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The role of cell-generated and externally applied force in tendon developmentKalson, Nicholas Stewart January 2012 (has links)
Tendons are collagen-based fibrous tissues that connect muscles to bones. Tendon injury is common, but currently available treatments for damaged tendons are limited, and often fail to restore pre-injury function. The cellular and molecular mechanisms underlying tendon formation are not completely understood, and a detailed understanding of the processes involved would facilitate new therapeutic strategies. Work presented in this thesis had two aims: to investigate the role and mechanisms of cell-generated force during tendon development, and to examine the effect of external force applied to tendon tissue. The rationale to investigate micro-mechanical aspects of tendon development came from observations that tendons are intrinsically tensioned, presumably to transmit force efficiently. Tension is also critical for the formation and regulation of the primary cell-matrix interaction structures seen in embryonic tendon, known as fibripositors. In initial experiments cell-generated force was disrupted with the non-muscle myosin II (NMII) inhibitor blebbistatin. NMII inhibition prevented formation of tendon tissue in a 3D cell culture system (tendon-construct). Cell-contraction was also shown to shape the ECM and generate a crimped collagen structure characteristic of tendon. These observations highlight the importance of cell-generated force in tendon development. Further investigation using novel microscopy techniques suggested that fibripositors contained newly formed collagen fibrils, and that fibrils in the ECM were internalised and fragmented in a NMII/MT1-MMP dependent pathway. It is proposed that these fragmented fibrils are re-secreted into the matrix, where they seed fibril growth. This pathway is disrupted in MT1-MMP deficient mice, which have half normal size tendons with fewer collagen fibrils. Investigations using a mechanical rig showed that external application of force to tendon-constructs stimulated matrix maturation that more closely mimicked embryonic development. Taken together these results show that both cell-generated force and external force have important roles during tendon development. Experiments in this thesis aimed to elucidate the role of force at the tissue level (generation of mechanical properties, crimp structure), the microscopic level (cell-matrix interactions) and the molecular level (role of NMII and MT1-MMP in ECM collagen fibril handling). Given the complexity of the developmental system being investigated the multi-level experimental approach used here is necessary to generate a holistic understanding of the important developmental processes involved. Therapeutic tissue regeneration may be facilitated by application of external force or modulation of cell-generated force. Furthermore, the identification of a fibril amplification pathway has implications for medical conditions characterised by excessive collagen fibril formation (fibrosis, some cancers) or by slow or inadequate fibril formation (tendon healing).
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Development of a Novel Single-Cell Attachment and Spreading Platform Utilizing Fused-Fiber NanonetsGill, Amritpal Singh 04 June 2015 (has links)
Initial attachment to the extracellular matrix (ECM) and consequent spreading is a necessary process in the cell cycle of which little is known. Cell spreading has been well-recognized in 2D systems, however, the native fibrous ECM presents cells with 3D biophysical cues. Thus, using suspended fibers as model systems, we present the development of a novel platform (Cell-STEPs) capable of capturing cell attachment dynamics and forces from the moment a cell in suspension contacts the fiber. Cell-STEPs comprises of a custom glass-bottom petri dish with a lid to deliver a constant supply of CO2 to maintain pH. Fibrous scaffolds are attached in the dish to allow cellular investigations over extended periods of time. We find that cell-fiber attachment occurs in three progressive phases: initial attachment of cell to fiber (phase 0), rapid drop in circularity (phase 1), and increase in cell spread area (phase 2). Furthermore, using iterative inverse methods, forces involved in cell spreading through deflection of fibers were estimated. Our findings provide new insights in attachment biomechanics, including initial sensing and latching of cell to fiber with a negligible or protrusive force, followed by rapid loss in circularity through protrusion sensing at nearly constant spread area and minimal force generation, transitioning to a final phase of increased contractile forces until spread area and force saturation is observed. Also, anisotropic spreading of cells on single and two-fibers are closely related, while cells attached to several fibers take longer and spread isotropically. The Cell-STEPs platform allows, for the first time, detailed interrogations in the discrete and orchestrated adhesion steps involved in cell-fibrous matrix recognition and attachment along with simultaneous measurements of forces involved in cell attachment. / Master of Science
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Cell-Fiber Interactions: A New Route to Mechano-Biological Investigations in Developmental and Disease BiologySheets, Kevin Tyler 03 November 2014 (has links)
