title: A SYSTEM TO SEGMENT  THE ROLE OF HYALURONIC ACID  DURING RE-EPITHELIALIZATION
creator: Dietrich, Franziska
subject: ddc-570
subject: 570 Life sciences
description: 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.
date: 2020
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/29012/1/Thesis-Franziska%20Dietrich.pdf
identifier: DOI:10.11588/heidok.00029012
identifier: urn:nbn:de:bsz:16-heidok-290128
identifier:   Dietrich, Franziska  (2020) A SYSTEM TO SEGMENT THE ROLE OF HYALURONIC ACID DURING RE-EPITHELIALIZATION.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/29012/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng