Quantification of 3D network geometry in collagen hydrogels

Cancer is one of the principal causes of death in the world. Despite of the fact that there are ways of fighting the disease, full understanding about cancer cells behaviour is still distant. Extracellular matrix (ECM) is the three-dimensional environment where cells live, and its arrangement is known to modulate cell fate. Study of how ECMcancer cell interactions affect tumour progression is key for developing more effective drugs to heal cancer. As collagen is the most abundant ECM component, collagen hydrogels can be used as ECM models for research. This bachelor thesis is focused on proposing a processing pipeline for the extraction and characterization of fibre network from collagen hydrogel’s reflection microscopy images. This pipeline is composed by an image binarization procedure and a fibre network extraction algorithm from which the network parameters (i.e., fibre length, fibre persistence length, and crosslink density) are computed. The whole pipeline was tested in order to ensure its consistency, and eventually used for the characterization of real collagen hydrogel reflection microscopy images at different gel concentrations introduced in two types of in vitro platforms, culture wells and microfluidic devices. The study showed that fibre length and persistence length are similar for the different concentrations, while the network pore size decreases as the collagen concentration of the hydrogel increases. The future final step of the project would be to introduce cancer cells along with collagen hydrogels in the two platforms, and observe how collagen (ECM) disposition and characteristics direct cancer cell behaviour.Ingeniería Biomédic

Cell contraction induces long-ranged stress stiffening in the extracellular matrix

Animal cells in tissues are supported by biopolymer matrices, which typically exhibit highly nonlinear mechanical properties. While the linear elasticity of the matrix can significantly impact cell mechanics and functionality, it remains largely unknown how cells, in turn, affect the nonlinear mechanics of their surrounding matrix. Here we show that living contractile cells are able to generate a massive stiffness gradient in three distinct 3D extracellular matrix model systems: collagen, fibrin, and Matrigel. We decipher this remarkable behavior by introducing Nonlinear Stress Inference Microscopy (NSIM), a novel technique to infer stress fields in a 3D matrix from nonlinear microrheology measurement with optical tweezers. Using NSIM and simulations, we reveal a long-ranged propagation of cell-generated stresses resulting from local filament buckling. This slow decay of stress gives rise to the large spatial extent of the observed cell-induced matrix stiffness gradient, which could form a mechanism for mechanical communication between cells

Synchrotron-based visualization and segmentation of elastic lamellae in the mouse carotid artery during quasi-static pressure inflation

This dataset contains images that were obtained during quasi-static pressure inflation of mouse carotid arteries. Images were taken with phase propagation imaging at the X02DA TOMCAT beamline of the Swiss Light Source synchrotron at the Paul Scherrer Institute in Villigen, Switzerland. Scans of n=12 left carotid arteries (n-6 Apoe-deficient mice, n=6 wild-type mice, all on a C57Bl6J background) were taken at pressure levels of 0, 10, 20, 30, 40, 50, 70, 90 and 120 mmHg. For analysis we selected 75 images from the center of each stack (starting at the center of the stack, and skipping 2 of every three images in both cranial and caudal axial directions) for each sample and for each pressure level, resulting in a total of 75 x 12 x 9 = 8100 analyzed images from 108 different scans. Segmentation, 3D visualization and geometric analysis is presented in the corresponding manuscript. Files are uploaded in 16bit .tif format and are named: mouseid_pressurelevel_stacknumber, with mouseid consisting of either Apoe (Apoe-deficient) or Bl (wild-type) and the mouse number, pressurelevel varies from P0 to P120 and stacknumber indicates which image from the stack has been uploaded

Microfluidic-based 3d fibroblast migration studies in biomimetic microenvironments

Cell migration in 3D is a fundamental process in many physiological and pathological phenomena. Indeed, migration through interstitial tissue is a multi-step process that turns out from the cell-ECM interaction. It is a dynamic and complex mechanism that depends on the physic-chemical balance between the cell and its surrounding. Early stage of deep dermal wound healing process is a relevant migratory example, in which the fibroblast is the epicenter: the recruitment of the fibroblasts -by chemotaxis of PDGF-BB- to the clotted wound occurs. Likewise, this work focuses on studying the major underlying mechanisms of 3D fibroblast migration and the main microenvironmental cues involved within. To do so, we have confined two physiologically relevant hydrogels, made of collagen and fibrin, within microfluidic platforms. Firstly, an integral comparative study of biophysical and biomechanical properties of both gels is presented. In these results, we have overcome the wide diversity of the existing data and special stress has been done in order to compare the microstructural arrangement, resistance to flow and elasticity. On the other hand, controlled chemical gradients have been generated and characterized within the microfluidic devices. Since biomolecules interact as purely diffusive factors or bound to the matrix proteins, in this work, distribution of PDGF-BB and TGF-ß1 across collagen and fibrin gels has been quantified. Finally, by taking advantage of the biophysico-chemical definition, we have characterized the migratory responses of human fibroblasts within the microsystems in the presence of a chemoattractant (PDGF-BB). Our results demonstrate that the local microarchitecture of the hydrogels determines the migratory properties of human fibroblasts in response to controlled chemotactic and haptotactic gradients, in a myosin II-dependent manner.La migración celular en 3D es fundamental en muchos fenómenos fisiológicos y patológicos. La migración, la cual resulta de la interacción célula-matriz, es un mecanismo dinámico y complejo que depende del equilibrio entre la célula y su entorno físico-químico. Concretamente, la etapa temprana del proceso de cicatrización de heridas profundas es un proceso migratorio ejemplar, en el cual el fibroblasto es el epicentro: se produce el reclutamiento de los fibroblastos -por quimiotaxis de PDGF-BB- del tejido circundante al coágulo. Este trabajo se centra en el estudio de los principales mecanismos subyacentes de la migración de fibroblastos en 3D y las principales señales microambientales involucradas en ella. Para ello, se han empleado modelos in vitro haciendo uso de plataformas microfluídicas para confinar dos hidrogeles fisiológicamente relevantes, compuestos por colágeno y fibrina. En primer lugar, se presenta un estudio comparativo integral de las propiedades biofísicas y biomecánicas de los hidrogeles. En estos resultados, se ha hecho especial hincapié en comparar la conformación microestructural, la resistencia al flujo de fluido y la elasticidad. Por otro lado, se han generado y caracterizado gradientes químicos dentro de los dispositivos. Puesto que las biomoléculas interactúan como factores puramente difusivos o adheridos a las proteínas de la matriz, en este trabajo se ha cuantificado la distribución de PDGF-BB y TGF-β1, en colágeno y fibrina. Finalmente, mediante esta definición físico-química, se ha caracterizado la respuesta migratoria de fibroblastos humanos dentro de los microdispositivos en presencia de un factor químico (PDGF-BB). Los resultados aquí mostrados demuestran que la microarquitectura local de los hidrogeles determina las propiedades migratorias de fibroblastos humanos en respuesta a gradientes quimiotácticos y haptotácticos, de manera dependiente de la miosina II

Flow dynamics control the effect of sphingosine-1-phosphate on endothelial permeability in a microfluidic vessel bifurcation model

Blood vessels are lined by endothelial cells that form a semipermeable barrier to restrict fluid flow across the vessel wall. The endothelial barrier is known to respond to various molecular mechanisms, but the effects of mechanical signals that arise due to blood flow remain poorly understood. Here, we report a microfluidic model that mimics the flow conditions and endothelial/extracellular matrix (ECM) architecture of a vessel bifurcation to enable systematic investigation of how flow dynamics that arise within bifurcating vessels guides the endothelial response to biochemical signals. Applying the strengths of our system, we further investigate the endothelial response to sphingosine-1-phosphate, a bioactive lipid that has demonstrated flow-dependent regulation of vascular permeability. We demonstrate that bifurcated fluid flow (BFF) that arises at the base of vessel bifurcations and laminar shear stress (LSS) that arises along downstream vessel walls induce a decrease in endothelial permeability. Furthermore, we identify that flow-dynamics and chaperone proteins regulate the endothelial response to S1P. Through pharmacological inhibition of S1P receptors 1 and 2, we report ligand-independent mechanical activation of S1P receptors 1 and 2, providing support for the role of G protein-coupled receptors as mechanosensors. These findings introduce BFF as an important regulator of vascular permeability, and establish flow dynamics as a determinant of the endothelial response to S1P.Pelotonia Fellowship ProgramBarry M. Goldwater Excellence in Education FoundationThe Ohio State University College of EngineeringA one-year embargo was granted for this item.Academic Major: Biomedical Engineerin

The use of mixed collagen-Matrigel matrices of increasing complexity recapitulates the biphasic role of cell adhesion in cancer cell migration: ECM sensing, remodeling and forces at the leading edge of cancer invasion

