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1.
Med Sci Monit ; 26: e919309, 2020 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-32146478

RESUMO

BACKGROUND Osteoblast differentiation is a critical process to maintain the stability of the bone homeostasis. Zingerone, 4-(4-hydroxy-3-methoxyphenyl)-2-butanone (ZG), isolated from ginger, performs a wide range of biological functions in human diseases. The objective of this paper was to clarify the role of ZG in human bone mesenchymal stem cells (hBMSCs) and associated mechanisms of ZG promoting osteoblast differentiation. MATERIAL AND METHODS The cytotoxicity of ZG was detected by MTT assay. The expression levels of miR-200c-3p, smad7, and osteoblast differentiation markers (alkaline phosphatase [ALP], osteocalcin [OC], osterix [OSX] and runt-related transcription factor 2 [RUNX2]) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of smad7, ALP, OC, OSX, and RUNX2 were quantified by western blot analysis. The target mRNAs were predicted by bioinformatics tools TargetScan. The interaction between miR-200c-3p and smad7 was verified by luciferase reporter assay and RIP assay. RESULTS ZG was nontoxic to hBMSCs, and it accelerated osteoblast differentiation by inducing the expression of ALP, OC, OSX, and RUNX2. MiR-200c-3p was upregulated, but smad7 was downregulated in hBMSCs treated with ZG at different concentrations at different periods. Besides, miR-200c-3p positively regulated the expression of ALP, OC, OSX, and RUNX2 in ZG-induced hBMSCs. Moreover, miR-200c-3p targeted smad7 and strengthened the expression of ALP, OC, OSX, and RUNX2 in ZG-induced hBMSCs by downregulating smad7. CONCLUSIONS ZG contributed to osteoblast differentiation via miR-200c-3p/smad7 regulatory axis by promoting the expression of ALP, OC, OSX, and RUNX2 in hBMSCs.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Guaiacol/análogos & derivados , Células-Tronco Mesenquimais/efeitos dos fármacos , MicroRNAs/genética , Osteoblastos/citologia , Osteogênese/efeitos dos fármacos , Proteína Smad7/genética , Fosfatase Alcalina/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Guaiacol/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia , Osteoblastos/efeitos dos fármacos , Osteocalcina/metabolismo , Fator de Transcrição Sp7/metabolismo
2.
Life Sci ; 248: 117455, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32088216

RESUMO

AIMS: Idiopathic scoliosis is a common deformity of the spine that has an especially high incidence rate in adolescents. Some studies have demonstrated a close relationship between idiopathic scoliosis and melatonin deficiency. Our team's previous research showed that melatonin can inhibit the proliferation of osteoblasts, but the mechanism remains unclear. This study aimed to determine the mechanism by which melatonin inhibits the proliferation of osteoblasts. MAIN METHODS: Cell viability experiment, DNA fragment detection and alkaline phosphatase (ALP) activity assays were performed to determine the effects of melatonin on the proliferation, apoptosis and differentiation of osteoblasts. We used immunofluorescence to detect the expression of STIM1 in melatonin-treated osteoblasts. STIM1 interference was achieved using a specific siRNA, and a TRPC inhibitor was used to block the influx of Ca2+. The mRNA expression was determined by RT-qPCR, and protein levels were measured by Western blot. KEY FINDINGS: In this study, we found that melatonin inhibited the proliferation, differentiation and apoptosis of osteoblasts in a concentration-dependent manner. Additional studies showed that melatonin elevated cytosolic calcium levels by upregulation of STIM1, leading to osteoblast apoptosis via the mitochondrial pathway. Finally, we demonstrated that the STIM1-mediated increase in cytosolic calcium levels induced apoptosis through the ERK pathway. SIGNIFICANCE: Melatonin induces mitochondrial apoptosis in osteoblasts by regulating the STIM1/cytosolic calcium elevation/ERK pathway. These basic findings provide a basis for further clinical studies on melatonin as a drug therapeutic for idiopathic scoliosis.


