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The aim of this project was to analyse the cellular and biomechanical changes after collagen cross-linking (CXL) treatment on postmortem eye-banked human corneas using different UVA intensities and repeated treatments, and to explore the effects of standard collagen cross-linking on keratoconic corneal buttons, in-vitro. Preliminary studies were conducted to assess the feasibility of using eye-banked corneas to assess the effects of collagen cross-linking, and the possibility of applying scanning acoustic microscopy (SAM) to measure the speed of sound/elasticity of corneal tissue. Eye-banked human corneas were successfully cross-linked allowing the effects of CXL to be studied in-vitro and SAM was used effectively to determine the mechanical properties of corneal tissue at different depths. The results of two experiments comparing UVA intensity suggested that no statistically significant difference was found in the histological changes or in the induced stiffness after applying low and high intensity cross-linking on normal human corneas. However, the number of apoptotic cells was found to be significantly less but deeper into the posterior stroma in the high intensity cross-linked corneas. Collectively, these results confirmed the safety and efficacy of both techniques with the advantage of reducing the treatment time using the higher-intensity treatment. In another in-vitro study, keratoconic corneal tissue was used. Different histological and biomechanical outcomes were found between the cross-linked and control keratoconic tissue. The effects of cross-linking were found to penetrate deeper in the keratoconic tissue compared to in the normal corneal tissue found in previous studies. This could be due to the altered collagens and extracellular matrix of the keratoconic corneas, as they were taken from patients in advanced stages of the disease. This study confirmed the importance of having corneal thickness of at least 400μm after epithelial debriding to maintain the endothelial cell density and integrity. Finally, further cross-links were induced when collagen cross-linking treatment was repeated. However, repeating cross-linking three times a deeper cell death close to the endothelium was noticed which suggests that multiple treatments could be unsafe. Additionally, lower speed of sound than the cross-linking twice. This could be due to elimination of the induced cross-links by longer exposure to UVA irradiation. In conclusion, eye-banked human corneas were successfully used to evaluate the effects of cross-linking treatment and repeated treatment. Additionally, keratoconic corneal buttons were used to study the effects of collagen cross-linking in-vitro. This model of using eye-banked human corneas and keratoconic corneal tissue enabled us to study the effects of cross-linking treatment using different protocols and the effects of repeated treatment, and it could ultimately be used to compare the results with in-vivo studies.

Schlüsselfaktoren der bemerkenswerten mechanischen Eigenschaften von Knochen sind seine komplexe hierarchische Struktur und chemische Zusammensetzung. Ziel dieser Dissertation war die simultane Untersuchung von Materialkomposition und 3D Struktur in Relation zu lokalen elastischen Eigenschaften von Knochengewebe unter Verwendung von neuen hochauflösenden experimentellen Konzepten. Im ersten Teil wurde polarisierte Raman Spektroskopie (PRS) eingesetzt um gesunden humanen kortikalen Knochen zu analysieren. Es konnte gezeigt werden, dass sich PRS eignet, um sowohl die chemische Zusammensetzung als auch die 3D Organisation der Kollagenfasern in einer Messung aufzuklären. Dominante Faserorientierungen ganzer Gewebedomänen konnten identifiziert und mit der Koexistenz zweier Faserorganisationsmuster verknüpft werden. Durch Kombination derPRS Experimente mit ko-lokalisierten Synchrotron-Phasenkontrast-Nano-Tomografie- undUltraschallmikroskopie-Messungen wurde eine komplementäre Untersuchung von Faserarchitektur, chemischer Komposition und elastischen Eigenschaften einzelner Knochenlamellen ermöglicht. Die multimodale Analyse ergab, dass die charakteristischen lamellären Ondulationen der Elastizität in erster Linie durch sich lokal ändernde Faserorientierungen bedingt werden und nicht durch Variationen der Materialzusammensetzung, Abweichungen der Mineralkristallpartikeleigenschaften oder durch Fluktuationen der Massendichte. Im letzten Teil wurde mittels akustischer Mikroskopie der Einfluss der Mutation des Neurofibromin 1 Genes auf die pathologische Entwicklung von mechanischen Knocheneigenschaften untersucht. Anhand zweier Knockout-Mausmodelle wurde festgestellt, dass nur eine Mutation in frühen mesenchymalen Vorläuferzellen die Steifigkeit der langen Röhrenknochen signifikant beeinträchtigt. Perspektivisch eignet sich der vorgestellte multimodale Ansatz für nicht-destruktive Charakterisierung eines breiten Spektrums biologischer und synthetischer Faserverbundwerkstoffe. / Key factors determining the remarkable mechanical performance of bone are its material composition and complex hierarchically structure. The aim of this thesis was the concurrent investigation of the chemical composition and 3D structure of bone tissue in relation to the local elastic properties by introducing novel high resolution experimental approaches. In the first part, polarized Raman spectroscopy (PRS) was applied to analyze healthy human cortical bone. In particular, it was demonstrated that PRS can be employed to simultaneously investigate the chemical composition and the 3D organization of collagen fibrils in a single experiment. Predominant fibril orientations in entire tissue domains were identified and linked to the coexistence of two fibril organization patterns. To further extend the analysis, PRS experiments were combined with synchrotron X-ray phase contrast nano tomography and scanning acoustic microscopy measurements in a site-matched study design. This multimodal approach enabled complementary imaging of the fibrillar architecture, tissue composition and resulting elastic properties of single bone lamellae. In line with earlier studies, crosscorrelation analysis strongly suggested that the characteristic elastic undulations of bone lamellae are the result of the twisting fibrillar orientation, rather than compositional variations, modulations of the mineral particle maturity, or mass density fluctuations. Finally, acoustic microscopy was applied to analyze the impact of the neurofibromin 1 gene mutation on the pathologic development of the mechanical properties of bone. Analysis of two knock-out mouse models revealed that only Nf1 ablation in early mesenchymal progenitor cells significantly impairs the elastic stiffness of long bones. In future studies, the presented multimodal methodology can be translated for non-destructive and high resolution characterization of a broad range of biological and synthetic fiber composite materials.

