br Data availability br Acknowledgments The work was support

Data availability
Acknowledgments The work was supported in Department of Biochemistry by grants from British Heart Foundation (SP/15/7/31561, FS/15/20/31335 and RG/15/4/31268). At the Department of Materials Science, Cambridge, funding was from the People Programme of the EU 7th Framework Programme (RAE no: PIIF-GA-2013-624904, to DVB), a Proof of Concept grant from the EPSRC Medical Technologies IKC, and an ERC Advanced Grant 320598 3D-E (to REC). At the National Heart and Lung Institute, London, funding was from MRC Doctoral training partnership PhD studentship, and Biomedicine and Bioengineering in Osteoarthritis (BBOA) studentship. We are grateful for help and advice from Dr Daniel Bax, Department of Materials Science, Cambridge, and Mr Douglas Sammon, National Heart and Lung Institute, London.
Introduction During endochondral ossification (EO), chondrocytes within the cartilage template undergo a highly co-ordinated sequence of proliferation, maturation and hypertrophy with the concomitant changes in the synthesis and deposition of extracellular matrix (ECM) components at each stage (Ortega et al., 2004). Many paracrine and endocrine factors are now known to be regulators of EO (Hering, 1999, de Crombrugghe et al., 2000, van der Eerden et al., 2003). Indian Hedgehog and parathyroid hormone related protein have been shown to regulate chondrocyte hypertrophy in a negative feedback control loop (Vortkamp et al., 1996), possibly acting in conjunction with several other regulatory factors including bone morphogenetic proteins and fibroblast growth factors (Minina et al., 2001, Minina et al., 2002). The ECM also regulates EO by mediating cell migration and shape changes, cell proliferation and differentiation either through direct cell–matrix interactions or by facilitating growth factor binding to (-)-Bicuculline methiodide (Shimazu et al., 1996, Bass and Humphries, 2002). It has been generally accepted that the hypertrophic ECM serves as a permissive matrix to vascularisation and mineralisation. It is therefore conceivable that collagen X, which exhibits unique temporal and spatial expression patterns in the hypertrophic zone, would have specific regulatory roles in EO besides the maintenance of tissue structure and integrity. Collagen X is a homotrimeric molecule of three α1(X) chains (Mr 59 kDa) comprising a 45 kDa triple-helical domain flanked by an N-terminal (NC2) and a larger C-terminal (NC1) non-collagenous domains (Shen, 2005). In the hypertrophic ECM, collagen X most likely forms an extended hexagonal network, as shown by in vitro studies (Kwan et al., 1991) and by electron microscopy on the murine growth plate (Jacenko et al., 2001), and is particularly abundant in the pericellular matrix of hypertrophic chondrocytes (LuValle et al., 1992, Tselepis et al., 1996). Mutations of the human Col10A1 gene are known to cause Schmid metaphyseal chondrodysplasia, an autosomal dominant disorder characterised by short stature, widened growth plates, bowing of the long bones and coxa vara (Warman et al., 1993, Wallis et al., 1994, Mäkitie et al., 2005) with the majority of mutations being found in the NC1 domain (Chan and Jacenko, 1998). Skeletal defects characteristic of spondylometaepiphyseal chondrodysplasia were reported in mice expressing a truncated collagen X transgene containing a large in-frame deletion (Jacenko et al., 1993). These studies indicate that either a reduction collagen X deposition due to haploinsufficiency or disruption of the normal collagen X network due to dominant interference can lead to aberrant EO. Furthermore, the complete lack of collagen X deposition in the matrix of Col10A1 −/− mice resulted in growth plate compression, displacement of proteoglycans, altered mineral deposition, and hematopoietic changes (Kwan et al., 1997, Gress and Jacenko, 2000, Jacenko et al., 2002). Based on the disease phenotypes observed in these transgenic mouse models, the pericellular collagen X network appears to be an important link between the hypertrophic chondrocytes and the interterritorial matrix especially in stabilising the proteoglycan network. We therefore propose that the interactions between hypertrophic chondrocytes and the collagen X network are important in maintaining the integrity of the hypertrophic matrix and regulate chondrocyte metabolism through cell adhesion molecules. This hypothesis is partially supported by our recent findings that hypertrophic chondrocytes can adhere and spread on a collagen X substrate (Luckman et al., 2003). Hypertrophic chondrocyte adhesion to collagen X is primarily mediated through the α2β1 integrin. In this study, we report the interactions between collagen X and a non-integrin collagen receptor, the discoidin domain receptor DDR2.