• Animal cells and systems
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• v.4 no.3
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• pp.273-278
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• 2000

Transforming growth factor-$\beta$ (IGF-$\beta$)s are multifunctional small polypeptides synthesized in most cell types. TGF-$\beta$ exerts pivotal effects on both bone formation and resorption. In addition, increasing lines of evidence implicate TGF-$\beta$ as a potential coupling factor between these two processes during bone remodeling. In the present study, the expression form and the activation mechanism of latent-TGF-$\beta$ were investigated using specific antibodies for each isoform. TGF-$\beta$s were observed to be synthesized and accumulated in a large amount in cultured osteoblastic cells. The estimated molecular weights of intracellular TGF-$\beta$2 and -$\beta$3 were 49 and 55 kDa, respectively. Based on proteolytic digestion study and immunofluorescence observation, these precursor forms seemed to be accumulated in distinct intracellular compartments. To examine whether the internal pool of TGF-$\beta$ was possiblely regulated by external signals, their biological activites were examined in a conditioned media of this cell. Although the intact conditioned media did not contain detectable TGF-$\beta$ activity, heat-treatment or acid-activation of the conditioned media revealed significant TGF-$\beta$ activity. Furthermore, in the presence of estrogen, this activity was dramatically diminished. It is known that activation of latent TGF-$\beta$ can be achieved by different chemical and enzymatic treatments, or by incubation with certain cell types. This extracellular activation was suggested as a key step in the regulation of TGF-$\beta$ activity. In addition to these extracellular activation, this study suggests that the synthesis and intracellular processing are important regulation steps for TGF-$\beta$ action. In addition, this regulation Is specific for TGF-$\beta$ type 2, because the change was not observed in TGF-$\beta$3 in osteoblastic cell line.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.6
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• pp.494-498
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• 2007

Pleiotrophin or osteoblast-specific factor 1(HOSF-1) is a growth-associated protein present in bone matrix. This study was designed to study pleiotrophin expression in osteoblastic cells. Pleiotrophin was expressed by osteoblast-like cell line. Pleiotrophin expression increased following the proliferative phase and was minimal at the terminal phases of the induced differentiation of cultured MC3T3-E1 cells. Pleiotrophin expression represents another autocrine factor that may contribute to the physiologic control of induced bone formation. In this study, induced osteogenesis will be examined in the context of the osteoblast expression of and regulation by PTN. I hypothesized that PDGF-BB stimulation of PTN expression represents an important paracrine signal during the induced osteogenesis associated with periodontal and implant surgeries. The possible mediation by PTN of anabolic effects attributed to PDGF-BB stimulation was examined in cell culture models of osteoblast differentiation. These studies will contribute fundamental insights to osteoblast biology and insights regarding the potential use of factors such as PTN in the clinical environment.

• International Journal of Oral Biology
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• v.34 no.1
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• pp.37-42
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• 2009

The attachment and adhesion of RAW 264.7 and MC3T3-E1 cells to titanium (Ti) discs with various degrees of roughness was investigated. The attachment, adhesion, and proliferation of these cells were evaluated after 4 hr, 24 hr and 7 day incubations. Both RAW 264.7 and MC3T3-E1 cells showed a time-dependant correlation between attachment and adhesion on the surface of the titanium discs. Both types of cells tended to have higher survival rate on these discs as the surface roughness increased. The percentage of adherent inflammatory RAW 264.7 cells was greater than MC3T3-E1 cells at 24 hr, but this was reversed at 7 days in culture. The morphology of osteoblastic MC3T3-E1 cells at 24 hr, determined using a surface emission microscope (SEM), appeared flattened and spread out while inflammatory RAW 264.7 cells were predominantly spherical in shape. The adhesion of both cell types on the titanium discs was dependant on the levels of fibronectin adsorbed on the disc surface, indicating that serum constituents modulate the efficient adhesion of these cells. Our data indicate that the cellular response to the titanium surface is dependent on the types of cells, surface roughness and serum constituents.

• International Journal of Oral Biology
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• v.38 no.3
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• pp.121-126
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• 2013

