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1.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33810030

RESUMO

This study evaluated the direct effect of a phytochemical, hesperidin, on pre-osteoblast cell function as well as osteogenesis and collagen matrix quality, as there is little known about hesperidin's influence in mineralized tissue formation and regeneration. Hesperidin was added to a culture of MC3T3-E1 cells at various concentrations. Cell proliferation, viability, osteogenic gene expression and deposited collagen matrix analyses were performed. Treatment with hesperidin showed significant upregulation of osteogenic markers, particularly with lower doses. Mature and compact collagen fibrils in hesperidin-treated cultures were observed by picrosirius red staining (PSR), although a thinner matrix layer was present for the higher dose of hesperidin compared to osteogenic media alone. Fourier-transform infrared spectroscopy indicated a better mineral-to-matrix ratio and matrix distribution in cultures exposed to hesperidin and confirmed less collagen deposited with the 100-µM dose of hesperidin. In vivo, hesperidin combined with a suboptimal dose of bone morphogenetic protein 2 (BMP2) (dose unable to promote healing of a rat mandible critical-sized bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a large dose of BMP2 (previously defined as optimal in healing the critical-sized defect, although of ectopic nature). PSR staining of newly formed bone demonstrated that hesperidin can promote maturation of bone organic matrix. Our findings show, for the first time, that hesperidin has a modulatory role in mineralized tissue formation via not only osteoblast cell differentiation but also matrix organization and matrix-to-mineral ratio and could be a potential adjunct in regenerative bone therapies.


Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Hesperidina/farmacologia , Osteogênese/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2/farmacologia , Regeneração Óssea , Linhagem Celular , Células Cultivadas , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Ratos
2.
Int J Nanomedicine ; 16: 2789-2801, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33880024

RESUMO

Objective: Gold nanorods (AuNRs) show great potential for versatile biomedical applications, such as stem cell therapy and bone tissue engineering. However, as an indispensable shape-directing agent for the growth of AuNRs, cetyltrimethylammonium bromide (CTAB) is not optimal for biological studies because it forms a cytotoxic bilayer on the AuNR surface, which interferes with the interactions with biological cells. Methods: Citrate-stabilized AuNRs with various aspect-ratios (Cit-NRI, Cit-NRII, and Cit-NRIII) were prepared by the combination of end-selective etching and poly(sodium 4-styrenesulfonate)-assisted ligand exchange method. Their effects on osteogenic differentiation of the pre-osteoblastic cell line (MC3T3-E1), rat bone marrow mesenchymal stem cells (rBMSCs), and human periodontal ligament progenitor cells (PDLPs) have been investigated. Potential signaling pathway of citrate-stabilized AuNRs-induced osteogenic effects was also investigated. Results: The experimental results showed that citrate-stabilized AuNRs have superior biocompatibility and undergo aspect-ratio-dependent osteogenic differentiation via expression of osteogenic marker genes, alkaline phosphatase (ALP) activity and formation of mineralized nodule. Furthermore, Wnt/ß-catenin signaling pathway might provide a potential explanation for the citrate-stabilized AuNRs-mediated osteogenic differentiation. Conclusion: These findings revealed that citrate-stabilized AuNRs with great biocompatibility could regulate the osteogenic differentiation of multiple cell types through Wnt/ß-catenin signaling pathway, which promote innovative AuNRs in the field of tissue engineering and other biomedical applications.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Ácido Cítrico/farmacologia , Ouro/farmacologia , Nanotubos/química , Osteogênese/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Calcificação Fisiológica/efeitos dos fármacos , Células Cultivadas , Cetrimônio/farmacologia , Endocitose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Nanotubos/ultraestrutura , Osteogênese/genética , Ligamento Periodontal/citologia , Ratos , Tiazolidinas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos
3.
Mol Med Rep ; 23(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33576449

RESUMO

Cirsium setidens (Dunn) Nakai, commonly known as gondre, is a perennial herb that grows predominantly in South Korea. It contains several bioactive phytochemicals with antioxidant, anti­cancer, anti­tumor and anti­inflammatory properties. The present study aimed to investigate the effects of methanolic extracts of gondre on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). As characterized by nuclear magnetic resonance spectroscopy and matrix­assisted laser deposition/ionization (time­of­flight) mass spectrometry, the methanol extract of gondre was found to be enriched with pectolinarin. After 48 h, enhanced viability of hPDLSCs was observed in the presence of gondre compared with under control conditions, suggesting the biocompatibility of gondre. Notably, biocompatibility was markedly affected by gondre concentration in cultured media. Relatively high cell viability was observed in medium containing 0.05% gondre. Furthermore, mineralization was significantly higher in hPDLSCs in the presence of gondre compared with that in control cells, indicating their mineralization potential. Increased expression of various transcription markers, such as collagen 1, runt­related transcription factor 2, bone sialoprotein and alkaline phosphatase, was also detected when hPDLSCs were stimulated with gondre compared with in the control groups, further confirming the superior osteogenic potential of gondre extract for tissue engineering applications, particularly in bone tissues.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Cirsium/química , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/citologia , Extratos Vegetais/farmacologia , Células-Tronco/efeitos dos fármacos , Adolescente , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Masculino , Extratos Vegetais/química , Células-Tronco/citologia , Células-Tronco/metabolismo , Adulto Jovem
4.
Int J Biol Macromol ; 172: 19-29, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33444651

RESUMO

The treatment and repair of large bone defects remains a major therapeutic challenge in the clinical setting. Nanofiber scaffolds fabricated via the electrospinning technique have been developed as a universal method for bone regeneration due to their suitable properties. However, traditional two-dimensional (2D) nanofiber mats are usually too dense, which may prevent cell infiltration and growth, thereby restricting their application. Herein, a three-dimensional (3D) polycaprolactone nanofiber scaffold was developed, modified by biomineralization and silk fibroin coating. The scaffold possessed a parallel array of nanofiber surfaces, mimicking the parallel structure of fibrils in natural bone tissue. Furthermore, the fabricated radially or laterally interconnected macrochannels were investigated to elucidate the effect of the scaffold structure on bone regeneration. In vitro studies revealed that the scaffolds could guide cell arrangement and that the radially aligned scaffold demonstrated a stronger ability to promote cell proliferation. In vivo results showed that the radially aligned scaffold could guide tissue arrangement and remodeling and support a significantly faster regeneration rate of bone tissue. Therefore, 3D-mineralized polycaprolactone nanofiber scaffolds with radially interconnected macrochannels and aligned nanofibers are expected to be used in tissue engineering, including in the repair of bone defects, cartilage or other composite tissues.


Assuntos
Biomineralização/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Fibroínas/química , Nanofibras/química , Poliésteres/química , Tecidos Suporte/química , Animais , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual/métodos
5.
Molecules ; 26(3)2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33503825

RESUMO

Several signalling pathways, including the JAK/STAT signalling pathway, have been identified to regulate the differentiation of human bone marrow skeletal (mesenchymal) stem cells (hBMSCs) into bone-forming osteoblasts. Members of the JAK family mediate the intracellular signalling of various of cytokines and growth factors, leading to the regulation of cell proliferation and differentiation into bone-forming osteoblastic cells. Inhibition of JAK2 leads to decoupling of its downstream mediator, STAT3, and the subsequent inhibition of JAK/STAT signalling. However, the crucial role of JAK2 in hBMSCs biology has not been studied in detail. A JAK2 inhibitor, Fedratinib, was identified during a chemical biology screen of a small molecule library for effects on the osteoblastic differentiation of hMSC-TERT cells. Alkaline phosphatase activity and staining assays were conducted as indicators of osteoblastic differentiation, while Alizarin red staining was used as an indicator of in vitro mineralised matrix formation. Changes in gene expression were assessed using quantitative real-time polymerase chain reaction. Fedratinib exerted significant inhibitory effects on the osteoblastic differentiation of hMSC-TERT cells, as demonstrated by reduced ALP activity, in vitro mineralised matrix formation and downregulation of osteoblast-related gene expression, including ALP, ON, OC, RUNX2, OPN, and COL1A1. To identify the underlying molecular mechanisms, we examined the effects of Fedratinib on a molecular signature of several target genes known to affect hMSC-TERT differentiation into osteoblasts. Fedratinib inhibited the expression of LIF, SOCS3, RRAD, NOTCH3, TNF, COMP, THBS2, and IL6, which are associated with various signalling pathways, including TGFß signalling, insulin signalling, focal adhesion, Notch Signalling, IL-6 signalling, endochondral ossification, TNF-α, and cytokines and inflammatory response. We identified a JAK2 inhibitor (Fedratinib) as a powerful inhibitor of the osteoblastic differentiation of hMSC-TERT cells, which may be useful as a therapeutic option for treating conditions associated with ectopic bone formation or osteosclerotic metastases.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Janus Quinase 2/antagonistas & inibidores , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Pirrolidinas/farmacologia , Sulfonamidas/farmacologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Fosfatase Alcalina/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
6.
Int J Biol Macromol ; 171: 185-197, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33412197

