Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.581
Filtrar
1.
J Cell Biol ; 223(11)2024 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-39212624

RESUMO

Osteocytes' response to dynamic loading plays a crucial role in regulating the bone mass but quickly becomes saturated such that downstream induction of bone formation plateaus. The underlying mechanisms that downregulate osteocytes' sensitivity and overall response to loading remain unknown. In other cell types, purinergic signaling through the P2Y2 receptor has the potential to downregulate the sensitivity to loading by modifying cell stiffness through actin polymerization and cytoskeleton organization. Herein, we examined the role of P2Y2 activation in regulating osteocytes' mechanotransduction using a P2Y2 knockout cell line alongside conditional knockout mice. Our findings demonstrate that the absence of P2Y2 expression in MLO-Y4 cells prevents actin polymerization while increasing the sensitivity to fluid flow-induced shear stress. Deleting osteocytes' P2Y2 expression in conditional-knockout mice enabled bone formation to increase when increasing the duration of exercise. Overall, P2Y2 activation under loading produces a negative feedback loop, limiting osteocytes' response to continuous loading by shifting the sensitivity to mechanical strain through actin stress fiber formation.


Assuntos
Mecanotransdução Celular , Camundongos Knockout , Osteócitos , Receptores Purinérgicos P2Y2 , Estresse Mecânico , Animais , Receptores Purinérgicos P2Y2/metabolismo , Receptores Purinérgicos P2Y2/genética , Osteócitos/metabolismo , Camundongos , Linhagem Celular , Osteogênese , Transdução de Sinais , Actinas/metabolismo , Camundongos Endogâmicos C57BL
2.
Acta Biomater ; 186: 141-155, 2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-39142531

RESUMO

Macrophages and osteocytes are important regulators of inflammation, osteogenesis and osteoclastogenesis. However, their interactions under adverse conditions, such as biomaterial-associated infection (BAI) are not fully understood. We aimed to elucidate how factors released from macrophages modulate osteocyte responses in an in vitro indirect 3D co-culture model. Human monocyte-derived macrophages were cultured on etched titanium disks and activated with either IL-4 cytokine (anti-inflammatory M2 phenotype) or Staphylococcus aureus secreted virulence factors to simulate BAI (pro-inflammatory M1 phenotype). Primary osteocytes in collagen gels were then stimulated with conditioned media (CM) from these macrophages. The osteocyte response was analyzed by gene expression, protein secretion, and immunostaining. M1 phenotype macrophages were confirmed by IL-1ß and TNF-α secretion, and M2 macrophages by ARG-1 and MRC-1.Osteocytes receiving M1 CM revealed bone inhibitory effects, denoted by reduced secretion of bone formation osteocalcin (BGLAP) and increased secretion of the bone inhibitory sclerostin (SOST). These osteocytes also downregulated the pro-mineralization gene PHEX and upregulated the anti-mineralization gene MEPE. Additionally, exhibited pro-osteoclastic potential by upregulating pro-osteoclastic gene RANKL expression. Nonetheless, M1-stimulated osteocytes expressed a higher level of the potent pro-osteogenic factor BMP-2 in parallel with the downregulation of the bone inhibitor genes DKK1 and SOST, suggesting a compensatory feedback mechanisms. Conversely, M2-stimulated osteocytes mainly upregulated anti-osteoclastic gene OPG expression, suggesting an anti-catabolic effect. Altogether, our findings demonstrate a strong communication between M1 macrophages and osteocytes under M1 (BAI)-simulated conditions, suggesting that the BAI adverse effects on osteoblastic and osteoclastic processes in vitro are partly mediated via this communication. STATEMENT OF SIGNIFICANCE: Biomaterial-associated infections are major challenges and the underlying mechanisms in the cellular interactions are missing, especially among the major cells from the inflammatory side (macrophages as the key cell in bacterial clearance) and the regenerative side (osteocyte as main regulator of bone). We evaluated the effect of macrophage polarization driven by the stimulation with bacterial virulence factors on the osteocyte function using an indirect co-culture model, hence mimicking the scenario of a biomaterial-associated infection. The results suggest that at least part of the adverse effects of biomaterial associated infection on osteoblastic and osteoclastic processes in vitro are mediated via macrophage-to-osteocyte communication.


Assuntos
Comunicação Celular , Macrófagos , Osteócitos , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos/microbiologia , Osteócitos/metabolismo , Osteócitos/efeitos dos fármacos , Técnicas de Cocultura , Infecções Relacionadas à Prótese/patologia , Infecções Relacionadas à Prótese/metabolismo , Infecções Relacionadas à Prótese/microbiologia , Meios de Cultivo Condicionados/farmacologia , Modelos Biológicos , Osteogênese/efeitos dos fármacos
4.
Exp Cell Res ; 442(1): 114211, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39147261

RESUMO

Blood vessel growth and osteogenesis in the skeletal system are coupled; however, fundamental aspects of vascular function in osteoblast-to-osteocyte transition remain unclear. Our study demonstrates that vascular smooth muscle cells (VSMCs), but not endothelial cells, are sufficient to drive bone marrow mesenchymal stromal cell-derived osteoblast-to-osteocyte transition via ß-catenin signaling and exosome-mediated communication. We found that VSMC-derived exosomes are loaded with transcripts encoding proteins associated with the osteocyte phenotype and members of the WNT/ß-catenin signaling pathway. In contrast, endothelial cell-derived exosomes facilitated mature osteoblast differentiation by reprogramming the TGFB1 gene family and osteogenic transcription factors osterix (SP7) and RUNX2. Notably, VSMCs express significant levels of tetraspanins (CD9, CD63, and CD81) and drive the intracellular trafficking of exosomes with a lower membrane zeta potential than those from other cells. Additionally, the high ATP content within these exosomes supports mineralization mechanisms, as ATP is a substrate for alkaline phosphatase. Osteocyte function was further validated by RNA sequencing, revealing activity in genes related to intermittent mineralization and sonic hedgehog signaling, alongside a significant increase in TNFSF11 levels. Our findings unveil a novel role of VSMCs in promoting osteoblast-to-osteocyte transition, thus offering new insights into bone biology and homeostasis, as well as in bone-related diseases. Clinically, these insights could pave the way for innovative therapeutic strategies targeting VSMC-derived exosome pathways to treat bone-related disorders such as osteoporosis. By manipulating these signaling pathways, it may be possible to enhance bone regeneration and improve skeletal health in patients with compromised bone structure and function.


Assuntos
Exossomos , Músculo Liso Vascular , Osteoblastos , Osteócitos , Osteogênese , beta Catenina , Osteoblastos/metabolismo , Osteoblastos/citologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Exossomos/metabolismo , Animais , beta Catenina/metabolismo , beta Catenina/genética , Osteócitos/metabolismo , Osteócitos/citologia , Camundongos , Osteogênese/genética , Osteogênese/fisiologia , Miócitos de Músculo Liso/metabolismo , Diferenciação Celular , Humanos , Via de Sinalização Wnt , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Células Cultivadas , Transdução de Sinais , Camundongos Endogâmicos C57BL
5.
Front Endocrinol (Lausanne) ; 15: 1359052, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39157681

RESUMO

Introduction: Changes to bone physiology play a central role in the development of osteoarthritis with the mechanosensing osteocyte releasing factors that drive disease progression. This study developed a humanised in vitro model to detect osteocyte responses to either interleukin-6, a driver of degeneration and bone remodelling in animal and human joint injury, or mechanical loading, to mimic osteoarthritis stimuli in joints. Methods: Human MSC cells (Y201) were differentiated in 3-dimensional type I collagen gels in osteogenic media and osteocyte phenotype assessed by RTqPCR and immunostaining. Gels were subjected to a single pathophysiological load or stimulated with interleukin-6 with unloaded or unstimulated cells as controls. RNA was extracted 1-hour post-load and assessed by RNAseq. Markers of pain, bone remodelling, and inflammation were quantified by RT-qPCR and ELISA. Results: Y201 cells embedded within 3D collagen gels assumed dendritic morphology and expressed mature osteocytes markers. Mechanical loading of the osteocyte model regulated 7564 genes (Padj p<0.05, 3026 down, 4538 up). 93% of the osteocyte transcriptome signature was expressed in the model with 38% of these genes mechanically regulated. Mechanically loaded osteocytes regulated 26% of gene ontology pathways linked to OA pain, 40% reflecting bone remodelling and 27% representing inflammation. Load regulated genes associated with osteopetrosis, osteoporosis and osteoarthritis. 42% of effector genes in a genome-wide association study meta-analysis were mechanically regulated by osteocytes with 10 genes representing potential druggable targets. Interleukin-6 stimulation of osteocytes at concentrations reported in human synovial fluids from patients with OA or following knee injury, regulated similar readouts to mechanical loading including markers of pain, bone remodelling, and inflammation. Discussion: We have developed a reproducible model of human osteocyte like cells that express >90% of the genes in the osteocyte transcriptome signature. Mechanical loading and inflammatory stimulation regulated genes and proteins implicated in osteoarthritis symptoms of pain as well as inflammation and degeneration underlying disease progression. Nearly half of the genes classified as 'effectors' in GWAS were mechanically regulated in this model. This model will be useful in identifying new mechanisms underlying bone and joint pathologies and testing drugs targeting those mechanisms.


Assuntos
Inflamação , Células-Tronco Mesenquimais , Osteoartrite , Osteócitos , Humanos , Osteócitos/metabolismo , Osteócitos/patologia , Osteoartrite/patologia , Osteoartrite/metabolismo , Inflamação/patologia , Inflamação/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Interleucina-6/metabolismo , Remodelação Óssea , Células Cultivadas , Diferenciação Celular
6.
Front Endocrinol (Lausanne) ; 15: 1342938, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39092287

