@phdthesis{Jihyoung2024, author = {Jihyoung, Choi}, title = {Development of an Add-On Electrode for Non-Invasive Monitoring in Bioreactor Cultures and Medical Devices}, doi = {10.25972/OPUS-35823}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358232}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Electrochemical impedance spectroscopy (EIS) is a valuable technique analyzing electrochemical behavior of biological systems such as electrical characterization of cells and biomolecules, drug screening, and biomaterials in biomedical field. In EIS, an alternating current (AC) power signal is applied to the biological system, and the impedance of the system is measured over a range of frequencies. In vitro culture models of endothelial or epithelial barrier tissue can be achieved by culturing barrier tissue on scaffolds made with synthetic or biological materials that provide separate compartments (apical and basal sides), allowing for further studies on drug transport. EIS is a great candidate for non-invasive and real-time monitoring of the electrical properties that correlate with barrier integrity during the tissue modeling. Although commercially available transendothelial/transepithelial electrical resistance (TEER) measurement devices are widely used, their use is particularly common in static transwell culture. EIS is considered more suitable than TEER measurement devices in bioreactor cultures that involve dynamic fluid flow to obtain accurate and reliable measurements. Furthermore, while TEER measurement devices can only assess resistance at a single frequency, EIS measurements can capture both resistance and capacitance properties of cells, providing additional information about the cellular barrier's characteristics across various frequencies. Incorporating EIS into a bioreactor system requires the careful optimization of electrode integration within the bioreactor setup and measurement parameters to ensure accurate EIS measurements. Since bioreactors vary in size and design depending on the purpose of the study, most studies have reported using an electrode system specifically designed for a particular bioreactor. The aim of this work was to produce multi-applicable electrodes and established methods for automated non-invasive and real-time monitoring using the EIS technique in bioreactor cultures. Key to the electrode material, titanium nitride (TiN) coating was fabricated on different substrates (materials and shape) using physical vapor deposition (PVD) and housed in a polydimethylsiloxane (PDMS) structure to allow the electrodes to function as independent units. Various electrode designs were evaluated for double-layer capacitance and morphology using EIS and scanning electron microscopy (SEM), respectively. The TiN-coated tube electrode was identified as the optimal choice. Furthermore, EIS measurements were performed to examine the impact of influential parameters related to culture conditions on the TiN-coated electrode system. In order to demonstrate the versatility of the electrodes, these electrodes were then integrated into in different types of perfusion bioreactors for monitoring barrier cells. Blood-brain barrier (BBB) cells were cultured in the newly developed dynamic flow bioreactor, while human umblical vascular endothelial cells (HUVECs) and Caco-2 cells were cultured in the miniature hollow fiber bioreactor (HFBR). As a result, the TiN-coated tube electrode system enabled investigation of BBB barrier integrity in long-term bioreactor culture. While EIS measurement could not detect HUVECs electrical properties in miniature HFBR culture, there was the possibility of measuring the barrier integrity of Caco-2 cells, indicating potential usefulness for evaluating their barrier function. Following the bioreactor cultures, the application of the TiN-coated tube electrode was expanded to hemofiltration, based on the hypothesis that the EIS system may be used to monitor clotting or clogging phenomena in hemofiltration. The findings suggest that the EIS monitoring system can track changes in ion concentration of blood before and after hemofiltration in real-time, which may serve as an indicator of clogging of filter membranes. Overall, our research demonstrates the potential of TiN-coated tube electrodes for sensitive and versatile non-invasive monitoring in bioreactor cultures and medical devices.}, subject = {Monitoring}, language = {en} } @phdthesis{Schlesinger2024, author = {Schlesinger, Tobias}, title = {Autolog zellbesiedelte Matrix zum Verschluss gastraler Inzisionen: Eine Machbarkeitsstudie im Schweinemodell}, doi = {10.25972/OPUS-30583}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-305832}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Einleitung: Strukturelle Defekte der gastrointestinalen Hohlorgane stellen ein allgegen-w{\"a}rtiges Problem im klinischen Alltag dar. Sie entstehen meist auf dem Boden einer ent-z{\"u}ndlichen oder tumor{\"o}sen Grunderkrankung und k{\"o}nnen außerdem traumatisch sowie durch medizinische Eingriffe hervorgerufen werden. In der Folge kommt es zur Kontami-nation des umliegenden Gewebes mit Magen- bzw. Darminhalt, wodurch delet{\"a}re Folgen wie eine systemische Infektion, also eine Sepsis mit Multiorganversagen drohen k{\"o}nnen. Vor diesem Hintergrund sind gastrointestinale Defekte immer als potenziell lebensbedroh-lich f{\"u}r den Patienten zu betrachten. Die ad{\"a}quate und kausale Behandlung erfolgt je nach {\"A}tiologie und Zustand des Patienten durch eine Operation oder eine endoskopische Inter-vention. Hierzu stehen zahlreiche etablierte, operative und interventionelle Therapieme-thoden zur Verf{\"u}gung. In manchen F{\"a}llen stoßen die etablierten Techniken jedoch an ihre Grenzen. Bei Patienten mit schwerwiegenden Komorbidit{\"a}ten oder im Rahmen neuer me-dizinischer Verfahren sind Innovationen gefragt. Die Grundidee der vorliegenden Arbeit ist die Entwicklung einer biotechnologischen Therapieoption zur Versorgung gastrointesti-naler Hohlorganperforationen. Methoden: Zur Durchf{\"u}hrung einer Machbarkeitsstudie wurden zehn G{\"o}ttinger Mi-nischweine in zwei Gruppen mit jeweils 5 Tieren aufgeteilt. Den Tieren der Experimental-gruppe wurden Hautbiopsien entnommen und daraus Fibroblasten isoliert, welche vo-r{\"u}bergehend konserviert wurden. Unter Verwendung von azellularisiertem Schweinedarm erfolgte die Herstellung von Implantaten nach den Prinzipien des Tissue Engineerings. Die Tiere beider Gruppen wurden einer Minilaparotomie und einer ca. 3cm-Inzision der Ma-genvorderwand unterzogen. Die anschließende Versorgung wurde in der Experimental-gruppe durch Implantation der neuartigen Konstrukte erzielt. In der Kontrollgruppe wur-de im Sinne des Goldstandards eine konventionelle Naht durchgef{\"u}hrt. Anschließend wurden die Tiere f{\"u}r vier Wochen beobachtet. Eine bzw. zwei Wochen nach dem pri-m{\"a}ren Eingriff wurde bei allen Tieren beider Gruppen eine Laparoskopie bzw. Gastrosko-pie durchgef{\"u}hrt. Am Ende der klinischen Observationsphase wurden die Versuchstiere get{\"o}tet und die entsprechenden Magenareale zur histologischen Untersuchung explantiert. Ergebnisse: Die Herstellung der Implantate konnte auf der Basis standardisierter zellbio-logischer Methoden problemlos etabliert werden. Alle Tiere beider Gruppen {\"u}berlebten den Prim{\"a}reingriff sowie das vierw{\"o}chige Nachbeobachtungsintervall und zeigten dabei keine klinischen Zeichen m{\"o}glicher Komplikationen. Die durchgef{\"u}hrten Laparoskopien und Gastroskopien ergaben bei keinem der Tiere Hinweise auf Leckagen oder lokale Infek-tionsprozesse. Die histologische Aufarbeitung zeigte im Bereich des urspr{\"u}nglichen De-fekts eine bindegewebige {\"U}berbr{\"u}ckung sowie ein beginnendes Remodeling der Magen-schleimhaut in beiden Gruppen. Schlussfolgerungen: Durch die Verkn{\"u}pfung von Einzelprozessen der Zellkultur und dem Großtier-OP konnte ein neues Verfahren zum Verschluss gastrointestinaler Defekt erfolgreich demonstriert und etabliert werden. Das Projekt konnte reibungslos durchge-f{\"u}hrt werden und lieferte Ergebnisse, die dem Goldstandard nicht unterlegen waren. Auf-grund der kleinen Fallzahl und weiterer methodischer Limitationen sind jedoch nur einge-schr{\"a}nkt Schlussfolgerungen m{\"o}glich, weshalb die Durchf{\"u}hrung gr{\"o}ßerer und gut geplan-ter Studien notwendig ist. Die Erkenntnisse dieser Pilotstudie liefern eine solide Basis f{\"u}r die Planung weiterf{\"u}hrender Untersuchungen.}, subject = {Magenkrankheit}, language = {de} } @phdthesis{Daeullary2024, author = {D{\"a}ullary, Thomas}, title = {Establishment of an infection model of the human intestinal epithelium to study host and pathogen determinants during the \(Salmonella\) Typhimurium infection process}, doi = {10.25972/OPUS-31154}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-311548}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {According to the WHO, foodborne derived enteric infections are a global disease burden and often manifest in diseases that can potentially reach life threatening levels, especially in developing countries. These diseases are caused by a variety of enteric pathogens and affect the gastrointestinal tract, from the gastric to the intestinal to the rectal tissue. Although the complex mucosal structure of these organs is usually well prepared to defend the body against harmful agents, specialised pathogens such as Salmonella enterica can overcome the intestinal defence mechanism. After ingestion, Salmonella are capable of colonising the gut and establishing their proliferative niche, thereby leading to inflammatory processes and tissue damage of the host epithelium. In order to understand these processes, the scientific community in the last decades mostly used cell line based in vitro approaches or in vivo animal studies. Although these approaches provide fundamental insights into the interactions between bacteria and host cells, they have limited applicability to human pathology. Therefore, tissue engineered primary based approaches are important for modern infection research. They exhibit the human complexity better than traditional cell lines and can mimic human-obligate processes in contrast to animal studies. Therefore, in this study a tissue engineered human primary model of the small intestinal epithelium was established for the application of enteric infection research with the exemplary pathogen Salmonella Typhimurium. To this purpose, adult stem cell derived intestinal organoids were used as a primary human cell source to generate monolayers on biological or synthetic scaffolds in a Transwell®-like setting. These tissue models of the intestinal epithelium were examined for their comparability to the native tissue in terms of morphology, morphometry and barrier function. Further, the gene expression profiles of organotypical mucins, tight junction-associated proteins and claudins were investigated. Overall, the biological scaffold-based tissue models showed higher similarity to the native tissue - among others in morphometry and polarisation. Therefore, these models were further characterised on cellular and structural level. Ultrastructural analysis demonstrated the establishment of characteristic microvilli and tight-junction connections between individual epithelial cells. Furthermore, the expression pattern of typical intestinal epithelial protein was addressed and showed in vivo-like localisation. Interested in the cell type composition, single cell transcriptomic profiling revealed distinct cell types including proliferative cells and stem cells, progenitors, cellular entities of the absorptive lineage, Enterocytes and Microfold-like cells. Cells of the secretory lineage were also annotated, but without distinct canonical gene expression patterns. With the organotypical polarisation, protein expression, structural features and the heterogeneous cell composition including the rare Microfold-like cells, the biological scaffold-based tissue model of the intestinal epithelium demonstrates key requisites needed for infection studies with Salmonella. In a second part of this study, a suitable infection protocol of the epithelial tissue model with Salmonella Typhimurium was established, followed by the examination of key features of the infection process. Salmonella adhered to the epithelial microvilli and induced typical membrane ruffling during invasion; interestingly the individual steps of invasion could be observed. After invasion, time course analysis showed that Salmonella resided and proliferated intracellularly, while simultaneously migrating from the apical to the basolateral side of the infected cell. Furthermore, the bacterial morphology changed to a filamentous phenotype; especially when the models have been analysed at late time points after infection. The epithelial cells on the other side released the cytokines Interleukin 8 and Tumour Necrosis Factor α upon bacterial infection in a time-dependent manner. Taken together, Salmonella infection of the intestinal epithelial tissue model recapitulates important steps of the infection process as described in the literature, and hence demonstrates a valid in vitro platform for the investigation of the Salmonella infection process in the human context. During the infection process, intracellular Salmonella populations varied in their bacterial number, which could be attributed to increased intracellular proliferation and demonstrated thereby a heterogeneous behaviour of Salmonella in individual cells. Furthermore, by the application of single cell transcriptomic profiling, the upregulation of Olfactomedin-4 (OLFM4) gene expression was detected; OLFM4 is a protein involved in various functions including cell immunity as well as proliferating signalling pathways and is often used as intestinal stem cell marker. This OLFM4 upregulation was time-dependent, restricted to Salmonella infected cells and seemed to increase with bacterial mass. Investigating the OLFM4 regulatory mechanism, nuclear factor κB induced upregulation could be excluded, whereas inhibition of the Notch signalling led to a decrease of OLFM4 gene and protein expression. Furthermore, Notch inhibition resulted in decreased filamentous Salmonella formation. Taken together, by the use of the introduced primary epithelial tissue model, a heterogeneous intracellular bacterial behaviour was observed and a so far overlooked host cell response - the expression of OLFM4 by individual infected cells - could be identified; although Salmonella Typhimurium is one of the best-studied enteric pathogenic bacteria. This proves the applicability of the introduced tissue model in enteric infection research as well as the importance of new approaches in order to decipher host-pathogen interactions with higher relevance to the host.