Electrospinning is a polymer processing technique which allows for the production of nano to micro size fibers and scaffolds which can be composed of numerous synthetic biodegradable materials and natural biopolymers. Natively, elastin and collagen are the main components of vascular tissue. Arranged in a tri-layered structure, they create a specific mechanical environment that can withstand the rigors of circulation. The goal of this study was to develop a mechanically ‘biomimicking’ vascular graft composed of three distinct layers through the process of electrospinning. We hypothesize that the use of bioactive agents such as elastin, collagen, and silk to supplement poly(caprolactone) at specified ratios for each layer would provide a finely tuned vascular replacement. This was accomplished by establishing cross-linking parameters for the biopolymer materials and then assessing the mechanical properties of individual materials and eventually a whole tri-layered graft. Additionally, while mechanical testing can lead to a good graft, a replacement graft requires excellent cellular properties as well to promote cell infiltration, proliferation, and migration. Therefore, the conclusion of this study examines the integrin binding characteristics of the electrospun biopolymers. First, the results from the preliminary cross-linking study examined the dissipation of soluble elastin when uncross-linked v. cross-linked. It was determined through this initial study that synthetic scaffolds blended with soluble proteins such as elastin require a fixation in order to retain their protein mass within the scaffold. Retaining this mass, incrementally changed the material properties of the blended scaffolds. This initial study was then carried further to establish optimal cross-linking parameters using two different types of reagents: carbodiimide and genipin. It was found that lower cross-linking molarities produced excellent results based on assays performed to assess cross-linking percentages and rate of reaction. Some differences in mechanical properties were seen, but they did not constitute a choice of one cross-linker over the other. The next portion of this study aimed to design a tri-layered graft. This was performed with the aid of mathematical analysis to observe circumferential wall stresses based on simple tensile properties. A series of tri-layered grafts were electrospun using poly(caprolactone), elastin, and collagen. The medial layers of these grafts were changed while the intima and adventitia remained constant. Differences were demonstrated as the elastin content of the medial layer decreased, proving that each layer had an affect on the overall graft properties and that it was possible to tune graft mechanics. A larger tri-layered study looked to evaluate changes in the adventitial and medial layers while keeping the intimal layer constant using poly(caprolactone), elastin, collagen, and silk fibroin. In this study, differences were exhibited under compliance and burst strength testing, narrowing the scope of material choices. Results from a 4 week degradation study with the best tri-layered grafts revealed no evidence of degradation, but did generate some positive compliance results for two of the grafts. Finally, integrin binding and protein analysis portrayed results that were indicative of the existence of ligand binding sites for collagen scaffolds and the possibility of a small amount of ligand sites on silk. Elastin, however, displayed low to non-existent adhesion. These studies produced results that allowed us to continuously narrow the scope of materials as the experiment progressed towards an optimized tri-layered vascular graft.

<p>The design and application of effective drug carriers is a fundamental concern in the delivery of therapeutics for the treatment of cancer and other vexing health problems. Traditionally utilized chemotherapeutics are limited in efficacy due to poor bioavailability as a result of their size and solubility as well as significant deleterious effects to healthy tissue through their inability to preferentially target pathological cells and tissues, especially in treatment of cancer. Thus, a major effort in the development of nanoscopic drug delivery vehicles for cancer treatment has focused on exploiting the inherent differences in tumor physiology and limiting the exposure of drugs to non-tumorous tissue, which is commonly achieved by encapsulation of chemotherapeutics within macromolecular or supramolecular carriers that incorporate targeting ligands and that enable controlled release. The overall aim of this work is to engineer a hybrid nanomaterial system comprised of protein and silica and to characterize its potential as an encapsulating drug carrier. The synthesis of silica, an attractive nanomaterial component because it is both biocompatible as well as structurally and chemically stable, within this system is catalyzed by self-assembled elastin-like polypeptide (ELP) micelles that incorporate of a class of biologically-inspired, silica-promoting peptides, silaffins. Furthermore, this methodology produces near-monodisperse, hybrid inorganic/micellar materials under mild reaction conditions such as temperature, pH and solvent. This work studies this material system along three avenues: 1) proof-of-concept silicification (i.e. the formation and deposition of silica upon organic materials) of ELP micellar templates, 2) encapsulation and pH-triggered release of small, hydrophobic chemotherapeutics, and 3) selective silicification of templates to potentiate retention of peptide targeting ability.</p> / Dissertation

