A engenharia de tecidos e a medicina regenerativa possuem como objetivo principal o restabelecimento morfológico/funcional de tecidos e órgãos lesionados com a utilização de células, matrizes celulares e células tronco, controlando as respostas imunológicas/bioquímicas promovidas pelo organismo. Adicionalmente, a ciência dos materiais busca desenvolver biomateriais biocompatíveis que não promovam reações imunológicas indesejadas e proporcionem o reestabelecimento das funções do tecido/órgão. Polímeros de origem natural destacam-se como biomateriais por assemelharem-se a macromoléculas biológicas, similaridade com a matriz extracelular, menor possibilidade de estimulação de inflamação crônica e baixa ou ausência de toxicidade. O presente trabalho teve como objetivo desenvolver matrizes macromoleculares originadas do óleo de soja epoxidado (OSE), analisando a relação estrutura química/atividade biológica das matrizes macromoleculares para uso como biomaterial na engenharia de tecidos. A síntese do OSE foi efetuada pela rota oleoquímica, cuja eficiência foi determinada por espectroscopia de infravermelho e o rendimento da reação de 85% determinado por ressonância magnética nuclear de prótons. A partir da análise por calorimetria exploratória diferencial, detectou-se uma diminuição da temperatura de transição vítrea do polímero do óleo de soja (POSE) em relação ao OSE, sugerindo aumento do crescimento das cadeias poliméricas do POSE. Através da análise termogravimétrica, foi possível definir o perfil de degradação do OSE, com degradação em duas etapas, e do POSE, que degrada em apenas uma etapa e demonstra maior estabilidade térmica do POSE pelo aumento das interações moleculares. A reticulação e a hidrofilicidade do POSE foram promovidas com a adição de metacrilato de 2-hidroxietila (HEMA) à formulação por enxertia do monômero pela irradiação gama. Os resultados obtidos identificaram aumento da estabilidade mecânica, da gelificação e da absorção de água com o aumento do conteúdo de HEMA. Por fim, o grau de cristalinidade estimado para esses polímeros enxertados com HEMA de 27,5% foi definido através da difratometria de raios-X. A segunda etapa caracterizou-se pelo (i) desenvolvimento de POSEs com a enxertia de HEMA nas proporções OSE/HEMA 90:10 e 65:35 com irradiação por raios gama nas doses de 50 e 100kGy, (ii) caracterização físico-química dos POSE-HEMA e (iii) análise biológica desses materiais. Através da espectroscopia de infravermelho, pode-se detectar as regiões epoxidadas do POSE, assim como o sucesso da enxertia do monômero HEMA em todas as concentrações e doses de radiação utilizadas. Através da calorimetria exploratória diferencial, calculou-se a energia de ativação (Ea) dos polímeros. A cristalinidade dos materiais foi definida por difratometria de raios-X, mostrando caráter amorfo do material, bem como um pequeno incremento na porcentagem da cristalinidade com o aumento da intensidade das doses de radiação durante a síntese e um decréscimo dessa cristalinidade com o aumento na concentração de HEMA. A análise da citotoxicidade das amostras mostrou a ausência de toxicidade dos POSE-HEMA, confirmando a eficiência das lavagens dos polímeros para retirada de resíduos do processamento. A análise da hemocompatibilidade mostrou ausência de adesão de plaquetas e os testes de crescimento celular nas matrizes foram positivos. Através dos resultados obtidos nesta pesquisa, pôde-se concluir pelo potencial de utilização dos POSE-HEMA na engenharia de tecidos. / Tissue engineering and regenerative medicine have as main objective the morphologic/functional reestablishment of injured tissues and organs using cells, scaffolds, stem cells and control of immunological/biochemical responses promoted by the body. In addition, materials science seeks to develop biocompatible biomaterials that do not promote unwanted immune responses and provide the re-establishment of the functions of the tissue/organ. Polymers of natural origin stand out as biomaterials to resemble biological macromolecules, similarity to the extracellular matrix, reduced chance of inflammation and chronic pacing low or no toxicity. This study aimed the development of macromolecular arrays originated from epoxidized soybean oil (OSE), analyzing the relationship between the chemical structure/biological activity of the macromolecular arrays for use as biomaterials in tissue engineering. The synthesis of OSE was performed through the oleochemical route, whose efficiency was determined by infrared spectroscopy and the reaction yield of 85%, determined by nuclear magnetic resonance spectroscopy. From the analysis by differential scanning calorimetry, it was detected a decrease of the glass transition temperature of the epoxidized soybean oil polymer (POSE) compared with OSE, suggesting an increase of the growth of polymer chains of POSE. Thermogravimetric analysis was performed to define the OSE degradation profile, which degrades in two steps. The POSE degrades in just one step and shows higher thermal stability by the increased molecular interactions. The hydrophilicity and crosslinking of POSE was promoted by the addition of 2-hydroxyethyl methacrylate (HEMA) with the monomer grafting by gamma irradiation. The results showed an increased mechanical stability, gelation and water absorption with the HEMA content increasing. Finally, the degree of crystallinity for such polymers grafted with HEMA was 27.5%, estimated by X-ray diffractometry. The second stage was characterized by (i) developing POSEs with the grafting of HEMA in the proportions OSE / HEMA 90:10 and 65:35 irradiated by gamma rays at doses of 50 and 100kGy, (ii) physico-chemical characterization of POSE-HEMA and (iii) analysis of biological materials. By infrared spectroscopy, it was detect the epoxidized regions of POSE, as well as the successful grafting of the monomer HEMA concentrations with all radiation doses. By differential scanning calorimetry, the activation energy was calculated (Ea) of the polymers. The crystallinity of the material was defined by X-ray diffraction, showing tendency of amorphous material as well as a small percentage of the increase in crystallinity with increasing intensity of radiation doses during this synthesis and a decrease in crystallinity with the increasing concentration of HEMA. The analysis of the samples did not show cytotoxicity on POSE-HEMA and confirmed the efficiency of polymer washings to remove the processing waste. The analysis of hemocompatibility showed any platelet adhesion and the cell growth on the scaffolds was positive. From the results obtained in this research, we concluded by the potential use of POSE-HEMA in tissue engineering.