}, subject = {Salmonella typhimurium}, language = {en} } @phdthesis{Andelovic2024, author = {Andelovic, Kristina}, title = {Characterization of arterial hemodynamics using mouse models of atherosclerosis and tissue-engineered artery models}, doi = {10.25972/OPUS-30360}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-303601}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Within this thesis, three main approaches for the assessment and investigation of altered hemodynamics like wall shear stress, oscillatory shear index and the arterial pulse wave velocity in atherosclerosis development and progression were conducted: 1. The establishment of a fast method for the simultaneous assessment of 3D WSS and PWV in the complete murine aortic arch via high-resolution 4D-flow MRI 2. The utilization of serial in vivo measurements in atherosclerotic mouse models using high-resolution 4D-flow MRI, which were divided into studies describing altered hemodynamics in late and early atherosclerosis 3. The development of tissue-engineered artery models for the controllable application and variation of hemodynamic and biologic parameters, divided in native artery models and biofabricated artery models, aiming for the investigation of the relationship between atherogenesis and hemodynamics Chapter 2 describes the establishment of a method for the simultaneous measurement of 3D WSS and PWV in the murine aortic arch at, using ultra high-field MRI at 17.6T [16], based on the previously published method for fast, self-navigated wall shear stress measurements in the murine aortic arch using radial 4D-phase contrast MRI at 17.6 T [4]. This work is based on the collective work of Dr. Patrick Winter, who developed the method and the author of this thesis, Kristina Andelovic, who performed the experiments and statistical analyses. As the method described in this chapter is basis for the following in vivo studies and undividable into the sub-parts of the contributors without losing important information, this chapter was not split into the single parts to provide fundamental information about the measurement and analysis methods and therefore better understandability for the following studies. The main challenge in this chapter was to overcome the issue of the need for a high spatial resolution to determine the velocity gradients at the vascular wall for the WSS quantification and a high temporal resolution for the assessment of the PWV without prolonging the acquisition time due to the need for two separate measurements. Moreover, for a full coverage of the hemodynamics in the murine aortic arch, a 3D measurement is needed, which was achieved by utilization of retrospective navigation and radial trajectories, enabling a highly flexible reconstruction framework to either reconstruct images at lower spatial resolution and higher frame rates for the acquisition of the PWV or higher spatial resolution and lower frame rates for the acquisition of the 3D WSS in a reasonable measurement time of only 35 minutes. This enabled the in vivo assessment of all relevant hemodynamic parameters related to atherosclerosis development and progression in one experimental session. This method was validated in healthy wild type and atherosclerotic Apoe-/- mice, indicating no differences in robustness between pathological and healthy mice. The heterogeneous distribution of plaque development and arterial stiffening in atherosclerosis [10, 12], however, points out the importance of local PWV measurements. Therefore, future studies should focus on the 3D acquisition of the local PWV in the murine aortic arch based on the presented method, in order to enable spatially resolved correlations of local arterial stiffness with other hemodynamic parameters and plaque composition. In Chapter 3, the previously established methods were used for the investigation of changing aortic hemodynamics during ageing and atherosclerosis in healthy wild type and atherosclerotic Apoe-/- mice using the previously established methods [4, 16] based on high-resolution 4D-flow MRI. In this work, serial measurements of healthy and atherosclerotic mice were conducted to track all changes in hemodynamics in the complete aortic arch over time. Moreover, spatially resolved 2D projection maps of WSS and OSI of the complete aortic arch were generated. This important feature allowed for the pixel-wise statistical analysis of inter- and intragroup hemodynamic changes over time and most importantly - at a glance. The study revealed converse differences of local hemodynamic profiles in healthy WT and atherosclerotic Apoe-/- mice, with decreasing longWSS and increasing OSI, while showing constant PWV in healthy mice and increasing longWSS and decreasing OSI, while showing increased PWV in diseased mice. Moreover, spatially resolved correlations between WSS, PWV, plaque and vessel wall characteristics were enabled, giving detailed insights into coherences between hemodynamics and plaque composition. Here, the circWSS was identified as a potential marker of plaque size and composition in advanced atherosclerosis. Moreover, correlations with PWV values identified the maximum radStrain could serve as a potential marker for vascular elasticity. This study demonstrated the feasibility and utility of high-resolution 4D flow MRI to spatially resolve, visualize and analyze statistical differences in all relevant hemodynamic parameters over time and between healthy and diseased mice, which could significantly improve our understanding of plaque progression towards vulnerability. In future studies the relation of vascular elasticity and radial strain should be further investigated and validated with local PWV measurements and CFD. Moreover, the 2D histological datasets were not reflecting the 3D properties and regional characteristics of the atherosclerotic plaques. Therefore, future studies will include 3D plaque volume and composition analysis like morphological measurements with MRI or light-sheet microscopy to further improve the analysis of the relationship between hemodynamics and atherosclerosis. Chapter 4 aimed at the description and investigation of hemodynamics in early stages of atherosclerosis. Moreover, this study included measurements of hemodynamics at baseline levels in healthy WT and atherosclerotic mouse models. Due to the lack of hemodynamic-related studies in Ldlr-/- mice, which are the most used mouse models in atherosclerosis research together with the Apoe-/- mouse model, this model was included in this study to describe changing hemodynamics in the aortic arch at baseline levels and during early atherosclerosis development and progression for the first time. In this study, distinct differences in aortic geometries of these mouse models at baseline levels were described for the first time, which result in significantly different flow- and WSS profiles in the Ldlr-/- mouse model. Further basal characterization of different parameters revealed only characteristic differences in lipid profiles, proving that the geometry is highly influencing the local WSS in these models. Most interestingly, calculation of the atherogenic index of plasma revealed a significantly higher risk in Ldlr-/- mice with ongoing atherosclerosis development, but significantly greater plaque areas in the aortic arch of Apoe-/- mice. Due to the given basal WSS and OSI profile in these two mouse models - two parameters highly influencing plaque development and progression - there is evidence that the regional plaque development differs between these mouse models during very early atherogenesis. Therefore, future studies should focus on the spatiotemporal evaluation of plaque development and composition in the three defined aortic regions using morphological measurements with MRI or 3D histological analyses like LSFM. Moreover, this study offers an excellent basis for future studies incorporating CFD simulations, analyzing the different measured parameter combinations (e.g., aortic geometry of the Ldlr-/- mouse with the lipid profile of the Apoe-/- mouse), simulating the resulting plaque development and composition. This could help to understand the complex interplay between altered hemodynamics, serum lipids and atherosclerosis and significantly improve our basic understanding of key factors initiating atherosclerosis development. Chapter 5 describes the establishment of a tissue-engineered artery model, which is based on native, decellularized porcine carotid artery scaffolds, cultured in a MRI-suitable bioreactor-system [23] for the investigation of hemodynamic-related atherosclerosis development in a controllable manner, using the previously established methods for WSS and PWV assessment [4, 16]. This in vitro artery model aimed for the reduction of animal experiments, while simultaneously offering a simplified, but completely controllable physical and biological environment. For this, a very fast and gentle decellularization protocol was established in a first step, which resulted in porcine carotid artery scaffolds showing complete acellularity while maintaining the extracellular matrix composition, overall ultrastructure and mechanical strength of native arteries. Moreover, a good cellular adhesion and proliferation was achieved, which was evaluated with isolated human blood outgrowth endothelial cells. Most importantly, an MRI-suitable artery chamber was designed for the simultaneous cultivation and assessment of high-resolution 4D hemodynamics in the described artery models. Using high-resolution 4D-flow MRI, the bioreactor system was proven to be suitable to quantify the volume flow, the two components of the WSS and the radStrain as well as the PWV in artery models, with obtained values being comparable to values found in literature for in vivo measurements. Moreover, the identification of first atherosclerotic processes like intimal thickening is achievable by three-dimensional assessment of the vessel wall morphology in the in vitro models. However, one limitation is the lack of a medial smooth muscle cell layer due to the dense ECM. Here, the utilization of the laser-cutting technology for the generation of holes and / or pits on a microscale, eventually enabling seeding of the media with SMCs showed promising results in a first try and should be further investigated in future studies. Therefore, the proposed artery model possesses all relevant components for the extension to an atherosclerosis model which may pave the way towards a significant improvement of our understanding of the key mechanisms in atherogenesis. Chapter 6 describes the development of an easy-to-prepare, low cost and fully customizable artery model based on biomaterials. Here, thermoresponsive sacrificial scaffolds, processed with the technique of MEW were used for the creation of variable, biomimetic shapes to mimic the geometric properties of the aortic arch, consisting of both, bifurcations and curvatures. After embedding the sacrificial scaffold into a gelatin-hydrogel containing SMCs, it was crosslinked with bacterial transglutaminase before dissolution and flushing of the sacrificial scaffold. The hereby generated channel was subsequently seeded with ECs, resulting in an easy-to-prepare, fast and low-cost artery model. In contrast to the native artery model, this model is therefore more variable in size and shape and offers the possibility to include smooth muscle cells from the beginning. Moreover, a custom-built and highly adaptable perfusion chamber was designed specifically for the scaffold structure, which enabled a one-step creation and simultaneously offering the possibility for dynamic cultivation of the artery models, making it an excellent basis for the development of in vitro disease test systems for e.g., flow-related atherosclerosis research. Due to time constraints, the extension to an atherosclerosis model could not be achieved within the scope of this thesis. Therefore, future studies will focus on the development and validation of an in vitro atherosclerosis model based on the proposed bi- and three-layered artery models. In conclusion, this thesis paved the way for a fast acquisition and detailed analyses of changing hemodynamics during atherosclerosis development and progression, including spatially resolved analyses of all relevant hemodynamic parameters over time and in between different groups. Moreover, to reduce animal experiments, while gaining control over various parameters influencing atherosclerosis development, promising artery models were established, which have the potential to serve as a new platform for basic atherosclerosis research.