The migration of cancer cells is highly regulated by the biomechanical properties of their local microenvironment. Using 3D scaffolds of simple composition, several aspects of cancer cell mechanosensing (signal transduction, EMC remodeling, traction forces) have been separately analyzed in the context of cell migration. However, a combined study of these factors in 3D scaffolds that more closely resemble the complex microenvironment of the cancer ECM is still missing. Here, we present a comprehensive, quantitative analysis of the role of cell-ECM interactions in cancer cell migration within a highly physiological environment consisting of mixed Matrigel-collagen hydrogel scaffolds of increasing complexity that mimic the tumor microenvironment at the leading edge of cancer invasion. We quantitatively show that the presence of Matrigel increases hydrogel stiffness, which promotes ß1 integrin expression and metalloproteinase activity in H1299 lung cancer cells. Then, we show that ECM remodeling activity causes matrix alignment and compaction that favors higher tractions exerted by the cells. However, these traction forces do not linearly translate into increased motility due to a biphasic role of cell adhesions in cell migration: at low concentration Matrigel promotes migration-effective tractions exerted through a high number of small sized focal adhesions. However, at high Matrigel concentration, traction forces are exerted through fewer, but larger focal adhesions that favor attachment yielding lower cell motility

Matrix architecture plays a pivotal role in 3D osteoblast migration: The effect of interstitial fluid flow

Osteoblast migration is a crucial process in bone regeneration, which is strongly regulated by interstitial fluid flow. However, the exact role that such flow exerts on osteoblast migration is still unclear. To deepen the understanding of this phenomenon, we cultured human osteoblasts on 3D microfluidic devices under different fluid flow regimes. Our results show that a slow fluid flow rate by itself is not able to alter the 3D migratory patterns of osteoblasts in collagen-based gels but that at higher fluid flow rates (increased flow velocity) may indirectly influence cell movement by altering the collagen microstructure. In fact, we observed that high fluid flow rates (1 µl/min) are able to alter the collagen matrix architecture and to indirectly modulate the migration pattern. However, when these collagen scaffolds were crosslinked with a chemical crosslinker, specifically, transglutaminase II, we did not find significant alterations in the scaffold architecture or in osteoblast movement. Therefore, our data suggest that high interstitial fluid flow rates can regulate osteoblast migration by means of modifying the orientation of collagen fibers. Together, these results highlight the crucial role of the matrix architecture in 3D osteoblast migration. In addition, we show that interstitial fluid flow in conjunction with the matrix architecture regulates the osteoblast morphology in 3D

Mechanisms of Interstitial Flow-Induced Remodeling of Fibroblast-Collagen Cultures

Interstitial fluid flow, critical for macromolecular transport, was recently shown to drive fibroblast differentiation and perpendicular cell and matrix alignment in 3D collagen cultures. Here we explore the mechanisms underlying this flow-induced cell and collagen alignment. Cell and matrix alignment was assessed from 3D confocal reflectance stacks using a Fast Fourier Transform method. We found that human dermal and lung fibroblasts align perpendicular to flow in the range of 5-13μm/s (0.1-0.3dyn/cm2) in collagen; however, neither cells nor matrix fibers align in fibrin cultures, which unlike collagen, is covalently cross-linked and generally degraded by cell fibrinolysis. We also found that even acellular collagen matrices align weakly upon exposure to flow. Matrix alignment begins within 12h of flow onset and continues, along with cell alignment, over 48h. Together, these data suggest that interstitial flow first induces collagen fiber alignment, providing contact guidance for the cells to orient along the aligned matrix; later, the aligned cells further remodel and align their surrounding matrix fibers. These findings help elucidate the effects of interstitial flow on cells in matrices and have relevance physiologically in tissue remodeling and in tissue engineering application

Characterization of Fibrin and Collagen Gels for Engineering Wound Healing Models

Hydrogels are used for 3D in vitro assays and tissue engineering and regeneration purposes. For a thorough interpretation of this technology, an integral biomechanical characterization of the materials is required. In this work, we characterize the mechanical and functional behavior of two specific hydrogels that play critical roles in wound healing, collagen and fibrin. A coherent and complementary characterization was performed using a generalized and standard composition of each hydrogel and a combination of techniques. Microstructural analysis was performed by scanning electron microscopy and confocal reflection imaging. Permeability was measured using a microfluidic-based experimental set-up, and mechanical responses were analyzed by rheology. We measured a pore size of 2.84 and 1.69 µm for collagen and fibrin, respectively. Correspondingly, the permeability of the gels was 1.00·10-12 and 5.73·10-13 m2. The shear modulus in the linear viscoelastic regime was 15 Pa for collagen and 300 Pa for fibrin. The gels exhibited strain-hardening behavior at ca. 10% and 50% strain for fibrin and collagen, respectively. This consistent biomechanical characterization provides a detailed and robust starting point for different 3D in vitro bioapplications, such as collagen and/or fibrin gels. These features may have major implications for 3D cellular behavior by inducing divergent microenvironmental cues

