3D microfabrication of biological machines

The burgeoning field of additive manufacturing, or “3D printing”, centers on the idea of creating three-dimensional objects from digital models. While conventional manufacturing approaches rely on modifying a base material via subtractive processes such as drilling or cutting, 3D printing creates three-dimensional objects through successive deposition of two- dimensional layers. By enabling rapid fabrication of complex objects, 3D printing is revolutionizing the fields of engineering design and manufacturing. This thesis details the development of a projection-based stereolithographic 3D printing apparatus capable of high- resolution patterning of living cells and cell signals dispersed in an absorbent hydrogel polymer matrix in vitro. This novel enabling technology can be used to create model cellular systems that lead to a quantitative understanding of the way cells sense, process, and respond to signals in their environment. The ability to pattern cells and instructive biomaterials into complex 3D patterns has many applications in the field of tissue engineering, or “reverse engineering” of cellular systems that replicate the structure and function of native tissue. While the goal of reverse engineering native tissue is promising for medical applications, this idea of building with biological components concurrently brings about a new discipline: “forward engineering” of biological machines and systems. In addition to rebuilding existing systems with cells, this technology enables the design and forward engineering of novel systems that harness the innate dynamic abilities of cells to self-organize, self-heal, and self-replicate in response to environmental cues. This thesis details the development of skeletal and cardiac muscle based bioactuators that can sense external electrical and optical signals and demonstrate controlled locomotive behavior in response to them. Such machines, which can sense, process, and respond to signals in a dynamic environment, have a myriad array of applications including toxin neutralization and high throughput drug testing in vitro and drug delivery and programmable tissue engineered implants in vivo. A synthesis of two fields, 3D printing and tissue engineering, has brought about a new discipline: using microfabrication technologies to forward engineer biological machines and systems capable of complex functional behavior. By introducing a new set of “building blocks” into the engineer’s toolbox, this new era of design and manufacturing promises to open up a field of research that will redefine our world

Biofunctional hydrogels for skeletal muscle constructs

Skeletal muscle tissue damage costs the US government hundreds of billions of dollars annually. Meanwhile, there is great potential to use skeletal muscle as a scalable actuator system, covering wide length scales, frequencies, and force regimes. Hence, the interest in soft robotics and regenerative medicine methods to engineer skeletal muscle has increased in recent years. The challenges to generate a functional muscle strip are typical to those of tissue engineering, where common issues such as cell source, material scaffold, bioreactor method or configuration play key roles. Specifically, it is important to translate the existing body of myogenesis knowledge into engineering muscle constructs by examining the impact of the cell microenvironment on growth, alignment, fusion, and differentiation of skeletal muscle cells. The main motivation behind this thesis was to generate a contractile 3D skeletal muscle construct utilizing organized biochemical and physical cues to guide muscle cell differentiation and maturation. Such a construct is expected to play an important role in medical applications and the development of soft robotics. To do this, 3D, swollen hydrogels were chosen to provide tailorable platforms that support cellular activities to similar extents as native matrices. For this work, we utilized an engineered bio-functionalized poly(ethylene glycol)-(PEG)-hydrogel with maleimide (MAL) cross-linking reaction chemistry that gels rapidly with high reaction efficiency under cytocompatible reaction conditions. PEG alone has been shown to have low protein adsorption, a minimal inflammatory profile, well established chemistry, and a long history of safety in vivo. The PEG-MAL system in particular allows “plug-and-play” design variation, control over polymerization time, and small degradation products. To develop an effective soft biomaterial for the development of an aligned, functional muscle construct, we (i) screened hydrogel properties for differentiation, (ii) recreated alignment of skeletal muscle cells, (iii) determined effective generated force upon action of an external agonist. The impact of this study in generating a controllable force actuator will be significant in the construction of biological machines. Concomitantly, this study will provide a unique regenerative solution for skeletal muscle tissue repair and regeneration.Ph.D

Exploration of torsional actuation and twist to writhe transition in nanostructured hydrogels

Torsional artificial muscles are a branch of actuators that react to a stimulus by rotating. This rotation is driven by a change in volume and mechanical properties such as modulus and was shown to be extremely large in the case of twisted fibers due to their helical geometry. The following thesis introduces a new method of fabrication of nanofiber yarns and nanocomposites with the aim of making hydrogel torsional catch actuators that combine responsiveness to pH changes and a high torsional output as well as a systematic approach to the modeling of their behavior using the single helix theory

