The role of extra cellular matrix in inducing complex human skin equivalent morphogenesis in vitro

Despite advances in the development of in vitro tissue models such as reconstructed human skin, the questions in dermatological research, which can be addressed with these models, are limited. This is mainly due to the lack of key extra-cellular components, which makes dermal compartment an incomplete approach to human ECM in vivo. Today there is a growing awareness of the fact that the ECM has a functional importance as a dynamic repository for morphogens, cytokines and growth factors, which in vivo regulate diverse cellular processes. We established a human skin equivalent by means of a tissue engineering process that induces the full morphogenesis of functional dermal and epidermal compartments. In our model dermal ECM presents laminin, fibronectin, hyaluronic acid, elastin and collagen arranged and organized as in the human counterpart. As proof of the physiological relevance of such tissue environment we demonstrate that -by using adult human skin cells-it is possible to generate follicle-like structures in vitro resembling what occurs in vivo in the fetal skin. Immunotypization evidences an inward-oriented differentiation of the follicular-like structures through immunopositivity for epithelial stem cell markers such as p63 and K19. Moreover we succeeded in innervating this human skin by inducing rat dorsal root ganglion neurons axon ingrowth and demonstrate the effective functionality of the nerve network. Neurofilaments network infiltrates the entire native dermis ECM until reaching the epidermis, as demonstrated by immunopositivity to neurofilament-M and second harmonic generation imaging. Calcium imaging demonstrates that electrical current travel in the neuronal network proving the its sensing functionality. Our results demonstrate the fundamental role of ECM in morphogenesis, corroborating the awareness of its importance in building up functional in vitro skin model for fundamental research applications.