| Summary: | Yanli Huang,1– 3 Qian Liu,1,2,4 Li Liu,1,2,4 Fangjun Huo,1,2 Shujuan Guo,1,2,4 Weidong Tian1,3 1State Key Laboratory of Oral Disease & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Oral Regenerative Medicine, West China School of Stomatology, Sichuan University, Chengdu, 610041, People’s Republic of China; 2Engineering Research Center of Oral Translational Medicine, Ministry of Education, West China School of Stomatology, Sichuan University, Chengdu, 610041, People’s Republic of China; 3Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, 610041, People’s Republic of China; 4Department of Periodontics, West China School of Stomatology, Sichuan University, Chengdu, Sichuan, 610041, People’s Republic of ChinaCorrespondence: Shujuan Guo; Weidong Tian, Tel/Fax +86 028 8550 3499, Email guo_shujuan@126.com; drtwd@sina.comPurpose: Lipopolysaccharide (LPS) pretreatment can enhance the therapeutic effect of dental follicle stem cells-derived small extracellular vesicles (DFC-sEV) for periodontitis, and this study aimed to investigate the underlying mechanisms and clinical application Of LPS-preconditioned DFC-sEV in periodontitis.Methods: The protein spectrum of DFC-sEV before and after LPS pretreatment was determined by liquid chromatography-tandem mass spectrometry and bioinformatic analysis. Their effects on inflammatory periodontal ligament stem cells (PDLSCs) and macrophages were investigated for cell proliferation, migration, type 2 macrophage (M2) polarization, and intracellular reactive oxygen species (ROS) levels separately. In addition, the regulation of ROS/Jun amino-terminal kinases (JNK) and ROS/extracellular signal-related kinases (ERK) signaling by LPS-preconditioned DFC-sEV was also studied to reveal the antioxidant mechanism. In vivo, two kinds of DFC-sEV loaded with 0.2% hyaluronic acid (HA) gel were applied for canine periodontitis to evaluate the therapeutic potential.Results: The proteomic analysis showed that thirty-eight proteins were differentially expressed in LPS-preconditioned DFC-sEV, and interestingly, the highly expressed proteins were mainly involved in antioxidant and enzyme-regulating activities. In addition to promoting PDLSCs and macrophage proliferation, LPS-preconditioned DFC-sEV inhibited intracellular ROS as an antioxidant. It reduced the RANKL/OPG ratio of PDLSCs by inhibiting ROS/JNK signaling under inflammatory conditions and promoted macrophages to polarize toward the M2 phenotype via ROS/ERK signaling. Furthermore, LPS-preconditioned DFC-sEV loaded with the HA injectable system could sustainably release sEV and enhance the therapeutic efficacy for periodontitis in canines.Conclusion: LPS-preconditioned DFC-sEV could be effectively used as an auxiliary method for periodontitis treatment via antioxidant effects in a subgingival environment, and loading it with HA is feasible and effective for clinical applications.Keywords: small extracellular vesicle, dental follicle stem cell, periodontitis, reactive oxygen species, antioxidant effect
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