scholarly journals Regulation of Osteogenic Markers at Late Stage of Osteoblast Differentiation in Silicon and Zinc Doped Porous TCP

2019 ◽  
Vol 10 (4) ◽  
pp. 48
Author(s):  
Gary A. Fielding ◽  
Naboneeta Sarkar ◽  
Sahar Vahabzadeh ◽  
Susmita Bose
Keyword(s):  
Trace Elements ◽  
Cell Differentiation ◽  
Late Stage ◽  
Osteoblast Differentiation ◽  
Bone Morphogenetic Protein 2 ◽  
Initial Time ◽  
Osteoblast Cell ◽  
Differentiation Markers ◽  
Receptor Activator ◽  
Highly Porous

Calcium phosphates (CaPs) are one of the most widely used synthetic materials for bone grafting applications in the orthopedic industry. Recent trends in synthetic bone graft applications have shifted towards the incorporation of metal trace elements that extend the performance of CaPs to have osteoinductive properties. The objective of this study is to investigate the effects of silicon (Si) and zinc (Zn) dopants in highly porous tricalcium phosphate (TCP) scaffolds on late-stage osteoblast cell differentiation markers. In this study, an oil emulsion method is utilized to fabricate highly porous SiO2 doped β-TCP (Si-TCP) and ZnO doped β-TCP (Zn-TCP) scaffolds through the incorporation of 0.5 wt.% SiO2 and 0.25 wt.% ZnO, respectively, to the β-TCP scaffold. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is utilized to analyze the mRNA expression of osteoprotegerin (OPG), receptor activator of nuclear kappa beta ligand (RANKL), bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (Runx2) at the later stage of osteoblast differentiation, day 21 and day 28. Results show that the addition of Si and Zn to the β-TCP structure inhibited the β to α-TCP phase transformation and enhance the density without affecting the dissolution properties. Normal BMP-2 and Runx2 transcriptions are observed in both Si-TCP and Zn-TCP scaffolds at the initial time point, as demonstrated by RT-qPCR. Moreover, the addition of both Si and Zn positively regulate the osteoprotegerin: receptor activator of nuclear factor k-β ligand (OPG:RANKL) ratio at 21-days for Si-TCP and Zn-TCP scaffolds. These results demonstrate the effects of Si and Zn doped porous β-TCP scaffolds on the upregulation of osteoblast marker gene expression including OPG, RANKL, BMP-2, and Runx2, indicating the role of trace elements on the effective regulation of late-stage osteoblast cell differentiation markers.

scholarly journals Food for Bone: Evidence for a Role for Delta-Tocotrienol in the Physiological Control of Osteoblast Migration

2020 ◽  
Vol 21 (13) ◽  
pp. 4661 ◽  
Author(s):  
Lavinia Casati ◽  
Francesca Pagani ◽  
Roberto Maggi ◽  
Francesco Ferrucci ◽  
Valeria Sibilia
Keyword(s):  
Wound Healing ◽  
Osteoblast Differentiation ◽  
Target Genes ◽  
Elaeis Guineensis ◽  
Signalling Pathway ◽  
Bone Morphogenetic Protein 2 ◽  
Luciferase Assay ◽  
Boyden Chamber ◽  
Differentiation Markers ◽  
Osteoblast Migration

Bone remodeling and repair require osteogenic cells to reach the sites that need to be rebuilt, indicating that stimulation of osteoblast migration could be a promising osteoanabolic strategy. We showed that purified δ-tocotrienol (δ-TT, 10 μg/mL), isolated from commercial palm oil (Elaeis guineensis) fraction, stimulates the migration of both MC3T3-E1 osteoblast-like cells and primary human bone marrow mesenchymal stem cells (BMSC) as detected by wound healing assay or Boyden chamber assay respectively. The ability of δ-TT to promote MC3T3-E1 cells migration is dependent on Akt phosphorylation detected by Western blotting and involves Wnt/β-catenin signalling pathway activation. In fact, δ-TT increased β-catenin transcriptional activity, measured using a Nano luciferase assay and pretreatment with procaine (2 µM), an inhibitor of the Wnt/β-catenin signalling pathway, reducing the wound healing activity of δ-TT on MC3T3-E1 cells. Moreover, δ-TT treatment increased the expression of β-catenin specific target genes, such as Osteocalcin and Bone Morphogenetic Protein-2, involved in osteoblast differentiation and migration, and increased alkaline phosphatase and collagen content, osteoblast differentiation markers. The ability of δ-TT to enhance the recruitment of BMSC, and to promote MC3T3-E1 differentiation and migratory behavior, indicates that δ-TT could be considered a promising natural anabolic compound.

scholarly journals Anti-Müllerian Hormone Negatively Regulates Osteoclast Differentiation by Suppressing the Receptor Activator of Nuclear Factor-κB Ligand Pathway

2021 ◽  
Vol 28 (3) ◽  
pp. 223-230
Author(s):  
Jung Ha Kim ◽  
Yong Ryoul Yang ◽  
Ki-Sun Kwon ◽  
Nacksung Kim
Keyword(s):  
Osteoblast Differentiation ◽  
Bone Cells ◽  
Osteoclast Differentiation ◽  
Nuclear Factor Κb ◽  
Bone Morphogenetic Protein 2 ◽  
Tartrate Resistant Acid Phosphatase ◽  
Nuclear Factor ◽  
Bone Homeostasis ◽  
Alizarin Red ◽  
Receptor Activator

Background: Multiple members of the transforming growth factor-β (TGF-β) superfamily have well-established roles in bone homeostasis. Anti-Müllerian hormone (AMH) is a member of TGF-β superfamily of glycoproteins that is responsible for the regression of fetal Müllerian ducts and the transcription inhibition of gonadal steroidogenic enzymes. However, the involvement of AMH in bone remodeling is unknown. Therefore, we investigated whether AMH has an effect on bone cells as other TGF-β superfamily members do.Methods: To identify the roles of AMH in bone cells, we administered AMH during osteoblast and osteoclast differentiation, cultured the cells, and then stained the cultured cells with Alizarin red and tartrate-resistant acid phosphatase, respectively. We analyzed the expression of osteoblast- or osteoclast-related genes using real-time polymerase chain reaction and western blot.Results: AMH does not affect bone morphogenetic protein 2-mediated osteoblast differentiation but inhibits receptor activator of nuclear factor-κB (NF-κB) ligand-induced osteoclast differentiation. The inhibitory effect of AMH on osteoclast differentiation is mediated by IκB-NF-κB signaling.Conclusions: AMH negatively regulates osteoclast differentiation without affecting osteoblast differentiation.

scholarly journals Phloretin Suppresses Bone Morphogenetic Protein-2-Induced Osteoblastogenesis and Mineralization via Inhibition of Phosphatidylinositol 3-kinases/Akt Pathway

2019 ◽  
Vol 20 (10) ◽  
pp. 2481 ◽  
Author(s):  
Ayumu Takeno ◽  
Ippei Kanazawa ◽  
Ken-ichiro Tanaka ◽  
Masakazu Notsu ◽  
Toshitsugu Sugimoto
Keyword(s):  
Osteoblast Differentiation ◽  
Glucose Transporter ◽  
Binding Protein ◽  
Bone Morphogenetic Protein 2 ◽  
Akt Pathway ◽  
Akt Inhibitor ◽  
Differentiation Markers ◽  
Uptake Inhibition ◽  
Fatty Acid Binding ◽  
Phosphatidylinositol 3

Phloretin has pleiotropic effects, including glucose transporter (GLUT) inhibition. We previously showed that phloretin promoted adipogenesis of bone marrow stromal cell (BMSC) line ST2 independently of GLUT1 inhibition. This study investigated the effect of phloretin on osteoblastogenesis of ST2 cells and osteoblastic MC3T3-E1 cells. Treatment with 10 to 100 µM phloretin suppressed mineralization and expression of osteoblast differentiation markers, such as alkaline phosphatase (ALP), osteocalcin (OCN), type 1 collagen, runt-related transcription factor 2 (Runx2), and osterix (Osx), while increased adipogenic markers, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid-binding protein 4, and adiponectin. Phloretin also inhibited mineralization and decreased osteoblast differentiation markers of MC3T3-E1 cells. Phloretin suppressed phosphorylation of Akt in ST2 cells. In addition, treatment with a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor, LY294002, suppressed the mineralization and the expression of osteoblast differentiation markers other than ALP. GLUT1 silencing by siRNA did not affect mineralization, although it decreased the expression of OCN and increased the expression of ALP, Runx2, and Osx. The effects of GLUT1 silencing on osteoblast differentiation markers and mineralization were inconsistent with those of phloretin. Taken together, these findings suggest that phloretin suppressed osteoblastogenesis of ST2 and MC3T3-E1 cells by inhibiting the PI3K/Akt pathway, suggesting that the effects of phloretin may not be associated with glucose uptake inhibition.

C2C12 myoblast/osteoblast transdifferentiation steps enhanced by epigenetic inhibition of BMP2 endocytosis

2002 ◽  
Vol 283 (1) ◽  
pp. C235-C243 ◽  
Author(s):  
Cyril Rauch ◽  
Anne-Christine Brunet ◽  
Julie Deleule ◽  
Emmanuel Farge
Keyword(s):  
Osteoblast Differentiation ◽  
Nuclear Translocation ◽  
Bone Morphogenetic Protein 2 ◽  
C2c12 Myoblast ◽  
Differentiation Markers ◽  
Signaling Protein ◽  
Mechanical Deformations ◽  
Morphogenetic Protein ◽  
Cell Genome ◽  
Differentiation Marker

We investigated the modulation of critical transcriptional steps of C2C12myoblast/osteoblast transdifferentiation triggered by the bone morphogenetic protein 2 (BMP2) signaling protein, in response to epigenetic inhibition of the endocytotic internalization of exogenous BMP2. BMP2 endocytosis was inhibited chemically with polyethylene glycol-50 (PEG-Chol) and cyclodextrin and mechanically by mild hyposmotic treatment. BMP2-dependent nuclear translocation of the mother against Dpp (Smad1) transcription factor was ten times faster if BMP2 endocytosis was inhibited. Smad1-dependent expression of the JunB gene, the first transcriptional step in myoblast dedifferentiation, was increased by a factor of three to four. JunB-dependent levels of myogenin repression, one of the critical markers of terminal myoblastic differentiation, was amplified by a factor of three. Smad1-dependent levels of alkaline phosphatase expression, one of the C2C12 osteoblast differentiation markers, were 3.5 to 5 times higher. The same behavior was observed for osteopontin, the other C2C12 osteoblast differentiation marker. These results suggest that the cell genome could “sense” tissue mechanical deformations by mechanical inhibition of signaling protein endocytosis, thereby translating mechanical strains into transcription events involved in cell differentiation.

scholarly journals Activation of p38 Mitogen-Activated Protein Kinase Is Required for Osteoblast Differentiation

Endocrinology ◽  
2003 ◽  
Vol 144 (5) ◽  
pp. 2068-2074 ◽  
Author(s):  
Yuanyu Hu ◽  
Emily Chan ◽  
Sherry X. Wang ◽  
Baojie Li
Keyword(s):  
Bone Marrow ◽  
P38 Mapk ◽  
Osteoblast Differentiation ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Bone Morphogenetic Protein 2 ◽  
Differentiation Markers ◽  
Mineral Deposition ◽  
Calvarial Osteoblast

p38 MAPK is a conserved subfamily of MAPKs involved in inflammatory response, stress response, cell growth and survival, as well as differentiation of a variety of cell types. In this report we demonstrated that p38 MAPK played an important role in osteoblast differentiation using primary calvarial osteoblast, bone marrow osteoprecursor culture, and a murine cell line, MC3T3-E1. We found that p38 MAPK was activated as calvarial osteoblast differentiates along with extracellular signal-regulated kinases (ERKs). When p38 MAPK is inhibited with a specific inhibitor, the expression of differentiation markers, such as alkaline phosphatase and mineral deposition, were significantly reduced. MC3T3-E1 cells expressing dominant negative p38 MAPK also displayed signs of delay in ALP and mineral deposition. Differentiation of the bone marrow osteoprecursors was also impeded by the p38 MAPK inhibitor, justified by the same markers. Yet the inhibitory effects observed in calvarial osteoblasts and bone marrow osteoprogenitor cells could be partially prevailed by bone morphogenetic protein-2. Inhibition of ERKs with a specific drug did not significantly affect osteoblast differentiation even though ERK1/2 were also activated during osteoblast differentiation. These results taken together indicate that p38 MAPK, but not ERKs, is necessary for osteoblast differentiation.

