The basement membrane microenvironment directs the normalization and survival of bioengineered human skin equivalents

Abstract

Epithelial-mesenchymal interactions promote the morphogenesis and homeostasis of human skin. However, the role of the basement membrane (BM) during this process is not well-understood. To directly study how BM proteins influence epidermal differentiation, survival and growth, we developed novel 3D human skin equivalents (HSEs). These tissues were generated by growing keratinocytes at an air-liquid interface on polycarbonate membranes coated with individual matrix proteins (Type I Collagen, Type IV Collagen or fibronectin) that were placed on contracted Type I Collagen gels populated with dermal fibroblasts. We found that only keratinocytes grown on membranes coated with the BM protein Type IV Collagen showed optimal tissue architecture that was similar to control tissues grown on de-epidermalized dermis (AlloDerm) that contained intact BM. In contrast, tissues grown on proteins not found in BM, such as fibronectin and Type I Collagen, demonstrated aberrant tissue architecture that was linked to a significant elevation in apoptosis and lower levels of proliferation of basal keratinocytes. While all tissues demonstrated a normalized, linear pattern of deposition of laminin 5, tissues grown on Type IV Collagen showed elevated expression of α6 integrin, Type IV Collagen and Type VII Collagen, suggesting induction of BM organization. Keratinocyte differentiation (Keratin 1 and filaggrin) was not dependent on the presence of BM proteins. Thus, Type IV Collagen acts as a critical microenvironmental factor in the BM that is needed to sustain keratinocyte growth and survival and to optimize epithelial architecture.

Rac1 Is Essential for Basement Membrane-Dependent Epiblast Survival

“…Also, mammary epithelial cells can survive for a long period of time when grown on a reconstituted basement membrane derived from Engelbreth-Holmof Swarm (EHS) tumor (Matrigel), but they die by apoptosis when grown on plastic, fibronectin, or type I collagen despite their firm attachment on these substrates (2,11,36). A similar response of keratinocytes to BM type IV collagen versus non-BM matrix proteins was observed in bioengineered human skin equivalents (40). These results suggest that the BM provides a unique microenvironment for the survival of associated epithelial cells.…”

Three-Dimensional Human Tissue Models That Incorporate Diabetic Foot Ulcer-Derived Fibroblasts Mimic In Vivo Features of Chronic Wounds

“…2,11 Briefly, fibroblasts (DFUF1, DFF6, or NFF10) were mixed with Type I Collagen (Organogenesis, Canton, MA) to a final concentration of 3 · 10 5 cells/mL. Submerged in epidermal growth media (containing 3.8 g/L glucose, 0.3% serum), 2,11 the fibroblasts contracted and remodeled the collagen matrices over the course of 7 days.…”

Skin extracellular matrix components accelerate the regenerative potential of Lin− cells

“…In our experimental groups, where the cells were cultivated in collagen type I and basement membrane matrixes, the proliferation activity increased by 29% after 72 h and by 30% after 96 h, respectively. Segal et al, [28] found that for optimal keratinocyte proliferation the basement membrane proteins are necessary. Experiments by Tsai et al [29] on the human mesenchymal stem cells (hMSC) analyzing the influence of different extracellular matrix proteins on cell proliferation properties in vitro proved that collagen type I might influence a later growth increase [29].…”