Bacterial polymertropism, the response to strain-induced alignment of polymers

In nature, bacteria often live in surface-associated communities known as biofilms. Biofilm-forming bacteria deposit a layer of polysaccharide on the surfaces they inhabit; hence, polysaccharide is their immediate environment on any surface. In this study, we examined how the physical characteristics of polysaccharide substrates influence the behavior of the biofilm-forming bacterium Myxococcus xanthus. M. xanthus colonies, and indeed those of the majority of biofilm-forming species tested, respond to the compression-induced deformation of polysaccharide substrates by preferentially spreading across the surface perpendicular to the axis of compression. This response is conserved across multiple distantly related phyla and is found in species with an array of distinct motility apparatuses.The birefringence and small angle X-ray scattering patterns of compressed polysaccharide substrates indicate that the directed surface movements of these bacteria consistently match the orientation of the long axes of aligned and tightly packed polysaccharide fibers in compressed substrates. Therefore, we refer to this behavior as polymertropism to denote that the directed movements are a response to the physical arrangement of the change in packing and alignment of the polymers in the substrate. In addition to altering the colony morphology we find the behavior of groups of cells, called flares, is also affected in several species resulting in increased flare speed, duration, and displacement on compressed gel substrates.We suggest that polymertropism, which requires a downward-facing motility apparatus in M. xanthus, may be responsible for the observed tendency of bacterial cells to follow trails of extruded and presumably aligned polysaccharides, which their neighbors secrete and deposit on the substrate as they move across it. Polymertropism may also play a role in the organization of bacteria in a biofilm, as the iterative process of polysaccharide trail deposition and following is proposed to yield aggregates of cells

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

Freezing-induced deformation of biomaterials in cryomedicine

Cryomedicine utilizes low temperature treatments of biological proteins, cells and tissues for cryopreservation, materials processing and cryotherapy. Lack of proper understanding of cryodamage that occurs during these applications remains to be the primary bottleneck for development of successful tissue cryopreservation and cryosurgery procedures. An engineering approach based on a view of biological systems as functional biomaterials can help identify, predict and control the primary cryodamage mechanisms by developing an understanding of underlying freezing-induced biophysical processes. In particular, freezing constitutes the main structural/mechanical origin of cryodamage and results in significant deformation of biomaterials at multiple length scales. Understanding of these freezing-induced deformation processes and their effects on post-thaw biomaterial functionality is currently lacking but will be critical to engineer improved cryomedicine procedures. This dissertation addresses this problem by presenting three separate but related studies of freezing-induced deformation at multiple length scales including nanometer-scale protein fibrils, single cells and whole tissues. A combination of rigorous experimentation and computational modeling is used to characterize post-thaw biomaterial structure and properties, predict biomaterial behavior and assess its post-thaw biological functionality. Firstly, freezing-induced damage on hierarchical extracellular matrix structure of collagen is investigated at molecular, fibril and matrix levels. Results indicate to a specific kind of fibril damage due to freezing-induced expansion of intrafibrillar fluid. This is followed by a study of freezing-induced cell and tissue deformation coupled to osmotically driven cellular water transport. Computational and semi empirical modeling of these processes indicate that intracellular deformation of the cell during freezing is heterogeneous and can interfere with cellular water transport, thereby leading to previously unconsidered mechanisms of cell freezing response. In addition, cellular water transport is identified as the critical limiting factor on the amount of freezing-induced tissue deformation, particularly in native tissues with high cell densities. Finally, effects of cryopreservation on post-thaw biological functionality of collagen engineered tissue constructs is investigated where cell-matrix interactions during fibroblast migration are considered as the functional response. Simultaneous cell migration and extracellular matrix deformation are characterized. Results show diminished cell-matrix coupling by freeze/thaw accompanied by a subtle decrease in cell migration. A connection between these results and freezing-induced collagen fibril damage is also suggested. Overall, this dissertation provides new fundamental knowledge on cryodamage mechanisms and a collection of novel multi-purpose engineering tools that will open the way for rational design of cryomedicine technologies