}, subject = {H{\"a}modynamik}, language = {en} } @phdthesis{Massih2024, author = {Massih, Bita}, title = {Human stem cell-based models to analyze the pathophysiology of motor neuron diseases}, publisher = {Frontiers in Cell and Developmental Biology}, doi = {10.25972/OPUS-34637}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-346374}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Motor neuron diseases (MNDs) encompass a variety of clinically and genetically heterogeneous disorders, which lead to the degeneration of motor neurons (MNs) and impaired motor functions. MNs coordinate and control movement by transmitting their signal to a target muscle cell. The synaptic endings of the MN axon and the contact site of the muscle cell thereby form the presynaptic and postsynaptic structures of the neuromuscular junction (NMJ). In MNDs, synaptic dysfunction and synapse elimination precede MN loss suggesting that the NMJ is an early target in the pathophysiological cascade leading to MN death. In this study, we established new experimental strategies to analyze human MNDs by patient derived induced pluripotent stem cells (iPSCs) and investigated pathophysiological mechanisms in two different MNDs. To study human MNDs, specialized cell culture systems that enable the connection of MNs to their target muscle cells are required to allow the formation of NMJs. In the first part of this study, we established and validated a human neuromuscular co-culture system consisting of iPSC derived MNs and 3D skeletal muscle tissue derived from myoblasts. We generated 3D muscle tissue by culturing primary myoblasts in a defined extracellular matrix in self-microfabricated silicone dishes that support the 3D tissue formation. Subsequently, iPSCs from healthy donors and iPSCs from patients with the progressive MND Amyotrophic Lateral Sclerosis (ALS) were differentiated into MNs and used for 3D neuromuscular co-cultures. Using a combination of immunohistochemistry, calcium imaging, and pharmacological stimulations, we characterized and confirmed the functionality of the 3D muscle tissue and the 3D neuromuscular co-cultures. Finally, we applied this system as an in vitro model to study the pathophysiology of ALS and found a decrease in neuromuscular coupling, muscle contraction, and axonal outgrowth in co-cultures with MNs harboring ALS-linked superoxide dismutase 1 (SOD1) mutation. In summary, this co-culture system presents a human model for MNDs that can recapitulate aspects of ALS pathophysiology. In the second part of this study, we identified an impaired unconventional protein secretion (UPS) of Sod1 as pathological mechanisms in Pleckstrin homology domain-containing family G member 5 (Plekhg5)-associated MND. Sod1 is a leaderless cytosolic protein which is secreted in an autophagy-dependent manner. We found that Plekhg5 depletion in primary MNs and NSC34 cells leads to an impaired secretion of wildtype Sod1, indicating that Plekhg5 drives the UPS of Sod1 in vitro. By interfering with different steps during the biogenesis of autophagosomes, we could show that Plekhg5-regulated Sod1 secretion is determined by autophagy. To analyze our findings in a clinically more relevant model we utilized human iPSC MNs from healthy donors and ALS patients with SOD1 mutations. We observed reduced SOD1 secretion in ALS MNs which coincides with reduced protein expression of PLEKHG5 compared to healthy and isogenic control MNs. To confirm this correlation, we depleted PLEKHG5 in control MNs and found reduced extracellular SOD1 levels, implying that SOD1 secretion depends on PLEKHG5. In summary, we found that Plekh5 regulates the UPS of Sod1 in mouse and human MNs and that Sod1 secretion occurs in an autophagy dependent manner. Our data shows an unreported mechanistic link between two MND-associated proteins.}, subject = {Tissue Engineering}, language = {en} } @phdthesis{Berberich2024, author = {Berberich, Oliver}, title = {Lateral Cartilage Tissue Integration - Evaluation of Bonding Strength and Tissue Integration \(in\) \(vitro\) Utilizing Biomaterials and Adhesives}, doi = {10.25972/OPUS-34602}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-346028}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Articular cartilage defects represent one of the most challenging clinical problem for orthopedic surgeons and cartilage damage after trauma can result in debilitating joint pain, functional impairment and in the long-term development of osteoarthritis. The lateral cartilage-cartilage integration is crucial for the long-term success and to prevent further tissue degeneration. Tissue adhesives and sealants are becoming increasingly more popular and can be a beneficial approach in fostering tissue integration, particularly in tissues like cartilage where alternative techniques, such as suturing, would instead introduce further damage. However, adhesive materials still require optimization regarding the maximization of adhesion strength on the one hand and long-term tissue integration on the other hand. In vitro models can be a valuable support in the investigation of potential candidates and their functional mechanisms. For the conducted experiments within this work, an in vitro disc/ring model obtained from porcine articular cartilage tissue was established. In addition to qualitative evaluation of regeneration, this model facilitates the implementation of biomechanical tests to quantify cartilage integration strength. Construct harvesting for histology and other evaluation methods could be standardized and is ethically less questionable compared to in vivo testing. The opportunity of cell culture technique application for the in vitro model allowed a better understanding of cartilage integration processes. Tissue bonding requires chemical or physical interaction of the adhesive material and the substrate. Adhesive hydrogels can bind to the defect interface and simultaneously fill the gap of irregularly shaped defect voids. Fibrin gels are derived from the physiological blood-clot formation and are clinically applied for wound closure. Within this work, comparisons of different fibrin glue formulations with the commercial BioGlue® were assessed, which highlighted the need for good biocompatibility when applied on cartilage tissue in order to achieve satisfying long-term integration. Fibrin gel formulations can be adapted with regard to their long-term stability and when applied on cartilage disc/ring constructs improved integrative repair is observable. The kinetic of repairing processes was investigated in fibrin-treated cartilage composites as part of this work. After three days in vitro cultivation, deposited extracellular matrix (ECM) was obvious at the glued interface that increased further over time. Interfacial cell invasion from the surrounding native cartilage was detected from day ten of tissue culture. The ECM formation relies on molecular factors, e.g., as was shown representatively for ascorbic acid, and contributes to increasing integration strengths over time. The experiments performed with fibrin revealed that the treatment with a biocompatible adhesive that allows cartilage neosynthesis favors lateral cartilage integration in the long term. However, fibrin has limited immediate bonding strength, which is disadvantageous for use on articular cartilage that is subject to high mechanical stress. The continuing aim of this thesis was to further develop adhesive mechanisms and new adhesive hydrogels that retain the positive properties of fibrin but have an increased immediate bonding strength. Two different photochemical approaches with the advantage of on-demand bonding were tested. Such treatment potentially eases the application for the professional user. First, an UV light induced crosslinking mechanism was transferred to fibrin glue to provide additional bonding strength. For this, the cartilage surface was functionalized with highly reactive light-sensitive diazirine groups, which allowed additional covalent bonds to the fibrin matrix and thus increased the adhesive strength. However, the disadvantages of this approach were the multi-step bonding reactions, the need for enzymatic pretreatment of the cartilage, expensive reagents, potential UV-light damage, and potential toxicity hazards. Due to the mentioned disadvantages, no further experiments, including long-term culture, were carried out. A second photosensitive approach focused on blue light induced crosslinking of fibrinogen (RuFib) via a photoinitiator molecule instead of using thrombin as a crosslinking mediator like in normal fibrin glue. The used ruthenium complex allowed inter- and intramolecular dityrosine binding of fibrinogen molecules. The advantage of this method is a one-step curing of fibrinogen via visible light that further achieved higher adhesive strengths than fibrin. In contrast to diazirine functionalization of cartilage, the ruthenium complex is of less toxicological concern. However, after in vitro cultivation of the disc/ring constructs, there was a decrease in integration strength. Compared to fibrin, a reduced cartilage synthesis was observed at the defect. It is also disadvantageous that a direct adjustment of the adhesive can only be made via protein concentration, since fibrinogen is a natural protein that has a fixed number of tyrosine binding sites without chemical modification. An additional cartilage adhesive was developed that is based on a mussel-inspired adhesive mechanism in which reactivity to a variety of substrates is enabled via free DOPA amino acids. DOPA-based adhesion is known to function in moist environments, a major advantage for application on water-rich cartilage tissue surrounded by synovial liquid. Reactive DOPA groups were synthetically attached to a polymer, here POx, to allow easy chemical modifiability, e.g. insertion of hydrolyzable ester motifs for tunable degradation. The possibility of preparing an adhesive hybrid hydrogel of POx in combination with fibrinogen led to good cell compatibility as was similarly observed with fibrin, but with increased immediate adhesive strength. Degradation could be adjusted by the amount of ester linkages on the POx and a direct influence of degradation rates on the development of integration in the in vitro model could be shown. Hydrogels are well suited to fill defect gaps and immediate integration can be achieved via adhesive properties. The results obtained show that for the success of long-term integration, a good ability of the adhesive to take up synthesized ECM components and cells to enable regeneration is required. The degradation kinetics of the adhesive must match the remodeling process to avoid intermediate loss of integration power and to allow long-term firm adhesion to the native tissue. Hydrogels are not only important as adhesives for smaller lesions, but also for filling large defect volumes and populating them with cells to produce tissue engineered cartilage. Many different hydrogel types suitable for cartilage synthesis are reported in the literature. A long-term stable fibrin formulation was tested in this work not only as an adhesive but also as a bulk hydrogel construct. Agarose is also a material widely used in cartilage tissue engineering that has shown good cartilage neosynthesis and was included in integration assessment. In addition, a synthetic hyaluronic acid-based hydrogel (HA SH/P(AGE/G)) was used. The disc/ring construct was adapted for such experiments and the inner lumen of the cartilage ring was filled with the respective hydrogel. In contrast to agarose, fibrin and HA-SH/P(AGE/G) gels have a crosslink mechanism that led to immediate bonding upon contact with cartilage during curing. The enhanced cartilage neosynthesis in agarose compared to the other hydrogel types resulted in improved integration during in vitro culture. This shows that for the long-term success of a treatment, remodeling of the hydrogel into functional cartilage tissue is a very high priority. In order to successfully treat larger cartilage defects with hydrogels, new materials with these properties in combination with chemical modifiability and a direct adhesion mechanism are one of the most promising approaches.}, subject = {Knorpel}, language = {en} } @phdthesis{Reuter2023, author = {Reuter, Christian Steffen}, title = {Development of a tissue-engineered primary human skin infection model to study the pathogenesis of tsetse fly-transmitted African trypanosomes in mammalian skin}, doi = {10.25972/OPUS-25114}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251147}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Many arthropods such as mosquitoes, ticks, bugs, and flies are vectors for the transmission of pathogenic parasites, bacteria, and viruses. Among these, the unicellular parasite Trypanosoma brucei (T. brucei) causes human and animal African trypanosomiases and is transmitted to the vertebrate host by the tsetse fly. In the fly, the parasite goes through a complex developmental cycle in the alimentary tract and salivary glands ending with the cellular differentiation into the metacyclic life cycle stage. An infection in the mammalian host begins when the fly takes a bloodmeal, thereby depositing the metacyclic form into the dermal skin layer. Within the dermis, the cell cycle-arrested metacyclic forms are activated, re-enter the cell cycle, and differentiate into proliferative trypanosomes, prior to dissemination throughout the host. Although T. brucei has been studied for decades, very little is known about the early events in the skin prior to systemic dissemination. The precise timing and the mechanisms controlling differentiation of the parasite in the skin continue to be elusive, as does the characterization of the proliferative skin-residing trypanosomes. Understanding the first steps of an infection is crucial for developing novel strategies to prevent disease establishment and its progression. A major shortcoming in the study of human African trypanosomiasis is the lack of suitable infection models that authentically mimic disease progression. In addition, the production of infectious metacyclic parasites requires tsetse flies, which are challenging to keep. Thus, although animal models - typically murine - have produced many insights into the pathogenicity of trypanosomes in the mammalian host, they were usually infected by needle injection into the peritoneal cavity or tail vein, bypassing the skin as the first entry point. Furthermore, animal models are not always predictive for the infection outcome in human patients. In addition, the relatively small number of metacyclic parasites deposited by the tsetse flies makes them difficult to trace, isolate, and study in animal hosts. The focus of this thesis was to develop and validate a reconstructed human skin equivalent as an infection model to study the development of naturally-transmitted metacyclic parasites of T. brucei in mammalian skin. The first part of this work describes the development and characterization of a primary human skin equivalent with improved mechanical properties. To achieve this, a computer-assisted compression system was designed and established. This system allowed the improvement of the mechanical stability of twelve collagen-based dermal equivalents in parallel through plastic compression, as evaluated by rheology. The improved dermal equivalents provided the basis for the generation of the skin equivalents and reduced their contraction and weight loss during tissue formation, achieving a high degree of standardization and reproducibility. The skin equivalents were characterized using immunohistochemical and histological techniques and recapitulated key anatomical, cellular, and functional aspects of native human skin. Furthermore, their cellular heterogeneity was examined using single-cell RNA sequencing - an approach which led to the identification of a remarkable repertoire of extracellular matrix-associated genes expressed by different cell subpopulations in the artificial skin. In addition, experimental conditions were established to allow tsetse flies to naturally infect the skin equivalents with trypanosomes. In the second part of the project, the development of the trypanosomes in the artificial skin was investigated in detail. This included the establishment of methods to successfully isolate skin-dwelling trypanosomes to determine their protein synthesis rate, cell cycle and metabolic status, morphology, and transcriptome. Microscopy techniques to study trypanosome motility and migration in the skin were also optimized. Upon deposition in the artificial skin by feeding tsetse, the metacyclic parasites were rapidly activated and established a proliferative population within one day. This process was accompanied by: (I) reactivation of protein synthesis; (II) re-entry into the cell cycle; (III) change in morphology; (IV) increased motility. Furthermore, these observations were linked to potentially underlying developmental mechanisms by applying single-cell parasite RNA sequencing at five different timepoints post-infection. After the initial proliferative phase, the tsetse-transmitted trypanosomes appeared to enter a reversible quiescence program in the skin. These quiescent skin-residing trypanosomes were characterized by very slow replication, a strongly reduced metabolism, and a transcriptome markedly different from that of the deposited metacyclic forms and the early proliferative trypanosomes. By mimicking the migration from the skin to the bloodstream, the quiescent phenotype could be reversed and the parasites returned to an active proliferating state. Given that previous work has identified the skin as an anatomical reservoir for T. brucei during disease, it is reasonable to assume that the quiescence program is an authentic facet of the parasite's behavior in an infected host. In summary, this work demonstrates that primary human skin equivalents offer a new and promising way to study vector-borne parasites under close-to-natural conditions as an alternative to animal experimentation. By choosing the natural transmission route - the bite of an infected tsetse fly - the early events of trypanosome infection have been detailed with unprecedented resolution. In addition, the evidence here for a quiescent, skin-residing trypanosome population may explain the persistence of T. brucei in the skin of aparasitemic and asymptomatic individuals. This could play an important role in maintaining an infection over long time periods.}, subject = {Trypanosoma brucei}, language = {en} } @phdthesis{Alzheimer2023, author = {Alzheimer, Mona}, title = {Development of tissue-engineered three-dimensional infection models to study pathogenesis of \(Campylobacter\) \(jejuni\)}, doi = {10.25972/OPUS-19344}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193440}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Infectious diseases caused by pathogenic microorganisms are one of the largest socioeconomic burdens today. Although infectious diseases have been studied for decades, in numerous cases, the precise mechanisms involved in the multifaceted interaction between pathogen and host continue to be elusive. Thus, it still remains a challenge for researchers worldwide to develop novel strategies to investigate the molecular context of infectious diseases in order to devise preventive or at least anti-infective measures. One of the major drawbacks in trying to obtain in-depth knowledge of how bacterial pathogens elicit disease is the lack of suitable infection models to authentically mimic the disease progression in humans. Numerous studies rely on animal models to emulate the complex temporal interactions between host and pathogen occurring in humans. While they have greatly contributed to shed light on these interactions, they require high maintenance costs, are afflicted with ethical drawbacks, and are not always predictive for the infection outcome in human patients. Alternatively, in-vitro two-dimensional (2D) cell culture systems have served for decades as representatives of human host environments to study infectious diseases. These cell line-based models have been essential in uncovering virulence-determining factors of diverse pathogens as well as host defense mechanisms upon infection. However, they lack the morphological and cellular complexity of intact human tissues, limiting the insights than can be gained from studying host-pathogen interactions in these systems. The focus of this thesis was to establish and innovate intestinal human cell culture models to obtain in-vitro reconstructed three-dimensional (3D) tissue that can faithfully mimic pathogenesis-determining processes of the zoonotic bacterium Campylobacter jejuni (C. jejuni). Generally employed for reconstructive medicine, the field of tissue engineering provides excellent tools to generate organ-specific cell culture models in vitro, realistically recapitulating the distinctive architecture of human tissues. The models employed in this thesis are based on decellularized extracellular matrix (ECM) scaffolds of porcine intestinal origin. Reseeded with intestinal human cells, application of dynamic culture conditions promoted the formation of a highly polarized mucosal epithelium maintained by functional tight and adherens junctions. While most other in-vitro infection systems are limited to a flat monolayer, the tissue models developed in this thesis can display the characteristic 3D villi and crypt structure of human small intestine. First, experimental conditions were established for infection of a previously developed, statically cultivated intestinal tissue model with C. jejuni. This included successful isolation of bacterial colony forming units (CFUs), measurement of epithelial barrier function, as well as immunohistochemical and histological staining techniques. In this way, it became possible to follow the number of viable bacteria during the infection process as well as their translocation over the polarized epithelium of the tissue model. Upon infection with C. jejuni, disruption of tight and adherens junctions could be observed via confocal microscopy and permeability measurements of the epithelial barrier. Moreover, C. jejuni wildtype-specific colonization and barrier disruption became apparent in addition to niche-dependent bacterial localization within the 3D microarchitecture of the tissue model. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D host environment deviated from those obtained with conventional in-vitro 2D monolayers but mimicked observations made in vivo. Furthermore, a genome-wide screen of a C. jejuni mutant library revealed significant differences for bacterial factors required or dispensable for interactions with unpolarized host cells or the highly prismatic epithelium provided by the intestinal tissue model. Elucidating the role of several previously uncharacterized factors specifically important for efficient colonization of a 3D human environment, promises to be an intriguing task for future research. At the frontline of the defense against invading pathogens is the protective, viscoelastic mucus layer overlying mucosal surfaces along the human gastrointestinal tract (GIT). The development of a mucus-producing 3D tissue model in this thesis was a vital step towards gaining a deeper understanding of the interdependency between bacterial pathogens and host-site specific mucins. The presence of a mucus layer conferred C. jejuni wildtype-specific protection against epithelial barrier disruption by the pathogen and prevented a high bacterial burden during the course of infection. Moreover, results obtained in this thesis provide evidence in vitro that the characteristic corkscrew morphology of C. jejuni indeed grants a distinct advantage in colonizing mucous surfaces. Overall, the results obtained within this thesis highlight the strength of the tissue models to combine crucial features of native human intestine into accessible in-vitro infection models. Translation of these systems into infection research demonstrated their ability to expose in-vivo like infection outcomes. While displaying complex organotypic architecture and highly prismatic cellular morphology, these tissue models still represent an imperfect reflection of human tissue. Future advancements towards inclusion of human primary and immune cells will strive for even more comprehensive model systems exhibiting intricate multicellular networks of in-vivo tissue. Nevertheless, the work presented in this thesis emphasizes the necessity to investigate host-pathogen interactions in infection models authentically mimicking the natural host environment, as they remain among the most vital parts in understanding and counteracting infectious diseases.}, subject = {Campylobacter jejuni}, language = {en} } @phdthesis{SchmidtgebSchmid2023, author = {Schmidt [geb. Schmid], Freia Florina}, title = {Ein dreidimensionales kutanes Melanommodell f{\"u}r den Einsatz in der pr{\"a}klinischen Testung}, doi = {10.25972/OPUS-32925}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-329255}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Das maligne Melanom nimmt als Tumorerkrankung mit hoher Metastasierungsrate und steigenden Inzidenzraten bei h{\"o}chster Mortalit{\"a}t aller Hauttumoren eine zunehmende Bedeutung in der modernen Onkologie ein. Fr{\"u}hzeitige Diagnosem{\"o}glichkeiten und moderne Behandlungen konnten das {\"U}berleben der Patienten bereits erheblich verbessern. Jedoch besteht nach wie vor Bedarf an geeigneten Modellen, um die Melanomprogression vollst{\"a}ndig zu verstehen und neue wirksame Therapien zu entwickeln. Hierf{\"u}r werden h{\"a}ufig Tiermodelle verwendet, diese spiegeln jedoch nicht die menschliche Mikroumgebung wider. Zweidimensionalen Zellkulturen fehlen dagegen entscheidende Elemente der Tumormikroumgebung. Daher wurde in dieser Arbeit ein dreidimensionales epidermales Tumormodell des malignen Melanoms, welches aus prim{\"a}ren humanen Keratinozyten und verschiedenen Melanomzelllinien besteht, entwickelt. Die eingesetzten Melanomzelllinien variieren in ihren Treibermutationen, wodurch das Modell in der Lage ist, Wirkstoffe zu untersuchen, die spezifisch auf diese Mutationen wirken. Mit Techniken des Tissue Engineerings konnte ein dreidimensionales Hautmodell aufgebaut werden, das alle charakteristischen Schichten der Epidermis aufweist und im Bereich des stratum basale Melanomcluster ausbildet. Diese reichen je nach Gr{\"o}ße und Ausdehnung bis in suprabasale Epidermisschichten hinein. Die Tumor-Histopathologie, der Tumorstoffwechsel sowie tumorassoziierte Proteinsekretionen ließen sich im in vitro Modell nachweisen. Dar{\"u}ber hinaus konnte ein Protokoll entwickelt werden, mit dem einzelne Zellen aus den Modellen reisoliert werden k{\"o}nnen. Dies erm{\"o}glichte es, den Proliferationszustand innerhalb des jeweiligen Modells zu charakterisieren und die Wirkung von Antitumortherapien gezielt zu bewerten. Die Anwendbarkeit als Testsystem im Bereich der Tumortherapeutika wurde mit dem in der Klinik h{\"a}ufig verwendeten v-raf-Maus-Sarkom-Virus-Onkogen-Homolog B (BRAF)-Inhibitor Vemurafenib demonstriert. Der selektive BRAF-Inhibitor reduzierte erfolgreich das Tumorwachstum in den Modellen mit BRAF-mutierten Melanomzellen, was durch eine Verringerung der metabolischen Aktivit{\"a}t, der proliferierenden Zellen und des Glukoseverbrauchs gezeigt wurde. F{\"u}r die Implementierung des Modells in die pr{\"a}klinische Therapieentwicklung wurde B-B-Dimethylacrylshikonin, ein vielversprechender Wirkstoffkandidat, welcher einen Zellzyklusarrest mit anschließender Apoptose bewirkt, im Modell getestet. Bei einer Anwendung der Modelle im Bereich der Testung topischer Behandlungen ist eine Barrierefunktion der Modelle notwendig, die der in vivo Situation nahe kommt. Die Barriereeigenschaften der Haut{\"a}quivalente wurden durch die Melanomzellen nachweislich nicht beeinflusst, sind aber im Vergleich zur in vivo Situation noch unzureichend. Eine signifikante Steigerung der Hautbarriere konnte durch die Bereitstellung von Lipiden und die Anregung hauteigener Regenerationsprozesse erreicht werden. {\"U}ber den Nachweis des transepidermalen Wasserverlusts konnte eine Messmethode zur nicht-invasiven Bestimmung der Hautbarriere etabliert und {\"u}ber den Vergleich zur Impedanzspektroskopie validiert werden. Hierbei gelang es, erstmals die Korrelation der Hautmodelle zur in vivo Situation {\"u}ber ein solches Verfahren zu zeigen. Das entwickelte epidermale Modell konnte durch die Integration eines dermalen Anteils und einer Endothelzellschicht noch weiter an die komplexe Struktur und Physiologie der Haut angepasst werden um Untersuchungen, die mit der Metastierung und Invasion zusammenh{\"a}ngen, zu erm{\"o}glichen. Die artifizielle Dermis basiert auf einem Kollagen-Hydrogel mit prim{\"a}ren Fibroblasten. Eine dezellularisierte Schweinedarmmatrix ließ sich zur Erweiterung des Modells um eine Endothelzellschicht nutzen. Dabei wanderten die prim{\"a}ren Fibroblasten apikal in die nat{\"u}rliche Schweindarmmatrix ein, w{\"a}hrend die Endothelzellen basolateral eine geschlossene Schicht bildeten. Die in dieser Arbeit entwickelten Gewebemodelle sind in der Lage, die Vorhersagekraft der in vitro Modelle und die in vitro - in vivo Korrelation zu verbessern. Durch die Kombination des Melanommodells mit einer darauf abgestimmten Analytik wurde ein neuartiges Werkzeug f{\"u}r die pr{\"a}klinische Forschung zur Testung von pharmazeutischen Wirkstoffen geschaffen.