Mixing Regime Controlled Kinetics of Amorphous Calcium Phosphate Formation and Transformation

Calcium phosphate precipitation plays a central role in biomineralization processes and the design of biomaterials, with amorphous calcium phosphate (ACP) often acting as a transient precursor to more stable crystalline phases such as hydroxyapatite (HA). Despite extensive research, the mechanistic pathways of ACP formation and transformation remain only partially understood, as they are highly system-specific and sensitive to dynamic changes in solution chemistry. Here, in situ potentiometric monitoring, combining pH and calcium ion-selective electrode (Ca-ISE) measurements, is used to monitor ACP precipitation and transformation under different mixing regimes and in the presence of model additives. Complementary solid-phase characterization is employed to validate phase identity and composition. All systems followed a two-step ACP-mediated pathway, characterized by an initial metastable period followed by ACP precipitation and subsequent transformation to poorly crystalline HA. However, significant differences in kinetics were observed: the P in Ca setup exhibited delayed ACP nucleation and extended ACP lifetime compared to Ca in P, while additive presence further prolonged ACP stability, with citrate exerting a stronger effect than BSA. These variations were linked to differences in pH evolution, transient supersaturation, and solution speciation established during precursor mixing. Our findings highlight in situ potentiometric measurements as a valuable, accessible approach for probing calcium phosphate crystallization, provided its limitations are recognized and interpretations are supported by complementary characterization methods.