• International Journal of Oral Biology
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• v.30 no.2
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• pp.47-57
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• 2005

Leptin, the product of the obese gene, is a circulating hormone secreted primarily from adipocytes. Several results suggest that leptin is important mediators of bone metabolism. The present study was undertaken to determine the effects of leptin on anti-osteoclastogenesis using murine precursors cultured on Ca-P coated plates and on the production of osteoprotegerin (OPG) in osteoblastic cells. Additionally, this study examined the possible involvement of prostaglandin $E_2\;(PGE_2)$/protein kinase C (PKC)-mediated signals on the effect of leptin on anti-osteoclastogenesis to various culture systems of osteoclast precursors. Osteoclast generation was determined by counting tartrate-resistant acid phosphatase positive [TRAP (+)] multinucleated cells (MNCs). Osteoclastic activity was determined by measuring area of resorption pits formed by osteoclasts on Ca-P coated plate. The number of 1,25-dihydroxycholecalciferol $(1,25[OH]_2D_3)$- or $PGE_2$-induced TRAP (+) MNCs in the mouse bone marrow cell culture decreased significantly after treatment with leptin. The number of receptor activator of NF-kB ligand (RANKL)-induced TRAP (+) MNCs in M-CSF dependent bone marrow macrophage (MDBM) cell or RAW264.7 cell culture decreased significantly with leptin treatment. Indomethacin inhibited osteoclast generation induced by $1,25[OH]_2D_3$ and dexamethasone, however, no significant differences were found in the leptin treated group when compared to the corresponding indomethacin group. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, inhibited osteoclast generation induced by $1,25[OH]_2D_3$. The number of TRAP (+) MNCs decreased significantly with treatment by PMA at concentrations of 0.01 and $0.1{\mu}M$ in culture. Leptin inhibited PMA-mediated osteoclast generation. Isoquinoline-5-sulfonic 2-methyl-1-piperazide dihydrochloride (H7) had no effect on osteoclast generation induced by $1,25[OH]_2D_3$. Cell culture treatment with leptin resulted in no significant differences in osteoclast generation compared to the corresponding H7 group. Indomethacin showed no significant effect on TRAP (+) MNCs formation from the RAW264.7 cell line. PMA inhibited TRAP (+) MNCs formation induced by RANKL in the RAW264.7 cell culture. H7 had no effect on osteoclast generation from the RAW264.7 cell line. There was no difference compared with the corresponding control group after treatment with leptin. $1,25[OH]_2D_3$- or $PGE_2$-induced osteoclastic activity decreased significantly with leptin treatment at a concentration of 100 ng/ml in mouse bone marrow cell culture. Indomethacin, PMA, and H7 significantly inhibited osteoclastic activity induced by $1,25[OH]_2D_3$ in mouse bone marrow cell culture. No significant differences were found between the leptin treated group and the corresponding control group. The secretion of OPG, a substance known to inhibit osteoclast formation, was detected from the osteoblasts. Treatment by leptin resulted in significant increases in OPG secretion by osteoblastic cells. Taken these results, leptin may be an important regulatory cytokines within the bone marrow microenvironment.

• 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.

• Proceedings of the Korean Vacuum Society Conference
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• 2011.02a
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• pp.20-20
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• 2011

It has been known that, under certain conditions, application of low-temperature atmospheric-pressure plasmas can enhance proliferation of cells. In this study, conditions for optimal cell proliferation were examined for various cells relevant for orthopaedic applications. Plasmas used in our experiments were generated by dielectric barrier discharge (DBD) with a helium flow (of approximately 3 litter/min) into ambient air at atmospheric pressure by a 10 kV~20 kHz power supply. Such plasmas were directly applied to a medium, in which cells of interest were cultured. The cells examined in this study were human synoviocytes, rat mesenchymal stem cells derived from bone marrow or adipose tissue, a mouse osteoblastic cell line (MC3T3-E1), a mouse embryonic mesenchymal cell line (C3H-10T1/2), human osteosarcoma cells (HOS), a mouse myoblast cell line (C2C12), and rat Schwann cells. Since cell proliferation can be enhanced even if the cells are not directly exposed to plasmas but cultured in a medium that is pre-treated by plasma application, it is surmised that long-life free radicals generated in the medium by plasma application stimulate cell proliferation if their densities are appropriate. The level of free radical generation in the medium was examined by dROMs tests and correlation between cell proliferation and oxidative stress was observed. Other applications of plasma medicine in orthopaedics, such as plasma modification of artificial bones and wound healing effects by direct plasma application for mouse models, will be also discussed. The work has been done in collaboration with Prof. H. Yoshikawa and his group members at the School of Medicine, Osaka University.

