As fraturas em equinos são responsáveis por grande prejuízo financeiro devido às dificuldades encontradas para instituir seu tratamento nessa espécie, principalmente em animais adultos. Entre as dificuldades técnicas no tratamento de fraturas em equinos estão a alta resistência óssea, que acarreta fraturas com grande transmissão de energia, e a escassez de materiais e técnicas de osteossíntese específicos para a espécie, gerando mau prognóstico em muitos casos. Estas fraturas, quando cominutivas acarretam em falhas ósseas, criando um espaço entre os fragmentos de maior tamanho e ainda extensa lesão de tecidos adjacentes, o que dificulta e prolonga o tempo de consolidação óssea. Atualmente a terapia com células tronco vem sendo bastante estudada em ortopedia, contudo o ambiente onde essas células são depositadas determina o caminho para onde elas vão se diferenciar. Para a utilização de células tronco em ortopedia, estas devem sem implantadas junto a um suporte tridimensional, por exemplo os biopolímeros, que além de fornecer um meio para a multiplicação e diferenciação dessas células, também deve apresentar características biomecânicas semelhantes ao tecido a ser reparado, que no caso da ortopedia é o tecido ósseo. O objetivo desse estudo foi avaliar as propriedades biomecânicas de dois biopolímeros a poliuretana de mamona com carbonato de cálcio e uma formulação de quitosana com fosfato de cálcio. Foram preparadas duas formulações diferentes de cada biopolímero, em corpos de prova cilíndricos de 12 mm de comprimento e 6 mm de diâmetro, sendo: poliuretana de mamona porosa e compacta, e quitosana com secagem a 38 e 60 graus Celsius. Essas formulações foram submetidas a ensaios compressivos nos momentos 3, 24, 48 e 72 horas após o preparo e avaliadas quanto sua resistência à compressão, deformação relativa e módulo de elasticidade. A poliuretana de mamona compacta apresentou o maior valor de resistência à compressão (45,805 N/mm2) após 48 horas. A fórmula de quitosana com secagem a 38oC apresentou a menor deformação relativa (3,952 %) após 72 horas de preparo e o maior valor de módulo de elasticidade encontrado foi na poliuretana de mamona compacta após 72 horas (1354,284 N/mm2). Sendo assim a poliuretana de mamona compacta apresenta maior resistência à compressão do que o osso esponjoso de terceiro metacarpiano equino e semelhante aos substitutos ósseos comerciais mais resistentes. A fórmula de quitosana 38oC apresentou valores similares aos observados no osso esponjoso equino. Podemos concluir com esses dados que a poliuretana de mamona compacta e a fórmula de quitosana 38oC apresentam características biomecânicas desejáveis nos materiais para enxerto ósseo. / Equine fractures are responsible for great economic losses due to difficulties in establishing their treatment, mainly regarding adult animals. Among technical difficulties faced in the equine fractures treatment, there are high bone strenght, which results in high energy fractures and the lack of materials and specific osteosynthesis techniques for the specimen, resulting bad prognostic in many cases. When cominutives, these fractures result in bone gaps, creating spaces between bigger fragments and still extensive surrounding tissue damages, which difficults and extends time for bone consolidation. Nowadays, therapy with steam cells is focused in orthopedy, but environment where these cells are established determines the path they will take. For the use of steam cells in orthopedy, they must be implanted together with a tridimensional support such as biopolymers which, besides offering conditions for replication and differentiation of these cells, they must present biomechanic characteristics similar to the tissue to be healed, which is the bone. The target of this study was to evaluate biomechanic properties of two biopolymers, a castor oil polyurethane with calcium carbonate and a formulation of chitosan with calcium phosphate. Two different formulations of each biopolymer were prepared, in cilindric parts of 12mm lenght and 6mm diameter: porous and compact castor oil polyurethane, and chitosan drying at 38oC and 60oC. These formulations were submitted to compressed tests at 3, 24 and 72 hours after preparation and evaluated for compressive strenght, relative deformation and modulus of elasticity. The compact castor oil polyurethane presented greater compressive value (45,805 N/mm2) after 48 hours. Chitosan formulation drying at 38oC presented lower relative deformation (3,952%) 72 hours after prepared, and the highest value for modulus of elasticity found was compact castor oil polyurethane after 72 hours (1354,284 N/mm2). Thus, compact castor oil polyurethane presents higher compressive strenght than trabecular bone of the third equine metacarpal and similar to strenghter comercial bone grafts. The formulation chitosan 38oC presented similar values to those observed in equine trabecular bone. With these data, we can conclude that the compact castor oil polyurethane and the formulation of chitosan 38oC present desirable biomechanic characteristics in materials for bone grafts.

