Department of Prosthodontics, Peking University School and Hospital of Stomatology, Beijing, China.
4th Division, Peking University Hospital of Stomatology, Beijing, China.
Cell Prolif. 2019 Sep;52(5):e12669. doi: 10.1111/cpr.12669. Epub 2019 Aug 5.
The present study aimed to investigate whether exosomes derived from miR-375-overexpressing human adipose mesenchymal stem cells (hASCs) could enhance bone regeneration.
Exosomes enriched with miR-375 (Exo [miR-375]) were generated from hASCs stably overexpressing miR-375 after lentiviral transfection and identified with transmission electron microscopy, nanosight and western blotting. The construction efficiency of Exo (miR-375) was evaluated with qRT-PCR and incubated with human bone marrow mesenchymal stem cells (hBMSCs) to optimize the effective dosage. Then, the osteogenic capability of Exo (miR-375) was investigated with ALP and ARS assays. Furthermore, dual-luciferase reporter assay and western blotting were conducted to reveal the underlying mechanism of miR-375 in osteogenic regulation. Finally, Exo (miR-375) were embedded with hydrogel and applied to a rat model of calvarial defect, and μ-CT analysis and histological examination were conducted to evaluate the therapeutic effects of Exo (miR-375) in bone regeneration.
miR-375 could be enriched in exosomes by overexpressing in the parent cells. Administration of Exo (miR-375) at 50 μg/mL improved the osteogenic differentiation of hBMSCs. With miR-375 absorbed by hBMSCs, insulin-like growth factor binding protein 3 (IGFBP3) was inhibited by binding to its 3'UTR, and recombinant IGFBP3 protein reduced the osteogenic effects triggered by Exo (miR-375). After incorporated with hydrogel, Exo (miR-375) displayed a slow and controlled release, and further in vivo analysis demonstrated that Exo (miR-375) enhanced the bone regenerative capacity in a rat model of calvarial defect.
Taken together, our study demonstrated that exosomes derived from miR-375-overexpressing hASCs promoted bone regeneration.
本研究旨在探讨过表达 miR-375 的人脂肪间充质干细胞（hASC）衍生的外泌体是否能增强骨再生。
通过慢病毒转染过表达 miR-375 的 hASC，生成富含 miR-375 的外泌体（Exo [miR-375]），并通过透射电子显微镜、纳米粒子跟踪分析和 Western blot 进行鉴定。通过 qRT-PCR 评估 Exo [miR-375] 的构建效率，并与骨髓间充质干细胞（hBMSC）共孵育以优化有效剂量。然后通过 ALP 和 ARS 测定来研究 Exo [miR-375] 的成骨能力。此外，通过双荧光素酶报告基因检测和 Western blot 研究 miR-375 调节成骨的潜在机制。最后，将 Exo [miR-375] 包埋在水凝胶中，应用于大鼠颅骨缺损模型，并通过 μ-CT 分析和组织学检查评估 Exo [miR-375] 在骨再生中的治疗效果。
通过在亲本细胞中过表达，miR-375 可被富集在外泌体中。50μg/ml 的 Exo [miR-375] 处理可改善 hBMSC 的成骨分化。miR-375 被 hBMSC 吸收后，通过结合其 3'UTR 抑制胰岛素样生长因子结合蛋白 3（IGFBP3），重组 IGFBP3 蛋白降低了 Exo [miR-375] 触发的成骨作用。与水凝胶结合后，Exo [miR-375] 显示出缓慢和可控的释放，进一步的体内分析表明，Exo [miR-375] 增强了颅骨缺损大鼠模型的骨再生能力。
综上所述，本研究表明，过表达 miR-375 的 hASC 衍生的外泌体促进了骨再生。