Assuntos
Antioxidantes/farmacologia , Cálcio/metabolismo , Sistema de Sinalização das MAP Quinases , Melatonina/farmacologia , Osteoblastos/efeitos dos fármacos , Molécula 1 de Interação Estromal/genética , Fosfatase Alcalina/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Transporte de Íons/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Molécula 1 de Interação Estromal/agonistas , Molécula 1 de Interação Estromal/antagonistas & inibidores , Molécula 1 de Interação Estromal/metabolismo , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo
3.
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
4.
Phytomedicine ; 68: 153146, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32028183

RESUMO

BACKGROUND: Dipsaci Radix has been clinically used for thousands of years in China for strengthening muscles and bones. Sweroside is the major active iridoid glycoside isolated from Dipsaci Radix. It has been reported that sweroside can promote alkaline phosphatase (ALP) activity in both the human osteosarcoma cell line MG-63 and rat osteoblasts. However, the underlying mechanism involved in these osteoblastic processes is poorly understood. PURPOSE: This study aimed to characterize the bone protective effects of sweroside and to investigate the signaling pathway that is involved in its actions in MC3T3-E1 cells. METHODS: Cell proliferation, differentiation and mineralization were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, ALP test and Alizarin Red S staining, respectively. The concentration of sweroside in intracellular and extracellular fluids was determined by ultra-performance liquid chromatography coupled to triple quadrupole xevo-mass spectrometry (UPLC/TQ-XS-MS). Proteins associated with the osteoblastic signaling pathway were analysed by western blot and immunofluorescence methods. RESULTS: Sweroside did not obviously affect the proliferation but significantly promoted the ALP activity and mineralization of MC3T3-E1 cells. The maximal absorption amount 0.465 ng/ml (1.3 × 10-9 M) of sweroside was extremely lower than the tested concentration of 358.340 ng/ml (10-6 M), indicating an extremely low absorption rate by MC3T3-E1 cells. Moreover, the ALP activity, the protein expression of ER-α and G protein-coupled receptor 30 (GPR30) induced by sweroside were markedly blocked by both the ER antagonist ICI 182780 and the GPR30 antagonist G15. In addition, sweroside also activated the phosphorylation of p38 kinase (p-p38), while the phosphorylation effects together with ALP and mineralization activities were completely blocked by a p38 antagonist, SB203580. Additionally, the phosphorylation of p38 induced by sweroside were markedly blocked by both the ER antagonist ICI 182780 and the GPR30 antagonist G15. CONCLUSIONS: The present study indicated that sweroside, as a potential agent in treatment of osteoporosis, might exert beneficial effects on MC3T3-E1 cells by interaction with the membrane estrogen receptor-α and GPR30 that then activates the p38 signaling pathway. This is the first study to report the specific mechanism of the effects of sweroside on osteoblastic differentiation and mineralization of MC3T3-E1 cells.


Assuntos
Receptor alfa de Estrogênio/metabolismo , Glucosídeos Iridoides/farmacologia , Osteoblastos/efeitos dos fármacos , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Camundongos , Osteoblastos/metabolismo , Fosforilação/efeitos dos fármacos
5.
Zhonghua Kou Qiang Yi Xue Za Zhi ; 55(2): 104-110, 2020 Feb 09.
Artigo em Chinês | MEDLINE | ID: mdl-32074671

RESUMO

Objective: To investigate the antibacterial properties and the osteoblast-compatibility of chlorhexidine (CHX)-modified porous titanium. Methods: Smooth pure titanium specimen with diameter of 10.0 mm and thickness of 1.5 mm treated with alkali heat method were set as control group. Those with covalent conjugation of aminosilane were set as silane group, and those with CHX grafted by glutaraldehyde were set as CHX group. Scanning electron microscope (SEM) was used to observe the surface morphology and element compositions were detected by X-ray photoelectron spectroscopy. Hydrophilicity was analyzed by surface water contact angle test (n=6), while surface amino/imine groups quantification were performed through acid orangeⅡ(n=5) and the CHX was quantified by optical densitometric method (n=5). Live/dead bacterial staining, the morphology of adherent bacteria by SEM, plate counting method and inhibition zone method were executed to evaluate the antibacterial property of the samples. Osteoblast compatibility was evaluated by methyl thiazolyl tetrazolium. Cell-bacterial co-culture was conducted to evaluated the cell viability on the samples under the circumstance with bacteria. Results: After CHX grafting, pores on the titanium surface were decreased, while the atom ratio of C, N, Cl increased and the water contact angle decreased to 37.5°±4.0°. The density of CHX on the surface was (5.07±0.39) µg/cm(2). The results of live/dead bacterial staining and the morphology of adherent bacteria showed that only little dead bacterial (bacterial wall rupture) adherent on the surface of CHX group, which proved that the modified surface could inhibit bacteria adhesion and even destroyed bacteria; the plate counting displayed sporadic colonies and a transparent inhibition zone could be observed, which demonstrated that CHX group could suppress bacteria multiplication from surrounding environment. When incubating for 1 and 3 days, the cell viability of CHX group showed no significant difference from that of control group (P>0.05) ; when incubating for 5 days, the value of cell viability of CHX group was 0.547±0.087, and this was significantly lower than that of the control group (0.751±0.056) (P<0.05), demonstrating a slight inhibition of cell proliferation by CHX. The results of bacteria-cell co-culture for 3 days showed that a mass of bacteria adhered on the surface of the control group while considerable cells adhered on the surface of CHX group and exhibited a good shape. Conclusions: Porous titanium surface grafted by CHX showed an excellent antibacterial properties and allowed cell adhesion in bacterial circumstance, providing immediate implantation options for patients with bad oral health.


