Mechanosensitivity describes the ability to respond to a mechanicalstimulus. The heart can respond to a mechanical stimulus through the action ofmechanosensitive ion channels (MSCs). MSCs provide a direct link betweenstretch and electrical activity. However, the heart does not react to stretch solelythrough the action of MSCs. The mammalian myocardium possesses a biphasicresponse to stretch: first, there is an immediately increase in the force of cardiaccontraction, known as the Frank-Starling response; secondly, there is a slowpositive inotropic response, known as the slow force response (SFR) that occursover the minutes following the initial stretch. Fish are unique amongst vertebrates as, with a few exceptions, they relymore heavily on changes in stroke volume than heart rate when regulatingcardiac output. Rainbow trout (Oncorhynchus mykiss) are particularly sensitiveto the Frank-Starling response and small increases in filling pressure lead to largeincreases in stroke volume (300 %) during strenuous exercise. The ability of fishhearts to undertake these large dilations makes them an ideal model whenlooking at the effect of stretch on cardiac muscle as they may exhibit morepronounced responses to mechanical stimuli. Despite this, the role of mechanicalregulation in the fish heart has undergone sparse investigation. The aim of this thesis was to investigate the mechanosensitivity of thefish heart at a number of resolutions. Chapter three looks at the effect of stretchon the isolated whole rainbow trout heart. I found that MSCs are activated atphysiological extremes of input and output pressures. The trout ortholog of acandidate MSC, TRPC1, was cloned and its presence in the heart was verified. Both MSCs and exaggerated cardiac transmural electrical heterogeneity cancause re-entrant arrhythmias in the mammalian heart. As the piscine heart hasshown resistance to these arrhythmias I examined the transmural electricalheterogeneity of the tuna heart in Chapter four. I found no evidence oftransmural electrical heterogeneity in the tuna heart which may explain thereduced susceptibility of the fish heart to re-enterant arrhythmias. In Chapter fiveI investigated the effect of stretch on ventricular trabecular bundle preparationsand isolated ventricular myocytes of the rainbow trout. This study was the first tofind a lack of a SFR in a vertebrate heart and provides evidence for theimportance of the Na+ /H+ -exchanger in the SFR. Finally the study in Chaptersix examined the length-dependent Ca2+ sensitivity of skinned ventricular rat andtrout myocytes. I show that the increased length-dependent Ca2+ sensitivity of thetrout myocytes may account for the extended functional limb of the piscinelength-tension relationship. Skinned trout myocytes were shown to develop ahigh passive tension that could not be explained by the trout titin isoform ratio,but may be explained by increased phosphorylation of titin in vivo. My PhD research has produced clear and novel evidence for theimportance of mechanosensitivity in the fish heart. From the level of the wholeheart to the level of the individual sarcomere, stretch induces physiologicalchanges in this vital organ. A greater understanding of piscine cardiacmechanosensitivity will greatly improve general knowledge ofmechanosensitivity in general and will provide an evolutionary point ofcomparison for the studies of mechanosensitivity in other organisms.

The actin cortex is an active biopolymer network that lines the inner side of the plasma membrane in most animal cells. It can both resist detrimental cell deformations and drive necessary cell shape changes. It is a highly dynamic system where actin filaments constantly polymerize and depolymerize and specific proteins form transient cross-links with lifetimes of a few tens of seconds at most. In addition, a particular type of cross-linkers, myosins, can also generate active stresses in the cortex. This dynamic nature allows for variations in the rheological properties of the cortex, which are very sensitive to its organization and in particular to the density and lifetime of cross-links. In vivo, the cortex is subject to variable levels of passive and active contractile stresses. At the molecular scale, these stresses result in tensile forces that can likely be sensed by the bonds between actin filaments and cross-linkers, and potentially other actin-bound cellular components. In this work, we investigated the mechanosensitive regulation of the organization of the cortex, focusing on the dynamics of the binding of cross-linkers to actin and on the variations in the amount of cortical actin filaments. To this aim, we carried out experiments on single HeLa cells, where we simultaneously measured their cortical tension using an atomic force microscope confinement method and assessed the spatial distribution of cross-linkers and actin filaments by confocal microscopy. On the one hand, we assessed the dependence of the actin-binding dynamics of filamins A and B, alpha-actinin-1 and nonmuscle myosin IIA to cortical tension. We measured how their residence time at the cortex and their contribution to the cortical connectivity depend on tension at steady state. Moreover, we also measured their response to peaks in passive cortical tension. The results of these experiments show that the two filamins and alpha-actinin-1 are catch-binding cross-linkers: the lifetime of their bonds to actin filaments increases under increasing tensile load. This leads to an increase in the connectivity of the cortex when the cortical tension is raised. On the other hand, we also probed the cortical response to peaks in active tension. We found that such tension peaks trigger a permanent increase in the amount of actin filaments at the cortex. In turn, this increase in cortical actin leads to a permanent increase in the cortical recruitment of all cross-linkers, in addition to the expected transient increase in the cortical recruitment of the catch-binding ones. We showed that this mechanosensitive process requires signaling by the cytosolic phospholipase A2 and the activity of the actin nucleator Arp2/3 complex. Together, our results show that two types of mechanosensitivity enable the reinforcement of the cortex in response to high mechanical stresses: a direct mechanosensitivity that relies on the catch-binding of cross-linkers, and an indirect mechanosensitivity mediated by biochemical signaling. / Der Aktinkortex ist ein aktives Biopolymer-Netzwerk, das in den meisten tierischen Zellen die Innenseite der Plasmamembran auskleidet. Er kann sowohl möglicherweise schädliche Zellverformungen abpuffern als auch notwendige Änderungen der Zellform vorantreiben. Es handelt sich um ein hochdynamisches System, in dem Aktinfilamente ständig polymerisieren und depolymerisieren und spezifische Proteine vorübergehende Quervernetzungen mit einer Lebensdauer von höchstens ein paar zehn Sekunden bilden. Darüber hinaus kann eine bestimmte Art von Quervernetzern, die Myosine, auch aktive Spannungen im Kortex erzeugen. Diese dynamische Natur ermöglicht Veränderungen der rheologischen Eigenschaften des Kortex, die sehr empfindlich auf seine Organisation und insbesondere auf die Dichte und Lebensdauer der Vernetzungen reagieren. In vivo ist der Kortex unterschiedlich starken passiven und aktiven kontraktilen mechanischen Spannungen ausgesetzt. Auf molekularer Ebene führen diese Spannungen zu Zugkräften, die wahrscheinlich von den Bindungen zwischen Aktinfilamenten und Quervernetzern und möglicherweise anderen aktingebundenen zellulären Komponenten wahrgenommen werden können. In dieser Arbeit haben wir die mechanosensitive Regulierung der Organisation des Kortex untersucht, wobei wir uns auf die Dynamik der Bindung von Quervernetzern an Aktin und die Schwankungen in der Menge der kortikalen Aktinfilamente konzentriert haben. Zu diesem Zweck haben wir Experimente an einzelnen HeLa-Zellen durchgeführt, bei denen wir gleichzeitig die kortikale Oberflächenspannung mit Hilfe einer rasterkraftmikroskopischen Confinement-Methode gemessen und die räumliche Verteilung von Quervernetzern und Aktinfilamenten mittels konfokaler Mikroskopie untersucht haben. Einerseits haben wir die Abhängigkeit der Aktinbindungsdynamik der Filamine A und B, des alpha-Actinin-1 und des nicht-muskulären Myosin IIA von der kortikale Spannung untersucht. Wir haben gemessen, wie ihre Verweildauer im Kortex und ihr Beitrag zur kortikalen Konnektivität von der Spannung im Gleichgewichtszustand abhängen. Wir haben auch ihre Reaktion auf Spitzen in der passiven kortikalen Spannung gemessen. Unsere Ergebnisse zeigen, dass die beiden Filamine und alpha-Actinin-1 'catch'-bindende Vernetzer sind: Die Lebensdauer ihrer Bindungen an Aktinfilamente nimmt mit zunehmender Zugbelastung zu. Dies führt zu einer Zunahme der Konnektivität des Kortex, wenn die kortikale Spannung erhöht wird. Andererseits haben wir auch die kortikale Reaktion auf aktive Spannungsspitzen untersucht. Wir fanden heraus, dass solche Spannungsspitzen einen permanenten Anstieg der Menge an Aktinfilamenten am Kortex auslösen. Dieser Anstieg des kortikalen Aktins wiederum führt zu einem permanenten Anstieg der kortikalen Rekrutierung aller Quervernetzer, zusätzlich zu dem erwarteten vorübergehenden Anstieg der kortikalen Rekrutierung der catch-bindenden Quervernetzer. Wir konnten zeigen, dass dieser mechanosensitive Prozess eine Signalisierung durch die zytosolische Phospholipase A2 und die Aktivität des Aktinnukleators Arp2/3-Komplexes erfordert. Zusammengenommen zeigen unsere Ergebnisse, dass zwei Arten von Mechanosensitivität die Verstärkung des Kortex als Reaktion auf hohe mechanische Belastungen ermöglichen: eine direkte Mechanosensitivität, die auf der Catch-Bindung von Quervernetzern beruht, und eine indirekte Mechanosensitivität, die durch biochemische Signalübertragung vermittelt wird.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The role of mechanical forces in the fate and function of adherent cells has been revealed to be a pivotal factor in understanding cell biology. Cells require certain physical cues to be present in their microenvironment or the cell will begin apoptosis. Mechanical signals from the environment are interpreted at the cellular level and biochemical responses are made due to the information from outside the cell, this process is known as mechanotransduction. Misinterpretation of physical cues has been indicated in many disease states, including heart disease and asthma. When a cell is bound to the ECM, proteins such as integrins are engaged at static and stable adhesion sites. These tight and static anchoring points found at the ECM exist in stark contrast to the dynamic conditions seen at intercellular junctions. Intercellular junctions, such as gap and adherens junctions, are formed between cells to act as a mechanism to relay information and exchange material. Due to the important role intercellular junctions play in processes of wound healing, epithelial-mesenchymal transition and cancer metastasis developing more sophisticated levels of understanding of these mechanisms would provide valuable insight. Complex biological processes, including immune cell signaling and cellular ECM adhesions, have been effectively replicated in model systems. These model systems have included the use of solid supported lipid bilayers and polymeric hydrogels that display cell adhesion molecules. Studies of cellular mechanotransduction at ECM adhesion sites has also been completed with covalently functionalized polymeric substrates of adjustable elasticity. However, developing model systems that allow the accurate reproduction of properties seen at intercellular junctions, while also allowing the investigation of cellular mechanosensitivity has proven to be a difficult task. Previous work has shown that polymer-tethered lipid bilayers (PTLBs) are a viable material to allow the replication of the dynamics and adhesion seen at intercellular junctions. Although efforts have been made to produce PTLBs with different mechanical properties, there is currently not a material with sufficient tunable elastic properties for the study of cellular mechanotransduction. To establish a system that allows the study of stiffness effects across a biologically relevant range (~0.50 – 40 kPa) while maintaining the dynamic properties seen at cell-to-cell junctions, polymer gel-tethered bilayers (PGTBs) were developed. A fabrication strategy was established to allow the incorporation of a hydrogel support with easily tunable stiffness and a tethered lipid bilayer coating, which produced a powerful platform to study the effects of stiffness at intercellular junctions. Careful attention was given to maintain the beneficial properties of membrane diffusion, and it was shown that on different linking architectures lipid bilayers could be established and diffusion was preserved. Microscopy-based FCS and FRAP methodology were utilized to measure lipid diffusion in these systems, while confocal microscopy was used to analyze cell spreading and adhesion. Three distinct architectures to link the lipid membrane to the underlying polyacrylamide hydrogel were pursued in this work, a non-covalent biotin-streptavidin system, a covalently linked design with fibronectin, and a direct covalent linkage utilizing crosslinker chemistry. In this work, it was shown that cells were able to spread and adhere on these substrates, with cell adhesion zones visualized under plated cells that demonstrate the capability of the cell to rearrange the presented linkers, while maintaining a stable material. Also confirmed is the tunability of the polymer hydrogel across a wide range of stiffness, this was shown by quantitative changes in cell spreading area in response to polymer properties.

