Nano- und mikrostrukturierte Polydimethylsiloxan-Substrate für biomedizinische Anwendungen

In an international comparison, Germany is one of the leading locations in the biomedical technology. This leading position is the result of a combination of the unique Know-How in fundamental research of excellent technological competence and medical excellence. Close collaboration between experts from a range of disciplines engineering, semiconductor and micro-electronic manufacturing technology, micro- and nano-technology, biomedical technology and medicine creates excellent preconditions for the research and development of innovative medical devices. This thesis represents results of a close interdisciplinary collaboration of micro- and nano-technology, electrically engineers, biologists and medical experts, respectively. Silicone elastomer polymers based on polydimethylsiloxane (PDMS) are well-known materials with extraordinary and multi-purpose properties that can be used in many different biological and medical applications. These materials are promising and widely applied in tissue engineering, lab-on-chip techniques, cell-based biosensors, and microfluidic devices, especially due to their biocompatibility, excellent optical transparency, and low cost. Additionally, PDMS is a promising biomaterial for generating artificial extracellular matrix, like patterned topographies, but its hydrophobic nature limits its applicability to cell-based approaches. Although plasma treatment can enhance the wettability of PDMS, the surface is known to be able to recover its hydrophobicity within a few hours of exposure to air. This study investigates the use of a novel PDMS-type (Q-branched siloxane X-PDMS) for in vitro-based applications of physiological cell properties and the use of thin-film micro-electrodes for neuronal stimulation. Thus, plane as well as nanometer- and micrometer-scaled patterned templates using the elastomer types S (soft)-, H (hard)-, and X-PDMS, which were made from commercially available components, were designed and fabricated. Most importantly, X-PDMS templates, which have not yet been investigated in this context, were compared with H-PDMS- as well as well-known S-PDMS-based substrates. Due to its applicability in the fabrication of nanometer-sized topographic features with high accuracy and pattern fidelity, this material may be of high relevance for specific biomedical applications. In order to assess the use of PDMS surfaces in cell-based applications, this study characterized them according to their wettability and topographical changes. Further, in order to characterize the cellular viability and adhesion of various cell types (e.g. melanoma cells, human foreskin fibroblasts, neuron cells), this study assessed various cell growth parameters (e.g. cell number, branching complexity). Additionally, deposited metal thin-films and thin-film micro-electrodes based on PDMS substrates were identified based on their adhesive, electrical, and electrochemical behavior. Finally, to assess the applicability of the thin-film micro-electrodes to neuronal stimulation based on PDMS substrates, an electrical stimulation chamber system was developed and fabricated. This thesis shows, for the first time, that different PDMS types are sensitive in different ways to plasma treatment (microwave and low frequency plasma excitation). X-ray photoelectron spectroscopy, atomic force microscopy, and water contact angle measurements were used to study the effect of various plasma treatments of PDMS substrates, as well as plasma gases, such as Ar, Ar/O2, O2, and N2/H2. After the modification, the topographical change and the wettability behavior exhibit a dependence on the applied plasma treatment and the PDMS type. Most importantly, results suggest that N2/H2 plasma treatment even leads to a super hydrophilic surface, which has been concluded to be caused by a combination of chemical change and the formation of a wavy surface topography. Further, for the first time, this work demonstrates the hydrophobic recovery behavior of plasma-treated S-, H-, and X-PDSM surfaces over a period of several years, stored under various conditions. Additionally, the study shows that surface hydrophobicity changes along with the changing height and width of the pillar- and groove-patterns. In the case of cellular adhesion behavior, the data indicates that plane and patterned X-PDMS exhibit adhesive properties comparable to the other elastomer types, S- and H-PDMS. In addition, X-PDMS material is used as a substrate for fabrication of titanium thin-film micro-electrodes. These electrodes together with the electrical stimulation chamber, which was developed and fabricated for this thesis, are successfully applied to neuronal stimulation experiments. The results demonstrate that nanometer-sized patterning of X-PDMS may serve as a powerful method for altering surface properties for the production of biomedical devices for cell-based applications.Im internationalen Vergleich ist Deutschland einer der führenden Standorte in der Biomedizintechnik. Diese Führungsposition wurde ermöglicht durch eine Kombination aus Know-how in der Grundlagenforschung von ausgezeichneter technologischer