FoxO1-Overexpressed Small Extracellular Vesicles Derived from hPDLSCs Promote Periodontal Tissue Regeneration by Reducing Mitochondrial Dysfunction to Regulate Osteogenesis and Inflammation

Qingru Niu,1– 3,* Chuanmiao Lin,1– 3,* Shuqing Yang,1– 3 Shuxuan Rong,1– 3 Junbin Wei,1– 3 Tingting Zhao,1– 3 Yingying Peng,1– 3 Zhilan Cheng,1– 3 Yunyi Xie,1– 3 Yan Wang1– 3 1Hospital of Stomatology, Guanghua School of Stomatology, Guangzhou, People’s Republic of China; 2Sun Yat-Sen University, Guangzhou, People’s Republic of China; 3Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yunyi Xie; Yan Wang, Email xieyy59@mail2.sysu.edu.cn; wang93@mail.sysu.edu.cnPurpose: Periodontitis is a chronic infectious disease characterized by progressive inflammation and alveolar bone loss. Forkhead box O1 (FoxO1), an important regulator, plays a crucial role in maintaining bone homeostasis and regulating macrophage energy metabolism and osteogenic differentiation of mesenchymal stem cells (MSCs). In this study, FoxO1 was overexpressed into small extracellular vesicles (sEV) using engineering technology, and effects of FoxO1-overexpressed sEV on periodontal tissue regeneration as well as the underlying mechanisms were investigated.Methods: Human periodontal ligament stem cell (hPDLSCs)-derived sEV (hPDLSCs-sEV) were isolated using ultracentrifugation. They were then characterized using transmission electron microscopy, Nanosight, and Western blotting analyses. hPDLSCs were treated with hPDLSCs-sEV in vitro after stimulation with lipopolysaccharide, and osteogenesis was evaluated. The effect of hPDLSCs-sEV on the polarization phenotype of THP-1 macrophages was also evaluated. In addition, we measured the reactive oxygen species (ROS) levels, adenosine triphosphate (ATP) production, mitochondrial characteristics, and metabolism of hPDLSCs and THP-1 cells. Experimental periodontitis was established in vivo in mice. HPDLSCs-sEV or phosphate-buffered saline (PBS) were injected into periodontal tissues for four weeks, and the maxillae were collected and assessed by micro-computed tomography, histological staining, and small animal in vivo imaging.Results: In vitro, FoxO1-overexpressed sEV promoted osteogenic differentiation of hPDLSCs in the inflammatory environment and polarized THP-1 cells from the M1 phenotype to the M2 phenotype. Furthermore, FoxO1-overexpressed sEV regulated the ROS level, ATP production, mitochondrial characteristics, and metabolism of hPDLSCs and THP-1 cells in the inflammatory environment. In the in vivo analyses, FoxO1-overexpressed sEV effectively promoted bone formation and inhibited inflammation.Conclusion: FoxO1-overexpressed sEV can regulate osteogenesis and immunomodulation. The ability of FoxO1-overexpressed sEV to regulate inflammation and osteogenesis can pave the way for the establishment of a therapeutic approach for periodontitis. Keywords: periodontal ligament stem cells, small extracellular vesicles, periodontitis, bone homeostasis, mitochondrial function