110 research outputs found
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
The CMS experiment at the CERN LHC
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
ENGINEERING COLLAGEN MIMETIC PEPTIDE AMPHIPHILE HYDROGELS BY TUNING MECHANICAL PROPERTIES FOR BIOMEDICAL APPLICATIONS
Ph.DDOCTOR OF PHILOSOPH
The Penobskan Porcupine Panic
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
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
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Self-assembled peptide as a long-acting drug formulation
Type 2 Diabetes Mellitus (T2DM) and obesity are widespread and associated metabolic diseases with worldwide rising prevalence [1]. The gut hormone Oxyntomodulin induces satiety and normalizes hyperglycaemia without risk of hypoglycaemic excursions [2]. However, native incretin hormones are quickly degraded by peptidases and renally excreted, which has so far impaired pharmaceutical exploitation of anorectic and glucose-homeostatic activities [3]. It is known that many – if not all – peptides can be converted to self-assembled nanostructures [4]. Amyloid fibrils are characterized by enhanced chemical, biological and mechanic stability compared to soluble peptides, and allow a controlled release by dissociation of monomers from the fibril termini [5]. Here I propose self-assembled peptides as a prolonged-activity, self-mediated drug delivery system for subcutaneous injection of pharmaceutically active drugs. Oxyntomodulin is used as a model peptide due to previously futile attempts at finding long-acting analogues, and Oxyntomodulin’s so far unreported self-assembly behaviour. This thesis shows a method to reproducibly form subcutaneously injectable Oxyntomodulin fibrils with high conversion yield and low polydispersity, as well as methods to characterize morphology, kinetics and thermodynamics of Oxyntomodulin self-assembly. Oxyntomodulin fibrils display amyloid-like characteristics and release soluble peptide in a peptide-deprived environment. Association and dissociation kinetics and thermodynamics are sensitively dependent on salts and temperature, with an association temperature optimum at room temperature that is unique in amyloid-type self-assembly. An alternative fibril type forms under different conditions and displays altered fibrillation kinetics and thermodynamics. The specificity of fibrillation to the peptide sequence is shown in presence of Oxyntomodulin’s sister peptide glucagon and the analogue Aib-2-Oxyntomodulin. Extended release of active peptide from a subcutaneous Oxyntomodulin fibril depot has been proved in rodent studies at MedImmune [6]. As amyloid-like self-assembly is a generic feature of the peptide chain, the strategies and methods described in this project can be applied to other pharmaceutically active peptides and proteins.This project was fully funded by MedImmune, LLC under grant number RG77937
Streptococcal collagen-like protein 1, Scl1, modulates group a Streptococcus adhesion, biofilm formation and virulence
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
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
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