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This chapter describes the isolation and culture of neonatal mouse calvarial osteoblasts. This primary cell population is obtained by sequential enzymatic digestion of the calvarial bone matrix and is capable of differentiating in vitro into mature osteoblasts that deposit a collagen extracellular matrix and form mineralized bone nodules. Maturation of the cultures can be monitored by gene expression analyses and staining for the presence of alkaline phosphatase or matrix mineralization. This culture system, therefore, provides a powerful model in which to test how various experimental conditions, such as the manipulation of gene expression, may affect osteoblast maturation and/or function.
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Calcificación Fisiológica/genética , Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Osteogénesis/genética , Animales , Animales Recién Nacidos , Matriz Ósea/crecimiento & desarrollo , Matriz Ósea/metabolismo , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , Osteoblastos/metabolismoRESUMEN
Recent in vitro evidence shows that glycosaminoglycans (GAGs) and proteoglycans (PGs) in bone matrix may functionally be involved in the tissue-level toughness of bone. In this study, we showed the effect of biglycan (Bgn), a small leucine-rich proteoglycan enriched in extracellular matrix of bone and the associated GAG subtype, chondroitin sulfate (CS), on the toughness of bone in vivo, using wild-type (WT) and Bgn deficient mice. The amount of total GAGs and CS in the mineralized compartment of Bgn KO mouse bone matrix decreased significantly, associated with the reduction of the toughness of bone, in comparison with those of WT mice. However, such differences between WT and Bgn KO mice diminished once the bound water was removed from bone matrix. In addition, CS was identified as the major subtype in bone matrix. We then supplemented CS to both WT and Bgn KO mice to test whether supplemental GAGs could improve the tissue-level toughness of bone. After intradermal administration of CS, the toughness of WT bone was greatly improved, with the GAGs and bound water amount in the bone matrix increased, while such improvement was not observed in Bgn KO mice or with supplementation of dermatan sulfate (DS). Moreover, CS supplemented WT mice exhibited higher bone mineral density and reduced osteoclastogenesis. Interestingly, Bgn KO bone did not show such differences irrespective of the intradermal administration of CS. In summary, the results of this study suggest that Bgn and CS in bone matrix play a pivotal role in imparting the toughness to bone most likely via retaining bound water in bone matrix. Moreover, supplementation of CS improves the toughness of bone in mouse models.
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Biglicano/genética , Matriz Ósea/crecimiento & desarrollo , Glicosaminoglicanos/metabolismo , Proteoglicanos/metabolismo , Animales , Densidad Ósea/efectos de los fármacos , Matriz Ósea/efectos de los fármacos , Matriz Ósea/metabolismo , Huesos/efectos de los fármacos , Huesos/metabolismo , Sulfatos de Condroitina/farmacología , Dermatán Sulfato/farmacología , Matriz Extracelular/genética , Glicosaminoglicanos/genética , Humanos , Ratones , Ratones Noqueados , Proteoglicanos/genética , AguaRESUMEN
OBJECTIVE: We aimed at investigating the effects of uniaxial static strain on osteoblasts in distraction osteogenesis (DO). METHODS: To simulate the mechanical stimulation of osteoblasts during DO, 10% uniaxial static strain was applied to osteoblasts using a homemade multiunit cell stretching and compressing device. Before and after applying strain stimulation, the morphological changes of osteoblasts were observed by inverted phase-contrast microscopy, Coomassie blue staining, and immunofluorescence. Alkaline phosphatase (ALP) activity, mRNA levels (proliferating cell nuclear antigen [PCNA], ALP, Runx2, osteocalcin [OCN], collagen type I, hypoxia-inducible factor- [HIF-] 1α, and vascular endothelial growth factor [VEGF]), and protein levels (Runx2, OCN, collagen type I, HIF-1α, and VEGF) were evaluated by using ALP kit, real-time quantitative reverse transcription-polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. RESULTS: After the mechanical stimulation, the cytoskeleton microfilaments were rearranged, and the cell growth direction of the osteoblasts became ordered, with their direction being at an angle of about 45° from the direction of strain. The proliferation of osteoblasts and the expression levels of mRNA and protein of ALP, Runx2, OCN, collagen type I, HIF-1α, and VEGF were significantly higher than in the nonstretch control groups. CONCLUSION: Our homemade device can exert uniaxial static strain and promote the proliferation of osteoblasts and bone matrix formation. It can be used to simulate the mechanical stimulation of osteoblasts during DO.
