TiO2 nanotubes grown on titanium alloy are known to increase the biocompatibility of the alloy when used in dental/orthopaedic implants. Furthermore, their nanotubular structures can act as antibacterial agent carrier and as a scaffold for tissue engineering with the aim of adding antibacterial properties to the implant. This study aims at fabricating an antibacterial and biocompatible nanocomposite coating on Ti-6Al-4V involving nano-ZnO and nano-Ag. Initially, TiO2 nanotubes were self-assembled on the polished surface of medical grade Ti-6Al-4V alloy discs using anodisation. First silver nanoparticles were chemically reduced from silver ammonia using delta-δ-gluconolactone for different duration on the nanotubes to form TiO2-Ag composite coating. Nano HA was added to the latter coating with the aim of reducing toxicity from silver, hence forming TiO2-Ag-HA coating. Secondly, nano-ZnO was thermo-chemically grown on the TiO2 nanotubes using zinc nitrate and hexamethylenetetramine. They were then annealed at 350-550 oC hence forming TiO2-ZnO. HA was grown on the latter coating by a biomimetic method whereby the coated discs were placed in a concentrated simulated body fluid at 37 oC forming TiO2-ZnO-HA. The stability of the 4 coatings, TiO2-Ag, TiO2-Ag-HA, TiO2-ZnO and TiO2-ZnO-HA were assessed using the dialysis method (n=3 each) and then exposed to S.aureus for 24 hours in BHI broth. Their antibacterial properties were assessed using different assays and microscopic imaging with respect to different controls (n=6 each for assays and n=3 for imaging). Their biocompatibility properties were assessed in the presence of primary human osteoblast cells in DMEM media with the help of biochemical assays, molecular gene expression and microscopic imaging (n=3). Both silver and zinc coated nanotubes showed significant level of antibacterial properties with silver coating being more bactericidal than the coating containing zinc. Nonetheless, the zinc oxide coatings were more biocompatible than the silver coating. Nano silver and zinc oxide containing composite coatings were successfully synthesised and tested in the presence of bacteria and human cells. The final conclusion was that nano-silver was still toxic and nano-ZnO coatings were more biocompatible.

In vivo, les cellules osseuses évoluent dans un microenvironnement complexe, tridimensionnel et interagissent avec celui-ci à de nombreuses échelles, depuis le nanomètre (tropocollagène) jusqu’à des structures de plusieurs centaines de micromètres (trabécules). Paradoxalement, la majeure partie de nos connaissances sur la physiologie cellulaire est issue d’expériences réalisées sur des cellules cultivées sur du plastique et en deux dimensions. Ces différences ne peuvent qu’avoir une influence sur le comportement des cellules, qui n’entretiennent plus les mêmes relations spatiales entre elles, ainsi qu’avec leur environnement. De plus, si ces dernières années, nombre d’études ont été réalisées sur l’influence de la topographie à des échelles nano et micrométriques, peu d’études ont montré le rôle de la géométrie du substrat à une échelle tissulaire, soit au sein de structures supérieures à 100 µm. Afin d’étudier l’influence de la macroarchitecture du substrat sur le comportement cellulaire, des céramiques en hydroxyapatite à architecture contrôlée ont été ensemencées avec des cellules primaires de calvaria de souris. Une première étude a été entreprise sur des substrats macroarchitecturés, présentant des sillons de différentes géométries : sillons semi-circulaires (Wave), sillons triangulaires à angle de 90° ou à angle de 45°. Plus la géométrie du substrat était refermée (45°>90°>Wave), plus la différenciation ostéoblastique était rapide. Cela s’est traduit par une augmentation des niveaux d’expression génique et protéique d’ostéocalcine et de sclérostine, indiquant la présence d’ostéocytes au sein de l’important tissu déposé par les cellules. De plus, au sein de la géométrie à l’angle le plus fermé (i.e. « 45° »), des structures fibreuses minéralisées, orientées parallèlement au fond du substrat ont été observées. Cette orientation s’est confirmée au niveau cellulaire, avec une orientation similaire des fibres de stress et un étirement des noyaux cellulaires. La géométrie du substrat influence donc le comportement des cellules en modifiant très probablement leur signalisation intracellulaire. Ces investigations ont été poursuivi par le développement d’un modèle d’ostéogénèse 3D sous perfusion au sein du bioréacteur BOSE ElectroForce® 5270 BioDynamic®de la plateforme Equipex IVTV, afin d’explorer les interactions cellulaires-substrat en réponse à des contraintes mécaniques (forces de cisaillement). Le dépôt tissulaire était particulièrement abondant au sein des pores triangulaires à angle de 45°, confirmant les données obtenues sur les substrats macroarchitecturés et laissant penser que ce type de pores est le plus à même de permettre une différenciation ostéoblastique optimale. Les résultats de ces travaux pourront permettre des avancées dans la compréhension de la biologie de l’os, mais également dans la conception d’implants innovants destinés à la réparation de défaux osseux, avec une ostéointégration stimulée via la présence de structures à géométrie fermée, tel que des sillons triangulaires à angles de 45°. / In vivo, cells reside in a complex and three-dimensional microenvironment, with which they interact at multiple scales, from the nanometer (tropocollagen) to structures of several hundred of micrometers (trabeculae). However, most of our knowledge on cell physiology has been obtained from cells grown in Petri dishes, on plastic and in two dimensions. In those conditions, the spatial relationships between cells and their environment can only be deeply modified. Moreover, if the impact of substrate closure at a cellular level is particularly well documented, very few studies have shown its role at a tissue level (i.e. greater than 100 µm), and thus focused mostly on the matrix deposition rather than on the osteoblastic differentiation. In order to study the effects of substrate macroarchitecture on cells, primary mouse calvarial cells were seeded on