Meniscal tears are the most common intra-articular injury of the knee joint. Due to the avascular zone with limited blood supply, treatment of the injury is a complex process. Today, research on the development of efficient treatments and meniscal replacements is of increasing interest. However, there are few alternatives of meniscal replacements available on the market and research has shown uncertain results in their ability to restore the natural biomechanics of the knee joint or prevent development of osteoarthritis. Furthermore there is no comparable method to evaluate tensile stresses caused by axial compressional load on a whole meniscus replacement. Therefore the possibility of knitted casing to contain, fixate and mechanically stabilize a cell seeded bioprinted gel and develop a methodology to characterize its compressional behaviour was analysed. By interlock knitting with segments of partial knit a 3D crescent-shaped biodegradable casing was produced mimicking the dimension of the medial meniscus. In the casing design, an Artelon® Flexband™ was incorporated functioning both as reinforcement at the peripheral rim and as fixation method. Moreover radial threads were added to the casing design by inclusion of weft inlays in the knitting pattern. In the non-destructive characterization of the compressional behaviour of the prototype, axial compressional forces of 10.82 N and 29.77 N were achieved. However the forces achieved were significantly lower if compared to the high force that is applied to the menisci in the knee joint. Furthermore a high influence of the coefficient of friction of the casing in the axial compressional force was concluded. Nevertheless refinements of the methodology are required to perform evaluation with comparable and reliable results.

Since the late 20th century, breakthroughs in technology have been occurring expeditiously. Indeed, technological innovations have provided the betterment of many aspects of life and ensured humans’ appropriate forms of evolution and civilization. It is safe to claim that medicine has advanced within the past few decades, especially with the upbringing of technological innovations. The world of medicine would not have experienced its recent breakthroughs and profound discoveries without utilizing the available technology. The improvements observed in medicine and technology resulted in better providing of healthcare. Customizing treatments for each patient is now possible. One method of applying customization is through 3D printing of materials such as artificial prosthetics, tissues, and organs. This literature review analyzes 3D printing by stating definitions, assessing its history, discussing its different applications and closing with evaluating future directions. 3D printing first appeared in the late 20th century, and its primary purpose was to design and manufacture products efficiently and accurately. Traditional production of structures involves subtractive manufacturing (carving, cutting, and other methods of reshaping materials) to achieve desired products, whereas 3D printers implement additive manufacturing (a layer-by-layer approach). This provides less time, greater accuracy, and labor-free fabrication of products. Computerized software is one of the essential parts of 3D printing, and functions include designing, scaling, visualizing, controlling production frequency, and many more. In medical applications, the software may require CT scans, cone beam computed tomography, and intraoral scanners (for dental applications). The 3D printing techniques identified in this review are generally applied in oral and maxillofacial procedures—stereolithography, which constructs a product layer-by-layer through curing liquid resin using a UV laser. Digital light projection is a method similar to stereolithography, with a few differences, such as using a UV light instead of a laser and using a liquid crystal display panel. Fused deposition modeling is a technique that melts plastic filaments and extrudes them through a nozzle to form a structure in a layer-by-layer fashion. Selective laser sintering is also similar to stereolithography, where it uses a laser to form an object layer by layer, but the material is a thin layer of plastic powder instead of liquid resin. The power binder printing technique applies droplets onto powdered materials, adhering and forming layers as designed via computerized software. Lastly, computed axial lithography is similar to digital light projection, except the light is projected from many angles at once instead of one layer at a time. The main objectives of this literature review are to investigate each technique, discuss the advantages and disadvantages, and list the commonly applied areas in medicine for each. Also, this review evaluates the current limitations experienced when using 3D printers and suggestions for overcoming them. Some limitations include, but are not limited to, excessive time allocated for producing specific structures, accurate capturing of surgical sites, use of appropriate materials that form printed structures, cost, and deficiencies of reported data. Lastly, this literature review assesses the future projections. The future holds promising breakthroughs in 3D printing technology, including the fabrication of dental stem cells, operating artificial organs, complex vascular tissues, customized artificial alveolar structures for oral and intracranial procedures, and regeneration of periodontal tissues. These projections may occur by overcoming the most reported limitations. Medicine is digitizing rapidly and will continue adapting to the latest technological inventions. The current efforts to advance 3D printing technology will likely positively impact the advancement of many fields, including healthcare, increase chances of positive postoperative outcomes, and potentially combat many health issues society faces today. Professionals across disciplines must come together to further research and educate curriculums to revolve around the innovative technologies to continuing education courses related to 3-D printing technologies.

