**4. Conclusion**

melanocyte culture and pigmented skin equivalent that melanocytes have a better capacity for surviving UVR than melanocytes in culture. These results suggest that keratinocytes and fibroblasts secrete factors that enhance melanocytes survival and migration, which could explain UVR-induced pigmentation by melanocytes. Unlike other teams, Freeman *et al*. elaborated another technique of culture with collagen gels [118]. They put a complete skin biopsy, which was affected by a melanoma, onto a collagen gel that contained fibroblasts. This technique allowed conservation of the *in vivo* properties of the melanoma and observation of the proliferation and other characteristics *in vitro*. All of these models composed with a collagen matrix are representative of human skin, and are effective for the study of pigmentation disorders. However, collagen models may not be useful for testing drugs, because they can be

Such as collagen gels, DED allowed the construction of pigmented skin equivalent that reproduced human skin for studying the pigmentary system. Unlike collagen gels, this method has a native extracellular matrix and a basal membrane that facilitates melanocyte adhesion. Principally, DED preparation is very similar. From a skin biopsy, the epidermis is removed and the dermis is incubated in saline solution or undergoes freezing-thawing cycles to kill cells [120]. After this treatment, the dermis remaining is called a dead de-epidermized dermis, and is ready to be seeded by keratinocytes and melanocytes. In 1993, Todd *et al*. used this model to demonstrate the effect of UV radiation on a pigmented equivalent [121]. They demonstrated that, after UV radiation, there is an increase of TRP1 activity, an increase of pigmentation and an increase of DOPA-positive melanocytes such as observed *in vivo*. In 2000, for a better understanding of melanoma invasion, Dekker *et al*. developed and characterized a DED skin equivalent with four types of melanoma cells [122]. They observed the expression of different integrins that play an important role in the behavior of melanoma cells. Their model is a useful tool for studying melanoma and other mechanisms involved in this cancer. In 2007, Cario-André *et al*. developed a DED skin model with normal and non-lesional vitiligo cells to understand if the loss of melanocytes in vitiligo is caused by a detachment of melanocytes due to stress factors [123]. The model with non-lesional vitiligo cells contains less melanocytes than the model with normal cells, and hydrogen peroxide and epinephrine could be the cause of the detachment of melanocytes. Todd, Dekker and Cario-André models demonstrated that deepidermized dermis is an interesting model for studying different pigmentation disorders and, in comparison with collagen gels, DED produced an epidermis, which is closer to the native

Commercial *in vitro* skin models have been developed to reduce tests on animals, replace animal models and refine methodologies. For the cosmetic industry, it allows testing of the toxicity of their products and their pharmaceutical effects on a complete human epidermis and dermis. Several companies produce skin models and some of them produce equally pigmented skin models that are useful for testing photoprotection, whitening agents and repigmentation

absorbed by the collagen and a higher quantity of drugs must be used [119].

*3.2.3. De-Epidermized Dermis (DED)*

774 Regenerative Medicine and Tissue Engineering

epidermis than collagen gels [124].

*3.2.4. Commercial models*

Melanocytes are underestimated cells that are more than pigmentation cells. They protect the skin against environmental factors and they are necessary for sight and hearing. However, melanocytes can also be the cause of some skin disorders and cancer. Even more mechanisms involved in pigmentation disorders remain unknown and need to be elucidated upon in order to give affected people a better quality of life. *In vitro* pigmented skin substitutes produced by tissue engineering and *in vivo* models are useful tools for understanding these mechanisms and developing appropriated treatments or drugs.
