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‐HFSCsGFP 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‐HFSCsGFP, 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‐HFSCsGFP, 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‐HFSCsGFP 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.


| INTRODUC TI ON
The skin, as the first protective barrier between the outside world and the body, is an important part of the immune system. 1,2 The skin consists of the epidermis, dermis, and subcutaneous tissues, and has defence, immunity, regulation of body temperature, and metabolism. [4][5][6] However, skin trauma or soft tissue injury from various clinical causes, such as trauma, burns, contusions, etc., is becoming common. With the increasing global trend of aging, the consequent problem of skin injury is gradually increasing, especially the trauma care and healing issues are thus receiving more attention.
Medical dressings and skin substitutes have significant advantages in accelerating skin wound healing and preventing and treating skin infections. It is estimated that the trade of medical dressings have reached nearly 23.8 billion yuan in 2021. [7][8][9] Tissue-engineered skin, as a skin substitute, is a biodegradable scaffolds prepared by engineering means, which is also loaded with cells and cytokine, etc. The scaffolds degrade themselves during the process of cell growth and differentiation and tissue formation to form an autologous skin. This provides a new method for constructing skin substitutes with good development prospects and also has very important clinical significance in producing more desirable therapeutic effects for people with difficult wound healing. First, screening for suitable seed cells is considered one of the simplest and most effective methods in tissue-engineered skin construction, [10][11][12] and hair follicle stem cells (HFSCs) are present in the ramus of the outer root sheath of the hair follicle, and their specific markers include cytokeratin 19 (CK19), 13,14 cell membrane protein CD200, 15,16 and integrin β1 (ITGβ1). 17,18 HFSCs have the ability of self-renewal and proliferation in vitro and have the potential to differentiate into hair follicle tissue, connective tissue, neuronal cells, epidermal layer cells and melanocytes, and vascular smooth muscle cells. 19 IPSCs have theoretically unlimited proliferative capacity and can differentiate into nearly all adult cell types, and are considered to be seed cells for the treatment of many diseases. Skin fibroblast is relatively easy to obtain and is a adult seed cell reprogrammed into iPSCs earlier.
The reprogramming technique, introducing specific transcription factors OCT4, SOX2, C-MYC, and KLF4 into adult seed cells to transform it into iPSCs, 24,25 overcomes the ethical hurdles of traditional pluripotent stem cell research and has the advantages of being widely available, easy to obtain, and the ability to obtain normal or disease models, and is useful in regenerative medicine for with great therapeutic promise. and EGF in turn differentiate iPSCs into ectodermal and mesodermal cells. [26][27][28] In our group, based on the reported protocols in the literature and the use of the above-mentioned cytokines in the early stage, although iPSCs induced CD200 + /ITGA6 + epithelial stem cells exhibited a hair follicle stem cell phenotype, the positive rate was still only about 20% and the induction protocol was relatively cumbersome.
The use of 3D printed technology and herbal monomers in skin wound repair has become a new hotspot. The skin bioactive scaffold can provide a suitable three-dimensional space for seed cells to grow, thus facilitating a good bioactive environment for skin wound healing. 29,30 Compared with traditional preparation techniques such as the freeze-drying method, electrostatic spinning method, and pore-making agent method, 3D printed bioactive scaffolds can be more efficiently prepared to meet the requirements of skin wound repair. 3D printed technology is highly flexible and reproducible and can be directly preset to meet the skin cell growth porosity for batch 3D structure printing. Astragalus polysaccharide has been found to have a strong pro-vascular regenerative effect, which accelerates trauma repair. 31,32 Collagen has low immunogenicity and can provide a microenvironment for cell apposition, growth, proliferation and directed differentiation. 33,34 Sodium alginate is water-soluble alginate that facilitates the maintenance of a moist wound environment and absorbs wound exudate, which also increases the formability of the scaffold. 35,36 Silk fibroin is a natural protein with good biocompatibility and mechanical properties. 37 Therefore, we constructed a normal human skin fibroblastderived iPSCs system. The iPSC-derived VEGF 165 gene-modified iPS-HFSCs GFP system was constructed using a modified induction method. Astragalus polysaccharide-collagen-sodium alginate-silk fibroin 3D printed degradable skin scaffolds were prepared and constructed with VEGF 165 gene-modified iPS-HFSCs GFP co-culture participate in vascular, collagen, and hair follicle regeneration in the skin, achieving more complete structural and functional skin regenerative repair.