Biological building blocks for 3D printed cellular systems

Advancements in the fields of tissue engineering, biomaterials, additive manufacturing, synthetic and systems biology, data acquisition, and nanotechnology have provided 21st-century biomedical engineers with an extensive toolbox of techniques, materials, and resources. These “building blocks” could include biological materials (such as cells, tissues, and proteins), biomaterials (bio-inert, -instructive, -compatible, or -degradable), soluble factors (growth factors or small molecules), and external signals (electrical, chemical, or mechanical). “Forward engineering” attempts to integrate these building blocks in different ways to yield novel systems and machines that, by promoting new relationships and interactions among their individual components, are greater than the sum of their parts. Drawing from an extensive reserve of parts and specifications, these bio-integrated forward-engineered cellular machines and systems could acquire the ability to sense, process signals, and produce force, and could also contain a countless array of applications in drug screening and delivery, programmable tissue engineering, and biomimetic machine design. An intuitive demonstration of a biological machine is one that can produce motion in response to controllable external signaling. In contrast to traditional machines that use external energy to produce an output, muscle cells can be fueled by glucose and other biomolecules. While cardiac cell driven biological actuators have been demonstrated, the requirements of these machines to respond to stimuli and exhibit controlled movement merit the use of skeletal muscle, the primary generator of actuation in animals, as a contractile power source. Here, we report the development of 3D printed hydrogel “bio-bots” powered by the actuation of an engineered mammalian skeletal muscle strip to result in net locomotion of the bio-bot upon applied electrical stimulation. The muscle strips were composed of differentiated skeletal myofibers in a matrix of natural proteins, including fibrin, that provide physical support and cues to the cells as an engineered basement membrane. The hierarchical organization, modularity, and scalable nature of mature skeletal muscle fibers (which can be combined in parallel to increase force production, for example), lends itself to “building with biology.” Few systems have shown net movement from an autonomous, freestanding biological machine composed of skeletal muscle, and even fewer have attempted to incorporate multiple cell types for greater functionality. Modular and flexible platforms for fabrication of such multi-cellular modules and their characterization have been lacking. We also present a modular heterotypic cellular system, made up of multi-layered tissue rings containing integrated skeletal muscle and motor neurons embedded in an extracellular matrix. Site-specific innervation of a group of muscle fibers in the multi-layered tissue rings allowed for muscle contraction via chemical stimulation of motor neurons with glutamate, a major excitatory mammalian neurotransmitter, with the frequency of contraction increasing with glutamate concentration. The addition of the nicotinic receptor antagonist tubocurarine chloride halted the contractions, indicating that muscle contraction was motor neuron-induced. We also present a thorough characterization and optimization of a co-culture system that harnesses the potential of engineered skeletal muscle tissue as the actuating component in a biological machine through the incorporation of motor neurons, and creates an environment that is amenable to both cell types and prime for functional neuromuscular formation. With a bio-fabricated system permitting controllable mechanical and geometric attributes on a range of length scales, our novel engineered cellular system can be utilized for easier integration of other modular “building blocks” in living cellular and biological machines. We are poised to design the next generation of complex biological machines with controllable function, specific life expectancy, and greater consistency. In the future, we envision that this system can be used for applications beyond bio-robotics and muscular actuators; as a functioning heterotypic co-culture, the muscle- neuron arrangement is also a highly relevant machine for the study of neuromuscular diseases and related drug toxicity studies. These results could prove useful for the study of disease-specific models, treatments of myopathies such as muscular dystrophy, and tissue engineering applications

Principles for the design of multicellular engineered living systems

Remarkable progress in bioengineering over the past two decades has enabled the formulation of fundamental design principles for a variety of medical and non-medical applications. These advancements have laid the foundation for building multicellular engineered living systems (M-CELS) from biological parts, forming functional modules integrated into living machines. These cognizant design principles for living systems encompass novel genetic circuit manipulation, self-assembly, cell–cell/matrix communication, and artificial tissues/organs enabled through systems biology, bioinformatics, computational biology, genetic engineering, and microfluidics. Here, we introduce design principles and a blueprint for forward production of robust and standardized M-CELS, which may undergo variable reiterations through the classic design-build-test-debug cycle. This Review provides practical and theoretical frameworks to forward-design, control, and optimize novel M-CELS. Potential applications include biopharmaceuticals, bioreactor factories, biofuels, environmental bioremediation, cellular computing, biohybrid digital technology, and experimental investigations into mechanisms of multicellular organisms normally hidden inside the “black box” of living cells

