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The Role of Clonal Evolution and Anti- Apoptotic Dependencies in Cancer Progression and Therapy Resistance: Implications for Personalized Medicine
No full textCancer is characterized by significant heterogeneity, both across and within individual tumors, posing challenges for treatment. This thesis investigates clonal evolution (CE) and its association with apoptotic mechanisms to enhance our understanding of therapy resistance and improve strategies for patient stratification and personalized therapeutic approaches. By integrating genetic and functional profiling, this work focuses on hematologic malignancies, but also provides an example in thymoma to advance personalized treatment approaches.
With the identification of distinct mechanisms conferring resistance to apoptotic cell death in cancer and emerging strategies to target these mechanisms, identification of targetable vulnerabilities within the apoptotic machinery is a specific focus of this thesis. In a case series of T-large granular lymphocytic leukemia (T-LGLL), we assessed targetable anti-apoptotic mechanisms of malignant T-LGLL clones versus the same patients’ healthy T-cells by flow-cytometry based selective BH3 profiling. This approach revealed functional heterogeneity across patients and uncovered a dominant dependence on the anti-apoptotic protein MCL-1 in individual cases, independent of the specific genetic background of the disease. The potential therapeutic relevance of this MCL-1 dependence was further corroborated by ex vivo treatment with an MCL-1 specific inhibitor and thus suggests an approach towards personalized treatment in T-LGLL.
The second study employed functional apoptosis profiling to assess apoptotic dependencies in thymic epithelial tumors and thus demonstrates the feasibility of applying functional apoptosis profiling in solid malignancies. Our findings revealed heterogeneous reliance on anti-apoptotic proteins such as MCL-1 and BCL-xL. Single-agent therapies were insufficient to induce effective apoptosis, but combination strategies successfully targeted tumor survival mechanisms, underscoring the value of personalized approaches for these chemoresistant tumors.
Next, functional apoptosis profiling was used in T-cell prolymphocytic leukemia (T-PLL) to support drug combination strategies and identified synergistic drug combinations, including cladribine and idasanutlin, which was validated in patient-derived xenograft models.
Together, these three studies provide examples of successful pre-clinical identification of targetable vulnerabilities across different entities and different genetic backgrounds.
Focusing on genetics in myeloid neoplasms, we characterized clonal dynamics (CD) in lower-risk myelodysplastic syndromes (LR-MDS) using comprehensive longitudinal genetic data. We identified diverse patterns of CE, ranging from stable clones to dynamic shifts in clonal architecture. Interestingly, CE did not directly correlate with adverse outcomes in LR-MDS, likely due to the absence of aggressive genetic alterations. Timely application of disease-modifying therapies mitigated the impact of CD, emphasizing the importance of early therapeutic intervention.
Ultimately, we analyzed treatment-associated clonal evolution and anti-apoptotic dependencies in patient samples of high-risk myelodysplastic neoplasms and acute myeloid leukemia. In patients treated with the hypomethylating agent azacitidine and the BCL-2 inhibitor venetoclax, we identified a highly significant correlation of pre-treatment BCL-2 dependence by BH3 profiling with molecular response and improved overall survival. Furthermore, this study characterized clonal dynamics during treatment and associated changes in anti-apoptotic dependencies. Importantly, this approach revealed targetable mechanisms of resistance in individual patients.
Overall, this thesis highlights the pivotal role of CE and apoptotic dependencies in therapy resistance, emphasizing the need for intensified and development of innovative and more effective personalized diagnostics and treatment strategies. Future studies should focus on expanding these approaches to validate their efficacy in larger, more comprehensive patient cohorts.2026-02-2
Antibody-mediated recognition of central nervous system antigen as therapeutic target
No full textThe discovery of autoantibodies directed against myelin oligodendrocyte glycoprotein (MOG) in a proportion of patients diagnosed with multiple sclerosis (MS) led to the definition of MOG antibody-associated disease (MOGAD) as a separate disorder. Although the pathogenesis of this demyelinating disorder of the central nervous system (CNS) is not yet fully understood, it is becoming increasingly clear that the mechanisms involved are different from those of MS. The discovery of the CNS lymphatic network together with the detection of myelin in cervical lymph nodes (cLN) of MS patients and healthy controls led to the hypothesis that degraded myelin might be drained to cLN, where antigen presentation and activation of autoreactive CNS-specific T cells through myeloid cells takes place. Furthermore, in an animal model mimicking MOGAD, where mice contain MOG-specific T cells, the addition of anti-MOG antibodies triggers the development of inflammatory CNS demyelination probably caused by enhanced antigen recognition by myeloid cells through opsonization.
