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1.
Life Sci ; 258: 118195, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32781073

RESUMO

AIMS: The estrogen-ERα axis participates in osteoblast maturation. This study was designed to further evaluated the roles of the estrogen-ERα axis in bone healing and the possible mechanisms. MAIN METHODS: Female ICR mice were created a metaphyseal bone defect in the left femurs and administered with methylpiperidinopyrazole (MPP), an inhibitor of ERα. Bone healing was evaluated using micro-computed tomography. Colocalization of ERα with alkaline phosphatase (ALP) and ERα translocation to mitochondria were determined. Levels of ERα, ERß, PECAM-1, VEGF, and ß-actin were immunodetected. Expression of chromosomal Runx2, ALP, and osteocalcin mRNAs and mitochondrial cytochrome c oxidase (COX) I and COXII mRNAs were quantified. Angiogenesis was measured with immunohistochemistry. KEY FINDINGS: Following surgery, the bone mass was time-dependently augmented in the bone-defect area. Simultaneously, levels of ERα were specifically upregulated and positively correlated with bone healing. Administration of MPP to mice consistently decreased levels of ERα and bone healing. As to the mechanisms, osteogenesis was enhanced in bone healing, but MPP attenuated osteoblast maturation. In parallel, expressions of osteogenesis-related ALP, Runx2, and osteocalcin mRNAs were induced in the injured zone. Treatment with MPP led to significant inhibition of the alp, runx2, and osteocalcin gene expressions. Remarkably, administration of MPP lessened translocation of ERα to mitochondria and expressions of mitochondrial energy production-related coxI and coxII genes. Furthermore, exposure to MPP decreased levels of PECAM-1 and VEGF in the bone-defect area. SIGNIFICANCE: The present study showed the contributions of the estrogen-ERα axis to bone healing through stimulation of energy production, osteoblast maturation, and angiogenesis.


Assuntos
Regeneração Óssea , Diferenciação Celular , Metabolismo Energético , Receptor alfa de Estrogênio/metabolismo , Neovascularização Fisiológica , Osteoblastos/citologia , Transdução de Sinais , Fosfatase Alcalina/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Diferenciação Celular/efeitos dos fármacos , Cromossomos de Mamíferos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacos
2.
Life Sci ; 257: 118033, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32621924

RESUMO

The present study aimed to investigate the effects of phosphatidylserine liposomes (PSLs) and phosphatidylserine liposomes containing alendronate (AL-PSLs) on the improvement of methylprednisolone (MP) induced osteoporosis in a rat model. AL-PSLs formulation was prepared, characterized, and evaluated in different pH media to simulate gastrointestinal condition. Osteoporosis was induced by 3 weeks oral administration of MP (10 mg/kg) and then treatment by PSLs, AL-PSLs, and alendronate (AL). Bone metabolic and biomechanical markers were measured in treated rat groups. Also, Tartrate-resistant acid phosphatase (TRAP) staining and histomorphometry were evaluated on bone tissues of treated rats. AL-PSLs were obtained in a size range of 155 nm and negatively surface charge with an entrapment efficiency of 42%. The AL leakage from AL-PSLs did not exhibit a significant difference in acidic or basic media in comparison with the neutral condition. The concentrations of calcium, osteocalcin, bone alkaline phosphatase, and osteoprotegerin (OPG) of serum were significantly increased in PSLs and AL-PSLs treated groups compared to the MP group. Also, PSLs and AL-PSLs significantly improved the thickness and volume of the cortical and trabecular bone mass in treated groups. In addition, TRAP staining indicated a significant decrease of osteoclast number in osteoporotic rats treated with AL-PSLs and PSLs. In this study, AL-PSLs and even PSLs alone made a potential bone mechanical strength in glucocorticoid-induced bone loss more than AL in rats. In conclusion, our findings suggest that PSLs consumption with or without an anti-osteoporotic drug might be an applicable choice in control of osteoporosis.


