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

• Journal of Periodontal and Implant Science
• /
• v.33 no.3
• /
• pp.359-372
• /
• 2003

Among objectives of periodontal therapy. the principal one is the morphological and functional reconstruction of lost periodontal supporting tissues. This includes de novo formation of connective tissue attachment and the regrowth of alveolar bone. The use of enamel matrix derivative(EMD) may be a suitable means of regeneration new periodontal attachment in the infrabony defects. Implant used to replace lost tooth but, implantitis occurred after installation. The purpose of this study was to investigate the effects of EMD on differentiation and growth of osteoblast in titanium disc. Twentyfive millimeter diameter and 1mm thick Ti disc which was coated 25, 50, 100, 200${\mu}g$/ml of EMD(Emdogain(R)) used as experimental group, 25, 50, 100, 200ng/d of rhBMP-2 as positive control group, and no coat as negative control group. A human osteosarcoma cell line Saos-2 was cultured in Ti disc and cell proliferation and Alkaline phosphatase (ALP) activity were measured at 1 and 6 days. PCR was performed at 2 and 8 hours. Semi-quantitative RT-PCR for mRNA expressions of various osteoblastic differentiation markers -type I collagen, ALP, osteopontin, and bone sialoprotein - were performed at appropriate concentrations based upon the results of MTT and ALP assay. Cultured cell-disc complexes were prepared for scanning electron microscopy (SEM) at 2 hour. Data were analyzed using Mann-Whitney and repeated- measures 1-way analysis of variance(SPSS software version 10,SPSS. Chicago. IL). After culture, there was more osteoblast in EMD100${\mu}g$/ml than in EMD50, 200${\mu}g$/ml on day 6. There was significant difference in experimental and positive control group compared control group, as times go by(1 and 6 days). Alkaline phosphatase activity was different significantly in EMD100, 200${\mu}g$/ml and BMP100, 200${\mu}g$/ml on day 6. The results of reverse transcriptase-polymerase chain reaction (RT-PCR) showed that expression of mRNA for ALPase, collagen type I, osteopontin. hone sialoprotein and BMP-2 was detected at 2 hour and 8 hour in EMI 200${\mu}g$/ml subgroup and BMP100ng/ml subgroup. The results of this study suggest that application of enamel matrix derivative on osteoblast attached to titanium surface facilitate the expression of bone specific protein and the differentiation and growth of osteoblast.

• Journal of Periodontal and Implant Science
• /
• v.36 no.4
• /
• pp.839-847
• /
• 2006

The aim of this study is to monitor reporter gene expression under osteocalcin gene promoter, using a real-time molecular imaging system, as tool to investigate osteoblast differentiation. The promoter region of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, was inserted in promoterless luciferase reporter vector. Expression of reporter gene was confirmed and relationship between the reporter gene expression and osteoblastic differentiation was evaluated. Gene expression according to osteoblstic differentiation on biomaterials, utilizing a real-time molecular imaging system, was monitored. Luciferase was expressed at the only cells transduced with pGL4/mOGP and the level of expression was statistically higher at cells cultured in mineralization medium than cells in growth medium. CCCD camera detected the luciferase expression and was visible differentiation-dependent intensity of luminescence. The cells produced osteocalcin with time-dependent increment in BMP-2 treated cells and there was difference between BMP-2 treated cells and untreated cells at 14days. There was difference at the level of luciferase expression under pGL4/mOGP between BMP-2 treated cells and untreated cells at 3days. CCCD camera detected the luciferase expression at cells transduced with pGL4/mOGP on Ti disc and was visible differentiation-dependent intensity of luminescence This study shows that 1) expression of luciferase is regulated by the mouse OC promoter, 2) the CCCD detection system is a reliable quantitative gene detection tool for the osteoblast differentiation, 3) the dynamics of mouse OC promoter regulation during osteoblast differentiation is achieved in real time and quantitatively on biomaterial. The present system is a very reliable system for monitoring of osteoblast differentiation in real time and may be used for monitoring the effects of growth factors, drug, cytokines and biomaterials on osteoblast differentiation in animal.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.38
• /
• pp.17.1-17.6
• /
• 2016

Background: The aim of this study is to verify the feasibility of using silk fibroin (SF) as a potential membrane for guided bone regeneration (GBR). Methods: Various cellular responses (i.e., cell attachment, viability, and proliferation) of osteoblast-like MG63 cells cultured on an SF membrane were quantified. After culturing on an SF membrane for 1, 5, and 7 days, the attachment and surface morphology of MG63 cells were examined by optical and scanning electron microscopy (SEM), cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell proliferation was quantified using 4',6-diamidino-2-phenylindole (DAPI) fluorescence staining. Results: Optical microscopy revealed that MG63 cells cultured on the SF membrane proliferated over the 7-day observation period. The viability of cells cultured on SF membranes (SF group) and on control surfaces (control group) increased over time (P < 0.05); however, at respective time points, cell viability was not significantly different between the two groups (P > 0.05). In contrast, cell proliferation was significantly higher in the SF membrane group than in the control group at 7 days (P < 0.05). Conclusions: These results suggest that silk fibroin is a biocompatible material that could be used as a suitable alternative barrier membrane for GBR.

