• Journal of the Korean Society of Food Culture
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• v.37 no.3
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• pp.248-255
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• 2022

Spinach (Spinacia oleracea L.), a green leafy vegetable, is well known as a functional food due to its biological activities. Vascular calcification is associated with several disease conditions including atherosclerosis, diabetes, and chronic kidney disease (CKD), and is known to raise the risk of cardiovascular diseases related morbidity and mortality. However, there are no previous studies that have investigated the effects of fermented spinach exract (FSE) against aortic and its underlying mechanisms. Therefore, this study investigated the effects and action of possible mechanisms of FSE on inorganic phosphate (PI)-induced vascular calcification in ex vivo mouse aortic rings. PI increased vascular calcification through calcium deposition in ex vivo aortic rings. FSE inhibited calcium accumulation and osteogenic key marker, runt-related transcription factor 2 (Runx2), and bone Morphogenetic Protein 2 (BMP-2) protein expression in ex vivo aortic rings. And, FSE inhibited PI-induced extracellular signal-regulated kinase (ERK) and p38 phosphorylation in ex vivo aortic rings. These results show that FSE can prevent vascular calcification which may be a crucial way for the prevention and treatment of vascular disease association with vascular calcification.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.34 no.4
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• pp.419-427
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• 2008

The present study aimed to investigate the osteogenic potentials of differentiated osteoblast-like cells (DOCs) induced from bone marrow-derived mesenchymal stem cells (MSCs) on ${\beta}-tricalcium$ phosphate (${\beta}-TCP$) with recombinant human bone morphogenetic protein (rhBMP-2) in vitro. Osteoblast differentiation was induced in confluent cultures by adding 100 nM dexamethasone, 10 mM ${\beta}$-glycerophosphate, 50 mM L-ascorbic acid. The Alizarin red S staining and reverse transcriptase-polymerase chain reaction (RT-PCR) were perfomed to examine the mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), receptor activator for nuclear factor ${\kappa}B$ ligand (RANKL), runt-related transcription factor 2 (RUNX2), collagen-Ⅰ (COL-Ⅰ). There were no significant differences in the osteogenic potentials of DOCs induced from MSCs on ${\beta}-TCP(+/-)$. According to the incubation period, there were significant increasing of Alizadin red S staining in the induction 3 weeks. The mRNA expression of ALP, RUNX2, and RANKL were higher in DOCs/${\beta}-TCP(-)$ than DOCs/${\beta}-TCP(+)$. According to rhBMP-2 concentrations, the mRNA expression of BSP was significantly increased in DOCs/${\beta}-TCP(+)$ compared to that of DOCs/${\beta}-TCP(-)$ on rhBMP 10 ng/ml. Our study presented the ${\beta}-TCP$ will have the possibility that calcium phosphate directly affect the osteoblastic differentiation of the bone marrowderived MSCs.

• CELLMED
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• v.8 no.3
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• pp.13.1-13.8
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• 2018

In the field of osteoporosis, there has been growing interest in anabolic agents that enhance bone formation. The purpose of this study was to examine the effects of NNMBS 246 osteoblastic differentiation with associated signaling pathways. NNMBS 246 markedly increased alkaline phosphatase (ALP) activity and calcium nodule formation. Stimulation with NNMBS 246 not only increased the differentiation markers (ALP, OPN, OCN) level and transcription markers (RUNX2, Osterix) mRNA expression but also upregulated the ECM molecules and OPG mRNA expression. Treatments of NNMBS 246 downregulated MMPs (MMP-1, MMP-2, MMP-9), but RANKL mRNA expression. Furthermore, NNMBS 246 activated osteoblastic differentiation markers and formed calcium nodules in human periodontal ligament cells (hPDLCs) and cementoblast cells. NNMBS 246 induced phosphorylation of MAPKs, Akt, nuclear p65 and IkB-${\alpha}$. BMP-2/Smad and ${\beta}$-catenin signaling pathways were activated by NNMBS 246. Sirtinol (SIRT1 inhibitor) inhibited NNMBS 246-induced osteoblastic differentiation markers mRNA expression. These results suggested that NNMBS 246 has the potential to enhance osteoblastogenesis probably through the activation of BMP/Smad and ${\beta}$-catenin signal pathways, and SIRT1 plays as critical mediator in bone anabolic effect of NNMBS 246.

