110 research outputs found

    Biological building blocks for 3D printed cellular systems

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    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

    The CMS experiment at the CERN LHC

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    The Compact Muon Solenoid (CMS) detector is described. The detector operates at the Large Hadron Collider (LHC) at CERN. It was conceived to study proton-proton (and leadlead) collisions at a centre-of-mass energy of 14 TeV (5.5 TeV nucleon-nucleon) and at luminosities up to 1034 cm-2s-1 (1027 cm-2s-1). At the core of the CMS detector sits a high-magnetic field and large-bore superconducting solenoid surrounding an all-silicon pixel and strip tracker, a lead-tungstate scintillating-crystals electromagnetic calorimeter, and a brass-scintillator sampling hadron calorimeter. The iron yoke of the flux-return is instrumented with four stations of muon detectors covering most of the 4π solid angle. Forward sampling calorimeters extend the pseudorapidity coverage to high values (|η| ≤ 5) assuring very good hermeticity. The overall dimensions of the CMS detector are a length of 21.6 m, a diameter of 14.6 m and a total weight of 12500 t

    The Penobskan Porcupine Panic

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    This creative writing thesis takes its origins from a ten-page story written for a fiction class in the spring of 2015 and inspired by the song Penobska Oakwalk from the band Quilt

    The Penobskan Porcupine Panic

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    This creative writing thesis takes its origins from a ten-page story written for a fiction class in the spring of 2015 and inspired by the song Penobska Oakwalk from the band Quilt

    Streptococcal collagen-like protein 1, Scl1, modulates group a Streptococcus adhesion, biofilm formation and virulence

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    Background: The collagens comprise a large family of versatile proteins found in all three domains of life. The streptococcal collagen-like protein 1, scl1, of group A Streptococcus (GAS) binds extracellular matrix components (ECM), cellular fibronectin and laminin, via the surface-exposed globular domain. GAS strains express scl1 and form biofilm in vitro, except for M3-type strains that are particularly invasive to humans. Hypothesis: Lack of scl1 adhesin in M3 GAS results in decreased adherence and biofilm formation, and increased virulence. Results and Discussion : First crystal structure of the globular domain revealed a unique six-helical bundle fold, consisting of three pairs of alpha helices connected by variable loops. ECM binding by Scl1 promotes the formation of stable tissue microcolonies, which was demonstrated in vitro during infection of wounded human skin equivalents. A conserved nonsense mutation was identified in the scl1 allele of the M3-type strains (scl1.3) that truncates the coding sequence, presumably resulting in a secreted Scl1 variant. Absence of Scl1 on the surface of M3-type GAS was demonstrated experimentally, as well as diminished expression of the scl1 transcript in M3 strains relative to other M-types. Therefore, M3-type strains have reduced biofilm capacity on ECM coatings relative to other M-types. Constructed full-length recombinant Scl1.3 protein displayed binding capacity to cellular fibronectin and laminin, and M3 strains complemented with functional Scl1.3 adhesin displayed increased biofilm formation. The isoallelic M3 strain, carrying a rare carrier allele encoding cell-associated Scl1.3 variant, showed decreased pathology in mice, compared to the invasive M3 strain. Similarly, scl1 inactivation in biofilm-capable M28- and M41-type GAS led to increased lesion size during subcutaneous infection. Conclusions: The studies presented here demonstrate the importance of surface Scl1 in modulating biofilm formation and virulence of GAS, and provide insight into the structure and function of Scl proteins

    Micro/nano-scale strategies for engineering in vitro the celular microenvironment using biodegradable biomaterials