Assuntos
Antibacterianos/farmacologia , Clorexidina/farmacologia , Nanoporos , Osteoblastos/efeitos dos fármacos , Titânio/farmacologia , Bactérias/efeitos dos fármacos , Aderência Bacteriana/efeitos dos fármacos , Células Cultivadas , Humanos , Propriedades de Superfície
6.
J Transl Med ; 18(1): 43, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996227

RESUMO

BACKGROUND: There are several effective therapies for osteoporosis but these agents might cause serious adverse events. Lycopene intake could prevent bone loss, however studies on its effects on bone are scarce. Our aim was to investigate the effects of lycopene on osteoblast cells as well as bone mineral density and bone turnover markers in postmenopausal women. METHODS: We investigated the effect of lycopene on the Wnt/ß-catenin and ERK 1/2 pathways, RUNX2, alkaline phosphatase, RANKL and COL1A of Saos-2. We also carried out a pilot controlled clinical study to verify the feasibility of an approach for bone loss prevention through the intake of a lycopene-rich tomato sauce in 39 postmenopausal women. RESULTS: Lycopene 10 µM resulted in higher ß-catenin and phERK1/2 protein Vs the vehicle (p = 0.04 and p = 0.006). RUNX2 and COL1A mRNA was induced by both 5 and 10 µM doses (p = 0.03; p = 0.03 and p = 0.03; p = 0.05) while RANKL mRNA was reduced (p < 0.05). A significant bone density loss was not detected in women taking the tomato sauce while the control group had bone loss (p = 0.002). Tomato sauce intake resulted in a greater bone alkaline phosphatase reduction than the control (18% vs 8.5%, p = 0.03). CONCLUSIONS: Lycopene activates the WNT/ß-catenin and ERK1/2 pathways, upregulates RUNX2, alkaline phosphatase, COL1A and downregulates RANKL Saos-2. These processes contributed to prevent bone loss in postmenopausal women.


Assuntos
Osso e Ossos/efeitos dos fármacos , Licopeno/farmacologia , Osteoblastos/efeitos dos fármacos , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Osteoblastos/citologia , Osteoblastos/metabolismo , Projetos Piloto , Estudos Prospectivos , RNA Mensageiro/genética , Via de Sinalização Wnt/efeitos dos fármacos
7.
Nat Commun ; 11(1): 87, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911667

RESUMO

Bone remodeling consists of resorption by osteoclasts followed by formation by osteoblasts, and osteoclasts are a source of bone formation-stimulating factors. Here we utilize osteoclast ablation by denosumab (DMAb) and RNA-sequencing of bone biopsies from postmenopausal women to identify osteoclast-secreted factors suppressed by DMAb. Based on these analyses, LIF, CREG2, CST3, CCBE1, and DPP4 are likely osteoclast-derived coupling factors in humans. Given the role of Dipeptidyl Peptidase-4 (DPP4) in glucose homeostasis, we further demonstrate that DMAb-treated participants have a significant reduction in circulating DPP4 and increase in Glucagon-like peptide (GLP)-1 levels as compared to the placebo-treated group, and also that type 2 diabetic patients treated with DMAb show significant reductions in HbA1c as compared to patients treated either with bisphosphonates or calcium and vitamin D. Thus, our results identify several coupling factors in humans and uncover osteoclast-derived DPP4 as a potential link between bone remodeling and energy metabolism.


