Carsten Werner

Fibronectin at Polymer Surfaces with Graduated Characteristics

Globular proteins adsorbed onto artificialmaterials often exhibit different functional characteristics due to an altered availability formolecular interactions. This effect is caused by the patterns of substrate-protein interactions and is attributable to conformational changes as well as to the orientation and/or the anchorage of the surface-confined proteins. To highlight this interrelation we report a detailed experimental investigation of the adsorption and displacement of fibronectin, a key protein of the extracellular matrix that enables cell adhesion, at a set of polymer thin films with various hydrophilicity, charge density and swelling characteristics. The patterns of protein displacement were analysed quantitatively for several substrates and adsorbed protein amounts, referring to a model recently suggested by Huetz et al. (Langmuir 11:3145–3152, 1995). The patterns of protein displacement were related to the substrate characteristics and the conditions applied during the formation of the protein layer (i. e. the solution concentration). These findings were compared with the reorganisation of adsorbed fibronectin on the compared polymer substrates by endothelial cells in culture. The results demonstrate that a certain binding strength of fibronectin is required to support the cell-driven formation of fibronectin fibrils, which, in turn, is an important prerequisite for the differentiation of the cells.

CHAPTER 3:Probing Biointerfaces: Electrokinetics

Unravelling interrelations between charge, structure and biomolecular interactions defines an important analytical challenge in the development of new biomaterials. Theory and experimental techniques have been extended to explore processes occurring upon contact of polymeric biomaterials with aqueous environments. Significant progress was achieved in the quantitative reconstruction of streaming current data applying recent theories for the electrohydrodynamics of diffuse soft planar interfaces. Here, we review experimental approaches and theory to analyze the charging and structural changes of soft planar films in dependence on solution pH, salt concentration or temperature. Furthermore, we present and discuss data for stimuli-responsive polymer films and biohybrid hydrogel layers that demonstrate how the combination of streaming current, surface conductivity and swelling measurements allows for a comprehensive understanding of electrostatic and structural properties of these materials. In a third example we show how the combined application of fluorescence microscopy and electrokinetics provides new insights into fluidity modulations of phospholipid bilayers by electrolyte ions. Finally, we discuss potential future developments in the investigation of electrosurface phenomena.

Bestimmung der Blutverträglichkeit von Materialoberflächen mit immobilisiertem Thrombininhibitor

A major complication with biomaterials in blood contact is blood coagulation followed by thrombus formation and stenosis. The immobilization of bioactive substances to either inhibit blood coagulation proteins or platelets are keypoints in research efforts to improve the hemocompatibility of biomaterials. We report herein a strategy based on the surface immobilization of small synthetic amidine derivatives known as selective and potent inhibitors acting directly on thrombins active site. The immobilization of biomimetic synthetic coagulation inhibitors to the surface of biomaterials could achieve a directed prevention of blood coagulation. Starting with 4-aminobenzamidine we sythesized an inhibitor and could show through molecular modeling that several of the relevant aminoacids participate in the interaction of the thrombin inhibitor with the binding pocket. A covalent immobilization was realized using thin films of poly(octadecene-alt-maleic anhydride) (PO-MA) attached to a solid support. We prepared four different surfaces. The first was attained through hydrolysis of the anhydride moieties of the copolymer film and resulted in a negatively charged surface (M1). The second was a polyethylene glycol (PEG) modified surface (M2). These two served as reference samples. The immobilization of the thrombin inhibitor was accomplished by either direct modification of thin films of PO-MA (M3) or using a bifunctional PEG spacer, linked to PO-MA (M4). The in vitro hemocompatibility assays were conducted using in-house-built incubation chambers with an advantageous relation of testing surface to blood volume. The generated materials (M1 4) were incubated with freshly drawn human blood anticoagulated with heparin for 2 hours at 37 °C. After incubation, the blood was analyzed with respect to changes in cell numbers and activation markers for coagulation (TAT), complement activation (C5a) and thrombogenicity (PF4) using commercially available ELISA assays. The surface of the incubated materials was analyzed for adherent platelets and fibrin deposition by SEM. We could show that the highest activation for all the parameters was attained for the highly negatively charged surface M1. The PEG-surface showed considerably less activation probably due to the well known inhibition of protein adsorption. The immobilization of thrombin inhibitor further reduced the activation of coagulation and complement compared ORIGINAL ARBEITEN Claudia Sperling: Bestimmung der Blutverträglichkeit von Materialoberflächen mit immobilisiertem Thrombininhibitor Abb. 9: REM Aufnahme der Probenoberfläche nach der Inkubation mit humanem Vollblut. M 1: Poly(octadecen-alt-maleinsäureanhydrid) hydrolysiert, M 2: PO-MA + Jeffamin, M 3: PO-MA + TI, M 4: PO-MA + PEG + TI