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1.
Int J Nanomedicine ; 15: 5061-5072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764936

RESUMO

Purpose: Zirconia is one of the most promising implant materials due to its favorable physical, mechanical and biological properties. However, until now, we know little about the mechanism of osseointegration on zirconia. The purpose of this study is to evaluate the effect of Syndecan (Sdc) on osteoblastic cell (MC3T3-E1) adhesion and proliferation onto zirconia materials. Materials and Methods: The mirror-polished disks 15 mm in diameter and 1.5 mm in thick of commercial pure titanium (CpTi), 3mol% yttria-stabilized tetragonal zirconia polycrystalline (3Y-TZP) and nano-zirconia (NanoZr) are used in this study. MC3T3-E1 cells were seeded onto specimen surfaces and subjected to RNA interference (RNAi) for Syndecan-1, Syndecan-2, Syndecan-3, and Syndecan-4. At 48h post-transfection, the cell morphology, actin cytoskeleton, and focal adhesion were observed using scanning electron microscopy or laser scanning confocal fluorescence microscopy. At 24h and 48h post-transfection, cell counting kit-8 (CCK-8) assay was used to investigate cell proliferation. Results: The cell morphology of MC3T3-E1 cells on CpTi, 3Y-TZP, and NanoZr changed into abnormal shape after gene silencing of Syndecan. Among the Syndecan family, Sdc-2 is responsible for NanoZr-specific morphology regulation, via maintenance of cytoskeletal conformation without affecting cellular attachment. According to CCK-8 assay, Sdc-2 affects the osteoblastic cell proliferation onto NanoZr. Conclusion: Within the limitation of this study, we suggest that Syndecan affects osteoblastic cell adhesion on CpTi, 3Y-TZP, and NanoZr. Sdc-2 might be an important heparin-sensitive cell membrane regulator in osteoblastic cell adhesion, specifically on NanoZr, through the organization of actin cytoskeleton and affects osteoblastic cell proliferation.


Assuntos
Osseointegração/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Sindecanas/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Adesão Celular/fisiologia , Membrana Celular/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Camundongos , Microscopia Eletrônica de Varredura , Osseointegração/genética , Propriedades de Superfície , Sindecana-2/genética , Sindecana-2/metabolismo , Sindecanas/genética , Titânio/química , Ítrio/química , Zircônio/química
2.
PLoS One ; 15(8): e0237660, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841254

RESUMO

This study evaluated the influence of type 2 diabetes mellitus on bone loss, bone repair and cytokine production in hyperglycemic rats, treated or not with metformin. The animals were distributed as follow: Non-Hyperglycemic (NH), Non Hyperglycemic with Ligature (NH-L), Treated Non Hyperglycemic (TNH), Treated Non Hyperglycemic with Ligature Treated (TNH-L), Hyperglycemic (H), Treated Hyperglycemic (TH), Hyperglycemic with Ligature (H-L), Treated Hyperglycemic with Ligature (TH-L). At 40th day after induction of hyperglycemia, the groups NH-L, TNH-L, H-L, TH-L received a ligature to induce periodontitis. On the 69th, the TNH, TNH-L, TH, TH-L groups received metformin until the end of the study. Bone repair was evaluated at histometric and the expression levels of Sox9, RunX2 and Osterix. Analysis of the ex-vivo expression of TNF-α, IFN-γ, IL-12, IL-4, TGF-ß, IL-10, IL-6 and IL-17 were also evaluated. Metformin partially reverse induced bone loss in NH and H animals. Lower OPG/RANKL, increased OCN and TRAP expression were observed in hyperglycemic animals, and treatment with metformin partially reversed hyperglycemia on the OPG/RANKL, OPN and TRAP expression in the periodontitis. The expression of SOX9 and RunX2 were also decreased by hyperglycemia and metformin treatment. Increased ex vivo levels of TNF-α, IL-6, IL-4, IL-10 and IL-17 was observed. Hyperglycemia promoted increased IL-10 levels compared to non-hyperglycemic ones. Treatment of NH with metformin was able to mediate increased levels of TNF-α, IL-10 and IL-17, whereas for H an increase of TNF-α and IL-17 was detected in the 24- or 48-hour after stimulation with LPS. Ligature was able to induce increased levels of TNF-α and IL-17 in both NH and H. This study revealed the negative impact of hyperglycemia and/or treatment with metformin in the bone repair via inhibition of transcription factors associated with osteoblastic differentiation.


