Abstract   Introduction Failure in the mechanism of self-tolerance in T or B cells can lead to autoimmunity. One of the autoimmune diseases is rheumatoid arthritis (RA), which is a chronic inflammatory disease of unknown cause and is characterized by systemic inflammation, autoantibodies and joint destruction. Serology is crucial for the classification of this disease. The first autoantibody found in RA patients was Rheumatoid factor (RF). However, anti-citrullinated peptide antibodies (ACPAs), a relatively new group of autoantibodies found in 70-90% of RA patients, are diagnostically more specific than RF. Type II collagen (CII) is the most abundant protein in human cartilage. In RA patients, immunity against CII leads to cartilage degradation and loss of joint function. Already from the 1970s, antibodies to CII (anti-CII) were found in RA sera, suggesting that CII autoimmunity might be pathogenetically important. Previous studies from our group show that a subgroup of patients with high levels of anti-CII at the time of diagnosis at the same time have high levels of inflammation in the joints. This is probably caused by anti-CII immune complexes (IC) inducing pro-inflammatory cytokines from macrophages. Although anti-CII positive patients have high inflammatory activity early on, as anti-CII levels decrease during the first year, the associated inflammation also diminishes. Thus, anti-CII positive patients have a rather good prognosis. Moreover, it is assumed that since anti-CII positive patients have a better prognosis than ACPA positive, patients with elevated anti-CII at the time of diagnosis might benefit from different and milder treatment. Previous studies from the group were performed on stored patient samples from the time before modern treatments with biologic agents (1995-2005). In this study, we aimed to investigate patients belonging to a more recent RA cohort, diagnosed between 2005-2014, with the aim to investigate whether patients with the anti-CII-associated RA phenotype would respond differently depending on the use of different modern RA therapies. Patients and Methods The primary cohort consisted of 2335 RA patients and 480 non-RA controls from the Epidermiological Investigations in Rheumatoid Arthritis (EIRA) case-control study. As we run into methodological problems two subgroups with 62 and 40 RA patients from the previous anti-CII studies were investigated when modifying the ELISA procedure, as well as a group of earlier investigated patients with non-specific ELISA reactivity. Totally 2776 RA patients were investigated. All investigated patients fulfilled the American College of Rheumatology classification criteria. To measure the anti-CII levels in RA patients and healthy controls anti-CII ELISA was performed. During the experiments, several different sources of CII from human, rat and bovine origin, and two different alternative coating buffers were used. The optical density (OD) was measured at 450 nm and anti-CII concentrations were calculated against the standard curve from an RA patient with high anti-CII levels.   Results My first analysis of the EIRA cohort showed that anti-CII are higher in RA patients than in controls, but could not confirm the association with acute onset RA. This was an unexpected finding and changed the focus of this master thesis project, to modify the measurement of anti-CII. Re-investigation of EIRA I showed that a proprietary coating buffer is important in the assay. Moreover, when different samples from RA patients were tested with bovine, rat and three different lots of human CII, correlation tests with clinical measures showed that bovine collagen and a new lot of human CII- prepared by the supplying company solely for this project- showed the strongest associations. Thereafter the EIRA cohort was re-investigated with two ELISAs, using bovine and human CII coated with the proprietary buffer. At the time of thesis writing almost all of the EIRA samples have been re-analysed, and results from both the modified ELISAs show the awaited clinical associations to early inflammation.   Conclusion Keeping the integrity of triple helical collagen is very important for the identification anti-native CII in RA patients. Our results show that the use of the proprietary coating buffer appears to be instrumental in this assay, irrespective of what source of CII was used. The new lot of human CII shows significant associations with the clinical measures, but associations are somewhat stronger with bovine CII. After finalising the re-investigations, we will be able to conclude which of the two analyses is most appropriate, and the corresponding dataset will then be merged with data from the first part of the EIRA study investigated previously by other group members. As anti-CII analysis shows the association to disease activity and prognosis, it can be used for predicting prognosis of RA and choosing the appropriate therapy in newly diagnosed RA patients, which might be clinically useful for rheumatologists. Our hypothesis is that as anti-CII positive patients have strong early inflammatory response but good long-term prognosis, they might benefit from other and perhaps short-term treatment compared to other RA patients.  If this is correct, our finding can have impact on the economy as it can define the patients who will not need expensive long-term medications. As modern anti-rheumatic therapies carry the risk of infections, such individualized therapies might also benefit anti-CII positive patients.

