Thesis (M.S.)--Florida State University, 2004. / Advisor: Dr. Teng Ma, Florida State University, College of Engineering, Dept. of Chemical and Biomedical Engineering. Title and description from dissertation home page (viewed Jan 12, 2005). Includes bibliographical references.

Thesis (doctoral)--Lund University, 1993. / Added t.p. with thesis statement inserted.

Suportes eletrofiados para crescimento celular são de interesse para a engenharia de tecidos, principalmente em função de sua estrutura em forma de rede tridimensional de fibras de diâmetro nanométrico. Esta arquitetura especial permite a geração de elevada área superficial e porosidade, características importantes para a adesão, proliferação e infiltração de células para o interior do suporte. A utilização de um suporte eletrofiado como enxerto vascular necessita ainda que este apresente excelentes propriedades mecânicas, associadas a uma elevada biocompatibilidade. Neste trabalho mostramos que estas propriedades podem ser alcançadas a partir da eletrofiação de uma co-solução de PET e colágeno gerando um material híbrido, visto que PET apresenta excelentes propriedades mecânicas e o colágeno é o principal componente da matriz extracelular. A obtenção dos suportes eletrofiados de PETcolágeno mostrou ser possível utilizando-se como solvente HFIP e HFIP/TFA 7:2. No entanto, neste último, o colágeno é completamente degradado durante o processo de solubilização. Fixando-se os parâmetros de eletrofiação, a morfologia da malha obtida mostrou ser dependente da relação massa PET/massa colágeno, concentração total da solução e solvente utilizado. Foram obtidos materiais com distribuição de diâmetros unimodal e bimodal, além de materiais com formato em fitas e teias entre as fibras. Ainda, PET e colágeno formam malhas de composição complexa, nas quais são encontradas fibras compostas de materiais puros, mas também formam blendas em que os dois materiais encontram-se misturados em uma mesma fibra. Os materiais S8,2 S4,6 foram caracterizados química-, mecânica- e biologicamente. Observou-se que, para filmes planos, estes materiais apresentaram energia de superfície mais próxima da do colágeno, o que justifica a melhor adesão celular em S8,2 e S4,6 do que no PET. S8,2 mostrou ter valores de módulo de elasticidade e elongação máxima próximos ao da artéria femoral, enquanto que S4,6 apresentou-se como um material quebradiço. Os ensaios de crescimento celular utilizando fibroblastos, um modelo de tecido conjuntivo (linhagem 3T3-L1) e células endoteliais, um modelo de tecido arterial e venoso (HUVECs) comprovaram a excelente adesão e proliferação celular nos suportes celulares. S8,2 apresentou-se como o melhor material frente às células HUVECS, enquanto que S4,6 foi o melhor material frente às células 3T3-L1. Propõe-se a utilização de S8,2 como um biomaterial para enxertia vascular e S4,6 como material de recobrimento de próteses já utilizadas. / Scaffolds obtained by electrospinning for cellular growth are of interest for materials engineering, especially considering its structure in the form of a three-dimensional fiber mesh of nanometric diameter. This special architecture allows the generation of larger surface areas and higher porosity structures, and also important characteristics for the adhesion, proliferation and infiltration of cells into the scaffold. The use of an electrospun scaffold as a vascular graft additionally requires excellent mechanical properties, associated with a high biocompatibility level. In this study we demonstrate that these properties can be achieved by means of electrospinning of PET and collagen co-solution producing a hybrid material, considering that PET possesses excellent mechanical properties and that collagen is the principal component of the extracellular matrix. The production of electrospun scaffolds of PET/collagen is shown to be possible using HFIP and HFIP/TFA 7:2 as solvents. However, in this last one, the collagen is completely degraded during the solubilization process. If the electrospinning parameters are maintained constant, the morphology of the mesh obtained was found to be dependent on the ratio of PET/collagen (w/w), total concentration of the solution and solvent employed. Materials were obtained with unimodal and bimodal diameter distribution, as well as material in the form of ribbons and mesh between the fibers. In addition, PET and collagen form a mesh of complex composition, in which fibers composed by pure and blended materials were found. The materials PET/collagen 80:20 (S8,2) and PET/collagen 40:60 (S4,6) were characterized chemically, mechanically and biologically. It was observed that, for spincoated films, these materials present a surface energy closer to that of collagen, explaining the better cellular adhesion in S8,2 e S4,6 than for PET. S8,2 presents very similar elasticity and elongation modulus values to the femoral artery, while S4,6 is a brittle material. The cellular growth experiments using fibroblasts as a model of conjunctive tissue (3T3-L1) and endothelial cells as a model of arterial and venous tissue (HUVEC) proved the excellent adhesion and cellular proliferation on the cellular PET/collagen scaffolds. S8,2 was shown to be the best material considering HUVEC cells, while S4,6 was the best material considering 3T3-L1 cells. According to the results obtained, the use of S8,2 is proposed as a biomaterial for vascular grafts and S4,6 as a material for a coating for vascular grafts prostheses.

