Im Rahmen der VDB-Fortbildungsveranstaltung für Fachreferenten der Ingenieurwissenschaften, die am 8. und 9. Dezember 2005 in der SLUB Dresden stattfand, referierte Prof. Dr.-Ing. Hartmut Worch vom Institut für Werkstoffwissenschaften der TU Dresden (siehe auch SLUB-Kurier, 2006, Heft 1).

The development of materials with tailored functionalities and with continuously shrinking linear dimensions towards (and below) the nanoscale is not only going to revolutionize state of the art fabrication technologies, but also the computational methodologies used to model the materials properties. Specifically, atomistic methodologies are becoming increasingly relevant in the field of materials science as a fundamental tool in gaining understanding on as well as for pre-designing (in silico material design) the behavior of nanoscale materials in response to external stimuli. The major long-term goal of atomistic modelling is to obtain structure-function relationships at the nanoscale, i.e. to correlate a definite response of a given physical system with its specific atomic conformation and ultimately, with its chemical composition and electronic structure. This has clearly its pendant in the development of bottom-up fabrication technologies, which also require a detailed control and fine tuning of physical and chemical properties at sub-nanometer and nanometer length scales. The current work provides an overview of different applications of atomistic approaches to the study of nanoscale materials. We illustrate how the use of first-principle based electronic structure methodologies, quantum mechanical based molecular dynamics, and appropriate methods to model the electrical and thermal response of nanoscale materials, provides a solid starting point to shed light on the way such systems can be manipulated to control their electrical, mechanical, or thermal behavior. Thus, some typical topics addressed here include the interplay between mechanical and electronic degrees of freedom in carbon based nanoscale materials with potential relevance for designing nanoscale switches, thermoelectric properties at the single-molecule level and their control via specific chemical functionalization, and electrical and spin-dependent properties in biomaterials. We will further show how phenomenological models can be efficiently applied to get a first insight in the behavior of complex nanoscale systems, for which first principle electronic structure calculations become computationally expensive. This will become especially clear in the case of biomolecular systems and organic semiconductors.

The development of bio-resorbable implant materials is rapidly going on. Sterilization of those materials is inevitable to assure the hygienic requirements for critical medical devices according to the medical device directive (MDD, 93/42/EG). Biopolymer-containing biomaterials are often highly sensitive towards classical sterilization procedures like steam, ethylene oxide treatment or gamma irradiation. Supercritical CO2 (scCO2) treatment is a promising strategy for the terminal sterilization of sensitive biomaterials at low temperature. In combination with low amounts of additives scCO2 treatment effectively inactivates microorganisms including bacterial spores. We established a scCO2 sterilization procedure under addition of 0.25% water, 0.15% hydrogen peroxide and 0.5% acetic anhydride. The procedure was successfully tested for the inactivation of a wide panel of microorganisms including endospores of different bacterial species, vegetative cells of gram positive and negative bacteria including mycobacteria, fungi including yeast, and bacteriophages. For robust testing of the sterilization effect with regard to later application of implant materials sterilization all microorganisms were embedded in alginate/agarose cylinders that were used as Process Challenge Devices (PCD). These PCD served as surrogate models for bioresorbable 3D scaffolds. Furthermore, the impact of scCO2 sterilization on mechanical properties of polysaccharide-based hydrogels and collagen-based scaffolds was analyzed. The procedure was shown to be less compromising on mechanical and rheological properties compared to established low-temperature sterilization methods like gamma irradiation and ethylene oxide exposure as well as conventional steam sterilization. Cytocompatibility of alginate gels and scaffolds from mineralized collagen was compared after sterilization with ethylene oxide, gamma irradiation, steam sterilization and scCO2 treatment. Human mesenchymal stem cell viability and proliferation were not compromised by scCO2 treatment of these materials and scaffolds. We conclude that scCO2 sterilization under addition of water, hydrogen peroxide and acetic anhydride is a very effective, gentle, non-cytotoxic and thus a promising alternative sterilization method especially for biomaterials.

Ziel dieser Arbeit war es, neue Methoden in der Rasterkraftmikroskopie (SFM) zu entwickeln und an polymeren Materialien zu demonstrieren. Die Nanotomographie ist eine moderne dreidimensionale Volumenabbildungsmethode auf der Nanometerskala basierend auf der Rasterkraftmikroskopie. In dieser Arbeit wird ein Ansatz zur voll automatisierten Nanotomographie mit einer Auflösung von ~ 10 nm am Beispiel des menschlichen Knochens demonstriert. Die nasschemische Abtragung der Probe und das Entfernen der Ätzrückstände erfolgt dabei automatisch und in situ in einer Flüssigkeitszelle des Rasterkraftmikroskops. Lineare Verschiebungen der aufgenommenen Schichten werden mit Hilfe eines implementierten Kreuzkorrelations-Algorithmus korrigiert. Darüber hinaus wird durch Kombination der Nanotomographie mit dem bimodalen Messprinzip die laterale Auflösung dieser Methode am Beispiel von elastomerem Polypropylen deutlich gesteigert. Die mechanischen Oberflächeneigenschaften dieses Polymers wurden mit dynamischen Indentationsexperimenten mit dem Rasterkraftmikroskop bestimmt. Die Auftragung der dissipierten Energie zwischen Spitze und Oberfläche als Funktion der Schwingungsamplitude der Spitze ergibt für die amorphen und kristallinen Bereiche charakteristische Dissipationskurven. Diese lassen Rückschlüsse auf den Dissipationsmechanismus zwischen Messspitze und Oberfläche zu. Damit können zusätzliche Informationen über die mechanische Eigenschaften der Oberfläche des Polymers gewonnen werden. Darüber hinaus werden Erkenntnisse über die lateralen mechanischen Oberflächeneigenschaften von Polymeren durch den Einsatz des frequenzmodulierten Torsionsmodus der Rasterkraftmikroskopie erlangt.

Im Rahmen der VDB-Fortbildungsveranstaltung für Fachreferenten der Ingenieurwissenschaften, die am 8. und 9. Dezember 2005 in der SLUB Dresden stattfand, referierte Prof. Dr.-Ing. Hartmut Worch vom Institut für Werkstoffwissenschaften der TU Dresden (siehe auch SLUB-Kurier, 2006, Heft 1).

The development of materials with tailored functionalities and with continuously shrinking linear dimensions towards (and below) the nanoscale is not only going to revolutionize state of the art fabrication technologies, but also the computational methodologies used to model the materials properties. Specifically, atomistic methodologies are becoming increasingly relevant in the field of materials science as a fundamental tool in gaining understanding on as well as for pre-designing (in silico material design) the behavior of nanoscale materials in response to external stimuli. The major long-term goal of atomistic modelling is to obtain structure-function relationships at the nanoscale, i.e. to correlate a definite response of a given physical system with its specific atomic conformation and ultimately, with its chemical composition and electronic structure. This has clearly its pendant in the development of bottom-up fabrication technologies, which also require a detailed control and fine tuning of physical and chemical properties at sub-nanometer and nanometer length scales. The current work provides an overview of different applications of atomistic approaches to the study of nanoscale materials. We illustrate how the use of first-principle based electronic structure methodologies, quantum mechanical based molecular dynamics, and appropriate methods to model the electrical and thermal response of nanoscale materials, provides a solid starting point to shed light on the way such systems can be manipulated to control their electrical, mechanical, or thermal behavior. Thus, some typical topics addressed here include the interplay between mechanical and electronic degrees of freedom in carbon based nanoscale materials with potential relevance for designing nanoscale switches, thermoelectric properties at the single-molecule level and their control via specific chemical functionalization, and electrical and spin-dependent properties in biomaterials. We will further show how phenomenological models can be efficiently applied to get a first insight in the behavior of complex nanoscale systems, for which first principle electronic structure calculations become computationally expensive. This will become especially clear in the case of biomolecular systems and organic semiconductors.