Kompetenz und medizinischer Exzellenz. Die enge Zusammenarbeit von Experten aus Ingenieur- wissenschaft, Halbleiter- und Mikroelektronikfertigungstechnik, Mikro- und Nanotechnologie, Biomedizintechnik und Medizin schaffte bislang hervorragende Ergebnisse und dadurch ideale Voraussetzungen für die Erforschung und Entwicklung innovativer Medizinprodukte. Diese von mir vorgestellte Arbeit ist also das Ergebnis einer sehr engen interdisziplinären Kollaboration von Mikro- und Nanotechnologie, Elektrotechnikern, Biologen und Medizinern. Silikonelastomere auf Basis von Polydimethylsiloxan (PDMS) sind bekannte Materialien mit außergewöhnlichen und vielseitigen Eigenschaften, die bei verschiedensten biologischen und medizinischen Anwendungen eingesetzt werden können. Diese Materialien sind aufgrund ihrer Biokompatibilität, ausgezeichneter optischer Transparenz und geringen Kosten perfekt geeignet und werden in der Gewebezüchtung, in Lab-on-Chip-Techniken, in zellbasierten Biosensoren und in mikrofluidischen Geräten häufig eingesetzt. Darüber hinaus ist PDMS ein vielversprechendes Biomaterial zur Erzeugung künstlicher, extrazellulärer Matrix wie zum Beispiel strukturierter Topographien. Leider beschränkt aber seine hydrophobe Natur deren Anwendbarkeit in den zellbasierten Systemen. Obwohl eine Plasmabehandlung die Benetzbarkeit von PDMS verbessern kann, ist hinreichend bekannt, dass die Oberfläche ihre Hydrophobizität innerhalb weniger Stunden nach dem Aussetzen an der Luft wiedererlangen kann. In dieser wissenschaftlichen Arbeit wird die Verwendung eines neuartigen PDMS-Typs (Q-verzweigtes Siloxan X-PDMS) als In-vitro Substrat mit bestimmten physiologischen Zelleigenschaften untersucht. Zudem wird das Material als Substrat für Dünnschicht-Mikroelektroden zur neuronalen Stimulation analysiert. Es wurden sowohl planare als auch nanometer- und mikrometergetreue strukturierte Substrate bestehend aus den Elastomertypen S (weich), H (hart) und X-PDMS entworfen und aus den handelsüblichen Komponenten hergestellt. Insbesondere wurden X-PDMS Substrate, die in diesem Zusammenhang noch nicht untersucht wurden, mit H-PDMS- sowie bekannten S-PDMS-basierten Substraten verglichen. Aufgrund seiner Anwendbarkeit bei der Herstellung von nanometergroßen, topografischen Elementen mit hoher Genauigkeit und Mustertreue, kann dieses Material für bestimmte biomedizinische Anwendungen von hoher Relevanz sein. Um die Verwendbarkeit von PDMS-Oberflächen in zellbasierten Anwendungsgebieten beurteilen zu können, wurden diese Substrate in der vorliegenden Arbeit in Bezug auf ihre Benetzbarkeit und topografischen Veränderungen charakterisiert. Um die Zell-Lebensfähigkeit und -adhäsion verschiedener Zelltypen (z. B. Melanom¬zellen, menschliche Vorhautfibroblasten, Neuronenzellen) charakterisieren zu können, wurden verschiedene Zellwachstumsparameter (z. B. Zellzahl, Verzweigungskomplexität) untersucht. Zusätzlich wurden abgeschiedene Metalldünnschichten und Dünnschicht-Mikroelektroden auf den PDMS-Substraten anhand ihres adhäsiven, elektrischen und elektrochemischen Verhaltens untersucht. Um die Anwendbarkeit der Dünnschicht-Mikroelektroden auf die neuronale Stimulation hin zu überprüfen, wurde hierfür ein elektrisches Stimulationskammersystem entwickelt. Diese Arbeit zeigt zum ersten Mal, dass verschiedene PDMS-Typen sich nach einer Plasmabehandlung auch unterschiedlich verhalten. Es wurden Röntgenphoto-elektronenspektroskopie, Rasterkraftmikroskopie und Wasserkontaktwinkelmessungen verwendet, um die Wirkung verschiedener Plasmabehandlungen von PDMS-Substraten sowie von Plasmagasen wie Ar, Ar/O2, O2 und N2/H2 zu untersuchen. Nach der Plasmabehandlung zeigen sowohl die topographischen Veränderungen, als auch deren Benetzbarkeitsverhalten eine Abhängigkeit von der angewandten Plasmabehandlung und dem PDMS-Typ. Am wichtigsten ist jedoch, dass die Ergebnisse darauf hindeuten, dass die N2/H2-Plasmabehandlung sogar zu einer superhydrophilen Oberfläche führt, was vermutlich auf eine Kombination aus chemischer Veränderung und der Bildung einer gewellten Oberflächentopographie zurückzuführen ist. Diese Arbeit demonstriert zum ersten Mal das hydrophobe Regenerationsverhalten von plasmabehandelten S-, H- und X-PDSM-Oberflächen über einen Zeitraum von mehreren Jahren, die unter verschiedenen Bedingungen gelagert wurden. Zusätzlich zeigt die Arbeit, dass sich die Oberflächenhydrophobizität mit der sich ändernden Höhe und Breite der Säulen- und Rillenmuster ändert. Im Falle des zellulären Adhäsionsverhaltens zeigen die Daten, dass unstrukturierte und struktu-rierte X-PDMS Substrate Adhäsionseigenschaften aufweisen, die mit den anderen Elastomertypen wie S- und H-PDMS vergleichbar sind. Darüber hinaus wird X-PDMS-Material als Substrat zur Herstellung von Titan-Dünnschicht-Mikroelektroden verwendet. Diese Elektroden werden zusammen mit der elektrischen Stimulationskammer, die für diese Arbeit entwickelt und hergestellt wurde, erfolgreich für neuronale Stimulationsexperimente eingesetzt. Die Ergebnisse zeigen, dass die Nanostrukturierung von X-PDMS als leistungsstarke Methode zur Änderung der Oberflächeneigenschaften für die Herstellung biomedizinischer Geräte für zellbasierte Anwendungen dienen kann