3D printed muscle-powered bio-bots

Complex biological systems sense, process, and respond to a range of environmental signals in real-time. The ability of such systems to adapt their functional response to dynamic external signals motivates the use of biological materials in other engineering applications. Recent advances in 3D printing have enabled the manufacture of complex structures from biological materials. We have developed a projection stereolithographic 3D printing apparatus capable of patterning cells and biocompatible polymers at physiologically relevant length scales, on the order of single cells. This enables reverse engineering in vitro model systems that recreate the structure and function of native tissue for applications ranging from high-throughput drug testing to regenerative medicine. While reverse engineering native tissues and organs has important implications in biomedical engineering, the ability to “build with biology” presents the next generation of engineers with both a unique design challenge and opportunity. Specifically, we now have the ability to forward engineer bio-hybrid machines and robots (bio-bots) that harness the adaptive functionalities of biological materials to achieve more complex functional behaviors than machines composed of synthetic materials alone. Perhaps the most intuitive demonstration of a “living machine” is a system that can generate force and produce motion. To that end, we have designed and 3D printed locomotive bio-bots, powered by external electrical and optical stimuli. In addition to being the first demonstrations of untethered locomotion in skeletal musclepowered soft robots, these bio-hybrid machines have served as meso-scale models for studying tissue self-assembly, maturation, damage, remodeling, and healing in vitro. Bio-hybrid machines that can dynamically sense and adaptively respond to a range of environmental signals have broad applicability in healthcare applications such as dynamic implants or targeted drug delivery. Advanced research in exoskeletons and hyper-natural functionality could even extend the useful application of such machines to national defense and environmental cleanup. We have developed a modular skeletal muscle bioactuator that can serve as a fundamental building block for such machines, setting the stage for future generations of bio-hybrid machines that can self-assemble, self-heal, and perhaps even self-replicate to target grand engineering challenges. Furthermore, we present a robust optimized protocol for manufacturing 3D printed muscle-powered biological machines, and a mechanism to incorporate biological “building blocks” into the toolbox of the next generation of engineers and scientists

Biohybrid swimmers at low Reynolds number powered by tissue-engineered neuromuscular units

Biohybrid machines are engineered systems which are built by integrating biological cells with synthetic materials and components. Development of biohybrid machines utilizes the classical engineering modalities of design, modeling, prototype fabrication, testing, and iteration, but also draws from a toolbox that includes biological cells and materials. This enables a range of exciting possibilities since biological systems can develop via self-organization, function autonomously, and monitor and adapt to their environments. Pioneering studies on biohybrid machines have demonstrated the development of devices powered by muscle cells, capable of locomotion, pumping, and micromanipulation. A currently emerging frontier in the field is the integration of neuronal control. A wide range of complex animal behaviors are orchestrated by the nervous system which interfaces the body with the environment through sensing, information processing, and coordinating motor activity. Hence, the integration of neurons may enable the development of autonomous biohybrid machines capable of higher-level functionalities such as sensing, memory, and adaptation. The focus of this dissertation is on the implementation of neuronal actuation in muscle powered biohybrid machines. Firstly, we develop an experimental bioactuator platform to study the in vitro development of neuromuscular units. Engineered skeletal muscle tissues, anchored to compliant pillars, are co-cultured on the platform with optogenetic stem cell-derived neuronal clusters containing motor neurons. The motor neurons extend axons and innervate the muscle fibers, forming functional neuromuscular units. Our study illustrates several outcomes of synergistic interactions between the muscles and neurons. Muscles co-cultured with neurons exhibit significantly higher contraction force and cytoskeletal maturation compared to muscles cultured alone. Neurons self-organize into networks which generate synchronous bursting patterns, the development of which is facilitated by muscle-secreted soluble factors. Next, we implement our neuron-muscle co-culture approach on a free-standing compliant scaffold containing slender flagella, to demonstrate the first example of a biohybrid swimmer powered by neuromuscular units. Optogenetic stimulation of motor neurons evokes periodic muscle contractions, and the swimmer is driven by the resulting time-irreversible deformations of the flagella, a common mechanism of propulsion at low Reynolds number. Lastly, we investigate potential design strategies for improving swimming performance, assisted by analytical and computational models. Our models predict that the swimming speed of our initial prototype can be improved by up to two orders of magnitude by redesigning the swimmer scaffold to reduce drag and increase actuation amplitude