}, subject = {Tissue Engineering}, language = {de} } @phdthesis{Altmann2023, author = {Altmann, Stephan}, title = {Characterization of Metabolic Glycoengineering in Mesenchymal Stromal Cells for its Application in thermoresponsive Bioinks}, doi = {10.25972/OPUS-29100}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-291003}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {This work developed during the first funding period of the subproject B05 in the framework of the interdisciplinary research consortium TRR 225 'From the Fundamentals of Biofabrication toward functional Tissue Models' and was part of a cooperation between the Orthopedic Department represented by Prof. Dr. Regina Ebert and the Institute of Organic Chemistry represented by Prof. Dr. J{\"u}rgen Seibel. This project dealed with cellular behavior during the bioprinting process and how to influence it by modifying the cell glycocalyx with functional target molecules. The focus was on the impact of potential shear stress, that cells experience when they get processed in thermoresponsive bioinks, and a way to increase the cell stiffness via metabolic glycoengineering to attenuate shear forces. For the characterization of the metabolic glycoengineering, four different peracetylated and four non-acetylated modified monosaccharides (two mannose and two sialic acid sugars) were tested in primary human mesenchymal stromal cells (hMSC) and telomerase-immortalized hMSC (hMSC-TERT). Viability results demonstrated a dose-dependent correlation for all sugars, at which hMSC-TERT seemed to be more susceptible leading to lower viability rates. The assessment of the incorporation efficiencies was performed by click chemistry using fluorescent dyes and revealed also a dose-dependent correlation for all mannose and sialic acid sugars, while glucose and galactose variants were not detected in the glycocalyx. However, incorporation efficiencies were highest when using mannose sugars in the primary hMSC. A subsequent analysis of the temporal retention of the incorporated monosaccharides showed a constant declining fluorescence signal up to 6 d for azido mannose in hMSC-TERT, whereas no signal could be detected for alkyne mannose after 2 d. Investigation of the differentiation potential and expression of different target genes revealed no impairment after incubation with mannose sugars, indicating a normal phenotype for hMSC-TERT. Following the successful establishment of the method, either a coumarin derivative or an artificial galectin 1 ligand were incorporated into the cell glycocalyx of hMSC-TERT as functional target molecule. The biophysical analysis via shear flow deformation cytometry revealed a slightly increased cell stiffness and lowered fluidity for both molecules. A further part of this project aimed to control lectin-mediated cell adhesion by artificial galectin 1 ligands. As that hypothesis was settled in the work group of Prof. Dr. J{\"u}rgen Seibel, this work supported with an initial characterization of galectin 1 as part of the hMSC biology. A stable galectin 1 expression at gene and protein level in both hMSC and hMSC-TERT could be confirmed, at which immunocytochemical stainings could detect the protein only in the glycocalyx. The treatment of hMSC-TERT with a galectin 1 ligand in different concentrations did not show an altered gene expression of galectin 1. However, these first data in addition to the investigation of stiffness confirmed the applicability of specific and artificial IV galectin 1 ligands in biofabrication approaches to alter cell properties of hMSC. To conclude, metabolic glycoengineering has been successfully implemented in hMSC and hMSC-TERT to introduce glycocalyx modifications which reside there for several days. A proof of concept was carried out by the increase of cell stiffness and fluidity by the incorporation of a coumarin derivative or an artificial galectin 1 ligand. For the characterization of shear stress impact on cells after printing in thermoresponsive bioinks, the processing of hMSC-TERT (mixing or additionally printing) with Pluronic F127 or Polyoxazoline-Polyoxazine (POx-POzi) polymer solution was investigated. While there were no changes in viability when using POx-POzi bioink, processing with Pluronic F127 indicated slightly lower viability and increased apoptosis activity. Assessment of cellular responses to potential shear stress showed no reorganization of the cytoskeleton independent of the bioink, but highly increased expression of the mechanoresponsive proto-oncogene c Fos which was more pronounced when using Pluronic F127 and just mixed with the bioinks. Interestingly, processing of the mechanoresponsive reporter cell line hMSC-TERT-AP1 revealed slightly elevated mechanotransduction activity when using POx-POzi polymer and just mixed with the bioinks as well. In conclusion, hMSC-TERT embedded in thermoresponsive bioinks might shortly experience shear stress during the printing process, but that did not lead to remarkable cell damage likely due to the rheological properties of the bioinks. Furthermore, the printing experiments also suggested that cells do not sense more shear stress when additionally printed.}, subject = {Glykobiologie}, language = {en} } @phdthesis{Gensler2023, author = {Gensler, Marius E.}, title = {Simultaneous printing of tissue and customized bioreactor}, doi = {10.25972/OPUS-28019}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-280190}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Additive manufacturing processes such as 3D printing are booming in the industry due to their high degree of freedom in terms of geometric shapes and available materials. Focusing on patient-specific medicine, 3D printing has also proven useful in the Life Sciences, where it exploits the shape fidelity for individualized tissues in the field of bioprinting. In parallel, the current systems of bioreactor technology have adapted to the new manufacturing technology as well and 3D-printed bioreactors are increasingly being developed. For the first time, this work combines the manufacturing of the tissue and a tailored bioreactor, significantly streamlining the overall process and optimally merging the two processes. This way the production of the tissues can be individualized by customizing the reactor to the tissue and the patient-specific wound geometry. For this reason, a common basis and guideline for the cross-device and cross-material use of 3D printers was created initially. Their applicability was demonstrated by the iterative development of a perfusable bioreactor system, made from polydimethylsiloxane (PDMS) and a lignin-based filament, into which a biological tissue of flexible shape can be bioprinted. Cost-effective bioink-replacements and in silico computational fluid dynamics simulations were used for material sustainability and shape development. Also, nutrient distribution and shear stress could be predicted in this way pre-experimentally. As a proof of functionality and adaptability of the reactor, tissues made from a nanocellulose-based Cellink® Bioink, as well as an alginate-based ink mixed with Me-PMeOx100-b-PnPrOzi100-EIP (POx) (Alginate-POx bioink) were successfully cultured dynamically in the bioreactor together with C2C12 cell line. Tissue maturation was further demonstrated using hMSC which were successfully induced to adipocyte differentiation. For further standardization, a mobile electrical device for automated media exchange was developed, improving handling in the laboratory and thus reduces the probability of contamination.}, subject = {3 D bioprinting}, language = {en} } @phdthesis{Malkmus2023, author = {Malkmus, Christoph}, title = {Establishment of a 3D \(in\) \(vitro\) skin culture system for the obligatory human parasite \(Onchocerca\) \(volvulus\)}, doi = {10.25972/OPUS-31717}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-317171}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Onchocerciasis, the world's second-leading infectious cause of blindness in humans -prevalent in Sub-Saharan Africa - is caused by Onchocerca volvulus (O. volvulus), an obligatory human parasitic filarial worm. Commonly known as river blindness, onchocerciasis is being targeted for elimination through ivermectin-based mass drug administration programs. However, ivermectin does not kill adult parasites, which can live and reproduce for more than 15 years within the human host. These impediments heighten the need for a deeper understanding of parasite biology and parasite-human host interactions, coupled with research into the development of new tools - macrofilaricidal drugs, diagnostics, and vaccines. Humans are the only definitive host for O. volvulus. Hence, no small-animal models exist for propagating the full life cycle of O. volvulus, so the adult parasites must be obtained surgically from subcutaneous nodules. A two-dimensional (2D) culture system allows that O. volvulus larvae develop from the vector-derived infective stage larvae (L3) in vitro to the early pre-adult L5 stages. As problematic, the in vitro development of O. volvulus to adult worms has so far proved infeasible. We hypothesized that an increased biological complexity of a three-dimensional (3D) culture system will support the development of O. volvulus larvae in vitro. Thus, we aimed to translate crucial factors of the in vivo environment of the developing worms into a culture system based on human skin. The proposed tissue model should contain 1. skinspecific extracellular matrix, 2. skin-specific cells, and 3. enable a direct contact of larvae and tissue components. For the achievement, a novel adipose tissue model was developed and integrated to a multilayered skin tissue comprised of epidermis, dermis and subcutis. Challenges of the direct culture within a 3D tissue model hindered the application of the three-layered skin tissue. However, the indirect coculture of larvae and skin models supported the growth of fourth stage (L4) larvae in vitro. The direct culture of L4 and adipose tissue strongly improved the larvae survival. Furthermore, the results revealed important cues that might represent the initial encapsulation of the developing worm within nodular tissue. These results demonstrate that tissue engineered 3D tissues represent an appropriate in vitro environment for the maintenance and examination of O. volvulus larvae.}, subject = {Tissue Engineering}, language = {en} } @phdthesis{Leikeim2022, author = {Leikeim, Anna}, title = {Vascularization Strategies for Full-Thickness Skin Equivalents to Model Melanoma Progression}, doi = {10.25972/OPUS-27295}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-272956}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Malignant melanoma (MM) is the most dangerous type of skin cancer with rising incidences worldwide. Melanoma skin models can help to elucidate its causes and formation or to develop new treatment strategies. However, most of the current skin models lack a vasculature, limiting their functionality and applicability. MM relies on the vascular system for its own supply and for its dissemination to distant body sites via lymphatic and blood vessels. Thus, to accurately study MM progression, a functional vasculature is indispensable. To date, there are no vascularized skin models to study melanoma metastasis in vitro, which is why such studies still rely on animal experimentation. In the present thesis, two different approaches for the vascularization of skin models are employed with the aim to establish a vascularized 3D in vitro full-thickness skin equivalent (FTSE) that can serve as a test system for the investigation of the progression of MM. Initially, endothelial cells were incorporated in the dermal part of FTSEs. The optimal seeding density, a spheroid conformation of the cells and the cell culture medium were tested. A high cell density resulted in the formation of lumen-forming shapes distributed in the dermal part of the model. These capillary-like structures were proven to be of endothelial origin by staining for the endothelial cell marker CD31. The established vascularized FTSE (vFTSE) was characterized histologically after 4 weeks of culture, revealing an architecture similar to human skin in vivo with a stratified epidermis, separated from the dermal equivalent by a basement membrane indicated by collagen type IV. However, this random capillary-like network is not functional as it cannot be perfused. Therefore, the second vascularization approach focused on the generation of a perfusable tissue construct. A channel was molded within a collagen hydrogel and seeded with endothelial cells to mimic a central, perfusable vessel. The generation and the perfusion culture of the collagen hydrogel was enabled by the use of two custom-made, 3D printed bioreactors. Histological assessment of the hydrogels revealed the lining of the channel with a monolayer of endothelial cells, expressing the cell specific marker CD31. For the investigation of MM progression in vitro, a 3D melanoma skin equivalent was established. Melanoma cells were incorporated in the epidermal part of FTSEs, representing the native microenvironment of the tumor. Melanoma nests grew at the dermo-epidermal junction within the well stratified epidermis and were characterized by the expression of common melanoma markers. First experiments were conducted showing the feasibility of combining the melanoma model with the vFTSE, resulting in skin models with tumors at the dermo-epidermal junction and lumen-like structures in the dermis. Taken together, the models presented in this thesis provide further steps towards the establishment of a vascularized, perfusable melanoma model to study melanoma progression and metastasis.