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In nature, cells reside within an extracellular matrix (ECM), consisting of 3-dimensional (3D) networks of collagen and elastin which provide biophysical/chemical signals to direct cellular development and behaviour. Traditional methods of studying cellular behaviour often involve using 2-dimensional in vitro models that are convenient and cost-effective, but may not be representative of 3D in vivo conditions. A synthetic polyampholyte system, poly(N-(3-aminopropyl)methacrylamide hydrochloride-co-acrylic acid) [p(APM-co-AA)], was used here for the first time to construct two types of 3-dimensionally structured cell supports to mimic the ECM: microstructured films and nanofibrous scaffolds. Microstructured films were fabricated using a shape-memory polymer structuring approach and nanofibrous scaffolds were formed using electrospinning. Both films and nanofibers were thermally crosslinked by reaction of initially formed anhydride groups with pendant amines. Film topography was tuned through polymer solution concentration, and the surface chemistry of the crosslinked p(APM-co-AA) scaffolds was tuned by reaction of residual anhydride groups with hydrophilic or hydrophobic amines. The effects of film surface topography and surface chemistry on fibroblast morphologies were explored using fluorescence microscopy. This work presents the fabrication and characterization of tunable, self-crosslinking p(APM-co-AA) scaffolds as promising ECM mimics. / Thesis / Master of Science (MSc) / The extracellular matrix (ECM), present within all biological tissues, consists of 3-dimensional (3D) networks of proteins which provide chemical and physical cues for cells. Abnormalities in the ECM is associated with a variety of disorders, such as coronary heart disease and tumours. Thus, the study of the ECM and how it affects cell behaviour is important both for a better understanding of ECM-related diseases, as well as for creating ECM-mimics for tissue engineering. The goal of this thesis is to explore the fabrication of 3D scaffolds using a synthetic polyampholyte that can be crosslinked under heat and chemically tuned using functionalization. This polyampholyte was electrospun into nanofibers and prepared into microstructured films, which were used to study the effects of surface topography and chemistry on murine fibroblast morphology and attachment. These polyampholyte scaffolds provide a new means to study cell behaviour in environments that mimic certain aspects of the ECM.

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Despite our understanding of the importance of the 3D environment on the behaviour of virtually every cell, most studies are still performed within 2D engineered cell culture devices. In this project, the main goal was to design and engineer tunable three-dimensional (3D) extracellular matrix (ECM)-mimicking scaffolds made of collagen and fibronectin (namely the two major building blocks of the ECM) that recapitulate the ECM structural and mechanical properties essential for wound healing and cancer research. Two different methods were implemented to fabricate 3D scaffolds. First, 3D collagen scaffolds with a ‘porous’ structure (fabricated by a previous student via an ice-templating technique) were used. It was shown that, by increasing collagen concentration to 1.25 wt.%, homogenous scaffolds with interconnected pores (needed for cell invasion through the entire scaffold) were obtained. Fibronectin (Fn) was then incorporated using thermal and mechanical gradients to modify protein content and tune scaffolds microarchitecture. The effect of Fn coating of the collagen underlying structure on cell behaviour such as cell adhesion, invasion and matrix deposition was studied. Results showed that overall more cells adhered to Fn-coated scaffolds with respect to pure collagen scaffolds. Furthermore, our findings indicated that cells were also able to sense the conformation of the Fn coating (as assessed by Fluorescence Resonance Energy Transfer, FRET) since they deposited a more compact ECM on compact Fn coating while a more unfolded and stretched ECM was deposited on unfolded Fn coating. Second, 3D more complex physiologically relevant scaffolds with a ‘fibrillar’ structure were fabricated via a cold/warm casting technique. Pure collagen scaffolds were first generated: in cold-cast scaffolds, clear thin and long collagen fibers were observed while warm-cast scaffolds were denser and comprised shorter collagen fibers. The effect of both collagen concentration and casting temperature on scaffolds’ microstructure was studied. Our results indicate a preponderant effect of temperature. We further engineered dual-protein fibronectin-collagen fibrillar scaffolds by incorporating Fn fibers using thermal gradient. Clear Fn fibers were observed in some conditions. FRET assessment of Fn fibers also showed significant difference of Fn conformation. In this more advanced casting technique, cells were initially embedded into the scaffolds, which provided a more homogeneous cell distribution and a better tissue-mimicking setting. In each case, the effect of resulting ECM properties was tested via cell viability assays. Our data indicate that cells were viable after 72 hours, they could proliferate inside the scaffolds and were able to spread in some conditions. Collectively, our 3D ECM-mimicking scaffolds represent a new tunable platform for biological and biomaterial research with many potential applications in tissue engineering and regenerative medicine. Investigating cell behaviour in 3D ECM-mimicking environment will provide valuable insights to understand cancer progression and approaches to limit the progression and ultimately prevent metastasis.

