Proteomische Analyse des nativen und fibrotischen humanen Lebermatrisoms für Organ Engineering

A persistent demand in suitable grafts has underscored the need for the development of bioartificial organs. Despite global effort to innovate novel alternatives, the creation of transplants yet faces fundamental challenges that are related to organ specific functionality and immunogenicity and hinder current approaches from clinical translation. A vital objective to successfully implement bioartificial organs into clinical practice is the creation of complex biomaterials that will mediate regenerative capacities for long-lasting performance of newly developed grafts. In this thesis, I present in-depth analysis of human derived liver extracellular matrices by utilizing a label-free shotgun proteomic approach. By applying various decellularization and defatting strategies to fabricate organotypic tissues for proteomic measurements, this work aims to provide insights into the native human liver matrisome to determine the overall complexity that needs to be taken into account when creating novel biomaterials to be functionalized in the development of bioartificial organs. Furthermore, by utilizing the devised experimental workflow for the analysis of fibrotic and cirrhotic human liver extracellular matrices, proteomic features were detected that can be potential targets for therapeutic exploitation. In addition to these two intertwining research threads, this thesis introduces a versatile platform to further functionalize the created human liver extracellular matrices by enabling a facile combination of these scaffolds with prevailing technology to successfully translate various bioengineering approaches from concept to therapeutic reality. The results obtained from human liver matrices support the necessity of including proteomic techniques in interpreting processes underlying tissue homeostasis and regeneration. Proteomic analysis of native tissues provides cues to be utilized in the creation of bioartificial products. This information shall bridge the gap between currently available digital fabrication technology and the clinical usage of functional organ replacements. Furthermore, by analyzing fibrotic and cirrhotic human derived liver scaffolds, specific characteristics were described that demand further investigation to fully understand signaling pathways underlying the emergence of fibrosis and cirrhosis.Eine anhaltende Nachfrage nach geeigneten Transplantaten hat die Notwendigkeit der Entwicklung bio-artifizieller Organe unterstrichen. Trotz globaler Bemühungen steht die Erzeugung von Transplantaten noch vor grundlegenden Herausforderungen, die mit der organspezifischen Funktionalität und Immunogenität zusammenhängen und aktuelle Ansätze an der klinischen Translation hindern. Ein wichtiges Ziel für die erfolgreiche Einführung bioartifizieller Organe in die klinische Praxis ist die Entwicklung und Herstellung komplexer Biomaterialien, welche zu Regeneration fähig sind und somit eine langanhaltende biologische Leistung ermöglichen. Im Rahmen dieser Dissertation führte ich eine eingehende Analyse von extrazellulären Lebermatrizes humanen Ursprungs unter Verwendung eines label-free shotgun proteomischen Ansatzes durch. Durch die Anwendung verschiedener Dezellularisierungs- und Entfettungsstrategien zur Herstellung organotypischer Gewebe für proteomische Analysen zielt diese Arbeit darauf ab, Einblicke in das native humane Lebermatrisom zu geben, um die gesamte Komplexität zu bestimmen, die bei der Herstellung neuartiger Biomaterialien zur Entwicklung bio-artifizieller Organe zum Einsatz kommen sollte. Des Weiteren wurde der entworfene experimentelle Arbeitsablauf verwendet, um proteomische Merkmale fibrotischer und zirrhotischer humaner Lebermatrizes zu identifizieren, die potenzielle Ziele für eine therapeutische Nutzung sein können. Zusätzlich zu diesen beiden miteinander verknüpften Forschungsfäden wird in dieser Arbeit eine vielseitig anwendbare Plattform zur umfangreichen Funktionalisierung der extrazellulären Lebermatrizes humanen Ursprungs vorgestellt. Hierdurch soll die Kombination dieser Matrizes mit vorherrschenden Technologien erleichtert werden, um die verschiedenen Ansätze des Bioengineerings erfolgreich vom Konzept zur therapeutischen Realität umzusetzen. Die in dieser Arbeit präsentierten Ergebnisse aus humanen Lebermatrizes unterstreichen die Relevanz proteomischer Techniken bei der Interpretation der Prozesse, die der Gewebehomöostase und -regeneration zugrunde liegen. Die Untersuchung der proteomischen Landschaft natürlicher Gewebe liefert Kenntnisse, die in der Erzeugung bioartifizieller Produckte verwendet werden können. Diese Informationen sollen die Lücke zwischen verfügbaren digitalen Fertigungstechnologien und dem klinischen Einsatz funktioneller Organersatzprodukte schließen. Darüber hinaus wurden durch die Analyse von fibrotischen und zirrhotischen humanen Lebermatrizes spezifische Eigenschaften beschrieben, die weitere Untersuchungen erfordern, um die der Entstehung von Fibrose und Zirrhose zugrunde liegenden Signalwege vollständig zu verstehen

