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La cellulose est l’une des bio-ressources les plus abondantes sur terre ; elle forme des microfibrilles où alternent régions cristallines, de taille nanométrique, et régions amorphes. L’utilisation des nanocristallites de cellulose, appelés nanowhiskers, comme renforts dans des composites à matrice polymère thermoplastique a fait l’objet de nombreuses publications. En revanche, l’utilisation de nanowhiskers comme éléments structurants d’hydrogels n’a pas été vraiment explorée, en dépit d’un intérêt potentiel pour la formulation d’hydrogels verts innovants. L’objectif de la thèse était d’étudier les relations entre état structural à différentes échelles et propriétés rhéologiques de systèmes hydrocolloïdaux constitués d’hydrogels d’agarose chargés de nanowhiskers de cellulose issus de tunicier, un animal marin. Les travaux expérimentaux menés au cours de cette thèse ont montré que l’effet renfort apporté par les nanowhiskers à la matrice agarose pouvait être marqué, et qu’il pouvait être modulé en faisant varier la densité de charges électriques à la surface des nanowhiskers. Aux fractions volumiques étudiées, inférieures à 0,2%, les résultats ont montré que les nanowhiskers ne percolaient pas et l’effet renfort a été attribué à des modifications topologiques du réseau d’agarose, et à un transfert des contraintes efficace entre la matrice et les charges, qui interagissent via des liaisons hydrogène. Les résultats obtenus au cours de cette thèse permettent d’améliorer la connaissance des mécanismes qui gouvernent les propriétés renfort apportées par l’ajout de nanocharges cellulosiques à une matrice hydrogel, et donnent des pistes réalistes pour une formulation maîtrisée d’hydrogels verts innovants ayant de bonnes propriétés mécaniques. / Cellulose is the most abundant organic compound on Earth; it is composed of microfibrils, containing nanocrystalline regions, and amorphous regions. Cellulose nanocrystallites, called nanowhiskers, have been studied as reinforcement agents in polymer composites with thermoplastic matrix in numerous publications. However, the use of nanowhiskers to reinforce hydrogels has not really been explored up to now, despite potential interest in the formulation of green innovative hydrogels. The aim of this PhD thesis was to study the relationship between the structural state, on different length scales, and the rheological properties of hydrocolloid systems consisting of agarose hydrogels filled by cellulose nanowhiskers from tunicate, amarine animal. The experimental work performed in this thesis has shown that the reinforcing effect provided by the nanowhiskers could be marked, and could also be modulated by varying the density of electrical charges on the surface of nanowhiskers. Within the volume fraction range studied, that is less than 0.2%, the results have shown that the nanowhiskers could not percolate, and reinforcement effect was attributed to topological modifications of the agarose network, and to an efficient stress transfer between the matrix and the fillers, which interact via hydrogen bonds.The results obtained in this thesis improve the understanding of the mechanisms that govern there inforcement effect provided by the addition of cellulose nanofillers in a hydrogel matrix; they could also help to design innovative green hydrogels having good mechanical properties.

Os hidrogéis foram obtidos a partir de material polimérico reticulado por processo de radiação ionizante de acordo com a técnica de Rosiak. Nos últimos 40 anos o uso dos hidrogéis têm sido investigado para diversas aplicações como curativos. Neste trabalho foram sintetizadas membranas de hidrogéis com poli(N-2- pirolidona) (PVP), poli(álcool vinílico) (PVAl), quitosana e argila laponita em encapsulamento do fármaco para liberação controlada de glucantime sobre a superfície cutânea de tecidos lesados por leishmaniose. O tratamento tradicional dos pacientes infectados pelos parasitas é feito com antimoniato pentavalente de forma injetável. Entretanto estes antimoniatos são muito tóxicos e provocam efeitos colaterais nestes pacientes, além disso, pacientes portadores de doenças cardíacas e renais não podem fazer uso deste tratamento. No tratamento com membranas de hidrogéis aplicadas na superfície de tecidos lesados pela leishmaniose, o fármaco é liberado diretamente no ferimento de forma controlada, diminuindo os efeitos colaterais. As membranas preparadas neste trabalho foram caracterizadas por difração de raios X (DRX), análise de termogravimetria (TG), intumescimento, fração gel, espectroscopia no infravermelho (FTIR), microscopia eletrônica de varredura (MEV) e microscopia de força atômica (AFM). As caracterizações funcionais foram feitas com teste de citotoxicidade e de liberação do fármaco in vitro e in vivo, de acordo com o protocolo de ética do Instituto de Medicina Tropical do Hospital das Clinicas da Faculdade de Medicina da USP. O teste \"in vivo\" dessas membranas provou ser eficiente na liberação controlada de fármacos diretamente nas superfícies lesadas pela leishmaniose. Nos testes \"in vivo\" as membranas de PVP/PVAl/argila 1,5% e glucantime apresentaram evidente contribuição para redução do ferimento chegando a uma cura clínica. / Polymeric Hydrogels formed by crosslinked polymeric chains were obtained by ionizing radiation process according to Rosiak technique. In the last 40 years the use of hydrogels has been investigated for various applications as curatives. In this work hydrogel membranes were synthesized with poly (N-2-pyrrolidone) (PVP), poly (vinyl alcohol) (PVA), chitosan and laponita clay for use as a vehicle for controlled glucantime release on the surface of skin tissues injured by leishmaniasis. Leishmaniasis is a disease caused by a protozoan parasite of the genus Leishmania transmitted by the bite of phlebotomies sandfly. The traditional treatment of patients infected by these parasites is done with pentavalent antimony in injectable form. However, these antimonates are highly toxic and cause side effects in these patients. In addition, patients with heart and kidney disease can not use this treatment. In treatment with drug delivery hydrogel membrane applied on the surface of leishmaniasis injured tissues the drug is released directly to the wound in a controlled manner, reducing the side effects. Membranes prepared in this study were characterized by X-ray diffraction (XRD), thermogravimetric analysis (TG), swelling, gel fraction, infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The characterizations of cytotoxicity and drug release were made \"in vitro\" and \"in vivo\" with functional test according to ethical protocol of the Division of Infectious and Parasitic Diseases at the Hospital of Clinics, São Paulo University-School of Medicine, University. The \"in vivo\" test of these membranes proved to be effective in controlled release of drugs directly into leishmaniasis damaged tissues. Results of \"in vivo\" tests using PVP/PVAl / clay 1,5% and glucantime membrane showed remarkable contribution to wound reduction and cure in clinical therapy.

<div>Vocal fold scarring is the fibrotic manifestation of most common pathological voice disorders. Voice disorders lead to direct healthcare costs of over $200 million annually and significantly reduce quality of life for patients. Despite advances in understanding the pathophysiology of vocal fold scarring, effective treatments for scarring and fibrosis remain elusive. The wound-healing cascade associated with vocal fold injury involves complex signaling interactions between cells and their extracellular matrix (ECM), which remain largely unexplored due to the lack of a physiologically relevant preclinical model to study them. Traditional preclinical models do not capture the complex 3D microenvironment of the vocal folds, and the use of stem cells or fibroblasts alone in models has resulted in poor reproducibility and predictability of in vitro models. Toward this end, this work describes the development of a preclinical model that strives to take into account cellular interactions between fibroblasts and epithelial cells and achieve a balance in the native vocal fold 3D environment to function as an in vitro model.