Made available in DSpace on 2015-12-06T23:47:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2008 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A estimulação elétrica de baixa intensidade (EE) tem sido utilizada para o reparo ósseo, mas pouco se sabe sobre seus efeitos no tecido ósseo após a menopausa. Os osteócitos provavelmente desempenham um importante papel mediando este estímulo físico, e poderiam agir como transdutores através da liberação de sinais bioquímicos como o óxido nítrico (NO). O objetivo deste trabalho foi investigar os efeitos da EE no metabolismo ósseo de ratas ovarectomizadas, e se o NO poderia ser um mediador destes efeitos. Sessenta ratas (200-220g) foram divididas em 6 grupos: SHAM; SHAM tratado com 6mg/d de L-NAME, um inibidor da NO sintase (SHAM-L-NAME); ovarectomizado (OVX); OVX tratado com L-NAME (OVX-L-NAME) ou submetido a uma estimulação elétrica no corpo todo do animal (OVX-EE) ou ambos (OVX-EE-L-NAME) por 12 semanas. A densidade mineral óssea (DMO) global, de coluna e membro posterior foram medidas através de densitometria óssea antes e ao final do protocolo. Após o sacrifício, as tíbias foram retiradas para análises histomorfométricas e imunohistoquímicas. A apoptose de osteócitos (técnicas de caspase-3 e TUNEL) e expressões da NO sintase endotelial (eNOS) e indutível (iNOS) foram analisadas por imunohistoquímica. As ratas OVX demonstraram significante (p<0.05 vs SHAM) diminuição da DMO final global, de coluna e de membro posterior, medidas por densitometria, redução de volume ósseo (10% vs 25%) e número de trabéculas (1.7 vs 3.9), e aumento das superfícies de reabsorção (4.7% vs 3.2%) e mineralização (15.9% vs 7.7%), medidas por histomorfometria. No entanto, após a EE, todos estes parâmetros foram semelhantes aos valores do grupo SHAM e significantemente diferentes de OVX. A EE não foi capaz de prevenir a redução de volume ósseo e número de trabéculas causadas pela OVX na presença de L-NAME (OVX-L-NAME vs OVXEE-L-NAME), como o fez na ausência deste inibidor da NOS. Porém, o L-NAME não bloqueou os efeitos da EE na reabsorção óssea (superfícies de reabsorção e de osteoclastos) em ratas OVX. A eNOS e iNOS foram: expressas de forma semelhante no córtex das tíbias de SHAM, não expressas em OVX, e similarmente expressas em OVX-EE quando comparadas ao SHAM. As expressões de eNOS e iNOS não foram detectadas no tecido ósseo de nenhum grupo tratado com L-NAME. Nas ratas OVX, a porcentagem de osteócitos apoptóticos (24%) foi significantemente maior do que em SHAM (11%) e OVX-EE (8%). Todos os grupos tratados com L-NAME tiveram uma diminuição na porcentagem de osteócitos apoptóticos. Assim, nosso estudo evidenciou que a EE previne alguns efeitos causados pela OVX no tecido ósseo preservando a DMO, estrutura e microarquitetura ósseas, expressão de eNOS e iNOS e viabilidade de osteócitos. Além disso, foi demonstrado que o L-NAME bloqueia parcialmente os efeitos da EE na estrutura óssea (mas não na reabsorção) e na expressão de eNOS e iNOS em ratas OVX, sugerindo que o NO possa ser um mediador dos efeitos positivos da EE no tecido ósseo. No entanto, não foi possível identificar se os efeitos positivos da EE na viabilidade de osteócitos foram mediados pelo NO, pois os efeitos do L-NAME nestas células foram semelhantes àqueles causados pela EE. / Low Intensity Electrical Stimulation (LIES) has been used for bone repair but little is known about its effects on bone after menopause. Osteocytes probably play a role in mediating this physical stimulus and they could act as transducers through the release of biochemical signals, such as nitric oxide (NO). The aim of the present study was to investigate the effects of LIES on bone metabolism in ovariectomized rats, and if NO could be a mediator of these effects. Sixty rats (200-220g) were divided into 6 groups: SHAM; SHAM treated with 6mg/d of LNAME, an inhibitor of NO synthase (SHAM-L-NAME); ovariectomized (OVX); OVX treated with L-NAME (OVX-L-NAME) or subjected to a whole body electrical stimulation (OVX-LIES) or both (OVX-LIES-L-NAME) for 12 weeks. Global, spine and posterior limb bone mineral density (BMD) were measured by bone densitometry before and at the end of protocol. After sacrifice, tibias were collected for histomorphometric and immunohistochemistry analysis. Osteocyte apoptosis (caspase-3 and TUNEL techniques) and expressions of endothelial NO synthase (eNOS) and inducible NOS (iNOS) were assessed by immunostaining. OVX rats showed significant (p<0.05 vs SHAM) decreased final global, spine and limb BMDs, measured by densitometry, decreased bone volume (10% vs 25%) and trabecular number (1.7 vs 3.9), and increased eroded surfaces (4.7% vs 3.2%) and mineralization surfaces (15.9% vs 7.7%), measured by histomorphometry. In contrast, after LIES, all these parameters tended to be similar to SHAM and significantly different from OVX. LIES was not able to prevent the reduction of bone volume and trabecular number caused by OVX in the presence of L-NAME (OVX-L-NAME vs OVX-LIES-L-NAME), as it did in the absence of this NOS inhibitor. However, L-NAME did not block the effects of LIES on bone resorption (eroded surface and osteoclasts surface) in OVX rats. eNOS and iNOS were: similarly expressed in tibiae cortices of SHAM, not expressed in OVX and correspondingly expressed in OVX+LIES when compared to SHAM. eNOS and iNOS expressions were not detected on bone of none of L-NAME treated groups. In OVX, the percentage of apoptotic osteocytes (24%) was significantly increased when compared to SHAM (11%) and OVX-LIES (8%). All LNAME treated groups showed a diminished percentage of apoptotic osteocytes. In conclusion, our study showed that LIES counteracts some effects of OVX on bone tissue preserving BMD, bone structure and microarchitecture, iNOS and eNOS expression and osteocyte viability. Moreover, it was demonstrated that L-NAME partially blocks the effects of LIES on bone structure (but not on bone resorption) and on iNOS and eNOS expressions in OVX rats, suggesting that NO may be a mediator of the positive effects of LIES on bone. However, it was not possible to identify if the known positive effects of LIES on osteocyte viability were mediated by NO, since the effects of L-NAME on these cells were similar to those caused by LIES. / BV UNIFESP: Teses e dissertações

Stroke is now a well-recognised risk factor for hip fracture. The aim of this study was to elucidate the pathophysiological mechanisms by which hip bone loss occurs in hemiplegia and to test the efficacy of a novel pharmaceutical strategy for preserving bone in stroke patients. Patients who were admitted acutely with a first-ever stroke and who remained unable to walk one week later were studied prospectively for 12 months, with a series of bone mineral density measurements of the hips (dual energy X-ray absorptiometry) in the context of a randomised controlled trial. Untreated patients (n=13) experienced a decline in bone mineral density at the hemiplegic hip that was rapid, with the greatest losses in the trochanteric region of the affected side. This bone loss was prevented by the administration of a single 4 mg dose of the intravenous bisphosphonate, zoledronate (n=14) within 35 days of stroke onset. Computed tomography of the hips in 8 untreated patients more than a year after stroke confirmed that the greatest difference between sides was in the trochanteric region. Serum vitamin D measurements in 44 patients with acute stroke were substantially lower than healthy elderly controls, with 77% of patients in the insufficient range, suggesting that vitamin D insufficiency preceded stroke. Histomorphometric analysis of iliac bone biopsies from hemiplegic patients 10 weeks following stroke showed normal erosion parameters, but a striking decrease in the surface extent of osteoid when compared with healthy reference values. Unexpectedly, treatment with zoledronate was associated with a significantly higher osteoid surface compared with placebo treated subjects in cancellous, endocortical and cortical bone. Sclerostin, a newly discovered osteocyte-derived protein was studied using immunohistochemical staining of the bone biopsies. Sclerostin is known to be an inhibitor of active osteoblasts, which led to the hypothesis that in stroke, the proportion of osteocytes expressing sclerostin would be inversely associated with the surface extent of bone formation. Histological analysis revealed widespread expression of sclerostin in osteocytes and their canaliculi in all subjects. However, examining individual osteocytes in relation to bone forming surfaces revealed that newly embedded osteocytes did not express sclerostin until after primary mineralisation. It is proposed that this precise pattern and timing of sclerostin expression by osteocytes allows bone formation to continue locally (during remodelling), but prevents excessive new bone formation elsewhere, as seen in the single gene disorder sclerosteosis.

Indiana University-Purdue University Indianapolis (IUPUI) / Bone is one of the most common sites for breast cancer metastasis. Osteocytes compose approximately 90% of the cell population in bone matrix. Osteocytes are very sensitive to mechanical stimulation, and physical activities play an essential role in maintaining bone's health. Mechanical stimulation can alter the gene expression profile in osteocytes. However, little is known about the effects of mechanical stimulation on tumor-bone interactions. In this thesis, this question has been addressed: Does applying mechanical stimulation to osteocytes change tumor-osteocytes interactions? The hypothesis is that mechanical stimulation can change osteocytes secreting signals and contribute to higher proliferation and migration of tumor cells. In this thesis, fluid flow-driven shear stress has been used as the mechanical stimulator for osteocytes, and the interactions of tumor-osteocytes, with and without mechanical stimulation has been investigated. Monolayer cultures and 3D spheroids of breast cancer cells, including TMD and 4T1 cells were cultured in the conditioned medium (CM) isolated from MLO-A5 osteocytes, and fluid flow-treated conditioned medium (FFCM), and their migratory behavior, proliferation, and protein expression have been evaluated. The results showed that in response to MLO-A5 FFCM, tumor cells behave differently in Src expression, proliferation, and migration compared to MLO-A5 CM. As opposed to MLO-A5 CM, FFCM promoted migration, reduced proliferation, and upregulated Src expression in tumor cells. Moreover, by plasmid and siRNA transfection it has been shown that Src is upstream of Snail and their upregulation is causing epithelial-mesenchymal transition(EMT) responses in tumor cells. Furthermore, ELISA concentration assessment showed the involvement of TGF-beta in Src upregulation. An in vivo study using seventeen mice was conducted to investigate the effect of mechanical stimulation on clinical conditions. Compressive loads were applied to tibia after intratibial injection of 4T1.2 cells. The results suggested that direct mechanical stimulation of metastasized bone, might not be advantageous, and cause more damage. Furthermore, the results indicated that direct mechanical loading can make the knee joint more fragile. This research showed mechanical stimulation can cause tumor cells to behave more migratory in bone microenvironment, and demonstrated its crucial role in tumor-osteocytes interactions.

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Human bone tissue has an inherent hierarchical structure, which is integral to its material properties. It is primarily composed of a collagen fiber matrix that is mineralized with hydroxyapatite. A comprehensive understanding of bone and the linkages between structural and cellular organization is imperative to developing fundamental knowledge that can be applied to better our understanding of bone disease manifestations and its interaction with implant devices. Herein, this thesis investigated non-traditional methods for evaluating bone structure across the nano- and meso-length scales. Firstly, due to the inhomogeneous organization of collagen fibrils and mineral platelets of bone ultrastructure, a suitable methodology for the investigation of both phases needed to be generated. In this work, focused ion beam (FIB) microscopy was employed to create site-specific scanning transmission electron microscopy (STEM) lift-outs of human osteonal bone that could be visualized with correlatively with STEM and small angle X-ray scattering (SAXS). Samples were successfully characterized using both techniques, and minimal visual damage was induced during data acquisition. This work is the first to demonstrate the potential for bone to be investigated correlatively using both STEM and SAXS. Secondly, this work is the first to employ a dual-beam plasma FIB (PFIB) equipped with a scanning electron microscope (SEM), to investigate bone tissue across the mesoscale. This equipment enables large volume three-dimensional (3D) imaging at nanoscale resolution across larger mesoscale volumes. This thesis aimed to reduce ion beam-based artifacts, which presents as curtain-like features by adjusting the composition of protective capping layers. Subsequently, large volume tomograms of bone tissue were acquired, demonstrating the effectiveness of the PFIB to reveal mesoscale features including the cellular network of bone tissue. Overall, this thesis has developed methods that allow for the application of advanced microscopy techniques to enhance the understanding of bone tissue across the nanoscale and mesoscale. / Thesis / Master of Applied Science (MASc) / Bone tissue has a unique structure that perplexes both biologists and materials scientists. The hierarchical structure of bone has garnered the interest of materials scientists since the body’s skeletal strength and toughness are governed by the nanoscale (millionth of centimetres) to macroscale (centimeters) organization of bone. In this work, the intricate organization of bone is investigated using advanced electron and ion beam microscopy techniques, which achieve high-resolution imaging of bone structure. Firstly, this work developed a sample preparation workflow to correlate electron and X-ray imaging of the same bone tissue. Secondly, this work was the first to apply serial-sectioning plasma focused ion beam tomography to human bone tissue to investigate its structure at high resolution across micron-sized volumes. Here, previously unexplored methodologies to image bone are demonstrated with the hopes of applying such techniques to investigate healthy and pathological bone tissue in the future.