RESUMO

Glucocorticoids (GC) and parathyroid hormone (PTH) are widely used therapeutic endocrine hormones where their effects on bone and joint arise from actions on multiple skeletal cell types. In osteocytes, GC and PTH exert opposing effects on perilacunar canalicular remodeling (PLR). Suppressed PLR can impair bone quality and joint homeostasis, including in GC-induced osteonecrosis. However, combined effects of GC and PTH on PLR are unknown. Given the untapped potential to target osteocytes to improve skeletal health, this study sought to test the feasibility of therapeutically mitigating PLR suppression. Focusing on subchondral bone and joint homeostasis, we hypothesize that PTH(1-34), a PLR agonist, could rescue GC-suppressed PLR. The skeletal effects of GC and PTH(1-34), alone or combined, were examined in male and female mice by micro-computed tomography, mechanical testing, histology, and gene expression analysis. For each outcome, females were more responsive to GC and PTH(1-34) than males. GC and PTH(1-34) exerted regional differences, with GC increasing trabecular bone volume but reducing cortical bone thickness, stiffness, and ultimate force. Despite PTH(1-34)'s anabolic effects on trabecular bone, it did not rescue GC's catabolic effects on cortical bone. Likewise, cartilage integrity and subchondral bone apoptosis, tartrate-resistant acid phosphatase (TRAP) activity, and osteocyte lacunocanalicular networks showed no evidence that PTH(1-34) could offset GC-dependent effects. Rather, GC and PTH(1-34) each increased cortical bone gene expression implicated in bone resorption by osteoclasts and osteocytes, including Acp5, Mmp13, Atp6v0d2, Ctsk, differences maintained when GC and PTH(1-34) were combined. Since PTH(1-34) is insufficient to rescue GC's effects on young female mouse bone, future studies are needed to determine if osteocyte PLR suppression, due to GC, aging, or other factors, can be offset by a PLR agonist.


Assuntos
Densidade Óssea , Remodelação Óssea , Glucocorticoides , Osteócitos , Hormônio Paratireóideo , Animais , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Hormônio Paratireóideo/farmacologia , Feminino , Masculino , Camundongos , Glucocorticoides/farmacologia , Remodelação Óssea/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Microtomografia por Raio-X
7.
JCI Insight ; 9(16)2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-39171528

RESUMO

Obesity can increase the risk of bone fragility, even when bone mass is intact. This fragility stems from poor bone quality, potentially caused by deficiencies in bone matrix material properties. However, cellular and molecular mechanisms leading to obesity-related bone fragility are not fully understood. Using male mouse models of obesity, we discovered TGF-ß signaling plays a critical role in mediating the effects of obesity on bone. High-carbohydrate and high-fat diets increase TGF-ß signaling in osteocytes, which impairs their mitochondrial function, increases cellular senescence, and compromises perilacunar/canalicular remodeling and bone quality. By specifically inhibiting TGF-ß signaling in mouse osteocytes, some of the negative effects of high-fat and high-carbohydrate diets on bones, including the lacunocanalicular network, perilacunar/canalicular remodeling, senescence, and mechanical properties such as yield stress, were mitigated. DMP1-Cre-mediated deletion of TGF-ß receptor II also blunted adverse effects of high-fat and high-carbohydrate diets on energy balance and metabolism. These findings suggest osteocytes are key in controlling bone quality in response to high-fat and high-carbohydrate diets. Calibrating osteocyte function could mitigate bone fragility associated with metabolic diseases while reestablishing energy balance.


Assuntos
Dieta Hiperlipídica , Obesidade , Osteócitos , Fator de Crescimento Transformador beta , Animais , Osteócitos/metabolismo , Dieta Hiperlipídica/efeitos adversos , Camundongos , Fator de Crescimento Transformador beta/metabolismo , Masculino , Obesidade/metabolismo , Transdução de Sinais , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Remodelação Óssea , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Osso e Ossos/metabolismo , Densidade Óssea/efeitos dos fármacos , Carboidratos da Dieta/efeitos adversos , Carboidratos da Dieta/administração & dosagem
8.
PLoS Biol ; 22(8): e3002753, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39163396

RESUMO

Cell-to-cell mitochondrial transfer has recently been shown to play a role in maintaining physiological functions of cell. We previously illustrated that mitochondrial transfer within osteocyte dendritic network regulates bone tissue homeostasis. However, the mechanism of triggering this process has not been explored. Here, we showed that stressed osteocytes in mice release adenosine diphosphate (ADP), resulting in triggering mitochondrial transfer from healthy osteocytes to restore the oxygen consumption rate (OCR) and to alleviate reactive oxygen species accumulation. Furthermore, we identified that P2Y2 and P2Y6 transduced the ADP signal to regulate osteocyte mitochondrial transfer. We showed that mitochondrial metabolism is impaired in aged osteocytes, and there were more extracellular nucleotides release into the matrix in aged cortical bone due to compromised membrane integrity. Conditioned medium from aged osteocytes triggered mitochondrial transfer between osteocytes to enhance the energy metabolism. Together, using osteocyte as an example, this study showed new insights into how extracellular ADP triggers healthy cells to rescue energy metabolism crisis in stressed cells via mitochondrial transfer in tissue homeostasis.


Assuntos
Difosfato de Adenosina , Homeostase , Mitocôndrias , Osteócitos , Animais , Osteócitos/metabolismo , Mitocôndrias/metabolismo , Camundongos , Difosfato de Adenosina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Consumo de Oxigênio , Metabolismo Energético , Camundongos Endogâmicos C57BL , Estresse Fisiológico
9.
J Bone Miner Res ; 39(9): 1253-1267, 2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39126373

RESUMO

Osteogenesis imperfecta (OI) is a group of severe genetic bone disorders characterized by congenital low bone mass, deformity, and frequent fractures. Type XV OI is a moderate to severe form of skeletal dysplasia caused by WNT1 variants. In this cohort study from southern China, we summarized the clinical phenotypes of patients with WNT1 variants and found that the proportion of type XV patients was around 10.3% (25 out of 243) with a diverse spectrum of phenotypes. Functional assays indicated that variants of WNT1 significantly impaired its secretion and effective activity, leading to moderate to severe clinical manifestations, porous bone structure, and enhanced osteoclastic activities. Analysis of proteomic data from human skeleton indicated that the expression of SOST (sclerostin) was dramatically reduced in type XV patients compared to patients with COL1A1 quantitative variants. Single-cell transcriptome data generated from human tibia samples of patients diagnosed with type XV OI and leg-length discrepancy, respectively, revealed aberrant differentiation trajectories of skeletal progenitors and impaired maturation of osteocytes with loss of WNT1, resulting in excessive CXCL12+ progenitors, fewer mature osteocytes, and the existence of abnormal cell populations with adipogenic characteristics. The integration of multi-omics data from human skeleton delineates how WNT1 regulates the differentiation and maturation of skeletal progenitors, which will provide a new direction for the treatment strategy of type XV OI and relative low bone mass diseases such as early onset osteoporosis.


