Cerium Oxide Nanoparticles Regulate Osteoclast Differentiation Bidirectionally by Modulating the Cellular Production of Reactive Oxygen Species

Abstract

Background

Cerium oxide nanoparticles (CeO ₂NPs) are potent scavengers of cellular reactive oxygen species (ROS). Their antioxidant properties make CeO ₂NPs promising therapeutic agents for bone diseases and bone tissue engineering. However, the effects of CeO ₂NPs on intracellular ROS production in osteoclasts (OCs) are still unclear. Numerous studies have reported that intracellular ROS are essential for osteoclastogenesis. The aim of this study was to explore the effects of CeO ₂NPs on osteoclast differentiation and the potential underlying mechanisms.

Methods

The bidirectional modulation of osteoclast differentiation by CeO ₂NPs was explored by different methods, such as fluorescence microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. The cytotoxic and proapoptotic effects of CeO ₂NPs were detected by cell counting kit (CCK-8) assay, TdT-mediated dUTP nick-end labeling (TUNEL) assay, and flow cytometry.

Results

The results of this study demonstrated that although CeO ₂NPs were capable of scavenging ROS in acellular environments, they facilitated the production of ROS in the acidic cellular environment during receptor activator of nuclear factor kappa-Β ligand (RANKL)-dependent osteoclast differentiation of bone marrow-derived macrophages (BMMs). CeO ₂NPs at lower concentrations (4.0 µg/mL to 8.0 µg/mL) promoted osteoclast formation, as shown by increased expression of Nfatc1 and C-Fos, F-actin ring formation and bone resorption. However, at higher concentrations (greater than 16.0 µg/mL), CeO ₂NPs inhibited osteoclast differentiation and promoted apoptosis of BMMs by reducing Bcl2 expression and increasing the expression of cleaved caspase-3, which may be due to the overproduction of ROS.

Conclusion

This study demonstrates that CeO ₂NPs facilitate osteoclast formation at lower concentrations while inhibiting osteoclastogenesis in vitro by inducing the apoptosis of BMMs at higher concentrations by modulating cellular ROS levels.