In most cases new technology emergence does not guarantee overnight success nor is it developed overnight, rather it is a result of industry expertise spanning over several fields of research. 3D bioprinting is an industry that promises revolutionary implications for healthcare and scholars are discussing application areas such as tissue for oncology research, tissue for patient specific drug development and lastly complex organs for implantation. The promises and strides of the industry pose new prospects in healthcare, for instance printing new skin for burn victims & drug development all while being patient specific. The promises of bioprinting are practices that will lead to improved quality of life whether it be in the form of medicine or a new organ. The industry is characterized by fast pace and rapid technological advancements, research has explored these technological advancements and explained them in detail, but information on the market remains scarce. To contribute to filling this gap the thesis explores what the state of the market currently is and what the stage of market development is.  In order to explore how the market has developed and the current state of it a longitudinal case study, in Sweden which is one of the most innovative countries in the world was conducted. Newspaper articles from three news outlets were collected, two of which are Swedens most reputable publishers regarding new technology and business and economic development, the third is an industry specific outlet to biotechnology advancement. This resulted in finding that while the dialog of bioprinting is promising, the degree of readiness remains the same, furthermore it shows that the industry is heavily dependent on external actors to further develop the market and technology. It was also found that there is no shortage in actors spanning across various industries that collaborate with bioprinting firms since not all industries need the full capacity of the technology that is necessary to print complex organ structures. It can be stated that while the bioprinting market is evolving the development of it is in early stages and is far from being established.

The interactions between cells and their microenvironment influence their morphological features and regulate important cellular processes. To understand deleterious neurological disorders such as Parkinson’s disease, there is an immense need to develop efficient in vitro 3D models that can recapitulate complex organs such as the brain. Ultrashort self- assembling peptides offer a revolutionary tool for generating tunable and well-defined 3D in vitro neural tissues capable of recreating complex cellular characteristics, and tissue-level responses. Herein, we describe the use of ultrashort self-assembling peptide-based scaffolds for the development of functional 3D neuronal models including an in vitro model for Parkinson’s disease. Both primary mouse embryonic dopaminergic neurons and human dopaminergic neurons derived from human embryonic stem cells were found biocompatible in our peptide-based models. Using microelectrode arrays, we recorded spontaneous activity in dopaminergic neurons encapsulated within these 3D peptide scaffolds for more than 1 month without a decrease in signal intensity. In addition, we demonstrate a 3D bioprinted model of dopaminergic neurons inspired by the mouse brain using an extrusion-based 3D robotic bioprinting technology. We used our 3D in vitro neuronal models to study the effect of both gabapentin and pregabalin on the development of dopaminergic neurons. Pregabalin and gabapentin are frequently regarded as first-line therapies for a variety of neuropathic pain syndromes, regardless of the underlying cause. Our results showed that both drugs can interfere with the neurogenesis and morphogenesis of ventral midbrain dopaminergic neurons during early brain development. Finally, to gain a better understanding of the influence of cell-cell and cell- matrix interactions on cellular behavior and function in 3D cultured cells within our peptide-based scaffolds compared to the ones cultured in 2D, we studied the metabolic and transcriptomic profiles of 2D and 3D cultured cells. 2D cultured cells exhibited distinct metabolic and transcriptomic profiles compared to the 3D cultured cells. Advancements in the fields of 3D in vitro modeling, 3D bioprinting, and biomaterials are of extreme value for the development of efficient models suitable for investigating disease-specific pathways, aiding the discovery of novel treatments, and promoting tissue regeneration.