For a considerable number of years the problem of revascularizing the myocardium after the development of coronary circulatory deficiency has stirred the imagination of physiologists, clinicians and surgeons. The obstruction in the coronary system brings a reduction in the coronary blood flow which is responsible for the symptoms and deterioration of the myocardial function In hearts otherwise normal or close to a standard normal state.

The role of matrix metalloproteinase (MMP)-9 and processing conditions of biodegradable polymer scaffolds has been investigated to optimize engineering vascular constructs. For a small diameter vascular construct, uniform 10 mm thickness of highly porous scaffolds were developed using a computer-controlled knife coater and exploiting phase transition properties of salts. The comparative study of fast vs. slow degrading three-dimensional scaffolds using a fast degrading poly D, L-lactic-glycolic acid co-polymer (PLGA) and a slow degrading poly e-caprolactone (PCL) indicated that fast degradation negatively affects cell viability and migration into the scaffold in vitro and in vivo, which is likely due to the fast polymer degradation mediated acidification of the local environment. MMP-9 was crucial for collagen remodeling process by smooth muscle cells (SMC). MMP-9 deficiency dramatically decreased inflammatory cell invasion as well as capillary formation within the scaffolds implanted in vivo. This study reports that the angiogenic response developed within the scaffolds in vivo was related to the presence of inflammatory response. Combinatorial polymer libraries fabricated from blended PLGA and PCL and processed at gradient annealing temperatures were utilized to investigate polymeric interactions with SMC. Surface roughness was also found to correlate with SMC adhesion. SMC aggregation, proliferation, and protein production, were highest in regions that exhibited increased surface roughness, reduced hardness, and decreased crystallinity of the PCL-rich phases. This study revealed a previously unknown processing temperature and blending compositions for two well-known polymers, which optimized SMC interactions.

Recombinant synthesis of elastin-mimetic proteins has been employed for several decades, however, long-term biocompatibility and biostability of such proteins was not fully defined. We present virtually crosslinked elastin-mimetic proteins which exhibit exceptional biocompatibility and long-term biostability over a period of at least seven months. This report is the first evidence of a non-chemically or ionically crosslinked system that exhibits long-term in vivo stability. Although, physically crosslinked protein-based materials possess a number of advantages over their chemically crosslinked counterparts, physical crosslinks and the related domains so formed may be deformed or damaged at applied stresses lower than those required to disrupt covalent crosslinks. In this regard, we have synthesized a new class of recombinant elastin-mimetic triblock copolymer capable of both physical and chemical crosslinking. We have demonstrated that chemical crosslinking provides an independent mechanism for control of protein mechanical responses. Specifically, elastic modulus was enhanced and creep strain reduced through the addition of chemical crosslinking sites. A number of reports have described the design of synthetic genes, which encode elastin-like proteins for bacterial expression in Escherichia coli. Although advantages with this expression system exist, significant limitations including the lack of eukaryotic post-translational systems, the tendency to sequester mammalian proteins into inclusion bodies, difficult purification protocols, and endotoxin contamination have been noted. We demonstrate the expression of a recombinant elastin-mimetic protein from P. pastoris. A novel synthetic strategy, monomer library concatamerization, was utilized in designing non-repetitive elastin genes for highly repetitive protein sequences. It is likely that this strategy will be useful for creating large, repetitive genes for a variety of expression systems in order to more closely approach the genetic diversity inherent to native DNA sequences. All told, elastin-based protein polymers are a promising class of material characterized by high degree of biocompatibility, excellent biostability, and a tunable range of mechanical properties from plastic to elastic. A variety of options facilitate the processing of these biopolymers into chemically crosslinked or non-crosslinked gels, films, or nanofibers for any of a number of implant applications including structural components of artificial organs and engineered living tissues, carriers for controlled drug release, or biocompatible surface coatings.

