Exosomes from Hypoxia Preconditioned BMSCs Improve Peri-implant Osteogenesis under Type II Diabetes Condition via miR-106b-5p/HIF-1α Signaling Axis

Rationale：Poor peri-implant osseointegration of dental implants in patients with type II diabetes has become a major clinical challenge in recent years. MSC (Mesenchymal stem cell)-derived exosomes may play an important role in peri-implant osseointegration, but the mechanism remains unclear. Enhancing the therapeutic effect of MSC-derived exosomes and exploring the potential mechanism can help provide a new therapeutic strategy to improve the clinical outcome of dental implant restorations in patients with type II diabetes. Methods：The exosomes derived from hypoxia (Hypo-exos) or normoxia (Nor-exos) preconditioned bone marrow mesenchymal stem cells (BMSCs) were co-cultured with BMSCs and human umbilical vein endothelial cells (HUVECs). The effect of exosomes on BMSCs cell proliferation was detected by CCK-8 assay and EdU assay, and the effect on angiogenesis ability of HUVECs was detected by wound healing assay, transwell migration assay, tube formation assay, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. A diabetic rat dental implant model was also established and the effect of exosomes on implant osseointegration was evaluated through micro-CT scanning and histological analysis. The differentially expressed miRNAs between Hypo-exos and Nor-exos were identified by high-throughput miRNA sequencing. Subsequently, the target genes and their roles in regulating angiogenesis were predicted and analyzed by bioinformatics analysis and dual luciferase reporter assay. Results：In vitro experiments indicated that hypoxia preconditioning could elevate exosome production and promote cell proliferation of BMSCs and angiogenesis of HUVECs. Moreover, Hypo-exos promoted peri-implant osteogenesis in rats with diabetes. Further investigation revealed the vital involvement of the miR-106b-5p/HIF-1α axis in promoting peri-implant osseointegration. Conclusion: Exosomes derived from hypoxia-preconditioned BMSCs could improve the peri-implant osseointegration in rats with diabetes by promoting cell proliferation and angiogenesis, and the miR-106b-5p/ HIF-1α axis could be the underlying mechanism.背景：近年来，Ⅱ型糖尿病患者口腔种植体周围骨结合不良已成为临床亟待攻克的重大挑战。间充质干细胞（Mesenchymal stem cell, MSC）来源的外泌体在种植体周围骨结合中或发挥关键调控作用，但其具体分子机制尚未明确。增强MSC来源外泌体的治疗效能并探索其潜在作用机制，可为改善Ⅱ型糖尿病患者口腔种植修复的临床结局提供全新治疗策略。 方法：将经缺氧预处理（Hypo-exos）或常氧预处理（Nor-exos）的骨髓间充质干细胞（Bone marrow mesenchymal stem cells, BMSCs）提取的外泌体，分别与BMSCs及人脐静脉内皮细胞（Human umbilical vein endothelial cells, HUVECs）共培养。采用CCK-8实验、EdU实验检测外泌体对BMSCs增殖能力的影响；通过划痕愈合实验、Transwell迁移实验、管形成实验、酶联免疫吸附实验（ELISA）、实时定量聚合酶链反应（qRT-PCR）及蛋白质印迹（western blot）检测外泌体对HUVECs血管生成能力的影响。此外，构建糖尿病大鼠口腔种植模型，通过显微计算机断层扫描（micro-CT）及组织学分析评估外泌体对种植体周围骨结合的干预效果。采用高通量miRNA测序鉴定Hypo-exos与Nor-exos间的差异表达miRNA，随后通过生物信息学分析及双荧光素酶报告实验预测并分析其靶基因及其在调控血管生成中的作用。 结果：体外实验表明，缺氧预处理可提升外泌体的分泌量，并促进BMSCs增殖及HUVECs血管生成。此外，Hypo-exos可促进糖尿病大鼠的种植体周围骨形成。进一步研究揭示，miR-106b-5p/缺氧诱导因子-1α（HIF-1α）轴在促进种植体周围骨结合中发挥关键调控作用。 结论：缺氧预处理BMSCs来源的外泌体可通过促进BMSCs增殖及HUVECs血管生成，改善糖尿病大鼠的种植体周围骨结合，且miR-106b-5p/HIF-1α轴或为其潜在作用机制。