RESUMEN
Mast cells may contribute to osteoporosis development, because patients with age-related or post-menopausal osteoporosis exhibit more mast cells in the bone marrow, and mastocytosis patients frequently suffer from osteopenia. We previously showed that mast cells crucially regulated osteoclastogenesis and bone loss in ovariectomized, estrogen-depleted mice in a preclinical model for post-menopausal osteoporosis and found that granular mast cell mediators were responsible for these estrogen-dependent effects. However, the role of the key regulator of osteoclastogenesis, namely, receptor activator of NFκB ligand (RANKL), which is secreted by mast cells, in osteoporosis development has, to date, not been defined. Here, we investigated whether mast-cell-derived RANKL participates in ovariectomy (OVX)-induced bone loss by using female mice with a conditional Rankl deletion. We found that this deletion in mast cells did not influence physiological bone turnover and failed to protect against OVX-induced bone resorption in vivo, although we demonstrated that RANKL secretion was significantly reduced in estrogen-treated mast cell cultures. Furthermore, Rankl deletion in mast cells did not influence the immune phenotype in non-ovariectomized or ovariectomized mice. Therefore, other osteoclastogenic factors released by mast cells might be responsible for the onset of OVX-induced bone loss.
Asunto(s)
Resorción Ósea , Osteoporosis Posmenopáusica , Osteoporosis , Humanos , Ratones , Femenino , Animales , Osteoclastos , Mastocitos , Osteoporosis Posmenopáusica/etiología , Ligandos , Osteogénesis , FN-kappa B/farmacología , Resorción Ósea/etiología , Osteoporosis/etiología , Estrógenos/farmacología , Ovariectomía/efectos adversos , Ligando RANK/genética , Ligando RANK/farmacologíaRESUMEN
Osteoporosis is a systemic metabolic skeletal disease characterized by low bone mass and strength associated with fragility fractures. Oxidative stress, which results from elevated intracellular reactive oxygen species (ROS) and arises in the aging organism, is considered one of the critical factors contributing to osteoporosis. Mitochondrial (mt)ROS, as the superoxide anion (O2-) generated during mitochondrial respiration, are eliminated in the young organism by antioxidant defense mechanisms, including superoxide dismutase 2 (SOD2), the expression and activity of which are decreased in aging mesenchymal progenitor cells, accompanied by increased mtROS production. Using a mouse model of osteoblast lineage cells with Sod2 deficiency, we observed significant bone loss in trabecular and cortical bones accompanied by decreased osteoblast activity, increased adipocyte accumulation in the bone marrow and augmented osteoclast activity, suggestive of altered mesenchymal progenitor cell differentiation and osteoclastogenesis. Furthermore, osteoblast senescence was increased. To date, there are only a few studies suggesting a causal association between mtROS and cellular senescence in tissue in vivo. Targeting SOD2 to improve redox homeostasis could represent a potential therapeutic strategy for maintaining bone health during aging.
Asunto(s)
Osteoblastos , Osteoporosis , Superóxido Dismutasa , Animales , Ratones , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoporosis/metabolismo , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Alterations in bone strength and structure were found in knockout (KO) mouse strains with deletion of several acetylcholine receptors. Interestingly, the expression of the nicotinic acetylcholine receptors (nAChR) subunit α10 was down-regulated in osteogenic differentiated mesenchymal stem cells of patients with osteoporosis whereas the expression of subunit α9 was not altered. Since nAChR subunits α9 and α10 are often combined in a functional receptor, we analyzed here the bone of adult female KO mice with single deletion of either nAChR alpha9 (α9KO) or alpha10 (α10KO). Biomechanical testing showed a significant decrease of bending stiffness and maximal breaking force in α9KO compared to their corresponding wild type mice. Furthermore, an increase in trabecular pattern factor (Tb.Pf) and structure model index (SMI) was detected by µCT in α9KO indicating reduced bone mass. On the mRNA level a decrease of Collagen 1α1 and Connexin-43 was measured by real-time RT-PCR in α9KO while no alteration of osteoclast markers was detected in either mouse strain. Using electron microcopy we observed an increase in the number of osteocytes that showed signs of degeneration and cell death in the α9KO compared to their wild type mice, while α10KO showed no differences. In conclusion, we demonstrate alterations in bone strength, structure and bio-marker expression in α9KO mice which imply the induction of osteocyte degeneration. Thus, our data suggest that nAChR containing the α9 subunit might be involved in the homeostasis of osteocytes and therefore in bone mass regulation.
Asunto(s)
Huesos/anatomía & histología , Eliminación de Gen , Receptores Nicotínicos/genética , Fosfatasa Alcalina/metabolismo , Animales , Animales Recién Nacidos , Huesos/fisiología , Hueso Esponjoso/anatomía & histología , Hueso Cortical/anatomía & histología , Femenino , Fémur/anatomía & histología , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteocitos/metabolismo , Osteocitos/ultraestructura , Receptores Nicotínicos/deficienciaRESUMEN
The terminal complement complex (TCC) is formed on activation of the complement system, a crucial arm of innate immunity. TCC formation on cell membranes results in a transmembrane pore leading to cell lysis. In addition, sublytic TCC concentrations can modulate various cellular functions. TCC-induced effects may play a role in the pathomechanisms of inflammatory disorders of the bone, including rheumatoid arthritis and osteoarthritis. In this study, we investigated the effect of the TCC on bone turnover and repair. Mice deficient for complement component 6 (C6), an essential component for TCC assembly, and mice with a knockout of CD59, which is a negative regulator of TCC formation, were used in this study. The bone phenotype was analyzed in vivo, and bone cell behavior was analyzed ex vivo. In addition, the mice were subjected to a femur osteotomy. Under homeostatic conditions, C6-deficient mice displayed a reduced bone mass, mainly because of increased osteoclast activity. After femur fracture, the inflammatory response was altered and bone formation was disturbed, which negatively affected the healing outcome. By contrast, CD59-knockout mice only displayed minor skeletal alterations and uneventful bone healing, although the early inflammatory reaction to femur fracture was marginally enhanced. These results demonstrate that TCC-mediated effects regulate bone turnover and promote an adequate response to fracture, contributing to an uneventful healing outcome.
Asunto(s)
Regeneración Ósea , Complejo de Ataque a Membrana del Sistema Complemento , Fracturas del Fémur , Curación de Fractura , Osteoclastos , Animales , Regeneración Ósea/genética , Regeneración Ósea/inmunología , Antígenos CD59/deficiencia , Técnicas de Cultivo de Célula , Complemento C6/deficiencia , Complejo de Ataque a Membrana del Sistema Complemento/genética , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Eritrocitos/inmunología , Eritrocitos/metabolismo , Eritrocitos/patología , Fracturas del Fémur/genética , Fracturas del Fémur/inmunología , Fracturas del Fémur/metabolismo , Fracturas del Fémur/patología , Curación de Fractura/genética , Curación de Fractura/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Noqueados , Osteoclastos/inmunología , Osteoclastos/metabolismo , Osteoclastos/patología , OvinosRESUMEN
The anaphylatoxin C5a is generated upon activation of the complement system, a crucial arm of innate immunity. C5a mediates proinflammatory actions via the C5a receptor C5aR1 and thereby promotes host defence, but also modulates tissue homeostasis. There is evidence that the C5a/C5aR1 axis is critically involved both in physiological bone turnover and in inflammatory conditions affecting bone, including osteoarthritis, periodontitis, and bone fractures. C5a induces the migration and secretion of proinflammatory cytokines of osteoblasts. However, the underlying mechanisms remain elusive. Therefore, in this study we aimed to determine C5a-mediated downstream signalling in osteoblasts. Using a whole-genome microarray approach, we demonstrate that C5a activates mitogen-activated protein kinases (MAPKs) and regulates the expression of genes involved in pathways related to insulin, transforming growth factor-ß and the activator protein-1 transcription factor. Interestingly, using coimmunoprecipitation, we found an interaction between C5aR1 and Toll-like receptor 2 (TLR2) in osteoblasts. The C5aR1- and TLR2-signalling pathways converge on the activation of p38 MAPK and the generation of C-X-C motif chemokine 10, which functions, among others, as an osteoclastogenic factor. In conclusion, C5a-stimulated osteoblasts might modulate osteoclast activity and contribute to immunomodulation in inflammatory bone disorders.
Asunto(s)
Quimiocina CXCL10/genética , Complemento C5a/genética , Inflamación/genética , Receptor de Anafilatoxina C5a/genética , Receptor Toll-Like 2/genética , Anafilatoxinas/genética , Anafilatoxinas/inmunología , Anafilatoxinas/metabolismo , Animales , Enfermedades Óseas/genética , Enfermedades Óseas/inmunología , Enfermedades Óseas/patología , Remodelación Ósea/genética , Complemento C5a/inmunología , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunidad Innata/genética , Inflamación/inmunología , Inflamación/patología , Ratones , Osteoblastos/inmunología , Osteoblastos/metabolismo , Osteoclastos/inmunología , Osteoclastos/metabolismo , Osteogénesis/genética , Osteogénesis/inmunología , Transducción de Señal , Factor de Crecimiento Transformador beta/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Patients with multiple injuries frequently suffer bone fractures and are at high risk to develop fracture healing complications. Because of its key role both in systemic posttraumatic inflammation and fracture healing, the pleiotropic cytokine interleukin-6 (IL-6) may be involved in the pathomechanisms of trauma-induced compromised fracture healing. IL-6 signals are transmitted by two different mechanisms: classic signaling via the membrane-bound receptor (mIL-6R) and trans-signaling via its soluble form (sIL-6R). Herein, we investigated whether IL-6 classic and trans-signaling play different roles in bone regeneration after severe injury. Twelve-week-old C57BL/6J mice underwent combined femur osteotomy and thoracic trauma. To study the function of IL-6, either an anti-IL-6 antibody, which inhibits both IL-6 classic and trans-signaling, or a soluble glycoprotein 130 fusion protein (sgp130Fc), which selectively blocks trans-signaling, were injected 30 min and 48 h after surgery. Bone healing was assessed using cytokine analyses, flow cytometry, histology, micro-computed tomography, and biomechanical testing. Selective inhibition of IL-6 trans-signaling significantly improved the fracture healing outcome after combined injury, as confirmed by accelerated cartilage-to-bone transformation, enhanced bony bridging of the fracture gap and improved mechanical callus properties. In contrast, global IL-6 inhibition did not affect compromised fracture healing. These data suggest that classic signaling may mediate beneficial effects on bone repair after severe injury. Selective inhibition of IL-6 trans-signaling might have therapeutic potential to treat fracture healing complications in patients with concomitant injuries.
Asunto(s)
Citocinas/antagonistas & inhibidores , Curación de Fractura/inmunología , Traumatismos Torácicos/inmunología , Heridas y Lesiones/inmunología , Animales , Anticuerpos/farmacología , Citocinas/inmunología , Fémur/diagnóstico por imagen , Fémur/cirugía , Curación de Fractura/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Osteotomía , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal , Microtomografía por Rayos XRESUMEN
Magnesium phosphate minerals have captured increasing attention during the past years as suitable alternatives for calcium phosphate bone replacement materials. Here, we investigated the degradation and bone regeneration capacity of experimental struvite (MgNH4PO4·6H2O) forming magnesium phosphate cements in two different orthotopic ovine implantation models. Cements formed at powder to liquid ratios (PLR) of 2.0 and 3.0â¯gâ¯ml-1 were implanted into trabecular bone using a non-load-bearing femoral drill-hole model and a load-bearing tibial defect model. After 4, 7 and 10â¯months the implants were retrieved and cement degradation and new bone formation was analyzed by micro-computed tomography (µCT) and histomorphometry. The results showed cement degradation in concert with new bone formation at both defect locations. Both cements were almost completely degraded after 10â¯months. The struvite cement formed with a PLR of 2.0â¯gâ¯ml-1 exhibited a slightly accelerated degradation kinetics compared to the cement with a PLR of 3.0â¯gâ¯ml-1. Tartrat-resistant acid phosphatase (TRAP) staining indicated osteoclastic resorption at the cement surface. Energy dispersive X-ray analysis (EDX) revealed that small residual cement particles were mostly accumulated in the bone marrow in between newly formed bone trabeculae. Mechanical loading did not significantly increase bone formation associated with cement degradation. Concluding, struvite-forming cements might be promising bone replacement materials due to their good degradation which is coupled with new bone formation. STATEMENT OF SIGNIFICANCE: Recently, the interest in magnesium phosphate cements (MPC) for bone substitution increased, as they exhibit high initial strength, comparably elevated degradation potential and the release of valuable magnesium ions. However, only few in vivo studies, mostly including non-load-bearing defects in small animals, have been performed to analyze the degradation and regeneration capability of MPC derived compounds. The present study examined the in vivo behavior of magnesiumammoniumphosphate hexahydrate (struvite) implants with different porosity in both mechanically loaded and non-loaded defects of merino sheep. For the first time, the effect of mechanical stimuli on the biological outcome of this clinically relevant replacement material is shown and directly compared to the conventional unloaded defect situation in a large animal model.