Assuntos
Alendronato/farmacologia , Osteoporose/tratamento farmacológico , Fosfatidilserinas/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacologia , Osso e Ossos/metabolismo , Modelos Animais de Doenças , Fêmur/efeitos dos fármacos , Lipossomos/farmacologia , Masculino , Metilprednisolona/farmacologia , Osteocalcina/metabolismo , Osteoclastos/metabolismo , Osteoporose/metabolismo , Fosfatidilserinas/metabolismo , Ratos , Ratos Wistar
3.
Gene ; 757: 144852, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32599019

RESUMO

Until now, various methods have been introduced to fabricate 3D scaffolds to provide a suitable substrate for cell growth and proliferation and subsequent use in tissue engineering to repair damaged tissues. The 3D scaffolds can simulate the natural cellular microenvironment well. Herein, the decellularized leaf spinach has been used which not only have no problems associated with artificial scaffolds, but they also do not cost significantly. Decellularized scaffolds surface properties were characterized by the investigation of scaffolds surface roughness, hydrophilicity, mechanical properties, size and shape of porosities and specific surface area. In the next step, osteogenic differentiation potential of bone marrow derived mesenchymal stem cells cultured on the scaffold and culture plate (as a control) was evaluated using alizarin staining and calcium content, alkaline phosphatase activity and bone related genes expression assays. The results indicated that the surface properties and shape of scaffold pores were effective in the stem cells binding, growth and proliferation. This higher biocompatibility due to the ideal surface hydrophilicity as well as high specific surface area due to the presence of a rough grid surface ultimately increased the efficiency of stem cell's bone differentiation. Taken together, it can be concluded that the decellularized spinach leaf scaffold, due to its easy availability, low prices and high efficiency, can be considered as a promising potential candidate for use as a proper substrate for stem cell growth and differentiation in bone tissue engineering.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteocalcina/genética , Folhas de Planta/química , Tecidos Suporte/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Biomineralização , Cálcio/metabolismo , Linhagem Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteocalcina/metabolismo , Spinacia oleracea/química
6.
J Bone Miner Metab ; 38(5): 631-638, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32350615

RESUMO

INTRODUCTION: Disuse-induced bone loss is caused by a suppression of osteoblastic bone formation and an increase in osteoclastic bone resorption. There are few data available for the effects of environmental conditions, i.e., atmospheric pressure and/or oxygen concentration, on osteoporosis. This study examined the effects of mild hyperbaric oxygen at 1317 hPa with 40% oxygen on unloading-induced osteoporosis. MATERIALS AND METHODS: Eighteen 8-week old male Wistar rats were randomly divided into three groups: the control for 21 days without unloading and mild hyperbaric oxygen (NOR, n = 6), the unloading for 21 days and recovery for 10 days without mild hyperbaric oxygen (HU + NOR, n = 6), and the unloading for 21 days and recovery for 10 days with mild hyperbaric oxygen (HU + MHO, n = 6). RESULTS: The cortical thickness and trabecular bone surface area were decreased in the HU + NOR group compared to the NOR group. There were no differences between the NOR and HU + MHO groups. Osteoclast surface area and Sclerostin (Sost) mRNA expression levels were decreased in the HU + MHO group compared to the HU + NOR group. These results suggested that the loss of the cortical and trabecular bone is inhibited by mild hyperbaric oxygen, because of an inhibition of osteoclasts and enhancement of bone formation with decreased Sost expression. CONCLUSIONS: We conclude that exposure to mild hyperbaric oxygen partially protects from the osteoporosis induced by hindlimb unloading.


Assuntos
Elevação dos Membros Posteriores/fisiologia , Oxigenação Hiperbárica , Osteoporose/fisiopatologia , Osteoporose/terapia , Animais , Peso Corporal , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Osso Esponjoso/patologia , Osso Esponjoso/fisiopatologia , Osso Cortical/patologia , Osso Cortical/fisiopatologia , Marcadores Genéticos/genética , Lâmina de Crescimento/patologia , Masculino , Osteocalcina/genética , Osteocalcina/metabolismo , Osteoclastos/patologia , Osteoporose/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar
7.
Life Sci ; 253: 117660, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32294474

