Abstract
The aim of this study was to determine the optimal preconditioning regimen for the wound healing therapeutic efficacy of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). To this end, we compared various preconditioning regimens for both the quantitative and qualitative production of MSC-derived EVs, and their therapeutic efficacy for proangiogenic activity in vitro and cutaneous wound healing in vivo. After preconditioning with thrombin (40 U), H2O2 (50 μM), lipopolysaccharide (1 μg/mL), or hypoxia (10% O2), EV secretion was assessed quantitatively by measuring production per cell and protein quantification, and qualitatively by measuring a proteome profiler and an enzyme-linked immunosorbent assay (ELISA) contained within EVs. The therapeutic efficacy of EVs was assessed in vitro by proliferation, migration and tube formation assays of human umbilical cord blood endothelial cells (HUVECs), and in vivo by quantification of cutaneous wound healing. Thrombin preconditioning optimally boosted EV production and enriched various growth factors including vascular endothelial growth factor and angiogenin contained within EVs compared to other preconditioning regimens. Thrombin preconditioning optimally enhanced proliferation, the migration and tube formation of HUVECs in vitro via pERK1/2 and pAKT signaling pathways, and cutaneous wound healing in vivo compared to other preconditioning regimens. Thrombin preconditioning exhibited optimal therapeutic efficacy compared with other preconditioning regimens in promoting proangiogenic activity in vitro and in enhancing cutaneous wound healing in vivo. These preconditioning regimen-dependent variations in therapeutic efficacy might be mediated by boosting EV production and enriching their cargo content.
Highlights
Cutaneous wound healing is a complex but well-orchestrated sequential process including coagulation and hemostasis, cell migration, inflammation, angiogenesis, proliferation and remodeling [1,2]
Stem cell therapy, especially using mesenchymal stem cells (MSCs), has emerged as a promising new and effective therapeutic strategy for treating chronic non-healing wounds, and this protective effect was predominantly mediated by paracrine rather than direct regenerative mechanisms [8,9]
Recent studies have demonstrated that the beneficial effects of MSCs for wound healing were only some mediated by the secretion of extracellular vesicles (EVs), nuclear membrane vesicles 40–100 nm in diameter and containing numerous proteins, lipids, mRNAs, and regulatory miRNAs [9,10]
Summary
Cutaneous wound healing is a complex but well-orchestrated sequential process including coagulation and hemostasis, cell migration, inflammation, angiogenesis, proliferation and remodeling [1,2]. Recent studies have demonstrated that the beneficial effects of MSCs for wound healing were only some mediated by the secretion of extracellular vesicles (EVs), nuclear membrane vesicles 40–100 nm in diameter and containing numerous proteins, lipids, mRNAs, and regulatory miRNAs [9,10]. Since this cell-free therapy can bypass concerns associated with viable MSC transplantation, the use of MSC-derived EVs might be a promising new, safe, and effective therapeutic modality for cutaneous wound healing. We evaluated whether preconditioning regimen-dependent variations in therapeutic efficacies were associated with or mediated by boosting EV production and enriching EV content by measuring the amount of EV secretion quantitatively and by measuring a proteome profiler and an enzyme-linked immunosorbent assay (ELISA) contained within EVs qualitatively
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