Assuntos
Osso e Ossos/metabolismo , Metabolismo Energético , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Remodelação Óssea , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Denosumab/administração & dosagem , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Pessoa de Meia-Idade , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Estudos Prospectivos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
8.
Cell Prolif ; 53(2): e12743, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31943455

RESUMO

OBJECTIVES: Alveolar bone osteoporosis has attracted more and more attention because of its profound impact on stomatognathic function and treatment, but current treatments have not been targeted to alveolar bone and might even cause severe side effects. Thus, identifying the effects of anti-osteoporosis agents on alveolar bone is essential. Icariin ameliorates metabolic dysfunction of long bones, but its effects on alveolar bone remain unclarified. MATERIALS AND METHODS: BMSCs were isolated from rat mandibles (mBMSCs). The osteogenic potential of mBMSCs and the signalling pathway involved under icariin treatment were measured by ALP and alizarin red staining, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence. Dual-luciferase assay, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation were used to investigate the molecular mechanism. Ovariectomized and sham-operated rats treated with or without icariin were analysed by micro-CT, TRAP staining and calcein double labelling. RESULTS: We found that icariin promoted osteoblast differentiation of mBMSCs. Furthermore, STAT3 was critical for icariin-promoted osteoblast differentiation, as indicated by increased phosphorylation levels in icariin-treated mBMSCs, while preventing STAT3 activation blocked icariin-induced osteoblast differentiation. Mechanistically, icariin-promoted transcription of the downstream osteogenic gene osteocalcin (Ocn) through STAT3 and STAT3 bound to the promoter of Ocn. Notably, icariin prevented the alveolar bone osteoporosis induced by oestrogen deficiency through promoting bone formation. CONCLUSIONS: For the first time, our work provides evidence supporting the potential application of icariin in promoting osteogenesis and treating alveolar bone osteoporosis.


Assuntos
Perda do Osso Alveolar/tratamento farmacológico , Estrogênios/metabolismo , Flavonoides/farmacologia , Osteogênese/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Perda do Osso Alveolar/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/efeitos dos fármacos , Osteocalcina/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
9.
Chem Biol Interact ; 315: 108875, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31669217

RESUMO

Endemic fluorosis is a serious problem in public health, affecting thousands of people. Abnormal proliferation and activation of osteoblasts in skeletal fluorosis lesions play a leading role and osteoblast proliferation is finely regulated by the cell cycle. There are a few reports on fluoride-induced DNA methylation. However, the role of DNA methylation of the cyclin/cyclin-dependent kinase (CDK)/cyclin-dependent kinase inhibitor (CKI) regulatory network in skeletal fluorosis has not been investigated. We used a population study and in vitro experiment to explore the relationship between the pathogenesis of skeletal fluorosis and methylation of Cyclin d1/CDK4/p21. The results showed a positive relationship between fluoride exposure and expression of Cyclin d1/CDK4, and a negative relationship between fluoride exposure and expression of P21. Hypermethylation of p21 was found in the fluoride-exposed population, and low expression of p21 attributed to promoter hypermethylation was confirmed in vitro. However, no changes in methylation levels of Cyclin d1 and CDK4 genes were observed in the population exposed to fluoride and NaF-treated osteoblasts. These results show that methylation of p21 gene has a significant impact on the proliferation of osteoblasts during the development of skeletal fluorosis. The present study was a first attempt to link the methylation of the Cyclin d1/CDK4/p21 regulatory network with osteoblast proliferation in skeletal fluorosis.


Assuntos
Quinase 4 Dependente de Ciclina/genética , Metilação de DNA/genética , Intoxicação por Flúor/genética , Fluoretos/efeitos adversos , Adulto , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Pré-Escolar , Inibidor de Quinase Dependente de Ciclina p21/genética , Metilação de DNA/efeitos dos fármacos , Feminino , Humanos , Masculino , Osteoblastos/efeitos dos fármacos , Adulto Jovem
10.
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
11.
Toxicol Lett ; 321: 122-130, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31874197

RESUMO

Our previous studies confirmed that prenatal caffeine exposure (PCE) could induce susceptibility to osteoarthritis in adult offspring rats due to poor chondrocyte differentiation, but its mechanism remains to be further investigated. This study aimed to explore whether subchondral bone dysplasia mediates susceptibility to osteoarthritis in adult offspring rats induced by PCE. Pregnant Wistar rats were treated with caffeine (120 mg/kg.d) or saline from gestational day (GD) 9 to 20. The female offspring were euthanized to collect femurs at GD20, postnatal week (PW) 6, and PW28 (non-ovariectomy and ovariectomy groups) to detect osteoarthritis-like phenotype, subchondral bone mass, ossification center development, and other evidence. The results showed that PCE increased the Mankin score of pathological articular cartilage, but decreased articular cartilage thickness and subchondral bone mass, which were more obvious after ovariectomy. Meanwhile, the correlation analysis results demonstrated that the Mankin score of articular cartilage was significantly negatively correlated with subchondral bone mass, and the thickness of articular cartilage was significantly positively correlated with subchondral bone mass. Further, the length and area of the primary and secondary ossification centers, the number of osteoblasts, and the related genes' expression of osteogenic differentiation (e.g., Runx2, BSP, ALP, and OCN) were all significantly decreased in the PCE group before and after birth. Taken together, PCE induced susceptibility to osteoarthritis in adult female offspring, which was likely related to the subchondral bone dysplasia and reduction of subchondral bone mass production due to developmental disorder of primary and secondary ossification centers caused by osteoblast differentiation disability before and after birth.


