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1.
J Bone Miner Res ; 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31269275

RESUMO

Osteoblast (OB) lineage cells are an important source of vascular endothelial growth factor (VEGF), which is critical for bone growth and repair. During bone development, pubertal differences in males and females exist, but little is known about whether VEGF signaling contributes to skeletal sexual dimorphism. We have found that in mice, conditional disruption of VEGF in osteocalcin-expressing cells (OcnVEGFKO) exerts a divergent influence on morphological, cellular, and whole bone properties between sexes. Furthermore, we describe an underlying sexual divergence in VEGF signaling in OB cultures in vitro independent of circulating sex hormones. High-resolution synchrotron computed tomography and backscattered scanning electron microscopy revealed, in males, extensive unmineralized osteoid encasing enlarged blood vessel canals and osteocyte lacunae in cortical bone after VEGF deletion, which contributed to increased porosity. VEGF was deleted in male and female long bone-derived OBs (OBVEGKO) in vitro and Raman spectroscopic analyses of mineral and matrix repertoires highlighted differences between male and female OBVEGFKO cells, with increased immature phosphate species prevalent in male OBVEGFKO cultures versus wild type (WT). Further sexual dimorphism was observed in bone marrow endothelial cell gene expression in vitro after VEGF deletion and in sclerostin protein expression, which was increased in male OcnVEGFKO bones versus WT. The impact of altered OB matrix composition after VEGF deletion on whole bone geometry was assessed between sexes, although significant differences between OcnVEGFKO and WT were identified only in females. Our results suggest that bone-derived VEGF regulates matrix mineralization and vascularization distinctly in males and females, which results in divergent physical bone traits.

2.
J Bone Miner Res ; 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31295366

RESUMO

Fibrous dysplasia of bone/McCune-Albright syndrome (Polyostotic FD/MAS; OMIM#174800) is a crippling skeletal disease caused by gain-of-function mutations of Gs α. Enhanced bone resorption is a recurrent histological feature of FD and a major cause of fragility of affected bones. Previous work suggests that increased bone resorption in FD is driven by RANKL and some studies have shown that the anti-RANKL monoclonal antibody, denosumab, reduces bone turnover and bone pain in FD patients. However, the effect of RANKL inhibition on the histopathology of FD and its impact on the natural history of the disease remain to be assessed. In this study, we treated the EF1α-Gs αR201C mice, which develop an FD-like phenotype, with an anti-mouse RANKL monoclonal antibody. We found that the treatment induced marked radiographic and microscopic changes at affected skeletal sites in 2-month-old mice. The involved skeletal segments became sclerotic due to the deposition of new, highly mineralized bone within developing FD lesions and showed a higher mechanical resistance compared to affected segments from untreated transgenic mice. Similar changes were also detected in older mice with a full-blown skeletal phenotype. The administration of anti-mouse RANKL antibody arrested the growth of established lesions and, in young mice, prevented the appearance of new ones. However, after drug withdrawal, the newly formed bone was remodelled into FD tissue and the disease progression resumed in young mice. Taken together, our results show that the anti-RANKL antibody significantly affected the bone pathology and natural history of FD in the mouse. Pending further work on the prevention and management of relapse after treatment discontinuation, our preclinical study suggests that RANKL inhibition may be an effective therapeutic option for FD patients. © 2019 American Society for Bone and Mineral Research.

