Made available in DSpace on 2014-07-29T15:07:56Z (GMT). No. of bitstreams: 1 Dissertacao Caroline Rocha de Oliveira Lima.pdf: 444731 bytes, checksum: f7805c76dff945571aca16f931a7c2a8 (MD5) Previous issue date: 2010-02-25 / In veterinary medicine, skin injuries represent a significant casuistry and require expensive therapies, considerable recovery time and often lead to unfavorable resolution. To establish an effective therapeutic protocol it is essential that the professional has a solid knowledge of the healing dynamic, as its phases, triggered events, features, as well as about the biological potential of the healing process coadjutants. Regarding these ones, it is known that phytotherapy has a wide range of botanical specimens with healing potential, and, among them, the barbatiman, native species of the Brazilian cerrado, by its antiseptic and astringent activities, becomes an attractive option for skin injures therapies. The chitosan biomaterial also has potential as coadjutant of the process, once it accelerates the triggering of the healing stages by its immunomodulatory property. To clarify the mechanism proposed by these agents, numerous tests are developed using laboratory animals, in order to give reliability to the therapy modality established. To follow clinical evolution in vivo, the clinical examination becomes essential, while morphometric analysis provide accurate data regarding the percentage of centripetal contraction of the lesions. Finally, the microscopic analysis is used to analyze the ultrastructure of the injured site, predominant kinds of cells in each stage and microscopic events triggered on the search for the morphofunctional restoration of the affected tissue. Considering it, with the development of this study it was aimed to evaluate the activity of 5% barbatiman cream (CB) and 5% chitosan cream (CQ) based therapeutic formulations, on the healing process of incisional skin wounds undergone healing by secondary intention, with rabbit as experimental model. In the first chapter it was performed a wide and detailed literature review about the topic of the study, which involved description of the target tissue, skin, hysiopathology of the healing process, barbatiman phytotherapic and chitosan biomaterial activities as healing coadjutants, use of laboratory animals in scientific research and evaluation methods of skin therapies. In the second chapter, it was tested the therapeutic activity of CB and CQ on healing of incisional cutaneous wounds with rabbit as experimental model, by daily clinical evaluation and morphometric analysis performed on days three, seven, 14 and 21 post-injury. There were performed four wounds on each animal, totaling 40 rabbits. Daily, all lesions of each animal received a specific topical therapy, which lasted for 21 consecutive days. First, we performed the antiseptic with 0.9% sodium chloride solution e and further applied treatment protocols themselves. In one of the protocols employed in cream barbatiman (CQ) in another cream chitosan (QC), and these were formulations tests. In the third protocol was the use of allantoin cream (positive control) and finally the cream neutral vehicle (negative control I). In other injury effected only without topical antiseptic and associated 0.9% sodium chloride solution (negative control II). The results showed that the CB and CQ contributed to the satisfactory progress of the healing process. Although there has been no statistical differences in the contraction percentage between the two treatments (CB and CQ), it was clinically observed that the wounds treated with CB showed better clinical responses than CQ. In the third chapter it was comparatively performed, by clinical and microscopic evaluations, the analysis of the action of CQ and CB in the wounds of the same animals that received the treatments from chapter two. For histological analysis, there were considered the predetermined period for morphometry and the samples were evaluated regarding the prevalence of polymorphic and mononuclear infiltrate, presence of necrosis, crust, granulation tissue, fibroplasia, angiogenesis, collagenization and skin reepithelialization. From the results obtained it can be affirmed that the activities attributed to barbatiman and chitosan contributed similarly to the satisfactory progress of the healing process, once both treatments did not differ significantly for any parameter considered in this study. It is suggested that therapeutic protocols chitosan and barbatiman based provided acute inflammation reduction, as much as promoted fibroblast activation, early development of connective tissue, neovascularization and tissue reepithelialization, providing proved effective and economically viable alternatives in the healing process. / Na clínica médica veterinária as injúrias cutâneas representam casuística significativa e cursam com terapias dispendiosas, tempo de recuperação considerável e resolução muitas vezes desfavorável. Para instituir um protocolo terapêutico eficaz é imprescindível que o profissional tenha conhecimento sólido da dinâmica cicatricial, como suas fases, eventos desencadeados, particularidades, bem como acerca do potencial biológico dos coadjuvantes do processo cicatricial. Quanto a estes últimos, sabe-se que a fitoterapia possui um leque variado de espécimes botânicos com potencial na cicatrização e dentre estes o barbatimão, espécie nativa do cerrado brasileiro, por suas atividades, antiséptica e adstringente, torna-se opção atraente quanto às terapias das injúrias cutâneas. O biomaterial quitosana também possui potencial como coadjuvante do processo, visto que por sua propriedade imunomoduladora acelera o desencadeamento das fases da cicatrização. Para esclarecer o mecanismo proposto por estes agentes, inúmeros ensaios são desenvolvidos, utilizando animais de laboratório, a fim de conferirem confiabilidade à modalidade terapêutica instituída. Para acompanhar a evolução clínica in vivo, o exame clínico torna-se essencial, enquanto que as análises morfométricas conferem dados precisos quanto ao percentual de contração centrípeta das lesões. Por último as análises microscópicas são empregadas para analisar a ultra-estrutura do sítio lesionado, tipos celulares predominantes em cada fase e eventos microscópicos desencadeados na busca do restabelecimento morfofuncional do tecido acometido. Diante do exposto, com o desenvolvimento desse estudo objetivou-se avaliar a atividade de formulações terapêuticas a base de creme de barbatimão a 5% (CB) e creme e quitosana a 5% (CQ) frente