Assuntos
Perda do Osso Alveolar/prevenção & controle , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/complicações , Hiperglicemia/complicações , Metformina/administração & dosagem , Periodontite/prevenção & controle , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/metabolismo , Processo Alveolar/citologia , Processo Alveolar/efeitos dos fármacos , Processo Alveolar/metabolismo , Processo Alveolar/patologia , Animais , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/genética , Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Hiperglicemia/induzido quimicamente , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Osteoblastos/fisiologia , Periodontite/etiologia , Periodontite/metabolismo , Ratos , Estreptozocina/toxicidade , Fatores de Transcrição/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(25): 14386-14394, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513693

RESUMO

We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine ß-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.


Assuntos
Disfunção Erétil/tratamento farmacológico , Osteogênese/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Inibidores da Fosfodiesterase 5/farmacologia , Envelhecimento/fisiologia , Animais , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/fisiologia , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Reposicionamento de Medicamentos , Disfunção Erétil/complicações , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Animais , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Osteoporose/complicações , Fraturas por Osteoporose/etiologia , Fraturas por Osteoporose/prevenção & controle , Inibidores da Fosfodiesterase 5/química , Inibidores da Fosfodiesterase 5/uso terapêutico , Cultura Primária de Células , Tadalafila/química , Tadalafila/farmacologia , Tadalafila/uso terapêutico , Dicloridrato de Vardenafila/química , Dicloridrato de Vardenafila/farmacologia , Dicloridrato de Vardenafila/uso terapêutico
4.
Arterioscler Thromb Vasc Biol ; 40(4): 885-900, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32160774

RESUMO

Aortic valve stenosis is the most prevalent heart valve disease worldwide. Although interventional treatment options have rapidly improved in recent years, symptomatic aortic valve stenosis is still associated with high morbidity and mortality. Calcific aortic valve stenosis is characterized by a progressive fibro-calcific remodeling and thickening of the aortic valve cusps, which subsequently leads to valve obstruction. The underlying pathophysiology is complex and involves endothelial dysfunction, immune cell infiltration, myofibroblastic and osteoblastic differentiation, and, subsequently, calcification. To date, no pharmacotherapy has been established to prevent aortic valve calcification. However, novel promising therapeutic targets have been recently identified. This review summarizes the current knowledge of pathomechanisms involved in aortic valve calcification and points out novel treatment strategies.


Assuntos
Estenose da Valva Aórtica/tratamento farmacológico , Estenose da Valva Aórtica/fisiopatologia , Animais , Estenose da Valva Aórtica/patologia , Comunicação Celular , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/fisiopatologia , Humanos , Inflamação/fisiopatologia , Lipoproteínas/metabolismo , Miofibroblastos/fisiologia , Osteoblastos/fisiologia , Osteoclastos/fisiologia , RNA não Traduzido/metabolismo , Calcificação Vascular/fisiopatologia
5.
Phytomedicine ; 69: 153195, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32200293

RESUMO

BACKGROUND: Areca nut has anti-inflammatory, antiparasitic, antihypertensive, and antidepressant properties. The pathological hallmarks of inflammatory joint diseases are an increased number of osteoclasts and impaired differentiation of osteoblasts, which may disrupt the bone remodeling balance and eventually lead to bone loss. PURPOSE: The present study assessed the effects of arecoline, the main alkaloid found in areca nut, on osteoclast and osteoblast differentiation and function. METHOD: M-CSF/RANKL-stimulated murine bone marrow-derived macrophages (BMMs) were incubated with several concentrations of arecoline, and TRAP staining and pit formation were assessed to monitor osteoclast formation. Quantitative real-time RT-PCR and western blot analyses were used to analyze the expression of osteoclast-associated genes and signaling pathways. The effects of arecoline on bone were investigated in an in vivo mouse model of lipopolysaccharide (LPS)-induced trabecular bone loss after oral administration of arecoline. Alizarin red S staining and assays to measure ALP activity and the transcription level of osteoblast-related genes were used to evaluate the effects of arecoline on osteoblast differentiation and bone mineralization. RESULTS: In a dose-dependent manner, arecoline at concentrations of 50-100 µM reduced both the development of TRAP-positive multinucleated osteoclasts and the formation of resorption pits in M-CSF/RANKL-stimulated BMMs. In M-CSF/RANKL-stimulated BMMs, arecoline also suppressed the expression and translocation of c-Fos and NFATcl, and osteoclast differentiated-related genes via interference with the AKT, MAPK, and NF-kB activation pathways. Femur bone loss and microcomputed tomography parameters were recovered by oral administration of arecoline in the mouse LPS-induced bone loss model. Lastly, arecoline increased ALP activity, bone mineralization, and the expression of osteoblast differentiation-related genes, such as ALP and Runx2, in MC3T3-E1 cells. CONCLUSION: Our data suggest that arecoline may attenuate or prevent bone loss by suppressing osteoclastogenesis and promoting osteoblastogenesis. These findings provide evidence supporting arecoline's use as a potential therapeutic agent in bone-loss disorders and diseases.