Hoje a criação de avestruz ((Struthio camelus, Linnaeus 1758)) é uma atividade de grande potencial, porém não existem padrões definidos sobre a histologia do seu fígado, que é um órgão de grande importância no metabolismo, o conhecimento de sua histologia e anatomia pode contribuir para a detecção de doenças e deficiências nutricionais que influenciam no crescimento e desenvolvimento do animal. Os objetivos desta pesquisa são: Estudar a anatomia e histologia do fígado e a ramificação de sua artéria hepática, veia porta hepática e ducto biliar. Para a realização da parte macroscópica foram utilizados quinze avestruzes com idades entre 12 e 18 meses (com peso médio em torno de 80 a 100 kg), provenientes do abatedouro Don Pig, situado próximo à cidade de Botucatu no estado de São Paulo. Os animais foram abatidos com pistola pneumática e posteriormente submetidos a sangria. Foram injetadas com látex a artéria hepática, o ducto biliar e a veia porta. O fígado dos avestruzes apresentam dois lobos (direito e esquerdo). No caso o direito é maior que o esquerdo e ambos são subdivididos em dorsal, intermédio e ventral. Além disso, amostras do fígado foram processadas para a observação em microscopia de luz e microscopia eletrônica de transmissão (MET). A hematoxilina e eosina (H.E), picrossírius, Gordon e Sweets, Sudan black e o ácido peródico de Schiff (PAS) são colorações usadas respectivamente para observar a morfologia do fígado, colágeno, fibras reticulares, gordura e glicogênio. Foram encontrados os espaços porta- hepáticos (artéria hepática, veia porta e ducto biliar e as veias centrolobulares). O glicogênio presente mostrou a média de 5,68%. O conteúdo lipídico, conferiu um aspecto goticular compatível com um quadro de esteatose hepática e a média obtida foi de 9,83%. Foi encontrado colágeno ao redor dos espaços porta-hepáticos, artérias centrolobulares e capilares sinusóides e a média foi de 14,71%. Encontrou-se também fibras reticulares ao redor dos capilares sinusóides e a média foi de 5,96%. Quanto à MET notou-se que no citoplasma dos hepatócitos desses animais existem numerosas mitocôndrias, glicogênio, muitas gotas de gordura, alguns lisossomos, retículo endoplasmático granular ao redor das mitocôndrias, algumas células estreladas, eritrócitos, núcleo, célula em degeneração e o canalículo biliar ao centro. Provavelmente o quadro sugestivo de esteatose é resultante do estado nutricional dos animais, já que nenhum outro aspecto relevante foi encontrado. Como estas aves são destinadas ao abate, sua alimentação é formulada para que os animais apresentem rápido desenvolvimento e alto ganho de peso, provavelmente com níveis de lipídios acima do necessário, causando uma deposição de micelas de gordura nos hepatócitos. Estes resultados demontraram que os hepatócitos dos avestruzes são muito simulares as outras aves apesar de também serem muito similares na estrutura das células do fígado de mamíferos. / At present the ostrich (Struthio camelus) breeding has been showing a great economical potential, although yet there are not distinct patterns about the histology of its liver, which is an organ of key importance in terms of metabolism. The knowledge of its histology and anatomy can help the detection of diseases and nutritional deficiencies that affect the growth and development of this bird. The aims of this work are to study the liver anatomical and histological structure and the branching of the hepatic artery, hepatic portal vein and bile duct. In the macroscopic study 15 ostriches with an average age of 12-18 months and average weight of 80-100 Kg, proceeding from Don Pig Abatteur, located next to Botucatu, São Paulo, were used. The birds were slaughtered with air gun and subsequently submitted to bleeding. The hepatic artery, the bile duct and the hepatic portal vein were injected with latex. The ostrich liver presents two lobules (right and left), being the right one larger than the left and both are subdivided into dorsal, intermediate and ventral. Liver samples were processed for light and electron transmission microscopic studies. Hematoxilin and eosin (HE), picrosirius, Gordon and Sweets, Sudan black and Schiff periodic acid (PAS) were respectively used to observe the liver morphology, collagen, reticular fibers, lipids and glycogen. The liver portal spaces were determined (hepatic artery, portal vein, bile duct and centrolobular veins). An average of 5.68% of glycogen was observed. The lipidic content provided a droplet aspect compatible to hepatic esteatosis, with an average of 9.83%. Collagen fibers around the liver portal spaces, centrolobular arteries and sinusoidal cappilaries were detected, at an average of 14.71%, as well as reticular fibers located in the vicinity of sinusoidal capillaries with an average of 5.96%. Through transmission electron microscopy we noticed in the hepaticyte cytoplasm the presence of numerous mithocondria, glycogen, several lipidic droplets, some lysosomes, granular endoplasmatic reticulum around the mithocondria, some stellate cells, erythrocytes, nucleus and degenerating cells, besides the central biliary canaliculus. The suggestive steatotic results might result from the animals nutritional status, once no other relevant aspect was detected. The birds are slaughtered for food comsumption and their ration is balanced striving for faster growth and higher weight gains, which are achieved through extra lipidic levels, motivating deposition of lipidic micelles in the hepatocytes. Our results demonstrated that ostrich and other birds hepatocytes are very similar.

