Neste trabalho foram estudados os efeitos causados pelo tratamento térmico na argila STx-1b-Li+ na condutividade de filmes argila/alginato de sódio. O argilomineral STx-1b foi submetido à substituição isomórfica do cátion Na+ por Li+ e posteriormente ao tratamento térmico em diferentes temperaturas. As argilas STx-1b-Li+ não tratada e tratada termicamente foram caracterizada por difração de raios-X (DRX) e termogravimetria (TG). As curvas TG mostraram que o argilomineral STx-1b-Li+ apresentou três perdas de massa que podem ser atribuídas à água adsorvida na argila, a água de hidratação dos íons Li+ e a água estrutural da argila. Para as argilas STx-1b-Li+ tratadas termicamente não foi observado a perda de massa associada à água quimicamente ligada aos contra-íons Li+. Estes resultados estão em concordância com os obtidos por DRX, onde observou-se uma diminuição no espaçamento interlamelar nas argilas tratadas termicamente. Em adição, foram preparados filmes de alginato de sódio com as argilas STx-1b-Li+ não tratada e tratada termicamente e estes foram caracterizados por calorimetria exploratória diferencia (DSC) e espectroscopia de impedância eletroquímica (EIE). As curvas DSC mostraram um aumento na temperatura de transição vítrea (Tg) dos filmes com o aumento da temperatura utilizada no tratamento das argilas. Este aumento nos valores de Tg é um indicativo que a mobilidade das cadeias diminuiu nos filmes polímero/argila que contém argilas tratadas em temperaturas mais elevadas. A espectroscopia de impedância eletroquímica (EIE) foi usada para avaliar-se a condutividade iônica dos filmes. Os resultados indicaram que os filmes contendo argilas que foram tratadas com temperaturas mais altas apresentaram menor condutividade. / In this work the effects caused by the heat treatment in STx-1b-Li+ clay on the conductivity of sodium alginate/clay were studied. The clay mineral STx-1b was submitted to isomorphous substitution of the Na+ for Li+ cation and subsequently to heat treatment at different temperatures. The STx-1b-Li+ clays untreated and heats treated were characterized by X-ray diffraction (XRD) and Thermogravimetric Analysis (TG). The TG curve showed that the clay mineral STL-1b-Li+ had three mass losses which can be attributed to adsorbed water in the clay, the water of hydration of the Li+ ions and structural clay water. To STx-1b-Li+ clays heat treated there were no mass loss associated to the water chemically bound to Li+ counter ions. These results are in agreement with those obtained by XRD, which it was observed a decrease in interlayer spacing in the heat treated clay. In addition, they were prepared sodium alginate films with STx-1b-Li+ clays untreated and heat treated and these films were characterized by Differential Scanning Calorimetry (DSC) and Electrochemical Impedance Spectroscopy (EIS). DSC curves showed an increase in glass transition temperature (Tg) of the films with temperature increasing used in the treatment of clays. This increase in Tg value is an indicative that the mobility of the chains had decrease in the polymer/clay films which contains heat treated clays at higher temperatures. The electrochemical impedance spectroscopy (EIS) was used to evaluate the ionic conductivity of the films. The results indicate that films containing clays which have been treated at higher temperatures had lower conductivity.