Die Analyse von Implantat-Gewebe-Wechselwirkungen basiert derzeit hauptsächlich auf histologischen Techniken. Der invasive Charakter der histologischen Präparation lässt allerdings keine Untersuchung am lebenden Tier zu. Dadurch ist es nicht möglich, den Prozess der Implantateinheilung wiederholt an einem Tier zu beobachten. Die Folgen sind eine hohe Anzahl aufzuwendender Versuchstiere und eine Vergrößerung der Messunsicherheit infolge der gestiegenen biologischen Variabilität. Nicht-invasive, bildgebende Verfahren spielen daher eine zunehmende Rolle für die Entwicklung neuer Biomaterialien. Während die Computertomographie (CT) häufig zur Untersuchung der knöchernen Implantateinheilung verwendet wird, hat sich die Nutzung der Magnetresonanztomographie (MRT) für diese Fragestellungen bisher nicht etabliert. Bei der Magnetresonanztomographie handelt es sich, analog zur Computertomographie, um ein bildgebendes Verfahren zur nicht-invasiven Erzeugung digitaler Schnittbilder. Im Gegensatz zur CT, die das Hartgewebe abbildet, wird bei der MRT das Weichgewebe detektiert, wobei keine ionisierende Strahlung verwendet wird. Der große Vorteil der MRT gegenüber anderen bildgebenden Methoden besteht darin, dass es möglich ist, das Weichgewebe auf den Schnittbildern anhand verschiedener Kontraste darzustellen. Zusätzlich können MR-spezifische Parameter quantifiziert werden, die einen direkten Rückschluss auf die Struktur zulassen. Mit diesen Kennzahlen ist es möglich, Veränderungen im Weichgewebe analysieren. Das Ziel der Arbeit war es deshalb, die Eignung und mögliche Anwendungen der Magnetresonanzto-mographie (MRT) zur Analyse der Implantat-Gewebe-Wechselwirkungen zu erörtern. Für die Untersu-chungen wurde ein NMR-Spektrometer inklusive Imaging-Zubehör verwendet. Die Dissertationsarbeit beinhaltete sowohl die Untersuchung verschiedener Materialsysteme hinsichtlich ihrer Eignung für die MRT und deren Biokompatibilität, als auch die Analyse der knöchernen Einheilung ausgewählter Biomaterialien. Diese umfasste Aussagen zur Darstellbarkeit und Abgrenzbarkeit von Strukturen und beinhaltete auch quantitativ gewonnene Messparameter. Die Ergebnisse wurden stets im Vergleich mit der Histologie diskutiert. In der Arbeit konnte dargestellt werden, dass die Überprüfung der Eignung des zu untersuchenden Materials für die MRT vor der Analytik erfolgen muss. Es wurde demonstriert, dass Metalle erheblich mit dem MR-System wechselwirken können, was in der Konsequenz zu drastischen Störungen der Bildqualität führt. Diese Effekte waren stark von den ausgewählten Messparametern abhängig. Als ein MRT-geeignetes Verbundmaterial wurde Titan-beschichtetes Polyetheretherketon (PEEK/Ti) vorgeschlagen. Die Beschichtung mit Titan führte zu einer signifikant verbesserten Biokompatibilität des Kunststoffes. Die erfolgreiche Analyse der knöchernen Einheilung mit der Magnetresonanztomographie wurde im Rahmen von zwei tierexperimentellen Studien an verschiedenen Biomaterialien gezeigt (die Analyse erfolg-te ex vivo). Die Untersuchung der knöchernen Integration eines Zahnimplantates aus PEEK/Ti hatte das Ziel, die Darstellbarkeit des Implantates und knöcherner Strukturen mit der Magnetresonanztomographie zu evaluieren. Außerdem wurde ebenfalls gezeigt, dass es anhand der MRT-Schnittbilder möglich ist, quantitative Messgrößen zur Beschreibung des Einheilprozesses zu gewinnen. Aufgrund der geringen Versuchstierzahl wurde jedoch eine breite Streuung der Messdaten festgestellt. Allerdings besitzt die Studie durch die Untersuchung eines Zahnimplantates aus Polyetheretherketon/Titan mit der MRT nicht nur Neuheitswert in der Biomaterialforschung, sondern schlägt gleichzeitig eine Brücke zur klinischen, dentalen Implantologie. Die Bewertung der Darstellbarkeit knöcherner Strukturen und der verwendeten (teils tissue-engineerten) Knochenersatzmaterialien mit MRT und Histologie und des klinischen Erfolges derselben bildeten einen Schwerpunkt der zweiten tierexperimentellen Studie (die Analyse erfolgte ex vivo). Es war möglich, mit beiden bildgebenden Verfahren zu zeigen, dass sich die verwendeten Knochenersatzmaterialien nicht für die vorgesehene Anwendung eigneten. Die Beurteilung der