Biochemical and mechanical properties of the extracellular matrix (ECM) are known to independently influence cell function. Given the complexity of cellular responses, I hypothesized that the integration of multiple matrix factors as opposed to their individual contribution is key to understanding and controlling cell function. The objective of this thesis was to systematically investigate matrix biochemical and mechanical regulation of multipotent stromal cell (MSC) osteogenesis. First, I demonstrated that substrate stiffness-dependent MSC spreading, proliferation and osteogenic response were differentially regulated by matrix protein type (collagen I vs. fibronectin) and concentration. Second, I developed and characterized a matrix microarray platform that enabled the efficient screening of multiple matrix-derived cues (substrate stiffness, ECM type and density). I implemented the matrix microarray platform together with parametric regression models to elucidate novel matrix interactions in directing mouse MSC osteogenic and adipogenic differentiation. Third, I extended the screening study to examine matrix-dependent human MSC osteogenesis. Non-parametric regression models were used to provide a nuanced description of the multi-factorial matrix regulation in MSC osteogenesis. The response surfaces revealed a biphasic relationship between osteogenesis and substrate stiffness, with the exact location and magnitude of the optimum contingent on matrix composition. Guided by the screening results and perturbation to key cytoskeletal regulators, I identified a novel pathway involving Cdc42 in matrix mechanical and biochemical regulation of MSC osteogenesis. Surprisingly, Cdc42 mediated stiffness-dependent MSC osteogenesis independent of ROCK, suggestive of a contractility-independent mechanism in matrix rigidity signal transduction. In summary, the integration of cell-based arrays and statistical modeling has enabled the systematic investigation of complex cell-matrix interactions. This generalizable approach is readily adaptable to other cellular contexts, complementing hypothesis-driven strategies to facilitate non-intuitive mechanistic discovery. Moreover, the improved understanding of matrix-dependent MSC function also has practical relevance to the development of biomaterials for tissue engineering applications.

Biochemical and mechanical properties of the extracellular matrix (ECM) are known to independently influence cell function. Given the complexity of cellular responses, I hypothesized that the integration of multiple matrix factors as opposed to their individual contribution is key to understanding and controlling cell function. The objective of this thesis was to systematically investigate matrix biochemical and mechanical regulation of multipotent stromal cell (MSC) osteogenesis. First, I demonstrated that substrate stiffness-dependent MSC spreading, proliferation and osteogenic response were differentially regulated by matrix protein type (collagen I vs. fibronectin) and concentration. Second, I developed and characterized a matrix microarray platform that enabled the efficient screening of multiple matrix-derived cues (substrate stiffness, ECM type and density). I implemented the matrix microarray platform together with parametric regression models to elucidate novel matrix interactions in directing mouse MSC osteogenic and adipogenic differentiation. Third, I extended the screening study to examine matrix-dependent human MSC osteogenesis. Non-parametric regression models were used to provide a nuanced description of the multi-factorial matrix regulation in MSC osteogenesis. The response surfaces revealed a biphasic relationship between osteogenesis and substrate stiffness, with the exact location and magnitude of the optimum contingent on matrix composition. Guided by the screening results and perturbation to key cytoskeletal regulators, I identified a novel pathway involving Cdc42 in matrix mechanical and biochemical regulation of MSC osteogenesis. Surprisingly, Cdc42 mediated stiffness-dependent MSC osteogenesis independent of ROCK, suggestive of a contractility-independent mechanism in matrix rigidity signal transduction. In summary, the integration of cell-based arrays and statistical modeling has enabled the systematic investigation of complex cell-matrix interactions. This generalizable approach is readily adaptable to other cellular contexts, complementing hypothesis-driven strategies to facilitate non-intuitive mechanistic discovery. Moreover, the improved understanding of matrix-dependent MSC function also has practical relevance to the development of biomaterials for tissue engineering applications.

<p>Intervertebral disc (IVD) disorders can cause pain and disability for affected individuals. A subset of IVD disorders are thought to originate in the nucleus pulposus (NP) region of the IVD, due to alterations in tissue structure and composition with age and injury. Cells of the NP undergo a phenotypic and behavioral shift with age, changes that are thought to disrupt tissue homeostasis and lead to disc degeneration. There is significant interest in developing biomaterials and strategies to revert adult degenerate NP cells to healthy, young NP cell phenotypes with increased biosynthesis and cell clustering. Studies demonstrate that healthy porcine NP cells interact with laminin proteins through specific integrin subunits on soft substrates in a process that regulates cell morphology and biosynthesis. The central hypothesis of this dissertation is that the engagement of cell-surface adhesion receptors, using laminin-mimetic peptides on a controlled stiffness material, can revert adult degenerate NP cellular phenotype and behaviors to their healthy, biosynthetically active form.</p><p>In the first part of this dissertation, integrin subunits used by adult degenerate human NP cells to attach to laminin were revealed using flow cytometric analyses, function blocking antibodies, and immunohistochemistry. Secondly, NP cell recognition peptides were identified by screening laminin-mimetic peptides for cell attachment. Finally, human NP cells were cultured on peptide functionalized polyacrylamide gels to examine the effect of ligand and substrate stiffness in regulating adult human NP cell phenotype and biosynthesis. </p><p>Findings reveal that adult human NP cells express and utilize integrin subunits α3, α5, and β1 to attach to laminins, in contrast to integrin α6β1 found previously for healthy porcine NP cells. This difference between current and previous findings likely arises from aging-associated difference in NP cells between human and porcine tissues. Select laminin-mimetic peptides, chosen from the literature and informed by NP cell integrin expression, were found to promote significant NP cell attachment in a concentration dependent manner. Finally, a subset of laminin mimetic peptides were found to promote a young NP cell phenotype by increasing cell clustering on soft (0.3 kPa) and stiff (14 kPa) substrates as well as increasing proteoglycan synthesis on soft substrates. </p><p>The studies presented in this dissertation demonstrate that adult degenerate human NP cells attach to laminin proteins in an integrin dependent manner. Furthermore, laminin-mimetic peptides are able to mediate human NP cell attachment at levels comparable to full-length laminin, increase cell clustering when presented on soft and stiff substrates, and can increase NP cell biosynthesis when presented on soft substrates. Utilizing laminin-mimetic peptides may allow for the design of biomaterials that promote a healthy young NP phenotype for a variety of disc therapies.</p> / Dissertation

Vascular endothelial growth factor (VEGF) is required for the development, growth and survival of blood vessels. Endothelial cell behavior is altered by cell substrate stiffness, suggesting that VEGF activity might also be influenced by cell-substrate mechanics. We studied VEGF binding, internalization, and signaling as a function of substrate stiffness using endothelial cells cultured on fibronectin (fn) linked polyacrylamide gels. Individual cell analysis of VEGF-induced calcium fluxes in endothelial cells on various stiffness extracellular matrices (ECM) revealed heterogeneity in our cell population that would have been lost using population based averaging. Cluster analysis of individual cells identified two key groups of reacting cells- a minor fraction of highly reactive cells and the bulk of the cells with minimal activation. At subsaturating VEGF doses, highly active cells were phenotypically smaller and thinner than the bulk population. Overall, cells on our softest substrates (4 kPa) were most sensitive to VEGF. To better understand the mechanisms underlying the changes in VEGF signaling due to stiffness, we explored how matrix binding of VEGF and tethering of cells to the matrix modulates VEGF processing. VEGF-ECM binding was enhanced with heparin pre-treatment, which exposed a cryptic VEGF binding site in the fn ECM. Cell produced ECM on the softest substrates were least responsive to heparin, but the cells internalized more VEGF and showed enhanced VEGF signaling compared to cells on all other substrates. Inhibiting VEGF-matrix binding with sucrose octasulfate decreased cell-internalization of VEGF in all conditions. β1 integrin, which connects cells to fn, modulated VEGF uptake in a stiffness dependent fashion. β1 protein levels were consistent with stiffness, yet cells on hard surfaces showed greater decreases in VEGF internalization than cells on softer matrices after β1 inhibition. Stiff matrices facilitate the unfolding of fn, which may reduce the binding capacity of β1 integrin. Thus a greater proportion of activated β1 integrin may be sensitive to inhibition in the stiff condition as compared to the soft. Ultimately, through analysis of individual and population-based VEGF-cell responses to stiffness, this study provides insight into how signaling dynamics, cell heterogeneity, and microenvironment influence tissue regeneration and response to injury and disease.

