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1.
Methods Mol Biol ; 2230: 325-335, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197022

RESUMO

Cell lineage tracing, an old technique which originated in the nineteenth century, regains popularity and relevance due to introduction of a more sensitive tomato fluorescent protein under the control of a ubiquitous promoter (Rosa 26 gene). In addition, various tissue specific CreERT2 mouse lines are widely available, making cell lineage tracing studies more specific and powerful. In this protocol, we provide a practical guide for researchers to map progeny of specific cells such as chondrocytes during development using a fluorescent reporter (tomato, red) and multiple chondrocyte Cre lines. Further, we provide valuable examples in which these tracing lines, combined with a bone reporter mouse line (2.3 Col 1a1-GFP) or costained with different immunofluorescent proteins, revealed how a chondrocyte transdifferentiates into a bone cell in vivo.


Assuntos
Linhagem da Célula/genética , Rastreamento de Células/métodos , Condrócitos/ultraestrutura , Crânio/ultraestrutura , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Condrócitos/metabolismo , Genes Reporter/genética , Camundongos , Camundongos Transgênicos , Osteócitos/metabolismo
2.
Nat Commun ; 11(1): 3282, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32612176

RESUMO

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/genética , Histona Desacetilases/genética , Mecanotransdução Celular/genética , Osteócitos/metabolismo , Transdução de Sinais/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Perfilação da Expressão Gênica/métodos , Histona Desacetilases/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteogênese/genética , Fosforilação
3.
J Vis Exp ; (160)2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32568236

RESUMO

The osteocyte, once thought to be a passive resident of the bone given the backstage function of sensing mechanical loading, is now brought to the spotlight and has been shown to have multiple major functions like actively modifying the extracellular matrix and forming an endocrine organ with the lacunocanalicular system that encloses it sending messages to distant sites. Owing to the methods that made it possible to test the osteocyte in vitro from isolating primary osteocytes to osteocyte-like cell lines, osteocytes are now experiencing a resounding interest and a surge of knowledge on structure and function. Many aspects of the osteocyte biology and interaction with other molecular components are yet to be discovered. In this protocol, we describe in detail the efficient isolation of primary osteocytes from dmp1-topaz neonatal mouse calvaria, which express the green fluorescent protein in osteocytes, through cell fractionation and subsequently acquiring cultures of primary osteocytes by FACS.


Assuntos
Fracionamento Celular , Proteínas de Fluorescência Verde/genética , Osteócitos/metabolismo , Crânio/citologia , Animais , Linhagem Celular , Matriz Extracelular/metabolismo , Expressão Gênica , Camundongos
4.
Cell Physiol Biochem ; 54(2): 271-286, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32233339

RESUMO

BACKGROUND/AIMS: Pericytes (PCs) are multipotent vascular precursors that play a critical physiological role in the development and maintenance of blood vessel integrity. In this study, we aim to characterize PCs isolated from human abdominal adipose tissue and develop an integration-free induced pluripotent stem cells (iPSCs) using episomal vectors. METHODS: The ultrastructure of adipose tissue-derived PCs was determined using scanning and transmission electron microscopy. The expression of mesenchymal stem cells (MSCs) and pericyte markers were examined using flow cytometry and PCR analysis. PCs were induced to adipogenic, osteogenic and myogenic lineages, and their angiogenic potential was determined using tube formation assay. We further established pericyte reprogramming protocol using episomal vectors. RESULTS: Our data showed that human adipose tissue-derived PCs uniformly expressed MSCs, CD105 and CD73, and PCs markers, desmin, and alpha smooth muscle actin (α-SMA), while lacked the expression of HLA-DR and the hematopoietic markers CD34, CD11b and CD45. Ultrastructure analysis showed typical internal structure for the PCs with a characteristic prominent eccentric nuclei and cytoplasmic invaginations forming a caveolar system. Functional analysis showed efficient differentiation into adipocytes, osteocytes, and myocyte-like cells. Adipose tissue-derived PCs showed angiogenic potential using tube-forming assay. To determine further application of these cells for personalized therapy, we reprogrammed PCs into induced pluripotent stem cells (iPSCs) using episomal vectors. Reprogrammed cells gradually lost their fusiform shape, acquired the epithelial cell morphology and formed colonies. Furthermore, reprogrammed cells successfully expressed the pluripotency markers OCT4, Nanog, SSEA-4, and ß-catenin, an early reprogramming marker. CONCLUSION: The accessibility and abundance of human fat supports the application of adipose derived PCs as a novel and promising source of cell therapy and regenerative medicine.


Assuntos
Tecido Adiposo/citologia , Técnicas de Reprogramação Celular/métodos , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Pericitos/citologia , 5'-Nucleotidase/metabolismo , Actinas/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo/ultraestrutura , Linhagem da Célula , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Desmina/metabolismo , Endoglina/metabolismo , Citometria de Fluxo , Proteínas Ligadas por GPI/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Células Musculares/citologia , Células Musculares/metabolismo , Desenvolvimento Muscular/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Osteogênese/genética , Pericitos/metabolismo , Pericitos/ultraestrutura , Antígenos Embrionários Estágio-Específicos/metabolismo , beta Catenina/metabolismo
5.
Toxicology ; 436: 152429, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32156525

RESUMO

Excessive systemic uptake of inorganic fluorides causes disturbances of bone homeostasis. The mechanism of skeletal fluorosis is still uncertain. This study aimed to study the effect of fluoride on osteocyte-driven osteoclastogenesis and probe into the role of PTH in this process. IDG-SW3 cells seeded in collagen-coated constructs were developed into osteocyte-like cells through induction of mineral agents. Then, osteocyte-like cells were exposed to fluoride in the presence or absence of parathyroid hormone (PTH). Cell viability and their capacity to produce receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and sclerostin (SOST) were detected by MTT and Western blot assays, respectively. Finally, a transwell coculture system using osteocyte-like cells seeded in the low compartment, and osteoclast precursors added in the inserts was developed to observe the osteocyte-driven osteoclasogenesis response to fluoride with or without PTH, and the expression of molecules involved in this mechanism were measure by real time RT-PCR. Results showed that osteocytes withstood a toxic dose of fluoride, and yet PTH administration significantly reduced osteocytes viability. PTH amplified the effect of fluoride on the expression of osteoclastogenesis-related molecules in osteocyte, but did not enlarged the stimulating effect of fluoride on osteoclastogenesis drove by osteocyte coculture. Gene expression levels of TRAP, RANK, JNK and NFAtc1 significantly increased in fluoride affected osteoclast precursor cocultured with osteocyte-like cells. The impact of fluoride on osteocyte-driven osteoclast differentiation was stronger than that of PTH. In conclusion, osteocyte played a pivotal role on the mechanism underlying fluoride-affected osteoclastogenesis in which RANK-JNK-NFATc1 signaling pathway was involved, and PTH had a significant impact in this process.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fluoreto de Sódio/toxicidade , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteócitos/metabolismo , Osteócitos/patologia , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Hormônio Paratireóideo/farmacologia , Ligante RANK/genética , Ligante RANK/metabolismo , Células RAW 264.7 , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais , Fosfatase Ácida Resistente a Tartarato/genética , Fosfatase Ácida Resistente a Tartarato/metabolismo
6.
Biomed Res Int ; 2020: 7149408, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32149126

RESUMO

Objective: Calprotectin is a heterocomplex of S100A8 and S100A9 and is mainly secreted from neutrophils, monocytes, and chondrocytes in inflammatory condition. Calprotectin binds to RAGE and TLR4 and induces the expression of proinflammatory chemokines and cytokines in various cells. Periodontitis is a chronic inflammatory disease that leads to gingival inflammation and alveolar bone resorption. Calprotectin levels in gingival crevicular fluid of periodontitis patients are higher than healthy patients. In the present study, the effects of S100A8 and S100A9 on the expressions of proinflammatory cytokines and bone metabolism-related factors in mouse osteocyte-like cells (MLO-Y4-A2) were investigated. Design: MLO-Y4-A2 cells were treated with S100A8 and S100A9, and the expressions of RAGE, TLR4, RANKL, and several inflammatory cytokines were analyzed by PCR and Western blotting or ELISA methods. To investigate the intracellular signaling pathways, phosphorylation of MAPK and STAT3 was determined by Western blotting, and chemical specific inhibitors and siRNAs were used. Results: Expressions of IL-6 and RANKL were increased by treatment with S100A9 but not S100A8. However, both S100A8 and S100A9 did not change expression of IL-1ß, IL-8, and TNF-α. Although RAGE and TLR4 expressions were not upregulated by S100A9 treatment, transfection of siRNA for RAGE and TLR4 significantly decreased IL-6 and RANKL expressions. In addition, S100A9 activated p38, ERK, and STAT3 signaling pathways, and inhibitors for these factors significantly decreased S100A9-induced IL-6 and RANKL expressions. Conclusions: These results indicated that S100A9 induces IL-6 and RANKL production via engagement with RAGE and TLR4 signalings in osteocytes and suggested that S100A9 may play important roles in the periodontal alveolar bone destruction.