}, subject = {Tissue Engineering}, language = {en} } @phdthesis{Fey2022, author = {Fey, Christina}, title = {Establishment of an intestinal tissue model for pre-clinical screenings}, doi = {10.25972/OPUS-24410}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-244107}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {The small intestine represents a strong barrier separating the lumen from blood circulation thereby playing a major role in the absorption and the transport of pharmacological agents prior to their arrival on the respective target site. In order to gain more knowledge about specialized uptake mechanisms and risk assessment for the patient after oral admission of drugs, intestinal in vitro models demonstrating a close similarity to the in vivo situation are needed. In the past, cell line-based in vitro models composed of Caco-2 cells cultured on synthetic cell carriers represented the "gold standard" in the field of intestinal tissue engineering. Expressive advantages of these models are a reproducible, cost-efficient and standardized model set up, but cell function can be negatively influenced by the low porosity or unwanted molecular adhesion effects of the artificial scaffold material. Natural extracellular matrices (ECM) such as the porcine decellularized small intestinal submucosa (SIS) are used as alternative to overcome some common drawbacks; however, the fabrication of these scaffolds is time- and cost-intensive, less well standardized and the 3Rs (replacement, reduction, refinement) principle is not entirely fulfilled. Nowadays, biopolymer-based scaffolds such as the bacterial nanocellulose (BNC) suggest an interesting option of novel intestinal tissue engineered models, as the BNC shows comparable features to the native ECM regarding fiber arrangement and hydrophilic properties. Furthermore, the BNC is of non-animal origin and the manufacturing process is faster as well as well standardized at low costs. In this context, the first part of this thesis analyzed the BNC as alternative scaffold to derive standardized and functional organ models in vitro. Therefore, Caco-2 cells were cultured on two versions of BNC with respect to their surface topography, the unmodified BNC as rather smooth surface and the surface-structured BNC presenting an aligned fiber arrangement. As controls, Caco-2 in vitro models were set up on PET and SIS matrices. In this study, the BNC-based models demonstrated organ-specific properties comprising typical cellular morphologies, a characteristic tight junction protein expression profile, representative ultrastructural features and the formation of a tight epithelial barrier together with a corresponding transport activity. In summary, these results validated the high quality of the BNC-based Caco-2 models under cost-efficient conditions and their suitability for pre-clinical research purposes. However, the full functional diversity of the human intestine cannot be presented by Caco-2 cells due to their tumorigenic background and their exclusive representation of mature enterocytes. Next to the scaffold used for the setup of in vitro models, the cellular unit mainly drives functional performance, which demonstrates the crucial importance of mimicking the cellular diversity of the small intestine in vitro. In this context, intestinal primary organoids are of high interest, as they show a close similarity to the native epithelium regarding their cellular diversity comprising enterocytes, goblet cells, enteroendocrine cells, paneth cells, transit amplifying cells and stem cells. In general, such primary organoids grow in a 3D Matrigel® based environment and a medium formulation supplemented with a variety of growth factors to maintain stemness, to inhibit differentiation and to stimulate cell migration supporting long term in vitro culture. Intestinal primary spheroid/organoid cultures were set up as Transwell®-like models on both BNC variants, which resulted in a fragmentary cell layer and thereby unfavorable properties of these scaffold materials under the applied circumstances. As the BNC manufacturing process is highly flexible, surface properties could be adapted in future studies to enable a good cell adherence and barrier formation for primary intestinal cells, too. However, the application of these organoid cultures in pre-clinical research represents an enormous challenge, as the in vitro culture is complex and additionally time- and cost-intensive. With regard to the high potential of primary intestinal spheroids/organoids and the necessity of a simplified but predictive model in pre-clinical research purposes, the second part of this thesis addressed the establishment of a primary-derived immortalized intestinal cell line, which enables a standardized and cost-efficient culture (including in 2D), while maintaining the cellular diversity of the organoid in vitro cultures. In this study, immortalization of murine and human intestinal primary organoids was induced by ectopic expression of a 10- (murine) or 12 component (human) pool of genes regulating stemness and the cell cycle, which was performed in cooperation with the InSCREENeX GmbH in a 2D- and 3D-based transduction strategy. In first line, the established cell lines (cell clones) were investigated for their cell culture prerequisites to grow under simplified and cost-efficient conditions. While murine cell clones grew on uncoated plastic in a medium formulation supplemented with EGF, Noggin, Y-27632 and 10\% FCS, the human cell clones demonstrated the necessity of a Col I pre coating together with the need for a medium composition commonly used for primary human spheroid/organoid cultures. Furthermore, the preceding analyses resulted in only one human cell clone and three murine cell clones for ongoing characterization. Studies regarding the proliferative properties and the specific gene as well as protein expression profile of the remaining cell clones have shown, that it is likely that transient amplifying cells (TACs) were immortalized instead of the differentiated cell types localized in primary organoids, as 2D, 3D or Transwell®-based cultures resulted in slightly different gene expression profiles and in a dramatically reduced mRNA transcript level for the analyzed marker genes representative for the differentiated cell types of the native epithelium. Further, 3D cultures demonstrated the formation of spheroid-like structures; however without forming organoid-like structures due to prolonged culture, indicating that these cell populations have lost their ability to differentiate into specific intestinal cell types. The Transwell®-based models set up of each clone exhibit organ-specific properties comprising an epithelial-like morphology, a characteristic protein expression profile with an apical mucus-layer covering the villin-1 positive cell layer, thereby representing goblet cells and enterocytes, together with representative tight junction complexes indicating an integer epithelial barrier. The proof of a functional as well as tight epithelial barrier in TEER measurements and in vivo-like transport activities qualified the established cell clones as alternative cell sources for tissue engineered models representing the small intestine to some extent. Additionally, the easy handling and cell expansion under more cost-efficient conditions compared to primary organoid cultures favors the use of these newly generated cell clones in bioavailability studies. Altogether, this work demonstrated new components, structural and cellular, for the establishment of alternative in vitro models of the small intestinal epithelium, which could be used in pre-clinical screenings for reproducible drug delivery studies.}, subject = {D{\"u}nndarm}, language = {en} } @phdthesis{WeigelgebSchneider2022, author = {Weigel [geb. Schneider], Verena}, title = {Entwicklung eines \(in\) \(vitro\) Modells f{\"u}r Verbrennungen ersten Grades zur Testung einer zellbasierten Wundauflage}, doi = {10.25972/OPUS-26151}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-261514}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Mit j{\"a}hrlich circa 11 Millionen F{\"a}llen weltweit, stellen schwere Brandwunden bis heute einen großen Anteil an Verletzungen dar, die in Kliniken behandelt werden m{\"u}ssen. W{\"a}hrend leichte Verbrennungen meist problemlos heilen, bedarf die Behandlung tieferer Verbrennungen medizinischer Intervention. Zellbasierte Therapeutika zeigen hier bereits große Erfolge, aufgrund der eingeschr{\"a}nkten {\"U}bertragbarkeit von Ergebnissen aus Tiermodellen ist jedoch sowohl die Testung neuer Produkte, als auch die Erforschung der Wundheilung bei Brandwunden noch immer schwierig. Aufgrund dessen wurden in dieser Arbeit zwei Ziele verfolgt: Die Etablierung von Methoden, um ein zellbasiertes Therapeutikum produzieren zu k{\"o}nnen und die Entwicklung eines Modells zur Untersuchung von Verbrennungswunden. Zun{\"a}chst wurden hierf{\"u}r die Kulturbedingungen und -protokolle zur Isolation und Expansion von Keratinozyten so angepasst, dass sie g{\"a}ngigen Regularien zur Produktion medizinischer Produkte entsprechen. Hier zeigten die Zellen auch in anschließenden Analysen, dass charakteristische Merkmale nicht verloren hatten. Dar{\"u}ber hinaus gelang es, die Zellen mithilfe verschiedener protektiver Substanzen erfolgreich einzufrieren und zu konservieren. Des Weiteren konnte ein Modell etabliert werden, das eine Verbrennung ersten Grades widerspiegelt. {\"U}ber einen Zeitraum von zwei Wochen wurde seine Regeneration hinsichtlich verschiedener Aspekte, wie der Histomorphologie, dem Metabolismus und der Reepithelialisierungsrate, untersucht. Die Modelle zeigten hier viele Parallelen zur Wundheilung in vivo auf. Um die Eignung der Modelle zur Testung von Wirkstoffen zu ermitteln wurde außerdem eine Behandlung mit 5\% Dexpanthenol getestet. Sie resultierte in einer verbesserten Histomorphologie und einer erh{\"o}hten Anzahl an proliferativen Zellen in den Modellen, beschleunigte jedoch die Reepithelialisierung nicht. Zusammengefasst konnten in dieser Arbeit zun{\"a}chst Methoden etabliert werden, um ein medizinisches Produkt aus Keratinozyten herzustellen und zu charakterisieren. Außerdem wurde ein Modell entwickelt, anhand dessen die Wundheilung und Behandlung von Verbrennungen ersten Grades untersucht werden kann und welches als Basis zur Entwicklung von Modellen von tieferen Verbrennungen dienen kann.