Extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (hUCMSCs) have emerged as promising candidates for cell-free therapy in various diseases, including chronic cutaneous wounds. However, the lack of standardized protocols for EVs’ preparation and identification poses a significant challenge to their clinical application. Thus, the objective was to develop a safe and efficient method for the large-scale production of hUCMSC-derived EVs while establishing a comprehensive identification protocol encompassing morphology, particle size distribution, protein expression, and purity. This study observed that most of the EVs acquired through the protocol exhibited either a cup-shaped or round-shaped structure, with a median diameter of ~73.25 nm. The proportions of EVs positive for CD9, CD63, and CD81 were 37.5%, 38.6%, and 19.8%, respectively. To enhance their therapeutic potential in wound treatment, EVs were incorporated into chitosan hydrogel, forming chitosan hydrogel-EVs (CS-EVs). Furthermore, it was demonstrated that CS-EVs exhibited continuous release of EVs into the surrounding environment and, importantly, that the released EVs were internalized by human umbilical vein endothelial cells (HUVECs), resulting in significant enhancement of cell migration and angiogenesis. Additionally, in a rat model of diabetic foot ulcers, CS-EVs demonstrated a robust therapeutic effect in promoting wound healing. Following a 15-day treatment period, the group treated with CS-EVs demonstrated an impressive 93.3% wound closure ability, accompanied by a high degree of re-epithelialization. In contrast, the control group exhibited only a 71.5% reduction in wound size. In summary, this study offers solutions for the purification, characterization, and application of EVs in clinical wound treatment. These results not only offer fresh perspectives on the involvement of hUCMSC-derived EVs in wound healing but also introduce a non-invasive approach for applying EVs that holds practical significance in skin repair.

Graphical Abstract

中文翻译：

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壳聚糖水凝胶内的间充质干细胞来源的细胞外囊泡增强治疗糖尿病足溃疡的治疗效果

源自人脐带间充质干细胞 (hUCMSC) 的细胞外囊泡 (EV) 已成为各种疾病（包括慢性皮肤伤口）无细胞疗法的有希望的候选者。然而，缺乏EV制备和鉴定的标准化方案对其临床应用构成了重大挑战。因此，我们的目标是开发一种安全有效的方法来大规模生产 hUCMSC 衍生的 EV，同时建立涵盖形态、粒径分布、蛋白质表达和纯度的综合鉴定方案。这项研究观察到，通过该协议获得的大多数 EV 表现出杯形或圆形结构，中位直径约为 73.25 nm。CD9、CD63和CD81阳性的EV比例分别为37.5%、38.6%和19.8%。为了增强其在伤口治疗中的治疗潜力，将 EV 纳入壳聚糖水凝胶中，形成壳聚糖水凝胶-EV（CS-EV）。此外，研究表明，CS-EV 能够持续将 EV 释放到周围环境中，重要的是，释放的 EV 被人脐静脉内皮细胞 (HUVEC) 内化，从而显着增强细胞迁移和血管生成。此外，在糖尿病足溃疡大鼠模型中，CS-EV 在促进伤口愈合方面表现出强大的治疗作用。经过 15 天的治疗期后，接受 CS-EV 治疗的组表现出令人印象深刻的 93.3% 伤口闭合能力，并伴有高度的上皮再化。相比之下，对照组的伤口尺寸仅减少了 71.5%。总之，本研究为 EV 的纯化、表征和在临床伤口治疗中的应用提供了解决方案。这些结果不仅为 hUCMSC 衍生的 EV 参与伤口愈合提供了新的视角，而且还介绍了一种应用 EV 的非侵入性方法，对皮肤修复具有实际意义。

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