3.
Bone ; 127: 427-435, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31306808

RESUMO

OBJECTIVE: Bone loss in anorexia nervosa and following bariatric surgery is associated with an elevated circulating concentration of the gastrointestinal, anorexigenic hormone, peptide YY (PYY). Selective deletion of the PYY receptor Y1R in osteoblasts or Y2R in the hypothalamus results in high bone mass, but deletion of PYY in mice has resulted in conflicting skeletal phenotypes leading to uncertainty regarding its role in the regulation of bone mass. As PYY analogs are under development for treatment of obesity, we aimed to clarify the relationship between PYY and bone mass. METHODS: The skeletal phenotype of Pyy knockout (KO) mice was investigated during growth (postnatal day P14) and adulthood (P70 and P186) using X-ray microradiography, micro-CT, back-scattered electron scanning electron microscopy (BSE-SEM), histomorphometry and biomechanical testing. RESULTS: Bones from juvenile and Pyy KO mice were longer (P < 0.001), with decreased bone mineral content (P < 0.001). Whereas, bones from adult Pyy KO mice had increased bone mineral content (P < 0.05) with increased mineralisation of both cortical (P < 0.001) and trabecular (P < 0.001) compartments. Long bones from adult Pyy KO mice were stronger (maximum load P < 0.001), with increased stiffness (P < 0.01) and toughness (P < 0.05) compared to wild-type (WT) control mice despite increased cortical vascularity and porosity (P < 0.001). The increased bone mass and strength in Pyy KO mice resulted from increases in trabecular (P < 0.01) and cortical bone formation (P < 0.05). CONCLUSIONS: These findings demonstrate that PYY acts as a negative regulator of osteoblastic bone formation, implicating increased PYY levels in the pathogenesis of bone loss during anorexia or following bariatric surgery.

4.
Am J Hum Genet ; 104(5): 925-935, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-30982609

RESUMO

Colony stimulating factor 1 receptor (CSF1R) plays key roles in regulating development and function of the monocyte/macrophage lineage, including microglia and osteoclasts. Mono-allelic mutations of CSF1R are known to cause hereditary diffuse leukoencephalopathy with spheroids (HDLS), an adult-onset progressive neurodegenerative disorder. Here, we report seven affected individuals from three unrelated families who had bi-allelic CSF1R mutations. In addition to early-onset HDLS-like neurological disorders, they had brain malformations and skeletal dysplasia compatible to dysosteosclerosis (DOS) or Pyle disease. We identified five CSF1R mutations that were homozygous or compound heterozygous in these affected individuals. Two of them were deep intronic mutations resulting in abnormal inclusion of intron sequences in the mRNA. Compared with Csf1r-null mice, the skeletal and neural phenotypes of the affected individuals appeared milder and variable, suggesting that at least one of the mutations in each affected individual is hypomorphic. Our results characterized a unique human skeletal phenotype caused by CSF1R deficiency and implied that bi-allelic CSF1R mutations cause a spectrum of neurological and skeletal disorders, probably depending on the residual CSF1R function.

5.
Methods Mol Biol ; 1914: 571-616, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30729487

RESUMO

This chapter describes methods for preparing samples of bone and bone cells for scanning electron microscopy (SEM). Backscattered electron (BSE) imaging is by far the most useful in the bone field, followed by secondary electrons (SE) and the energy dispersive X-ray (EDX) analytical modes. Samples may have 3D detail in a 3D surface, or be topography-free, polished or micromilled, resin-embedded block surfaces, or resin casts of space compartments surrounded by bone matrix. Methods for cells include fixation, drying, looking at undersides of bone cells, and metallic conductive coating. Maceration with alkaline bacterial pronase, hypochlorite, hydrogen peroxide, and sodium or potassium hydroxide to remove cells and unmineralized matrix is described in detail. Attention is given especially to methods for 3D BSE SEM imaging of bone samples. Recommendations are made for the types of resin embedding for BSE SEM imaging. Correlated confocal and SEM imaging of PMMA embedded bone requires the use of glycerol to coverslip. Cathodoluminescence (CL) mode SEM imaging is an alternative for visualizing fluorescent mineralizing front labels such as calcein and tetracyclines. Making spatial casts from PMMA or other resin-embedded samples is an important use of this material. Correlation with other imaging means, including microradiography and microtomography is important. Shipping wet bone samples between labs is best done in glycerol. Control of the vacuum pressure in the SEM sample chamber (now generally available) can be used to eliminate "charging" problems which were common, for example, with large, complex, cancellous bone samples.