Assuntos
Arecolina/farmacologia , Reabsorção Óssea/tratamento farmacológico , Osteoclastos/efeitos dos fármacos , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos DBA , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteoclastos/citologia , Osteoclastos/fisiologia , Osteogênese/efeitos dos fármacos , Osteoporose/induzido quimicamente , Osteoporose/tratamento farmacológico , Ligante RANK/metabolismo , Ligante RANK/farmacologia , Microtomografia por Raio-X
6.
J Appl Oral Sci ; 28: e20190156, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32049134

RESUMO

OBJECTIVE: The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). METHODOLOGY: Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. RESULTS: FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. CONCLUSIONS: Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Osteoblastos/efeitos dos fármacos , Quinolonas/farmacologia , Sulfonas/farmacologia , Titânio/química , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteína-Tirosina Quinases de Adesão Focal/análise , Proteína-Tirosina Quinases de Adesão Focal/química , Expressão Gênica , Integrinas/análise , Microscopia Eletrônica de Varredura , Osseointegração/efeitos dos fármacos , Osteoblastos/fisiologia , Quinolonas/química , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Sulfonas/química , Propriedades de Superfície
7.
Medicine (Baltimore) ; 99(8): e19120, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32080087

RESUMO

Osteoporosis (OP) is a disease characterized by bone mass loss, bone microstructure damage, increased bone fragility, and easy fracture. The molecular mechanism underlying OP remains unclear.In this study, we identified 217 genes associated with OP, and formed a gene set [OP-related genes gene set (OPgset)].The highly enriched GOs and pathways showed OPgset genes were significantly involved in multiple biological processes (skeletal system development, ossification, and osteoblast differentiation), and several OP-related pathways (Wnt signaling pathway, osteoclast differentiation, steroid hormone biosynthesis, and adipocytokine signaling pathway). Besides, pathway crosstalk analysis indicated three major modules, with first module consisted of pathways mainly involved in bone development-related signaling pathways, second module in Wnt-related signaling pathway and third module in metabolic pathways. Further, we calculated degree centrality of a node and selected ten key genes/proteins, including TGFB1, IL6, WNT3A, TNF, PTH, TP53, WNT1, IGF1, IL10, and SERPINE1. We analyze the K-core and construct three k-core sub-networks of OPgset genes.In summary, we for the first time explored the molecular mechanism underlying OP via network- and pathway-based methods, results from our study will improve our understanding of the pathogenesis of OP. In addition, these methods performed in this study can be used to explore pathogenesis and genes related to a specific disease.


Assuntos
Osso e Ossos/patologia , Fraturas Ósseas/etiologia , Osteoporose/genética , Adipocinas/genética , Densidade Óssea/genética , Osso e Ossos/metabolismo , Osso e Ossos/ultraestrutura , Diferenciação Celular/genética , Biologia Computacional/métodos , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Humanos , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteogênese/genética , Osteoporose/complicações , Osteoporose/epidemiologia , Prevalência , Via de Sinalização Wnt/genética
8.
Life Sci ; 245: 117389, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32007573

RESUMO

Osteogenesis is the process of new bone formation where transcription factors play an important role in controlling cell proliferation and differentiation. Runt-related transcription factor 2 (Runx2), a key transcription factor, regulates the differentiation of mesenchymal stem cells into osteoblasts, which further mature into osteocytes. Runx2 acts as a modulator such that it can either stimulate or inhibit the osteoblast differentiation. A defect/alteration in the expression/activity of this gene may lead to skeletal dysplasia. Runx2 thus serves as the best therapeutic model gene for studying bone and bone-related diseases. In this review, we briefly outline the regulation of Runx2 and its activity at the post-translational levels by the virtue of phosphorylation, acetylation, and ubiquitination in controlling the bone homeostasis.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Osteoblastos/fisiologia , Osteogênese/fisiologia , Animais , Humanos , Processamento de Proteína Pós-Traducional
9.
Plast Reconstr Surg ; 145(3): 552e-562e, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32097313

