Fibrotic Remodeling of Tissue-Engineered Skin with Deep Dermal Fibroblasts Is Reduced by Keratinocytes

Abstract

Two-thirds of burn patients with deep dermal injuries are affected by hypertrophic scars, and currently, there are no clinically effective therapies. Tissue-engineered skin is a very promising model for the elucidation of the role of matrix microenvironment and biomechanical characteristics and could help in the identification of new therapeutic targets for hypertrophic scars. Conventionally, tissue-engineered skin is made of heterogeneous dermal fibroblasts and keratinocytes; however, recent work has shown that superficial and deep dermal fibroblasts are antifibrotic and profibrotic, respectively. Furthermore, keratinocytes are believed to regulate the development and remodeling of fibrosis in skin. This study aimed to assess the influence of keratinocytes and layered fibroblasts on the characteristics of tissue-engineered skin. Layered fibroblasts and keratinocytes isolated from superficial and deep dermis and epidermis, respectively, of the lower abdominal tissue were independently co-cultured on collagen-glycosaminoglycan scaffolds, and the resulting tissue-engineered skin was assessed for differences in tissue remodeling based on the underlying specific dermal fibroblast subpopulation. Collagen production by deep fibroblasts but not by superficial fibroblasts was significantly reduced upon co-culture with keratinocytes. Also, keratinocytes in the tissue-engineered skin resulted in significantly reduced expression of profibrotic connective tissue growth factor and fibronectin, and increased expression of the antifibrotic matrix metalloproteinase-1 by deep fibroblasts but not by superficial fibroblasts. Tissue-engineered skin made of deep fibroblasts and keratinocytes had lower levels of small proteoglycans, decorin, and fibromodulin, and higher levels of large proteoglycan, versican, compared to tissue-engineered skin made of superficial fibroblasts and keratinocytes. Tissue-engineered skin made of deep fibroblasts and keratinocytes had lower expression of transforming growth factor (TGF)-α, interleukin (IL)-1, and keratinocyte growth factor but higher expression of platelet-derived growth factor and IL-6, compared to tissue-engineered skin made of superficial fibroblasts and keratinocytes. Furthermore, co-culture with keratinocytes reduced TGF-β1 production of deep but not superficial fibroblasts. Additionally, keratinocytes reduced the differentiation of deep fibroblasts to myofibroblasts in tissue-engineered skin constructs, but not that of superficial fibroblasts. Taken together, keratinocytes reduce fibrotic remodeling of the scaffolds by deep dermal fibroblasts. Our results therefore demonstrate that tissue-engineered skin made specifically with a homogeneous population of superficial fibroblasts and keratinocytes is less fibrotic than that with a heterogeneous population of fibroblasts and keratinocytes.