• 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.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.

• Journal of Periodontal and Implant Science
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• v.35 no.2
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• pp.461-474
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• 2005

The ultimate goal of periodontal therapy is the regeneration of periodontal tissue and the repair of function. For more than a decade there have been many efforts to develop materials and methods of treatment to promote periodontal tissue regeneration. Recently many efforts are concentrated on the regeneration potential of material used in traditional medicine. Safflower(Carthamus tinctorius L.) seed extract(SSE) have long clinically used in Korea to promote bone formation and prevent osteoporosis. The purpose of this study was to examine the effects of SSE on bone formation in human osteoblastic cell line. Human fetal osteoblastic cell line(hFOB 1.19) was cultured with DMEM and SSE($1{\mu}g/ml$, $10{\mu}g/ml$, $100{\mu}g/ml$, $1mg/ml$) at $34^{\cdot}C$ with 5% $CO_2$ in 100% humidity. The proliferation, differentiation of the cell was evaluated by several experiments. Cell proliferation was significantly increased at $10{\mu}g/ml$, $100{\mu}g/ml$, 1mg/ml of SSE after 3 and 7 days incubation(p<0.05). Cell spreading assay was significantly increased at $100{\mu}g/ml$ of SSE after 3 days and $1{\mu}g/ml$, $10{\mu}g/ml$, $100{\mu}g/ml$, 1mg/ml of SSE after 7 days(p<0.05). Alkaline Phosphatase(ALP) level was significantly increased in $10{\mu}g/ml$, $100{\mu}g/ml$, 1mg/ml of SSE(p<0.05). Collagen synthesis was significantly increased at $10{\mu}g/ml$, $100{\mu}g/ml$, 1mg/ml of SSE(p<0.05). A quantified calcium accumulation was significantly increased at $10{\mu}g/ml$, $100{\mu}g/ml$ of SSE(p<0.05). ALP and osteocalcin mRNA was expressed in $100{\mu}g/ml$ of SSE by RT-PCR. These results indicate that SSE are capable of increasing osteoblasts mineralization and may play an important role in bone formation.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.6
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• pp.3751-3755
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• 2013

Backgroud and Aims: MicroRNA-206 has proven to be down-regulated in many human malignancies in correlation with tumour progression. Our study aimed to characterize miR-206 contributions to initiation and malignant progression of human osteosarcoma. Methods: MiR-206 expression was detected in human osteosarcoma cell 1ine MG63, human normal osteoblastic cell line hFOB 1.19, and paired osteosarcoma and normal adjacent tissues from 65 patients using quantitative RT-PCR. Relationships of miR-206 levels to clinicopathological characteristics were also investigated. Moreover, miR-206 mimics and negative control siRNA were transfected into MG63 cells to observe effects on cell viability, apoptosis, invasion and migration. Results: We found that miR-206 was down-regulated in the osteosarcoma cell line MG63 and primary tumor samples, and decreased miR-206 expression was significantly associated with advanced clinical stage, T classification, metastasis and poor histological differentiation. Additionally, transfection of miR-206 mimics could reduce MG-63 cell viability, promote cell apoptosis, and inhibit cell invasion and migration. Conclusions: These findings indicate that miR-206 may have a key role in osteosarcoma pathogenesis and development. It could serve as a useful biomarker for prediction of osteosarcoma progression, and provide a potential target for gene therapy.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.38
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• pp.48.1-48.8
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• 2016

