This thesis focuses on testing and improving Ti-6Al-4V ELI biomaterials, which are currently one of the most used titanium alloys in biomedicine (predominantly in orthopaedics and dentistry), in cooperation with research institutions and private companies developing and producing such materials. The metallic samples were previously modified by plasma electrolytic oxidation (PEO) with use of electrolytes of a different composition to induce development of a homogeneous TiO2 layer on its surface. In vitro interactions of human osteoblast-like cell line Saos-2 with the surface of Ti-6Al-4V ELI alloy samples are investigated. Initial cell attachment, spreading, morphology, cell population density, viability, calcium deposition and expression of selected osteogenic markers, e.g. collagen type I, alkaline phosphatase and osteocalcin, were evaluated on cultured cells. The cells behavior were then correlated with physicochemical properties of the material surface, such as its topography, roughness, wettability, surface layer chemical composition etc. The results are also compared with those obtained in cells cultured on control samples of untreated alloys as well as microscopic glass coverslips and bottom of standard polystyrene cell culture wells. The aim of this thesis is to select the most promising...

Interactions of cells with their environment regulate important cellular functions and are required for the organization of cells into tissues and complex organisms. These interactions involve different types of adhesion receptors. Interactions with extracellular matrix (ECM) proteins are mainly mediated by the integrin family of adhesion molecules. Situations in which integrin-ECM interactions are deregulated cause diseases and play a crucial role in cancer cell invasion. Thus, the mechanisms underlying integrin-binding and regulation are of high interest, particularly at the molecular level. How can cell-ECM interactions be studied? While there are several methods to analyze cell adhesion, few provide quantitative data on adhesion forces. One group, single-cell force spectroscopy (SCFS), quantifies adhesion at the single-cell level and can therefore differentiate the adhesive properties of individual cells. One implementation of SCFS is based on atomic force microscopy (AFM); this technique has been employed in the presented work. Advantageously AFM-SCFS combines high temporal and spatial cell manipulation, the ability to measure a large range of adhesion forces and sufficiently high-force resolution to allow the study of single-molecule binding events in the context of a living cell. Since individual adhesion receptors can be analyzed within their physiological environment, AFM-SCFS is a powerful tool to study the mechanisms underlying integrin-regulation. The presented work is split into six chapters. Chapter one gives background information about cell-ECM interactions. In chapter two, different adhesion assays are compared and contrasted. The theoretical Bell-Evans model which is used to interpret integrin-mediated cell adhesion is discussed in chapter three. Thereafter, the three projects that form the core of the thesis are detailed in chapters four through six. In the first project (chapter 4), α2β1-integrin mediated cell adhesion to collagen type I, the most abundant structural protein in vertebrates, was quantified using CHO cells. Firstly, α2β1-collagen interactions were investigated at the single-molecule level. Dynamic force spectroscopy permitted calculation of bond specific parameters, such as the bond dissociation rate koff (1.3 ± 1.3 sec-1) and the barrier width xu (2.3 ± 0.3 Å). Next, α2β1-integrin mediated cell adhesion to collagen type I was monitored over contact times between 0 and 600 sec. Thereby the kinetics of α2β1-integrin mediated interactions was explored and insights into the underlying binding mechanisms were gained. In the second project (chapter five), effects of cryptic integrin binding sites within collagen type I exerted on pre-osteoblasts were investigated. Collagen type I matrices were thermally denatured which lead to exposure of cryptic RGD (Arg-Gly-Asp)-motifs. As a consequence pre-osteoblasts enhanced their adhesion to denatured collagen. Compared to native collagen type I, adhesion to denatured collagen was mediated by a different set of integrins, including αv- and α5β1-integrins. Cells grown on denatured collagen showed enhanced spreading and motility, which correlated with increased focal adhesion kinase phosphorylation levels. Moreover, osteogenic differentiation kinetics and differentiation potential were increased on denatured collagen. The findings of this project open new perspectives for optimization of tissue engineering substrates. In the third part (chapter six), the effect of the fusion protein BCR/ABL, a hallmark of chronic myeloid leukemia, on adhesion of myeloid progenitor cells was studied. Adhesion between BCR/ABL transformed progenitor cells to bone marrow derived stromal cells and to different ECM proteins was quantitatively compared to that of control cells. The tyrosine kinase activity of BCR/ABL enhanced cell adhesion, which was blocked by imatinib mesylate, a drug interfering with BCR/ABL activity. BCR/ABL-enhanced adhesion correlated with increased β1-integrin cell surface concentrations. Since adhesion of leukemic cells to the bone marrow compartment is critical for the development of drug resistance, the reported results may provide a basis for optimized target therapies. In the three described projects AFM-based SCFS was applied to investigate early steps of integrin-mediated adhesion at the molecular level. Taken together, the results demonstrate that AFM-SCFS is a versatile tool that permits monitoring of cell adhesion from single-molecule interactions to the formation of more complex adhesion sites at the force level. / Interaktionen zwischen Zellen und ihrer Umgebung sind