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Matriz Ósea/crecimiento & desarrollo , Osteoblastos/citología , Osteogénesis por Distracción/métodos , Fosfatasa Alcalina/metabolismo , Animales , Animales Recién Nacidos , Fenómenos Biomecánicos , Western Blotting , Matriz Ósea/citología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Diseño de Equipo , Regulación de la Expresión Génica , Osteoblastos/fisiología , Osteogénesis por Distracción/instrumentación , Reacción en Cadena de la Polimerasa , Ratas Sprague-Dawley , Sincalida/metabolismoRESUMEN
Bone homeostasis requires continuous remodeling of bone matrix to maintain structural integrity. This involves extensive communication between bone-forming osteoblasts and bone-resorbing osteoclasts to orchestrate balanced progenitor cell recruitment and activation. Only a few mediators controlling progenitor activation are known to date and have been targeted for intervention of bone disorders such as osteoporosis. To identify druggable pathways, we generated a medaka (Oryzias latipes) osteoporosis model, where inducible expression of receptor-activator of nuclear factor kappa-Β ligand (Rankl) leads to ectopic formation of osteoclasts and excessive bone resorption, which can be assessed by live imaging. Here we show that upon Rankl induction, osteoblast progenitors up-regulate expression of the chemokine ligand Cxcl9l. Ectopic expression of Cxcl9l recruits mpeg1-positive macrophages to bone matrix and triggers their differentiation into osteoclasts. We also demonstrate that the chemokine receptor Cxcr3.2 is expressed in a distinct subset of macrophages in the aorta-gonad-mesonephros (AGM). Live imaging revealed that upon Rankl induction, Cxcr3.2-positive macrophages get activated, migrate to bone matrix, and differentiate into osteoclasts. Importantly, mutations in cxcr3.2 prevent macrophage recruitment and osteoclast differentiation. Furthermore, Cxcr3.2 inhibition by the chemical antagonists AMG487 and NBI-74330 also reduced osteoclast recruitment and protected bone integrity against osteoporotic insult. Our data identify a mechanism for progenitor recruitment to bone resorption sites and Cxcl9l and Cxcr3.2 as potential druggable regulators of bone homeostasis and osteoporosis.
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Matriz Ósea/metabolismo , Quimiocina CXCL9/metabolismo , Proteínas de Peces/metabolismo , Oryzias/metabolismo , Osteoclastos/metabolismo , Osteoporosis/metabolismo , Receptores CXCR3/metabolismo , Células Madre/metabolismo , Animales , Matriz Ósea/crecimiento & desarrollo , Diferenciación Celular , Quimiocina CXCL9/genética , Modelos Animales de Enfermedad , Proteínas de Peces/genética , Humanos , Macrófagos/metabolismo , Oryzias/genética , Oryzias/crecimiento & desarrollo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoclastos/citología , Osteoporosis/genética , Osteoporosis/fisiopatología , Unión Proteica , Receptores CXCR3/genética , Células Madre/citologíaRESUMEN
Osteoblasts secrete collagen and isolate bone matrix from extracellular space. In the matrix, alkaline phosphatase generates phosphate that combines with calcium to form mineral, liberating 8 H+ per 10 Ca+2 deposited. However, pH-dependent hydroxyapatite deposition on bone collagen had not been shown. We studied the dependency of hydroxyapatite deposition on type I collagen on pH and phosphate by surface plasmon resonance in 0-5 mM phosphate at pH 6.8-7.4. Mineral deposition saturated at <1 mM Ca2+ but was sensitive to phosphate. Mineral deposition was reversible, consistent with amorphous precipitation; stable deposition requiring EDTA removal appeared with time. At pH 6.8, little hydroxyapatite deposited on collagen; mineral accumulation increased 10-fold at pH 7.4. Previously, we showed high expression Na+/H+ exchanger (NHE) and ClC transporters in osteoblasts. We hypothesized that, in combination, these move protons across osteoblasts to the general extracellular space. We made osteoblast membrane vesicles by nitrogen cavitation and used acridine orange quenching to characterize proton transport. We found H+ transport dependent on gradients of chloride or sodium, consistent with apical osteoblast ClC family Cl-,H+ antiporters and basolateral osteoblast NHE family Na+/H+ exchangers. Little, if any, active H+ transport, supported by ATP, occurred. Major transporters include cariporide-sensitive NHE1 in basolateral membranes and ClC3 and ClC5 in apical osteoblast membranes. The mineralization inhibitor levamisole reduced bone formation and expression of alkaline phosphatase, NHE1, and ClC5. We conclude that mineral deposition in bone collagen is pH-dependent, in keeping with H+ removal by Cl-,H+ antiporters and Na+/H+-exchangers. Periodic orientation hydroxyapatite is organized on type I collagen-coiled coils.