hydroxyapatite-based bioceramics, made from wax molds by 3D printing. A first study was conducted on macroarchitectured substrates. These bioceramics have three patterns of different degrees of closure: semi-circular grooves (Wave), triangular grooves with 90° angle and triangular grooves with 45° angle. The tighter was the substrate geometry (45°> 90°> Wave), the faster was osteoblastic differentiation. This resulted in increased levels of gene and protein expression of osteocalcin and sclerostin, indicating the presence of osteocytes inside the tissue layed by cells. Moreover, in the tightest geometry (i.e. 45°), mineralized fibrous structures, oriented parallel to the bottom substrate were observed. This orientation was confirmed at the cellular level, with a similar orientation of stress fibers and a stretch of cell nuclei. Thus, the substrate macroarchitecture influences the cellular behavior by, most likely, modifying the intracellular signaling. These investigations were pursued with the development of a 3D model of osteogenesis under perfusion, in the BOSE 5270 ElectroForce® BioDynamic® bioreactor of the IVTV Equipex platform, to explore cell-substrate interactions in response to mechanical stress (shear forces). Tissue deposition was particularly abundant in the triangular pores with 45° angles, confirming our previous observations and suggesting that this geometry was able to promote osteoblast differentiation.Our results could lead to breakthroughs in the understanding of the bone biology but also in the design of innovative implants for the repair of bone defects, with a stimulated osseointegration throught the presence of structures with closed geometries, such as triangular grooves with 45° angles.

Este estudo analisou a influência de diferentes superfícies de titânio na adesão, proliferação e diferenciação de células semelhantes a osteoblastos de rato (osteo-1) em culturas, na presença ou não da proteína morfogenética óssea recombinante-2 (rhBMP-2). As células osteo-1 foram cultivadas sobre as seguintes superfícies de titânio: 1. superfície lisa, 2. superfície desgastada com partículas de areia e condicionamento ácido (SLA) e 3. superfície desgastada com partículas de areia e condicionamento ácido sob proteção de nitrogênio e armazenadas em solução isotônica de cloreto de sódio (SLActive), na presença ou não de 20 ng/ml de rhBMP-2. Foram analisadas a adesão celular em 24 horas, o conteúdo total de proteínas, o conteúdo de colágeno e a atividade de fosfatase alcalina em 7, 14 e 21 dias e a formação de nódulos calcificados em 21 dias. Os resultados mostraram que a adesão não foi influenciada nem pelo tipo de superfície nem pelo tratamento com rhBMP-2 (p=0,0936). Quando relacionamos o conteúdo total de proteínas ao número total de células, percebemos que a proliferação não foi influenciada pelo tipo de superfície de titânio, porém a adição de rhBMP-2 levou a uma redução estatisticamente significante na superfície SLA aos 21 dias (p=0,0000). Em relação à diferenciação, pudemos observar que o tipo de superfície não influenciou o conteúdo total de proteínas, o conteúdo de colágeno e a formação de nódulos calcificados em quaisquer dos períodos analisados. A atividade de fosfatase alcalina somente foi influenciada pelo tipo de superfície aos 14 dias, onde o grupo C/SLAactive apresentou valores inferiores ao grupo C/Liso (p=0,0000). A adição de rhBMP-2 promoveu uma maior influência sobre o processo de diferenciação, levando a uma redução estatisticamente significante no conteúdo total de proteínas na superfície SLA aos 21 dias (p=0,0000), a um aumento estatisticamente significante no conteúdo de colágeno na superfície SLActive no período de 7 dias (p=0,0005) e a uma diminuição estatisticamente significante na atividade de fosfatase alcalina na superfície lisa nos períodos de 14 e 21 dias, na superfície SLA aos 14 dias e na superfície SLActive aos 21 dias (p=0,0000). Somente a formação de nódulos calcificados não sofreu influência da adição de rhBMP-2. / This study has analyzed the influence of different titanium surfaces in the adhesion, proliferation and differentiation of rat osteoblast-like cells (osteo-1 culture), in the presence or not, of the recombinant bone morphogenetic protein-2 (rhBMP-2). The osteo-1 cells were grown on the following titanium surfaces: 1. smooth surface; 2. coarse grit-blasted and acid-etched surface (SLA); and 3. coarse grit-blasted and acid-etched surface under nitrogen protection, and stored in sodium chloride isotonic solution (SLActive), in the presence or not, of 20 ng/ml of rhBMP-2. It was analyzed the cell adhesion in 24 hours, the total protein content, the collagen content, and the alkaline phosphatase in 7, 14 and 21-day periods, and also the formation of calcified nodules in 21 days. The results showed that the adhesion was neither influenced by the surface type, nor by the treatment with rhBMP-2 (p=0.0936). When we related the total protein content to the total number of cells, we noticed that the proliferation was not influenced by the titanium surface type; however, the addition of rhBMP-2 led to a statistically significant reduction on the SLA surface at 21 days (p=0.0000). Concerning the differentiation, we could observe that the surface type did not influence the total content of proteins, the collagen content and the formation of calcified nodules in any of the analyzed periods. The alkaline phosphatase activity was only influenced by the surface type at 14 days, where the group C/SLActive presented lower values than the group C/Smooth (p=0.0000). The addition of rhBMP- 2 promoted a bigger influence over the differentiation process, thus leading to a statistically significant reduction in the total protein content on the SLA surface at 21 days (p=0.0000), a statistically significant increase in the collagen content on the surface SLActive in the 7-day period (p=0.0005), a statistically significant reduction in the alkaline phosphatase activity on the smooth surface in the 14 and 21-day periods, on the SLA surface at 14 days, and on the SLActive surface at 21 days (p=0.0000). Only the formation of calcified nodules did not undergo influence of the rhBMP-2 addition.

A osteoporose, uma doença óssea progressiva, é considerada um grave problema de saúde pública, sendo uma das condições mais importantes associadas ao envelhecimento e que afeta milhões de pessoas no mundo. Esta doença multifatorial é caracterizada pela densidade óssea reduzida e deterioração da microarquitetura óssea. O objetivo deste trabalho foi investigar as mudanças comportamentais em células mesenquimais da medula óssea e células osteoblásticas da calvária de ratas induzidas à osteoporose. Após aprovação da Comissão de Ética no Uso de Animais, 18 ratas Wistar foram utilizadas e divididas em grupos controle e ovariectomizadas. Após 150 dias, as ratas de ambos os grupos foram sacrificadas para coleta dos fêmures e fragmentos da calvária. As células recolhidas a partir da medula óssea e calvária foram cultivadas em placas de 24 poços (n = 5) para avaliação da proliferação e viabilidade celular, atividade de fosfatase alcalina (ALP), detecção e quantificação de nódulos mineralizados e análise da expressão gênica por meio de PCR em tempo real. Os dados foram submetidos ao testes de Kruskal-Wallis e Mann-Whitney, com nível de significância de 5%. As células da medula óssea do grupo controle (MC) mostraram uma diminuição significativa na proliferação quando comparado com as células do grupo controle da calvária (CC) em todos os períodos (p < 0,05). Por outro lado, as células da medula óssea de ratas com osteoporose (MO) revelaram um aumento significativo na taxa de proliferação após 7 e 10 dias (p < 0,01) em comparação às células da calvária de ratas ovariectomizadas (CO). A viabilidade celular foi maior em todos os períodos estudados dos grupos CC e CO em relação aos grupos MC e MO (p < 0,05). A atividade de fosfatase alcalina não foi significativamente diferente após 7 dias de cultura entre os grupos estudados; por outro lado, após 10 e 14 dias, observou-se uma diminuição da sua atividade no grupo MC quando comparado ao grupo CC (p < 0,01). Nas ratas com osteoporose, as células da medula óssea mostraram um aumento desta atividade quando comparada às células de calvária (p < 0,01). A análise dos nódulos mineralizados após 14 e 21 dias revelou que os grupos controle CC e MC não apresentaram diferenças significativas, ao passo que no grupo MO observou-se um aumento da mineralização quando comparado ao grupo CO nos mesmos períodos experimentais. Os resultados obtidos na análise de expressão gênica mostraram que para os genes Runx2, Oc, Alpl, Rank-l e Er&alpha; a expressão no grupo MO foi maior que no grupo MC (p < 0,05), enquanto que para as células da calvária, CC teve maior expressão gênica que CO (p < 0,05). Os genes Opg e Er&beta; apresentaram variações de acordo com o grupo avaliado. Frente aos resultados obtidos, sugere-se que existam alterações no metabolismo das células progenitoras e células diferenciadas após a indução da osteoporose e que as células da medula óssea apresentam um aumento da sua função como uma resposta compensatória neste modelo animal ovariectomizado. / Osteoporosis, a progressive bone disease, is considered a serious public health problem, being one of the most important conditions associated with aging, affecting millions of people worldwide. This multifactorial disease is characterized by reduced bone density and deterioration of bone microarchitecture. The objective of this work was to investigate the behavioral changes in mesenchymal cells from bone marrow and osteoblastic cells from calvaria bone of female rats induced to osteoporosis. After institutional review board approval, 18 Wistar female rats were used and divided into control and ovariectomized groups. After 150 days, the rats of both groups were sacrificed for collection of femurs and calvariae fragments. The cells collected from bone marrow and calvaria were cultured in 24-well plates (n=5) for the assessment of cell proliferation and viability, alkaline phosphatase (ALP) activity and detection and quantification of mineralized nodules. The data were submitted to Kruskal-Wallis and Mann-Whitney tests, with significance level set at 5%. The cells from bone marrow control group (MC) showed a significantly decrease in proliferation when compared to the cells from calvaria bone control group (CC) in all periods evaluated (p < 0,05). On the other hand, bone marrow cells from osteoporotic rats (MO) revealed a significant increase in their proliferation rate after 7 and 10 days (p < 0,01) compared to calvaria cells from ovariectomized rats (CO). Cell viability was higher in all investigated periods of CC and CO groups compared to MC and MO groups (p < 0,05). Alkaline phosphatase activity was not significantly different after 7 days of culture among the studied