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    Programa doutoral em BioengenhariaBiological tissues result of a specific spatial organization of cells, extracellular matrix (ECM) molecules, and soluble factors. These micro and nanoscaled biological entities organize into regional tissue architectures, creating highly complex and heterogeneous cellular microenvironments. To generate functional tissue equivalents in vitro, engineered biomaterials should mimic the structural, chemical and cellular complexity by recapitulating the unique native microenvironments. Thus, the main goal of this thesis was to engineer biodegradable polymers using various micro and nanofabrication techniques, with specific structural, biochemical and cellular cues for improved performance. The main governing hypotheses of this thesis were: 1) substrates with improved structural properties can be engineered using biodegradable polymers that have previously shown good results in in vivo studies, 2) biochemical cues can be incorporated into biodegradable polymers, yielding biomaterials with integrated chemical cues for improved cellular performance, and 3) these structural and biochemical cues can be incorporated into a single system. To develop biomaterials with structural cues, micromolding of poly(butylene succinate) (PBS) was performed to engineer surfaces with features at a microscale that induced the alignment of human adipose stem cells. Although this polymeric material has been processed at a macroscale into scaffolds, this was the first report on the engineering of this material at a microscale, demonstrated by the development of twenty features with different dimensions. Improved substrates with structural cues were also engineered using the polysaccharide gellan gum (GG), which has been extensively studied at 3B’s Research Group. Microcapsules of GG, aimed at being used as drug or cell carriers and/or delivery agents, were engineered using a two-phase system. The principle of hydrophobic-hydrophilic repulsion forces was combined with a microfabrication process by means of a needle/syringe pump system. Microcapsules with different diameters were produced by varying the system parameters. As an original proof-of-concept, fluorescent beads, cell suspensions and cell aggregates were encapsulated within this microfabrication system. To develop biomaterials with enhanced biochemical cues, GG was chemically modified with ester bonds, yielding novel hydrogels crosslinkable by ultraviolet (UV) light. Methacrylated GG (MeGG) hydrogels were formed using physical and chemical mechanisms resulting in hydrogels with tunable mechanical properties, matching those of natural tissues from soft to hard, as the brain or collagenous bone. In a subsequent step, this material was combined with chitosan (CHT), a natural polysaccharide, resulting in a polyelectrolyte complex (PEC) hydrogel that combined the most advantageous properties of CHT and MeGG. PEC hydrogels are commonly formed by the interaction between the chains of oppositely charged polymers and are thus held together by ionic forces, which can be disrupted by changes in physiological conditions. However, in our new system, the biochemical cues earlier introduced in GG, allowed to crosslink the MeGG-CHT hydrogel using UV light, stabilizing the structure of the hydrogel. This rather important property also enabled for the development of microgels by photolithography. The encapsulation of rat cardiac fibroblasts within MeGG before PEC hydrogel production, led to the fabrication of microgels with combined biochemical, structural and cellular cues. The developed MeGG-CHT hydrogel was further engineered into a multi-hierarchical fibrous hydrogel by means of combining fluidics technology and chemistry principles of the interaction of two oppositely charged polymers. Two converging fluidic channels were used to extrude the MeGG-CHT hydrogel, formed by the assembly of the polymeric chains at the location where the channels converged. The resulting hydrogel closely mimicked the architecture of natural collagen fibers not only at a micro but also at a nanoscale. The developed hydrogel with relevant biological structural properties was enhanced by incorporating cell adhesive motifs (RGD peptides) into the MeGG backbone before processing. The research work described in this thesis addresses strategies to mimic several parameters of the native microenvironment of tissues. Biochemical and cellular cues were incorporated into biomaterials that were microprocessed with relevant biological micro and nanoscale features. In summary, the works reported in this thesis show the importance of combining different areas of knowledge into the development of improved systems for biomedical engineering applications. Undoubtfully, chemistry and micro and nanofabrication technologies are two areas of knowledge that allow the fabrication of micro and nanostructured materials. Herein, this synergy was achieved with a top-down approach (by micromolding, photolithography or fluidics technologies) and/or with a bottom-up approach (by the assembly of polymer chains). The last work of this thesis is the result of the original combination of both approaches for the development of enhanced micro and nanostructured biomaterials, thus presenting significant improved features compared to currently developed systems to be successfully used in several regenerative medicine approaches.A funcionalidade dos tecidos biológicos está associada à organização espacial de células, à composição e distribuição de moléculas da matriz extracelular e a outros componentes solúveis. Estas entidades