RESUMO
Recent studies have revealed that the effects of estrogen deficiency are not restricted to osteoclasts and bone resorption, but that bone matrix composition is altered and osteoblasts exhibit an impaired response to mechanical stimulation. In this study, we test the hypothesis that estrogen depletion alters osteogenic differentiation and matrix production by mechanically stimulated osteoblasts in vitro. MC3T3-E1 cells were pre-treated with estrogen for 14 days, after which estrogen was withdrawn or inhibited with Fulvestrant up to 14 days. Fluid shear stress (FSS) was applied using an orbital shaker. Under estrogen depletion in static culture, osteogenic marker (ALP) and gene expression (Runx2) were decreased at 2 and after 7 days of estrogen depletion, respectively. In addition, up to 7 day the inhibition of the estrogen receptor significantly decreased fibronectin expression (FN1) under static conditions. Under estrogen depletion and daily mechanical stimulation, changes in expression of Runx2 occurred earlier (4 days) and by 14 days, changes in matrix production (Col1a1) were reported. We propose that changes in osteoblast differentiation and impaired matrix production during estrogen depletion may contribute to the altered quality of the bone and act as a contributing factor to increased bone fragility in postmenopausal osteoporosis.
Assuntos
Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Estrogênios/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Animais , Reabsorção Óssea/tratamento farmacológico , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Diferenciação Celular/fisiologia , Estrogênios/metabolismo , Humanos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Osteoporose Pós-Menopausa/tratamento farmacológico , Osteoporose Pós-Menopausa/metabolismo , Estresse MecânicoRESUMO
OBJECTIVE/BACKGROUND: Arterial calcification, a process that mimics bone formation, is an independent risk factor of cardiovascular morbidity and mortality, and has a significant impact on surgical and endovascular procedures and outcomes. Research efforts have focused mainly on the coronary arteries, while data regarding the femoral territory remain scarce. METHODS: Femoral endarterectomy specimens, clinical data, and plasma from a cohort of patients were collected prospectively. Histological analysis was performed to characterize the cellular populations present in the atherosclerotic lesions, and that were potentially involved in the formation of bone like arterial calcification known as osteoid metaplasia (OM). Enzyme linked immunosorbent assays and cell culture assays were conducted in order to understand the cellular and molecular mechanisms underlying the formation of OM in the lesions. RESULTS: Twenty-eight of the 43 femoral plaques (65%) displayed OM. OM included osteoblast and osteoclast like cells, but very few of the latter exhibited the functional ability to resorb mineral tissue. As in bone, osteoprotegerin (OPG) was significantly associated with the presence of OM (p = .04). Likewise, a high plasma OPG/receptor activator for the nuclear factor kappa B ligand (RANKL) ratio was significantly associated with the presence of OM (p = .03). At the cellular level, there was a greater presence of pericytes in OM+ compared with OM- lesions (5.59 ± 1.09 vs. 2.42 ± 0.58, percentage of area staining [region of interest]; p = .04); in vitro, pericytes were able to inhibit the osteoblastic differentiation of human mesenchymal stem cells, suggesting that they are involved in regulating arterial calcification. CONCLUSION: These results suggest that bone like arterial calcification (OM) is highly prevalent at femoral level. Pericyte cells and the OPG/RANK/RANKL triad seem to be critical to the formation of this ectopic osteoid tissue and represent interesting potential therapeutic targets to reduce the clinical impact of arterial calcification.
Assuntos
Artéria Femoral/metabolismo , Osteoprotegerina/metabolismo , Pericitos/metabolismo , Doença Arterial Periférica/metabolismo , Calcificação Vascular/metabolismo , Idoso , Células Cultivadas , Endarterectomia , Inglaterra/epidemiologia , Feminino , Artéria Femoral/patologia , Artéria Femoral/cirurgia , Humanos , Masculino , Pericitos/patologia , Doença Arterial Periférica/epidemiologia , Doença Arterial Periférica/patologia , Doença Arterial Periférica/cirurgia , Placa Aterosclerótica , Prevalência , Estudos Prospectivos , Ligante RANK/metabolismo , Calcificação Vascular/epidemiologia , Calcificação Vascular/patologiaRESUMO
Recent studies have demonstrated increased bone mineral heterogeneity following estrogen withdrawal in vivo. Such changes likely contribute to fracture risk during post-menopausal osteoporosis since tissue mineralization is correlated with bone strength and stiffness. However, the cellular mechanisms responsible for increased mineral variability have not yet been distinguished. The objective of this study is to elucidate how alterations in mineral distribution are initiated during estrogen depletion. Specifically, we tested two separate hypotheses; (1) estrogen deficiency directly alters osteoblast mineralization and (2) estrogen deficiency increases bone cell apoptosis. Osteoblast-like cells (MC3T3-E1) and osteocyte-like cells (MLO-Y4) were pretreated with or without estrogen (17ß-estradiol) for 14 days. Estrogen deficiency was subsequently induced by either withdrawing estrogen from cells or blocking estrogen receptors using an estrogen antagonist, fulvestrant (ICI 182,780). Cell number (Hoechst DNA), alkaline phosphatase activity (p-NPP), mineralization (alizarin red) and apoptosis (Caspase 3/7) were evaluated. Whether estrogen withdrawal altered apoptosis rates in the presence of an apoptosis promoting agent (etoposide) was also determined. Interestingly, estrogen withdrawal from cells accustomed to estrogen exposure caused significantly increased osteoblast mineralization and osteocyte apoptosis compared with continued estrogen treatment. In contrast, blocking estrogen receptors with fulvestrant abrogated the mineralization induced by estrogen treatment. When apoptosis was induced using etoposide, cells undergoing estrogen withdrawal increased apoptosis compared to cells with continued estrogen treatment. Recognizing the underlying mechanisms regulating bone cell mineralization and apoptosis during estrogen deficiency and their consequences is necessary to further our knowledge of osteoporosis.
Assuntos
Apoptose/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Estrogênios/farmacologia , Osteoblastos/citologia , Osteócitos/citologia , Fosfatase Alcalina/metabolismo , Animais , Caspase 3/metabolismo , Caspase 7/metabolismo , Contagem de Células , Linhagem Celular , Estrogênios/deficiência , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Osteócitos/efeitos dos fármacos , Osteócitos/enzimologiaRESUMO
BACKGROUND: The clinical impact of stent fractures is still controversial. This study analyzed the incidence and the clinical impact of stent fractures after stenting of long femoropopliteal lesions. METHODS: From November 2008 to October 2009, 58 patients (62 limbs) were treated in a single center with a primary nitinol self-expanding stent for Trans-Atlantic Inter-Consensus (TASC) C and D de novo femoropopliteal lesions. Patients were prospectively followed by medical and duplex scan examinations. Stent fractures were assessed by biplane X-rays at 12 months. Logistic regression analysis was performed. RESULTS: At 1 year a complete follow-up was obtained in 42 limbs/90 stents. The median length of the stented segment was 240 ± 180 cm with a mean of 2.1 (1-4) stents per patient. Sixteen stents (17.8%) were fractured: one type I (asymptomatic); seven type II (2 restenosis); five type III (asymptomatic), and three type IV stent fractures (1 restenosis). Stent diameter (p = .04) and stent implantation in the distal part of the superficial femoral artery (p = .05) were positively associated with stent fractures. Stent fracture had no influence on restenosis. CONCLUSION: This study suggests that the high stent fracture rate associated with endovascular treatment of long femoropopliteal lesions should be balanced with its low clinical impact.