maßgeblich an der Regulierung zellulärer Funktionen beteiligt und daher notwendig für die Organisation von Zellen in Geweben und komplexen Organismen. Zellinteraktionen mit der extrazellulären Matrix (EZM) werden hauptsächlich durch Integrine vermittelt. Situationen, in denen Integrin- EZM Interaktionen verändert sind, können Krankheiten verursachen und spielen zudem eine wichtige Rolle bei der Invasion von Krebszellen. Daher besteht ein großes Interesse darin, die molekularen Mechanismen, die Integrin-EZM Interaktionen regulieren, besser zu verstehen. Wie können Zell-EZM Interaktionen untersucht werden? Obwohl es mehrere Methoden gibt, mit denen Zelladhäsion untersucht werden kann, sind die wenigsten dazu geeignet, Zelladhäsionskräfte zu quantifizieren. Einzelzellspektroskopie erfasst die Adhäsionskräfte einzelner Zellen quantitativ und ermöglicht dadurch eine differenzierte Betrachtung der Adhäsion individueller Zellen. Eine Variante der Einzelzellspektroskopie basiert auf der Rasterkraftmikroskopie (AFM); diese Technik wurde in der vorliegenden Arbeit verwendet. Ein Vorteil von AFM- Einzelzellspektroskopie besteht darin, dass Zellen mit hoher zeitlicher und räumlicher Präzision manipuliert werden können. Zelladhäsionskräfte können zudem über einen großen Kraftbereich hinweg untersucht werden. Dabei ermöglicht es die hohe Kraftauflösung, einzelne Integrin-Ligandenbindungen in lebenden Zellen zu untersuchen. Die vorliegende Arbeit gliedert sich in sechs Kapitel. Kapitel eins gibt Hintergrundinformationen über Zell-EZM Wechselwirkungen. In Kapitel zwei werden verschiedene Adhäsionsassays einander gegenüber gestellt. Das theoretische Bell-Evans Modell, mit dessen Hilfe die gewonnenen Daten interpretiert wurden, wird in Kapitel drei diskutiert. Im Anschluss werden drei Projekte, welche das Herzstück dieser Doktorarbeit bilden, in Kapiteln vier bis sechs näher ausgeführt. Im ersten Projekt (Kapitel vier) wurde die Adhäsion von α2β1-Integrin exprimierenden CHO Zellen zu Kollagen I, dem häufigsten strukturellen Protein in Wirbeltieren, quantitativ untersucht. Zunächst wurden α2β1-Kollagen-Interaktionen auf Einzelmolekülebene analysiert. Mithilfe der dynamischen Kraftspektroskopie wurden für diese Bindung Dissoziationsrate koff (1.3 ± 1.3 sec-1) und Potentialbarrierenbreite xu (2.3 ± 0.3 Å) bestimmt. Daraufhin wurde die α2β1-vermittelte Adhäsion über einen Zeitraum von zehn Minuten untersucht. Dadurch konnten Einblicke in die Kinetik von α2β1-integrin vermittelter Zelladhäsion sowie in die zugrunde liegenden Regulationsmechanismen gewonnen werden. Im zweiten Projekt (Kapitel fünf) wurde die Rolle von kryptischen Integrin-Bindungsstellen in Kollagen I untersucht. Die zuvor verwendeten Kollagenoberflächen wurden thermisch denaturiert, wodurch versteckte RGD (Arg-Gly-Asp)-Sequenzen freigelegt wurden. Die partielle Denaturierung hatte- verglichen mit nativem Kollagen I- eine erhöhte Adhäsion von Präosteoblasten (MC3T3-E1) zur Folge, was auf das Binden zusätzlicher Integrine zurückgeführt wurde. Im Unterschied zu nativem Kollagen wurde die Zelladhäsion zu denaturiertem Kollagen I u.a. durch αv- and α5β1-Integrine vermittelt. Präosteoblasten zeigten verstärktes Zellspreiten sowie höhere Motilität auf denaturiertem Kollagen I; zudem wurde ein erhöhtes Differenzierungpotential der Präosteoblasten festgestellt. Die in diesem Projekt erhaltenen Einblicke bilden eine hilfreiche Basis für die Entwicklung optimierter Oberflächen für diverse Zell- und Gewebekulturanwendungen. Im dritten Projekt (Kapitel sechs) wurde der Einfluss des Fusionproteins BCR/ABL, charakteristisch für chronische myeloische Leukämie, auf die Adhäsion von myeloischen Vorläuferzellen untersucht. Dazu wurde die Adhäsion von BCR/ABL transformierten Vorläuferzellen (32D Zellen) bzw. Kontrollzellen zu Stromazellen (M2-10B4) sowie verschiedenen EZM Proteinen untersucht. BCR/ABL erhöhte die Zelladhäsion der myeloischen Vorläuferzellen signifikant. Dieser Effekt wurde durch die Zugabe von Imatinib, welches die Tyrosinkinaseaktivität von BCR/ABL inhibiert, aufgehoben. Die BCR/ABL-verstärkte Zelladhäsion korrelierte mit erhöhten β1-Integrin-konzentrationen. Da die Adhäsion von Leukämiezellen im Knockenmark bekanntermaßen kritisch für die Entwicklung von Resistenzen gegenüber verschiedenen Wirkstoffen ist, könnten die Ergebnisse dieser Studie eine Grundlage für die Entwicklung optimierter Target-Therapien sein. In den drei beschriebenen Projekten wurde AFM Einzelzellspektroskopie verwendet, um Integrin- vermittelte Adhäsion auf molekularer Ebene zu untersuchen. Die Ergebnisse zeigen, dass AFM-Einzelzellspektroskopie ein vielseitiges Werkzeug darstellt, das überaus geeignet dazu ist, Zelladhäsion- ausgehend von Einzelmolekülinteraktionen bis hin zur Entstehung komplexerer Adhäsionsstellen- auf der Kraftebene zu verfolgen.

The aim of this pilot study was to evaluate the bioactive, surface-coated polycaprolactone-co-lactide scaffolds as bone implants in a tibia critical size defect model. Polycaprolactone-co-lactide scaffolds were coated with collagen type I and chondroitin sulfate and 30 piled up polycaprolactone-co-lactide scaffolds were implanted into a 3 cm sheep tibia critical size defect for 3 or 12 months (n¼5 each). Bone healing was estimated by quantification of bone volume in the defects on computer tomography and microcomputer tomography scans, plain radiographs, biomechanical testing as well as by histological evaluations. New bone formation occurred at the proximal and distal ends of the tibia in both groups. The current pilot study revealed a mean new bone formation of 63% and 172% after 3 and 12 months, respectively. The bioactive, surface coated, highly porous three-dimensional polycaprolactone-co-lactide scaffold stack itself acted as a guide rail for new bone formation along and into the implant. These preliminary data are encouraging for future experiments with a larger group of animals.

