**2. Challenges for the development of pigmented skin models**

#### **2.1. Tissue engineering**

It is known that there is a real need for human organs available for transplantations [79]. Each year, people die while they are waiting for a compatible organ. Just in Canada, in 2011, a person needing an organ had a 30 to 40% probability of not receiving it [80]. Moreover, there are some problems with allogeneic grafts, such as problems of incompatibility and reject preoccupa‐ tions. To get rid of these problems, a new approach emerged at the end of the 1980s [81]. This approach, called tissue engineering, is a science that combines both biology and engineering expertise. Its goal is to develop biological substitutes for maintaining, repairing or regenerating human organs or tissue, such as skin [82-83].

#### **2.2. Skin substitutes**

Skin substitutes are useful in different fields. They can be grafted onto patients suffering from severe burns or chronic wounds such as skin ulcer [84-85]. They can also be used for funda‐ mental research to analyze skin functional mechanisms. Moreover, skin substitutes can be used for cosmetic testing to replace animal testing [86]. While skin is the interface tissue between human body and exterior environment, it is an organ particularly exposed to chemical and mechanical wounds and to pathological agents. Furthermore, it is an important organ in area and complexity both in structural and functional ways [87]. Consequently, to regenerate a human skin with all its functionality is a big challenge.

#### *2.2.1. Skin substitute characteristics*

In recent years, many skin substitutes have been reported, becoming more and more similar to natural human skin, but different from each others, and still not perfectly simulating skin functionality. Those substitutes can be characterized by different factors. Provenance of cells used to produce the substitute can be either allogeneic, xenogeneic or autologous. Presence of autologous cells allows reduction of incompatibility and graft reject problems [88]. The purpose of substitutes can also vary between a permanent, semi-permanent or temporary use [83]. Depending on the use wanted, complexity of substitutes will change and can be either monolayered or bilayered. As there is no perfect substitute that has been developed to this day, research and development on skin substitutes are still very current. Many aspects must be considered in the elaboration of a skin substitute so that it will be as close as possible to human skin. First, histological properties must be evaluated. Indeed, a perfect skin substitute must have both dermis and epidermis, with an epidermis well-differentiated and the presence of all its four principal layers: *stratum basale, stratum spinosum, stratum granulosum* and *stratum corneum*. Moreover, skin structure is important and can be evaluated by the expression or not of different markers and their localization. Furthermore, skin plays an important role as a protective barrier against chemical, biological and mechanical aggression. Consequently, the presence of this barrier functionality is important. A method to evaluate the barrier efficacy is to perform permeability analysis. Finally, *stratum corneum* lipid organization is also important in skin barrier functionality. This organization must be well-structured and features can be analyzed by ATR-FTIR [88]. One of the lacks in the currently available skin substitutes is the absence of melanocytes that lead to a hypopigmentation of the skin where the substitute is grafted. While melanocytes are part of the skin protection against UV irradiations, the lack of this molecule in skin substitutes can become problematic for the patient, beyond the cosmetic preoccupations [89]. Therefore, there is a need for developing skin substitutes with melanocyte cells. Moreover, incorporation of ill melanocytes in skin models allows the study of melanocyte skin diseases such as melanoma. While tests on animal skin are not always representative of human skin and can be ethically problematic, the development of pigmented skin models becomes a real need.