Assuntos
Osso e Ossos/diagnóstico por imagem , Técnicas de Preparação Histocitológica/métodos , Imagem Tridimensional/métodos , Microscopia Eletrônica de Varredura/métodos , Imagem Multimodal/métodos , Animais , Osso e Ossos/ultraestrutura , Células Cultivadas , Técnicas de Preparação Histocitológica/instrumentação , Humanos , Imagem Tridimensional/instrumentação , Microrradiografia/instrumentação , Microrradiografia/métodos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia Eletrônica de Varredura/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Imagem Multimodal/instrumentação , Osteoclastos , Osteócitos , Software , Microtomografia por Raio-X/instrumentação , Microtomografia por Raio-X/métodos
6.
Hum Mol Genet ; 26(23): 4572-4587, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28973168

RESUMO

Bone remodeling is a balanced process between bone synthesis and degradation, maintaining homeostasis and a constant bone mass in adult life. Imbalance will lead to conditions such as osteoporosis or hyperostosis. Osteoblasts build bone, becoming embedded in bone matrix as mature osteocytes. Osteocytes have a role in sensing and translating mechanical loads into biochemical signals, regulating the differentiation and activity of osteoblasts residing at the bone surface through the secretion of Sclerostin (SOST), an inhibitor of WNT signaling. Excessive mechanical load can lead to activation of cellular stress responses altering cell behavior and differentiation. The unfolded protein response (UPR) is a shared pathway utilized by cells to cope with stress stimuli. We showed that in a transgenic mouse model, activation of the UPR in early differentiating osteocytes delays maturation, maintaining active bone synthesis. In addition, expression of SOST is delayed or suppressed; resulting in active WNT signaling and enhanced periosteal bone formation, and the combined outcome is generalized hyperostosis. A clear relationship between the activation of the unfolded protein response was established and the onset of hyperostosis that can be suppressed with a chemical chaperone, sodium 4-phenobutyrate (4-PBA). As the phenotype is highly consistent with craniodiaphyseal dysplasia (CDD; OMIM 122860), we propose activation of the UPR could be part of the disease mechanism for CDD patients as these patients are heterozygous for SOST mutations that impair protein folding and secretion. Thus, therapeutic agents ameliorating protein folding or the UPR can be considered as a potential therapeutic treatment.


Assuntos
Anormalidades Craniofaciais/metabolismo , Hiperostose/metabolismo , Osteocondrodisplasias/metabolismo , Osteócitos/metabolismo , Resposta a Proteínas não Dobradas , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Remodelação Óssea/fisiologia , Osso e Ossos/metabolismo , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , Marcadores Genéticos/genética , Humanos , Hiperostose/genética , Hiperostose/patologia , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Osteócitos/patologia , Osteogênese/fisiologia , Fenilbutiratos/farmacologia , Estresse Mecânico , Via de Sinalização Wnt
7.
J Anat ; 231(2): 298-308, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28737011

RESUMO

The phosphatase PHOSPHO1 is involved in the initiation of biomineralisation. Bones in Phospho1 knockout (KO) mice show histological osteomalacia with frequent bowing of long bones and spontaneous fractures: they contain less mineral, with smaller mineral crystals. However, the consequences of Phospho1 ablation on the microscale structure of bone are not yet fully elucidated. Tibias and femurs obtained from wild-type and Phospho1 null (KO) mice (25-32 weeks old) were embedded in PMMA, cut and polished to produce near longitudinal sections. Block surfaces were studied using 20 kV backscattered-electron (BSE) imaging, and again after iodine staining to reveal non-mineralised matrix and cellular components. For 3D characterisation, we used X-ray micro-tomography. Bones opened with carbide milling tools to expose endosteal surfaces were macerated using an alkaline bacterial pronase enzyme detergent, 5% hydrogen peroxide and 7% sodium hypochlorite solutions to produce 3D surfaces for study with 3D BSE scanning electron microscopy (SEM). Extensive regions of both compact cortical and trabecular bone matrix in Phospho1 KO mice contained no significant mineral and/or showed arrested mineralisation fronts, characterised by a failure in the fusion of the calcospherite-like, separately mineralising, individual micro-volumes within bone. Osteoclastic resorption of the uncalcified matrix in Phospho1 KO mice was attenuated compared with surrounding normally mineralised bone. The extent and position of this aberrant biomineralisation varied considerably between animals, contralateral limbs and anatomical sites. The most frequent manifestation lay, however, in the nearly complete failure of mineralisation in the bone surrounding the numerous transverse blood vessel canals in the cortices. In conclusion, SEM disclosed defective mineralising fronts and extensive patchy osteomalacia, which has previously not been recognised. These data further confirm the role of this phosphatase in physiological skeletal mineralisation.