}, subject = {Tissue Engineering}, language = {de} } @phdthesis{Schmidt2021, author = {Schmidt, Stefanie}, title = {Cartilage Tissue Engineering - Comparison of Articular Cartilage Progenitor Cells and Mesenchymal Stromal Cells in Agarose and Hyaluronic Acid-Based Hydrogels}, doi = {10.25972/OPUS-25171}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251719}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Articular cartilage damage caused by sports accidents, trauma or gradual wear and tear can lead to degeneration and the development of osteoarthritis because cartilage tissue has only limited capacity for intrinsic healing. Osteoarthritis causes reduction of mobility and chronic pain and is one of the leading causes of disability in the elderly population. Current clinical treatment options can reduce pain and restore mobility for some time, but the formed repair tissue has mostly inferior functionality compared to healthy articular cartilage and does not last long-term. Articular cartilage tissue engineering is a promising approach for the improvement of the quality of cartilage repair tissue and regeneration. In this thesis, a promising new cell type for articular cartilage tissue engineering, the so-called articular cartilage progenitor cell (ACPC), was investigated for the first time in the two different hydrogels agarose and HA-SH/P(AGE-co-G) in comparison to mesenchymal stromal cells (MSCs). In agarose, ACPCs´ and MSCs´ chondrogenic capacity was investigated under normoxic (21 \% oxygen) and hypoxic (2 \% oxygen) conditions in monoculture constructs and in zonally layered co-culture constructs with ACPCs in the upper layer and MSCs in the lower layer. In the newly developed hyaluronic acid (HA)-based hydrogel HA-SH/P(AGE-co-G), chondrogenesis of ACPCs and MSCs was also evaluated in monoculture constructs and in zonally layered co-culture constructs like in agarose hydrogel. Additionally, the contribution of the bioactive molecule hyaluronic acid to chondrogenic gene expression of MSCs was investigated in 2D monolayer, 3D pellet and HA-SH hydrogel culture. It was shown that both ACPCs and MSCs could chondrogenically differentiate in agarose and HA-SH/P(AGE-co-G) hydrogels. In agarose hydrogel, ACPCs produced a more articular cartilage-like tissue than MSCs that contained more glycosaminoglycan (GAG), less type I collagen and only little alkaline phosphatase (ALP) activity. Hypoxic conditions did not increase extracellular matrix (ECM) production of ACPCs and MSCs significantly but improved the quality of the neo-cartilage tissue produced by MSCs. The creation of zonal agarose constructs with ACPCs in the upper layer and MSCs in the lower layer led to an ECM production in zonal hydrogels that lay in general in between the ECM production of non-zonal ACPC and MSC hydrogels. Even though zonal co-culture of ACPCs and MSCs did not increase ECM production, the two cell types influenced each other and, for example, modulated the staining intensities of type II and type I collagen in comparison to non-zonal constructs under normoxic and hypoxic conditions. In HA-SH/P(AGE-co-G) hydrogel, MSCs produced more ECM than ACPCs, but the ECM was limited to the pericellular region for both cell types. Zonal HASH/P(AGE-co-G) hydrogels resulted in a native-like zonal distribution of ECM as MSCs in the lower zone produced more ECM than ACPCs in the upper zone. It appeared that chondrogenesis of ACPCs was supported by hydrogels without biological attachment sites such as agarose, and that chondrogenesis of MSCs benefited from hydrogels with biological cues like HA. As HA is an attractive material for cartilage tissue engineering, and the HA-based hydrogel HA-SH/P(AGE-co-G) appeared to be beneficial for MSC chondrogenic differentiation, the contribution of HA to chondrogenic gene expression of MSCs was investigated. An upregulation of chondrogenic gene expression was found in 2D monolayer and 3D pellet culture of MSCs in response to HA supplementation, while gene expression of osteogenic and adipogenic transcription factors was not upregulated. MSCs, encapsulated in a HA-based hydrogel, showed upregulation of gene expression for chondrogenic, osteogenic and adipogenic differentiation markers as well as for stemness markers. In a 3D bioprinting process, using the HA-based hydrogel, gene expression levels of MSCs mostly did not change. Nevertheless, expression of three tested genes (COL2A1, SOX2, CD168) was downregulated in printed in comparison to cast constructs, underscoring the importance of closely monitoring cellular behaviour during and after the printing process. In summary, it was confirmed that ACPCs are a promising cell source for articular cartilage engineering with advantages over MSCs when they were cultured in a suitable hydrogel like agarose. The performance of the cells was strongly dependent on the hydrogel environment they were cultured in. The different chondrogenic performance of ACPCs and MSCs in agarose and HA-SH/P(AGE-co-G) hydrogels highlighted the importance of choosing suitable hydrogels for the different cell types used in articular cartilage tissue engineering. Hydrogels with high polymer content, such as the investigated HA-SH/P(AGE-co-G) hydrogels, can limit ECM distribution to the pericellular area and should be developed further towards less polymer content, leading to more homogenous ECM distribution of the cultured cells. The influence of HA on chondrogenic gene expression and on the balance between differentiation and maintenance of stemness in MSCs was demonstrated. More studies should be performed in the future to further elucidate the signalling functions of HA and the effects of 3D bioprinting in HA-based hydrogels. Taken together, the results of this thesis expand the knowledge in the area of articular cartilage engineering with regard to the rational combination of cell types and hydrogel materials and open up new possible approaches to the regeneration of articular cartilage tissue.}, subject = {Hyaliner Knorpel}, language = {en} } @phdthesis{Lodes2021, author = {Lodes, Nina Theresa}, title = {Tissue Engineering f{\"u}r seltene Erkrankungen mit St{\"o}rungen des mukozili{\"a}ren Transports}, doi = {10.25972/OPUS-20017}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200178}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Bei der zystischen Fibrose (CF) sowie der prim{\"a}ren Ziliendyskinesie (PCD) handelt es sich um zwei seltene Erkrankungen, die unter anderem den mukozili{\"a}ren Transport beeintr{\"a}chtigen. CF geh{\"o}rt hierbei zu den am h{\"a}ufigsten vorkommenden angeborenen Stoffwechselerkrankungen, wobei Betroffene unter einem Defekt des Cystic Fibrosis Transmembrane Conductor Regulator (CFTR)-Gens leiden, der durch die Produktion von hochviskosem Sekret in muzinproduzierenden Organen, wie dem gastrointestinalen Trakt und der Lunge, gekennzeichnet ist. Patienten, die an PCD leiden, weisen Defekte in, zum jetzigen Zeitpunkt, ca. 38 bekannten und PCD-assoziierten Genen auf, die in strukturellen Defekten des zili{\"a}ren Apparats und somit in dysfunktionalen Kinozilien resultieren. Da aktuell weder f{\"u}r die CF noch f{\"u}r die PCD eine Heilung m{\"o}glich ist, steht bei der Therapie vor allem die Linderung der Symptome im Fokus. Grundlegendes Ziel ist der langfristige Erhalt der Lungenfunktion sowie die Pr{\"a}vention bakterieller Infekte. Als bisherige Modellsysteme zur Erforschung m{\"o}glicher Therapeutika gelten Tiermodelle, die den humanen Ph{\"a}notyp aufgrund von Speziesdiversit{\"a}t nicht vollst{\"a}ndig abbilden k{\"o}nnen. Als vielversprechende Testsysteme f{\"u}r die zystische Fibrose gelten humane intestinale Organoidkulturen. Nachdem allerdings vorwiegend respiratorische Symptome f{\"u}r die Mortalit{\"a}t der Patienten verantwortlich sind, stellen CF-Atemwegsmodelle bessere Testsysteme f{\"u}r zuk{\"u}nftige Therapeutika dar. Atmungsorganoidkulturen wurden verwendet, um die CFTR-Funktionalit{\"a}t zu untersuchen, repr{\"a}sentieren aber nicht vollst{\"a}ndig die in vivo Situation. Deshalb werden zur Entwicklung neuer Therapiestrategien patientenspezifische 3D in vitro Testsysteme der humanen Atemwege ben{\"o}tigt, die insbesondere im Hinblick auf personalisierte Medizin ihren Einsatz finden. In der vorliegenden Arbeit wurde eine f{\"u}r den Lehrstuhl neue Methode zur Zellgewinnung aus nasalen Schleimhautabstrichen etabliert, die eine standardisierte Versorgung mit humanem Prim{\"a}rmaterial garantiert. Zur Generierung einer krankheitsspezifischen Zelllinie, wie beispielsweise einer PCD-Zelllinie mit Hilfe des CRISPR/Cas9-Systems, ist eine Atemwegszelllinie erforderlich, die die in vivo Situation vollst{\"a}ndig repr{\"a}sentiert. So wurden vier verschiedene respiratorische Epithelzelllinien (HBEC3-KT, Calu-3, VA10 und Cl-huAEC) auf ihren mukozili{\"a}ren Ph{\"a}notyp hin untersucht, wobei lediglich die Zelllinie HBEC3-KT in zilientragende Zellen differenzierte. Diese zeigten jedoch nur auf ca. 5 \% der Modelloberfl{\"a}che Kinozilien, wodurch die humane respiratorische Mukosa nicht komplett abgebildet werden konnte und die HBEC3-KT-Zelllinie keine geeignete Zelllinie zur Generierung einer PCD-Zelllinie darstellte. Mit Hilfe des Tissue Engineering war es m{\"o}glich, 3D in vitro Testsysteme basierend auf zwei unterschiedlichen Matrices, der biologischen SIS (small intestinal submucosa) und der synthetischen Polyethylenterephthalat (PET)-Membran, aufzubauen. Es wurden 3D Atemwegstestsysteme mit humanen prim{\"a}ren nasalen und tracheobronchialen Epithelzellen generiert. Erg{\"a}nzend zu histologischen Untersuchungen und zur Charakterisierung spezifischer Marker des respiratorischen Systems mittels Immunfluoreszenz, wurde die Ultrastruktur der Modelle, mit speziellem Fokus auf zili{\"a}re Strukturen, analysiert. Um R{\"u}ckschl{\"u}sse auf die zili{\"a}re Funktionalit{\"a}t ziehen zu k{\"o}nnen und somit eine hohe in vivo Korrelation zu best{\"a}tigen, wurde im Rahmen dieser Arbeit am Lehrstuhl f{\"u}r Tissue Engineering und Regenerative Medizin die Methode der Hochgeschwindigkeitsvideomikroskopie etabliert, welche die Analyse der Zilienschlagfrequenz sowie des mukozili{\"a}ren Transports erm{\"o}glicht. Ebenfalls wurde der Einfluss von isotoner Kochsalzl{\"o}sung und des � 2-adrenergen Agonisten Salbutamol, das vor allem als Bronchodilatator bei Asthmapatienten eingesetzt wird, auf die Zilienschlagfrequenz analysiert. Es konnte gezeigt werden, dass beide Substanzen den Zilienschlag im Atemwegsmodell erh{\"o}hen. Zur Generierung der Testsysteme der beiden seltenen Erkrankungen CF und PCD wurden Epithelzellen der betroffenen Patienten zun{\"a}chst mittels nicht-invasiver Raman-Spektroskopie auf einen potentiellen Biomarker untersucht, welcher Einsatz in der Diagnostik der beiden Krankheiten finden k{\"o}nnte. Es konnte jedoch weder f{\"u}r die CF noch f{\"u}r die PCD ein Biomarker aufgedeckt werden. Jedoch zeigten PCD-Zellen eine geringe Auftrennung gegen{\"u}ber nicht-PCD Zellen. Anschließend wurden 3D-Atemwegstestsysteme basierend auf Patientenzellen aufgebaut. Der Ph{\"a}notyp der CF-Modelle wurde mittels immunhistologischer F{\"a}rbung und der Analyse des gest{\"o}rten mukozili{\"a}ren Transports verifiziert. Strukturelle zili{\"a}re Defekte konnten durch die ultrastrukturelle Analyse von Zilienquerschnitten in drei donorspezifischen PCD-Modellen identifiziert werden. Dar{\"u}ber hinaus konnte die zili{\"a}re Funktionalit{\"a}t mit Hilfe der Hochgeschwindigkeitsvideomikroskopie nicht nachgewiesen werden. Zusammenfassend ist es in dieser Arbeit gelungen, eine neue Methode zur vollst{\"a}ndigen Charakterisierung von 3D-Atemwegstestsystemen zu etablieren, die die Analyse der Zilienschlagfrequenz sowie des mukozili{\"a}ren Transports erm{\"o}glicht. Es konnte erstmalig gezeigt werden, dass mit Hilfe des Tissue Engineering ein personalisiertes Krankheitsmodell f{\"u}r die PCD auf Segmenten eines dezellularisierten porzinen Jejunums generiert werden kann, das zuk{\"u}nftig ein Testsystem f{\"u}r potentielle Therapeutika darstellen kann.}, subject = {In-vitro-Kultur}, language = {de} } @phdthesis{Wallstabe2020, author = {Wallstabe, Julia}, title = {Induktion von GvHD-artigen Gewebesch{\"a}den an humanen artifiziellen Hautmodellen}, doi = {10.25972/OPUS-15396}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153966}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Graft-versus-Host Disease (GvHD) stellt einen h{\"a}ufigen, den Gesamterfolg einer allogenen h{\"a}matopoetischen Stammzelltransplantation limitierenden Faktor dar. Bei dieser Komplikation attackieren vor allem alloreaktive T-Lymphozyten des Stammzellspenders gesunde K{\"o}rperzellen des Patienten. Infolgedessen kommt es zu Gewebesch{\"a}den in den Zielorganen Haut, Leber und Darm. Die Behandlung der GvHD erfordert eine effektive Immunsuppression, was wiederum Graft-versusTumor-Effekte kompromittiert und den R{\"u}ckfall der malignen Grunderkrankung bedingen kann. Viele Patienten sprechen aus bisher ungekl{\"a}rten Gr{\"u}nden nicht auf die klassische immunsuppressive Therapie mit Steroiden oder second-line Therapien an. Neue zellul{\"a}re Therapien zur Behandlung der refrakt{\"a}ren GvHD sind auf dem Vormarsch, bed{\"u}rfen aber einer weiterf{\"u}hrenden klinischen Testung, auch um die exakten Wirkungsmechanismen zu verstehen. Idealerweise k{\"o}nnten neue Testsysteme das GvHD-Potential von allogenen Stammzellpr{\"a}paraten oder aber das immunsuppressive Potential von neuen GvHD-Therapien vorhersagen, bevor diese in klinischen Studien eingesetzt werden. Ziel der vorliegenden Arbeit war es, ein erstes, in multiplen Replikaten einsetzbares, humanes organotypisches Gewebemodell zur Simulation einer GvHD-Reaktion am Beispiel der Haut zu etablieren. Zu diesem Zweck wurden artifizielle humane Hautmodelle unter statischen (KollagenHautmodelle) und dynamischen Kulturbedingungen (vaskularisierte Hautmodelle) generiert. Die Injektion unstimulierter PBMCs (engl. peripheral blood mononuclear cells) f{\"u}hrte zu keinen histomorphologischen Ver{\"a}nderungen in den KollagenHautmodellen. Im Gegensatz dazu hatte die Injektion vorstimulierter allogener PBMCs eine Zerst{\"o}rung der epidermalen Strukturen der Kollagen-Hautmodelle zur Folge, welche vergleichbar waren mit Gewebesch{\"a}den bei einer akuten GvHD der Haut. Dieselben Sch{\"a}digungen der Epidermis wurden durch die Injektion von Medium{\"u}berst{\"a}nden vorstimulierter PBMCs in die Kollagen-Hautmodelle erreicht. Im Kulturmedium der Kollagen-Hautmodelle wurden hohe Konzentrationen von Interleukin 2 und 17, Interferon gamma sowie Tumornekrosefaktor alpha gemessen, wodurch auf die Beteiligung von Zytokinen an der inflammatorischen Reaktion geschlossen werden konnte. Auch im komplexeren vaskularisierten Hautmodell verursachte die Injektion vorstimulierter PBMCs histomorphologische Ver{\"a}nderungen entsprechend einer akuten Haut-GvHD sowie einen zeitabh{\"a}ngigen Anstieg proinflammatorischer Zytokine. Zusammenfassend zeigen die Resultate dieser Arbeit, dass die Induktion einer starken Inflammations- und Immunreaktion in artifiziellen humanen Hautmodellen, welche histomorphologisch eine GvHD imitiert, m{\"o}glich ist. Dieses Modell k{\"o}nnte als Grundlage f{\"u}r die Entwicklung eines klinisch relevanten Testsystems zur Bestimmung des GvHD-Restpotentials oder zur Festlegung der immunsuppressiven Kapazit{\"a}t innovativer Zellpr{\"a}parate dienen. Somit k{\"o}nnten humane artifizielle GvHDModelle in klinischen Studien eingesetzt werden und die Erfahrungen aus Tiermodellen erg{\"a}nzen sowie erste in vitro Ergebnisse im humanen System liefern, welche dann mit dem tats{\"a}chlichen klinischen Resultat verglichen werden k{\"o}nnten.