Tissue engineered heart valve (TEHV) is believed to be a promising candidate for curative heart valve replacements. Collagen, elastin and chondroitin-4-sulfate (C4S) comprise the extra-cellular matrix (ECM) of native heart valves and therefore are suitable materials for TEHV scaffolds. Freeze-drying technique was able to produce scaffolds with relative densities of 0.3%-2.0% and pore sizes of 33.2µm-201.5µm, without having any major effects on the ultra-structures on the scaffold materials. Subsequent dehydrothermal (DHT) treatment and ultra-violet (UV) irradiation introduced inter- or intra-molecular crosslinks in the scaffolds in forms of ester and amide bonds, as well as the accompanying denaturation of the proteins (i.e. ultra-structure transition from helices to random coils). The collagen-based scaffolds had tensile, compressive and effective bending moduli ranging from 39.8kPa to 1082kPa, from 2.4kPa to 213.9kPa, and from 11.0kPa to 415.8kPa, respectively. The different behaviours of the wall stretching and the wall buckling in the individual pores of the scaffolds contributed to the different tensile, compressive and bending moduli. The mechanical properties could be tailored through controlling the freezing temperature, the relative density and the composition of the scaffolds. A lower freezing temperature might lead to lower mechanical properties because different pore structures were introduced. When the the relative density of the scaffold increased, the values of the moduli increased exponentially, with an exponential dependence factor larger for the compressive modulus than for the tensile modulus. Adding elastin or C4S into the collagen scaffolds lowered the mechanical properties due to the decrease in the collagen content. Layered structures that combined collagen-rich layers with elastin-rich and/or C4S -rich layers allowed the scaffolds to make use of the different mechanical properties of different layers, and hence to show anisotropic bending behaviour depending on the loading directions. The lower effective bending modulus (9.6 to 25.0kPa) in the with curvature (WC) direction than that (18.1kPa to 39.3kPa) in the against curvature (AC) direction mimicked the characteristic behaviour of the native heart valves and would be beneficial for a mechanically desirable TEHV. The DHT treatment and UV irradiation were able to increase the mechanical properties of the scaffolds to up to 2.5 times of the original values, by reinforcing the scaffold materials with more crosslinks. In the hydrated status, the hydrophilic C4S improved the water uptake ability of the scaffold and the hydrophobic elastin reduced it. The hydrated layered scaffolds still exhibited bending anisotropy despite much lower effective bending modulus. Finite element models of the scaffolds produced results that were in agreement with the experiments, and enabled us to perform distributed loading and internal stress analysis on the scaffolds. The collagen-based scaffolds were seeded with cardiosphere-derived cells (CDCs), and they attached to the scaffolds and showed visible cell division, proliferation and migration. The CDCs exhibited preferred proliferation behaviours on the collagen-C4S scaffolds to that on the collagen-elastin scaffolds because of the cell affinity to the C4S, as well as the elastin-induced contractile cell phenotype and scaffold volume shrinkage. This difference seemed to be less evident in the layered scaffolds due to the cell communication between the layers. The crosslinking process also had effects on the cell proliferation in the ways that it induced ultra-structure changes or volume shrinkage in the scaffolds. The layered scaffold-cell constructs designed and produced in this study served as a forwarding step towards a mechanically desirable and biologically active TEHV.