To date, there is no approved therapeutic option for the treatment of MOGAD available. Emerging strat-egies are mainly focused on reducing anti-MOG antibodies in order to reduce their pathogenic function. However, to have a more lasting therapeutic effect, it might be important to interfere with pathogenic B cells before they differentiate into antibody-producing plasma cells. Furthermore, targeting antigen-presenting myeloid cells to reduce the activation of CNS-specific T cells might also be an interesting therapeutic approach. Hence, the inhibition of the Bruton´s tyrosine kinase (BTK) is an emerging treat-ment strategy that has been shown to fulfill a pleiotropic immunomodulatory effect on both antibody-producing B cells and antibody-recognizing myeloid cells. Based on this, its immunomodulatory effects were investigated as a therapeutic option for antibody-mediated diseases such as MOGAD.
The modulatory effects of the BTK inhibitor evobrutinib was evaluated both in an in vivo setting with and without an inflammatory environment. The obtained results showed a general modulatory effect on BTK-expressing B cells by preventing their maturation while fostering regulatory properties. Furthermore, evobrutinib treatment limited the pro-inflammatory activity of monocytes and macrophages. These im-mune modulatory effects were also maintained in the inflammatory context.
Furthermore, we were interested in understanding the underlying mechanisms of BTK inhibition. Anti-bodies enhance antigen internalization by myeloid cells in a Fc receptor (FcR)-dependent manner via a BTK-dependent signaling pathway. Therefore, the impact of various BTK inhibitors on the phenotype and function of human monocyte-derived macrophages were investigated following FcR-stimulation. Indeed, all BTK inhibitors reduced the production of the pro-inflammatory cytokines TNF-α and IL-6, while in our setting no effect could be observed on the expression of molecules associated with antigen presentation. In conjunction, our findings highlight BTK inhibitors as a promising therapeutic approach for MOGAD, especially by limiting IL-6 production while fostering regulatory properties in B cells.2026-03-1
Real-time MRI for the evaluation of respiratory impairment in patients with Pompe disease
No full textRespiratory dysfunction is a major contributor to morbidity and mortality in patients suffering from Pompe disease. Standard diagnostic methods, which aim to evaluate diaphragm weakness, often diagnose it at a later stage due to compensatory mechanisms that can conceal early symptoms of respiratory involvement. This dissertation aims to evaluate real-time MRI (RT-MRI) for characterizing breathing patterns in patients with late-onset Pompe disease (LOPD) compared to standard diagnostic tools such as spirometry, body plethysmography, and diaphragm sonography.
Ten patients with LOPD and ten matched controls underwent 3 Tesla RT-MRI at 20 frames per second. Natural breathing mechanisms and dynamic respiratory maneuvers (sniff) were characterized and quantified manually as well as via U-Net based segmentation and subsequent analysis. MRI measurements were compared with pulmonary function tests and ultrasound of the diaphragm.
The novel RT-MRI method allowed for the precise quantification of diaphragmatic weakness in LOPD patients, as well as compensatory mechanisms such as increased thoracic movement during the sniff maneuver. U-Net-based segmentation enabled the analysis of diaphragmatic/thoracic synchronicity, diaphragmatic/thoracic velocity during sniff maneuvers, and paradoxical diaphragm movement.
