Implanted tissue engineered substitutes constitute dynamic systems, with remodeling mediated by both the implanted cells and the host. Thus, there exists a significant need for methods to monitor the function and morphology of tissue engineered constructs. Noninvasive monitoring using 1H Nuclear Magnetic Resonance (NMR) spectroscopy and imaging can prove to be the solution to this problem. Spectroscopy allows for assessment of cellular function through the monitoring of inherent metabolic markers, such as total-choline, while high resolution imaging enables the evaluation of construct morphology and interfacial remodeling. We applied these 1H NMR methods to monitor betaTC3 mouse insulinoma cells within hydrogel-based materials as a model pancreatic tissue substitute. In vitro research established a strong correlation between total-choline, measured by 1H NMR spectroscopy, and viable betaTC3 cell number, measured by MTT. Extending these methods to in vivo monitoring, however, was met with additional challenges. First, the implanted cells needed to be contained within a planar construct above a threshold density to allow for adequate quantification of the total-choline peak. Secondly, cell-free buffer zones between the implanted cells and the host tissue needed to be incorporated to prevent host tissue signal contamination. Finally, quantitative techniques needed to be developed to accurately account for contaminating signal from diffusing molecules. To overcome these challenges, a disk-shaped agarose construct, initially containing a minimum of 4 million betaTC3 cells and coated with an outer layer of pure agarose, was fabricated. Mathematical simulations aided the implant design by characterizing diffusive transport of nutrients and metabolites into and out of the construct. In vivo 1H NMR studies of these constructs implanted in mice established a strong correlation between total-choline, measured noninvasively using 1H NMR spectroscopy, and viable cell number, measured invasively using MTT. This study establishes total-choline as a reliable marker for noninvasively quantifying dynamic changes in viable betaTC3 cell number in vivo. 1H NMR imaging was used to monitor the implants structural integrity over time, while also assessing the hosts fibrotic response. We expect these studies to establish quantitative criteria for the capabilities and limitations of NMR methodologies for monitoring encapsulated insulinomas, as well as other tissue implants.

The development of a bioartificial pancreas (BAP) has the potential to substantially improve the treatment of insulin-dependent diabetes. Composed of insulin-secreting cells encapsulated in a hydrogel material, a BAP may provide superior glycemic regulation compared with conventional exogenous insulin-delivery therapies. Towards this goal, β- cells or islets encapsulated in alginate microcapsules remain a promising approach. Due to the limited supply of human islets, alternative cell sources are under investigation for incorporation into a BAP, including porcine islets and β- cell lines. Several challenges remain to clinical implementation, including loss of islet or β- cell function and viability following transplantation and host response to the transplanted microcapsules. The objective of this work was to evaluate strategies to improve a BAP by supporting the function and survival of encapsulated islets and β -cells. Towards this goal, two areas were explored: 1) the provision of pro-survival and insulinotropic factors, namely, CXCL12 and GLP-1 (or a GLP-1 analog, Exendin-4), to encapsulated islets and β-cells and 2) modification of the alginate microcapsule to confer long-term resistance to host cell adhesion. To achieve the first objective, methods to deliver both pro-survival and insulinotropic factors to a BAP were developed and their effects on encapsulated β-cells and porcine islets were studied, both in vitro and in vivo. Results demonstrate that delivery of pro-survival and insulinotropic factors is a promising strategy to prolong the survival and function of a BAP. To reduce host cell adhesion to the microcapsule, we employed covalent conjugation of PEG to the surface of alginate-PLL capsules to replace the un-crosslinked layer of alginate used in traditional alginate-PLL-alginate (APA) microcapsules. Results demonstrate that while PEGylation of alginate-PLL microcapsules initially reduced host cell adhesion over 2 weeks in vivo compared with APA capsules, the PEG coating did not provide long-term protection over 3 months. Taken together, these studies represent a multipronged approach towards improving the duration of BAP function, with the ultimate goal of advancing this technology to the clinic.

