Marine biofouling is the accumulation of marine species on surfaces submerged in seawater leading to unwanted problems for man-made surfaces such as hulls of ships and aquaculture nets. Historically, the amount of biofouling was regulated using metal based coatings whose usage have been disused lately due to adverse toxic effects. Alternative environmentally friendly coatings are currently avidly being pursued. Nanopatterned polymer thin films were investigated as potential candidates for marine antifouling coatings. Polystyrene-block-poly(2-vinyl pyridine) and polystyrene-block-poly(methyl methacrylate) diblock copolymer thin films self-assembled using vapor solvent annealing into cylinders perpendicular to the substrate composed of poly(2-vinyl pyridine) or poly(methyl methacrylate) respectively with diameters between 30 nm to 82 nm and center-to-center spacing between 46 nm to 113 nm in a polystyrene matrix on various substrates such as silicon or nylon. Polystyrene-block-poly(2-vinyl pyridine) copolymers were also mixed with the photoinitiator benzophenone and irradiated with ultraviolet light to crosslink the polymer chains and decrease the surface hydrophobicity. In the case of polystyrene-block-poly(methyl methacrylate), the yield of these nanopatterned films increased with the modification of the vapor annealing method. A low temperature vapor annealing technique was developed in which the annealing occurs at 2 °C. In another strategy, polystyrene and poly(2-vinyl pyridine) homopolymers were nanopatterned with alternating lines and grooves with widths between 200 nm and 900 nm and depths between 15 nm to 100 nm using Thermal Nanoimprint Lithography. Poly(2-vinyl pyridine) films were synthesized as brushes using surface initiated Atom Transfer Radical Polymerization to produce robust polymer films. The chemical and/or the topographical heterogeneity of the polymer surfaces influenced the settlement of Ulva linza algae zoospores. Overall, the incorporation of nanoscale features enhanced the antifouling properties of the samples. Further exploration of these types of coatings is highly encouraged.

Marine biofouling is the accumulation of marine species on surfaces submerged in seawater leading to unwanted problems for man-made surfaces such as hulls of ships and aquaculture nets. Historically, the amount of biofouling was regulated using metal based coatings whose usage have been disused lately due to adverse toxic effects. Alternative environmentally friendly coatings are currently avidly being pursued. Nanopatterned polymer thin films were investigated as potential candidates for marine antifouling coatings. Polystyrene-block-poly(2-vinyl pyridine) and polystyrene-block-poly(methyl methacrylate) diblock copolymer thin films self-assembled using vapor solvent annealing into cylinders perpendicular to the substrate composed of poly(2-vinyl pyridine) or poly(methyl methacrylate) respectively with diameters between 30 nm to 82 nm and center-to-center spacing between 46 nm to 113 nm in a polystyrene matrix on various substrates such as silicon or nylon. Polystyrene-block-poly(2-vinyl pyridine) copolymers were also mixed with the photoinitiator benzophenone and irradiated with ultraviolet light to crosslink the polymer chains and decrease the surface hydrophobicity. In the case of polystyrene-block-poly(methyl methacrylate), the yield of these nanopatterned films increased with the modification of the vapor annealing method. A low temperature vapor annealing technique was developed in which the annealing occurs at 2 °C. In another strategy, polystyrene and poly(2-vinyl pyridine) homopolymers were nanopatterned with alternating lines and grooves with widths between 200 nm and 900 nm and depths between 15 nm to 100 nm using Thermal Nanoimprint Lithography. Poly(2-vinyl pyridine) films were synthesized as brushes using surface initiated Atom Transfer Radical Polymerization to produce robust polymer films. The chemical and/or the topographical heterogeneity of the polymer surfaces influenced the settlement of Ulva linza algae zoospores. Overall, the incorporation of nanoscale features enhanced the antifouling properties of the samples. Further exploration of these types of coatings is highly encouraged.

