Biological nucleation of apatite mineral involves a local increase in calcium and/or phosphate concentration. In 1923, Robison postulated that alkaline phosphatase (ALP) activity resulted in an increased local inorganic phosphate (Pi) concentration. In the presence of calcium, carbonate and at neutral pH, this biochemically controlled increase in Pi concentration could provide the chemical potential to nucleate a carbonated apatite. The substrate for ALP that results in skeletal apatite nucleation has not yet been identified. The hypotheses that a calcium-carbonate-polyphosphate species is a substrate for ALP, and that an apatite mineral is a possible product of ALP activity was tested. Amorphous materials produced by mixing synthetic sodium polyphosphate with solutions containing calcium, magnesium and carbonate with a final total P (as polyphosphate) concentration of 0.5 M resulted in a hydrogel. Similar precipitations with sodium phosphate instead of sodium polyphosphate resulted in mineral precipitation. The hydrogel or Pi precipitate were filtered and incubated in a background electrolyte buffered at pH 9 and 37°C to optimise ALP activity. After 1 week, the precipitates were characterised by powder X-ray diffraction and Raman spectroscopy. Initial and final solution pH and phosphate concentration (by colorimetry) were measured. Raman spectroscopy identified a Pi peak with a Raman shift of ~960 cm−1 for precipitates formed from Pi, and from polyphosphate incubated with ALP. Precipitates incubated without ALP maintained a distinct polyphosphate shift at ~1170 cm-1. Powder X-ray diffraction patterns from precipitates formed in the presence of ALP exhibited many peaks, including peaks characteristic of carbonated apatite, among other peaks. Colorimetric analysis for phosphate indicated an increase in Pi concentration over the initial buffer solutions, which contained no Pi. This represents preliminary in vitro evidence that the combination of polyphosphate anions as the sole phosphorus source, and active ALP can result in apatite mineral nucleation.
Disclosure: The authors declared no competing interests. This work was supported by the Volkswagen Foundation Experiment! Program.