Assuntos
Calgranulina B/metabolismo , Calgranulina B/farmacologia , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Fator de Transcrição STAT3/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Calgranulina A/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos
7.
Arch Biochem Biophys ; 685: 108333, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32194044

RESUMO

This study summarizes the available evidence from systematic reviews on the in vitro effects of photobiomodulation on the proliferation and differentiation of human bone and stromal cells by appraising their methodological quality. Improvements for future studies are also highlighted, with particular emphasis on in vitro protocols and cell-related characteristics. Six reviews using explicit eligibility criteria and methods selected in order to minimize bias were included. There was no compelling evidence on the cellular mechanisms of action or treatment parameters of photobiomodulation; compliance with quality assessment was poor. A rigorous description of laser parameters (wavelength, power, beam spot size, power density, energy density, repetition rate, pulse duration or duty cycle, exposure duration, frequency of treatments, and total radiant energy), exposure conditions (methods to ensure a uniform irradiation and to avoid cross-irradiation, laser-cell culture surface distance, lid presence during irradiation) and cell-related characteristics (cell type or line, isolation and culture conditions, donor-related factors where applicable, tissue source, cell phenotype, cell density, number of cell passages in culture) should be included among eligibility criteria for study inclusion. These methodological improvements will maximize the contribution of in vitro studies on the effects of photobiomodulation on human bone and stromal cells to evidence-based translational research.


Assuntos
Terapia com Luz de Baixa Intensidade , Osteócitos/metabolismo , Células Estromais/metabolismo , Animais , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Humanos , Osteócitos/efeitos da radiação , Células Estromais/efeitos da radiação , Revisões Sistemáticas como Assunto
8.
Int J Mol Sci ; 21(3)2020 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-32050469

RESUMO

The skeleton adapts to mechanical loading to promote bone formation and remodeling. While most bone cells are involved in mechanosensing, it is well accepted that osteocytes are the principal mechanosensory cells. The osteocyte cell body and processes are surrounded by a fluid-filled space, forming an extensive lacuno-canalicular network. The flow of interstitial fluid is a major stress-related factor that transmits mechanical stimulation to bone cells. The long dendritic processes of osteocytes form a gap junction channel network connecting not only neighboring osteocytes, but also cells on the bone surface, such as osteoblasts and osteoclasts. Mechanosensitive osteocytes also form hemichannels that mediate the communication between the cytoplasmic and extracellular microenvironment. This paper will discuss recent research progress regarding connexin (Cx)-forming gap junctions and hemichannels in osteocytes, osteoblasts, and other bone cells, including those richly expressing Cx43. We will then cover the recent progress regarding the regulation of these channels by mechanical loading and the role of integrins and signals in mediating Cx43 channels, and bone cell function and viability. Finally, we will summarize the recent studies regarding bone responses to mechanical unloading in Cx43 transgenic mouse models. The osteocyte has been perceived as the center of bone remodeling, and connexin channels enriched in osteocytes are a likely major player in meditating the function of bone. Based on numerous studies, connexin channels may present as a potential new therapeutic target in the treatment of bone loss and osteoporosis. This review will primarily focus on Cx43, with some discussion in other connexins expressed in bone cells.