Data_Sheet_1_Nano- and Micro-Patterned S-, H-, and X-PDMS for Cell-Based Applications: Comparison of Wettability, Roughness, and Cell-Derived Parameters.pdf

<p>Polydimethylsiloxane (PDMS) is a promising biomaterial for generating artificial extracellular matrix (ECM) like patterned topographies, yet its hydrophobic nature limits its applicability to cell-based approaches. Although plasma treatment can enhance the wettability of PDMS, the surface is known to recover its hydrophobicity within a few hours after exposure to air. To investigate the capability of a novel PDMS-type (X-PDMS) for in vitro based assessment of physiological cell properties, we designed and fabricated plane as well as nano- and micrometer-scaled pillar-patterned growth substrates using the elastomer types S-, H- and X-PDMS, which were fabricated from commercially available components. Most importantly, we compared X-PDMS based growth substrates which have not yet been investigated in this context with H- as well as well-known S-PDMS based substrates. Due to its applicability to fabricating nanometer-sized topographic features with high accuracy and pattern fidelity, this material may be of high relevance for specific biomedical applications. To assess their applicability to cell-based approaches, we characterized the generated surfaces using water contact angle (WCA) measurement and atomic force microscopy (AFM) as indicators of wettability and roughness, respectively. We further assessed cell number, cell area and cellular elongation as indirect measures of cellular viability and adhesion by image cytometry and phenotypic profiling, respectively, using Calcein and Hoechst 33342 stained human foreskin fibroblasts as a model system. We show for the first time that different PDMS types are differently sensitive to plasma treatment. We further demonstrate that surface hydrophobicity changes along with changing height of the pillar-structures. Our data indicate that plane and structured X-PDMS shows cytocompatibility and adhesive properties comparable to the previously described elastomer types S- and H-PDMS. We conclude that nanometer-sized structuring of X-PDMS may serve as a powerful method for altering surface properties toward production of biomedical devices for cell-based applications.</p