Cardiovascular disease is the main cause of death in the United States. Many of these conditions require the grafting or bypassing of compromised blood vessels. To this effect, biological vascular grafts (autografts and allografts) are the first line of action. However, when the patient lacks vasculature suitable for grafting use, several synthetic grafting options are available. The search for an inert biomaterial for vascular grafts has proven to be unsuccessful. This makes the interaction taking place on the blood-biomaterial interface critical for the success of the grafts. This thesis introduces a new bio-inspired approach to tackle the mechanical and biological challenges of vascular material design. The hypothesis of this research is that recombinant fibronectin protein can be stably incorporated onto elastin-mimetic polymers to increase endothelialization. Recombinant elastin, designed to recreate the mechanical properties of natural elastin as a candidate material for vascular graft fabrication, was used as a model surface. Recombinant fibronectin-functionalized elastin-mimetic polymer displayed significant improvement in cell adhesion. Quantification of surface bound recombinant fibronectin verified the concentration dependence of this cell adhesive behavior. Modified elastin-mimetic polymer also demonstrated an enhanced ability to support endothelial cell proliferation. Furthermore, the stability of recombinant fibronectin-modified polymers was assessed. These studies provide the foundation for fabricating elastin-mimetic vascular grafts with improved endothelialization and subsequent biological performance.

Biomimetic materials that recapitulate the complex mechanical and biochemical cues in load-bearing tissues are of significant interest in regenerative medicine and tissue engineering applications. Several investigators have endeavored to not only emulate the mechanical properties of the vasculature, but to also mimic the biologic responsiveness of the blood vessel in creating vascular substitutes. Previous studies in our lab generated the elastin-like protein polymer LysB10, which was designed with the capability of physical and chemical crosslinks, and was shown to display a range of elastomeric properties that more closely matched those of the native artery. While extensive validation of the mechanical properties of elastin-mimetic polymers has demonstrated their functionality in a number of tissue engineering applications, limited cell growth on the surfaces of the polymers has motivated further optimization for biological interaction. Recent biologically-inspired surface strategies have focused on functionalizing material surfaces with extracellular matrix molecules and bioactive motifs in order to encourage integrin-mediated cellular responses that trigger precise intracellular signaling processes, while limiting nonspecific biomaterial interactions. Consequently, this dissertation addresses three approaches to modulating cellular behavior on elastin-mimetic analogs with the goal of promoting vascular wall healing and tissue regeneration: genetic engineering of elastin-like protein polymers (ELPs) with cell-binding domains, biofunctionalization of elastin-like protein polymers via chemoselective ligation of bioactive ligands, and incorporation of matrix protein fibronectin for engineering of cell-seeded multilamellar collagen-reinforced elastin-like constructs. The synthesis of recombinant elastin-like protein polymers that integrate biologic functions of the extracellular matrix provides a novel design strategy for generating clinically durable vascular substitutes. Ultimately, the synthesis of model protein networks provides new insights into the relationship between molecular architecture, biomimetic ligand presentation, and associated cellular responses at the cell-material interface. Understanding how each of these design parameters affects cell response will contribute significantly to the rational engineering of bioactive materials. Potential applications for polymer blends with enhanced mechanical and biological properties include surface coatings on vascular grafts and stents, as well as composite materials for tissue engineered scaffolds and vascular substitutes.

