Lombardi, Bernadette (2016) Development of a three-dimensional organotypic skin model for in vitro study of skin disease state. [Tesi di dottorato]

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The skin functions as a protective physical barrier against the outside and has a primary role to protect body from external influences such as pathogenic microorganisms and mechanical injuries. Moreover in the skin, the interaction of keratinocytes, fibroblasts and melanocytes is tightly controlled by various factors and cascades. Under normal conditions, there is a balance between cell types via cell–cell contact and the extracellular matrix (ECM). The ECM provides structural scaffolding for cells, as well as contextual information. The disruption of these equilibria can result in an uncontrolled stroma degeneration (as involved in scarring) or uncontrolled proliferation of epithelial cells (as in malignant melanoma). Wound healing is a highly organized series of processes resulting in tissue integrity and function of the damaged tissue; this process needs a complex microenvironement to be studied. Carcinogenesis is defined as complex, adaptive process, controlled by intricate communications between the host and the tissue microenvironment. Thus, the microenvironment and the stroma play an important role in both wound healing process and tumour development. In this perspective, in the present PhD thesis a tissue engineering bottom-up approach was used to fabricate a 3D dermis tissue. This model was composed by a cell-synthesized and responsive extracellular matrix that resembles the in vivo dermis, and it was used as living platform to study in vitro skin alteration and diseases. The first model of skin alteration dealt with the wound healing process, by exploiting its self-repairing capability. Interestingly, the relationship between cell migration, differentiation marker and ECM production and remodeling during repair process followed the same in vivo timing. Moreover, the presence of a responsive dermis allowed possibility to evaluate granulation tissue and to study and understand processes involved in scarring. Indeed, due to the endogenous nature of the stroma, the model proposed could represent a valuable tool to in vitro study tissue status at both cellular and extracellular level after a physical damage. At least, once demonstrated dermis responsivity, we investigated epidermal counterpart. In this perspective, we fabricated a 3D human skin equivalent (3D-HSE) model with the same endogenous stroma as dermis component to study cell-ECM – with native basement membrane (BM) – and cell–cell communications, in the presence of an aggressive form of skin cancer: melanoma. As a fact, carcinogenesis can disrupt these forms of communication, thus altering cell biology of human skin. Consequently, we investigated the role of skin cells and BM components on melanoma biology and invasive ability in reconstructed human skin equivalent. We first made-up and characterized a human skin model that resembled the architecture of skin in situ, than we carried out an analogous procedure for the equivalent engineered tumor model. On the basis of our results, we can assert that there is communication between skin cells and melanoma cells and the outcome is dictated by the nature of the melanoma cells. Thus, the bioengineered 3D melanoma skin model may become a valuable tool to investigate the underlying mechanics of melanoma infiltration. The proposed study does not recapitulate yet melanoma metastasis process as a whole; however, the present engineered 3D tissue represents a reliable model for investigating the phenotype and behavior of melanoma cells derived from primary sites. Indeed, the 3D melanoma skin model is suitable to study the biological properties of radial growth phase invasion. This study represents a preliminary model for investigating all aspects of melanoma metastasis and it has great potential for improving our understanding of the interactive biology between melanoma cells and their immediate surroundings and evaluating melanoma cells influence on epidermis structure and differentiation. In conclusion, the present 3D engineered skin model represents a valid platform to study scar formation and a valuable tool for studying healthy and disease skin or for screening test in vitro. Moreover, this platform could provide an in vivo skin substitute in clinical applications.

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