Calcium phosphate materials are widely investigated for the study of biomineralization processes and for the development of new biomaterials. It has been proposed that the crystallization of hydroxyapatite in aqueous media occurs in steps and not directly by ion-by-ion accretion. Prenucleation nanoclusters aggregate to form amorphous spheres and further crystallize into hydroxyapatite. However, conventional analytical techniques cannot visualize phase transformations over time continuously, and the nonclassical crystallization of hydroxyapatite is still under debate. In situ liquid-phase transmission electron microscopy (TEM) is a novel technique that allows us to follow reactions occurring during TEM in real time and thus to visualize the formation of calcium phosphate crystals from the formation of the first mineral nucleus to the final crystallized phase. In this work, in situ TEM was performed, and videos showed the exact moment of hydroxyapatite nucleation and crystallization. During the experiment, different amorphous calcium phosphate nanoparticles were identified prior to the formation of the plate/needle-like hydroxyapatite particles. Crystallization was achieved only after particle elongation. A combined kinetic model that considered the thermodynamic nonideality of the electrolyte solution was applied. Sensitivity analyses were performed to interpret the thermodynamic environment of the liquid cell and the possible species formed over time.
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