Department of Spine Surgery, Shandong University Qilu Hospital, Jinan, China.
Department of Radiology, Shandong University Qilu Hospital, Qingdao, Qingdao, China.
Stem Cell Res Ther. 2020 Jan 28;11(1):38. doi: 10.1186/s13287-020-1562-9.
As important players in cell-to-cell communication, exosomes (exo) are believed to play a similar role in promoting fracture healing. This study investigated whether exosomes derived from bone marrow mesenchymal stem cells (BMMSC-Exos) could improve fracture healing of nonunion.
BMMSC-Exos were isolated and transplanted into the fracture site in a rat model of femoral nonunion (Exo group) every week. Moreover, equal volumes of phosphate-buffered saline (PBS) and exosome-depleted conditioned medium (CM-Exo) were injected into the femoral fracture sites of the rats in the control and CM-Exo groups. Bone healing processes were recorded and evaluated by radiographic methods on weeks 8, 14 and 20 after surgery. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative week 20. The expression levels of osteogenesis- or angiogenesis-related genes were evaluated in vitro by western blotting and immunohistochemistry. The ability to internalize exosomes was assessed using the PKH26 assay. Altered proliferation and migration of human umbilical vein endothelial cells (HUVECs) and mouse embryo osteoblast precursor cells (MC3TE-E1s) treated with BMMSC-Exos were determined by utilizing EdU incorporation, immunofluorescence staining, and scratch wound assay. The angiogenesis ability of HUVECs was evaluated through tube formation assays. Finally, to explore the effect of exosomes in osteogenesis via the BMP-2/Smad1/RUNX2 signalling pathway, the BMP-2 inhibitors noggin and LDN193189 were utilized, and their subsequent effects were observed.
BMMSC-Exos were observed to be spherical with a diameter of approximately 122 nm. CD9, CD63 and CD81 were expressed. Transplantation of BMMSC-Exos obviously enhanced osteogenesis, angiogenesis and bone healing processes in a rat model of femoral nonunion. BMMSC-Exos were taken up by HUVECs and MC3T3-E1 in vitro, and their proliferation and migration were also improved. Finally, experiments with BMP2 inhibitors confirmed that the BMP-2/Smad1/RUNX2 signalling pathway played an important role in the pro-osteogenesis induced by BMMSC-Exos and enhanced fracture healing of nonunion.
Our findings suggest that transplantation of BMMSC-Exos exerts a critical effect on the treatment of nonunion by promoting osteogenesis and angiogenesis. This promoting effect might be ascribed to the activation of the BMP-2/Smad1/RUNX2 and the HIF-1α/VEGF signalling pathways.
作为细胞间通讯的重要参与者，外泌体（exo）被认为在促进骨折愈合方面发挥着相似的作用。本研究探讨了骨髓间充质干细胞（BMMSC-Exos）衍生的外泌体是否可以改善骨不连的骨折愈合。
分离并每周向大鼠股骨骨不连模型的骨折部位移植 BMMSC-Exos（Exo 组）。此外，在对照组和 CM-Exo 组的大鼠股骨骨折部位注射等量的磷酸盐缓冲盐水（PBS）和外泌体耗尽的条件培养基（CM-Exo）。术后第 8、14 和 20 周，通过影像学方法记录和评估骨愈合过程。术后第 20 周，通过影像学和组织学方法评估骨折部位的成骨和血管生成。通过 Western blot 和免疫组织化学法在体外评估成骨和成血管相关基因的表达水平。通过 PKH26 测定评估外泌体的内化能力。通过 EdU 掺入、免疫荧光染色和划痕伤口试验测定用 BMMSC-Exos 处理的人脐静脉内皮细胞（HUVEC）和小鼠胚胎成骨前体细胞（MC3TE-E1）的增殖和迁移能力。通过管形成试验评估 HUVEC 的血管生成能力。最后，为了探讨外泌体通过 BMP-2/Smad1/RUNX2 信号通路在成骨中的作用，利用 BMP-2 抑制剂 noggin 和 LDN193189，并观察其后效。
观察到 BMMSC-Exos 呈球形，直径约为 122nm。表达 CD9、CD63 和 CD81。BMMSC-Exos 的移植明显增强了大鼠股骨骨不连模型中的成骨、血管生成和骨愈合过程。BMMSC-Exos 在体外被 HUVEC 和 MC3T3-E1 摄取，其增殖和迁移也得到改善。最后，BMP2 抑制剂实验证实，BMP-2/Smad1/RUNX2 信号通路在 BMMSC-Exos 诱导的成骨作用中发挥重要作用，并增强了骨不连的骨折愈合。
我们的研究结果表明，BMMSC-Exos 的移植通过促进成骨和血管生成对骨不连的治疗具有重要作用。这种促进作用可能归因于 BMP-2/Smad1/RUNX2 和 HIF-1α/VEGF 信号通路的激活。