The development of sustainable replacements for fossil fuels has been spurred by concerns over global warming effects. Biofuels are typically produced through fermentation of edible crops, or forest or agricultural residues requiring cost-intensive pretreatment. An alternative is to use photosynthetic cyanobacteria to directly convert CO2 and sunlight into fuel. In this thesis, the cyanobacterium Synechocystis sp. PCC 6803 was genetically engineered to produce the biofuel n­-butanol. Several metabolic engineering strategies were explored with the aim to increase butanol titers and tolerance. In papers I-II, different driving forces for n-butanol production were evaluated. Expression of a phosphoketolase increased acetyl-CoA levels and subsequently butanol titers. Attempts to increase the NADH pool further improved titers to 100 mg/L in four days. In paper III, enzymes were co-localized onto a scaffold to aid intermediate channeling. The scaffold was tested on a farnesene and polyhydroxybutyrate (PHB) pathway in yeast and in E. coli, respectively, and could be extended to cyanobacteria. Enzyme co-localization increased farnesene titers by 120%. Additionally, fusion of scaffold-recognizing proteins to the enzymes improved farnesene and PHB production by 20% and 300%, respectively, even in the absence of scaffold. In paper IV, the gene repression technology CRISPRi was implemented in Synechocystis to enable parallel repression of multiple genes. CRISPRi allowed 50-95% repression of four genes simultaneously. The method will be valuable for repression of competing pathways to butanol synthesis. Butanol becomes toxic at high concentrations, impeding growth and thus limiting titers. In papers V-VI, butanol tolerance was increased by overexpressing a heat shock protein or a stress-related sigma factor. Taken together, this thesis demonstrates several strategies to improve butanol production from cyanobacteria. The strategies could ultimately be combined to increase titers further.

The integration of functional and structural properties makes genetically engineered proteins appealing in tissue engineering. Silk-elastinlike proteins (SELPs), containing tandemly repeated polypeptide sequence derived from natural silk and elastin, are recently under active study due to the interesting structure. The biological, chemical, physical properties of SELPs have been extensively investigated for their possible applications in drug/gene delivery, surgical tissue sealing and spine repair surgery. However, the mechanical aspect has rarely been looked into. Moreover, many other biomaterials have been fabricated into fibers in micrometer and nanometer scale to build extracellular matrix-mimic scaffolds for tissue regeneration, but many have one or mixed defects such as: poor strength, mild toxicity or immune repulsion etc. The SELP fibers, with the intrinsic primary structures, have novel mechanical properties that can make them defects-minimized scaffolds in tissue engineering.In this study, one SELP (SELP-47K) was fabricated into microfibers and nanofibers by the techniques of wet-spinning and electrospinning. Microfibers of meters long were formed and collected from a methanol coagulation bath, and later were crosslinked by glutaraldehyde (GTA) vapor. The resultant microfibers displayed higher tensile strength up to 20 MPa and higher deformability as high as 700% when tested in hydrated state. Electrospinnig of SELP-47K in formic acid and water resulted in rod-like and ribbon-like nanofibrous scaffolds correspondingly. Both chemical (methanol and/or GTA) and physical (autoclaving) crosslinking methods were utilized to stabilize the scaffolds. The chemical crosslinked hydrated scaffolds exhibit elastic moduli of 3.4-13.2 MPa, ultimate tensile strength of 5.7-13.5 MPa, and deformability of 100-130%, closely matching or exceeding the native aortic elastin; while the autoclaved one had lower numbers: 1.0 MPa elastic modulus, 0.3 MPa ultimate strength and 29% deformation. However, the resilience was all above 80%, beyond the aortic elastin, which is 77%. Additionally, Fourier transform infrared spectra showed clear secondary structure transition after crosslinking, explaining the phenomenon of scaffold water-insolubility from structural perspective and showed a direct relationship with the mechanical performance. Furthermore, the in vitro biocompatibility of SELP-47K nanofibrous scaffolds were verified through the culture of NIH 3T3 mouse embryonic fibroblast cells.