K E Y W O R D S
3D printed degradable scaffold, astragalus polysaccharide, hair follicle stem cells, induced pluripotent stem cells, regeneration and repair, skin defect system. This tissue-engineered skin was transplanted into the nude mice with dorsal whole skin defect wounds to explore and analyse its role effectively, form of action and mechanism of action in accelerating wound repair.

| Experimental animals
Thirty-six SPF-grade BALB/c nude mice, both male and female,

| Reagents
The main reagents include DMEM/F12

| Generation and identification of IPSC
The frozen human skin fibroblasts from our group were revived and induction of iPSCs refers to early mature methods of our group. 38,39 Briefly, four plasmids (PCXLE-hOCT3/4-shp53-F, PCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1) were transfected into human skin   14 days later, using the proven system for the expansion and purification of HFSCs passaged by our group, the single suspension of induced cells obtained by digestion and centrifugation was inoculated into type IV collagen-coated culture dishes, and the cells were purified and collected using the differential apposition method. The changes in cell morphology after induction were observed under an inverted microscope and photographed and recorded.
Immunofluorescence detection of HFSCs target proteins and RT-qPCR detection of target genes was performed as described previously.
Then, the induced hair follicle stem cells were compared with three target genes (CK19, Integrinβ1, CD200) of human hair follicle stem cells. After fixation, live nude mice were imaged in vivo using an Olympus orthotopic two-photon microscope, and the FV1000 driver software was used to acquire images of traumatic blood vessels (red) and VEGF 165 gene-modified iPS-HFSCs GFP (green), and the images were synthesized in three dimensions by Imaris software.

| Statistical analysis
Each experiment was repeated at least three times. Results are shown as mean ± standard deviation (s), and the Wilcoxon test and t-test were used for comparison between groups. Differences were considered statistically significant at p < 0.05.

| Generation and identification of iPSC
Clone clusters appeared in the electrotransformed cells after 20 days.
The clone clusters were palted onto feeder layer MEF and Matrigel gel, and microscopically, the cell morphology was similar to that of embryonic stem cells (ESCs), with clone clusters present in aggregates, in which the cells were closely arranged, with large nuclei, in good condition, with high clonogenic capacity and good viability ( Figure 1A). iPSCs clone clusters with high ALP expression suggested that the cells were in the same undifferentiated state as embryonic stem cells, with high proliferative potential ( Figure 1B). Immunofluorescence staining results showed positive expression of the allosteric specific proteins Nanog and OCT4 (red fluorescence). The specific markers cytosolic protein SOX2, and cell membrane protein SSEA-4 were positively expressed (red fluorescence). This further confirmed that we have obtained human skin fibroblast-derived iPSCs ( Figure 1C). In vitro differentiation of embryoid bodies showed that the cells grew in suspension, in clusters, and gradually aggregated and grew larger on the second day. After 7 days, the embryoid bodies grew against the wall, and cells kept crawling out of the cell clusters with high proliferation capacity ( Figure 1D). The results of teratoma formation identification experiments showed that the histological structures of the three embryonic layers could be observed under the microscope, especially the representative endoderm: intestinal epithelial cells; mesoderm: smooth muscle cells; and ectoderm: retinal pigment epithelial cells ( Figure 1E). The above results demonstrated that we established a human skin fibroblast-derived iPSCs system.  The above results indicate that we have obtained cells with typical morphological characteristics of HFSCs.

| Immunofluorescence assay results
HFSCs markers cytoplasmic protein CK19 was positively expressed (red fluorescence), cell membrane protein integrinβ1 and CD200 were positively expressed (red fluorescence), while the allosteric gene Nanog was not expressed in the induced iPSCs (faint green fluorescence). The identification of specific markers further demonstrated that we have obtained cells with HFSCs phenotypes ( Figure 3A).

| Results of RT-qPCR assay
The RT-qPCR results showed that the expression of HFSCs-specific related genes (CK19, integrinβ1 and CD200) started to be upregulated

| Astragalus polysaccharide on cell proliferation ability assay and analysis of optimal concentration
After 72 h culture effect, when the concentration of astragalus polysaccharide was between 25 and 400 μg/mL, the proliferation of VEGF 165 gene modified iPS-HFSCs GFP were obvious, and the cell proliferation efficiency was positively correlated with the concentration of astragalus polysaccharide, especially the proliferation reached the peak at the concentration of 200 μg/mL, and the OD value was 0.80 ± 0.07. The above results suggest that our astragalus polysaccharide can effectively promote the proliferation of VEGF 165 gene-modified iPS-HFSCs GFP , and 200 μg/ mL astragalus polysaccharide is the relative optimal concentration, which can be prepared for the preparation of astragalus polysaccharide-containing 3D printed degradable skin scaffolds.

