Die Knochenregeneration während der Frakturheilung beinhaltet das Zusammenspiel von Wachstumsfaktoren. In einigen Patienten kommt es zu einer verzögerten oder unvollständigen Heilung. Die Gründe hierfür sind bisher nicht komplett verstanden. Neutralisierende Autoantikörper (aAB) gegen Wachstumsfaktoren oder deren Rezeptoren könnten den Heilungsprozess verzögern und potentiell beeinträchtigen In dieser Arbeit wurden 265 Frakturpatienten analysiert. Autoantikörper gegen IGF1 Rezeptor, Insulin Rezeptor, BMP7, BMP2, IGF1 und (Pro)Insulin wurden in den Seren dieser Frakturpatienten gemessen. In Frakturpatienten wurden in 5% der Seren aAB gegen den IGF1R und in 6% gegen den IR gefunden. Das Auftreten von IGF1R- und IR-aAB wurde nicht induziert und war nicht mit dem Heilungsergebnis assoziiert. BMP7-aAB wurden in 1-2,5% gesunder Probanden und Frakturpatienten, die nicht mit rhBMP7 behandelt wurden, detektiert. Patienten, die mit rhBMP7 behandelt wurden, zeigten ein höheres Auftreten der BMP7-aAB Positivität mit 6% zum Zeitpunkt der Operation und 18% vier Wochen nach der Operation. BMP2-aAB wurden in 2% der gesunden Kontrollen und 6% der mit rhBMP7-behandelten Frakturpatienten entdeckt. Bei der Charakterisierung des biologischen Effekts der BMP7-aAB durch einen zell-basierten Reporter-Assay, zeigte sich ein neutralisierender Effekt in Proben mit hohem BMP7-aAB Titer. Als das wichtigste Kriterium für klinische Relevanz wurde die Konsolidierung untersucht. Das Vorhandensein von BMP-aAB wurde nicht signifikant mit der Konsolidierung in Zusammenhang gebracht. Zusammenfassend wurden neue diagnostische Assays zur Detektion von aAB gegen Wachstumsfaktoren und deren Rezeptoren generiert und angewandt um aAB in Seren von Frakturpatienten zu messen. Keiner der identifizierten aAB war negativ mit dem Heilungsprozess assoziiert. Bedenken bezüglich der Sicherheit von rhBMP7 Behandlungen sind berechtigt, da die Anwendung aAB gegen BMP7 induziert, die den BMP7-Signalweg blockieren. / Regeneration of bone during fracture healing includes concerted actions of growth factors. Some fractures show delayed healing or non-union due to as yet unknown reasons. Neutralizing autoantibodies (aAB) against growth factors or their receptors might influence and potentially impair the bone healing capacity. In this study, a cohort of 265 fracture patients with different treatment regimen and healing outcomes were analysed. Autoantibodies against IGF1 receptor, insulin receptor, BMP7, BMP2, IGF1 and (pro)insulin were measured in sera of these fracture patients. The prevalence of aAB against IGF1R and IR was 5% and 6% in fracture patients, respectively. The appearance of IGF1R- and IR-aAB was not induced by the surgical intervention and was unrelated to the healing outcome. BMP7-aAB were found in 1-2.5% of healthy subjects and in fracture patients that were not treated with rhBMP7. Patients that had received rhBMP7 treatment showed a higher incidence of BMP7-aAB positivity of 6% at surgery and 18% four weeks post surgery. BMP2-aAB were found in 2% of healthy controls and 6% of the fracture patients that were treated with rhBMP7. Characterizing the biological effect of BMP7-aAB in a cell-based reporter assay, a neutralizing effect was observed for samples with high titres. As the most relevant clinical outcome, the criterion consolidation was analysed defining whether the fracture gap was closed after six months or not. The presence of BMP-aAB was not significantly associated with the healing outcome. In summary, novel diagnostic assays for the detection and quantification of growth factor and receptor aAB were generated and used to determine aAB in sera from fracture patients. None of the identified aAB were negatively associated with the regeneration process or healing outcome. Ongoing concerns regarding the safety of rhBMP7 treatment are justified as the biological treatment induces aAB against BMP7 that block the BMP signal transduction.