Hybrid bio-robotics: from the nanoscale to the macroscale

[eng] Hybrid bio-robotics is a discipline that aims at integrating biological entities with synthetic materials to incorporate features from biological systems that have been optimized through millions of years of evolution and are difficult to replicate in current robotic systems. We can find examples of this integration at the nanoscale, in the field of catalytic nano- and micromotors, which are particles able to self-propel due to catalytic reactions happening in their surface. By using enzymes, these nanomotors can achieve motion in a biocompatible manner, finding their main applications in active drug delivery. At the microscale, we can find single-cell bio-swimmers that use the motion capabilities of organisms like bacteria or spermatozoa to transport microparticles or microtubes for targeted therapeutics or bio-film removal. At the macroscale, cardiac or skeletal muscle tissue are used to power small robotic devices that can perform simple actions like crawling, swimming, or gripping, due to the contractions of the muscle cells. This dissertation covers several aspects of these kinds of devices from the nanoscale to the macro-scale, focusing on enzymatically propelled nano- and micromotors and skeletal muscle tissue bio-actuators and bio-robots. On the field of enzymatic nanomotors, there is a need for a better description of their dynamics that, consequently, might help understand their motion mechanisms. Here, we focus on several examples of nano- and micromotors that show complex dynamics and we propose different strategies to analyze their motion. We develop a theoretical framework for the particular case of enzymatic motors with exponentially decreasing speed, which break the assumptions of constant speed of current methods of analysis and need different strategies to characterize their motion. Finally, we consider the case of enzymatic nanomotors moving in complex biological matrices, such as hyaluronic acid, and we study their interactions and the effects of the catalytic reaction using dynamic light scattering, showing that nanomotors with negative surface charge and urease-powered motion present enhanced parameters of diffusion in hyaluronic acid. Moving towards muscle-based robotics, we investigate the application of 3D bioprinting for the bioengineering of skeletal muscle tissue. We demonstrate that this technique can yield well-aligned and functional muscle fibers that can be stimulated with electric pulses. Moreover, we develop and apply a novel co-axial approach to obtain thin and individual muscle fibers that resemble the bundle-like organization of native skeletal muscle tissue. We further exploit the versatility of this technique to print several types of materials in the same process and we fabricate bio-actuators based on skeletal muscle tissue with two soft posts. Due to the deflection of these cantilevers when the tissue contracts upon stimulation, we can measure the generated forces, therefore obtaining a force measurement platform that could be useful for muscle development studies or drug testing. With these applications in mind, we study the adaptability of muscle tissue after applying various exercise protocols based on different stimulation frequencies and different post stiffness, finding an increase of the force generation, especially at medium frequencies, that resembles the response of native tissue. Moreover, we adapt the force measurement platform to be used with human-derived myoblasts and we bioengineer two models of young and aged muscle tissue that could be used for drug testing purposes. As a proof of concept, we analyze the effects of a cosmetic peptide ingredient under development, focusing on the kinematics of high stimulation contractions. Finally, we present the fabrication of a muscle-based bio-robot able to swim by inertial strokes in a liquid interface and a nanocomposite-laden bio-robot that can crawl on a surface. The first bio-robot is thoroughly characterized through mechanical simulations, allowing us to optimize the skeleton, based on a serpentine or spring-like structure. Moreover, we compare the motion of symmetric and asymmetric designs, demonstrating that, although symmetric bio-robots can achieve some motion due to spontaneous symmetry breaking during its self-assembly, asymmetric bio-robots are faster and more consistent in their directionality. The nanocomposite-laden crawling bio-robot consisted of embedded piezoelectric boron nitride nanotubes that improved the differentiation of the muscle tissue due to a feedback loop of piezoelectric effect activated by the same spontaneous contractions of the tissue. We find that bio-robots with those nanocomposites achieve faster motion and stronger force outputs, demonstrating the beneficial effects in their differentiation. This research presented in this thesis contributes to the development of the field of bio-hybrid robotic devices. On enzymatically propelled nano- and micromotors, the novel theoretical framework and the results regarding the interaction of nanomotors with complex media might offer useful fundamental knowledge for future biomedical applications of these systems. The bioengineering approaches developed to fabricate murine- or human-based bio-actuators might find applications in drug screening or to model heterogeneous muscle diseases in biomedicine using the patient’s own cells. Finally, the fabrication of bio-hybrid swimmers and nanocomposite crawlers will help understand and improve the swimming motion of these devices, as well as pave the way towards the use of nanocomposite to enhance the performance of future actuators.[spa] La bio-robótica híbrida es una disciplina cuyo objetivo es la integración de entidades biológicas con materiales sintéticos para superar los desafíos existentes en el campo de la robótica blanda, incorporando características de los sistemas biológicos que han sido optimizadas durante millones de años de evolución natural y no son fáciles de reproducir artificialmente. Esta tesis cubre varios aspectos de este tipo de dispositivos desde la nanoescala a la macroescala, enfocándose en nano- y micromotores propulsados enzimáticamente y bio-actuadores y bio-robots basados en tejido muscular esquelético. En el campo de nanomotores enzimáticos, existe la necesidad de encontrar mejores modelos que puedan describir la dinámica de su movimiento para llegar a entender sus mecanismos de propulsión subyacentes. Aquí, nos enfocamos en diversos ejemplos de nano- y micromotores que muestran dinámicas de movimiento complejas y proponemos diferentes estrategias que se pueden utilizar para analizar y caracterizar este movimiento. Moviéndonos hacia robots basados en células musculares, investigamos la aplicación de la técnica de bioimpresión en 3D para la biofabricación de músculo esquelético. Demostramos que esta técnica puede producir fibras musculares funcionales y bien alineadas que puede ser estimuladas y contraerse con pulsos eléctricos. Investigamos la versatilidad de esta técnica para imprimir varios tipos de materiales en el mismo proceso y fabricamos bio-actuadores basados en músculo esquelético. Debido a los movimientos de unos postes gracias a las contracciones musculares, podemos obtener medidas de la fuerza ejercida, obteniendo una plataforma de medición de fuerzas que podría ser de utilidad para estudios sobre el desarrollo del músculo o para testeo de fármacos. Finalmente, presentamos la fabricación de un bio-robot basado en músculo esquelético capaz de nadar en la superficie de un líquido y un bio-robot con nanocompuestos incrustados que puede arrastrarse por una superficie sólida. El primer de ellos es minuciosamente caracterizado a través de simulaciones mecánicas, permitiéndonos optimizar su esqueleto, basado en una estructura tipo serpentina o muelle. El segundo bio-robot contiene nanotubos piezoeléctricos incrustados en su tejido, los cuales ayudan en la diferenciación del músculo debido a una retroalimentación basada en su efecto piezoeléctrico y activada por las contracciones espontáneas del tejido. Mostramos que estos bio-robots pueden generar un movimiento más rápido y una mayor generación de fuerza, demostrando los efectos beneficiales en la diferenciación del tejido

