Extracellular vesicles from 3D cultured dermal papilla cells improve wound healing via Krüppel-like factor 4/vascular endothelial growth factor A-driven angiogenesis

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 andα-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.