RESUMO

Alhagi pseudalhagi, commonly known as camel thorn, is used as an indigenous medicinal plant in China. The present study was designed to elucidate the structure of a novel polysaccharide, APP90-2, isolated from Alhagi pseudalhagi and evaluate its osteogenic activity. A homogeneous polysaccharide (APP90-2) was obtained from A. pseudalhagi via DEAE-52 and Sephacryl S-100 columns, with a molecular weight of 5.9 kDa. Monosaccharide, GC-MS, and NMR analyses showed that APP90-2 consisted of α-l-Rhap-(1→, →3)-α-l-Araf-(1→, →5)-α-l-Araf-(1→, →4)-ß-d-Xylp-(1→, α-d-Glcp-(1→, →3,5)-α-l-Araf-(1→, →4)-ß-d-GlcAp-(1→, →4)-3-OAc-α-d-Glcp-(1→, →3)-α-d-Galp-(1→, →3)-ß-d-GalAp-(1→, →4)-α-d-Galp-(1→, →6)-α-d-Manp-(1→, →4,6)-ß-d-Galp-(1→, and →3,6)-ß-d-Glcp-(1→ with relative molar ratios of 4.1:1.8:6.1:6.7:1.7:1.0:1.5:2.7:2.4:1.1:2.3:2.6:1.4:2.0. Morphological analyses revealed that APP90-2 interacted with Congo-red and had an obvious honeycomb structure. Additionally, APP90-2 significantly promoted proliferation, differentiation, and mineralization of MC3T3-E1 cells, indicating that APP90-2 exhibited pronounced osteogenic activity. Therefore, our findings suggest that A. pseudalhagi may be used as an alternative medicine or health supplement for the prevention and treatment of osteoporosis.


Assuntos
Conservadores da Densidade Óssea/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Fabaceae/química , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Polissacarídeos/farmacologia , Animais , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/isolamento & purificação , Sequência de Carboidratos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , China , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Plantas Medicinais , Polissacarídeos/química , Polissacarídeos/isolamento & purificação
7.
Int J Mol Sci ; 21(24)2020 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-33371439

RESUMO

Transforming growth factor ß1 (TGFß1) is a major mediator in the modulation of osteoblast differentiation. However, the underlying molecular mechanism is still not fully understood. Here, we show that TGFß1 has a dual stage-dependent role in osteoblast differentiation; TGFß1 induced matrix maturation but inhibited matrix mineralization. We discovered the underlying mechanism of the TGFß1 inhibitory role in mineralization using human osteoprogenitors. In particular, the matrix mineralization-related genes of osteoblasts such as osteocalcin (OCN), Dickkopf 1 (DKK1), and CCAAT/enhancer-binding protein beta (C/EBPß) were dramatically suppressed by TGFß1 treatment. The suppressive effects of TGFß1 were reversed with anti-TGFß1 treatment. Mechanically, TGFß1 decreased protein levels of C/EBPß without changing mRNA levels and reduced both mRNA and protein levels of DKK1. The degradation of the C/EBPß protein by TGFß1 was dependent on the ubiquitin-proteasome pathway. TGFß1 degraded the C/EBPß protein by inducing the expression of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (SMURF1) at the transcript level, thereby reducing the C/EBPß-DKK1 regulatory mechanism. Collectively, our findings suggest that TGFß1 suppressed the matrix mineralization of osteoblast differentiation by regulating the SMURF1-C/EBPß-DKK1 axis.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular , Matriz Extracelular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osteoblastos/citologia , Fator de Crescimento Transformador beta1/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Idoso , Proteína beta Intensificadora de Ligação a CCAAT/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese , Ubiquitina-Proteína Ligases/genética
8.
Nat Commun ; 11(1): 4278, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855388

RESUMO

Activation and migration of endogenous mesenchymal stromal cells (MSCs) are critical for bone regeneration. Here, we report a combinational peptide screening strategy for rapid discovery of ligands that not only bind strongly to osteogenic progenitor cells (OPCs) but also stimulate osteogenic cell Akt signaling in those OPCs. Two lead compounds are discovered, YLL3 and YLL8, both of which increase osteoprogenitor osteogenic differentiation in vitro. When given to normal or osteopenic mice, the compounds increase mineral apposition rate, bone formation, bone mass, and bone strength, as well as expedite fracture repair through stimulated endogenous osteogenesis. When covalently conjugated to alendronate, YLLs acquire an additional function resulting in a "tri-functional" compound that: (i) binds to OPCs, (ii) targets bone, and (iii) induces "pro-survival" signal. These bone-targeted, osteogenic peptides are well suited for current tissue-specific therapeutic paradigms to augment the endogenous osteogenic cells for bone regeneration and the treatment of bone loss.