Assuntos
Osso e Ossos/patologia , Osso e Ossos/ultraestrutura , Osteomalacia/patologia , Monoéster Fosfórico Hidrolases/deficiência , Animais , Calcificação Fisiológica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
8.
Endocrinology ; 158(9): 3055-3066, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28637283

RESUMO

T3 is an important regulator of skeletal development and adult bone maintenance. Thyroid hormone action requires efficient transport of T4 and T3 into target cells. We hypothesized that monocarboxylate transporter (MCT) 8, encoded by Mct8 on the X-chromosome, is an essential thyroid hormone transporter in bone. To test this hypothesis, we determined the juvenile and adult skeletal phenotypes of male Mct8 knockout mice (Mct8KO) and Mct8D1D2KO compound mutants, which additionally lack the ability to convert the prohormone T4 to the active hormone T3. Prenatal skeletal development was normal in both Mct8KO and Mct8D1D2KO mice, whereas postnatal endochondral ossification and linear growth were delayed in both Mct8KO and Mct8D1D2KO mice. Furthermore, bone mass and mineralization were decreased in adult Mct8KO and Mct8D1D2KO mice, and compound mutants also had reduced bone strength. Delayed bone development and maturation in Mct8KO and Mct8D1D2KO mice is consistent with decreased thyroid hormone action in growth plate chondrocytes despite elevated serum T3 concentrations, whereas low bone mass and osteoporosis reflects increased thyroid hormone action in adult bone due to elevated systemic T3 levels. These studies identify an essential physiological requirement for MCT8 in chondrocytes, and demonstrate a role for additional transporters in other skeletal cells during adult bone maintenance.


Assuntos
Osso e Ossos/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Animais , Desenvolvimento Ósseo/genética , Osso e Ossos/embriologia , Osso e Ossos/fisiologia , Condrócitos/fisiologia , Desenvolvimento Fetal/genética , Iodeto Peroxidase/genética , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Doenças da Glândula Tireoide/genética , Doenças da Glândula Tireoide/metabolismo , Glândula Tireoide/metabolismo , Glândula Tireoide/fisiologia , Hormônios Tireóideos/sangue
9.
Skeletal Radiol ; 46(10): 1435-1439, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28660402

RESUMO

Bisphosphonates (BPs) are currently used in the treatment of diverse bone diseases including fibrous dysplasia of bone (FD). In pediatric patients, a radiographic consequence of cyclical administration of BPs is the development of apo-, epi-, and meta-physeal sclerotic bands, otherwise known as zebra lines, which result from the temporary inhibition of osteoclastic activity at the time of drug treatment. We report here on a child with McCune-Albright syndrome (FD in addition to hyperfunctioning endocrinopathies and skin hyperpigmentation) treated with cyclical intravenous infusions of pamidronate in which conventional radiography, contact microradiography, histology, and backscattered electron image analysis demonstrated that zebra lines formed only where bone was normal, were arrested at the boundary between FD-unaffected and FD-affected bone where bone is sclerotic, and were absent within the undermineralized FD bone. Moreover, in spite of the treatment, the FD lesions continued to expand. This case report is unique because no previously published studies correlated the radiographic and the histologic features of BP-induced zebra lines in the metaphysis of an FD-affected long bone of the limbs.


Assuntos
Difosfonatos/efeitos adversos , Fêmur/efeitos dos fármacos , Fêmur/diagnóstico por imagem , Displasia Fibrosa Poliostótica/diagnóstico por imagem , Displasia Fibrosa Poliostótica/tratamento farmacológico , Adolescente , Progressão da Doença , Feminino , Displasia Fibrosa Poliostótica/cirurgia , Humanos , Pamidronato
10.
Int J Mol Sci ; 18(1)2016 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-28035954

