Background:

Extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (UC-MSCs) have emerged as promising cell-free therapy candidates in various diseases, including chronic cutaneous wounds. The goal of this study was to create a safe and efficient method for large-scale production of MSC-derived EVs and establish an EVs identification protocol to advance the clinical application of EVs in wound therapy.

Methods:

Polyethylene glycol (PEG) precipitation and ultracentrifugation were used to isolate EVs from the culture supernatant of UC-MSCs. Transmission electron microscopy (TEM), Western blot, and a high-sensitivity flow cytometer (HSFCM) were used to examine the size distribution, particle concentration, and phenotype of EVs. Chitosan hydrogel-EVs were created to improve the use of EVs in wound treatment by incorporating EVs into chitosan hydrogel (CS-EVs). The Transwell method was used to assess the release behavior and uptake dynamics of EVs from chitosan hydrogel in vitro. The scratch wound assay and tube formation assay were used to determine whether EVs altered the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs). Every five days, CS-EVs were transplanted onto the wound site of a diabetic rat model. On days 0, 5, 10, and 20 following surgery, wound healing processes were graphically recorded and evaluated. Finally, after 15 days of surgery, all rats were sacrificed, and the wound skin was collected for hematoxylin and eosin (H&E) staining.

Results:

The majority of the EVs obtained in our protocol had a cup-shaped or round-shaped morphology with a diameter of about 80 nm. CD9, CD63, and CD81 were all found. In the experiment, the chitosan hydrogel was liquid at 4 °C and was gelatinized for 10 minutes at 37 °C. The CS-EVs continuously released EVs into the environment, and the EVs released by the CS-EVs were internalized by human umbilical vein endothelial cells (HUVECs), resulting in significant cell migration and angiogenesis promotion. Furthermore, we discovered that CS-EVs have a good therapeutic function in the promotion of wound healing in a rat model of diabetic foot ulcers.

Conclusions:

Our findings suggest that PEG precipitation and ultracentrifugation can be used to isolate EVs on a large scale. Chitosan hydrogel-EVs (CS-EVs) were formed by incorporating EVs into chitosan hydrogels, which is an effective application scheme for EVs in wound therapy. Overall, our research could help with the purification, characterization, and application of EVs in wound care.