Au sein des cellules, les voies enzymatiques sont souvent organisées spatialement sous forme de complexes, sur des structures protéiques ou dans des micro-compartiments. Cette organisation spatiale aide au déroulement optimal des réactions enzymatiques en limitant les pertes d’intermédiaires métaboliques, en isolant les voies de signalisations et en augmentant le rendement des réactions enzymatiques. Dans ce travail de thèse nous avons étudié la possibilité de créer des outils permettant de contrôler et optimiser de novo l’organisation spatiale de voies métaboliques in vivo.Nous avons dessiné et assemblé des structures d’ARN non codants utilisées comme support pour organiser le métabolisme bactérien. Ces ARN s’assemblent spontanément in vivo en des structures à une ou deux dimensions avec des sites distincts d’attachement protéique. Nous démontrons l’utilité de cette approche via l’optimisation d’une voie enzymatique de synthèse de biohydrogène et démocratisons l’utilisation de ces structures d’ARN en développant un protocole simplifié. Nous étendons cette étude à d’autres stratégies d’organisation, notamment via l’ingénierie des cellules spécialisées dans la fixation de l’azote atmosphérique de la cyanobactérie Anabaena PCC7120, les hétérocystes. Ce travail de thèse ouvre de nouvelles portes à la biologie de synthèse à la biologie structurale et aux nanotechnologies / In cells bio-enzymatic pathways are often spatially organized into complexes, into organelles or onto protein scaffolds. Spatial organization limits diffusion and helps channels substrates between enzymatic cores, limiting competing reactions, insulating and increasing yields of sequential metabolic reactions. In this PhD thesis work, we engineered new tools to control the precise spatial organization of enzymes and increase the titer of specific pathways. We design and engineer “artificial organelles” made of assembling RNA nanostructures. These scaffolds are made out of assembling non-coding RNA molecules we specifically design to polymerize into multi-dimensional nanostructures inside bacterial cells. These structures have docking sites to target enzymes onto them and control their respective distance and stochiometry. We demonstrate the validity of our approach by optimizing and improving the production of biohydrogen and designing a protocol to simplify and standardize the use of RNA scaffold. Moreover, we develop a new synthetic biology “chassis” by developing strategies to engineer AnabaenaPCC7120 and control the spatial localization of metabolic pathway at the cellular level. By targeting specific enzymes into oxygen-depleting heterocysts, metabolic engineers can now implement oxygen-sensitive pathways into oxygen evolving cyanobacteria. This PhD work opens the door to an array of new applications spanning synthetic biology, structural biology to nanotechnology

O desenvolvimento de scaffolds para a aplicação em biomateriais, seja na produção de tecidos biomiméticos ou mesmo em sistemas para liberação de drogas, tem sido fundamental tanto para o entendimento dos mecanismos de crescimento de tecidos biológicos e seu funcionamento, quanto para o desenvolvimento de biomateriais que possam ser incorporados aos tecidos naturais para seu reparo e para a efetiva aplicação de agentes terapêuticos. Dentre as várias técnicas para a produção destes scaffolds, a técnica de Electrospinning (ES) foi utilizada neste trabalho para a confecção de scaffolds poliméricos com a incorporação moléculas de interesse biotecnológico. Foram produzidos scaffolds e scaffolds compósitos pela adição de nanopartículas de óxido de cério, nanoargila haloisita e protoporfirina IX complexada à nanoargila haloisita, os quais foram estudados quanto à sua morfologia e propriedades tênseis, além de terem sidos testados quanto a sua viabilidade como sistemas biomiméticos de tecidos. Os scaffolds compósitos mostraram um ganho em ordenamento e homogeneidade, e os scaffolds compósitos contendo óxido de cério mostraram um leve aumento em sua capacidade elástica, além de terem sido viáveis para o crescimento de células HCat / The development of scaffolds for biomaterials applications, in biomimetic tissues production and drug-delivery systems, have been a fundamental tool for the understanding of biological tissues growing and repair mechanisms and for the development of biomaterials that can be incorporated to the natural tissues for both repair and effective application of therapeutic agents. Amongst the several techniques for scaffolds production, the Electrospinning (ES) methodology was applied in this work for developing polymeric scaffolds with the encapsulation of molecules of biotechnological interest. Scaffolds and blend scaffolds by cerium oxide nanoparticles and haloisite nanoclay addiction were produced and studied regarding its morphology, tensile properties and cell viability as biomimetic tissues. The blend scaffolds shoed an enhancement in order and homogeneity, and those within cerium oxide showed also an increase in elastic capacity and viable physical base for HCat cells

Thesis (M.S.)--University of Alabama at Birmingham, 2008. / Title from PDF t.p. (viewed July 21, 2010). Includes bibliographical references.

