Effect of adipose-derived stem cells exosomes cross-linked chitosan-αβ-glycerophosphate thermosensitive hydrogel on deep burn wounds

Figure 1 Physical characterization of adipose-derived stem cell exosomes and chitosan-αβ-glycerophosphate hydrogel. A: Electron microscopy showing vesicle-like structures (30-150 nm) with double-concave bilayer membranes at 15 k and 40 k magnification; B: Western blot confirmation of exosome markers CD9, CD63, and CD81; C: Enhanced porosity of chitosan-αβ-glycerophosphate hydrogel vs chitosan hydrogel; D: Temporal release profile of cross-linked vs non-cross-linked adipose-derived stem cell exosomes; E: Temperature-dependent storage (G’) and loss (G’’) moduli; F: In vitro degradation profile. ^cP < 0.001. ASC-Exos: Adipose-derived stem cell exosomes; CS: Chitosan; CS-αβ-GP: Chitosan-αβ-glycerophosphate.

Figure 2 The effect of chitosan-αβ-glycerophosphate hydrogel crosslinked with adipose-derived stem cell exosomes on the healing rate of deep burn wounds in rats. A: Wound healing images of the four groups at 4, 7, and 14 days after wound treatment; B: Wound healing rates (%) of the four groups at 4, 7, and 14 days after wound treatment. ^aP < 0.05, ^bP < 0.01. ASC-Exos: Adipose-derived stem cell exosomes; CS: Chitosan.

Figure 3 Histological and immunohistochemical analysis of wound healing. A: Hematoxylin and eosin: Chitosan (CS) + adipose-derived stem cell exosomes (ASC-Exos) treatment reduced lymphocyte infiltration and vascular density compared to normal tissue. ASC-Exos alone showed similar effects but with prominent glands. CS group displayed increased smooth muscle, fibroblasts, and vascular structures. Control group exhibited extensive fibroblast presence, disorganized vasculature, and marked lymphocyte infiltration; B: Masson: CS+ASC-Exos: In the wound, numerous muscle fibers are interspersed with a small amount of collagen fibers, and the quantity and distribution of collagen fibers and muscle fibers are consistent with those in normal tissue. ASC-Exos group showed mild collagen increase. CS and control groups demonstrated excessive collagen deposition and muscle fiber formation. Collagen volume fraction was significantly lower in CS + ASC-Exos group; C: Immunohistochemical: CS + ASC-Exos group showed elevated interleukin (IL)-10, transforming growth factor-β, and epidermal growth factor expression, with reduced tumor necrosis factor-α, IL-1α, and IL-6 levels compared to other groups. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. ASC-Exos: Adipose-derived stem cell exosomes; CS: Chitosan; TNF: Tumor necrosis factor; IL: Interleukin; TGF: Transforming growth factor; EGF: Epidermal growth factor.

Figure 4 Impact of chitosan-αβ-glycerophosphate hydrogel cross-linked with adipose-derived stem cell exosome on wound macrophages. A: The expression of interleukin-1α and CD86 in the chitosan + adipose-derived stem cell exosomes group is lower compared to the control group, while the expression of C-C motif chemokine ligand 22 and CD163 is higher; B: Western blotting analysis of nuclear factor κB pathway-related protein expression; C: Quantification of nuclear factor κB pathway-related proteins. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. ASC-Exos: Adipose-derived stem cell exosomes; CS: Chitosan; IL: Interleukin; CCL22: C-C motif chemokine ligand 22; IκBα: Inhibitor of kappa Balpha.