Assuntos
Anabolizantes/farmacologia , Fraturas Ósseas/tratamento farmacológico , Osteogênese/efeitos dos fármacos , Peptídeos/farmacologia , Células-Tronco/efeitos dos fármacos , Anabolizantes/química , Animais , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Fraturas Ósseas/patologia , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Orquiectomia , Osteogênese/fisiologia , Ovariectomia , Peptídeos/química , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Técnicas de Síntese em Fase Sólida , Células-Tronco/citologia
9.
Life Sci ; 257: 118044, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32622944

RESUMO

AIMS: High-dose glucocorticoid (GC) administration causes osteoporosis. Many previous studies from our group and other groups have shown that melatonin participates in the regulation of osteoblast proliferation and differentiation, especially low concentrations of melatonin, which enhance osteoblast osteogenesis. However, the role of melatonin in glucocorticoid-induced osteoblast differentiation remains unknown. MATERIALS AND METHODS: An examination of the expression of osteoblast differentiation markers (ALP, OCN, COLL-1), as well as alkaline phosphatase staining and alkaline phosphatase enzymatic activity assay to measure osteoblast differentiation and quantifying Alizarin red S staining to measure mineralization, were performed to determine the effects of dexamethasone (Dex) and melatonin on the differentiation of MC3T3-E1 cells. We used immunofluorescence staining to detect the expression of Runx2 in melatonin-treated MC3T3-E1 cells. The expression of mRNA was determined by qRT-PCR, and protein levels were measured by western blotting. KEY FINDINGS: In the present study, we found that 100 µM Dex significantly reduced osteoblast differentiation and mineralization in MC3T3-E1 cells and that 1 µM melatonin attenuated these inhibitory effects. We found that only inhibition of PI3K/AKT (MK2206) and BMP/Smad (LDN193189) signalling abolished melatonin-induced differentiation and mineralization. Meanwhile, MK2206 decreased the expression of P-AKT and P-Smad1/5/9 and LDN193189 decreased the expression of P-Smad1/5/9 but had no obvious effect on P-AKT expression in melatonin-treated and Dex-induced MC3T3-E1 cells. SIGNIFICANCE: These findings suggest that melatonin rescues Dex-induced inhibition of osteoblast differentiation in MC3T3-E1 cells via the PI3K/AKT and BMP/Smad signalling pathways and that PI3K/AKT signalling may be the upstream signal of BMP/Smad signalling.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Melatonina/metabolismo , Osteoblastos/metabolismo , Animais , Biomineralização/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Linhagem Celular , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/efeitos adversos , Glucocorticoides/farmacologia , Melatonina/farmacologia , Camundongos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo
10.
Poult Sci ; 99(5): 2595-2607, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32359595

RESUMO

Effects of dietary available phosphorus (aP) and Ca levels and an Escherichia coli 6-phytase supplementation were studied in Lohmann LSL-Lite hens from 25 to 37 wk of age. Eighty-four hens were used in a completely randomized design with 7 treatments. The treatments were a positive control (PC) diet with 0.45% aP, 3.70% Ca, and 0.16% Na from 25 to 28 wk and 0.38% aP, 3.73% Ca, and 0.15% Na from 29 to 37 wk; a negative control (NC) diet, similar to the PC diet, with 0.22% aP, 3.00% Ca, and 0.13% Na from 25 to 28 wk and 0.19% aP, 3.02% Ca, and 0.13% Na from 29 to 37 wk; the NC diets supplemented with phytase at 150 (NC + 150), 300 (NC + 300), 600 (NC + 600), or 1,200 (NC + 1,200) phytase unit (FTU)/kg; and the PC diet supplemented with phytase at 1,200 (PC + 1,200) FTU/kg. Hen performance, eggshell, and bone quality were measured on a 4-wk basis. Bone breaking strength and ash and apparent ileal digestibility (AID) of P and Ca were determined at 37 wk. One- and 2-way ANOVA were conducted, and Tukey's range test was used to compare multiple means where P ≤ 0.05. No differences in hen performance, eggshell quality, bone breaking strength, bone ash, and P digestibility were observed between the PC and the NC treatments. The NC hens had lower cortical (P < 0.001) and trabecular + medullary bone mineral density (P = 0.004) and total bone mineral content (P < 0.001) than the PC hens. The PC + 1,200 increased cortical bone mineral density (P < 0.001). The reductions of aP and Ca in the NC diet were not deficient for performance but had a minor impact on bone mineralization. The NC + 600 and NC + 1,200 increased AID of P (P = 0.024), and all phytase treatments except the NC + 150 increased AID of Ca (P = 0.010) compared with the NC diet.


