Title of the PhD-Thesis: From porcine skin samples in situ to three-dimensional human skin constructs in vitro. Studying skin with a focus on the 3R principles. Chapter 1 gives a general introduction to the importance of animal models, focusing on porcine models as well as on in vitro models in skin research. Chapter 2 presents a literature review of research on pigs as animal models for human skin in skin research and of in vitro models of skin research. Furthermore, the principle of the 3Rs is reviewed. Chapter 3 states the objectives and hypotheses of this thesis. Chapters 4 and 5 cover the two main publications for this thesis. Chapter 4 reports on the comparison between abdominal human skin and four different areas of pig skin (the ear, the flank, the back, and the caudal abdomen). The skin samples were examined by light microscope (LM), transmission electron microscope (TEM) and immunohistochemistry. In addition, skin permeability studies were performed. The results of this study show both the similarities and differences in histological and ultrastructural features. The epidermis of the examined regions in both pigs and humans, except for the stratum corneum, is of similar thickness. Although the number of corneocyte layers was not different in the two species, the human corneocyte layer was thicker than that of the pig. The dermal structure of humans and pigs is similar, the thickness of the human one is more similar to the one of the porcine flank and back region. The ear and caudal abdomen of the pig showed a thinner dermis when compared with those of humans. The epidermal-dermal thickness ratio of the porcine back, flank and abdomen was similar to that of humans. The dermo-epidermal length was not found to be different in humans and pigs. This is significant since the skin´s barrier function depends on this interface. The number of hair follicles and arrector pili muscles of the porcine abdominal and ear skin was different from that of the human skin. The number of sweat glands of the four areas of pig skin was not significantly different from that of the human skin. The number of blood vessels in the human dermis was significantly lower than in the pig dermis. Chapter 5 reports on the comparison between the two types of in-house skin constructs and human skin as examined by light and transmission electron microsopy. Data were analyzed by semi-quantitative and qualitative methods. The two types of skin constructs included a construct consisting of keratinocytes and fibroblasts only (type 1, KCFB) and another construct made of keratinocytes as well as fibroblasts and vascular endothelial cells (type 2, KCFB_EC). Both types of skin constructs showed all strata as known from human skin. When compared with the skin constructs without EC or with normal human skin, the skin constructs with endothelial cells were characterized by unorganized epidermal layers, significantly more mitotic cells in the stratum spinosum, more layers of the stratum granulosum and more keratohyaline granules. Chapter 6 discusses whether specific skin regions of domestic pigs were suitable for human skin replacement. According to the results reported in the first section, the back region, particularly of the German Landrace breed, had superior skin characteristics to those from the flank, the abdominal and the caudal ear regions. The dermal thickness of the back region was comparable to the thickness of human abdominal skin. It is possible that the caudal ear skin could be used as a model in studies of chemicals utilizing the trans-follicular route for their absorption. The second section addressed the morphology and ultrastructure, and the effect of endothelialization on epidermal differentiation in two types of skin constructs. The epidermis in both the KCFB and KCFB-EC skin models had a similar overall architecture and ultrastructure to native skin. When the KCFB-EC and KCFB models were compared, however, there were several differences. The KCFB-EC constructs featured more mitotic cells, a considerable epidermal differentiation, and a significantly more disorganized epidermal architecture. The data supported the hypothesis that VEGF promotes epidermal thickness and keratinocyte proliferation. VEGF was obtained from both culture medium and keratinocyte culture. Increased VEGF levels may play a crucial role in these issues. In addition, no vasculogenesis was observed in the fibroblast matrix of the KCFB-EC constructs. Due to a lack of interaction between endothelial cells and fibroblasts, endothelial cells failed to form capillaries. To build a stable dermis, fibroblasts and endothelial cells quantity must be optimized. Thus, the epidermis may be initiated by seeding keratinocytes on top of the optimal dermis.
