Branched Polymeric Prenucleation Assemblies Initiate Calcium Phosphate Precipitation

The formation of crystalline calcium phosphate (CaP) has recently gained ample attention as it does not follow the classic nucleation-and-growth mechanism of solid formation. Instead, the precipitation mechanisms can involve numerous intermediates, including soluble prenucleation species. However, structural features, stability, and transformation of such solution-state precursors remain largely undisclosed. Herein, we report a detailed and comprehensive characterization of the sequential events involved in calcium phosphate crystallization starting from the very early prenucleation stage. We integrated an extensive set of time-resolved methods, including NMR, turbidimetry, SAXS, cryo-TEM, and calcium-potentiometry to show that CaP nucleation is initiated by the transformation of "branched" polymeric prenucleation assemblies into amorphous calcium phosphate spheres. Such a mineralization process starts with the spontaneous formation of so-called nanometric prenucleation clusters (PNCs) that later assemble into those branched polymeric assemblies without calcium ion uptake from the solution. Importantly, the branched macromolecular species are invisible to many techniques (NMR, turbidity, calcium-potentiometry) but can readily be evidenced by time-resolved SAXS. We find that these polymeric assemblies constitute the origin of amorphous calcium phosphate (ACP) precipitation through an unexpected process: spontaneous dissolution is followed by local densification of 100-200 nm wide domains leading to ACP spheres of similar size. Finally, we demonstrate that the timing of the successive events involved in the CaP mineralization pathway can be kinetically controlled by the Ca²⁺/P_i molar ratio, such that the lifetime of the soluble transient species can be increased up to hours when decreasing it.