O grau de acetilação (GA) da quitosana é um parâmetro que influi nas interações inter e intramoleculares deste polímero e, portanto, em suas propriedades. É comum a incorporação de compostos a filmes de quitosana para melhora de suas propriedades, contudo, não há relatos da incorporação da resina de jatobá, composto natural com potencial atividade biológica, a estes filmes. Portanto, esta pesquisa buscou estudar a influência do grau de acetilação da quitosana e da inclusão da resina de jatobá sobre as propriedades de filmes de quitosana/gelatina, visando uma possível aplicação em cicatrização de feridas cutâneas. Foram produzidas amostras de quitosana a partir de gládios de lula com três diferentes valores de GA, variáveis de 7 a 35%. As soluções formadoras de filme apresentaram redução no valor de concentração inibitória mínima contra Staphylococcus aureus tanto com a redução no GA quanto com a incorporação da resina de jatobá, apesar da resina isoladamente não ter apresentado atividade antimicrobiana nas concentrações estudadas. Os ensaios reológicos demonstraram que a inclusão da resina de jatobá e maiores valores de GA reduziram os valores de viscosidade das soluções, enquanto a temperatura de gelificação também diminuiu com a inclusão da resina de jatobá. Os filmes apresentaram uma superfície lisa e compacta para as blendas quitosana/gelatina e uma morfologia rugosa e irregular após adição da resina de jatobá, quando analisados por MEV. A adição de resina de jatobá levou também a redução na estabilidade térmica dos filmes. Nos testes de absorção em PBS ficou evidente o efeito do GA da quitosana sobre a propriedade, exibindo valores de intumescimento consideravelmente maiores para os filmes conforme o aumento no GA, fator relacionado ao aumento do número de grupos acetila. Por outro lado, a adição de resina de jatobá e o processo de neutralização reduziram consideravelmente o grau de intumescimento dos filmes. Foi observado também que a redução no GA e a inclusão da resina de jatobá tanto aumentaram a permeação ao vapor de água dos filmes, quanto levaram a uma redução da solubilidade destes materiais. / The degree of acetylation (DA) of chitosan is a parameter that influences the inter and intramolecular interactions of this polymer and, therefore, its properties. In addition, it is common the incorporation of other compounds to chitosan films to improve their properties. However, there are no reports of the incorporation of jatoba resin, a natural compound with potential biological activity. Therefore, this research aimed to study the influence of the degree of chitosan acetylation and the inclusion of the jatoba resin on the properties of chitosan/gelatin films, with potential applications in wound healing. Samples of chitosan were produced from squid pens with three different DA values, ranging from 7 to 35%. The reduction in DA of chitosan and incorporation of the jatoba resin into the film-forming solutions resulted in reductions in the minimum inhibitory concentration against Staphylococcus aureus, although the isolated resin did not exhibit antimicrobial activity at the concentrations studied. The rheological tests showed that the inclusion of the jatoba resin and higher DA values reduced the viscosity of the solutions, while gelation temperature values decreased with the jatoba resin addition. The films presented a smooth and compact surface for the chitosan/gelatin blends and a rough and irregular morphology after addition of the jatoba resin. The addition of jatoba resin also led to the reduction in thermal stability of the films. The effect of the DA of chitosan on the absorption of PBS was evident, exhibiting considerably higher swelling values for the films as a function of the DA increase, a factor related to the increase in the number of acetyl groups. Moreover, the addition of jatoba resin and neutralization process considerably reduced the degree of swelling of the films. The water vapor permeability of the films increased due to both the reduction in DA and the inclusion of the jatoba resin, whereas these changes led to the reduction of the solubility of these materials.

