Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin

Abstract

Bone sialoprotein (BSP) and osteopontin (OPN) are two major non-collagenous proteins in bone that have similar biochemical properties and can mediate cell attachment through an RGD (Arg-Gly-Asp) motif that recognizes the vitronectin receptor. To facilitate evaluations of the biological functions of BSP and OPN in bone formation, affinity-purified rabbit polyclonal antibodies against porcine BSP and OPN were used, together with a high-resolution protein A-gold immunocytochemical technique to reveal the ultrastructural localization of these proteins in undermineralized sections of 50-day fetal porcine calvarial bone. In addition, 35S-labelled antisense riboprobes were prepared to demonstrate the cellular expression of BSP and OPN in the same tissues using in situ hybridization. Immunolocalization for both BSP and OPN revealed the highest density of gold particles associated with electron-dense organic material found at the mineralization front and in 'cement lines'. Labelling was also observed in the mineralized matrix over electron-dense material between collagen fibrils. In the osteoid of newly-formed bone, immunogold labelling for BSP and OPN was associated with loci of mineralization, which were often characterized by feathery clusters of fine needle-like crystals. Results of in situ hybridization on the same tissues demonstrated that BSP mRNA expression was restricted to differentiated osteoblasts with particularly strong signals evident at sites of de novo bone formation. More moderate expression of BSP was observed in 'older' osteoblasts and in some of the newly-entrapped osteocytes. Although expression of OPN mRNA was also observed in osteoblasts and osteocytes, the level of hybridization was similar for most bone cells and not markedly stronger than the signal observed in some stromal cells. While it is evident from these and other studies that both BSP and OPN are associated with bone formation, the differences observed in cellular expression indicate distinct roles for these proteins in bone formation.

Immunocytochemistry of matrix proteins in calcified tissues: functional biochemistry on section

“…There is also an apparent accumulation of BSP and OPN in cement lines and laminae limitantes (Chen et al 1994;Riminucci et al 1995;McKee and Nanci 1996b). It has been proposed, using en bloc staining with the cationic dye malachite green to retain and stain polyanionic molecules, that cement lines and the interfibrillar patches form a continuous network (Riminucci et al 1995).…”

Changes of Osteopontin Distribution and Matrix Mineralization during Remodeling in Experimental Bone Formation.

“…The collagen fibers in lamellar bone are usually oriented in a preferential direction and closely packed, whereas in woven bone they appear as irregular bundles and have comparatively wide interfiber spaces. NCPs have been demonstrated in the spaces between collagen fibers [5,13,17,18]. Biologically, these matrix proteins mediate various events involving cells and the bone matrix, including mineralization during bone formation, and subsequent bone remodeling.…”

Calcified tissue histochemistry: from microstructures to nanoparticles

“…The main ones found in bone are osteocalcin, osteonectin, osteopontin (OPN), bone sialoprotein (BSP) and bone acidic glycoprotein-75 (BAG-75). Except for the first, these are all phosphorylated glycoproteins.Their role in calcification is still under discussion; it is interesting, however, that three of them, OPN, BSP and BAG-75 co-localize in calcification nodules, i.e., areas of initial calcification which develop in the osteoid tissue (Bianco et al, 1993;Riminucci et al, 1995;Nanci 1999).They are also found in cement lines (Chen et al, 1994).…”