ao processo de cicatrização de feridas cutâneas incisionais submetidas à cicatrização por segunda intenção, tendo o coelho com o modelo experimental. No primeiro capítulo efetuou-se revisão de literatura ampla e detalhada do tema do estudo que envolveu descrição do tecido alvo, pele, fisiopatologia do processo cicatricial, atividade do fitoterápico barbatimão e do biomaterial quitosana como coadjuvantes da cicatrização, emprego de animais de laboratório em pesquisas científicas e métodos de avaliação das terapias cutâneas. No segundo capítulo, testou-se a atividade terapêutica dos CB e CQ na cicatrização de feridas cutâneas incisionais, por meio da avaliação clínica efetuada diariamente e morfométrica nos dias três, sete, 14 e 21 pós-lesão. Foram realizadas quatro feridas em cada animal, totalizando 40 coelhos. Diariamente, todas as lesões de cada animal receberam uma terapia tópica específica, que se estendeu durante 21 dias consecutivos. Primeiramente, realizava-se a antissepsia com solução de cloreto de sódio a 0,9% e na sequência aplicavam-se os protocolos terapêuticos propriamente ditos. Em um dos protocolos empregou-se creme de barbatimão (CQ), em outro creme de quitosana (CQ), sendo estas as formulações testes. No terceiro protocolo fez uso do creme de alantoína (controle positivo) e por ultimo o creme base (controle negativo I). Em outra lesão efetuou-se apenas antissepsia sem terapia tópica associada com solução de cloreto de sódio a 0,9% (controle negativo II). Os resultados apontaram que os CB e o CQ contribuíram para que o processo de cicatrização evoluísse satisfatoriamente. Embora não tenham ocorrido diferenças estatísticas no percentual de contração entre os dois tratamentos (CB e CQ), clinicamente, notouse que as feridas tratadas com CB apresentaram respostas clínicas superiores ao CQ. No terceiro capítulo efetuou-se comparativamente, por meio da avaliação clínica e microscópica, a ação dos CQ e CB nas feridas dos mesmos animais submetidas aos tratamentos do capítulo dois. Para as análises histológicas foram considerados os períodos pré-definidos para morfometria e as amostras foram avaliadas quanto à predominância de infiltrado polimorfo e mononuclear, presença de crosta-necrose, tecido de granulação, fibroplasia, angiogênese, colagenização e reepitelização cutânea. A partir dos resultados obtidos pode-se afirmar que as atividades implicadas ao barbatimão e a quitosana contribuíram de forma semelhante para que o processo de cicatrização evoluísse satisfatoriamente, visto que os dois tratamentos não diferiram significativamente para nenhum parâmetro considerado neste estudo. Sugere-se que os protocolos terapêuticos à base de barbatimão e quitosana proporcionaram redução da inflamação aguda, bem como promoveram ativação fibroblástica, desenvolvimento precoce de tecido conjuntivo, neovascularização e reepitelização tecidual, conferindo alternativas comprovadamente eficazes e economicamente viáveis em relação ao processo de cicatrização.

Made available in DSpace on 2014-07-29T15:13:55Z (GMT). No. of bitstreams: 1 Marcelo Seixo - digitalizados tese.pdf: 3279535 bytes, checksum: 24af83caa54ca7c77d0f55b99fbb329f (MD5) Previous issue date: 2009-01-16 / Synthetic materials are the leading choice for the correction of defects in abdominal wall, but the formation of adhesions are a problem encountered when placing implants in contact with the abdominal viscera. With In order to avoid its formation, various materials and mechanisms are used in an attempt to prevent contact with peritoneal surfaces of the prostheses. In this sense, we investigated the possibility of the hydrogel poly (2-hydroxyethyl methacrylate) (polyHEMA) coating a face of polypropylene mesh and thereby preventing peritoneal adhesion in dogs and rats, and, in the latter species, also performed a histological evaluation of implanted materials. For this, an abdominal defect was created in the rectus abdominis of 16 bitches were divided into two groups of eight animals and the defect was corrected with screen polypropylene and polypropylene mesh coated with hydrogel, respectively. The presence of adhesions was evaluated after 20 days by laparoscopy. As to rats, the surgical procedure was similar and the evaluations were performed at 15 and 30 days, with euthanasia and collection of fragments for histology with picrosirius red staining and HE The hydrogel coating the polyHEMA polypropylene mesh implanted in the abdominal wall prevented completely visceral adhesions and omentum remaining on the surface the screen when compared to polypropylene mesh in dogs and isolated rats. Was found greater amount of adhesions in the suture zone in the group a mesh coated with polyHEMA. The physical structure of the biomaterial interfered the organization and persistence of tissue response, but not in the same type. / Os materiais sintéticos são a principal escolha para a correção de defeitos na parede abdominal, mas a formação de aderências é um problema encontrado quando da colocação de próteses em contato com as vísceras abdominais. Com o objetivo de evitar a sua formação, vários materiais e mecanismos são utilizados na tentativa de impedir o contato das superfícies peritoniais com as próteses. Neste sentido, investigou-se a possibilidade do hidrogel de poli(2-hidroxietil metacrilato) (poliHEMA) revestir uma face de tela de polipropileno e, com isso, prevenir a aderência peritonial em cadelas e ratas, sendo, nesta última espécie, também realizada a avaliação histológica dos materiais implantados. Para isso, um defeito abdominal foi produzido no músculo reto abdominal de 16 cadelas que foram divididas em dois grupos de oito animais e, o defeito foi corrigido com tela de polipropileno e tela de polipropileno revestida de hidrogel respectivamente. A presença de aderências foi avaliada após 20 dias por videolaparoscopia. Quanto às ratas, o procedimento cirúrgico foi semelhante e as avaliações ocorreram aos 15 e 30 dias, com eutanásia dos animais e colheita de fragmentos para histologia com as colorações de picrossirius e H.E. O hidrogel de poliHEMA revestindo a tela de polipropileno implantada na parede abdominal impediu totalmente a formação de aderências viscerais e do omento remanescente sobre a superfície da tela quando comparada à tela de polipropileno isolada em cadelas e ratas. Foi encontrada maior quantidade de aderências na zona de sutura no grupo que recebeu a tela revestida com poliHEMA. A estrutura física do biomaterial interferiu na organização e persistência da resposta tissular, mas não no tipo da mesma.