Novos materiais obtidos a partir de polímeros biodegradáveis são uma alternativa para a redução do impacto ambiental causado pelo uso excessivo de polímeros derivados do petróleo. Atualmente, vários estudos têm sido realizados na busca de matéria-prima para o desenvolvimento de filmes biodegradáveis, com boa viabilidade técnica e econômica. Dentre estas matérias-primas, destacam-se as que são provenientes de fontes renováveis, de baixo custo e que tenham grande importância econômica e ambiental, como, por exemplo, o amido, as argilas, e a quitosana. Nos filmes que preparamos, adicionamos como plastificante a glicerina, um subproduto do biodiesel, e que contribui para maior estabilidade térmica dos filmes, em conjunto com o amido. O propósito deste trabalho foi o preparo de um biopolímero a base de quitosana e argila com propriedades de nanocompósitos, pois estes materiais costumam exibir propriedades físico-químicas diferenciadas em relação a outros materiais, devido à redução no seu tamanho. Sendo assim, através do estudo e comparação de duas argilas esmectíticas sódicas naturais, Bentogel e Corral, pôde-se observar o comportamento dos filmes formados em presença de amido e glicerina. Os filmes obtidos, através do método de dispersão em solução do polímero, foram caracterizados através das técnicas de DRX, MEV, IV, TG e DSC. Os resultados obtidos mostraram a formação de filmes nanocompósitos esfoliados de boa estabilidade térmica. / New materials made from biodegradable polymers are an alternative to reducing the environmental impact caused by excessive use of polymers derived from petroleum. Currently, several studies have been conducted in search of raw material for the development of biodegradable films, with good technical and economic feasibility. Among these materials, we highlight those that are from renewable resources, low cost and have great economic and environmental importance, such as, starch, clays, and chitosan. In preparing films, we added glycerol as a plasticizer, a byproduct of biodiesel, and contributes to better thermal stability of the films, together with starch. The purpose of this study was the preparation of a biopolymer based on chitosan and clay nanocomposites properties, because these materials tend to display different physico-chemical properties compared to other materials because of the reduction in size. Thus, through the study and comparison of two natural sodium smectite clays, Bentogel and Corral, it was observing the behavior of films formed in the presence of starch and glycerol. The films obtained by the method of dispersion in the polymer solution, were characterized by techniques of XRD, SEM, FTIR, TG and DSC. The results obtained showed the formation of exfoliated nanocomposites films and good thermal stability.

Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is a polycationic, biocompatible and biodegradable polymer. In addition, chitosan has different functional groups that can be modified with a wide array of ligands. Because of its unique physicochemical properties, chitosan has great potential in a range of biomedical applications, including tissue engineering, non-viral gene delivery and enzyme immobilization. In our work, the primary amine groups of chitosan were utilized for chitosan modification through biotinylation using N-hydroxysuccinimide chemistry. This was followed by the addition of avidin which strongly binds to biotin. Biotinylated ligands such as polyethylene glycol (PEG) and RGD peptide sequence, or biotinylated enzymes such as trypsin, were then added to modify the surface properties of the chitosan for a variety of purposes. Modified chitosans were formulated into nano-sized particles or cast into films. Different factors affecting fabrication of chitosan particles, such as the pH of the preparation, the inclusion of polyanions, the charge ratios and the degree of deacetylation and the molecular weight of chitosan were studied. Similarly, parameters affecting the fabrication of chitosan films, such as cross-linking, were investigated for potential applications in tissue engineering and enzyme immobilization. It was found that the inclusion of dextran sulfate resulted in optimum interaction between chitosan and DNA, as shown by the high stability of these nanoparticles and their high in vitro transfection efficiencies in HEK293 cells. When applying these formulations as DNA vaccines in vivo, chitosan nanoparticles loaded with the ovalbumin antigen and the plasmid DNA encoding the same antigen resulted in the highest antibody response in C57BL/6 mice. Furthermore, engineering of the surface of chitosan nanoparticles was done by utilizing the avidin-biotin interaction for attaching PEG and RGD. The modified formulations were tested for their in vitro gene delivery properties and it was found that these ligands improved gene transfection efficiencies significantly. Chitosan nanoparticles were optimized further for enzyme immobilization purposes using sodium sulfate and glutaraldehyde as physical and chemical cross-linking agents, respectively. These particles and chitosan films were used for immobilizing trypsin utilizing several techniques. Enzyme immobilization via avidin-biotin interaction resulted in high immobilization efficiency and high enzymatic activity in different reaction conditions. Additionally, the immobilized trypsin systems were stable and amenable to be regenerated for multiple uses. Finally, glutaraldehyde cross-linked chitosan films were modified with PEG and RGD for their cell repellant and cell adhesion properties, respectively, using avidin-biotin interaction. This method was again effective in engineering chitosan surfaces for modulating cell adhesion and proliferation. In conclusion, using avidin-biotin technique to modify biotinylated chitosan surfaces is a facile method to attach a wide variety of ligands in mild reaction conditions, while preserving the functionality of these ligands.

