Migratory cells in vivo move within a predominantly fibrous microenvironment through the action of forces. These dynamic interactions facilitate mechanosensing, critical to fundamental biological processes in pathophysiology. Naturally, the field of mechanobiology has evolved over the past several decades to decipher the role of forces in mechanotransduction using a variety of force-measurement platforms. A central challenge that has yet to be overcome in the field is connecting forces with the interplay between cell shape and ever-changing environment. Here, through design of specific fibrous architectures, a mechanobiological understanding of force feed-forward loop accounting for shape shifting of the environment and cells is developed. Using the non-electrospinning Spinneret Tunable Engineered Parameters (STEP) technique, two complementary force measurement platforms of varying physical attributes are developed to investigate how the force feed-forward loop impacts cell fate. Nanonet Force Microscopy (NFM) comprised of aligned nanonets is designed to study anisotropic cell shapes, while Crosshatch Force Microscopy (CM) comprised of orthogonal arrangement of fibers is designed to study cell bodies of broad shapes. The combination of shapes achieved on these networks recapitulate mesenchymal shapes observed in vivo, which are used to describe cell behaviors not reported before. The new findings include (i) discovery of a new biological structure, termed 3D-perpendicular lateral protrusions (3D-PLPs) which is proposed to be the missing biophysical link in the remodeling of the ECM and perpetuation of desmoplasia. Using NFM, seven discreet steps in formation of force-exerting PLPs anywhere along the cell body is documented, which allow cells to spread laterally and increase in contractility. Using a variety of fiber networks, it is shown that aligned fibers are necessary for PLP formation and suitable environments for myofibroblast activation, and (ii) a force dipole that links matrix deformability with cell contractility. Aided by machine learning, CFM automates the process of fiber feature recognition to measure forces as cells change shapes during migration and differentiate to osteogenic and adipogenic lineages. The force platforms are applied to investigate (i) the bioenergetic contributors fueling cellular migration and a surprisingly overwhelming impact of glycolytic energetic pathway over the traditionally thought mitochondrial energy production is found. However, neither pathway has substantial impact over the cellular force production, and (ii) quantitate the migratory and contractile response of enucleated cytoplasmic fragments naturally shed by cells. A peculiar contractility driven oscillatory migratory phenotype is found, capable of lasting over tens of hours, and absent in intact cells. Overall, new high spatiotemporal capabilities are developed in mechanobiology to quantitate the force-feed forward loops between cell shape and ECM in pathophysiology. / Doctor of Philosophy / Pathophysiology is the study of abnormal changes in the regular body functions of an organism that are causes or consequences of disease onset. Research in this area is mainly focused on identifying the different factors that cause and propagate the disease states such as cancer. Central to many of these processes are events such as cell migration and remodeling of their surrounding environment. The native microenvironment surrounding cells is highly complex and is composed of many classes of macromolecules, with fibrous components being one of the most important. How cells interact with these environments through application of forces and how this further regulates cellular behavior is vital to advancing our understanding of many of these pathophysiological processes. Currently, there is a lack in our understanding of how this dynamic process referred to as the "force feed-forward loop", is perpetuated. This limitation in our understanding can be attributed to the lack of an in vivo mimicking platform that captures this dynamic interaction and is capable of measuring the forces. To this end, the development of two novel single cell force measurement platforms: Nanonet Force Microscopy (NFM) and Crosshatch Force Microscopy (CFM) is presented. These platforms are fiber based systems, generated with the utilization of previously established non-electrospinning technique of Spinneret based Tunable Engineered Parameters (STEP) technique. Using NFM and CFM, forces were computed in wide range of cell shapes from anisotropic to all other spread morphologies. These platforms were applied to identify a new biological structure called perpendicular lateral protrusions and shown to have potential role in the spreading of tumor microenvironment. Furthermore, the force dynamics in physiological processes such as stem cell differentiation into fat cells or bone cells is also identified. How cellular processes such as migration and force production is fueled is also investigated and found to be not heavily reliant on the commonly understood mitochondrial activity. Finally, sub-cellular components known as cell fragments, which are devoid of nucleus, are also observed to be contractile and migratory in nature, independent of parent cell body. These platforms and findings can be further utilized to advance our current knowledge of the progression of these physiological and pathological processes and serve as diagnostic tools for the early identification of disease onset. Furthermore, based on these findings, strategies can be developed for early intervention to inhibit disease progression or devise bioengineered scaffolds for applications in tissue engineering.