POLYMERIC IMPULSIVE ACTUATION MECHANISMS: DEVELOPMENT, CHARACTERIZATION, AND MODELING

Recent advances in the field of biomedical and life-sciences are increasingly demanding more life-like actuation with higher degrees of freedom in motion at small scales. Many researchers have developed various solutions to satisfy these emerging requirements. In many cases, new solutions are made possible with the development of novel polymeric actuators. Advances in polymeric actuation not only addressed problems concerning low degree of freedom in motion, large system size, and bio-incompatibility associated with conventional actuators, but also led to the discovery of novel applications, which were previously unattainable with conventional engineered systems. This dissertation focuses on developing novel actuation mechanisms for soft polymeric gel systems with easily adjustable mechanochemical properties and applicability to various environmental conditions. Inspired by stunning examples in nature which exhibit extremely fast motion in a repeatable manner, termed impulsive motion, we have developed polymeric gel actuators applicable for small-scale, self-contained impulsive systems. In particular, we focused on the effect of geometry and the mechanics of surface-mediated stresses on the dynamic shape-change of polymer gel actuators. We found new opportunities from observation of transient deformations which occur during swelling, or deswelling, of asymmetric gels. We described the development of time-dependent three-dimensional deformation mechanism (4D fabrication) by the utilization of transient inhomogeneous swelling state of the asymmetric polymer gel. We discussed the mechanism and the application of the new deformations mechanism for the development of a novel functionality: chemical gradient sensor. In addition, we developed a high-rate and large-strain reversible actuation mechanism for sub-micrometer scale polymeric gel actuators by utilizing balanced effects of two surface-mediated phenomena, surface diffusion and interfacial-tension, and elasticity of soft and small-scale hydrogels. These new findings were harnessed for developing autonomously controlled power amplified polymeric gel devices. Utilizing deswelling induced transient deformation of gel, we developed design principles for generating meta-stable structures and inducing self-regulating transition forces for repeated snap-through buckling transition of polymeric gel devices. In parallel, we deconvoluted the effect of material properties and geometry on dynamic deformations by establishing simulation models and conducting analyses on the performances of actual synthetic systems. The systematic approach will serve to broaden the application spectrum and manufacturing possibilities of polymeric actuator systems