Thus, RT-MRI has proven to be an efficient and detailed tool for quantifying respiratory muscle function. It holds promising potential for improving the detection and monitoring of breathing impairment in patients suffering from neuromuscular diseases.2026-03-3
Evaluation of the Scapholunate Joint Space Width Using Real-Time MRI
No full textThe scapholunate interosseous ligament plays a crucial role in maintaining wrist stability, and its dysfunction can lead to instability and progressive joint degeneration. This study investigates the use of real-time MRI to dynamically evaluate the scapholunate joint during wrist motion, aiming to assess its diagnostic utility and effectiveness in comparison to static imaging modalities. Ten healthy participants underwent real-time MRI scans during wrist abduction/adduction and fist-clenching maneuvers. Measurements were performed at proximal, medial, and distal landmarks on both dynamic and static images. Statistical analyses were conducted to assess the reliability of measurements at each landmark and to compare dynamic data with established static imaging results. Additionally, inter- and intraobserver variabilities were analyzed. Among the landmarks, medial measurements showed the highest concordance with static images and the lowest measurement variability. Distal measurements demonstrated a similar level of agreement but exhibited higher variability. Proximal measurements, however, showed significant deviations along with greater scatter. While no significant differences were found between abduction/adduction and fist-clenching maneuvers, notable inter- and intraobserver variabilities were observed across all measurement points. This study underscores the potential of real-time MRI for dynamic evaluation of the scapholunate joint, particularly at the medial landmark. Nevertheless, challenges such as variability in measurements persist. Future efforts focused on standardization and the integration of advanced image processing techniques could improve the precision and reliability of real-time MRI, promoting its adoption in clinical applications for dynamic wrist imaging.2025-02-2
Coping mechanisms and dynamics as predictors of quality of life and somatic outcome in subjects with heart failure or risk factors for heart failure
No full textChronic heart failure is a highly prevalent disease. For the patients affected heart failure is associated with poor prognosis and, in most cases, a reduced quality of life. Some widespread diseases are considered risk factors for the development of heart failure. To deal with the ongoing stress of a chronic illness, coping strategies are used. The aim of this study was to analyze the prognostic value of different coping strategies and the variability within coping for quality of life and somatic outcome.
The work is based on data from the longitudinal, observational DIAST-CHF study. The analysis included 1536 subjects who either had cardiovascular risk factors or were diagnosed with heart failure. Data was collected at baseline, after one, two, five and ten years. Coping was investigated using the Freiburg Questionnaire for Coping with Illness. To quantify coping variability, a contrast value was calculated from the scores of the individual strategies. Depending on the change in this contrast value over the first three timepoints, the subjects were divided into groups with and without coping variability. In addition, the maximum delta of the contrast value at the first three timepoints was used to quantify the coping variability. Health-related quality of life was assessed using Short Form-36 Health Survey. Somatic outcome was quantified by incidence of major adverse cardiac and cerebrovascular events and 6-minute-walking-distance as a measure of physical performance.
Correlation and multiple linear regression analyses showed a significant prediction of health-related quality of life after five and ten years by coping strategies of the first two years of observation. Changes in the psychosocial quality of life could be explained to a greater extent by coping than variances in somatic quality of life.
The results showed correlations between depressive coping and coping by minimization and wishful thinking at the first three timepoints with lower health-related quality of life over the course of the illness. After controlling for the initial quality of life depressive coping predicted a worse psychosocial and somatic quality of life after five – but not ten – years. After adjustment, minimization and wishful thinking predicted psychosocial quality of life after five and ten years - but not somatic quality of life. Active problem-orientated coping could not significantly predict health-related quality of life after adjustment.
Minimization and wishful thinking was the only coping mechanism that significantly predicted an increased risk for cardiac and cerebrovascular events in the Cox regression and reduction in 6-minute-walking-dsitance at five years. The other coping strategies did not significantly predict somatic outcome.
Coping variability showed no favorable or unfavorable correlation with the health-related quality of life or somatic outcome. Mixed linear models showed no significant differences in health-related quality of life or 6-minute-walking distance between the groups with and without coping variability. Also, incidence of cardiac and cerebrovascular events, which was analyzed using Cox regression analysis with the addition of covariates, did not differ between variable and non-variable coping groups. The maximum delta of the contrast value also showed no significant predictive value for quality of life or event risk in multiple linear regression.