Fibronectin is an important protein that is able to bind to other fibronectin molecules and to cell surface receptors. In doing so, the interactions fibronectin can perform is important for the processes of cell migration and tissue formation. Understanding the properties of fibronectin and fibril assembly is useful for areas such as wound healing, where fibronectin molecules are assembled to protect the tissue and to perform other tasks. Because of these reasons, it is important to understand how fibronectin is assembled and how its properties affect the fibril assembly, which in return affects the way the cell matrix operates. Previously published papers illustrate that the properties of fibronectin affect the mechanotransduction process, the cell conversion of mechanical stimulus to chemical, and this leads to various changes of the fibril assembly. However, the question that now comes to focus is what variables affect the fibril assembly? The two main variables that come into question is the substrate stiffness (ksub) (pN/nm) and the actin velocity (Vu) (nm/s). In order to test this hypothesis, several fibril assembly simulations were performed via MATLAB based upon the Weinberg-Mair-Lemmon Fibronectin Model. These simulations were performed by varying the parameters of substrate stiffness and actin velocity as well as fibril size, which affect the various measurements of the fibronectin, such as stretched length, relaxed length, etc. Through these various experiments, it was determined that the actin velocity and fibril size had the greatest impacts in affecting the fibronectin’s properties and its assembly.

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ABSTRACT INTERACTION BETWEEN MICRO AND NANO PATTERNED POLYMERIC SURFACES AND DIFFERENT CELL TYPES &Ouml / z&ccedil / elik, Hayriye Ph.D., Department of Biology Supervisor: Prof. Dr. Vasif Hasirci Co-Supervisor: Dr. Celestino Padeste August 2012, 139 pages Micro and nanopatterned surfaces are powerful experimental platforms for investigating the mechanisms of cell adhesion, cell orientation, differentiation and they enable significant contributions to the fields of basic cell and stem cell biology, and tissue engineering. In this study, interaction between micro and nanopatterned polymeric surfaces and different cell types was investigated. Three types of micropillars were produced by photolithography (Type 1-3), while nanometer sized pillars were produced in the form of an array by electron beam lithography (EBL). Replica of silicon masters were made of polydimethylsiloxane (PDMS). Polymeric [P(L-D,L)LA and a P(L-D,L)LA:PLGA blend] replica were prepared by solvent casting of these on the PDMS template and used in in vitro studies. The final substrates were characterized by various microscopic methods such as light microscopy, atomic force microscopy (AFM) and scanning electron microscopy (SEM). In order to investigate deformation of the nucleus in response to the physical restrictions imposed by micropillars, Type 1 and Type 2 pillars were used. These substrates were covered with pillars with different interpillar distances. While Type 1 is covered with symmetrically (in X-Y directions) distributed pillars, Type 2 pillars were distributed asymmetrically and the inter-pillar distances were increased. Nuclei deformation of five cell v types, two cancer cell lines (MCF7 and Saos-2), one healthy bone cell (hFOB1.19), one stem cell (bone marrow origined mesemchymal stem cells, BMSCs) and one standard biomaterial test cell type, (L929) fibroblasts was examined by using fluorescence microscopy and SEM. The nuclei of Saos-2 and MCF7 cells were found to be deformed most drastically. Nucleus deformation and intactness of nuclear membrane was examined by Anti- Lamin A staining. The interaction of the cells with micropillars was visualized by labelling focal adhesion complexes (FAC). Wettabilities