National and Regional United Engineering Laboratory of Tissue Engineering, Department of Orthopedics, Southwest Hospital, Army Medical University (Third Military Medical University), Chongqing, China (mainland).
Department of Orthopedics, The First People's Hospital of Chenzhou, Chenzhou, Hunan, China (mainland).
Med Sci Monit. 2019 Jul 27;25:5572-5579. doi: 10.12659/MSM.916838.
BACKGROUND The aim of this study was to investigate the effect of using osteoprotegerin (OPG) to treat bone defects mediated by endothelial progenitor cell (EPC) recruitment and migration through the CXCR4 signaling pathway. MATERIAL AND METHODS The EPCs extracted from human peripheral blood were cultured in vitro and the expression of CXCR4 and its downstream p-AKT was monitored by the Western blot analysis after OPG treatment. Using the scratch wound healing test and Transwell assay, we assessed the variables influencing the effect of OPG on EPCs after pre-treatment with CXCR4 blocker (AMD3100) and PI3K blocker (Ly294002). After 4 weeks, the bone defect repair condition was estimated via micro-CT and staining with HE and Masson trichrome. Then, immunofluorescence staining was performed to assess angiogenesis in bone defects, while the expression of EPC marker and vascular endothelial growth factor receptor 2 (VEGFR2) was detected by immunohistochemical staining. RESULTS The EPCs treated with OPG had increased levels of CXCR4 and p-AKT. Moreover, the difference in EPC levels among groups in the scratch wound healing experiment and migration experiment indicated that the OPG treatment promoted cell migration and AMD3100 and LY294002 inhibited the function of OPG. In addition, OPG promoted angiogenesis and repair of bone defect in rats, and these effects were abolished by AMD3100 and LY294002 administration. CONCLUSIONS OPG enhanced the proliferation and migration of EPCs through the CXCR4 pathway and promoted angiogenesis and bone formation at bone defect sites.
本研究旨在探讨通过 CXCR4 信号通路募集和迁移内皮祖细胞（EPC）来治疗骨缺损，使用骨保护素（OPG）的效果。
体外培养人外周血来源的 EPC，经 OPG 处理后，通过 Western blot 分析监测 CXCR4 及其下游 p-AKT 的表达。采用划痕愈合试验和 Transwell 试验，评估 CXCR4 阻滞剂（AMD3100）和 PI3K 阻滞剂（Ly294002）预处理对 OPG 作用于 EPC 的影响。4 周后，通过 micro-CT 评估骨缺损修复情况，并用 HE 和 Masson 三色染色。然后，通过免疫荧光染色评估骨缺损中的血管生成，免疫组织化学染色检测 EPC 标志物和血管内皮生长因子受体 2（VEGFR2）的表达。
OPG 处理的 EPCs 中 CXCR4 和 p-AKT 的水平升高。此外，划痕愈合试验和迁移试验中各组 EPC 水平的差异表明，OPG 处理促进了细胞迁移，AMD3100 和 LY294002 抑制了 OPG 的功能。此外，OPG 促进了大鼠骨缺损处的血管生成和骨修复，AMD3100 和 LY294002 的给药则消除了这些作用。
OPG 通过 CXCR4 途径增强了 EPC 的增殖和迁移，并促进了骨缺损部位的血管生成和骨形成。
