Branched Polymeric\nPrenucleation Assemblies Initiate\nCalcium Phosphate Precipitation

The formation of crystalline calcium phosphate (CaP)\nhas recently\ngained ample attention as it does not follow the classic nucleation-and-growth\nmechanism of solid formation. Instead, the precipitation mechanisms\ncan involve numerous intermediates, including soluble prenucleation\nspecies. However, structural features, stability, and transformation\nof such solution-state precursors remain largely undisclosed. Herein,\nwe report a detailed and comprehensive characterization of the sequential\nevents involved in calcium phosphate crystallization starting from\nthe very early prenucleation stage. We integrated an extensive set\nof time-resolved methods, including NMR, turbidimetry, SAXS, cryo-TEM,\nand calcium-potentiometry to show that CaP nucleation is initiated\nby the transformation of “branched” polymeric prenucleation\nassemblies into amorphous calcium phosphate spheres. Such a mineralization\nprocess starts with the spontaneous formation of so-called nanometric\nprenucleation clusters (PNCs) that later assemble into those branched\npolymeric assemblies without calcium ion uptake from the solution.\nImportantly, the branched macromolecular species are invisible to\nmany techniques (NMR, turbidity, calcium-potentiometry) but can readily\nbe evidenced by time-resolved SAXS. We find that these polymeric assemblies\nconstitute the origin of amorphous calcium phosphate (ACP) precipitation\nthrough an unexpected process: spontaneous dissolution is followed\nby local densification of 100–200 nm wide domains leading to\nACP spheres of similar size. Finally, we demonstrate that the timing\nof the successive events involved in the CaP mineralization pathway\ncan be kinetically controlled by the Ca<sup>2+</sup>/P<sub>i</sub> molar ratio, such that the lifetime of the soluble transient species\ncan be increased up to hours when decreasing it.