Biaxial Nematic Order in Liver Tissue

Understanding how biological cells organize to form complex functional tissues is a question of key interest at the interface between biology and physics. The liver is a model system for a complex three-dimensional epithelial tissue, which performs many vital functions. Recent advances in imaging methods provide access to experimental data at the subcellular level. Structural details of individual cells in bulk tissues can be resolved, which prompts for new analysis methods. In this thesis, we use concepts from soft matter physics to elucidate and quantify structural properties of mouse liver tissue. Epithelial cells are structurally anisotropic and possess a distinct apico-basal cell polarity that can be characterized, in most cases, by a vector. For the parenchymal cells of the liver (hepatocytes), however, this is not possible. We therefore develop a general method to characterize the distribution of membrane-bound proteins in cells using a multipole decomposition. We first verify that simple epithelial cells of the kidney are of vectorial cell polarity type and then show that hepatocytes are of second order (nematic) cell polarity type. We propose a method to quantify orientational order in curved geometries and reveal lobule-level patterns of aligned cell polarity axes in the liver. These lobule-level patterns follow, on average, streamlines defined by the locations of larger vessels running through the tissue. We show that this characterizes the liver as a nematic liquid crystal with biaxial order. We use the quantification of orientational order to investigate the effect of specific knock-down of the adhesion protein Integrin ß-1. Building upon these observations, we study a model of nematic interactions. We find that interactions among neighboring cells alone cannot account for the observed ordering patterns. Instead, coupling to an external field yields cell polarity fields that closely resemble the experimental data. Furthermore, we analyze the structural properties of the two transport networks present in the liver (sinusoids and bile canaliculi) and identify a nematic alignment between the anisotropy of the sinusoid network and the nematic cell polarity of hepatocytes. We propose a minimal lattice-based model that captures essential characteristics of network organization in the liver by local rules. In conclusion, using data analysis and minimal theoretical models, we found that the liver constitutes an example of a living biaxial liquid crystal.:1. Introduction 1 1.1. From molecules to cells, tissues and organisms: multi-scale hierarchical organization in animals 1 1.2. The liver as a model system of complex three-dimensional tissue 2 1.3. Biology of tissues 5 1.4. Physics of tissues 9 1.4.1. Continuum descriptions 11 1.4.2. Discrete models 11 1.4.3. Two-dimensional case study: planar cell polarity in the fly wing 15 1.4.4. Challenges of three-dimensional models for liver tissue 16 1.5. Liquids, crystals and liquid crystals 16 1.5.1. The uniaxial nematic order parameter 19 1.5.2. The biaxial nematic ordering tensor 21 1.5.3. Continuum theory of nematic order 23 1.5.4. Smectic order 25 1.6. Three-dimensional imaging of liver tissue 26 1.7. Overview of the thesis 28 2. Characterizing cellular anisotropy 31 2.1. Classifying protein distributions on cell surfaces 31 2.1.1. Mode expansion to characterize distributions on the unit sphere 31 2.1.2. Vectorial and nematic classes of surface distributions 33 2.1.3. Cell polarity on non-spherical surfaces 34 2.2. Cell polarity in kidney and liver tissues 36 2.2.1. Kidney cells exhibit vectorial polarity 36 2.2.2. Hepatocytes exhibit nematic polarity 37 2.3. Local network anisotropy 40 2.4. Summary 41 3. Order parameters for tissue organization 43 3.1. Orientational order: quantifying biaxial phases 43 3.1.1. Biaxial nematic order parameters 45 3.1.2. Co-orientational order parameters 51 3.1.3. Invariants of moment tensors 52 3.1.4. Relation between these three schemes 53 3.1.5. Example: nematic coupling to an external field 55 3.2. A tissue-level reference field 59 3.3. Orientational order in inhomogeneous systems 62 3.4. Positional order: identifying signatures of smectic and columnar order 64 3.5. Summary 67 4. The liver lobule exhibits biaxial liquid-crystal order 69 4.1. Coarse-graining reveals nematic cell polarity patterns on the lobulelevel 69 4.2. Coarse-grained patterns match tissue-level reference field 73 4.3. Apical and basal nematic cell polarity are anti-correlated 74 4.4. Co-orientational order: nematic cell polarity is aligned with network anisotropy 76 4.5. RNAi knock-down perturbs orientational order in liver tissue 78 4.6. Signatures of smectic order in liver tissue 81 4.7. Summary 86 5. Effective models for cell and network polarity coordination 89 5.1. Discretization of a uniaxial nematic free energy 89 5.2. Discretization of a biaxial nematic free energy 91 5.3. Application to cell polarity organization in liver tissue 92 5.3.1. Spatial profile of orientational order in liver tissue 93 5.3.2. Orientational order from neighbor-interactions and boundary conditions 94 5.3.3. Orientational order from coupling to an external field 99 5.4. Biaxial interaction model 101 5.5. Summary 105 6. Network self-organization in a liver-inspired lattice model 107 6.1. Cubic lattice geometry motivated by liver tissue 107 6.2. Effective energy for local network segment interactions 110 6.3. Characterizing network structures in the cubic lattice geometry 113 6.4. Local interaction rules generate macroscopic network structures 115 6.5. Effect of mutual repulsion between unlike segment types on network structure 118 6.6. Summary 121 7. Discussion and Outlook 123 A. Appendix 127 A.1. Mean field theory fo the isotropic-uniaxial nematic transition 127 A.2. Distortions of the Mollweide projection 129 A.3. Shape parameters for basal membrane around hepatocytes 130 A.4. Randomized control for network segment anisotropies 130 A.5. The dihedral symmetry group D2h 131 A.6. Relation between orientational order parameters and elements of the super-tensor 134 A.7. Formal separation of molecular asymmetry and orientation 134 A.8. Order parameters under action of axes permutation 137 A.9. Minimal integrity basis for symmetric traceless tensors 139 A.10. Discretization of distortion free energy on cubic lattice 141 A.11. Metropolis Algorithm for uniaxial cell polarity coordination 142 A.12. States in the zero-noise limit of the nearest-neighbor interaction model 143 A.13. Metropolis Algorithm for network self-organization 144 A.14. Structural quantifications for varying values of mutual network segment repulsion 146 A.15. Structural quantifications for varying values of self-attraction of network segments 148 A.16. Structural quantifications for varying values of cell demand 150 Bibliography 152 Acknowledgements 17