<p>Osteoporosis causes over two million skeletal fractures in the United States every year in people over 50 years of age. Age-related bone loss results from imbalanced bone turnover mainly caused by decreases in sex hormones and skeletal mechanobiology. Estrogen receptor β (ERβ) in osteocytes (Ot) has been proposed to mediate skeletal structural adaptations in response to estrogen and mechanical stimuli. However, direct <em>in vivo</em> studies on Ot-ERβ are lacking, and relevant <em>in vitro</em> studies are mostly made in two-dimensional (2D) culture models, whose cellular environment restricts Ot morphology and biology. To better understand the mechanisms of estrogen-ERs in age-related bone loss, it is important to investigate the role of Ot-ERβ in skeletal turnover in response to sex hormonal and mechanical cues and develop a novel 3D culture model that can reproduce Ot morphology for future <em>in vitro</em> ER studies. The role of Ot-ERβ in bone turnover and skeletal adaptive response to mechanical load were examined in male and female mice at 12wk and 30wk old. Ot-ERβ shows age- and sex-dependent effects on bone morphology. Young male mice with Ot-ERβ deletion (ERβ-dOT) showed increased vertebral cancellous bone, whereas decreased cortical and cancellous vertebral bone mass appeared in adult male ERβ-dOT mice. No difference in bone mass occurred in female mice between genotypes. Ot-ERβ mediates tibial mechanoadaptation in cortical but not cancellous in young and adult male mice but plays an inhibitory role in young female mice during cortical mechanoadaptation. Gonadectomy studies on young adult mice revealed that deletion of Ot-ERβ inhibits the sex hormone withdrawal-induced decreases in bone mass and skeletal strength for male mice but did not play a major role for female mice. Lastly, a novel 3D <em>in vitro</em> culture system was developed using collagen-mineral composites for investigating culture mineralization, osteocyte biology, and osteocyte-osteoblast interaction. Cell viability and cellular differentiation were validated after 3 days and 56 days of culture. Optimal PSC-HA culture conditions were determined based on osteocyte differentiation, gene expression analyses, and tissue mineralization. Overall, this work takes novel steps to demonstrate the <em>in vivo</em> role osteocyte-ERβ plays in skeletal morphology and mechanobiology and develops a novel <em>in vitro</em> 3D culture using PSC-HA composites. These advances will contribute to future mechanistic studies of sex hormone receptors in osteoblasts and osteocytes in age-related bone loss using controlled <em>in vitro</em> environments. </p>

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Dans ce travail de thèse nous nous sommes intéressés aux phénomènes de transport au sein du réseau lacuno-canaliculaire (RLC) et de l'ostéon dans le tissu osseux cortical. Pour étudier la mécanotransduction ostéocytaire amenant au remodelage osseux, nous avons développé un modèle à trois échelles où sont pris en : l'électrcompte ostatique (modélisée par l'équation de Poisson Boltzmann), l'écoulement du fluide (représenté textit{via} une équation de Stokes modifiée et la conservation de la masse fluide) et le transport ionique (régi par l'équation de Nernst-Planck). L'étude de la distribution du potentiel électrique, a mis en exergue l'importance des double-couches électriques au voisinage des parois chargées des pores. Ces double-couches électriques, ainsi que la composition chimique du fluide donnent lieu à des phénomènes d'osmose et d'électroosmose intervenant dans l'écoulement du fluide interstitiel, et influençant la diffusion efficace des ions dans les pores. L'étude a démarré à l'échelle du pore canaliculaire pour être propagée à l'échelle du canalicule puis de l'ostéon, en utilisant une procédure d'homogénéisation périodique asymptotique. Une étude paramétrique nous a permis de cibler les paramètres agissant sur les phénomènes de transport et pouvant faire réagir les ostéocytes. Il est ressorti de cette étude que les effets électro-chimiques jouent rôle important. Nous avons donc choisi de nous focaliser sur la chimie et plus particulièrement sur les effets des flux ioniques physiologiques sur les ostéocytes dans le RLC. Des expériences, mises en place pour étayer ces aspects ont souligné l'importance des échanges chimiques entre les cellules et le fluide qui les entoure. Finalement, nous avons montré que les phénomènes de transports ayant lieu dans le RLC et dans l'ostéon interagissent les uns les autres, parachevant ainsi la description à trois échelles du tissu cortical / Transport phenomena