Assuntos
Angioplastia com Balão/instrumentação , Artéria Femoral , Doença Arterial Periférica/terapia , Artéria Poplítea , Falha de Prótese , Stents , Idoso , Idoso de 80 Anos ou mais , Ligas , Angioplastia com Balão/efeitos adversos , Distribuição de Qui-Quadrado , Constrição Patológica , Feminino , Artéria Femoral/diagnóstico por imagem , Artéria Femoral/fisiopatologia , Humanos , Estimativa de Kaplan-Meier , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/fisiopatologia , Artéria Poplítea/diagnóstico por imagem , Artéria Poplítea/fisiopatologia , Valor Preditivo dos Testes , Estudos Prospectivos , Desenho de Prótese , Radiografia , Recidiva , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Ultrassonografia Doppler Dupla , Grau de Desobstrução VascularRESUMO
Although osteoporosis reduces overall bone mass causing bone fragility, recent studies report that the remaining bone tissue is significantly stiffer. Preliminary studies indicate that alterations in bone tissue mineral content might explain these changes, albeit that other studies report conflicting observations. The objective of this study is to quantify whether the distribution of bone tissue mineral is altered during oestrogen deficiency. Individual trabeculae were harvested from the proximal femur of 7 ovariectomised sheep (OVX), sacrificed 12 months post-surgery, and 5 age-matched controls. Mineral content (wt% Ca) was determined using a quantitative backscattered scanning electron microscopy imaging approach. Mineral heterogeneity within individual trabeculae was compared by calculating the full width at half maximum (FWHM) of mineral density distributions. Mean calcium content, the spatial distribution of mineral within trabeculae and the inter-trabecular variation between regions of proximal femora were also compared. Oestrogen deficiency increased mineral heterogeneity within individual trabeculae compared to healthy controls, as measured by FWHM (3.57 ± 0.68 vs. 3.17 ± 0.36 wt% Ca, p = 0.04). In particular mineral variability increased between superficial and deep regions of trabeculae of OVX animals (p = 0.04). Interestingly, mineralisation variability between greater and lesser trochanters (i.e. intertrochanteric fracture line) was increased in OVX compared to CON, as indicated by a greater % difference in the standard deviation of trabecular mineral content (77.11 ± 11.70 vs. 45.64 ± 23.70 %, p = 0.03). Such changes are undetectable by evaluating the mean mineral content of bone tissue, but may contribute to changes in bone mechanical strength following osteoporotic bone loss.
Assuntos
Densidade Óssea , Estrogênios/deficiência , Fêmur/metabolismo , Animais , Cálcio/metabolismo , Fêmur/fisiopatologia , Osteoporose/metabolismo , Osteoporose/fisiopatologia , Ovariectomia , OvinosRESUMO
Bone is the most transplanted tissue human with 1 million procedures every year in Europe. Surgical interventions for bone repair are required for varied reasons such as trauma resulting non-union fractures, or diseases including osteoporosis or osteonecrosis. Autologous bone grafting is the gold standard in bone regeneration but it requires a second surgery with associated pain and complications, and is also limited by harvested bone quantity. Synthetic bone substitutes lack the osteoinductive properties to heal large bone defects. Cell therapies based on bone marrow or ex vivo expanded mesenchymal stromal stem cells (MSCs) in association with synthetic calcium phosphate (CaP) bone substitutes may be alternatives to autologous bone grafting. This manuscript reviews the different conventional biological and synthetic bone grafting procedures as well as the more recently introduced cell therapy approaches used in orthopaedic surgery for bone regeneration. Some clinical studies have demonstrated safety and efficacy of these approaches but regeneration of large bone defects remain challenging due to the absence of rapid and adequate vascularisation. Future directions in the field of bone regeneration are presented, such as testing alternative cell sources or in situ fabrication of vascularized bone grafts in patients.