O desenvolvimento de uma matriz de colágeno associado à gelatina tem potencial como biomaterial devido sua alta biocompatibilidade, capacidade de alterar suas propriedades físico-químicas e estruturais por modificações químicas e a habilidade de formar géis estáveis. Porém, um ponto negativo de sua aplicação está na biodegradabilidade. Desta forma, para reduzir esta degradação, agentes de reticulação, como a proantocianidina (PA), que age formando ligações de hidrogênio as quais estabilizam o complexo proteína-PA, podem ser empregados. Este trabalho teve como objetivo a obtenção de hidrogéis neutros de colágeno aniônico:gelatina:extrato de semente de uva. Para tanto, foram utilizadas duas proporções de extrato, 0,25 e 0,50% e três diferentes tempos de hidrólise alcalina do colágeno, 24, 72 e 120 horas, gerando nove diferentes biomateriais, incluindo os hidrogéis sem tratamento com o extrato. O colágeno foi extraído de tendão bovino e o agente reticulante foi o extrato de semente de uva cujo componente majoritário é a proantocianidina. A caracterização foi feita por termogravimetria (TG), calorimetria exploratória diferencial (DSC), espectroscopia de absorção no infravermelho (FTIR), microscopia eletrônica de varredura (MEV), cinética de absorção de água e ensaios de citotoxicidade in vitro, pelos métodos de difusão em ágar e difusão de extrato em solução (MTT). Estudos de TG mostraram a perda de água em um processo único, representando em média 95% do hidrogel. As curvas DSC mostraram que quanto maior a concentração de extrato, maior é a temperatura de desnaturação, aumentando em média 6,8°C com a adição de 0,50% de extrato, o que indica a eficácia da proantocianidina na reticulação do colágeno. Além disso, notaram-se temperaturas menores para maiores tempos de hidrólise alcalina do colágeno, sendo de 58,4°C, 49,7°C e 46,5°C para hidrogéis preparados com colágeno 24, 72 e 120 horas, respectivamente. A presença do extrato não causou alterações significativas nos espectros FTIR, apenas surgimento das bandas em 1118 e 1288 cm-1 referentes ao anel aromático da proantocianidina, mas gerou mudanças nas estruturas internas dos hidrogéis, visualizadas por MEV, como aumento do número de poros e interconectividade entre eles. A cinética de absorção de água mostrou que o equilíbrio é atingido em aproximadamente 10 minutos, indicando vantagem para a aplicação do hidrogel, já que é obtido rapidamente a partir de sua forma liofilizada, adequada para o armazenamento. Também foi observado que quanto menor é o tempo de hidrólise, maiores são as absorções, variando de 540 até 1360%. Com os ensaios de citotoxicidade foi possível concluir que o hidrogel C24GE50 mostrou-se mais adequado para uma aplicação como biomaterial, com um índice de 92,7% de sobrevivência celular. / The development of a collagen matrix associated with gelatin has potential as biomaterial due to its high compatibility, ability to change its physical-chemical and structural properties by chemical modifications and also the ability to form stable gels. However, a negative point of its application is the biodegradability. Thus, to reduce this degradation, crosslinking agents, such as proanthocyanidin (PA), which acts forming hydrogen bonds which stabilize the PA protein complex may be employed. This project aimed to obtain neutral hydrogels prepared by mixture of anionic collagen: gelatin: grape seed extract. It was used two extract proportions (0.25 and 0.50%) and three different periods of time for collagen alkaline hydrolysis (24, 72, 120 hours), giving nine different biomaterials, including hydrogels without treatment with the extract. The collagen was extracted from bovine tendon and the crosslinking agent was grape seed extract whose major component is the proanthocyanidin. The characterization was done by thermogravimetry (TG), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy, scanning electron microscopy (SEM), water absorption kinetics and in vitro cytotoxicity assays, by agar diffusion method and diffusion of extract in solution (MTT). TG results showed loss of water in a single process, representing about 95% of the hydrogel.DSC curves showed that the higher the concentration of the extract, the higher the melting temperature, increasing on average 6.8 °C with the addition of 0.50% extract, which indicates the effectiveness of the proanthocyanidin crosslinking of the collagen. Moreover, lower temperatures were observed for longer periods of alkaline hydrolysis of collagen, being 58.4°C, 49.7°C and 46.5°C for preparations with 24, 72 and 120 hours, respectively. The presence of the extract did not cause significant changes in the FTIR spectra, only appearance of bands at 1118 and 1288 cm-1 related to the aromatic ring of proanthocyanidin but led to changes in internal structures of the hydrogels viewed by SEM, showing increased number of pores and interconnectivity between them. The water absorption kinetics showed that the equilibrium is achieved in approximately 10 minutes, indicating advantages in using this hydrogel, which is quickly obtained from its lyophilized form, a suitable form for storage. It was also observed that the shorter the time of hydrolysis, the greater the absorptions, ranging from 540 to 1360%. It was possible to conclude from the cytotoxicity analysis that the C24GE50 is more suitable for application as a biomaterial, with an index of 92.7% cell survival.

A quitina é um biopolímero abundante na natureza e seu derivado, a quitosana é tido como excelente biomaterial na engenharia tecidual por sua versatilidade e propriedades, assim como colágeno que por sua presença natural no organismo e seu papel biológico fazem com que seja amplamente utilizado na medicina. As blendas destes biopolímeros possuem excelentes propriedades mecânicas devido às interações eletrostáticas e pontes de hidrogênio, e propriedades biológicas pelos materiais que as compõe, já demonstrando eficácia na regeneração tecidual. O extrato de Garcinia mangostna L., ou extrato de mangostão, é pouco estudado no ocidente, mas no oriente é amplamente utilizado para fins medicinais, com propriedades promissoras na área de engenharia tecidual. A interação deste com as blendas de quitosana/colágeno ainda não foram estudadas. Este trabalho teve como objetivo a obtenção e estudo de scaffolds de quitosana/colágeno em diferentes proporções (1:1, 2:1 e 3:1) com extrato de mangostão em variadas concentrações (10, 20 e 30%). A quitosana foi extraída por desacetilação da &#946-quitina de gládios de lula, o colágeno aniônico foi obtido de tendão bovino por hidrólise alcalina e o extrato de mangostão foi obtido da casca do fruto. A caracterização da quitosana e assim como colágeno, extrato e misturas foram analisados por absorção na região do infravermelho (FT-IR), efetuou-se um ensaio reológico dos géis e os scaffolds foram submetidos à calorimetria exploratória diferencial (DSC), microscopia eletrônica de varredura (MEV), ensaios de intumescimento e liberação do extrato. Ensaios reológicos indicam um comportamento pseudoplástico para todas as amostras e se feito um tratamento matemático, o melhor modelo às quais estas se adaptam seria o modelo de Carreau. Os DSC dos scaffolds mostram que a adição de extrato tende à elevar a temperatura de desnaturação (Td) do colágeno. Espectros FT-IR caracterizaram o extrato e não mostraram resultados conclusivos sobre a incorporação dele aos filmes. Fotomicrografias por MEV mostram alteração no tamanho médio de poros e canais com a adição de extrato. Ensaios de intumescimento dos scaffolds mostram uma diminuição de capacidade de absorção com adição de extrato, os ensaios de liberação feitos para os scaffolds das misturas mostram uma liberação entre 25-35% do extrato sendo as cinéticas de liberação modeladas pelo modelo de Kosmeyer-Peppas para a maioria das amostras. Obtendo-se assim géis e scaffolds que mostram resultados inicias promissores e com acréscimo de estudos complementares podem vir a ser utilizados no campo da engenharia tecidual. / Chitosan is an abundant biopolymer in nature, and it is an excellent biomaterial for tissue engineering due to its versatility and properties. Collagen, by the natural presence in the organism and to the biological role makes it widely used in medicine. These biopolymers blends have excellent mechanical properties due to electrostatic interactions and hydrogen bonds, and biological properties, demonstrating good efficiency in tissue engineering. The Garcinia mangostana L. extract, or mangosteen extract, is not very well studied in the western culture, but