Bio-Engineering a Prevascularized Human Tri-Layered Skin Substitute Containing a Hypodermis

Severe injuries to skin including hypodermis require full-thickness skin replacement. Here, we bio-engineered a tri-layered human skin substitute (TLSS) containing the epidermis, dermis, and hypodermis. The hypodermal layer was generated by differentiation of human adipose stem cells (ASC) in a collagen type I hydrogel and combined with a prevascularized dermis consisting of human dermal microvascular endothelial cells and fibroblasts, which arranged into a dense vascular network. Subsequently, keratinocytes were seeded on top to generate the epidermal layer of the TLSS. The differentiation of ASC into adipocytes was confirmed in vitro on the mRNA level by the presence of adiponectin, and by the expression of perilipin and FABP-4 proteins. Moreover, functional characteristics of the hypodermis in vitro and in vivo were evaluated by Oil Red, BODIPY, and AdipoRed stainings visualizing intracellular lipid droplets. Further, the enhanced secretion of TGF-β1 by hypodermal layer affected the epidermal morphogenesis as assessed by the expression of key proteins involved in the epidermal differentiation - cytokeratin 1, 10, 19, markers for cornified envelope formation -involucrin, and components of dermo-epidermal junction – laminin 332 in vivo. Here, we propose a novel functional hypodermal-dermo-epidermal tri-layered skin substitute containing blood capillaries that efficiently promote regeneration of skin defects.