This study showed an association of depressive coping and minimization and wishful thinking with worse long-term quality of life in people at risk of heart failure or with manifest heart failure. Minimization and wishful thinking also predicted worse somatic outcome. Early screenings for depressive coping or coping by minimization and wishful thinking as well as interventions to reduce the use of these strategies could therefore contribute to a long-term improvement in mental and physical outcome. Further studies which examine the predictive value of minimization and wishful, which investigate the underlying pathophysiological mechanisms, or which investigate the benefit of interventions reducing minimization and wishful thinking and depressive coping could contribute to a deeper understanding of the role of coping in cardiological populations.2025-03-1
Studies on the influence of shear stress, inflammation and BET inhibition on human epithelial cells
No full textAtherosclerosis is an important cause of some of the most common causes of death, cardiovascular disease. In addition to dyslipidemia, inflammation is one of the key causes of remodeling processes in the endothelium, which can ultimately lead to the final stage of the disease with plaque formation and atherothrombotic occlusion. One possible therapeutic approach, which is being considered for a number of diseases with an inflammatory component, is the inhibition of bromodomain and extraterminal domain (BET) proteins with corresponding inhibitors (BETi). BET proteins are important transcriptional regulator proteins. They bind to histones and transcription factors and serve as transcriptional cofactors. In addition, lipid-lowering properties have already been described for a BETi and it has already been clinically tested. Although BETis are in clinical trials for various diseases, their exact effect has not been fully characterized due to the broad influence that BET proteins have on transcriptional control and the inflammatory cascade. It is therefore useful to identify proteins and signaling pathways relevant to the pathogenesis of atherosclerosis and the effects of BETis. This promotes understanding of the disease and enables new therapeutic approaches.In this way, potential targets for precision therapies can be identified in order to replicate the positive effects of BETi administration, but also potential weaknesses and side effects of the therapy can be identified.
The aim of this work was therefore to create an experimental basis to test and better understand the therapy with BETis in an in vitro model of atherosclerosis. To achieve this, a model was established in which endothelial shear stress (ESS) could be applied using a viscometer in order to approximate the intravascular conditions in the arterial vascular system. Human umbilical vein cells (HUVECs) were chosen for this purpose. In this setting, an inflammation model was established to simulate an environment typical in the initial phase of atherosclerotic diseases. The cells were then treated with the non-selective BETi JQ1 under these conditions. The model was validated in different steps using microscopic morphology, immunofluorescence microscopy, quantitative real-time PCR (qRT-PCR) and electrochemiluminescence immunoassay (ECLIA). Finally, the different established treatment methods were combined and tested against each other in eight different treatment groups. Two control groups with and without ESS were created, as well as two groups in which the cells were put into an inflammatory state, with and without ESS. Furthermore, two groups were treated with JQ1 only, with and without ESS, and finally cells in two groups were treated with JQ1 and an inflammatory state was induced, with and without ESS.
To gain a comprehensive insight into the changes in the treated cells, the use of data-independent acquisition-mass spectrometry (DIA-MS) was chosen. After creating a library, this makes it possible to make a reliable quantitative statement about changes in the overall proteome of the cells.
4037 proteins were identified, of which 3316 were available for further statistical analysis after rigorous statistical filtering. For the first time, global, unbiased proteomic data of HUVEC cells under the influence of ESS, BET inhibition with JQ1 and inflammation were generated. Significantly differentially expressed proteins were found when comparing the above treatment groups. Using ANOVA-like tests, 1815 differentially expressed proteins were identified across all treatment groups. When comparing the two untreated control groups with and without ESS, 35 differentially expressed proteins were found, among which the overexpressed sodium-hydrogen exchanger 3 regulator 2 (SLC9A3R2), a scaffold protein thought to have a protective function against hypertension, stood out. In addition, the significantly overrepresented signaling pathways fluid shear stress and atherosclerosis and complement and coagulation cascades were identified. A comparison of the non-sheared control group with the group treated with JQ1 alone revealed 962 differentially expressed proteins. In particular, the inhibition of Ras-related protein Rab-5B (Rab5B), a protein that plays a role in angiogenesis, was described. In addition, the treatment showed a particular influence on the signaling pathways regulated exocytosis, response to wounding and cell activation.When comparing the group in which inflammation was induced under ESS with the group in which an attempt was made to suppress this effect under ESS with JQ1, 955 differentially expressed proteins were found. In particular, an additive effect of ESS and JQ1 on the expression of SLC9A3R2 was found, which underlines the atheroprotective potential of BET inhibition in vessels. Furthermore, 186 significantly enriched signaling pathways affecting cell cycle, transcription and metabolic processes were identified. In particular, prolyl 4-hydroxylase subunit alpha-1 (P4HA1), whose increased suppression under TNFα and JQ1 and ESS could possibly lead to dysregulation of the extracellular matrix and inhibition of angiogenesis. Furthermore, the overexpression of high mobility group protein B1 (HMGB1) under TNFα and JQ1 and ESS should be noted, which may support an inflammatory state and induce increased cell death. These are potentially negative findings. The suppression of Sorbin And SH3 Domain Containing 2 (SORBS2) should also be noted. Together with other studies, this may illustrate the therapeutic potential of BETis in diabetics.