3D & 4D printing for health application: new insights into printed formulations and bioprinted organs is the topical route on this way?: topical printed formulations and skin bioprinting 3D and 4D printing

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2021, Universidade de Lisboa, Faculdade de Farmácia.A impressão 3D é uma tecnologia inovadora com um vasto campo de aplicações. Engloba um grupo de tecnologias versáteis capazes de revolucionar várias áreas ao oferecer soluções a certos desafios. A sua versatilidade é devida à natureza de additive mannufacturing, fácil acesso, vasta variedade de técnicas e materiais que podem ser utilizados. Aqui as técnicas de impressão 3D e 4D e de bioimpressão são revistas com o objectivo de providenciar as mais recentes aplicações na área da saúde. Tanto a indústria médica como a farmacêutica partilham desvantagens, tais como baixa adesão à terapêutica devida à polimedicação, falta de formulações farmacêuticas para populações específicas que levam a um baixo sucesso terapêutico, escassez e falta de dadores de órgãos, próteses à medida do doente, modelos de estudo insuficientes e o decréscimo no processo de cicatrização de feridas. Com a tecnologia de impressão 3D, a formulação de medicação personalizada que inclui comprimidos, hidrogeles, entre outras formulações farmacêuticas contendo uma ou mais substâncias activas, é possível com o uso de tintas específicas onde as substâncias activas e os excipientes são combinados. Evita o uso de vários equipamentos enquanto providencia uma variedade de perfis de libertação através da manipulação da forma das formulações. Juntamente com a impressão 3D, novas tecnologias surgem, tais como a impressão 4D e a bioimpressão 3D, uma evolução da primeira. A técnica de bioimpressão permite a impressão de componentes biológicos ao usar bioinks, abrindo novos caminhos na engenharia de tecidos e medicina regenerativa. A impressão de tecidos e órgãos tornou-se uma realidade com uma taxa de sucesso promissora. Em particular, a bioimpressão de pele é um dos investimentos mais promissores para o melhoramento do tratamento e cicatrização de lesões e queimaduras, ao oferecer uma solução à medida com uma taxa de sucesso aumentada e com menos sofrimento. Em resumo, esta revisão sobre o uso de impressão 3D e bioimpressão no desenvolvimento de formulações farmacêuticas e bioimpressão claramente evidencia o impacto positivo desta tecnologia de ponta nesta área e os seus resultados promissores no bem-estar.3D printing is an innovative technology with a broad range of applications. It encompasses a group of versatile technologies capable of revolutionizing various fields by offering new solutions for major challenges. Its versatility is due to the additive manufacturing nature, easy access, wide variety of techniques, and a vast range of processable materials. Hereby, the 3D and 4D printing and bioprinting techniques are overviewed aiming to provide a comprehensive state of the art of their main applications in the health field. Both medical and pharmaceutical industries share some drawbacks such as low patient compliance due to the poly-medication, lack of appropriate pharmaceutical formulations for specific populations leading to a poor therapeutic success, scarcity and shortage of organ donors, patient-specific prosthetics, insufficient study models, and decreased success in wound healing. With 3D printing technology, the formulation of personalized medication including tablets, hydrogels, among other pharmaceutical formulations containing single or multiple drugs, is possible by using specific inks where a defined composition of drugs and excipients are combined. It avoids the use of much equipment while providing a variety of release rates through the shape manipulation of those formulations. Alongside 3D printing, newer technologies arise, such as 4D printing as well as 3D bioprinting, an evolution of the first one. The bioprinting technique allows the fabrication of biologically functional tissue constructs and in vitro tissue models by the controlled deposition of bioinks, thus opening new avenues in tissue engineering and regenerative medicine. Tissue and organ printing have become a reality with promising rate of success. In particular, skin bioprinting is one of the most promising investments to improve the treatment and healing of skin injuries, including chronic wounds and burns, by providing a tailored solution with greater chance of success and less suffering. In summary, this overview about the use of 3D printing and bioprinting on the development of pharmaceutical formulations and skin bioprinting clearly evidenced the positive impact of this cut edge technology in this crucial field and the promising outcomes in wellness