groups; in spite of that, after 10 and 14 days, there was a decrease in its activity in MC group when compared to CC (p < 0,01). In the osteoporotic rats, bone marrow cells showed an increase in this activity when compared to calvaria cells (p < 0,01). The analysis of mineralized nodules after 14 and 21 days revealed that control groups (CC and MC) did not have significant differences, whereas it was observed in MO group an increase in the mineralization when compared to CO in the same experimental periods. The data obtained in the analysis of gene expression showed that for Runx2, Oc, Alpl, Rank-l and Er&alpha; genes, the expression in MO group was higher than in MC (p < 0,05), whereas for the calvaria cells, CC group had higher gene expression than CO group (p < 0,05). The Opg and Er&beta; genes showed variations according to the evaluated group. Thus, it is suggested that there are changes in the metabolism of progenitor and differentiated cells after osteoporosis induction and that bone marrow cells present an enhancement of their function as a compensatory response in this ovariectomized rat model.

Orientador: Carmen Verissima Ferreira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T15:16:22Z (GMT). No. of bitstreams: 1 ChavesNeto_AntonioHernandes_D.pdf: 8240508 bytes, checksum: 293949f75ca619cdc0fa61d29d1703ba (MD5) Previous issue date: 2009 / Resumo: Riboflavina (Rb - Vitamina B2) é o precursor das flavocoenzimas essenciais flavina mononucleotídeo (FMN) e flavina adenina dinucleotídeo (FAD). Estas coenzimas participam de processos enzimáticos dependentes das reações de transferências de elétrons, que ocorrem nas vias de produção de energia, biossíntese, desintoxicação e sequestro de elétrons. O aumento dietético da riboflavina e piridoxina foi associado com maiores densidades minerais em mulheres e homens idosos. Fotoderivados da riboflavina demonstraram efeitos citotóxicos em células cancerosas de próstata e leucemias, entretanto, o efeito direto da Rb e seus fotoderivados em osteoblastos não foram examinados. Neste trabalho os efeitos biológicos da Rb e riboflavina irradiada (IRb) foram investigados na linhagem de pré-osteoblastos MC3T3-E1, um modelo bem aceito de osteogênese in vitro caracterizado pela indução de genes específicos associados com o fenótipo osteoblástico quando tratados com ácido ascórbico e ß-glicerofosfato. A viabilidade celular foi avaliada através da redução do MTT, da incorporação do corante vermelho neutro e do conteúdo de ácidos nucléicos. Marcadores de diferenciação osteoblástica foram analisados através do RT-PCR semi-quantitativo (osteopontina e osteocalcina) e através de análises colorimétricas de atividade da fosfatase alcalina (FAL) e síntese de colágeno pela coloração de picrosirius. As atividades das metaloproteinases (MMP) -9 e -2 foram avaliadas pela zimografia de gelatina. Microarranjos de peptídeos com subtratos específicos para quinases e imunoblotting foram usados para identificar os efeitos na sinalização celular. As atividades de enzimas antioxidantes (superóxido dismutase, catalase, glutationa peroxidase e glutationa S-transferase) foram determinadas em lisados celulares usando métodos espectrofotométricos. As atividades das caspases-8, -9 e -3 foram analisadas através de métodos colorimétricos. Na primeira análise Rb e IRb causaram a parada do ciclo celular na fase G0/G1 e também a inibição da quinase AKT, um mediador da proliferação. Flavinas causaram a diferenciação de pré-osteoblastos, evidenciada pelo aumento da expressão de osteocalcina, osteopontina e BMP-2. Atividades mais elevadas de MMP-9 e MMP-2 também foram observadas. A capacidade das flavinas em engatilhar a diferenciação de osteoblastos foi reforçada pelo aumento da conexina 43, diminuição da caveolina-1 e repressão da sinalização Notch. Na segunda análise, nós encontramos que as interações entre Rb, em sua forma irradiada e não-irradiada, e indutores osteogênicos (ácido ascórbico e ß-glicerofosfato) afetaram significativamente a proliferação de osteoblastos, a atividade de FAL, biossíntese de colágeno, expressão de osteocalcina e osteopontina, a atividade das MMP-2 e MMP-9 e a expressão de fatores osteoclastogênicos (RANKL e osteoprotegerina). Nós também encontramos que os efeitos das flavinas em osteoblastos nesta segunda etapa foram independentes das suas propriedades antioxidantes. A atividade biológica da combinação de indutores osteogênicas com Rb e seus fotoprodutos foi associada com a ativação de diferentes vias de sinalização (AKT, FAK, CaMKII), caspases -8, -9 e -3 e aumento da expressão e/ou estabilização de fatores de transcrição osteoblásticos (Runx2 e ß-catenin). Este estudo nos trouxe fortes evidências que