It is estimated that approximately 2 million fires which occur in United States each year result in 1.2 million burn victims. Fibroblasts are responsible for responding to this tissue damage by breaking down the damaged extracellular matrix (ECM) and secreting a new ECM which aids in wound repair and supports the migration of immune cells. Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with health-care infections (HCAIs) due to its ability to take advantage of immunocompromised hosts. However, little research has investigated how wound invading P. aeruginosa interacts with wound repairing fibroblasts. To address this lack of understanding, this thesis focuses on quantifying changes in fibroblast morphology, migratory behavior, and force exertion to investigate this host cell's response to representative cytotoxic (PAO1) and invasive (PA14) strains of P. aeruginosa. These assays study host cell-pathogen interactions on highly aligned nanofibers of varied spacing and diameter, which mimic the fibroblast deposited ECM and dictate fibroblast morphology. We discovered that the cytotoxic strain of P. aeruginosa induced significantly shorter fibroblast death times. Furthermore, two modes of death, sharp and gradual, were identified and found to be dependent on both fiber configuration and strain of P. aeruginosa. In addition, fibroblasts exposed to PAO1 migrating on the parallel formation were found to be significantly slower and less persistent than those exposed to PA14, however, fibroblasts exposed to both strains of bacteria were shown to exert similar forces. Lastly, exposure to PA14 led to the greatest change in actin, evident by increased actin punctae and less prominent actin stress fiber formation. / Master of Science / It is estimated that approximately 2 million fires which occur in United States each year result in 1.2 million burn victims. Fibroblasts respond to burn wounds by breaking down the damaged tissue fibers, termed extracellular matrix (ECM), and secreting a new ECM. Unfortunately, severe thermal injuries place hospitalized burn victims at high risk of infection. Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with health-care infections (HCAIs) due to its ability to take advantage of immunocompromised hosts. However, little research has investigated how wound invading P. aeruginosa interacts with wound healing fibroblasts. To address this knowledge gap, this thesis focuses on quantifying changes in fibroblast shape, migratory behavior, and force exertion to investigate this host cell's response to two strains of P. aeruginosa, which employ different mechanisms of invasion. These interactions are studied on a platform of suspended nanofibers with controlled spacing and diameter, to dictate fibroblast shape and mimic the fibroblast deposited ECM. We discovered that the two strain of P. aeruginosa induced significantly different fibroblast death times. During death, it was observed that fibroblasts either balled up quickly, termed sharp death, or remained spread out, termed gradual death, dependent upon fibroblast shape and strain of P. aeruginosa introduced. In addition, significant differences in migration speed and persistence were found between fibroblasts exposed to the two strains of bacteria, however, both groups were shown to exert similar forces. Lastly, the fibrous proteins which make up the cytoskeleton of the cell, actin stress fibers, were found to vary among the control and bacteria treated cells.