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Calcificación Fisiológica/genética , Canales de Cloruro/genética , Intercambiador 1 de Sodio-Hidrógeno/genética , Adenosina Trifosfato/metabolismo , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Matriz Ósea/crecimiento & desarrollo , Matriz Ósea/metabolismo , Calcio/metabolismo , Diferenciación Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Colágeno Tipo I/química , Colágeno Tipo I/genética , Durapatita/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Transporte Iónico/genética , Levamisol/farmacología , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Fosfatos/metabolismo , Sodio/metabolismo , Resonancia por Plasmón de Superficie , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
The mineralization process is crucial to the load-bearing characteristics of the bone extracellular matrix. In this work, we have studied the spatiotemporal dynamics of mineral deposition by human bone marrow mesenchymal stem cells differentiating toward osteoblasts promoted by the presence of exogenous hydroxyapatite nanoparticles. At the molecular level, the added nanoparticles positively modulated the expression of bone-specific markers and enhanced calcified matrix deposition during osteogenic differentiation. The nucleation, growth and spatial arrangement of newly deposited hydroxyapatite nanocrystals have been evaluated using scanning micro X-ray diffraction and scanning micro X-ray fluorescence. As leading results, we have found the emergence of a complex scenario where the spatial organization and temporal evolution of the process exhibit heterogeneous and self-organizing dynamics. At the same time the possibility of controlling the differentiation kinetics, through the addition of synthetic nanoparticles, paves the way to empower the generation of more structured bone scaffolds in tissue engineering and to design new drugs in regenerative medicine.
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Matriz Ósea/crecimiento & desarrollo , Durapatita/farmacología , Células Madre Mesenquimatosas/citología , Nanopartículas , Osteogénesis , Ingeniería de Tejidos , Diferenciación Celular , Células Cultivadas , Humanos , Andamios del TejidoRESUMEN
Background: The study of osteoblasts and their osteogenic functions is essential in order to understand them and their applications in implantology. In this sense, this study try to study BMP-2 production and bone matrix deposition, in addition to other biological variables, in osteoblasts cultured on a rough double acid-etched titanium surface (Osseotite®, Biomet 3i, Palm Beach Garden, Florida, USA) in comparison to a smooth titanium surface (machined) and a control Petri dish. Material and Methods: An in vitro prospective study. NHOst human osteoblasts from the femur were cultured on three different surfaces: Control group: 25-mm methacrylate dish (n = 6); Machined group: titanium discs with machined surface (n = 6) and Experimental group: titanium discs with a double acid-etched nitric and hydrofluoric Osseotite® acid surface (n = 6). A quantification of the mitochondrial membrane potential, and studies of apoptosis, mobility and adhesion, bone productivity (BMP-2) and cellular bone synthesis were carried out after culturing the three groups for forty-eight hours. Results: A statistically significant difference was observed in the production of BMP-2 between the experimental group and the other two groups (22.33% ± 11.06 vs. 13.10% ± 5.51 in the machined group and 3.88% ± 3.43 in the control group). Differences in cellular bone synthesis were also observed between the groups (28.34% ± 14.4% in the experimental group vs. 20.03% ± 6.79 in the machined group and 19.34% ± 15.93% in the control group). Conclusions: In comparison with machined surfaces, Osseotite® surfaces favor BMP-2 production and bone synthesis as a result of the osteoblasts in contact with it (AU)