Osteoarthritis (OA) is a progressive cartilage degeneration disease with a complex pathologic mechanism. Although OA has devastating effects on patient quality of life and places a significant burden on the healthcare system, no disease-modifying drugs have been found, and surgical treatment options are often unsustainable. 3D bioprinting is a novel field within tissue engineering that focuses on developing biocompatible constructs that can be implanted to replace an organ or tissue. Such constructs have a great potential to become treatments for OA. Understanding cell mobility within hydrogels could play a vital role in advancing the development of biocompatible constructs. However, due to the novelty of bioprinting, limited research on cell mobility within hydrogels is available. Therefore, this project aims to fill the gap in existing research regarding cell mobility within bioprinted constructs with varying mechanical properties. To achieve this goal, green fluorescent protein-tagged mesenchymal stromal cells (MSCs) were developed to assess progenitor cell mobility in bioprinted hydrogel constructs. Constructs were printed with three zones: hydrogel with embedded chondrocytes or MSCs; hydrogel spacer; and chemoattractant. Designed constructs were bioprinted (BioAssemblyBot, Advanced Solutions) using GelMA:HAMA bioinks containing photoinitiator with varying bioink percentages. Cell viability and directional mobility within constructs were assessed by fluorescence viability assay and time-lapse fluorescence microscopy. The protocol to evaluate cell mobility in bioprinted constructs and optimized bioprinting settings for GelMA:HAMA bioinks were gained through this project. Overall, this project allowed us to fill the gap in existing knowledge regarding MSC and chondrocyte mobility in hydrogels and contribute to developing a novel treatment method for OA.

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.

Chondrozyten stellen die zelluläre Komponente von hyalinem Knorpel dar, der die Gelenkflächen diarthrotischer Gelenke bedeckt. Über die perizelluläre Matrix (PZM) sind sie mit der extrazellulären Matrix des Knorpelgewebes, die im Wesentlichen aus Wasser, Kollagen-Typ-II (Koll-II) und Glykosaminoglykan (GAG) gebildet wird, verbunden. Die PZM gilt als wichtiges modulatorisches und protektives Element in der Signal- und Mechanotransduktion sowie für die Homöostase innerhalb des Knorpelgewebes. Degenerative und inflammatorische Prozesse führen zu irreparablen Schäden der Gewebearchitektur und -funktionalität. Die Regenerative Medizin strebt den Ersatz destruierter Gelenkflächen durch mittels Tissue Engineering hergestellten Neoknorpel an. 3D-Bioprinting gilt hier als attraktive Methode, nimmt jedoch über Scherkräfte während des Druckvorgangs auch schädigenden Einfluss auf das Überleben oder die Funktionalität der Zellen. Zielsetzung dieser Arbeit war es, den möglichen protektiven Einfluss der PZM während des Druckvorgangs zu untersuchen. Aus porcinem Frischknorpel isolierte Chondrozyten wurden nach cast bzw. 3D-Bioprinting in Agarose-Biotinte hinsichtlich ihres Überlebens und ihrer Syntheseleistung von knorpelspezifischem Koll-II und GAG untersucht. Chondrozyten ohne PZM wurden mit Chondrozyten verglichen, die nach enzymatischer Isolation noch perizellulär Kollagen-Typ-VI als Marker der PZM aufwiesen. Chondrozyten mit PZM zeigten allgemein eine stärkere Produktion von Koll-II als Chondrozyten ohne PZM. Nach 3D-Bioprinting konnte für Chondrozyten ohne PZM eine signifikant geringere Produktion von GAG nachgewiesen werden als in der cast-Vergleichsgruppe, während dies für Chondrozyten mit PZM nicht gezeigt werden konnte. Der gezeigte protektive Einfluss der PZM gegenüber Scherkräften während des Druckvorgangs eröffnet neue Methoden für das Cartilage Tissue Engineering. Weitere Untersuchungen sind notwendig, um dies zu bestätigen und die Translation in die klinische Forschung ermöglichen. / Chondrocytes are the cellular component of the hyaline cartilage that lines the articular surfaces of diarthrotic joints. They are bound to the extracellular matrix of the cartilage tissue by the pericellular matrix (PCM), which consists mainly of water, collagen type II (coll-II) and glycosaminoglycan (GAG). PCM is considered to be an important modulatory and protective element in signalling, mechanotransduction and homeostasis within cartilage tissue. Degenerative and inflammatory processes cause irreparable damage to tissue architecture and functionality. Regenerative medicine aims to replace damaged joint surfaces with neocartilage produced by tissue engineering. 3D bioprinting is considered to be an attractive method for this purpose, but also has a detrimental effect on the survival or functionality of the cells due to shear forces during the printing process. The aim of this study was to investigate the potential protective effect of PZM during the printing process. Chondrocytes isolated from fresh porcine cartilage were analysed after casting or 3D bioprinting in agarose bioprinting for their survival and their ability to synthesise cartilage-specific Coll-II and GAG. Chondrocytes without PCM were compared with chondrocytes that still had pericellular collagen type VI as a marker of PCM after enzymatic isolation. Chondrocytes with PCM generally showed a higher production of Coll-II than chondrocytes without PCM. After 3D bioprinting, chondrocytes without PCM showed significantly lower GAG production than the control group, whereas chondrocytes with PCM did not. The demonstrated protective effect of PCM against shear forces during the printing process opens up new possibilities for cartilage tissue engineering. Further studies are needed to confirm this and to enable translation into clinical research.

Additive manufacturing (AM) has received much attention from researchers over the past decades because of its diverse applications in various industries. AM is an advanced manufacturing process that facilitates the fabrication of complex geometries represented by computer-aided design (CAD) models. Traditionally, designed parts are fabricated by extruding material layer-by-layer using a tool path planning obtained from slicing programs by using CAD models as an input. Recently, there has been a growing interest in non-planar AM technologies, which offer the ability to fabricate multilayer constructs conforming to freeform surfaces. Non-planar AM processes have been utilized in various applications and involved objects of varying material properties and geometric characteristics. Although the current state of the art suggests AM can provide novel opportunities in conformal manufacturing, several challenges remain to be addressed. The identified challenges in non-planar AM fall into three categories: 1) conformal 3D printing on substrates with complex topography of which CAD model representation is not readily available, 2) understanding the relationship between the tool path planning and the quality of the 3D-printed construct, and 3) conformal 3D printing in the presence of mechanical disturbances. An open-loop non-planar tool path planning algorithm based on point cloud representations of object geometry and a closed-loop non-planar tool path planning algorithm based on position sensing were proposed to address these limitations and enable conformal 3D printing and spatiotemporal 3D sensing on objects of near-arbitrary organic shape. Three complementary studies have been completed towards the goal of improving the conformal tool path planning capabilities in various applications including fabrication of conformal electronics, in situ bioprinting, and spatiotemporal biosensing: i. A non-planar tool path planning algorithm for conformal microextrusion 3D printing based on point cloud data representations of object geometry was presented. Also, new insights into the origin of common conformal 3D printing defects, including tool-surface contact, were provided. The impact and utility of the proposed conformal microextrusion 3D printing process was demonstrated by the fabrication of 3D spiral and Hilbert-curve loop antennas on various non-planar substrates, including wrinkled and folded Kapton films and origami. ii. A new method for closed-loop controlled 3D printing on moving substrates, objects, and unconstrained human anatomy via real-time object position sensing was proposed. Monitoring of the tool position via real-time sensing of nozzle-surface offset using 1D laser displacement sensors enabled conformal 3D printing on moving substrates and objects. The proposed control strategy was demonstrated by microextrusion 3D printing on oscillating substrates and in situ bioprinting on an unconstrained human hand. iii. A reverse engineering-driven collision-free path planning program for automated inspection of macroscale biological specimens, such as tissue-based products and organs, was proposed. The path planning program for impedance-based spatiotemporal biosensing was demonstrated by the characterization of meat and fruit tissues using two impedimetric sensors: a cantilever sensor and a multifunctional fiber sensor. / Doctor of Philosophy / Additive Manufacturing (AM), commonly referred to as 3D printing, is a computer-aided manufacturing process that facilitates the fabrication of personalized and customized models, tissues, devices, and wearables. AM has several advantages over traditional manufacturing processes. For example, directing computer-driven robotics enables control over spatial structure and composition of parts. While 3D printing is typically performed using layer-by-layer planar tool paths generated by slicing programs, non-planar 3D printing is an emerging area that has recently been examined for various post-processing applications. Processes that enable material deposition conforming to complex geometric and freeform objects (e.g., anatomical structures), are central to various industries, including additive manufacturing, electronics manufacturing, and biomanufacturing. In this dissertation, tool path planning methods and real-time control strategies for non-planar 3D printing onto stationary and moving arbitrary surfaces, and various conformal electronics and in situ bioprinting applications will be presented. In addition to the tool path planning methods for 3D printing, a collision-free path planning program will be proposed for the inspection of large tissues and organs. The utility of the proposed method will be demonstrated through electrical impedance-based biosensing of meat and fruit to characterize their compositional and physiochemical properties which are used for quality assessment.