Integrated Mathematical and Experimental Study of Cell Migration and Shape

Cell migration plays an essential role in many of physiological and pathological processes, including morphogenesis, inflammation, wound healing, and tumor metastasis. It is a complex process that involves multi-scale interactions between the cell and the extracellular matrix (ECM). Cells migrate through stromal ECM with native and cell-derived curvature at micron-meter scale are context-specific. How does the curvature of ECM mechanically change cell morphology and motility? Can the diverse migration behaviors from genetically identical cells be predictively using cell migrating data? We address these questions using an integrated computational and experimental approach: we developed three-dimensional biomechanical cell model and measured and analyzed a large number of cell migration images over time. Our findings suggest that 1. substrate curvature determines cell shape through contact and regulating protrusion dynamics; 2. effective cell migration is characterized with long cellular persistence time, low speed variation, spatial-temporally coordinated protrusion and contraction; 3. the cell shape variation space is low dimensional; and 4. migration behavior can be determined by a single image projected in the low dimensional cell shape variation space

New strategies for tissue regeneration

My PhD project is divided in two parts, focusing on the development of new strategies for orthopedic tissue regeneration. In particular, the first part is about cartilage regeneration using human lipoaspirate as autologous injectable active scaffold for one-step repair of cartilage defects, the second part is about bone regeneration, through an injectable medicated graft substitute active on bone tissue regeneration. I. Cartilage regeneration Research on mesenchymal stem cells from adipose tissue (ASC) shows promising results for cell-based therapy in cartilage lesions: in these studies cells have been isolated, expanded, and differentiated in vitro, before transplantation into the damaged cartilage, or onto materials used as scaffolds to deliver cells to the impaired area. The present study employed in vitro assays to investigate the potential of intra-articular injection of micro-fragmented lipoaspirate, as a one-step repair strategy; it aimed to determine whether adipose tissue can act as a scaffold for cells naturally present at their anatomical site. Cultured clusters of lipoaspirate showed a spontaneous outgrowth of cells with mesenchymal phenotype and with multilineage differentiation potential. Transduction of lipoaspirate clusters by lentiviral vectors expressing GFP underlined the propensity of the outgrown cells to repopulate fragments of damaged cartilage. On the basis of the results, which showed an induction of proliferation and extracellular matrix (ECM) production of human primary chondrocytes, it was hypothesized that lipoaspirate may play a paracrine role. Moreover, the structure of a floating culture of lipoaspirate, treated for three weeks with chondrogenic growth factors, changed: tissue with a high fat component was replaced by a tissue with a lower fat component and connective tissue rich in glycosaminoglycan (GAG) and in collagen type I, increasing the mechanical strength of the tissue. From these promising in vitro results, it may be speculated that an injectable autologous biologically-active scaffold (lipoaspirate), employed intra-articularly, may: 1) become a fibrous tissue that provides mechanical support for the load on the damaged cartilage; 2) induce host chondrocytes to proliferate and produce ECM; 3) provide cells at the site of injury, which could regenerate or repair the damaged or missing cartilage. II. Bone regeneration With the aim to obtain an injectable medicated scaffold, which speeds bone formation in sinus lift augmentation, in bony void and in fracture repair, we have developed a three-dimensional (3D) jelly collagen containing Lysophosphatidic acid (LPA) and 1a,25-Dihydroxyvitamin D3 (1,25D3) using soluble native collagen prepared from rat tail tendons. We have demonstrated with an in vitro 3D culture model of bone fracture an osteoblasts\u2019Rho-kinase mediated contraction of the collagen that causes an approach of human bone trabecular fragments with the formation of new union tissue within 3 weeks of organ culture. The contraction was faster in LPA medicated collagen while 1,25D3 enhanced the mineralization of the new formed tissue that showed also increased tensile strength. LPA was shown to modulate gel contraction rate not only mechanically, working in cytoskeleton reorganization, but also osteoconductively evidencing activity on proliferation, differentiation and migration of human primary osteoblasts (hOB). When LPA was used in combination with 1,25D3 a synergism on hOB\u2019s activity in term of alkaline phosphatase and mineralization was seen. On the basis of these data, collagen can be considered as an injectable natural scaffold that allows the migration of cells from the side of bone defect and its enrichment with LPA and 1,25D3 could be used in vivo to accelerate bone growth and fracture healing