altas concentrações de Rb e IRb geraram um microambiente osteogênico através da modulação de diferentes vias de sinalização, além de promover um efeitos aditivo durante a diferenciação das células pré-osteoblasticas MC3T3 induzida por ácido ascórbico e ß- glicerofosfato. Em resumo, este estudo aponta para uma potencial aplicação da Rb e seus fotoprodutos no desenvolvimento do fenótipo osteoblástico e, consequentemente, uma alternativa terapêutica coadjuvante para osteoporose. / Abstract: Riboflavin (Rb-Vitamin B2) is the precursor of essential flavocoenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These coenzymes participate in numerous enzymatic processes dependent on electron transfer reactions that occur in energyproducing, biosynthetic, and detoxifying and electron-scavenging pathways. Increase dietary riboflavin and pyridoxine intake has been associated with higher bone mineral density in elderly men and women. Photoderivatives of riboflavin have been shown strong activity in haematological malignancy and prostate cancer cells, however, the direct effect of Rb and its photoderivatives on osteoblast has not been examined. In this work, the biologic effects of Rb and irradiated riboflavin (IRb) were investigated in the MC3T3-E1 pre-osteoblastic cell line, a well-accepted model of osteogenesis in vitro characterized for the induction of specific genes associated with the osteoblastic phenotype when treated with ascorbic acid and ß-glycerophosphate. Cell viability was assessed by MTT reduction, neutral red uptake and nucleic acids content. Osteoblastic differentiation markers were analyzed by semiquantitative RT-PCR (osteopontin and osteocalcin), alkaline phosphatase (ALP) activity measured colorimetrically and collagen synthesis by Sirius red staining. Metalloproteinases (MMP) -9 and -2 activities were assayed by gelatin zymography. Peptide microarray of substrate specificity to kinases and immunoblotting were used to identify the effects on signal transduction pathways. Antioxidant enzyme activities (superoxide dismutase, catalase, glutathione peroxidase and glutathione Stransferase) were determined in cellular lysate using spectrophotometric methods. Caspase-8, -9 and -3 activation were measured by a colorimetric assay. In the first analysis Rb and IRb caused cell cycle arrest at G0/G1 phase and accordingly inhibited AKT kinase, a proliferation mediator. Flavins caused differentiation of preosteoblast cells as evidenced by increase of osteocalcin, osteopontin and BMP2 expressions. In addition, higher MMP-9 and -2 activities were observed. Importantly, the capacity of flavins to trigger osteoblasts differentiation was also reinforced by upregulation of connexin 43, down regulation of caveolin-1 and negative modulation of Notch cascade. In the second analysis, we found that the interaction between Rb and IRb and osteogenic inductors (ascorbic acid and ß-glycerophosphate) significantly affected the osteoblast proliferation, alkaline phosphatase activity, collagen biosynthesis, osteopontin and osteocalcin mRNA expression, MMP-2 and MMP-9 activities and the expression of osteoclastogenesis factors (RANKL and OPG). We also showed that the effects of flavins in osteoblasts cells were independent on flavins antioxidant property. The biological activity of the combination of osteogenic medium with riboflavin and its photoderivatives was associated with the activation of different signaling pathways (AKT, FAK, CaMKII), caspases -8, -9 and -3, and up-regulation and/or stabilization of osteoblastic transcription factors (Runx2 and ß-catenin). This study brought out strong evidences that high concentration of Rb and IRb generates an osteogenic microenvironment through modulating different mediators of signaling pathways, besides of the additive effect of riboflavin and its photoproducts during the ascorbate and ß-glycerophosphateinduced osteoblast differentiation of MC3T3-E1 cells. In summary, this study pointed out the potential application of Rb and its photoproducts in osteoblasts phenotype development and, consequently, it is possible use as an alternative therapeutic adjuvant of osteoporosis. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