Knochen ist ein multifunktionales Organ und zugleich ein biologisches Material. In dieser Arbeit wird der Heilungsverlauf eines Knochenbruchs (als biologisches Material) näher untersucht mit Hilfe von Computermodellen. Im menschlichen Körper kommt es nach einem Bruch zu einer vollständigen Regeneration des Knochens, ohne dass eine Narbe nach der Heilung zurückbleibt. In grob 10% der Frakturen kommt es jedoch zu Komplikationen bis zu einem Nicht-Heilen des Bruches. Das Ziel von intensiver interdisziplinärer Forschung ist es daher, nicht nur die medikamentöse Behandlung solcher Komplikationen zu verbessern, sondern auch durch externe, biophysikalische Stimulation die Heilung anzuregen. Gewöhnlich heilt ein Knochenbruch nicht direkt (Primäre Knochenheilung), das heißt durch Bildung von neuem Knochen im Knochenspalt, sondern über Sekundäre Knochenheilung. Während der sekundären Heilung bildet sich vorübergehend zusätzliches Gewebe außerhalb des Frakturspaltes, der so genannte Kallus, der die Aufgabe hat, den Bruch zu stabilisieren. Im Kallus werden im Laufe der Heilung verschiedene Gewebearten gebildet (z.B. Bindegewebe, Knorpel und Knochen). Die Gewebe werden von spezialisierten biologischen Zellen gebildet. Die spezialisierten Zellen entwickeln sich aus mesenchymalen Stammzellen (d.h. sie differenzieren), die in den Kallus wandern. Hauptziel der Arbeit ist das bessere Verständnis der mechano-biologischen Regulation der Gewebeformation während der Heilung eines normalen Knochenbruches. Dazu wurden Computersimulationen durchgeführt und mit experimentellen Daten eines Schafmodels verglichen. / Bone is a multifunctional organ, a biological material and is able to fully restore bone fractures without leaving a scar. However, in about 10% of the bone fractures, healing does not lead to a successful reunion of the broken bone ends. Intensive interdisciplinary research therefore looks for new ways to promote healing not only by medication, but also by external biophysical stimulation. Usually, bone fractures do not heal by a direct bridging of the fracture gap with newly formed bone (primary bone healing). Instead, secondary bone healing proceeds indirectly via the formation of an external callus (additional tissue). Within the callus, intricate tissue type patterns are formed, which evolve during the healing progression. Stem cells differentiate into specialized cells, which lay down different tissues such as fibrous tissue, cartilage and bone. This cell differentiation can be biophysically stimulated, e.g. by mechanical deformation of the cytoskeleton. The main aim of this thesis was to connect the microscopic cell response to mechanical stimulation with the macroscopic healing progression. Simple rules for cell behaviour were implemented in a computer model, the progression of healing was simulated and the outcome of the simulations was compared to results from animal experiments. In comparison to existing simulations of bone healing, this study approached the problem from a more physical viewpoint and linked experimental in vivo data and computer modelling.

Fluid pressures regulate endothelial cell (EC) tubulogenic activity involving fibroblast growth factor 2 (FGF-2) and its receptor, FGF receptor 2 (FGFR2). Our lab has recently shown that sustained 20 mmHg hydrostatic pressure (HP) upregulates EC sprout formation in a FGF2-dependent fashion. This upregulation of sprout formation may be due to enhanced FGF-2 / FGFR2 interactions in the presence of 20 mmHg HP. We hypothesize that exposure of ECs to 20 mmHg sustained HP enhances FGF-2 binding kinetics. We used a custom hydrostatic pressure system, immunofluorescence, and FACS to quantify FGF-2 binding by ECs in the absence or presence of a range of HPs for 30 minutes. Relative to cells maintained under control pressure, ECs exposed to 20, but neither 5 nor 40 mmHg, displayed a significant increase in binding affinity to FGF-2. EC binding of VEGF-A, another angiogenic growth factor, was unaffected by similar pressure stimuli. Additional studies showed that pressure-selective FGF-2 binding was independent of FGFR2 surface expression. These results implicate the FGF-2 axis in the pressure-sensitive, magnitude-dependent angiogenic processes which we have previously described. The present study provides novel insight regarding the involvement of FGF-2 signaling and interstitial pressure changes in various microvascular physiological and pathobiological processes.