For clinical translation of a pancreatic substitute, long-term storage is essential, and cryopreservation is a promising means to achieve this goal. The two main cryopreservation methods are conventional freezing and vitrification, or ice-free cryopreservation. However, as both methods have their potential drawbacks for cryopreservation of a pancreatic substitute, they must be systematically evaluated in order to determine the appropriate method of cryopreservation. Furthermore, previous studies have indicated benefits to encapsulation in 3-D adhesive environments for pancreatic substitutes and that adhesion affects cell response to cryopreservation. Thus, the overall goal of this thesis was to investigate cryopreservation effects on model pancreatic substitutes consisting of cells encapsulated in non-adhesive and adhesive 3-D alginate hydrogels. Murine insulinoma betaTC-tet cells encapsulated in unmodified alginate hydrogels were chosen as the model pancreatic substitute in a non-adhesive 3-D environment. Murine myoblast C2C12 cells, stably transfected to secrete insulin, encapsulated in partially oxidized, RGD-modified alginate hydrogels were chosen as the model pancreatic substitute in a 3-D adhesive environment. With respect to cryopreservation effects on intermediary metabolism of betaTC-tet cells encapsulated in unmodified alginate, results indicate that relative carbon flow through the tricarboxylic acid cycle pathways examined is unaffected by cryopreservation. Additionally, insulin secretory function is maintained in Frozen constructs. However, vitrification by a cryopreservation cocktail referred to as DPS causes impairment in insulin secretion from encapsulated betaTC-tet cells, possibly due to a defect in late-stage insulin secretion. Results from Stable C2C12 cells encapsulated in RGD vs. RGE-alginate indicate that up to one day post-warming, cell-matrix interactions do not affect cellular response to cryopreservation after vitrification or freezing. Although there are differences in metabolic activity and insulin secretion immediately post-warming for DPS-vitrified RGD-encapsulated Stable C2C12 cells relative to Fresh controls, metabolic activity and insulin secretion are maintained at all time points assayed for Frozen constructs. Overall, due to results comparable to Fresh controls and simplicity of procedure, conventional freezing is appropriate for cryopreservation of betaTC-tet cells encapsulated in unmodified alginate or Stable C2C12 cells encapsulated in partially oxidized, RGD-modified alginate.

Cette thèse présente un travail sur l'obtention d'aérogels d'HDL par séchage en conditions CO2 supercritique et l'élaboration de nouveaux bionanocomposites formés par la coprécipitation d'hydroxydes doubles lamellaire (HDL) dans l'espace confiné des billes d'alginate. Grâce à la combinaison d'une synthèse par coprécipitation Flash et d'un séchage en conditions supercritiques au CO2, des aérogels d'HDL possédant des surfaces spécifiques élevées sont élaborés. Parallèlement, l'alginate est utilisé comme une matrice de confinement pour la précipitation inorganique d'HDL. D'une part, les billes d'alginate sont synthétisées par complexation des ions Ca2+ et la coprécipitation des phases HDL s'effectue en réalisant des imprégnations successives de réactifs. D'autre part, des billes d'alginate sont formées directement en présence des cations divalents (Mg2+, Ni2++, Co2+, ...) et des cations des métaux trivalents (Al3+), précurseurs des composés inorganiques. La coprécipitation des HDL se produit dans ce cas lors d'une étape d'imprégnation dans une solution d'hydroxyde de sodium. Tous les composés HDL, aérogels ou encore bionanocomposites sont caractérisés en détail par DRX, spectroscopie IR, MEB/MET, adsorption/désorption d'azote et ATG/DTG, pour obtenir un meilleur aperçu de la structure des particules, de leur taille et de leur morphologie. Des études menées sur l'adsorption de la trypsine pour les aérogels ou encore sur les performances d'électrodes modifiées HDL-alginate ont permis de montrer qu'il était possible d'améliorer les performances des HDL en augmenter leur porosité et en élaborant des bionanocomposites.