In summary, this work was the first to investigate the impact of shear stress, inflammation and BET inhibition on human endothelial cells in a very broad, non-targeted proteomic approach. A library of 4037 proteins was generated. Significantly altered proteins were also identified for different treatment groups and possible induced or suppressed signaling pathways can be identified. These data may form the basis for identifying potential candidates for diagnostic or prognostic biomarkers or for possible therapeutic approaches in the treatment of atherosclerosis and/or for understanding the pathophysiology of shear stress and JQ1 treatment on endothelial cells. Finally, potentially deleterious effects of JQ1 could be demonstrated, which in turn calls for further, more detailed research on the impact of BETi therapy on cardiovascular disease.2025-02-1
Sportliche Aktivität: Häufigkeiten und Assoziationen mit kardiovaskulären Risikofaktoren, Ernährungsverhalten und psychischem Wohlbefinden in einer Nordwest-Deutschen Kohortenstudie
No full textPhysical activity is closely linked to cardiovascular risk factors, dietary behavior, and psychological well-being, as demonstrated by the northwestern German ELITE study involving over 4,600 participants. The aim of the study was to reduce health risk factors and improve quality of life through individualized prevention recommendations. Regular exercise proved to be crucial, as it was associated with improved health parameters such as lower blood pressure and better cholesterol levels. Active participants were less likely to suffer from obesity, diabetes, and nicotine use, consumed more fruits and less meat, and experienced reduced stress and depression.
Based on their activity levels, participants were divided into three groups: highly active, moderately active, and minimally active. Only 41% met the WHO’s exercise recommendations. Less active individuals were significantly more likely to exhibit multiple risk factors simultaneously. The findings highlight the importance of physical activity for both the prevention of cardiovascular diseases and the promotion of psychological well-being. They emphasize the need to develop innovative approaches, such as team sports and integrating movement into daily routines, to motivate less active individuals and make the health benefits of exercise more widely accessible.2025-03-2
Exploring the role of Cytoglobin (CYGB) in melanoma pathobiology
No full textSkin cancer is one of the most common malignancies worldwide, with melanoma being the deadliest form. Although both targeted therapies inhibiting the MAPK pathway and immunotherapy have improved the prognosis of patients significantly, disease relapse resulting from drug resistance is still the major challenge in melanoma treatment. Therefore, the identification and functional classification of new therapeutic targets in melanoma is essential. In this study, we identified Cytoglobin (CYGB), a protein from the globin family which is commonly downregulated during melanocyte malignant transformation, as a potential therapeutic target for melanoma treatment. We characterized the role of CYGB in melanoma by inducing CYGB knock-down in three different melanoma cell lines. Our results indicate that upon CYGB depletion melanoma cells display decreased colony formation, diminished global translation and increased susceptibility to the thioredoxin reductase (TRXR) inhibitor Auranofin. However, CYGB expression had no effect on the efficacy pf BRAF and MEK inhibitors Vemurafenib and Trametinib as well as NK cell- mediated cytotoxicity. Taken together, our findings indicate that CYGB controls melanoma cell metabolic state as well as melanoma cell phenotype. Further studies are warranted in elucidating the therapeutic potential of CYGB in melanoma and other cancer entities.2026-02-1
Suizidalität bei Depressionen unter repetitiver transkranieller Magnetstimulation
