Gelatin-chondroitin-6-sulfate-hyaluronic acid scaffold seeded with vascular endothelial growth factor 165 modified hair follicle stem cells as a three-dimensional skin substitute

Abstract

Introduction: In the field of skin tissue engineering, gelatin-chondroitin-6-sulfate-hyaluronic acid (Gel-C6S-HA) stents are a suitable bio skin substitute. The purpose was to investigate the effect of genetically-modified hair follicle stem cells (HFSCs), combined with Gel-C6S-HA scaffolds, on the vascularization of tissue-engineered skin.

Methods: Three-dimensional (3D) Gel-C6S-HA scaffolds were prepared by freeze-drying. Vascular endothelial growth factor (VEGF) 165 gene-modified rat HFSCs (rHFSCs) were inoculated into the scaffolds and cultured for 7 days. Two bilateral full-thickness skin defects were created on the back of 18 Sprague-Dawley rats. Rats were randomly divided into four groups: Group A, HFSCs transduced with VEGF165 seeded onto Gel-C6S-HA scaffolds; Group B, HFSCs transduced with empty vector seeded onto Gel-C6S-HA scaffolds; Group C, Gel-C6S-HA scaffold only; Group D, Vaseline gauze dressing. These compositions were implanted onto the defects and harvested at 7, 14 and 21 days. Wound healing was assessed and compared among groups according to hematoxylin-eosin staining, CD31 expression, alpha smooth muscle actin (α-SMA) and major histocompatibility complex class I (MHC-I) immunohistochemistry, and microvessel density (MVD) count, to evaluate the new blood vessels.

Results: SEM revealed the Gel-C6S-HA scaffold was spongy and 3D, with an average pore diameter of 133.23 ± 43.36 μm. Cells seeded on scaffolds showed good adherent growth after 7 days culture. No significant difference in rHFSC morphology, adherence and proliferative capacity was found before and after transfection (P >0.05). After 14 and 21 days, the highest rate of wound healing was observed in Group A (P <0.05). Histological and immunological examination showed that after 21 days, MVD also reached a maximum in Group A (P <0.05). Therefore, the number of new blood vessels formed within the skin substitutes was greatest in Group A, followed by Group B. In Group C, only trace amounts of mature subcutaneous blood vessels were observed, and few subcutaneous tissue cells migrated into the scaffolds.

Conclusions: Tissue-engineered skin constructs, using 3D Gel-C6S-HA scaffolds seeded with VEGF165-modified rHFSCs, resulted in promotion of angiogenesis during wound healing and facilitation of vascularization in skin substitutes. This may be a novel approach for tissue-engineered skin substitutes.

Figure 1

Observation of Gel-C6S-HA scaffold characteristics. (a), (b) The scaffold formed a three-dimensional sponge-like structure, with connective transport holes between pores, observed using scanning electron microscopy. Pores were circular or polygonal in microstructure. (c) Pore sizes were not uniform, with an average pore diameter of 133.23 ± 43.36 μm. (d) Macroscopic appearance of the gelatin–chondroitin-6-sulfate–hyaluronic acid (Gel-C6S-HA) scaffold.

Figure 2

Isolation and biological characteristics of rat hair follicle stem cells. Primary cell culture on (a) day 3, (b) day 7 and (c), (d) day 14. Rat hair follicle stem cells (HFSCs; P2) (e) before and (f) after purification. (g) Quantitative polymerase chain reaction results of six correlated genes in rat HFSCs; ACTB was used as the reference gene. (h) Immunofluorescence staining for expression of cytokeratin (CK) 15, integrin α6 and integrin β1. (i) Growth curves of different generations of HFSCs. Scale bars: 100 μm (a to c, e, f, h); 250 μm (d). Ct, cycle threshold; P, passage.

Figure 3

VEGF165 gene-modified rat hair follicle stem cells. (a) Fluorescein isothiocyanate image using fluorescence microscopy. Green staining indicates that the target plasmid pLV–VEGF165–IRES–EGFP was successfully transfected into the hair follicle stem cells (HFSCs). (b) Phase contrast image. (c) Reverse transcription-polymerase chain reaction for VEGF165 expression after transfection. (d) Western blot for expression of VEGF165 protein. Scale bars: 100 μm (a, b). EGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; VEGF, vascular endothelial growth factor.