Assuntos
Remodelação Óssea , Osso e Ossos/fisiologia , Conexinas/metabolismo , Animais , Fenômenos Biomecânicos , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Homeostase , Humanos , Mecanotransdução Celular , Osteócitos/metabolismo , Estresse Mecânico , Suporte de Carga
9.
Exerc Sport Sci Rev ; 48(2): 59-66, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32004169

RESUMO

It is commonly assumed that beneficial adaptations in bone occur with vigorous exercise, yet any adaptive re/modeling in bone undergoing persistent overloading can be counteracted by superimposed inflammatory, compressive, and tensile loading-induced damage responses above thresholds of tissue fatigue failure and repair. This leads to a tenuous balance between achieving bone accrual and loss.


Assuntos
Reabsorção Óssea , Ocupações , Osteogênese/fisiologia , Esforço Físico/fisiologia , Acidentes de Trabalho , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Densidade Óssea/fisiologia , Osso e Ossos/inervação , Transtornos Traumáticos Cumulativos/fisiopatologia , Exercício Físico/fisiologia , Humanos , Modelos Animais , Osteócitos/metabolismo , Ligante RANK/metabolismo , Estresse Mecânico
10.
Int J Mol Sci ; 21(3)2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31973091

RESUMO

Forkhead box class O family member proteins (FoxOs) are evolutionarily conserved transcription factors for their highly conserved DNA-binding domain. In mammalian species, all the four FoxO members, FoxO1, FoxO3, FoxO4, and FoxO6, are expressed in different organs. In bone, the first three members are extensively expressed and more studied. Bone development, remodeling, and homeostasis are all regulated by multiple cell lineages, including osteoprogenitor cells, chondrocytes, osteoblasts, osteocytes, osteoclast progenitors, osteoclasts, and the intercellular signaling among these bone cells. The disordered FoxOs function in these bone cells contribute to osteoarthritis, osteoporosis, or other bone diseases. Here, we review the current literature of FoxOs for their roles in bone cells, focusing on helping researchers to develop new therapeutic approaches and prevent or treat the related bone diseases.


Assuntos
Osso e Ossos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Osteócitos/metabolismo , Fatores de Transcrição/metabolismo , Doenças Ósseas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Condrogênese/fisiologia , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O3/metabolismo , Fatores de Transcrição Forkhead/classificação , Fatores de Transcrição Forkhead/genética , Células-Tronco Hematopoéticas , Osteoartrite/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/fisiologia , Osteoporose/metabolismo , Transdução de Sinais
11.
Commun Biol ; 3(1): 45, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988398

RESUMO

Intraflagellar transport (IFT) proteins are essential for cilia assembly and function. IFT protein mutations lead to ciliopathies, which manifest as variable skeletal abnormalities. However, how IFT proteins regulate cell alignment during bone development is unknown. Here, we show that the deletion of IFT20 in osteoblast lineage using Osterix-Cre and inducible type I Collagen-CreERT cause a compromised cell alignment and a reduced bone mass. This finding was validated by the disorganized collagen fibrils and decreased bone strength and stiffness in IFT20-deficient femurs. IFT20 maintains cilia and cell alignment in osteoblasts, as the concentric organization of three-dimensional spheroids was disrupted by IFT20 deletion. Mechanistically, IFT20 interacts with the ceramide-PKCζ complex to promote PKCζ phosphorylation in cilia and induce the apical localization of ß-catenin in osteoblasts, both of which were disrupted in the absence of IFT20. These results reveal that IFT20 regulates polarity and cell alignment via ceramide-pPKCζ-ß-catenin signaling during bone development.


Assuntos
Desenvolvimento Ósseo/genética , Proteínas de Transporte/metabolismo , Ceramidas/metabolismo , Cílios/metabolismo , Osteócitos/metabolismo , Proteína Quinase C/metabolismo , beta Catenina/metabolismo , Animais , Proteínas de Transporte/genética , Diferenciação Celular/genética , Linhagem da Célula , Polaridade Celular/genética , Fêmur/metabolismo , Camundongos Transgênicos , Fosforilação/genética , Proteína cdc42 de Ligação ao GTP/metabolismo
12.
FASEB J ; 34(2): 2579-2594, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908007