published_or_final_version / Anatomy / Master / Master of Philosophy

A desmineralização óssea superficial tem se demonstrado favorável à consolidação de enxertos e ao comportamento celular, entretanto os mecanismos envolvidos ainda não estão esclarecidos. Os subsídios para o embasamento biológico da desmineralização, proporcionado por publicações anteriores, sugeriram que modificações na superfície óssea teriam influenciado o comportamento de pré-osteoblastos em cultura. Assim, este estudo objetivou comparar o efeito de duas concentrações de ácido cítrico na desmineralização de superfícies ósseas onde foram cultivadas células pré-osteoblásticas (MC3T3-E1), e analisar parâmetros de superfície comparando superfícies desmineralizadas a não desmineralizadas. Setenta amostras ósseas bicorticais foram removidas das calvárias de 35 ratos e divididas em grupos para as análises: 1) Microscopia Eletrônica de Varredura (MEV) para avaliação da área de recobrimento e espessura da camada de células sobre as amostras (n = 15) durante 24, 48 e 72 horas: Grupo AC.10 amostras desmineralizadas por 30 segundos com ácido cítrico 10 %; Grupo AC.50 amostras desmineralizadas por 30 segundos com ácido cítrico 50 %; e Grupo C (controle) amostras não desmineralizadas; 2) Microscopia Confocal para análise da área de expressão e intensidade de fluorescência das BMP-2, -4 e -7: AC.10 seis amostras desmineralizadas conforme item 1); AC.50 seis amostras desmineralizadas conforme item 1); C três amostras não desmineralizadas; 3) Microscopia Confocal para análise da rugosidade superficial média (Ra e Sa): Grupos AC.10 e AC.50 com cinco amostras cada, desmineralizadas conforme o item 1), sendo cada amostra seu próprio controle (análises antes e depois da desmineralização). Também foram avaliadas as distâncias entre picos (P-P) e entre picos e vales (P-V) antes e depois da desmineralização; 4) Microscopia Eletrônica de Varredura / Espectroscopia de Energia Dispersiva (MEV / EDS) para análise da composição superficial: mesmas amostras do item 3) foram avaliadas antes e depois da desmineralização quanto à porcentagem atômica (%A) de carbono, oxigênio, magnésio, fósforo, enxofre e cálcio. Análises estatísticas foram feitas adotando nível de significância de 95 %. Amostras desmineralizadas apresentaram células morfologicamente em estágios mais avançados de diferenciação do que as não desmineralizadas. A área de recobrimento superficial foi significantemente maior após 24 horas de cultura nos grupos teste do que no controle e a espessura da camada de células também foi maior nos grupos teste às 48 e 72 horas. Houve significantemente maior expressão de BMP-2 e -7 nos grupos teste do que no controle e, apenas AC.10 demonstrou maior expressão de BMP-4 do que os demais grupos, sem significância em relação a AC.50. Os parâmetros de superfície Ra e Sa foram inconclusivos, mas P-P e P-V diminuíram consideravelmente após a desmineralização para distâncias compatíveis com superfícies favoráveis à adesão e diferenciação celular. A análise da composição química superficial revelou diminuição da %A de enxofre e magnésio nos grupos teste. A concentração do ácido, embora não tenha apresentado diferença significante para a maioria das análises, pareceu ter influência positiva nos resultados para o ácido cítrico 10 %. Concluiu-se que a desmineralização superficial parece promover a proliferação e diferenciação celular, proporcionando superfícies com características de composição e topografia que favorecem o comportamento celular verificado. / The superficial bone demineralization has proved to be a favorable procedure for bone grafts consolidation and cell behavior, however the underlying mechanisms have not been clarified yet. Therefore, this study aimed to compare the effect of two concentrations of citric acid on demineralization of bone surfaces where pre-osteoblastic cells (MC3T3-E1) were cultivated, and analyze surface parameters comparing demineralized bone