Osteogenesis imperfecta is a rare disease characterized by low bone mass, frequent fractures, and long bone deformity. Type XV osteogenesis imperfect is an autosomal recessive disorder caused by WNT1 variants, while heterozygous variants of WNT1 result in early onset osteoporosis. In this cohort study, we summarized the clinical features of 25 patients diagnosed with type XV osteogenesis imperfect. The WNT1 variants were confirmed by genetic test. Molecular assays were conducted to reveal the impact of variants on WNT1 protein activity and bone structure. We then compared the protein levels in bone tissues isolated from the type XV patients and patients with mild deformity using proteomic method, and found that the expression of SOST, mainly produced by mature osteoblasts and osteocytes, was dramatically reduced in type XV patients. We further compared the global mRNA expression levels in the skeletal cells using single-cell RNA sequencing. Analyses of these data indicated that more immature progenitors were identified and maturation of osteocytes was impaired with WNT1 loss-of-function. Our study helps to understand the underlying pathogenesis of type XV osteogenesis imperfecta.


Assuntos
Diferenciação Celular , Osteogênese Imperfeita , Proteína Wnt1 , Humanos , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Proteína Wnt1/metabolismo , Proteína Wnt1/genética , Masculino , Feminino , Criança , Mutação com Perda de Função , Pré-Escolar , Animais , Proteômica , Adolescente , Osteócitos/metabolismo , Osteócitos/patologia , Fenótipo , Camundongos , Multiômica
10.
Biofabrication ; 16(4)2024 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-39116896

RESUMO

Osteoporosis is the most common bone disorder, which is a highly dangerous condition that can promote bone metastases. As the current treatment for osteoporosis involves long-term medication therapy and a cure for bone metastasis is not known, ongoing efforts are required for drug development for osteoporosis. Animal experiments, traditionally used for drug development, raise ethical concerns and are expensive and time-consuming. Organ-on-a-chip technology is being developed as a tool to supplement such animal models. In this study, we developed a bone-on-a-chip by co-culturing osteoblasts, osteocytes, and osteoclasts in an extracellular matrix environment that can represent normal bone, osteopenia, and osteoporotic conditions. We then simulated bone metastases using breast cancer cells in three different bone conditions and observed that bone metastases were most active in osteoporotic conditions. Furthermore, it was revealed that the promotion of bone metastasis in osteoporotic conditions is due to increased vascular permeability. The bone-on-a-chip developed in this study can serve as a platform to complement animal models for drug development for osteoporosis and bone metastasis.


Assuntos
Neoplasias Ósseas , Dispositivos Lab-On-A-Chip , Osteoporose , Osteoporose/patologia , Osteoporose/tratamento farmacológico , Neoplasias Ósseas/secundário , Neoplasias Ósseas/patologia , Animais , Humanos , Osteoblastos/metabolismo , Técnicas de Cocultura , Camundongos , Osteoclastos/patologia , Osteoclastos/metabolismo , Osteócitos/patologia , Osteócitos/metabolismo , Osso e Ossos/patologia , Linhagem Celular Tumoral , Feminino
11.
Mol Metab ; 88: 102000, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39074536

RESUMO

OBJECTIVE: The skeleton is one of the largest organs in the body, wherein metabolism is integrated with systemic energy metabolism. However, the bioenergetic programming of osteocytes, the most abundant bone cells coordinating bone metabolism, is not well defined. Here, using a mouse model with partial penetration of an osteocyte-specific PPARG deletion, we demonstrate that PPARG controls osteocyte bioenergetics and their contribution to systemic energy metabolism independently of circulating sclerostin levels, which were previously correlated with metabolic status of extramedullary fat depots. METHODS: In vivo and in vitro models of osteocyte-specific PPARG deletion, i.e. Dmp1CrePparγflfl male and female mice (γOTKO) and MLO-Y4 osteocyte-like cells with either siRNA-silenced or CRISPR/Cas9-edited Pparγ. As applicable, the models were analyzed for levels of energy metabolism, glucose metabolism, and metabolic profile of extramedullary adipose tissue, as well as the osteocyte transcriptome, mitochondrial function, bioenergetics, insulin signaling, and oxidative stress. RESULTS: Circulating sclerostin levels of γOTKO male and female mice were not different from control mice. Male γOTKO mice exhibited a high energy phenotype characterized by increased respiration, heat production, locomotion and food intake. This high energy phenotype in males did not correlate with "beiging" of peripheral adipose depots. However, both sexes showed a trend for reduced fat mass and apparent insulin resistance without changes in glucose tolerance, which correlated with decreased osteocytic responsiveness to insulin measured by AKT activation. The transcriptome of osteocytes isolated from γOTKO males suggested profound changes in cellular metabolism, fuel transport, mitochondria dysfunction, insulin signaling and increased oxidative stress. In MLO-Y4 osteocytes, PPARG deficiency correlated with highly active mitochondria, increased ATP production, and accumulation of reactive oxygen species (ROS). CONCLUSIONS: PPARG in male osteocytes acts as a molecular break on mitochondrial function, and protection against oxidative stress and ROS accumulation. It also regulates osteocyte insulin signaling and fuel usage to produce energy. These data provide insight into the connection between osteocyte bioenergetics and their sex-specific contribution to the balance of systemic energy metabolism. These findings support the concept that the skeleton controls systemic energy expenditure via osteocyte metabolism.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Metabolismo Energético , Osteócitos , PPAR gama , Animais , Osteócitos/metabolismo , Camundongos , Masculino , Feminino , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , PPAR gama/metabolismo , PPAR gama/genética , Estresse Oxidativo , Camundongos Knockout , Mitocôndrias/metabolismo , Camundongos Endogâmicos C57BL
12.
Biochem Biophys Res Commun ; 727: 150315, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-38950493