Adherent microbial communities, known as biofilms, are a major contributing factor in the incidence of healthcare-associated infections (HCAIs). HCAIs are responsible for annually causing 100,000 deaths and medical expenses estimated to be $35-45 billion. Physical and chemical surface modification techniques are thought to be critical in the fight against biofilm formation within medical settings. Nanoscale structural features have been found to have significant effects on bacterial adhesion and biofilm formation, but their effects on fungal pathogens are less explored. This thesis systematically explores the effect of surface topography in the form of nano and microscale polymeric fibers (~0.4-1.2 µm in diameter) on biofilm formation and virulence of a common HCAI-causing fungal pathogen, Candida albicans. We show that both C. albicans attachment density and differentiation to its virulent phenotype significantly vary with fiber diameter and spacing on polymeric fiber-coated surfaces. We further show that high throughput and high content techniques, such as Raman spectroscopy, can be used to track environmental and physical effects on the organism's resulting morphology and associated virulence. Findings from this thesis will inform the design of antifouling surfaces including implantable medical devices. In a prototypical example, we demonstrate the use of fiber coating to modulate C. albicans attachment on polyurethane, silicone, and latex catheters. / Master of Science / Microbial communities that adhere to surfaces, known as biofilms, are largely associated with incidence of healthcare-associated infections (HCAIs). HCAIs are responsible for annually causing 100,000 deaths and medical expenses estimated to be $35-45 billion. Modification of surfaces using physical and chemical techniques is believed to be critical in the fight against biofilm formation on surfaces within medical settings. Nanoscale structural features have been found to have significant effects on bacterial adhesion and biofilm formation, but their effects on fungal pathogens have not been extensively studied. This thesis focuses on the effect of surface topography, or textures, in the form of nano and microscale polymeric fibers (~0.4-1.2 µm in diameter) on biofilm formation and disease-causing ability, or virulence, of a common HCAI-causing fungal pathogen, Candida albicans. We show that both C. albicans attachment density and differentiation to its virulent form significantly vary with fiber diameter and spacing on polymeric fiber-coated surfaces. We also show that high throughput and detailed imaging techniques, such as Raman spectroscopy, can be used to track environmental and physical effects on the organism's resulting morphology and associated virulence. Findings from this thesis can be used to aid in the design of surfaces that discourage biofilm formation, including implantable medical devices. We demonstrate the use of fiber coating to vary C. albicans attachment on polyurethane, silicone, and latex catheters in one of our studies.

Biofilm formation on medical devices is responsible for a substantial portion of healthcare associated infections with approximately 99,000 deaths and estimated financial burden of $28-$45 billion annually. Given the long-standing challenges of biofilm eradication, physical and chemical surface modifications to prevent biofilm formation from the early adhesion stage, continue to gain momentum. Nanoscale structural features, ubiquitous in both natural and synthetic surfaces, are increasingly recognized to have wide-ranging effects on microorganism adhesion and biofilm development. In this thesis, bio-inspired nanofiber-coated polystyrene surfaces were developed to systematically investigate how highly ordered surface nanostructures (200nm-2000nm in size) impact adhesion and proliferation of model fungal pathogen, Candida albicans. A theoretical model for cell-textured surface interaction was also developed using thermodynamic principles to demonstrate that single cell adhesion to surface can be used to describe the population behavior. The trend for adhesion density of C. albicans on nanofiber-textured surfaces of varying diameters correlates with our theoretical finding of adherent single-cell energetic state. Findings from this thesis can be used for enhanced ab initio design of antifouling surfaces for medical applications and beyond. We demonstrate a successful prototypical example of reduction in biofilm formation by optimally designed nanofiber coating of urinary catheters. / Master of Science

