Live Visualization of the Nucleation and Growth of Needle-Like Hydroxyapatite Crystals in Solution by In Situ TEM

Abstract

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.

Counterintuitive Crystallization: Rate Effects in Calcium Phosphate Nucleation at Near-Physiological pH

“…Although it is not possible to unequivocally determine which phase forms first using our methods, we believe it to be reasonable that an amorphous precursor to PC-HA exists, possibly forming alongside DCPD once the addition rate is in the “transition region”; this precursor results in the “bump” and eventual plateau post-nucleation. The presence of amorphous precursors preceding the formation of PC-HA is supported by earlier works . The “bump” strongly indicates that two phases are present due to its downward slope, which indicates a transformation is occurring; if one stable phase were present, then a definite plateau would be seen.…”

“…The presence of amorphous precursors preceding the formation of PC-HA is supported by earlier works. 29 The "bump" strongly indicates that two phases are present due to its downward slope, which indicates a transformation is occurring; if one stable phase were present, then a definite plateau would be seen. It is possible that multiple processes are superimposed in such a manner; multimechanism pathways have been proposed previously for this system.…”

“…Other groups have also confirmed the importance of ACP in in situ TEM studies. Dalmonico et al suggested that multiple mechanisms exist, seeing two types of ACP followed by growth driven by addition of ions or nanoclusters, or ACP nanoparticles either aggregating or attaching to previously nucleated HA . Other authors have also supported the possibility of multiple nucleation mechanisms coexisting, declaring that both classical and nonclassical nucleation pathways exist in liquid cell TEM .…”

Competition and Neutralization: Thermally Induced Crystallization and Phase Evolution of Amorphous Calcium Phosphate with Cosubstitution of Larger and Smaller Divalent Cations

“…Compared with crystalline phases, amorphous calcium phosphates (ACPs) draw great attention from researchers due to their unique characteristics. − Among its wide range (1.2–2.2) of Ca/P, the specific ACP under 1.5 have been widely studied for its thermally crystalline analogue is tricalcium phosphate (TCP), a universally utilized biomaterial. Benefiting from the short-range ordered structure, ACPs could transform into their crystalline analogue under appropriate thermal treatment and α-TCP was believed to be the first crystalline phase despite the fact that α-TCP was the high-temperature phase corresponding to β-TCP.…”

Liquid Transmission Electron Microscopy for Probing Collagen Biomineralization

“…41 Liquid imaging is often performed in one of two ways: with a commercial liquid TEM holder or by other, typically custom- made, encapsulation methods. Both approaches have been used previously to probe simple CaP reactions, revealing intricate dynamic pathways behind the formation of apatite; 34,37,43,44 however, neither approach has been used to observe collagen mineralization processes.…”