• Journal of Dental Rehabilitation and Applied Science
• /
• v.19 no.3
• /
• pp.185-193
• /
• 2003

The object of this study is to observe the effects of magnetism on the osteoblasts using a neodymium magnet. The osteoblasts was cultured under magnetic fields of varying intensities to evaluate the effect of magnetism on the activity and alkaline phosphatase acitivty of the osteoblasts. Osteoblasts were cultured in the cell density of $10^4$ for the evaluation of cell proliferation and 105/ml for the evaluation of ALP activity under 0. 10, 100, 500, 1000, 2000, 4000 gauss for 24 hour. For evaluation of osteoblast morphologic changes under magnetic, osteoblasts were observed by inverted microscope and TEM. To elucidate if IGF-receptors are increased under the magnetic field, we investigated osteoblasts by immunofluoroscence staining. The results were as follows: In the varying intensities of magnetic fields, the degree of cell proliferation was the highest in the magnetic field of 10 gauss and this gradually decreased up to 1000 gauss. In the magnetic fields stronger than 1000 gauss, the degree of the cell proliferation decreased to an even lower level than that of the control group. The ALP activity and protein synthesis showed a similar increase pattern as the degree of cell proliferation compared to the control group but showed little difference. Under the microscope, morphological change of the cells ( decrease in length and increase in roundness) were observed but no peculiarity of cell distribution could be found according to the magnetic field line. In the proper intensity of magnetic fields (10 gauss), the cultured cells showed increase in number of IGF Receptors compared to that of the control group.

• Bulletin of the Korean Chemical Society
• /
• v.28 no.4
• /
• pp.652-656
• /
• 2007

Hydroxyapatite [Ca10(PO4)6(OH)2, HAp] and titanium(Ti) metal are known to be excellent materials with high affinities for natural bone through the apatite. Tricalcium phosphate [Ca3(PO4)2, TCP] is a promising alternative material because it is similar to HAp in its physical properties and biocompatibility. To examine the influence of hydroxyapatite, tricalcium phosphate, pure titanium and pre-treated titanium on osteopontin expression in osteoblasts, RNA was extracted from proliferated and cultured osteoblast cells and OPN mRNA expression was observed by RT-PCR.

• Journal of Dental Rehabilitation and Applied Science
• /
• v.19 no.2
• /
• pp.87-96
• /
• 2003

The purposes of this study were to find out the optimum intensity of magnetic field where magnetism could promote the activity of osteoblast, and to discover the possibility of clinical application in the areas of dental implants and bone grafts by confirming the effect of clinically increasing bone formation. In this experiment, we used the Neodymium magnet, which had magnetic power six times as strong as the current ones and enabled the resistances against the demagnetization up to 20 to 50 times to be minimized with the size of 1mm in sight. In order to culture cells, a specially designed device was used. It was made to adjust the distance and accordingly to control the intensity of the magnetic field, by placing the cell culture plate in the center with a magnet of 1mm long and thick installed on the both ends. Using MC3T3-E1 cell, a kind of osteoblast-like cell, we cultured, for 24 hours, not only the test group which had been cultured under the magnetic fields with different intensity of 5, 10, 50, 100, 500, and 1000 Gauss, but also the control group excluding the influences of the magnetic field. After observing the cell's form and the density of the culture medium through an inverted microscope, we made a series of proceedings needed for the immunofluoroscence staining, such as fixation, normal serum reaction, primary antibody reaction, and secondary antibody reaction. And with a fluorescence microscope, we observed those-above and compared the frequency of expression of IFG-1 receptor. To make a Western immunoblotting analysis, the cells cultured under the same condition as the above had the procedure of the lysis buffer and the acrylamide gel electrophoresis was carried out. Protein transferred into the nitrocellulose membrane and tested on the primary and the secondary antibody reactions was observed and compared. The results were as follows: When observed through an inverted microscope, the nuclear divisions of the cells under the magnetic field of 10 Gauss were the most active, and the density of the cells could be observed the most enormously. As the result of an immunofluoroscence staining of IGF-1 receptor, the expression of IFG-1 was the most frequently observed under the magnetic field of 10 Gauss. On the other hand, few differences of consideration were made between the test group cultured under the magnetic fields of 5, 500, and 1000 Gauss and the control group. In respect of the expression of IFG-1 receptor, the test group cultured under the magnetic fields of 50 and 100 Gauss were higher than the control group, and lower than that cultured under the magnetic field of 10 Gauss.(p<0.05) According to the Western immunoblotting analysis, the band of IFG-1 receptor which had 85KDa of molecular weight was the darkest. Judging from the above-mentioned results, the growth factor receptor of an osteoblast cell which was an important criterion for the bone formation was increased in maximum under the magnetic field of 10 Gauss. Moreover it was observed that the optimum intensity of magnetic field in which magnetism made the activity of the osteoblast cell increase was about 10 Gauss.