Übereinstimmung der quantitativ gewonnenen Parameter beider Analysenmethoden bildete den Abschluss der Arbeit. Es wurde festgestellt, dass zwischen den Messdaten stets ein syste-matischer Unterschied bestand. Nachweislich war dieser aber weniger das Resultat der ungleichen lateralen Auflösungen oder der unterschiedlichen Darstellbarkeit von Gewebestrukturen der beiden Verfahren, sondern konnte auf den Einfluss der Analyse verschiedener Schichtebenen und individueller Unterschiede bei der digitalen Quantifizierung der auswertenden Personen zurückgeführt werden. / Currently, histological techniques are used to analyse implant-tissue-interactions. However, these methods are destructive and do not allow for the investigation of living animals. Therefore, it is not possible to study the integration of biomaterials repeatedly with one animal, resulting in a large number of animals and an increase of biological variability. Non-invasive imaging techniques have gained interest in the field of biomaterials. Whereas Computed Tomography (CT) was often used to evaluate the osseous integration, the assessment using Magnetic Resonance Imaging (MRI) has not been established, yet. MRI is a non-invasive medical imaging method that detects soft tissue. In contrast to CT the method does not require individuals to be exposed to radiation. The most important benefit of MRI is the possibility to acquire different soft tissue contrasts in situ because the various tissues have different signal intensities on MR images that can be altered by using different experimental parameters. Furthermore, it is possible to gain MR-specific properties that allow conclusions to the tissue structure. Thus, the objective of the doctoral thesis has been to investigate the suitability of MRI for the use in biometerial research and to show potential areas of application. The examinations were performed using a laboratory NMR-spectrometer inclusive imaging accessory. The thesis included an evaluation of the MR compatibility of different materials and their biocompati-bility and an analysis of the ingrowth of chosen biomaterials into bone. For that, the detection and identification of tissue structures and biomaterials was investigated with both, MRI and histology. Additionally, quantitative parameters were acquired and their comparability was assessed. It was clearly demonstrated, that metals interacted with the MR system and provoked large image distortions. These effects were strongly dependent on experimental parameters chosen. Polyetheretherketone with titanium coating (PEEK/Ti) was investigated and has been found to be MR safe. Above all, it was demonstrated that the biocompatibility of the polymer was significantly enhanced by coating with titanium. Within two animal studies the successful analysis of the osseous healing of different biomaterials with MRI was presented. To demonstrate the visibility of bony structures and biomaterials a dental implant made of PEEK/Ti was analysed. The ability to measure quantitative data in analogy to histomorphometry was shown, ditto. A large variation of the values was detected due to the limited number of animals used for the pilot study. Evaluating the displayability of bone and (to some extent tissue engineered) bone substitutes and assessing the clinical success of these materials was one main focus of the second animal study. Both, MRI and histological analysis could undeniably illustrate that all of the bone substitutes were not suitable for the chosen application. The thesis was completed with the determination of the agreement of quantitative values from both analysing methods. It was concluded that all values gained from the animal study were significantly different. It was proven that the chosen slice position and the image interpretation with two evaluators had a larger share to disagreement than the different lateral resolution of MRI and histological images or the diverging displayability of bone and bone substitutes. By investigating a MR suitable dental PEEK implant the doctoral thesis fulfils the criteria of novelty in biomaterial research. Moreover, it forges links between preclinical research and dental implantology.