No full textSuicidality is a serious global public health problem and is closely associated with depression. There is a need for effective anti-suicidal therapies. rTMS shows promising potential as a modern form of therapy. Due to the still very limited evidence on this connection, there is a need for further research. Thus, the primary research objective of the present project was to investigate the effect of rTMS on suicidality in depressive patients. Two research questions were to be answered. Firstly, how the suicidality of test subjects changes after the intervention compared to the initial situation. Secondly, whether the use of rTMS shows greater anti-suicidal effectiveness than the use of conventional pharmacotherapy in a direct sample comparison. As part of a crossover study, a sample of 54 depressives underwent an iTBS intervention (5Hz trains with 50Hz bursts). The data was collected in clinical interviews using standardized measurement instruments. The suicidality of the participants was significantly reduced. However, there were no significant differences between the two study arms. In a similarly composed sample of 33 depressive subjects undergoing conventional antidepressant pharmacotherapy, a comparable anti-suicidal effect was shown. The secondary research objective of this project was to identify associations between an anti-suicidal treatment effect after rTMS intervention in depressive subjects deep clinical risk factors for suicidality. Lifetime suicidality (suicidal thoughts and behavior), certain sociodemographic characteristics (family status, income and social support) as well as anxiety, impulsivity and sleep disturbances were found to be potentially suitable factors. The analysis of the 54 depressive subjects undergoing rTMS therapy showed significant positive correlations between the reduction in suicidality and the reduction in impulsivity and sleep disorders, a trend towards a positive association with the reduction in anxiety and significant group differences according to income. Overall, the results of the present project showed a significant reduction in suicidality in depressive patients after rTMS intervention compared to the initial situation, which initially confirms the assumed potential for anti-suicidal effectiveness. However, this is put into perspective by the fact that the results did not allow a reliable differentiation from placebo effects, nor did the observed effect prove to be superior to that of conventional pharmacotherapy. Furthermore, the results obtained suggest that the change in impulsivity and sleep disturbances as well as possibly that of anxiety and income level with regard to the reduction of suicidality in depressive subjects undergoing rTMS therapy could be of clinical benefit. There is still a need for further research in this area. On one hand, in order to outline the anti-suicidal effectiveness of rTMS even more clearly in the future, and on the other hand, in order to be able to identify and specify further factors for estimating the success of therapy.2025-04-0
Chondrogenic differentiation of mesenchymal stem cells: The influence of hyaluronan and collagen type II on chondrogenesis in PEGDA hydrogels
No full textDie Therapie von Knorpeldefekten ist herausfordernd, da Knorpel als bradytrophem und avaskulärem Gewebe die nötige Selbstregenerationskapazität fehlt, um degenerative, entzündliche oder traumatische Defekte adäquat reparieren zu können. Tissue Engineering stellt eine neue Möglichkeit dar, hyalinen Knorpel mithilfe von Ersatzgeweben reparieren, ersetzen oder unterstützen zu können.
Die chondrogene Differenzierung mesenchymaler Stammzellen (MSCs) bietet die Möglichkeit, aus den Vorläuferzellen von Knorpelgewebe die zelluläre Grundlage solcher Ersatzgewebe zu schaffen. In diesem Kontext kann die Chondrogenese durch physikalische oder biologisch-chemische Differenzierungsreize initiiert und die Entwicklung von Stammzellen mithilfe solcher Differenzierungstrigger gezielt gelenkt werden. Lösliche Komponenten biologisch-chemischer Einflussfaktoren wie Hormone, aber auch physikalische Faktoren wie ein geringer Sauerstoffpartialdruck sind bekannte Stimuli, die die chondrogene Differenzierung beeinflussen. Die Einflüsse der Extrazellulären Matrix (EZM) auf die Differenzierung sind bisher nur in den Grundzügen bekannt, weshalb es weitere Forschung benötigt, um die Interaktion mesenchymaler Stammzellen mit ihrer Umgebung zu verstehen. Um diese Interaktionen im physiologischen Umfeld untersuchen zu können, sind 3D-Zellkulturen notwendig.
Das Ziel der vorliegenden Arbeit war es, den Einfluss von Hyaluronan und Kollagen-Typ-II als häufigste EZM-Bestandteile auf die chondrogene Differenzierung humaner MSCs zu untersuchen. Hierzu erfolgte die Kultivierung in dreidimensionalen PEGDA-Hydrogel-Kulturen. Des Weiteren sollten Stammzellen aus dem Knochenmark von Patient*innen der Universitätsmedizin Göttingen isoliert und als Zelllinien etabliert werden.
Insgesamt konnten drei humane Stammzelllinien (hBM MSCs) gewonnen werden, deren MSC-typische Oberflächenmarker mittels Durchflusszytometrie nachgewiesen wurden. Das multipotente Differenzierungspotenzial in einen adipogenen, einen chondrogenen und einen osteogenen Genotyp wurde an einer hBM-MSC-Linie bestätigt und im gleichen Zug wurden selbst hergestellte adipogene und chondrogene Differenzierungsmedien etabliert. Darüber hinaus zeigte sich in 3D-Sphäroid-Kulturen Hypoxie als wirksamer Trigger der Chondrogenese, der die Wirkung des Differenzierungsmediums zusätzlich verstärkte.
Für die herausfordernde Isolation von Nukleinsäuren aus Sphäroid- und PEGDA-Kulturen wurde aus zahlreichen Isolationsmethoden die wirksamste Vorgehensweise bestimmt und anschließend eingesetzt. Ein Protokoll zur Erstellung von PEGDA-Gefrierschnitten zur histologischen Aufbereitung wurde ebenfalls erarbeitet.
Um den Einfluss von Hyaluronan und Kollagen-Typ-II auf die chondrogene Differenzierung zu untersuchen, wurden drei verschiedene PEGDA-Hydrogele erstellt: ein reines Hydrogel (P-PEGDA), ein hyaluronanhaltiges Hydrogel (H-PEGDA) und ein Kollagen-Typ-II-haltiges Hydrogel (K-II-PEGDA-Hydrogel). In physikalischen Analysen zeigte sich, dass die reinen P-PEGDA-Hydrogele einen höheren Quellindex als die H- und K-II-PEGDA-Hydrogele besaßen und dass der Elastizitätsmodul der H-PEGDA-Proben deutlich über dem der anderen Hydrogelarten lag. Das Überleben der Zellen in PEGDA-Kulturen wurde mittels Lebend-Tot-Färbung über 21 Tage nachgewiesen, wobei sich PEGDA-6000 als das geeignetste Hydrogel erwies.
Die Analyse der Genexpression ergab, dass die H-PEGDA-Kulturen nach 14-tägiger Differenzierung unabhängig vom Zellkulturmedium die stärkste chondrogene Genexpression aufwiesen. Bei den P- und K-II-PEGDA-Kulturen wurden gleich starke Expressionen beobachtet, die beide signifikant über dem Niveau der Kontrollgruppe lagen.
Die Histologie offenbarte keine wesentliche Produktion von knorpeltypischer extrazellulärer Matrix, wohingegen sich in der Immunhistochemie das Vorkommen von Aggrecan und Chondroitinsulfat zeigte. Dieses war am deutlichsten in den H-PEGDA-Kulturen zu sehen.
Insgesamt konnte diese Arbeit einerseits die stark chondrogene Wirkung von Hyaluronan auf MSCs beweisen und andererseits verdeutlichen, dass knorpeltypische Kulturbedingungen wie beispielsweise Hypoxie und knorpeltypische Zelldichten die Chondrogenese von MSCs auch ohne weitere Fremdeinflüsse initiieren können. Die Frage nach der bisher nicht eindeutigen Rolle von Kollagen-Typ-II im Differenzierungsprozess bleibt unbeantwortet, was verdeutlicht, dass die Chondrogenese ein komplexes Zusammenspiel zahlreicher Faktoren ist. Um bessere Konstrukte für den Ersatz von Knorpel zu finden, wird derzeit an zahlreichen Hydrogelen und anderen Ersatzgeweben geforscht, die sich in ihrer Grundstruktur, Mikroarchitektur und genauen Zusammensetzung stark unterscheiden. Die Entschlüsselung der Matrix-Zell-Interaktion im Rahmen der Gewebedifferenzierung läuft somit weiter, wie auch die Suche nach einem idealen Ersatzgewebe zur Therapie von Knorpeldefekten.The treatment of cartilage defects is challenging since cartilage, as a bradytrophic and avascular tissue, lacks the necessary self-regeneration capacity to adequately repair degenerative, inflammatory or traumatic defects. Tissue engineering presents a new possibility for repairing, replacing or supporting hyaline cartilage with the aid of artificial tissues.
