Introduction

Tissue-engineered human skin equivalents (HSEs) have contributed significantly to the understanding of biological processes and skin diseases 1 2 3_{. Wound healing has been}

studied before in HSEs in which wounds were created by burning, freezing, or punching the epithelium 4 5 _{: however to the best of our knowledge a few of information on dermal healing}

are reported in literature. Indeed, the central question debated in the literature in skin wound healing are often process and mechanism of the creation and extension of the epidermal tongue 6 5–8_{. Dermal substitutes function as an optimized wound bed to support outgrowth of}

keratinocytes from the wound margins, or from an epidermal component such as a skin graft, cultured skin or an artificial epidermal component containing autologous or allogeneic keratinocytes 9–11_{. Following any injury,various intracellular and intercellular pathways must be}

activated and coordinated if tissue integrity need to be restored. Relevant processes regards dermal matrix 12_{. In dermal wound healing, after a skin injury, several interacting events are}

initiated including inflammation, tissue formation, angiogenesis, tissue contraction and tissue remodeling 13_{. Regarding the phase of tissue formation and contraction (third phase of wound}

healing process), the crosstalking between cells and extracellular matrix (ECM) is crucial. After the blood clot has formed, white blood cells invade the wound region by migrating through the ECM 14_{. Fibroblasts subsequently differentiate into myofibroblasts and migrate}

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into the region and begin to replace the blood clot with provisional matrix 15,16_{. The formation}

of this provisional matrix – granulation tissue – and regulation of collagen fibers and bundles formation by the fibroblasts, as well as the organization of these elements into an endogenous stroma after wounding, was the key issue of these two stages 17 18_.

Regarding the last phase of a wound repair – remodeling – recent studies confirmed that the natural evolution of an adult wound healing process is scar formation. Scarring can be assessed clinically by using the Vancouver scar scale, or the Manchester scar proforma or the Visual Analogue Scale 19_{. Scarring involves high collagen deposition in response to tissue}

injury, for this reason a variety of treatment modalities have been attempted. Moreover, with the introduction of new treatments for in vivo injury, as the development of skin replacement materials and grafting with autologous split thickness grafts, scar question is more and more relevant. In spite of dermal substitutes are important in treating full thickness skin defects, relevant problems associated to their use because usually severe scarring occurred after grafting 10,20,21_{. For this reason in the last few years there was an increase of focusing on the}

mechanism of scar formation and progression. It is really important to in vivo track the wound status at the molecular, cellular and tissue levels, helping to understand the scarring response mechanisms and prevent it. Recent works have shown that molecular biology at the wound margin is important for scarring 22_{, and the advancing or receding of a wound margin may be}

potentially used for prevention 14_{. Although several of the ECM–fibroblast interactions have}

been experimentally studied, this area remains poorly understood 23,24 25_{. This is partly}

because all interactions have not been found, but mainly because the processes involved interact in a complex manner. A major limitation in the progress of scar management is the lack of physiologically relevant human models to explore the pathogenesis of scar formation

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and to test new therapeutics 26 10_{. Today, human models, animal models, and in vitro cell}

culture models are used to study skin scar formation. The possibilities to use patient as in vivo models are limited, due to obvious ethical considerations 27_{. Current and common}

alternative is the use of animal models 28_{. Despite the large number of studies describing}

mice, pigs, rabbits, rats and other animals as models to investigate scar and hypertrophic scarring, the wound healing process in these species presents significant differences and is extremely difficult compared with human tissue specially because of lack of specific biomarkers, differences in skin structure and physiology with consequently differences in scar formation, presence of aberrant scars formation 29 30 31_{. Moreover, with increasing pressure}

from the EU (Directive 2010/63/EU) for the replacement, reduction, and refinement of the use of animal models, there is an urgent need to develop a physiologically relevant and responsive in vitro human wound model to investigate the matrix response and the pathogenesis of scar formation 32–3426_{. In current in vitro models, is possible to study only cell}

migration, contraction and proliferation, cell–cell interaction and protein synthesis 35 28 26_.

Accurate study it is not possible to evaluate granulation tissue and scar formation. Moreover it was seen that an in vitro model of burn wound healing in human skin did not induce the differentiation of myofibroblasts 36 10_{. This shows that a human skin equivalent model – with}

just a stratified and differentiated epithelium – isn’t enough to correctly mimic in vivo situation. To date, no gold standard exists for the treatment of scar tissue.

In this perspective, the development of biologically relevant three-dimensional human dermal equivalent (3D-HDE) as in vitro model can emerge as new challenge in tissue engineering to evaluate dermal remodeling during healing process. The fabrication of an endogenous stroma that interact in a complex manner with cells population allows possibility to confirm the

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relationship between cell migration, differentiation marker and ECM production during a wound healing process and also demonstrated the same in vivo timing into a highly responsive endogenous matrix. Also, the presence of a responsive dermis – that mimic in vivo response occurred during a repair that take place in the stroma – allows possibility to evaluate scar formation and to study and understand processes involved during scarring. This should be a great achievement considering difficulties to understand mechanism of scarring and the high request of new strategies for prevention.