No disponible
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Humanos , Calcificación Fisiológica , Osteoblastos , Proteína Morfogenética Ósea 2/farmacocinética , Técnicas In Vitro , Estudios Prospectivos , Proteínas del Citoesqueleto/fisiología , Apoptosis/fisiología , Matriz Ósea/crecimiento & desarrollo , Supervivencia Celular/fisiologíaRESUMEN
OBJECTIVE: To evaluate bone formation in human extraction sockets with absorbed surrounding walls augmented with Bio-Oss and Bio-Gide after a 6-month healing period by histologic and histomorphometric analyses. METHODS: Six fresh molar tooth extraction sockets in 6 patients who required periodontally compromised moral tooth extraction were included in this study. The six fresh extraction sockets were grafted with Bio-Oss particle covered with Bio-Gide. The 2.8 mm×6.0 mm cylindric bone specimens were taken from the graft sites with aid of stent 6 months after the surgery. Histologic and histomorphometric analyses were performed. RESULTS: The histological results showed Bio-Oss particles were easily distinguished from the newly formed bone, small amounts of new bone were formed among the Bio-Oss particles, large amounts of connective tissue were found. Intimate contact between the newly formed bone and the small part of Bio-Oss particles was present. All the biopsy cylinders measurement demonstrated a high inter-individual variability in the percentage of the bone, connective tissues and Bio-Oss particles. The new bone occupied 11.54% (0-28.40%) of the total area; the connective tissues were 53.42% (34.08%-74.59%) and the Bio-Oss particles were 35.04% (13.92%-50.87%). The percentage of the particles, which were in contact with bone tissues, amounted to 20.13% (0-48.50%). CONCLUSION: Sites grafted with Bio-Oss particles covered with Bio-Gide were comprised of connective tissues and small amounts of newly formed bone surrounding the graft particles.
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Matriz Ósea/anatomía & histología , Matriz Ósea/crecimiento & desarrollo , Colágeno/farmacología , Colágeno/uso terapéutico , Tejido Conectivo/anatomía & histología , Tejido Conectivo/efectos de los fármacos , Tejido Conectivo/crecimiento & desarrollo , Minerales/farmacología , Minerales/uso terapéutico , Alveolo Dental/anatomía & histología , Alveolo Dental/efectos de los fármacos , Alveolo Dental/crecimiento & desarrollo , Matriz Ósea/efectos de los fármacos , Sustitutos de Huesos/uso terapéutico , Humanos , Diente Molar , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Extracción Dental , Alveolo Dental/lesiones , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiologíaRESUMEN
PURPOSE: To observe, histologically, bone induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) in onlay grafted and sinus lifted alveolaris. MATERIAL AND METHODS: Eighteen patients were treated with rhBMP-2 at concentration 1.5 mg/mL with an absorbable collagen sponge (ACS). The treated bone was harvested with small trephine bur at 5 or 7 months after surgery for the micro Computer Scanning (CT) and light microscopic observation. RESULTS: Micro CT showed clearly 3-dimensional trabecular bone structure. New bone formation and bone marrow structure were observed in the observed area. Osteoblastic cells existed along the new bone, and osteopontin was localized in the bone matrix weakly. In the connective tissue around the new bone, many CD34-positive blood vessel cells were present. Some tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells were observed around bone at this stage. CONCLUSION: The application of rhBMP-2 with ACS induced a new bone accompanied by blood vessels in atrophied alveolaris. This suggests that rhBMP-2 is capable of osteoinductivity in human jaw.
