Extracellular Vesicles Derived from H2O2-Stimulated Adipose-Derived Stem Cells Alleviate Senescence in Diabetic Bone Marrow Mesenchymal Stem Cells and Restore Their Osteogenic Capacity

Yu Li,1 Guangren Yue,1 Shuying Yu,1 Zheng Liu,1 Yilin Cao,1– 3 Ximei Wang1 1Department of Plastic and Reconstructive Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China; 2Shanghai Key Laboratory of Tissue Engineering, Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 3National Tissue Engineering Center of China, Shanghai, People’s Republic of ChinaCorrespondence: Ximei Wang, Department of Plastic and Reconstructive Surgery, The First Affiliated Hospital of Zhengzhou University, 1st Jianshe Eastern Road, Zhengzhou, 450052, People’s Republic of China, Tel +86 371-66278102, Email tracywang@zzu.edu.cnIntroduction: Autologous stem cell transplantation has emerged as a promising strategy for bone repair. However, the osteogenic potential of mesenchymal stem cells derived from diabetic patients is compromised, possibly due to hyperglycemia-induced senescence. The objective of this study was to assess the preconditioning effects of extracellular vesicles derived from H2O2-stimulated adipose-derived stem cells (ADSCs) and non-modified ADSCs on the osteogenic potential of diabetic bone marrow mesenchymal stem cells (BMSCs).Methods: Sprague-Dawley (SD) rats were experimentally induced into a diabetic state through a high-fat diet followed by an injection of streptozotocin, and diabetic BMSCs were collected from the bone marrow of these rats. Extracellular vesicles (EVs) were isolated from the conditioned media of ADSCs, with or without hydrogen peroxide (H2O2) preconditioning, using density gradient centrifugation. The effects of H2O2 preconditioning on the morphology, marker expression, and particle size of the EVs were analyzed. Furthermore, the impact of EV-pretreatment on the viability, survivability, migration ability, osteogenesis, cellular senescence, and oxidative stress of diabetic BMSCs was examined. Moreover, the expression of the Nrf2/HO-1 pathway was also assessed to explore the underlying mechanism. Additionally, we transplanted EV-pretreated BMSCs into calvarial defects in diabetic rats to assess their in vivo bone formation and anti-senescence capabilities.Results: Our study demonstrated that pretreatment with EVs from ADSCs significantly improved the viability, senescence, and osteogenic differentiation potential of diabetic BMSCs. Moreover, in-vitro experiments revealed that diabetic BMSCs treated with H2O2-activated EVs exhibited increased viability, reduced senescence, and enhanced osteogenic differentiation compared to those treated with non-modified EVs. Furthermore, when transplanted into rat bone defects, diabetic BMSCs treated with H2O2-activated EVs showed improved bone regeneration potential and enhanced anti-senescence function t compared to those treated with non-modified EVs. Both H2O2-activated EVs and non-modified EVs upregulated the expression of the Nrf2/HO-1 pathway in diabetic BMSCs, however, the promoting effect of H2O2-activated EVs was more pronounced than that of non-modified EVs.Conclusion: Extracellular vesicles derived from H2O2-preconditioned ADSCs mitigated senescence in diabetic BMSCs and enhanced their bone regenerative functions via the activation of the Nrf2/HO-1 pathway.Keywords: extracellular vesicles, ADSCs, BMSCs, diabetes mellitus, bone regeneration