RESUMO

AIMS: Osteoporosis has been known to generally result from an imbalance between bone formation and resorption. Osteogenesis is the process of differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Sirtuin6 (SIRT6) has been reported to mediate osteogenic differentiation (OD) in rat bone MSCs (rBMSCs). The present study aimed to assess the influence of microRNA miR-186 on the proliferation and OD potential of rBMSCs. MAIN METHODS: OD was performed and evaluated through Alizarin red S staining, alkaline phosphatase (ALP) activity, and specific marker expression. KEY FINDINGS: miR-186 downregulation was observed during OD. rBMSCs with miR-186 overexpression were generated via transfection. Compared with vehicle negative controls, miR-186 upregulation significantly repressed rBMSCs' OD, as evidenced by a reduced ALP activity and decreased mRNA levels of osteogenic markers [osteocalcin, Runx2, BSP, and ALP]. Furthermore, bioinformatic prediction and dual-luciferase reporter assay demonstrated that miR-186 targeted SIRT6 3'-UTR for silencing. SIRT6 overexpression reversed the inhibitory effect of miR-186 on the OD of rBMSCs. Additionally, further examination showed that the activation of nuclear factor-kappa B (NFκB) pathway was involved in the miR-186/SIRT6 signal axis, and phorbol 12-myristate 13-acetate, a NFκB activator, also inhibited the OD of rBMSCs. SIGNIFICANCE: The present study results may demonstrate a novel mechanism of rBMSCs OD via miR-186-SIRT6 interaction.


Assuntos
MicroRNAs/metabolismo , Osteoblastos/metabolismo , Osteogênese/genética , Sirtuínas/genética , Fosfatase Alcalina/metabolismo , Animais , Sequência de Bases , Regeneração Óssea/genética , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Lentivirus/genética , Células-Tronco Mesenquimais/citologia , NF-kappa B/metabolismo , Osteocalcina/metabolismo , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais , Sirtuínas/metabolismo , Transfecção
8.
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
9.
Gene ; 740: 144534, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32145328

RESUMO

The function of tissue cells is strongly depends on the extracellular matrix (ECM) that can guide and support cell structure. This support plays a crucial role in the process of cell proliferation and differentiation. Herein, three different nanofibrous scaffolds that are highly attractive for tissue engineering were selected and then osteogenic related genes and protein expression patterns of human adipose-derived mesenchymal stem cells (AT-MSCs) were investigated when grown on substrates. Polycaprolactone, Poly (L-lactic acid) and Polyvinylidene-fluoride nanofibrous scaffolds were fabricated using Electrospinning method and then AT-MSCs viability and osteogenic differentiation were evaluated while cultured on them. The highest AT-MSCs survival rate when grown on the scaffolds was detected when grown on Polyvinylidene-fluoride. In addition, the highest ALP activity and mineralization were also observed in differentiated AT-MSCs has grown on Polyvinylidene-fluoride. The expression levels of Runx2, osteonectin and osteocalcin genes and osteocalcin protein in the AT-MSCs has grown on the Polyvinylidene-fluoride were also significantly higher than the rest of the scaffolds. Based on the results, it seems that since the studied substrate have a similar structural characteristics, their nature may have an important role in the stem cell's osteogenesis process, where the Polyvinylidene-fluoride piezoelectricity was a most distinguished characteristic.


Assuntos
Células-Tronco Mesenquimais , Nanofibras , Osteogênese , Engenharia Tecidual/métodos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Nanofibras/química , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Osteonectina/metabolismo , Poliésteres , Polivinil
10.
Ann Agric Environ Med ; 27(1): 66-75, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32208582