The role of Ca2+ influx mechanisms in the regulation of human cardiac fibroblasts in 2D- and 3D- culture models

Cardiac fibrosis is a serious health problem commonly associated with cardiovascular diseases and remains an increasing health burden around the globe. The development of cardiac fibrosis involves the activation and differentiation of mainly resident cardiac fibroblasts (CF) by biochemical factors including angiotensin II (Ang II) and transforming growth factor β (TGF-β). As both factors were shown to interfere with the Ca2+ handling in CF, the role of Ca2+ influx mechanisms were studied in 2D and 3D cultures of CF. In a first step, the influence of the inhibition of the store-operated calcium entry (SOCE) and the transient receptor potential channel canonical 3 (TRPC3) by BTP2 (YM-58483) and Pyr3 (Pyrazole 3), respectively, on the Ca2+ handling were determined in 2D cultured normal human ventricular cardiac fibroblasts (NHCF-V). Both inhibitors were shown to reduce the basal and Ang II-dependent Ca2+ oscillations, as well as the Ang II-induced Ca2+ transients. Moreover, BTP2 was demonstrated to reduce the TGF-β-dependent Ca2+ transient and the ER calcium content under basal condition. Long-term treatment indicated that BTP2 and Pyr3 inhibited the proliferation of NHCF-V and induced cytotoxic effects in 2D cultured cells. An important difference between both inhibitors were identified for their effects on the pro-fibrotic gene expression. BTP2 blunted the expression of the major cardiac collagen isoform Col1a1, but Pyr3 was without effect. BTP2 also down-regulated the matricellular protein connective tissue growth factor (CTGF). Importantly, BTP2 increased the expression of important effectors of the unfolded protein response (UPR). In the next step, the NHCF-V were used to generate human engineered connective tissues (hECT). To induce cell activation a co-treatment with Ang II and TGF-β (AT) was applied. This treatment was shown to increase hECT compaction, contraction, stiffness, and strength, and to decrease extensibility. In the following, the anti-fibrotic potential of BTP2 was shown by a significant reduction of the AT-induced compaction, contraction, stiffness, and strength, and by an increase in elasticity and extensibility of the fibrotic hECT. Molecular analysis revealed that BTP2 treatment resulted in enhanced cell loss within the initial culture period and a downregulation of Col1a1. Mechanistically, BTP2 induced mild ER-stress indicated by an up-regulation of the UPR mediator DDIT3. Finally, Pyr3 also significantly reduce the AT-induced contraction and stiffness but surprisingly, none of the other biomechanical parameters. In summary, the BTP2-dependent Ca2+ influx inhibition induced ER-stress which interfered with ECM protein expression and fibrotic processes in hECT, whereas, Pyr3 interfered only with the contractile behavior of NHCF-V.2021-12-1

Collagen-Based Biomimetic Systems to Study the Biophysical Tumour Microenvironment

The extracellular matrix (ECM) is a pericellular network of proteins and other molecules that provides mechanical support to organs and tissues. ECM biophysical properties such as topography, elasticity and porosity strongly influence cell proliferation, differentiation and migration. The cell’s perception of the biophysical microenvironment (mechanosensing) leads to altered gene expression or contractility status (mechanotransduction). Mechanosensing and mechanotransduction have profound implications in both tissue homeostasis and cancer. Many solid tumours are surrounded by a dense and aberrant ECM that disturbs normal cell functions and makes certain areas of the tumour inaccessible to therapeutic drugs. Understanding the cell-ECM interplay may therefore lead to novel and more effective therapies. Controllable and reproducible cell culturing systems mimicking the ECM enable detailed investigation of mechanosensing and mechanotransduction pathways. Here, we discuss ECM biomimetic systems. Mainly focusing on collagen, we compare and contrast structural and molecular complexity as well as biophysical properties of simple 2D substrates, 3D fibrillar collagen gels, cell-derived matrices and complex decellularized organs. Finally, we emphasize how the integration of advanced methodologies and computational methods with collagen-based biomimetics will improve the design of novel therapies aimed at targeting the biophysical and mechanical features of the tumour ECM to increase therapy efficacy

The development of a stratified keratinocyte model for chlamydia trachomatis pathogenesis studies.