Follicular dendritic cells (FDCs) reside in primary B-cell follicles and in the light zones of germinal centers (GCs) in secondary follicles, where their dendrites interdigitate forming extensive networks intimately interacting with B-cells. In GCs, FDCs can be found at the edges attached to the supporting reticular fibers. They trap and arrange immune complexes (ICs) in vivo and in vitro in a periodic manner with 200–500Å spacing and provide both antigen-specific and non-specific accessory signals to B-cells. FDCs exist in resting and activated states, with two characteristically different phenotypes. In their activated state, FDCs upregulate the expression of accessory molecules and cytokines important in the FDC-B cell interaction in GCs. We sought to determine the mechanisms influencing the transition of FDCs from a resting to an activated state in GCs and their impact on T-cell dependent (TD) and independent (TI)-GC reactions (GCRs). We found that IC-FDC interactions via FDC-FcgammaRIIB induce the upregulation of FDC-FcgammaRIIB, -ICAM-1, and -VCAM-1, at both the protein and mRNA levels. We also reported for the first time the expression of TLR-4 on FDCs. Moreover, engagement of FDC-TLR4 with LPS activated NF-kappaB, up-regulated expression of important FDC-accessory molecules, including FcgammaRIIB, ICAM-1, and VCAM-1, and enhanced FDC accessory activity in promoting recall IgG responses. Moreover, IC-activated FDCs produced IL-6 and FDC-IL-6 promoted GCRs, somatic hypermutation (SHM) and IgG production. Further, we reported that binding of FDCs to collagen coated surfaces induced restoration of their dendritic processes and networks in vitro. In addition, we designed an FDC-supported in vitro model capable of induction and assessment of primary human antibody responses to protein antigens characterized by class-switching and affinity maturation. Uniquely, we generated TI immune responses to TD protein Ags in the complete absence of T cell help in vivo and in vitro. In the presence of FDC-associated second signals such as BAFF and C4BP, FDC- FcgammaRIIB-periodically trapped-ICs induced the production of Ag-specific IgM, GC-development and plasmablast-differentiation in anti-Thy-1-pretreated nude mice. Purified murine and human B cells cultured in vitro with IC-bearing FDCs also showed the production of antigen–specific IgM within just 48 h.

A alveolite é uma complicação pós-operatória de carácter inflamatório que acomete alvéolos de dentes recém-extraídos. A incidência dessa complicação varia de 1 a 4% e pode chegar a 30%. O objetivo deste estudo foi analisar os mecanismos biológicos envolvidos no processo de reparo de alvéolos intencionalmente infectados, em ratos; comparar diferentes modalidades de tratamento e correlacionar os resultados encontrados através de duas análises (microscópica e molecular). Foram utilizados 84 ratos, divididos nos grupos: I: alvéolo não infectado; II: alvéolo infectado sem nenhum tratamento; III: alvéolo infectado tratado com irrigação de solução de iodeto de sódio a 2% e peróxido de hidrogênio a 3% na proporção de 1:1; e IV: alvéolo infectado submetido à curetagem, irrigação com soro fisiológico e preenchimento com uma pasta à base de metronidazol. Os animais foram eutanasiados aos 6, 15 e 28 dias pós-operatório. Foi realizada a análise quantitativa da expressão de genes envolvidos no processo de reparo [colágeno tipo I (COL-I), fator de crescimento do endotélio vascular (VEGF), osteocalcina (OCN), fosfatase alcalina (ALP), runt-related transcription factor 2 (RUNX2) e fator de necrose tumoral alfa (TNF-\'alfa\')], através da RealTimePCR, correlacionando sua expressão com as características microscópicas observadas qualitativa e quantitativamente. Com base nos resultados da análise microscópica e molecular, podemos concluir que os marcadores RUNX2, OCN e TNF-\'alfa\' podem ser usados como indicadores para avaliar a neoformação óssea e a quantidade de infiltrado inflamatório em alveolite. Os marcadores ALP e VEGF não representaram adequadamente o que se observou microscopicamente. Embora o tratamento da alveolite com a pasta à base de metronidazol promova maior densidade de neoformação óssea aos 28 dias, não há diferenças entre os tratamentos. / Dry socket is an inflammatory postoperative complication that undertakes sockets of recently extracted teeth. The incidence of such complication varies from 1 to 4% and might reach up to 30%. The objective of this study was to analyze the biological mechanisms involved in the repair process of intentionally infected sockets in mice; compare different treatment conditions and correlate the results of two different analysis (microscopic and molecular). 84 mice were used in this study, divided according the following groups: I: uninfected socket; II: infected socket without any treatment; III: infected socket treated with irrigation of 2% sodium iodide and 3% hydrogen peroxide solution at 1:1 proportion; and IV: infected socket submitted to curettage, physiological saline solution irrigation and fulfillment with metronidazole base paste. The animals were killed at a postoperative period of 6, 15 and 28 days. A quantitative analysis was performed using a RealTimePCR to evaluate the genes expression involved [Collagen Type I (COL-I), vascular endothelial growth factor (VEGF), osteocalcin (OCN), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and tumor necrosis factor-alpha (TNF-\'alpha\')], in the repair process, correlating its expression with the microscopic characteristics observed in both qualitative and quantitative manner. Based in the results of the microscopic and molecular analysis, it can be concluded that the RUNX2, OCN and TNF-\'alpha\' markers can be used as indicators to evaluate the dry socket bone neoformation and inflammatory infiltrate quantity. The ALP and VEGF markers did not represented appropriately what was observed microscopically. Although the dry socket treatment with metronidazole base paste promotes an increase in the bone neoformation density at 28 days, no difference was found among the treatments.