Assuntos
6-Fitase/metabolismo , Calcificação Fisiológica/fisiologia , Cálcio na Dieta/metabolismo , Digestão , Casca de Ovo/fisiologia , Fósforo na Dieta/metabolismo , 6-Fitase/administração & dosagem , Ração Animal/análise , Animais , Calcificação Fisiológica/efeitos dos fármacos , Cálcio/deficiência , Galinhas , Dieta/veterinária , Suplementos Nutricionais/análise , Digestão/efeitos dos fármacos , Relação Dose-Resposta a Droga , Casca de Ovo/efeitos dos fármacos , Feminino , Íleo/fisiologia , Fósforo/deficiência , Distribuição Aleatória
11.
Eur J Endocrinol ; 183(2): 181-189, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32454455

RESUMO

Objective: Long-term androgen deprivation therapy (ADT) negatively influences bone. The short-term effects on bone and mineral homeostasis are less known. Therefore, we aimed to investigate the early effects of ADT on calcium/phosphate homeostasis and bone turnover. Design: Prospective cohort study. Methods: Eugonadal adult, male sex offenders, who were referred for ADT to the endocrine outpatient clinic, received cyproterone acetate. Changes in blood markers of calcium/phosphate homeostasis and bone turnover between baseline and first follow-up visit were studied. Results: Of 26 screened patients, 17 were included. The median age was 44 (range 20-75) years. The median time interval between baseline and first follow-up was 13 (6-27) weeks. Compared to baseline, an 81% decrease was observed for median total testosterone (to 3.4 nmol/L (0.4-12.2); P < 0.0001) and free testosterone (to 0.06 nmol/L (0.01-0.18); P < 0.0001). Median total estradiol decreased by 71% (to 17.6 pmol/L (4.7-35.6); P < 0.0001). Increased serum calcium (P < 0.0001) and phosphate (P = 0.0016) was observed, paralleled by decreased PTH (P = 0.0156) and 1,25-dihydroxyvitamin D3 (P = 0.0134). The stable calcium isotope ratio (δ44/42Ca) decreased (P = 0.0458), indicating net calcium loss from bone. Bone-specific alkaline phosphatase and osteocalcin decreased (P < 0.0001 and P = 0.0056, respectively), periostin tended to decrease (P = 0.0500), whereas sclerostin increased (P < 0.0001), indicating suppressed bone formation. Serum bone resorption markers (TRAP, CTX) were unaltered. Conclusions: In adult men, calcium release from the skeleton occurs early following sex steroid deprivation, reflecting early bone resorption. The increase of sclerostin and reduction of bone formation markers, without changes in resorption markers, suggests a dominant negative effect on bone formation in the acute phase.


Assuntos
Antagonistas de Androgênios/farmacologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/fisiologia , Calcificação Fisiológica/efeitos dos fármacos , Acetato de Ciproterona/farmacologia , Adulto , Idoso , Bélgica , Remodelação Óssea/efeitos dos fármacos , Cálcio/sangue , Estudos de Coortes , Homeostase/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Fosfatos/sangue , Estudos Prospectivos , Delitos Sexuais , Testosterona/sangue
12.
PLoS One ; 15(5): e0234009, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470038

RESUMO

One of the potential contributing factors for iron overload-induced osteoporosis is the iron toxicity on bone forming cells, osteoblasts. In this study, the comparative effects of Fe3+ and Fe2+ on osteoblast differentiation and mineralization were studied in UMR-106 osteoblast cells by using ferric ammonium citrate and ferrous ammonium sulfate as Fe3+ and Fe2+ donors, respectively. Effects of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and iron chelator deferiprone on iron uptake ability of osteoblasts were examined, and the potential protective ability of 1,25(OH)2D3, deferiprone and extracellular calcium treatment in osteoblast cell survival under iron overload was also elucidated. The differential effects of Fe3+ and Fe2+ on reactive oxygen species (ROS) production in osteoblasts were also compared. Our results showed that both iron species suppressed alkaline phosphatase gene expression and mineralization with the stronger effects from Fe3+ than Fe2+. 1,25(OH)2D3 significantly increased the intracellular iron but minimally affected osteoblast cell survival under iron overload. Deferiprone markedly decreased intracellular iron in osteoblasts, but it could not recover iron-induced osteoblast cell death. Interestingly, extracellular calcium was able to rescue osteoblasts from iron-induced osteoblast cell death. Additionally, both iron species could induce ROS production and G0/G1 cell cycle arrest in osteoblasts with the stronger effects from Fe3+. In conclusions, Fe3+ and Fe2+ differentially compromised the osteoblast functions and viability, which can be alleviated by an increase in extracellular ionized calcium, but not 1,25(OH)2D3 or iron chelator deferiprone. This study has provided the invaluable information for therapeutic design targeting specific iron specie(s) in iron overload-induced osteoporosis. Moreover, an increase in extracellular calcium could be beneficial for this group of patients.