RESUMO

Mice harbouring a dentin matrix protein 1 (Dmp1) promoter-driven human diphtheria toxin (DT) receptor (HDTR) transgene (Tg) have recently been used to attain targeted ablation of osteocytes by diphtheria toxin (DT) treatment in order to define osteocyte function. Use of these Tg mice has asserted mechano- and novel paracrine regulatory osteocyte functions. To explore osteocyte roles fully, we sought to confirm the selectivity of DT effects in these transgenic mice. However, our findings revealed incomplete DT-induced osteocyte ablation, prevalent HDTR misexpression, as well as more prominent histopathological DT-induced changes in multiple organs in Tg than in wild-type (WT) littermate mice. Mechanistic evidence for DT action, via prominent regulation of phosphorylation status of elongation factor-2 (EF-2), was also found in many non-skeletal tissues in Tg mice; indicative of direct "off-target" DT action. Finally, very rapid deterioration in health and welfare status in response to DT treatment was observed in these Tg when compared to WT control mice. Together, these data lead us to conclude that alternative models for osteocyte ablation should be sought and caution be exercised when drawing conclusions from experiments using these Tg mice alone.


Assuntos
Proteínas da Matriz Extracelular/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Transgenes , Animais , Osso e Ossos/metabolismo , Encéfalo/metabolismo , Toxina Diftérica/toxicidade , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Rim/metabolismo , Camundongos , Miocárdio/metabolismo , Especificidade de Órgãos , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Regiões Promotoras Genéticas
12.
Curr Opin Pharmacol ; 22: 107-14, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25978274

RESUMO

Studying severe phenotypes of rare syndromes can elucidate disease mechanisms of more common disorders and identify potential therapeutic targets. Lessons from rare bone diseases contributed to the development of the most successful class of bone active agents, the bisphosphonates. More recent research on rare bone diseases has helped elucidate key pathways and identify new targets in bone resorption and bone formation including cathepsin K and sclerostin, for which drugs are now in clinical trials. By contrast, there has been much less focus on rare cartilage diseases and osteoarthritis (OA) remains a common disease with no effective therapy. Investigation of rare cartilage syndromes is identifying new potential targets in OA including GDF5 and lubricin. Research on the arthropathy of the ultra-rare disease alkaptonuria has identified several new features of the OA phenotype, including high density mineralized protrusions (HDMPs) which constitute a newly identified mechanism of joint destruction.


Assuntos
Doenças Ósseas/fisiopatologia , Doenças das Cartilagens/fisiopatologia , Doenças Raras/fisiopatologia , Animais , Conservadores da Densidade Óssea/farmacologia , Conservadores da Densidade Óssea/uso terapêutico , Doenças Ósseas/tratamento farmacológico , Doenças Ósseas/epidemiologia , Doenças das Cartilagens/tratamento farmacológico , Doenças das Cartilagens/epidemiologia , Difosfonatos/farmacologia , Difosfonatos/uso terapêutico , Desenho de Drogas , Humanos , Terapia de Alvo Molecular , Osteoartrite/tratamento farmacológico , Osteoartrite/fisiopatologia , Fenótipo , Doenças Raras/tratamento farmacológico
13.
Int J Mol Sci ; 16(5): 9600-11, 2015 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-25927581

RESUMO

We studied changes in articular calcified cartilage (ACC) and subchondral bone (SCB) in the third carpal bones (C3) of Standardbred racehorses with naturally-occurring repetitive loading-induced osteoarthritis (OA). Two osteochondral cores were harvested from dorsal sites from each of 15 post-mortem C3 and classified as control or as showing early or advanced OA changes from visual inspection. We re-examined X-ray micro-computed tomography (µCT) image sets for the presence of high-density mineral infill (HDMI) in ACC cracks and possible high-density mineralized protrusions (HDMP) from the ACC mineralizing (tidemark) front (MF) into hyaline articular cartilage (HAC). We hypothesized and we show that 20-µm µCT resolution in 10-mm diameter samples is sufficient to detect HDMI and HDMP: these are lost upon tissue decalcification for routine paraffin wax histology owing to their predominant mineral content. The findings show that µCT is sufficient to discover HDMI and HDMP, which were seen in 2/10 controls, 6/9 early OA and 8/10 advanced OA cases. This is the first report of HDMI and HDMP in the equine carpus and in the Standardbred breed and the first to rely solely on µCT. HDMP are a candidate cause for mechanical tissue destruction in OA.