Amyloidogenic amyloid-β-peptide variants induce microbial agglutination and exert antimicrobial activity

Amyloid-β (Aβ) peptides are the main components of the plaques found in the brains of patients with Alzheimer’s disease. However, Aβ peptides are also detectable in secretory compartments and peripheral blood contains a complex mixture of more than 40 different modified and/or N- and C-terminally truncated Aβ peptides. Recently, anti-infective properties of Aβ peptides have been reported. Here, we investigated the interaction of Aβ peptides of different lengths with various bacterial strains and the yeast Candida albicans. The amyloidogenic peptides Aβ1-42, Aβ2-42, and Aβ3p-42 but not the non-amyloidogenic peptides Aβ1-40 and Aβ2-40 bound to microbial surfaces. As observed by immunocytochemistry, scanning electron microscopy and Gram staining, treatment of several bacterial strains and Candida albicans with Aβ peptide variants ending at position 42 (Aβx-42) caused the formation of large agglutinates. These aggregates were not detected after incubation with Aβx-40. Furthermore, Aβx-42 exerted an antimicrobial activity on all tested pathogens, killing up to 80% of microorganisms within 6 h. Aβ1-40 only had a moderate antimicrobial activity against C. albicans. Agglutination of Aβ1-42 was accelerated in the presence of microorganisms. These data demonstrate that the amyloidogenic Aβx-42 variants have antimicrobial activity and may therefore act as antimicrobial peptides in the immune system

Nano- and Micro-Patterned S-, H-, and X-PDMS for Cell-Based Applications: Comparison of Wettability, Roughness, and Cell-Derived Parameters

Polydimethylsiloxane (PDMS) is a promising biomaterial for generating artificial extracellular matrix (ECM) like patterned topographies, yet its hydrophobic nature limits its applicability to cell-based approaches. Although plasma treatment can enhance the wettability of PDMS, the surface is known to recover its hydrophobicity within a few hours after exposure to air. To investigate the capability of a novel PDMS-type (X-PDMS) for in vitro based assessment of physiological cell properties, we designed and fabricated plane as well as nano- and micrometer-scaled pillar-patterned growth substrates using the elastomer types S-, H- and X-PDMS, which were fabricated from commercially available components. Most importantly, we compared X-PDMS based growth substrates which have not yet been investigated in this context with H- as well as well-known S-PDMS based substrates. Due to its applicability to fabricating nanometer-sized topographic features with high accuracy and pattern fidelity, this material may be of high relevance for specific biomedical applications. To assess their applicability to cell-based approaches, we characterized the generated surfaces using water contact angle (WCA) measurement and atomic force microscopy (AFM) as indicators of wettability and roughness, respectively. We further assessed cell number, cell area and cellular elongation as indirect measures of cellular viability and adhesion by image cytometry and phenotypic profiling, respectively, using Calcein and Hoechst 33342 stained human foreskin fibroblasts as a model system. We show for the first time that different PDMS types are differently sensitive to plasma treatment. We further demonstrate that surface hydrophobicity changes along with changing height of the pillar-structures. Our data indicate that plane and structured X-PDMS shows cytocompatibility and adhesive properties comparable to the previously described elastomer types S- and H-PDMS. We conclude that nanometer-sized structuring of X-PDMS may serve as a powerful method for altering surface properties toward production of biomedical devices for cell-based applications