RESUMO

The skeleton of type 1 diabetes mellitus (T1DM) has deteriorated mechanical integrity and increased fragility, whereas the mechanisms are not fully understood. Load-induced microdamage naturally occurs in bone matrix and can be removed by initiating endogenous targeted bone remodeling. However, the microdamage accumulation in diabetic skeleton and the corresponding bone remodeling mechanisms remain poorly understood. Herein, streptozotocin-induced T1DM rats and age-matched non-diabetic rats were subjected to daily uniaxial ulnar loading for 1, 4, 7, and 10 days, respectively. The SPECT/CT and basic fuchsin staining revealed significant higher-density spatial accumulation of linear and diffuse microdamage in diabetic ulnae than non-diabetic ulnae. Linear microcracks increased within 10-day loading in diabetic bone, whereas peaked at Day 7 in non-diabetic bone. Moreover, diabetic fatigued ulnae had more severe disruptions of osteocyte canaliculi around linear microcracks. Immunostaining results revealed that diabetes impaired targeted remodeling in fatigued bone at every key stage, including increased apoptosis of bystander osteocytes, decreased RANKL secretion, reduced osteoclast recruitment and bone resorption, and impaired osteoblast-mediated bone formation. This study characterizes microdamage accumulation and abnormal remodeling mechanisms in the diabetic skeleton, which advances our etiologic understanding of diabetic bone deterioration and increased fragility from the aspect of microdamage accumulation and bone remodeling.


Assuntos
Remodelação Óssea/fisiologia , Reabsorção Óssea/metabolismo , Diabetes Mellitus/metabolismo , Osteoclastos/metabolismo , Animais , Apoptose/fisiologia , Reabsorção Óssea/fisiopatologia , Masculino , Osteócitos/metabolismo , Ratos Sprague-Dawley , Estresse Mecânico , Ulna/fisiopatologia , Suporte de Carga/fisiologia
13.
Sci Rep ; 10(1): 254, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937885

RESUMO

The recent discovery of bone flexoelectricity (strain-gradient-induced electrical polarization) suggests that flexoelectricity could have physiological effects in bones, and specifically near bone fractures, where flexoelectricity is theoretically highest. Here, we report a cytological study of the interaction between crack stress and bone cells. We have cultured MC3T3-E1 mouse osteoblastic cells in biomimetic microcracked hydroxyapatite substrates, differentiated into osteocytes and applied a strain gradient to the samples. The results show a strong apoptotic cellular response, whereby mechanical stimulation causes those cells near the crack to die, as indicated by live-dead and caspase staining. In addition, analysis two weeks post-stimulation shows increased cell attachment and mineralization around microcracks and a higher expression of osteocalcin -an osteogenic protein known to be promoted by physical exercise. The results are consistent with flexoelectricity playing at least two different roles in bone remodelling: apoptotic trigger of the repair protocol, and electro-stimulant of the bone-building activity of osteoblasts.


Assuntos
Fraturas Ósseas/patologia , Células 3T3 , Animais , Apoptose , Impedância Elétrica , Fraturas Ósseas/metabolismo , Camundongos , Minerais/metabolismo , Modelos Biológicos , Osteócitos/metabolismo , Osteócitos/patologia , Estresse Mecânico
14.
Acta Orthop ; 91(1): 115-120, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31762353

RESUMO

Background and purpose - Insufficient initial fixation or early micromotion of an implant is associated with a thin layer of fibrous tissue at the peri-implant interface. It is unknown if bone loss is induced by the fibrous tissue interface acting as an active biological membrane, or as a membrane that will produce supraphysiologic fluid flow conditions during gait, which activates the mechanosensitive osteocytes to mediate osteoclast differentiation. We investigated whether mechanically induced osteolysis is dependent on the fibrous tissue interface as a biologically active scaffold, or if it merely acts as a conduit for fluid flow, affecting the mechanosensitive osteocytes in the peri-prosthetic bone.Methods - Using a rat model of mechanically instability-induced aseptic loosening, we assessed whether the induction of osteoclast differentiation was dependent on the presence of a peri-implant fibrous interface. We analyzed the amount of osteoclast differentiation, osteocyte apoptosis, pro-resorptive cytokine expression and bone loss using immunohistochemistry, mRNA expression and micro-CT.Results - Osteoclast differentiation and bone loss were induced by mechanical instability but were not affected by the presence of the fibrous tissue membrane or associated with osteocyte apoptosis. There was no increased mRNA expression of any of the cytokines in the fibrous tissue membrane compared with the peri-implant bone.Interpretation - Our data show that the fibrous tissue membrane in the interface plays a minor role in inducing bone loss. This indicates that the peri-implant bone adjacent to loose bone implants might play an important role for osteoclast differentiation.