of patterned and smooth surfaces were determined. As the patterns become denser (closer micropillars, Type 1) the hydrophobicity increased. Similar to water droplets, the cells were mostly spread at the top of the Type 1 pillars. The number of cells spread on the substrate surface was much higher on Type 2 patterned films. In order to support these qualitative findings, nucleus deformation was quantified by image analysis. Frequency of nucleus deformation was determined as the ratio of deformed to the total number of nuclei (%). In order to quantify the intensity of nuclei deformation, their circularity was evaluated. In addition to nucleus deformation, alterations in the ratio of cell area-to-nucleus area in response to micropillars were determined by image analysis. The results indicated that cancerous cells were more deformable. The qualitative microscopic evaluation and the data obtained by quantification of the nucleus and cellular deformation were in good agreement. In addition, the findings were consistent with expectations which suggest that cancerous cells are &ldquo / softer&rdquo / . In the second part of the research the force applied by the cells on arrays of micropillars with high aspect ratios (Type 3 substrates) during tugging at the pillars was investigated. Micropillars were produced using P(L-D,L)LA as well as a 60:40 blend of P(L-D,L)LA with PLGA. The blend is a material with lower stiffness than P(L-D,L)LA. The mechanical properties of the two materials were determined by tensile testing of solvent cast films. Deformation of Type 3 micropillars by the cellular tugging force of Saos-2 and L929 was studied by fluorescence and SEM microscopy, both on stiff and softer substrates. Displacements of the centers nodes of the pillars were evaluated from SEM micrographs. On the stiff surface, the two cell types bent the pillars to the same extent. On the other softer substrate (blends), however, the maximum displacements observed with Saos-2 cells were higher than the ones caused on the stiffer substrate or the ones caused by L929 cells. It is reported that stiffness of the substrate can determine stem cell lineage commitment. In order to examine the effects of change of substrate stiffness on osteogenic differentiation of BMSCs, osteopontin (OPN) expression was determined microscopically. It was found that osteogenic differentiation is enhanced when BMSCs are cultured on P(L-D,L)LA Type 3 pillars. vi In the last part of research, arrays of nanopillars whose interpillar distances systematically varied to form different fields were examined in terms of adhesion and alignment in order to determine the differential adhesion of BMSCs and Saos-2 cells. The difference in their adhesion preference on nanopillar arrays was quantified by image analysis. It was observed that BMSCs and Saos-2 cells behaved in an opposite manner with respect to each other on the fields with the highest density of nanopillars. The BMSCs avoided the most densely nanopillar covered fields and occupied the pattern free regions. The Saos-2, on the other hand, occupied the most densely nanopillar covered fields and left the pattern free regions almost unpopulated. It was also found that both BMSCs and Saos-2 cells aligned in the direction of the shorter distance between the pillars. Both BMSCs and Saos-2 cells started to align on the pillars if the distance in any direction was &gt / 1.5 &mu / m. To better understand the effects of chemical and physical cues, protein coating and material stiffness were tested as two additional parameters. After fibronectin coating, the surfaces of P(L-D,L)LA films with the highly dense pillar covered fields, which were avoided when uncoated, were highly populated by the BMSC. Similarly, decreasing the stiffness of a surface which was normally avoided by the BMSCs made it more acceptable for the cells to attach.

No description available.

Macrophages are innate immune cells responsible for engulfing microbes and cell debris through phagocytosis and orchestrating immune responses to maintain homeostasis. While conducting immune surveillance over all types of organs and tissues, macrophages face inherently heterogeneous microenvironments with unique biophysical features. For instance, microglia residing in the brain, Kupffer cells living in the skin and bone osteoclasts are exposed to very distinct tissue stiffnesses. Despite the research done in the last decade clearly indicates that macrophages are sensitive to physical factors, how mechanical cues modulate their inflammatory response remains poorly understood. The present study aims at investigating how microenvironment stiffness influences the pro-inflammatory behaviour of macrophages. Besides characterising the regulatory effect on pro-inflammatory gene expression and cytokine production, this work examines the impact of stiffness on the inflammasome, one of the main macrophage signalling platforms. For this, an in vitro system based in 2D polyacrylamide hydrogels whose stiffness can be independently tuned was established. Using substrates with an elastic moduli between 0.2 and 33.1 kPa, bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff polyacrylamide. Upon priming with lipopolysaccharide, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels, yet there were no significant differences in the expression of other major pro-inflammatory genes. Additionally stimulating macrophages with the ionophore nigericin revealed higher secreted protein levels of IL-1β and IL-6 on compliant substrates. Interestingly, macrophages challenged on compliant polyacrylamide also displayed an enhanced formation of the NLRP3 inflammasome as well as increased levels of pyroptotic cell death. The upregulation of inflammasome assembly on compliant hydrogels was not primarily attributed to the reduced cell spreading, since spatially confining cells on micropatterns led to a decrease of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, these results show that substrate stiffness affects the pro-inflammatory response of macrophages and for the first time describe that the NLRP3 inflammasome is one of the signalling components affected by stiffness mechanosensing. The work presented here expands our understanding of how microenvironment stiffness affects macrophage behaviour and which elements of their machinery might contribute to integrate mechanical cues into the regulation of their inflammatory functions. The onset of pathological processes or the implant of foreign bodies represent immune challenges in which macrophages can face a mechanically changing environment. Therefore, a better insight on how macrophages detect and process biophysical signals could potentially provide a basis for new strategies to modulate inflammatory responses.:INTRODUCTION 1.1 Macrophage cell biology 1.1.1 The origin of macrophages 1.1.2 The macrophage: a swiss army knife 1.1.3 The macrophage pro-inflammatory response 1.2 Immunobiophysics: the force of the immune system 1.2.1 Exertion of immune cell forces 1.2.2 Immune cell mechanosensing 1.3 Cellular mechanosensing and mechanotransduction 1.3.1 Cell adhesions to the extracellular matrix 1.3.2 Nuclear mechanotransduction 1.3.3 Membrane mechanosensing elements 1.4 Macrophage mechanosensing AIMS AND SCOPE OF THE THESIS RESULTS 3.1 Morphol. characterisation of macrophages cultured on substrates of varying stiffness 3.1.1 BMDMs adhere and can be cultured on polyacrylamide hydrogels 3.1.2 Macrophage morphology is influenced by substrate stiffness 3.1.3 PEG-Hep hydrogels induce similar morphological differences as PAA substrates but do not constitute a suitable macrophage culture platform 3.1.4 Substrate stiffness affects membrane architecture 3.2 Impact of substrate stiffness on the pro-inflammatory response of macrophages 3.2.1 The morphol. differences induced by different stiffness persist after macrophage priming 3.2.2 Tuning substrate stiffness does not cause major changes in the expression of pro-inflammatory genes 3.2.3 Lower substrate stiffness upregulates the secretion of