The development of small diameter tissue engineered blood vessels (TEBVs) with low thrombogenicity, low immunogenicity, suitable mechanical properties, and a capacity to remodel to their environment could significantly advance the treatment of coronary and peripheral artery disease. Despite significant advances in the field of tissue engineering, autologous vessels are still primarily utilized as grafts during bypass surgeries. However, undamaged autologous tissue may not always be available due to disease or prior surgery. TEBVs lack long-term efficacy due to a variety of types of failures including aneurysmal dilations, thrombosis, and rupture; the mechanisms of these failures are not well understood. In vitro mechanical testing may help the understanding of these failure mechanisms. The typical mechanical tests lack standardized methodologies; thus, results vary widely. The overall goal of this study is to develop novel experimental and mathematical models to study the mechanical properties and failure mechanisms of TEBVs. Our results suggest that burst pressure tests, the current standard, are not sufficient to assess a TEBVs’ suitability as a coronary substitute; creep and/or cyclic loading tests are also required. Results from this model can help identify the most insightful experiments and quantities to be measured – ultimately reducing the overall number of experimental iterations. Improving the testing and characterization of TEBVs is critically important in decreasing the time necessary to validate the mechanical and functional responses of TEBVs over time, thus quickly moving TEBVs from the benchtop to the patient.

Compliance mismatch is a significant hurdle to long-term patency in small-diameter arterial bypass grafts. Vascular tissue engineering has the potential to produce compliant, non-thrombogenic small-diameter grafts. However, current engineered grafts are relatively non-compliant, resulting in intimal hyperplasia and graft occlusion when subjected to arterial pressures. This research investigates the mechanical and biological properties of engineered constructs based on a biodegradable synthetic elastomer, poly(glycerol sebacate) (PGS). Several methods for fabricating porous PGS scaffolds in a tubular geometry were developed and compared. Adult baboon vascular cells were cultured in the scaffolds under various in vitro experimental conditions, including variations in initial cell seeding density, the type of scaffold used for culture, culture time, scaffold material, and hydrostatic pressure, and properties of the resultant constructs were compared. Scaffold fabrication using heat-shrinkable mandrels and glass tubes coated with hyaluronic acid significantly decreased tolerances of wall thickness and mechanical properties, improved handling, and decreased culture time required to reach luminal cellular confluence compared to scaffolds made with other fabrication techniques. Altering scaffold material from PGS to poly(lactide-co-glycolide) (PLGA), a benchmark biomaterial, did not affect scaffold yield, porosity, or luminal cellular confluence. Extracellular matrix (ECM) deposition increased with SMC-only culture time, and ECM deposition and remodeling during culture influenced construct compliance. Compared to PLGA scaffolds, PGS scaffolds promoted elastin crosslinking by SMCs and elastic tissue properties but attenuated collagen deposition. Hydrostatic pressure promoted ECM synthesis and deposition by SMCs and decreased construct compliance. Collagen and crosslinked elastin content in constructs correlated positively with construct burst pressure, and a negative correlation dependent on scaffold type was found between collagen content and construct compliance at low pressures. The systematic investigation of culture conditions in this research provides insights into the control of engineered blood vessel properties. The central hypothesis of this work, that grafts engineered from PGS scaffolds and adult vascular cells under biomimetic in vitro culture conditions can possess compliance comparable to autologous vessels, is true at pressures below 60 mmHg and demonstrates potential for PGS-based vascular tissue engineering. Overall, this work provides tools for engineering tubular soft tissues based on porous PGS scaffolds.