Organic Extracellular Matrix Components at the Bone Cell/Substratum Interface

We have recently shown that differentiating bone cells are capable of elaborating, in vitro, a calcified matrix at the interface with several non-biological substrata which is similar to cement lines found in remodelling bone tissue. From previous morphological observations, this matrix appeared free of assembled collagen fibres which indicated that osteogenic cells produce an initial mineralized matrix before overt collagen fibre production. In the work reported herein, we used six antibodies to extracellular matrix components and two conjugates of secondary antibody, fluorescein and colloidal gold, to initiate a preliminary characterization of the organic components at the bone cell substratum interface at early periods of culture. The labels were visualized by transmitted fluorescence light and immuno-gold scanning electron microscopy, respectively. Both secondary and backscattered modes were employed in the latter . The results showed that while chondroitin sulphate was ubiquitous in this culture system, osteopontin labeled discretely at the ends of cell pseudopodia, initially, following which the substratum surface was strongly labeled. The distribution of fibronectin was significantly different to that of osteopontin and indicated that this protein was primarily involved in cell/cell rather than cell/substratum adhesion. Although Type I collagen was not present in the extracellular matrix at early periods of culture, it was evident intracellularly at 3 days and extracellularly after the formation of the initial cement-like matrix. However, Type III collagen displayed the inverse sequence. In less mature cultures, it exhibited a ubiquitous distribution, while it was un-detectable at later stages of culture. Finally, osteocalcin was the only antibody employed which showed no labeling at early culture periods while at later stages, both the cellular compartment and the previously formed interfacial matrix labeled positively.