ABSTRACT INTERACTION BETWEEN MICRO AND NANO PATTERNED POLYMERIC SURFACES AND DIFFERENT CELL TYPES &Ouml / z&ccedil / elik, Hayriye Ph.D., Department of Biology Supervisor: Prof. Dr. Vasif Hasirci Co-Supervisor: Dr. Celestino Padeste August 2012, 139 pages Micro and nanopatterned surfaces are powerful experimental platforms for investigating the mechanisms of cell adhesion, cell orientation, differentiation and they enable significant contributions to the fields of basic cell and stem cell biology, and tissue engineering. In this study, interaction between micro and nanopatterned polymeric surfaces and different cell types was investigated. Three types of micropillars were produced by photolithography (Type 1-3), while nanometer sized pillars were produced in the form of an array by electron beam lithography (EBL). Replica of silicon masters were made of polydimethylsiloxane (PDMS). Polymeric [P(L-D,L)LA and a P(L-D,L)LA:PLGA blend] replica were prepared by solvent casting of these on the PDMS template and used in in vitro studies. The final substrates were characterized by various microscopic methods such as light microscopy, atomic force microscopy (AFM) and scanning electron microscopy (SEM). In order to investigate deformation of the nucleus in response to the physical restrictions imposed by micropillars, Type 1 and Type 2 pillars were used. These substrates were covered with pillars with different interpillar distances. While Type 1 is covered with symmetrically (in X-Y directions) distributed pillars, Type 2 pillars were distributed asymmetrically and the inter-pillar distances were increased. Nuclei deformation of five cell v types, two cancer cell lines (MCF7 and Saos-2), one healthy bone cell (hFOB1.19), one stem cell (bone marrow origined mesemchymal stem cells, BMSCs) and one standard biomaterial test cell type, (L929) fibroblasts was examined by using fluorescence microscopy and SEM. The nuclei of Saos-2 and MCF7 cells were found to be deformed most drastically. Nucleus deformation and intactness of nuclear membrane was examined by Anti- Lamin A staining. The interaction of the cells with micropillars was visualized by labelling focal adhesion complexes (FAC). Wettabilities of patterned and smooth surfaces were determined. As the patterns become denser (closer micropillars, Type 1) the hydrophobicity increased. Similar to water droplets, the cells were mostly spread at the top of the Type 1 pillars. The number of cells spread on the substrate surface was much higher on Type 2 patterned films. In order to support these qualitative findings, nucleus deformation was quantified by image analysis. Frequency of nucleus deformation was determined as the ratio of deformed to the total number of nuclei (%). In order to quantify the intensity of nuclei deformation, their circularity was evaluated. In addition to nucleus deformation, alterations in the ratio of cell area-to-nucleus area in response to micropillars were determined by image analysis. The results indicated that cancerous cells were more deformable. The qualitative microscopic evaluation and the data obtained by quantification of the nucleus and cellular deformation were in good agreement. In addition, the findings were consistent with expectations which suggest that cancerous cells are &ldquo / softer&rdquo / . In the second part of the research the force applied by the cells on arrays of micropillars with high aspect ratios (Type 3 substrates) during tugging at the pillars was investigated. Micropillars were produced using P(L-D,L)LA as well as a 60:40 blend of P(L-D,L)LA with PLGA. The blend is a material with lower stiffness than P(L-D,L)LA. The mechanical properties of the two materials were determined by tensile testing of solvent cast films. Deformation of Type 3 micropillars by the cellular tugging force of Saos-2 and L929 was studied by fluorescence and SEM microscopy, both on stiff and softer substrates. Displacements of the centers nodes of the pillars were evaluated from SEM micrographs. On the stiff surface, the two cell types bent the pillars to the same extent. On the other softer substrate (blends), however, the maximum displacements observed with Saos-2 cells were higher than the ones caused on the stiffer substrate or the ones caused by L929 cells. It is reported that stiffness of the substrate can determine stem cell lineage commitment. In order to examine the effects of change of substrate stiffness on osteogenic differentiation of BMSCs, osteopontin (OPN) expression was determined microscopically. It was found that osteogenic differentiation is enhanced when BMSCs are cultured on P(L-D,L)LA Type 3 pillars. vi In the last part of research, arrays of nanopillars whose interpillar distances systematically varied to form different fields were examined in terms of adhesion and alignment in order to determine the differential adhesion of BMSCs and Saos-2 cells. The difference in their adhesion preference on nanopillar arrays was quantified by image analysis. It was observed that BMSCs and Saos-2 cells behaved in an opposite manner with respect to each other on the fields with the highest density of nanopillars. The BMSCs avoided the most densely nanopillar covered fields and occupied the pattern free regions. The Saos-2, on the other hand, occupied the most densely nanopillar covered fields and left the pattern free regions almost unpopulated. It was also found that both BMSCs and Saos-2 cells aligned in the direction of the shorter distance between the pillars. Both BMSCs and Saos-2 cells started to align on the pillars if the distance in any direction was &gt / 1.5 &mu / m. To better understand the effects of chemical and physical cues, protein coating and material stiffness were tested as two additional parameters. After fibronectin coating, the surfaces of P(L-D,L)LA films with the highly dense pillar covered fields, which were avoided when uncoated, were highly populated by the BMSC. Similarly, decreasing the stiffness of a surface which was normally avoided by the BMSCs made it more acceptable for the cells to attach.

We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α2 were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α2 was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α2 expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α2-mediated matrix binding was verified by preincubation with an α2-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α2 were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α2 was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α2 expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α2-mediated matrix binding was verified by preincubation with an α2-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.