La synthèse de glycomonomères à base d'oligoalginate (AlgiMERs) et leur polymérisations conventionnelles et RAFT en solution aqueuse ont été étudiés. Premièrement, l'oligoalginates de départ ont été transformés soit dans le glycosylamines correspondant ou en amino alditols (via une amination réductrice). A cette étape, l'optimisation des protocoles d'amination ont été identifiées par la réalisation d'une étude systématique sur un simple acide uronique (acide D-glucuronique). Deuxièmement, les sucres aminés ont été obtenus a réagi avec une électrophile portant un groupe vinyle polymérisable à céder AlgiMERs. Le glycomonomères résultant n'a pas homopolymérisé même en haute force ionique et pour temps de réaction longs, mais leur copolymérisations radicalaire conventionnelles avec N-(2-hydroxyéthyl)méthacrylamide (HEMAm) donne de glycopolymères avec de haute mass molaires (Mw ≈ 1.500.000 Da) contenant jusqu'à 50% en masse de oligoalginate. Une étude cinétique a confirmé que la consommation des deux monomères suivi une cinétique de premier ordre et que les AlgiMERs ont été intégrées tôt dans le processus de polymérisation. Basé sur ces résultats, l'enquête a été étendue à la copolymérisation radicalaire vivante en milieu aqueuse et glycopolymères gradient bien définies ont été obtenues (Mn = 12 000 Da - 90 000 Da; PDI ≤ 1,20). Enfin, j'ai pu prouver qu'un polymère synthétique portant des résidus d'oligo (1→4)-α-L-guluronan conduit des gels en présence d'ions Ca2+ et offre un hydrogel transparent et stable.

In this study, polyelectrolyte membranes were prepared by layer-by-layer self-assembly on top of an interfacially polymerized polyamide substrate, and these thin-film-composite membranes were studied for pervaporative dehydration of ethylene glycol, ethanol and isopropanol. The performance of composite membranes based on polyethylenimine/poly(acrylic acid) (PEI/PAA) multilayers on a polyamide substrate showed good selectivity and stability for ethylene glycol dehydration. In order to understand the formation process of the polyelectrolyte multilayers, the growth of polyelectrolyte multilayers fabricated on the inner surface of cuvette was investiagted. The membrane surface became increasingly hydrophilic with an increase in the number of polyelectrolyte double layers, which favored water permeation for pervaporative dehydration of organic solvents. Water contact angle on the membrane surface decreased from 68?? to 20?? when 7 polyelectrolyte bilayers were deposited on the polyamide substrate. Although the (PEI/PAA) based polyelectrolyte membranes showed good performance for dehydration of ethylene glycol, these membranes did not perform well for the dehydration of ethanol and isopropanol at relatively high feed alcohol concentrations. This was found to be caused by insufficient stability of PEI/PAA bilayers and the polyamide substrate in the ethanol and isopropanol. To improve the performance of the composite membranes for dehydration of ethanol and isopropanol, the outermost surface layer was deposited with PEI, followed by crosslinking. A further improvement in the membrane selectivity was accomplished by substituting the PEI with partially protonated chitosan in the last few polyelectrolyte bilayers during membrane fabrication. It was demonstrated that using interfacially polymerized polyamide membrane as a substrate, polyelectrolyte membranes with less than 8 bilayers could be fabricated for the dehydration of alcohol and diol. This represents a siginificant advancement as a large number of polyelectrolyte bilayers (as many as 60) are often needed. Glutaraldehyde crosslinked polyelectrolyte self-assembled membranes comprising of chitosan and PAA were also prepared for isopropanol/water separation. The resulting membrane showed stable performance with good permeation flux and separation factor. The effects of crosslinking conditions (e.g., concentration and temperature of crosslinking agent, and crosslinking time) on the membrane performance were studied. Alternatively, using PEI as polycation, when anionic PAA was substituted with alginate in the last few polyelectrolyte bilayers during membrane fabrication, stable membranes with a good performance were obtained without the need of chemical crosslinking. The polyethylenimine/alginate self-assembly membranes showed good selectivity and stability for dehydration of ethanol. For instance, a permeation flux of 0.24 kg/(??? h) and a separation factor of 206 were obtained at room temperature at 10 wt% feed water concentration with a membrane comprising of 10 double layers of polyelectrolytes.