Cells in the body interact with a predominantly fibrous microenvironment and constantly adapt to changes in their neighboring physiochemical environment, which has implications in developmental and disease biology. A myriad of in vitro platforms including 2D flat and 3D gel substrates with and without anisotropy have demonstrated cellular alterations to subtle changes in topography. Recently, our work using suspended fibers as a new in vitro biological assay has revealed that cells are able to sense and respond to changes in fiber curvature and structural stiffness as evidenced by alterations to cytoskeleton arrangement, including focal adhesion cluster lengths and nucleus shape indices, leading to altered migration speeds. It is hypothesized that these behaviors occur due to modulation of cellular inside-out forces in response to changes in the external fibrous environment (outside-in). Thus, in this study, we investigate the role of fiber curvature and structural stiffness in force modulation of single cells attached to suspended fibers. Using our previously reported non-electrospinning Spinneret based Tunable Engineered Parameters (STEP) fiber manufacturing platform, we present our findings on single cell inside-out and outside-in forces using fibers of three diameters (250 nm, 400 nm and 800 nm) representing a wide range of structural stiffness (3-45 nN/μm). To investigate cellular adaptability to external perturbation, we present the development of a first-of-its-kind force measurement 'nanonet' platform capable of investigating cell adhesion forces in response to symmetric and non-symmetric (injury model) loading. Our combined findings are multi-fold: (i) Cells on suspended fibers are able to form focal adhesion clusters approximately four times longer than those on flat substrates, which gives them potential to double their migration speeds, (ii) Nanonets as force probes show that the contractility-based inside-out forces are nearly equally distributed on both sides of the cell body, and that overall force magnitudes are dependent on fiber structural stiffness, and (iii) External perturbation can evenly (symmetric) or unevenly (non-symmetric) distribute forces within the cell, and the resulting bias causes diameter-dependent outside-in adhesion force response. Finally, we demonstrate the power of the developed force measurement platform by extending our studies to cell-cell junctional forces as well as single-cell disease models including cancer and aortic aneurysm. / Ph. D.
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Cell Traction Force Mapping in MG63 and HaCaTsSoon, Chin Fhong, Genedy, Mohamed A., Youseffi, Mansour, Denyer, Morgan C.T. January 2013 (has links)
No / The ability of a cell to adhere and transmit traction forces to a surface reveals the cytoskeleton integrity of a cell. Shear sensitive liquid crystals were discovered with new function in sensing cell traction force recently. This liquid crystal has been previously shown to be non-toxic, linear viscoelastic and sensitive to localized exerted forces. This paper reports the possibility of extending the application of the proposed liquid crystal based cell force sensor in sensing traction forces of osteoblast-like (MG-63) and human keratinocyte (HaCaT) cell lines exerted to the liquid crystal sensor. Incorporated with cell force measurement software, force distributions of both cell types were represented in force maps. For these lowly contractile cells, chondrocytes expressed regular forces (10 – 90 nN, N = 200) around the circular cell body whereas HaCaT projected forces (0 – 200 nN, N = 200) around the perimeter of poly-hedral shaped body. These forces are associated with the organisation of the focal adhesion expressions and stiffness of the LC substrate. From the results, liquid crystal based cell force sensor system is shown to be feasible in detecting forces of both MG63 and HaCaT.
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Single Cell Force Spectroscopy for Quantification of Cellular Adhesion on SurfacesJanuary 2016 (has links)
abstract: Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is αMβ2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering quantitative insights. / Dissertation/Thesis / Doctoral Dissertation Physics 2016
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Cellular locomotion and adhesion in the context of different substrate propertiesBaronsky, Thilo 10 June 2016 (has links)
No description available.
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Hierarchical Assembly of Polymeric Nanofibers for Advanced Material ApplicationsWang, Ji 27 March 2015 (has links)
Polymer nanofibers are gaining importance due to their wide applicability in diverse fields, such as tissue engineering, fuel cells, photonics and sensors. For these applications, manufacturing aligned polymer nanofibers with precisely controlled morphology and well characterized mechanical properties in a bottom up configuration is essential. In this work, we developed an isodiametric design space for fabrication of aligned polystyrene nanofibers (diameter 60-800nm) using non-electrospinning Spinneret based Tunable Engineered Parameter (STEP) technique. By adjusting the processing parameters such as relative humidity, solvent volatility and polarity, porous polymer tubes are demonstrated having large specific surface areas. Combining STEP with sol-gel process, aligned inorganic nanofibers, such as Titanium Oxide (TiO2) with varied morphologies can be conveniently obtained. Mechanical properties of aligned polymer nanofibers (diameter from 50nm to several hundred nanometers) with fixed-fixed boundary conditions were estimated using a lateral force microscope (LFM). We find that the tension in the fiber caused during fabrication process scales with fiber diameter and it dominates fiber stiffness. Our studies demonstrate isotropic arrangement of polymer chains in the fibers and anisotropic arrangement in the necking region for fibers undergone deformation. Finally, this study demonstrates development of force sensors capable of measuring single cell forces, which scale with the fiber structural stiffness. The ability to measure cell forces during cell division, migration and apoptosis provides new insights in cell mechanobiology. / Ph. D.
