Abstract
IntroductionIn 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.MethodsThree-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.ResultsSEM 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.ConclusionsTissue-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.
Highlights
In the field of skin tissue engineering, gelatin-chondroitin-6-sulfate-hyaluronic acid (Gel-C6S-HA) stents are a suitable bio skin substitute
Poor angiogenesis capability can lead to a limited vascular system and insufficient supply of nutrients to the early grafted skin, which in turn can lead to necrosis of the skin substitute and graft failure
Primary culture of rat hair follicle stem cell (HFSC) and their biological characteristics By 3 days, a small number of cells had migrated from the periphery of the follicle bulge (Figure 2a)
Summary
In the field of skin tissue engineering, gelatin-chondroitin-6-sulfate-hyaluronic acid (Gel-C6S-HA) stents are a suitable bio skin substitute. Current tissue-engineered skin repair approaches are often complicated by wound infection, nonunion and other complications, and treatment efficacy is unsatisfactory. This is closely related to the extent of vascularization of the repaired wound [1,2,3]. Incorporation of chondroitin6-sulfate into gelatin scaffolds resulted in a scaffold with higher resistance to collagenase degradation, higher elastic modulus and a more porous structure than gelatin scaffolds [17,18]. Hyaluronic acid can be further modified by hydroxyl and carboxyl functional groups with specific cell or extracellular matrix components, to enhance its biological function [20]
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