Chez les vertébrés, la douleur est perçue par des neurones spécialisés, les nocicepteurs, dont le corps cellulaire est localisé dans les ganglions de la racine dorsale (DRG) et qui présentent une grande hétérogénéité. Nous cherchons donc à identifier de nouveaux marqueurs des sous populations de nocicepteurs afin de pouvoir comprendre cette diversité et d’attribuer des fonctions physiologiques précises à ces différentes sous-populations neuronales. Nous avons identifié le gène GINIP, spécifiquement exprimé dans une sous populations de nocicepteurs non peptidergiques et définissant deux classes particulières de neurones : les neurones MRGPRD+ et les C-Low Threshold MecanoReceptors (C-LTMRs). Durant ma thèse, j’ai cherché à savoir quelles modalités sensorielles sont détectées et transmises par la population GINIP+. Pour cela, j’ai tiré avantage d’un modèle murin ginip généré au laboratoire, permettant d’éliminer spécifiquement les neurones GINIP+ au sein des neurones du DRG. J’ai pu démontrer que l’ablation ciblée de ces neurones entraine une diminution de la douleur induite par l’injection de formaline, cela sans affecter la sensibilité thermique ou mécanique. Sachant que les neurones MRGPRD positifs ne sont pas impliqués dans la réponse douloureuse induite par l’injection de formaline, mais jouent un rôle primordial dans la mécano sensibilité en condition normale et pathologique, notre étude montre que la réponse douloureuse induite par l’injection de formaline est due à l’activation des C-LTMRs. En conclusion, notre étude révèle que les C-LTMRs agissent en tant que puissants modulateurs des douleurs chimiques et mécaniques. / Primary sensory neurons are heterogeneous by myriad of molecular criteria. However, the functional significance of this remarkable heterogeneity is just emerging. Here we used our recently generated ginip mouse model to selectively ablate the cutaneous free nerve endings MRGPRD+ neurons and the C-Low threshold mechanoreceptors (C-LTMRs). Ablation of GINIP-expressing neurons led to a significant decrease of formalin-evoked first pain and a complete absence of the second phase pain response, without affecting thermal or mechanical sensitivity. Knowing that MRGPRD+ neurons are dispensable for formalin-evoked pain and that these neurons play a critical role in acute and injury-induced mechanical pain, our data demonstrate that formalin-induced pain hypersensitivity is primarily transduced via C-LTMRs, and suggest that C-LTMRs and MRGPRD+ neurons play antagonistic roles in transduction of acute and injury-induced mechanical pain. Therefore, our results suggest that C-LTMRs act as strong modulators of chemical and mechanical pain signals.

<p>The role of mechanical forces in the fate and function of adherent cells has been revealed to be a pivotal factor in understanding cell biology. Cells require certain physical cues to be present in their microenvironment or the cell will begin apoptosis. Mechanical signals from the environment are interpreted at the cellular level and biochemical responses are made due to the information from outside the cell, this process is known as mechanotransduction. Misinterpretation of physical cues has been indicated in many disease states, including heart disease and asthma. When a cell is bound to the ECM, proteins such as integrins are engaged at static and stable adhesion sites. These tight and static anchoring points found at the ECM exist in stark contrast to the dynamic conditions seen at intercellular junctions. Intercellular junctions, such as gap and adherens junctions, are formed between cells to act as a mechanism to relay information and exchange material. Due to the important role intercellular junctions play in processes of wound healing, epithelial-mesenchymal transition and cancer metastasis developing more sophisticated levels of understanding of these mechanisms would provide valuable insight.</p> <p>Complex biological processes, including immune cell signaling and cellular ECM adhesions, have been effectively replicated in model systems. These model systems have included the use of solid supported lipid bilayers and polymeric hydrogels that display cell adhesion molecules. Studies of cellular mechanotransduction at ECM adhesion sites has also been completed with covalently functionalized polymeric substrates of adjustable elasticity. However, developing model systems that allow the accurate reproduction of properties seen at intercellular junctions, while also allowing the investigation of cellular mechanosensitivity has proven to be a difficult task. Previous work has shown that polymer-tethered lipid bilayers (PTLBs) are a viable material to allow the replication of the dynamics and adhesion seen at intercellular junctions. Although efforts have been made to produce PTLBs with different mechanical properties, there is currently not a material with sufficient tunable elastic properties for the study of cellular mechanotransduction.