O objetivo desse estudo foi investigar a participação de integrina &alpha;1&beta;1 e microRNAs (miRs) no potencial osteogênico de superfícies de titânio (Ti) com nanotopografia. Discos de Ti previamente polidos foram tratados quimicamente com H2SO4/H2O2 para obtenção de nanotopografia, que foi observada por microscopia eletrônica de varredura. Para o estudo da participação da integrina &alpha;1&beta;1, células-tronco mesenquimais (CTMs) de ratos foram cultivadas em condições osteogênicas e não osteogênicas sobre superfícies de Ti com nanotopografia e sem tratamento químico (controle). O resultados mostraram que a nanotopografia de Ti aumentou a proliferação celular, a atividade de fosfatase alcalina (Alp) e regulou positivamente a expressão gênica de marcadores da diferenciação osteoblástica em CTMs cultivadas tanto em condições osteogênicas quanto em condições não osteogênicas. Além disso, uma maior expressão gênica para as integrinas &alpha;1 e &beta;1 foi observada em culturas crescidas sobre nanotopografia em condições não osteogênicas em relação ao Ti controle. O uso de obtustatina, um inibidor de integrina &alpha;1&beta;1, reduziu os efeitos da nanotopografia sobre os marcadores osteoblásticos, indicando a participação da via de sinalização dessa integrina nos efeitos da nanotopografia sobre CTMs. Para investigar a participação de miRs no efeito osseoindutor da nanotopografia de Ti, foram utilizadas CTMs humanas e células préosteoblásticas de camundongos da linhagem MC3T3-E1. A análise em larga escala da expressão de miRs revelou que 60 miRs foram regulados positivamente (no mínimo, 2x maior), enquanto 58 miRs foram regulados negativamente (no mínimo, 2x menor) em CTMs crescidas sobre a nanotopografia. Três desses miRs, miR-4448, -4708 e -4773, cuja expressão foi significativamente reduzida pela nanotopografia de Ti (no mínimo, 5x menor), afetaram a diferenciação osteoblástica de CTMs. Esses miRs atuam diretamente sobre SMAD1 e SMAD4, proteínas transdutoras da sinalização da proteína óssea morfogenética 2 (Bmp-2), conhecida por sua capacidade osseoindutora. Além disso, verificou-se que a sobreexpressão de miR-4448, -4708 e -4773 em células pré-osteoblásticas MC3T3-E1 inibiu a expressão gênica e proteica de SMAD1 e SMAD4 e, consequentemente, a expressão gênica de marcadores ósseos. Esses dados sugerem a influência do circuito miR-SMAD-Bmp-2 sobre o efeito osseoindutor da nanotopografia. Conjuntamente, os achados do presente estudo mostraram que o efeito da nanotopografia de Ti sobre a diferenciação osteoblástica resulta de um mecanismo regulatório complexo, do qual fazem parte as vias de sinalização da integrina &alpha;1&beta;1 e da Bmp-2, com a participação de miRs. Esses resultados podem representar um avanço para o desenvolvimento de novas modificações de superfície, com o objetivo de acelerar e/ou melhorar o processo de osseointegração. / The aim of this study was to investigate the role of the &alpha;1&beta;1 integrin and microRNAs (miRs) on the osteogenic potential of titanium (Ti) with nanotopography. Polished Ti discs were chemically treated with H2SO4/H2O2 to generate nanotopography, which was observed under scanning electron microscopy. For the study related to the &alpha;1&beta;1 integrin, rat mesenchymal stem cells (MSCs) were cultured under osteogenic and non-osteogenic conditions on Ti with nanotopography and non-treated Ti discs (control). Nanotopography increased cell proliferation and alkaline phosphatase (Alp) activity and upregulated the gene expression of bone markers in cells cultured under osteogenic and non-osteogenic conditions. Furthermore, the gene expression of &alpha;1 and &beta;1 integrins was higher in cells cultured on nanotopography under non-osteogenic conditions compared with control. Obtustatin, an inhibitor of &alpha;1&beta;1 integrin, reduced the higher gene expression of the bone markers induced by nanotopography. These results indicate that &alpha;1&beta;1 integrin signaling pathway determines the osteoinductive effect of nanotopography on MSCs. The role of miRs in the osteogenic potential of Ti with nanotopography was evaluated using human MSCs and MC3T3-E1 mouse pre-osteoblastic cells. The miR sequencing analysis revealed that 60 miRs were upregulated (> 2 fold), while 58 miRs were downregulated (< 2 fold) in MSCs grown on nanotopography. Three miRs, miR-4448, -4708 and -4773, which were significantly downregulated (< 5 fold) by nanotopography, affected the osteoblast differentiation of MSCs. These miRs directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (Bmp-2) osteogenic signal, which were upregulated by nanotopography. Overexpression of miR-4448 - 4708 and 4773 in MC3T3-E1 cells noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and by targeting them, these miRs repressed gene expression of key bone markers. These results suggest that a miR-SMAD-Bmp-2 circuit acts in the Ti nanotopography-mediated osteoblast differentiation. Taken together, our data showed that the osteoblast differentiation induced by Ti with nanotopography is governed by a complex regulatory network involving a crosstalk between &alpha;1&beta;1 integrin and Bmp-2 signaling pathways with participation of miRs.