The chondrogenic differentiation of mesenchymal stem cells (MSCs) offers the possibility to create the cellular basis of such artificial tissues from the precursor cells of cartilage tissue. In this context, chondrogenesis can be initiated by physical or biochemical differentiation stimuli and the development of stem cells can be directed with the help of such differentiation cues. Soluble biochemical factors such as hormones, but also physical factors such as low oxygen partial pressure are known stimuli that influence chondrogenic differentiation. The influence of the extracellular matrix (ECM) on differentiation is only known in its basic principles, which is why further research is needed to understand the interaction of mesenchymal stem cells with their environment. In order to study these interactions in a physiological environment, 3D cell cultures are necessary.
The aim of this thesis was to investigate the influence of hyaluronan and collagen type II as the most common ECM components on the chondrogenic differentiation of human MSCs. For this purpose, MSCs were cultivated in three-dimensional PEGDA hydrogels. Furthermore, stem cells were isolated from the bone marrow of patients operated at the University Medical Center Göttingen and established as cell lines.
A total of three human bone marrow mesenchymal stem cell lines (hBM MSCs) were obtained and their MSC-typical surface markers were detected by flow cytometry. The multipotent differentiation potential into an adipogenic, a chondrogenic and an osteogenic genotype was confirmed on one hBM MSC line and, at the same time, self-mixed adipogenic and chondrogenic differentiation media were established. In addition, hypoxia proved to be an effective trigger of chondrogenesis in 3D spheroid cultures, which further enhanced the effect of the differentiation medium.
The most effective procedure for the challenging isolation of nucleic acids from spheroid and PEGDA cultures was determined from numerous isolation methods and subsequently applied. A protocol for the preparation of PEGDA frozen sections for histological processing was also developed.
To investigate the influence of hyaluronan and collagen type II on chondrogenic differentiation, three different PEGDA hydrogels were prepared: a pure hydrogel (P PEGDA), a hydrogel containing hyaluronan (H PEGDA) and a hydrogel containing collagen type II (K II PEGDA hydrogel). Physical analyses showed that the pure P PEGDA hydrogels had a higher swelling ratio than the H and K II PEGDA hydrogels and that the Young's modulus of the H PEGDA samples was significantly higher than the one of the other hydrogel types. Cell viability in PEGDA cultures was shown by live dead staining over a period of 21 days, with PEGDA 6000 proving to be the most suitable hydrogel.
The analysis of gene expression showed that the H PEGDA cultures exhibited the strongest chondrogenic gene expression after 14 days of differentiation, regardless of the cell culture medium. Equal levels of expression were observed in the P and K II PEGDA cultures, both of which were significantly higher than in the control group.
Histology revealed no significant production of extracellular matrix typical for cartilage tissue, whereas immunohistochemistry confirmed the presence of aggrecan and chondroitin sulfate. This was most clearly seen in the H PEGDA culture.
Overall, this project was able to prove the strong chondrogenic effect of hyaluronan on MSCs. Furthermore, it illustrated that culture conditions typical for cartilage, such as hypoxia and cartilage-typical cell densities, can initiate the chondrogenesis of MSCs even without further external influences. The question of the still quite ambiguous role of collagen type II in the differentiation process remains unanswered, which emphasizes that chondrogenesis is a complex interaction of numerous factors. In order to find better constructs for the replacement of cartilage, research is currently being conducted on numerous hydrogels and other artificial tissues, which differ greatly in their basic structure, microarchitecture and exact composition. The decoding of matrix-cell interaction in the context of tissue differentiation is therefore continuing, as is the search for an ideal artificial tissue for the treatment of cartilage defects.2025-03-0