the cytokines IL-6 and IL-1β 3.2.4 Stiffer substrates diminish macrophage pyroptotic cell death 3.2.5 Compliant substrates enhance NLRP3 inflammasome formation 3.3 Investigation of macrophage mechanotransducing elements 3.3.1 Limiting cell spreading alone does not recapitulate the effects induced by stiffness on inflammasome formation 3.3.2 Actomyosin contractility may play a role in transducing the mechanical cues given by substrate stiffness DISCUSSION AND CONCLUSIONS 4.1 Compliant substrates enhance the macrophage pro-inflammatory response 4.2 Substrate stiffness influences the formation of the NLRP3 inflammasome 4.3 Exclusively altering cell spreading does not explain the differences induced by substrate stiffness 4.4 Actomyosin contractility as a potential macrophage mechanotransducer element 4.5 Potential impact of the study in the context of cancer 4.6 Potential impact of the study in the context of implant design 4.7 Conclusions of the study MATERIALS AND METHODS 5.1 Production of polyacrylamide (PAA) hydrogels 5.2 Production of polyethylenglycol-heparin (PEG-Hep) hydrogels 5.3 Mechanical characterisation of hydrogels and macrophages 5.4 Isolation and culture of bone marrow-derived macrophages (BMDMs) 5.5 Fluorescence confocal microscopy 5.6 Scanning electron microscopy (SEM) 5.7 Gene expression analysis using quantitative real-time PCR (qRT-PCR) 5.8 Cytokine quantification assays 5.9 Cell viability assay 5.10 Culture of BMDMs on micropatterns 5.11 Optical diffraction tomography (ODT) 5.12 Statistical analysis and data visualisation APPENDIX LIST OF ACRONYMS AND ABBREVIATIONS LIST OF FIGURES BIBLIOGRAPHY ACKNOWLEDGEMENTS / Als Teil des angeborenen Immunsystems sind Makrophagen dafür verantwortlich Pathogene und Zellrückstände durch Phagozytose zu beseitigen. Sie orchestrieren Immunantworten um homöostatische Bedingungen von Organen und Geweben aufrechtzuerhalten. Dabei sind sie extrem heterogenen Mikroumgebungen ausgesetzt, welche sich jeweils durch eine einzigartige Kombination von (bio)chemischen und mechanischen Eigenschaften, vor allem Gewebesteifigkeiten, auszeichnen. Dies veranschaulichen beispielsweise im Gehirn residierende Mikroglia, Kupffer-Zellen in der Haut und Osteoklasten in Knochen. Obwohl diverse Studien aus dem letzten Jahrzehnt eindeutig zeigen, dass Makrophagen auf mechanische Signale reagieren, ist der zugrunde liegende Mechanismus, wie diese Signale eine Entzündungsreaktion modulieren, noch immer unzureichend verstanden. Die vorliegende Studie beinhaltet die systematische Untersuchung, wie die Steifigkeit der Mikroumgebung das proinflammatorische Verhalten von Makrophagen beeinflusst. Neben der Charakterisierung der regulatorischen Wirkung auf die proinflammatorische Genexpression und Zytokinproduktion untersucht diese Arbeit auch den Einfluss der Steifigkeit auf das Inflammasom; eine der wichtigsten Signalplattformen für Makrophagen. Zu diesem Zweck wurde zunächst ein Zellkultursystem mit 2D-Polyacrylamid-Hydrogelen als Zellsubstrat entwickelt, bei dem das Elastizitätsmodul der Gelsubstrate gezielt eingestellt werden kann. Unter Verwendung von Substraten mit einem Elastizitätsmodul zwischen 0,2 kPa und 33,1 kPa zeigt die mikroskopische Analyse, dass aus Knochenmark stammende Makrophagen im Vergleich zu steifem Polyacrylamid eine weniger ausgebreitete und rundere Morphologie annehmen. Nach dem Primen mit Lipopolysaccharid waren die Expressionsniveaus des Gens, das für TNF-α kodiert, auf deformierbareren Hydrogelen höher, jedoch gab es keine signifikanten Unterschiede in der Expression anderer wichtiger pro-inflammatorischer Gene. Eine zusätzliche Stimulierung von Makrophagen mit dem Ionophor Nigericin bewirkte höhere sekretierte Proteinspiegel von IL-1β und IL-6 auf deformierbaren Substraten. Makrophagen, die deformierbarem Polyacrylamid ausgesetzt waren, zeigten auch eine verstärkte Bildung des NLRP3-Inflammasoms sowie ein erhöhtes Ausmaß an pyroptotischem Zelltod. Die Hochregulierung der Inflammasom-Assemblierung auf deformierbaren Hydrogelen wurde nicht primär auf die reduzierte Zellausbreitung zurückgeführt, da räumlich begrenzte Zellen auf Mikromustern zu einer Abnahme von