Background: Silk fibroin (SF), as a natural biomacromolecule, has good biocompatibility, degradability, and potentiality for use as a tissue engineering scaffold material. Previous work suggested that adult human dermal fibroblasts(HDFs) can adhere to a novel type of 3D silk fibroin nonwovens (3D-SFnws) and release exosomes promoting neoangiogenesis. By combining a layer of electrospun SF nanofibers with the original 3D-SFnws scaffolds, we obtained the novel 3D-SFnws/ES hybrid scaffolds. The present study used cocultures of HDFs and human keratinocytes (HaCaT cells) on this new scaffold to assess whether the released exosomes contained factors promoting neoangiogenesis and regulating inflammatory responses. Method: The features of novel 3D-SFnws/ES hybrids were defined by electron microscopy imaging and specific physical tests. HDFs and HaCaT cells were separately cultured on the opposite sides of this scaffolds and on polystyrene plates using a medium with exosome-depleted FBS. The growth and metabolic activities of the two types of cells on the scaffold were explored by tests assessing DNA amounts and D-glucose consumption. The total exosomes were isolated respectively from conditioned media of HDFs and HaCaT cells cocultured on 3D-SFnws/ES hybrids and on Transwell 1 2 plates. The exosomes’ expression of their specific surface markers CD9 and CD81 was verified using two ELISA kits. Analyses using human cytokines antibody arrays assessed the expression of Inflammation factors (IFs) and Angiogenic growth factors (AGFs) transported by equal quantities of exosomes from the two groups. A tube formation assay was also used to evaluate the exosomes’ angiogenic ability by using in vitro cultures of human dermal microvascular endothelial cells (HDMVECs). Results: Not only the novel 3D-SFnws/ES hybrids imitated the epithelial-mesenchymal structure of normal skin, but also met the typical biomechanical requirements of human soft tissues implants. Compared with experimental day 3, at day experimental 15 in vitro HDFs adhering to the 3D-SFnws side of the new scaffold had increased by 4.7-fold in numbers and metabolized 5.3-fold more D-glucose. Also HaCaT cells growing on the scaffold's ES side had increased by 6.5-fold in numbers and metabolized 6.2-fold more D-glucose between day 3 and 15 in vitro. Exosomes from HDFs and HaCaT cells cocultured on 3D-SFnws/ES hybrids carried significantly higher amounts of IFs than the Transwell group, such as Interleukin-8 (IL-8), IL-10, Monocyte Chemoattractant Protein-1 (MCP-1), Macrophage Inflammatory Protein-1α (MIP-1α), Eosinophil chemotactic protein 2 (Eotaxin-2), Macrophage Colony-Stimulating Factor (M-CSF), and Tissue Inhibitor of MetalloProteinase-2 (TIMP-2). And the amounts of AGFs, such as Growth-Regulated Oncogene (GRO), Monocyte Chemoattractant Protein-1 (MCP-1), TIMP-1, TIMP-2, and Matrix MetalloPeptidase 9 (MMP-9), were significantly increased in the exosomes isolated from the 3D-SFnws/ES hybrids group. At concentrations from 0.5 to 5 µg/mL, the latter exosomes showed their angiogenic power by inducing HDMVECs to form tubes in vitro. However, an alike effect (P > 0.05) brought about the exosomes from the Transwell group. Conclusions: The novel scaffold we obtained by combining a layer of electrospun SF nanofibers with the 3D-SFnws scaffolds not only kept the good physical features of original ones, but also allowed HDFs and HaCaT cells to adhere and proliferate on their opposite sides having different structures. HDFs and HaCaT cells co-grown on these scaffolds released exosomes carrying several AGFs which swiftly induced HDMVECs to form tubes in vitro. The same exosomes conveyed IFs capable of promoting and orderly regulating inflammatory response. Thus, we posit that once implanted in vivo this new composite SF scaffold could promote skin wound healing by promoting human keratinocytes and HDFs growth and metabolism, advancing vascularization, and modulating local inflammation.