A eletrofiação é uma celebrada técnica de processamento de polímeros, capaz de produzir fibras de diâmetro nanométrico. A montagem comum do sistema de eletrofiação permite a captação de fibras aleatórias sob a forma de um não-tecido. Diversas modificações nessa montagem permitem a obtenção de diferentes morfologias de fibras. Tais modificações são revisadas e discutidas neste trabalho. Na produção de suportes de crescimento de células neurais, é interessante que seja incorporada alguma anisotropia no meio. Assim, um aparato de eletrofiação, capaz de produzir fibras alinhadas, foi construído e a variação dos parâmetros de seu processamento permitiu a obtenção de diferentes qualidades de alinhamento das fibras para dois polímeros biodegradáveis. Diversos parâmetros influenciaram a qualidade desse alinhamento, porém a velocidade de captação das fibras mostrou ser o mais impactante, em acordo com dados reportados na literatura. A morfologia das fibras foi avaliada quanto ao seu diâmetro, com o auxílio de micrografias de MEV e do software de edição de imagens ImageJ. Adicionalmente buscou-se avaliar a qualidade do alinhamento de tais fibras. Para tanto, foi desenvolvida uma metodologia de quantificação de qualidade de alinhamento de fibras, baseado nas micrografias e na ferramenta de FFT do ImageJ. A metodologia proposta foi capaz de ordenar de maneira objetiva e consistente a qualidade do alinhamento das fibras obtidas, mesmo quando a análise visual (usada como referência) se provava ineficiente. A metodologia proposta foi incorporada num plugin para ImageJ, via algoritmo computacional escrito em Java. Com o uso do plugin, foi possível processar diversas micrografias, obtidas em diferentes pontos das malhas eletrofiadas e com variadas magnificações, a fim de se criar uma estatística dos resultados obtidos para qualidade de alinhamento das fibras, algo inédito na literatura. Malhas eletrofiadas com diferentes qualidades de alinhamento de suas fibras foram utilizadas como substrato na cultura de células precursoras neurais, provenientes de neuroesferas. Foi feita a cultura de células progenitoras neurais, provenientes de neuroesferas, tendo como substrato malhas eletrofiadas com diferentes qualidades de alinhamento, a fim de se avaliar o impacto dos contatos físicos das fibras sobre a migração e diferenciação de tais células. / Electrospinning is a celebrated technique of polymer processing, able to produce fibers with nanometric diameter. Common assembly of electrospinning apparatus allows collection of random fibers in a non-woven matt. Several modifications on this assembly enable different fiber morphologies to be obtained. Such modifications are revised and discussed in this work. In the production of cell growth scaffolds, its interesting that some anisotropy is incorporated in the medium. Therefore, an electrospinning apparatus capable of producing aligned fibers was constructed. Variation of processing parameters of said apparatus enabled different alignment qualities of fibers to be attained for two biodegradable polymers. Many parameters influenced on the quality of said alignment; fiber collection speed, however, proved more impacting, in accordance with literature data. Fiber morphology was assessed in regard to its diameter with the aid of MEV micrographs and ImageJ software. Furthermore, assessment of fiber alignment quality was sought. For this matter, it has been developed a quantification methodology for fiber alignment quality, based on micrographs and ImageJ\'s FFT tool. The proposed methodology was able to objectively and consistently rank fiber alignment quality, even when visual analysis (used as reference) failed to do so. This methodology was incorporated in a plugin for ImageJ, via Java script algorithm. With the aid of this plugin it was feasible to process several micrographs, taken from electrospun mats at different spots and magnifications. This helped create statistics about obtained results of fiber alignment quality, on an unprecedented approach in written literature. Electrospun mats with varying quality in fiber alignment were used as substrate in the culture of neural precursor cells from neurospheres to assess the influence of contact guidance on migration and differentiation of such cells