Assuntos
Apoptose , Diferenciação Celular , Citocinas/metabolismo , Instabilidade Articular/metabolismo , Osteoclastos/metabolismo , Osteócitos/metabolismo , Falha de Prótese , Tíbia/metabolismo , Animais , Reabsorção Óssea/diagnóstico por imagem , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Interface Osso-Implante/diagnóstico por imagem , Citocinas/genética , Modelos Animais de Doenças , Imuno-Histoquímica , Instabilidade Articular/diagnóstico por imagem , Instabilidade Articular/genética , Osteoclastos/citologia , Osteócitos/citologia , RNA Mensageiro/metabolismo , Ratos , Tíbia/diagnóstico por imagem , Microtomografia por Raio-X
15.
Ann N Y Acad Sci ; 1460(1): 68-76, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31646646

RESUMO

The biological effect of ultrasound on bone regeneration has been well documented, yet the underlying mechanotransduction mechanism is largely unknown. In relation to the mechanobiological modulation of the cytoskeleton and Ca2+ influx by short-term focused acoustic radiation force (FARF), the current study aimed to visualize and quantify Ca2+ oscillations in real-time of in situ and in vivo osteocytes in response to focused low-intensity pulsed ultrasound (FLIPUS). For in situ studies, fresh mice calvaria were subjected to FLIPUS stimulation at 0.05, 0.2, 0.3, and 0.7 W. For the in vivo study, 3-month-old C57BL/6J Ai38/Dmp1-Cre mice were subjected to FLIPUS at 0.15, 1, and 1.5 W. As observed via real-time confocal imaging, in situ FLIPUS led to more than 80% of cells exhibiting Ca2+ oscillations at 0.3-0.7 W and led to a higher number of Ca2+ spikes with larger values at >0.3 W. In vivo FLIPUS at 1-1.5 W led to more than 90% of cells exhibiting Ca2+ oscillations. Higher FLIPUS energies led to larger Ca2+ spike magnitudes. In conclusion, this study provided a pilot study of both in situ and in vivo osteocytic Ca2+ oscillations under noninvasive FARF, which aids further exploration of the mechanosensing mechanism of the controlled bone cell motility response to the stimulus.


Assuntos
Acústica , Sinalização do Cálcio , Mecanotransdução Celular , Osteócitos/metabolismo , Radiação , Ultrassom , Estimulação Acústica , Animais , Feminino , Camundongos Endogâmicos C57BL , Crânio/diagnóstico por imagem
16.
J Bone Miner Metab ; 38(2): 172-178, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31501981

RESUMO

INTRODUCTION: Lactation inevitably leads to a state of rapid bone loss; however, maternal bone undergoes rapid remineralization after weaning. Sclerostin, encoded by the gene SOST, is exclusively secreted from osteocytes and plays important roles in bone remodeling. However, there are few studies about the effect of sclerostin during lactation and weaning on bone microstructures. Therefore, we conducted the study to demonstrate any possible association of sclerostin with bone metabolism and skeletal changes during lactation and after weaning. MATERIALS AND METHODS: We analyzed bone mineral density (BMD) by dual-energy X-ray absorptiometry at the spine and femur, bone microstructure by micro-computed tomography (µCT) at the distal and mid-shaft of the femur and biochemical markers such as sclerostin and bone turnover markers at 1 week and 3 weeks of lactation and 2 weeks post-weaning in osteocyte-specific sclerostin-overexpressed transgenic mice, and compared them with wild type. RESULTS: Lactation significantly resulted in decreased spine and femur BMD at day 7 and day 21 of breastfeeding; specifically, cortical microstructure (cross-sectional thickness and cross-sectional area) at the mid-shaft of the femur had significantly deteriorated. At day 14 after weaning, femur BMD and cortical microstructure at the mid-shaft of the femur in both the wild and DMP-SOST mice had incompletely recovered; however, spine BMD and trabecular microstructures at the distal femur recovered in wild type mice. CONCLUSIONS: Sclerostin, secreted by osteocytes, played a role in bone loss during lactation and also in the recovery of trabecular bone compartment by activating bone formation after weaning.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Osso e Ossos/fisiologia , Fêmur/fisiologia , Lactação/fisiologia , Osteócitos/metabolismo , Desmame , Proteínas Adaptadoras de Transdução de Sinal/sangue , Animais , Densidade Óssea , Osso e Ossos/diagnóstico por imagem , Feminino , Fêmur/diagnóstico por imagem , Humanos , Lactação/sangue , Camundongos Transgênicos , Osteogênese , Coluna Vertebral/metabolismo , Microtomografia por Raio-X
17.
Biol Trace Elem Res ; 193(1): 226-233, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30877522