surfaces with non-demineralized surfaces. Seventy bicortical bone samples were harvested from the calvaria of 35 rats and divided into groups as follows: 1) Scanning Electron Microscopy (SEM) to evaluate the coating area and thickness of cells layers cultured on the samples (n = 15) for 24, 48, and 72 hours: Group CA.10 samples demineralized for 30 seconds with 10 % citric acid; Group CA.50 samples demineralized for 30 seconds with 50 % citric acid, and Group C (control) non-demineralized samples; 2) Confocal Microscopy for analysis of expression area and intensity of fluorescence of BMP-2, -4, and -7: CA.10 six samples demineralized as item 1); CA.50 six samples demineralized as item 1); Group C three non-demineralized samples; 3) Confocal Microscopy for surface mean roughness analysis (Ra and Sa): Groups CA.10 and CA.50 made up of five samples each and demineralized according to item 1), each sample was its own control (analysis before and after demineralization). The distances between peaks (P-P) and between peaks and valleys (P-V) were also evaluated before and after demineralization; 4) Scanning Electron Microscopy / Energy dispersive Spectroscopy (SEM / EDS) to analyze the surface composition: the same samples of item 3) were evaluated before and after demineralization for atomic percentage (%A) of carbon, oxygen, magnesium, phosphorus, sulfur and calcium. Statistical test was made by adopting the 95 % significance level. Demineralized samples showed cells with morphology in the later stages of differentiation than non-demineralized ones. The coating surface area by cells was significantly higher after 24 hours of culture in the test groups than in the control and the thickness of the layers were also greater in the test groups at 48 and 72 hours of evaluation. There was significantly higher expression of BMP-2 and -7 in test groups than in the control group, and only the CA.10 group showed higher BMP-4 expression than the other groups, but the difference was not statistically significant compared to the CA.50 group. Ra and Sa surface parameters were inconclusive, however P-P and P-V decreased considerably after demineralization to distances compatible with surfaces favorable to cell adhesion and cell differentiation. The chemical composition analysis of the surfaces revealed a decrease in the %A for sulfur and magnesium in test groups. Although the acid concentration did not shown significant difference for most analysis, it seemed to have a positive influence for the results with citric acid 10 %. It was concluded that the surface demineralization seems to promote cell proliferation and differentiation, providing surfaces with composition and topography that can favor observed cell behavior.

Orientador: Eduardo Hochuli Vieira / Banca: Osvaldo Magro Filho / Banca: Idelmo Rangel Garcia Júnior / Banca: Michel Reis Messora / Banca: Thallita Pereira Queiroz / Resumo: OBJETIVOS: A proposta deste estudo foi avaliar a expressão de proteínas que participam da fase de osteoindução (VEGF, BMP-2 e CBFA1) durante o processo de regeneração óssea de defeitos criados em calvária de ratos e preenchidos com enxerto autógeno em bloco. MATERIAIS E MÉTODOS: Para o presente estudo foram utilizados 10 ratos adultos machos (Rattus norvegicus albinus, Wistar) que receberam dois defeitos ósseos de 5 mm cada, em calvária. Os defeitos ósseos constituiram dois grupos experimentais (n=10): Grupo controle (CONT) (defeitos preenchidos com o próprio coágulo); Grupo enxerto (ENX) (defeitos preenchidos com osso autógeno removido do defeito contralateral). Os animais foram submetidos a eutanásia nos períodos de 7 e 30 dias pós-operatórios. RESULTADOS: A análise quantitativa demonstrou formação óssea significativamente maior no Grupo ENX, no entanto, a presença das proteínas estudadas foi significativamente maior no Grupo CONT em ambos os períodos de observação. CONCLUSÃO: O enxerto ósseo autógeno cortical em bloco não expressou de forma