biológicas à escala micro/nanométrica organizam-se em arquitecturas locais específicas, criando micro-ambientes celulares complexos e heterogéneos. Existe portanto um grande interesse no desenvolvimento de equivalentes funcionais dos tecidos humanos usando biomateriais de modo a mimetizar a complexidade química, estrutural e celular. Acredita-se que estes biomateriais poderão recapitular as características únicas dos micro-ambientes dos tecidos, favorecendo a sua regeneração funcional. O objectivo principal desta tese consistiu em produzir e desenvolver polímeros biodegradáveis com estímulos químicos, estruturais e celulares de modo a obter uma elevada funcionalidade, usando para isso diferentes técnicas de micro/nano-fabricação. As hipóteses científicas que estão na base do trabalho descrito nesta tese são: 1) é possível desenvolver substratos com estímulos estruturais usando polímeros biodegradáveis que já tenham demonstrado resultados promissores in vivo, 2) é possível incorporar estímulos bioquímicos em sistemas baseados em polímeros biodegradáveis, produzindo biomateriais com sinais bioquímicos integrados para o melhor desempenho biológico dos materiais, e 3) é possível combinar estes sinais estruturais e bioquímicos num único sistema. O polímero polibutileno succinato foi micro-moldado de modo a desenvolver superfícies com topografias à escala micrométrica, visando o desenvolvimento de biomateriais com sinais estruturais, capazes de induzir o alinhamento de células do tecido adiposo humano. Embora este material tenha sido processado anteriormente sob a forma de estrutura 3D porosa, esta foi a primeira vez que foi descrito o processamento deste material à escala micrométrica, demonstrado pelo desenvolvimento de vinte padrões com diferentes dimensões. O polissacarídeo goma gelana (GG), extensivamente estudado no Grupo de Investigação 3B’s, foi usado para desenvolver substratos com sinais estruturais. Micro-cápsulas de GG foram fabricadas usando um sistema de duas fases, com o intuito de serem usadas para o transporte ou libertação de drogas ou células. O princípio de repulsão entre soluções hidrofóbicas e hidrófilas foi combinado com um processo de micro-fabricação, usando uma bomba de injecção. De modo a demonstrar o conceito, partículas fluorescentes, suspensões celulares e agregados celulares foram encapsulados usando este sistema. Para desenvolver biomateriais com sinais bioquímicos, a GG foi modificada quimicamente com ligações éster, produzindo hidrogéis reticuláveis por radiação ultravioleta (UV). Os hidrogéis de GG metacrilada (MeGG) são formados com mecanismos físicos e químicos, resultando em géis com propriedades mecânicas ajustáveis numa gama que se situa próximo da dos tecidos humanos moles e duros, como o cérebro e o osso. Este material foi posteriormente combinado com quitosano, um polissacarídeo de origem natural, resultando num complexo polieletrolítico (PEC) que combina as melhores propriedades do quitosano e da MeGG. A formação de hidrogéis de PECs resulta da interacção entre cadeias de polímeros com cargas opostas, sendo o mecanismo de ligação dependente de forças iónicas, as quais podem ser perturbadas por mudanças na composição da solução. Os sinais bioquímicos introduzidos anteriormente permitiram reticular o hidrogel MeGG-CHT com a radiação UV, estabilizando a estrutura do hidrogel. Este material permitiu também o desenvolvimento de micro-géis por fotolitografia. O encapsulamento de fibroblastos do coração de ratos na MeGG previamente à produção dos hidrogéis conduziu à fabricação de micro-géis com sinais bioquímicos, estruturais e celulares integrados num mesmo sistema. O sistema de hidrogel MeGG-CHT foi usado para obter um hidrogel fibroso hierárquico, através da combinação de microfluídica e complexação polieletrolitica. Extrudiu-se o MeGGCHT em dois canais convergentes com o objectivo de obter a complexação das cadeias poliméricas na forma de fibra. O hidrogel desenvolvido mimetiza a arquitectura das fibras de colagénio existentes no corpo humano, não só ao nível micrométrico mas também à escala nanométrica. O hidrogel desenvolvido foi funcionalizado através da incorporação de moléculas adesivas (péptidos RGD) na MeGG antes do seu processamento. O trabalho de investigação descrito nesta tese demonstra o potencial de diferentes estratégias para mimetizar várias características do micro-ambiente existente nos tecidos. Sinais bioquímicos e celulares foram incorporados em biomateriais que foram posteriormente processados para obter estruturas biológicas relevantes à escala micro/nanométrica. Esta tese demonstra a importância de combinar diferentes áreas do conhecimento para o desenvolvimento de sistemas funcionais para aplicações biomédicas. É inquestionável que a química e as tecnologias de micro e nano-fabricação são duas áreas de conhecimento que se complementam e permitem a fabricação de materiais micro e nanoestruturados. Esta sinergia foi alcançada usando para o efeito uma abordagem top-down (através de fotolitografia, micro-moldação ou microfluídica) e/ou uma abordagem bottom-up (através da complexação de cadeias poliméricas). No último trabalho da tese estas duas abordagens convergem para o desenvolvimento de biomateriais micro e nano-estruturados. Este tipo de sistemas permitem a funcionalização de biomateriais até níveis de aproximação dos tecidos biológicos não tem paralelo nos sistemas convencionais, o que se traduz no desenvolvimento de sistemas de elevado desempenho para diferentes abordagens em engenharia de tecidos

    Topical Workshop on Electronics for Particle Physics

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