[ES] La ingeniería tisular hepática ofrece una herramienta propicia de cribado toxicológico y modelización de enfermedades, que también podría proporcionar una terapia alternativa al trasplante de hígado para tratar enfermedades hepáticas en fase terminal. Se han propuesto varios modelos celulares para estudiar la fisiología hepática y el desarrollo de enfermedades. Los sistemas in vitro clásicos se basan en cultivos en monocapa de hepatocitos humanos primarios o líneas celulares de hepatoma, aunque en los últimos años se ha fomentado el uso de sistemas más complejos que también tienen en cuenta la organización tridimensional. El objetivo principal de esta Tesis Doctoral es desarrollar un sistema hepático tridimensional que recapitule la organización tridimensional del hígado utilizando hidrogeles y constructos porosos basados en la matriz extracelular hepática, y su aplicación para el estudio y tratamiento de enfermedades hepáticas. Para ello, primero optimizamos la composición utilizando hidrogeles de gelatina y ácido hialurónico, buscando las propiedades mecánicas más parecidas a las del hígado humano. Para determinar la idoneidad de los hidrogeles desarrollados para el cultivo de células hepáticas, se utilizó la reticulación in situ de células HepG2, una línea celular de hepatoma ampliamente utilizada en Hepatología, y se analizó tanto la viabilidad como la funcionalidad de las células. La caracterización mecánica de los hidrogeles y la evaluación de la funcionalidad de las células HepG2, nos llevaron a seleccionar la composición 20-80 gelatina-ácido hialurónico como la óptima para el cultivo de células hepáticas. En segundo lugar, se prepararon constructos porosos de gelatina-ácido hialurónico con porosidad interconectada y se utilizaron para el cultivo de hepatocitos humanos primarios y la evaluación de las funciones hepáticas clave. Los hepatocitos humanos primarios cultivados en constructos porosos de gelatina-ácido hialurónico mostraron una mayor secreción de albúmina y urea, y una mayor capacidad metabólica (niveles de actividad citocromo P450 y UDP-glucuronosiltransferasa) en comparación con los cultivos en monocapa estándar. El trasplante del constructo poroso con hepatocitos humanos produjo una mejora de la función hepática (niveles de transaminasas, necrosis) y mejoró el daño en un modelo de ratón de insuficiencia hepática inducida por acetaminofeno. Además, el estudio in vivo también proporcionó una comprensión mecanicista de la lesión hepática inducida por acetaminofeno y el impacto del trasplante mediante el análisis de la producción de citoquinas y la inducción de estrés oxidativo para encontrar biomarcadores adecuados de la eficacia de la terapia celular. Finalmente, en la última parte de esta tesis doctoral hemos explorado el uso de la bioimpresión como método para aumentar el rendimiento de los sistemas de ensayo que mejor imitan el entorno y la complejidad del tejido hepático. Para transformar los hidrogeles desarrollados en biotintas, se aumentó el peso molecular del ácido hialurónico. A continuación, se estudió la idoneidad de biotintas híbridas de gelatina y ácido hialurónico mediante la determinación de la viscosidad, la uniformidad y el factor de poros. La bioimpresión de células HepG2 demostró la idoneidad de las biotintas seleccionadas para el cultivo de células hepáticas y la mejora de su producción, lo que sugiere la conveniencia de automatizar y escalar la fabricación de hidrogeles. / [CA] L'enginyeria tissular hepàtica ofereix una ferramenta propícia de cribratge toxicològic i modelització de malalties, que també podria proporcionar una teràpia alternativa al trasplantament de fetge per a tractar malalties hepàtiques en fase terminal. S'han proposat diversos models cel·lulars per a estudiar la fisiologia hepàtica i el desenvolupament de malalties. Els sistemes in vitro clàssics es basen en cultius en monocapa d'hepatòcits humans primaris o línies cel·lulars de hepatoma, encara que en els últims anys s'ha fomentat l'ús de sistemes més complexos que també tenen en compte l'organització tridimensional. L'objectiu principal d'esta Tesi Doctoral és desenvolupar un sistema hepàtic tridimensional que recapitula l'organització tridimensional del fetge utilitzant hidrogels i bastides basades en la matriu extracel·lular hepàtica, i la seua aplicació per a l'estudi i tractament de malalties hepàtiques. Per a això, primer optimitzem la composició utilitzant hidrogels de gelatina