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Bone is a dynamic tissue that in some cases, due to fractures, infection or interruption of blood supply, does not repair completely, leading to bone loss; therefore it is necessary to recur to bone grafts. However, bone grafts (i.e.autografts) may require additional surgery and present risks associated with potential disease transmission from donor to recipient (i.e.allografts). The limitations of these grafts have encouraged the pursuit of engineered alternatives that are based on the synchronous interplay between biomaterials, biological macromolecules and cells. 3-D gelatin-based scaffolds were prepared and evaluated for their ability to promote osteogenesis. Three types of gelatin based scaffolds were prepared via the crosslinking of gelatin B with glutaraldehyde or EDC/NHS in the presence or absence of PLG . The porosity and pore size of the scaffolds were controlled by varying the freeze-drying temperature (-20°C and -80°C). To promote osteogenesis, human stromal MIAMI cells were incorporated in the scaffolds. Results demonstrated MIAMI cells grew and spread actively throughout gelatin and gelatin/PLG scaffolds after 14 days of incubation. The rate of osteogenic activity was confirmed through histochemical staining for alkaline phosphatase and calcium. Mineral deposition was increased in the gelatin scaffold as opposed to the gelatin/PLG scaffold after at day 35.

Tissue engineering is an emerging area with an aim to create artificial tissues or organs by employing methods of biology, engineering and material science. In tissue engineering, scaffolds are three-dimensional (3D) structure made from biomaterials with highly interconnected pore networks or microstructure, and are used to provide the mechanical and biological cues to guide cell differentiation in order to form desired three-dimensional tissues or functional organs. Hence, tissue scaffold plays a critical role in tissue engineering. However, fabrication of such scaffolds has proven to be a challenge task. One important barrier is the inability to fabricate scaffolds with designed pore size and porosity to mimic the microstructure of native tissue. Another issue is the prediction of process-induced cell damage in the cell-involved scaffold fabrication processes. By addressing these key issues involved in the scaffold fabrication, this research work is aimed at developing methods and models to represent the dispensing-based solid free form scaffold fabrication process with and without the presence of living cells.<p> The microstructure of scaffolds, featured by the pore size and porosity, has shown to significantly affect the biological and mechanical properties of formed tissues. As such, during fabrication process the ability to predict and determine scaffold pore size and porosity is of great importance. In the first part of this research, the flow behaviours of the scaffold materials were investigated and a model of the flow rate of material dispensed during the scaffold fabrication was developed. On this basis, the pore size and porosity of the scaffolds fabricated were represented by developing a mathematical model. Scaffold fabrication experiments using colloidal gels with different hydroxylapatite volume fractions were carried out and the results obtained agreed with those from model simulations, indicating the effectiveness of the models developed. The availability of these models makes it possible to control the scaffold fabrication process rigorously, instead of relying upon a trial and error process as previously reported.<p> In the scaffold fabrication process with the presence of living cells, cells are continuously subjected to mechanical forces. If the forces exceed certain level and/or the forces are applied beyond certain time periods, cell damage may result. In the second part of this research, a method to quantify the cell damage in the bio-dispensing process is developed. This method consists of two steps: one step is to establish cell damage models or laws to relate cell damage to the hydrostatic pressure / shear stress that is applied on cells; and the second step is to represent the process-induced forces that cells experience during the bio-dispensing process and then apply the established cell damage law to model the percent cell damage in the process. Based on the developed method, the cell damage percents in the scaffold fabrication processes that employ two types of dispensing needles, i.e., tapered and cylindrical needles, respectively, were investigated and compared. Also, the difference in cell damage under the high and low shear stress conditions was investigated, and a method was developed to establish the cell damage law directly from the bio-dispensing process. To validate the aforementioned methods and models, experiments of fabricating scaffolds incorporating Schwann cells or 3T3 fibroblasts were carried out and the percent cell damage were measured and compared with the simulation results. The validated models allow one to determine of the influence of process parameters, such as the air pressure applied to the process and the needle geometry, on cell damage and then optimize these values to preserve cell viability and/or achieve the desired cell distribution within the scaffolds.