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2008. / Submitted by Caroline Silva (krol_bilhar@hotmail.com) on 2012-09-16T23:58:31Z No. of bitstreams: 1 diss2008fernandagermanoalves.pdf: 800800 bytes, checksum: 497362ef874efddd275ccc67dcdf0d1e (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-09-22T15:51:30Z (GMT) No. of bitstreams: 1 diss2008fernandagermanoalves.pdf: 800800 bytes, checksum: 497362ef874efddd275ccc67dcdf0d1e (MD5) / Made available in DSpace on 2012-09-22T15:51:30Z (GMT). No. of bitstreams: 1 diss2008fernandagermanoalves.pdf: 800800 bytes, checksum: 497362ef874efddd275ccc67dcdf0d1e (MD5) Previous issue date: 2008 / A β-galactosidase é amplamente distribuída na natureza, podendo ser encontrada em plantas, órgãos de animais e microrganismos. A hidrólise da lactose via enzimática vem sendo uma alternativa para as indústrias alimentícias, visto que os açúcares resultantes deste processo, glicose e galactose, são mais solúveis e doces, o que proporciona melhorias nas características sensoriais de produtos lácteos e, desenvolvimento de alimentos com baixo teor desse carboidrato, tornando-os ideais a consumidores intolerantes a este açúcar. O aumento da demanda industrial da β-galactosidase resulta na necessidade do estudo da agitação e da aeração, visando obter um produto de elevada atividade. A utilização de enzimas na indústria alimentícia é muitas vezes limitada devido a sua baixa estabilidade. Uma das alternativas é o emprego de enzimas imobilizadas para reduzir os problemas causados pela utilização de enzimas solúveis em aplicações industriais. A presente dissertação teve por objetivo geral o estudo das condições de produção da β-galactosidase de Kluyveromyces marxianus CCT 7082 e caracterização parcial da enzima livre e imobilizada. No primeiro estudo foi avaliada a influência da agitação e da aeração na produção da enzima por fermentação submersa em fermentador Biostat B de 2 L, utilizando a técnica de planejamento experimental, através de um planejamento experimental 22 (4 ensaios e 3 pontos centrais), onde as condições estudadas foram: 200; 350; 500 rpm e 0,5; 1,0; 1,5 vvm para a agitação e a aeração, respectivamente. Neste estudo verificou-se que a agitação e a aeração, exerceram influência na produção da enzima, sendo a condição mais favorável 500 rpm e 1,5 vvm, respectivamente, atingindo uma produtividade de 1,2 U.mL-1.h-1, uma atividade enzimática de 17 U.mL-1 e uma concentração celular de 11 mg.mL-1. Em um segundo estudo foi realizada a imobilização da β-galactosidase empregando a técnica de inclusão em gel de alginato de cálcio, seguida da caracterização das enzimas livre e imobilizada, quanto ao pH e temperatura ótimos, parâmetros cinéticos e estabilidade térmica. Para o estudo da influência do pH na atividade enzimática foram testados valores de pH entre 4,6 e 8,6. A influência da temperatura na reação enzimática foi determinada pela atividade de β-galactosidase nas temperaturas de 25 a 60ºC. Os parâmetros cinéticos foram determinados utilizando como substrato o-nitrofenil-b-D-galactopiranosídeo (ONPG) e lactose. A estabilidade térmica foi estudada determinando-se a constante cinética de desnaturação térmica, o tempo de meia vida e a energia de ativação da reação de desnaturação, incubando-se a enzima nas temperaturas de 30 a 45ºC. Os valores ótimo de pH e temperatura não foram alterados quando a enzima foi imobilizada, obtendo como resultados pH 6,6 e 37ºC, respectivamente, para ambas as formas enzimáticas. Os resultados dos parâmetros cinéticos, Km e Vmax, para a enzima livre foram 15,1 mM e 18,9 U.mL-1; 93,71 mM e 43,9 U.mL-1 para os substratos ONPG e lactose, respectivamente. Para a enzima na forma imobilizada os resultados para Km e Vmax foram 18,5 mM e 3,9 U.mL-1; 115,7 mM e 3,7 U.mL-1, respectivamente, utilizando ONPG e lactose. Com relação à estabilidade térmica enzimática, a equação de Arrhenius pôde ser aplicada para estabelecer uma relação entre a constante cinética de desnaturação térmica e a temperatura. Pela equação de Arrhenius determinou-se as energias da reação de ativação (9,4 e 2,1 Kcal.mol-1) e de ativação da reação de desnaturação (100 e 106 Kcal.mol-1), respectivamente, para b-galactosidase livre e imobilizada. / Production of b-galactosidase from Kluyveromyces marxianus CCT 7082 in fermenter and partial characterization of the soluble and immobilized enzyme β-galactosidase is widely distributed in nature and it can be found in plants, organs of animals and microorganisms. The enzymatic hydrolysis of lactose has been an alternative to the food industries, because the result sugars of this process, glucose and galactose, are more soluble and sweet, providing improvements in sensory characteristics of dairy products, and development of products without lactose, ideal to consumers intolerant to this sugar. The increased of the industrial demand of β-galactosidase