Cell traction forces in 3-D microenvironments

Las células son capaces de sentir y responder activamente frente a los estímulos mecánicos de su entorno. Los estímulos mecánicos que provienen de la matriz extracelular, tales como la rigidez, la topología de la superficie o la deformación, son traducidos en señales bioquímicas a través de las interacciones entre la célula y la matriz. Para poder sobrevivir y crecer las células necesitan adherirse y propagarse sobre el sustrato que las rodea. Una vez adheridas, las células generan fuerzas contráctiles a través de la interacción actina-miosina, ejerciendo de este modo tracción sobre el sustrato subyacente. Es por ello, que las fuerzas de tracción ejercidas por las células son reguladores críticos de la adhesión, la señalización y la función celular, y por tanto son muy importantes en numerosos procesos biológicos tales como la inflamación, la cicatrización de heridas, la angiogénesis e incluso la metástasis. Pese a su importancia, la medición de las fuerzas celulares en un contexto fisiológico así como entender su contribución en los procesos biológicos sigue siendo todavía un reto. Además, debido a que las interacciones célula-matriz varían considerablemente entre ambientes bidimensionales y tridimensionales, entender su influencia sobre las respuestas celulares normales y patológicas en sistemas tridimensionales es esencial para poder traducir de manera eficiente dichos conocimientos en terapias médicas. El principal objetivo de esta Tesis es, por tanto, el desarrollo de modelos computacionales enfocados al estudio de diferentes aspectos de las interacciones célula-matriz, que permitan entender mejor los fenómenos específicos y que sirvan como referencia para el desarrollo de nuevos experimentos y de técnicas de modelado in vitro. Además, todos los modelos y experimentos contenidos en esta tesis se centran en el estudio de células individuales. En primer lugar, debido a la complejidad y a las grandes diferencias que presentan con respecto a la migración celular colectiva, y en segundo lugar debido a la importancia que supone el estudio de la migración celular individual en procesos tan importantes como es la invasión de células tumorales. Además, debido a la relevancia que suponen fisiológicamente los entornos tridimensionales, en la mayoría de los modelos in silico desarrollados en esta Tesis, se han considerado aproximaciones tridimensionales para poder así imitar mejor las condiciones in vivo de células y tejidos.En primer lugar, se ha investigado la dinámica de unión de los sitios de adhesión célula-matriz, más en particular cómo las células transmiten las fuerzas a través de estas uniones a la matriz extracelular. Para ello, se ha desarrollado un modelo numérico mediante el uso del método de los elementos finitos [1]. En segundo lugar, se ha desarrollado un modelo in vitro para el estudio de las interacciones célula-matriz tanto a nivel celular como a nivel de tejido. En particular, se presentan diferentes dispositivos de microfluídica, los cuales están siendo utilizados en la actualidad para el estudio de diferentes procesos biológicos. Estos han sido utilizados para estudiar los procesos de formación de gradientes químicos a través de una matriz tridimensional [2]. Investigaciones recientes han indicado que las fuerzas de tracción celular son reguladores críticos de la invasión de las células tumorales, las cuales dependen en gran medida de las propiedades mecánicas tanto de las células como de la matriz que las rodea. Debido a que surge la necesidad de tener un conocimiento mucho más profundo sobre este mecanismo, la segunda parte de esta Tesis se ha centrado en el desarrollo de diferentes experimentos para cuantificar las fuerzas celulares, así como en el desarrollo de un modelo in silico basado en elementos finitos para reconstruir las fuerzas ejercidas por las células durante su migración, permitiendo de este modo estudiar la dependencia de las propiedades mecánicas de las células sobre la solución de fuerzas obtenida [3]. En resumen, una mejor comprensión de los mecanismos subyacentes a las interacciones célula-matriz, aportados en parte por la aparición de nuevas tecnologías para estudiar la mecánica celular a alta resolución espacial y temporal, no sólo resulta en una mejor comprensión del comportamiento de células normales, sino que también conduce al desarrollo de terapias novedosas para tratar enfermedades relacionadas con los defectos en las interacciones mecánicas celulares.<br /