• International Journal of Oral Biology
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• v.39 no.3
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• pp.145-151
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• 2014

During bone remodeling, there is requirement of differentiation of osteoblastic cells. Previously, we identified proteins differentially expressed in soft tissue during bone healing. Of these proteins, we focused the effect of LTF on differentiation of osteoblast. In order to analyze the osteogenic ability of LTF, we treated conditioned media collected from human LTF-stably transfected HEK293T cells into osteoblastic MC3T3-E1. The results showed that the activity and expression of alkaline phosphatase were increased in MC3T3-E1 cells treated with conditioned media containing LTF in dose- and time-dependent manner. At the same time, we observed the significant increase of the expression of osteoblastic genes, such as ALP, BSP, COL1A1, and OCN, and along with matrix mineralization genes, such as DMP1 and DMP2, in LTF conditioned media-treated groups. Moreover, the result of treating recombinant human LTF directly into osteoblastic MC3T3-E1 showed the same pattern of treating conditioned media containing LTF. Our study demonstrated that LTF constitutively enhances osteoblastic differentiation via induction of osteoblastic genes and activation of matrix mineralization in MC3T3-E1 cells.

• The korean journal of orthodontics
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• v.28 no.1 s.66
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• pp.155-163
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• 1998

To evaluate the effect of a static magnetic field on the bone producing potential of MC3T3-E1 cells, the alkaline phosphatase activity was measured after the cells having been cultured under 76.4mT static magnetic field using a $SmCo_5$ magnets for 5days, 7days, 11days, 15days and 21days for each cell culture group. Also, the amount of bone nodule stained with Alizarin red S was observed. The results were as follows . The alkaline phosphatase activity of the 7, 11, and 15 days group among the experimental groups was decreased as compared with the control groups, and the decrease of alkaline phosphatase activity in the 11 days group was the most evident among them. . Any stained bone nodules of both groups had not been observed until the 11th day. The stained bone nodules in the control groups were found on the 15th day, but not in the experimental groups. The stained bone nodules were observed in both groups on the 21st day, but the control groups have more bone nodules than the experimental groups.

• Journal of dental hygiene science
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• v.20 no.4
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• pp.221-229
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• 2020

Background: The purpose of this study was to investigate the anti-oral microbial activity and anti-inflammatory effects of rosmarinic acid (RA) in lipopolysaccharide (LPS)-stimulated MC3T3-E1 osteoblastic cells on a titanium (Ti) surface during osseointegration, and to confirm the possibility of using RA as a safe natural substance for the control of peri-implantitis (PI) in Ti-based dental implants. Methods: A disk diffusion test was conducted to confirm the antimicrobial activity of RA against oral microorganisms. In order to confirm the anti-inflammatory effects of RA, inflammatory conditions were induced with 100 ng/ml of LPS in MC3T3-E1 osteoblastic cells on the Ti surface treated with or without 14 ㎍/ml of RA. The production of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated MC3T3-E1 osteoblastic cells on the Ti surface was confirmed using an NO assay kit and PGE2 enzyme-linked immunosorbent assay kit. Reverse transcription polymerase chain reaction and western blot analysis were performed to confirm the expression of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in total RNA and protein. Results: RA showed weak antimicrobial effects against Streptococcus mutans and Escherichia coli, but no antimicrobial activity against the bacteria Aggregatibacter actinomycetemcomitans and the fungus Candida albicans. RA reduced the production of pro-inflammatory mediators, NO and PGE2, and proinflammatory cytokines, TNF-α and IL-1β, in LPS-stimulated MC3T3-E1 osteoblastic cells on the Ti surface at the protein and mRNA levels. Conclusion: RA not only has anti-oral microbial activity, but also anti-inflammatory effects in LPS-stimulated MC3T3-E1 osteoblasts on the Ti surface, therefore, it can be used as a safe functional substance derived from plants for the prevention and control of PI for successful Ti-based implants.

• Imaging Science in Dentistry
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• v.39 no.3
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• pp.121-132
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• 2009

Purpose : To investigate the effects of 2-deoxy-D-glucose (2-DG) and quercetin (QCT) on gene expression of bone sialoprotein (BSP) and osteocalcin (OC) during the differentiation in irradiated MC3T3-E1 osteoblastic cells. Materials and Methods : When MC3T3-E1 osteoblastic cells had reached 70-80% confluence, cultures were transferred to a differentiating medium supplemented with 5 mM 2-DG or $10{\mu}M$ QCT, and then irradiated with 2, 4, 6, and 8 Gy. At various times after irradiation, the cells were analyzed for the synthesis of type I collagen, and expression of BSP and OC. Results : The synthesis of type I collagen in cells exposed to 2 Gy of radiation in the presence of 2-DG or QCT showed no significant difference compared with the control group within 15 days post-irradiation. When the cells were irradiated with 8 Gy, 2-DG facilitated the irradiation mediated decrease of type I collagen synthesis, whereas such decrease was inhibited by treating with QCT. During MC3T3-E1 osteoblastic cell differentiation, the mRNA expression of BSP and OC showed the peak value at 14 days and 21 days, respectively. 2-DG or QCT treatment alone decreased the level of BSP mRNA, but increased the OC mRNA level only at early time of differentiation (day 7). In the cells irradiated with 2, 4, 8 Gy, the mRNA expression of BSP and OC decreased at 7 days after the irradiation. The cells were treated with various dose of radiation in the presence of 2-DG or QCT, the mRNA level of both BSP and OC increased although this increase was observed at low dose of radiation (2 Gy) and at the early stage of differentiation. However, when the cells were exposed to 4, 6, or 8 Gy, the increase of BSP and OC mRNAs was detected only in cells co-incubated with QCT. Conclusion : This study demonstrates that 2-DG and QCT affect differently the expression of bone formation related factors, type I collagen, BSP, and OC in the irradiated MC3T3-E1 osteoblasic cells, according to the dose of radiation and the times of differentiation. Overall, the present findings suggest that 2-DG and QCT could have the regulatory roles as radiation-sensitizer and -protector, respectively.