Assuntos
Calcinose/complicações , Ossos do Carpo/patologia , Cartilagem Articular/patologia , Osteoartrite/complicações , Osteoartrite/patologia , Estresse Mecânico , Animais , Calcinose/diagnóstico por imagem , Calcinose/patologia , Ossos do Carpo/diagnóstico por imagem , Cartilagem Articular/diagnóstico por imagem , Cavalos , Osteoartrite/diagnóstico por imagem , Interpretação de Imagem Radiográfica Assistida por Computador , Microtomografia por Raio-X
14.
Endocrinology ; 156(9): 3098-113, 2015 09.
Artigo em Inglês | MEDLINE | ID: mdl-26018249

RESUMO

The ancestral glycoprotein hormone thyrostimulin is a heterodimer of unique glycoprotein hormone subunit alpha (GPA)2 and glycoprotein hormone subunit beta (GPB)5 subunits with high affinity for the TSH receptor. Transgenic overexpression of GPB5 in mice results in cranial abnormalities, but the role of thyrostimulin in bone remains unknown. We hypothesized that thyrostimulin exerts paracrine actions in bone and determined: 1) GPA2 and GPB5 expression in osteoblasts and osteoclasts, 2) the skeletal consequences of thyrostimulin deficiency in GPB5 knockout (KO) mice, and 3) osteoblast and osteoclast responses to thyrostimulin treatment. Gpa2 and Gpb5 expression was identified in the newborn skeleton but declined rapidly thereafter. GPA2 and GPB5 mRNAs were also expressed in primary osteoblasts and osteoclasts at varying concentrations. Juvenile thyrostimulin-deficient mice had increased bone volume and mineralization as a result of increased osteoblastic bone formation. However, thyrostimulin failed to induce a canonical cAMP response or activate the noncanonical Akt, ERK, or mitogen-activated protein kinase (P38) signaling pathways in primary calvarial or bone marrow stromal cell-derived osteoblasts. Furthermore, thyrostimulin did not directly inhibit osteoblast proliferation, differentiation or mineralization in vitro. These studies identify thyrostimulin as a negative but indirect regulator of osteoblastic bone formation during skeletal development.


Assuntos
Osso e Ossos/metabolismo , Glicoproteínas/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Animais , Densidade Óssea , Células CHO , Calcificação Fisiológica , Cricetinae , Cricetulus , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Comunicação Parácrina , Fenótipo , Receptores da Tireotropina/metabolismo , Tireotropina/metabolismo
16.
J Bone Miner Res ; 30(6): 1030-43, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25487351

RESUMO

We recently reported the generation and initial characterization of the first direct model of human fibrous dysplasia (FD; OMIM #174800), obtained through the constitutive systemic expression of one of the disease-causing mutations, Gsα(R201C) , in the mouse. To define the specific pathogenetic role(s) of individual cell types within the stromal/osteogenic system in FD, we generated mice expressing Gsα(R201C) selectively in mature osteoblasts using the 2.3kb Col1a1 promoter. We show here that this results in a striking high bone mass phenotype but not in a mimicry of human FD. The high bone mass phenotype involves specifically a deforming excess of cortical bone and prolonged and ectopic cortical bone remodeling. Expression of genes characteristic of late stages of bone cell differentiation/maturation is profoundly altered as a result of expression of Gsα(R201C) in osteoblasts, and expression of the Wnt inhibitor Sost is reduced. Although high bone mass is, in fact, a feature of some types/stages of FD lesions in humans, it is marrow fibrosis, localized loss of adipocytes and hematopoietic tissue, osteomalacia, and osteolytic changes that together represent the characteristic pathological profile of FD, as well as the sources of specific morbidity. None of these features are reproduced in mice with osteoblast-specific expression of Gsα(R201C) . We further show that hematopoietic progenitor/stem cells, as well as more mature cell compartments, and adipocyte development are normal in these mice. These data demonstrate that effects of Gsα mutations underpinning FD-defining tissue changes and morbidity do not reflect the effects of the mutations on osteoblasts proper.


Assuntos
Osso e Ossos , Displasia Fibrosa Óssea , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Mutação de Sentido Incorreto , Osteoblastos , Substituição de Aminoácidos , Animais , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Cromograninas , Modelos Animais de Doenças , Displasia Fibrosa Óssea/genética , Displasia Fibrosa Óssea/metabolismo , Displasia Fibrosa Óssea/patologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Tamanho do Órgão , Osteoblastos/metabolismo , Osteoblastos/fisiologia
17.
J Bone Miner Res ; 30(5): 786-95, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25418329

RESUMO

Bone is a strong and tough material composed of apatite mineral, organic matter, and water. Changes in composition and organization of these building blocks affect bone's mechanical integrity. Skeletal disorders often affect bone's mineral phase, either by variations in the collagen or directly altering mineralization. The aim of the current study was to explore the differences in the mineral of brittle and ductile cortical bone at the mineral (nm) and tissue (µm) levels using two mouse phenotypes. Osteogenesis imperfecta model, oim(-/-) , mice have a defect in the collagen, which leads to brittle bone; PHOSPHO1 mutants, Phospho1(-/-) , have ductile bone resulting from altered mineralization. Oim(-/-) and Phospho1(-/-) were compared with their respective wild-type controls. Femora were defatted and ground to powder to measure average mineral crystal size using X-ray diffraction (XRD) and to monitor the bulk mineral to matrix ratio via thermogravimetric analysis (TGA). XRD scans were run after TGA for phase identification to assess the fractions of hydroxyapatite and ß-tricalcium phosphate. Tibiae were embedded to measure elastic properties with nanoindentation and the extent of mineralization with backscattered electron microscopy (BSE SEM). Results revealed that although both pathology models had extremely different whole-bone mechanics, they both had smaller apatite crystals, lower bulk mineral to matrix ratio, and showed more thermal conversion to ß-tricalcium phosphate than their wild types, indicating deviations from stoichiometric hydroxyapatite in the original mineral. In contrast, the degree of mineralization of bone matrix was different for each strain: brittle oim(-/-) were hypermineralized, whereas ductile Phospho1(-/-) were hypomineralized. Despite differences in the mineralization, nanoscale alterations in the mineral were associated with reduced tissue elastic moduli in both pathologies. Results indicated that alterations from normal crystal size, composition, and structure are correlated with reduced mechanical integrity of bone.


Assuntos
Minerais/metabolismo , Tíbia/fisiologia , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Matriz Óssea/efeitos dos fármacos , Matriz Óssea/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Fosfatos de Cálcio/farmacologia , Cristalização , Elasticidade/efeitos dos fármacos , Camundongos , Pós , Termogravimetria , Tíbia/efeitos dos fármacos , Difração de Raios X
18.
Bone ; 69: 139-47, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25260930

RESUMO

Ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyse nucleotide triphosphates to the corresponding nucleotide monophosphates and the mineralisation inhibitor, pyrophosphate (PPi). This study examined the role of NPP1 in osteocytes, osteoclasts and cortical bone, using a mouse model lacking NPP1 (Enpp1(-/-)). We used microcomputed tomography (µCT) to investigate how NPP1 deletion affects cortical bone structure; excised humerus bones from 8, 15 and 22-week old mice were scanned at 0.9 µm. Although no changes were evident in the cortical bone of 8-week old Enpp1(-/-) mice, significant differences were observed in older animals. Cortical bone volume was decreased 28% in 22-week Enpp1(-/-) mice, whilst cortical porosity was reduced 30% and 60% at 15 and 22-weeks, respectively. This was accompanied by up to a 15% decrease in closed pore diameter and a 55% reduction in the number of pores. Cortical thickness was reduced up to 35% in 15 and 22-week Enpp1(-/-) animals and the endosteal diameter was increased up to 23%. Thus, the cortical bone from Enpp1(-/-) mice was thinner and less porous, with a larger marrow space. Scanning electron microscopy (SEM) revealed a decrease in the size and number of blood vessel channels in the cortical bone as well as a 40% reduction in the mean plan area of osteocyte lacunae. We noted that the number of viable osteocytes isolated from the long bones of Enpp1(-/-) mice was decreased ≤50%. In contrast, osteoclast formation and resorptive activity were unaffected by NPP1 deletion. µCT and histological analysis of Enpp1(-/-) mice also revealed calcification of the joints and vertebrae as well as soft tissues including the whisker follicles, ear pinna and trachea. This calcification worsened as the animals aged. Together, these data highlight the key role of NPP1 in regulating calcification of both soft and skeletal tissues.