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Proceso Alveolar/crecimiento & desarrollo , Proteína Morfogenética Ósea 2/farmacología , Proceso Alveolar/anatomía & histología , Proceso Alveolar/química , Proceso Alveolar/diagnóstico por imagen , Matriz Ósea/anatomía & histología , Matriz Ósea/diagnóstico por imagen , Matriz Ósea/crecimiento & desarrollo , Humanos , Osteopontina/análisis , Proteínas Recombinantes/farmacología , Elevación del Piso del Seno Maxilar/métodos , Microtomografía por Rayos XRESUMEN
The aim of the study was to assess the interaction of of octacalcium phosphate (OCP) with bone matrix and cells and its impact on the process of bone generation. The survey was conducted on animal model: critical hipbone defect was created in 12 230-250 g Wister rats. The animals were then divided in two groups. In group 1 (6 animals) defect was left to heal under blood clot and in group 2 (6 animals) it was filled with OCP. Three animals with no defect served as a control group. It was showed significant (p<0.05) increase of the area of the newly formed bone tissue and its direct correlation with duration of observation.
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Matriz Ósea/efectos de los fármacos , Sustitutos de Huesos/farmacología , Fosfatos de Calcio/farmacología , Osteogénesis/efectos de los fármacos , Animales , Matriz Ósea/citología , Matriz Ósea/crecimiento & desarrollo , Fémur/citología , Fémur/efectos de los fármacos , Fémur/lesiones , Ratas , Ratas WistarRESUMEN
Objective: To produce bionic bone material that is consistent with human bone in chemical composition and molecular structure using rat tail tendon collagen type â . Methods: The type â collagen derived from rat tail was extracted by acetic acid to form collagen fibers. The reconstructed collagen fibers were placed in the mineralized solution to mimic bone mineralization for 2-6 days. Bone mineralization was observed by transmission electron microscopy and electron diffraction.Results: Collagen fibers with characteristic D-Band structure were reconstructed by using rat tail tendon collagen type â extracted with acid hydrolysis method. Transmission electron microscopy and electron diffraction showed that calcium hydroxyapatite precursor infiltrated into the collagen fibers, and the collagen fibers were partially mineralized after 2 days of mineralization; the collagen fibers were completely mineralized and bionic bone material of typeâ collagen/calcium hydroxyapatite was formed after 6 days of mineralization.Conclusion: The collagen type â can be extracted from rat tail tendon by acid hydrolysis method, and can be reformed and mineralized to form the bionic bone material which mimics human bone in chemical composition and the molecular structure.
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Materiales Biocompatibles/síntesis química , Sustitutos de Huesos/síntesis química , Calcificación Fisiológica , Colágeno Tipo I/química , Tendones/química , Ingeniería de Tejidos/métodos , Animales , Matriz Ósea/química , Matriz Ósea/crecimiento & desarrollo , Huesos/anatomía & histología , Huesos/química , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/ultraestructura , Humanos , Hidroxiapatitas/química , Ratas , Cola (estructura animal) , Tendones/ultraestructuraRESUMEN
As a determinant of skeletal fragility, the organic matrix is responsible for the post-yield and creep behavior of bone and for its toughness, while the mineral apatite acts on stiffness. Specific to the fibula and ulna in children, greenstick fractures show a plastic in vivo mechanical behavior before bone fracture. During growth, the immature form of collagen enzymatic cross-links gradually decreases, to be replaced by the mature form until adolescence, subsequently remaining constant throughout adult life. However, the link between the cortical bone organic matrix and greenstick fractures in children remains to be explored. Here, we sought to determine: 1) whether plastic bending fractures can occur in vitro, by testing cortical bone samples from children's fibula and 2) whether the post-yield behavior (ωp plastic energy) of cortical bone before fracture is related to total quantity of the collagen matrix, or to the quantity of mature and immature enzymatic cross-links and the quantity of non-enzymatic cross-links. We used a two-step approach; first, a 3-point microbending device tested 22 fibula machined bone samples from 7 children and 3 elderly adults until fracture. Second, biochemical analysis by HPLC was performed on the sample fragments. When pooling two groups of donors, children and elderly adults, results show a rank correlation between total energy dissipated before fracture and age and a linear correlation between plastic energy dissipated before fracture and ratio of immature/mature cross-links. A collagen matrix with more immature cross-links (i.e. a higher immature/mature cross-link ratio) is more likely to plastically deform before fracture. We conclude that this ratio in the sub-nanostructure of the organic matrix in cortical bone from the fibula may go some way towards explaining the variance in post-yield behavior. From a clinical point of view, therefore, our results provide a potential explanation of the presence of greenstick fractures in children.