• The Journal of Korean Academy of Prosthodontics
• /
• v.43 no.4
• /
• pp.511-518
• /
• 2005

Purpose: The purpose of this study is to find the effect of rare earth magnet's magnetic field of to the osteoblast around the implant by the means of observation number, and distribution around the implant which is connected to the permanent magnet but not, counted and compared by the number of cells attached to the surface of the implant. Material and method: The permanent magnets, made in the healing cap form, were connected to the implant future, and placed on the culture plate, The osteoblast-like cell: MC3T3-E1 were used for cell culture. As the control group, the implant were connected to normal healing cap, and cultured in the same conditions. 48 hours later, using inverted microscope, the number and distribution of osteoblast around the implant were observed, and 72 hours later, the number of the cells attached to the implant were counted. Results: As a result, the implant connected to the permanent magnet had proved to have a more concentrated cell distribution rate than the control group. The implant connected to the permanent magnet, neck area : which has about 10 gauss magnetic force, had more cells than apex area. The implant connected to the permanent magnet had proven to attach to the osteoblast more productively than control group's implant. Conclusions: This research showed that the magnetic field of the permanent magnet affected the distribution and growth rate of the osteoblast around the implant. In order to support this study, it also had need to monitor the progress of the permanent magnet specifically shown on the neck area, which has10 gauss magnetic force. So after additional research on the distribution and attachment of the cells, and further more, on bone formation, it will be concluded that the clinical applications ,such as immediate loading of implant treatment are possible.

• IMMUNE NETWORK
• /
• v.2 no.3
• /
• pp.166-174
• /
• 2002

Background: Human umbilical cord bloods, which could be taken during the delivery are utilized as a source of hematopoietic stem cells. Also in cord blood, there are several kinds of stem cells such as endothelial and mesenchymal stem cells. Methods: We isolated the mesenchymal stem cells from human umbilical cord bloods and confirmed the differentiation of these cells into osteoblast progenitor cells. The mesenchymal stem cells derived from umbilical cord blood have the ability to differentiate into specific tissue cells, which is one of characteristics of stem cells. These cells were originated from the multipolar shaped cells out of adherent cells of the umbilical cord blood mononuclear cell culture. Results: The mesenchymal stem cells expressed cell surface antigen CD13, CD90, CD102, CD105, ${\alpha}$-smooth muscle actin and cytoplasmic antigen vimentine. Having cultrued these cells in bone formation media, we observed the formation of extracellular matrix and the expression of alkaline phosphatase and of mRNA of cbfa-1, ostoecalcin and type I collagen. Conclusion: From these results we concluded that the cells isolated from the umbilical cord blood were mesenchymal stem cells, which we could differentiate into osteoblast when cultured in bone formation media. In short, it is suggested that these cells could be used as a new source of stem cells, which has the probability to alternate the embryonic stem cells.

• Journal of Periodontal and Implant Science
• /
• v.33 no.1
• /
• pp.49-60
• /
• 2003

Several growth factors and polypeptides are not commonly yet used for regenerators of bone tissue or alveolar bone because of the insufficiency of studies on their side effects, genetic engineering for mass production and stability for clinical application. Recently, many herbal medicines, which have advantage of less side effects and possibility of long-term use, have been studied for their capacity and effects of anti-bacterial, antiinflammatory and regenerative potential of periodontal tissues. Morindae Radix, Cibotium Barometz (L.), Albizziae Cortex, Cistandhis Herba have been traditionally used as medicines for treatment of bone disease in Eastern medicine. The objective of the present study is to examine the ability of alkaline phosphatase (ALP) activity of human fetal osteoblast (hFOB1) when several natural medicines were supplemented. hFOB1 were cultured with Dulbecuo's Modified Eagle's Medium Nutrient Mixture F-12 HAM ( DMEM/F-12 1:1 Mixture, Sigma, USA) and negative control, dexamethasone (positive control), and each natural medicines for 3 days. And then ALP activity was measured by spectrophotometer for enzyme activity and Alizarin red S staining for morphometry. Among the natural medicines of this study, Morindae Radix, Cibotium Barometz (L.) and Cistanchis Herba induced higher activity of ALP synthesis than negative controls in all experimental group. Albizziae Cortex showed mild increases than negative control group. According to measurement of positively stained area, all of the natural medicines of this study increased compared to negative control. Especially, Cibotium Barometz (L.) and Cistanchis Herba showed statistical significance compared to negative control (p<0.05). These results indicate that Morindae Radix, Cibotium Barometz (L.), Albizziae Cortex, Cistandhis Herba have an inducing ability of ALP synthesis on osteoblast.