Exosomes from human fibroblasts and HaCaT cells cocultured on 3D silk fibroin nonwovens electrospun hybrids stimulate neoangiogenesis and regulate inflammation-related cytokines release

HU PENG
2022-01-01

Abstract

Background: Silk fibroin (SF), as a natural biomacromolecule, has good biocompatibility, degradability, and potentiality for use as a tissue engineering scaffold material. Previous work suggested that adult human dermal fibroblasts(HDFs) can adhere to a novel type of 3D silk fibroin nonwovens (3D-SFnws) and release exosomes promoting neoangiogenesis. By combining a layer of electrospun SF nanofibers with the original 3D-SFnws scaffolds, we obtained the novel 3D-SFnws/ES hybrid scaffolds. The present study used cocultures of HDFs and human keratinocytes (HaCaT cells) on this new scaffold to assess whether the released exosomes contained factors promoting neoangiogenesis and regulating inflammatory responses. Method: The features of novel 3D-SFnws/ES hybrids were defined by electron microscopy imaging and specific physical tests. HDFs and HaCaT cells were separately cultured on the opposite sides of this scaffolds and on polystyrene plates using a medium with exosome-depleted FBS. The growth and metabolic activities of the two types of cells on the scaffold were explored by tests assessing DNA amounts and D-glucose consumption. The total exosomes were isolated respectively from conditioned media of HDFs and HaCaT cells cocultured on 3D-SFnws/ES hybrids and on Transwell 1 2 plates. The exosomes’ expression of their specific surface markers CD9 and CD81 was verified using two ELISA kits. Analyses using human cytokines antibody arrays assessed the expression of Inflammation factors (IFs) and Angiogenic growth factors (AGFs) transported by equal quantities of exosomes from the two groups. A tube formation assay was also used to evaluate the exosomes’ angiogenic ability by using in vitro cultures of human dermal microvascular endothelial cells (HDMVECs). Results: Not only the novel 3D-SFnws/ES hybrids imitated the epithelial-mesenchymal structure of normal skin, but also met the typical biomechanical requirements of human soft tissues implants. Compared with experimental day 3, at day experimental 15 in vitro HDFs adhering to the 3D-SFnws side of the new scaffold had increased by 4.7-fold in numbers and metabolized 5.3-fold more D-glucose. Also HaCaT cells growing on the scaffold's ES side had increased by 6.5-fold in numbers and metabolized 6.2-fold more D-glucose between day 3 and 15 in vitro. Exosomes from HDFs and HaCaT cells cocultured on 3D-SFnws/ES hybrids carried significantly higher amounts of IFs than the Transwell group, such as Interleukin-8 (IL-8), IL-10, Monocyte Chemoattractant Protein-1 (MCP-1), Macrophage Inflammatory Protein-1α (MIP-1α), Eosinophil chemotactic protein 2 (Eotaxin-2), Macrophage Colony-Stimulating Factor (M-CSF), and Tissue Inhibitor of MetalloProteinase-2 (TIMP-2). And the amounts of AGFs, such as Growth-Regulated Oncogene (GRO), Monocyte Chemoattractant Protein-1 (MCP-1), TIMP-1, TIMP-2, and Matrix MetalloPeptidase 9 (MMP-9), were significantly increased in the exosomes isolated from the 3D-SFnws/ES hybrids group. At concentrations from 0.5 to 5 µg/mL, the latter exosomes showed their angiogenic power by inducing HDMVECs to form tubes in vitro. However, an alike effect (P > 0.05) brought about the exosomes from the Transwell group. Conclusions: The novel scaffold we obtained by combining a layer of electrospun SF nanofibers with the 3D-SFnws scaffolds not only kept the good physical features of original ones, but also allowed HDFs and HaCaT cells to adhere and proliferate on their opposite sides having different structures. HDFs and HaCaT cells co-grown on these scaffolds released exosomes carrying several AGFs which swiftly induced HDMVECs to form tubes in vitro. The same exosomes conveyed IFs capable of promoting and orderly regulating inflammatory response. Thus, we posit that once implanted in vivo this new composite SF scaffold could promote skin wound healing by promoting human keratinocytes and HDFs growth and metabolism, advancing vascularization, and modulating local inflammation.
Silk fibroin, Nonwovens, Electrospun, Fibroblast, Keratinocyte, HaCaT, Exosome, Angiogenesis, Inflammation, Regeneration, Tissue engineering
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/1063475
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