Outcomes of Liver Resections after Liver Transplantation at a High-Volume Hepatobiliary Center

Although more than one million liver transplantations have been carried out worldwide, the literature on liver resections in transplanted livers is scarce. We herein report a total number of fourteen patients, who underwent liver resection after liver transplantation (LT) between September 2004 and 2017. Hepatocellular carcinomas and biliary tree pathologies were the predominant indications for liver resection (n = 5 each); other indications were abscesses (n = 2), post-transplant lymphoproliferative disease (n = 1) and one benign tumor. Liver resection was performed at a median of 120 months (interquartile range (IQR): 56.5-199.25) after LT with a preoperative Model for End-Stage Liver Disease (MELD) score of 11 (IQR: 6.75-21). Severe complications greater than Clavien-Dindo Grade III occurred in 5 out of 14 patients (36%). We compared liver resection patients, who had a treatment option of retransplantation (ReLT), with actual ReLTs (excluding early graft failure or rejection, n = 44). Bearing in mind that late ReLT was carried out at a median of 117 months after first transplantation and a median of MELD of 32 (IQR: 17.5-37); three-year survival following liver resection after LT was similar to late ReLT (50.0% vs. 59.1%; p = 0.733). Compared to ReLT, liver resection after LT is a rare surgical procedure with significantly shorter hospital (mean 25, IQR: 8.75-49; p = 0.034) and ICU stays (mean 2, IQR: 1-8; p < 0.001), acceptable complications and survival rates

Proteomic analysis of decellularized mice liver and kidney extracellular matrices

Abstract Background The extracellular matrix (ECM) is a three-dimensional network of proteins that encases and supports cells within a tissue and promotes physiological and pathological cellular differentiation and functionality. Understanding the complex composition of the ECM is essential to decrypt physiological processes as well as pathogenesis. In this context, the method of decellularization is a useful technique to eliminate cellular components from tissues while preserving the majority of the structural and functional integrity of the ECM. Results In this study, we employed a bottom-up proteomic approach to elucidate the intricate network of proteins in the decellularized extracellular matrices of murine liver and kidney tissues. This approach involved the use of a novel, perfusion-based decellularization protocol to generate acellular whole organ scaffolds. Proteomic analysis of decellularized mice liver and kidney ECM scaffolds revealed tissue-specific differences in matrisome composition, while we found a predominantly stable composition of the core matrisome, consisting of collagens, glycoproteins, and proteoglycans. Liver matrisome analysis revealed unique proteins such as collagen type VI alpha-6, fibrillin-2 or biglycan. In the kidney, specific ECM-regulators such as cathepsin z were detected. Conclusion The identification of distinct proteomic signatures provides insights into how different matrisome compositions might influence the biological properties of distinct tissues. This experimental workflow will help to further elucidate the proteomic landscape of decellularized extracellular matrix scaffolds of mice in order to decipher complex cell–matrix interactions and their contribution to a tissue-specific microenvironment

Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided

Outcomes of Liver Resections after Liver Transplantation at a High-Volume Hepatobiliary Center

Although more than one million liver transplantations have been carried out worldwide, the literature on liver resections in transplanted livers is scarce. We herein report a total number of fourteen patients, who underwent liver resection after liver transplantation (LT) between September 2004 and 2017. Hepatocellular carcinomas and biliary tree pathologies were the predominant indications for liver resection (n = 5 each); other indications were abscesses (n = 2), post-transplant lymphoproliferative disease (n = 1) and one benign tumor. Liver resection was performed at a median of 120 months (interquartile range (IQR): 56.5&ndash;199.25) after LT with a preoperative Model for End-Stage Liver Disease (MELD) score of 11 (IQR: 6.75&ndash;21). Severe complications greater than Clavien&ndash;Dindo Grade III occurred in 5 out of 14 patients (36%). We compared liver resection patients, who had a treatment option of retransplantation (ReLT), with actual ReLTs (excluding early graft failure or rejection, n = 44). Bearing in mind that late ReLT was carried out at a median of 117 months after first transplantation and a median of MELD of 32 (IQR: 17.5&ndash;37); three-year survival following liver resection after LT was similar to late ReLT (50.0% vs. 59.1%; p = 0.733). Compared to ReLT, liver resection after LT is a rare surgical procedure with significantly shorter hospital (mean 25, IQR: 8.75&ndash;49; p = 0.034) and ICU stays (mean 2, IQR: 1&ndash;8; p &lt; 0.001), acceptable complications and survival rates

Additional file 2 of Proteomic analysis of decellularized mice liver and kidney extracellular matrices

Supplementary material 2

Additional file 1 of Proteomic analysis of decellularized mice liver and kidney extracellular matrices

Supplementary material 1