[Influence of human skin organoid-derived extracellular vesicles composite hydrogels on wound healing of full-thickness skin defects in mice].

Objective: To investigate the influence of human skin organoid-derived extracellular vesicles (SOEVs) composite hydrogels on wound healing of full-thickness skin defects in mice. Methods: This study was an experimental study using a group design and a repeated-measures design. HaCaT cells, human skin fibroblasts, and human umbilical vein endothelial cells (HUVECs) were mixed in a ratio of 2∶1∶1 and cultured three-dimensionally in 6-well ultra-low attachment plates to generate skin organoids. The formation of skin organoids was observed on days 1, 3, 7, and 14 of culture. On day 7 of culture, the expressions of epidermal marker cytokeratin 14 (CK14), vascular marker CD31, and dermal marker vimentin in skin organoids were detected by immunofluorescence method. On day 7 of culture, SOEVs were isolated from the culture supernatant of skin organoids by sequential differential ultracentrifugation. The ultrastructure of SOEVs was observed using transmission electron microscopy, and the particle size of SOEVs was measured using a nanoparticle tracking analyzer. HaCaT cells and HUVECs were collected, and both cell types were divided into a SOEV group cultured with 30 μg/mL SOEV and a control group cultured under conventional condition. A scratch test was performed, and cell migration rate was calculated at 24 h after scratching (n=6). Gelatin methacrylate anhydride (GelMA) hydrogel and GelMA hydrogel containing 30 μg/mL SOEV (i.e. SOEV composite hydrogel) were prepared. Eighteen 6-week-old male C57BL/6J mice were divided into a control group, a GelMA group, and a SOEV@GelMA group using a random number table method, with 6 mice in each group. A full-thickness skin defect wound was created on the back of each mouse. Immediately after injury (day 0), phosphate-buffered saline, GelMA hydrogel, and SOEV composite hydrogel were applied to the wounds of mice in control group, GelMA group, and SOEV@GelMA group, respectively. Wound healing was observed at post-injury day (PID) 0, 3, 7, 10, and 14, and the wound healing rate was calculated at PID 3, 7, 10, and 14. At PID 14, wound tissue was collected. Hematoxylin-eosin staining was performed to observe the degree of wound epithelial regeneration; Masson staining was performed to observe collagen fiber deposition, and the proportion of collagen fiber-positive area was calculated. Results: After 1 to 14 days of culture, the skin organoids gradually matured, with the structures becoming increasingly compact and dense, and the spheroids exhibiting clear boundaries. On day 7 of culture, the skin organoids expressed CK14, CD31, and vimentin. SOEVs extracted from the culture supernatant of skin organoids on day 7 of culture exhibited a typical disc-shaped vesicular structure, with an average particle size of 70.1 nm. At 24 h post-scratching, the migration rates of HaCaT cells and HUVECs in SOEV group were significantly higher than those in corresponding control group (with t values of 16.73 and 7.71, respectively, P<0.05). From PID 0 to 14, the wound area in all three groups of mice gradually decreased. At PID 3, 7, 10, and 14, the wound healing rates of mice in SOEV@GelMA group were (56.47±9.26)%, (73.87±6.02)%, (92.69±3.74)%, and (98.92±0.26)%, respectively, which were significantly higher than (28.18±15.63)%, (49.21±11.96)%, (72.53±7.93)%, and (87.39±0.83)% in control group (P<0.05) and (34.51±14.43)%, (58.30±8.00)%, (79.16±4.15)%, and (90.16±0.80)% in GelMA group (P<0.05). At PID 14, the area of neo-epithelial coverage in the wounds of mice in GelMA group and SOEV@GelMA group was greater than that in control group; some wounds of mice in GelMA group and control group still showed separation of the epidermis and dermis, while the wounds of mice in SOEV@GelMA group achieved almost complete re-epithelialization. At PID 14, collagen fiber deposition was observed in the wound tissue in all three groups of mice, with the collagen fibers in the dermal layer of the wound tissue of mice in SOEV@GelMA group arranged in a more orderly manner. The proportion of collagen fiber-positive area in the wound tissue of mice in GelMA group and SOEV@GelMA group was significantly higher than that in control group (P<0.05), and the proportion of collagen fiber-positive area in the wound tissue of mice in SOEV@GelMA group was significantly higher than that in GelMA group (P<0.05). Conclusions: The human SOEV composite hydrogel can promote collagen fiber deposition in full-thickness skin defect wounds of mice and accelerate the wound repair process, significantly improving the efficacy and quality of wound healing.