Combined Bone Marrow Mesenchymal Stem Cells-derived Nanovesicles and Low-Level Laser Therapy Potentiate Proliferation and Osteogenesis of Bone Marrow Mesenchymal Stem Cells

Jingwei Zhang¹Tonghao Yao¹Qi Han²Yongqiang Mo¹Dailuo Li¹Zhengye Zhang¹Lihuang Cui¹Zhibin Geng¹Weitao He¹Jingtao Chen¹Xin Liu¹Xintao Wang^1*

• ¹The Second Affiliated Hospital of Harbin Medical University, Harbin, China
• ²The Frist Affiliated Hospital of Shandong Second Medical University, Weifang, China

Abstract Addressing the persistent challenge of bone defect repair requires innovative bioengineering strategies. Enhancing the biological activity of bone marrow mesenchymal stem cells (BMSCs) is pivotal for effective bone regeneration. This study pioneers a novel combinatorial bioengineering approach leveraging two distinct biotechnological modalities: low-level laser therapy (LLLT) and BMSCs-derived nanovesicles (BMSC-NVs). LLLT, a non-invasive biophysical stimulation technique with defined light parameters, is known to prime cellular responses. Concurrently, BMSC-NVs represent an emerging engineered cell-free therapeutic platform with significant promise for tissue regeneration. Thus, we hypothesize that combining LLLT's direct regulatory effects on BMSCs with the bioactive cargo of BMSC-NVs will synergistically enhance BMSCs function. This study presents the first evaluation of the combined impact of LLLT irradiation and BMSC-NVs on the proliferation and osteogenic differentiation of rat BMSCs in vitro. Cell proliferation was quantified using CCK-8 assay, while osteogenic differentiation was assessed through alkaline phosphatase staining, alizarin red staining, and real-time quantitative polymerase chain reaction (osteogenic gene expression). Results demonstrate that the LLLT+BMSC-NVs combinatorial strategy effectively enhances BMSC proliferation capacity (as indicated by increased OD values measured via CCK-8 assay), ALP activity, mineralized nodule formation, and upregulation of key osteogenic genes (ALP, RUNX2), showing superior effects on both proliferation and osteogenic differentiation compared to individual LLLT or BMSC-NVs treatments. In conclusion, this study pioneers a novel cell-free therapeutic paradigm by synergistically integrating LLLT with BMSC-NVs, establishing a potent and promising bioengineering strategy for for bone defect repair.