RESUMO

OBJECTIVE: The aim of the study was to determine the effect of nesfatin-1 on bone properties in female rats in the conditions of developing osteopenia induced by ovariectomy (OVX). MATERIAL AND METHODS: The experiment was performed on 21 female Wistar rats assigned to 3 groups receiving intraperitoneally physiological saline (SHO, OVX-PhS) and nesfatin-1 in dose 2 µg/kg BW of (OVX-NES) once a day for 8 wks. At the end of the experiment, the rats were scanned using the DXA method to determine the body composition, tBMC, and tBMD. The isolated femora and tibia were tested with the DXA method for BMD and BMC, and with the pQCT method for separate analysis of the cortical and trabecular bone tissue. The bone strength parameters were also determined. The immunohistochemical method was used for determination of nesfatin-1 localization in growth cartilage. Bone metabolism markers (osteocalcin, bALP, and NTx) were identified using an ELISA kit. RESULTS: OVX exerts a negative effect on bone tissue. The nesfatin-1 administration influenced positively the DXA parameters of tibia. TvBMD and TbvBMD measured by pQCT in metaphysis of bones were significantly higher in the OVX-NES group than in OVX-PhS. No differences were found in the values of bone strength parameters between SHO and OVX-NES females. Extra- and intracellular immunohistochemical reaction for nesfatin-1 was observed in all zones of growth cartilage, with the strongest reaction detected in the calcifying zone. Nesfatin-1 administration caused a significant increase in the osteocalcin and bALP concentration in relation to the OVX-PhS animals. CONCLUSIONS: The results of the experiment indicate that nesfatin-1 exerts a protective effect on bone tissue properties and can be used in the prevention of osteoporosis.


Assuntos
Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/tratamento farmacológico , Nucleobindinas/farmacologia , Absorciometria de Fóton , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Doenças Ósseas Metabólicas/diagnóstico por imagem , Doenças Ósseas Metabólicas/metabolismo , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Feminino , Fêmur/diagnóstico por imagem , Fêmur/efeitos dos fármacos , Osteocalcina/metabolismo , Ovariectomia , Ratos Wistar , Tíbia/diagnóstico por imagem , Tíbia/efeitos dos fármacos
11.
Biomed Res Int ; 2020: 9467683, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32149147

RESUMO

Yishen Bugu Ye (YSBGY), a traditional Chinese medicine comprising 12 types of medicinal herbs, is often prescribed in China to increase bone strength. In this study, the antiosteoporotic effects of YSBGY were investigated in C57BL/6 mice afflicted with dexamethasone- (Dex-) induced osteoporosis (OP). The results showed that YSBGY reduced the interstitial edema in the liver and kidney of mice with Dex-induced OP. It also increased the number of trabecular bone elements and chondrocytes in the femur, promoted cortical bone thickness and trabecular bone density, and modulated the OP-related indexes in the femur and tibia of OP mice. It also increased the serum concentrations of type I collagen, osteocalcin, osteopontin, bone morphogenetic protein-2, bone morphogenetic protein receptor type 2, C-terminal telopeptide of type I collagen, and runt-related transcription factor-2 and reduced those of tartrate-resistant acid phosphatase 5 and nuclear factor of activated T cells in these mice, suggesting that it improved osteoblast differentiation and suppressed osteoclast differentiation. The anti-inflammatory effect of YSBGY was confirmed by the increase in the serum concentrations of interleukin- (IL-) 33 and the decrease in concentrations of IL-1, IL-7, and tumor necrosis factor-α in OP mice. Furthermore, YSBGY enhanced the serum concentrations of superoxide dismutase and catalase in these mice, indicating that it also exerted antioxidative effects. This is the first study to confirm the antiosteoporotic effects of YSBGY in mice with Dex-induced OP, and it showed that these effects may be related to the YSBGY-induced modulation of the osteoblast/osteoclast balance and serum concentrations of inflammatory factors. These results provide experimental evidence supporting the use of YSBGY for supporting bone formation in the clinical setting.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Animais , Densidade Óssea , Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Osso Esponjoso , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osso Cortical/metabolismo , Osso Cortical/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteocalcina/metabolismo , Osteoclastos/metabolismo , Osteopontina/metabolismo , Osteoporose/diagnóstico por imagem , Osteoporose/patologia , Peptídeos , Superóxido Dismutase/sangue , Fosfatase Ácida Resistente a Tartarato/metabolismo
12.
Arch Oral Biol ; 112: 104681, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32070866

