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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8562-8565, September 1993


Nucleation of hydroxyapatite by bone sialoprotein
(sepontn/sedy stte)

Faculty of Dentistry, University of Western Ontario, London, ON N6A 5C1, Canada

Communicated by Heinz A. Lowenstamt, May 27, 1993

ABSTRACT Bone sdaloprotein (BSP) and osteopontin, the majorphosphorylated proteins of mammalin bone, have been proposed to finctIon in the initiation of mineralization. To test this hypothes, the effects ofBSP and osteopontin on hydroxyaatite crystdal formation were determied by usig a steadystate agarose gel system. At low calcium phosphate concentrations, no accmulatio of calcium and phosphate ocurred in control gels or gels cotinin osteopontin. Gelscontaining BSP at 1-5 pg/mn, however, exhibited a vidble precipitation band and sg ay elevated Ca + P04 contents. By powder x-ray diffrcion, the precipitate formed in the presence of BSP was shown to be hydroxyapatite. These ings es that bone ialoprotein may be involved in the nucleation of hydroxyapatite at the mineralzation front of bone.
The mineralization of bone occurs by deposition ofcarbonated hydroxyapatite (HA) crystals in an extracellular matrix consisting of type I collagen and a variety of noncollagenous proteins. The means by which bone mineralization is achieved is not understood, but several authors have suggested that HA could be nucleated by a complex composed of a polyanionic protein, probably a phosphoprotein, and the large highly ordered fibrils of type I collagen (1-4).The major phosphoproteins of mammalian bone are bone sialoprotein (BSP) and osteopontin (5). Both are phosphorylated sialoproteins containing tyrosine sulfates, regions enriched in acidic amino acids, and an Arg-Gly-Asp cell attachment sequence. Although osteopontin and BSP do not appear to be evolutionarily related, they have similar amino acid compositions and post-transcriptional modifications.However, BSP contains more sialic acid and sulfate, whereas osteopontin contains more phosphate (5). These proteins also differ in their biological properties. By immunocytochemistry, in situ hybridization, and Northern blotting, it has been shown that osteopontin is expressed at high levels in mineralized connective tissues but also at lower levels in other tissues, including kidney, nervoustissue, and uterus; BSP, in contrast, is specific to mineralized tissues (6-8). During embryogenesis, BSP is first expressed at the onset of bone formation, whereas osteopontin expression is a later event (7). In mineralizingbone cell and organ cultures, BSP associates with the osteoid matrix, whereas osteopontin is released into the culture medium (9, 10). For these reasons, Sodek and coworkers (7)have suggested that BSP is a strong candidate for a role in mineral nucleation. In the present study, the effects of BSP and osteopontin on HA formation have been studied by using a steady-state agarose gel system.

calcium reservoir

phosphate reservoir

dialysis membrane





FiG. 1. Steady-state agarose gel system for growth of HA.METHODS Purfication of BSP and Osteopontin from Porcine Calvaria. BSP and osteopontin were purified from adult porcine calThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

varia by fast protein liquid chromatography using ionexchange,HA, and gel-fltration columns as previously described (11). The identity and purity of the protein preparations used were ascertained by gel electrophoresis, Western blotting using affimity-purified antibodies, and amino acid analysis. Growth of HA Crystals in Steady-State Agarose Gels. SeaPlaque agarose (Mandel Scientific, Guelph, ON) was dissolved at 3% in deionized water by heating at 100°C,...
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