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MYCELIUM MILLENNIUM imagines a new era in which biological resources, specifically Fungi and Mycelium, are used to grow a collection of objects for everyday domestic rituals, merging biofabrication and traditional craft. The project is an effort to demystify the transformation of an organism to a biomaterial and at the same time raise questions of aesthetics and cultural acceptance. The fungal mycelium material qualities –  antibacterial, fire-resistant, heat isolating and water-resistant – are incorporated in the function of each design.  The objects adopt antique symbols embedding them in a longer material history, as well as a scale and form that introduces the fungal material to the context of the home. While the collection attempts to stress longevity and resilience, it is integrated into a circular vision, where the material is sourced from nature and returns to nature.   MYCELIUM MILLENNIUM is an invitation to raise awareness for a material revolution, an opportunity to learn from nature and its potential, where products and objects could match the planet's needs.

<p>Additive manufacturing (AM) of hydrogels has gained increasing interest across various fields. Drop-on-demand (DOD) printing (also known as inkjet printing) shows the great potential to construct 3D hydrogels with spatially controlled properties and compositions. However, a limited mechanistic understanding of the behavior of printed polymer drops makes it challenging to design and optimize DOD printing protocols for a wide variety of hydrogels. Here, we have demonstrated an extensive and in-depth study from the theoretical and experimental research of drop-wise structure to the development of functional materials by DOD printing of polymer inks. First, computational and experimental studies are performed to establish a mechanism of the water-matrix interaction within printed polymer drops. The results ultimately enabled a dimensionless parameter that characterizes water transport during the dehydration process of printed polymer drops. Next, as particles are suspended in polymer inks to add functionality, this dimensionless parameter was further extended to characterize particle movement and distribution patterns in the printed particle-laden hydrogels. By correlating the intra-drop particle distribution to the similarity parameter, a scaling law is confirmed to guide ink formulation and printing protocol that enables advanced materials with spatially digitized functionality (i.e., digital hydrogels). Finally, cells that serve as active particles are embedded in the hydrogels to mimic the native tissues. A "digital cell printing" method based on DOD printing of "two colors" cell-laden (i.e., cancer cells and CAFs) polymer inks is developed to rapidly (< 1 day) create 3D tumor models with tumor-stroma interface (i.e., tumoroids) and high cell density (~108 cells/cm3) that closely recapitulate the tumor microenvironment in vivo. Overall, DOD printing of particulate-laden polymer inks showed the great potential to construct 3D functional hydrogels with spatially controlled properties and compositions.</p>

<p>The untethered transfer of energy and scalability of magnetic actuators enables functionality to an otherwise passive system. For example, wireless magnetic actuation can turn static 2D and 3D cell cultures into a more physiologically-relevant dynamic environment while limiting contamination. Moreover, indwelling catheters and implantable sensors are typically stationary devices that are notorious for their short lifespan when implanting into the body due to immune responses. Magnetic microactuators may be used for wireless actuation for in situ removal of biological materials accumulated on chronically implanted devices. In this dissertation, I will demonstrate examples of novel biomedical microdevices enabled by magnetic actuation for added functional benefits. First, I will describe a soft polymer magnetic actuator that can facilitate the study of a physiologically relevant cell culturing system. By cyclically stretching an extracellular matrix protein in a 3D cell culture, this system can elucidate the process by which breast cancer cells respond to a dynamic environment in the lungs. The fibrillar fibronectin suspended across the body of the magnetic actuator provides a matrix representative of early metastasis for 3D cell culture that has not yet been recapitulated in vitro until now. Our results demonstrate a clear suppressive cellular response due to cyclic stretching that has implications for a mechanical role in the dormancy and reactivation of disseminated breast cancer cells to macrometastases. As a second application, I will demonstrate the use of magnetic microactuators to remove biofouling on an implantable biosensor in order to prolong its functionality. The results of our work suggest that the motion of the actuator on the sensor surface can maintain biosensor signal integrity and prevents the downstream effects of the foreign body response. Additionally, I will present the design and proof of concept testing of a novel aspiration thrombectomy catheter meant to improve the engagement between the catheter and the blood clot being removed. Preliminary results demonstrate the added benefit of incorporating a microstructure in the inner diameter of the catheter meant to increase the retraction force aspiration catheters have when retrieving corked emboli at the catheter tip. </p> <p><br></p>

Two-dimensional (2D) in vitro models of tissues and organs have long been used as one of the main tools to understand human physiology and for applications such as drug discovery. But there is a huge disparity between in vivo conditions and these models which has created the need for better models. It has been shown that making three-dimensional models with dynamic environments that provide proper physical and chemical cues for cells, can bridge this gap between 2D models and in vivo conditions but the toolbox for creating such models has been imperfect and rudimentary. Introduction of tissue engineering concept and advent of biofabrication tools to meet its demands has provided new possible avenues for in vitro modeling but many of these tools are specifically designed to create tissue and organ replacements and lack features such as the ability to investigate cellular behavior with ease that are necessary for in vitro modeling purposes. The objective of this doctoral thesis was to introduce a novel toolbox of biofabrication techniques, based on bioprinting and bioassembly, that together are capable of recapitulating anatomical and physiological requirements of different tissue in in vitro setups in a more relevant way while creating the possibility of investigating cellular behavior. A bioprinting technique was developed that allowed formation of large constructs with proper mechanical stability, perfusion, and direct access to cells in different locations. The second technique was based on bioassembly of collagenous grafts in micro-molds and cells from different tissues with the ability to control cell positioning and create tissue-relevant cell densities with higher degree of similarity to human tissues compared to previous techniques. The third technique was based on bioassembled stand alone and dense cell-sheets for cells capable of fusion. These techniques were subsequently used for modeling a few chosen biological phenomenon to showcase the advantages of the techniques over previously developed ones and to further shed light on possible shortcomings of each of the techniques in their application for those specific tissues. In conclusion, our techniques may serve as valuable and easy to use tools for researchers, specifically biologists to investigate different aspects of human biology and disease mechanism in more details. / Thesis / Doctor of Philosophy (PhD) / Experimentation on humans is unethical, therefore in order to understand how human body works and test new therapeutic drugs researchers have used animals and cells isolated from animals or humans. Animals are inherently different from humans and isolated cells are culture in conditions different than human body, therefore a huge gap exists between the knowledge derived from these models and what happens in human body. Since there is no one-size-fits-all technique to model all of the human tissues, the objective of current study was set to build a toolbox of techniques that each could create better environment in the lab for cells isolated from different tissues and organs with more similarity to original tissues, to bridge the gap and eliminate the need to use animal models entirely. During the course of this PhD studies, three different techniques that can be used to make such models for different tissues and organs, as well as different diseases, were developed and characterized. These techniques were also used to shed light on some of the cellular behavior that are already observed in human body but either are not explained or aren’t re-created in the lab for mechanistic studies. Certain questions regarding selected tissues were chosen and the technique most compatible with that tissue was used for the modeling purposes. For example, one investigated niche was the origin of the bone sensory cells which could be important to heal damaged bones or prevent osteoporosis. The first technique was deemed most suitable for this question while for the next question, how the fat and muscle cells are affecting each other that can be useful to better understand conditions such as diabetes and obesity, the second technique was the best option. Overall, a variety of tools were developed that can be used by biologists to create better models of human tissues in the lab as platforms to study human physiology and as media for developing treatments for different diseases.

Afin de résoudre la problématique des substituts osseux faiblement vascularisés, un des challenges majeurs en ingénierie tissulaire osseuse est de favoriser le développement précoce d’une microvascularisation. La reproduction du microenvironnement local et l’organisation cellulaire in situ sont des approches innovantes pour optimiser la formation osseuse. En Biofabrication, la Bioimpression Assistée par Laser (LAB) est une technologie émergente permettant l’impression de cellules et de biomatériaux avec une résolution micrométrique. L’objectif de ce travail était d’étudier l’effet de l’organisation de la pré-vascularisation par LAB sur la régénération osseuse. La station de bioimpression Novalase a été utilisée pour imprimer des motifs de cellules endothéliales sur un « biopaper » constitué de collagène et de cellules souches issues de la papille apicale. Les paramètres d’impression, densités cellulaires et conditions de recouvrement ont été optimisés afin de favoriser la formation d’un réseau microvasculaire avec une architecture définie in vitro. Ce modèle a ensuite été transposé in vivo, grâce à la bioimpression in situ de cellules endothéliales au niveau de défauts osseux critiques chez la souris, afin d’évaluer si la prévascularisation organisée par LAB permettait de promouvoir et contrôler spatialement le processus de régénération osseuse. Les résultats ont montré que la bioimpression permettait d’augmenter la densité de vaisseaux dans les défauts osseux et de favoriser la régénération osseuse. / In order to solve the issue of poorly vascularized bone substitutes, development of a microvasculature into tissue-engineered bone substitutes represents a current challenge. The reproduction of local microenvironment and in situ organization of cells are innovating approaches to optimize bone formation. In Biofabrication, Laser-Assisted Bioprinting (LAB) has emerged as a relevant method to print living cells and biomaterials with micrometric resolution. The aim of this work was to study the effect of prevascularization organized by LAB on bone regeneration. The laser workstation Novalase was used to print patterns of endothelial cells onto a « biopaper » of collagen hydrogel seeded with stem cells from the apical papilla. Printing parameters, cell densities and overlay conditions were optimized to enhance the formation of microvascular networks with a defined architecture in vitro. This model was then transposed in vivo, through in situ bioprinting of endothelial cells into mouse calvarial bone defects of critical size, to investigate if prevascularization organized by LAB can promote and spatially control bone regeneration. The results showed that bioprinting allowed to increase blood vessel density in bone defects and promote bone regeneration.

<p>  </p> <p>Traumatic Brain Injuries (TBI) are a major cause of disability and death in the United States. One of the greatest consequences of the disease is the resulting long-term damage, especially in milder injury cases where the damage is initially subclinical and thus lacking acutely observable manifestations that over time can compound significantly. Among these chronic issues, Alzheimer’s Disease (AD) is one of the most serious. While multiple studies demonstrate an increased likelihood of developing neurodegenerative diseases in response to TBI, the underlying mechanisms remain undefined and no current treatment options are available. Multiple hypotheses have been postulated based on various animal and clinical models, which have contributed a great deal to our current knowledge base and implicated several targets of interest in this pathway (i.g. oxidative stress, inflammation, disruptions in proteostasis). While extremely valuable, these <em>in vivo</em> procedures and analyses are physiologically and ethically complex: there is currently no model capable of separating and visualizing TBI-induced sub-cellular damage in the moments (seconds) immediately following injury, and the subsequent associated long-term changes (AD). In addition, no mechanistic study has been performed to link mechanical-trauma independently with neurodegeneration initiation via protein aggregation. It is clear that additional investigative tools are needed to rectify these intricate issues, and while <em>in vitro </em>methodologies generally offer the type of resolution required, no such model replicates these phenomena. Therefore, we introduce the “TBI-on-a-chip” <em>in vitro </em>concussive model, with a series of concomitant targeted-experiments to address this urgent, currently unmet need. This dissertation work describes the development of our cellular trauma model, featuring a multi-disciplinary approach that provides investigatory opportunities into cellular mechanics, molecular biology, functional alterations (electrophysiology), and morphology, in both primary and secondary injury. Utilizing this model, we directly observe evidence of impact-induced electrical/functional and biochemical consequences, in addition to isolating oxidative stress as a key, contributing component. Taken together, these collective efforts suggest that oxidative stress may be a viable target for both acute and chronic potential therapeutic interventions.</p>