results in the necessity to study the agitation and aeration, to obtain a product of high activity. The use of enzymes in the food industries is sometimes limited due to the low stability. One of the alternatives is the use of immobilized enzymes to reduce the problems caused by the use of soluble enzymes in industrial applications. The present dissertation had as main goal the study of the conditions of β-galactosidase production from Kluyveromyces marxianus CCT 7082 and the partial characterization of the free and immobilized enzyme. In the first study, the influence of the agitation and the aeration was evaluated in the enzyme production by submerged fermentation in Biostat B fermenter of 2 L, using experimental design 22 (4 assays with three replicates at the center point); the study conditions were: 200, 350, 500 rpm and 0.5, 1.0, 1.5 vvm for agitation and aeration, respectively. In this study the agitation and aeration, influenced in the β-galactosidase production, and the favorable condition was 500 rpm and 1.5 vvm, respectively, obtaining 1.2 U.mL-1.h-1 for productivity, 17 U.mL-1 for the enzymatic activity and a cellular concentration of 11 mg.mL-1. In a second study of β-galactosidase was immobilized employing the technique of calcium alginate gel inclusion, followed by the characterization of this enzyme, and comparison between free and immobilized enzymes, as for optimum pH and temperature, kinetic parameters and thermal stability. For the study of the influence of pH on the enzymatic activity, values of pH between 4.6 and 8.6 were tested. The influence of temperature on the enzyme reaction was determined at 25 to 60°C. The kinetic parameters were determined employing o-nitrophenyl-b-D-galactopyranoside (ONPG) and lactose. The thermal stability was studied in order to determinate the deactivation rate constant, the half-life and the deactivation energy, incubating the suspension enzyme at 30 to 45°C. The optimum values for pH and temperature weren’t changed by the immobilization process, obtaining as results pH 6.6 and 37°C, respectively. The results for the Km and Vmax for soluble enzyme were 15.1 mM and 18.9 U.mL-1; 93.7 mM and 43.9 U.mL-1 for ONPG and lactose, respectively; and for immobilized enzyme the results for Km and Vmax were 18.5 mM and 3.9 U.mL-1; 115.7 mM and 3.7 U.mL-1, respectively, for ONPG and lactose. In relation to the enzymatic thermal stability, the Arrhenius equation could be applied to establish a relation between deactivation rate constant and temperature. By the Arrhenius equation were determinated the activation energy (9.4 e 2.1 Kcal.mol-1) e deactivation energy (100 e 106 Kcal.mol-1), respectively, for the soluble and immobilized enzyme.

Orientador: Marisa Masumi Beppu / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-13T07:18:21Z (GMT). No. of bitstreams: 1 Paiva_RafaelGoncalvesde_M.pdf: 2354207 bytes, checksum: c86d978c811175888a0c4b73a90c0a52 (MD5) Previous issue date: 2009 / Resumo: Estudos envolvendo membranas têm atraído muito interesse nos últimos anos, uma vez que, além da aplicação clássica em processos de separação, estas têm sido usadas também em áreas como medicina, microeletrônica, tratamento de água, etc. À medida que o número de aplicações cresce, a necessidade de criação de estruturas elaboradas também aumenta. Neste contexto, este trabalho realizou a adaptação da técnica "layer-by-layer" de produção de membranas multicamadas, porém visando a produção de membranas com espessuras de ordem micrométricas. Foi realizada, também, a subseqüente incorporação de íons cobre a estas membranas. Caracterizações morfológicas, químicas e cristalinas, além de testes de difusão de íons e avaliação de capacidade bacteriostática das membranas foram realizados. As análises químicas mostraram a interação entre as cadeias de quitosana e alginato, garantindo a estabilidade dimensional da membrana. Foi possível observar também mudanças na cristalinidade das membranas, que se tornaram mais amorfas após a incorporação de íons cobre em sua estrutura. Análises de EXAFS mostraram que estes átomos se ligam preferencialmente aos átomos de oxigênio e nitrogênio constituintes dos grupos funcionais de alginato e quitosana, respectivamente. Além disso, a incorporação de cobre às membranas causou mudanças notáveis na difusividade destas, tornando a difusão de cloreto de sódio mais lenta do que nas membranas sem cobre. A capacidade bacteriostática foi sensivelmente influenciada pela presença do metal nas membranas: as amostras com cobre, independente de sua composição, proporcionaram inibição do crescimento bacteriano, tanto para E. coli quanto para S. aureus, ao passo que as amostras sem cobre não apresentaram capacidade bacteriostática. Esses resultados mostram potencial aplicabilidade destas membranas em situações onde este tipo de capacidade é requerida. / Abstract: Study of membranes