RESUMO

BACKGROUND: Several studies have verified that bone morphogenetic proteins (BMPs) may be involved in the development of craniosynostosis; little attention has been focused on the role of BMP9 in cranial suture biology. The authors investigated the role of BMP9 in suture progenitor cells. METHODS: The authors isolated and cultured prematurely fused and internal control patent suture progenitor cells from patients with nonsyndromic craniosynostosis. Overexpression of BMP9 was mediated by adenoviral vectors. Osteoblast and osteoclast differentiation-related markers were evaluated by staining techniques and touchdown quantitative polymerase chain reaction analysis. In vivo analysis of BMP9-induced suture progenitor cell osteogenesis was performed in an ectopic bone formation model. RESULTS: The authors demonstrated that the prematurely fused sutures have a higher endogenous expression of the osteogenic differentiation-related genes than patent sutures, whereas the same pattern of gene expression exists between fused and patent suture progenitor cells. Importantly, both patent and fused suture progenitor cells undergo osteogenic differentiation and express multiple lineage regulators and NELL-1 on BMP9 stimulation, whereas fused suture progenitor cells have a higher basal osteogenic potential than patent suture progenitor cells. BMP9 regulates the expression of osteoclast differentiation-related genes in suture progenitor cells. Forced BMP9 expression enhances the mineralization and maturity of ectopic bone formation of suture progenitor cells implanted in vivo. CONCLUSIONS: The authors' findings suggest that fused suture progenitor cells have elevated osteogenic potential. BMP9 could regulate the expression of multiple osteoblast and osteoclast differentiation-related genes, and NELL-1, in both suture progenitor cells, indicating that BMP9 may play a role in craniosynostosis.


Assuntos
Suturas Cranianas/patologia , Craniossinostoses/genética , Fator 2 de Diferenciação de Crescimento/metabolismo , Células-Tronco Mesenquimais/patologia , Osteogênese/genética , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular/genética , Suturas Cranianas/citologia , Suturas Cranianas/cirurgia , Craniossinostoses/patologia , Craniossinostoses/cirurgia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Lactente , Masculino , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Cultura Primária de Células , Procedimentos Cirúrgicos Reconstrutivos
10.
Carbohydr Polym ; 230: 115612, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31887952

RESUMO

This work reports on the preparation and systematic testing of a novel multi-layered coating, comprised of the non-steroid anti-inflammatory drug diclofenac and biopolymer carboxymethyl cellulose. Drug release testing was performed on an Automated Transdermal Diffusion Cells Sampling System in combination with UV-VIS spectroscopy as the released drug concentration determination method. The results showed that most of the drug is released in the first six hours, whereas the overall released amount could be tailored through changes in the multi-layered coating composition. Biocompatibility tests performed on human osteoblast cells, showed cell viability improvement between 7% and 17% compared to the control sample. The expression of proteins playing important roles in extracellular matrix production and functioning was performed in order to obtain additional proof of the prepared materials' osteointegration boosting capacity. Finally, electrochemical measurements confirmed that the coatings do not influence the corrosion susceptibility of AISI 316LVM stainless steel.


Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Carboximetilcelulose Sódica/química , Diclofenaco/administração & dosagem , Liberação Controlada de Fármacos , Osteoblastos/efeitos dos fármacos , Osteogênese , Aço/química , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Materiais Biocompatíveis/química , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Proliferação de Células , Células Cultivadas , Diclofenaco/química , Diclofenaco/farmacologia , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Humanos , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Proteoglicanas/genética , Proteoglicanas/metabolismo
11.
Mol Cells ; 43(1): 34-47, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-31896234

RESUMO

The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Reverbs, comprising Rev-erbα and Rev-erbß, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erbα in osteoclast precursor cells enhanced receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erbα leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erbα in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erbα interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Reverbα in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erbα negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo . Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.


Assuntos
Reabsorção Óssea/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteogênese/genética , Animais , Remodelação Óssea , Reabsorção Óssea/genética , Diferenciação Celular , Células Cultivadas , Relógios Circadianos , Modelos Animais de Doenças , Mutação com Ganho de Função/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
FASEB J ; 34(2): 3399-3412, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31925860

RESUMO

Osteoporotic osteoarthritis (OPOA) is a common bone disease mostly in the elderly, but the relationship between Osteoporotic (OP) and osteoarthritis (OA) is complex. It has been shown that knee loading can mitigate OA symptoms. However, its effects on OPOA remain unclear. In this study, we characterized pathological linkage of OP to OA, and evaluated the effect of knee loading on OPOA. We employed two mouse models (OA and OPOA), and conducted histology, cytology, and molecular analyses. In the OA and OPOA groups, articular cartilage was degenerated and Osteoarthritis Research Society International score was increased. Subchondral bone underwent abnormal remodeling, the differentiation of bone marrow mesenchymal stem cells (BMSCs) to osteoblasts and chondrocytes was reduced, and migration and adhesion of pre-osteoclasts were enhanced. Compared to the OA group, the pathological changes of OA in the OPOA group were considerably aggravated. After knee loading, however, cartilage degradation was effectively prevented, and the abnormal remodeling of subchondral bone was significantly inhibited. The differentiation of BMSCs was also improved, and the expression of Wnt/ß-catenin was elevated. Collectively, this study demonstrates that osteoporosis aggravates OA symptoms. Knee loading restores OPOA by regulating subchondral bone remodeling, and may provide an effective method for repairing OPOA.


