Deciphering the biology of axon stretch-growth

Traditional nerve regeneration strategies focus on growth cone-mediated growth, a form of nerve growth that occurs primarily during embryogenesis. Early axons continue to grow from the end distal to the soma, seeking targets on which to synapse. It is believed that once the axons synapse, stretch-growth takes over and is responsible for the great lengths achieved by nerves of the central and peripheral nervous systems. Recent work has demonstrated stretch-growth in vitro resulting in dramatically increased growth rates compared to the growth cone. Here, the aim was to decipher the underlying biology associated with axon stretch-growth using two approaches. First, a device was created for live imaging of stretch-growth as it occurs on the stage of a microscope. Morphology changes and cytoskeletal transport were captured live at up to 600x magnification over six days of culturing. Second, the RNA species produced during stretch-growth were isolated in order to reveal the regulatory genes involved in this process. Successive RNA quantifications have revealed up to a three-fold increase in RNA population of stretch- grown tissue when compared to controls

Microfluidic Organ/Body-on-a-Chip Devices at the Convergence of Biology and Microengineering

Recent advances in biomedical technologies are mostly related to the convergence of biology with microengineering. For instance, microfluidic devices are now commonly found in most research centers, clinics and hospitals, contributing to more accurate studies and therapies as powerful tools for drug delivery, monitoring of specific analytes, and medical diagnostics. Most remarkably, integration of cellularized constructs within microengineered platforms has enabled the recapitulation of the physiological and pathological conditions of complex tissues and organs. The so-called organ-on-a-chip technology, which represents a new avenue in the field of advanced in vitro models, with the potential to revolutionize current approaches to drug screening and toxicology studies. This review aims to highlight recent advances of microfluidic-based devices towards a body-on-a-chip concept, exploring their technology and broad applications in the biomedical field.European Regional Development Fund-Project FNUSA-ICRC [CZ.1.05/1.1.00/02.0123]; Fundacao para a Ciencia e a Tecnologia (FCT), Portugal [UID/BIM/04773/2013]; Internal Research Grant Program, Universita Campus Bio-Medico di Romainfo:eu-repo/semantics/publishedVersio

Bioreactors as engineering support to treat cardiac muscle and vascular disease

Cardiovascular disease is the leading cause of morbidity and mortality in the Western World. The inability of fully differentiated, load-bearing cardiovascular tissues to in vivo regenerate and the limitations of the current treatment therapies greatly motivate the efforts of cardiovascular tissue engineering to become an effective clinical strategy for injured heart and vessels. For the effective production of organized and functional cardiovascular engineered constructs in vitro, a suitable dynamic environment is essential, and can be achieved and maintained within bioreactors. Bioreactors are technological devices that, while monitoring and controlling the culture environment and stimulating the construct, attempt to mimic the physiological milieu. In this study, a review of the current state of the art of bioreactor solutions for cardiovascular tissue engineering is presented, with emphasis on bioreactors and biophysical stimuli adopted for investigating the mechanisms influencing cardiovascular tissue development, and for eventually generating suitable cardiovascular tissue replacements

Development of a Biaxial Stretch Bioreactor and Finite Element Models for Mechanobiological Study of Aortic Valve Leaflets

Aortic heart valve disease is a significant cause of mortality worldwide; and replacement surgery is necessary in 70% of cases. Tissue engineered heart valves (TEHVs) are biocompatible and biodegradable, with ability to grow with the patient. However, to date, TEHVs mostly lack ability to withstand native mechanical forces since they are unable to mimic the heterogeneous and anisotropic structure of extracellular matrix (ECM) in native valves. Cyclic stretch is known to modulate ECM fiber synthesis and alignment. However, little tools are available for studying the interaction between aortic tissues and stretch condition. Finite element method is a powerful tool to simulate the complex structure of aortic valve, however, most current simulations modeled the leaflet as a homogenous material, and none of them took the distinctions between two surface layers into account, which were critical for the proper function of the aortic valve.To study the effects of cyclic stretch on extracellular matrix remodeling, the heterogeneous properties of the aortic leaflet, and the effects of heterogeneity on the function of valve, we have 1) Designed, fabricated and validated a biaxial stretch bioreactor; 2) Analyzed train patterns of native aortic leaflets using digital image correlation method; 3) Designed and validated an anisotropic and heterogeneous finite element (FE) model for leaflets. These studies provided insights into the interaction between aortic valve tissue and the mechanical environment, anisotropy and heterogeneity of aortic leaflets ECM due to the distribution of collagen fibers, and detailed distinct strain patterns in fibrosa vs. ventricularis sides and 3 aortic leaflets. Our novel biaxial stretch bioreactor and refined FE model of aortic leaflet will pave path for other scientists to study mechanobiology, design and condition engineered tissues and simulate engineered aortic valve grafts or pathology of calcium deposition

Development of Tethered Aligned Engineered Neural Tissue Containing Elongated Neurons for Peripheral Nerve Regeneration

Following peripheral nerve injury, the axons in the distal nerve between the injury site and the muscle degenerate. When the injured site is very proximal, functional recovery from nerve repair is a clinical challenge since neuronal regeneration rate is limited, resulting in muscle atrophy due to the delay in reinnervation, even where the ‘gold standard’ autograft is used. Much research focuses on developing biomaterial scaffolds that mimic the autograft and promote host neurite regeneration from proximal to distal stump, whereas here, we aim to improve long distance repair by populating constructs with functional neurons and glial cells. With an engineered living scaffold populated with neurons exhibiting long neurite extensions supported by glial cells, the gap between proximal stump and muscle could potentially be reconnected promptly once the challenge of integration is overcome. To test the concept, a method was developed using tethered aligned engineered neural tissue (TaeNT) formed from simultaneous self-alignment of Schwann cells and collagen fibrils in a fully-hydrated tethered gel resulting in an anisotropic tissue-like structure. The in vitro results showed neurite elongation and alignment in the co-culture of neurons and Schwann cells in TaeNT, indicating that TaeNT could be an appropriate substrate for growing long neurites with a view to generating therapeutic constructs containing long functional neurons. The implantation of TaeNT containing neurons and Schwann cells in a 10mm-gap rat sciatic nerve for 3 weeks provided information about host-transplant cell interaction including Schwann cell migration and alignment inside the conduit, and neurite elongation across the conduit interface. Furthermore, in an attempt to induce longer neurite growth, TaeNT was proposed as a substrate that could be combined with mechanical tension application using a 3D-printed mould developed to stretch the cellular gels in a controlled manner. A series of newly designed protocols for mechanical tension application to induce growth response for enhanced neural regeneration was developed and discussed correspondingly. In summary, the findings represent the development and investigation of the regenerative potential for engineered living scaffolds containing neurons and Schwann cells suitable for stretch-growth to provide an elongated functional nerve graft. With a view to translation for clinical use, investigating the source of therapeutic cells in the conduit and the functional integration of host and transplanted cells is an important step towards optimising the regenerative potential of the engineered living scaffold

Innervation: The Missing Link for Biofabricated Tissues and Organs

Innervation plays a pivotal role as a driver of tissue and organ development as well as a means for their functional control and modulation. Therefore, innervation should be carefully considered throughout the process of biofabrication of engineered tissues and organs. Unfortunately, innervation has generally been overlooked in most non-neural tissue engineering applications, in part due to the intrinsic complexity of building organs containing heterogeneous native cell types and structures. To achieve proper innervation of engineered tissues and organs, specific host axon populations typically need to be precisely driven to appropriate location(s) within the construct, often over long distances. As such, neural tissue engineering and/or axon guidance strategies should be a necessary adjunct to most organogenesis endeavors across multiple tissue and organ systems. To address this challenge, our team is actively building axon-based living scaffolds that may physically wire in during organ development in bioreactors and/or serve as a substrate to effectively drive targeted long-distance growth and integration of host axons after implantation. This article reviews the neuroanatomy and the role of innervation in the functional regulation of cardiac, skeletal, and smooth muscle tissue and highlights potential strategies to promote innervation of biofabricated engineered muscles, as well as the use of living scaffolds in this endeavor for both in vitro and in vivo applications. We assert that innervation should be included as a necessary component for tissue and organ biofabrication, and that strategies to orchestrate host axonal integration are advantageous to ensure proper function, tolerance, assimilation, and bio-regulation with the recipient post-implant

Evolution of biochip technology : a review from lab-on-a-chip to organ-on-a-chip

ABSTRACT: Following the advancements in microfluidics and lab-on-a-chip (LOC) technologies, a novel biomedical application for microfluidic based devices has emerged in recent years and microengineered cell culture platforms have been created. These micro-devices, known as organ-on-a-chip (OOC) platforms mimic the in vivo like microenvironment of living organs and offer more physiologically relevant in vitro models of human organs. Consequently, the concept of OOC has gained great attention from researchers in the field worldwide to offer powerful tools for biomedical researches including disease modeling, drug development, etc. This review highlights the background of biochip development. Herein, we focus on applications of LOC devices as a versatile tool for POC applications. We also review current progress in OOC platforms towards body-on-a-chip, and we provide concluding remarks and future perspectives for OOC platforms for POC applications

Genetically engineered hydrogels based on elastin-like recombinamers for cardiovascular applications