</div><div><br></div><div>Since a major shortcoming of current in vitro models is the lack of a standardized epithelial fibroblast coculture, initial work focused on developing a coculture system between commercially available tracheal epithelial cells and vocal fold fibroblasts in an in vitro setting that would provide more accurate information about the disease pathophysiology and help design better targeted treatments. We designed a healthy and disease state coculture model that can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1. We also demonstrated that both cell types maintained phenotype in the healthy and disease state coculture models.</div><div><br></div><div>To further transfer this model in a physiologically relevant 3D system, follow-up research characterized 3D matrices to mimic the native ECM of the vocal folds by using natural biomimetic materials found in the vocal folds such as hyaluronic acid, type I collagen, and type III collagen. We hypothesized that the ability to control the viscoelastic and structuralcharacteristics of the scaffold in combination with presenting relevant biological cues to cells will result in a better biomimetic scaffold. This research is expected to lay effective groundwork for developing a functional tissue engineered 3D coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform.</div>

There is an important need to immobilise enzymes for use in industry, to do this I have the promising idea that by conjugating the enzyme to a hydrogel network, thus fabricating a stable biocatalytic network would be a potential method for immobilising enzymes for the manufacture of fine chemicals, this has not been done before for octapeptide systems. Hydrogels have been previously shown as a viable way of immobilising and stabilising enzymes. In this thesis the octapeptide VKVKVEVK (V is valine, K is lysine and E is glutamic acid) is used to immobilise enzymes tagged with VKVKVEVK. This peptide sequence is chosen as it forms stable hydrogels at enzyme appropriate conditions (pH 7). The enzymes chosen are; PETNR as it is well understood and is therefore a good starting point, CDH and CHMO were also chosen as they could combine with PETNR to form a cascade reaction. PETNR was both chemically conjugated to VKVKVEVK (SpepPETNR) and also genetically modified to express the peptide tag (CpepPETNR), whilst CDH and CHMO were genetically modified to express the tag (NpepCDH and CpepCHMO respectively). For S/CpepPETNR retention within the hydrogels was superior to retention for untagged PETNR. NpepCDH was found to not precipitate within the hydrogel whilst untagged was found to do so. CpepCHMO functionalised hydrogels were found to be heterogeneous. Characterisation of CpepPETNR functionalised hydrogels was undertaken using micro differential scanning calorimetry (µDSC), rheology, small angle neutron scattering (SANS) and atomic force microscopy (AFM). From the µDSC evidence of 'protective immobilisation' was observed by the increase in denaturation energy (+253 kJ mol-1) in the hydrogel in comparison to in solution (+18 kJ mol-1). The ability of S/CpepPETNR functionalised hydrogels to perform the ketoisophorone to levodione biotransformation reaction was explored with yields of 86%. S/CpepPETNR within VKVKVEVK hydrogels was found to retain ~90% conversion for at least 9 months at room temperature. Incubation overnight at 90°C resulted in a yield of 84% of levodione. These two results added more evidence for 'protective immobilisation'. Hydrogels functionalised with NpepCDH or CpepCHMO were characterised using rheology and atomic force microscopy. The biotransformation ability of NpepCDH was elucidated; the overall yield of carvone was a maximum of 54% from the hydrogel phase. NpepCDH was used alongside CpepPETNR for the cascade reaction producing dihydrocarvone in low yields; however, an improvement from 2% to 13% in yield is presented. The yield of lactone products from CpepCHMO functionalised hydrogel was low at 15%. The CpepPETNR/ CpepPETNR cascade reaction proceeded with a yield of 36%. The initial activities of CpepPETNR, NpepCDH and CpepCHMO were assayed in both solution and in gel phase using a modified method. The activities were assessed with varying conditions; temperature, pH, quantity of ethanol and incubation at high and low temperatures. Generally, it was found that immobilisation within the hydrogel phase resulted in 'protective immobilisation' against non-optimal conditions. This work will be of benefit to those who are interesting immobilising enzymes within hydrogels in the future.