Atresia de vias biliares (AVB) é uma hepatopatia colestática específica da criança, de etiologia desconhecida, com evolução para fibrose hepática precoce. AVB é a principal causa de cirrose na infância e principal indicação de transplante hepático pediátrico (Tx). Compreender os fatores envolvidos na progressão da fibrose é fundamental para estabelecer tratamentos efetivos nas hepatopatias crônicas. Identificar padrões histopatológicos associados ao prognóstico da AVB permitiria melhor planejamento dos centros de transplante e adequado aconselhamento familiar. OBJETIVO: Estabelecer padrões de marcadores histopatológicos e de imunofluorescência para colágenos em biópsias hepáticas iniciais e finais de pacientes com AVB submetidos a tratamento cirúrgico. Correlacionar esses marcadores com o prognóstico da doença, definido com base no tempo de evolução até realização do Tx. MÉTODO: Avaliação histológica de alterações biliares e fibrose hepática e histomorfometria da fibrose marcada por picrossírius e da deposição dos colágenos tipos I, III, IV e V marcados por imunofluorescência indireta (IF), em biópsias hepáticas iniciais e finais de 36 pacientes com AVB submetidos à hepatoportoenterostomia de Kasai (KPE) e ao Tx nos últimos 20 anos em nossa instituição. RESULTADOS: A mediana das idades de realização da KPE foi de 12,5 semanas (6-20) e do Tx foi de 27 meses (6-120). Reação ductular e malformação de placa ductal foram mais intensas nas biópsias iniciais (p < 0,05), enquanto fibrose hepática e ductopenia apresentaram padrão progressivo (p < 0,001), sem correlações com a idade de realização da KPE nem com o tempo de evolução até Tx. A morfometria da fibrose hepática marcada pelo picrossírius nas biópsias iniciais apresentou correlação positiva com a idade da KPE (p = 0,01), mas não com a idade do Tx (p = 0,24). A deposição perissinusoidal dos colágenos dos tipos III e V foi mais intensa nas biópsias iniciais (p < 0,01), enquanto os colágenos dos tipos I e IV apresentaram padrão de deposição progressiva (p < 0,01). Pacientes com maior deposição perissinusoidal de colágeno tipo I nas biópsias iniciais apresentaram curva de tempo de evolução até Tx sugerindo pior prognóstico (p = 0,04). CONCLUSÃO: Marcadores histopatológicos de alterações biliares, fibrose hepática e deposição de colágenos apresentaram características evolutivas distintas nas fases inicial e final da AVB, sem correlação com o tempo de evolução até Tx. A morfometria da deposição perissinusoidal de colágeno tipo I em biopsias iniciais marcadas por IF pode ser correlacionada ao tempo de evolução até Tx em pacientes com AVB operada / Biliary atresia (BA) is a specific cholestatic liver disease of unknown etiology that affects children and progresses to early hepatic fibrosis. BA is the main indication of pediatric liver transplantation (LTx). Understanding the factors involved in the progress of fibrosis is essential to establish effective treatment to chronic liver disease. Histopathological markers in liver biopsies could be useful to predict progression to end stage disease and to make it possible to improve planning in transplantation centers and parental orientation. OBJECTIVE: To establish histopathological or immunohistochemical markers in initial or final liver biopsies of BA patients and correlate those markers with prognosis, as defined by progression time lapse until LTx. METHOD: Histological analysis of multiple parameters of biliary alterations and morphometrical assessment of liver fibrosis were performed, besides indirect immunofluorescence assays (IF) for type I, III, IV and V collagens in initial and final liver biopsies of 36 patients with BA submitted to Kasai hepatoportoenterostomy (KPE) and LTx in the last 20 years in a single center. RESULTS: The median of the ages at KPE was 12.5 weeks (6-20) and at LTx was 27 months (6-120). Ductular reaction and ductal plate malformation were more severe in the initial biopsies (p < 0.05), while ductopenia and liver fibrosis were more severe in final biopsies (p < 0.001), though without correlation with age at KPE nor with progression time lapse until LTx. Morphometrical assessment of liver fibrosis marked by picrosirius red in initial biopsies demonstrated positive correlation with age at KPE (p = 0.01) but not with age at LTx (p = 0.24). The perisinusoidal deposition of type III and V collagens was more extended in the initial biopsies (p < 0.01), while type I and IV collagens deposition indicated progression (p < 0.01). Patients with large amounts of perisinusoidal type I collagen in the initial biopsies had worse progression time curve until LTx (p = 0.04). CONCLUSION: Biliary alterations, liver fibrosis and collagens deposition demonstrated distinctive progression findings in the initial or final phases of the BA, without prognostic correlation. Morphometrical assessment of perisinusoidal deposition of type I collagen by IF in the initial biopsies can be correlated with progression time until LTx in patients with post-surgical BA

A alveolite é uma complicação pós-operatória de carácter inflamatório que acomete alvéolos de dentes recém-extraídos. A incidência dessa complicação varia de 1 a 4% e pode chegar a 30%. O objetivo deste estudo foi analisar os mecanismos biológicos envolvidos no processo de reparo de alvéolos intencionalmente infectados, em ratos; comparar diferentes modalidades de tratamento e correlacionar os resultados encontrados através de duas análises (microscópica e molecular). Foram utilizados 84 ratos, divididos nos grupos: I: alvéolo não infectado; II: alvéolo infectado sem nenhum tratamento; III: alvéolo infectado tratado com irrigação de solução de iodeto de sódio a 2% e peróxido de hidrogênio a 3% na proporção de 1:1; e IV: alvéolo infectado submetido à curetagem, irrigação com soro fisiológico e preenchimento com uma pasta à base de metronidazol. Os animais foram eutanasiados aos 6, 15 e 28 dias pós-operatório. Foi realizada a análise quantitativa da expressão de genes envolvidos no processo de reparo [colágeno tipo I (COL-I), fator de crescimento do endotélio vascular (VEGF), osteocalcina (OCN), fosfatase alcalina (ALP), runt-related transcription factor 2 (RUNX2) e fator de necrose tumoral alfa (TNF-\'alfa\')], através da RealTimePCR, correlacionando sua expressão com as características microscópicas observadas qualitativa e quantitativamente. Com base nos resultados da análise microscópica e molecular, podemos concluir que os marcadores RUNX2, OCN e TNF-\'alfa\' podem ser usados como indicadores para avaliar a neoformação óssea e a quantidade de infiltrado inflamatório em alveolite. Os marcadores ALP e VEGF não representaram adequadamente o que se observou microscopicamente. Embora o tratamento da alveolite com a pasta à base de metronidazol promova maior densidade de neoformação óssea aos 28 dias, não há diferenças entre os tratamentos. / Dry socket is an inflammatory postoperative complication that undertakes sockets of recently extracted teeth. The incidence of such complication varies from 1 to 4% and might reach up to 30%. The objective of this study was to analyze the biological mechanisms involved in the repair process of intentionally infected sockets in mice; compare different treatment conditions and correlate the results of two different analysis (microscopic and molecular). 84 mice were used in this study, divided according the following groups: I: uninfected socket; II: infected socket without any treatment; III: infected socket treated with irrigation of 2% sodium iodide and 3% hydrogen peroxide solution at 1:1 proportion; and IV: infected socket submitted to curettage, physiological saline solution irrigation and fulfillment with metronidazole base paste. The animals were killed at a postoperative period of 6, 15 and 28 days. A quantitative analysis was performed using a RealTimePCR to evaluate the genes expression involved [Collagen Type I (COL-I), vascular endothelial growth factor (VEGF), osteocalcin (OCN), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and tumor necrosis factor-alpha (TNF-\'alpha\')], in the repair process, correlating its expression with the microscopic characteristics observed in both qualitative and quantitative manner. Based in the results of the microscopic and molecular analysis, it can be concluded that the RUNX2, OCN and TNF-\'alpha\' markers can be used as indicators to evaluate the dry socket bone neoformation and inflammatory infiltrate quantity. The ALP and VEGF markers did not represented appropriately what was observed microscopically. Although the dry socket treatment with metronidazole base paste promotes an increase in the bone neoformation density at 28 days, no difference was found among the treatments.