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Matriz Ósea/crecimiento & desarrollo , Matriz Ósea/fisiopatología , Fracturas Óseas/fisiopatología , Anciano , Anciano de 80 o más Años , Fenómenos Biomecánicos , Matriz Ósea/química , Niño , Preescolar , Cromatografía Líquida de Alta Presión , Peroné/fisiología , Humanos , Estrés MecánicoRESUMEN
OBJECTIVES: This article aims to study differences in the bone formation and the graft resorption of two bone graft substitutes (BGS). Besides, it is our attempt to observe possible qualitative and quantitative differences in the bone reparation of the outer layer covered by collagen membrane and the uncovered inner layer in close contact with dura mater. MATERIAL AND METHODS: Twelve rabbits were employed. Deproteinized bovine bone (DBB) and ß-tricalcium phosphate (BTCP) were used as BGS. Four subcritical round defects (7 mm) were drilled in the cranial vault, removing both cortical walls. One of the holes was filled with DBB, and other was filled with BTCP. Each symmetrical position to DBB and BTCP was left empty. The whole defect set was covered with a collagen membrane. Histological and morphometric analysis was performed for 1, 4, 8, 16, 32 and 52 weeks. Morphometry measurements were carried out taking into account the whole defect and splitting inner and outer areas. RESULTS: In DBB sites, a rapid bone growth is observed, linking the remaining particles and integrating them into the bone matrix. Permanence of these DBB particles from week 16 onwards restrains the growth of bone fraction. A greater bone growth appears in areas repaired with BTCP than in those repaired with DBB, both in the outer layer (under-membrane) and the inner layer (over dura mater). In DBB sites, a slower growth is observed in the inner layer, with no significant differences in the final bone fraction at both strata. CONCLUSIONS: Both materials favour the closure of the defects provoked. In both cases, a synergistic effect with the collagen membrane is observed. DBB remains integrated in the bone matrix, while BTCP displays a pattern of highly developed progressive resorption with an outstanding bone fraction development.
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Matriz Ósea/crecimiento & desarrollo , Regeneración Ósea/fisiología , Sustitutos de Huesos/uso terapéutico , Fosfatos de Calcio/uso terapéutico , Regeneración Tisular Dirigida/métodos , Osteogénesis/fisiología , Pérdida de Hueso Alveolar/prevención & control , Pérdida de Hueso Alveolar/cirugía , Animales , Bovinos , Estudios Longitudinales , Conejos , Cráneo/cirugíaRESUMEN
Craniofacial sutures and synchondroses form the boundaries among bones in the human skull, providing functional, developmental and evolutionary information. Bone articulations in the skull arise due to interactions between genetic regulatory mechanisms and epigenetic factors such as functional matrices (soft tissues and cranial cavities), which mediate bone growth. These matrices are largely acknowledged for their influence on shaping the bones of the skull; however, it is not fully understood to what extent functional matrices mediate the formation of bone articulations. Aiming to identify whether or not functional matrices are key developmental factors guiding the formation of bone articulations, we have built a network null model of the skull that simulates unconstrained bone growth. This null model predicts bone articulations that arise due to a process of bone growth that is uniform in rate, direction and timing. By comparing predicted articulations with the actual bone articulations of the human skull, we have identified which boundaries specifically need the presence of functional matrices for their formation. We show that functional matrices are necessary to connect facial bones, whereas an unconstrained bone growth is sufficient to connect non-facial bones. This finding challenges the role of the brain in the formation of boundaries between bones in the braincase without neglecting its effect on skull shape. Ultimately, our null model suggests where to look for modified developmental mechanisms promoting changes in bone growth patterns that could affect the development and evolution of the head skeleton.