Osteoarthritis is a degenerative joint disease characterized by progressive erosion of the articular cartilage. Epidemiological studies have established a relationship between osteoarthritis and menopause suggesting that estrogen may be important in the development of cartilage regeneration therapies. The overall goal of this research project was to advance the field of cartilage tissue regeneration by investigating the role of 17 ß -estradiol (E2), an active estrogen metabolite, on the chondrocyte phenotype. The central hypothesis was that E2 plays an important and sex-specific role in regulating chondrogenesis. Specific Aim-1 focused on establishing and characterizing a primary human articular chondrocyte (HAC) cell source, and then examining the response of the cells in culture to E2. It was demonstrated that the response of HACs to E2 treatment was sex-specific despite both male and females cells expressing estrogen receptors. Female HACs showed changes in proliferation, matrix production, and differentiation while male cells did not. In addition, the female response was regulated through a rapid membrane signaling pathway mediated by protein kinase C. Specific Aim-2 involved establishing an ovariectomized animal model to investigate the effects of E2 on orthopaedic tissue implants. Human demineralized bone matrix (DBM) was implanted intramuscularly into female nude mice and rats. Ovariectomy was shown to reduce the ability of DBM to induce the formation of cartilage and bone tissue. Moreover, the inductive properties of DBM were reestablished with subcutaneous E2 supplementation. Specific Aim-3 entailed developing and characterizing a microencapsulation method for in vitro culture and in vivo delivery of chondrocytes to study the effects of E2 on chondrogenesis. Rat growth plate chondrocytes and HACs were microencapsulated in alginate using an extrusion method in conjunction with high electrostatic potential. Chondrocytes maintained their phenotype in alginate suspension but were unable to form cartilage tissue when implanted into our animal model. Further optimization of the system is required before the role of E2 on chondrogenesis of tissue engineered constructs can be determined. In summary, our results suggest that the successful production of tissue engineered therapies will likely depend on understanding and manipulating the actions of sex hormones in both the in vitro and in vivo environment.

Content removed from thesis due to copyright restrictions: Winger, R.J. and L. Ren (2009). "Solubility of sodium and potassium iodates in saturated salt solutions." Food Chemistry 113: 600-601. / This paper was to improve the production of calcium-induced alginate gels manufactured by a company in Auckland. Problems encountered included yield and syneresis of the beads post-gelation. Essentially the alginate, sugars and other ingredients were dissolved in water at 80ºC. The pH of the solution was adjusted and the alginate beads were extruded into a 5% CaCl2 bath before being drained and dried. The chemical reaction between sodium alginate and calcium ions is dependent upon the solubility and availability of calcium ions. Some calcium salts (e.g., CaCl2, calcium lactate) were readily soluble and fully dissociated in water and resulted in an immediate gelation of the alginate. Dicalcium phosphate (DCP) was sparingly soluble at pH 7 and calcium ions were not released significantly until the pH reached about pH 4.2. Sodium hexametaphosphate (SHMP) is a chelating agent and this was used to soak up small quantities of Ca+2 to ensure no gelation occured while the alginate was being mixed. The optimum quantities of alginate, DCP and SHMP were defined in the laboratory trials. The use of SHMP, maltodextrin, and gums significantly affected the hardness and stickiness of gel beads. It was found that the combination of xanthan and alginate Protanal LF 120 gave the best results in terms of minimal stickiness and maximum yield after drying. Key words: alginate gel beads, syneresis, formula, pH, citric acid, gelation time, SHMP, setting time, yield rate, drying, hardness, stickiness, maltodextrin, xanthan gum, guar gum, stickiness by touching, leakage, apparent viscosity.