Atresia de vias biliares (AVB) é uma hepatopatia colestática específica da criança, de etiologia desconhecida, com evolução para fibrose hepática precoce. AVB é a principal causa de cirrose na infância e principal indicação de transplante hepático pediátrico (Tx). Compreender os fatores envolvidos na progressão da fibrose é fundamental para estabelecer tratamentos efetivos nas hepatopatias crônicas. Identificar padrões histopatológicos associados ao prognóstico da AVB permitiria melhor planejamento dos centros de transplante e adequado aconselhamento familiar. OBJETIVO: Estabelecer padrões de marcadores histopatológicos e de imunofluorescência para colágenos em biópsias hepáticas iniciais e finais de pacientes com AVB submetidos a tratamento cirúrgico. Correlacionar esses marcadores com o prognóstico da doença, definido com base no tempo de evolução até realização do Tx. MÉTODO: Avaliação histológica de alterações biliares e fibrose hepática e histomorfometria da fibrose marcada por picrossírius e da deposição dos colágenos tipos I, III, IV e V marcados por imunofluorescência indireta (IF), em biópsias hepáticas iniciais e finais de 36 pacientes com AVB submetidos à hepatoportoenterostomia de Kasai (KPE) e ao Tx nos últimos 20 anos em nossa instituição. RESULTADOS: A mediana das idades de realização da KPE foi de 12,5 semanas (6-20) e do Tx foi de 27 meses (6-120). Reação ductular e malformação de placa ductal foram mais intensas nas biópsias iniciais (p < 0,05), enquanto fibrose hepática e ductopenia apresentaram padrão progressivo (p < 0,001), sem correlações com a idade de realização da KPE nem com o tempo de evolução até Tx. A morfometria da fibrose hepática marcada pelo picrossírius nas biópsias iniciais apresentou correlação positiva com a idade da KPE (p = 0,01), mas não com a idade do Tx (p = 0,24). A deposição perissinusoidal dos colágenos dos tipos III e V foi mais intensa nas biópsias iniciais (p < 0,01), enquanto os colágenos dos tipos I e IV apresentaram padrão de deposição progressiva (p < 0,01). Pacientes com maior deposição perissinusoidal de colágeno tipo I nas biópsias iniciais apresentaram curva de tempo de evolução até Tx sugerindo pior prognóstico (p = 0,04). CONCLUSÃO: Marcadores histopatológicos de alterações biliares, fibrose hepática e deposição de colágenos apresentaram características evolutivas distintas nas fases inicial e final da AVB, sem correlação com o tempo de evolução até Tx. A morfometria da deposição perissinusoidal de colágeno tipo I em biopsias iniciais marcadas por IF pode ser correlacionada ao tempo de evolução até Tx em pacientes com AVB operada / Biliary atresia (BA) is a specific cholestatic liver disease of unknown etiology that affects children and progresses to early hepatic fibrosis. BA is the main indication of pediatric liver transplantation (LTx). Understanding the factors involved in the progress of fibrosis is essential to establish effective treatment to chronic liver disease. Histopathological markers in liver biopsies could be useful to predict progression to end stage disease and to make it possible to improve planning in transplantation centers and parental orientation. OBJECTIVE: To establish histopathological or immunohistochemical markers in initial or final liver biopsies of BA patients and correlate those markers with prognosis, as defined by progression time lapse until LTx. METHOD: Histological analysis of multiple parameters of biliary alterations and morphometrical assessment of liver fibrosis were performed, besides indirect immunofluorescence assays (IF) for type I, III, IV and V collagens in initial and final liver biopsies of 36 patients with BA submitted to Kasai hepatoportoenterostomy (KPE) and LTx in the last 20 years in a single center. RESULTS: The median of the ages at KPE was 12.5 weeks (6-20) and at LTx was 27 months (6-120). Ductular reaction and ductal plate malformation were more severe in the initial biopsies (p < 0.05), while ductopenia and liver fibrosis were more severe in final biopsies (p < 0.001), though without correlation with age at KPE nor with progression time lapse until LTx. Morphometrical assessment of liver fibrosis marked by picrosirius red in initial biopsies demonstrated positive correlation with age at KPE (p = 0.01) but not with age at LTx (p = 0.24). The perisinusoidal deposition of type III and V collagens was more extended in the initial biopsies (p < 0.01), while type I and IV collagens deposition indicated progression (p < 0.01). Patients with large amounts of perisinusoidal type I collagen in the initial biopsies had worse progression time curve until LTx (p = 0.04). CONCLUSION: Biliary alterations, liver fibrosis and collagens deposition demonstrated distinctive progression findings in the initial or final phases of the BA, without prognostic correlation. Morphometrical assessment of perisinusoidal deposition of type I collagen by IF in the initial biopsies can be correlated with progression time until LTx in patients with post-surgical BA