</p> <p>To establish a system that allows the study of stiffness effects across a biologically relevant range (~0.50 – 40 kPa) while maintaining the dynamic properties seen at cell-to-cell junctions, polymer gel-tethered bilayers (PGTBs) were developed. A fabrication strategy was established to allow the incorporation of a hydrogel support with easily tunable stiffness and a tethered lipid bilayer coating, which produced a powerful platform to study the effects of stiffness at intercellular junctions. Careful attention was given to maintain the beneficial properties of membrane diffusion, and it was shown that on different linking architectures lipid bilayers could be established and diffusion was preserved. Microscopy-based FCS and FRAP methodology were utilized to measure lipid diffusion in these systems, while confocal microscopy was used to analyze cell spreading and adhesion. Three distinct architectures to link the lipid membrane to the underlying polyacrylamide hydrogel were pursued in this work, a non-covalent biotin-streptavidin system, a covalently linked design with fibronectin, and a direct covalent linkage utilizing crosslinker chemistry. In this work, it was shown that cells were able to spread and adhere on these substrates, with cell adhesion zones visualized under plated cells that demonstrate the capability of the cell to rearrange the presented linkers, while maintaining a stable material. Also confirmed is the tunability of the polymer hydrogel across a wide range of stiffness, this was shown by quantitative changes in cell spreading area in response to polymer properties.</p>

Gewebe sind nicht nur durch ihre biochemische Zusammensetzung definiert, sondern auch durch ihre individuellen mechanischen Eigenschaften. Inzwischen ist es weithin akzeptiert, dass Zellen ihre mechanische Umgebung spüren und darauf reagieren. Zum Beispiel werden Zellmigration und die Differenzierung von Stammzellen durch die Umgebungssteifigkeit beeinflusst. Um diese Effekte in vitro zu untersuchen, wurden viele Zellkulturstudien auf 2D Hydrogelsubstraten durchgeführt. Zusätzlich dazu steigt die Anzahl von Studien an, die hydrogelbasierte 3D-Scaffolds nutzen, um 2D Studien zu validieren und die experimentellen Bedingungen der Situation in vivo anzunähern. Jedoch erweist es sich weiterhin als schwierig den Effekt von Mechanik in 3D in vitro zu untersuchen, da in den gemeinhin genutzten 3D Hydrogelsystemen immer eine Kopplung zwischen Gelporosität und Steifigkeit besteht. Zusätzlich hängt die Konzentration der biologisch aktiven Bindungsstellen für Zellen oft ebenfalls von der Steifigkeit ab. Diese Arbeit präsentiert die Entwicklung und Optimierung neuer 3D Hydrogelkugel-Scaffolds, in denen die Steifigkeit von der Porosität schließlich entkoppelt wird. Mit Hydrogelkugeln als Scaffold-Bausteine ist es nun möglich 3D Scaffolds mit definierten mechanischen Eigenschaften und konstanter Porengröße zu generieren. Während der Methodenentwicklung wurden verschiedene Prinzipien und Kultivierungskammern konstruiert und überarbeitet, gefolgt von der theoretischen Betrachtung der Sauerstoffdiffusion, um die Eignung der gewählten Kammer hinsichtlich Zellvitalität und Zellwachstum zu überprüfen. Eine Kombination aus mehreren getesteten Filtern wurde ausgewählt um HydrogelkugelScaffolds erfolgreich in der ausgewählten Kammer zu generieren. Im Weiteren wurden verschiedene Hydrogelmaterialien untersucht hinsichtlich der erfolgreichen Produktion monodisperser Hydrogelkugeln und der Erzeugung stabiler Scaffolds. Hydrogelkugeln aus Polyacrylamid (PAAm) wurden als Scaffold-Bausteine ausgewählt um damit die Eignung des entwickelten Systems zu demonstrieren lebende Zellen zu mikroskopieren. Außerdem wurde das Überleben von Fibroblasten über vier Tage in unterschiedlich steifen HydrogelkugelScaffolds erfolgreich gezeigt. Weiterhin war es möglich erste Zellmigrationsexperimente durchzuführen. Dafür wurden sowohl einfache PAAm-Hydrogelkugeln als auch mit Adhäsionsmolekülen funktionalisierte Hydrogelkugeln genutzt, um unterschiedlich steife Schichten in einem Scaffold zu erzeugen. Dadurch war es möglich nicht nur Zellmigration anhand von