Assuntos
Cartilagem Articular/metabolismo , Osteoartrite/terapia , Osteoporose Pós-Menopausa/terapia , Suporte de Carga , Via de Sinalização Wnt , Animais , Cartilagem Articular/patologia , Adesão Celular , Diferenciação Celular , Movimento Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/fisiologia , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteoartrite/etiologia , Osteoartrite/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Osteoporose Pós-Menopausa/complicações , Osteoporose Pós-Menopausa/metabolismo
13.
Arch Oral Biol ; 110: 104594, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31733582

RESUMO

OBJETIVE: It has been claimed that micro-pulse vibration can accelerate the rate of tooth movement during orthodontic treatment; however, the underlying cellular mechanism has yet to be elucidated. The purpose of this study was to understand the mechanisms underlying tooth movement acceleration by measuring alterations in a panel of intercellular signalling molecules and markers of osteoblast/osteoclast function following micro-pulse vibration for 20 min at 30 Hz. DESIGN: Primary BALB/c mouse calvarial osteoblasts were cultivatedin vitro and subjected to micro-pulse vibration (0.25 N; 30 Hz) with the AcceleDent® Aura appliance for 20 min and assayed for IL-4, IL-13, IL-17, OPG, soluble RANKL and TGF-ß protein by ELISA; for PCNA in osteoblasts and caspase 3/7 in osteoclasts by immunohistochemistry; for IL-4, IL-13, and Il-17 in osteoclasts by ELISA; and for cathepsin K by flow cytometry. RESULTS: After micro-pulse vibration, the murine osteoblast culture supernatant showed increased IL-4, IL-13, IL-17, OPG and TGF-ß levels and decreased RANKL levels; PCNA in osteoblasts and caspase 3/7 in osteoclasts were also upregulated. The osteoclast culture supernatant had increased levels of IL-4, IL-13 and IL-17, and cathepsin K was upregulated in the treatment group compared with the control group. CONCLUSIONS: Micro-pulse vibration promotes the production of soluble factors that inhibit osteoclasts, promote apoptosis and activate osteoblasts in vitro, which could increase bone mineral density. Further studies should be conducted in order to understand the biological mechanism of how micro-vibration might influence tooth movement during orthodontic treatment.


Assuntos
Diferenciação Celular , Osteoblastos , Osteoclastos , Vibração , Animais , Camundongos , Camundongos Endogâmicos BALB C , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteoprotegerina , Ligante RANK/metabolismo
14.
Phytomedicine ; 66: 153107, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31790903

RESUMO

BACKGROUND: Gomisin A is a lignan isolated from the hexane of Schisandra chinensis fruit extract with antioxidant properties. Oxidative stress mediated by high glucose is one of the major complications of diabetes mellitus. PURPOSE: This study investigates the role of gomisin A in osteoblast differentiation under high glucose-induced oxidative stress in MC3T3 E1 cells and determines its relationship with heme oxygenase-1 (HO-1) and mitochondrial biogenesis. METHODS: MC3T3 E1 cells were treated by gomisin A following induced by high glucose levels and glucose oxidase to investigate the inhibitory effect of gomisin A against high glucose oxidative stress. Western blot analysis, alizarin red staining, alkaline phosphatase (ALP) activity, analysis of reactive oxygen species (ROS) and confocal microscopy were used to determine mitochondrial biogenesis, oxidative stress, osteoblast differentiation and mineralization. To analyze the role of HO-1, the MC3T3 E1 cells were treated with the HO-1 inhibitor zinc protoporphyrin IX (ZnPP). RESULTS: Gomisin A enhanced the expression of HO-1, increased mitochondrial biogenesis factors (peroxisome proliferator-activated receptor gamma coactivator 1-alpha, nuclear respiratory factor-1, and mitochondrial transcription factor A), antioxidant enzymes (copper-zinc superoxide dismutases and manganese superoxide dismutase), osteoblast differentiation molecules (bone morphogenic protein-2/7, osteoprotegerin and Runt-related transcription factor-2) and mineralization by upregulation of ALP and alizarin red staining, which were decreased by ZnPP and high glucose oxidative stress. Similarly, gomisin A inhibited ROS which was increased by ZnPP and the high glucose-mediated oxidative stress. CONCLUSIONS: The findings demonstrated the antioxidative effects of gomisin A, and its role in mitochondrial biogenesis and osteoblast differentiation. It potentially regulated osteoblast differentiation under high glucose-induced oxidative stress via upregulation of HO-1 and maintenance of mitochondrial homeostasis. Thus, gomisin A may represent a potential therapeutic agent for prevention of bone fragility fractures and implant failure triggered by diabetes.


