The International Journal of Biochemistry & Cell Biology 42 (2010) 880–889 Contents lists available at ScienceDirect The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel Periostin expression distinguishes between light and dark hypertrophic chondrocytes Kuan-Sheng Chen, Liliana Tatarczuch, Michiko Mirams, Yasser A. Ahmed, Charles N. Pagel, Eleanor J. Mackie ∗ School of Veterinary Science, University of Melbourne, Parkville, Victoria 3010, Australia article info Article history: Received 6 November 2009 Received in revised form 8 January 2010 Accepted 13 January 2010 Available online 18 January 2010 Keywords: Endochondral ossification Hypertrophic chondrocyte Light chondrocyte Dark chondrocyte Periostin abstract Hypertrophic chondrocytes exist in two forms detectable by electron microscopy, light and dark chondro- cytes; the functional implications of the heterogeneous morphology are unknown. The aims of the study were to establish a method for separating light from dark hypertrophic chondrocytes and to identify genes differentially expressed between the two populations. Three-dimensional pellet cultures of chondrocytes from cartilage of neonatal rats were induced to undergo hypertrophy by treatment with triiodothyro- nine. Cultures were dissociated and subjected to density gradient centrifugation. The cell fraction with the lowest density comprised predominantly light hypertrophic chondrocytes, and the fraction with the highest density comprised predominantly dark hypertrophic chondrocytes. An Affymetrix GeneChip ® rat expression array was used to compare expression between dark cell-containing pellets and the light cell-enriched fraction. Genes identified on the array as putative dark cell-selective genes included genes encoding extracellular matrix proteins and enzymic modulators thereof. Expression of a subset of genes (Col1a1, periostin, osteoglycin, tPA/Plat, and Chst11) was confirmed as dark cell-selective using quanti- tative reverse transcriptase polymerase chain reaction. The most highly differentially expressed dark cell-selective gene was periostin. In immunocytochemical studies of light and dark cell-enriched frac- tions, periostin staining was detectable in dark, but not light hypertrophic chondrocytes. The results provide insight into molecular differences between light and dark hypertrophic chondrocytes. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Most of the bones in the mammalian skeleton develop through the process of endochondral ossification, whereby a cartilage model of the future bone is formed then progressively replaced by bone tissue (Mackie et al., 2008). The longitudinal expansion of the bone results from proliferation of chondrocytes within the growth plate, a cartilaginous structure that persists until growth has ceased. Following proliferation, growth plate chondrocytes undergo hypertrophy, dramatically expanding their volume while partially degrading the extracellular matrix (ECM) surrounding them. Hypertrophic chondrocytes then die, and blood vessels and osteogenic cells invade the remaining cartilage tissue, depositing bone ECM on the cartilage remnants. Hypertrophic chondrocytes are generally considered to com- prise a single population that undergoes progressive morphological and molecular changes during the process of terminal differentia- tion and death. Ultrastructural studies have, however, described ∗ Corresponding author. Tel.: +61 3 8344 7360; fax: +61 3 8344 7374. E-mail address: ejmackie@unimelb.edu.au (E.J. Mackie). two morphologically distinct types of chondrocytes, ‘light’ and ‘dark’, in the growth plate of several species (Ahmed et al., 2007; Anderson, 1964; Erenpreisa and Roach, 1998; Hwang, 1978; Roach and Clarke, 2000; Wilsman et al., 1981). Dark hypertrophic chondrocytes are irregularly shaped with cytoplasmic processes; they contain electron-dense cytoplasm, abundant rough endo- plasmic reticulum (RER) and Golgi apparatus and their nucleus contains patches of condensed chromatin (heterochromatin). Light hypertrophic chondrocytes, in contrast, are typically round; the cytoplasm is electron-lucent with sparse RER, and the nuclear chromatin is not condensed (i.e. euchromatin). We have recently provided strong evidence that, in the horse, light and dark hyper- trophic chondrocytes constitute two distinct post-proliferative populations, rather than different stages of differentiation of a single population (Ahmed et al., 2007). Dark and light chondro- cytes can be observed at all levels of the growth plate, from the zone of proliferative chondrocytes to the ossification front, each undergoing distinct cell type-specific morphological changes during hypertrophy and death. In three-dimensional chondrocyte pellet culture, the ratio of light: dark cells can be manipulated using different treatments, but once the cells have started to undergo hypertrophy and can be distinguished as light or dark, the ratio 1357-2725/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2010.01.018