Assuntos
Calcitriol/farmacologia , Deferiprona/farmacologia , Espaço Extracelular/química , Sobrecarga de Ferro/metabolismo , Ferro/farmacologia , Osteoblastos/citologia , Animais , Biomarcadores/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Cálcio/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo
13.
Gene ; 749: 144703, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32339623

RESUMO

The repair of segmental bone defects and bone fractures is a clinical challenge involving high risk and postsurgical morbidity. Bone injury and partial bone tumor resection via traditional bone grafting result in high complications. Growth factors have been proposed as alternatives to promote bone repair and formation and circumvent these limitations. In this study, we classified different lengths of mechano growth factor (MGF) E peptides in different species and analyzed their effects on MC3T3-E1 cell proliferation, cell cycle, alkaline phosphatase (ALP) activity, differentiation-related factor expression, and cell mineralization. A rabbit bone injury model was constructed, and the repair function of MGF E peptide was verified by injecting the candidate MGF E peptide. We analyzed 52 different MGF-E peptides and classified them into the following four categories: T-MGF-25E, M-MGF-25E, T-MGF-19E, and M-MGF-19E. These peptides were synthesized for further study. T-MGF-19E peptide obviously promoted cell proliferation by regulating cell cycle after MGF E peptide treatment at 72 h. T-MGF-25E and T-MGF-19E peptide significantly promoted the differentiation of osteoblasts on day 14, and M-MGF-25E peptide promoted cell differentiation on day 7. T-MGF-19E, T-MGF-25E, and M-MGF-19E significantly promoted osteoblast mineralization, with T-MGF19E showing the most significant effect. These results implied that T-MGF19E peptide could remarkably promote MC3T3-E1 cell proliferation, differentiation, and mineralization. The rabbit bone defect model showed that the low-dose T-MGF-19E peptide significantly promoted bone injury healing, suggesting its promoting effect on the healing of bone injury.


Assuntos
Osso e Ossos/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Osteogênese/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Osso e Ossos/fisiologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/genética
14.
Biochem Biophys Res Commun ; 525(3): 576-580, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32115151

RESUMO

Coral calcification is intricately linked to the chemical composition of the fluid in the extracellular calcifying medium (ECM), which is situated between the calcifying cells and the skeleton. Here we demonstrate that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) is expressed in calcifying cells of the coral Stylophora pistillata. Furthermore, pharmacological inhibition of sAC in coral microcolonies resulted in acidification of the ECM as estimated by the pH-sensitive ratiometric indicator SNARF, and decreased calcification rates, as estimated by calcein labeling of crystal growth. These results indicate that sAC activity modulates some of the molecular machinery involved in producing the coral skeleton, which could include ion-transporting proteins and vesicular transport. To our knowledge this is the first study to directly demonstrate biological regulation of the alkaline pH of the coral ECM and its correlation with calcification.


Assuntos
Equilíbrio Ácido-Base , Adenilil Ciclases/metabolismo , Antozoários/enzimologia , Antozoários/fisiologia , Calcificação Fisiológica , Equilíbrio Ácido-Base/efeitos dos fármacos , Inibidores de Adenilil Ciclases/farmacologia , Álcalis/metabolismo , Animais , Antozoários/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Concentração de Íons de Hidrogênio , Solubilidade
15.
Poult Sci ; 99(2): 926-935, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32029169

