Fractional carbon dioxide laser-induced photothermal activation of mesenchymal stem cell-derived exosomes accelerates diabetic wound healing by enhancing angiogenesis

Figure 1 Characterization of adipose-derived mesenchymal stem cells. A: Morphology of third-passage adipose-derived mesenchymal stem cells (Ad-MSCs). Scale bar = 200 μm; B: Osteogenic and adipogenic differentiation potential of Ad-MSCs. Scale bar = 100 μm; C: Flow cytometry analysis of surface markers on Ad-MSCs, including CD105, CD90, CD44, CD106, CD31, and CD34. Ad-MSCs: Adipose-derived mesenchymal stem cells.

Figure 2 Low-energy fractional CO₂ laser-induced heat stress enhances heat shock protein 90 expression in adipose-derived mesenchymal stem cells. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Data are presented as mean ± SD (n = 3). A: Infrared thermal images of cell culture dishes following CO₂ laser irradiation; B: Photothermal curve; C and D: Western blot analysis of heat shock protein 90 protein expression; E and F: Flow cytometric analysis of apoptosis. HSP90: Heat-shock protein 90; NC: Negative control; V-FITC: Vascular fluorescein isothiocyanate.

Figure 3 Characterization of exosomes. A: Transmission electron microscopy images showing the morphology of exosomes (Exos) and CO₂ laser-Exos. Scale bar = 100 nm; B: Western blot analysis of exosomal markers CD63 and tumor susceptibility gene 101 in Exos and CO₂ laser-Exos; C: Nanoparticle tracking analysis of size distribution of Exos and CO₂ laser-Exos; D: Confocal laser scanning microscopy showing the uptake of PKH26-labeled Exos and CO₂ laser-Exos by human umbilical vein endothelial cells. Exos and nuclei were stained red and blue, respectively. Scale bar = 10 μm. Exos: Exosomes; CO₂ laser-Exos: Exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells; TSG101: Tumor susceptibility gene 101.

Figure 4 CO₂ laser-exosomes promote angiogenesis in human umbilical vein endothelial cells. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Data are presented as mean ± SD (n = 3). A and B: 5-ethynyl-2’-deoxyuridine incorporation assay to assess the proliferative capacity of human umbilical vein endothelial cells (HUVECs). Scale bar = 100 μm; C: Cell Counting Kit-8 assay to evaluate the viability of HUVECs after treatment with high glucose and exosomes; D and E: Transwell migration assay to evaluate the migratory ability of HUVECs. Scale bar = 200 μm; F and G: In vitro wound healing assay to assess cell migration. Scale bar = 200 μm; H and I: Tube formation assay to evaluate the ability of HUVECs to form capillary-like structures. Scale bar = 200 μm; J and K: Western blot analysis of the expression levels of phosphorylated protein kinase B, protein kinase B, hypoxia-inducible factor-1α, and vascular endothelial growth factor-A. NG: Normal glucose; HG: High glucose; Exos: Exosomes; CO₂ laser-Exos: Exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells; EdU: 5-Ethynyl-2’-deoxyuridine; AKT: Protein kinase B; p-AKT: Phosphorylated protein kinase B; HIF-1α: Hypoxia-inducible factor-1α; VEGF-A: Vascular endothelial growth factor-A.

Figure 5 The sphingosine-1-phosphate/sphingosine-1-phosphate receptor signaling axis mediates exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells-induced angiogenesis. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Data are presented as mean ± SD (n = 3). A: Enzyme-linked immunosorbent assay analysis of sphingosine-1-phosphate concentrations in activated supernatant from non-irradiated adipose-derived mesenchymal stem cells (Ad-MSCs), activated supernatant from laser-irradiated Ad-MSCs, exosomes, and exosomes derived from CO₂ laser-preconditioned Ad-MSCs; B: Relative mRNA expression levels of sphingosine-1-phosphate receptor (S1PR1)-3 in human umbilical vein endothelial cells (HUVECs); C and D: Western blot and quantitative analysis of the relative protein expression of S1PR1-3 in HUVECs; E and F: 5-Ethynyl-2’-deoxyuridine incorporation assay to evaluate HUVEC proliferation. Scale bar = 100 μm; G: Cell Counting Kit-8 assay to assess HUVEC viability; H and I: Transwell migration assay to evaluate HUVEC motility. Scale bar = 200 μm; J and K: In vitro wound healing assay to assess migration. Scale bar = 200 μm; L and M: Tube formation assay to examine capillary-like network formation by HUVECs. Scale bar = 200 μm; N and O: Western blot analysis of the expression levels of phosphorylated protein kinase B, protein kinase B, hypoxia-inducible factor-1α, vascular endothelial growth factor-A, and S1PR1. NC: Activated supernatant from non-irradiated adipose-derived mesenchymal stem cells; NG: Normal glucose; HG: High glucose; HP: High permeability; CO₂ laser-AS: Activated supernatant from laser-irradiated adipose-derived mesenchymal stem cells; Exos: Exosomes; CO₂ laser-Exos: Exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells; EdU: 5-Ethynyl-2’-deoxyuridine; S1P: Sphingosine-1-phosphate; S1PR: Sphingosine-1-phosphate receptor; shS1PR: Short hairpin RNA targeting sphingosine-1-phosphate receptor; AKT: Protein kinase B; p-AKT: Phosphorylated protein kinase B; HIF-1α: Hypoxia-inducible factor-1α; VEGF-A: Vascular endothelial growth factor-A.