O tratamento da erosão dental transformou-se no novo desafio do século XXI e a restauração direta com resina composta é uma das opções de tratamento para lesões severas, em que há comprometimento estético e funcional. Dentro desse contexto, novos materiais restauradores têm sido avaliados, como materiais contendo flúor em sua composição. Desta forma, o presente estudo teve como por objetivo avaliar in vitro a resistência de união da resina composta contendo ou não biomaterial S-PRG (Ionômero de Vidro com Superfície Pré-Ativada) à dentina erodida. A dentina oclusal planificada de 30 molares humanos (n=15) teve metade de sua face protegida com verniz ácido resistente (dentina hígida - grupo controle), enquanto na outra metade foi produzida uma lesão de erosão através da ciclagem em ácido cítrico 0,05 M (pH 2,3, 10 minutos, 6x/dia) e solução supersaturada (pH 7,0, 60 minutos entre os ataques ácidos). Em cada substrato, foram testados dois sistemas adesivos (um autocondicionante e outro com condicionamento prévio) e duas resinas compostas - uma com nanopartículas (Z350-3M/ESPE) e uma que contém biomaterial S-PRG(Ionômero de Vidro com Superfície Pré-Ativada) em sua composição (Beautifil II -Shofu Inc.). Foram, então, confeccionados cilindros de resina composta, os quais foram submetidos à avaliação da Resistência de União através do ensaio de microtração, após armazenamento em água por 24 horas. A análise do padrão de fratura foi realizada em microscópio ótico (40x). Os valores obtidos de resistência de união (MPa) foram submetidos aos testes de ANOVA (dois fatores) e de comparações múltiplas de Tukey (p<0,05). De acordo com os resultados, não houve diferença estatisticamente significativa entre a interação substrato e materiais restauradores (p=0.711). No entanto, houve diferença entre os substratos (p<0,001) e os materiais restauradores (p=0,002) avaliados. Quanto à resistência de união, os valores obtidos foram: G1 (47,5 ± 12,2 MPa), G2 (34,1 ± 15,8 MPa), G3(31,0 ± 8,3 MPa) e G4 (15,5 ± 13,6 MPa). Os resultados, revelaram uma diferença estatisticamente significante nos substratos avaliados e materiais restauradores. Dentro dos limites do presente estudo, conclui-se que a resistência de união no substrato erodido é inferior ao substrato hígido e que o sistema adesivo autocondicionante contendo biomaterial S-PRG(Ionômero de Vidro com Superfície Pré-Ativada) resulta em menor resistência de união da resina composta à dentina hígida ou erodida. / The treatment of dental erosion has become the new challenge of the 21st century and direct restoration with composite resin is one of the treatment options for severe injuries, in which there is aesthetic and functional impairment. Within this context, new restorative materials have been introduced containing fluoride in their composition. In this way, the objective of the present in vitro study was to evaluate the bond strength of composite resin containing or not biomaterial (S-PRG- Surface Pre-Reacted Glass ) to the eroded dentine. Planned occlusal dentin of 30 human molars (n = 15) had half of its surface protected with acid resistant varnish (sound dentin - control group), while on the other half an erosion lesion was produced by cycling in 0.05 citric acid M (pH 2.3, 10 minutes, 6x/day) and supersaturated solution (pH 7.0, 60 minutes between acid attacks). On both eroded and non-eroded substrates, two adhesive systems (one self-etching and one with pre-conditioning) and two composite resins (one with nanoparticles (Z350-3M / ESPE) and one containing biomaterial S-PRG(Glass Ionomer with Pre-Activated Surface) (Beautifil II-Shofu Inc.)) were tested. Then, composite resin cylinders were built and, after storage in water for 24 hours, were submitted to shear bond strength evaluation under microtensile bond test. The analysis of the fracture pattern was performed under an optical microscope (40x). The obtained values of shear bond strength (MPa) were submitted to ANOVA (two factors) and Tukey multiple comparisons tests (p<0.05). According to the results, there was no statistically significant difference between substrate interaction and restorative materials (p = 0.711). However, there was a difference between the substrates (p <0.001) and the restorative materials (p = 0.002) evaluated. The values obtained were G1 (47.5 ± 12.2 MPa), G2 (34.1 ± 15.8 MPa), G3 (31.0 ± 8.3 MPa) and G4 (15 , 5 ± 13.6 MPa), with a statistically significant difference in the evaluated substrates and restorative materials. The results, revealed a statistically significant difference in the evaluated substrates and restorative materials. According to the limits of the present study, it was concluded that the bond strength in the eroded substrate is inferior to the sound substrate and that the self-etching adhesive system containing S-PRG(Surface Pre-Reacted Glass) biomaterial results in lower bond strength of the composite resin to the sound or eroded dentin.