Assuntos
Osso e Ossos/diagnóstico por imagem , Calcificação Fisiológica/genética , Osteócitos/patologia , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Animais , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Colágeno , Tecido Conjuntivo/patologia , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Osteoclastos/citologia , Diester Fosfórico Hidrolases/deficiência , Pirofosfatases/deficiência , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Microtomografia por Raio-X
19.
Microsc Res Tech ; 77(12): 1044-51, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25219801

RESUMO

Iodine imparts strong contrast to objects imaged with electrons and X-rays due to its high atomic number (53), and is widely used in liquid form as a microscopic stain and clinical contrast agent. We have developed a simple technique which exploits elemental iodine's sublimation-deposition state-change equilibrium to vapor stain specimens with iodine gas. Specimens are enclosed in a gas-tight container along with a small mass of solid I2 . The bottle is left at ambient laboratory conditions while staining proceeds until empirically determined completion (typically days to weeks). We demonstrate the utility of iodine vapor staining by applying it to resin-embedded tissue blocks and whole locusts and imaging them with backscattered electron scanning electron microscopy (BSE SEM) or X-ray microtomography (XMT). Contrast is comparable to that achieved with liquid staining but without the consequent tissue shrinkage, stain pooling, or uneven coverage artefacts associated with immersing the specimen in iodine solutions. Unmineralized tissue histology can be read in BSE SEM images with good discrimination between tissue components. Organs within the locust head are readily distinguished in XMT images with particularly useful contrast in the chitin exoskeleton, muscle and nerves. Here, we have used iodine vapor staining for two imaging modalities in frequent use in our laboratories and on the specimen types with which we work. It is likely to be equally convenient for a wide range of specimens, and for other modalities which generate contrast from electron- and photon-sample interactions, such as transmission electron microscopy and light microscopy.


Assuntos
Iodo/química , Microscopia Eletrônica de Varredura/métodos , Microtomografia por Raio-X/métodos , Animais , Gafanhotos/ultraestrutura , Humanos , Ratos , Coloração e Rotulagem
20.
Endocrinology ; 155(9): 3699-712, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24914936

RESUMO

A new genetic disorder has been identified that results from mutation of THRA, encoding thyroid hormone receptor α1 (TRα1). Affected children have a high serum T3:T4 ratio and variable degrees of intellectual deficit and constipation but exhibit a consistently severe skeletal dysplasia. In an attempt to improve developmental delay and alleviate symptoms of hypothyroidism, patients are receiving varying doses and durations of T4 treatment, but responses have been inconsistent so far. Thra1(PV/+) mice express a similar potent dominant-negative mutant TRα1 to affected individuals, and thus represent an excellent disease model. We hypothesized that Thra1(PV/+) mice could be used to predict the skeletal outcome of human THRA mutations and determine whether prolonged treatment with a supraphysiological dose of T4 ameliorates the skeletal abnormalities. Adult female Thra1(PV/+) mice had short stature, grossly abnormal bone morphology but normal bone strength despite high bone mass. Although T4 treatment suppressed TSH secretion, it had no effect on skeletal maturation, linear growth, or bone mineralization, thus demonstrating profound tissue resistance to thyroid hormone. Despite this, prolonged T4 treatment abnormally increased bone stiffness and strength, suggesting the potential for detrimental consequences in the long term. Our studies establish that TRα1 has an essential role in the developing and adult skeleton and predict that patients with different THRA mutations will display variable responses to T4 treatment, which depend on the severity of the causative mutation.


Assuntos
Doenças do Desenvolvimento Ósseo/genética , Receptores alfa dos Hormônios Tireóideos/genética , Tiroxina/metabolismo , Animais , Tamanho Corporal , Densidade Óssea , Doenças do Desenvolvimento Ósseo/metabolismo , Doenças do Desenvolvimento Ósseo/fisiopatologia , Calcificação Fisiológica , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Receptores alfa dos Hormônios Tireóideos/metabolismo
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