Low-intensity ultrasound stimulation promotes differentiation of bone marrow mononuclear cells to nucleus pulposus cells for matrix synthesis

Objective: To investigate the role of low-intensity ultrasound stimulation (LIUS) in facilitating the differentiation of bone marrow mononuclear cells (BMMNCs) into nucleus pulposus cells (NPCs) for matrix synthesis, offering a possible new therapeutic approach for intervertebral disc degeneration. Methods: Human BMMNCs and NPCs were cultured, and exosomes were extracted from NPCs using differential ultracentrifugation, followed by characterization. LIUS was utilized to evaluate exosome uptake, induce cell differentiation, measure apoptosis, and track DNA synthesis by EdU assays. Various experimental conditions were tested, including different LIUS intensities and differentiation durations. A range of detection techniques, such as RT-qPCR, western blotting, and cellular staining, were employed to monitor relevant indicators. Results: Exosomes were successfully isolated from NPCs, and their purity was confirmed using nanoparticle tracking analysis (NTA), transmission electron microscopy, and western blot. PKH67-labeled exosomes were internalized by BMMNCs during co-incubation. LIUS treatment at different intensities revealed that the LIUS-100 group exhibited the most significant cell proliferation, as shown by EdU assays. Flow cytometry revealed that the LIUS-100 and LIUS-150 groups demonstrated the most pronounced inhibition of apoptosis. In NPC exosome-induced differentiation experiments, the expression of relevant marker mRNA and protein levels increased over time under standard conditions, with even greater upregulation observed under LIUS-100 stimulation. Moreover, LIUS-100 enhanced the intracellular accumulation of glycosaminoglycans and proteoglycans, suggesting its role in promoting BMMNC differentiation into NPCs and matrix component synthesis. Conclusion: NPC exosomes and LIUS are essential for guiding the differentiation of BMMNCs into NPCs, representing a promising therapeutic strategy for intervertebral disc degeneration. However, further in vivo studies are needed to refine LIUS technique, ensure safety, and evaluate long-term efficacy.