scholarly journals Effect of Cyanobacteria (Nostoc species) Extracts on Osteogenesis Activities

2020 ◽  
Vol 17 (6) ◽  
pp. 620-630
Author(s):  
Phatthilakorn CHAMNANPUEN ◽  
Veena SATITPATIPAN ◽  
Sophon SIRISATTHA ◽  
Thanchanok MUANGMAN
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Differentiation ◽  
Chlorophyll A ◽  
Osteoblast Differentiation ◽  
Osteoblast Cell ◽  
Treatment Groups ◽  
Positive Effects ◽  
Alp Activity ◽  
Crude Extracts ◽  
Β Carotene

This study focused on osteogenesis activities from Cyanobacteria (Nostoc species), including cytotoxicity, cell proliferation, cell differentiation and cell mineralization with osteoblast cell (MC3T3-E1). The four Nostoc species; N. parmelioides, N punctiforme, N. muscorum and N. paludosum were made axenic and cultured for a month. The ethanolic crude extracts were obtained by maceration extraction; the yield was in the range of 7.00 - 10.00 % (w/w). By using the TLC technique, it was observed that in all of the samples, there were phytochemical bands corresponding with β-carotene and chlorophyll a which were used as standard compounds. As a result, all crude extracts had no toxic effect on osteoblast cells after being incubated for 24 and 48 h with various concentrations (1 - 1,000 µg/mL), although they did not significantly promote osteoblast proliferation. In addition, it was found that 3 crude extracts (N. parmelioides, N punctiforme, and N. paludosum) also promoted osteoblast differentiation significantly after being induced for 9 days. Similarly, there was no difference between the relative cellular mineralization among treatment groups after being induced for 25 days. The potential ethanolic extracts on osteoblast cell differentiation were further partitioned by liquid-liquid partition chromatography (hexane, dichloromethane and aqueous fractions), and were analyzed for osteoblast differentiation with alkaline phosphatase (ALP) activity. All of hexane and dichloromethane fractions from N. parmelioides (181.72±6.75 % and 195.11±3.27 %), N punctiforme (194.34±2.36 % and 168.69±1.08 %), and N. paludosum (191.54±7.66 % and 167.12±9.46 %) had a significantly higher effect of ALP activity than their aqueous fractions. In conclusion, our data suggested that the nonpolar potential major compound, which might correspond with carotenoids (β-carotene, xanthophyll and chlorophyll a) from Nostoc species, exhibited positive effects on osteoblast differentiation by increasing the alkaline phosphatase enzyme.

scholarly journals Bone Morphogenetic Protein 2-Induced Osteoblast Differentiation Requires Smad-Mediated Down-Regulation of Cdk6

2004 ◽  
Vol 24 (15) ◽  
pp. 6560-6568 ◽  
Author(s):  
Toru Ogasawara ◽  
Hiroshi Kawaguchi ◽  
Shigeki Jinno ◽  
Kazuto Hoshi ◽  
Keiji Itaka ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Differentiation ◽  
Bone Morphogenetic Protein ◽  
Transcriptional Repression ◽  
Osteoblast Differentiation ◽  
Inhibitory Effect ◽  
Bone Morphogenetic Protein 2 ◽  
Down Regulation ◽  
Potent Inducer ◽  
Morphogenetic Protein

ABSTRACT Because a temporal arrest in the G1 phase of the cell cycle is thought to be a prerequisite for cell differentiation, we investigated cell cycle factors that critically influence the differentiation of mouse osteoblastic MC3T3-E1 cells induced by bone morphogenetic protein 2 (BMP-2), a potent inducer of osteoblast differentiation. Of the G1 cell cycle factors examined, the expression of cyclin-dependent kinase 6 (Cdk6) was found to be strongly down-regulated by BMP-2/Smads signaling, mainly via transcriptional repression. The enforced expression of Cdk6 blocked BMP-2-induced osteoblast differentiation to various degrees, depending on the level of its overexpression. However, neither BMP-2 treatment nor Cdk6 overexpression significantly affected cell proliferation, suggesting that the inhibitory effect of Cdk6 on cell differentiation was exerted by a mechanism that is largely independent of its cell cycle regulation. These results indicate that Cdk6 is a critical regulator of BMP-2-induced osteoblast differentiation and that its Smads-mediated down-regulation is essential for efficient osteoblast differentiation.