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates voltage-dependent calcium channel function. Given its expression in both excitable and non-excitable cell types, the control mechanisms for Rad regulation and the potential for novel functions for Rad beyond calcium channel modulation are open questions. Here we report a novel interaction between Rad and Enigma, a scaffolding protein that also binds to the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1). Overexpression of Smurf1, but not of a catalytically inactive mutant enzyme, results in ubiquitination of Rad and down regulation of Rad protein levels. The Smurf1-mediated decrease in Rad levels is sensitive to proteasome inhibition and requires the ubiquitination site Lys204, suggesting that Smurf1 targets Rad for degradation. Rad protein levels, but notably not mRNA levels, are increased in the hearts of Enigma-/- mice, leading to the hypothesis that Enigma may function as a scaffold to enhance Smurf1 regulation of Rad. In addition to ubiquitination, phosphorylation of RGK proteins represents another potential means of regulation. Indeed, Rem phosphorylation has been shown to abolish calcium channel inhibition. We demonstrate that b-adrenergic signaling promotes Rad phosphorylation at Ser39. Rad Ser39 phosphorylation is correlated with a decrease in the interaction between Rad and the CaVb subunit of the calcium channel and an increase in Rad binding to 14-3-3. Interestingly, Enigma overexpression promotes an increase in Rad Ser39 phosphorylation as well. Despite an interaction between Enigma and the CaV1.2 calcium channel subunit, overexpression of Enigma had no effect on Rad-mediated channel inhibition. Thus, Rad Ser39 phosphorylation alters its association with the calcium channel, but its impact on calcium channel regulation has yet to be determined. Finally, we report a novel function for Rad in the regulation of bone homeostasis. Rad deletion in mice results in a significant decrease in bone mass. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression is not altered in Rad-/- osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher bone marrow adipose tissue (BMAT) levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of WT osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity. In summary, this dissertation expands our understanding of Rad regulation through identification of a novel binding partner and characterization of post-translational regulatory mechanisms for Rad function. This work also defines a new role for Rad that may not depend upon its calcium channel regulatory properties: regulation of the bone-fat balance. These findings suggest that the regulation of Rad GTPase is likely more complex than guanine nucleotide cycling and that functions of Rad in non-excitable tissues warrant further study.

The use of nanoparticles for disease treatment is an increasingly popular area of research. The potential for multi-functionality allows nanoparticles to be used as transport and delivery vehicles for drugs and as diagnostic aides, among other applications, to address the unmet needs of many disease treatments. One such class of disease is osteoporosis including severe disorders, like Paget’s disease, Osteogenesis Imperfecta and Legg Calve Perthes disease. In this dissertation, we discuss a nanoparticle system consisting of gold nanoparticles surface functionalized with primary amine bisphosphonates, which is a classification of pharmaceuticals that is common in the treatment of osteoporosis. Functionalized nanoparticles allow for greater intracellular concentrations of pharmaceutical, while the properties of the gold nanoparticles provide the ability to track the pharmaceutical and enhance imaging. We have synthesized and characterized bisphosphonate functionalized gold nanoparticles of controlled size of approximately 15 nm, which are suitable for cellular uptake, and functionalized the surface using self-assembly with pamidronate and alendronate. In one major finding of this study, inductively coupled plasma mass spectrometry was used to estimate approximate surface density of the bisphosphonates on the gold nanoparticles. This resulted in concentrations of approximately 0.65 molecules per nm2 (approximately 154 Å2/molecule) for pamidronate functionalized on gold, and approximately 2.6 molecules per nm2 (approximately 39 Å2/molecule) for alendronate functionalized on gold. This allows for more accurate estimates of pharmaceutical concentrations, during in vitro and in vivo studies. Additionally, we investigated the effects of bisphosphonate functionalized gold nanoparticles on the viability and morphology of osteoclast and osteoblast cells in vitro. We found that attaching the bisphosphonates to the surface of the nanoparticles leads to increased apoptotic effects of the bisphosphonates on the osteoclast cells compared to free bisphosphonates. Further, we showed bisphosphonate functionalized gold nanoparticles may have an effect on nuclei morphology that may provide an additional means of modulating bone resorption rather than just through influencing viability. Further we showed that it may be possible to target concentrations that are safe for osteoblasts, which is critical in determining potential treatment concentrations. These viability results bring to light a number of potential considerations into the optimization of potential treatments, such as dosing concentrations. Finally, detailed results are given on effects of bisphosphonate functionalized gold nanoparticles on important behavior and activity of osteoclast and osteoblast cells in vitro. We showed that while using concentrations below the toxicity threshold, some of the normal activity of the cells could be maintained. RANKL and ALP expression in osteoblasts were maintained when removing viability as a variable. Additionally, bone nodule formation was also maintained for osteoblasts and co-cultured in vitro systems. Finally, we showed that the introduction of bone in the in vitro studies adds a new degree of consideration as to the interaction of the bisphosphonates with the hydroxyapatite surface. This strong interaction with bone is an important consideration in further developing potential treatments for osteoporotic disease. This dissertation provides insights into the use of bisphosphonate functionalized gold nanoparticles as a potential treatment and means of study for bone remodeling disorders.