RESUMO

In response to mechanical loading of bone, osteocytes produce nitric oxide (NO•) and decrease sclerostin protein expression, leading to an increase in bone mass. However, it is unclear whether NO• production and sclerostin protein loss are mechanistically linked, and, if so, the nature of their hierarchical relationship within an established mechano-transduction pathway. Prior work showed that following fluid-shear stress (FSS), osteocytes produce NOX2-derived reactive oxygen species, inducing calcium (Ca2+) influx. Increased intracellular Ca2+ results in calcium-calmodulin dependent protein kinase II (CaMKII) activation, which regulates the lysosomal degradation of sclerostin protein. Here, we extend our discoveries, identifying NO• as a regulator of sclerostin degradation downstream of mechano-activated CaMKII. Pharmacological inhibition of nitric oxide synthase (NOS) activity in Ocy454 osteocyte-like cells prevented FSS-induced sclerostin protein loss. Conversely, short-term treatment with a NO• donor in Ocy454 cells or isolated murine long bones was sufficient to induce the rapid decrease in sclerostin protein abundance, independent of changes in Sost gene expression. Ocy454 cells express all three NOS genes, and transfection with siRNAs targeting eNOS/Nos3 was sufficient to prevent FSS-induced loss of sclerostin protein, while siRNAs targeting iNOS/Nos2 mildly blunted the loss of sclerostin but did not reach statistical significance. Similarly, siRNAs targeting both eNOS/Nos3 and iNOS/Nos2 prevented FSS-induced NO• production. Together, these data show iNOS/Nos2 and eNOS/Nos3 are the primary producers of FSS-dependent NO•, and that NO• is necessary and sufficient for sclerostin protein control. Further, selective inhibition of elements within this sclerostin-controlling mechano-transduction pathway indicated that NO• production occurs downstream of CaMKII activation. Targeting Camk2d and Camk2g with siRNA in Ocy454 cells prevented NO• production following FSS, indicating that CaMKII is needed for NO• production. However, NO• donation (1min) resulted in a significant increase in CaMKII activation, suggesting that NO• may have the ability to tune CaMKII response. Together, these data support that CaMKII is necessary for, and may be modulated by NO•, and that the interaction of these two signals is involved in the control of sclerostin protein abundance, consistent with a role in bone anabolic responses.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Óxido Nítrico , Osteócitos , Óxido Nítrico/metabolismo , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Osteócitos/metabolismo , Camundongos , Estresse Mecânico , Camundongos Endogâmicos C57BL , Mecanotransdução Celular , Linhagem Celular , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo
13.
Curr Osteoporos Rep ; 22(4): 396-415, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38980532

RESUMO

PURPOSE OF REVIEW: Quantification of the morphology of osteocyte lacunae has become a powerful tool to investigate bone metabolism, pathologies and aging. This review will provide a brief overview of 2D and 3D imaging methods for the determination of lacunar shape, orientation, density, and volume. Deviations between 2D-based and 3D-based lacunar volume estimations are often not sufficiently addressed and may give rise to contradictory findings. Thus, the systematic error arising from 2D-based estimations of lacunar volume will be discussed, and an alternative calculation proposed. Further, standardized morphological parameters and best practices for sampling and segmentation are suggested. RECENT FINDINGS: We quantified the errors in reported estimation methods of lacunar volume based on 2D cross-sections, which increase with variations in lacunar orientation and histological cutting plane. The estimations of lacunar volume based on common practice in 2D imaging methods resulted in an underestimation of lacunar volume of up to 85% compared to actual lacunar volume in an artificial dataset. For a representative estimation of lacunar size and morphology based on 2D images, at least 400 lacunae should be assessed per sample.