scholarly journals Probiotic Propionibacterium freudenreichii MJ2 Enhances Osteoblast Differentiation and Mineralization by Increasing the OPG/RANKL Ratio

2021 ◽  
Vol 9 (4) ◽  
pp. 673
Author(s):  
Jiah Yeom ◽  
Seongho Ma ◽  
Young-Hee Lim
Keyword(s):  
Signaling Pathway ◽  
Osteoblast Differentiation ◽  
Surface Proteins ◽  
Bone Morphogenetic Protein 2 ◽  
Ovariectomized Rats ◽  
Osteoblastic Cell ◽  
Enhancement Effect ◽  
Propionibacterium Freudenreichii ◽  
Alizarin Red ◽  
Osteoblastic Cell Line

Osteoblast differentiation is important for the development of bone and the maintenance of bone density. Propionibacterium freudenreichii is a probiotic with an anti-inflammatory property. The aim of this study was to investigate the enhancement effect of P. freudenreichii MJ2 (MJ2) isolated from raw milk on osteoblast differentiation, mineralization, and its signaling pathway. For in vitro and in vivo experiments, human fetal osteoblastic cell line hFOB 1.19 and an ovariectomized rat model were used, respectively. Expression levels of genes and proteins related to osteoblast differentiation and mineralization were measured by real-time polymerase chain reaction (qPCR) and Western blotting, respectively. Alizarin red S staining was performed to measure osteoblast mineralization. Heat-killed MJ2 (hkMJ2)-treated cells showed significantly increased osteoblast differentiation via an increase in the osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL) ratio and significantly increased osteoblast mineralization by stimulating the expression of bone morphogenetic protein 2 and runt-related transcription factor 2. Additionally, oral administration of live or heat-killed MJ2 to ovariectomized rats inhibited osteoporosis-induced bone loss. Specifically, surface proteins isolated from MJ2 promoted osteoblast differentiation and mineralization. In conclusion, MJ2 enhanced osteoblast differentiation and mineralization through the OPG/RANKL signaling pathway and the effective component of MJ2 might be its surface proteins.

scholarly journals Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway

2015 ◽  
Vol 112 (41) ◽  
pp. 12711-12716 ◽  
Author(s):  
Andrea M. Brum ◽  
Jeroen van de Peppel ◽  
Cindy S. van der Leije ◽  
Marijke Schreuders-Koedam ◽  
Marco Eijken ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Osteoblast Differentiation ◽  
Target Genes ◽  
Focal Adhesions ◽  
Bone Fragility ◽  
Bone Morphogenetic Protein 2 ◽  
Marker Genes ◽  
Connectivity Map ◽  
Morphogenetic Protein

Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs). Gene expression profiling was performed in hMSCs differentiated toward osteoblasts (at 6 h). Significantly regulated genes were analyzed in silico, and the Connectivity Map (CMap) was used to identify candidate bone stimulatory compounds. The signature of parbendazole matches the expression changes observed for osteogenic hMSCs. Parbendazole stimulates osteoblast differentiation as indicated by increased alkaline phosphatase activity, mineralization, and up-regulation of bone marker genes (alkaline phosphatase/ALPL, osteopontin/SPP1, and bone sialoprotein II/IBSP) in a subset of the hMSC population resistant to the apoptotic effects of parbendazole. These osteogenic effects are independent of glucocorticoids because parbendazole does not up-regulate glucocorticoid receptor (GR) target genes and is not inhibited by the GR antagonist mifepristone. Parbendazole causes profound cytoskeletal changes including degradation of microtubules and increased focal adhesions. Stabilization of microtubules by pretreatment with Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Cotreatment with the BMP-2 antagonist DMH1 limits, but does not block, parbendazole-induced mineralization. Using the CMap we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteogenic differentiation through a combination of cytoskeletal changes and increased BMP-2 activity.

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