Assuntos
Doenças do Desenvolvimento Ósseo/induzido quimicamente , Cafeína/toxicidade , Cartilagem Articular/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/toxicidade , Osteoartrite/induzido quimicamente , Osteogênese/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Fatores Etários , Animais , Doenças do Desenvolvimento Ósseo/genética , Doenças do Desenvolvimento Ósseo/metabolismo , Doenças do Desenvolvimento Ósseo/patologia , Cartilagem Articular/patologia , Diferenciação Celular/efeitos dos fármacos , Feminino , Fêmur/efeitos dos fármacos , Fêmur/metabolismo , Fêmur/patologia , Idade Gestacional , Osteoartrite/genética , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese/genética , Ovariectomia , Gravidez , Ratos Wistar , Fatores Sexuais , Tíbia/efeitos dos fármacos , Tíbia/metabolismo , Tíbia/patologia
12.
Mater Sci Eng C Mater Biol Appl ; 107: 110306, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31761228

RESUMO

Hydroxyapatite (HA) coatings onto Ti6Al4V alloy substrates were obtained by several thermal spray technologies: atmospheric plasma spray (APS) and high velocity oxy fuel (HVOF), together with the cold spray (CS) technique. A characterization study has been performed by means of surface and microstructure analyses, as well as biological performance. In-vitro tests were performed with primary human osteoblasts at 1, 7 and 14 days of cell culture on substrates. Cell viability was tested by MTS and LIVE/DEAD assays, cell differentiation by alkaline phosphatase (ALP) quantification, and cell morphology was analyzed by scanning electron microscopy. The HA coatings showed an increase of HA crystallinity from 62,4% to 89%, but also an increase of hydrophilicity from ∼32° to 0°, with the decrease of the operating temperature of the thermal spray techniques (APS > HVOF > CS). Additionally, APS HA coatings showed more surface micro-features than HVOF and CS HA coatings; cells onto APS HA coatings showed faster attachment by acquiring osteoblastic morphology in comparison with the rounded cell morphology observed onto CS HA coatings at 1 day of cell culture. HVOF HA coatings also showed proper cell adherence but did not show extended filopodia as cells onto APS HA coatings. However, at 14 days of cell culture, higher cell proliferation and differentiation was detected on HA coatings with higher crystallinity (HVOF and CS techniques). Cell attachment is suggested to be favoured by surface micro-features but also moderate surface wettability whereas cell proliferation and differentiation is suggested to be highly influenced by HA crystallinity and crystal size.


Assuntos
Materiais Revestidos Biocompatíveis/química , Durapatita/química , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Durapatita/farmacologia , Humanos , Teste de Materiais , Nanopartículas/química , Osteoblastos/efeitos dos fármacos , Propriedades de Superfície
13.
J Craniofac Surg ; 30(8): e776-e780, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31689739

RESUMO

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) is the critical regulator of the proliferation, differentiation, and survival of granulocytes. Recently, it has been shown that G-CSF can adversely affect bone health in both animal models and patients. Here, the authors aimed to investigate whether G-CSF could inhibit the growth of osteoblasts and osteocytes by regulating nitric oxide. METHODS: The C57BL/6 mice were divided into the control group, G-CSF treatment group and recovery group (G-CSF+L-NAME). The morphology of femurs was assessed by histology and immunohistochemistry. The expression of apoptosis-related molecules in femurs was detected by immunohistochemistry and quantitative RT-PCR, respectively. To examine if neutrophil-secreted factors can induce apoptosis in osteoblasts, Gr1-positive (Gr1+) neutrophils from the bone marrow of wild-type mice were sorted and co-cultured with MC3T3 pre-osteoblasts for 2 days. RESULTS: The number of osteoblasts and newly embedding osteocytes significantly decreased and markers related to osteoblasts and osteocytes were downregulated in the G-CSF treatment compared to the control group. Moreover, G-CSF treatment did not change proliferation markers but induced apoptosis in osteoblast-lineage cells. The combined treatment of mice with G-CSF and a nitric oxide inhibitor partially restored the number of osteoblasts and osteocyte parameters. CONCLUSIONS: The G-CSF can inhibit osteoblasts and osteocytes by upregulating nitric oxide.


Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Neutrófilos/efeitos dos fármacos , Óxido Nítrico/biossíntese , Osteoblastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/metabolismo , Osteoblastos/citologia , Osteócitos/citologia , Osteócitos/metabolismo , Regulação para Cima/efeitos dos fármacos
14.
Molecules ; 24(20)2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31623098

RESUMO

Oleoyl serine (OS), an endogenous fatty acyl amide (FAA) found in bone, has been shown to have an anti-osteoporotic effect. OS, being an amide, can be hydrolyzed in the body by amidases. Hindering its amide bond by introducing adjacent substituents has been demonstrated as a successful method for prolonging its skeletal activity. Here, we tested the therapeutic efficacy of two methylated OS derivatives, oleoyl α-methyl serine (HU-671) and 2-methyl-oleoyl serine (HU-681), in an ovariectomized mouse model for osteoporosis by utilizing combined micro-computed tomography, histomorphometry, and cell culture analyses. Our findings indicate that daily treatment for 6 weeks with OS or HU-671 completely rescues bone loss, whereas HU-681 has only a partial effect. The increased bone density was primarily due to enhanced trabecular thickness and number. Moreover, the most effective dose of HU-671 was 0.5 mg/kg/day, an order of magnitude lower than with OS. The reversal of bone loss resulted from increased bone formation and decreased bone resorption, as well as reversal of bone marrow adiposity. These results were further confirmed by determining the serum levels of osteocalcin and type 1 collagen C-terminal crosslinks, as well as demonstrating the enhanced antiadipogenic effect of HU-671. Taken together, these data suggest that methylation interferes with OS's metabolism, thus enhancing its effects by extending its availability to its target cells.


Assuntos
Adiposidade/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Ácidos Oleicos/química , Osteoporose/etiologia , Osteoporose/metabolismo , Serina/análogos & derivados , Serina/farmacologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Medula Óssea/diagnóstico por imagem , Modelos Animais de Doenças , Feminino , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoporose/diagnóstico , Ovariectomia/efeitos adversos , Serina/química , Microtomografia por Raio-X
15.
Artigo em Inglês | MEDLINE | ID: mdl-31597358

RESUMO

Estrogen deficiency frequently leads to a fall in estrogen receptor- (ER) numbers and then reduces the skeletal response to mechanical strain. It, however, is still unclear whether phytoestrogen administration will enhance the effects of exercise on the estrogen-deficient bone loss. This study aimed to determine the effect of Icariin treatment on the response of osteogenic formation to exercise in ovariectomized (OVX) rats. Thirty-two 3-month old female Sprague-Dawley rats were randomly allocated into four groups: (1) Sham-operated (SO); (2) OVX; (3) OVX plus exercise (EX); and (4) OVX plus exercise and Icariin (EI). After 8-week interventions, the rats were killed and samples were collected for bone morphometry, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analyses. EI interventions showed a greater improvement for the OVX-induced bone loss and the elevated serum tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) compared with EX only. Both EX and EI interventions bettered the OVX-related reduction of BV/TV and trabecular number and thickness, and decreased the enlargement of trabecular bone separation (Tb. Sp); the improvement for BV/TV and Tb. Sp was greater in EI group. Furthermore, EX and EI treatment significantly increased the number of ALP+ cells and mineralized nodule areas compared with OVX group; the change was higher in EI group. Additionally, in comparison to OVX rats, the protein and mRNA expression of -catenin, phosphorylated-Akt (p-Akt) or Akt, ER, and Runt-related transcription factor 2 (Runx2) in osteoblasts were elevated in EX and EI intervention rats, with greater change observed in EI group. The upregulated -catenin and Akt mRNA levels in EX and EI groups was depressed by ICI182780 treatment, and the difference in -catenin and Akt mRNA levels between EX and EI groups was no longer significant. Conclusively, the combination of Icariin and exercise significantly prevent OVX-induced bone loss and increase osteoblast differentiation and the ability of mineralization compared with exercise alone; the changes might be regulated partly by ER/Akt/-catenin pathway.


Assuntos
Densidade Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Flavonoides/uso terapêutico , Osteoblastos/efeitos dos fármacos , Osteoporose/prevenção & controle , Condicionamento Físico Animal/fisiologia , Receptores Estrogênicos/efeitos dos fármacos , Animais , Ratos , Ratos Sprague-Dawley
16.
Int J Nanomedicine ; 14: 7309-7322, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571855

RESUMO

Introduction: The only treatment for aseptic loosening is the replacement of the prosthesis through revision surgery. A preventive approach, achieved through anti-inflammatory drugs released from the device, has shown to be a viable strategy; however, the performance of these devices is not yet satisfactory thus further improvements are necessary. Methods: We used titanium nanoparticles as a model for implant surfaces and developed a coating containing dexamethasone (DEX) using layer-by-layer deposition. Results: The amount of deposited drug depended on the number of layers and the release was sustained for months. The efficiency of the released DEX in reducing inflammation markers (tumor necrosis factor alpha and IL-6) produced by human monocytes and macrophages was similar to the pure drug at the same concentration without negative impacts on the viability and morphology of these cells. Conclusion: These coatings were not inferior to medical grade titanium (the standard material used in uncemented devices) regarding their ability to sustain osteoblasts and fibroblasts growth.


Assuntos
Anti-Inflamatórios/farmacologia , Cimentos para Ossos/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Liberação Controlada de Fármacos , Nanopartículas/química , Falha de Prótese , Linhagem Celular , Forma Celular/efeitos dos fármacos , Dexametasona/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Monócitos/efeitos dos fármacos , Nanopartículas/ultraestrutura , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Tamanho da Partícula , Termogravimetria
17.
Mol Cell Biochem ; 462(1-2): 173-183, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31620952

RESUMO

Osteoblasts and osteoclasts play essential and opposite roles in maintaining bone homeostasis. Osteoblasts fill cavities excavated by osteoclasts. The mevalonate pathway provides essential prenyl pyrophosphates for the activities of GTPases that promote differentiation of osteoclasts but suppress that of osteoblasts. Preclinical and clinical studies suggest that mevalonate suppressors such as statins increase bone mineral density and reduce risk of bone fracture. Tocotrienols down-regulate 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate-limiting enzyme in the mevalonate pathway. In vivo studies have shown the bone-protective activity of tocotrienols. We hypothesize that d-δ-tocotrienol, a mevalonate suppressor, induces differentiation of murine MC3T3-E1 preosteoblasts. Alizarin staining showed that d-δ-tocotrienol (0-25 µmol/L) induced mineralized nodule formation in a concentration-dependent manner in MC3T3-E1 preosteoblasts. d-δ-Tocotrienol (0-25 µmol/L), but not D-α-tocopherol (25 µmol/L), significantly induced alkaline phosphatase activity, an indicator of preosteoblast differentiation. The expression of differentiation marker genes including BMP-2 and VEGFα was stimulated dose dependently by d-δ-tocotrienol (0-25 µmol/L). Concomitantly, Western blot analysis showed that d-δ-tocotrienol down-regulated HMG CoA reductase. d-δ-Tocotrienol (0-25 µmol/L) had no impact on the viability of MC3T3-E1 preosteoblasts following 48-h incubation, suggesting lack of cytotoxicity at these doses. Tocotrienols and other mevalonate suppressors have potential in maintaining bone health.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Osteoblastos/citologia , Vitamina E/análogos & derivados , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/metabolismo , Ácido Mevalônico/metabolismo , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Vitamina E/farmacologia , Proteínas ras/metabolismo
18.
Life Sci ; 239: 116975, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31654748

RESUMO

AIMS: Previous study indicated that the increase of local bio-availability of 3'3'5-triiodothyronine (T3) influenced osteoarthritis (OA) initiation. We aimed to investigate the role of thyroid hormone receptors (THRs) signaling in OA osteoblasts. MATERIALS AND METHODS: THRs expression in OA was detected by immunohistochemistry, immunofluorescence, RT-qPCR and western blotting. These effects on the expression of angiogenesis-related factors were examined after THRα or THRß knockdown in OA osteoblasts. Fluorescence in situ hybridization was used to confirm the leading receptor for regulating angiogenesis-related factors. Co-culture model was utilized to observe the MMPs expression in chondrocytes after THRα knockdown in osteoblasts. The in vivo effects were also studied after intra-articular injection with THRα siRNA in OA model mice. Micro-CT and immunohistochemistry were employed to evaluate the changes of subchondral bone. KEY FINDINGS: THRs expression and nuclear translocation were upregulated in human OA osteoblasts. Immunohistochemistry showed that angiogenic activities were increased in OA subchondral bone of human and mice. VEGF, HIF-1α and IGF-1, these THR downstream genes were downregulated after THRα knockdown in OA osteoblasts. Fluorescence in situ hybridization further indicated that THRα signaling mainly regulated VEGF expression. Intra-articular injection with THRα siRNA reduced angiogenic activities in OA model mice subchondral bone and ameliorated cartilage degradation. Micro-CT analysis displayed that the aberrant subchondral bone formation in OA was promoted. SIGNIFICANCE: The microangiogenesis in subchondral bone may be partly attributed to abnormal THRα signaling in osteoblasts, and local inhibition of the THRα could be a potential target to treat OA.


Assuntos
Neovascularização Fisiológica/fisiologia , Osteoblastos/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Osso e Ossos/metabolismo , China , Condrócitos/metabolismo , Feminino , Humanos , Injeções Intra-Articulares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Osteoartrite do Joelho/tratamento farmacológico , Osteoblastos/efeitos dos fármacos , Receptores dos Hormônios Tireóideos/fisiologia , Transdução de Sinais/fisiologia , Tiroxina/análise , Tiroxina/sangue , Tri-Iodotironina/análise , Tri-Iodotironina/sangue
19.
Phytother Res ; 33(11): 2948-2959, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31478281

RESUMO

The balance between the osteoblasts and the osteoclasts is important for the maintenance of the skeleton of the human body. The osteoclasts absorb bone after differentiated into polymorphonuclear cells by the fusion of monocytes/macrophages. We have found that 6,7,4'-Trihydroxyflavone (THF), a compound from the heartwood of Dalbergia Odorifera inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, actin ring formation, and bone resorption in RAW 264.7 cells and bone marrow macrophage. THF significantly inhibited the c-Jun-N-terminal kinase signaling pathway without affecting extracellular signal-regulated kinase, p38, and AKT signaling. Moreover, THF inhibited the expression of c-Fos, nuclear factor-activated T cells cytoplasm 1, cathepsin K, and c-src by RANKL. We used a lipopolysaccharide (LPS)-induced bone loss model in mice. Consequently, bone volume per tissue volume, trabecular number's reduction was recovered in THF-treated mice, and trabecular separation's augmentation was also attenuated by THF administration. In summary, THF inhibits RANKL-induced osteoclast differentiation by MAPK signaling pathway and inhibits bone resorption by destroying the actin ring in mature osteoclasts. THF also prevented LPS-induced bone loss in a mice model. Thus, THF may be useful in the treatment of bone diseases associated with excessive osteoclast differentiation and bone resorption.


Assuntos
Reabsorção Óssea/prevenção & controle , Diferenciação Celular/efeitos dos fármacos , Isoflavonas/farmacologia , Osteoclastos/efeitos dos fármacos , Animais , Células Cultivadas , Dalbergia/química , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos
20.
Mater Sci Eng C Mater Biol Appl ; 105: 109985, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31546404

RESUMO

Ceramic/polymer-based biocomposites have emerged as potential biomaterials to fill, replace, repair or regenerate injured or diseased bone, due to their outstanding features in terms of biocompatibility, bioactivity, injectability, and biodegradability. However, these properties can be dependent on the amount of ceramic component present in the polymer-based composite. Therefore, in the present study, the influence of nanohydroxyapatite content (30 to 70 wt%) on alginate-based hydrogels was studied in order to evaluate the best formulation for maximizing bone tissue regeneration. The composite system was characterized in terms of physic-chemical properties and biological response, with in vitro cytocompatibility assessment with human osteoblastic cells and ex vivo functional evaluation in embryonic chick segmental bone defects. The main morphological characteristics of the alginate network were not affected by the addition of nanohydroxyapatite. However, physic-chemical features, like water-swelling rate, stability at extreme pH values, apatite formation, and Ca2+ release were nanoHA dose-dependent. Within in vitro cytocompatibility assays it was observed that hydrogels with nanoHA 30% content enhanced osteoblastic cells proliferation and expression of osteogenic transcription factors, while those with higher concentrations (50 and 70%) decreased the osteogenic cell response. Ex vivo data underlined the in vitro findings, revealing an enhanced collagenous deposition, trabecular bone formation and matrix mineralization with Alg-nanoHA30 composition, while compositions with higher nanoHA content induced a diminished bone tissue response. The outcomes of this study indicate that nanohydroxyapatite concentration plays a major role in physic-chemical properties and biological response of the composite system and the optimization of the components ratio must be met to maximize bone tissue regeneration.


Assuntos
Alginatos/farmacologia , Regeneração Óssea/efeitos dos fármacos , Durapatita/farmacologia , Hidrogéis/farmacologia , Nanopartículas/química , Animais , Cálcio/análise , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Íons , Células-Tronco Mesenquimais , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , Água/química
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