Assuntos
Antioxidantes/farmacologia , Ciclo-Octanos/farmacologia , Diabetes Mellitus/tratamento farmacológico , Dioxóis/farmacologia , Glucose/efeitos adversos , Lignanas/farmacologia , Osteogênese/efeitos dos fármacos , Schisandra/química , Animais , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Heme Oxigenase-1/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Biogênese de Organelas , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Protoporfirinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo
15.
Ann Anat ; 227: 151427, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31614180

RESUMO

Mandibular/alveolar (m/a) bone, as a component of the periodontal apparatus, allows for the proper tooth anchorage and function of dentition. Bone formation around the tooth germs starts prenatally and, in the mouse model, the mesenchymal condensation turns into a complex vascularized bone (containing osteo-blasts, -cytes, -clasts) within only two days. This very short but critical period is characterized by synchronized cellular and molecular events. The m/a bone, as others, is subjected to endocrine regulations. This not only requires vasculature to allow the circulation of active molecules (ligands), but also the expression of corresponding cell receptors to define target tissues. This contribution aimed at following the dynamics of calciotropic receptors´ expression during morphological transformation of a mesenchymal condensation into the initial m/a bone structure. Receptors for all three calciotropic systemic regulators: parathormone, calcitonin and activated vitamin D (calcitriol), were localized on serial histological sections using immunochemistry and their relative expression was quantified by q-PCR. The onset of calciotropic receptors was followed along with bone cell differentiation (as checked using osteocalcin, sclerostin, RANK and TRAP) and vascularization (CD31) during mouse prenatal/embryonic (E) days 13-15 and 18. Additionally, the timing of calciotropic receptor appearance was compared with that of estrogen receptors (ESR1, ESR2). PTH receptor (PTH1r) appeared in the bone already at E13, when the first osteocalcin-positive cells were detected within the mesenchymal condensation forming the bone anlage. At this stage, blood vessels were only lining the condensation. At E14, the osteoblasts started to express the receptor for activated vitamin D (VDR). At this stage, the vasculature just penetrated the forming bone. On the same day, the first TRAP-positive (but not yet multinucleated) osteoclastic cells were identified. However, calcitonin receptor was detected only one day later. The first Sost-positive osteocytes, present at E15, were PTH1r and VDR positive. ESR1 almost copied the expression pattern of PTH1r, and ESR2 appearance was similar with VDR with a significant increase between E15 and E18. This report focuses on the in vivo situation and links morphological transformation of the mesenchymal cell condensation into a bone structure with dynamics of cell differentiation/maturation, vascularization and onset of receptors for calciotropic endocrine signalling in developing m/a bone.


Assuntos
Mandíbula/crescimento & desenvolvimento , Osteogênese/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diferenciação Celular , Imuno-Histoquímica , Camundongos , Osteoblastos/fisiologia , Osteocalcina/análise , Osteocalcina/genética , Osteoclastos/fisiologia , Osteócitos/fisiologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Calcitonina/metabolismo
16.
J Cell Physiol ; 235(1): 210-220, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31188489

RESUMO

Lateral meningocele syndrome (LMS), a genetic disorder characterized by meningoceles and skeletal abnormalities, is associated with NOTCH3 mutations. We created a mouse model of LMS (Notch3tm1.1Ecan ) by introducing a tandem termination codon in the Notch3 locus upstream of the proline (P), glutamic acid (E), serine (S) and threonine (T) domain. Microcomputed tomography demonstrated that Notch3tm1.1Ecan mice exhibit osteopenia. The cancellous bone osteopenia was no longer observed after the intraperitoneal administration of antibodies directed to the negative regulatory region (NRR) of Notch3. The anti-Notch3 NRR antibody suppressed the expression of Hes1, Hey1, and Hey2 (Notch target genes), and decreased Tnfsf11 (receptor activator of NF Kappa B ligand) messenger RNA in Notch3tm1.1Ecan osteoblast (OB) cultures. Bone marrow-derived macrophages (BMMs) from Notch3tm1.1Ecan mutants exhibited enhanced osteoclastogenesis in culture, and this was increased in cocultures with Notch3tm1.1Ecan OB. Osteoclastogenesis was suppressed by anti-Notch3 NRR antibodies in Notch3tm1.1Ecan OB/BMM cocultures. In conclusion, the cancellous bone osteopenia of Notch3tm1.1Ecan mutants is reversed by anti-Notch3 NRR antibodies.