91f. / Submitted by Suelen Reis (suziy.ellen@gmail.com) on 2013-04-23T13:36:16Z No. of bitstreams: 1 Dissertacao Ana Emilia.pdf: 3842407 bytes, checksum: 06b8d72eed292f7ebdf42b0a223ed0f6 (MD5) / Approved for entry into archive by Rodrigo Meirelles(rodrigomei@ufba.br) on 2013-05-08T11:44:00Z (GMT) No. of bitstreams: 1 Dissertacao Ana Emilia.pdf: 3842407 bytes, checksum: 06b8d72eed292f7ebdf42b0a223ed0f6 (MD5) / Made available in DSpace on 2013-05-08T11:44:00Z (GMT). No. of bitstreams: 1 Dissertacao Ana Emilia.pdf: 3842407 bytes, checksum: 06b8d72eed292f7ebdf42b0a223ed0f6 (MD5) Previous issue date: 2010 / A bioengenharia tecidual tem possibilitado o desenvolvimento de biomateriais e técnicas inovadoras capazes de restituir a qualidade de vida aos pacientes com perdas ósseas, após a remoção de grandes tumores ósseos, traumas e em algumas doenças metabólicas. Dentre os biomateriais empregados nas terapias regenerativas destacam-se as biocerâmicas sintéticas, na forma de microesferas. Estas microesferas constituem um arcabouço que possibilita, no interstício formado entre as esferas, a difusão de fatores de crescimento e angiogênicos, a migração de cálulas osteogênicas essenciais à regeneração deste tecido. Ainda apresentam características físico-químicas semelhantes ao componente inorgânico do tecido ósseo e biocompatibilidade. O objetivo deste trabalho foi avaliar a regeneração de defeitos ósseos críticos, em calvária de rato. Para tanto, utilizou-se uma amostra composta de 60 animais, dividida em quatro grupos: Gl - defeito sem biomaterial, preenchido por coágulo; Gll - microesferas de hidroxiapatita e alginato (HA - Alg); Glll - microeferas de hidroxiapatita dopadas com estrôncio (HASr); GlV - microesferas de hidroxiapatita dopadas com estrôncio e alginato (HASr -Alg), avaliados aos 15, 45 e 120 dias de pós operatório. As análises histológicas, por microscopia de luz, demonstraram neoformação óssea mais evidente no grupo Gll em todos os pontos biológicos quando comparado aos demais grupos. As microesferas implantadas nos grupos lll e lV apresentaram maior fragmentação quando comparadas com as do grupo ll, provavelmente devido à adição de estrôncio à estrutura da HA. A neoformação óssea caracterizada pela osteocondução foi observada de permeio a esses fragmentos do biomaterial. No grupo lll, a ausência do alginato tornou as microesferas mais porosas, com formação de septos de tecido conjuntivo e células fusiformes no interior das microesferas. Em conclusão, os biomateriais apresentaram potencial osteogênico e osteocondutor formando um arcabouço tridimensional que favoreceu a neoformação óssea. A HA associada ao alginato apresentou características histológicas mais evidentes quanto a biocompatibilidade e a osteogênese, quando comparada a HA dopada com estrôncio. / Salvador

Esta Dissertação teve como objetivo,a síntese de hidrogéis à base de alginato e nanopartículas magnéticas (maghemita) preparadas in situ. Os hidrogéis foram preparados em diferentes concentrações de alginato de sódio (2 e 3% m/v), FeSO4 (0,3 e 0,5 mol L-1) e CaCl2 (0,1 e 0,3 mol L-1). As propriedades físico-químicas dos hidrogéis foram analisadas e, posteriormente, foram avaliados quanto à capacidade de remoção de íons Ni2+ e Mn2+ de soluções aquosas. Para caracterização das amostras foram utilizadas diversas técnicas de análises, tais como, análise granulométrica, microscopia óptica (OM), microscopia eletrônica de varredura (SEM), microscopia eletrônica de transmissão (TEM), magnetometria de amostra vibrante (VSM), espectroscopia na região do infravermelho por transformada de Fourier (FTIR), difratometria de raios-X (XRD), espectroscopia Mössbauer, e análise termogravimétrica (TGA). Foram preparados hidrogéis com morfologia predominantemente esférica