Bone undergoes constant turnover throughout life and has the capacity to regenerate itself. However, the repair of critical size defects, caused by bone diseases such as osteoporosis, can be more problematic. Therefore, there is a clinical need for a bone graft substitute that can be used at sites of surgical intervention to enhance bone regeneration. Poly(vinylphosphonic acid-co-acrylic acid) (PVPA-co-AA) has recently been identified as a potential candidate for use in bone tissue scaffolds. It is hypothesised that PVPA-co-AA can mimic the action of bisphosphonates – a class of drugs used in the treatment of osteoporosis – by binding to calcium ions from bone mineral surfaces. In this way, bisphosphonates can affect bone turnover by increasing the activity of osteoblasts and reducing osteoclast activity. Although PVPA-co-AA has been shown to improve bone formation, the mechanism of action has so far not been fully elucidated. Therefore, this work aims to understand the effect of copolymer composition on the properties of PVPA-co-AA, and thus to determine its effect on osteoblast adhesion and proliferation. PVPA-co-AA copolymers have been synthesised with a range of monomer feed ratios. It was found that a VPA content of 30 mol % led to the greatest calcium binding affinity of the copolymer and is thus expected to lead to enhanced bone formation and mineralisation of the matrix produced by osteoblast cells. The release profile of PVPA-co-AA from electrospun PCL scaffolds was investigated. It was shown that all of the PVPA-co-AA was released into aqueous media within 8 h of immersion. It was also found that the calcium chelation from osteogenic differentiation media significantly increased within the first 8 h. Therefore, it was concluded that PVPA-co-AA is released from the scaffolds, where it can then bind to calcium ions from the bone mineral surface to promote mineralisation, thus acting as a mimic of non-collagenous proteins, which are present in the extracellular matrix (ECM) of bone. Hydrogels of PVPA-co-AA have been produced and the effect of monomer feed ratio (0-50 mol % VPA) on the properties of the gels was explored. It was found that an increase in VPA content led to greater hydrogel swelling and increased porosities. Hydrogels that contained 30 and 50 mol % VPA were shown to have similar morphologies to the native ECM of bone. Rheological testing showed that hydrogels with higher VPA contents were more flexible and could be deformed to a large extent without permanent deformation of their structure. An increase in osteoblast adhesion and proliferation was observed for hydrogels with 30 and 50 mol % VPA content as well as superior cell spreading. Osteoblast cell metabolic activity also increased as a function of VPA content in the hydrogels. This work indicates that hydrogels of PVPA-co-AA, with VPA contents of 30 or 50 mol %, are ideal for use as bone tissue scaffolds. Furthermore, the mechanical and cell adhesion properties of the gels can be tuned by altering the copolymer composition. Finally, composite hydrogels of PVPA-co-AA and hydroxyapatite (HA) have been produced and investigated for their ability to remove fluoride ions from groundwater. It was found that the fluoride uptake ability of PVPA-HA hydrogels was significantly enhanced when compared with HA powder alone. Furthermore, the fluoride uptake was dependent on many factors, including pH, contact time and the presence of competing ions. It was possible to regenerate the hydrogel to remove the fluoride ions, and thus it was shown that the material can be used a number of times with only a slight reduction in its fluoride uptake capacity.

Os hidrogéis foram obtidos a partir de material polimérico reticulado por processo de radiação ionizante de acordo com a técnica de Rosiak. Nos últimos 40 anos o uso dos hidrogéis têm sido investigado para diversas aplicações como curativos. Neste trabalho foram sintetizadas membranas de hidrogéis com poli(N-2- pirolidona) (PVP), poli(álcool vinílico) (PVAl), quitosana e argila laponita em encapsulamento do fármaco para liberação controlada de glucantime sobre a superfície cutânea de tecidos lesados por leishmaniose. O tratamento tradicional dos pacientes infectados pelos parasitas é feito com antimoniato pentavalente de forma injetável. Entretanto estes antimoniatos são muito tóxicos e provocam efeitos colaterais nestes pacientes, além disso, pacientes portadores de doenças cardíacas e renais não podem fazer uso deste tratamento. No tratamento com membranas de hidrogéis aplicadas na superfície de tecidos lesados pela leishmaniose, o fármaco é liberado diretamente no ferimento de forma controlada, diminuindo os efeitos colaterais. As membranas preparadas neste trabalho foram caracterizadas por difração de raios X (DRX), análise de termogravimetria (TG), intumescimento, fração gel, espectroscopia no infravermelho (FTIR), microscopia eletrônica de varredura (MEV) e microscopia de força atômica (AFM). As caracterizações funcionais foram feitas com teste de citotoxicidade e de liberação do fármaco in vitro e in vivo, de acordo com o protocolo de ética do Instituto de Medicina Tropical do Hospital das Clinicas da Faculdade de Medicina da USP. O teste \"in vivo\" dessas membranas provou ser eficiente na liberação controlada de fármacos diretamente nas superfícies lesadas pela leishmaniose. Nos testes \"in vivo\" as membranas de PVP/PVAl/argila 1,5% e glucantime apresentaram evidente contribuição para redução do ferimento chegando a uma cura clínica. / Polymeric Hydrogels formed by crosslinked polymeric chains were obtained by ionizing radiation process according to Rosiak technique. In the last 40 years the use of hydrogels has been investigated for various applications as curatives. In this work hydrogel membranes were synthesized with poly (N-2-pyrrolidone) (PVP), poly (vinyl alcohol) (PVA), chitosan and laponita clay for use as a vehicle for controlled glucantime release on the surface of skin tissues injured by leishmaniasis. Leishmaniasis is a disease caused by a protozoan parasite of the genus Leishmania transmitted by the bite of phlebotomies sandfly. The traditional treatment of patients infected by these parasites is done with pentavalent antimony in injectable form. However, these antimonates are highly toxic and cause side effects in these patients. In addition, patients with heart and kidney disease can not use this treatment. In treatment with drug delivery hydrogel membrane applied on the surface of leishmaniasis injured tissues the drug is released directly to the wound in a controlled manner, reducing the side effects. Membranes prepared in this study were characterized by X-ray diffraction (XRD), thermogravimetric analysis (TG), swelling, gel fraction, infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The characterizations of cytotoxicity and drug release were made \"in vitro\" and \"in vivo\" with functional test according to ethical protocol of the Division of Infectious and Parasitic Diseases at the Hospital of Clinics, São Paulo University-School of Medicine, University. The \"in vivo\" test of these membranes proved to be effective in controlled release of drugs directly into leishmaniasis damaged tissues. Results of \"in vivo\" tests using PVP/PVAl / clay 1,5% and glucantime membrane showed remarkable contribution to wound reduction and cure in clinical therapy.

Estudos recentes têm demonstrado o efeito das isoflavonas da soja, especialmente da genisteína (GEN), administrada topicamente, na prevenção do fotoenvelhecimento e fotocarcinogênesis. Esse efeito tem sido relacionado com a sua atividade inibidora de tirosina quinase e antioxidante. Recentemente, demonstramos um lento perfil de permeação da GEN in vitro (usando células de difusão de Franz) a partir de nanoemulsões através de pele de orelha suína (SILVA et al., Pharmazie, v. 64, p. 32- 35, 2009). Entretanto, estudos relativos à distribuição da GEN através das camadas da pele ainda não foram realizados. Neste contexto, o principal objetivo deste estudo foi avaliar a distribuição da GEN através da pele suína a partir de nanoemulsões, antes e após incorporação em hidrogéis de ácido acrílico, a fim de ajustar a viscosidade das mesmas para aplicação tópica. Nanoemulsões contendo GEN apresentaram valores de potencial  negativos e baixa viscosidade. Os hidrogéis derivados contendo as nanoemulsões de GEN demonstraram um comportamento não-Newtoniano pseudoplástico. Um método isocrático de cromatografia líquida para determinar a GEN nas camadas da pele de orelha suína foi desenvolvido e validado. O método mostrou ser específico, linear, preciso e exato para a determinação da GEN nas camadas da pele de orelha suína (i.e. stratum corneum, epiderme e derme). Nas condições validadas, o perfil de permeação/retenção da GEN foi claramente influenciado pela natureza do núcleo oleoso empregado. Nanoemulsões constituídas de um núcleo oleoso de triglicerídeos de cadeia média apresentaram uma permeação mais lenta e uma maior retenção da GEN na pele quando comparado com nanoemulsões contendo octildodecanol. A incorporação das nanoemulsões de GEN em hidrogéis aumenta a retenção da GEN, especialmente na epiderme. Tal resultado pode estar relacionado com o aumento da hidratação da epiderme como sugerido nas observações histológicas. Em conclusão, o conjunto dos resultados demonstra o efeito das formulações sobre a distribuição da GEN através das camadas da pele de orelha suína. / Recent studies have shown the effect of soy isoflavones, especially genistein (GEN), topically administrated, in preventing skin photoaging and photocarcinogenesis. This effect has been related to tyrosine kinase inhibition and antioxidant activities. Recently, we have demonstrated a slow permeation profile of GEN in vitro (using Franz type diffusion cells) from nanoemulsions through porcine ear skin (SILVA et al., Pharmazie, v. 64, p. 32-35, 2009). However, studies concerning the distribution of GEN in the skin layers have yet to be performed. In this context, the main purpose of this study was to evaluate the GEN distribution through the porcine skin from topical nanoemulsions, before and after their incorporation into acrylic acid-hydrogels, in order to have their viscosity adjusted to topical applications. Nanoemulsions containing GEN exhibit negative -potential and low viscosity. The derived hydrogels containing GEN-loaded nanoemulsions presents non-Newtonian pseudoplastic behavior. An isocratic liquid chromatographic method to determine GEN in porcine ear skin layers was developed and validated. The method proved to be specific, linear, precise and accurate to determine GEN in the porcine skin layers (i.e. stratum corneum, epidermis and dermis). In the validated conditions, the GEN permeation/ retention profile was clearly influenced by the nature of the oil core used. Nanoemulsions composed by a medium chain triglycerides oil core exhibit both a slower permeation and a higher retention profile of GEN as compared to octyldodecanol nanoemulsions. The incorporation of GEN-loaded nanoemulsion into hydrogels improved the GEN retention, especially in the epidermis. Such a result might be related to the increased hydration of epidermis as suggested in histological observations. In conclusion, the overall results show the effect of the formulation on distribution of GEN through the porcine skin layers.

abstract: Hydrogel polymers have been the subject of many studies, due to their fascinating ability to alternate between being hydrophilic and hydrophobic, upon the application of appropriate stimuli. In particular, thermo-responsive hydrogels such as N-Isopropylacrylamide (NIPAM), which possess a unique lower critical solution temperature (LCST) of 32°C, have been leveraged for membrane-based processes such as using NIPAM as a draw agent for forward osmosis (FO) desalination. The low LCST temperature of NIPAM ensures that fresh water can be recovered, at a modest energy cost as compared to other thermally based desalination processes which require water recovery at higher temperatures. This work studies by experimentation, key process parameters involved in desalination by FO using NIPAM and a copolymer of NIPAM and Sodium Acrylate (NIPAM-SA). It encompasses synthesis of the hydrogels, development of experiments to effectively characterize synthesized products, and the measuring of FO performance for the individual hydrogels. FO performance was measured using single layers of NIPAM and NIPAM-SA respectively. The values of permeation flux obtained were compared to relevant published literature and it was found to be within reasonable range. Furthermore, a conceptual design for future large-scale implementation of this technology is proposed. It is proposed that perhaps more effort should focus on physical processes that have the ability to increase the low permeation flux of hydrogel driven FO desalination systems, rather than development of novel classes of hydrogels / Dissertation/Thesis / Masters Thesis Mechanical Engineering 2019

Decellularized extracellular matrices have been a growing area of interest in the biomedical engineering fields of tissue engineering and regenerative medicine.As these materials move toward clinical applications, the immune response to these materials will be a driving force toward their success in clinical approaches. Fully digested decellularized matrix constructs derived from porcine liver, muscle and lung were created to test the adaptive immune response. Hydrogel characterization ensured that the materials had relatively similar stiffness levels to reduce variability, and in vitro studies were conducted. Each individual construct as well as a gelatin control were plated with a co-culture of macrophages and T-cells to measure T-cell proliferation. In addition standard markers of inflammation through qPCR were measured in the macrophage group. Constructs were then placed into animals for 3 and 7 days in addition to a second group that received constructs for 21 days before secondary constructs were placed. These groups were then sacrificed following 3, 7 and 14 days to measure the residual and memory-like response of the constructs. Our results showed that t-cell proliferation was increased with decellularized constructs, particularly in tissue with higher DNA content. In vivo, animals with secondary treatments showed extended inflammatory response, driven by Th1 and Th17 polarization suggesting a memory-like response due to recognition of peptides in the constructs from secondary placements.