Synthetic biomaterials are constantly being developed and play central roles in contemporary strategies in regenerative medicine and tissue engineering as artificial extracellular microenvironments. Such scaffolds provide 2D- and 3D-support for interaction with cells and thus convey spatial and temporal control over their function and multicellular processes, such as differentiation and morphogenesis. A model fibrillar system with tunable viscoelastic properties, comprised of 2 native ECM components like collagen type I and the GAG heparin, is presented here. Although the individual components comply with the adhesive, mechanical and bioinductive requirements for artificial reconstituted ECMs, their interaction and structural characterization remains an intriguing conundrum. The aim of the work was to analyze and structurally characterize a xenogeneic in vitro cell culture scaffold reconstituted from two native ECM components, collagen type I and the highly negatively charged glycosaminoglycan heparin. Utilizing a broad spectrum of structural analysis it could be shown that pepsin-solubilized collagen type I fibrils, reconstituted in vitro in the presence of heparin, exhibit an unusually thick and straight shape, with a non-linear dependence in size distribution, width-to-length ratio, and morphology over a wide range of GAG concentrations. The experiments imply a pronounced impact of the nucleation phase on the cofibril morphology as a result of the strong electrostatic interaction of heparin with atelocollagen. Heparin is assumed to stabilize the collagen-GAG complexes and to enhance their parallel accretion during cofibrillogenesis, furthermore corroborated by the heparin quantitation data showing the GAG to be intercalated as a linker molecule with a specific binding site inside the cofibrils. In addition, the exerted morphogenic effect of the GAG, appears to be influenced by factors as degree of sulfation, charge, and concentration. Further detailed structural analysis of the PSC-heparin gels using TEM and SFM showed a hierarchy involving 3 different structural levels and banding patterns in the system: asymmetric segment longspacing (SLS) fibrils and symmetric segments with an average periodicity (AP) of 250 - 260 nm, symmetric fibrous longspacing (FLS IV) nanofibrils with AP of 165 nm, and cofibrils exhibiting an asymmetric D-periodicity of 67 nm with a striking resemblance to the native collagen type I banding pattern. The intercalation of the high negatively charged heparin in the cofibrils was suggested as the main trigger for the hierarchical formation of the polymorphic structures. We also proposed a model explaining the unexpected presence of a symmetric and asymmetric form in the system and the principles governing the symmetric or asymmetric fate of the molecules. The last section of the experiments showed that the presence of telopeptides and heparin both had significant effects on the structural and mechanical characteristics of in vitro reconstituted fibrillar collagen type I. The implemented structural analysis showed that the presence of telopeptides in acid soluble collagen (ASC) impeded the reconstitution of D-periodic collagen fibrils in the presence of heparin, leaving behind only a symmetric polymorphic form with a repeating unit of 165 nm (FLS IV). Further x-ray diffraction analysis of both telopeptide-free and telopeptide-intact collagen fibrils showed that the absence of the flanking non-helical termini in pepsin-solubilized collagen (PSC) resulted in a less compact packing of triple helices of atelocollagen with an increase of interhelical distance from 1.0 to 1.2 nm in dried samples. The looser packing of the triple helices was accompanied by a decrease in bending stiffness of the collagen fibrils, which demonstrated that the intercalated heparin cannot compensate for the depletion of telopeptides. Based on morphological, structural and mechanical differences between ASC and PSC-heparin fibrils reported here, we endorsed the idea that heparin acts as an intrafibrillar cross-linker which competed for binding sites at places along the atelocollagen helix that are occupied in vivo by telopeptides in the fibrillar collagen type I. The performed studies are of particular interest for understanding and gaining control over a rather versatile and already exploited xenogeneic cell culture system. The reconstituted cofibrils with their unusual morphology and GAG intercalation – a phenomenon not reported in vivo – are expected to exhibit interesting biochemical behavior as a biomaterial for ECM scaffolds. Varying the experimental conditions, extent of telopeptide removal, and heparin concentration provides powerful means to control the kinetics, structure, dimensions, as well as mechanical properties of the system which is particularly important for predicting a certain cell behavior towards the newly developed matrix. The GAG intercalation could be interesting for studies with required long-term 'release upon demand' of the GAG, as well as native binding and stabilization of growth factors, cytokines, chemokines, thus providing a secondary tool to control cell signaling and fate, and later on tissue morphogenesis. / Synthetische Biomaterialien werden stetig weiterentwickelt und spielen als künstliche Mikroumgebungen eine zentrale Rolle in den modernen Strategien der regenerativen Medizin und des Tissue Engineerings. Solche