Introduction: Bone sialoprotein (BSP) is an RGD (Arginine-Glycine-Aspartate) containing non-collagenous extracellular matrix (ECM) protein that is extensively post-translationally modified, predominantly with glycosylated and phosphorylated residues. BSP plays a major role in bone mineralisation and this is thought to exert RGD and non-RGD effects on bone cells. In vivo studies have shown that BSP induces new bone formation in rat critical calvarial defects and that the state of phosphorylation of BSP and OPN changes during the healing of bone defects. We hypothesised that variable BSP phosphorylation was a determinant of bone turnover.

Methods: We adopted an ex vivo approach utilising neonatal mouse calvarial organ culture systems to test this hypothesis and utilised PTH (parathormone) treated mouse calvarial organ cultures to assess the effect of native BSP (phosphorylated) and dephosphorylated BSP on osteoclastogenesis. Seven day old outbred CD1 mice calvarial bone explants were incubated in culture media with 10nM PTH containing native or dephosphorylated BSP. Arsenazo III microplate calcium assays on the media and alkaline phosphatase and tartrate resistant acid phosphatase (TRAP) microplate assays were performed on calvarial lysates. At the end of culture, calvaria were fixed in 10% neutral buffered formalin and stained with H& E, von Kossa or Neutral Red.

Results: The cumulative release of calcium in response to PTH did not change significantly, in the presence of native BSP whereas in the presence of dephosphorylated BSP the calcium release was significantly (p< 0.001) inhibited, 3.1 μmol/ml +/− 0.2 and 1.5 μmol +/− 0.1. TRAP enzyme activity measurements on calvarial lysates were consistent with the above results. Histology showed readily apparent differences in osteoclastic activity on H& E, von Kossa and Neutral Red staining.

Discussion: BSP can inhibit osteoclast formation and activity, and this appears to be dependent on its state of phosphorylation. The dephosphorylation of ECM proteins by bone phosphatases is proposed as an auto-regulatory mechanism during bone turnover. Loss of organic phosphate with aging or disease may disturb auto-regulatory mechanisms of bone turnover leading to pathologic bone disease.