Chondrogenic ATDC5 cells: An optimised model for rapid and physiological matrix mineralisation

Abstract

The development of chondrogenic cell lines has led to major advances in the understanding of how chondrocyte differentiation is regulated, and has uncovered many signalling pathways and gene regulatory mechanisms required to maintain normal function. ATDC5 cells are a well established in vitro model of endochondral ossification; however, current methods are limited for mineralisation studies. In this study we demonstrate that culturing cells in the presence of ascorbic acid and 10 mM β-glycerophosphate (βGP) significantly increases the rate of extracellular matrix (ECM) synthesis and reduces the time required for mineral deposition to occur to 15 days of culture. Furthermore, the specific expression patterns of Col2a1 and Col10a1 are indicative of ATDC5 chondrogenic differentiation. Fourier transform-infrared spectroscopy analysis and transmission electron microscopy (TEM) showed that the mineral formed by ATDC5 cultures is similar to physiological hydroxyapatite. Additionally, we demonstrated that in cultures with βGP, the presence of alkaline phosphatase (ALP) is required for this mineralisation to occur, further indicating that chondrogenic differentiation is required for ECM mineralisation. Together, these results demonstrate that when cultured in the presence of ascorbic acid and 10 mM βGP, ATDC5 cells undergo chondrogenic differentiation and produce a physiological mineralised ECM from Day 15 of culture onwards. The rapid and novel method for ATDC5 culture described in this study is a major improvement compared with currently published methods and this will prove vital in the pursuit of underpinning the molecular mechanisms responsible for poor linear bone growth observed in a number of chronic diseases such as cystic fibrosis, chronic kidney disease, rheumatological conditions and inflammatory bowel disease.

Expression of Sulf1 and Sulf2 in cartilage, bone and endochondral fracture healing

“…Unlike the immunolabelling analysis of human and rat articular cartilage showing comparable levels of both SULF1 and SULF2 in most samples as shown in later figures, these two transcripts changed in opposite direction in ATDC5 cells concomitant with the onset of matrix mineralisation (Newton et al 2012). Sulf1 under these conditions thus showed very low level expression up to day 8 after which it gradually increased up to day 34.…”

“…Cells were incubated in ALP staining solution for 30 min and washed with deionized water before imaging. ATDC5 cell culture: Chondrogenic ATDC5 cells (Riken Cell Bank, Ibaraki, Japan) were cultured in a differentiation medium [DMEM/F-12 (1:1) with GlutaMAX I containing 5% fetal bovine serum (FBS), 1% insulin transferrin and selenium, 1% sodium pyruvate and 0.5% gentamicin (Invitrogen, Paisley, UK)] at a density of 6,000 cells/cm 2 in multi-well plates (Iwaki Cell Biology; Sterilin, Feltham, UK) as described previously (Newton et al 2012;Staines et al 2012). Cells were left for 6 days to reach confluency at which point the medium was supplemented with 10 mM βGP and 50μg/ml L-ascorbate-2-phosphate (ascorbic acid).…”

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

“…16 Alcian blue staining of cultured cells Alcian blue staining and quantification was performed as described previously. 40 Quantification of colonies was performed using ImageJ by setting color and size thresholds.…”

MMP and TIMP temporal gene expression during osteocytogenesis

“…Cells were fixed with 4% paraformaldehyde (PFA) for 5 min at 4 o C. Cell layers were stained with aqueous 2% (w/v) Alizarin red solution (Sigma) at pH 4.2, for 5min at room temperature as previously described (Newton et al, 2012;Staines et al, 2012). The bound stain was subsequently solubilized in 10% cetylpyridinium chloride (Sigma) and the optical density of the resultant solution determined at 570 nm by spectrophotometry (Thermo Multiskan Ascent).…”