Assuntos
Imageamento Tridimensional , Osteócitos , Humanos , Osteócitos/citologia , Imageamento Tridimensional/métodos , Densidade Óssea , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Processamento de Imagem Assistida por Computador/métodos
14.
Biochem Biophys Res Commun ; 730: 150391, 2024 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-39002199

RESUMO

Glucocorticoid-induced osteoporosis serves as a primary cause for secondary osteoporosis and fragility fractures, representing the most prevalent adverse reaction associated with prolonged glucocorticoid use. In this study, to elucidate the impact and underlying mechanisms of fluid shear stress (FSS)-mediated Piezo1 on dexamethasone (Dex)-induced apoptosis, we respectively applied Dex treatment for 6 h, FSS at 9 dyne/cm2 for 30 min, Yoda1 treatment for 2 h, and Piezo1 siRNA transfection to intervene in MLO-Y4 osteocytes. Western blot analysis was used to assess the expression of Cleaved Caspase-3, Bax, Bcl-2, and proteins associated with the PI3K/Akt pathway. Additionally, qRT-PCR was utilized to quantify the mRNA expression levels of these molecules. Hoechst 33258 staining and flow cytometry were utilized to evaluate the apoptosis levels. The results indicate that FSS at 9 dyne/cm2 for 30 min significantly upregulates Piezo1 in osteocytes. Following Dex-induced apoptosis, the phosphorylation levels of PI3K and Akt are markedly suppressed. FSS-mediated Piezo1 exerts a protective effect against Dex-induced apoptosis by activating the PI3K/Akt pathway. Additionally, downregulating the expression of Piezo1 in osteocytes using siRNA exacerbates Dex-induced apoptosis. To further demonstrate the role of the PI3K/Akt signaling pathway, after intervention with the PI3K pathway inhibitor, the activation of the PI3K/Akt pathway by FSS-mediated Piezo1 in osteocytes was significantly inhibited, reversing the anti-apoptotic effect. This study indicates that under FSS, Piezo1 in MLO-Y4 osteocytes is significantly upregulated, providing protection against Dex-induced apoptosis through the activation of the PI3K/Akt pathway.


Assuntos
Apoptose , Dexametasona , Canais Iônicos , Osteócitos , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Estresse Mecânico , Osteócitos/metabolismo , Osteócitos/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Camundongos , Canais Iônicos/metabolismo , Canais Iônicos/genética , Transdução de Sinais/efeitos dos fármacos , Dexametasona/farmacologia , Linhagem Celular
15.
Acta Biomater ; 184: 210-225, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38969078

RESUMO

Osteocytes perceive and process mechanical stimuli in the lacuno-canalicular network in bone. As a result, they secrete signaling molecules that mediate bone formation and resorption. To date, few three-dimensional (3D) models exist to study the response of mature osteocytes to biophysical stimuli that mimic fluid shear stress and substrate strain in a mineralized, biomimetic bone-like environment. Here we established a biomimetic 3D bone model by utilizing a state-of-art perfusion bioreactor platform where immortomouse/Dmp1-GFP-derived osteoblastic IDG-SW3 cells were differentiated into mature osteocytes. We evaluated proliferation and differentiation properties of the cells on 3D microporous scaffolds of decellularized bone (dBone), poly(L-lactide-co-trimethylene carbonate) lactide (LTMC), and beta-tricalcium phosphate (ß-TCP) under physiological fluid flow conditions over 21 days. Osteocyte viability and proliferation were similar on the scaffolds with equal distribution of IDG-SW3 cells on dBone and LTMC scaffolds. After seven days, the differentiation marker alkaline phosphatase (Alpl), dentin matrix acidic phosphoprotein 1 (Dmp1), and sclerostin (Sost) were significantly upregulated in IDG-SW3 cells (p = 0.05) on LTMC scaffolds under fluid flow conditions at 1.7 ml/min, indicating rapid and efficient maturation into osteocytes. Osteocytes responded by inducing the mechanoresponsive genes FBJ osteosarcoma oncogene (Fos) and prostaglandin-endoperoxide synthase 2 (Ptgs2) under perfusion and dynamic compressive loading at 1 Hz with 5 % strain. Together, we successfully created a 3D biomimetic platform as a robust tool to evaluate osteocyte differentiation and mechanobiology in vitro while recapitulating in vivo mechanical cues such as fluid flow within the lacuno-canalicular network. STATEMENT OF SIGNIFICANCE: This study highlights the importance of creating a three-dimensional (3D) in vitro model to study osteocyte differentiation and mechanobiology, as cellular functions are limited in two-dimensional (2D) models lacking in vivo tissue organization. By using a perfusion bioreactor platform, physiological conditions of fluid flow and compressive loading were mimicked to which osteocytes are exposed in vivo. Microporous poly(L-lactide-co-trimethylene carbonate) lactide (LTMC) scaffolds in 3D are identified as a valuable tool to create a favorable environment for osteocyte differentiation and to enable mechanical stimulation of osteocytes by perfusion and compressive loading. The LTMC platform imitates the mechanical bone environment of osteocytes, allowing the analysis of the interaction with other cell types in bone under in vivo biophysical stimuli.