in the eastern, it is widely used in local medicine, with promising properties in tissue engineering field. The interaction between chitosan/collagen blends and mangosteen extract has not yet been studied. This study aims to obtain scaffolds from chitosan/collagen blends in different ratios, such as 1:1, 2:1 and 3:1 with different extract concentrations (10, 20 and 30%). Chitosan was obtained by deproteinization and deacetylation of &#946-chitin from squid\'s pens, anionic collagen was obtained from bovine tendon by alkaline hydrolysis, and the mangosteen extract was obtained from the hulls of fruits. Materials characterization was done by FT-IR, DSC, SEM, swelling and extract release in buffer solution. Rheological measurements were made with the gels formed from the mixture of the components. The rheological studies indicates a pseudo plastic behavior to all samples and by a mathematical treatment, the Carreau mathematical model was the best fitting. The DSC analysis for the collagen mixtures indicated a tendency to elevate the denaturation temperature with addition of extract. With the FT-IR results, we could obtain mangosteen extract spectrum but the mixtures spectrum did not show the extract incorporation. The photomicrographies obtained by SEM showed pore and channels alteration with extract addition. Swelling studies showed a decrease in swelling capacity after extract addition, release studies showed a 25-35% extract release by scaffolds and in most cases the kinetic was fitted by Kosmeyer-Peppas. Thus obtaining promising gels and scaffolds in the field of tissue engineering, but needing of further development.

Orientador: Eloi Dezan Junior / Banca: Francine Benetti / Banca: Renato de Toledo Leonardi / Resumo: Além da atividade antimicrobiana e biocompatibilidade de um curativo de demora, é oportuno que ele tenha também a capacidade de estimular a biomineralização e reparo dos tecidos periapicais. O objetivo deste estudo foi avaliar a resposta inflamatória, a capacidade de induzir biomineralização e reparo tecidual em tecido subcutâneo de ratos, causada pelas pastas de iodofórmio e hidróxido de cálcio. Foram utilizados 18 ratos Wistar albinos (n=6), que receberam implantes subcutâneos de tubos de polietileno com os seguintes materiais: hidróxido de cálcio + propilenoglicol (1:1) [Ca(OH)2+P], hidróxido de cálcio + propilenoglicol + iodofórmio (2:1:1) [Ca(OH)2+P+Iodo], iodofórmio + Carbowax (5:1) [Iodo+Carbow] and Carbowax [Carbow]. Tubos vazios extras foram utilizados como grupo controle. Após 7, 15 e 30 dias, os implantes foram removidos conjuntamente com tecido circundante. Foram utilizadas as colorações de hematoxilina-eosina (HE), Von Kossa (VK), técnica de luz polarizada (LP) e Picrosirius red (PSR). Para HE foram utilizados os scores de 0, poucas células inflamatórias; 1, menos de 25 células inflamatórias - reação leve; 2, entre 25 e 125 células inflamatórias - reação moderada; e 3, 125 ou mais células inflamatórias - reação severa (400x). A cápsula fibrosa foi considerada fina quando menor que 150 μm e espessa quando maior que 150 μm. A capacidade de mineralização foi analisada como positivo ou negativo com VK e como presente ou ausente sob LP. Para PSR as fibras imaturas apa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The antimicrobial activity and biocompatibility of a root canal dressing are important requirement, but it is also interesting that it has the ability of stimulate biomineralization and repair of the periapical tissues. The objective of this study was to evaluate the inflammatory response, biomineralization and tissue repair induction in subcutaneous tissue of rats caused by iodoform and calcium hydroxide. Eighteen Wistar rats (n=6) received subcutaneous implants with iodoform and calcium hydroxide pastes. Extra empty tubes were used as control group. After 7, 15 and 30 days, the implants were removed with surrounding tissue. Kruskal-Wallis test was used with a significance level of 5%. At 7 days, all groups excluding carbowax, showed moderate inflammatory reaction and thick fibrous capsule. At 15 days, all groups excluding control decrease their fibrous capsule thickness. At 30 days all groups presented mild reaction and fine fibrous capsule. Only the groups containing calcium hydroxide presented mineralized tissue in all periods analyzed. At 7 days all groups showed a higher proportion of immature fibers. At 15 days, only [Ca(OH)2 + P] and [Carbow] groups increased their proportion of mature / immature fibers. At 30 days, the [Ca(OH)2 + P] group was the only that presented prevalence of mature collagen fibers, with significant difference (p <0.05). The null hypothesis was partially rejected. All groups showed biocompatibility. Only the groups containing Ca(OH)2 induced the ... (Complete abstract click electronic access below) / Mestre

Rupture of atherosclerotic plaque can cause acute life-threatening events such as myocardial infarction and ischemic stroke; therefore, there is much interest in developing therapies aimed at increasing plaque stability. More stable lesions are characterized as having high collagen content and containing a large number of vascular smooth muscle cells (SMCs) of contractile/differentiated phenotype. In our previous studies using an apolipoprotein E-deficient (Apoe-/-) mouse model of atherosclerosis, we found that insulin-like growth factor-1 (IGF-1)-infusion not only reduced total plaque burden, but also increased collagen expression and the number of alpha-smooth muscle actin (αSMA)-positive cells in plaque. In this study, we identify cellular mechanisms responsible for these observations. We found that in human aortic smooth muscle cells (HASMCs) grown in culture, IGF-1 post-transcriptionally upregulated expression of the procollagen type I alpha-1 subunit (pro-α1(I)) as well as contractile proteins, αSMA and smooth muscle 22-alpha (SM22α), via a PI3K-dependent but Erk1/2- and mTOR-independent signaling mechanism. Furthermore, experiments using an inhibitor of collagen synthesis or a blocking antibody against the alpha2beta1-integrin (α2β1) suggested that interaction with collagen type I promotes HASMC contractile phenotype. To elucidate mechanisms underlying IGF-1 upregulation of collagen synthesis we investigated the effect of IGF-1 on the mRNA-binding protein, la ribonucleoprotein domain family member 6 (LARP6), which had been shown to bind a conserved stem-loop secondary motif in the 5’UTR of COL1a1 and COL1a2 mRNA. IGF-1 rapidly increased LARP6 expression in HASMCs leading to increased COL1a1 and COL1a2 mRNA bound LARP6 and increased synthesis of collagen type I. Mutation of the 5’stem-loop of Col1a1 mRNA (that inhibited binding by LARP6) or overexpression of a 5’stem-loop RNA molecular decoy (that sequesters LARP6) both prevented the ability of IGF-1 to increase pro-α1(I) synthesis as well as mature α1(I) expression in cultured medium. Furthermore, IGF-1-infusion in Apoe-/- mice increased LARP6 and pro-α1(I) expression in aortic lysates, and SMC-specific IGF-1-overexpression in transgenic mice robustly increased collagen fibrillogenesis in atherosclerotic plaque. In conclusion, this work identifies LARP6 as a critical mediator by which IGF-1 augments synthesis of collagen type I in vascular smooth muscle, and uncovers key mechanisms whereby IGF-1 promotes atherosclerotic plaque stability. / acase@tulane.edu

Traumatic injuries to peripheral nerves often leave gaps that cannot be repaired by direct suture methods. In such instances, repair with a tubular nerve guide, allows connection of the nerve ends, provides directional guidance, and concentrates endogenous trophic factors for regenerating axons. We hypothesized that collagen nerve guides containing longitudinally oriented channels would further improve the outcome of nerve repair by increasing the surface area available for cell migration. We restored the continuity of a 10mm peripheral nerve gap (rat sciatic nerve) by suturing the nerve stumps into a type I collagen nerve guide (1.5 mm ID), which contained longitudinal channels. Two different channel designs were tested. They were compared to empty nerve guides and autografts. One channel design contained five longitudinally-oriented collagen microtubes (0.4 mm ID) and the other contained 32 longitudinally-oriented collagen filaments (90 micro m diameter). Nerve regeneration was examined at 6 weeks and 12 weeks post repair by a determination of the number and diameter of myelinated axons in the middle sections of the nerve guides. Sciatic function Indices were calculated from walking