Carboxymethyl cellulose-based cryogels as scaffolds for pancreatic and skeletal muscle tissue engineering

[eng] Diabetes incidence highly increased in the last years. According to IDF (International Diabetes Federation), 463 million people suffered this disease in 2019. The estimations of diabetic people highly increase in the upcoming years, rising approximately to 700 million diabetic patients in 2045 [1]. Type 2 diabetes (T2D) is the most common type of diabetes, representing 90% of diabetic patients. It occurs when the body becomes resistant to insulin. Body insulin resistance confirms that T2D is not only a pancreatic disease, as there are many other tissues involved, like liver, adipose tissue, or skeletal muscle. This last has a significant implication in glucose-insulin homeostasis as it is one of the main glucose-consuming organs in the body. Nowadays, to study how two tissues crosstalk between them, animal testing is the gold standard. However, the unmatching physiological behaviors compared to humans, the variability between different animals, ethical dilemmas, and the need to go for more personalized medicine activates the search for other suitable alternatives. At this point, Organs-on-a-chip appeared as a valid alternative. Organs-on-a-chip (OOC) are 3D bioengineered microfluidic cell culture platforms to simulate microphysological environments of an organ or its specific functions. Nowadays, to engineer the tissues for OOC applications, encapsulating cells inside hydrogels is the most common technique. Its beneficial properties include high water content, mechanical adjustability, and moldability to generate the desired architectures [2]. However, its small porosity limits nutrient and oxygen diffusion through it [3]. This problem is a significant limitation when pancreatic islets are encapsulated inside hydrogels due to their size (~100 μm of diameter). Pancreatic islets are cell aggregations composed of many different cells as insulin-secreting cells (Beta-cells) or glucagon-secreting cells (alpha-cells). Similarly, skeletal muscle tissue is generally encapsulated in small bundles. Skeletal muscle is a highly aligned and multinucleated tissue formed from the fusion of single cells, called myoblasts, into multinucleated cells, called myotubes. Cryogels have been proposed as a valid alternative to overcome these limitations. Cryogels are fabricated by crosslinking a prepolymer solution at sub-zero temperatures, so while the material crosslinks, water freezes, generating the desired micropore architecture. After thawing, cryogels are sponge-like scaffolds with microporous structure, high interconnected porosity, high diffusivity, fine-tuned properties, and desired internal pore architecture. This thesis developed two cryogel scaffolds made of gelatin and carboxymethylcellulose with different pore architectures to engineer pancreatic and skeletal muscle tissues. Here, we proved that the achieved pore architecture fits with the prerequisites to engineer each tissue. Moreover, the mechanical and physical properties of each scaffold highly resemble the 3D microenvironment of each tissue. In pancreatic tissue, we generate a random pore cryogel to aggregate beta-cells to form pseudoislets. We proved that these engineered pseudoislets are viable, functional responding correctly to the glucose and improving insulin response compared to monolayer results. In the skeletal muscle approach, we could develop a highly aligned pore architecture to prompt cell alignment and cell fusion. Moreover, we incorporate carbon nanotubes to enhance the electrical conductivity of the scaffold, so by applying electrical pulse stimulation, we could improve the early steps of the myogenic maturation.