• The Journal of Korean Academy of Prosthodontics
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• v.46 no.3
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• pp.227-237
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• 2008

STATEMENT OF PROBLEM: Zirconium oxide can be a substitute to titanium as implant materials to solve the esthetic problems of dark color in the gingival portion of implant restorations. PURPOSE: This study was performed to define attachment and growth behavior of osteoblast- like cells cultured on grooved surfaces of zirconium oxide and evaluate the genetic effect of zirconium oxide surfaces using the reverse transcriptase-polymerase chain reaction (RT-PCR). MATERIAL AND METHODS: MC3T3-E1 cells were cultured on (1) commercially pure titanium discs with smooth surface (T group), (2) yttrium-stabilized tetragonal zirconia polycrystal (Y-TZP) with machined surface (ZS group), and (3) Y-TZP with $100{\mu}m$ grooves (ZG group). Cell proliferation activity was evaluated through MTT assay and cell morphology was examined by SEM. The mRNA expression of Runx2, alkaline phosphatase, osteocalcin, TGF-${\beta}1$, IGF-1, G3PDH in E1 cells were evaluated by RT-PCR. RESULTS: From the MTT assay, after 48 hours of adhesion of MC3T3-E1 cells, the mean optical density value of T group and ZG group significantly increased compared to the ZS group. SEM images of osteoblast-like cells showed that significantly more cells were observed to attach to the grooves and appeared to follow the direction of the grooves. After 24 hours of cell adhesion, more spreading and flattening of cells with active filopodia formation occurred. Results of RT-PCR suggest that T group, ZS group, and ZG group showed comparable osteoblast-specific gene expression after 24 hours of cell incubation. CONCLUSION: Surface topography and material of implants can play an important role in expression of osteoblast phenotype markers. Zirconia ceramic showed comparable biological responses of osteoblast-like cells with titanium during a short-time cell culture period. Also, grooves influence cell spreading and guide the cells to be aligned within surface grooves.

• Tissue Engineering and Regenerative Medicine
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• v.15 no.6
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• pp.793-801
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• 2018

BACKGROUND: The aim of this study was to evaluate the combined effect of low-level laser treatment (LLLT) and recombinant human bone morphological protein-2 (rhBMP-2) applied to hypoxic-cultured MC3T3-E1 osteoblastic cells and to determine possible signaling pathways underlying differentiation and mineralization of osteoblasts under hypoxia. METHODS: MC3T3-E1 cells were cultured under 1% oxygen tension for 72 h. Cell cultures were divided into four groups: normoxia control, low-level laser (LLL) alone, rhBMP-2 combined with LLLT, and rhBMP-2 under hypoxia. Laser irradiation was applied at 0, 24, and 48 h. Cells were treated with rhBMP-2 at 50 ng/mL. Alkaline phosphatase activity was measured at 3, 7, and 14 days to evaluate osteoblastic differentiation. Cell mineralization was determined with Alizarin red S staining at 7 and 14 days. Western blot assays were performed to evaluate whether p38/protein kinase D (PKD) signaling was involved. RESULTS: The results indicate that LLLT and rhBMP-2 synergistically increased alkaline phosphatase (ALP) activity and mineralization. Western blot analyses showed that expression of type I collagen, runt-related transcription factor 2 (RUNX2), and Osterix (Osx), increased and expression of hypoxia-inducible factor 1-alpha ($HIF-1{\alpha}$), decreased more in the LLLT and rhBMP-2 combined group than in the rhBMP-2 or LLL alone groups. Moreover, LLLT and rhBMP-2 stimulated p38 phosphorylation and rhBMP-2 and LLLT increased Prkd1 phosphorylation. CONCLUSION: Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxic-cultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.