RESUMO

OBJECTIVE: The aim of the present study was to investigate the effect of palmitate on human periodontal ligament stem cells (PDLSCs). DESIGN: PDLSCs were isolated from the third molars of healthy adult donors, and cultured in normal or osteogenic medium supplemented with palmitate (0, 100, or 250 µM) for 21 days. Cell proliferation was evaluated by measuring the amount of formazan at 6, 24, 48, and 72 h. Apoptosis was detected by ELISA and terminal deoxynucleotidyl transferase dUTP nick end labeling assay at days 3 and 7. Osteogenic differentiation was evaluated by measuring the alkaline phosphatase (ALP) activity, production of procollagen type I C-peptide and osteocalcin, mineralization, and mRNA expression of Runx2 at days 3, 7, 14, and 21. In addition, mRNA expression of IL-6 and IL-8 was measured at day 3. RESULTS: Palmitate inhibited the proliferation, ALP activity, production of procollagen type I C-peptide and osteocalcin, mineralization, and mRNA expression of Runx2 in the cultured PDLSCs. Palmitate also induced apoptosis and mRNA expression of IL-6 and IL-8 in the PDLSCs. CONCLUSIONS: The results of the present study demonstrate that palmitate induces apoptosis and inhibits osteogenic differentiation of PDLSCs. These findings may help clarify the relationship between palmitate and periodontal tissue regeneration.


Assuntos
Osteogênese , Palmitatos/farmacologia , Ligamento Periodontal/citologia , Células-Tronco/efeitos dos fármacos , Adulto , Fosfatase Alcalina/metabolismo , Apoptose , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Interleucinas/metabolismo , Osteocalcina/metabolismo , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno/metabolismo , Células-Tronco/citologia
13.
Cell Prolif ; 53(2): e12758, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31922317

RESUMO

OBJECTIVE: The aim of this study is to investigate the role and potential mechanism of p75NTR in mineralization in vivo using p75NTR-knockout mice and in vitro using ectomesenchymal stem cells (EMSCs). MATERIALS AND METHODS: Femur bone mass and daily incisor mineralization speed were assessed in an in vivo p75NTR-knockout mouse model. The molecular signatures alkaline phosphatase (ALP), collagen type 1 (Col1), melanoma-associated antigen (Mage)-D1, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), distal-less homeobox 1 (Dlx1) and Msh homeobox 1 (Msx1) were examined in vitro in EMSCs isolated from p75NTR+/+ and p75NTRExIII-/- mice. RESULTS: p75NTR-knockout mice were smaller in body size than heterozygous and wild-type mice. Micro-computed tomography and structural quantification showed that the osteogenic ability of p75NTRExIII -knockout mice was significantly decreased compared with that of wild-type mice (P < .05). Weaker ALP and alizarin red staining and reduced expression of ALP, Col1, Runx2, BSP, OCN and OPN were also observed in p75NTRExIII-/- EMSCs. Moreover, the distance between calcein fluorescence bands in p75NTRExIII -knockout mice was significantly smaller than that in wild type and heterozygous mice (P < .05), indicating the lower daily mineralization speed of incisors in p75NTRExIII -knockout mice. Further investigation revealed a positive correlation between p75NTR and Mage-D1, Dlx1, and Msx1. CONCLUSION: p75NTR not only promotes osteogenic differentiation and tissue mineralization, but also shows a possible relationship with the circadian rhythm of dental hard tissue formation.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Calcificação Fisiológica/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Colágeno Tipo I/metabolismo , Feminino , Sialoproteína de Ligação à Integrina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Osteocalcina/metabolismo , Osteogênese/fisiologia , Osteopontina/metabolismo
14.
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
15.
PLoS One ; 15(1): e0225589, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923243

RESUMO

Carbon nanotubes (CNTs) have desirable mechanical properties for use as biomaterials in orthopedic and dental area such as bone- and tooth- substitutes. Here, we demonstrate that a glass surface densely coated with single-walled carbon nanotubes (SWNTs) stimulate the osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs). MSCs incubated on SWNT- and multi-walled carbon nanotube (MWNT)-coated glass showed high activities of alkaline phosphatase that are markers for early stage osteogenic differentiation. Expression of Bmp2, Runx2, and Alpl of MSCs showed high level in the early stage for MSC incubation on SWNT- and MWNT-coated surfaces, but only the cells on the SWNT-coated glass showed high expression levels of Bglap (Osteocalcin). The cells on the SWNT-coated glass also contained the most calcium, and their calcium deposits had long needle-shaped crystals. SWNT coating at high density could be part of a new scaffold for bone regeneration.