O pedúnculo de caju é um pseudofruto carnoso, suculento, de ótimo aroma, apresenta altos teores de ácido ascórbico e compostos fenólicos. Apesar da condição nutricional, a vida útil e a comercialização in natura do pedúnculo é limitada principalmente devido a sua alta perecibilidade e susceptibilidade ao ataque de microrganismos patogênicos. O objetivo do trabalho foi estudar métodos de conservação pós-colheita de pedúnculos de caju, entre eles: radiação gama, quitosana e atmosfera modificada passiva, de forma primeiramente individual para avaliar o efeito na conservação dos principais atributos físico-químicos, nutricionais e nos aspectos fisiológicos e depois em combinação para verificar efeitos sobre a vida útil, na atividade antioxidante e no perfil de compostos voláteis. No experimento de irradiação a dose de 2,0 kGy reduziu drasticamente a incidência de podridão mas ocasionou alta perdas de firmeza, ácido ascórbico, fenólicos e de pigmentos. A dose de 0,5 kGy proporcionou a melhor qualidade dos pedúnculos, pois além de reduzir podridões, reduziu perda de massa, manteve valores adequados de firmeza e, diferente da maior dose, não interferiu na pigmentação da epiderme, além de manter altos os níveis de compostos fenólicos e reduzir a adstringência. Com relação ao uso de quitosana, as concentrações de 1 e 2% foram eficientes na conservação dos atributos de qualidade de pedúnculos de caju, sendo que a concentração de 2% foi mais eficiente na redução de podridões, além de reduzir a adstringência do pedúnculo. O uso da atmosfera modificada, por meio do filme de PEBD, que proporcinou atmosfera de 11% de O2 e 8% de CO2, foi a mais eficiente na conservação dos atributos da qualidade visual e nutricional de pedúnculos de caju até o 20º dia após a colheita. Mudanças mais dráticas da atmosfera, como observadas em PP e BOPP não causaram anaerobiose, porém, a maior barreira aos gases e ao vapor de água, levou a perda de alguns atributos de qualidade. Os tratamentos de combinação de irradiação+quitosana e irradiação+atmosfera modificada foram eficientes na conservação dos pedúnculos de caju por estender a vida útil até o 25º dia após a colheita, reduzindo a incidência de podridão e perda de massa e mantendo adequada a firmeza da polpa, fato não alcançado com os tratamentos individuais. Além disso, a combinação irradiação + quitosana manteve também altos níveis de atividade antioxidante e perfil de voláteis característico de um pedúnculo agradável, sendo desta forma a combinação mais indicada. A irradiação e a quitosana quando utilizadas como tratamentos individuais também preservam a qualidade volátil, porém até o 20º dia após a colheita. / Cashew apple is a fleshy, juicy pseudofruit of great aroma, presents high levels of ascorbic acid and phenolic compounds. Despite the nutritional condition, the shelf life and in natura commercialization of the peduncle is limited mainly due to its high perishability and susceptibility to the attack of pathogenic microorganisms. The objective of this research was to study post-harvest methods of cashew peduncles among them: gamma radiation, chitosan and passive modified atmosphere, in a first individual way to evaluate the effect on the conservation of the main physical-chemical, nutritional and physiological aspects and then in combination to check effects on shelf life, antioxidant activity and the volatile compound profile. In the irradiation experiment the dose of 2.0 kGy drastically reduced the incidence of rot but caused high losses of firmness, ascorbic acid, phenolics and pigments. The dose of 0.5 kGy provided the best quality of the peduncles, because besides reducing rot, reduced mass loss, maintained adequate values of firmness and, unlike the higher dose, did not interfere in the pigmentation of the epidermis, besides maintaining high levels of phenolic compounds and reduce astringency. Regarding the use of chitosan, the concentrations of 1 and 2% were efficient in the conservation of the attributes of cashew peduncles, being that the concentration of 2% was more efficient in the reduction of rot, besides reducing the astringency of the peduncle. The use of the modified atmosphere through the LDPE film, which provided an atmosphere of 11% O2 and 8% CO2, was the most efficient in preserving the visual and nutritional quality attributes of cashew peduncles until the 20th day after harvest. More dramatic changes of the atmosphere, as observed in PP and BOPP did not cause anaerobiosis, however, the greater barrier to gases and water vapor, led to the loss of some quality attributes. The treatments of irradiation+chitosan and irradiation+modified atmosphere treatments were efficient in the conservation of the cashew peduncles by extending the shelf life until the 25th day after harvesting, reducing the incidence of rot and loss of mass and maintaining the firmness of the pulp. A fact not reached with individual treatments. In addition, the combination of irradiation+chitosan also maintained high levels of antioxidant activity and volatile profile characteristic of a pleasant peduncle, thus being the most indicated combination. Irradiation and chitosan when used as individual treatments also preserve the volatile quality, but until the 20th day after harvest.

Gene therapy is the process of delivering genetic material, such as DNA (encoding for an important protein) into a patient’s cells in order to treat a particular disease such as a genetic disorder or heart disease. This process of DNA delivery into cells is known as “transfection” and it is important that the efficiency of transfection be optimized such that a patient can obtain maximum therapeutic benefit from such a treatment. DNA is susceptible to being destroyed by harsh physiological environments prior to reaching its target. This problem can be diminished with the use of vectors that not only protect against harsh conditions but also encourage entry into cells. By mixing 1) DNA with 2) positively charged polymers, “polyplexes” form which protect DNA from degradation and increase transfection efficiency. The development of effective polyplex formulations requires optimization. In the work presented here, it was discovered that when polyplexes contained specific sequences within the DNA called “CpG”, this lowered transfection efficiencies and increased inflammatory responses compared to DNA without CpG, as measured using a mouse lungs model. Thus, DNA composition played an important role in influencing DNA transfection efficiency of polyplexes. Another aspect to take into account is the degree of positive charge of the polymer. We tested a new polymer called poly(galactaramidoamine) or PGAA. We found that this PGAA can form polyplexes with DNA and could be used in gene therapy. At the present time, mechanisms by which the polyplexes get inside and transfect the cells are still unclear. We also introduced a new system called high-content screening to the gene delivery field. This system offers automated measurements of transfection efficiency and cytotoxicity and could be used to reveal the polyplexes trafficking inside cells.

Nesta tese, foram produzidas e estudadas membranas de gelatina e quitosana com nanopartículas de prata, visando obter sinergia em suas propriedades para aplicações em engenharia de tecidos. Os materiais foram escolhidos por suas propriedades e aplicações similares. A gelatina é um polipeptídio biocompatível e biodegradável, obtido do colágeno de animais. A quitosana, um polímero encontrado na parede celular de alguns organismos do reino fungi, pode ser obtida da desacetilação da quitina. É biocompatível, biodegradável, e pode ter efeito antimicrobiano. Nanopartículas de prata também têm efeito antimicrobiano. As membranas foram preparadas por método de casting, e as nanopartículas de prata (AgNPs) foram sintetizadas com o método de Turkevich adaptado (TK-AgNPs) e outro similar com adição de ácido tânico (AT-AgNPs). Membranas com diferentes composições foram fabricadas: de gelatina apenas, de quitosana, e blendas de gelatina com quitosana (GQ), com adição de diferentes concentrações de AT-AgNPs ou TK-AgNPs, reticuladas com glutaraldeído (Gta) ou não. O objetivo era identificar uma composição com boa capacidade antimicrobiana e baixa toxicidade para células humanas. As membranas eram visualmente homogêneas, e a incorporação de nanopartículas foi comprovada por difração de raios X e espectroscopia no infravermelho. As AT-AgNPs eram menores e mais monodispersas do que as TK-AgNPs. As membranas contendo apenas quitosana e gelatina não apresentaram atividade antimicrobiana. As que continham AgNPs foram tóxicas para a bactéria Gram- positiva Staphylococcus aureus segundo testes de halo de inibição, com toxicidade dependente da concentração de