has attracted much interest in recent years due to their applicability in areas such as medicine, microelectronics, water treatment, etc. rather than in traditional separation processes. As the application range arises, the need for establishing more elaborated structures also increases. In this context, the present study adapts the layer-by-layer technique for production of micro-scale multilayer membranes. Subsequent incorporation of copper ions is performed n these membranes. Morphological, chemical and crystalline characterizations, besides ion diffusion tests and evaluation of bacteriostatic ability were performed. The chemical analysis showed the interaction between the chains of chitosan and alginate, ensuring the dimensional stability of the membrane. Changes in crystallinity of membranes were also observed, as they became amorphous after the incorporation of copper ions in its structure. EXAFS analysis indicated that these atoms are linked preferentially to oxygen and nitrogen atoms, constituents of alginate and chitosan functional groups, respectively. Moreover, the amount of copper to the membrane caused remarkable changes in its diffusivity, reducing its value in comparison to membranes without copper. The bacteriostatic capacity was significantly influenced by the presence of metal in membranes: the samples with copper, regardless of its composition, provided inhibition of bacterial growth, both for E. coli and for S. aureus, while the untreated control samples showed no bacteriostatic capacity. These results show the potential of using these membranes in applications where this kind of capacity is required. / Mestrado / Engenharia de Processos / Mestre em Engenharia Química

Orientador: Marisa Masumi Beppu / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-08T12:50:27Z (GMT). No. of bitstreams: 1 Lima_DanielOliveirade_M.pdf: 10551325 bytes, checksum: b19eaea79808ec2adad2fc6b3728f8de (MD5) Previous issue date: 2006 / Resumo: Não informado / Abstract: Natural systems are able to create organic-inorganic composites of remarkable properties. Bone, for instance, presents properties that are found both in ceramics and polymers. This effect is related to the organic matrix, which controls nucleation and growth of calcium phosphate crystals, besides composite's organization and architecture in nanometric levels, in order to achieve a better understanding of this process, precipitation of calcium phosphate was carried out in a medium which contained small amounts of alginate and chitosan, seeking to emulate the matrix effect. Calcium phosphates precipitates were obtained using two different concentrations of the biopolymer and one control sample. The experiments were divided in two groups, depending on the system's initial pH. In the first group, the starting pH was acid. However, part of the reaction medium was separated and had its pH adjusted to an alkaline range. In the second group, the initial pH was calculated in order to be in the range of hydroxyapatite stability. Characterization was accomplished by scanning electron microscopy, X-Ray diffraction and Fourier-transform infrared spectroscopy. The powders obtained in the first group were compose mainly by brushite (CaHPO4.2H2O) and its anhydrous phase, monetite, CaHP04. In the first group of experiments with alginate, monetite concentration was related mainly to the reaction medium pH, suggesting that alginate-calcium interaction was strongly dependent on the formation of complexes among carboxyl radicals and calcium, which avoided water bonding. Chitosan-doped samples, on the other hand, seemed to be affected mainly by the biopolymer concentration. Chitosan and monetite concentration seemed to be inversely proportional. This is attributed to the ionotropic effect, which reduced auto-diffusion and consequently, calcium phosphate-chitosan interaction. These samples also possessed an apparent degree of orientation, supposedly due to a coherent growth of calcium phosphate crystals over heterogeneous nuclei. Alginate-doped samples obtained in the second group of experiments presented reduced cristal Unity, which was related to reprecipitation of the crystals over biopolymers nuclei. Chitosan-doped samples, however, presented an increase in crista!Iinity. This may be attributed to a process of nucleation inside the gel, implying in crystalline growth controlled by sterical effects / Mestrado / Engenharia de Processos / Mestre em Engenharia Química