appearing within the cortical bone lacuno-canalicular network (LCN) and the osteon were the objective of this study. We developped a three-scale model to investigate the osteocyte mechanotranduction which is at the origin of the bone remodeling process. This model took into account three physical phenomena : the electrostatics (through the Poisson-Boltzman equation), the interstitial fluid flow (modeled by a modified Stokes equation) and the ionic transport (governed by a Nernst-Planck equation). Analysis of the electrical potential distribution highlighted the importance of the electrical double layers close to the pore charged surface. These electrical double layers, as well as the interstitial fluid chemical composition, induce osmotic and electroosmotic fluid flows and affect the ionic effective diffusion within the pores. Using a periodic asymptotic homogeneisation procedure, the model at the canalicular pore scale was upscaled at the canalicular scale and then at the osteonal scale. A parametric study pointed out the relevant parameters acting on the transport phenomena and possibly affecting osteocyte mecanosensitivity. Our results emphasized the importance of the electro-chemical effects. We thus focused on the chemistry and more especially on the effects of the physiological ionic fluxes on the osteocyte. In vitro experiments and numrical simulations were performed to elucidate these questions. Our results underlined the importance of the chemical exchanges between the osteocyte and the surrounding fluid. Finally, we showed that fluid flow and chemical transport occuring within the LCN and the osteon interact with each other, thus achieving a three-scale description of the transport phenomena in the osteon

Le ranelate de strontium (RS) est un traitement anti-ostéoporotique agissant sur la formation osseuse via les ostéoblastes et la résorption osseuse via les ostéoclastes grâce au Calcium Sensing Receptor (CaSR). L’activité physique (EXE) est bien connue pour améliorer les propriétés osseuses. Dans ce travail, nous avons étudié ① l’effet de différentes activités physiques (tapis roulant et impact). Nous avons observé que 10 impacts par jour pendant 8 semaines à 45cm de hauteur ont eu des effets bénéfiques sur l’os (paramètres de microarchitecture et biomécanique, marqueurs du remodelage). ② L’étude du RS et de l’EXE sur l’os de rate ovariectomisée a montré que le RS a des effets comparables à l’EXE et que le RS+EXE ont des effets cumulatifs sur l’os (paramètres de microarchitecture et biomécanique, marqueurs du remodelage). ③ Enfin, une étude in vivo sur des MLO-Y4 a montré la présence des CaSR sur la membrane des ostéocytes et leur nombre est modulé en fonction de la concentration en RS. De plus, le RS a un effet sur la différenciation des CSM lors d’une différenciation ostéogénique en favorisant la différenciation ostéocytaire et elle est modulée par la concentration en RS. En conclusion, ce travail a démontré l’importance d’une pratique d’un exercice physique et du traitement du RS contre l’ostéoporose : maintien de la balance du remodelage osseux du côté de la formation. L’effet cumulatif du RS+EXE s’explique par le fait que le RS agit sur les ostéoblastes, ostéocytes et ostéoclastes via les CaSR et l’EXE sur les mécanorécepteurs des ostéocytes. / Strontium ranelate (SR) is an anti-osteoroporotic treatment acting on bone formation via osteoblasts and bone resorption via osteoclasts thanks to Calcium Sensing Receptor (CaSR). Physical activity is well known to improve bone properties. In this work, we studied ① different physical acticities (treadmill and impact). We observed that 10 impacts per day during 8 weeks from 45cm of height had beneficial effects on bone (microarchitecture and biomechanical parameters, bone remodeling markers). ② The study of SR and EXE effects on bone ovariectomized rats showed that RS had similar effects to EXE and SR+EXE had cumulative effects on bone (microarchitecture and biomechanical parameters, bone remodeling markers). ③ Finally, an in vivo study on MLO-Y4 showed CaSR presence on osteocyte with their number depending on SR concentration. Moreover, RS had positive effects on CSM differentiation in favor of osteocyte differentiation and it is modulated by SR concentration. In conclusion, this work has demonstrated the importance of taking physical exercise as well as SR treatment for osteoporosis: maintaining the bone remodeling in favor of bone formation. The cumulative effect of SR+EXE is explained by the fact the SR acts on osteoblasts, osteocytes and osteoclasts via CaSR and the EXE on osteocyte mechanoreceptors.