Insight by Cryo-TEM into the growth and crystallization processes of calcium phosphate nanoparticles in aqueous medium

Abstract

Hydroxyapatite (HA) is the main constituent of bone mineral in vertebrates and is widely used as a biomaterial in biomedical studies and medical clinics. The synthesis of this calcium phosphate phase is a crucial factor in its potential use in various applications as well as in understanding the nucleation and growth processes in biological mineralization (biomineralization). In this work, a sequential analysis of the different steps of HA synthesis in aqueous media was performed by using cryo-transmission electron microscopy (Cryo-TEM), which allows preservation of the specimens at different stages of the synthesis process, preventing their modification during drying. X-ray energy dispersive spectroscopy and electron diffraction, in addition to high-resolution imaging, were performed on vitrified samples. We showed that drying a specimen from the synthesis solution induces the crystallization of nanoparticles into an HA phase after 30 min of reaction, while when analyzed by Cryo-TEM, crystalline nanoparticles were detected only after 15 h. Calcium phosphate nanoparticles presented three different morphologies: irregular shapes characterized by low contrast in TEM (5 min), isotropic shapes with well-defined contrast (10 min) and needle-like shapes (after 30 min). The long axis of the HA needle-like nanoparticles can be associated with the c-axis direction of the hexagonal unit cell, which can thus be assigned to the preferential crystallization direction. However, a diffraction signal of the nanoparticles in vitreous ice that “mimics” the original solution was detected by Cryo-TEM only after 15 h of synthesis. These apparently controversial results are discussed in this manuscript considering various aspects of the use of Cryo-TEM in relation to the current concepts of calcium phosphate nucleation and growth.