Assuntos
Diferenciação Celular , Nanotubos de Carbono/química , Osteogênese , Tecidos Suporte/química , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/classificação , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Cálcio/química , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Vidro/química , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Ratos , Ratos Endogâmicos F344
16.
Med Sci Monit ; 26: e918541, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31911574

RESUMO

BACKGROUND Osteoporosis is an osteolytic disease resulted from imbalance in bone homeostasis. Studies indicated that N-myc downstream-regulated gene 2 (NDRG2) could affect the osteoclast differentiation. However, the effect of NDRG2 on osteoblastic differentiation and calcification remains unknown. Hence, we aimed to analyze the effect of NDRG2 on the proliferation and differentiation of osteoblasts. MATERIAL AND METHODS The differentiation of bone morphogenetic protein 2 (BMP2) induced MC3T3-E1 cells was observed by the microscope. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis detected the expression of BMP2, NDRG2, runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), osterix (OSX), and osteocalcin (OCN). Alkaline phosphatase (ALP) activity assay was detecting the ALP activity and alizarin red staining assay was analyzing intracellular calcium salt deposition. The cell transfection was also verified by RT-qPCR analysis. RESULTS The results demonstrated that BMP2 promoted the osteoblastic differentiation with the increasing expression of Runx2, OPG, OSX, and OCN. NDRG2 expression was upregulated during osteogenic differentiation. NDRG2 overexpression promoted the expression of Runx2, OPG, OSX, and OCN, and increased the ALP activity while NDRG2 inhibition reversed the changes. NDRG2 overexpression increased the intracellular calcium salt deposition and NDRG2 inhibition reversed the changes. The role of NDRG2 in osteoblastic differentiation and calcification was played through the JAK3/STAT3 signal pathway. CONCLUSIONS The presented data indicated that NDRG2 promoted BMP2-induced osteoblastic differentiation and calcification by activating the JAK3/STAT3 signal pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Janus Quinase 3/metabolismo , Osteoblastos/metabolismo , Fator de Transcrição STAT3/metabolismo , Células 3T3 , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Calcificação Fisiológica/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Camundongos , Osteocalcina/metabolismo , Osteogênese/fisiologia , Transdução de Sinais , Fator de Transcrição Sp7/metabolismo
17.
Am J Physiol Endocrinol Metab ; 318(3): E381-E391, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31935114

RESUMO

Osteocalcin (OCN) is a bone-derived hormone involved in the regulation of glucose metabolism. In serum, OCN exists in carboxylated and uncarboxylated forms (ucOCN), and studies in rodents suggest that ucOCN is the bioactive form of this hormone. Whether this is also the case in humans is unclear, because a reliable assay to measure ucOCN is not available. Here, we established and validated a new immunoassay (ELISA) measuring human ucOCN and used it to determine the level of bioactive OCN in two cohorts of overweight or obese subjects, with or without type 2 diabetes (T2D). The ELISA could specifically detect ucOCN concentrations ranging from 0.037 to 1.8 ng/mL. In a first cohort of overweight or obese postmenopausal women without diabetes (n = 132), ucOCN correlated negatively with fasting glucose (r = -0.18, P = 0.042) and insulin resistance assessed by the homeostatic model assessment of insulin resistance (r = -0.18, P = 0.038) and positively with insulin sensitivity assessed by a hyperinsulinemic-euglycemic clamp (r = 0.18, P = 0.043) or insulin sensitivity index derived from an oral glucose tolerance test (r = 0.26, P = 0.003). In a second cohort of subjects with severe obesity (n = 16), ucOCN was found to be lower in subjects with T2D compared with those without T2D (2.76 ± 0.38 versus 4.52 ± 0.06 ng/mL, P = 0.009) and to negatively correlate with fasting glucose (r = -0.50, P = 0.046) and glycated hemoglobin (r = -0.57, P = 0.021). Moreover, the subjects with ucOCN levels below 3 ng/mL had a reduced insulin secretion rate during a hyperglycemic clamp (P = 0.03). In conclusion, ucOCN measured with this novel and specific assay is inversely associated with insulin resistance and ß-cell dysfunction in humans.


