Molecular and biochemical approach for understanding the transition of amorphous to crystalline calcium phosphate deposits in human teeth

Calcium phosphate (CaP) deposition during bone mineralization starts with the aggregation of Posner's clusters Ca₉(PO₄)₆ into amorphous Ca-phosphate (ACP), which then transforms into crystalline CaP and finally maturates to hydroxyapatite (HA). Using dentin/enamel of human teeth as a model system, we show that the physiological inorganic polymer polyphosphate (polyP), a phosphate donor in mineralization, prevents the transition from amorphous to crystalline CaP at concentrations> 15 wt%. Stabilization of the amorphous phase of CaP by polyP is reversed by hydrolysis of the polymer by alkaline phosphatase (ALP), an enzyme that releases phosphate for mineralization. It is still present in calcified enamel and dentin, as shown here by immunostaining and enzyme activity measurements. The phase transfer into crystalline CaP can be prevented by the ALP inhibitor levamisole. Besides TEM and SEM, the modulating effects of polyP and ALP on the kinetics of the phase transition from amorphous to crystalline CaP are demonstrated and confirmed by XRD and FTIR analyses. Molecular modeling studies show that the polyP chains, due to their dimensions, are able to penetrate into the channels between the Posner molecules, preventing cluster association to ACP and impairing HA crystal formation.