Bone Abstracts

Polyphosphates (polyPs) are inorganic phosphate chains found in many cell types with higher concentrations in bone cells. As a source of inorganic phosphate (Pi) and an effective calcium reservoir due to chelation, PolyPs enable total Ca²⁺ and PO₄²⁻ concentrations above those required for apatite saturation. Alkaline phosphatase (ALP) cleaves Pi from polyP, thus polyPs may be involved in apatite mineralization.

Aim: To investigate the role of exogenous polyP as a Pi source for mineralization.

Methods: We conducted experiments with osteoblastic cells expressing different endogenous ALP levels and also utilized lentiviral vectors (LV) to overexpress the ALP transgene. SAOS-2 cells (high ALP), MC3T3-E1 (typical ALP) and MC3T3-E1 LV-ALP (ALP overexpression) were cultured in the presence of either β-glycerophosphate (βGP) alone or polyP alone.

Results: Control (βGP–treated) SAOS-2 cells were von Kossa (VK, Pi staining) and alizarin red (AlzR, Ca-staining) positive. Despite an endogenously high level of ALP expression, SAOS-2 cells treated with polyP did not mineralize, as demonstrated by negative VK and AlzR. PolyP-treated MC3T3s and LV-ALP MC3T3s similarly displayed negative VK staining. However both MC3T3s cell types (control and LV-ALP), when treated with polyP yielded a uniform AlzR stain atypical of the standard punctate AlzR pattern observed with βGP-treated cells. Scanning electron microscopy and energy dispersive X-ray spectroscopy suggest that in these polyP-treated cultures AlzR binds to residual Ca-polyP, not mineral, resulting in non-specific, ‘false positive’ staining.

Conclusions: Our results highlight the caution required when evaluating mineralization with AlzR. They also demonstrate that, under standard cell culture conditions, exogenous polyP does not promote extracellular mineralization, suggesting incomplete metabolism of polyP by ALP and/or their involvement in inhibitory events. Understanding the role(s) of polyP in physiological nucleation and subsequent regular mineral deposition may provide perspective for regulation of normal and pathological mineralization. Supported by CIHR, FRQ20, NSERC and RSBO-FRQ20.