AgNPs. As mais eficazes foram as membranas quitosana/TK- AgNPs/Gta, GQ 0,05% AT-AgNPs e GQ 0,1% AT-AgNPs. Nenhuma das membranas apresentou toxicidade contra células de fibroblastos humano. Tais resultados confirmam a possibilidade de aplicação dos materiais estudados como curativos para regeneração tecidual. / In this thesis, gelatin and chitosan membranes with silver nanoparticles were produced and studied, aiming at obtaining synergy in their properties for tissue engineering applications. The materials were chosen for their similar properties and applications. Gelatin is a biocompatible and biodegradable polypeptide derived from animal collagen. Chitosan, a polymer found in the cell wall of some fungi, can be obtained from the deacetylation of chitin. It is biocompatible, biodegradable, and may have antimicrobial effect. Silver nanoparticles also have antimicrobial effect. The membranes were prepared using the casting method, and the silver nanoparticles (AgNPs) were synthesized with an adapted Turkevich method (TK- AgNPs) and a similar one with addition of tannic acid (AT-AgNPs). Membranes with different compositions were made: gelatin only, chitosan, and blends of gelatin and chitosan (GQ), with the addition of different concentrations of AT-AgNPs or TK-AgNPs, crosslinked with glutaraldehyde (Gta) or not. The objective was to identify a composition with good antimicrobial capacity and low toxicity to human cells. The membranes were visually homogeneous, and the incorporation of nanoparticles was confirmed by X-ray diffraction and infrared spectroscopy. The AT-AgNPs were smaller and more monodisperse than the TK- AgNPs. Membranes containing only chitosan and gelatin had no antimicrobial activity. Those containing AgNPs were toxic to the Gram-positive Staphylococcus aureus bacterium according to halo tests, with AgNP concentration-dependent toxicity. The most effective were the chitosan/TK-AgNPs/Gta membranes, 0.05% GQ AT-AgNPs and 0.1% GQ AT-AgNPs. None of the membranes showed toxicity against human fibroblast cells. These results confirm the possibility of application of the membranes as curatives for tissue regeneration.

Thesis (M.S.) -- Mississippi State University. Department of Agricultural and Biological Engineering. / Title from title screen. Includes bibliographical references.

<p>In this thesis, chitosan, a biocompatible polysaccharide that has been approved as a food additive was selected as a platform for the development of safe, efficient non-viral gene delivery systems to mammalian cells. Previously, chitosan-based gene formulations had been generally associated with high molecular weight chitosans, which were poorly characterised in terms of molecular weight distribution and degree of acetylation. Therefore, in order to improve the properties of chitosan-based gene formulations, the research associated with this thesis focused on establishing the structure-property relationships of well-defined, low molecular weight chitosans (chitosan oligomers) as delivery systems for nucleic acids (pDNA and siRNA)<i> in vitro</i> and after lung administration <i>in vivo</i>. pDNA dissociated more easily from chitosan oligomers than from conventional high molecular weight chitosans, resulting in a faster onset and higher levels of<i> in vivo</i> gene expression, comparable to those mediated by polyethyleneimine (PEI), one of the most efficient non-viral delivery systems. Coupling of a trisaccharide branch to the chitosan oligomers so as to target extracellular lectins resulted in a significant improvement in transfection efficiency because of enhanced cellular uptake and colloidal stability. In contrast to pDNA, longer linear chitosan oligomers were required to form physically-stable nanoparticles with siRNA that mediated efficient, sustained gene silencing <i>in vitro</i>. Finally, the use of an optimised catheter device for the nebulisation of small volumes of pDNA formulations resulted in improved dose precision and lung distribution<i> in vivo</i> compared with conventional intratracheal instillation. In conclusion, chitosan oligomers are interesting and viable alternatives to other non-viral gene delivery systems.</p>

Contents: Chitosan -- Controlled drug delivery -- Indomethacin -- Inotropic gelation -- Tripolyphosphate (TPP) -- Explotab® -- Ac-Di-Sol® -- Vitamin C / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.