Background: This study investigates the effect of alendronate-treated osteoblasts, as well as the effect of low-level laser therapy (LLLT) on the alendronate-treated osteoblasts. Bisphosphonate decreases the osteoblastic activity. Various treatment modalities are used to enhance the bisphosphonate-treated osteoblasts; however, there were no cell culture studies conducted using a low-level laser. Methods: Human fetal osteoblastic (hFOB 1.19) cells were treated with $50{\mu}M$ alendronate. Then, they were irradiated with a $1.2J/cm^2$ low-level Ga-Al-As laser (${\lambda}=808{\pm}3nm$, 80 mW, and 80 mA; spot size, $1 cm^2$; NDLux, Seoul, Korea). The cell survivability was measured with the MTT assay. The three cytokines of osteoblasts, receptor activator of nuclear factor ${\kappa}B$ ligand (RANKL), osteoprotegerin (OPG), and macrophage colony-stimulating factor (M-CSF) were analyzed. Results: In the cells treated with alendronate at concentrations of $50{\mu}M$ and higher, cell survivability significantly decreased after 48 h (p < 0.05). After the applications of low-level laser on alendronate-treated cells, cell survivability significantly increased at 72 h (p < 0.05). The expressions of OPG, RANKL, and M-CSF have decreased via the alendronate. The RANKL and M-CSF expressions have increased, but the OPG was not significantly affected by the LLLT. Conclusions: The LLLT does not affect the OPG expression in the hFOB cell line, but it may increase the RANKL and M-CSF expressions, thereby resulting in positive effects on osteoclastogenesis and bone remodeling.

• Journal of Oral Medicine and Pain
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• v.26 no.1
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• pp.39-50
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• 2001

In order to investigate the effect of Transforming growth factor ${\beta}1$(below TGF-${\beta}1$) and osteogenic protein-1(below Op-1) onto the myogenic differentiation, C2C12 satellite myoblastic cell line was cultured and treated with both growth factors. At first morphological changes with microscopical examination were examined, and isolated total RNA to analyse mRNA expression of bone marker proteins, muscle regulatory proteins, TGF-${\beta}$ receptor and their ligands by Northern blot analysis. And cellular proliferative inducibility of both growth factors was also tested to C2C12 cells. Incubating the cell with $5ng/m{\ell}$ of TGF-${\beta}1$ until 4 days almost inhibited multinucleated myotube formation expressing muscular regulatory proteins, and induced decreasing Id proteins. However, no osteoblastic phenotypes was induced by TGF-${\beta}1$ in C2C12 cells. The mRNA expression of TGF-${\beta}$ receptors with TGF-${\beta}1$ was conversed after 48 hours cultured. Type I TGF-${\beta}$ receptor was seemed to play a role in negative signalling for inhibition of myogenic differentiation. OP-1 dose dependently induced ALP activity, osteopontine production and bone sialoprotein production at concentrations above $100ng/m{\ell}$ and osteocalcin production at concentrations above $300ng/m{\ell}$. The concentration of OP-1 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubation with above $100ng/m{\ell}$ OP-1 suppressed the expression of mRNA for muscular egulatory proteins from 2 days after incubation. Expression of Id-1, 2, 3 mRNA were stimulated by OP-1 at concentration above $300ng/m{\ell}$. When C2C12 cells were treated with both growth factors, TGF-${\beta}1$ potentiated the inhibitory effect of OP-1 on myotube formation and expression of mRNA for myogenin at 12 days. And TGF-${\beta}1$ reduced osteocalcin and bone sialoprotein production induced by OP-1 at 12 days in C2C12 cells. Both growth factor had no mitogenic effect. These results indicate that OP-1 converts the differentiation pathway of C2C12 myoblasts into that of osteoblastic lineage cells and it's not heritable, but TGF-${\beta}1$ does not and has reversible inhibitory activity on the myogenic differentiation. TGF-${\beta}1$ and OP-1 play a role in myogenic differentiation via different mechanism between them.