Assuntos
Reatores Biológicos , Diferenciação Celular , Osteócitos , Osteócitos/citologia , Osteócitos/metabolismo , Animais , Alicerces Teciduais/química , Camundongos , Perfusão , Estresse Mecânico , Linhagem Celular , Proliferação de Células , Força Compressiva , Modelos Biológicos
16.
Biomater Adv ; 163: 213939, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38954876

RESUMO

The bone turnover capability influences the acquisition and maintenance of osseointegration. The architectures of osteocyte three-dimensional (3D) networks determine the direction and activity of bone turnover through osteocyte intercellular crosstalk, which exchanges prostaglandins through gap junctions in response to mechanical loading. Titanium nanosurfaces with anisotropically patterned dense nanospikes promote the development of osteocyte lacunar-canalicular networks. We investigated the effects of titanium nanosurfaces on intercellular network development and regulatory capabilities of bone turnover in osteocytes under cyclic compressive loading. MLO-Y4 mouse osteocyte-like cell lines embedded in type I collagen 3D gels on titanium nanosurfaces promoted the formation of intercellular networks and gap junctions even under static culture conditions, in contrast to the poor intercellular connectivity in machined titanium surfaces. The osteocyte 3D network on the titanium nanosurfaces further enhanced gap junction formation after additional culturing under cyclic compressive loading simulating masticatory loading, beyond the degree observed on machined titanium surfaces. A prostaglandin synthesis inhibitor cancelled the dual effects of titanium nanosurfaces and cyclic compressive loading on the upregulation of gap junction-related genes in the osteocyte 3D culture. Supernatants from osteocyte monolayer culture on titanium nanosurfaces promoted osteocyte maturation and intercellular connections with gap junctions. With cyclic loading, titanium nanosurfaces induced expression of the regulatory factors of bone turnover in osteocyte 3D cultures, toward higher osteoblast activation than that observed on machined surfaces. Titanium nanosurfaces with anisotropically patterned dense nanospikes promoted intercellular 3D network development and regulatory function toward osteoblast activation in osteocytes activated by cyclic compressive loading, through intercellular crosstalk by prostaglandin.


Assuntos
Osteoblastos , Osteócitos , Titânio , Titânio/farmacologia , Titânio/química , Animais , Osteócitos/metabolismo , Osteócitos/fisiologia , Osteócitos/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Linhagem Celular , Propriedades de Superfície , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/fisiologia , Junções Comunicantes/metabolismo , Nanoestruturas
17.
Zhongguo Zhong Yao Za Zhi ; 49(11): 2981-2990, 2024 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-39041158

RESUMO

This study aims to investigate the role and mechanism of Gusong Qianggu Decoction(GSQG) in attenuating bone loss in ovariectomized mice by targeting the endoplasmic reticulum stress(ERS)-induced apoptosis of osteocytes. After the modeling of osteoporosis in mice with bilateral ovary removal(OVX), 60 mice were randomized by the random number method into six groups: sham,model, low-, medium-, and high-dose GSQG(GSQG-L, GSQG-M, and GSQG-H, respectively), and estradiol(E_2), with 10 mice in each group. The mice in each group were administrated with corresponding drugs by gavage one month after surgery and the administration lasted for 3 months. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the serum levels of osteocalcin(OCN), procollagen type Ⅰ N-terminal propeptide(PINP), carboxy-terminal cross-linked telopeptide of type Ⅰ collagen(CTX),and anti-tartarte acid phosphatase 5b(TRAcP-5b). Micro-CT was employed to observe the changes in bone microstructure of the distal femur. Hematoxylin-eosin(HE) staining was employed to observe the morphology of the bone tissue. RT-qPCR was conducted to determine the m RNA levels of tibial stem osteogenesis-associated genes [type Ⅰ collagen(Col-Ⅰ), alkaline phosphatase(ALP), Runtrelated transcription factor-2(Runx2), bone sialoprotein(BSP), and OCN] and bone-breaking related genes [tartrate-resistant acid phosphatase(TRAP), nuclear factor-activated T cell 1(NFATc1), and cathepsin K(CATK)]. TUNEL staining and immunohistochemistry were employed to detect the apoptosis of osteoblasts. Western blot was employed to measure the expression of ERS-related proteins glucose-regulated protein 78( Grp78), protein kinase RNA-like endoplasmic reticulum kinase( PERK), phosphorylated PERK(p-PERK),eukaryotic translation initiation factor 2 alpha(eIF2α), phosphorylated e IF2α(p-eIF2α), inositol-requiring enzyme 1 alpha(IRE1α), phosphorylated IRE1α(p-IRE1α), and activating transcription factor 6(ATF6) in the proximal tibial bone tissue. The results showed that GSQG significantly recovered the levels of OCN, PINP, TRAc P-5b, and CTX in the serum of ovariectomized mice, and Micro-CT showed that GSQG improved the bone microstructure of distal femur in a dose-dependent manner. Compared with the model group, GSQG widened and increased the bone trabeculae, restored the reticular structure with neat arrangement and enlarged interstitial gaps, and reduced the number of TUNEL-positive cells(P<0. 05, P<0. 01). Furthermore, GSQG down-regulated the expression levels of cysteine aspartate protease-3( caspase-3) and factor Bcl-2-associated X protein( Bax)(P< 0. 05,P<0. 01) and up-regulated the expression level of Bcl-2(P<0. 05, P<0. 01). The GSQG groups showed up-regulated m RNA levels of Col-Ⅰ, ALP, Runx2, BSP, and OCN(P< 0. 01) and down-regulated m RNA levels of TRAP, NFATc1, and CATK(P< 0. 05,P<0. 01). In addition, GSQG, especially GSQG-H, down-regulated the protein levels of Grp78, p-PERK, p-eIF2, p-IRE1α, and ATF6(P< 0. 05, P< 0. 01). In conclusion, GSQG can inhibit the apoptosis of osteocytes by inhibiting the Grp78/PERK/e IF2α/IRE1α/ATF6 signaling pathway in the proximal tibia tissue, thus reducing bone loss in ovariectomized mice.