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Desarrollo Óseo , Matriz Ósea/crecimiento & desarrollo , Modelos Biológicos , Cráneo/crecimiento & desarrollo , Algoritmos , Evolución Biológica , Suturas Craneales/crecimiento & desarrollo , Huesos Faciales/crecimiento & desarrollo , Humanos , Cráneo/anatomía & histologíaRESUMEN
Although the regulation of osteoblast and adipocyte differentiation from mesenchymal stem cells has been studied for some time, very little is known about what regulates their appearance in discrete regions of the embryo. Here we show that, as in other vertebrates, zebrafish osteoblasts and adipocytes originate in part from cephalic neural crest (CNC) precursors. We investigated the roles that the retinoic acid (RA) and Peroxisome proliferator-activated receptor gamma (Pparg) pathways play in vivo and found that both pathways act on CNC to direct adipocyte differentiation at the expense of osteoblast formation. In addition, we identify two distinct roles for RA in the osteoblast lineage: an early role in blocking the recruitment of osteoblasts and a later role in mature osteoblasts to promote bone matrix synthesis. These findings might help to increase our understanding of skeletal and obesity-related diseases and aid in the development of stem cell-based regenerative therapies.
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Cresta Neural/citología , PPAR gamma/fisiología , Tretinoina/fisiología , Adipocitos/citología , Animales , Matriz Ósea/crecimiento & desarrollo , Diferenciación Celular , Linaje de la Célula , Osteoblastos/citología , Células Madre/citología , Pez CebraRESUMEN
The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.
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Matriz Ósea/crecimiento & desarrollo , Expresión Génica/efectos de la radiación , Terapia por Luz de Baja Intensidad , Oseointegración/efectos de la radiación , Osteoblastos/efectos de la radiación , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/genética , Análisis de Varianza , Proteína Morfogenética Ósea 7/biosíntesis , Proteína Morfogenética Ósea 7/genética , Células Cultivadas/efectos de la radiación , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Humanos , Sialoproteína de Unión a Integrina/biosíntesis , Sialoproteína de Unión a Integrina/genética , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Láseres de Semiconductores/uso terapéutico , Osteoblastos/metabolismo , Osteocalcina/biosíntesis , Osteocalcina/genética , Osteopontina/biosíntesis , Osteopontina/genética , Osteoprotegerina/biosíntesis , Osteoprotegerina/genética , Ligando RANK/biosíntesis , Ligando RANK/genética , Estadísticas no Paramétricas , TitanioRESUMEN
The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.
Este estudo teve como objetivo investigar o efeito do laser diodo de gálio-alumÃnio-arsênio (GaAlAs) em células osteoblásticas humanas cultivadas sobre discos de Ti. Para tanto, células osteoblásticas foram obtidas por digestão enzimática de osso alveolar humano e cultivadas sobre discos de Ti por 17 dias. As células foram submetidas à irradiação no 3º e 7º dias na dose de 3 J/cm2 e comprimento de onda de 780 nm e células não irradiadas foram usadas como controle. A irradiação não alterou a proliferação celular, atividade de ALP e formação de matriz mineralizada. Microscopia por epifluorescência indicou que após 24 h da aplicação do laser, as culturas irradiadas apresentaram áreas sem células, que mais tarde foram repovoadas por células em fase de proliferação e menos diferenciadas. O laser aumentou a expressão gênica relativa da ALP, OC, BSP e BMP-7 e reduziu a de RUNX2, OPN e OPG. Os resultados indicam que a terapia com laser modula de forma complexa as respostas celulares, estimulando a diferenciação osteoblástica. Assim, é possÃvel sugerir possÃveis benefÃcios do laser na osseointegração de implantes de Ti apesar do efeito deletério à s células imediatamente após a irradiação.
Asunto(s)
Humanos , Matriz Ósea/crecimiento & desarrollo , Expresión Génica/efectos de la radiación , Terapia por Luz de Baja Intensidad , Oseointegración/efectos de la radiación , Osteoblastos/efectos de la radiación , Análisis de Varianza , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/genética , /biosíntesis , /genética , Células Cultivadas/efectos de la radiación , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Sialoproteína de Unión a Integrina/biosíntesis , Sialoproteína de Unión a Integrina/genética , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Láseres de Semiconductores/uso terapéutico , Osteoblastos/metabolismo , Osteocalcina/biosíntesis , Osteocalcina/genética , Osteopontina/biosíntesis , Osteopontina/genética , Osteoprotegerina/biosíntesis , Osteoprotegerina/genética , Ligando RANK/biosíntesis , Ligando RANK/genética , Estadísticas no Paramétricas , TitanioRESUMEN
OBJECTIVE: Periradicular healing involves osteoblasts that are dependent on the Runt-related transcription factor 2 (Runx2). The purpose of this study was to determine if mineral trioxide aggregate (MTA) root-end filling materials support Runx2 expression in osteoblasts. STUDY DESIGN: Human alveolar bone cells were grown on alternative formulations of MTA. Cell-surface interactions were visualized by scanning electron microscopy. Gene expression was examined by reverse-transcription polymerase chain reaction and Western blot analysis. RESULTS: Cells attached to and spread out on MTA surfaces within 24 hours and formed a collagenous matrix overlay within 1 week of growth. Runx2 expression increased from low levels in the 24-hour cultures to an abundance during 2 weeks of growth and differentiation on MTA surfaces and on tissue culture plastic controls. The cells responded similarly to ProRoot, Tooth-Colored MTA, and MTA mixed with local anesthetic solution. CONCLUSION: Mineral trioxide aggregate materials support cell attachment and Runx2 expression in osteoblasts.
Asunto(s)
Compuestos de Aluminio/farmacología , Compuestos de Calcio/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Óxidos/farmacología , Materiales de Obturación del Conducto Radicular/farmacología , Silicatos/farmacología , Compuestos de Aluminio/química , Proceso Alveolar/citología , Matriz Ósea/citología , Matriz Ósea/crecimiento & desarrollo , Compuestos de Calcio/química , Adhesión Celular , Diferenciación Celular , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Combinación de Medicamentos , Expresión Génica , Humanos , Ensayo de Materiales , Óxidos/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Materiales de Obturación del Conducto Radicular/química , Silicatos/químicaRESUMEN
Collagen XXIV is an ill-characterized fibrillar collagen that is predominantly expressed in the forming skeleton of the mouse embryo. Here we report that the Col24al gene is constitutively transcribed in the trabecular bone and periosteum of the newborn mouse as well. The bone specificity of Col24al was further documented using three well-characterized cell culture models of osteoblast differentiation. These in vitro analyses indicated that Col24al transcription is activated at about the same time as that of the osteocalcin gene, and gradually increases to eventually plateau as osteoblasts begin to deposit a mineralizing matrix. These findings lend further support to the hypothesis that collagen XXIV may be implicated in the formation of a mineralization-competent bone matrix.
Asunto(s)
Desarrollo Óseo/genética , Diferenciación Celular/genética , Colágeno/genética , Regulación del Desarrollo de la Expresión Génica/genética , Osteoblastos/metabolismo , Osteogénesis/genética , Animales , Matriz Ósea/crecimiento & desarrollo , Matriz Ósea/metabolismo , Calcificación Fisiológica/genética , Línea Celular Tumoral , Marcadores Genéticos/genética , Ratones , Células 3T3 NIH , Osteocalcina/genética , Periostio/crecimiento & desarrollo , Periostio/metabolismo , RatasRESUMEN
Articular cartilage repair is still a challenge in orthopaedic surgery. Although many treatment options have been developed in the last decade, true regeneration of hyaline articular cartilage is yet to be accomplished. In vitro experiments are useful for evaluating cell-matrix interactions under controlled parameters. When introducing new treatment options into clinical routine, adequate animal models are capable of closing the gap between in vitro experiments and the clinical use in human beings. We developed an animal model in the Göttingen minipig (GMP) to evaluate the healing of osteochondral or full-thickness cartilage defects. The defects were located in the middle third of the medial portion of the patellofemoral joint at both distal femurs. Chondral defects were 6.3 mm, osteochondral defects either 5.4 or 6.3 mm in diameter and 8 or 10 mm deep. In both defects the endogenous repair response showed incomplete repair tissue formation up to 12 months postoperatively. Based on its limited capability for endogenous repair of chondral and osteochondral defects, the GMP is a useful model for critical assessment of new treatment strategies in articular cartilage tissue engineering.