Feasibility of repairing skin defects by VEGF165 gene-modified iPS-HFSCs seeded on a 3D printed scaffold containing astragalus polysaccharide

Abstract

The preparation of biodegradable scaffolds loaded with cells and cytokine is a feature of tissue-engineered skin. IPSCs-based tissue-engineered skin treatment for wound repair is worth exploring. Healthy human skin fibroblasts were collected and reprogrammed into iPSCs. After gene modification and induction, CK19⁺ /Integrinβ1⁺ /CD200⁺ VEGF₁₆₅ gene-modified iPS-HFSCs^GFP were obtained and identified by a combination of immunofluorescence and RT-qPCR. Astragalus polysaccharide-containing 3D printed degradable scaffolds were prepared and co-cultured with VEGF₁₆₅ gene-modified iPS-HFSCs^GFP , and the biocompatibility and spatial structure of the tissue-engineered skin was analysed by cell counting kit-8 (CCK8) assay and scanning electron microscopy. Finally, the tissue-engineered skin was transplanted onto the dorsal trauma of nude mice, and the effect of tissue-engineered skin on the regenerative repair of total skin defects was evaluated by a combination of histology, immunohistochemistry, immunofluorescence, RT-qPCR, and in vivo three-dimensional reconstruction under two-photon microscopy. CK19⁺ /Integrinβ1⁺ /CD200⁺ VEGF₁₆₅ gene-modified iPS-HFSCs^GFP , close to the morphology and phenotype of human-derived hair follicle stem cells, were obtained. The surface of the prepared 3D printed degradable scaffold containing 200 μg/mL astragalus polysaccharide was enriched with honeycomb-like meshwork, which was more conducive to the proliferation of the resulting cells. After tissue-engineered skin transplantation, combined assays showed that it promoted early vascularization, collagen and hair follicle regeneration and accelerated wound repair. VEGF₁₆₅ gene-modified iPS-HFSCs^GFP compounded with 3D printed degradable scaffolds containing 200 μg/mL astragalus polysaccharide can directly and indirectly participate in vascular, collagen, and hair follicle regeneration in the skin, achieving more complete structural and functional skin regenerative repair.

Keywords: 3D printed degradable scaffold; astragalus polysaccharide; hair follicle stem cells; induced pluripotent stem cells; regeneration and repair; skin defect.

FIGURE 1

(A) Morphology of iPSCs on mouse embryo fibroblast feeder layers and in Martrigel gel‐coated Petri dishes. Scale bar, 100 μm. (B) Alkaline phosphatase staining of iPSCs. Scale bar, 100 μm. (C) Immunofluorescence staining of iPSCs‐specific markers. Scale bar, 200 μm. (D) Differentiation of iPSCs to mimic embryos. Scale bar, 100 μm. (E) H&E staining of teratoma showing the structure of the three germ layers. Scale bar, 100 μm.

FIGURE 2

(A) Fluorescence microscopy results of GFP‐VEGF₁₆₅ gene‐modified iPSCs. Scale bar, 100 μm. (B, C) Expression of VEGF₁₆₅ gene and protein after iPSCs gene modification. (D) Flow chart of the differentiation system of GFP‐VEGF₁₆₅ gene‐modified iPSCs to HFSCs. (E) Cell morphology at different differentiation stages. Scale bar, 100 μm.

FIGURE 3

(A) Immunofluorescence staining of HFSCs specific markers. Scale bar, 50 μm. (B) Expression of relevant target genes after iPSCs induction at different time points. (C) Expression of relevant target genes after 14 days of iPSCs induction compared with human hair follicle stem cells.

FIGURE 4

(A) Proliferation inhibition of VEGF₁₆₅ gene‐modified iPS‐HFSCs^GFP by different concentrations of astragalus polysaccharide. (B) Naked eye view of astragalus polysaccharide‐collagen‐sodium alginate‐silk fibroin 3D printed degradable skin scaffolds. (C) Microscopic observation of astragalus polysaccharide‐containing 3D printed degradable scaffolds co‐cultured with VEGF₁₆₅ gene‐modified iPS‐HFSCs^GFP, scale bar, 100 μm. (D) Growth curves of different groups of VEGF₁₆₅ gene‐modified iPS‐HFSCs^GFP. (E) Scanning electron microscopy observation of astragalus polysaccharide‐containing 3D printed scaffold and co‐culture with VEGF₁₆₅ gene‐modified iPS‐HFSCs^GFP.

FIGURE 5

(A) Whole skin defect models and interventions in nude mice. (B) Visualization of wound healing after 7 and 14 days in the three different intervention models. (C) Comparison of wound healing rates after 7 and 14 days in the three groups with different intervention modes. (D) Histological examination of wounds after 14 days in the three groups with different intervention modalities. Scale bar, 100 μm. (E) Immunohistochemical detection of wounds after 14 days in the three groups with different intervention modes. Scale bar, 100 μm.

FIGURE 6

(A) Trauma‐related genes detected after 14 days in the three groups with different intervention modes. (B, C) Immunofluorescence detection of trauma in the tissue‐engineered skin group after 14 days. Scale bars, 200 μm and 100 μm. (D) Three‐dimensional reconstruction of neovascularization and VEGF₁₆₅ gene‐modified iPS‐HFSCs^GFP in the healing skin area of living nude mice in the tissue‐engineered skin group.

FIGURE 7

Schematic diagram of repairing full‐thickness skin defects by CK19⁺/Integrinβ1⁺/CD200⁺ VEGF₁₆₅ gene modified iPS‐HFSCs^GFP seeded on a 3D printed degradable scaffold containing 200 μg/mL astragalus polysaccharide.