Textile structures made of biocompatible, osteoconductive and resorbable chitosan-filaments provide excellent preconditions as scaffolds for Bone Tissue Engineering applications. The novel Net Shape Nonwoven (NSN) technique that enables short fibers to be processed into three-dimensional net-shaped nonwoven structures with adjustable pore size distributions is described. NSN scaffolds made of pure chitosan fibers were fabricated. NSN hybrid scaffolds for improved initial cell adhesion were realized by combining the NSN technique with electrospinning and dip-coating with collagen, respectively. Scanning electron microscopy and liquid displacement porosimetry revealed an interconnecting open porous scaffold structure. The novel chitosan-hybrid scaffolds provide proper conditions for adhesion, proliferation and differentiation of the seeded human bone marrow stromal cells, proving that they are suitable for usage in hard-tissue regeneration.

Impaired wound healing and the resulting chronic wounds may cause significant morbidity and mortality. In these pathogenic wound environments, the ratio of inflammatory and anti-inflammatory cytokines is highly biased to the pro-inflammatory side. While the inflammatory process is an essential step in healthy wound healing, chronic wounds remain in a constant self-sustaining state of inflammation. Thus, decreased cell proliferation, reduced matrix deposition and delayed wound closure are the results. Although various cytokine-based therapies have shown promising results on skin regeneration in preliminary studies, their overall clinical use has been considerably limited by the short half-life time of the signaling molecules due to rapid dilution and degradation in the protease-rich chronic wound environment. In this work, we explored the ability of starPoly(ethylene glycol)-GAG hydrogels to modulate the hallmarks of chronic wound development, such as the prolonged inflammation, increased cell influx and delayed proliferative phase. Therefore, different strategies were developed to shape the cytokine levels in the wound towards a more pro-regenerative direction, finally promoting the natural repair process in chronic skin wounds. By biomimetically utilizing the interactions between cytokines and the tissue ECM in a GAG-based biohybrid hydrogel, we could engineer the concentrations of various signaling factors involved in the regulation of the repair process. More in detail, we utilized customized functionalized starPEG-GAG hydrogels to (1) reduce the extensive levels of inflammatory chemokines by scavenging them via GAG component of the hydrogel and thus diminish immune cell influx in a mouse wound model; (2) locally deliver the immunomodulatory IL-4 and IL-10 to shift the signaling balance into the pro-regenerative direction and thus resolve inflammation and (3) administer pre-conjugated TGF-β to enhance myofibroblast differentiation and extracellular matrix deposition. We believe that the presented hydrogel platform may become a promising tool in the management of cytokines in regenerative applications, which can be translated towards the clinical use for the treatment of chronic wounds and other diseases characterized by uncontrolled inflammation.:1 introduction 1.1 Motivation 1.2 Current state of biomaterial-based concepts in dermal wound healing 1.3 Objective 2 fundamentals 2.1 The physiological process of wound healing 2.1.1 The role of macrophages in wound healing 2.1.2 The role of fibroblasts in wound healing 2.1.3 The role of cytokines and their interaction with the ECM 2.2 The pathophysiology of chronic wounds 2.3 Strategies for treatment of chronic wounds 2.4 Biomaterials in medicine 2.4.1 Polymers in medicine 2.4.2 Mechanical properties 2.4.3 Cellular adhesion 2.4.4 Interaction with cytokines 2.4.5 Scaffold degradability 2.4.6 StarPEG-GAG hydrogels as potential material in wound healing 3 materials & methods 3.1 Preparation of hydrogels 3.1.1 Functionalization of glass surfaces 3.1.2 Hydrogel formation with EDC - NHS chemistry 3.1.3 Hydrogel formation with thiol - maleimide chemistry 3.1.4 Rheometric measurement of hydrogel discs 3.1.5 Characterization of cytokine uptake and release 3.2 Culture of human & murine cells 3.2.1 Isolation and differentiation of murine dermal fibroblasts 3.2.2 Isolation & differentiation of murine macrophages 3.2.3 Culture of human & murine cell lines 3.3 In vitro methods 3.3.1 Enzyme-linked immunosorbent assay (ELISA) 3.3.2 Bead-based multiplex immunoassay 3.3.3 Live/Dead Staining 3.3.4 Crystal violet staining 3.3.5 Cell proliferation assay 3.3.6 RNA extraction & analysis 3.3.7 cDNA synthesis 3.3.8 Quantitative real time rt-PCR 3.4 Statistical analysis 3.5 Software use 4 scavenging inflammatory chemokines to control immune cell influx in the wound 4.1 Results 4.1.1 Engineering heparin-based hydrogels to scavenge chemokines 4.1.2 Heparin-based hydrogels reduce migration of immune cells 4.1.3 Heparin-based hydrogels decrease wound immune cell influx and inflammatory signaling 4.2 Discussion 5 promotion of regenerative macrophage polarizationin inflammatory environments 5.1 Results 5.1.1 Reversible complexation of IL-4 & IL-10 to starPEG-heparin gels 5.1.2 Stabilizing effects of starPEG-heparin gels on IL-4 5.1.3 IL-4 & IL-10-laden starPEG-heparin hydrogels modulate macrophage polarization 5.1.4 IL-4-laden starPEG-heparin induce collagen deposition in dermal fibroblasts 5.2 Discussion 6 modulation of human dermal fibroblast proliferation and differentiation 6.1 Results 6.1.1 Reversible complexation of TGF-b to starPEG heparin gels 6.1.2 Cell attachment, spreading and proliferation 6.1.3 Matrix deposition by fibroblasts grown on starPEG-heparin hydrogels 6.1.4 Degradation of starPEG-heparin hydrogels 6.1.5 TGF-b-laden starPEG-heparin that efficiently induces myofibroblast differentiation 6.2 Discussion 7 general discussion 7.1 Summary and conclusion 7.2 Future perspective Appendix 8 supplementary materials & methods 9 declaration of authorship 10 publications and conference contributions bibliography list of figures list of tables nomenclature selbstständigkeitserklärung

Renal tissue engineering and regenerative medicine represent a significant clinical objective because of the very limited prospect of cure after classical kidney treatment. Thus, approaches to isolate, manipulate and reintegrate structures or stimulating the selfregenerative potential of renal tissue are of special interest. Such new strategies go back to knowledge and further outcome of developmental biological research. An understanding of extracellular matrix (ECM) structure and composition forms thereby a particularly significant aspect in comprehending the complex dynamics of tissue regeneration. Consequently the reconstruction of these structures offers beneficial options for advanced cell and tissue culture technology and tissue engineering. In an effort to investigate the influence of natural extracellular structures and components on embryonic stem cell and renal embryonic tissue, methodologies which allow the easy application of exogenous signals on tissue in vitro on the one hand and the straight forward evaluation of decellularization methods on the other hand, were developed. Both systems can be used to investigate and modulate behaviour of biological systems and represent novel interesting tools for tissue engineering. The novel technique for culturing tissue in vitro allows the growing of embryonic renal explants in very low volumes of medium and optimized observability, which makes it predestined for testing additives. In particular, this novel culture set up provides an ideal opportunity to investigate renal development and structure formation. Further studies indicated that the set is universally applicable on all kinds of (embryonic) tissue. Following hereon, more than 20 different ECM components were tested for their impact on kidney development under 116 different culture conditions, including different concentrations and being either bound to the substrate or dissolved in the culture medium. This allowed to study the role of ECM constituents on renal structure formation. In ongoing projects, kidney rudiments are exposed to aligned matrix fibrils and hydrogels with first promising results. The insights gained thereof gave rise to a basis for the rational application of exogenous signals in regenerative kidney therapies. Additionally new strategies for decellularization of whole murine adult kidneys were explored by applying different chemical agents. The obtained whole matrices were analysed for their degree of decellularization and their residual content and composition. In a new straight forward approach, a dependency of ECM decellularization efficiency to the different agents used for decellularization could be shown. Moreover the capability of the ECM isolated from whole adult kidneys to direct stem cell differentiation towards renal cell linage phenotypes was proved. The data obtained within this thesis give an innovative impetus to the design of biomaterial scaffolds with defined and distinct properties, offering exciting options for tissue engineering and regenerative kidney therapies by exogenous cues.