Background Non-healing wounds are an intractable problem of major clinical relevance. Evidence has shown that dermal papilla cells (DPCs) may regulate the wound-healing process by secreting extracellular vesicles (EVs). However, low isolation efficiency and restricted cell viability hinder the applications of DPC-EVs in wound healing. In this study, we aimed to develop novel 3D-DPC spheroids (tdDPCs) based on self-feeder 3D culture and to evaluate the roles of tdDPC-EVs in stimulating angiogenesis and skin wound healing. Methods To address the current limitations of DPC-EVs, we previously developed a self-feeder 3D culture method to construct tdDPCs. DPCs and tdDPCs were identified using immunofluorescence staining and flow cytometry. Subsequently, we extracted EVs from the cells and compared the effects of DPC-EVs and tdDPC-EVs on human umbilical vein endothelial cells (HUVECs) in vitro using immunofluorescence staining, a scratch-wound assay and a Transwell assay. We simultaneously established a murine model of full-thickness skin injury and evaluated the effects of DPC-EVs and tdDPC-EVs on wound-healing efficiency in vivo using laser Doppler, as well as hematoxylin and eosin, Masson, CD31 and α-SMA staining. To elucidate the underlying mechanism, we conducted RNA sequencing (RNA-seq) of tdDPC-EV- and phosphate-buffered saline-treated HUVECs. To validate the RNA-seq data, we constructed knockdown and overexpression vectors of Krüppel-like factor 4 (KLF4). Western blotting, a scratch-wound assay, a Transwell assay and a tubule-formation test were performed to detect the protein expression, cell migration and lumen-formation ability of KLF4 and vascular endothelial growth factor A (VEGFA) in HUVECs incubated with tdDPC-EVs after KLF4 knockdown or overexpression. Dual-luciferase reporter gene assays were conducted to verify the activation effect of KLF4 on VEGFA. Results We successfully cultured tdDPCs and extracted EVs from DPCs and tdDPCs. The tdDPC-EVs significantly promoted the proliferation, lumen formation and migration of HUVECs. Unlike DPC-EVs, tdDPC-EVs exhibited significant advantages in terms of promoting angiogenesis, accelerating wound healing and enhancing wound-healing efficiency both in vitro and in vivo. Bioinformatics analysis and further functional experiments verified that the tdDPC-EV-regulated KLF4/VEGFA axis is pivotal in accelerating wound healing. Conclusions 3D cultivation can be utilized as an innovative optimization strategy to effectively develop DPC-derived EVs for the treatment of skin wounds. tdDPC-EVs significantly enhance wound healing via KLF4/VEGFA-driven angiogenesis.

中文翻译：

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来自 3D 培养真皮乳头细胞的细胞外囊泡通过 Krüppel 样因子 4/血管内皮生长因子 A 驱动的血管生成改善伤口愈合。

背景 不愈合的伤口是一个具有重要临床意义的棘手问题。有证据表明，真皮乳头细胞（DPC）可以通过分泌细胞外囊泡（EV）来调节伤口愈合过程。然而，低分离效率和有限的细胞活力阻碍了 DPC-EV 在伤口愈合中的应用。在本研究中，我们旨在开发基于自饲养 3D 培养的新型 3D-DPC 球体 (tdDPC)，并评估 tdDPC-EV 在刺激血管生成和皮肤伤口愈合中的作用。方法为了解决 DPC-EV 目前的局限性，我们之前开发了一种自饲养 3D 培养方法来构建 tdDPC。使用免疫荧光染色和流式细胞术鉴定 DPC 和 tdDPC。随后，我们从细胞中提取了 EV，并使用免疫荧光染色、划痕实验和 Transwell 实验比较了 DPC-EV 和 tdDPC-EV 在体外对人脐静脉内皮细胞（HUVEC）的影响。我们同时建立了小鼠全层皮肤损伤模型，并使用激光多普勒以及苏木精和伊红、Masson、CD31和α-SMA染色评估了DPC-EV和tdDPC-EV对体内伤口愈合效率的影响。为了阐明潜在机制，我们对 tdDPC-EV 和磷酸盐缓冲盐水处理的 HUVEC 进行了 RNA 测序 (RNA-seq)。为了验证 RNA-seq 数据，我们构建了 Krüppel 样因子 4 (KLF4) 的敲低和过表达载体。采用Western blotting、划痕实验、Transwell实验和小管形成实验检测tdDPC-培养的HUVEC中KLF4和血管内皮生长因子A（VEGFA）的蛋白表达、细胞迁移和管腔形成能力。 KLF4 敲低或过度表达后的 EV。采用双荧光素酶报告基因检测验证KLF4对VEGFA的激活作用。结果我们成功培养了 tdDPC 并从 DPC 和 tdDPC 中提取了 EV。tdDPC-EVs显着促进HUVECs的增殖、管腔形成和迁移。与DPC-EVs不同，tdDPC-EVs在促进血管生成、加速伤口愈合和提高伤口愈合效率方面在体外和体内表现出显着的优势。生物信息学分析和进一步的功能实验证实，tdDPC-EV 调节的 KLF4/VEGFA 轴对于加速伤口愈合至关重要。结论 3D 培养可以作为一种创新的优化策略，有效开发 DPC 衍生的 EV 来治疗皮肤伤口。tdDPC-EV 通过 KLF4/VEGFA 驱动的血管生成显着增强伤口愈合。