i àcid hialurònic, buscant les propietats mecàniques més semblants a les del fetge humà. Per a determinar la idoneïtat dels hidrogels desenvolupats per al cultiu de cèl·lules hepàtiques, es va utilitzar la reticulació in situ de cèl·lules HepG2, una línia cel·lular de hepatoma àmpliament utilitzada en hepatologia, i es va analitzar tant la viabilitat com la funcionalitat de les cèl·lules. La caracterització mecànica dels hidrogels i l'avaluació de la funcionalitat de les cèl·lules HepG2, ens van portar a seleccionar la composició 20-80 gelatina-àcid hialurònic com l'òptima per al cultiu de cèl·lules hepàtiques. En segon lloc, es van preparar bastides de gelatina-àcid hialurònic amb porositat interconnectada i es van utilitzar per al cultiu d'hepatòcits humans primaris i l'avaluació de les funcions hepàtiques clau. Els hepatòcits humans primaris cultivats en bastides de gelatina-àcid hialurònic van mostrar una major secreció d'albúmina i urea, i una major capacitat metabòlica (nivells d'activitat citocrom P450 i UDP-glucuronosiltransferasa) en comparació amb els cultius en monocapa estàndard. El trasplantament de la bastida amb hepatòcits humans va produir una millora de la funció hepàtica (nivells de transaminases, necrosis) i va millorar el mal en un model de ratolí d'insuficiència hepàtica induïda per acetaminofèn. A més, l'estudi in vivo també va proporcionar una comprensió mecanicista de la lesió hepàtica induïda per acetaminofèn i l'impacte del trasplantament mitjançant l'anàlisi de la producció de citocines i la inducció d'estrès oxidatiu per a trobar biomarcadors adequats de l'eficàcia de la teràpia cel·lular. Finalment, en l'última part d'esta tesi doctoral hem explorat l'ús de la bioimpresió com a mètode per a augmentar el rendiment dels sistemes d'assaig que millor imiten l'entorn i la complexitat del teixit hepàtic. Per a transformar els hidrogels desenvolupats en bioimpresions, es va augmentar el pes molecular de l'àcid hialurònic. A continuació, es va estudiar la idoneïtat de la gelatina híbrida i l'àcid hialurònic com biotintes mitjançant la determinació de la viscositat, la uniformitat i el factor de porus. La bioimpresió de cèl·lules HepG2 va demostrar la idoneïtat de les biotintes seleccionades per al cultiu de cèl·lules hepàtiques i la millora del seu rendiment, la qual cosa suggereix la conveniència d'automatitzar i ampliar la fabricació d'hidrogels. / [EN] Liver tissue engineering offers an advantageous toxicological screening and disease-modelling tool and could also provide an alternative therapy to liver transplantation to treat end-stage liver diseases. Several cell-based models have been proposed to study liver physiology and disease development. The classical in vitro systems rely on monolayer cultures of primary human hepatocytes or hepatoma cell lines, although the use of more complex systems that also consider the three-dimensional organization has been encouraged in the last years. The main objective of this PhD Thesis is to develop a three-dimensional hepatic system that recapitulates the three-dimensional organization of the liver using hydrogels and scaffolds based on liver's extracellular matrix composition, and their application for the study and treatment of liver diseases. To this purpose, we first optimized the composition using gelatin-hyaluronic acid hydrogels, looking for the mechanical properties closer to the human liver. In order to determine the suitability of the developed hydrogels for hepatic cell culture, in situ crosslinking of HepG2 cells, a hepatoma cell line widely used in Hepatology, was used and both viability and functionality of the cells were analyzed. Mechanical characterization of hydrogels and the evaluation of HepG2 cells functionality, led us to select the 20-80 gelatin-hyaluronic acid composition as the optimal for hepatic cell culture. Secondly, gelatin-hyaluronic acid scaffolds with interconnected porosity were prepared and used for the culture of primary human hepatocytes and the evaluation of key hepatic functions. Primary human hepatocytes cultured in gelatin-hyaluronic acid scaffolds exhibited increased albumin and urea secretion and metabolic capacity (cytochrome P450 and UDP-glucuronosyltransferase activity levels) compared to standard monolayer cultures. The transplant of the scaffold containing human hepatocytes led to an improvement in liver function (transaminase levels, necrosis) and ameliorated damage in a mouse model