Vascularization of skin substitute constructed by vascular endothelial growth factor 165 gene-modified hair follicle stem cells compounded with three-dimensional gelatin-chondroitin sulfate-hyaluronic acid

Abstract

Objective To investigate the effect of three-dimensional gelatin-chondroitin sulfatehyaluronic acid (Gel-C6S-HA) composite scaffold seeded with genetically modified hair follicle stem cells (HFSCs) on tissue engineering skin angiogenesis.Methods Three-dimensional scaffolds composed of Gel-C6S-HA were fabricated by freeze-lyophilizing.VEGF165 modified HFSCs were seeded on those scaffolds and cellular morphology and adhesion were observed using scanning electron microscope.Eighteen rats were subjected to the full-thickness skin defects with dimensions of 1.2 cm × 1.2 cm on the bilateral sides of the back and covered with VEGF165 transduced HFSCs/Gel-C6S-HA scaffold (Group A),empty-vector transduced HFSCs/Gel-C6S-HA scaffold (Group B),Gel-C6S-HA scaffold (Group C),and vaseline gauze (Group D) respectively according to the random number table.At days 7,14,and 21 after surgery,wound healing was observed,HE staining and immunohistochemistry of CD31 and alpha smooth muscle actin (α-SMA) were performed,and microvessel density (MVD) was used to measure new blood vessel growth.Results Electron microscopy exhibited three-dimensional spongy structure of the scaffold with round or polygon apertures connecting mutually and the scaffold pore size of (133.2 ± 43.4) μm.Cells seeded on the scaffolds spread thoroughly out and anchored firmly after being cultured for 7 days.There were no obvious inflammation reactions on the wounds for all groups at day 7 after operation.Wound healing and scaffold degradation were faster in Group A than in other groups at days 14 and 21 after operation.Histological and immunological detections showed microvasculariztion of the scaffold in Groups A and B with fluffy three-dimensional structure and evenly distributed cells,but scaffolds remained sharp at the edge and there were small cells aggregating at subcutaneous tissue junction area in Group C.At 14 and 21 days after operation,new blood vessels were large and rich in Group A; scaffolds in Groups A and group B were filled with cells,partial of which gathered in the epidermal layer and the scaffold materials were assimilated differentially,whereas a few subcutaneous tissue cells migrated to scaffolds in Group C.MVD was significantly higher in Group A than in Groups B and C at each time point (P ＜ 0.05).Conclusion VEGF165 modified HFSCs compounded with Gel-C6S-HA composite scaffold can facilitate the growth of blood vessels and promote the angiogenesis in wound healing and hence is a promising skin substitute in clinical applications. Key words: Soft tissue injuries ; Hair follicle; Stem cells; Vascularization