tracks and static stance images, and electrophysiological assessments were performed. Compound muscle action potentials of the gastrocnemius and intrinsic muscles of the foot were recorded from animals in each group at 12 weeks, indicating that axons regenerated through the nerve repair site, into the distal nerve stump, and successfully reinnervated peripheral targets. At 6 weeks, there was no significant difference between the mean number of myelinated axons with the mid sections of the 3 types of nerve guides (P = 0.488). At 12 weeks, the nerve guide that contained 5 microtubes within its lumen had significantly more axons than the nerve guide that contained 32 filaments in its lumen (P = 0.008). The mean myelinated axon number in the microtube group is larger than the empty nerve guide group but this difference was not statistically significance (P < 0.05). Autografts at both 6 and 12 weeks had significantly more myelinated axons in the mid section of the repair site than either of the nerve guide repairs at the respective time points (P < 0.05).

Bibliography: leaves 243-267. / xxiv, 268, [32] leaves, [25] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Special methods for the ultrastructural localization of basal lamina laminin and type IV collagen in animal oral mucosa were developed in a series of experiments aimed at determining optimim methods for tissue fixation, dehydration, embedding and immunoincubation. The results provide a tool enabling further understanding of the molecular organization of normal oral mucosal basal lamina and bassal lamina in squamous cell carcinomas. / Thesis (Ph.D.)--University of Adelaide, Dept. of Dentistry, 1994?

Extrinsic repair techniques for the treatment of acute chondral injuries continue to yield suboptimal repair. The inability of these techniques to produce hyaline cartilage underscores the limitations in our understanding of basic chondrocyte biology. Conversely, intrinsic repair tissue has not been extensively studied despite the fact that it can yield hyaline-like cartilage and is commonly observed in osteoarthritis. Attempts at extrinsic repair could therefore benefit from a better understanding of the successes and failures inherent in the intrinsic repair process. Chondrocyte culture has typically been conducted under non-physiologic conditions whereby chondrocytes readily dedifferentiate. Consequently, much of the knowledge gained about chondrocytes has been misleading thus hindering advancements in chondrocyte biology and attempts at extrinsic articular cartilage (AC) repair. Hypoxic culture conditions, which are beneficial towards the preservation of the chondrocyte phenotype, remain insufficient due to elevated collagen I gene expression. As such, an appropriate model system does not yet exist in which to study physiologically-relevant chondrocyte biology. The presence and prevalence of collagen I in both degenerate and de novo osteoartritic tissue was examined immunohistochemically. Collagen I deposition during osteoarthritic progression was compared against IHC staining for collagen II and aggrecan. A novel model system was also evaluated for chondrocytic phenotype retention. To this end, hypoxic, high-density-monolayer-chondrocyte (HDMC) cultures were compared to freshly isolated chondrocytes for their ability to maintain a chondrocytic extracellular matrix (ECM) gene expression profile. HDMC culture conditions prevented the severe loss of the phenotype typically associated with conventional monolayer culture. Moreover, prolonged HDMC culture resulted in the formation of a complex ECM and a marked suppression of collagen I expression. This study also demonstrated that collagen I deposition occurs in osteoarthritic AC at the onset of structural damage and increases in response to increasing structural damage. Collagen I deposition was also found in different types of de novo cartilage associated with osteoarthritic joints and suggests that it plays an important role in intrinsic cartilage repair. Taken together, this work demonstrates that collagen I is a common feature in the ECM of structurally immature and structurally damaged AC and hence may play a role in tissue stabilization. / Experimental Surgery

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 137-153).