| Preparation results of astragalus polysaccharide-collagen-sodium alginate-silk fibroin 3D printed degradable skin scaffold
The whole scaffold was transparent, with good formability, consistent scaffold pore size, uniform spacing, consistent alignment, uniform thickness, and certain mechanical properties, which could withstand the extrusion and stretching of forceps without deformation and fracture ( Figure 4B). It indicates that we successfully prepared good astragalus polysaccharide-containing 3D printed degradable skin scaffolds.

| Scanning electron microscopy detection of scaffold and cell ultrastructure results
Scanning electron microscopy showed uniform pores of the astragalus polysaccharide-containing 3D printed scaffolds with uniform pore spacing, intact scaffolds and fine texture (magnification ×30, Figure 4E). Further local magnification observation showed that the scaffold was enriched with a honeycomb-like mesh structure, which was porous and dense and interconnected, and the internal distribution was three-dimensional, which was conducive to cell adhesion growth (magnification ×500 and ×1000, Figure 4E). Under high magnification, the VEGF 165 gene-modified iPS-HFSCs GFP were seen to adhere to the surface of the astragalus polysaccharidecontaining 3D printed scaffold material and grew in a polygonal spread with synapses between the cells, spanning the pores on the surface of the material, with good cellular status and excellent refractive index (magnification ×1300, Figure 4E). The above results indicate that the scaffolds have good biocompatibility.

| Wound healing
The visualization of the trauma on the back of the nude mice showed that the trauma in the tissue-engineered skin group shrank more significantly after 7 days, and the trauma was dry and crusted while new skin tissue was formed. 14 days later, the skin repair of the trauma in all groups improved significantly, and almost all of the trauma in the tissue-engineered skin group was healed and new skin tissue was formed ( Figure 5B). The wound healing rate of the tissue-engineered skin group increased the fastest after 7 days. After 14 days, the wound healing rate of each group increased substantially and was close to complete healing, with the tissue-engineered skin group > empty scaffold group > blank control group. These results indicated that the tissue-engineered skin covering the wound further accelerated wound healing ( Figure 5C).

| Histological test results
The tissue-engineered skin group showed proliferation of epithelial cells and fibroblasts, dense arrangement of collagen fibres, abundant myofibers, presence of intact new hair follicles, and the moderate thickness of the new dermis, which was better than the other two groups. This indicates that tissue-engineered skin can increase collagen formation on the traumatic surface, increase the thickness of the new dermis, and reconstruct skin hair follicles to improve the quality of regenerative repair of defective skin ( Figure 5D).

| Immunohistochemical test results
The expression of α-SMA and CD31 in the tissue-engineered skin group was significantly better than that in the empty scaffold group and the blank control group in the new skin. It indicated that the tissue-engineered skin group had higher intertissued collagen and vascular neovascularization than the other two groups and abundant vascular maturation ( Figure 5E).

| RT-qPCR related gene detection results
The To verify the effect of iPS-HFSCs compounded with astragalus polysaccharide-containing 3D printed degradable scaffold on the regenerative repair of total skin defects, to elucidate its form of action in promoting vascular neovascularization in nude mice, and to explore its mechanism of action in promoting regenerative repair of total skin defects, we designed the VEGF 165 gene-modified iPS-HFSCs GFP compounded with 200 μg/mL astragalus polysaccharidecontaining 3D printed degradable scaffold. Polysaccharide 3D printed degradable scaffolds containing 200 μg/mL astragalus were implanted into the dorsal whole skin defect area of nude mice for the study.
Histopathologically, the tissue-engineered skin in this study was found to increase trabecular collagen formation, increase the thickness of the new dermis, and reconstruct skin follicles, improving the quality of regenerative repair of the defective skin. The results of RT-qPCR and Western blot assay of neonatal skin tissue samples also suggested that VEGF 165 gene-modified iPS-HFSCs GFP were most directly involved in intertissued collagen neogenesis, and a small portion was directly involved in angiogenesis, which is consistent with the results of 3D reconstruction assay of neovascular and VEGF 165 gene-modified iPS-HFSCs GFP . We also found that VEGF 165 gene-modified iPS-HFSCs GFP were mainly distributed around the new hair follicles and a small portion was distributed in the basal bulge of the new hair follicles, suggesting that VEGF 165 gene-modified iPS-HFSCs GFP could also directly participate in the formation of new hair follicles with bulges, thus promoting the reconstruction of new hair follicles in the host skin.

| CON CLUS ION
In summary, as shown in Figure

CO N FLI C T O F I NTE R E S T S TATE M E NT
All the authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated and/or analysed during this study are included in this published article.