Tumor necrosis factor alpha ($TNF{\alpha}$) is a multifunctional inflammatory cytokine that regulates various cellular and biological processes. Increased levels of $TNF{\alpha}$ have been implicated in a number of human diseases including diabetes and arthritis. Sympathetic nervous system stimulation via the beta2-adrenergic receptor (${\beta}2AR$) in osteoblasts suppresses osteogenic activity. We previously reported that $TNF{\alpha}$ upregulates ${\beta}2AR$ expression in murine osteoblastic cells and that this modulation is associated with $TNF{\alpha}$ inhibition of osteoblast differentiation. In our present study, we explored whether $TNF{\alpha}$ induces ${\beta}2AR$ expression in human osteoblasts and then identified the downstream signaling pathway. Our results indicated that ${\beta}2AR$ expression was increased in Saos-2 and C2C12 cells by $TNF{\alpha}$ treatment, and that this increase was blocked by the inhibition of NF-${\kappa}B$ activation. Chromatin immunoprecipitation and luciferase reporter assay results indicated that NF-${\kappa}B$ directly binds to its cognate elements on the ${\beta}2AR$ promoter and thereby stimulates ${\beta}2AR$ expression. These findings suggest that the activation of $TNF{\alpha}$ signaling in osteoblastic cells leads to an upregulation of ${\beta}2AR$ and also that $TNF{\alpha}$ induces ${\beta}2AR$ expression in an NF-${\kappa}B$-dependent manner.

• Imaging Science in Dentistry
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• v.33 no.1
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• pp.51-57
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• 2003

Purpose: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-El osteoblastic cell line, particularly an the expression of type I collagen and alkaline phosphatase mRNA. Materials and Methods: Cells were irradiated with a single dose of 0.5, 1, 2, 4, and 8 Gy at a dose rate of 5.38 Gy/min using a cesium 137 irradiator. The specimens were then harvested and RNA extraction was carried out at 1 and 3 days after irradiation. The extracted RNA strands were reverse-transcribed and the resulting cDNA fragments were amplified by PCR. Results: The irradiated cells demonstrated a dose-dependent increase in type I collagen mRNA expression relative to the control group, with a maximum level of type I collagen mRNA expression occurring at 8 Gy. The degree of type I collagen mRNA expression increased significantly at 1 day after irradiation, but little differences were found between the control group and at the 3rd day. The amount of alkaline phosphatase mRNA expression increased significantly at land 3 days after irradiation in the 1 Gy exposed group compared with the control group. Conclusion: The amount of type I collagen and alkaline phosphatase mRNA expression increased significantly 1 day after irradiation when compared with the control group.

• Journal of Acupuncture Research
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• v.22 no.2
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• pp.181-190
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• 2005

Background & Object : The differentiation of osteoblasts controlled by various growth factors and matrix proteins expression in bone. The aim of this study was to identify the Astragalus membranaceus that may induce the osteogenic activity in human osteoblast-like SaOS-2 cells. Methods : The osteogenic activity of Astragalus membranaceus were evaluated by WST-8 assay, ALP activity, RT-PCR analysis of VEGF, OCN, OPN, Col I mRNA, and ELISA or colorimetric analysis, and mineralization by Alizarin red staining in SaOS-2 cells. Results : Astragalus membranaceus had no effect on viability of osteoblastic cells, and dose dependently increased alkaline phosphatase (ALP) activity. Astragalus membranaceus markedly increased mRNA expression for vascular endothelial growth factor (VEGF), osteocalcin (OCN), osteopontin (OPN), and type I collagen (Col 1) in SaOS-2 cells. Extracellular accumulation of proteins such as VEGF, and Col I was increased in a dose-dependent manner. Also, Astragalus membranaceus significantly induced mineralization in the culture of SaOS-2 cells. Conclusion : This study showed that Astragalus membranaceus not affect on viability, but it enhanced ALP activity, VEGF, bone matrix proteins such as OCN, OPN and Col I, and mineralization in SaOS-2 cells. These results propose that Astragalus membranaceus plays an important role in osteoblastic bone formation, and possibly lead to the development of bone-forming drug.

• International Journal of Oral Biology
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• v.39 no.2
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• pp.121-128
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• 2014

Sambucus sieboldiana (SS) is a member of the family Caprifoliaceae and has been recommended as a functional material because of its several bioactivities. Although numerous literatures are available on the pharmacological and biological activities, the biological activity of SS in bone regeneration process has not yet been well-defined. Therefore, in this study, the effect of SS was investigated in the proliferation and differentiation of MC3T3-E1 osteoblastic cell line. The treatment of SS did not significantly affect the cell proliferation in MC3T3-E1 cells. SS significantly accelerated the mineralization and significantly increased the expression of alkaline phosphatase (ALP) and osteocalcin (OC) mRNAs, compared to the control, in the differentiation of MC3T3-E1 cells. SS significantly accelerated the decrease of osteonectin (ON) mRNA expression as compared with the control in a time-dependent manner in the differentiation of MC3T3-E1 cells. These results suggest that the SS facilitate the osteoblast differentiation and mineralization in MC3T3-E1 osteoblastic cells. Therefore, there may be potential properties for development and clinical application of bone regeneration materials.