Osteoporose und gestörte Heilungsverläufe von Knochenbrüchen verursachen immer noch beachtliche klinische Komplikationen. Ein vielversprechender Ansatz für die nichtinvasive und nichtionisierende Abschätzung des Frakturrisikos und der Bildgebung von Frakturheilung ist quantitativer Ultraschall (QUS). Dennoch liegt die derzeitige Akzeptanz für die Knochenqualitätsabschätzung noch weit hinter herkömmlichen röntgenbasierten Anwendungen. Es wurden akustische Mikroskopie und Synchrotronstrahlen-Mikrotomographie für die Anatomie und altersabhängige Erfassung von strukturellen und elastischen Variationen auf der mikroskopischen Ebene von humanen Femora verwendet. Die gewonnenen Daten dienten als Grundlage für die Erstellung mikromechanischer Modelle von Knochen für numerische Simulationen der Schallausbreitung im humanen Femurhals. Dabei wurde der Aufbau eines US-basierten Femur-Scanners in transversaler Transmission (TT) nachempfunden. Im letzten Abschnitt der Arbeit wurde QUS in TT in in vitro Experimenten am Rattenfrakturmodell auf eine Anwendung für die Bildgebung der Frakturheilung getestet. Die Studien konnten zeigen, dass ein Großteil der adaptiven Fähigkeiten von Knochen auf mikroskopischer Ebene auf eine Kombination von extrazellulärer Matrixelastizität und Gewebeporosität zurückzuführen ist. Die Simulationen des zweiten Teils konnten die Existenz von geführten Wellen im humanen Femurhals bestätigen. Die sensitive Abhängigkeit von US-parametern von frakturrelevanten Knocheneigenschaften zeigt das hohe Potential von QUS für die Frakturrisikoabschätzung. Der zweite Teil der Arbeit konnte erfolgreich die Möglichkeit von QUS in TT zur Diskriminierung von zeitigen Heilungsstadien demonstrieren. Zusammenfassend bestätigt die Studie das hohe Potential von QUS für die Frakturrisikoabschätzung und die Bildgebung der Frakturheilung. / Osteoporosis and impaired bone healing are of high relevance. A promising non-invasive, non-ionizing candidate for fracture risk prediction and monitoring fracture healing is quantitative ultrasound (QUS). However, the acceptance of QUS for bone quality assessment is still not comparable to X-ray based methods. Scanning acoustic microscopy (SAM) and Synchrotron Radiation micro-computer tomography (SRµCT) has been used to investigate anatomical and age dependent variations of micro elastic, structural and mineralization parameters at the tissue level of human femoral bone. Femoral neck models were created based on these data for numerical sound propagation simulations emulating a transverse transmission (TT) setup of an in vivo QUS prototype. In the last part of the project the TT approach has been tested in ex vivo experiments in a rat healing model. The power of QUS, to discriminate two early healing stages has been compared to µCT measurements at the same specimens. It was found that the major contributor to bone adaptation is related to a combination of extracellular matrix elasticity and tissue porosity. It is hypothesized that these parameters are likely to have a considerable impact on the reliability of in silico models. The simulations of the second part confirmed the existence of guided wave propagation in the cortical shell and a high dependency of US parameters on fracture relevant bone properties. The results demonstrate the high potential for bone fracture risk prediction at the femoral neck using QUS. Finally, it was successfully demonstrated that early healing stage discrimination of QUS in TT was superior compared to µCT. In summary these investigations not only show the importance for a precise estimation of micro mechanical properties for numerical modelling but also demonstrate the feasibility and high potential of QUS for bone quality assessment and monitoring of fracture healing.