• Journal of Periodontal and Implant Science
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• v.42 no.3
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• pp.95-104
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• 2012

Purpose: The purpose of this study was to determine whether increasing the Ti6Al4V surface oxide negative charge through heat ($600^{\circ}C$) or radiofrequency plasma glow discharge (RFGD) pretreatment, with or without a subsequent coating with fibronectin, stimulated osteoblast gene marker expression in the MC3T3 osteoprogenitor cell line. Methods: Quantitative real-time polymerase chain reaction was used to measure changes over time in the mRNA levels for osteoblast gene markers, including alkaline phosphatase, bone sialoprotein, collagen type I (${\alpha}1$), osteocalcin, osteopontin and parathyroid hormone-related peptide (PTH-rP), and the osteoblast precursor genes Runx2 and osterix. Results: Osteoprogenitors began to differentiate earlier on disks that were pretreated with heat or RFGD. The pretreatments increased gene marker expression in the absence of a fibronectin coating. However, pretreatments increased osteoblast gene expression for fibronectin-coated disks more than uncoated disks, suggesting a surface oxide-mediated specific enhancement of fibronectin's bioactivity. Heat pretreatment had greater effects on the mRNA expression of genes for PTH-rP, alkaline phosphatase and osteocalcin while RFGD pretreatment had greater effects on osteopontin and bone sialoprotein gene expression. Conclusions: The results suggest that heat and RFGD pretreatments of the Ti6Al4V surface oxide stimulated osteoblast differentiation through an enhancement of (a) coated fibronectin's bioactivity and (b) the bioactivities of other serum or matrix proteins. The quantitative differences in the effects of the two pretreatments on osteoblast gene marker expression may have arisen from the unique physico-chemical characteristics of each resultant oxide surface. Therefore, engineering the Ti6Al4V surface oxide to become more negatively charged can be used to accelerate osteoblast differentiation through fibronectin-dependent and independent mechanisms.

• Journal of the Korean Society of Food Science and Nutrition
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• v.42 no.7
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• pp.1015-1021
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• 2013

Glycyrrhiza inflata Batal, an important species of licorice, is one of the most widely used medicinal plants for over 4000 years. Glycyrrhiza plant species has been well known for its various therapeutic activities such as anti-inflammatory, anti-allergic, and anti-ulcer. The purpose of this study was to determine the effects of Glycyrrhiza inflata Batal ethanol extracts (GBE) on adipocyte and osteoblast differentiation. Mesenchymal C3H10T1/2 cells were treated with sub-cytotoxic doses of GBE, and its effects on adipocyte differentiation were assessed. We found that GBE dose-dependently increased lipid accumulation and also induced the expression of adipocyte markers, such as $PPAR{\gamma}$ and its target genes, aP2, and adiponectin, in C3H10T1/2 cells. Consistently, similar effects of GBE on lipid accumulation were also observed in preadipocyte 3T3-L1 cells that further supports the pro-adipogenic activities of GBE. We also investigated the effects of GBE on osteoblast differentiation of mesenchymal C3H10T1/2 cells. As a results, we found that GBE increased the activity of alkaline phosphatase in a dose-dependent manner and also promoted the expression of osteoblast markers, such as ALP and RUNX2, during osteoblast differentiation of C3H10T1/2 cells. Similar pro-osteogenic effects of GBE were also observed in preosteoblast MC3T3-E1 cells. Finally, our data show that a major bioactive compound found in Glycyrrhiza inflata Batal, licochalcone A (LA) but not glycyrrhizic acid (GA), can mediate the pro-adipogenic and pro-osteogenic effects of GBE. Taken together, this study provides data to show the possibility of GBE and its bioactive component LA as putative strategies for type 2 diabetes and bone diseases.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.27 no.5
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• pp.424-430
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• 2005

