the periphery of the cells as compared to untreated controls), more proliferation as noted by bromo‐deoxyuridine (BrdU) labeling, decreases in terminal deoxynucleotidyl transferase (Tdt)‐mediated dUTP nick‐end labeling (TUNEL) staining, and fewer apoptotic bodies in electron microscopy. Stimulation of apoptosis with 40–120 nM staurosporine prior to the onset of calcification resulted in inhibition of calcium accretion, with the extent of total calcium uptake significantly decreased, the amount of matrix deposition impaired, and the formation of abnormal mineral crystals. These results indicate that chondrocyte apoptosis is not a pre‐requisite for calcification in this culture system. J. Cell. Biochem. 100: 43–57, 2007. © 2006 Wiley‐Liss, Inc.