Figure 6 LY294002 suppresses exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells -induced angiogenesis. ^bP < 0.01; ^cP < 0.001. Data are presented as mean ± SD (n = 3). A and B: Western blot analysis of phosphorylated protein kinase B, protein kinase B, hypoxia-inducible factor-1α, and vascular endothelial growth factor-A protein expression levels in human umbilical vein endothelial cells (HUVECs) treated with LY294002 vs negative control and LY294002 + exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells vs exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells; C and D: 5-Ethynyl-2’-deoxyuridine incorporation assay to assess HUVEC proliferation. Scale bar = 100 μm; E: Cell Counting Kit-8 assay to evaluate HUVEC viability; F and G: Transwell migration assay to assess the migratory capacity of HUVECs. Scale bar = 200 μm; H and I: In vitro wound healing assay to evaluate cell migration. Scale bar = 200 μm; J and K: Tube formation assay to examine the formation of capillary-like structures by HUVECs. Scale bar = 200 μm. EdU: 5-Ethynyl-2’-deoxyuridine; NC: Negative control; CO₂ laser-Exos: Exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells; AKT: Protein kinase B; p-AKT: Phosphorylated protein kinase B; HIF-1α: Hypoxia-inducible factor-1α; VEGF-A: Vascular endothelial growth factor-A.

Figure 7 SC79 enhances exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells-induced angiogenesis. ^bP < 0.01; ^cP < 0.001. Data are presented as mean ± SD (n = 3). A and B: Western blot analysis of phosphorylated protein kinase B, protein kinase B, hypoxia-inducible factor-1α, and vascular endothelial growth factor-A protein levels in human umbilical vein endothelial cells (HUVECs) treated with SC79 vs negative control and SC79 + short hairpin RNA targeting sphingosine-1-phosphate receptor vs short hairpin RNA targeting sphingosine-1-phosphate receptor; C and D: 5-Ethynyl-2’-deoxyuridine incorporation assay to assess cell proliferation. Scale bar = 100 μm; E: Cell Counting Kit-8 assay to evaluate the viability of HUVECs; F and G: Transwell assay to assess HUVEC migration. Scale bar = 200 μm; H and I: In vitro wound healing assay to assess the migratory capacity of HUVECs. Scale bar = 200 μm; J and K: Tube formation assay to visualize the capillary-like structure formation in HUVECs. Scale bar = 200 μm. EdU: 5-Ethynyl-2’-deoxyuridine; NC: Negative control; shS1PR1: Short hairpin RNA targeting sphingosine-1-phosphate receptor; AKT: Protein kinase B; p-AKT: Phosphorylated protein kinase B; HIF-1α: Hypoxia-inducible factor-1α; VEGF-A: Vascular endothelial growth factor-A.

Figure 8 Exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells accelerate angiogenesis in diabetic wounds. ^bP < 0.01; ^cP < 0.001. Data are presented as mean ± SD (n = 6). A and B: Representative images of full-thickness skin defects in non-diabetic mice and diabetic mice treated with phosphate-buffered saline, exosomes, or exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells on 0 day, 3 days, 7 days, 10 days, and 14 days post-surgery; C and D: Quantification of hematoxylin and eosin staining and skin thickness on day 14. Scale bar = 100 μm; E and F: Quantification of Masson’s trichrome staining and collagen deposition on day 14. Scale bar = 100 μm; G and H: Representative immunohistochemical staining images for CD31. Scale bar = 100 μm. NC: Negative control; PBS: Phosphate-buffered saline; Exos: Exosomes; CO₂ laser-Exos: Exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells.

Figure 9 Schematic illustration showing that low-energy fractional CO₂ laser enhances heat-shock protein 90 expression in adipose-derived mesenchymal stem cells via photothermal effects, thereby improving the biological activity of their derived exosomes. These exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells promote angiogenesis and accelerate diabetic wound healing by activating the sphingosine-1-phosphate receptor/protein kinase B/hypoxia-inducible factor-1α signaling pathway. Ad-MSCs: Adipose-derived mesenchymal stem cells; CO₂ laser-Exos: Exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells; HSP90: Heat-shock protein 90; AKT: Protein kinase B; HIF-1α: Hypoxia-inducible factor-1α; VEGF-A: Vascular endothelial growth factor-A; S1P: Sphingosine-1-phosphate; S1PR: Sphingosine-1-phosphate receptor; HUVECs: Human umbilical vein endothelial cells.