Inflammasom-positiven Zellen im Vergleich zu stark ausgebreiteten Zellen führten. Schließlich verringerte eine Störung der Aktomyosin-Kontraktilität die Unterschiede in der Inflammasombildung zwischen deformierbaren und steifen Substraten. Zusammenfassend zeigen diese Ergebnisse, dass die Substratsteifigkeit die proinflammatorische Reaktion von Makrophagen beeinflusst und beschreiben erstmalig, dass das NLRP3-Inflammasom eine der Signalkomponenten ist, die von der zellulären Steifheitswahrnehmung beeinflusst werden. Die hier vorgestellte Arbeit erweitert unser Verständnis davon, wie die Steifigkeit der Mikroumgebung das Verhalten von Makrophagen beeinflusst und welche Elemente ihrer Maschinerie dazu beitragen könnten mechanische Signale in die Regulierung ihrer Entzündungsfunktionen zu integrieren. Das Einsetzen pathologischer Prozesse oder die Implantation von Fremdkörpern stellen Immunherausforderungen dar, bei denen Makrophagen einer sich mechanisch verändernden Umgebung ausgesetzt sein können. Daher könnte ein besserer Einblick in die Art und Weise, wie Makrophagen biophysikalische Signale erkennen und verarbeiten, möglicherweise eine Grundlage für neue Strategien zur Modulation von Entzündungsreaktionen bieten.:INTRODUCTION 1.1 Macrophage cell biology 1.1.1 The origin of macrophages 1.1.2 The macrophage: a swiss army knife 1.1.3 The macrophage pro-inflammatory response 1.2 Immunobiophysics: the force of the immune system 1.2.1 Exertion of immune cell forces 1.2.2 Immune cell mechanosensing 1.3 Cellular mechanosensing and mechanotransduction 1.3.1 Cell adhesions to the extracellular matrix 1.3.2 Nuclear mechanotransduction 1.3.3 Membrane mechanosensing elements 1.4 Macrophage mechanosensing AIMS AND SCOPE OF THE THESIS RESULTS 3.1 Morphol. characterisation of macrophages cultured on substrates of varying stiffness 3.1.1 BMDMs adhere and can be cultured on polyacrylamide hydrogels 3.1.2 Macrophage morphology is influenced by substrate stiffness 3.1.3 PEG-Hep hydrogels induce similar morphological differences as PAA substrates but do not constitute a suitable macrophage culture platform 3.1.4 Substrate stiffness affects membrane architecture 3.2 Impact of substrate stiffness on the pro-inflammatory response of macrophages 3.2.1 The morphol. differences induced by different stiffness persist after macrophage priming 3.2.2 Tuning substrate stiffness does not cause major changes in the expression of pro-inflammatory genes 3.2.3 Lower substrate stiffness upregulates the secretion of the cytokines IL-6 and IL-1β 3.2.4 Stiffer substrates diminish macrophage pyroptotic cell death 3.2.5 Compliant substrates enhance NLRP3 inflammasome formation 3.3 Investigation of macrophage mechanotransducing elements 3.3.1 Limiting cell spreading alone does not recapitulate the effects induced by stiffness on inflammasome formation 3.3.2 Actomyosin contractility may play a role in transducing the mechanical cues given by substrate stiffness DISCUSSION AND CONCLUSIONS 4.1 Compliant substrates enhance the macrophage pro-inflammatory response 4.2 Substrate stiffness influences the formation of the NLRP3 inflammasome 4.3 Exclusively altering cell spreading does not explain the differences induced by substrate stiffness 4.4 Actomyosin contractility as a potential macrophage mechanotransducer element 4.5 Potential impact of the study in the context of cancer 4.6 Potential impact of the study in the context of implant design 4.7 Conclusions of the study MATERIALS AND METHODS 5.1 Production of polyacrylamide (PAA) hydrogels 5.2 Production of polyethylenglycol-heparin (PEG-Hep) hydrogels 5.3 Mechanical characterisation of hydrogels and macrophages 5.4 Isolation and culture of bone marrow-derived macrophages (BMDMs) 5.5 Fluorescence confocal microscopy 5.6 Scanning electron microscopy (SEM) 5.7 Gene expression analysis using quantitative real-time PCR (qRT-PCR) 5.8 Cytokine quantification assays 5.9 Cell viability assay 5.10 Culture of BMDMs on micropatterns 5.11 Optical diffraction tomography (ODT) 5.12 Statistical analysis and data visualisation APPENDIX LIST OF ACRONYMS AND ABBREVIATIONS LIST OF FIGURES BIBLIOGRAPHY ACKNOWLEDGEMENTS