Human amniotic mesenchymal stem cell-derived small extracellular vesicles repair of jaw osteoradionecrosis

Figure 1 Identification of human amniotic mesenchymal stem cell-derived small extracellular vesicles. A: Morphology of human amniotic mesenchymal stem cell-derived small extracellular vesicles (hAMSCs-sEVs) by transmission electron microscopy (TEM); scale bar = 100 nm; B: Expression of hAMSCs-sEVs surface proteins CD63 and tumor susceptibility gene 101 by western blot; C and D: Intensity and concentration of hAMSCs-sEVs particles by nanoparticle tracking analysis. hAMSCs-sEVs: Human amniotic mesenchymal stem cell-derived small extracellular vesicles; TSG101: Tumor susceptibility gene 101.

Figure 2 Human amniotic mesenchymal stem cell-derived small extracellular vesicles promoted bone healing in rats with osteoradionecrosis of the jaw. A: Healing of tooth sockets at 7 days and 14 days post-extraction in the control, osteoradionecrosis of the jaw (ORNJ) and human amniotic mesenchymal stem cell-derived small extracellular vesicles (hAMSCs-sEVs) groups; B: Micro computed tomography examination of tooth sockets at 7 days and 14 days post-extraction in the control, ORNJ and hAMSCs-sEVs groups; C-F: Hematoxylin and eosin and Masson trichrome staining of tooth sockets at 7 days and 14 days post-extraction in the control, ORNJ and hAMSCs-sEVs groups. Scale bar = 100 μm or 50 μm; G-J: Representative immunohistochemical images of collagen-1, ALP, CD31 and vascular endothelial growth factor in tooth sockets at 14 days post-extraction in the control, ORNJ and hAMSCs-sEVs groups. Scale bar = 100 μm or 50 μm. ORNJ: Osteoradionecrosis of the jaw; sEVs: Small extracellular vesicles.

Figure 3 Human amniotic mesenchymal stem cell-derived small extracellular vesicles alleviated radiation-induced bone marrow mesenchymal stem cells osteogenesis inhibition. A: Human amniotic mesenchymal stem cell-derived small extracellular vesicles (hAMSCs-sEVs) uptake by bone marrow mesenchymal stem cells (BMSCs). Scale bar = 50 μm; B and C: Alkaline phosphatase staining and rate in the control, 12 Gy and 12 Gy + 10⁸/10⁹/10¹⁰ hAMSCs-sEVs groups on day 14. Scale bar = 200 μm; D and E: Alizarin red staining in the control, 12 Gy and 12 Gy + 10⁸/10⁹/10¹⁰ hAMSCs-sEVs groups on day 21. Scale bar = 200 μm; F: Proliferation of BMSCs in the control, 12 Gy and 12 Gy + 10⁸/10⁹/10¹⁰ hAMSCs-sEVs groups; G-K: Polymerase chain reaction and western blot analyses of collagen-1, alkaline phosphatase, runt-related transcription factor 2, and osteopontin expression in BMSCs in the control, 12 Gy and 12 Gy + 10⁸/10⁹/10¹⁰ hAMSCs-sEVs groups. ^aP < 0.05, ^bP < 0.01. sEVs: Small extracellular vesicles; OD: Optical density; ALP: Alkaline phosphatase; ARS: Alizarin Red staining; COL-1: Collagen-1; RUNX2: Runt-related transcription factor 2; OPN: Osteopontin.

Figure 4 Human amniotic mesenchymal stem cell-derived small extracellular vesicles activated phosphorylated-protein kinase B expression in irradiated bone marrow mesenchymal stem cells. A: Kyoto Encyclopedia of Genes and Genomes pathway analysis illustrating the top 20 downregulated genes in bone marrow mesenchymal stem cells (BMSCs) between the 12 Gy and control groups; B: Kyoto Encyclopedia of Genes and Genomes pathway analysis illustrating the top 20 upregulated genes in BMSCs between the 12 Gy + 10¹⁰ human amniotic mesenchymal stem cell-derived small extracellular vesicles (hAMSCs-sEVs) and 12 Gy groups; C and D: Western blot analysis of protein kinase B and phosphorylated-protein kinase B expression in BMSCs in the control, 12 Gy and 12 Gy + 10¹⁰ hAMSCs-sEVs groups; E and F: Alkaline phosphatase staining and rate in the control, 12 Gy and 12 Gy + 10⁸/10⁹/10¹⁰ hAMSCs-sEVs groups treated with or without the selective inhibitor Ly294002. Scale bar = 200 μm; G and H: Alizarin Red staining in the control, 12 Gy and 12 Gy + 10¹⁰ hAMSCs-sEVs groups treated with or without the selective inhibitor Ly294002. Scale bar = 200 μm; I: Western blot analyses of collagen-1, alkaline phosphatase, runt-related transcription factor 2, and osteopontin expression in BMSCs in the control, 12 Gy, 12 Gy + 10¹⁰ hAMSCs-sEVs and 12 Gy + 10¹⁰ hAMSCs-sEVs + selective inhibitor Ly294002 groups. ^bP < 0.01. KEGG: Kyoto Encyclopedia of Genes and Genomes; PI3K: Phosphatidylinositol 3-kinase; AKT: Protein kinase B; p-AKT: Phosphorylated-protein kinase B; sEVs: Small extracellular vesicles; ALP: Alkaline phosphatase; ARS: Alizarin Red staining; COL-1: Collagen-1; RUNX2: Runt-related transcription factor 2; OPN: Osteopontin.