Assuntos
Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Osteocalcina/análise , Osteocalcina/metabolismo , Testes de Função Pancreática , Adolescente , Adulto , Idoso , Animais , Glicemia , Estudos de Coortes , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Técnica Clamp de Glucose , Hemoglobina A Glicada/análise , Humanos , Imunoensaio/métodos , Resistência à Insulina , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Obesidade/metabolismo , Sobrepeso/metabolismo
18.
J Periodontal Res ; 55(1): 141-151, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31539178

RESUMO

BACKGROUND AND OBJECTIVES: Strontium ranelate is a medication indicated for the treatment of osteoporosis that presents concomitant anti-resorptive and osteoanabolic dual biological activity. However, the effects of strontium ranelate on alveolar bone have been poorly explored. Furthermore, to date, there are no data on the effects of this medication on alveolar bone loss (BL) during conditions of estrogen deficiency. Therefore, the aim of this study was to evaluate the effects of strontium ranelate on ligature-induced periodontitis in estrogen-deficient and estrogen-sufficient rats. METHODS: Ninety-six rats were assigned to one of the following groups: sham-surgery + water (estrogen-sufficient; n = 24); ovariectomy + water (estrogen-deficient; n = 24), sham-surgery + strontium ranelate (ranelate/estrogen-sufficient; n = 24) and; ovariectomy + strontium ranelate (ranelate/estrogen-deficient; n = 24). The rats received strontium ranelate or water from the 14th day after ovariectomy until the end of the experiment. On the 21st day after ovariectomy, one first mandibular molar received a ligature, while the contralateral tooth was left unligated. Eight rats per group were killed at 10, 20, and 30 days after ligature placement. Bone loss (BL) and trabecular bone area (TBA) were analyzed in the furcation area of ligated and unligated teeth at all experimental times by histometry. Tartrate-resistant acid phosphatase (TRAP) positive cells and immunohistochemical staining for osteocalcin (OCN), osteopontin (OPN), osteoprotegerin (OPG), and receptor activator of NF-КB ligand (RANKL) were assessed in the ligated teeth at 30 days after ligature placement. RESULTS: At 10 and 30 days, ligated teeth of the estrogen-deficient group exhibited higher BL, when compared to all other groups (P < .05). At 10 days, TBAs were higher in the unligated teeth of strontium ranelate-treated groups, when compared to those of untreated groups (P < .05). At 30 days, the ligated teeth of the estrogen-deficient group exhibited lower TBA than the other groups (P < .05). There were no differences among groups regarding the number of TRAP-stained cells (P < .05). The strontium ranelate-treated groups exhibited lower expressions of OCN and RANKL than the untreated groups (P < .05). The estrogen-sufficient group presented higher staining for OPG than both treated and untreated estrogen-deficient groups (P < .05). CONCLUSIONS: Strontium ranelate prevented ligature-induced BL in an estrogen-deficiency condition and, to a certain extent, increased TBA in the presence and absence of periodontal collapse in states of estrogen deficiency and estrogen sufficiency. Furthermore, strontium ranelate also affected the expression of bone markers, appearing to have acted predominantly as an anti-resorptive agent.