• Restorative Dentistry and Endodontics
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• v.39 no.3
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• pp.187-194
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• 2014

Objectives: The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. Materials and Methods: MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). Results: Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). Conclusions: These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.

• Food Science and Biotechnology
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• v.16 no.5
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• pp.807-811
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• 2007

Diabetes is marked by high glucose levels and is associated with decreased bone mass and increased fracture rates. To determine if [6]-gingerol could influence osteoblast dysfunction induced by 2-deoxy-D-ribose (dRib), osteoblastic MC3T3-E1 cells was treated with dRib and [6]-gingerol and markers of osteoblast function and oxidized protein were examined. [6]-Gingerol ($10^{-7}\;M$) significantly increased the growth of MC3T3-E1 cells in the presence of 30 mM dRib (p<0.05). [6]-Gingerol ($10^{-7}\;M$) caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and osteocalcin secretion in the cells. We then examined the effect of [6]-gingerol on the production of osteoprotegerin and protein carbonyl in osteoblasts. Treatment with [6]-gingerol ($10^{-9}$ and $10^{-7}\;M$) increased osteoprotegerin secretion in osteoblastic cells. Moreover, [6]-gingerol ($10^{-9}$ and $10^{-7}\;M$) decreased protein carbonyl contents of osteoblastic MC3T3-E1 cells in the presence of 30 mM dRib. Taken together, these results demonstrate that [6]-gingerol inhibits dRib-induced damage and may be useful in the treatment of diabetes related bone diseases.

• Natural Product Sciences
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• v.11 no.3
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• pp.139-144
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• 2005

Bioflavone quercetin is believed to play an important role preventing bone loss by affecting osteoclastogenesis and regulating many systemic and local factors including hormones and cytokines. This study examined how quercetin acts on tumor necrosis factor-alpha ($TNF-{\alpha}$)-mediated apoptosis in MC3T3-E1 osteoblastic cells. Apoptosis assays revealed the dose-dependent acceleration of quercetin on $TNF-{\alpha}-induced$ apoptosis in MC3T3-E1 cells, which was demonstrated by the increased number of positively stained cells in the trypan blue staining and TUNEL assay, and the migration of many cells to the $sub-G_0/G_1$ phase in flow cytometric analysis. In particular, quercetin treatment alone increased the expression of p53 and p21 proteins in the cells. Consequently, this study showed that quercetin accelerates the $TNF-{\alpha}-induced$ apoptosis in MC3T3-E1 osteoblastic cells.

• 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.

• BMB Reports
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• v.44 no.10
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• pp.659-664
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• 2011

As part of the search for biologically active anti-osteoporotic agents that enhance differentiation and mineralization of osteoblastic MC3T3-E1 cells, we identified the ginsenoside Rh2(S), which is an active component in ginseng. Rh2(S) stimulates osteoblastic differentiation and mineralization, as manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and Alizarin Red staining, respectively. Rh2(S) activates p38 mitogen-activated protein kinase (MAPK) in time- and concentration-dependent manners, and Rh2(S)-induced differentiation and mineralization of osteoblastic cells were totally inhibited in the presence of the p38 MAPK inhibitor, SB203580. In addition, pretreatment with Go6976, a protein kinase D (PKD) inhibitor, significantly reversed the Rh2(S)-induced p38 MAPK activation, indicating that PKD might be an upstream kinase for p38 MAPK in MC3T3-E1 cells. Taken together, these results suggest that Rh2(S) induces the differentiation and mineralization of MC3T3-E1 cells through activation of PKD/p38 MAPK signaling pathways, and these findings provide a molecular basis for the osteogenic effect of Rh2(S).

• Nutritional Sciences
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• v.8 no.4
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• pp.242-249
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• 2005

It is well established that zinc plays an important role in bone metabolism and mineralization. The role of zinc in bone formation is well documented in animal models, but not much reported in cell models. In the present study, we evaluated zinc deficiency effects on osteoblastic cell proliferation, alkaline phosphatase activity and expression, and extracellular matrix bone nodule formation and bone-related gene expression in osteoblastic MC3T3-E1 cells. To deplete cellular zinc, chelexed-FBS and interpermeable zinc chelator TPEN were used. MC3T3-E1 cells were cultured in zinc concentration-dependent (0-15 ${\mu}M\;ZnCl_2$) and time-dependent (0-20 days) manners. MC3T3-E1 cell proliferation by MTT assay was increased as medium zinc level increased (p<0.05). Cellular Ca level and alkaline phosphatase activity were increased as medium zinc level increased (p<0.05). Alkaline phosphatase expression, a marker of commitment to the osteoblast lineage, measured by alkaline phosphatase staining was increased as medium zinc level increased. Extracellular calcium deposits measured by von Kossa staining for nodule formation also appeared higher in Zn+(15 ${\mu}M\;ZnCl_2$) than in Zn-(0 ${\mu}M\;ZnCl_2$). Bone formation marker genes, alkaline phosphatase and osteocalcin, were also expressed higher in Zn+ than in Zn-. The current work supports the beneficial effect of zinc on bone mineralization and bone-related gene expression. The results also promote further study as to the molecular mechanism of zinc deficiency for bone formation and thus facilitate to design preventive strategies for zinc-deficient bone diseases.