Assuntos
Anormalidades Múltiplas/genética , Anormalidades Múltiplas/terapia , Anticorpos/uso terapêutico , Meningocele/genética , Meningocele/terapia , Receptor Notch3/imunologia , Animais , Osso e Ossos/anormalidades , Feminino , Predisposição Genética para Doença , Macrófagos/fisiologia , Masculino , Camundongos , Mutação , Osteoblastos/fisiologia , Microtomografia por Raio-X
17.
Phytomedicine ; 65: 153103, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31805425

RESUMO

BACKGROUND: Regulation of the survival and differentiation of bone marrow mesenchymal stem cells is an essential consideration in the development of targeted drugs for treatment of osteoporosis. PURPOSE: The present study aimed to evaluate the combined effect of wedelolactone and oleonuezhenide, two compounds from Chinese formula Er-Zhi-Wan, on osteoblastogenesis and the underlying molecular mechanisms. METHODS: MTT assay was taken to evaluate cell proliferation. The alkaline phosphatase (ALP) activity assay was used to determine the activity of ALP. Alizarin red S (ARS) staining was taken to indicate the intensity of the calcium deposits. Quantitative real-time PCR and Western blot were performed to the levels of Runx2, Osteocalcin, and Osterix expression in mouse bone marrow mesenchymal stem cells (BMSCs). Ovariectomized mouse model and bone histomorphometric analysis were also used to research the effects of wedelolactone and oleonuezhenide on bone loss caused by ovariectomy. RESULTS: Wedelolactone combined with oleonuezhenide enhanced osteoblast differentiation and bone mineralization. Osteoblastogenesis-related marker genes including osteocalcin, Runx2, and osteorix were upregulated in the presence of wedelolactone and oleonuezhenide. At the molecular level, oleonuezhenide did not affect GSK-3ß phosphorylation induced by wedelolactone, but elevated casein kinase 2-alpha (CK2α) expression, resulting in ß-catenin and Runx2 nuclear translocation. In addition, 30 µM wedelolactone-induced cytotoxicity in bone marrow mesenchymal stem cells was relieved by 9 µM oleonuezhenide. These cells were protected by oleonuezhenide and maintained osteoblastic activity. Oleonuezhenide increased Wnt5a and CK2α expression. Wedelolactone-reduced extracellular signal-regulated kinase (ERK) phosphorylation was reversed by oleonuezhenide. In ovariectomized mice, administration of wedelolactone and oleonuezhenide prevented ovariectomy-induced bone loss by enhancing osteoblastic activity. CONCLUSION: These results suggested that oleonuezhenide enhanced the effects of wedelolactone on osteoblastogenesis. These two compounds could be developed as a combined therapeutic agent for osteoporosis.


Assuntos
Cumarínicos/farmacologia , Glucosídeos Iridoides/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Quinase 3 da Glicogênio Sintase/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos Endogâmicos BALB C , Osteoblastos/fisiologia , Osteoporose/etiologia , Osteoporose/prevenção & controle , Ovariectomia , Via de Sinalização Wnt/efeitos dos fármacos
18.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801223

RESUMO

Matrix metalloproteinase (MMP)-2 and MMP-9 are well-known gelatinases that disrupt the extracellular matrix, including gelatin. However, the advantages of modulating MMP expression in gelatin-based materials for applications in bone regenerative medicine have not been fully clarified. In this study, we examined the effects of epigallocatechin gallate (EGCG), a major polyphenol catechin isolated from green tea, on MMP expression in gelatin sponges and its association with bone formation. Four gelatin sponges with or without EGCG were prepared and implanted into bone defects for up to 4 weeks. Histological and immunohistological staining were performed. Micro-computed tomography was used to estimate the bone-forming capacity of each sponge. Our results showed that EGCG integration attenuated MMP-2 (70.6%) and -9 expression (69.1%) in the 1 week group, increased residual gelatin (118.7%), and augmented bone formation (101.8%) in the 4 weeks group in critical-sized bone defects of rat calvaria compared with vacuum-heated gelatin sponges without EGCG. Moreover, vacuum-heated gelatin sponges with EGCG showed superior bone formation compared with other sponges. The results indicated that integration of EGCG in gelatin-based materials modulated the production and activity of MMP-2 and -9 in vivo, thereby enhancing bone-forming capacity.