}, subject = {Graft-versus-host-disease}, language = {de} } @phdthesis{Wiesner2020, author = {Wiesner, Miriam}, title = {Stem Cell-based Adipose Tissue Engineering - Engineering of Prevascularized Adipose Tissue Constructs In Vitro \& Investigation on Gap Junctional Intercellular Communication in Adipose-derived Stem Cells}, doi = {10.25972/OPUS-18500}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-185005}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {In reconstructive and plastic surgery, there exists a growing demand of adequate tissue implants, since currently available strategies for autologous transplantation are limited by complications including transplant failure and donor site morbidity. By developing in vitro and in vivo autologous substitutes for defective tissue sites, adipose tissue engineering can address these challenges, although there are several obstacles to overcome. One of the major limitations is the sufficient vascularization of in vitro engineered large constructs that remains crucial and demanding for functional tissues. Decellularized jejunal segments may represent a suitable scaffolding system with preexisting capillary structures that can be repopulated with human microvascular endothelial cells (hMVECs), and a luminal matrix applicable for the adipogenic differentiation of human adipose-derived stem cells (hASCs). Hence, co-culture of these cells in jejunal segments, utilizing a custom-made bioreactor system, was characterized in terms of vascularization and adipose tissue development. Substantial adipogenesis of hASCs was demonstrated within the jejunal lumen in contrast to non-induced controls, and the increase of key adipogenic markers was verified over time upon induction. The development of major extracellular matrix components of mature adipose tissue, such as laminin and collagen IV, was shown within the scaffold in induced samples. Successful reseeding of the vascular network with hMVECs was demonstrated in long-term culture and co-localization of vascular structures and adipogenically differentiated hASCs was observed. Therefore, these results represent a novel approach for in vitro engineering of vascularized adipose tissue constructs that warrants further investigations in preclinical studies. Another still existing obstacle in adipose tissue engineering is the insufficient knowledge about the applied cells, for instance the understanding of how cells can be optimally expanded and differentiated for successful engineering of tissue transplants. Even though hASCs can be easily isolated from liposuction of abdominal fat depots, yielding low donor site morbidity, huge numbers of cells are required to entirely seed complex and large 3D matrices or scaffolds. Thus, cells need to be large-scale expanded in vitro on the premise of not losing their differentiation capacity caused by replicative aging. Accordingly, an improved differentiation of hASCs in adipose tissue engineering approaches remains still desirable since most engineered constructs exhibit an inhomogeneous differentiation pattern. For mesenchymal stem cells (MSCs), it has been shown that growth factor application can lead to a significant improvement of both proliferation and differentiation capacity. Especially basic fibroblast growth factor (bFGF) represents a potent mitogen for MSCs, while maintaining or even promoting their osteogenic, chondrogenic and adipogenic differentiation potential. As there are currently different contradictory information present in literature about the applied bFGF concentration and the explicit effect of bFGF on ASC differentiation, here, the effect of bFGF on hASC proliferation and differentiation capacity was investigated at different concentrations and time points in 2D culture. Preculture of hASCs with bFGF prior to adipogenic induction showed a remarkable effect, whereas administration of bFGF during culture did not improve adipogenic differentiation capacity. Furthermore, the observations indicated as mode of action an impact of this preculture on cell proliferation capacity, resulting in increased cellular density at the time of adipogenic induction. The difference in cell density at this time point appeared to be pivotal for increased adipogenic capacity of the cells, which was confirmed in a further experiment employing different seeding densities. Interestingly, furthermore, the obtained results suggested a cell-cell contact-mediated mechanism positively influencing adipogenic differentiation. As a consequence, subsequently, studies were conducted focusing on intercellular communication of these cells, which has hardly been investigated to date. Despite the multitude of literature on the differentiation capacity of ASCs, little is reported about the physiological properties contributing to and controlling the process of lineage differentiation. Direct intercellular communication between adjacent cells via gap junctions has been shown to modulate differentiation processes in other cell types, with connexin 43 (Cx43) being the most abundant isoform of the gap junction-forming connexins. Thus, in the present study we focused on the expression of Cx43 and gap junctional intercellular communication (GJIC) in hASCs, and its significance for adipogenic differentiation of these cells. Cx43 expression in hASCs was demonstrated histologically and on the gene and protein expression level and was shown to be greatly positively influenced by cell seeding density. Functionality of gap junctions was proven by dye transfer analysis in growth medium. Adipogenic differentiation of hASCs was shown to be also distinctly elevated at higher cell seeding densities. Inhibition of GJIC by 18α-glycyrrhetinic acid significantly compromised adipogenic differentiation, as demonstrated by histology, triglyceride quantification, and adipogenic marker gene expression. Flow cytometry analysis showed a lower proportion of cells undergoing adipogenesis when GJIC was inhibited, further indicating the importance of GJIC in the differentiation process. Altogether, these results demonstrate the impact of direct cell-cell communication via gap junctions on the adipogenic differentiation process of hASCs and may contribute to further integrate direct intercellular crosstalk in rationales for tissue engineering approaches.}, subject = {Tissue Engineering}, language = {en} } @phdthesis{Nelke2019, author = {Nelke, Lena}, title = {Establishment and optimization of 3-dimensional mamma carcinoma models for therapy simulation and drug testing}, doi = {10.25972/OPUS-17228}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172280}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Breast cancer is the most common cancer among women worldwide and the second most common cause of cancer death in the developed countries. As the current state of the art in first-line drug screenings is highly ineffective, there is an urgent need for novel test systems that allow for reliable predictions of drug sensitivity. In this study, a tissue engineering approach was used to successfully establish and standardize a 3-dimensional (3D) mamma carcinoma test system that was optimized for the testing of anti-tumour therapies as well as for the investigation of tumour biological issues. This 3D test system is based on the decellularised scaffold of a porcine small intestinal segment and represents the three molecular subsets of oestrogen receptor-positive, HER2/Neu-overexpressing and triple negative breast cancer (TNBC). The characterization of the test system with respect to morphology as well as the expression of markers for epithelial-mesenchymal transition (EMT) and differentiation indicate that the 3D tumour models cultured under static and dynamic conditions reflect tumour relevant features and have a good correlation with in vivo tumour tissue from the corresponding xenograft models. In this respect, the dynamic culture in a flow bioreactor resulted in the generation of tumour models that exhibited best reflection of the morphology of the xenograft material. Furthermore, the proliferation indices of 3D models were significantly reduced compared to 2-dimensional (2D) cell culture and therefore better reflect the in vivo situation. As this more physiological proliferation index prevents an overestimation of the therapeutic effect of cytostatic compounds, this is a crucial advantage of the test system compared to 2D culture. Moreover, it could be shown that the 3D models can recapitulate different tumour stages with respect to tumour cell invasion. The scaffold SISmuc with the preserved basement membrane structure allowed the investigation of invasion over this barrier which tumour cells of epithelial origin have to cross in in vivo conditions during the process of metastasis formation. Additionally, the data obtained from ultrastructural analysis and in situ zymography indicate that the invasion observed is connected to a tumour cell-associated change in the basement membrane in which matrix metalloproteinases (MMPs) are also involved. This features of the model in combination with the mentioned methods of analysis could be used in the future to mechanistically investigate invasive processes and to test anti-metastatic therapy strategies. The validation of the 3D models as a test system with respect to the predictability of therapeutic effects was achieved by the clinically relevant targeted therapy with the monoclonal antibody trastuzumab which induces therapeutic response only in patients with HER2/Neu-overexpressing mamma carcinomas due to its specificity for HER2. While neither in 2D nor in 3D models of all molecular subsets a clear reduction of cell viability or an increase in apoptosis could be observed, a distinct increase in antibody-dependent cell-mediated cytotoxicity (ADCC) was detected only in the HER2/NEU-overexpressing 3D model with the help of an ADCC reporter gene assay that had been adapted for the application in the 3D model in the here presented work. This correlates with the clinical observations and underlines the relevance of ADCC as a mechanism of action (MOA) of trastuzumab. In order to measure the effects of ADCC on the tumour cells in a direct way without the indirect measurement via a reporter gene, the introduction of an immunological component into the models was required. This was achieved by the integration of peripheral blood mononuclear cells (PBMCs), thereby allowing the measurement of the induction of tumour cell apoptosis in the HER2/Neu-overexpressing model. Hence, in this study an immunocompetent model could be established that holds the potential for further testing of therapies from the emergent field of cancer immunotherapies. Subsequently, the established test system was used for the investigation of scientific issues from different areas of application. By the comparison of the sensitivity of the 2D and 3D model of TNBC towards the water-insoluble compound curcumin that was applied in a novel nanoformulation or in a DMSO-based formulation, the 3D test system was successfully applied for the evaluation of an innovative formulation strategy for poorly soluble drugs in order to achieve cancer therapy-relevant concentrations. Moreover, due to the lack of targeted therapies for TNBC, the TNBC model was applied for testing novel treatment strategies. On the one hand, therapy with the WEE1 kinase inhibitor MK 1775 was evaluated as a single agent as well as in combination with the chemotherapeutic agent doxorubicin. This therapy approach did not reveal any distinct benefits in the 3D test system in contrast to testing in 2D culture. On the other hand, a novel therapy approach from the field of cellular immunotherapies was successfully applied in the TNBC 3D model. The treatment with T cells that express a chimeric antigen receptor (CAR) against ROR1 revealed in the static as well as in the dynamic model a migration of T cells into the tumour tissue, an enhanced proliferation of T cells as well as an efficient lysis of the tumour cells via apoptosis and therefore a specific anti-cancer effect of CAR-transduced T cells compared to control T cells. These results illustrate that the therapeutic application of CAR T cells is a promising strategy for the treatment of solid tumours like TNBC and that the here presented 3D models are suitable for the evaluation and optimization of cellular immunotherapies. In the last part of this work, the 3D models were expanded by components of the tumour stroma for future applications. By coculture with fibroblasts, the natural structures of the intestinal scaffold comprising crypts and villi were remodelled and the tumour cells formed tumour-like structures together with the fibroblasts. This tissue model displayed a strong correlation with xenograft models with respect to morphology, marker expression as well as the activation of dermal fibroblasts towards a cancer-associated fibroblast (CAF) phenotype. For the integration of adipocytes which are an essential component of the breast stroma, a coculture with human adipose-derived stromal/stem cells (hASCs) which could be successfully differentiated along the adipose lineage in 3D static as well as dynamic models was established. These models are suitable especially for the mechanistic analysis of the reciprocal interaction between tumour cells and adipocytes due to the complex differentiation process. Taken together, in this study a human 3D mamma carcinoma test system for application in the preclinical development and testing of anti-tumour therapies as well as in basic research in the field of tumour biology was successfully established. With the help of this modular test system, relevant data can be obtained concerning the efficacy of therapies in tumours of different molecular subsets and different tumour stages as well as for the optimization of novel therapy strategies like immunotherapies. In the future this can contribute to improve the preclinical screening and thereby to reduce the high attrition rates in pharmaceutical industry as well as the amount of animal experiments.}, subject = {Brustkrebs}, language = {en} }