Assuntos
Apoptose , Medicamentos de Ervas Chinesas , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Osteócitos , Ovariectomia , Animais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Camundongos , Apoptose/efeitos dos fármacos , Feminino , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/administração & dosagem , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Humanos , Osteocalcina/genética , Osteocalcina/metabolismo , Densidade Óssea/efeitos dos fármacos
18.
Biomed Pharmacother ; 177: 117139, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39018871

RESUMO

The notion that obesity can be a protective factor for bone health is a topic of ongoing debate. Increased body weight may have a positive impact on bone health due to its mechanical effects and the production of estrogen by adipose tissue. However, recent studies have found a higher risk of bone fracture and delayed bone healing in elderly obese patients, which may be attributed to the heightened risk of bone immune regulation disruption associated with obesity. The balanced functions of bone cells such as osteoclasts, osteoblasts, and osteocytes, would be subverted by aberrant and prolonged immune responses under obese conditions. This review aims to explore the intricate relationship between obesity and bone health from the perspective of osteoimmunology, elucidate the impact of disturbances in bone immune regulation on the functioning of bone cells, including osteoclasts, osteoblasts, and osteocytes, highlighting the deleterious effects of obesity on various diseases development such as rheumatoid arthritis (RA), osteoarthritis (AS), bone fracture, periodontitis. On the one hand, weight loss may achieve significant therapeutic effects on the aforementioned diseases. On the other hand, for patients who have difficulty in losing weight, the osteoimmunological therapies could potentially serve as a viable approach in halting the progression of these disease. Additional research in the field of osteoimmunology is necessary to ascertain the optimal equilibrium between body weight and bone health.


Assuntos
Osso e Ossos , Obesidade , Humanos , Obesidade/imunologia , Obesidade/complicações , Animais , Osso e Ossos/imunologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Osteócitos/metabolismo , Osteócitos/imunologia , Osteoclastos/imunologia , Osteoclastos/metabolismo , Osteoblastos/imunologia , Osteoblastos/metabolismo , Remodelação Óssea/imunologia
19.
Adv Protein Chem Struct Biol ; 142: 397-420, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39059992

RESUMO

Considering the importance, bone physiology has long been studied to understand what systematic and cellular impact its cells and functions have. Exploring more questions is a substantially solid way to improve the understanding of bone physiological functions in/out sides. In adult bone, osteocytes (Ots) form about 95% of bone cells and live the longest lifespan inside their mineralized surroundings. Ots are the endocrine cells and originate from blood vessel's endothelial cells. In this work, we discussed the vital role of the "Ots". To determine the association between osteocytes' network with metabolic parameters in healthy mice, the experiments were performed on ten (10) adult C57BL6 male mice. Fasting blood and bone samples were collected weekly from mice for measurement of metabolic parameters and bone morphology. Scanning electron microscopy (SEM) revealed a 2D fine morphology of the bone which indicates a strong functional interconnection with bone nano/micro, and macro components of the organs. The long-branched canaliculi look like neurocytes in structure. The morphology and quantitative measurements of the osteocyte lacunal-canalicular system showed its wide spectrum spatial resolution of the positive and negative relationship within this system or metabolite parameters, confirming a strong cross connection between osteocyte lacunal-canalicular system and metabolism. We believe that the findings of this study can deliver a strategy about the potential roles of metabolic relation among osteocytes, insulin, and lipid in management of bone and metabolic diseases.


Assuntos
Osteócitos , Osteócitos/metabolismo , Osteócitos/citologia , Animais , Camundongos , Masculino , Camundongos Endogâmicos C57BL , Osso e Ossos/metabolismo
20.
Autoimmunity ; 57(1): 2364686, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38946534

RESUMO

BACKGROUND: Chondrocyte viability, apoptosis, and migration are closely related to cartilage injury in osteoarthritis (OA) joints. Exosomes are identified as potential therapeutic agents for OA. OBJECTIVE: This study aimed to investigate the role of exosomes derived from osteocytes in OA, particularly focusing on their effects on cartilage repair and molecular mechanisms. METHODS: An injury cell model was established by treating chondrocytes with IL-1ß. Cartilage repair was evaluated using cell counting kit-8, flow cytometry, scratch test, and Western Blot. Molecular mechanisms were analyzed using quantitative real-time PCR, bioinformatic analysis, and Western Blot. An OA mouse model was established to explore the role of exosomal DLX2 in vivo. RESULTS: Osteocyte-released exosomes promoted cell viability and migration, and inhibited apoptosis and extracellular matrix (ECM) deposition. Moreover, exosomes upregulated DLX2 expression, and knockdown of DLX2 activated the Wnt pathway. Additionally, exosomes attenuated OA in mice by transmitting DLX2. CONCLUSION: Osteocyte-derived exosomal DLX2 alleviated IL-1ß-induced cartilage repair and inactivated the Wnt pathway, thereby alleviating OA progression. The findings suggested that osteocyte-derived exosomes may hold promise as a treatment for OA.


Assuntos
Condrócitos , Exossomos , Proteínas de Homeodomínio , Osteoartrite , Via de Sinalização Wnt , Animais , Humanos , Masculino , Camundongos , Apoptose , Cartilagem/metabolismo , Cartilagem/patologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Movimento Celular , Sobrevivência Celular , Condrócitos/metabolismo , Modelos Animais de Doenças , Exossomos/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Interleucina-1beta/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteócitos/metabolismo , Fatores de Transcrição/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA