From microfluidics to hierarchical hydrogel materials

Over the past two decades, microfluidics has made significant contributions to material and life sciences, particularly via the design of nano-, micro- and mesoscale materials such as nanoparticles, micelles, vesicles, emulsion droplets, and microgels. Unmatched in control over a multitude of material parameters, microfluidics has also shed light on fundamental aspects of material design such as the early stages of nucleation and growth processes as well as structure evolution. Exemplarily, polymer hydrogel particles can be formed via microfluidics with exact control over size, shape, functionalization, compartmentalization, and mechanics that is hardly found in any other processing method. Interestingly, the utilization of microfluidics for material design largely focuses on the fabrication of single entities that act as reaction volume for organic and cell-free biosynthesis, cell mimics, or local environment for cell culturing. In recent years, however, hydrogel design has shifted towards structures that integrate a large variety of functions, e.g., to address the demands for sensing tasks in a complex environment or more closely mimicking architecture and organization of tissue by multiparametric cultures. Hence, this review provides an overview of recent literature that explores microfluidics for fabricating hydrogel materials that go well beyond common length scales as well as the structural and functional complexity of microgels necessary to produce hierarchical hydrogel structures. We focus on examples that utilize microfluidics to design microgel-based assemblies, on microfluidically made polymer microgels for 3D bioprinting, on hydrogels fabricated by microfluidics in a continuous fashion, like fibers, and on hydrogel structures that are shaped by microchannels

Development and characterization of a biopolymer direct-write process for 3D microvascular structures formation.

Engineering of bulk tissues has been limited by the lack of nutrient and waste exchange in these tissues without an adjacent capillary network. To produce microvasculature, a scaffold must be produced that provides temporary mechanical support and stimulate endothelial cell adhesion, growth, and morphogenesis into a vessel. However, current well-established techniques for producing microvasculature, such as electrospinning, are limited since they lack both the precision to control fiber placement in three-dimensional space and the ability to create fiber networks with predefined diameters to replicate the physiological microvascular progression from arteriole to capillary to venule. Our group has developed a “Direct-write” technique using a 3-Axis robotic dispensing system to process polymers into precisely positioned, three-dimensional, suspended fibers with controlled diameters. Within this dissertation, a conceptual scaffold-covering strategy is presented for the formation of the precisely positioned, three-dimensional microvascular structure with a controlled diameter in vitro. This study considers ways to extend the 3-Axis robotic dispensing system by incorporating new biodegradable materials into micro-fibers. First, a number of different biopolymers (natural, synthetic, composites, and copolymers) were used for demonstrating the capability of direct-writing micro-fibers and branched structures with microvascular-scale diameter through the 3-Axial robotic dispensing system. Then, the fabrication process was characterized by a design of experiments and a generalized mathematical model was developed through dimensional analysis. The empirical model determined the correlation between polymer fiber diameter and intrinsic properties of the polymer solution together with the processing parameters of the robotic dispensing system and allows future users the ability to employ the 3-Axis robotic dispensing system to direct-write micro-fibers without trial-and-error work. This study also considers ways to broaden the pre-vascularization methods by covering Human Dermal Microvascular Endothelial Cells (HDMECs) on the fabricated scaffold to generate the microvascular structure. HDMECs cultured on the produced micro-fiber scaffolds were observed to form a confluent monolayer spread along the axis and around the circumference of the fibers within two days of seeding. Once confluency was reached, the cell-covered scaffold was embedded into a collagen gel and a hybrid structure was formed. Through these experiments, we demonstrate the ability to obtain a cell-viable, flexible, and free-standing “modular tissue”, which could be potentially assembled to a three-dimensional microvascular network through angiogenesis mechanism

The stress granule response in wild-type and ALS-mutant neurons

Stress granules (SGs) are cytoplasmic ribonucleoprotein aggregates which form in response to cellular stress and disassemble following stress cessation. Mutations in human SG proteins cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and have been demonstrated to alter granule dynamics. Further, SGs have a compositional protein overlap with the post-mortem cytoplasmic inclusions that characterise ALS, suggesting that SGs may act as a precursor to, or seed, these inclusions. For this thesis, I first optimised the use of different SG inducers in mouse primary fibroblast and neuronal cultures. Following this, I cultured primary neurons in compartmentalised devices, applying the oxidative stressor sodium arsenite to either the somal or axonal compartment, to investigate the kinetics of SG formation. I observed a delayed SG assembly response in neuronal somas when arsenite was applied axonally, for both cortical and sensory neurons. This response is decreased by inhibition of dynein or protein translation in the axonal compartment. Further, I investigated the effect of ALS-causing mutations on the SG response, using a novel FUS-mutant mouse model. This model expresses a humanised Cterminal disease-causing mutation that results in a frameshifted amino acid sequence downstream of the mutation, eradicating the nuclear localisation signal of the FUS protein. This frameshift sequence allowed for the generation of antibodies able to distinguish between wild-type and mutant FUS. Using these antibodies, I observed that the normally predominately nuclear FUS protein mislocalised to the cytoplasm in neurons heterozygous and homozygous for the mutation. Additionally, I demonstrated that the mutant protein is present in SGs at a higher level than the wild-type protein. Finally, I optimised a method for sorting neuronal somas following labelling with fluorescently-tagged retrograde toxin subunits. These results demonstrate the ability of neuronal axons to respond to exogeneous oxidative stressors and highlight the importance of the SG response in ALS-mutant cell