significativa as proteínas osteoindutoras estudadas durante o processo de reparo / Abstract: AIMS: The proposal of this study was to evaluate the expression of proteins that act in osteoinduction (VEGF, BMP-2, CBFA1) phase during the bone defects regeneration created in rat calvaria and filled with autogenous bone graft in block. METHODS: For the present study, 10 adult male rats (Rattus norvegicus albinus, Wistar) had two 5mm- bone defects in calvaria. Bone defects constituted two experimental groups: CONTROL Group (defects filled with blood clot); GRAFT Group (defects filled with bone graft). Animals were sacrificed at 7 and 30 days post operative. RESULTS: Quantitative analysis showed significantly higher bone formation in Graft Group, however, the presence of studied proteins was significantly higher in Control Group in both observation periods. CONCLUSION: Autogenous cortical bone graft in block did not express the studied osteoinductive proteins during bone repair / Doutor

Modifications of biomaterial surface properties are employed to increase osteoblast differentiation and bone formation. Microtextured metallic surfaces promote osteoblast differentiation and high surface energy- achieved by controlling surface hydrocarbon contamination- increases osteoblast differentiation and peri-implant bone formation. Recombinant human bone morphogenic protein 2 (BMP2) is approved to induce bone formation in a number of applications. It is used clinically in combination with biomaterials to improve peri-implant bone formation and osseointegration. The amount of BMP2 that is required is large and inflammatory (swelling/seroma) and bone-related (ectopic bone/bone resorption) complications have been reported after BMP2 treatment. The aim of this study was to examine potential deleterious effects of BMP2 on the inflammatory environment and apoptosis of osteoblasts. Surface roughness and energy decreased pro-inflammatory interleukins and increased anti-inflammatory interleukins. In contrast, BMP2 abolished the surface effect, increasing pro-inflammatory interleukin (IL) 6, IL8, and IL17 in a surface roughness-dependent fashion and decreasing anti-inflammatory IL10 on rough surfaces. 5Z-7-Oxozeaenol and Dorsomorphin, but not H-8, blocked the effect of BMP2 on IL1A expression. There was an increase in expression of IL6 when treated with BMP2 for the control and H-8 groups, but both 5Z-7-Oxozeaenol and Dorsomorphin blocked the effect. Both 5Z-7-Oxozeaenol and H-8 blocked the effect of BMP2 on IL10 expression. BMP2 treatment had little effect on apoptosis in human mesenchymal stem cells (MSCs). Exogenous BMP2 had no effect on TUNEL. Caspase-3 activity was increased only at 200ng/ml BMP2. BAX/BCL2 decreased in MSCs treated with 50 and 100ng/ml BMP2. In contrast, BMP2 increased caspase-3 activity and TUNEL at all doses in normal human osteoblasts (NHOst). BAX/BCL2 increased in NHOst treated with BMP2 in a dose-dependent manner. Cells treated with 200 ng/ml BMP2 had an 8-fold increase in BAX/BCL2 expression in comparison with untreated cells. Similarly, BMP2 increased DNA fragmentation in NHOst cells. The BMP2-induced increase in DNA fragmentation was eliminated by 5-Z7-Oxozeaenol and Dorsomorphin. The results suggest that while surface features modulate an initial controlled inflammatory response, the addition of BMP2 induces a pro-inflammatory response. The effect of BMP2 on apoptosis depends on cell maturation state, inducing apoptosis in committed osteoblasts. BMP2 together with microtextured orthopaedic and dental implants may increase inflammation and possibly delay bone formation. Dose, location, and delivery strategies are important considerations in BMP2 as a therapeutic and must be optimized to minimize complications.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 180-205). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 180-205). Also available online.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences