A NEW HUMAN SKIN EQUIVALENT MODEL AS IN VITRO TESTING PLATFORM FOR BIOACTIVE MOLECULES
Creators: name: Scamardella, Sara
Publisher: name: Università degli Studi di Napoli Federico II
Year: 2015

description: The aim of this PhD thesis is the realization of an in vitro human skin model and its validation as platform for testing bioactive molecules. Due to its endogenous nature  the human skin equivalent realized was able to recapitulate both physiological and pathological status of the human skin. As consequence it has been used for recapitulating the typical UVA-induced skin alterations and to test the efficacy of photoprotectants. The thesis has been organized as follow: chapter 1 deals with a critical analysis of the literature reporting an overview of the existing human skin equivalent model highlighting their importance as alternative to animal model for testing the irritancy and toxicity of several bioactive compounds. This is a very important point, since the EU Directive prohibits the use of animals in cosmetic testing for certain endpoints, and in general strongly dictate the reduction of animal testing.
In the chapter 2 the realization of 3D dermis equivalent completely made up of endogenous extracellular matrix has been reported. The dermis is the connective part of the skin, we realized it by assembling functional microtissues precursor and demonstrate the presence of all dermis matrix components by histological and immunohistochemical analyses.
In the chapter 3 the 3D human dermis equivalent model has been used to quantify photoaging damage induced by UVA irradation to cells and extra-cellular matrix (ECM) and to evaluate bioactivity of cosmetic compounds. To this aim, matrix metalloproteinases (MMPs), collagen and hyaluronic acid (HA) synthesis -as well as collagen organization remodeling- have been investigated by immunofluorescence, histological and second harmonic generation (SHG) imaging. The results indicate that, despite to the existing model consisting in fibroblasts-populated exogenous matrix, the endogenous dermal equivalent developed is a unique model to investigate and quantify in vitro, the alterations in the structure and assembly of the ECM due to photoaging.
In chapter 4 is reported the bio-fabrication of a skin full thickness in vitro model. After a first morphological characterization, through histological and immunofluorescence analysis, the model has been used as in vitro screening tool. A photodamage due to UVA has been induced as reported in the chapter 4, and the damage on both dermal and epidermal part of the skin has been assessed. Finally the effect of a cosmetic compound has been tested. Our results reported that the model is able to recapitulate several events typical of in vivo photoaging (collagen reduction, MMP overexpression and cellular damage such as reduction of Ki67 and P63 expression).  We speculated that due to its endogenous nature the skin model realized is able to recapitulate a more physiological microenviroment for cell and tissue development compared to existing fibroblasts-populated exogenous matrix. 
Moreover we hypothesized that the human skin model realized could be used not only in vitro but also in vivo as skin substitute in clinical application