Microtechnologies for Cardiovascular Tissue Engineering

Cardiovascular disease is a rising epidemic worldwide, and curative therapies remain elusive. Heart and vascular disease remain some of the hardest to cure due to the limited capacity of the heart to repair itself, necessitating a cell or organ based therapy to cure the inevitable descent into heart failure. Tissue engineering is uniquely poised to significantly alter this disease burden though the fabrication of cardiac and vascular tissues in vitro. However, the challenges for achieving these aims are significant - for cardiac tissues, the therapy must adhere to strict requirements of adequate perfusion and functional integration with the damaged heart. Vascular tissues are required to be amenable to surgical anastomosis while at the same time provide nutrient transport on the cellular level. Recently, a new set of technologies based from the semiconductor industry, have enabled micron level control over the cellular environment and cells themselves and may enable novel approaches to fulfill these tissue engineering requirements. In this dissertation, these microtechnologies will be leveraged to address some of the current obstacles that limit the use of tissue engineering approaches for functional therapy. Specifically, microtechnologies were used to screen the effect of electrical stimulation on the function and maturation of human embryonic stem cell derived cardiomyocytes, which resulted in the ability to program specific individual beating frequencies of the cells while improving contractile function and led to the identification of a channel specific effect for frequency modulation. These technologies were also used to distinguish the vasculogenic potential of different mesenchymal stem cell sources for nascent vessel stabilization, and enabled the development of a powerful hydrogel docking platform with the novel capability to spatially pattern any number of cells, cytokines or drugs on the microscale, while permitting scale up for larger tissue generation without the loss of precision. Finally, these technologies were used to create vascular networks with hierarchical branching patterns that could be implanted and used in vivo fulfilling a major criterion of vascular tissue function - surgical compatibility with microscale architecture for tissue perfusion. Therefore, these microtechnologies support novel interrogation of cell function and enable new methods to engineer cardiovascular tissues

A New Dimension of Cancer Treatment: Analysing and Targeting High-Grade Glioma Invasion Using Three-Dimensional In Vitro Models

High-Grade Gliomas (HGG) are an extremely aggressive form of brain cancer with limited targeted treatments and dire prognosis. Despite global improvements in overall cancer survival, HGG survival rates have stagnated over the last three decades. This can be partly attributed to HGG’s characteristically invasive progression into the brain, where far-invaded cells are often missed by first-line therapies leading to tumour recurrence. The use of anti-invasive agents may limit extensive cell spread and improve efficacy of adjuvant anti-cancer therapies, thus, this study has investigated the novel anti-invasive PTU (p-tolyl-ureidopalmitic acid), an omega-3-derivative with the ability to cross the blood brain barrier. PTU was tested in primary patient-derived HGG cells cultured in 3D models designed to replicate discrete aspects of the 3D in vivo brain microenvironment. Across models, PTU treatment decreased the number of invading HGG cells and mitigated both single and collective invasion phenotypes. Importantly, this data demonstrates that 3D in vitro culture can reliably represent a whole-picture of HGG invasion. Additionally, this study involved the development of a dynamic, temporal reporter of transcriptional co-activators Yes-associated Protein and Transcriptional co-Activator with PDZ-binding motif (YAP/YAZ), which are mediators of a number of malignant processes and regulated by bio-mechanical signalling. This was achieved with an Adeno-associated-virus (AAV)-mediated gene delivery method, which also revealed a number of AAV capsid variants with tropism to primary patient-derived HGG cells. This reporter has potential future uses in revealing YAP/TAZ activity within the 3D biologically-relevant in vitro models. Together, these findings reveal potential successful therapeutic strategies for HGG and provide multiple insights into future research possibilities in the hopes of mitigating the extent of this truly debilitating and devastating disease

Gold nanoparticles and chitosan encapsulation for therapy and sensing

Un sinfín de aplicaciones en el campo de la biomedicina se verían beneficiadas por el futuro uso de nanopartículas de oro (AuNPs). Sin embargo, a pesar de las miles de publicaciones científicas que destacan cada año las fabulosas propiedades de estas nanopartículas en este área, su empleo más allá del ámbito científico es testimonial, especialmente cuando hablamos de su uso con finalidades terapéuticas en el ámbito clínico. El objetivo principal de esta tesis consiste en mejorar la aplicabilidad de las nanopartículas de oro con diferentes geometrías mediante dos líneas de acción: 1) el estudio de la relación entre sus propiedades fisicoquímicas y su rendimiento para las aplicaciones de bio-detección y terapia fototérmica y 2) la búsqueda de una manera complementaria de mejorar sus propiedades intrínsecas para aplicaciones terapéuticas mediante su encapsulación en hidrogeles de quitosano. En la primera parte de esta tesis la inexistencia de una metodología clínica adecuada para la detección de miRNA nos ha llevado a buscar nuevas estrategias para su detección mediante el empleo de nanopartículas de oro como amplificadores de la señal de detección gracias a sus idóneas propiedades: elevada densidad, tamaño adecuado y facilidad para su funcionalización con diferentes proteínas. Además, para descubrir su utilidad en la detección de miRNA en condiciones similares a las necesarias en el ámbito clínico, se ha llevado a cabo un estudio comparativo empleando nanopartículas de oro con diversas geometrías y diferentes estrategias de biofuncionalización para su interacción con los miRNA. En la segunda parte de esta tesis se ha continuado con el estudio de la aplicación de diferentes geometrías de nanopartículas de oro para su uso en terapia fototérmica, centrándonos en sus propiedades ópticas, su interacción celular y eficacia, ya que hay una falta de conocimiento acerca de la importancia que tiene la forma de las nanopartículas en este tema. Gracias a las conclusiones extraídas durante este estudio concernientes a la baja internalización celular de los nanorods, hemos propuesto una estrategia innovadora consiste en su encapsulación en hidrogeles de quitosano motivada por las propiedades de adhesión celular del quitosano que han permitido una mejora remarcable de su eficacia. Finalmente se ha desarrollado una nueva metodología para la encapsulación de nanopartículas de oro en hidrogeles de quitosano empleando la tecnología de eyección automática de tinta o inkjet para mejorar su encapsulación, la cual permite que el proceso sea más fácil de escalar, efectivo y controlable. Esta tecnología ofrece una metodología automática para la producción continua de microcápsulas de quitosano que contengan las nanopartículas de oro en su interior, proporcionando biomateriales con propiedades muy interesante para las aplicaciones biomédicas. Como resultado, la investigación llevada a cabo durante esta tesis doctoral contribuye a ampliar nuestro conocimiento en la importancia del empleo de diferentes geometrías de nanopartículas de oro para las aplicaciones terapéuticas y de detección a la vez que proporciona nuevas herramientas para aumentar su aplicabilidad gracias a la funcionalización de su superficie y a su encapsulación en hidrogeles de quitosano. <br /

Bio-Inspired Soft Artificial Muscles for Robotic and Healthcare Applications

Soft robotics and soft artificial muscles have emerged as prolific research areas and have gained substantial traction over the last two decades. There is a large paradigm shift of research interests in soft artificial muscles for robotic and medical applications due to their soft, flexible and compliant characteristics compared to rigid actuators. Soft artificial muscles provide safe human-machine interaction, thus promoting their implementation in medical fields such as wearable assistive devices, haptic devices, soft surgical instruments and cardiac compression devices. Depending on the structure and material composition, soft artificial muscles can be controlled with various excitation sources, including electricity, magnetic fields, temperature and pressure. Pressure-driven artificial muscles are among the most popular soft actuators due to their fast response, high exertion force and energy efficiency. Although significant progress has been made, challenges remain for a new type of artificial muscle that is easy to manufacture, flexible, multifunctional and has a high length-to-diameter ratio. Inspired by human muscles, this thesis proposes a soft, scalable, flexible, multifunctional, responsive, and high aspect ratio hydraulic filament artificial muscle (HFAM) for robotic and medical applications. The HFAM consists of a silicone tube inserted inside a coil spring, which expands longitudinally when receiving positive hydraulic pressure. This simple fabrication method enables low-cost and mass production of a wide range of product sizes and materials. This thesis investigates the characteristics of the proposed HFAM and two implementations, as a wearable soft robotic glove to aid in grasping objects, and as a smart surgical suture for perforation closure. Multiple HFAMs are also combined by twisting and braiding techniques to enhance their performance. In addition, smart textiles are created from HFAMs using traditional knitting and weaving techniques for shape-programmable structures, shape-morphing soft robots and smart compression devices for massage therapy. Finally, a proof-of-concept robotic cardiac compression device is developed by arranging HFAMs in a special configuration to assist in heart failure treatment. Overall this fundamental work contributes to the development of soft artificial muscle technologies and paves the way for future comprehensive studies to develop HFAMs for specific medical and robotic requirements

Electrospun Nanofibers for Biomedical Applications

Electrospinning is a versatile and effective technique widely used to manufacture nanofibrous structures from a diversity of materials (synthetic, natural or inorganic). The electrospun nanofibrous meshes’ composition, morphology, porosity, and surface functionality support the development of advanced solutions for many biomedical applications. The Special Issue on “Electrospun Nanofibers for Biomedical Applications” assembles a set of original and highly-innovative contributions showcasing advanced devices and therapies based on or involving electrospun meshes. It comprises 13 original research papers covering topics that span from biomaterial scaffolds’ structure and functionalization, nanocomposites, antibacterial nanofibrous systems, wound dressings, monitoring devices, electrical stimulation, bone tissue engineering to first-in-human clinical trials. This publication also includes four review papers focused on drug delivery and tissue engineering applications