Osteogenic Cell Cultures Cannot Utilize Exogenous Sources of Synthetic Polyphosphate for Mineralization

Abstract

Phosphate is critical for mineralization and deficiencies in the regulation of free phosphate lead to disease. Inorganic polyphosphates (polyPs) may represent a physiological source of phosphate because they can be hydrolyzed by biological phosphatases. To investigate whether exogenous polyP could be utilized for mineral formation, mineralization was evaluated in two osteogenic cell lines, Saos-2 and MC3T3, expressing different levels of tissue non-specific alkaline phosphatase (tnALP). The role of tnALP was further explored by lentiviral-mediated overexpression in MC3T3 cells. When cells were cultured in the presence of three different phosphate sources, there was a strong mineralization response with β-glycerophosphate (βGP) and orthophosphate (Pi) but none of the cultures sustained mineralization in the presence of polyP (neither chain length 17-Pi nor 42-Pi). Even in the presence of mineralizing levels of phosphate, low concentrations of polyP (50 μM) were sufficient to inhibit mineral formation. Energy-dispersive X-ray spectroscopy confirmed the presence of apatite-like mineral deposits in MC3T3 cultures supplemented with βGP, but not in those with polyP. While von Kossa staining was consistent with the presence or absence of mineral, an unusual Alizarin staining was obtained in polyP-treated MC3T3 cultures. This staining pattern combined with low Ca:P ratios suggests the persistence of Ca-polyP complexes, even with high residual ALP activity. In conclusion, under standard culture conditions, exogenous polyP does not promote mineral deposition. This is not due to a lack of active ALP, and unless conditions that favor significant processing of polyP are achieved, its mineral inhibitory capacity predominates.

Polyphosphate: A Morphogenetically Active Implant Material Serving as Metabolic Fuel for Bone Regeneration

“…While Na + can form only one ion bond to phosphate groups, Ca 2+ is able to link two polyP chains together via its two positive charges. PolyP, with chain lengths between 17 and 50 P i units, has been described as a polymer that either activates or stimulates or has no effect or even inhibits bone, or bone‐like (SaOS‐2 or MC3T3) cells in vitro with respect to their mineralizing potential . In a recent thorough study, the potential reasons for those discrepancies have been discussed and attributed to different cellular as well as physical interference; the most likely explanation had been seen in the integrity of the polyP – differential hydrolytic cleavage of the polymer by ALP – as well as the morphology of the insoluble Ca 2+ deposits .…”

“…PolyP, with chain lengths between 17 and 50 P i units, has been described as a polymer that either activates or stimulates or has no effect or even inhibits bone, or bone‐like (SaOS‐2 or MC3T3) cells in vitro with respect to their mineralizing potential . In a recent thorough study, the potential reasons for those discrepancies have been discussed and attributed to different cellular as well as physical interference; the most likely explanation had been seen in the integrity of the polyP – differential hydrolytic cleavage of the polymer by ALP – as well as the morphology of the insoluble Ca 2+ deposits . Surely also the differences in the composition of the media used, e.g., McCoy's medium containing 0.1 g · L −1 of CaCl 2 and 0.5 g · L −1 of Na‐phosphate (monobasic; anhydrous) versus Dulbecco's modified essential media composed of 0.2 g · L −1 of CaCl 2 and 0.1 g · L −1 of Na‐phosphate (monobasic; anhydrous) might become determining.…”

Amorphous Ca2+ polyphosphate nanoparticles regulate the ATP level in bone-like SaOS-2 cells

“…One prerequisite for this activating function of polyP is that the soluble Na + salt of polyP (Na-polyP) is complexed with Ca 2+ in order to prevent Ca 2+ deprivation in the medium (Müller et al, 2011). Very recently, a compelling report has proposed that the metabolic effect of polyP might be dependent on the morphology of the polyP Ca 2+ salt (CapolyP) polymer added (Ariganello et al, 2014). A solution to those diverging results came after the introduction of the standardized protocol for the preparation of Ca-polyP (Müller et al, 2015a).…”

Polyphosphate as a metabolic fuel in Metazoa: A foundational breakthrough invention for biomedical applications

“…[79,117,118]. However, recently also conflicting data have been presented [119]. As pointed out by this group [119] one reason for those differences might be seen in the different chain length of the polymer and the morphology of Ca-polyP complexes [119].…”

“…However, recently also conflicting data have been presented . As pointed out by this group one reason for those differences might be seen in the different chain length of the polymer and the morphology of Ca‐polyP complexes . A further affecting parameter could be the endogenous ALP, present in the culture sera used; ALP is one component of the different sera (e.g.…”