RESUMO

The present study evaluated the effects of butyric acid supplementation and Saccharomyces boulardii (alone or in combination) on growth performance, nutrient digestibility, bone mineralization, and blood hormones of male broiler chickens fed a diet including reduced levels of nonphytate phosphorus (NPP). The chickens were allocated to 6 dietary treatments: 1) positive control diet with adequate amounts of NPP (PC; 0.48, 0.43, and 0.39% in the starter, grower, and finisher period, respectively); 2) negative control diet with low amounts of NPP (NC; 0.38, 0.33, and 0.29% in the starter, grower, and finisher period, respectively); 3) NC plus 500 FTU/kg microbial phytase (PHY); 4) NC plus 0.2% butyric acid (BA); 5) NC plus 1 × 108 cfu/kg S. boulardii (SB); 6) NC plus butyric acid and S. boulardii (BA+SB). Each treatment had 5 pen replicates of 25 birds. After 6 wk, the body weight and ADG in birds fed with any of the diets were higher (P < 0.001) than those in birds fed with the NC diet, where the birds fed with the PHY and BA+SB diets had the highest values. However, only the PHY diet improved (P = 0.041) overall F:G. All diets, except the SB diet, resulted in the increased apparent ileal digestibility coefficient (AIDC) of CP, AMEn, and tibia ash content and decreased serum alkaline phosphatase level compared with the NC diet (P < 0.05). Broiler chickens fed with the PHY, SB, and BA+SB diets also had increased AIDC of phosphorus (P = 0.017) than those fed with the NC and PC diets. Feeding PC, PHY, and BA+SB diets increased (P = 0.007) the tibia phosphorus content but decreased (P = 0.033) serum parathyroid hormone concentration. Overall, the present data indicate that the simultaneous inclusion of butyric acid plus S. boulardii in the low-NPP diets was beneficial for improving growth rate and bone mineralization, but not for feed efficiency.


Assuntos
Ácido Butírico/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Galinhas/fisiologia , Digestão/efeitos dos fármacos , Hormônios/sangue , Saccharomyces boulardii/química , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Ácido Butírico/administração & dosagem , Cálcio/metabolismo , Galinhas/crescimento & desenvolvimento , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Nutrientes/metabolismo , Fósforo/deficiência , Fósforo/metabolismo , Probióticos/farmacologia , Distribuição Aleatória
16.
Exp Cell Res ; 389(1): 111883, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32014443

RESUMO

Melatonin has been demonstrated to protect against calcification in cyclosporine nephrotoxicity. Autophagy may affect vascular calcification by inhibiting apoptosis and the transdifferentiation process. This study sought to explore whether melatonin attenuates vascular calcification by regulating autophagy via the AMP-activated protein kinase/mammalian target of rapamycin/Unc-51-like kinase 1 (AMPK/mTOR/ULK1) signaling pathway. The effects of melatonin on vascular calcification were investigated in vascular smooth muscle cells (VSMCs). Calcium deposits were visualised by Alizarin red staining, while calcium content and alkaline phosphatase (ALP) activity were used to evaluate osteogenic differentiation. Western blots were used to measure expression of runt-related transcription factor 2 (Runx2, an osteogenic transcription factor), light chain 3 (LC3) II/I, and cleaved caspase 3. Melatonin markedly reduced calcium deposition and ALP activity. Runx2 and cleaved caspase 3 were downregulated, whereas LC3 II/I was increased in response to melatonin, and was accompanied by decreased apoptosis. An immunofluorescence assay revealed that melatonin treatment markedly decreased Runx2 expression and upregulated LC3 expression. Treatment with the autophagy inhibitor 3-methyladenine reversed this phenomenon. Melatonin significantly increased expression of p-AMPK and p-ULK1, and decreased mTOR expression. Treatment with compound C (an inhibitor of AMPK) or MHY1485 (an agonist of mTOR) ablated the observed benefits of melatonin treatment. Melatonin protects VSMCs against calcification by activating autophagy via the AMPK/mTOR/ULK1 pathway.


Assuntos
Autofagia/efeitos dos fármacos , Melatonina/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Calcificação Vascular/prevenção & controle , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Células Cultivadas , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/efeitos dos fármacos
17.
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
18.
Mater Sci Eng C Mater Biol Appl ; 108: 110374, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31924043

RESUMO

The development of highly biomimetic scaffolds in terms of composition and structures, to repair or replace damaged bone tissues, is particularly relevant for tissue engineering. This paper investigates a 3D printed porous scaffold containing aligned multi-walled carbon nanotubes (MWCNTs) and nano-hydroxyapatite (nHA), mimicking the natural bone tissue from the nanoscale to macroscale level. MWCNTs with similar dimensions as collagen fibres are coupled with nHA and mixed within a polycaprolactone (PCL) matrix to produce scaffolds using a screw-assisted extrusion-based additive manufacturing system. Scaffolds with different material compositions were extensively characterised from morphological, mechanical and biological points of views. Transmission electron microscopy and polarised Raman spectroscopy confirm the presence of aligned MWCNTs within the printed filaments. The PCL/HA/MWCNTs scaffold are similar to the nanostructure of native bone and shows overall increased mechanical properties, cell proliferation, osteogenic differentiation and scaffold mineralisation, indicating a promising approach for bone tissue regeneration.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Durapatita/farmacologia , Nanotubos de Carbono/química , Osteogênese/efeitos dos fármacos , Poliésteres/farmacologia , Tecidos Suporte/química , Fosfatase Alcalina/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Humanos , Nanotubos de Carbono/ultraestrutura , Osteocalcina/metabolismo , Análise Espectral Raman
19.
J Anim Sci ; 98(2)2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31974567

