Interactions between myofibroblast differentiation and epidermogenesis in constructing human living skin equivalents

During skin wounding and healing, skin homeostasis is interrupted. How the altered epithelial-mesenchymal interactions influence scar formation and epidermogenesis should be investigated using three-dimensional models that are similar to in vivo structures.In this study, we assessed the effects of epithelial-mesenchymal interactions on myofibroblast differentiation and how myofibroblasts influence epidermogenesis using a human living skin equivalent (LSE) model.We constructed a fibroblast-populated type I collagen gel upon which LSEs were formed by seeding with normal human keratinocytes. Samples of the collagen gel and LSEs were collected at different time points. Myofibroblast differentiation, epidermal differentiation, and proliferation status were investigated immunohistochemically. Several measures were taken to suppress α-smooth muscle actin (α-SMA) expression to determine the effects of myofibroblasts on epidermogenesis, including the addition of basic fibroblast growth factor or a transformation growth factor-β (TGF-β) kinase inhibitor to the culture medium and the inclusion of an amniotic membrane (AM) in the dermal matrix.The myofibroblast/fibroblast ratio in the fibroblast-populated collagen gel kept rising during culture. In the LSEs, most fibroblasts were α-SMA-negative, except for those along the dermal-epidermal junction. The suppression of α-SMA expression enhanced epidermal differentiation and decreased TGF-β1 expression in the epidermis. The inhibition of TGF-β kinase completely suppressed α-SMA expression in the dermal matrix.Epidermogenesis suppressed α-SMA expression in the fibroblast-rich dermal matrix, except near the dermal-epidermal junction. The α-SMA-positive cells at the dermal-epidermal junction contributed to the hyperproliferative phenotype of the epidermis. In contrast, the hyperproliferative epidermis expressed more TGF-β1, which is responsible for myofibroblast differentiation.