of acetaminophen-induced liver failure. Additionally, the in vivo study also provided a mechanistic understanding of acetaminophen-induced liver injury and the impact of transplantation by analyzing cytokine production and oxidative stress induction to find suitable biomarkers of cell therapy's effectiveness. Finally, in the last part of this doctoral thesis we have explored the use of bioprinting as a method for increasing the throughput of the test systems that best mimic the environment and complexity of liver tissue. To transform the developed hydrogels into bioinks, the molecular weight of hyaluronic acid was increased. Then, the suitability of hybrid gelatin and hyaluronic acid as bioinks was studied by determining viscosity, uniformity, and pore factor. Bioprinting of HepG2 cells demonstrated the fitness of the selected bioinks for culturing hepatic cells and improving their performance, suggesting the suitability for automatising and scaling-up hydrogel's manufacturing. / Rodríguez Fernández, J. (2024). Biomimetic injectable hydrogels of gelatin and hyaluronic acid for hepatic cell culture [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/207128

Les maladies cardiovasculaires sont une des causes de mortalités les plus élevées mondialement. Parmi celles-ci, on retrouve l’infarctus du myocarde, qui n’a pour traitement que la transplantation cardiaque. Or, dû à la faible quantité de donneur, une solution alternative est recherchée. De ce fait, l’ingénierie tissulaire permet le développement de tissus et d’implants thérapeutiques tels les patchs cardiaques, qui peuvent être bioimprimés. Or, une des limitations actuelles de l’utilisation d’une telle stratégie est la vascularisation de tissu bioimprimés. Dans cette étude, la bioimpression 3D a été utilisée afin de bioimprimer des patchs perfusables de gélatine méthacrylate (GelMA) à utiliser potentiellement pour le tissu cardiaque. Il a été possible de développer une bioencre pouvant être utilisée pour une application dans le tissu cardiaque, d’évaluer l’imprimabilité de l’encre et de bioimprimer de patchs standards et perfusables. Pour ce faire, GelMA a été synthétisé et les propriétés mécaniques ont été évaluées pour finalement sélectionner une encre de 10 % GelMA, ayant un module de Young approprié pour le tissu cardiaque, de 23,7±5,1 kPa. Par la suite, les processus d’impression, standard et coaxial, de patchs standards et perfusables ont pu être optimisés. Finalement, des patchs perfusables de GelMA 10% et gélatine 2% ont pu être imprimés avec une viabilité cellulaire élevée, jusqu’à 79,7±8,7 % et 83,5±5,7 % obtenue aux jours 1 et 7 de culture respectivement, avec des fibroblastes 3T3. La présence de canaux vides et la perfusabilité des patchs démontrent le potentiel de cette méthode pour éventuellement bioimprimer des patchs cardiaques vascularisés épais. / Cardiovascular diseases are a leading cause of death worldwide. Myocardial infarction captures a significant segment of this population, and the end-stage myocardial infarction can only be treated by heart transplantation. However, due to the scarcity donors, tissue engineering has been considered as an alternative solution. Tissue engineering allows the development of tissues and therapeutic implants such as cardiac patches. However, one of the main hurdles in the use of such a strategy is the vascularization of bioprinted tissue. In this study, 3D bioprinting was used to bioprint perfusable gelatin methacrylate (GelMA) patches for a potential use in cardiac tissue. This work consists in the development of a bioink that can be used for the cardiac tissue, the evaluation of the printability of the ink, and the final bioprinting of standard and perfusable patches. For this purpose, GelMA was synthesized and a final concentration of 10 % was selected as it showed an appropriate Young's modulus for cardiac tissue, of 23.7±5.1 kPa, while maintaining high biocompatibility. Subsequently, the printing process of standard and perfusable patches could be optimized with the use of GelMA and gelatin inks. Finally, 10% GelMA and 2% gelatin vascularized patches could be printed with high cell viability, of up to 79,7±8,7 % and 83,5±5,7 % on days 1 and 7 of culture respectively for 3T3 fibroblasts. Additionally, the presence of hollow channels of the perfusable patches demonstrates the potential of this method to be eventually applied to the bioprinting of thick vascularized cardiac patches.