Turhan Ertan, Goldberga Ieva, Pötzl Christopher, Keil Waldemar, Guigner Jean-Michel, Haßler Martin F T, Peterlik Herwig, Azaïs Thierry, Kurzbach Dennis
Institute of Biological Chemistry, Faculty of Chemistry, University of Vienna, Währinger Str. 38, Vienna 1090, Austria.
Vienna Doctoral School in Chemistry (DoSChem), University of Vienna, Währinger Str. 42, Vienna 1090, Austria.
J Am Chem Soc. 2024 Sep 18;146(37):25614-25624. doi: 10.1021/jacs.4c07325. Epub 2024 Sep 3.
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 Ca/P molar ratio, such that the lifetime of the soluble transient species can be increased up to hours when decreasing it.
结晶磷酸钙（CaP）的形成近来备受关注，因为它并不遵循固体形成的经典成核-生长机制。相反，沉淀机制可能涉及众多中间体，包括可溶性预成核物种。然而，此类溶液态前体的结构特征、稳定性和转变情况在很大程度上仍未被揭示。在此，我们报告了从极早期预成核阶段开始的磷酸钙结晶过程中一系列事件的详细而全面的表征。我们整合了一系列广泛的时间分辨方法，包括核磁共振（NMR）、比浊法、小角X射线散射（SAXS）、冷冻透射电子显微镜（cryo-TEM）和钙电位分析法，以表明CaP成核是由“分支状”聚合物预成核聚集体转变为无定形磷酸钙球引发的。这样一个矿化过程始于所谓的纳米级预成核簇（PNCs）的自发形成，这些预成核簇随后组装成那些分支状聚合物聚集体，且在此过程中未从溶液中摄取钙离子。重要的是，这些分支状大分子物种在许多技术（NMR、浊度法、钙电位分析法）下是不可见的，但可以通过时间分辨SAXS轻易地证明其存在。我们发现这些聚合物聚集体通过一个意想不到的过程构成了无定形磷酸钙（ACP）沉淀的起源：自发溶解之后是100 - 200纳米宽区域的局部致密化，从而形成类似尺寸的ACP球。最后，我们证明了CaP矿化途径中连续事件的时间可以通过Ca/P摩尔比进行动力学控制，这样当降低该摩尔比时，可溶性瞬态物种的寿命可以延长至数小时。