Assuntos
Materiais Biocompatíveis/síntese química , Regeneração Óssea/efeitos dos fármacos , Reabsorção Óssea/prevenção & controle , Catequina/análogos & derivados , Gelatina/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Engenharia Tecidual/métodos , Implantes Absorvíveis , Aldeídos/antagonistas & inibidores , Aldeídos/metabolismo , Animais , Reabsorção Óssea/diagnóstico por imagem , Catequina/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Ratos , Ratos Sprague-Dawley , Crânio/diagnóstico por imagem , Crânio/efeitos dos fármacos , Crânio/lesões , Crânio/fisiologia , Tecidos Suporte , Microtomografia por Raio-X
19.
Mol Med ; 25(1): 56, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31847800

RESUMO

BACKGROUND: There are two isoforms of sphingomyelin synthase (SMS): SMS1 and SMS2. SMS1 is located in the Golgi apparatus only while SMS2 is located in both the plasma membrane and the Golgi apparatus. SMS1 and SMS2 act similarly to generate sphingomyelin (SM). We have undertaken the experiments reported here on SMS and osteoblast differentiation in order to better understand the role SMS plays in skeletal development. METHODS: We analyzed the phenotype of a conditional knockout mouse, which was generated by mating a Sp7 promoter-driven Cre-expressing mouse with an SMS1-floxed SMS2-deficient mouse (Sp7-Cre;SMS1f/f;SMS2-/- mouse). RESULTS: When we compared Sp7-Cre;SMS1f/f;SMS2-/- mice with C57BL/6, SMS2-deficient mice (SMS1f/f;SMS2-/-) and SP7-Cre positive control mice (Sp7-Cre, Sp7-Cre;SMS1+/+;SMS2+/- and Sp7-Cre;SMS1+/+;SMS2-/-), we found that although cartilage formation is normal, Sp7-Cre;SMS1f/f;SMS2-/- mice showed reduced trabecular and cortical bone mass, had lower bone mineral density, and had a slower mineral apposition rate than control mice. Next, we have used a tamoxifen-inducible knockout system in vitro to show that SMS1 plays an important role in osteoblast differentiation. We cultured osteoblasts derived from ERT2-Cre;SMS1f/f SMS2-/- mice. We observed impaired differentiation of these cells in response to Smad1/5/8 and p38 that were induced by bone morphogenic protein 2 (BMP2). However, Erk1/2 phosphorylation was unaffected by inactivation of SMS1. CONCLUSIONS: These findings provide the first genetic evidence that SMS1 plays a role in bone development by regulating osteoblast development in cooperation with BMP2 signaling. Thus, SMS1 acts as an endogenous signaling component necessary for bone formation.


Assuntos
Diferenciação Celular/genética , Osteoblastos/fisiologia , Osteogênese/genética , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
20.
J Orthop Surg Res ; 14(1): 366, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727100

RESUMO

BACKGROUND: Bone marrow mesenchymal stem cells (BMMSCs) can be used for bone regeneration in the specified condition. Osteogenic differentiation of BMMSCs is controlled by microRNAs (miRNAs) and other factors. This study was aimed to identify the role and mechanism of miR-889 in regulating the osteogenic differentiation of BMMSCs. METHODS: Osteoporosis patients and normal control bone tissues were collected and used PCR techniques to identify the change of miR-889 and WNT7A. Moreover, the dynamic change of miR-889 and WNT7A during osteogenic differentiation of BMMSCs was also measured. Bioinformatic analysis was performed to identify the target genes and potential pathways of miR-889. Then, we constructed miR-889 mimic and inhibitor, ALP staining, ARS, osteoblastic-related protein, and Wnt ß-catenin signaling pathway-related protein were also measured. WNT7A siRNA was also used to verify the function of miR-889. RESULTS: In the present study, we showed that miR-889 expression was upregulated in osteoporosis patients than healthy control. However, the miR-889 expression was downregulated during osteogenic differentiation. Bioinformatics analysis found that miR-889 targets 666 genes and mainly through Wnt ß-catenin signaling pathway. Administrated miR-889 mimic, the ALP activity, and calcium deposition were decreased than the control group, while miR-889 inhibitor shown the opposite trend. And miR-889 could bind the 3'UTR of WNT7A. We further used WNT7A siRNA to explore the function of miR-889, and the results revealed that co-cultured with miR-889 inhibitor and WNT7A siRNA was associated with a reduction of ALP activity and calcium deposition and osteoblastic-related proteins than miR-889 inhibitor alone. CONCLUSION: Our results revealed that miR-889 plays a negative role in inducing osteogenic differentiation of BMSCs through Wnt ß-catenin signaling pathway.


Assuntos
Células da Medula Óssea/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Osteoporose/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Feminino , Humanos , Osteoblastos/fisiologia , Via de Sinalização Wnt
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