Zelladhäsion in 3D Scaffolds mit Steifigkeitsgradienten zu beobachten, sondern auch Zellmigration ohne Zelladhäsion.:1 Introduction 1.1 Mechanics play a role in biology 1.2 3D cultures and scaffolds 1.3 3D hydrogel systems to study effects of mechanics 1.4 Decoupling stiffness and porosity in 3D scaffolds 2 Materials 3 Methods 3.1 Laser scanning microscopy and microscopy data processing 3.2 Atomic force microscopy (AFM) 3.3 Refractive index matching of PMMA beads 3.4 Regular PMMA bead scaffolds for developing analysis algorithm 3.5 Cell culture standards 3.6 Fluorescent labelling of ULGP agarose 3.7 Production of polydisperse ULGP agarose beads 3.8 Hydrogel bead production via microfluidics 3.9 PAAm bead functionalization 3.10 Real-time fluorescence and deformability cytometry (RT-fDC) 3.11 3D scaffolds made from hydrogel beads 3.12 Statistics 4 Results 4.1 Design of a suitable scaffold device 4.2 Theoretical oxygen supply in 3D culture system is sufficient for cell survival and proliferation 4.3 Further optimization of 3D scaffold device 4.3.1 PMMA beads can be arranged in stable scaffolds 4.3.2 Regular PMMA bead scaffolds can be achieved and analysed 4.3.3 PMMA bead scaffolds and agarose bead scaffolds act as combined filter to stack up hydrogel beads 4.4 PAAm hydrogel beads produced by microfluidics are suitable to create compliant 3D scaffolds 4.5 Reproducible, regular and stable 3D scaffolds made of hydrogel beads 4.6 NIH-3T3/GFP cell migration within 3D hydrogel bead scaffolds 5 Discussion and Concluding Remarks 6 Bibliography List of Figures List of Tables Eigenständigkeitserklärung Appendix A Appendix B FIJI macro for FFT analysis maxima Python script to determine regularity of PMMA bead scaffolds Excel macro to determine number of peaks for regularity analysis / Tissues are defined not only by their biochemical composition, but also by their distinct mechanical properties. It is now widely accepted that cells sense their mechanical environment and respond to it. For example, cell migration and stem cell differentiation is affected by stiffness. To study these effects in vitro, many cell culture studies have been performed on 2D hydrogel substrates. Additionally, the amount of 3D studies based on hydrogels as 3D scaffold is increasing to validate 2D in vitro studies and adjust experimental conditions closer to the situation in vivo. However, studying the effects of mechanics in vitro in 3D is still challenging as commonly used 3D hydrogel assays always link gel porosity with stiffness. Additionally, the concentration of biologically active adhesion sides often also depends on the stiffness. This work presents the development and optimization of novel 3D hydrogel bead scaffolds where the stiffness is finally decoupled from porosity. With hydrogel beads as scaffold building blocks it was possible to generate 3D scaffolds with defined mechanical properties and a constant pore size. During the method development, different culture devices were constructed and revised, followed by oxygen diffusion simulations to proof the suitability of the chosen device for cell survival and growth. A combination of different filter approaches was selected to generate hydrogel bead scaffolds in the culture device. Furthermore, different hydrogel materials were investigated regarding successful production of monodisperse beads and stable scaffold generation. Polyacrylamide (PAAm) hydrogel beads were chosen as scaffold building blocks to demonstrate live-cell imaging and successful cell survival over four days in differently compliant hydrogel bead scaffolds. Moreover, first cell migration experiments were performed by using plain PAAm hydrogel beads as well as PAAm hydrogel beads functionalized with adhesion molecules with differently stiff layers in one scaffold. Thereby fibroblast migration was observed not only in adhesion-dependent migration manner, but also in an adhesion-independent mode .:1 Introduction 1.1 Mechanics play a role in biology 1.2 3D cultures and scaffolds 1.3 3D hydrogel systems to study effects of mechanics 1.4 Decoupling stiffness and porosity in 3D scaffolds 2 Materials 3 Methods 3.1 Laser scanning microscopy and microscopy data processing 3.2 Atomic force microscopy (AFM) 3.3 Refractive index matching of PMMA beads 3.4 Regular PMMA bead scaffolds for developing analysis algorithm 3.5 Cell culture standards 3.6 Fluorescent labelling of ULGP agarose 3.7 Production of polydisperse ULGP agarose beads 3.8 Hydrogel bead production via microfluidics 3.9 PAAm bead functionalization 3.10 Real-time