Cancer is lacking early detection methods and treatment specificity. In order to increase the sensitivity and specificity towards cancer cells, we propose the use of multivalent interactions targeting specific receptor combinations at the cancer cell surface. In this thesis, we explored the design of multimers, which could provide such interactions. The design was investigated and revisited based on specific parameters, essential for the creation of multivalent interactions such as thermodynamics. The synthesis was designed so that libraries of homo- and hetero-multimers of different valencies can be obtained efficiently with good yields. The established synthetic scheme is empowered by its modularity, necessary to investigate different essential factors. Trimers composed of micromolar affinity MSH(4) targeting the MC1-R, overexpressed in melanoma, were investigated on a model cell line and resulted in the creation of nanomolar affinity constructs with up to 350 fold increase in affinity. Different multimers such as hexavalent and nonavalent dendrimers were synthesized and studied for their properties. All constructs had nanomolar affinity and showed to be non-toxic up to micromolar concentrations and imaging studies also confirmed their internalization, which overall demonstrate the potential for these compounds to be used as markers for cancer cells and as delivery agents. Trimers targeting the CCK2-R were similarly investigated for their potential as pancreatic cancer markers. However, those constructs did not seem to result in the expected enhancements in affinity, but the affinity of the initial monovalent agonist was in the 10-50 nanomolar range. As we were unable to design micromolar affinity agonist we investigated the use of antagonists. This study, revealed the importance of thermodynamics in the creation of multivalent interaction. Heterotrivalent ligands (CCK and MSH) were investigated for their potential in cross-linking different receptors and the study demonstrated the subtility to detect cross-linking. Finally, the different attempts toward the efficient synthesis of a tetra-orthogonal scaffold, a key feature needed to generate multimers that could target up to 3 different receptors was investigated and showed promising results. It is our hypothesis that such an approach will ultimately lead to specific markers of tumor cells, which could be used as diagnosis agents when modified with an imaging moiety and as a therapeutic agent when modified with a drug.

No description available.

Common therapy for diabetes mellitus is subcutaneous administration of insulin that is subject to serious disadvantages, such as patient noncompliance and occasional hypoglycemia. Hence, oral administration of insulin could be more convenient and serve as a desired route. However, oral administration of insulin is severely limited by the low bioavailability of insulin through the gastrointestinal tract. In this study, a semi-interpenetrating network gelatin fiber scaffold (sIPN GF) was fabricated for oral mucosal delivery of insulin as an alternative route. This sIPN GF was engineered from an electrospun gelatin fiber scaffold (GF), which was further crosslinked with polyethylene glycol diacrylate (PEG-DA) to enhance its stability. Within the crosslinking process, eosin Y served as a photoinitiator, and the ratio of PEG-DA to eosin Y was optimized with respect to cytocompatibility and degradation rate. The results showed that the fabricated scaffold morphology, mechanical properties, and degradation rate were significantly enhanced after the crosslinking process. This optimized formulation was used to fabricate sIPN gelatin-co-insulin fiber scaffold (sIPN GIF). Enzyme-linked immunosorbent assay (ELISA) was used to monitor the insulin releasing kinetics of sIPN GIF. Western blot analysis showed that sIPN GIF activated intracellular AKT phosphorylation in a releasing time-dependant manner. Oil red O staining confirmed the released insulin was able to induce 3T3-L1 preadipocyte differentiation. The permeability of insulin from sIPN GIF was determined on the order of 10^-7 cm/s using a vertical Franz diffusion cell system mounted with porcine buccal mucosa. These findings suggest that sIPN GIF holds a great potential for oral mucosal delivery of insulin.

The purpose of this work is to identify the parameters of scaffold's mechanical properties by observing/monitoring their response to defined external mechanical strain. The first part of the work is summarizing the knowledge about the required properties of scaffolds, their production and the factors influencing production. The practical part of the work concerns itself with measurement, analysis and evaluation of data based on proprietary methodology. Based on the results at this stage of the research, we can confirm, that scaffolds have viscoelastic, or viscoplastic character and its response depends on the magnitude of deformation, state of hydration, ratio of solutions and period of networking. Keywords: scaffod, tensile test, rheologic model

Orientador: Fernando Zambuzzi Willian / Resumo: Artigo 1 Dos transplantes intestinais convencionais à Engenharia de Tecidos: estado da arte Resumo O intestino delgado é responsável pela realização de múltiplas funções, incluindo motilidade, digestão e absorção. Nos distúrbios gastrointestinais, algumas dessas funções são prejudicadas ou perdidas. A ressecção do segmento doente é uma abordagem comum. No entanto, os pacientes sofrem de complicações e de baixa qualidade de vida. Substituições funcionais são, portanto, necessárias para restaurar, reparar ou substituir partes danificadas do intestino. A Engenharia de Tecidos e a Medicina Regenerativa fornecem uma abordagem alternativa para reconstruir diferentes segmentos do trato gastrointestinal. Os desafios são grandes e a área é extremamente promissora. Artigo 2 Avaliação e caracterização de scaffolds de matriz extracelular para aplicações na Engenharia de Tecidos do trato gastrointestinal Resumo Várias estratégias têm sido exploradas para aumentar o número de enxertos de intestino delgado para transplantes e, principalmente, para diminuir as taxas de rejeição deste órgão transplantado. Avanços recentes na Medicina Regenerativa estão fornecendo novas abordagens para os estudos de reparação ou substituição funcional do intestino afetado, seja por traumas ou por doenças. Neste contexto, a Engenharia de Tecidos pode ser uma opção para produzir scaffolds biocompatíveis para a produção de segmentos de intestino delgado em laboratório. Desta forma, o objetivo deste estudo foi ava... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: From conventional intestinal transplants to Tissue Engineering: state of the art ABSTRACT The small intestine is responsible for performing multiple functions including motility, digestion and absorption of food and nutrients. In gastrointestinal disorders, some of these functions are impaired or lost. Resection of the diseased segment is a common approach; however, patients suffer from complications and poor quality of life. Functional replacements are therefore necessary to restore, repair or replace damaged parts of the gut. Tissue Engineering and Regenerative Medicine provide an alternative approach to reconstruct different segments of the gastrointestinal tract. There are many challenges and the research field is extremely promising. Evaluation and characterization of extracellular matrix scaffolds for applications in Tissue Engineering of the gastrointestinal tract ABSTRACT Several strategies have been explored to increase the number of small bowel grafts intended for transplants and, mainly, to decrease the rejection rates of this transplanted tissue. Recent advances in Regenerative Medicine are providing new approaches for studies of functional repair or replacement of the affected bowel, whether by trauma or disease. In this context, Tissue Engineering has become an option to produce biocompatible scaffolds in order to produce segments of the small intestine in the laboratory, mainly through the application of ex-vivo tissue decellularization methodologies. Thus, the... (Complete abstract click electronic access below) / Doutor