Multi-Scale Design of Ink Formulations for the 3D Bioprinting of Soft and Elastic Tissue-Mimetic Structures

Three-dimensional (3D) engineered tissue and organ models with adjustable biochemical and physical properties are needed in regenerative medicine. As such, 3D printing has emerged as a potential strategy to create more complex tissue-like constructs compared to conventional microfabrication techniques. It allows for precise positioning of biomaterials to create tissue constructs that imitate natural tissues and organs. Among 3D printing strategies, including extrusion-based, light-induced, and inkjet-based methods, the extrusion-based technique is broadly employed owing to its ease of use and compatibility with multi-material printing. However, the fabrication of accurate and precise human-mimetic functional 3D constructs still remains challenging. This thesis presents an investigation on the bioink material preparation from nano-, micro- to macro-scale to provide high structural resolution and cell viability and functionality. Chapter 3 presents a strong shear-thinning, solid-like bioink employing alginate (1%), cellulose nanocrystals (CNCs) (3%), and gelatin methacryloyl (GelMA) (5%) (namely 135ACG hybrid ink) for the direct printing of cell-laden and acellular architectures. After crosslinking, the ACG gel can also provide a stiff extracellular matrix (ECM) ideal for stromal cell growth. By controlling the polymer concentration, a GelMA (4%) bioink was designed to encapsulate hepatoma cells (hepG2), as GelMA gel possesses the desired low mechanical stiffness matched human liver tissue. Four different versions of to-scale liver lobule-mimetic constructs were fabricated via ME bioprinting, with precise positioning of two different cell types (NIH/3T3 and hepG2) embedded in matching ECMs (135ACG and GelMA, respectively). The four versions allowed us to exam effects of mechanical cues and intercellular interactions on cell behaviors. Fibroblasts thrived in stiff 135ACG matrix and aligned at the 135ACG/GelMA boundary due to durotaxis, while hepG2 formed spheroids exclusively in the soft GelMA matrix. Elevated albumin production was observed in the bicellular 3D co-culture of hepG2 and NIH/3T3, both with and without direct intercellular contact, indicating that improved hepatic cell function can be attributed to soluble chemical factors. Overall, our results showed that complex constructs with multiple cell types and varying ECMs can be bioprinted and potentially be useful for both fundamental biomedical research and translational tissue engineering. Chapter 4 presents a supporting bath-based embedded 3D printing strategy to fabricate large-scale architectures. Novel inks composed of 10 wt% glycidyl methacrylated poly(vinyl alcohol) (PVAGMA) with a different degree of substitution (DOS) and 4 wt% CNCs (PVAGMA(DOS)/CNC) with strong shear-thinning properties were developed. By controlling the DOS of PVAGMA and the crosslinking method, inks with the desired mechanical stiffness and toughness mimicking that of healthy arteries and arteries with atherosclerosis were designed to construct vascular phantoms. Cyclic tensile tests and an in-vitro hemodynamic study were performed on the hydrogels, illustrating that our ink compositions, namely PVAGMA2/CNC and PVAGMA4/CNC, possessed strong mechanical stability, durability, and recovery property. The burst circumferential tensile stress obtained from an in-vitro hemodynamic study and rupture tensile stress acquired under uniaxial test of PVAGMA2/CNC and PVAGMA4/CNC were comparable suggesting the optimal layer integrity and the mechanical strength was not influenced by the printing direction. Additionally, the burst pressure of our hydrogels was found to be about 90 mm Hg and the printed vascular phantoms were able to withstand a carotid pulse-mimetic pulsatile flow (60 beats/min with a peak flow rate of 27 mL/s) for over 10 days. Furthermore, with acoustic properties of PVAGMA/CNC hydrogels similar to those of human arteries, we demonstrated their capability in ultrasonics by embedded printing three different versions of straight vascular phantoms. B-mode imaging were performed on the phantoms to confirm the vessel dimensions and analyze vessel strain of the softer PVAGMA2/CNC and stiffer PVAGMA4/CNC. It was found that the vessel strain was 24.6% and 8.9% for PVAGMA2/CNC and PVAGMA4/CNC, respectively, demonstrating that the two bioink materials can be used for vessel-mimetic materials. It is anticipated that this artery phantom can serve as a platform to evaluate local arterial stiffness estimation algorithms. Overall, our results elucidated the great potential of the PVAGMA-based inks and granular supporting material to create biofunctional heterogeneous vasculature via embedded printing strategy for regenerative medicine and tissue engineering applications. Taken all together, this thesis presents a comprehensive study on developing and characterizing various bioink materials and bioprinting cellular and acellular constructs via free-form or embedded printing strategy. Our developed bioink materials and ME-based printing methods offered new approaches to address the central challenges in tissue engineering to create heterogeneous constructs with varying ECMs to recapitulate biological functions

Resolving the liver sinusoidal endothelial phenotype in health and disease

The burden of liver disease is continuously increasing globally, and this emphasises the need for the development of therapeutics. In order for this to be achieved, potential cellular and molecular targets need to be identified. Liver sinusoidal endothelial cells (LSECs) play a key role in maintaining liver homeostasis and their dysfunction drives liver disease pathophysiology and this role needs to be further elucidated. In order to identify phenotypic differences in LSECs in health and disease, a combination of analytical techniques such as immunohistochemistry and qPCR was applied on human tissue specimens. To confirm whether these changes are recapitulated in vitro, I isolated LSECs from human healthy and cirrhotic tissue specimens for the establishment of culture model of human LSECs. Validation of functional and phenotypic characteristics of LSECs in vitro was carried out using immunocytochemistry and qPCR. Furthermore, the development and optimisation of a super-resolution imaging protocol for the visualisation of LSEC fenestrations was performed. Altered expression and downregulation of scavenger receptors in LSECs was identified in diseased human tissue specimens compared to healthy specimens and this confirmed capillarisation of sinusoidal endothelial cells in liver disease. Expression of scavenger receptors and key regulatory molecules was maintained in LSECs in vitro. The phenotypic changes in LSECs identified in liver tissue specimens were partially recapitulated in LSECs in vitro. The application of pharmaceutical molecules for the enhancement of nitric oxide (NO) signalling in LSECs revealed an altered genotype in healthy and cirrhotic LSECs. Finally, fenestrations were visualised on the LSEC membrane using the developed super-resolution imaging protocol and improvement in LSEC porosity following the application of sildenafil citrate. Hence these findings emphasise the relevance of appropriate culture models and imaging approaches to study phenotypic changes in LSECs in relation to disease and highlight the therapeutic potential of sildenafil citrate in improving LSECs porosity

