Wound healing activity of bioactive compound and application of composite scaffold

Abstract

Wound sites in skin must be immediately repaired because the skin isolates and protects from the outside. Wound sites become healing with three processes such as inflammation, proliferation and tissue remodeling. Treatment in wound sites is aiming to accelerate the wound healing, to protect the wound sites and to reduce the pain. A skin substitute, one of the treatment methods in wound sites, is used in complete section that requires epidermis and dermis. A skin substitute used commercially is made of the extracellular matrix (ECM), the living cells, the cytokines and the growth factors. However, these materials have some problems to induct the over expression of matrix metalloproteinase in wound sites and to require the cell culture times and the less validity date. In this study, we investigated the biodegradable scaffolds as a skin substitute. The scaffolds made of poly (lactic-co-glycolic acid) (PLGA) what is one of the most used biodegradable polymers was fabricated using electrospinning. Electrospinning easily fabricates the multi-fiber structure with the polymers. This structure has the advantages to provide the large surface area for cell attachment and the fluid passages. The fabricated scaffolds were investigated to composite with natural bioactive compounds instead of the cytokines and the growth factors what accelerate the wound healing. The natural bioactive compounds instead of them were used (1,3)-(1,6)-β-D-glucans (BGs) and epigallocatechin-3-O-gallate (EGCG). BGs, a natural product of glucose polymers, have immune stimulatory activity that is especially effective in wound healing. EGCG is the major polyphenolic compound in green tea, and has been investigated to regulate the secretion of cytokines and the activation of skin cells during wound healing. First of all, the porous PLGA scaffolds were investigated by electrospinning using different

process parameters. The electrospun scaffolds showed the uniformity structures when the scaffolds were fabricated with PLGA (PLA:PGA = 75:25) in HFIP or THF/DMF at 20% (w/v). These process parameters had been used for this study because the scaffolds as a skin substitute were demanded the uniformity shapes. The scaffolds processed with dry ice had a loose connection between the fibers and showed large pore sizes. The infiltration of the adult human fibroblasts was directly improved with the increase of the pore sizes of the scaffolds. Biodegradable scaffolds were sometimes deformed by heat, high temperature, pressure and moisture during sterilization. In this study, the electrospun scaffolds were treated with direct current (DC) for sterilization. During sterilization with DC treatment, the fiber structures were maintained and the microorganism sterilized with a short time. Therefore, DC treatment has the possibility of applying in the sterilization method for biodegradable scaffolds. The scaffolds composed of PLGA and BGs (BGs/PLGA scaffold) or EGCG (EGCG/PLGA scaffold) was investigated using electrospinning. The growth rate of human dermal fibroblasts (HDFs) increased in 50BGs/PLGA scaffolds (PLGA scaffolds included 50 wt% BGs) because BGs improved the HDFs proliferation. HDFs showed the cytotoxicity in PLGA scaffolds included 5 wt% EGCG (5EGCG/PLGA scaffolds) because EGCG induced caspase-3 activity to enhance that caused apoptosis. Therefore, 50BGs/PLGA scaffolds and 1EGCG/PLGA scaffolds (PLGA scaffolds included 1 wt% EGCG) were examined the wound healing effect in the animal study. According to H&E staining results, the re-epithelialization in wound sites and the cell infiltration in scaffolds were significantly accelerated the sites treated with the scaffolds included BGs or EGCG at 2 weeks after scaffold implantation. At 2 weeks, the proliferating cells (Ki-67 staining) in basal layers and the angiogenesis (CD 31 staining) in new dermis were also improved with the scaffolds included BGs or EGCG.In conclusion, BGs/PLGA scaffolds and EGCG/PLGA scaffolds should be applied as a skin substitute for accelerated wound healing. Before application to a skin substitute, the EGCG concentration in a skin substitute should be regulated because the EGCG has cytotoxicity at high concentration.