fluorescence and deformability cytometry (RT-fDC) 3.11 3D scaffolds made from hydrogel beads 3.12 Statistics 4 Results 4.1 Design of a suitable scaffold device 4.2 Theoretical oxygen supply in 3D culture system is sufficient for cell survival and proliferation 4.3 Further optimization of 3D scaffold device 4.3.1 PMMA beads can be arranged in stable scaffolds 4.3.2 Regular PMMA bead scaffolds can be achieved and analysed 4.3.3 PMMA bead scaffolds and agarose bead scaffolds act as combined filter to stack up hydrogel beads 4.4 PAAm hydrogel beads produced by microfluidics are suitable to create compliant 3D scaffolds 4.5 Reproducible, regular and stable 3D scaffolds made of hydrogel beads 4.6 NIH-3T3/GFP cell migration within 3D hydrogel bead scaffolds 5 Discussion and Concluding Remarks 6 Bibliography List of Figures List of Tables Eigenständigkeitserklärung Appendix A Appendix B FIJI macro for FFT analysis maxima Python script to determine regularity of PMMA bead scaffolds Excel macro to determine number of peaks for regularity analysis

No / We demonstrate the synergistic benefits of using multiple technologies to investigate complex multi-scale biological responses. The combination of reductionist and integrative methodologies can reveal novel insights into mechanisms of action by tracking changes of in vivo phenomena to alterations in protein activity (or vice versa). We have applied this approach to electrical and mechanical remodelling in right ventricular failure caused by monocrotaline-induced pulmonary artery hypertension in rats. We show arrhythmogenic T-wave alternans in the ECG of conscious heart failure animals. Optical mapping of isolated hearts revealed discordant action potential duration (APD) alternans. Potential causes of the arrhythmic substrate; structural remodelling and/or steep APD restitution and dispersion were observed, with specific remodelling of the Right Ventricular Outflow Tract. At the myocyte level, [Ca2+]i transient alternans were observed together with decreased activity, gene and protein expression of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Computer simulations of the electrical and structural remodelling suggest both contribute to a less stable substrate. Echocardiography was used to estimate increased wall stress in failure, in vivo. Stretch of intact and skinned single myocytes revealed no effect on the Frank-Starling mechanism in failing myocytes. In isolated hearts acute stretch-induced arrhythmias occurred in all preparations. Significant shortening of the early APD was seen in control but not failing hearts. These observations may be linked to changes in the gene expression of candidate mechanosensitive ion channels (MSCs) TREK-1 and TRPC1/6. Computer simulations incorporating MSCs and changes in ion channels with failure, based on altered gene expression, largely reproduced experimental observations.

Many preterm infants require mechanical ventilation as life-saving therapy. However, ventilation-induced overpressure can result in lung diseases. Considering the lung as a viscoelastic material, positive pressure inside the lung results in increased hydrostatic pressure and tissue compression. To elucidate the effect of positive pressure on lung tissue mechanics and cell behavior, we mimic the effect of overpressure by employing an uniaxial load onto fetal and adult rat lungs with different deformation rates. Additionally, tissue expansion during tidal breathing due to a negative intrathoracic pressure was addressed by uniaxial tension. We found a hyperelastic deformation behavior of fetal tissues under compression and tension with a remarkable strain stiffening. In contrast, adult lungs exhibited a similar response only during compression. Young’s moduli were always larger during tension compared to compression, while only during compression a strong deformation-rate dependency was found. In fact, fetal lung tissue under compression showed clear viscoelastic features even for small strains. Thus, we propose that the fetal lung is much more vulnerable during inflation by mechanical ventilation compared to normal inspiration. Electrophysiological experiments with different hydrostatic pressure gradients acting on primary fetal distal lung epithelial cells revealed that the activity of the epithelial sodium channel (ENaC) and the sodium-potassium pump (Na,K-ATPase) dropped during pressures of 30 cmH2O. Thus, pressures used during mechanical ventilation might impair alveolar fluid clearance important for normal lung function.

No description available.