Assuntos
Perda do Osso Alveolar/tratamento farmacológico , Estrogênios/deficiência , Periodontite/tratamento farmacológico , Tiofenos/farmacologia , Animais , Osteocalcina/metabolismo , Osteopontina/metabolismo , Osteoprotegerina/metabolismo , Ovariectomia , Ligante RANK/metabolismo , Ratos , Ratos Wistar
19.
Mol Cell Biochem ; 463(1-2): 91-100, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31606864

RESUMO

Baicalin (BAI), a sort of flavonoid monomer, acquires from Scutellaria baicalensis Georgi, which was forcefully reported in diversified ailments due to the pleiotropic properties. But, the functions of BAI in osteoblast differentiation have not been addressed. The intentions of this study are to attest the influences of BAI in the differentiation of osteoblasts. MC3T3-E1 cells or rat primary osteoblasts were exposed to BAI, and then cell viability, ALP activity, mineralization process, and Runx2 and Ocn expression were appraised through implementing CCK-8, p-nitrophenyl phosphate (pNPP), Alizarin red staining, western blot, and RT-qPCR assays. The microRNA-217 (miR-217) expression was evaluated in MC3T3-E1 cells or rat primary osteoblasts after BAI disposition; meanwhile, the functions of miR-217 in BAI-administrated MC3T3-E1 cells were estimated after miR-217 inhibitor transfection. The impacts of BAI and miR-217 inhibition on Wnt/ß-catenin and MEK/ERK pathways were probed to verify the involvements in BAI-regulated the differentiation of osteoblasts. BAI accelerated cell viability, osteoblast activity, and Runx2 and Ocn expression in MC3T3-E1 cells or rat primary osteoblasts, and the phenomena were mediated via activations of Wnt/ß-catenin and MEK/ERK pathways. Elevation of miR-217 was observed in BAI-disposed MC3T3-E1 cells or rat primary osteoblasts, and miR-217 repression annulled the functions of BAI in MC3T3-E1 cell viability and differentiation. Additionally, the activations of Wnt/ß-catenin and MEK/ERK pathways evoked by BAI were both restrained by repression of miR-217. These explorations uncovered that BAI augmented the differentiation of osteoblasts via activations of Wnt/ß-catenin and MEK/ERK pathways by ascending miR-217 expression.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Flavonoides/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , MicroRNAs/biossíntese , Osteoblastos/metabolismo , Animais , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Camundongos , MicroRNAs/genética , Osteoblastos/citologia , Osteocalcina/genética , Osteocalcina/metabolismo
20.
Am J Physiol Cell Physiol ; 318(1): C73-C82, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31577514

RESUMO

Our objective was to investigate the role of primary cilia in low-magnitude, high-frequency vibration (LMHFV) treatment of MC3T3-E1 osteoblasts (OBs). We used chloral hydrate (CH), which has a well-characterized function in chemically removing primary cilia, to elucidate the role of primary cilia in LMHFV-induced OB osteogenic responses through cell viability assay, Western blot analysis, real-time quantitative RT-PCR, and histochemical staining methods. We observed a significant, 30% decrease in the number of MC3T3-E1 OBs with primary cilia (reduced from 64.3 ± 5%) and an approximately 50% reduction in length of primary cilia (reduced from 3 ± 0.8 µm) after LMHFV stimulation. LMHFV stimulation upregulated protein expression of the bone matrix markers collagen 1 (COL-1), osteopontin (OPN), and osteoclacin(OCN) in MC3T3-E1 OBs, indicating that LMHFV induces osteogenesis. High-concentration or long-duration CH exposure resulted in inhibition of MC3T3-E1 OB survival. In addition, Western blot analysis and RT-PCR revealed that CH treatment prevented LMHFV-induced osteogenesis. Furthermore, decreased alkaline phosphate activity, reduced OB differentiation, mineralization, and maturation were observed in CH-pretreated and LMHFV-treated OBs. We showed that LMHFV induces morphological changes in primary cilia that may fine-tune their mechanosensitivity. In addition, we demonstrated the significant inhibition by CH of LMHFV-induced OB mineralization, maturation, and differentiation, which might reveal the critical role of primary cilia in the process.


Assuntos
Diferenciação Celular , Cílios/metabolismo , Mecanotransdução Celular , Osteoblastos/metabolismo , Osteogênese , Vibração , Células 3T3 , Animais , Diferenciação Celular/genética , Hidrato de Cloral/toxicidade , Cílios/efeitos dos fármacos , Cílios/patologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Regulação da Expressão Gênica , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Osteocalcina/metabolismo , Osteogênese/genética , Osteopontina/metabolismo , Fatores de Tempo
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