Mesenchymal Stem Cell-Derived Extracellular Vesicles Protect Rat Nucleus Pulposus Cells from Oxidative Stress.

Abstract

Oxidative stress (OS) is mainly associated with the pathogenesis of intervertebral disc (IVD) degeneration; it causes nucleus pulposus cells (NPCs) to undergo senescence and triggers autophagy and apoptosis. This study aims to evaluate the regeneration potential of extracellular vesicles (EVs) derived from human umbilical cord-mesenchymal stem cells (hUC-MSCs) in an in vitro rat NPC-induced OS model. NPCs were isolated from rat coccygeal discs, propagated, and characterized. OS was induced by hydrogen peroxide (H2O2), which is confirmed by 2,7-dichlorofluorescein diacetate (H2DCFDA) assay. EVs were isolated from hUC-MSCs and characterized by analyzing the vesicles using fluorescence microscope, scanning electron microscope (SEM), atomic force microscope (AFM), dynamic light scattering (DLS), and Western blot (WB). The in vitro effects of EVs on migration, uptake, and survival of NPCs were determined. SEM and AFM topographic images revealed the size distribution of EVs. The phenotypes of isolated EVs showed that the size of EVs was 403.3 ± 85.94 nm, and the zeta potential was -0.270 ± 4.02 mV. Protein expression analysis showed that EVs were positive for CD81 and annexin V. Treatment of NPCs with EVs reduced H2O2-induced OS as evidenced by a decrease in reactive oxygen species (ROS) levels. Co-culture of NPCs with DiI-labeled EVs showed the cellular internalization of EVs. In the scratch assay, EVs significantly increased NPC proliferation and migration toward the scratched area. Quantitative polymerase chain reaction analysis showed that EVs significantly reduced the expression of OS genes. EVs protected NPCs from H2O2-induced OS by reducing intracellular ROS generation and improved NPC proliferation and migration.