Realization a complex skin equivalent tissue

The aim of this work is the realization of a three-dimensional human skin equivalent tissue (3D-HSE) completely made up by endogenous extra cellular matrix (ECM). The realization of 3D-HSE has been pursued by means of a bottom-up tissue engineering strategy that firstly comprises the fabrication of functional micro-metric tissue precursors (mTPs) through dynamic seeding of fibroblasts on biodegradable porous microbeads with a controlled and tunable degradation rate. Then mTPs have been assembled in a 3D dermis tissue and the epidermal cells are lastly seeded on the top of dermal equivalent. In the development of epithelial stratum is important to maintein the culture submerged to allow complete epidermal cells coverage, melanocytes organization along epidermal basal membrane and early keratinocytes stratification, and later raising the developing culture to the air-liquid interface thus promoting epidermal differentiation. Epidermal differentiation markers have been investigated by means of the immunofluorescence analysis while collagen was assesses by multiphoton microscopy analysis. The functional endogenous dermis has a crucial role in the morphogenesis of a pluristratified epidermis. Transmission electron microscopy well show the fine structure of the dermal-epidermal junction confirming that epidermal layer is well anchored to the dermis. Moreover dermis-epidermis cross-talking is guaranteed as proved by the presence of basal membrane and this structure has a characteristic rete ridge morphology with typical epidermis appendages going deep through the underlying dermis resembling bulge-like structure. In view of its complexity, this skin model can be specifically used for tackling various problems in the chemical/pharmaceutical and cosmetics industry. Since an endogenous ECM is present it is possible to study not only the cellular response but also the ECM response in terms of change in assembly as well as composition to a drug.