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Tracking Traction Force Changes of Single Cells on the Liquid Crystal SurfaceSoon, Chin Fhong, Tee, K.S., Youseffi, Mansour, Denyer, Morgan C.T. 02 December 2014 (has links)
Yes / Cell migration is a key contributor to wound repair. This study presents findings indicating that the liquid crystal based cell traction force transducer (LCTFT) system can be used in conjunction with a bespoke cell traction force mapping (CTFM) software to monitor cell/surface traction forces from quiescent state in real time. In this study, time-lapse photo microscopy allowed cell induced deformations in liquid crystal coated substrates to be monitored and analyzed. The results indicated that the system could be used to monitor the generation of cell/surface forces in an initially quiescent cell, as it migrated over the culture substrate, via multiple points of contact between the cell and the surface. Future application of this system is the real-time assaying of the pharmacological effects of cytokines on the mechanics of cell migration.
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Cell adhesion and cell mechanics during zebrafish development / Zelladhäsion und Zellmechanik während der ZebrafischentwicklungKrieg, Michael 11 January 2010 (has links) (PDF)
During vertebrate development, gastrulation leads to the formation of three distinct germlayers. In zebrafish a central process is the delamination and the ingression of single cells from a common ancestor tissue - that will lead to the formation of the germlayers. Several molecules have been identified to regulate this process but the precise cellular mechanisms are poorly understood. Differential adhesiveness, a concept first introduced by Steinberg over 40 years ago, has been proposed to represent a key phenomena by which single hypoblast cells separate from the epiblast to form the mesendoderm at later stages. In this work it is shown that differential adhesion among the germlayer progenitor cells alone cannot predict germlayer formation. It is a combination of several mechanical properties such as cell cortex tension, cell adhesion and membrane mechanical properties that influence the migratory behavior of the constituent cells.
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Development of a novel cell traction force transducer based on cholesteryl ester liquid crystals. Characterisation, quantification and evaluation of a cholesteryl ester liquid crystal based single cell force transducer system.Soon, Chin Fhong January 2011 (has links)
In biomechano-transducing, cellular generated tension can be measured by soft substrates based on polymers but these techniques are limited either by spatial resolution or ability to detect localised cell traction forces (CTF) due to their non-linear viscous behaviour under shear rates. A newly developed cell traction force transducer system based on cholesteryl ester lyotropic liquid crystals (LCTFT) was developed to sense localised traction forces of human keratinocyte cell lines (HaCaTs), in which the length of the deformation line induced represents the intensity of the CTF exerted. The physical properties of the cholesteryl ester based lyotropic liquid crystals (LLC) were characterised by using polarising microscopy, rheology, atomic force microscopy (AFM) based nano-indentation, spherical indentation, and micro-tensile tests. The interactions of LLC with cells were studied by using cell viability studies, cytochemical treatments, widefield surface plasmon resonance (WSPR) microscopy and various immuno-staining techniques. The results show that LLC is thermally stable (0 - 50 oC) and linearly viscoelastic below 10 % shear strain at shear rates of < 1 s-1. AFM nano and spherical indentations show a good agreement on the Young¿s modulus of both determined at ~110 kPa which is close to the elastic modulus of the epidermis. The Poisson¿s ratio of LLC was determined at ~0.58 by using micro tensile tests. The biophysical interaction studies indicated that LLC is biocompatible and allowed cell attachment. Cell relaxation technique by cytochalasin-B treatment suggested that the attachment and contraction of cells on LLC was due to the contractile activity of actin cytoskeletons that are mediated by focal adhesions. The staining experiments showed that cells consistently expressed the same suites of integrins (¿2, ¿3, ¿5 and ¿1) and ECM proteins (collagen type IV, laminin and fibronectin) on both glass and LLC coated substrates. Interfacial interaction of cells with LLC observed via the staining of actin and vinculin, and WSPR imaging suggest the association of marginal actin filaments and focal adhesions in attaching HaCaT cells to the LLC. Linear static analysis applied in the Finite Element model of focal adhesion-LC confirmed the compressive force patterns induced by cells. By applying cell relaxation techniques and Hooke¿s theorem, the force-deformation relationships of the LLC were derived and used for direct quantification of CTF in culture. The sensitivity of the LCTFT was implied by a wide range of CTF (10 - 140 nN) measured at high resolutions (~2 ¿m). Nonetheless, a custom-built cell traction force measurement and mapping software (CTFM) was developed to map CTF of single cells. Reliability of the LCTFT was evaluated by using a known pharmacological active cytokine, TGF-¿1, in inducing contraction of human keratinocytes. This study inferred internal consistency and repeatability of the LCTFT in sensing contraction responses of HaCaT cells in a concentration dependent manner of TGF-¿1. The overall LCTFT and CTFM software had shown good potential for use in the study of contraction and migration of keratinocytes. / Malaysia Ministry of Higher Education
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