The oral cavity is humid environment mainly due to the continuous salivary flow. The reaction of oral mucosa to fluid flow is important for homeostasis and pathogenesis. The objective of this study is the screening the change of gene expression after the application of fluid induced shear stress (FISS) on the gingival fibroblast using cDNA microarray assay. The immortalized human gingival fibroblasts were grown and FISS was applied using a cone viscometer at a rotational velocity of 40 rpm, respectively for periods of 2 and 4 hours. The synthesis of cDNA was done from the extracted total RNA and cDNA microarray assay was done subsequently. The genes that showed over 1.6 in the Cy3/Cy5 or the Cy5/Cy3 value were regarded as genes influenced significantly by the FISS application ion (/M/>0.7). The " RUNX-1" was increased its expression in 2 hours group and " RUN and SH3 domain containing 1" was increased its expression in 4 hours group. The "CC020415", "cyclin L1", "interferon regulatory factor1", "early growth response 1", "immediate early response 2", and "immediate early response 3" genes were increased their expression in 2 and 4 hours after FISS application. In conclusion, we could find many genes that were probably related to the FISS application. Interestingly, most of them were placed in similar molecular pathways and these findings improve the reliability of chip data and usefulness in overall screening. From this experiment, we could find many items for further study and it will make improvement in the understanding of intracellular events in response to FISS.

• Molecules and Cells
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• v.43 no.1
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• pp.58-65
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• 2020

Fat mass and obesity-associated (FTO) gene helps to regulate energy homeostasis in mammals by controlling energy expenditure. In addition, FTO functions in the regulation of obesity and adipogenic differentiation; however, a role in osteogenic differentiation is unknown. This study investigated the effects of FTO on osteogenic differentiation of C3H10T1/2 cells and the underlying mechanism. Expression of osteogenic and endoplasmic reticulum (ER) stress markers were characterized by reverse-transcriptase polymerase chain reaction and western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity. BMP2 treatment increased mRNA expression of osteogenic genes and FTO. Overexpression of FTO increased expression of the osteogenic genes distal-less homeobox5 (Dlx5) and runt-related transcription factor 2 (Runx2). Activation of adenosine monophosphate-activated protein kinase (AMPK) increased FTO expression, and there was a positive feedback loop between FTO and p-AMPK. p-AMPK and FTO induced mild ER stress; however, tunicamycin-induced severe ER stress suppressed FTO expression and AMPK activation. In summary, FTO induces osteogenic differentiation of C3H10T1/2 cells upon BMP2 treatment by inducing mild ER stress via a positive feedback loop with p-AMPK. FTO expression and AMPK activation induce mild ER stress. By contrast, severe ER stress inhibits osteogenic differentiation by suppressing FTO expression and AMPK activation.

• Advances in nano research
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• v.12 no.3
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• pp.301-317
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• 2022

Many studies have shown that Mg-Nd-Zn-Zr (abbreviated as JDBM) alloy has good biocompatibility and biodegradability as well as promotion of cell adhesion, proliferation and differentiation, and Wnt/β-catenin signaling pathway may play a unique role in joint tissue by controlling the function of chondrocytes, osteoblasts and synoviocytes. However, it is not clear whether the JDBM alloy induces osteochondral repair through Wnt/β-catenin signaling pathway. This study aims to verify that JDBM alloy can repair osteochondral defects in rats, which is realized by Wnt/β-catenin signaling pathway. In this study, the osteochondral defect model of the right femoral condyle non-weight-bearing area in rats was established and randomly divided into three groups: Control group, JDBM alloy implantation group and JDBM alloy implantation combined with signaling pathway inhibitor drug ICRT3 injection. It was found that after JDBM alloy implantation, the bone volume fraction (BVF) became larger, the bone trabeculae were increased, the relative expression of osteogenesis gene Runx2, Bmp2, Opn, Ocn and chondrogenesis gene Collagen II, Aggrecan were increased, and the tissue repair was obvious by HE and Masson staining, which could be inhibited by ICRT3.