3D RECONSTRUCTED SKIN EQUIVALENT MODELS FOR IRRITANT TESTING.

Acute skin irritation is the reversible inflammatory response of the epidermis to a topically applied irritant substance. A tissue engineered model of the epidermis is used to test chemicals. The degree of development of the model needs to be carefully judged in order to get the correct proportion of proliferating through to differentiated phenotypes for normal function. This judgement typically necessitates over sensitive models with an underdeveloped barrier functionality, as opposed to an insensitive model due to terminal differentiation and low numbers of basal keratinocytes. It has been reported that the lack of proliferative epidermal cells in cultures may be due to the absence of fibroblasts. Paracrine signalling in response to potential irritants is required for propagating an acute inflammatory response. The aim of this thesis is to develop a skin model using a Three Dimensional scaffold that accurately mimics the micro-environment at the DEJ, for supporting keratinocyte and fibroblast self-organisation. We hypothesise that it takes a full thickness skin model with a complete cascade of inflammatory stimuli and cytokine signalling to provide a real indication of irritation. Initial studies focused on Alvetex® (Reinnervate Ltd.), a highly porous polystyrene scaffold, with the aim of developing a skin model using the immortalised cell line HaCaT (human adult low calcium high temperature) keratinocytes or NhKs, in co-culture with dermal fibroblasts. Skin models using electrospun biodegradable polymer scaffolds made of Poly L-lactide (PLLA) and a Poly L-lactide/Polyhydroxybutyrate-co-hydroxyvalerate/Poly L-lactide (PLLA/PHBV/PLLA) composites were then developed. Issues with achieving epidermal-dermal separation in the Alvetex® scaffold due to keratinocyte entrapment lead to an Alvetex®-PHBV Bilayer. Concentration of the SDS needed to illicit an irritant response was deduced at 2D to be 0.1-0.15mM, 3D submerged to be 0.33-0.5mM and for 3D air-liquid models were at best unaffected by 8mM SDS with a Bilayer scaffold of PHBV-PLLA.