Tissue engineering and regenerative medicine (TERM) is a prominent field of research that aims to repair or replace damaged tissues or organs, by the development of scaffolds with essential features, such as biocompatibility and functionality. Nowadays, recombinant polypeptides arise as promising candidates due to their tunability at the genetic level, affording exquisite control over the final physico-chemical properties and bioactivities. In particular, elastin-like recombinamers (ELRs) are genetically engineered polypeptides based on the repetition of the pentapeptide Val-Pro-Gly-X-Gly, found in the hydrophobic domains of tropoelastin, where X can be any amino acid except L-proline. These, ELRs exhibit a reversible phase transition in aqueous environments and their recombinant nature allows the inclusion of specific epitopes, such as cell adhesion, proteolytic sequences, and biologically active molecules such as growth factors. Interestingly, they can be chemically modified to obtain covalently cross-linked hydrogels through orthogonal and cytocompatible &#8220;click chemistry&#8221; reactions. The first chapter of this thesis is dedicated to the spatiotemporal control of angiogenesis, which has been proven essential for the correct integration and long-term stability of the implant. To this end, we designed a three-dimensional (3D) model consisting of a coaxial binary ELR tubular construct that displays proteolytic sequences with fast and slow cleavage kinetics towards the urokinase plasminogen activator protease on its inner and outer part respectively. The ELRs further included the universal cell-adhesion domain (RGD) and a VEGF-mimetic tethered peptide (QK) to induce angiogenesis. In vitro studies evidenced the effect of the QK peptide on endothelial cell extension and anastomosis. The subcutaneous implantation of the 3D models in mice showed a guided cell infiltration and capillary formation in the pre-designed spatiotemporal arrangement of the construct. Furthermore, the ELR hydrogels induced a mild macrophage response that resolved over time, supporting the potential integration of the resorbable scaffold within the host tissue. The second chapter study the preferential guidance of angiogenesis and neurogenesis in a spatiotemporal manner. In particular, we designed a 3D model ELR scaffold comprising two internal cylinders, with the pro-angiogenic peptide (QK) in one of them, and the neuronal cell adhesive peptide (IKVAV) in the vicinal one, both covalently tethered. In addition, these cylinders contain proteolytic sequences with fast cleavage kinetics towards the urokinase plasminogen activator enzyme and RGD cell adhesive domains. On the other hand, the outer part displays a slow-resorbable or non-protease-sensitive ELR hydrogel. In vitro studies demonstrated the effect of IKVAV epitope on neurite extension. The subcutaneous implantation of the 3D model ELR constructs in mice showed a guided cell infiltration accompanied by preferential angiogenesis or innervation on the respective QK and IKVAV containing cylinders, with a faster integration within the host tissue for the slow-resorbable scaffold. The third chapter describes the development of a ready-to-use bi-leaflet transcatheter venous valve for the treatment of chronic venous insufficiency (CVI), a leading worldwide vascular disease. For this purpose, we combined (i) ELRs, (ii) a textile mesh reinforcement and (iii) a bioabsorbable magnesium stent. Burst strength analysis demonstrated mechanical properties suitable for vascular pressures, whereas equibiaxial analysis confirmed the anisotropic performance equivalent to the native saphenous vein valves. In vitro studies identified the non-thrombogenic, minimal hemolysis and self-endothelialization properties endowed by the ELR hydrogel. The hydrodynamic testing under pulsatile conditions revealed minimal regurgitation (< 10%) and pressure drop (< 5 mmHg) in accordance with values stated for functional venous valves, and no stagnation points. Furthermore, in vitro simulated transcatheter delivery showed the ability to withstand the implantation procedure. In summary, the thesis presented herein provide new insights in the design and development of novel ELR-forming hydrogels to be used in tissue engineering and regenerative medicine applications.La ingeniería de tejidos y la medicina regenerativa (TERM) es un campo de investigación cuyo objetivo es reparar o reemplazar tejidos u órganos dañados, mediante el desarrollo de andamios biocompatibiles y funcionalizados. Hoy en día, los polipéptidos recombinantes, permiten un control exquisito sobre las propiedades fisicoquímicas y bioactividades. En particular, los elastin-like recombinamers (ELRs) son polipéptidos modificados genéticamente basados en la repetición del pentapéptido Val-Pro-Gly-X-Gly, que se encuentra en los dominios hidrófobos de la tropoelastina, donde X puede ser cualquier aminoácido excepto L-prolina. Estos ELR exhiben una transición de fase reversible en medios acuosos y su naturaleza recombinante permite la inclusión de epítopos específicos, como la adhesión celular, secuencias proteolíticas y moléculas bioactivas como factores de crecimiento. Curiosamente, pueden modificarse químicamente para obtener hidrogeles entrecruzados covalentemente a través de reacciones de "química de clic" ortogonales y citocompatibles. El primer capítulo está dedicado al control espaciotemporal de la angiogénesis, la cual es fundamental para la correcta integración y estabilidad del implante. Para ello, diseñamos un modelo tridimensional (3D) que consiste en una construcción binaria coaxial de hidrogeles de ELR, que lleva secuencias proteolíticas con cinética de escisión rápida y lenta sensibles a la proteasa del activador del plasminógeno tipo uroquinasa (uPA) en su parte interna y externa respectivamente, y un péptido mimético de VEGF (QK) anclado para inducir la angiogénesis. Los estudios in vitro evidenciaron el efecto del péptido QK sobre la extensión y anastomosis de las células endoteliales. La implantación subcutánea del modelo 3D en ratones mostró una infiltración celular guiada. Además, los hidrogeles ELR indujeron una respuesta leve de macrófagos que se resolvió con el tiempo, lo que respalda la integración de estos andamios reabsorbibles. El segundo capítulo estudia la orientación preferencial de la angiogénesis y la neurogénesis de manera espaciotemporal. En particular, diseñamos un modelo 3D de ELR que comprende dos cilindros internos, con el péptido proangiogénico (QK) en uno de ellos, y el péptido adhesivo de células neuronales (IKVAV) en el vecinal, ambos unidos covalentemente. Además, estos cilindros contienen secuencias proteolíticas con una cinética de escisión rápida frente a la enzima uPa y los dominios adhesivos RGD. Por otro lado, la parte exterior presenta un hidrogel ELR de reabsorción lenta o no sensible a las proteasas. Los estudios in vitro demostraron el efecto del epítopo IKVAV sobre la extensión de axones. La implantación subcutánea de las construcciones en ratones mostró una infiltración celular guiada acompañada de angiogénesis o inervación preferencial en los respectivos, con una integración más rápida dentro del tejido hospedador para el andamio con reabsorción lenta. El tercer capítulo describe el desarrollo de una válvula venosa transcatéter biválvula lista para usar para el tratamiento de la insuficiencia venosa crónica (IVC), una enfermedad vascular predominante en todo el mundo. Para ello, combinamos (i) ELR, (ii) un refuerzo de malla textil y (iii) un stent de magnesio bioabsorbible. El análisis de resistencia a rotura demostró propiedades mecánicas adecuadas para las presiones vasculares, mientras que el análisis equibiaxial confirmó el rendimiento anisotrópico equivalente a las válvulas de vena safena nativa. Los estudios in vitro identificaron las propiedades no trombogénicas, de hemólisis mínima y de autoendotelización que otorga el hidrogel ELR. Las pruebas hidrodinámicas en condiciones pulsátiles revelaron regurgitación mínima (< 10 %) y caída de presión (< 5 mmHg) de acuerdo con los valores establecidos para válvulas venosas funcionales y sin puntos de estancamiento. Además, el suministro transcatéter simulado in vitro mostró la capacidad de soportar el procedimiento de implantación. En resumen, la tesis presentada proporciona nuevos conocimientos en el diseño y desarrollo de nuevos hidrogeles ELR para su uso en ingeniería de tejidos y medicina regenerativa.Escuela de DoctoradoDoctorado en Físic