sogenannten Scaffolds liefern eine 2D- und 3D-Struktur zur Interaktion mit Zellen und üben somit eine räumliche und zeitliche Kontrolle auf ihre Funktion und multizelluläre Prozesse aus, wie die Differenzierung und Morphogenese. Obwohl häufig die adhäsiven, mechanischen und bioinduzierenden Eigenschaften von Einzelkomponenten aus natürlichen Bestandteilen der extrazellulären Matrix (ECM) rekonstituierten Trägerstrukturen bekannt sind, bleiben die funktionalen und strukturellen Auswirkungen in Mehrkomponentensystemen eine faszinierende Fragestellung. Das Ziel der Arbeit war die Analyse und die strukturelle Charakterisierung einer xenogenen in vitro Zellkultur-Trägerstruktur, die aus den zwei nativen ECM Komponenten Kollagen Typ I und das stark negativ geladene Glykosaminoglykan (GAG) Heparin rekonstituiert wurde. Unter Nutzung eines breiten Spektrums von Methoden zur strukturellen Analyse konnte gezeigt werden, dass im Beisein von Heparin rekonstituierte Pepsin-gelöste Kollagen Typ I Fibrillen eine ungewöhnlich dicke und gerade Form, mit nichtlinearen Abhängigkeiten der Größenverteilung, des Breite-zu-Länge Verhältnises und der Morphologie für eine Reihe von GAG Konzentrationen, aufweisen. Die Experimente deuten auf eine besondere Wirkung der Nukleierungsphase auf die Kofibrillmorphologie hin, als Folge der starken elektrostatischen Inteaktionen Heparins mit Atelokollagen. Es wird angenommen, dass Heparin die Komplexe aus Kollagen-GAG stabilisiert, die parallele Anlagerung während der Kofibrillogenese verbessert und dass überdies, belegt durch Heparin Quantitätsdaten, als Verbindungsmolekül mit einer spezifischen Anbindungsstelle innerhalb der Kofibrillen eingelagert wird. Darüber hinaus scheint der ausgeübte morphogene Effekt des GAGs Heparins von Faktoren wie Grad der Sulfatierung, Ladung und Konzentration abzuhängen. Weitere detailierte Strukturanalysen der PSC - Heparin Gele mit TEM und SFM zeigten eine Hierarchie mit drei unterschiedlichen strukturellen Ebenen und Bandmustern im System: asymmetrisch segmentierte, weitabständige Fibrillen (SLS) und symmetrische Segmente mit einem AP von 250-260 nm, symmetrische fibrose weitabständige (FLS IV) Nanofibrillen mit einem AP von von 165 nm und Kofibrillen asymmetrischer D-Periodizität von 67 nm, die eine erstaunliche Ähnlichkeit zum natürlichen Kollagen Typ I Bandmuster haben. Die Einlagerung des sehr negativ geladenen Heparins in die Kofibrillen wurde als Hauptauslöser der hierarchischen Formation der polymorphen Strukturen betrachtet. Wir schlugen ebenso ein Model vor, welches sowohl das unerwartete Vorhandensein symmetrischer und asymmetrischer Formen im System als auch die Regeln erklärt, die das symmetrische oder asymmetrische Schicksal der Moleküle steuern. Der letzte Abschnitt der Experimente zeigte, dass die Anwesenheit der Telopeptide und Heparins eine signifikante Wirkung auf die strukturellen und mechanischen Charakteristika der in vitro rekonstituierten Kollagen Typ I Fibrillen hatte. Die durchgeführten Strukturanalysen zeigten außerdem, dass die Anwesenheit der Telopeptide in säurelöslichem Kollagen (ASC) die Rekonstitution D-periodischer Kollagenfibrillen mit Heparin verhinderte, sodass nur symmetrisch polymorphe Formen mit einer Wiederholeinheit von 165 nm möglich waren (FLS IV). Weitere Messungen der Telopeptid-freien und Telopeptid-intakten Kollagenfibrillen mit Röntgendiffraktometrie ergaben, dass die Abwesenheit der nicht-helix-strukturierten Enden in Pepsin-gelöstem Kollagen (PSC) zu einer weniger kompakten Anordnung der Tripelhelices von Atelokollagen führte. Der interhelix Abstand erhöhte sich von 1,0 zu 1,2 nm für getrocknete Proben. Das zeigt, dass die losere Anordnung der Tripelhelices einhergeht mit der Verringerung der Biege-Elastizitäts-module der Kollagenfibrillen,. Basierend auf den hier vorgestellten morphologischen, strukturellen und mechanischen Unterschieden zwischen ASC und PSC-Heparin Fibrillen wird die Idee unterstützt, dass Heparin als intrafibrillärer Vernetzer fungiert und an Bindungsstellen der Helix bindet, welche in vivo bei Kollagen Typ I Fibrillen durch Telopeptide besetzt sind. Die durchgeführten Studien sind von besonderem Interesse für das Verständnis und die Steuerung eines sehr vielseitigen und bereits verwendeten xenogenes Zellkultursystem für das Tissue Engineering. Von den rekonstituierten Kofibrillen mit ihrer ungewöhnlichen Morphologie und GAG Einlagerung - ein in vivo nicht bekanntes Phänomen - erwartet man, dass sie ein intressantes biochemisches Verhalten als Biomaterial für ECM Scaffolds zeigen. Variationen der experimentellen Bedingungen, des Ausmaßes der Telopeptidentfernung und der Heparinkonzentration liefern vielfältige Möglichkeiten um die Kinetik, Struktur, Dimension sowie die mechanischen Eigenschaften des Systems zu kontrollieren. Damit sollte es möglich sein, ein bestimmtes Zellverhalten gegenüber der neu entwickelten Matrix vorherzusagen. Die GAG-Einlagerung bietet interessante Optionen für eine langfristige Freisetzung des GAGs 'on demand', sowie die native Bindung und Stabilisierung von Wachstumsfaktoren, Cytokinen, Chemokinen, womit zusätzlich Zellsignalisierung und -schicksal und später Gewebemorphogenese kontrolliert werden kann.