Masters Degree. University of KwaZulu-Natal, Durban.A number of different methods to generate stratified keratinocyte layers have been published. These involved the use of normal human epidermal keratinocytes (NHEKs/NEKS), which have a better ability to stratify compared to HaCaT keratinocytes, which usually require supplemented growth factors or stromal interactions with fibroblasts to do so. This study aimed to generate a model of stratified keratinocytes, closely resembling in vivo skin, using HaCaT cells and to demonstrate the effect that C. trachomatis has on these layered keratinocytes, allowing us to gain insight on the pathophysiology of this organism. All cells and bacteria were propagated and titrated according to conventional protocols. HaCaT cells were subcultured upon confluence, seeded (1x106 cells/ml) onto collagen-coated PTFE Transwell membrane inserts and incubated at 33°C and 37°C for 24 days to allow differentiation and stratification. Once cells became confluent they were exposed to the air-liquid interface and fed with KGM Gold (Lonza) supplemented with 10% FBS and additional calcium. Thereafter, cells were fixed in 3.7% phosphate-buffered formaldehyde, embedded in a paraffin block, sectioned, stained and viewed. Hematoxylin and Eosin (H&E) staining was used to determine the resemblance to in vivo human skin. Immunofluorescence was used to detect keratin 10, keratin 14 and involucrin which are markers of keratinocyte differentiation. Stratified keratinocyte layers were infected with C. trachomatis and this was confirmed using the MicroTrak ® C. trachomatis Culture Confirmation Test Kit. Subsequent changes to the layers were also observed and recorded. It was shown that HaCaT cells grown at the air-liquid interface on collagen-coated PTFE Transwell membrane inserts were able to stratify at 33°C. However, more layers of keratinocytes were seen at 37°C after the same duration of incubation (24 days). Keratin 10, keratin 14 and involucrin were all detected in the layers grown at both temperatures, suggesting that the keratinocytes had committed to differentiation. However, the fluorescence seen at 33°C for keratin 10 and involucrin was more intense as compared to that seen at 37°. This suggests that although stratification was faster at 37°C, differentiation was quicker at 33°C. C. trachomatis was able to infect layered keratinocytes grown at both temperatures although not all layers formed at 33°C were infected. Degradation of keratinocyte layers after infection with C. trachomatis was more prominent in those grown at 37°C, which is in keeping with previous findings that the optimum growth temperature of the C. trachomatis LGV biovar is 37°C. This study provided a novel insight in suggesting the manner in which C. trachomatis is able to infect and migrate through in vivo skin, leaving room for further studies in which similar methods of generating stratified keratinocytes may be used to better understand the pathophysiology of various other organisms that affect keratinocytes

Reconstruction and Simulation of Cellular Traction Forces

Biological cells are able to sense the stiffness, geometry and topography of their environment and sensitively respond to it. For this purpose, they actively apply contractile forces to the extracellular space, which can be determined by traction force microscopy. Thereby cells are cultured on elastically deformable substrates and cellular traction patterns are quanti- tatively reconstructed from measured substrate deformations, by solving the inverse elastic problem. In this thesis we investigate the influence of environmental topography to cellular force generation and the distribution of intracellular tension. For this purpose, we reconstruct traction forces on wavy elastic substrates, using a novel technique based on finite element methods. In order to relate forces to single cell-matrix contacts and different structures of the cytoskeleton, we then introduce another novel variant of traction force microscopy, which introduces cell contraction modeling into the process of cellular traction reconstruction. This approach is robust against experimental noise and does not need regularisation. We apply this method to experimental data to demonstrate that different types of actin fibers in the cell statistically show different contractilities. We complete our investigation by simulation studies considering cell colonies and single cells as thermoelastically contracting continuum coupled to an elastic substrate. In particular we examined the effect of geometry on cellular behavior in collective cell migration and tissue invasion during tumor metastasis

ACTIVE MANIPULATION OF EXTRACELLULAR MATRIX STIFFNESS

Ph.DDOCTOR OF PHILOSOPH