Revisiting whitlockite, the second most abundant biomineral in bone: Nanocrystal synthesis in physiologically relevant conditions and biocompatibility evaluation

The synthesis of pure whitlockite (WH: Ca₁₈Mg ₂(HPO₄)₂(PO₄)₁₂) has remained a challenge even though it is the second most abundant inorganic in living bone. Although a few reports about the precipitation of WH in heterogeneous phases have been published, to date, synthesizing WH without utilizing any effects of a buffer or various other ions remains difficult. Thus, the related research fields have encountered difficulties and have not been fully developed. Here, we developed a large-scale synthesis method for pure WH nanoparticles in a ternary Ca(OH)₂-Mg(OH)₂-H ₃PO₄ system based on a systematic approach. We used excess Mg²⁺ to impede the growth of hydroxyapatite (HAP: Ca ₁₀(PO₄)₆(OH)₂) and the formation of other kinetically favored calcium phosphate intermediate phases. In addition, we designed and investigated the synthesis conditions of WH under the acidic pH conditions required to dissolve HAP, which is the most thermodynamically stable phase above pH 4.2, and to incorporate the HPO₄^2- group into the chemical structure of WH. We demonstrated that pure WH nanoparticles can be precipitated under Mg²⁺-rich and acidic pH conditions without any intermediate phases. Interestingly, this synthesized nano-WH showed comparable biocompatibility with HAP. Our methodology for determining the synthesis conditions of WH could provide a new platform for investigating other important precipitants in aqueous systems.