Introdução: A obstrução infravesical (OIV) de longo prazo secundária a hiperplasia prostática benigna (HPB) pode causar alterações funcionais e morfológicas na bexiga. Um dos principais eventos consiste no aumento da deposição de colágeno e perda de complacência vesical, levando a alteração de armazenamento e esvaziamento urinário. O aumento da deposição de colágeno na matriz extracelular (MEC) da musculatura detrusora é a principal razão para a diminuição da complacência vesical. Na bexiga, assim como em outros órgãos, este fenômeno depende da atividade equilibrada de enzimas proteolíticas, incluindo as metaloproteinases (MMP) e os seus inibidores endógenos (inibidores teciduais de metaloproteinases-TIMPs). Como estes fenômenos são desconhecidos na bexiga obstruída, o objetivo deste estudo foi avaliar a expressão gênica de colágeno, MMPs e seus inibidores na bexiga de pacientes com obstrução infravesical. Material e Métodos: Foi realizada uma análise prospectiva e controlada de 43 pacientes com OIV devido a HPB, que foram submetidos à ressecção transuretral da próstata (RTUP) entre 2011 e 2012. Como grupo controle foram selecionados espécimes de músculo detrusor de 10 pacientes que foram submetidos a prostatectomia radical retropúbica devido adenocarcinoma de próstata. Todos estes pacientes tinham idade menor que 60 anos, tamanho de próstata menor que 30 gramas ao ultra-som e escore internacional de sintomas prostáticos (IPSS) menor que 7. Todos os pacientes foram submetidos a estudo urodinâmico pré e pós operatório (após 6 meses). A biópsia de fragmento de músculo da bexiga foi realizada ao final da RTUP e colocada em solução estabilizadora de RNA para quantificação da expressão de colágenos I e III, metaloproteinases de matriz 1, 2 e 9, e inibidores de MMPs (TIMP1, TIMP2 e RECK) na bexiga de pacientes com HPB. Os genes descritos foram avaliados através da técnica de reação em cadeia da polimerase quantitativa em tempo real (qRT-PCR). Resultados: Todos os pacientes com HPB tinham confirmado OIV, através da análise do estudo urodinâmico (média de pressão detrusora no fluxo máximo de 78,5 cmH2O e fluxo urinário máximo de 7,7 ml / s). O gene MMP1 mostrou-se superexpresso em pacientes com HPB (mediana = 1,87). MMP9, TIMP1 e RECK estavam subexpressos na maioria dos casos, enquanto TIMP2, colágeno I e III foram superexpressos (1,5, 4,4 e 1,9 vezes, respectivamente). No que diz respeito às características clínicas e urodinâmicas encontramos que MMP2 foi mais expresso entre pacientes com um baixo IPSS global (0,005) e sem urgência (p=0,035). Colágeno III foi mais expresso em pacientes com contrações vesicais não inibidas (p = 0,049). Os outros genes não mostraram nenhuma correlação estatística com quaisquer características clínicas ou urodinâmicas. Após 6 meses de RTU, pacientes que possuíam expressão aumentada de duas ou mais MMPs, apresentaram resolução da CNI em 66,6% dos casos, contra 14,0% quando apenas uma ou nenhuma MMP estava aumentada (p=0,038) Conclusões: Encontramos um perfil de superexpressão de MMP1, TIMP2, colágenos I e III, e expressão baixa de MMP9, TIMP1 e RECK nos pacientes com OIV. Considerando o escore de sintomas prostáticos e a urgência miccional, encontramos curiosamente uma maior expressão de MMP2 em pacientes menos sintomáticos e sem urgência miccional. Encontramos uma associação entre a maior expressão de colágeno III com HD. A expressão aumentada de duas ou mais MMPs está relacionada à maiores taxas de resolução das CNIs. / Introduction: Long-term Bladder outlet obstruction (BOO) secondary to Benign prostatic Hyperplasia (BPH) can cause functional and morphological abnormalities in the bladder, such as increased collagen deposition and loss of compliance, leading to urinary storage and voiding symptoms. A decrease in bladder compliance is known to be correlated with deterioration of renal function. Increased deposition of collagen in the extracellular matrix (ECM) is the primary reason for a decreased compliance. In the bladder, as in other organs, this phenomenon is dependent on the balanced activity of proteolytic enzymes, including matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs). The imbalance between MMPs and TIMPs is a key regulator in ECM turnover. Since these mechanisms are unknown in the obstructed bladder, the objective of this study was to evaluate gene expression of collagen, MMPs and their inhibitors in patients with bladder outlet obstruction due to BPH. Material and Methods: We performed a prospective and controlled analysis of 43 patients with BOO due to BPH who underwent transurethral resection of the prostate (TURP) from 2011 to 2012. The control group was comprised of 10 bladder specimens from patients with < 60 years who underwent radical prostatectomy with an International Prostatic Symptom Score (IPSS) < 8 and prostate volume < 30 grams. All patients underwent urodynamic analysis pre and post operatively after 6 months. A biopsy of the bladder muscle was performed at the end of TURP for analysis of collagen, metalloproteinases and TIMPs gene expressions. For this purpose we used the quantitative real time polymerase chain reaction method (qRT-PCR). Results: All patients with BPH had confirmed BOO confirmed through urodynamic analysis (mean detrusor pressure at maximun flow 78.5 cmH2O and mean maximun flow 7.7 ml/s). MMP1 gene showed an important an overexpression in patients with BPH (median = 1.87). A similar phenomenon occurred in a lesser extent to MMP2, to which 13 of 23 subjects had under-expression (mean = 1.2). MMP9, TIMP1 and RECK were under-expressed in the majority of cases, while TIMP2, colagen I and III were over-expressed (1.5, 4.4 and 1.9x respectively) (figure). With regard to clinical and urodynamic characteristics we found that MMP2 was more over-expressed among patients with a low global IPSS (0.005) and without urgency (p=0.035). Colagen III was more over-expressed in patients with non-inhibited bladder contractions (p=0.049), RECK was more over-expressed in patients with a decreased complacence (p=0.049). The other genes showed no statistical correlation with any clinical or urodynamic characteristics. After 6 months of TURP, patients with non-inhibited bladder contractions showed resolution in 66.6% of cases, when had increased expression of two or more (> 02) MMPs in patients compared with 14.0% when only 01 MMP was increased (p = 0.038) Conclusions: BOO is related with an over-expression of MMP1, TIMP2, colagens I and III, and with an under-expression of MMP9, TIMP1 and RECK. Detrusor overactivity is related with higher collagen III expression, this fact may be due to a lower MMP1 expression. A lower global IPSS and no urgency were related to a higher expression of MMP2, sugesting that this gene may be inhibiting collagen deposition in the bladder. The increased expression of two or more MMPs isrelated to greater rates of resolution of non-inhibited bladder contractions