Advances in microfluidic in vitro systems for neurological disease modeling

Neurological disorders are the leading cause of disability and the second largest cause of death worldwide. Despite significant research efforts, neurology remains one of the most failure‐prone areas of drug development. The complexity of the human brain, boundaries to examining the brain directly in vivo, and the significant evolutionary gap between animal models and humans, all serve to hamper translational success. Recent advances in microfluidic in vitro models have provided new opportunities to study human cells with enhanced physiological relevance. The ability to precisely micro‐engineer cell‐scale architecture, tailoring form and function, has allowed for detailed dissection of cell biology using microphysiological systems (MPS) of varying complexities from single cell systems to “Organ‐on‐chip” models. Simplified neuronal networks have allowed for unique insights into neuronal transport and neurogenesis, while more complex 3D heterotypic cellular models such as neurovascular unit mimetics and “Organ‐on‐chip” systems have enabled new understanding of metabolic coupling and blood–brain barrier transport. These systems are now being developed beyond MPS toward disease specific micro‐pathophysiological systems, moving from “Organ‐on‐chip” to “Disease‐on‐chip.” This review gives an outline of current state of the art in microfluidic technologies for neurological disease research, discussing the challenges and limitations while highlighting the benefits and potential of integrating technologies. We provide examples of where such toolsets have enabled novel insights and how these technologies may empower future investigation into neurological diseases