• Journal of Periodontal and Implant Science
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• v.36 no.2
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• pp.449-459
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• 2006

DFDBA(Decalcified freeze-dried bone allograft) is one of the allograft materials for periodontal bone regeneration. DFDBA provides an osteoconductive surface and osteoinductive factors. Therefore, DFDBA have been used successfully to regenerate the attachment apparatus during periodontal treatment. But recent studies was reported that wide variations in commercial bone bank preparations of DFDBA do exist, including the ability to induce new bone formation. DFDBA was experimental materials that was recovered, processed, tested, shipped and invoiced through Musculoskeletal Transplant Foundation. MTF(Musculoskeletal Transplant Foundation) is the world largest, non-profit, AATB(American Association of Tissue Banks) accredited tissue bank. The objective of this study was to determine the effects of serial dilutions of a DFDBA on human fetal osteoblastic cell proliferation and their potential to form and mineralize bone nodules. Human fetal osteoblastic cell line(hFOB 1.19) was cultured with DMEM and SSE($1{\mu}g/m{\ell}$,$10{\mu}g/m{\ell}$, $100{\mu}g/m{\ell}$, $1mg/m{\ell}$) at $34^{\circ}C$ with 5% CO2 in 100% humidity. Cell proliferation was significantly increased at $1mg/m{\ell}$, $100{\mu}g$, $10{\mu}g/m{\ell}$, $1{\mu}g/m{\ell}$, $100ng/m{\ell}$, $10ng/m{\ell}$, $1ng/m{\ell}$ of DFDBA after 5 days incubation (p<0.05). Alkaline Phosphatase(ALP) level was significantly increased in $100ng/m{\ell}$, $10ng/m{\ell}$, $1ng/m{\ell}$ of DFDABA(p<0.05). A quantified calcium accumulation was significantly increased at $1ng/m{\ell}$, $10ng/m{\ell}$ of MTF(p<0.05). These results indicated that DFDBA has an inductive effect on bone formation in vitro.

• Preventive Nutrition and Food Science
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• v.10 no.4
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• pp.353-356
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• 2005

It has been recently reported that boron affects bone metabolism in humans and animals. In this study we examined whether boron affects the proliferation on various cell types, MG-63, HOS, Raw 264.7 and SK-N-SH. When treated with different concentrations of boron $(1,\;10,\;100{\mu}M)$ for 24 and 48 hr, the proliferation of MG-63 cells was enhanced at $10{\mu}M\;(p<0.05)$, for 24 hr. In HOS cells, boron had no effect on cell proliferation at 24 or 48 hr. In addition, treatment of pre-osteoclastic cells (Raw 264.7) with 1, 10, $100{\mu}M$ boron resulted in no effect on cell proliferation. Proliferation of neuronal cells (SK-N-SH) was enhanced by boron in a concentration dependent manner at low concentrations (0.1, 0.5, $1{\mu}M$). Besides proliferation activity, boron has an effect on the enhancement of NO production in SK-N-SH cells in a concentration-dependent manner. These studies showed that boron enhances proliferation of osteoblastic cells (especially MG-63), depending upon the concentration of boron. These results also provide further evidence of the positive effects of boron in neuronal disease.

• Journal of Nutrition and Health
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• v.51 no.1
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• pp.23-30
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• 2018

Purpose: Runx2 (runt-related transcription factor 2), a bone-specific transcription factor, is a key regulator of osteoblast differentiation and its expression is induced by the activation of BMP-2 signaling. This study examined whether zinc modulates BMP-2 signaling and therefore stimulates Runx2 and osteoblast differentiation gene expression. Methods: Two osteoblastic MC3T3-E1 cell lines (subclones 4 as a high osteoblast differentiation and subclone 24 as a low osteoblastic differentiation) were cultured in an osteogenic medium (OSM) as the normal control, Zn-($1{\mu}M$ Zn) or Zn+($15{\mu}M$ Zn) for 24 h. The genes and proteins for BMP-2 signaling (BMP-2, Smad-1/p-Smad-1), transcription factors (Runx2, osterix), and osteoblast differentiation marker proteins were assessed. Results: In both cell lines, BMP-2 mRAN and protein expression and extracellular BMP-2 secretion all decreased in Zn-. The expression of Smad-1 (downstream regulator of BMP-2 signaling) and p-Smad-1 (phosphorylated Smad-1) also downregulated in Zn-. Furthermore, the expression of the bone-specific transcription factors, Runx2 and osterix, decreased in Zn-, which might be due to the decreased BMP-2 expression and Smad-1 activation (p-Smad-1) by Zn-, because Runx2 and osterix both are downstream in BMP-2 signaling. Bone marker gene expression, such as alkaline phosphatase (ALP), collagen type I (COLI), osteocalcin, and osteopontin were also downregulated in Zn-. Conclusion: The results suggest that a zinc deficiency in osteoblasts suppresses the BMP-2 signaling pathway via the suppression of Smad-1 activation, and this suppressed BMP-2 signaling can cause poor osteoblast differentiation.