Assuntos
Regeneração Óssea , Transplante Ósseo/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Animais , Células da Medula Óssea/citologia , Regeneração Óssea/fisiologia , Substitutos Ósseos/uso terapêutico , Osso e Ossos/irrigação sanguínea , Fosfatos de Cálcio/uso terapêutico , Separação Celular/métodos , Células Cultivadas , Durapatita/uso terapêutico , Necrose da Cabeça do Fêmur/terapia , Previsões , Consolidação da Fratura/fisiologia , Fraturas não Consolidadas/terapia , Humanos , Injeções Intralesionais , Neovascularização Fisiológica , Alicerces Teciduais , Transplante AutólogoRESUMO
Questionnaires on exercise practices, reasons for exercising, factors that would increase exercise, and demographics were mailed to a stratified random sample of 19,510 active duty personnel in all of the United States military services who were stationed at 38 large military installations and a number of remote locations. Responses were returned by 8,572 (55%). Approximately 63% exercised three times a week for 15-20 minutes, and 15% did not exercise at all. Mean BMI was 25.2. The top five reasons for exercising were to improve physical conditioning, have fun, manage weight, improve appearance and manage stress.
Assuntos
Índice de Massa Corporal , Exercício Físico , Militares/psicologia , Militares/estatística & dados numéricos , Estudos Transversais , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Masculino , Motivação , Inquéritos e Questionários , Fatores de Tempo , Estados UnidosRESUMO
Satisfaction with fitness facilities has long been accepted as a positive contributing factor to physical activity, readiness, and overall quality of life for military families. Our findings are based on a random sample of military families surveyed at 38 installations worldwide and at remote locations. A total of 8,572 service member and 3,493 spouse (55% and 32% response rate, respectively) questionnaires were completed and returned. Overall, members were satisfied with fitness facilities and programs, but spouses were less satisfied and more unfamiliar with these facilities. Most valued programs were fitness centers and swimming pools. Members reported that elimination of fitness facilities would greatly decrease the quality of life on installations, particularly at sites outside the continental United States, whereas spouses reported that this would not have a dramatic effect. The results of this study could be used to direct funding allocations, improve the health and fitness of military families, and suggest areas for further research.
Assuntos
Atitude Frente a Saúde , Academias de Ginástica/normas , Militares/psicologia , Cônjuges/psicologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Qualidade de Vida , Inquéritos e Questionários , Piscinas , Estados UnidosRESUMO
The quantity and distribution of bone tissue mineral are key determinants of bone strength. Recent research revealed altered mineral distribution within sheep femora following estrogen deficiency. Rapid increases in bone remodeling occur at the onset of estrogen deficiency and abate over time. Therefore, altered tissue mineralization might be a transient characteristic of osteoporosis. Bisphosphonates reduce fracture incidence by 40-60% but increases in bone mineral density are insufficient to explain such changes. In this study the hypotheses that bone tissue mineralization is altered over prolonged estrogen depletion and bisphosphonate treatment were tested. Quantitative backscattered imaging (qBEI) was used to quantify bone mineral density distribution (BMDD) parameters (mean, FWHM) in trabeculae from the proximal femora of an ovariectomized sheep model that underwent estrogen deficiency for 31 months, an ovariectomized group administered with Zoledronic acid and age-matched controls. To assess the effects of normal ageing and prolonged estrogen deficiency, data were compared to BMDD data from sheep that were estrogen deficient for 12 months and age-matched controls. This study reports that normal ageing increases mean mineralization and mineral heterogeneity at a trabecular level. In contrast, prolonged estrogen deficiency leads to significantly decreased mean mineralization and further exacerbates increases in mineral heterogeneity. Interestingly, ZOL treatment of OVX sheep significantly reduced tissue mineral variability, both at a trabecular level and between femoral regions. Together, these findings indicate that ZOL treatment acts to reverse the increased mineral heterogeneity occurring during estrogen deficiency, which may contribute to its capacity to reduce osteoporotic fractures.
Assuntos
Envelhecimento/fisiologia , Conservadores da Densidade Óssea/farmacologia , Densidade Óssea/efeitos dos fármacos , Difosfonatos/farmacologia , Estrogênios/deficiência , Fêmur/efeitos dos fármacos , Fêmur/fisiologia , Imidazóis/farmacologia , Animais , Calcificação Fisiológica/efeitos dos fármacos , Fêmur/citologia , Ovinos , Fatores de Tempo , Ácido ZoledrônicoRESUMO
Extracellular mechanical cues have been shown to have a profound effect on osteogenic cell behaviour. However, it is not known precisely how these cues alter intracellular mechanics to initiate changes in cell behaviour. In this study, a combination of in vitro culture of MC3T3-E1 cells and finite-element modelling was used to investigate the effects of passive differences in substrate stiffness on intracellular mechanics. Cells on collagen-based substrates were classified based on the presence of cell processes and the dimensions of various cellular features were quantified. Focal adhesion (FA) density was quantified from immunohistochemical staining, while cell and substrate stiffnesses were measured using a live-cell atomic force microscope. Computational models of cell morphologies were developed using an applied contraction of the cell body to simulate active cell contraction. The results showed that FA density is directly related to cell morphology, while the effect of substrate stiffness on internal cell tension was modulated by both cell morphology and FA density, as investigated by varying the number of adhesion sites present in each morphological model. We propose that the cells desire to achieve a homeostatic stress state may play a role in osteogenic cell differentiation in response to extracellular mechanical cues.
Assuntos
Diferenciação Celular , Adesões Focais/metabolismo , Modelos Biológicos , Osteoblastos , Osteogênese , Estresse Fisiológico , Animais , Adesão Celular , Linhagem Celular , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismoRESUMO
We provide evidence that Salmonella typhimurium kills phagocytes by an unusual proinflammatory mechanism of necrosis that is distinguishable from apoptosis. Infection stimulated a distinctly diffuse pattern of DNA fragmentation in macrophages, which contrasted with the marked nuclear condensation displayed by control cells undergoing chemically induced apoptosis. In apoptotic cells, DNA fragmentation and nuclear condensation result from caspase-3-mediated proteolysis; caspases also subvert necrotic cell death by cleaving and inactivating poly ADP-ribose polymerase (PARP). Caspase-3 was not activated during Salmonella infection, and PARP remained in its active, uncleaved state. Another hallmark of apoptosis is sustained membrane integrity during cell death; yet, infected macrophages rapidly lost membrane integrity, as indicated by simultaneous exposure of phosphatidylserine with the uptake of vital dye and the release of the cytoplasmic enzyme lactate dehydrogenase. During experimentally induced necrosis, lethal ion fluxes through the plasma membrane can be prevented by exogenous glycine; similarly, glycine completely blocked Salmonella-induced cytotoxicity. Finally, inhibition of the interleukin (IL)-1-converting enzyme caspase-1 blocked the death of infected macrophages, but not control cells induced to undergo apoptosis or necrosis. Thus, Salmonella-infected macrophages are killed by an unusual caspase-1-dependent mechanism of necrosis.
Assuntos
Apoptose/fisiologia , Caspase 1/fisiologia , Macrófagos/microbiologia , Salmonella typhimurium/fisiologia , Membrana Celular/metabolismo , Marcação In Situ das Extremidades Cortadas , Transporte de Íons , NecroseRESUMO
Co-ordinate expression of genes associated with pathogenicity in Vibrio cholerae requires two transcription activators, ToxR and ToxT. Work carried out to date suggests that ToxR activates transcription of the toxT gene and that ToxT directly activates transcription of several genes whose products play a role in colonization or CT production by V. cholerae. Previous work also suggests that ToxR can directly activate transcription of the CT operon (ctxAB) independently of ToxT, thereby implying a degree of complexity in control of the cixAB operon not found with other genes of the ToxR regulon. We tested the regulatory cascade model of virulence gene expression by constructing strains of classical and El Tor V. cholerae deleted for the coding sequence for the putative DNA-binding domain of toxT. Phenotypic analysis of these strains suggests that V. cholerae has ToxT-dependent and ToxT-independent branches of its virulence regulon. The results also raise questions about the precise role for ToxR in activation of ctxAB transcription.