Most animal cells adopt an approximately spherical shape when entering mitosis. This process has been termed mitotic rounding. It ensures the correct morphogenesis of the mitotic spindle and, in turn, successful cell division. When cells acquire a round shape at the entry of mitosis, they need to mechanically deform the surrounding tissue to do so. Previous studies suggest that the forces necessary for this deformation emerge from the contractility of the mitotic actin cortex. In fact, at the onset of mitosis, cortical contractility was found to be upregulated giving rise to an increased cell surface tension which drives the mitotic cell into a spherical shape. In a growing tumor, an increasing cell density generates a compressive mechanical stress which would likely lead to an increasing mechanical obstacle for mitotic rounding. Indeed, mechanical confinement or external pressure have been shown to hamper cell proliferation in tumor spheroids. Thus, it has been hypothesized that the actin cortex of cancer cells exhibits oncogenic adaptations that allow for ongoing mitotic rounding and division inside tumors. In fact, it was shown that the human oncogene Ect2 contributes to mitotic rounding through RhoA activation and that Ras overexpression promotes mitotic rounding. Epithelial-mesenchymal transition (EMT) is a cellular transformation in which epithelial cells loose epithelial polarity and intercellular adhesiveness gaining migratory potential. EMT, a hallmark in cancer progression, is commonly linked to early steps in metastasis promoting cancer cell invasiveness. Moreover, EMT was connected to cancer stem cells and the outgrowth of secondary tumors, suggesting that EMT may also be important for cell proliferation in a tumor. In this work, I investigated the role of EMT in actin cortex mechanics and mitotic rounding. To assess cortex mechanics, I measured the mechanical properties of the actin cortex in mitosis, in particular cortical stiffness and contractility before and after EMT. Furthermore, I also determined the mechanical changes of the actin cortex of interphase cells upon EMT; mechanics of interphase cells may critically influence mitotic rounding as interphase cells are a major constituent of the surrounding of a mitotic cell which needs to be deformed in the process of rounding. For our cortex-mechanical measurements, I used an established dynamic cell confinement assay based on atomic force microscopy. I show striking cortex- mechanical changes upon EMT that are opposite in interphase and mitosis. They are accompanied by a strong change in the activity of the actomyosin master regulators Rac1 and RhoA. Concomitantly, I characterize cortex-mechanical changes induced by Rac1 and RhoA signaling. In particular, I show that Rac1 inhibition restores epithelial cortex mechanics in post-EMT cells. Furthermore, I give evidence that EMT, as well as Rac1 activity changes induce actual changes in mitotic rounding in spheroids embedded in mechanically confining, covalently crosslinked hydrogels. Overall, I give evidence that EMT-induced changes results in a softer and less contractile cortex in interphase and a stiffer and more contractile cortex in mitotic cells, and it correlates with increased proliferation in confined environment.:Summary Zusammenfassung Acknowledgements 1-Introduction 1.1-The actin cortex 1.1.1-Regulation of actin cortex polymerization 1.1.2-Rho-GTPases in actin cortex regulation 1.1.3-The actin cortex in cell shape regulation and mitotic rounding 1.1.4-Experimental approaches to measure actin cortex mechanics 1.1.5-AFM cell confinement assay – a new tool for actin cortex-mechanical measurements 1.2-Epithelial-mesenchymal transition in cancer progression and metastasis 1.2.1-EMT effects on cell proliferation 1.2.2-EMT effects on Rho-GTPases activities 1.2.3-EMT effects on transcription factors 1.3-Outline of the thesis 2-Pharmacological induction of EMT 3-Mechanical changes of actin cortex mechanics upon EMT 3.1-Cell volume change during AFM confinement 3.2-Interphase and mitotic actin cortex mechanical changes upon EMT 3.3- Rho-GTPases activity changes upon EMT 4- Molecular perturbations of the cortex and their impact on cortex mechanics 5-Mitotic rounding in confined cell spheroids before and after EMT 5.1-The effect of cortex regulators on confined spheroids upon EMT 6-Time-dependence of actin cortex mechanics in breast epithelial cells 6.1-Rheology of actin cortex as a thin active film 6.2-Viscoelasticity of the actin cortex in relation to malignancy 7-Discussion 8-Outlook 8.1-Mitosis duration and quiescence in confined spheroids 8.2-Signalling cascades that trigger EMT-induced cortex-mechanical phenotype 8.2-Membrane tension upon EMT 9-Bibliography 10-Appendix 10.1-Abbreviations 10.2-Symbols / Die meisten tierischen Zellen nehmen beim Eintritt in die Mitose eine annähernd kugelförmige Form an. Dieser Vorgang wird als mitotische Aufrundung bezeichnet. Sie sorgt für die korrekte Morphogenese der mitotischen Spindel und damit für eine erfolgreiche Zellteilung. Wenn Zellen beim Eintritt der Mitose eine runde Form annehmen, müssen sie das umgebende Gewebe mechanisch verformen. Frühere Studien legen nahe, dass die für diese Verformung erforderlichen Kräfte aus der Kontraktilität des mitotischen Aktin-Cortexes resultieren; zu Beginn der Mitose führt ein Anstieg der kortikalen Kontraktilität zu einer erhöhten Zelloberflächenspannung, die die mitotische Zelle in eine kugelförmige Form treibt. Bei einem wachsenden Tumor erzeugt eine zunehmende Zelldichte einen Kompressionsdruck, der vermutlich ein zunehmendes mechanisches Hindernis für die mitotische Aufrundung darstellt. Es wurde gezeigt, dass mechanische Begrenzung oder äußerer Druck die Zellproliferation in Tumorsphäroiden hemmen. Es wurde daher die Hypothese aufgestellt, dass der Aktinkortex von Krebszellen onkogene Anpassungen aufweist, die eine fortlaufende mitotische Aufrundung und Zellteilung innerhalb von Tumoren ermöglichen. Weiterhin wurde gezeigt, dass das humane Onkogen Ect2 durch RhoA-Aktivierung zur mitotischen Aufrundung beiträgt und dass die Überexpression von Ras die mitotische Aufrundung fördert. Die epithelial-mesenchymale Transition (EMT) ist eine zelluläre Transformation, bei der Epithelzellen die epitheliale Polarität und die interzelluläre Adhäsivität verlieren und Migrationspotential gewinnen. EMT, ein Kennzeichen für das Fortschreiten von Krebs, ist häufig mit frühen Schritten der Metastasierung und einer Steigerung der Invasivität von Krebszellen verbunden. Darüber hinaus wird die EMT mit Krebsstammzellen und der Entstehung von Sekundärtumoren in Verbindung gebracht, was darauf hindeutet, dass die EMT auch für die Zellproliferation in einem Tumor wichtig sein könnte. In dieser Arbeit wurde die Bedeutung der EMT für die Mechanik des Aktinkortex und die mitotische Aufrundung untersucht. Die mechanischen Eigenschaften des Zellkortexes, insbesondere die kortikale Steifheit und Kontraktilität, wurden in mitotischen und nicht-adhärenten Interphasezellen gemessen vor und nach der EMT. Die Mechanik von Interphasenzellen kann die mitotische Aufrundung entscheidend beeinflussen, da Interphasenzellen ein Hauptbestandteil der Umgebung einer mitotischen Zelle sind, die während des Aufrundungsprozesses deformiert werden muss. Für meine kortexmechanischen Messungen verwendete ich einen etablierten Assay, der auf Rasterkraftmikroskopie basiert. Ich konnte ausgeprägte kortexmechanische Veränderungen durch die EMT feststellen, die in Interphase und Mitose entgegengesetzt sind. Diese kortikalen Veränderungen gehen mit einer starken Modifikation der Aktivitäten der Actomyosin-Hauptregulatoren Rac1 und RhoA einher. Weiterhin konnte ich kortexmechanische Veränderungen charakterisieren, die durch Rac1- und RhoA- Signale induziert werden. Insbesondere zeige ich, dass die Rac1-Hemmung die epitheliale Kortexmechanik in Post-EMT-Zellen wiederherstellt. Darüber hinaus fand ich Hinweise darauf, dass EMT- und Rac1-Aktivitätsänderungen zu einer Änderung der mitotischen Aufrundung in eingebetteten Sphäroiden führen. Insgesamt zeigen die Daten in dieser Arbeit klare Hinweise darauf, dass EMT-induzierte Veränderungen zu einem weicheren und weniger kontraktilen Kortex in der Interphase und einem steiferen und kontraktileren Kortex in mitotischen Zellen führen und mit einer erhöhten Proliferation in mechanisch begrenzten Zellumgebungen korrelieren.:Summary Zusammenfassung Acknowledgements 1-Introduction 1.1-The actin cortex 1.1.1-Regulation of actin cortex polymerization 1.1.2-Rho-GTPases in actin cortex regulation 1.1.3-The actin cortex in cell shape regulation and mitotic rounding 1.1.4-Experimental approaches to measure actin cortex mechanics 1.1.5-AFM cell confinement assay – a new tool for actin cortex-mechanical measurements 1.2-Epithelial-mesenchymal transition in cancer progression and metastasis 1.2.1-EMT effects on cell proliferation 1.2.2-EMT effects on Rho-GTPases activities 1.2.3-EMT effects on transcription factors 1.3-Outline of the thesis 2-Pharmacological induction of EMT 3-Mechanical changes of actin cortex mechanics upon EMT 3.1-Cell volume change during AFM confinement 3.2-Interphase and mitotic actin cortex mechanical changes upon EMT 3.3- Rho-GTPases activity changes upon EMT 4- Molecular perturbations of the cortex and their impact on cortex mechanics 5-Mitotic rounding in confined cell spheroids before and after EMT 5.1-The effect of cortex regulators on confined spheroids upon EMT 6-Time-dependence of actin cortex mechanics in breast epithelial cells 6.1-Rheology of actin cortex as a thin active film 6.2-Viscoelasticity of the actin cortex in relation to malignancy 7-Discussion 8-Outlook 8.1-Mitosis duration and quiescence in confined spheroids 8.2-Signalling cascades that trigger EMT-induced cortex-mechanical phenotype 8.2-Membrane tension upon EMT 9-Bibliography 10-Appendix 10.1-Abbreviations 10.2-Symbols

Extracellular matrix stiffening of breast tissues has been clinically correlated with malignant transformation and poor prognosis. An increase of collagen fibril diameter and lysyl-oxidase mediated crosslinking has been observed in advanced tumor stages. Many current reports suggest that the local mechanical properties of single fibrillar components dominantly regulate cancer cell behavior. Here, we demonstrate by an independent control of fibril diameter and intrafibrillar crosslinking of threedimensional (3D) collagen matrices that fibril bending stiffness instructs cell behavior of invasive and non-invasive breast cancer cells. Two types of collagen matrices with fibril diameter of either 650 nm or 800 nm at a similar pore size of 10 µm were reconstituted and further modified with the zero-length crosslinker 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide at concentrations of 0, 20, 100 and 500 mM. This approach yields a set of collagen matrices with overlapping variation of matrix elasticity. Within this set of matrices we could prove the common assumption that matrix elasticity of collagen networks is bending dominated with a linear dependence on fibril bending stiffness. We derive that the measured variation of matrix elasticity is directly correlated to the variation of fibril bending stiffness, being independently controlled either by fibril diameter or by intrafibrillar crosslinking. We use these defined matrices to demonstrate that the adjustment of fibril bending stiffness allows to instruct the behavior of two different breast cancer cell lines, invasive MDA-MB-231 (human breast carcinoma) and non-invasive MCF-7 cells (human breast adenocarcinoma). Invasiveness and spreading of invasive MDA-MB-231 cells as well as clustering of non-invasive MCF-7 cells is thereby investigated over a broad parameter range. Our results demonstrate and quantify the direct dependence of cancer cell phenotypes on the matrix mechanical properties on the scale of single fibrils.

Das menschliche Skelett besteht aus einem dynamischen Material welches in der Lage ist zu heilen, sowie sich durch strukturellen Umbau an mechanische Beanspruchung anzupassen. In dieser Arbeit ist die mechanische Regulierung dieser Prozesse untersucht worden. Hierfür ist ein Computermodell, sowie die dreidimensionale Abbildung des Knochens und die Auswertung dieser Bilder benutzt worden. An dem Heilungsprozesses von Knochen sind verschiedene Gewebetypen beteiligt. Dabei hängt die räumliche und zeitliche Anordnung dieser Gewebe von der mechanischen Belastung ab. Ein Computermodell, welches den vollständigen Verlauf der Heilung beschreibt, wurde mit der dokumentierten Gewebeentwicklung eines Tierexperimentes verglichen. Verschiedene Hypothesen, wie die mechanische Stimulation die Bildung verschiedene Gewebe beeinflusst, wurden getestet. Zwar ließen sich durch den Vergleich mit dem Experiment keine der Hypothesen verwerfen, jedoch konnten wir Vorschläge machen, worauf bei zukünftigen Experimenten verstärkt geachtet werden soll. Es wird angenommen dass der Umbauprozesses des Knochens vom dichten Netzwerk der Osteozyten mechanisch reguliert wird. Diese Zellen sind in den Knochen eingebettet und über ein dichtes Netzwerk aus engen Kanälen, den sogenannten Canaliculi, miteinander verbunden. Dieses Netzwerk mittels konfokaler Mikrokopie dreidimensional abgebildet. Spezielle Routinen zur Auswertung der Netzwerkorientierung sowie dessen Dichte wurden entwickelt. Die Hauptorientierung des Netzwerkes entspricht der Richtung in der Knochengewebe aufgebaut wird. Die Orientierung des zu dieser Richtung senkrechten Anteils des Netzwerkes rotiert abhängig von der Position entlang der Aufbaurichtung. Dies verdeutlicht den Zusammenhang zwischen der Netzwerkorientierung und der Vorzugsrichtung des Kollagens, dem faserigen Bestandteils des Knochens. Darüber hinaus zeigt die Auswertung der Daten weitere strukturelle Unterschiede im Netzwerk. / Our skeleton is composed of a dynamic material that is capable of healing and of adapting to changing mechanical loads through structural remodeling. In this thesis the mechano-regulation of these dynamic processes are addressed using computer modeling and 3-dimensional imaging and image analysis. During bone healing an intricate pattern of different newly formed tissues around the fracture site evolves in time and is influenced by the mechanical loading. Using a computer model which is describing this temporal-spatial evolution of tissue types for the full time-course of healing, this evolution is compared to the documented evolution of an animal experiment. Different hypotheses were tested how the mechanical stimulation results in the formation of different tissues. While the comparison with the outcome of the animal experiments does not allow to falsify any of the hypotheses, it suggests a different design of future animal experiments. Bone remodeling is thought to be mechano-regulated by the dense network of osteocytes. These osteocytes are embedded in bone and are connected to each other via a network of narrow canaliculi. The 3-dimensional structure of the network was imaged using rhodamine staining and laser scanning confocal microscopy. Image analysis tools were developed to determine the network topology and to analyze its density and orientation. The analysis focused on osteons, the building blocks of cortical bone. Within an osteon we found a large variability of the network density with extensive regions without network. Most of the network is oriented radially towards the center of the osteon, i.e.\ parallel to the direction in which the bone material is deposited. The network perpendicular to this direction twists when moving along the direction of bone deposition. A correlation with the main orientation the fibrous constituent of bone, collagen, was detected. Furthermore indicates our data additional structural changes in the network alignment.

The skin provides protection and maintains homeostasis, making it essential for survival. Additionally, skin has the impressive ability to grow, as observed in children as they grow into adults. However, skin functions are compromised in large skin defects, a serious problem that can be fatal. The gold standard treatment is to use an autologous skin graft; however, due to donor site morbidity and limited availability, when full-thickness defects surpass 2% total body surface area (TBSA), skin substitutes are preferred. Unfortunately, current skin substitutes on the market: are slow to revascularize (2+ weeks), have low graft survival rates (<50% take), and lead to significant scarring and contracture. Fortunately, a promising solution is to prevascularize engineered skin substitutes in vitro, which has been shown to facilitate rapid tissue integration upon grafting by providing an intact vascular network that readily connects to the host’s circulation. However, current approaches for prevascularizing tissue constructs require long in vitro culture times or implement low extracellular matrix (ECM) density tissue constructs – both which are problematic in a clinical setting. To address this, we implemented a novel multitissue interface culture model to define the design parameters that were essential for rapid vascularization of soft tissue constructs in vitro. Here, we identified endothelial colony forming cell (ECFC) density and maintenance of cell-matrix tensional forces as important factors for rapid in vitro tissue vascularization (18% vessel volume percentage after 3 days of culture). We then applied these parameters to achieve rapid in vitro vascularization of dense, oligomer tissue constructs (12, 20, and 40 mg/mL). We demonstrated, for the first time, rapid in vitro vascularization at 3 days within dense matrices (ECM concentration > 10 mg/mL). Lastly, a rat full-thickness excisional wound model was developed to determine the acellular densified oligomer’s (20 and 40 mg/mL) ability to resist wound contraction and facilitate a wound healing response (recellularization and vascularization) when grafted into wounds. Future work will implement the vascularized, dense tissue constructs into the developed animal model to assess the vascularized graft’s efficacy on treating wounds to reduce scarring and contracture outcomes.

L’ingénierie tissulaire est une stratégie médicale qui repose sur la régénération de tissu par les cellules avec ou sans matériaux. Pour maîtriser cette synthèse, il faut comprendre la cellule comme une part intégrante du tissu. Hormis ses interactions biochimiques avec son support, la cellule interagit également mécaniquement avec son environnement. Elle s’accroche à ce dernier et évalue sa dureté pour adapter sa réponse biologique. Dans cette étude, j’ai développé des modèles numériques pour analyser l’influence de la rigidité du substrat sur le comportement mécanique de la cellule, sur sa structure contractile interne et les efforts qu’elle génère. En d’autres termes, j’ai essayé de comprendre comment la cellule ressent la rigidité de son environnement. De plus, au lieu de me focaliser sur les propriétés mécaniques quantitatives, j’ai cherché à développer un modèle conceptuel simplifié plus proche de la structure cellulaire. / Tissue engineering is a medical strategy based on utilizing cells and materials to regenerate a new tissue. Yet, it involves intertwined interactions that allow cells to act as integrated parts of an organ. In addition to chemical reactions, the cell interacts mechanically with its environment by sensing its rigidity. Here, we used several computational models to understand how substrate rigidity affects a cell’s structure as it adheres and spreads on it. In other words we tried to understand the way a cell feels how soft or hard it surrounding is, how it affects its internal structure and the forces that transit within it. In addition, instead of focusing on mechanical properties, we developed a simplified, yet coherent conceptual understanding of the cellular structure.

Le récepteur low-density lipoprotein receptor-related protein 1 (LRP-1) est capable d’internaliser des protéases impliquées dans la progression du cancer, et constitue donc une cible thérapeutique prometteuse. Cependant, LRP-1 peut également réguler certaines protéines membranaires. Son ciblage dans une stratégie de modulation de la protéolyse pourrait donc affecter l’adhésion et la dynamique du cytosquelette. Dans ce travail, nous avons étudié l’influence de l’invalidation de LRP-1 sur des paramètres originaux corrélés au potentiel invasif de cellules cancéreuses par microscopie à force atomique (AFM). Cette invalidation induit des changements dans la dynamique d’adhérence des cellules et dans la morphologie, tels qu’un renforcement des fibres de stress et un étalement plus prononcé, causant une augmentation de la surface et de la circularité cellulaires. L’analyse des propriétés mécaniques par AFM a montré que ces différences sont acccompagnées par une augmentation du module d’Young. De plus, les mesures montrent une diminution globale de la motilité cellulaire et une perturbation de la persistance directionnelle. Une augmentation de la force d’adhésion entre cellules invalidées pour LRP-1 et une bille fonctionnalisée à la gélatine a également été observée. Enfin, nos données de spectroscopie de force enregistrées à l’aide d’une pointe fonctionnalisée par un anticorps anti-sous-unité d’intégrine β1 montrent que l’invalidation de LRP-1 modifie la dynamique des intégrines. Dans leur ensemble, nos résultats montrent que des techniques classiquement utilisées dans l’investigation de cellules cancéreuses peuvent être couplées à l’AFM pour ouvrir l’accès à des paramètres complémentaires, pouvant faciliter la discrimination entre différents degrés de potentiel invasif. / The low-density lipoprotein receptor-related protein 1 (LRP-1) can internalize proteases involved in cancer progression and is thus considered a promising therapeutic target. However, it has been demonstrated that LRP-1 is also able to regulate membrane-anchored proteins. Thus, strategies that target LRP-1 to modulate proteolysis could also affect adhesion and cytoskeleton dynamics. Here, we investigated the effect of LRP-1 silencing on parameters reflecting cancer cells’ invasiveness by atomic force microscopy (AFM). The results show that LRP-1 silencing induces changes in the cells’ adhesion behavior, particularly the dynamics of cell attachment. Clear alterations in morphology, such as more pronounced stress fibers and increased spreading, leading to increased area and circularity, were also observed. The determination of the cells’ mechanical properties by AFM showed that these differences are correlated with an increase in Young’s modulus. Moreover, the measurements show an overall decrease in cell motility and modifications of directional persistence. An overall increase in the adhesion force between the LRP-1-silenced cells and a gelatin-coated bead was also observed. Ultimately, our AFM-based force spectroscopy data, recorded using an antibody directed against the β1 integrin subunit, provide evidence that LRP-1 silencing modifies integrin dynamics. Together, our results show that techniques traditionally used for the investigation of cancer cells can be coupled with AFM to gain access to complementary phenotypic parameters that can help discriminate between specific phenotypes associated with different degrees of invasiveness.