Blendas poliméricas são misturas físicas de dois ou mais polímeros. O interesse de obtenção de blendas poliméricas, com características físicas e químicas desejadas para as mais diversas aplicações, é crescente em todo o mundo. Neste trabalho foram desenvolvidos filmes de blendas de quitosana e poli(N-vinil-2-pirrolidona) (PVP) em várias proporções. A irradiação destes filmes com radiação UV254nm gerou hidrogéis estáveis. Foram também obtidos hidrogéis a partir de co-soluções aquosas destes polímeros, em diversas proporções, com reticulação de seus componentes induzida pela radiação ultravioleta. Em ambos os casos, os materiais foram caracterizados e avaliados quanto a sua potencialidade de uso como biomaterial. Para análise e caracterização das blendas e hidrogéis utilizaram-se as técnicas de espectroscopia no infravermelho (IV), microscopia eletrônica de varredura (MEV), calorimetria exploratória diferencial (DSC), análise termogravimétrica (TGA), difratometria de raios X e transmissão de vapor de água (WVT). Os resultados mostraram que a forte interação entre PVP e quitosana leva a um aumento da insolubilidade da blenda em relação à PVP pura, mesmo com baixas quantidades de quitosana. A presença de PVP aumenta a hidrofilicidade da blenda e também ocasiona a diminuição da cristalinidade da quitosana. Outro aspecto importante foi o aumento da estabilidade térmica das quitosanas, também influenciada pelo aumento da parcela de PVP nas blendas. A análise dos dados parece indicar que uma SIPN foi formada, onde a rede básica é constituída de PVP reticulada, enquanto a quitosana é mantida \"presa\" nesta rede, sem participar da mesma, mas impedida de ser extraída. O estudo da irradiação de co-soluções aquosas de PVP e quitosana gerou hidrogéis estáveis, porém restritos a baixa porcentagem de quitosana na mistura - até 30%. Este estudo revelou, também, que a presença de ácido acético na solução facilita o processo de reticulação, incluindo solução de PVP pura. A permeabilidade à água dos filmes de blendas de quitosana/PVP não irradiados e irradiados indica sua potencialidade de aplicações desses materiais em áreas medicinal e alimentícia. A literatura descreve vários métodos de produção de hidrogéis de quitosana, porém, todos baseados no uso de agentes reticulantes externos ou da enxertia de grupos reativos na cadeia de quitosana. Deve-se ressaltar que o sistema de reticulação aqui proposto não faz uso de agentes de reticulação, fotoiniciadores nem incorporação de algum grupo fotorreativo nas cadeias dos polímeros. De modo geral, os resultados obtidos dos materiais poliméricos irradiados e não-irradiados sugerem que possuem potencialidade para várias aplicações: como biomaterial, por exemplo, matrizes poliméricas para liberação controlada de fármacos, curativos para ferimentos mais leves, outros. / Polymer blends are physical mixtures of two or more polymers. The interest in obtaining polymer blends, with desired physical and chemical characteristics for the most diverse applications, is increasing in the whole world. In this work, chitosan and poly(N-vinyl-2-pyrrolidone) (PVP) blend films were developed at several ratios. Hydrogels were also obtained from co-solutions of these polymers, at diverse ratios, through crosslinking induced by ultraviolet radiation. In both cases, the materiais were characterized and evaluated in terms of their potentiality of use as biomaterial. For analysis and characterization of blends and hydrogels were utilized the techniques of infrared spectroscopy (IR), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray diffractometry (XRD), and water vapor transmission (WVT). The results have shown that the strong interaction between PVP and chitosan leads to an increase of blend insolubility in relation to pure PVP, even with low amounts of chitosan. PVP presence increases blend hydrophilicity and causes reduction of chitosan crystallinity, as well. Another important aspect was the increase of chitosan thermal stability, influenced by the increase of PVP portion in blends. Analysis of the data seems to indicate that a SIPN was formed, where the basic net is constituted of crosslinked PVP, while chitosan is kept \"imprisoned\" in this net, without participating of the same, but hindered of being extracted. The study of irradiation of PVP and chitosan aqueous co-solutions generated stable hydrogels, however restricted low chitosan percentage in the mixture - up to 30%. This study also disclosed that the acetic acid presence in the solution facilitates the crosslinking process, including pure PVP solution. Permeability to water of non-irradiated and irradiated blend films of chitrosan/PVP indicates application potentiality of these materiais in medicinal and food areas. Literature describes several methods of chitosan hydrogel production, however, all basing on the use of external crosslinkers or graft of reactive groups on chitosan chain. It must be pointed out that the crosslinking system considered here does not make use of crosslinkers, photoinitiators nor incorporation of some photoreactive group into the chains of the polymers. In a general way, the results obtained from irradiated and non-irradiated polymer materials suggest they possess potentiality for some applications: as biomaterial, for example, polymer matrices for controlled drug release, dressings for simpler wounds, others.

A hidroxiapatita (HA) tem sido amplamente utilizada como material de implante por possuir alta similaridade com a composição dos ossos e ter capacidade de formar ligações químicas fortes com o tecido ósseo. Entretanto, a adsorção fácil de moléculas orgânicas (proteínas, aminoácidos e polissacarídeos) pela HA também favorece a adsorção e replicação de bactérias, aumentando os casos de infecções relacionados a esse biomaterial. Visando contribuir para o avanço de novos biomateriais com propriedades antibacterianas, foram produzidas neste trabalho hidroxiapatitas contendo nanopartículas de prata (AgNPs) com diferentes proporções (0,01; 0,05; 0,10 e 0,25% m/m). A primeira etapa do trabalho consistiu na obtenção do pó de HA por precipitação química e na produção de AgNPs em suspensões coloidais por meio de síntese que utiliza a quitosana como agente redutor e estabilizante. Na segunda etapa, as AgNPs foram adsorvidas na HA pelo método de imersão do pó usando um dos coloides sintetizados, obtendo-se quatro hidroxiapatitas contendo AgNPs (HA-AgNPs) nas formas de pó e disco. Na terceira e última etapa, os materiais produzidos foram caracterizados utilizando diversas técnicas e os pós (HA e HA-AgNPs) foram submetidos a avaliações in vitro. O efeito antibacteriano foi avaliado frente a cepas de S. aureus e E. coli utilizando métodos qualitativos e quantitativos. A adesão bacteriana sobre a superfície dos discos foi observada por microscopias eletrônica de varredura (MEV) e confocal de varredura a laser. A citotoxicidade in vitro foi avaliada utilizando cultura de células contendo linhagem de osteoblastos (MG-63). Nesse ensaio, a viabilidade dos osteoblastos foi estudada usando o teste da resazurina; a atividade enzimática foi investigada através da fosfatase alcalina (ALP); e a adesão dos osteoblastos foi observada por MEV. Os resultados mostraram que a HA produzida é nanométrica e que as AgNPs sintetizadas são estáveis e esféricas, com diâmetros variando entre 5-10 nm. Técnicas de MEV, EDS e ICP-AES confirmaram a presença de prata nas HA-AgNPs. As demais caracterizações não mostraram alterações relevantes na estrutura, morfologia e características de superfície (hidrofobicidade e carga líquida). No entanto, a atividade antibacteriana dos materiais mostrou-se eficiente para ambas as cepas e com uma redução de 99,9% das colônias bacterianas em quase todos os materiais já nas primeiras 4 h. Também foi encontrado que a eliminação foi maior tanto para E. coli quanto utilizando materiais em pó. O estudo da adesão bacteriana confirmou os resultados anteriores mostrando efeito superior para quase todas HA-AgNPs produzidas comparado a HA, exceto para o material com menor proporção de AgNPs (0,01% m/m). Por outro lado, os testes de viabilidade, ALP e adesão demonstraram que a HA-AgNPs com maior quantidade de AgNPs (0,25% m/m) tem um efeito negativo sobre os osteoblastos. Considerando ambos os efeitos de citotoxicidade e antibacteriano, aliado a metodologia simples e de baixo custo envolvido na produção desses materiais, sugere-se que a HA contendo entre 0,05 0,10% m/m das AgNPs sintetizada pode ser a mais favorável para o desenvolvimento proposto visando futuras aplicações biomédicas. / Hydroxyapatite (HA) has been widely used as implant material due to its high similarity with the bone composition and its capacity to form strong chemical bonds with the bone tissue. However, the easy adsorption of organic molecules (proteins, amino acids and polysaccharides) by the HA also favors bacteria adsorption and replication, increasing the cases of infection related to this biomaterial. Aiming to contribute to the advancement of new biomaterials with antibacterial properties, were produced in this work hydroxyapatites containing silver nanoparticles (AgNPs) with different ratios (0.01, 0.05, 0.10 and 0.25% m/m). The first step of this work consisted in obtaining the HA powder by chemical precipitation and production of AgNPs colloidal suspensions by synthesis using chitosan as a reducing agent and stabilizer. In the second step, the AgNPs were adsorbed to the HA matrix by the powder immersion method using onde of the synthesized colloids, resulting in four hydroxyapatites containg AgNPs (HA-AgNPs) in disk and powder forms. In the third and final step, the materials produced were characterized using several techniques, with the powders (HA and HA-AgNPs) evaluated in vitro. The antibacterial effect was evaluated against strains of S. aureus and E. coli using qualitative (ágar diffusion test) and quantitative (bacterial colony counts) methods. The bacterial adhesion over the disks surface were observed by Scanning Electron Microscopy (SEM) and confocal laser scanning microscopy. Cytotoxicity was evaluated in vitro using cell culture containing the osteoblast lineage (MG- 63). In this assay, osteoblasts viability was studied using the resazurin test; the enzyme activity was investigated by alkaline phosphatase (ALP), and osteoblast adhesion was observed by SEM. The results showed that the HA produced is nanometric and the synthesized AgNPs are stable and spherical with diameters ranging from 5-10 nm. SEM, EDS and ICP-AES techniques confirmed the presence of silver in the HA-AgNPs. The others characterizations showed no significant changes in the structure, morphology and surface characteristics (hydrophobicity and net charge). However, the materials antibacterial activity was effective for both strains and with reduction of 99.9% of bacterial colonies in almost all materials within the first 4 h. It was also found that the removal was as great for E. coli as for powder materials. The bacterial adhesion study confirmed previous results showing superior effect for almost all HA-AgNPs produced compared to HA, except for the material with the lowest proportion of AgNPs (0.01% m/m). On the other hand, viability tests, ALP and adhesion demonstrated that HA-AGNPS with higher AGNPS quantities (0.25% m/m) has a negative effect on osteoblasts. Considering both cytotoxicity and antibacterial effects, combined with the simple methodology and low cost involved in the production of these materials, it is suggested that the HA containing synthesized AgNPs ranging from 0.05 to 0.10% m/m can be the most favorable for the development of the proposed material for future biomedical applications.

Für die Behandlung von Knochendefekten überkritischer Größe stehen seit einigen Jahren zahlreiche resorbierbare Materialien zur Verfügung, die eine Defektheilung bis hin zur vollständigen knöchernen Regeneration erlauben. Im Fall systemischer Knochenerkrankungen, insbesondere im osteoporotischen Knochen, ist jedoch die Selbstheilungskapazität des Gewebes stark eingeschränkt, was neben der Defektbehandlung eine knochenanabole sowie resorptionshemmende Therapie erfordert. Diese kann beispielsweise durch die Gabe von Strontium-haltigen Präparaten erreicht werden, da der duale Wirkmechanismus der Strontium-Ionen zu vermehrter Knochenneubildung bei gleichzeitig verminderter Knochenresorption führt. Ziel der vorliegenden Arbeit war die Entwicklung eines Strontium-haltigen Knochenzements, welcher eine Freisetzung von Strontium-Ionen spezifisch im jeweiligen Knochendefekt und somit eine lokale Stimulation der Knochenneubildung ermöglicht. Basierend auf einem etablierten Calciumphosphat-Knochenzement wurden Strontium-haltige Zementvarianten hergestellt und ausgiebig charakterisiert. Im Gegensatz zu den meisten bislang verfolgten Methoden konnten Zemente mit deutlich verbesserten mechanischen Eigenschaften hergestellt werden, welche weiterhin Strontium-Ionen in physiologisch relevanten Konzentrationen freisetzen. Durch Zellkulturuntersuchungen an humanen Zellen sowohl der osteoblastären- als auch osteoklastären Linie konnte eine Stimulation der für den Knochenaufbau verantwortlichen Zellen sowie eine Hemmung der den Knochen resorbierenden Zellen durch die entwickelten Zemente nachgewiesen werden.

Mechanical stimulation plays a crucial role in promoting cell differentiation. However, applying physical force directly to cells requires complex equipment and a sterile environment, posing challenges. To overcome this, stimuli-responsive biomaterials or 4D scaffolds can serve as an alternative platform for mechanical stimulation. These scaffolds, fabricated using advanced 3D printing techniques, can apply the necessary force to cells. To optimize their functionality, bioactive molecules or extracellular matrices can be incorporated or decorated on their surfaces. This thesis proposal focuses on developing a versatile material platform that allows customization through systematic composition adjustment and on-demand printing, while also offering surface modification capabilities. The primary objective is to create a novel cell carrier platform using thermo-responsive polymers. By manipulating the additive monomer compositions, we can finely adjust properties such as the transition temperature of the polymers, tailoring them to specific requirements. Furthermore, this platform will enable the fabrication of complex three-dimensional biomaterial structures with controllable porosity, a critical aspect of biomaterial design. Leveraging the capabilities of three-dimensional printing technology, we can program and achieve desired porosity levels in the printed structures, providing enhanced flexibility for biomaterial design. The development of thermo-responsive scaffolds involved three distinct stages aimed at designing an optimized platform that effectively operates within the physiological range while ensuring cell viability. One of the key challenges was to achieve a balance between thermoresponsive behavior and biocompatibility. In the initial stage, we investigated the interplay between a crosslinkable three-armed macromer (trimethylolpropane triacrylate-TMPTA) and various monomers (N-isopropylacrylamide-NiPAAm, methyl methacrylate-MMA, dimethylaminoethyl acrylate-DMAEA, 4-acryloylmorpholine-AMO) using thermally induced solution polymerization. NiPAAm, known for its thermoresponsive properties, was selected despite its limited biocompatibility. DMAEA was chosen to adjust the polymer network transition temperature by introducing cationic charge, which disrupts the coil-globule effect of PNiPAAm and provides cell adhesiveness of the composition. Additionally, the hydrophilic monomer AMO was incorporated to further fine-tune the polymeric network. We examined the behavior of these components within the physiological range and their integration into the PNiPAAm network, establishing significant correlations between the transition temperature of the polymer and the crosslinker and monomers in their soluble condition. In the second stage of our research, we introduced photo-induced polymerization to enhance the crosslinking ratio. By utilizing this method, we successfully fabricated photo-polymerized mixtures (photoresists) into thermo-responsive discs, enabling us to study their swelling behavior between 37℃ and 25℃. Our findings revealed that the swelling behavior could be adjusted by varying the ratios of the crosslinker and monomers in the experimental groups. Through careful experimentation, we identified a suitable composition (3% w/w TMPTA, 80% w/w NiPAAm, 15% w/w DMAEA, 5% w/w AMO, and 4% w/w photo-initiator(PI)) that required minimal crosslinking incorporation while still retaining thermo-responsiveness. Furthermore, we conducted a preliminary biocompatibility study by fabricating the mixture into thin-films and cultivating them with L929 fibroblast cells. In the third and final stage, we utilized the optimized formulations from the previous stage to build thermo-responsive 3D scaffolds using continuous Digital Light Processing (cDLP) printing. We investigated the effects of various parameters, such as curing time and monomer composition, on the swelling property of the scaffolds. Additionally, we introduced glycofurol (GF) as a photo-polymerization solvent, which allowed us to produce scaffolds with improved resolution and reduced printing time. The resulting optimized scaffolds, with a composition of 3% w/w TMPTA, 80% w/w NiPAAm, 15% w/w DMAEA, 5% w/w AMO, 4% w/w PI, and 10 seconds per layer, exhibited the desired thermo-responsiveness. To further understand the mechanical properties and thermal dependencies of these scaffolds, we conducted rheological analysis. This analysis helped establish a relationship between the mechanical properties of the scaffolds and their response to temperature changes. To investigate the potential of cell stimulation through periodic