Exosomes secreted by endothelial progenitor cells accelerate bone regeneration during distraction osteogenesis by stimulating angiogenesis

BACKGROUND: Distraction osteogenesis (DO) is an effective but lengthy procedure to fully induce bone regeneration in large bone defects. Accumulating evidence supports the role of exosomes secreted by endothelial progenitor cells (EPC-Exos) in stimulating angiogenesis, which is closely coupled with osteogenesis. This study aimed to investigate whether EPC-Exos promote bone regeneration during DO in rats. METHODS: Exosomes were isolated from the supernatants of rat bone marrow EPCs via ultracentrifugation and characterized via transmission electron microscopy, tunable resistive pulse sensing analysis, and western blot analysis. Unilateral tibial DO models were generated using 68 Sprague-Dawley rats with a distraction rate of 0.5 mm per day for 10 days. After local injection of EPC-Exos into the distraction gaps after distraction, the therapeutic effects of EPC-Exos on bone regeneration and angiogenesis were assessed via X-ray, micro-computed tomography (micro-CT), and biomechanical and histological analyses. Pro-angiogenic effects and the potential mechanism underlying the effects of EPC-Exos on human umbilical vein endothelial cells were subsequently evaluated via in vitro assays including Cell Counting Kit-8, wound healing, tube formation, and western blot assays. RESULTS: EPC-Exos were spherical or cup-shaped vesicles ranging from 50 to 150 nm in diameter and expressed markers including CD9, Alix, and TSG101. X-ray, micro-CT, and histological analyses revealed that bone regeneration was markedly accelerated in rats treated with EPC-Exos. The distracted tibias from the Exos group also displayed enhanced mechanical properties. Moreover, vessel density was higher in the Exos group than in the control group. In addition, in vitro analyses revealed that EPC-Exos enhanced the proliferation, migration, and angiogenic capacity of endothelial cells in an miR-126-dependent manner. Further, EPC-Exos downregulated SPRED1 and activated Raf/ERK signaling. CONCLUSIONS: The present results show that EPC-Exos accelerate bone regeneration during DO by stimulating angiogenesis, suggesting their use as a novel method to shorten the treatment duration of DO.