Ultrastructural organisation and molecular interactions in the hypertrophic cartilage extracellular matrix

Type X collagen is a member of the family of network-forming collagens, it contains a triple helical domain flanked by two non-collagenous (NC) domains, NC2 at the N-terminal and NCI at the C-terminal. It is expressed and synthesised by hypertrophic chondrocytes of the epiphyseal growth plates during the process of endochondral ossification (EO). This process involves replacement of a cartilaginous anlagen by bone, the coordination of chondrocyte proliferation, maturation and hypertrophy are followed by calcification of hypertrophic cartilage, vascular invasion and deposition of a bone matrix. A precise functional role for type X collagen has not been defined, although its temporal and spatial expression has linked it to endochondral ossification. A family of Small Leucine Rich Proteoglycans (SLRPs) have been demonstrated to be important for collagen biology. In particular, the well characterised decorin and biglycan, interact with numerous collagen types, non-collagenous proteins and growth factors. Decorin and biglycan have also been linked to the mineralisation process. This led to the hypothesis that type X collagen interacts with decorin and biglycan in the hypertrophic cartilage extracellular matrix. Interactions of type X collagen with decorin and biglycan were investigated using a solid phase assay and surface plasmon resonance (SPR). The interaction of type X collagen with decorin and biglycan was found to be of high affinity, with dissociation constants in the nanomolar range. Through using different domains of the type X collagen molecule the NCI domain, the triple helical region or whole type X collagen, it was demonstrated that the interactions are likely to be mediated by the NCI domain. The interaction of type X collagen with decorin and biglycan was found to be independent of the presence of the glycosaminoglycan chain(s) on decorin and biglycan, indicating the protein cores of decorin and biglycan are involved. Negative staining and transmission electron microscopy were used to visualise the interactions of type X collagen with decorin and biglycan labelled gold particles. The localisation of gold particles to the ends of type X collagen molecules supports the finding that the interactions are mediated by NCI. Recombinant fragments of the NCI domain were synthesised in an attempt to further determine regions of NC 1 important for the interactions with decorin and biglycan, these fragments have to date provided no additional information. Using RT-PCR decorin was demonstrated to be co-expressed with type X collagen by hypertrophic chondrocytes. Immunohistochemistry was utilised to study the localisation of type X collagen, decorin and biglycan in the hypertrophic cartilage extracellular matrix. All of the interacting components were shown to co-localise. The co-expression and co-localisation studies indicate that these interactions could occur in vivo. The characterised interactions of type X collagen with decorin and biglycan are likely to have functional roles in EO. Potentially they may be involved in regulation and assembly of a type X collagen pericellular matrix, they may adopt a structural role providing mechanical stability to an extracellular matrix undergoing dynamic remodelling, they could be involved in sequestering growth factors in the hypertrophic cartilage and hence be involved in modulating interactions of growth factors with their signalling receptors. These postulated functions are all possible in the normal development and growth of a long bone. Type X collagen, decorin and biglycan have all been implicated in pathology the interactions could alternatively be involved in pathological calcification. Molecular interactions that contribute to the molecular assembly of the growth plate are fundamental to its functions. Characterisation of such interactions will aid in defining precise roles for molecules such as type X collagen, decorin and biglycan during the process of EO.