changes, we conducted an experiment involving the seeding of L929 fibroblasts and C2C12 myoblasts on thermo-responsive 3D scaffolds. Our objective was to assess the ability of cells to proliferate on scaffolds with different compositions. Specifically, we examined two types of scaffolds: lattice scaffolds, characterized by a porous structure with a periodic network that enables cells to inhabit a 3D environment, and raft scaffolds, which feature a dense 3D structure designed for cells to reside on the surface for observation and evaluation. The lattice scaffolds were composed of ≥2% w/w DMAEA, while the raft scaffolds consisted of ≥5% w/w DMAEA. To evaluate cell proliferation, we conducted direct contact experiments and employed live/dead assays, subjecting the scaffolds to temperature switching conditions at 31℃ and 37℃. These experimental setups aimed to provide insights into the response and behavior of cells in the presence of thermo-responsive scaffolds with varying compositions. The results revealed favorable adhesion and spreading of the cells on the scaffolds. Interestingly, in our dynamic temperature experiment, we observed that myoblasts seeded on the scaffolds exhibited both proliferation and spreading, whereas myoblasts subjected to constant-temperature conditions did not show the same behavior. This suggests that the expansion and contraction of the scaffold, observed in previous experiments, may impact cell viability. Further investigation is needed to better understand this phenomenon. Additionally, we enhanced cell adhesiveness of the scaffolds by impregnating the scaffolds with poly-L-lysine and tested them with hASCs (human adipose-derived stem cells). Significant differences were observed between scaffolds with and without poly-L-lysine, highlighting the effectiveness of this approach. In conclusion, we have successfully developed a thermo-responsive 3D scaffold that exhibits a transition temperature within the physiological range, ensuring cell survival, and provides mechanical stimulation to the cells through the coil-globule effect without causing cell detachment. Among the formulations tested, the GF-printed formulation (3% w/w TMPTA, 80% w/w NiPAAm, 15% w/w DMAEA, 5% w/w AMO, and 4% w/w photo-initiator) with an exposure time of 10 seconds per layer showed the most promising results for cell cultivation under periodic changes in temperature, with a transition temperature of 36.3 °C ± 0.9 °C. Furthermore, we conducted direct cell contact experiments and confirmed the biocompatibility of the thermo-responsive macromer-based scaffolds. These findings demonstrate that this material platform offers a versatile and responsive material for mechanical stimulation of cells on three-dimensional scaffolds. These promising results suggest that this approach holds significant potential for tissue engineering applications and can be utilized to develop mechanical stimulation devices for various biomedical applications.:CHAPTER 1……………………..……………...…………………………..…4 Introduction CHAPTER 2……………………..…………………………..……………….29 Material and Methods CHAPTER 3……………………………………..…..……………………….52 Thermo-Responsive Polymer from Thermal Synthesis Studies CHAPTER 4…………………………………..……………………………...70 An Adjustable Thermo-Responsive Polymer from Photo Synthesis CHAPTER 5……………………………………………………………....….88 Fabrication of Thermo-Responsive Scaffolds from DLP Printing CHAPTER 6…………………...…………………………………………....107 3D Scaffold Biocompatibility Studies CHAPTER 7…………………...……………………………………………139 Discussions CHAPTER 8…………………...……………………………………………161 Summery APPENDIX…………………...………………………………………….…166 Bibliography, List of Publications, CV, Declaration of Authorship, Acknowledgements, Related publication

Understanding the lignin effect and related structural parameters relevant to the recalcitrance of the plant cell wall and the individual and cooperative effects on enzymatic saccharification are vital for improving current processing and conversion methods for cellulosic biofuels. Data were collected from several pretreatment technologies (Hot-water, organo-solv, lime, lime-oxidant, dilute acid, and dilute acid-oxidant pretreatments) on cellulose ultrastructure, partial delignification followed by dilute acid pretreatment, dilute acid pretreatment of enzymatic isolated lignin, and melt rheology test of organo-solv lignin. Results showed minimal cellulose ultrastructural changes occurred due to lime and lime-oxidant pretreatments, which however especially at short residence time displayed relatively high enzymatic glucose yield. Dilute acid and dilute acid-oxidant pretreatments resulted in the largest increase in cellulose crystallinity, para-crystalline, and cellulose-Iβ allomorph content as well as the largest increase in cellulose microfibril or crystallite size. Organo-solv pretreatment generated the highest glucose yield, which was accompanied by the most significant increase in cellulose microfibril or crystallite size and decrease in relatively lignin contents. Lignin acted as a barrier which restricted cellulose crystallinity increase and cellulose crystallite growth during dilute acid pretreatment, and that partial delignification instead of complete lignin removal during DAP would benefit the increase of sugar yield. Furthermore, a deeper understanding of the structural change of lignin in the absence of cellulose-hemicellulose matrix during dilute acid pretreatment confirmed that delignification had the most beneficial effect in poplar, but for switchgrass was the xylan removal. In addition, investigation on the structural change of organo-solv lignin during melt rheology test indicated that high purity lignin isolated from plant biomass with the lowest S/G (syringyl/guaiacyl) ratios will exhibit superior processing performance characteristics to produce high-quality carbon fibers. These findings can aid both in the development of improved enzymes that contain activities to decompose recalcitrant structures and in the design of various processing conditions that efficiently convert specific biomass feedstock into sugars. They can also help in the design of new chemical modifications on lignin and innovative biosynthesis strategies for producing linear-fiber-forming lignin with high-performance.