e dimensões micrométricas (500 a 850 m), com átomos de Fe e Ca dispersos uniformemente em sua estrutura. Os hidrogéis apresentaram boa resistência térmica e comportamento superparamagnético. As amostras foram intumescidas em água deionizada durante um intervalo de tempo a fim de avaliar o grau de intumescimento (Q) para determinar a amostra com a melhor resposta para posterior aplicação em solução aquosa contendo íons metálicos (Ni2+ e Mn2+). Os resultados revelaram que a amostra cuja concentração de 3% m/v de alginato de sódio, 0,3 mol L-1 de FeSO4 e 0,3 mol L-1 de CaCl2 obteve maior Q (50%). Em consequência deste resultado, optou-se por utilizar estaamostra, na remoção de metais pesados presentes em soluções aquosas e em efluentes industriais. Vários parâmetros,tais como: tempo de contato,pH, concentração inicial do íon e massa de hidrogel foram estudados.Os resultados, para efluente sintético, revelaram que o tempo de equilíbrio foi de 60 minutos; a capacidade de remoção dos metais melhora com o aumento de pH (3 a 9), sendo máxima em pH 7;quanto menor a concentração inicial da solução iônica (50 a 500 mg L-1), maior a capacidade de remoção, 52% de Ni2+ e 49% de Mn2+ (concentração inicial de 50 mg L-1). No efluente industrial, a remoção foi de 61% de Ni2+ e 57% de Mn2+(300 mg de hidrogel). Os resultados encontrados revelaram que os hidrogéis magnéticos produzidos à base de alginato têm potencial uso no tratamento de efluentes industriais contaminados com metais pesados / This Dissertation aims thesynthesis of hydrogels based on alginate and magnetic nanoparticles (maghemite) prepared in situ. Hydrogels were prepared at different concentrations of sodium alginate (2 and 3% w/v), FeSO4 (0.3 and 0.5 mol L-1) and CaCl2 (0.1 and 0.3 mol L-1). The physicochemical properties of the hydrogels were analyzed and, subsequently, evaluated for their ability to remove ions Ni2+ and Mn2+ from aqueous solutions. In orderto characterize the samples,several techniques were used, such as, granulometric analysis, optical microscopy (OM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), vibrating sample magnetometry (VSM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Mössbauer spectroscopy and thermogravimetry analysis (TGA). Hydrogels with predominant spherical morphology and micrometric dimension were prepared (500 and 850 m) with atoms of Fe and Ca dispersed uniformly in their structure. The hydrogels presented good thermal resistance and superparamagnetic behavior. The samples were swollen in deionized water, for a period of time, to evaluate the swelling degree (Q) and determine the sample with the best result for subsequent application in an aqueous solution containing metallic ions (Ni2+ and Mn2+). The results revealed that the sample with concentration of 3% w/v of sodium alginate, 0.3 mol L-1 of FeSO4, and 0.3 mol L-1 of CaCl2 presented the higher Q (50%). In consequence of this result, we decided to use thissample, in the removal of heavy metals from aqueous solution and industrial wastewater.Several parameters, such as: contact time, pH, initial concentration of ionic solution and hydrogel mass were studied. The results, to the synthetic solution, revealed that the equilibrium time was 60 minutes; the capacity of metals removal improves with the pH increasing (3 to 9), and was maximum at pH 7; the lower the initial concentration of ionic solution (50 to 500 mg L-1), the higher the removal capacity, 52% of Ni2+ and 49% of Mn2+ (initial concentration of 50 mg L-1). In the industrial wastewater, the removal was 61% of Ni2+ and 57% de Mn2+ (300 mg of hydrogel). The results showed that magnetic hydrogels based on alginate synthesized have potential use in the treatment of industrial wastewater contaminated with heavy metals