[cat] La incidència de la diabetis ha augmentat considerablement en els últims anys. Segons l’IDF (International Diabetes Federation), al 2019 hi havia 463 milions de persones que patien diabetis i les estimacions estimen un augment considerable de casos, arribant als 700 milions de persones diabètiques cap al 2045 [1]. Entre els diferents tipus de diabetis, la diabetis tipus 2, és la que té major incidència en la població, corresponent al 90% dels casos de pacients amb diabetis. Aquest tipus de diabetis, succeeix quan el cos es torna resistent a la insulina. Aquesta resistència a la insulina per part dels teixits perifèrics ens prova que la diabetis no és només una malaltia del pàncreas, sinó que hi ha altres teixits relacionats, com el fetge, el teixit adipós o el múscul esquelètic. Aquest últim té un factor molt rellevant en la homeòstasi de la insulina i la glucosa, ja que és un dels principals teixits consumidors de glucosa. La interacció, però, entre aquest dos teixits encara presenta molts interrogants. Actualment, per estudiar com dos teixits interactuen entre ells, el testeig animal és el mètode més confiable. No obstant, presenta certes limitacions, com la poca similitud en quan a l’activitat dels illots, la variabilitat fisiològica entre diferents animals, dilemes ètics o la necessitat d’encarar la recerca cap a una medicina més personalitzada. Aquesta finalitat és el que ha portat als científics a buscar alternatives a l’experimentació animal. Entre moltes, una de les més prometedores són els anomenats Òrgans-en-un-xip, plataformes 3D de cultiu cel·lular combinades amb microfluídica i biomaterials que permeten simular les funcions específiques d’un òrgan. Per tal de generar el teixit dins d’aquesta plataforma, l’encapsulació de cèl·lules dins de hidrogels és la tècnica més utilitzada, degut al seu alt contingut d’aigua, la seva adaptabilitat mecànica o la possibilitat de generar una certa estructura geomètrica [2]. No obstant, la seva petita porositat, limita la difusió homogènia d’oxigen i de nutrients dins seu [3]. Aquest problema creix quan es volen encapsular illots pancreàtics en bastides d’hidrogel, degut a la seva mida (~100 μm de diàmetre). Els illots pancreàtics són agregacions de varis tipus diferent de cèl·lules, on destaquen les cèl·lules secretores de insulina (cèl·lules beta) i les secretores de glucagó (cèl·lules alfa). Per altre costat, el teixit muscular s’encapsula en petits constructes per tal d’imitar l’estructura d’aquest. El múscul esquelètic és un teixit altament alineat, amb cèl·lules multi nucleades, anomenades miotubs, que s’obtenen a partir de la fusió de cèl·lules soles, anomenades mioblasts. Per tal de solucionar aquests problemes, els criogels han aparegut com a alternativa. Els criogels, estan fabricats a temperatures sota zero, així mentres el polímer crosslinca es formen cristalls de gel. Un cop formada la matriu, la bastida es descongela i aquests cristalls es desfaran, deixant pas a espais buits, anomenats pors. Aquests, seran els que posteriorment li donaran la l’estructura porosa, altament interconnectada, amb alta permeabilitat i amb una arquitectura de pors determinada a la nostra bestida. En aquesta tesi s’han desenvolupat dos bastides de cel·lulosa carboxymetilada diferents seguint la tècnica de la criogelificació. Cada bastida ha estat dissenyada per tenir una distribució i una arquitectura de pors diferent d’acord amb la necessitat i propietat del teixit que es vulgui generar. A més, les propietats físiques i mecàniques de les dos bastides tenen alta semblança amb les propietats físiques i mecàniques de la matriu extracel·lular de cada teixit. Per el teixit pancreàtic, s’ha generat una bastida amb un diàmetre de pors similar als illots pancreàtics, per tal que, sembrant cèl·lules beta, aquestes formin pseudoillots similars als illots fisiològics. A més, s’ha demostrat que aquests illots tenen el diàmetre i la arquitectura desitjada, són viables i capaços de respondre a diferents nivells de glucosa. A més, s’ha demostrat que aquestes cèl·lules agregades en forma de pseudoillots responen millor a la glucosa que les cèl·lules configurades en distribució dispersa. En el cas del múscul esquelètic, s’ha desenvolupat una bastida amb una arquitectura de pors altament alineada per promoure l’alineament cel·lular i la fusió cel·lular. A més, s’han pogut incorporar nanotubs de carboni per millorar les propietats elèctriques de la vestida. D’aquesta manera, aplicant pulsos elèctrics per estimular el teixit, s’han pogut millorar les etapes primerenques de la maduració miogènica