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One of the key challenges in osteochondral tissue engineering is to define specified zones with varying material properties, cell types and biochemical factors supporting locally adjusted differentiation into the osteogenic and chondrogenic lineage, respectively. Herein, extrusion-based core–shell bioprinting is introduced as a potent tool allowing a spatially defined delivery of cell types and differentiation factors TGF-β3 and BMP-2 in separated compartments of hydrogel strands, and, therefore, a local supply of matching factors for chondrocytes and osteoblasts. Ink development was based on blends of alginate and methylcellulose, in combination with varying concentrations of the nanoclay Laponite whose high affinity binding capacity for various molecules was exploited. Release kinetics of model molecules was successfully tuned by Laponite addition. Core–shell bioprinting was proven to generate well-oriented compartments within one strand as monitored by optical coherence tomography in a non-invasive manner. Chondrocytes and osteoblasts were applied each in the shell while the respective differentiation factors (TGF-β3, BMP-2) were provided by a Laponite-supported core serving as central factor depot within the strand, allowing directed differentiation of cells in close contact to the core. Experiments with bi-zonal constructs, comprising an osteogenic and a chondrogenic zone, revealed that the local delivery of the factors from the core reduces effects of these factors on the cells in the other scaffold zone. These observations prove the general suitability of the suggested system for co-differentiation of different cell types within a zonal construct.

La fabrication de tissus biologiques a été largement étudiée pour ses applications dans la recherche, la transplantation d'organes et le dépistage de drogues. Bien que des tissus minces ou avasculaires aient été fabriqués avec succès auparavant, le maintien de la viabilité des tissus épais nécessite la présence d'un réseau capillaire tout au long de la construction pour permettre l'apport de nutriments et l'élimination des déchets cellulaires par le sang. En plus des cellules endothéliales, l'incorporation de types de cellules de soutien dans le réseau capillaire est nécessaire pour favoriser la survie et la maturation. Comparée à d'autres méthodes de biofabrication, la bioimpression est une technologie prometteuse qui permet la fabrication précise de motifs 3D complexes à haute résolution spatiale. Nous avons conçu de nouveau notre procédé technique de bio-impression laser nommé LIST (de l'anglais \textit{laser-induced side transfer}) dans laquelle la bioencre de la suspension cellulaire passe à travers un capillaire horizontal avec un orifice face à l'échafaudage. Lorsque le laser frappe la bioencre, une bulle se forme qui propulse une gouttelette à travers l'orifice. Nous avons mené une étude détaillée pour caractériser cette bio-impression technique et validé sa cytocompatibilité par l'évaluation de la viabilité de HUVECs imprimés grâce à LIST. Nous avons incorporé des fibroblastes et des péricytes dans nos échantillons et observé le recrutement progressif de ces cellules par les structures de type capillaire HUVEC imprimées sur Matrigel. Des images fluorescentes ont été analysées pour quantifier le recrutement de fibroblastes/péricytes au fil du temps. / The fabrication of biological tissues in laboratory settings has been widely investigated for its applications in research, organ transplantation, and drug screening. Although several previous attempts to generate avascular or thin tissues have been successful, there remains the challenge to create thick functional tissues. Maintaining the viability of thick tissues requires the presence of a capillary network throughout the construct to allow the intake of nutrients and the discard of cellular waste through blood. In addition to endothelial cells, the incorporation of supporting cell types is necessary to promote survival, maturation, and acquire in vivo-like functionality. Compared to other biofabrication methods, bioprinting is a promising technology that enables the precise fabrication of complex 3D patterns at high spatial resolution. We have come up with a new configuration of our in-house laser-based bioprinting technique called laser-induced side transfer (LIST) in which the bioink passes through a horizontal glass capillary with an orifice facing the receiving substrate. When the laser beam causes bubble formation in the bioink, a liquid jet exits through the orifice that will eventually form a droplet. We have conducted a detailed study to characterize this bioprinting technique and validated its cytocompatibility through viability assessment of LIST-printed human umbilical vein endothelial cells (HUVECs). In an effort to generate physiological blood vessels, we incorporated fibroblasts and pericytes in our samples and observed the gradual recruitment of these cells by the printed HUVEC capillary-like structures on Matrigel. Fluorescent images were taken and analyzed to quantify the fibroblast/pericyte recruitment over time.