RESUMO

Fluoride accumulates and is toxic to bones. Clinical bone lesions occur in a phased manner, being less severe early in the natural course of skeletal fluorosis. Previous research rarely focused on osteocyte, osteoclast, and osteoblast at the same time, although these three types of cells are involved in the process of fluorosis. In this study, commitment of bone cells was performed according to their respective characteristics. Osteocyte-like cells were verified by protein expression of sclerostin (SOST) in IDG-SW3 cell culture with mineral medium. Positive tartrate-resistant acid phosphatase (TRACP) staining, characteristic of osteoclasts, is observed in RAW264.7 cells after administration of RANKL. We successfully purified a high percentage (94%) of bone mesenchymal stem cells (BMSCs) co-expressing CD34 and CD44. Parallel studies were performed to observe cell viability and apoptosis rates in osteocyte, osteoclast, and osteoblast like cells by using MTT and Annexin V FITC assays. Our results demonstrated that osteocytes have a strong tolerance to high fluoride concentrations, while osteoclasts are more sensitive to changes of fluoride dose. The range of anabolic action of fluoride concentration on osteoblast was narrow. Notably, fluoride exposure aggravated apoptosis of osteocyte and osteoclast induced by administration of PTH and TGF-ß, respectively. In short, three types of bone cells display disparate responses to fluoride exposure and to PTH- and TGF-ß-induced apoptosis.


Assuntos
Células da Medula Óssea/metabolismo , Fluoretos/toxicidade , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Células da Medula Óssea/patologia , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos ICR , Osteoblastos/patologia , Osteócitos/patologia , Ligante RANK/farmacologia , Células RAW 264.7
18.
Biochem Biophys Res Commun ; 521(3): 806-813, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31708103

RESUMO

Osteocytes function as critical regulators of bone homeostasis by coordinating the functions of osteoblasts and osteoclasts, and are constantly exposed to mechanical force. However, the molecular mechanism underlying the mechanical signal transduction in osteocytes is not well understood. Here, we found that Yoda1, a selective Piezo1 agonist, increased intracellular calcium mobilization and dose-dependently decreased the expression of Sost (encoding Sclerostin) in the osteocytic cell line IDG-SW3. We also demonstrated that mechanical stretch of IDG-SW3 suppressed Sost expression, a result which was abrogated by treatment with the Piezo1 inhibitor GsMTx4, and the deficiency of Piezo1. Furthermore, the suppression of Sost expression was abolished by treatment with an Akt inhibitor. Taken together, these results indicate that the activation of the Piezo1-Akt pathway in osteocytes is required for mechanical stretch-induced downregulation of Sost expression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Canais Iônicos/metabolismo , Mecanotransdução Celular , Osteócitos/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Regulação para Baixo , Camundongos , Osteócitos/metabolismo , Transdução de Sinais
19.
Histol Histopathol ; 35(1): 111-124, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31268167

