Real-Time Visualization of Calcium Phosphate Formation on Titanium Dioxide Nanoparticles Using Liquid Transmission Electron Microscopy

Abstract The integration of titanium dioxide (TiO 2 ) with calcium phosphate (CaP)-based hydroxyapatite (HAP) is a promising strategy for enhancing the bioactivity of bone implants. However, a fundamental understanding of the interfacial reactions governing CaP mineralization on TiO 2 remains limited due to lack of characterization techniques with sufficient spatial and temporal resolution in hydrated state. In this study, we combined in situ liquid TEM imaging and correlative ex situ TEM analyses to investigate the nucleation, aggregation, and crystallization of the CaP layer on TiO 2 nanoparticle surfaces. Our findings reveal a three-step mineralization process: (1) initial aggregation of TiO 2 nanoparticles in solution, (2) formation of an amorphous calcium phosphate-like (ACP-like) layer on the TiO 2 surface, leading to an ACP-like coated TiO 2 nanoparticle structure, and (3) progressive crystallization of ACP into HAP, forming a HAP-like coated TiO 2 nanoparticle structure. Liquid-TEM imaging captured dynamic transformations, including nanoparticle aggregation, structural evolution, and phase transitions, providing unprecedented insights into the physicochemical interactions underlying mineralization. Additionally, we evaluated the effects of electron beam exposure on TiO 2 nanoparticles, demonstrating that high electron flux densities can induce morphological instability. This study advances our understanding of CaP-TiO 2 interfacial mineralization and offers valuable guidance for optimizing bioactive coatings to improve osseointegration and the long-term stability of bone implants.