This work presents the development of entropy-elastic gelatin based networks in the form of films or scaffolds. The materials have good prospects for biomedical applications, especially in the context of bone regeneration. Entropy-elastic gelatin based hydrogel films with varying crosslinking densities were prepared with tailored mechanical properties. Gelatin was covalently crosslinked above its sol gel transition, which suppressed the gelatin chain helicity. Hexamethylene diisocyanate (HDI) or ethyl ester lysine diisocyanate (LDI) were applied as chemical crosslinkers, and the reaction was conducted either in dimethyl sulfoxide (DMSO) or water. Amorphous films were prepared as measured by Wide Angle X-ray Scattering (WAXS), with tailorable degrees of swelling (Q: 300-800 vol. %) and wet state Young’s modulus (E: 70 740 kPa). Model reactions showed that the crosslinking reaction resulted in a combination of direct crosslinks (3-13 mol.-%), grafting (5-40 mol.-%), and blending of oligoureas (16-67 mol.-%). The knowledge gained with this bulk material was transferred to the integrated process of foaming and crosslinking to obtain porous 3-D gelatin-based scaffolds. For this purpose, a gelatin solution was foamed in the presence of a surfactant, Saponin, and the resulting foam was fixed by chemical crosslinking with a diisocyanate. The amorphous crosslinked scaffolds were synthesized with varied gelatin and HDI concentrations, and analyzed in the dry state by micro computed tomography (µCT, porosity: 65±11–73±14 vol.-%), and scanning electron microscopy (SEM, pore size: 117±28–166±32 µm). Subsequently, the work focused on the characterization of the gelatin scaffolds in conditions relevant to biomedical applications. Scaffolds showed high water uptake (H: 630-1680 wt.-%) with minimal changes in outer dimension. Since a decreased scaffold pore size (115±47–130±49 µm) was revealed using confocal laser scanning microscopy (CLSM) upon wetting, the form stability could be explained. Shape recoverability was observed after removal of stress when compressing wet scaffolds, while dry scaffolds maintained the compressed shape. This was explained by a reduction of the glass transition temperature upon equilibration with water (dynamic mechanical analysis at varied temperature (DMTA)). The composition dependent compression moduli (Ec: 10 50 kPa) were comparable to the bulk micromechanical Young’s moduli, which were measured by atomic force microscopy (AFM). The hydrolytic degradation profile could be adjusted, and a controlled decrease of mechanical properties was observed. Partially-degraded scaffolds displayed an increase of pore size. This was likely due to the pore wall disintegration during degradation, which caused the pores to merge. The scaffold cytotoxicity and immunologic responses were analyzed. The porous scaffolds enabled proliferation of human dermal fibroblasts within the implants (up to 90 µm depth). Furthermore, indirect eluate tests were carried out with L929 cells to quantify the material cytotoxic response. Here, the effect of the sterilization method (Ethylene oxide sterilization), crosslinker, and surfactant were analyzed. Fully cytocompatible scaffolds were obtained by using LDI as crosslinker and PEO40 PPO20-PEO40 as surfactant. These investigations were accompanied by a study of the endotoxin material contamination. The formation of medical-grade materials was successfully obtained (<0.5 EU/mL) by using low-endotoxin gelatin and performing all synthetic steps in a laminar flow hood. / Diese Arbeit beschreibt die Entwicklung