Skeletal metastasis is a lethal component of many advanced cancers including prostate, the second most common cancer among men. Patients whose prostate cancer is localized and detected early benefit from multiple treatment options ranging from active surveillance to radiation and surgery, resulting in a 5-year survival rate of nearly 100%. Unfortunately, the prognosis and survival for patients with advanced metastatic disease is much worse due to the highly aggressive nature of the disease and a paucity of treatment options. Understanding the mechanisms and interactions that occur between metastatic cancer cells and the bone will enable the future treatment landscape for bone metastatic prostate cancer to expand, thereby improving patient outcomes. Our current knowledge of how metastatic prostate cancer cells interact with the bone is summarized in a model known as the “vicious cycle.” Numerous fundamental vicious cycle factors have been identified, including parathyroid hormone-related protein (PTHrP), while additional elements, such as matrix metalloproteinases (MMPs), are progressively being discovered and added to the model. PTHrP is a critical regulator of bone resorption and augments osteolysis in skeletal malignancies. In Chapter 2, we report that the mature PTHrP1-36 hormone is processed by MMPs to yield a stable product, PTHrP1-17. PTHrP1-17 retains the ability to signal through PTH1R to induce calcium flux and ERK phosphorylation but not cyclic AMP production or CREB phosphorylation. Notably, PTHrP1-17 promotes osteoblast migration and mineralization in vitro, and systemic administration of PTHrP1-17 augments ectopic bone formation in vivo. Further, in contrast to PTHrP1-36, PTHrP1-17 does not affect osteoclast formation/function in vitro or in vivo. Finally, immunoprecipitation-mass spectrometry analyses using PTHrP1-17-specific antibodies establish that PTHrP1-17 is indeed generated by cancer cells. Thus, MMP-directed processing of PTHrP disables the osteolytic functions of the mature hormone to promote osteogenesis, indicating important roles for this mechanism in bone remodeling in normal and disease contexts. MMPs have traditionally been associated with cancer progression based on their extracellular matrix degrading activities. However, it has become evident that their regulation of non-extracellular matrix substrates can exert both contributive and protective effects during tumorigenesis. Previous studies of matrix metalloproteinase-3 (MMP-3) have demonstrated tissue dependent pro- and anti-tumorigenic effects, but despite elevated expression, its roles have not been explored in bone metastatic prostate cancer. In Chapter 3, we show that tumor-derived MMP-3 contributes to prostate tumor growth in bone. In vitro, we observe that silencing MMP-3 reduces prostate cancer cell proliferation. Further, we found increased levels of IGFBP3, a known MMP-3 substrate, and decreased IGF-1R, ERK, and AKT phosphorylation in the MMP-3 silenced cells. Notably, we also observe reduced tumor growth and proliferation in in vivo intratibial models when tumor-derived MMP-3 expression is silenced. These data suggest that increased MMP-3 expression by prostate cancer cells contributes to their proliferation in bone by regulating the activity of the IGF/IGF-1R signaling axis. Taken together, our studies indicate that MMPs possess important functional roles in bone metastatic prostate cancer. We believe that elucidation of these mechanisms and their contributions to the vicious cycle of bone metastasis will offer novel opportunities to design effective therapeutic treatment options.

Abstract Type XIII collagen is a type II transmembrane protein which is expressed in many tissues throughout development and adult life. It is located in focal adhesions of cultured cells and in the adhesive structures of tissues such as the myotendinous junctions in muscle, intercalated discs in the heart and the cell-basement membrane interphases. To further characterize the function of this protein, we generated transgenic mice overexpressing it in normal and mutant forms. A large in-frame deletion in the COL2 domain of type XIII collagen led to synthesis of truncated α1(XIII) chains in transgenic mice, disrupting the assembly of normal type XIII collagen trimers. Fibroblasts derived from the mutant mice expressed shortened α1(XIII) chains, and no intracellular accumulation of the mutant protein was detected, suggesting that the mutant molecules were expressed on the cell surface. Transgene expression led to an embryonally lethal phenotype in offspring from heterozygous mating at two distinct stages of development. The early phenotype fetuses died due to the lack of chorioallantoic fusion and functioning placenta at 10.5 dpc, while the death of the late phenotype fetuses was caused by cardiac and placental defects around 13.5 dpc. The phenotype resembles closely several other cell adhesion molecule mutants, indicating that type XIII collagen has an essential role in certain adhesive interactions that are necessary for normal development. Mice overexpressing type XIII collagen with or without a point mutation developed postnatally an unexpected skeletal phenotype marked by a massive increase in bone mass. The cortical bone cross-sectional area and volumetric bone mineral density were highly increased, but trabecular bone volume was not significantly altered. The bone formation rate was several times higher in the mutant mice than in their normal littermates, while the osteoclast number and resorption activity were normal. Type XIII collagen was expressed highly in primary osteoblasts derived from the transgenic mice. Overexpression of type XIII collagen in osteoblasts enhanced both cell proliferation and differentiation while lack of it had opposite effects. Furthermore, mutant cells responded to mechanical strain differently than wild-type cells. The findings suggest that type XIII collagen has an important role in bone modeling, and it may in particular have a function in coupling the regulation of bone mass to mechanical usage.