Os alginatos de metais alcalinos (Li+, Na+ e K+), alcalino-terrosos (Mg2+, Ca2+, Sr2+ e Ba2+), de amônio, mono-, di- e trietanolamônio foram sintetizados por neutralização do ácido algínico com os respectivos hidróxidos, ou carbonatos, e com as aminas. Os sais foram caracterizados por análise elementar e espectroscopia na região do infravermelho, confirmando as sínteses. Após caracterização, os compostos foram submetidos à análise térmica (TG/DTG, DTA e DSC) para avaliar seu comportamento térmico e seus resíduos de decomposição, que foram caracterizados por IR e XRD. Os alginatos de amônio e etanolamônio se decompuseram com liberação de amônia ao final do experimento. Os alginatos de metais alcalinos foram convertidos nos seus respectivos carbonatos, enquanto os alginatos de metais alcalino-terrosos se decompuseram formando carbonatos, em seguida óxidos. Foi feita uma avaliação de procedimentos de secagem por estufa a vácuo a 40 ºC e liofilização; e o último método mostrou-se mais eficaz. A água residual, não-congelável, sai associada à decomposição do material e não foi possível definir exatamente seu teor. Um método de medir o grau de substituição nos sais das aminas foi desenvolvido com base em 13C ? NMR em fase sólida. / The alkaline (Li+, Na+ and K+), earth-alkaline (Mg2+, Ca2+, Sr2+ and Ba2+), ammonium, mono-, di- and triethanolammonium alginic acid salts were obtained from the neutralization reaction between alginic acid and the respective hydroxides or carbonates, and the amines, The salts were characterized by elemental analysis and infrared spectroscopy, confirming the synthesis. After the characterization, the compounds were submitted to thermal analysis (TG/DTG, DTA and DSC), in order to evaluate their thermal behavior. The thermal decomposition residues were characterized by IR and/or XRD. The NH4+ and ethanolammonium alginates decomposed via NH3 release without residue in the crucible at the end of the experiment. The alkaline alginates were converted to the respective carbonates, and the earth-alkaline decomposed with production of the carbonates followed by convertion to the oxides. An evaluation of drying procedures involving heating under vaccum up to 40°C and lyophilization were performed, pointing better results in the last case. The residual water, of the non-freezing type, was completely released only during the decomposition of the biopolymer, and it was not possible to define its exact content in the samples. An attempt to estimate the substitution degree in the ethanolammonium salts using 13C ? NMR data, in solid state, was also described.

A indústria têxtil busca recuperar a diminuição do ritmo dos negócios, notado principalmente em países desenvolvidos devido ao cenário da economia mundial, por meio da elaboração de têxteis com maior valor agregado. A microencapsulação é uma técnica versátil e flexível que apresenta diversas vantagens, como evitar que o princípio ativo reaja com outros compostos presentes no sistema e possibilitar a liberação controlada, que aumenta potencialmente a eficiência do produto. O principal objetivo deste trabalho foi desenvolver microcápsulas de quitosana e alginato com incorporação de Triclosan, que possui propriedades bactericida e fungicida, para aplicação em substratos têxteis para utilizações médicas. As microcápsulas foram produzidas a partir do método de emulsificação e reticulação, e caracterizadas por Termogravimetria (TG), Calorimetria Exploratória Diferencial (DSC), Espectroscopia no Infravermelho com Transformada de Fourier (FTIR), capacidade de absorção de água e perda de massa, Microscopia Eletrônica de Varredura (MEV), ensaio de atividade bactericida e liberação in vitro. Após caracterizadas, as microcápsulas foram impregnadas em tecidos 100% algodão com ligamentos tela e sarja. Estes foram submetidos a testes físicos e análise de resistência à lavagem. As microcápsulas produzidas apresentaram forma esférica e tiveram 80,78% de eficiência de encapsulação do fármaco. Os ensaios de liberação mostraram que o fármaco não foi liberado em 24h, entretanto, o material apresentou atividade bactericida contra a bactéria gram-positiva S. aureus, com halo de inibição de até 60 mm e também contra a bactéria gram-negativa E. coli, com halo de até 25 mm. Os resultados de resistência à lavagem avaliados por MEV mostraram que as microcápsulas não permenceram no substrato. Entretanto, o material apresentou atividade antibacteriana podendo ser interessante para aplicação em materiais têxteis descartáveis, como bandagens utilizadas na área médica / The textile industry seeks to recover the decrease of the pace of business, noted mainly in developed countries due to the scenario of the world economy, through the development of textiles with higher added value. The microencapsulation is a versatile and flexible technique that presents several advantages such as to avoid that the active ingredient react with other compounds present in the system, and allow controlled release that potentially increases the efficiency of the product. The main objective of this work was to develop microcapsules of chitosan and alginate with incorporation of triclosan, which has bactericidal and fungicide properties, for use in textile substrates for medical uses. The microcapsules were produced from the method of emulsification and crosslinking, and characterized by Thermogravimetry (TG), Differential Scanning Calorimetry (DSC), Infrared Spectroscopy Fourier Transform (FTIR), water absorption capacity and mass loss, Scanning Electron Microscopy (SEM), bactericidal activity assay and in vitro release. After characterized, the microcapsules were impregnated in 100% cotton twill and taffeta woven. Physical tests and analysis of resistance to washing were carried out. The microcapsules produced presented spherical shape and had 80.78% of drug encapsulation efficiency. Release tests showed that the drug was not released in 24 hours, however, the material presented bactericidal activity against the gram-positive bacterium S. aureus, with inhibition halo up to 60 mm and also against the gram-negative bacterium E. coli, with halo of up to 25 mm. The results of washing resistance evaluated by SEM showed that the microcapsules did not remain in the substrate. However, the material showed antibacterial activity and may be interesting for application in disposable textiles, such as bandages used in the medical field

In Canada, one in five people suffers from arthritis, of which the most common type is osteoarthritis (OA). OA is a group of joint diseases that cause pain and loss of range of motion and for which there is currently no cure. OA can be caused by numerous factors such as aging, genetics, environmental elements, and abnormal joint biomechanics (e.g., injury, obesity). These diseases are degenerative and lead to the progressive breakdown of joint cartilage, as well as changes in the underlying bone and other tissues of the joint over a period of years to decades. Articular cartilage incorporates a single type of resident cells, termed chondrocyte cells. These cells are entrapped within a dense extracellular matrix that limits their ability to proliferate and migrate to a site of injury, while the absence of blood vessels in the cartilage, amongst other factors, hinders the ability of progenitor cells to reach the site of injury, contributing to a limited capacity for intrinsic regeneration of the damaged tissue following an injury. As such, efforts to develop tissue engineering strategies that combine a biomaterial with bioactive signals to induce cells with the chondrogenic potential to regenerate tissue have been pursued actively. In this thesis, we investigate the potential of one such cartilage tissue engineering approach, whereby chondrocytes are encapsulated with alginate hydrogels incorporating inorganic polyphosphate (polyP), a promising chondrogenic signal. The driving hypothesis of the work was that polyanionic polyP would crosslink within the alginate hydrogel meshwork by ionic bonds with the multivalent cations used to form the hydrogel. Initial efforts focussed on optimizing the sterile chondrocyte encapsulation protocol for alginate beads, chondrocyte culture conditions to reduce proliferation – a response that is associated with dedifferentiation and a pathological state – and protocol for the incorporation of polyP in alginate bead when using calcium as a cationic crosslinker. We observed that polyP release from the calcium-alginate bead exhibited an important burst release to nearly 80% of the initial polyP loading within 24 hours of incubation in the culture medium. Increasing the alginate concentration led to approximately a 2.5-fold increase in polyP retention following the burst release. Subsequent incubation showed a more controlled release for at least 1 week. Efforts to reduce hydrogel swelling and increase its stability by substituting Ca2+ by Sr2+ as a crosslinker did not reduce the release rate during the burst release phase, nor did it increase the polyP retention following this initial stage. Other divalent cations including Mg2+ and Co2+, and pre-loading the polyP-alginate solution with a small concentration of Ca2+ did not impact the release profile either. Chondrocytes encapsulated in calcium- and strontium-alginate beads showed decreased DNA content and increased sulfated glycosaminoglycan accumulation at 1 week when polyP was incorporated in the beads compared to controls without polyP; however, this effect was lost at longer time points. These results suggest that this new material may find applications as a vehicle for the short-term delivery of polyP in joints and other tissues. Further efforts to improve the polyP release profile from alginate beads lead to promising results with the use of polyethylenimine (PEI) as a cationic tethering molecule between polyP and alginate. This thesis aims to generate novel biomaterials that can be used to stimulate cartilage tissue regeneration and to eventually develop a treatment strategy for OA. The work presented here will serve as a basis for continued efforts to ensure the prolonged retention of exogenous polyP into the joint.

Alginate is a widely used biomaterial for a variety of biomedical applications ranging from drug delivery to cell transplantation. The unique polysaccharide backbone endows the material with a number of useful properties such as hydrophilicity, biocompatibility, and gelation ability. Despite these advantages, one limitation for alginate is the lack of a tunable degradation rate, and its gels may only partially degrade and implants are not fully cleared from the body long after their purpose is fulfilled. To further extend the utility of this biomaterial, we hypothesized that by creating a polymer chimera between polylactic acid (PLA) and alginate we can integrate tunable degradation properties into alginate hydrogels. The alginate-b-PLA diblock copolymers were synthesized by utilizing an inverse electron demand Diels-Alder reaction, and were then fabricated into hydrogels using two approaches: doping with low viscosity alginate (LWA) and direct gelation. These hydrogel chimeras exhibited degradation rates that could be tuned from days to weeks. Morphologically, the combination of different domain sizes of alginate and PLA contributed to different microstructures within the hydrogel matrix that contributes to its degradability. Drug release was not impacted by matrix degradation rate, as four different encapsulated payloads of variable hydrophobicity and molecular weight were encapsulated with the chimeric hydrogels showed comparable release rates to non-degradable alginates. These new degradable alginates could have future utility as degradable drug-eluting implants. / 2022-09-24T00:00:00Z

The use of 3D printing in biological applications is a new field of study given that 3D printing technology has become more available and user friendly. Possible uses include using existing 3D printing polymers to use in extracorporeal or in vitro devices, like Lab-on-a-Chip, and the development of new biologically derived materials to print cell-containing constructs. The latter concept is what is more commonly known as bioprinting. Our research had the goal of developing a bioprinting system including the printer, a bioink, and a feedback system for printing parameter optimization which could be done cheaply and within the reach of nearly any research lab. To make the bioprinter, we were able to take a popular plastic 3D printer and convert it to a bioprinter with 3D printed parts and the addition of a new motherboard. This came with great contribution from Carnegie Melon University. We were also able to improve upon the original design and, along with the new bioprinting capabilities, maintain the original capabilities of the plastic 3D printer. A new bioink was developed to work in coordination with this bioprinting system. Our lab has the luxury of having access to decellularized tissue, which provided a unique material to create a bioink which is derived from the extra-cellular matrix of porcine hearts. The final bioink protocol allows the users to make their own bioink, from easily obtainable tissue and determine their own concentration of the extra-cellular matrix/collagen within a range. Lastly, a feedback system was developed using a Raspberry Pi and camera module to provide real-time visual feedback of the bioprinting process which is otherwise very difficult to see and optimize parameters from. A protocol was developed to sequentially optimize the parameters for an open-source slicing software which governs the resolution of the bioprinter itself. In related research, the cytotoxicity and cell adherence properties of a printing resin for a microfluidic 3D printer were evaluated for use in Lab-on-a-Chip applications. The existing resin was tested and determined to be cytotoxic to cells and therefore not suitable for biological applications. We showed that a simple ethanol washing step and plasma treatment pulled the cytotoxic elements out of the polymer and modified the surface such that cells could attach and proliferate on the printed resin. Another printed resin was also tested which was determined to have no natural cytotoxicity, but the same plasma treatment was needed to allow for cell adherence.

Background: Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T(σ22)to initiate transcription of the alginate biosynthetic operon. Results: In the current study, we mapped the mucE transcriptional start site, and determined that P mucEactivity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in P mucEactivity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE. Conclusions: Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.