Efficient Wound Healing Using a Synthetic Nanofibrous Bilayer Skin Substitute in Murine Model.

Treatment of full-thickness skin wounds with minimal scarring and complete restoration of native tissue properties still exists as a clinical challenge. A bilayer skin substitute was fabricated by coating human amniotic membrane (AM) with electrospun silk fibroin nanofibers, and its in vivo biological behavior was studied using murine full-thickness skin wound model. Donut-shaped silicon splints were utilized to prevent wound contraction in mouse skin and simulate re-epithelialization, which is the normal path of human wound healing. Skin regeneration using the bilayer scaffold was compared with AM and untreated defect after 30 d. Tissue samples were taken from healed wound areas and investigated through histopathological and immunohistochemical staining to visualize involucrin (IVL), P63, collagen I, CD31, and vascular endothelial growth factor. In addition, mRNA expression of IVL, P63, interleukin-6, and cyclooxygenase-2 was studied. The application of bilayer scaffold resulted in the best epidermal and dermal regeneration, demonstrated by histopathological examination and molecular analysis. In regenerated wounds of the bilayer scaffold group, the mRNA expression levels of inflammatory markers (interleukin-6 and cyclooxygenase-2) were downregulated, and the expression pattern of keratinocyte markers (IVL and P63) at both mRNA and protein levels was more similar to native tissue in comparison with AM and no-treatment groups. There was no significant difference in the expression level of collagen I, CD31, and vascular endothelial growth factor among different groups. Conclusively, these promising results serve as a supporting evidence for proceeding to clinical phase to examine the capacity of this bilayer scaffold for human skin regeneration.