Figure 4

Hair follicle stem cell morphology, adherence and proliferation on scaffolds. (a), (b), (c) After 1 day of culture, no significant difference was observed between all groups with respect to cell morphology, with few adherent cells, the majority being spherical. (d), (e), (f) After 7 days of culture, all cells were firmly adhered to the scaffold and were growing three-dimensionally along the scaffold. (g) After transfection of VEGF165, hair follicle stem cells (HFSCs) adhered efficiently to the scaffold wall. There was no significant difference in proliferative capacity with the control group. (a), (d) HFSCs after VEGF165 transfection (Group A). (b), (e) Control group with empty support (Group B). (c), (f) Control group (Group C). HFSCs/pLV-VEGF165-IRES-EGFP, HFSCs transduced with VEGF165 seeded on Gel-C6S-HA scaffolds; HFSCs/pLV-IRES-EGFP, HFSCs transduced with empty vector seeded on Gel-C6S-HA scaffolds: HFSCs, HFSCs seeded on Gel-C6S-HA scaffolds. EGFP, enhanced green fluorescent protein; Gel-C6S-HA, gelatin–chondroitin-6-sulfate–hyaluronic acid; IRES, internal ribosome entry site; OD, optical density; VEGF, vascular endothelial growth factor.

Figure 5

Transplantation of the cell-seeded scaffold. (a) After anesthesia with 1% (w/v) sodium pentobarbital, disinfection was carried out with iodine and hair removal treatment was performed in the surgical area. (b), (c) The skin subcutaneous superficial fascia was incised and the skin substitute transplanted. (d) The specially designed flexible protective coat was used to prevent damage to the affected area from rat bites, preventing contamination and destruction.

Figure 6

Postoperative skin wound. (a) to (l) After 7, 14 and 21 days, there were no visible signs of wound inflammation in all four groups, the graft was in close contact with the wound, and the wound of Group D was dry and clean with red granulation. (m) Wound healing rates of Groups A to C were fast after 7 days, with the graft absorption speed in Group A being fastest at 14 and 21 days. In Group A, the graft combined solidly with its surrounding tissue, and the wound healing rate was significantly higher than in other groups. *P <0.05. Gel-C6S-HA, gelatin–chondroitin-6-sulfate–hyaluronic acid; HFSC, hair follicle stem cell; VEGF, vascular endothelial growth factor.

Figure 7

Hematoxylin and eosin staining of the graft. (a), (b), (c) After 7 days, skin grafts in Groups A and B had formed microvessels, the three-dimensional morphology of the scaffold was loosely structured and cell distribution was uniform; conversely, the Group C scaffold was clear. (d), (e), (f) After 14 days, the newly formed vessels in Group A were significantly increased with relatively fewer in Group B. Scaffolds in Group A and B were full of uniformly distributed cells, with varying degrees of degradation and absorption. Subcutaneous tissue cells tended to migrate into the Group C scaffold material, but numbers remained limited. (g), (h), (i) After 21 days, new blood vessels with uniform distribution could be found within the full layer of Group A, and these vessels were large and abundant. Vascularization in Group B was different from that in Group A, with only a few blood vessels formed at the junctions between the subcutaneous tissue and scaffold in Group C. Scale bars: 100 μm. Gel-C6S-HA, gelatin–chondroitin-6-sulfate–hyaluronic acid; HFSC, hair follicle stem cell; VEGF, vascular endothelial growth factor.

Figure 8

Markers of new blood vessels. (a), (d), (g) At each time point after surgery, positive expression (brown) of CD31 in Group A was most abundant. (b), (e), (h) In Group B, CD31 expression was lower than Group A, and the vessels were relatively small. (c), (f), (i) In Group C, CD31 was significantly lower than that in Groups A and B, with only trace expression. (j) Vessel density results. Scale bars: 100 μm. Group A was compared with Groups B and C, *P <0.05. Gel-C6S-HA, gelatin–chondroitin-6-sulfate–hyaluronic acid; HFSC, hair follicle stem cell; VEGF, vascular endothelial growth factor.

Figure 9

Markers of mature blood vessels. (a), (d), (g) At each time point after surgery, vessels were larger and uniformly distributed with the highest vascular maturity in Group A. (b), (e), (h) Mature vessels were smaller in Group B. (c), (f), (i) There were almost no vessels in the upper layer of the scaffold in Group C, and the positive expression (brown) zone was confined to the junction of the subcutaneous tissue. There were more blood vessels growing into the scaffold when it was closer to the subcutaneous tissue, but the number remained small and fewer than in the experimental group (Group A). Scale bars: 100 μm. Gel-C6S-HA, gelatin–chondroitin-6-sulfate–hyaluronic acid; HFSC, hair follicle stem cell; VEGF, vascular endothelial growth factor.

Figure 10

Major histocompatibility complex class I immunofluorescence staining. Yellow boxes and arrows highlight major histocompatibility complex class I (MHC-I) antibody expressed on the cytomembrane. Within the 21 days, almost no significant difference was detected in the expression of MHC-I antibodies in the three groups. (a), (b), (d), (e) There were a few red dots within the first 14 days in Groups A and B. (c), (f), (i) Postoperative at 21 days, there was no visible MHC-I expression in all cells migrating into the scaffold in Group C. Scale bars: 50 μm. Gel-C6S-HA, gelatin–chondroitin-6-sulfate–hyaluronic acid; HFSC, hair follicle stem cell; VEGF, vascular endothelial growth factor.