A engenharia tecidual visa repor, reparar ou ajudar a regenerar tecidos e órgãos danificados por meio da combinação de biomateriais, biomoléculas e células. Scaffolds de nanofibras biodegradáveis mimetizam a matriz extracelular natural fornecendo uma estrutura ideal para o crescimento celular. Blendas de policaprolactona (PCL) e gelatina são biodegradáveis e proporcionam uma combinação de boas propriedades mecânicas, do PCL, com a hidrofilicidade e caráter que promove a adesão celular, da gelatina. Neste contexto, o objetivo deste trabalho é avaliar a importância das diferentes espessuras de scaffolds eletrofiados em relação às suas propriedades principais. Quatro conjuntos de scaffolds de PCL/gelatina com diferentes espessuras foram produzidos sob as mesmas condições apenas aumentando o tempo de duração do processo de eletrofiação. Os resultados indicam que as espessuras aumentaram proporcionalmente ao tempo de eletrofiação, variando de 100 nm a 300 nm nos períodos de 1 a 3 horas, enquanto a densidade aparente e a porosidade mantiveram-se constantes. As micrografias das membranas revelaram fibras lisas com diâmetros maiores para os scaffolds de menor espessura, e fibras irregulares com diâmetros menores e regiões fundidas ou ligadas para os scaffolds de maior espessura. Além disso, o aumento da espessura melhorou a resistência mecânica e a molhabilidade dos scaffolds. A esterilização por peróxido de hidrogênio não modificou quimicamente a composição das membranas de PCL/gelatina, embora algumas amostras tenham se deformado. As membranas também apresentaram bons resultados de citotoxicidade, melhorando a viabilidade celular, apesar desses valores diminuírem minimamente para os scaffolds de maior espessura, provavelmente devido à maior quantidade de PCL. O teste de adesão não foi conclusivo e deverá ser repetido. / Tissue engineering aims to replace, repair, or helping regenerate damaged tissues and organs through the combination of biomaterials, biomolecules and cells. Biodegradable nanofibrous scaffolds mimic the natural extracellular matrix providing an ideal structure to cellular growth. Blends of polycaprolactone (PCL) and gelatin are biodegradable and provide a combination of good mechanical properties, from PCL, with the hydrophilicity and cell adhesion promoter character, from gelatin. The aim of this work was to evaluate the importance of the thickness of electrospun scaffolds on their key properties. Four sets of PCL/gelatin scaffolds with different thicknesses were produced under the same conditions by simply increasing the time length of electrospinning process. Results indicate that the thickness increases proportionally to the electrospinning time, varying from 100 nm to 300 nm in periods of 1 to 3 hours, while the apparent density and porosity remained constant. Micrographs from the nonwoven mats revealed smooth fibers with larger diameters in the thinner scaffold, and irregular fibers with smaller diameters and molten or bonded regions as the thickness increased. Furthermore, the increase of thickness improved mechanical resistance and wettability of the scaffolds. Plasma sterilization did not modify chemical composition of PCL/gelatin membranes, although some samples have been deformed. Membranes also presented good results for cytotoxicity, improving cell viability, despite these values decreased minimally to the thicker scaffolds, probably due to the higher amount of PCL. Adhesion test was not conclusive and might be repeat.

Cardiovascular disease is the leading cause of death in the United States, accounting for approximately 25% of total deaths. Myocardial infarction (MI) is an extreme case of cardiovascular disease where ischemia leads to irreversible tissue necrosis. As the heart lacks the capacity to endogenously regenerate, the infarcted region is negatively remodeled, reducing cardiac function. Current therapies are not able to regenerate cardiac function post-MI, requiring novel approaches such as tissue engineering. However, there are three major pitfalls that are currently limiting the clinical translation of a tissue engineered cardiac patch: lack of proper vascularization within the tissues; biocompatible material; and lack of electrical integration between engineered tissue and host. The research within this dissertation aimed to engineer solutions to overcome these three pitfalls. Plants and animals exploit fundamentally different approaches to transporting fluids, yet there are surprising structural similarities. To take advantage of these similarities, we looked across different kingdoms and investigated whether plants and their innate vasculature could serve as perfusable scaffolds for tissue engineering. Standard perfusion decellularization techniques were adapted and applied to spinach leaves, which were found to be fully devoid of DNA following processing. Leaf vasculature remained patent post-decellularization and supported transport of various sized microparticles. Human cells successfully seeded onto and inside the plant scaffolds. Decellularized leaves were found to be nearly void of any cytotoxic affects. Leaf biocompatibility was then investigated in vivo through subcutaneous implantation in a rat model. Leaf scaffolds were found to be biocompatible after 4 weeks of implantation. Furthermore, leaves that were pre-functionalized with an RGD-dopamine peptide were fully integrated into the host tissue within one week. This shows the leaf scaffold’s potential to be an immuno-modulatory material, depending upon the intended application. Electrically conducting biofibers were engineered through the combination of fibrin microthreads and engineered conductive HEK293 cells. Biofibers could act as a modular platform to allow for electrical integration between the host tissue and any engineered cardiac patch. Biofibers directionally carried electrical current and were found capable of bridging electrical signal between two separate clusters of cardiomyocytes. In vivo investigation bridging a biofiber from the left atria to the left ventricle was accomplished in a rat model. Electrical maps demonstrated a visible accessory pathway that created a feedback electrical signal from the ventricle to the atria through the implanted biofiber. These results demonstrate electrical integration in vivo between host myocardium and the engineered biofiber.