O objetivo deste estudo foi investigar os efeitos do hipotiroidismo experimental sobre o leito vascular da aorta torácica. Para a análise histológica foram realizadas colorações de hematoxilina-eosina, picrosirius e Weigert. Na análise de expressão proteica, foram realizadas as quantificações para colágeno I e III, elastina, MMP-9 e MMP-2, TIMP-1 e TIMP-2. As análises histológicas demonstraram uma diminuição da AST das aortas dos animais hipo e juntamente a esta alteração, foi constatada a diminuição da expressão proteica de colágeno do tipo I. Em relação à elastina, foi possível observar aumento da expressão deste elemento nas aortas dos animais hipotiroideos. Na avaliação da expressão proteica para MMP-9, foi possível verificar uma redução desta proteína no grupo hipotiroideo, assim como um aumento da expressão de TIMP-2. Frente aos presentes resultados, é possível sugerir que o estado hipometabólico desencadeado pelo hipotiroidismo afeta as CMLVs comprometendo mecanismos de síntese/degradação, alterando o importante arranjo da MEC presente na aorta torácica. / The aim of this study was to investigate hypothyroidism effects on thoracic aorta wall. For histological analyses were performed stains like hematoxilin-eosin, picrosirius and Weigert. In the protein expression assays were performed quantification for collagen I and III, elastin, MMP-9, MMP-2, TIMP-1 and TIMP-2. The histological analyses showed a decrease in aortas CSA and also a decrease in protein expression of collagen I in the hypo group. As regards to elastin, was possible to see an increase of this protein expression in hypo animals. In the evaluation for MMP-9 expression, was found a decrease in this protein and for TIMP-2 an increase in hypothyroidism group. Facing to these results, is possible to suggest that the hypometabolic state triggered by hypothyroidism, affects the VSMCs compromising mechanisms of synthesis/degradation and changing the important constitution of thoracic aorta ECM.

Blood contacting devices are frequently limited by complications such as surface-induced thrombosis. This thesis investigated the feasibility of using a family of recombinant elastin-like polypeptides (ELPs), namely ELP1, ELP2 and ELP4 that differ by molecular weight and sequence length, as potential thromboresistant coatings. The ELP coatings were prepared by physical adsorption onto the surface of Mylar, with surface modification confirmed by goniometry, X-ray photoelectron spectroscopy (XPS), and chemical force microscopy (CFM). Both surface wettability and hydrophilic adhesion force increased as the ELP sequence length decreased. The ELP adsorption process monitored by using quartz crystal microbalance with dissipation (QCM-D) showed that the ELPs adsorbed within a monolayer. Additionally, ELP surface coverage was found to increase with the polypeptide sequence length. The QCM-D studies also revealed that the longer polypeptides (ELP2 and ELP4) exhibited higher specific dissipation values indicating that they established adsorbed layers with greater structural flexibility and associated water content compared to ELP1. Exposure of the ELP coatings to flowing reconstituted blood demonstrated that both the ELP2 and ELP4 coatings reduced the quantity of adsorbed fibrinogen (Fg), with the ELP4 coating resulting in the lowest levels of adherent platelets. Energy dissipation versus frequency shift plots obtained from QCM-D studies indicated that adsorbed Fg on the ELP4 coating maintained a softer, more flexible film then on the other ELPs. The ELP4 coating also demonstrated an altered binding activity for GPIIb/IIIa where only the AGDV motif in the adsorbed Fg gamma-chain appeared to be exposed and bioactive. Conversely, on the other ELP coatings both the AGDV and RGD motifs (found within the Fg alpha-chain) were available for binding, suggesting that a different Fg conformational state exists on the ELP1 and ELP2 coatings. Moreover, both the ELP2 and ELP4 coatings displayed minimal bulk platelet reactivity following extended whole blood shear exposure (up to an hour) compared to Mylar. This was not observed with the ELP1 coating. Overall, the results suggest that the structural flexibility and associated water content of the ELP coatings appear to be important criteria influencing their thrombogenicity, with ELP4 displaying the most favourable blood-material response compared to ELP1 and ELP2.

