Intracellular pathway and subsequent transformation of hydroxyapatite nanoparticles in the SAOS-2 osteoblast cell line.

Internalization of hydroxyapatite nanoparticles in SAOS-2 osteoblasts for 2 and 24 h was investigated in vitro using 5 and 50 µg/mL nanoparticles in culture medium. No cytotoxic effects were observed in a PrestoBlue viability assay. Focused ion beam-scanning electron microscopy and transmission electron microscopy were used to study nanoparticle trafficking inside cells and to characterize the physicochemical properties of the remodeled nanoparticles. Nanoparticles were actively internalized by cells and maintained in intracellular membrane-bound compartments. Dissolution of hydroxyapatite nanoparticles was observed inside phagolysosome in all samples. After 24 h of internalization in cell culture assays, reprecipitation of calcium phosphate minerals was observed in membrane-bound compartments in 5 and 50 µg/mL samples. Compared to the original nanoparticles, the reprecipitated calcium phosphate phase presented a different morphology, structure, and chemical composition. Two sample preparation methods were used and confirmed that reprecipitation of the calcium phosphate crystallites occurred in the intracellular environment and not during electron microscopy sample preparation. Reprecipitation of calcium phosphate prevented the release of large amounts of calcium and phosphate ions inside the cells. This phenomenon may be linked to physiological processes in the cell that control the concentration and trafficking of intracellular calcium ions, which are highly controlled by cells.