Figure 5 Human amniotic mesenchymal stem cell-derived small extracellular vesicles alleviated radiation-induced human umbilical vein endothelial cells proliferation, migration and angiogenesis inhibition. A: Human amniotic mesenchymal stem cell-derived small extracellular vesicles (hAMSCs-sEVs) uptake by human umbilical vein endothelial cells (HUVECs). Scale bar = 25 μm; B: Proliferation of HUVECs in the control, 12 Gy and 12 Gy + 10⁸/10⁹/10¹⁰ hAMSCs-sEVs groups; C and D: Western blot and polymerase chain reaction analyses of vascular endothelial growth factor expression in HUVECs in the control, 12 Gy and 12 Gy + 10⁸/10⁹/10¹⁰ hAMSCs-sEVs groups; E and F: Tube formation assay in the control, 12 Gy and 12 Gy + 10⁸/10⁹/10¹⁰ hAMSCs-sEVs groups. Scale bar = 100 μm; G and H: Transwell assay in the control, 12 Gy and 12 Gy + 10⁸/10⁹/10¹⁰ hAMSCs-sEVs groups. Scale bar = 100 μm; I and J: Scratch wound healing assay in the control, 12 Gy and 12 Gy + 10⁸/10⁹/10¹⁰ hAMSCs-sEVs groups. Scale bar = 100 μm. ^aP < 0.05, ^bP < 0.01. OD: Optical density; VEGF: Vascular endothelial growth factor; sEVs: Small extracellular vesicles.

Figure 6 Human amniotic mesenchymal stem cell-derived small extracellular vesicles activated Wnt/β-catenin expression in irradiated human umbilical vein endothelial cells. A: Kyoto Encyclopedia of Genes and Genomes pathway analysis illustrating the top 30 downregulated genes in human umbilical vein endothelial cells (HUVECs) between the 12 Gy and control groups; B: Kyoto Encyclopedia of Genes and Genomes pathway analysis illustrating the top 30 upregulated genes in HUVECs between the 12 Gy + 10¹⁰ human amniotic mesenchymal stem cell-derived small extracellular vesicles (hAMSCs-sEVs) and 12 Gy groups; C-E: Western blot and polymerase chain reaction analysis of β-catenin expression in HUVECs in the control, 12 Gy and 12 Gy + 10¹⁰ hAMSCs-sEVs groups; F-I: Western blot and polymerase chain reaction analysis of β-catenin and vascular endothelial growth factor expression in HUVECs in the control, 12 Gy and 12 Gy + 10¹⁰ hAMSCs-sEVs groups treated with or without the selective inhibitor MSAB; J-L: Tube formation assay in the control, 12 Gy and 12 Gy + 10¹⁰ hAMSCs-sEVs groups treated with or without the selective inhibitor MSAB. Scale bar = 100 μm; M and N: Transwell assay in the control, 12 Gy and 12 Gy + 10¹⁰ hAMSCs-sEVs groups treated with or without the selective inhibitor MSAB. Scale bar = 100 μm; O and P: Scratch wound healing assay in the control, 12 Gy and 12 Gy + 10¹⁰ hAMSCs-sEVs groups treated with or without the selective inhibitor MSAB. ^aP < 0.05, ^bP < 0.01. VEGF: Vascular endothelial growth factor; sEVs: Small extracellular vesicles.

Figure 7 Schematic diagram of the biological effects of human amniotic mesenchymal stem cell-derived small extracellular vesicles on promoting bone healing in osteoradionecrosis of the jaw rats. hAMSCs: Human amniotic mesenchymal stem cells; sEVs: Small extracellular vesicles; HUVECs: Human umbilical vein endothelial cells; AKT: Protein kinase B; P-AKT: Phosphorylated-protein kinase B; BMSCs: Bone marrow mesenchymal stem cells; ORNJ: Osteoradionecrosis of the jaw.