The extracellular matrix is a vital component of the cardiovascular system, in that, it not only provides structural support but also plays a critical role in the maintenance of cellular stability. One of the major components of the vascular matrix is elastin, which confers vessels with the specialized property of stretch and recoil. Elastin deficiency has been implicated in many vascular diseases and determined experimentally to be a negative regulator of smooth muscle cell proliferation. In zebrafish, two elastin genes have been identified, which are actively expressed during development. Based on this finding, protein production and spatial localization for the two elastin proteins was studied by immunohistochemistry with specific antibodies. Results revealed a global distribution for elastin 1 in the ventral aorta and swim bladder, whereas elastin 2 was preferentially localized to the bulbus arteriosus indicating a possible specialized function of elastin 2 in this structure. This observation, and the unique physiological property of this structure, suggests a possible reason for the preservation of both elastin genes during evolution. / In the second part of this study, elastin-null mice were studied to uncover the impact of the loss of elastin on the expression of other elastic fiber-associated proteins. The expression of fibrillin-1, the major component of microfibrils, was not altered in the absence of elastin, implying that elastin is not necessary for the formation of microfibrils. On the other hand, both fibulin-2 and -5 were upregulated in the absence of elastin, suggesting that expression of these genes are controlled by elastin. Overall, this study highlights the importance of elastin in evolution, as well as its potential role in the regulation of expression of other matrix molecules.

Blood contacting devices are frequently limited by complications such as surface-induced thrombosis. This thesis investigated the feasibility of using a family of recombinant elastin-like polypeptides (ELPs), namely ELP1, ELP2 and ELP4 that differ by molecular weight and sequence length, as potential thromboresistant coatings. The ELP coatings were prepared by physical adsorption onto the surface of Mylar, with surface modification confirmed by goniometry, X-ray photoelectron spectroscopy (XPS), and chemical force microscopy (CFM). Both surface wettability and hydrophilic adhesion force increased as the ELP sequence length decreased. The ELP adsorption process monitored by using quartz crystal microbalance with dissipation (QCM-D) showed that the ELPs adsorbed within a monolayer. Additionally, ELP surface coverage was found to increase with the polypeptide sequence length. The QCM-D studies also revealed that the longer polypeptides (ELP2 and ELP4) exhibited higher specific dissipation values indicating that they established adsorbed layers with greater structural flexibility and associated water content compared to ELP1. Exposure of the ELP coatings to flowing reconstituted blood demonstrated that both the ELP2 and ELP4 coatings reduced the quantity of adsorbed fibrinogen (Fg), with the ELP4 coating resulting in the lowest levels of adherent platelets. Energy dissipation versus frequency shift plots obtained from QCM-D studies indicated that adsorbed Fg on the ELP4 coating maintained a softer, more flexible film then on the other ELPs. The ELP4 coating also demonstrated an altered binding activity for GPIIb/IIIa where only the AGDV motif in the adsorbed Fg gamma-chain appeared to be exposed and bioactive. Conversely, on the other ELP coatings both the AGDV and RGD motifs (found within the Fg alpha-chain) were available for binding, suggesting that a different Fg conformational state exists on the ELP1 and ELP2 coatings. Moreover, both the ELP2 and ELP4 coatings displayed minimal bulk platelet reactivity following extended whole blood shear exposure (up to an hour) compared to Mylar. This was not observed with the ELP1 coating. Overall, the results suggest that the structural flexibility and associated water content of the ELP coatings appear to be important criteria influencing their thrombogenicity, with ELP4 displaying the most favourable blood-material response compared to ELP1 and ELP2.

Erratum pages inserted inside back cover. / Bibliography: leaves 163-200. / xvi, 215 leaves, [13] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Measurement of elastin derived peptides may be a powerful tool to evaluate mechanisms of elastin breakdown in vivo and in vitro. In human studies EDP levels may provide an early indicator of subjects undergoing increased elastin degradation that may lead to emphysema, and may serve as a biological marker of the effectiveness of therapeutic antielastases. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 1994

Bibliography: leaves 266-303 / xviii, 303 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 1985

Erratum pages inserted inside back cover. / Bibliography: leaves 163-200. / xvi, 215 leaves, [13] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Measurement of elastin derived peptides may be a powerful tool to evaluate mechanisms of elastin breakdown in vivo and in vitro. In human studies EDP levels may provide an early indicator of subjects undergoing increased elastin degradation that may lead to emphysema, and may serve as a biological marker of the effectiveness of therapeutic antielastases. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 1994

Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Nerem, Robert; Committee Member: Chaikof, Elliot; Committee Member: Taylor, W. Robert; Committee Member: Vito, Raymond; Committee Member: Wight, Thomas.