RESUMO

In this study, we identified cadmium (Cd) as a potential endocrine disruptor that impairs laying performance, egg quality, and eggshell deposition and induces oxidative stress and inflammation in the eggshell glands of laying hens. A total of 480 38-wk-old laying hens were randomly assigned into 5 groups that were fed a basal diet (control) or a basal diet supplemented with Cd (provided as CdCl2·2.5 H2O) at 7.5, 15, 30, and 60 mg Cd per kg feed for 9 wk. The results showed that, when compared with the control group, a low dose of dietary Cd (7.5 mg/kg) had positive effects on egg quality by improving albumen height, Haugh unit, yolk color, and shell thickness at the third or ninth week. However, with the increase in the dose and duration of Cd exposure, the laying performance, egg quality, and activities of eggshell gland antioxidant enzymes (catalase [CAT], glutathione peroxide [GSH-Px]), and ATPase (Na+/K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase) deteriorated, and the activity of total nitric oxide synthase (T-NOS) and the level of malondialdehyde (MDA) increased significantly (P < 0.05). The histopathology and real-time quantitative PCR results showed that Cd induced endometrial epithelial cell proliferation accompanied by upregulation of the mRNA levels of progesterone receptor (PgR) and epidermal growth factor receptor (EGFR), downregulation of the mRNA levels of estrogen receptor α (ERα) and interleukin 6 (IL6), and inflammation of the eggshell gland accompanied by significantly increased expression of complement C3 and pro-inflammatory cytokine tumor necrosis factor α (TNFα) (P < 0.05). In addition, the ultrastructure of the eggshell showed that dietary supplementation with 7.5 mg/kg Cd increased the palisade layer and total thickness of the shell, but with the increase in dietary Cd supplementation (30 and 60 mg/kg) the thickness of the palisade layer and mammillary layer decreased significantly (P < 0.05), and the outer surface of the eggshell became rougher. Correspondingly, the expression of calbindin 1 (CALB1), ovocalyxin-32 (OCX-32), ovocalyxin-36 (OCX-36), osteopontin (SPP1), and ovocledidin-17 (OC-17) decreased significantly (P < 0.05) with increasing dietary Cd supplementation. Conclusively, the present study demonstrates that dietary supplementation with Cd negatively affects laying performance, egg quality, and eggshell deposition by disturbing the metabolism of eggshell glands in laying hens but has a positive effect on egg quality at low doses.


Assuntos
Cloreto de Cádmio/toxicidade , Calcificação Fisiológica/efeitos dos fármacos , Galinhas , Casca de Ovo/metabolismo , Ração Animal/análise , Animais , Antioxidantes/farmacologia , Cloreto de Cádmio/administração & dosagem , Dieta/veterinária , Casca de Ovo/química , Feminino
20.
Int J Biol Macromol ; 145: 558-567, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31883888

RESUMO

Glycosaminoglycans (GAGs) play an important role in various biological activities. A lot of them are present in fish processing discards from abattoirs and fish processing industries which can serve as a valuable source of GAGs. We have, in this paper, isolated and characterized GAGs from fish processing discard (head) generated from the processing of Labeo rohita (L. rohita) and Piaractus brachypomus (P. brachypomus) and have determined their ability to promote osteogenic activity. Isolated GAGs showed higher amounts of chondroitin sulfate/dermatan sulfate (CS/DS) than heparan sulfate (HS). CS/DS from both the fish have a distinct disaccharide composition indicating differences in their structure. Biological activity, in terms of promoting osteogenesis, evaluated in MC3T3-E1 cells and primary cells of the calvaria showed that early mineralization, characterized by alkaline phosphatase staining and activity, and late mineralization, was supported by both the GAGs.


Assuntos
Peixes , Glicosaminoglicanos/química , Glicosaminoglicanos/isolamento & purificação , Animais , Biomarcadores , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dissacarídeos/química , Água Doce , Glicosaminoglicanos/farmacologia , Hidrólise , Camundongos , Peso Molecular , Osteogênese/efeitos dos fármacos , Análise Espectral
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