Osteoclasts Resorb Protein-free Mineral (Osteologic™ Discs) Efficiently in the Absence of Osteopontin

Abstract

Osteopontin (OPN) is both a matrix protein in mineralized tissues and a cytokine, and it has a pivotal role in osteoclast-mediated bone resorption. Here, using a proprietary hydroxyapatite substitute for bone mineral (Osteologic™ discs), we investigated the requirement for OPN in mineral resorption. Resorption pits formed in the Osteologic™ discs, revealed by staining with silver nitrite (Von Kossa stain), were analyzed using the NIH Image J program, which can determine the number of pits formed per unit area, their average size, and the fractional area resorbed. After a preincubation of bone marrow cells from OPN^-/- and OPN^+/+ mice with M-CSF to allow the multiplication of osteoclast precursors on cell culture plastic, osteoclast formation on both Osteologic™ discs and standard cell culture plates was induced with soluble receptor activator of NFÎ B ligand, sRANKL. We did not detect a dramatic difference in osteoclast formation between OPN^+/+ and OPN^-/- cells, as judged by staining for tartrate-resistant acid phosphatase in osteoclasts formed on cell culture plastic, nor was there a significant difference in the ability of the osteoclasts to form resorption pits in the Osteologic™ discs. Additionally, none of six different anti-OPN monoclonal antibodies had a significant and reproducible effect on the formation or subsequent functioning of the OPN^+/+ osteoclasts. These studies suggest that, in contrast to what has been found for normal bone, the efficiency of dissolution of a ceramic, protein-free (excepting protein adsorbed from the culture medium) hydroxyapatite/tri-calcium phosphate substrate by osteoclasts is not substantially enhanced by endogenous or exogenous OPN.