Entropie-elastischer Gelatine-basierter Netzwerke als Filme und Scaffolds. Mögliche Anwendungen für die entwickelten Materialien liegen im biomedizinischen Bereich, insbesondere der Knochenregeneration. Im ersten Schritt der Arbeit wurden Entropie-elastische, Gelatine-basierte Hydrogel-Filme entwickelt, deren mechanische Eigenschaften durch die Veränderung der Quervernetzungsdichte eingestellt werden konnten. Dazu wurde Gelatine in Lösung oberhalb der Gel-Sol-Übergangstemperatur kovalent quervernetzt, wodurch die Ausbildung helikaler Konformationen unterdrückt wurde. Als Quervernetzer wurden Hexamethylendiisocyanat (HDI) oder Lysindiisocyanat ethylester (LDI) verwendet, und die Reaktionen wurden in Dimethylsulfoxid (DMSO) oder Wasser durchgeführt. Weitwinkel Röntgenstreuungs Spektroskopie (WAXS) zeigte, dass die Netzwerke amorph waren. Der Quellungsgrad (Q: 300-800 vol. %) und der Elastizitätsmodul (E: 70 740 kPa) konnten dabei durch die systematische Veränderung der Quervernetzungsdichte eingestellt werden. Die Analyse der Quervernetzungsreaktion durch Modellreaktionen zeigte, dass die Stabilisierung der Hydrogele sowohl auf kovalente Quervernetzungen (3-13 mol.-%) als auch auf Grafting von (5-40 mol.-%) und Verblendung mit Oligoharnstoffen (16-67 mol.-%) zurückgeführt werden kann. Die Erkenntnisse aus dem Umgang mit dem Bulk-Material wurden dann auf einen integrierten Prozess der Verschäumung und chemischen Quervernetzung transferiert, so dass poröse, dreidimensionale Scaffolds erhalten wurden. Dafür wurde eine wässrige Gelatinelösung in Gegenwart eines Tensids, Saponin, verschäumt, und durch chemische Quervernetzung mit einem Diisocyanat zu einem Scaffold fixiert. Die Scaffolds hergestellt mit unterschiedlichen Mengen HDI und Gelatine, wurden im trockenen Zustand mittels Mikro Computertomographie (µCT, Porosität: 65±11–73±14 vol.-%) und Rasterelektronenmikroskopie (SEM, Porengröße: 117±28–166±32) charakterisiert. Anschließend wurden die Scaffolds unter Bedingungen charakterisiert, die für biomedizinische Anwendungen relevant sind. Die Scaffolds nahmen große Mengen Wasser auf (H: 630 1680 wt.-%) bei nur minimalen Änderungen der äußeren Dimensionen. Konfokale Laser Scanning Mikroskopie zeigte, dass die Wasseraufnahme zu einer verminderten Porengröße führte (115±47–130±49 µm), wodurch die Formstabilität erklärbar ist. Eine Formrückstellung der Scaffolds wurde beobachtet, wenn Scaffolds im nassen Zustand komprimiert wurden und dann entlastet wurden, während trockene Proben in der komprimierten Formen blieben (kalte Deformation). Dieses Entropie-elastische Verhalten der nassen Scaffolds konnte durch die Verminderung der Glasübergangstemperatur des Netzwerks nach Wasseraufnahme erklärt werden (DMTA). Die zusammensetzungsabhängigen Kompressionsmoduli (Ec: 10 50 kPa) waren mit den mikromechanischen Young’s moduli vergleichbar, die mittels Rasterkraftmikroskopie (AFM) gemessen wurden. Das hydrolytische Degradationsprofil konnte variiert werden, und während des Abbaus kam es nur zu kontrolliert-graduellen Änderungen der mechanischen Eigenschaften. Während der Degradation konnte ein Anstieg der mittleren Porengröße beobachtet werden, was durch das Verschmelzen von Poren durch den Abbau der Wände erklärt werden kann. Die Endotoxinbelastung und die Zytotoxizität der Scaffolds wurden untersucht. Humane Haut-Fibroblasten wuchsen auf und innerhalb der Scaffolds (bis zu einer Tiefe von 90 µm). Indirekte Eluat-Tests mit L929 Mausfibroblasten wurden genutzt, um die Zytotoxizität der Materialien, insbesondere den Einfluss des Quervernetzertyps und des Tensids, zu bestimmen. Vollständig biokompatible Materialien wurden erzielt, wenn LDI als Quervernetzer und PEO40 PPO20-PEO40 als Tensid verwendet wurden. Durch den Einsatz von Gelatine mit geringem Endotoxin-Gehalt, und die Synthese in einer Sterilarbeitsblank konnten Materialien für medizinische Anwendungen (Endotoxin-Gehalt < 0.5 EU/mL) hergestellt werden.

Los estímulos mecánicos son uno de los factores que afectan a la diferenciación celular en el proceso de regeneración del tejido óseo, por lo tanto, en el desarrollo de andamios para ingeniería de tejidos, se pueden aplicar las cargas mecánicas con el fin de inducir la actividad de las células. Cuando se aplican cargas mecánicas, los estímulos mecánicos específicos transmitidos a las células a nivel microscópico pueden estudiarse mediante técnicas numéricas. El objetivo de esta tesis fue estudiar la mecanoregulación de la diferenciación de tejido en andamios de fosfato de calcio utilizando modelos de elementos finitos basados en micro tomografía axial computarizada.Dos muestras de materiales porosos basados en fosfato de calcio fueron utilizadas. Se desarrollaron mallas de elementos finitos congruentes, discretizando la fase sólida y los macro poros interconectados, con el fin de tener en cuenta la morfología irregular de los andamios.En primer lugar, se estudió la distribución de los estímulos mecánicos. La fase sólida y el fluido intersticial se simularon como material elástico lineal y como fluido Newtoniano, respectivamente. Se simuló una compresión del 0.5% en el sólido y un fluido con velocidades de entrada de 1, 10 y 100 µm/s en los poros. Se encontraron distribuciones de deformación similares en las paredes ambos materiales, con valores máximos de 1.6% en compresión y de 0.6% en tracción. En algunos poros, la velocidad del fluido aumentó a 100 y 1000 veces la velocidad de entrada. Este estudio mostró como estímulos mecánicos macroscópicos pueden causar distintos niveles de estímulos mecánicos microscópicos dentro los andamios, debido a la morfología.A continuación se realizó un estudio en el tiempo de la diferenciación de tejido en un andamio sometido a condiciones in vitro. La compresión y la perfusión se modelaron como en el estudio anterior. Se simularon una compresión del 0.5% y una velocidad de entrada de fluido constante de 10 µm/s o una presión de entrada de fluido constante de 3 Pa. La deformación cortante octaédrica y el esfuerzo cortante del fluido se utilizaron como estímulos mecano-regulatorios basándose en la teoría de Prendergast et al. (1997). Al aplicar velocidad constante, se predijeron fluctuaciones entre los estímulos equivalentes a la formación de tejido y a la muerte celular, debido al aumento en el esfuerzo cortante del fluido cuando el tejido comienza a llenar los poros. Sin embargo, al aplicar presión constante, se predijo estímulo equivalente a la diferenciación de tejido óseo en la mitad del volumen de los poros. Estos resultados sugieren que para permitir la diferenciación de tejido, la velocidad del fluido debe disminuirse cuando el tejido empieza a mineralizarse.Finalmente, se llevó acabo un estudio en el tiempo de la angiogénesis y de la diferenciación de tejido en un andamio bajo condiciones in vivo. La deformación cortante octaédrica y la velocidad relativa del fluido se utilizaron como estímulos mecano-regulatorios. Las fases sólida y porosa fueron tratadas como materiales poroelásticos. Se simuló la actividad individual de las células. Compresiones de 0.5 y 1% fueron simuladas. La mayoría de los vasos crecieron en los poros de la periferia del andamio y se bloquearon por las paredes. Se formaron redes capilares similares independientemente de la magnitud de deformación utilizada. Al aplicar 0.5% de compresión, estímulos correspondientes a la formación de hueso se predijeron en el 70% del volumen de los poros, sin embargo, sólo el 40% del volumen se llenó de osteoblastos debido a la falta de oxigeno. Este estudio mostró el efecto de la falta de vascularización en el centro del andamio en la diferenciación de tejido.Ese tipo de estudios, combinados con estudios in vitro, deberían contribuir a la comprensión del proceso de diferenciación de los tejidos dentro de los andamios y por lo tanto a la mejora de los métodos de diseño de andamios. / Mechanical stimuli are one of the factors that affect cell differentiation in the process of bone tissue regeneration; therefore, in the development of scaffolds for tissue engineering, mechanical loads can be applied in order to induce cell activity. The specific mechanical stimuli transmitted to cells at a microscopic level when mechanical loads are applied can be studied using numerical techniques. The objective of this thesis was to study the mechanoregulation of tissue differentiation within calcium phosphate scaffolds using micro computed tomographed based finite element models.Two samples of porous calcium phosphate based materials were used. Congruent finite element meshes, with the solid phase and the interconnected pores discretized, were developed in order to account for the scaffold irregular morphology.First, a study of the distribution of mechanical stimuli was performed. The solid phase and the fluid flow within the pores were modeled as linear elastic solid material and Newtonian fluid respectively. Compressive strains of 0.5% of total deformation applied to the solid and interstitial fluid flows with inlet velocities of 1, 10 and 100 µm/s applied to the pores were simulated. Similar strain distributions for both materials were found, with compressive and tensile strain maximal values of 1.6% and 0.6% respectively. For the fluid flow models, the fluid velocity in some of the scaffold pores increased to 100 and 1000 times the inlet velocity. This study showed how mechanical loads and fluid flow applied to the scaffolds caused different levels of mechanical stimuli within the samples according to the morphology of the materials.Next, a study of the mechanoregulation of tissue differentiation over time in a scaffold subjected to in vitro loads was performed. The solid phase and the fluid flow were modeled as in the study described above. Compressive strain of 0.5% and fluid flow with constant inlet velocity of 10 µm/s or constant inlet pressure of 3 Pa were applied. Octahedral shear strain and fluid shear stress were used as mechano-regulatory stimuli based on the theory of Prendergast et al. (1997). When a constant velocity was simulated, fluctuations between stimuli equivalent to tissue formation and cell death were predicted due to the increase in the fluid shear stress when tissue started to fill the pores. However, when constant pressure was applied, stimuli equivalent to bone formation were predicted in about half of the pore volume. These results suggest that in order to allow tissue differentiation within a scaffold, the fluid velocity should be decreased when tissue starts mineralizing.Finally, a study of the angiogenesis and the mechanoregulation of tissue differentiation over time in a scaffold subjected to in vivo conditions was performed. Octahedral shear strain and relative fluid velocity were used as mechano-regulatory stimuli. The solid and pore phases were treated as poroelastic materials. Individual cell activity was simulated within the pore domain. Compressive strains of 0.5 and 1% of total deformation were simulated. Most vessels grew in the pores at the periphery of the scaffolds and were blocked by the scaffold walls. Similar capillary networks were formed independently of the magnitude of the mechanical strain applied. When 0.5% of strain was applied, 70% of the pore volume was affected by mechano-regulatory stimuli corresponding to bone formation; however, because of the lack of oxygen, only 40% of the volume was filled with osteoblasts. This study showed the effect of the lack of vascularization in the center of the scaffold on the tissue differentiation.Such kind of studies, combined with in vitro studies, should contribute to the understanding of the process of tissue differentiation within the constructs and therefore to the improvement of scaffold design methods.

In this work, a new polyurethane (PU) chain extender was developed to incorporate a Glycine-Leucine (Gly-Leu) dipeptide, the cleavage site of several matrix metalloproteinases. PUs were synthesized with either the Gly-Leu-based chain extender (Gly-Leu PU) or a phenylalanine-based chain extender (Phe PU). Both PUs had high molecular weight averages (Mw > 125,000 g/mol) and were phase segregated, semi-crystalline polymers (Tm ~ 42°C) with a low soft segment glass transition temperature (Tg < -50°C). Uniaxial tensile testing of PU films revealed that the polymers could withstand high ultimate tensile strengths (~ 8-13 MPa) and were flexible with breaking strains of ~ 870-910% but the two PUs exhibited a significant difference in mechanical properties. The Phe and Gly-Leu PUs were electrospun into porous scaffolds for degradation and cell-based studies. Fibrous Phe and Gly-Leu PU scaffolds were formed with randomly organized fibers and an average fiber diameter of approximately 3.6 µm. In addition, the Phe PU was electrospun into scaffolds of varying architecture to investigate how fiber alignment affects the orientation response of cardiac cells. To achieve this, the Phe PU was electrospun into aligned and unaligned scaffolds and the physical, thermal, and mechanical properties of the scaffolds were investigated. The degradation of the Phe and Gly-Leu PU scaffolds was investigated in the presence of active MMP-1, active MMP-9, and a buffer solution over 28 days to test MMP-mediated and passive hydrolysis of the PUs. Mass loss and structural assessment suggested that neither PU experienced significant hydrolysis to observe degradation over the course of the experiment. In cell-based studies, Phe and Gly-Leu PU scaffolds successfully supported a high density of viable and adherent mouse embryonic fibroblasts (MEFs) out to at least 28 days. Culturing murine embryonic stem cell-derived cardiomyocytes (mESCDCs) alone and with MEFs on aligned and unaligned Phe PU scaffolds revealed both architectures supported adherent and functionally contractile cells. Importantly, fiber alignment and coculture with MEFs improved the organization and differentiation of mESCDCs suggesting these two parameters are important for developing engineered myocardial constructs using mESCDCs and PU scaffolds.

In this work, a new polyurethane (PU) chain extender was developed to incorporate a Glycine-Leucine (Gly-Leu) dipeptide, the cleavage site of several matrix metalloproteinases. PUs were synthesized with either the Gly-Leu-based chain extender (Gly-Leu PU) or a phenylalanine-based chain extender (Phe PU). Both PUs had high molecular weight averages (Mw > 125,000 g/mol) and were phase segregated, semi-crystalline polymers (Tm ~ 42°C) with a low soft segment glass transition temperature (Tg < -50°C). Uniaxial tensile testing of PU films revealed that the polymers could withstand high ultimate tensile strengths (~ 8-13 MPa) and were flexible with breaking strains of ~ 870-910% but the two PUs exhibited a significant difference in mechanical properties. The Phe and Gly-Leu PUs were electrospun into porous scaffolds for degradation and cell-based studies. Fibrous Phe and Gly-Leu PU scaffolds were formed with randomly organized fibers and an average fiber diameter of approximately 3.6 µm. In addition, the Phe PU was electrospun into scaffolds of varying architecture to investigate how fiber alignment affects the orientation response of cardiac cells. To achieve this, the Phe PU was electrospun into aligned and unaligned scaffolds and the physical, thermal, and mechanical properties of the scaffolds were investigated. The degradation of the Phe and Gly-Leu PU scaffolds was investigated in the presence of active MMP-1, active MMP-9, and a buffer solution over 28 days to test MMP-mediated and passive hydrolysis of the PUs. Mass loss and structural assessment suggested that neither PU experienced significant hydrolysis to observe degradation over the course of the experiment. In cell-based studies, Phe and Gly-Leu PU scaffolds successfully supported a high density of viable and adherent mouse embryonic fibroblasts (MEFs) out to at least 28 days. Culturing murine embryonic stem cell-derived cardiomyocytes (mESCDCs) alone and with MEFs on aligned and unaligned Phe PU scaffolds revealed both architectures supported adherent and functionally contractile cells. Importantly, fiber alignment and coculture with MEFs improved the organization and differentiation of mESCDCs suggesting these two parameters are important for developing engineered myocardial constructs using mESCDCs and PU scaffolds.

Currently growth plate fractures account for nearly 18.5% of fractures in children and can lead to stunted bone growth or angular deformation. If the body is unable to heal itself a bony bar forms, preventing normal bone growth. Clinical treatment involves removing the bony bar and replacing it with a filler substance, which causes poor results 60% of the time. Using primarily poly(lactic-co-glycolic acid) (PLGA) as the scaffold material, the goal was to develop an implant that would support to the implant site, allow for cell ingrowth, and degrade away over time. Porous scaffolds were fabricated from PLGA microspheres using the salt leaching method. The first part of this work investigated the effect of sintering the microspheres by studying the mechanical properties, degradation and morphology and their potential applications for hard and soft tissue implants. Growth factor or drugs can be encapsulated into PLGA microspheres, which was the second part of this work. Encapsulated insulin-like growth factor I (IGF-I) was able to withstand the scaffold fabrication process without compromising it’s bioactivity and promoted cell proliferation. The next part of this work experimented with the addition of a hydrogel porogen. Porogen particles were made using a quick degrading poly(beta-amino ester) (PBAE) hydrogel and loaded with ketoprofen. The addition of the porogen creates a dual drug-releasing scaffold with a localized delivery system. The final step of this work involved animal studies to determine the effectiveness of the scaffolds in growth plate regeneration and how they compare to the current clinical treatment option. Gross observation, microCT analysis, angular measurement of bone growth and histological methods were employed to evaluate the scaffolds. The goal was to develop a versatile scaffold that could be used for a wide range of tissue engineering applications. The mechanical properties, degradation profiles and drug delivery capabilities can be all tailored to meet the specific needs of an implant site. One specific application was regenerating the native growth plate that can also encourage the endogenous mesenchymal stem cells to follow the desire linage. By regenerating the native growth plate, angular deformation and stunted limb growth were greatly reduced.

In situ forming implants (ISIs) are popular due to their ease of use and local drug delivery potential, but they suffer from high initial drug burst, and release behavior is tied closely to solvent exchange and polymer properties. Additionally, such systems are traditionally viewed purely as drug delivery devices rather than potential scaffold materials due to their poor mechanical properties and minimal porosity. The aim of this research was to develop an injectable ISI with drug release, mechanical, and microstructural properties controlled by micro- and nanoparticle additives. First, an injectable ISI was developed with appropriate drug release kinetics for orthopedic applications. Poly(β-amino ester) (PBAE) microparticles were loaded with simvastatin or clodronate, and their loading efficiency and drug retention after washing was quantified. Drug-loaded PBAE microparticles and hydroxyapatite (HA) microparticles were added to a poly(lactic-co-glycolic acid) (PLGA)–based ISI. By loading simvastatin into PBAE microparticles, release was extended from 10 days to 30 days, and burst was reduced from 81% to 39%. Clodronate burst was reduced after addition of HA, but was unaffected by PBAE loading. Scaffold mass and porosity fluctuated as the scaffolds swelled and then degraded over 40 days. Next, the mechanical properties of these composite ISIs were quantified. Both micro- and nanoparticulate HA as well as PBAE microparticle content were varied. Increasing HA content generally improved compressive strength and modulus, with a plateau occurring at 30% nano-HA. Injectability remained clinically acceptable for up to 10% w/w PBAE microparticles. Ex vivo injections into trabecular bone improved both strength and modulus. Lastly, HA-free ISIs were investigated for drug delivery into the gingiva to treat periodontitis. Doxycycline and simvastatin were co-delivered, with delivery of doxycycline over 1 week accompanied by simvastatin release over 30 days. PBAE-containing ISIs exhibited higher initial and progressive porosity and accessible volume than PBAE-free ISIs over the course of degradation. Additionally, PBAE-containing ISIs provided superior tissue retention within a simulated periodontal pocket. The ISIs investigated here have a wide range of potential applications due to their flexible material and drug release properties, which can be controlled by both the chemistry and concentration of various particulate additives.

Tissue engineering is a multidisciplinary field that is rapidly emerging as a promising approach for tissue repair and regeneration. In this approach, scaffolds which allow cells to invade the construct and guide the cells grow into specific tissue play a pivotal role. Electrospinning has gained popularity recently as a simple and versatile method to produce fibrous structures with nano- to microscale dimensions. These electrospun fibers have been extensively applied to create nanofiber scaffolds for tissue engineering applications. Specifically for bone and cartilage tissue engineering, polymeric materials have some attractive properties such as the biodegradability. Ceramic scaffolds and implant coatings, such as hydroxyapatite and silica-based bioglass have also been considered as bone graft substitutes for bone repair because of their bioactivity and, in some cases, tunable resorbability. Besides tissue engineering scaffolds, for clinical application, especially for load-bearing artificial implants, metallic materials such as titanium are the most commonly used material. Osseointegration between bone and implants is very essential for implant success. To achieve better osseointegration between bone and the implant surface, three dimensional porous structures can provide enhanced fixation with bone by allowing tissue to grow into the pores. In this study, pre-3D electrospun polymer and ceramic scaffolds with peptide conjugation and 3D titanium scaffolds with different surface morphology were fabricated to testify the osteoblast and mensechymal stem cell attachment and differentiation. The overall goal of this thesis is to determine if the peptide functionalization of polymeric scaffolds and physical parameters of ceramic and metallic scaffold can promote osteoblast maturation and mesenchymal stem cell differentiation in vitro to achieve an optimal scaffold design for greater osseointegration. The results of the studies showed with functionalization of MSC- specific peptide, polymer scaffolds behaved with higher biocompatibility and MSC affinity. For the ceramic and metallic scaffolds, microstructures and nanostructures can synergistically promote osteoblast maturation and 3D micro-environment with micro-roughness is a promising design for osteoblast maturation and MSC differentiation in vitro compared to 2D surfaces.

Cartilage tissue engineering has been emerging as a promising therapeutic approach, where engineered constructs or scaffolds are used as temporary supports to promote regeneration of functional cartilage tissue. Hybrid constructs fabricated from cells, hydrogels, and solid polymeric materials show the most potential for their enhanced biological and mechanical properties. However, fabrication of customized hybrid constructs with impregnated cells is still in its infancy and many issues related to their structural integrity and the cell functions need to be addressed by research. Meanwhile, it is noticed that nowadays monitoring the success of tissue engineered constructs must rely on animal models, which have to be sacrificed for subsequent examination based on histological techniques. This becomes a critical issue as tissue engineering advances from animal to human studies, thus raising a great need for non-invasive assessments of engineered constructs in situ. To address the aforementioned issues, this research is aimed to (1) develop novel fabrication processes to fabricate hybrid constructs incorporating living cells (hereafter referred as “construct biofabrication”) for cartilage tissue regeneration and (2) develop non-invasive monitoring methods based on synchrotron X-ray imaging techniques for examining cartilage tissue constructs in situ. Based on three-dimensional (3D) printing techniques, novel biofabrication processes were developed to create constructs from synthetic polycaprolactone (PCL) polymer framework and cell-impregnated alginate hydrogel, so as to provide both structural and biological properties as desired in cartilage tissue engineering. To ensure the structural integrity of the constructs, the influence of both PCL polymer and alginate was examined, thus forming a basis to prepare materials for subsequent construct biofabrication. To ensure the biological properties, three types of cells, i.e., two primary cell populations from embryonic chick sternum and an established chondrocyte cell line of ATDC5 were chosen to be incorporated in the construct biofabrication. The biological performance of the cells in the construct were examined along with the influence of the polymer melting temperature on them. The promising results of cell viability and proliferation as well as cartilage matrix production demonstrate that the developed processes are appropriate for fabricating hybrid constructs for cartilage tissue engineering. To develop non-invasive in situ assessment methods for cartilage and other soft tissue engineering applications, synchrotron phase-based X-ray imaging techniques of diffraction enhanced imaging (DEI), analyzer based imaging (ABI), and inline phase contrast imaging (PCI) were investigated, respectively, with samples prepared from pig knees implanted with low density scaffolds. The results from the computed-tomography (CT)-DEI, CT-ABI, and extended-distance CT-PCI showed the scaffold implanted in pig knee cartilage in situ with structural properties more clearly than conventional PCI and clinical MRI, thus providing information and means for tracking the success of scaffolds in tissue repair and remodeling. To optimize the methods for live animal and eventually for human patients, strategies with the aim to reduce the radiation dose during the imaging process were developed by reducing the number of CT projections, region of imaging, and imaging resolution. The results of the developed strategies illustrate that effective dose for CT-DEI, CT-ABI, and extended-distance CT-PCI could be reduced to 0.3-10 mSv, comparable to the dose for clinical X-ray scans, without compromising the image quality. Taken together, synchrotron X-ray imaging techniques were illustrated promising for developing non-invasive monitoring methods for examining cartilage tissue constructs in live animals and eventually in human patients.