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Abstract

Wound healing is a complex, multi-step process. To be able to comprehensively examine the role of hyaluronic acid (HA) on re-epithelialization it is crucial to isolate re-epithelialization as the process where keratinocytes migrate and proliferate from the other processes during wound healing. The effect of HA on re-epithelialization is controversially discussed in literature and comparability between independent studies is hampered as applied HA concentrations, molecular weight (MW) of HA as well as cell systems, substrate nature (glass, plastic) and extracellular matrix (ECM) composition all differ from publication to publication. Therefore, in this thesis I comprehensively compared for the first time the effect of apical presented HA of different MW (5, 60, 700 and 1800 kDA) and concentrations (5, 50, 500, 1000 μg/mL) within the framework of re-epithelialization using a human keratinocyte cell line (HaCaT). I showed that HA hampers migration on collagen I-coated glass surfaces in a concentrationdependent manner whereas in terms of proliferation HA has a pro-proliferative effect under conditions of low density on cell culture plastic. With the aim to further reduce the complexity of the interplay of HA with other ECM molecules it is crucial to have a wound healing system comprising of a minimal number of components with the possibility to stepwise increase complexity. Following a bottom-up approach I employed bifunctionalized glass surfaces and altered their functional groups: Only HA and a second, integrinaddressing binding ligand were presented on an otherwise passivated background. In order to address different integrin-binding families I proved the established cRGD as well as a novel peptide, EPDIM to be suited to mediate HaCaT cell adhesion and subsequent spreading. Additionally, I investigated whether end-alkylated HA of 5, 10 and 60 kDA can be bound to azide-presenting surfaces via click chemistry. Surface saturation with HA was determined via QCM-D experiments and accessibility of surface-bound 5 and 10 kDA HA by the hyaladherin aggrecan proved bioactivity of immobilized HA. Surfaces presenting HA without additional adhesive peptides did not mediate HaCaT cell adhesion. Cell adhesion and subsequent spreading was observed on bi-functionalized glass surfaces with cRGD or EPDIM as a binding ligand together with three different concentrations of immobilized HA. Furthermore, indirect immunofluorescence revealed a direct correlation between immobilized HA and the degree of focal adhesion formation. In order to mimic the dynamic changes in ECM composition during wound healing (e.g. when the provisional matrix evolves into granulation tissue) I developed a two-stage wound model system. I showed that formation of confluent monolayers on polycarbonate membranes was achieved within 24 h. By cutting such HaCaT-cultivated membranes (2D epidermal models) a real wound site was introduced. Customized molds enabled the mechanical fixation of membrane-cultivated HaCaTs on any desired surface without the need of collagen I glue thus ensuring a clean and well-controllable ECM environment without unintentional insertion of collateral ECM molecules. I demonstrated that such vegetated membranes can be used to observe migrational activities of keratinocytes in time-lapse VI imaging on collagen I gels, collagen I-coated glass surfaces as well as on minimalistic, highly-defined ECM models, where only one single binding motif is presented. Furthermore, migration was also mediated upon providing bi-functionalized glass surfaces where the binding ligands cRGD or EPDIM were presented orthogonally with HA of varying concentration. In contrast to systems in which cells form a confluent layer on the same surface they migrate on after wounding, this novel two-stage ECM system ensures that the migration interface is not harmed by the wounding procedure and can be chemically different than the interface on which the cell monolayer has been cultivated. Combining the two-stage ECM model with bi-functionalized surfaces creates a two-stage ECM wound healing model, which can be applied in 2D cell culture as well as 3D tissue systems and has the potential to reveal HA’s signaling role in cooperation with other ECM molecules on re-epithelialization in future application.