Assembly of living building blocks to engineer complex tissues

The great demand for tissue and organ grafts, compounded by an aging demographic and a shortage of available donors, has driven the development of bioengineering approaches that can generate biomimetic tissues in vitro. Despite the considerable progress in conventional scaffold‐based tissue engineering, the recreation of physiological complexity has remained a challenge. Bottom‐up tissue engineering strategies have opened up a new avenue for the modular assembly of living building blocks into customized tissue architectures. This Progress Report overviews the recent progress and trends in the fabrication and assembly of living building blocks, with a key highlight on emerging bioprinting technologies that can be used for modular assembly and complexity in tissue engineering. By summarizing the work to date, providing new classifications of different living building blocks, highlighting state‐of‐the‐art research and trends, and offering personal perspectives on future opportunities, this Progress Report aims to aid and inspire other researchers working in the field of modular tissue engineering

Cell encapsulation systems toward modular tissue regeneration: from immunoisolation to multifunctional devices

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Nanotechnology in the Regeneration of Complex Tissues.

This is the final published version. It first appeared at http://www.la-press.com/nanotechnology-in-the-regeneration-of-complex-tissues-article-a4503.Modern medicine faces a growing crisis as demand for organ transplantations continues to far outstrip supply. By stimulating the body's own repair mechanisms, regenerative medicine aims to reduce demand for organs, while the closely related field of tissue engineering promises to deliver "off-the-self" organs grown from patients' own stem cells to improve supply. To deliver on these promises, we must have reliable means of generating complex tissues. Thus far, the majority of successful tissue engineering approaches have relied on macroporous scaffolds to provide cells with both mechanical support and differentiative cues. In order to engineer complex tissues, greater attention must be paid to nanoscale cues present in a cell's microenvironment. As the extracellular matrix is capable of driving complexity during development, it must be understood and reproduced in order to recapitulate complexity in engineered tissues. This review will summarize current progress in engineering complex tissue through the integration of nanocomposites and biomimetic scaffolds.JWC was previously funded by a Scholarship from the University of Glasgow and is now in receipt of a Cancer Research UK Scholarship

Design considerations and analysis of a bioreactor for application in a bio-artificial liver support system

Acute Liver Failure (ALF) is a devastating ailment with a high mortality rate and limited treatment alternatives. This study presents a methodology for the design and development of a bio-artificial bioreactor to be used in a Bio-Artificial Liver Support System. The system will ultimately be used either to bridge a patient to orthotopic liver transplant (OLT), the only current cure for end stage ALF, or spontaneous recovery. Methods to optimize and visualize the flow and related mass transfer in the BR are presented. The use of magnetic resonance imaging (MRI), scanning electron microscopy (SEM) and simple testing methodology is applied with emphasis on modeling the flow conditions in the BR. The bioreactor (BR) used in the Bio-Artificial Liver Support System (BALSS), currently under-going animal trials at the University of Pretoria, was modeled and simulated for the flow conditions in the device. Two different perfusion steps were modeled including the seeding of hepatocyte cells and later the clinical perfusion step. It was found that the BR geometry was not optimal with “dead spots” and regions of retarded flow. This would restrict the effective transport of nutrients and oxygen to the cells. The different perfusion rates for the seeding and clinical perfusion steps allowed for different velocity contours with cells seeing inconsistent flow patterns and mass transfer gradients. An optimized BR design is suggested and simulated, that effectively reduces the areas of retarded flow (dead spots) and increases the flow speed uniformly through the BR to an order of magnitude similar to that found in the sinusoidal range. The scaffolding volume was also decreased to allow a larger local cell density promoting cell-cell interaction. Finally a summarized design table for the design of a hepatic BR is presented.Dissertation (MEng (Mechanical))--University of Pretoria, 2008.Mechanical and Aeronautical Engineeringunrestricte

Lab-on-a-Chip Fabrication and Application

The necessity of on-site, fast, sensitive, and cheap complex laboratory analysis, associated with the advances in the microfabrication technologies and the microfluidics, made it possible for the creation of the innovative device lab-on-a-chip (LOC), by which we would be able to scale a single or multiple laboratory processes down to a chip format. The present book is dedicated to the LOC devices from two points of view: LOC fabrication and LOC application