RESUMO

Hypoxia response pathways have a central role in normal and abnormal bone biology but the effect of systemic hypoxia-reoxygenation on bone is not clear. Following hypoxic exposure, aberrant synthesis, folding and trafficking of proteins has been reported to occur, which can result in endoplasmic reticulum (ER) stress and may finally cause cell death. This study aimed to examine the effect of systemic hypoxia-reoxygenation injury on bone biology in postnatal rats. Immunoexpression of HIF-1α and VEGF was upregulated in femurs of newborn Wistar rats in response to systemic hypoxia-reoxygenation. Along with that, increased apoptosis of osteoblast precursors, osteoblasts, osteocytes and endothelial cells was observed in comparison to femurs of control animals by transmission electron microscopy, TUNEL staining and immunoexpression of cleaved caspase-3. The viability of osteoclasts was not affected. After hypoxia-reoxygenation, ER stress was observed in the osteoblasts and osteocytes as indicated by dilatation of the ER and enhanced immunoexpression of the ER stress marker GRP78. Localisation of collagen α1 immunoreaction was widespread in the bone matrix of control femurs but was confined to the osteoblasts and osteocytes in response to hypoxia-reoxygenation. In support of these findings, in vitro work showed reduced viability of osteoblast-like SaOs-2 cells and upregulation of GRP78 protein expression in them by western blotting following exposure to hypoxia. This suggests that systemic hypoxia-reoxygenation may disturb bone biology in postnatal Wistar rats by inducing ER stress and apoptosis in osteoblasts and osteocytes, without affecting the viability of osteoclasts. More in-depth research is needed to confirm causality between ER stress and apoptosis of osteoblasts and osteocytes.


Assuntos
Osso e Ossos/metabolismo , Estresse do Retículo Endoplasmático , Hipóxia/metabolismo , Osteoblastos/metabolismo , Oxigênio/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Retículo Endoplasmático/metabolismo , Células Endoteliais , Feminino , Masculino , Microscopia Eletrônica de Transmissão , Osteócitos/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Regulação para Cima
20.
Aging Cell ; 19(2): e13095, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31880094

RESUMO

To determine whether 1,25-dihydroxyvitamin D (1,25(OH)2 D) can exert an anti-osteoporosis role through anti-aging mechanisms, we analyzed the bone phenotype of mice with 1,25(OH)2 D deficiency due to deletion of the enzyme, 25-hydroxyvitamin D 1α-hydroxylase, while on a rescue diet. 1,25(OH)2 D deficiency accelerated age-related bone loss by activating the p16/p19 senescence signaling pathway, inhibiting osteoblastic bone formation, and stimulating osteoclastic bone resorption, osteocyte senescence, and senescence-associated secretory phenotype (SASP). Supplementation of exogenous 1,25(OH)2 D3 corrected the osteoporotic phenotype caused by 1,25(OH)2 D deficiency or natural aging by inhibiting the p16/p19 pathway. The proliferation, osteogenic differentiation, and ectopic bone formation of bone marrow mesenchymal stem cells derived from mice with genetically induced deficiency of the vitamin D receptor (VDR) were significantly reduced by mechanisms including increased oxidative stress, DNA damage, and cellular senescence. We also demonstrated that p16 deletion largely rescued the osteoporotic phenotype caused by 1,25(OH)2 D3 deficiency, whereas 1,25(OH)2 D3 could up-regulate the enzyme Ezh2 via VDR-mediated transcription thereby enriching H3K27me3 and repressing p16/p19 transcription. Finally, we demonstrated that treatment with 1,25(OH)2 D3 improved the osteogenic defects of human BM-MSCs caused by repeated passages by stimulating their proliferation and inhibiting their senescence via the VDR-Ezh2-p16 axis. The results of this study therefore indicate that 1,25(OH)2 D3 plays a role in preventing age-related osteoporosis by up-regulating Ezh2 via VDR-mediated transcription, increasing H3K27me3 and repressing p16 transcription, thus promoting the proliferation and osteogenesis of BM-MSCs and inhibiting their senescence, while also stimulating osteoblastic bone formation, and inhibiting osteocyte senescence, SASP, and osteoclastic bone resorption.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Receptores de Calcitriol/metabolismo , Vitamina D/análogos & derivados , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Envelhecimento/genética , Animais , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/fisiopatologia , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Dano ao DNA/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Histonas/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Osteogênese/genética , Osteoporose/enzimologia , Osteoporose/metabolismo , Osteoporose/fisiopatologia , Estresse Oxidativo/genética , Receptores de Calcitriol/genética , Vitamina D/farmacologia , Vitamina D/uso terapêutico
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