T. pallidum can survive a primary immune response and continue growing in the host for an extended period of time. T. pallidum is thought to bind serum fibronectin (FN) through Tp0483 on the surface to obscure antigens. A Tp0483 fragment (rTp0483) was adsorbed onto functionalized self-assembled monolayers (SAMs) with FN. FN capture by adsorbed rTp0483 depended greatly on surface chemistry with COO- groups being best for FN binding. Hemocompatibility was determined by analysis of plasma protein adsorption, intrinsic pathway activation, and platelet activation. rTp0483+FN bound an equal or lesser amount of fibrinogen (Fg), human serum albumin (HSA), and factor XII (FXII) compared to rTp0483 or FN alone and adsorption of rTp0483 prior to FN greatly decreased platelet activation. Inhibition of protein binding and platelet activation suggested an attenuated hematological response. Biocompatibility of rTp0483 and FN coated surfaces was characterized by macrophage uptake of protein coated polystyrene microspheres (PSMs), macrophage adsorption onto protein coated surfaces, cytotoxic effects of adsorbed rTp0483 and FN, and TNF-α and NO2- release in macrophages stimulated with rTp0483 and FN adsorbed and in solution. Addition of FN to rTp0483 on plain and COO- PSMs reduced phagocytosis compared to rTp0483 alone and on plain PSMs compared to FN alone. On plain PSMs addition of FN to adsorbed rTp0483 decreased TNF-α generation. Adsorption of rTp0483 before FN on large, flat COO- surfaces decreased macrophage adsorption and TNF-α and NO2- generation. High concentrations of rTp0483 were mildly cytotoxic to macrophages. FN binding by Tp0483 on T. pallidum likely plays a role in antigenic disguise and rTp0483+FN coatings may potentially inhibit FN and rTp0483 specific interactions with macrophages. Molecularly imprinted polymer coatings were also examined for biomaterial development. Fouling resistant 2-methacryloyloxyethyl phosphorylcholine (MPC) was imprinted with bovine serum albumin (BSA) protein templates to facilitate BSA specific binding. The BSA template was constructed and verified and BSA specific binding quantified using quartz crystal microbalance (QCM) and enzyme linked immunosorbent assay (ELISA). BSA imprinted coatings were determined to bind significantly more BSA than nonfouling MPC controls demonstrating the feasibility of targeted protein capture.

Oligonucleotides are popular recognition-based biomaterials assembly and disassembly tools due to their specificity and ease of control. Their susceptibility to degradation by nucleases and false positive signals under certain conditions, however, has led to great interest in chemically modified oligonucleotides such as locked nucleic acids (LNA) that enhance both nuclease resistance and target specificity. This dissertation extends prior work with DNA sequences to investigate incorporating locked nucleic acid (LNA), a synthetic oligonucleotide, in isothermal colloidal assembly and disassembly schemes as well as on hybridization kinetics between single-stranded and double-stranded probes immobilized on microspheres. Incorporation of LNA nucleotides into DNA sequences is of particular interest as a means of enhancing the performance of DNA in a biomaterials context due to the increased resistance of LNA to nuclease degradation, its greater intrinsic affinity for oligonucleotide targets, and low cytotoxicity effects. The effects of LNA modification, target sequence length, sequence fidelity, and salt concentration are key variables explored. After providing an overview of DNA and its properties, synthetic oligonucleotides, colloidal particles, and previous applications of DNA and LNA in colloidal assembly schemes, this work then discusses the selection and characteristics of appropriate pairs of hybridization partners for reversible colloidal assembly scenarios. A comparative investigation of the in situ primary hybridization kinetics between select LNA or DNA targets and single-stranded probes immobilized on colloidal surfaces is performed. To support the disassembly studies, the in situ competitive displacement kinetics of hybridized LNA primary targets by either LNA or DNA secondary targets is discussed. For these in situ studies, flow cytometry was used to quantify the hybridization reactions as they occur on microsphere surfaces. While comparable rate constants were typically observed between target and single-stranded probes, LNA typically exhibited more extensive primary and secondary hybridization activity. Optimizing hybridization parameters, such as duplex concentration, sequence fidelity, and LNA content in the probe and target strands, allows for the extent of colloidal disassembly to be tuned, an important step in developing a multifunctional colloid-based biomaterial system.