• Korean Journal of Food Science and Technology
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• v.40 no.6
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• pp.674-679
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• 2008

Scutellaria radix (SR) has been utilized as a traditional medicine for a variety of diseases including Rheumatoid arthritis and its major flavonoids - baicalein, baicalin, and wogonin - have been reported to exert beneficial health effects, including anti-bacterial, anti-viral, anti-inflammatory, and free-radical scavenging. However, the mechanisms underlying this effect remain poorly understood. The principal objective of this study was to determine the effect of SR on osteoblast and osteoclast cells. SR extract was prepared using 70% ethanol solvent. Osteoblastic MC3T3-E1 cells and osteoclast precursor Raw 264.7 macrophage cells were utilized. SR extract increased MC3T3-E1 cell proliferation and stimulated alkaline phosphatase activity dose-dependently, 152.0% of the control at concentration $1{\mu}g/mL$. Additionally, SR extract ($1{\mu}g/mL$) stimulated Bone nodule formation activity in MC3T3-E1 cells, approximately 223.3% of the control, 20 days after the exposure. In addition, SR extract significantly reduced the number of tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cells from Raw 264.7 cells. In conclusion, SR extract stimulates the proliferation and bioactivities of boneforming osteoblasts, and inhibits the activities of bone-resorbing osteoclasts to a certain degree.

• Journal of the korean academy of Pediatric Dentistry
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• v.30 no.3
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• pp.391-405
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• 2003

Craniosynostosis, known as a premature fusion of cranial sutures, is a developmental disorder characterized by precocious differentiation and mineralization of osteoblasts in the calvarial sutures. Recent genetic studies have demonstrated that mutation in the homeobox gene Msx2 causes Boston-type human craniosynostosis. Additionally, the phenotype of Dlx5 homozygote mutant mouse presents craniofacial abnormalities including a delayed ossification of calvarial bone. Furthermore transcription of osteocalcin, a mature osteoblast marker, is reciprocally regulated by the homeodomain proteins Msx2 and Dlx5. These facts suggest important roles of osteocalcin, Msx2 and Dlx5 genes in the calvarial bone growth and suture morphogenesis. To elucidate the function of these molecules in the early morphogenesis of mouse cranial sutures, we have first analyzed by in situ hybridization the expression of osteocalcin, Msx2 and Dlx5 genes in the developing parietal bone and sagittal suture of mouse calvaria during the embryonic (E15-E18) stage. Osteocalcin mRNA was found in the periosteum of parietal bones from E15, and gradually more highly expressed with aging. Msx2 mRNA was intensely expressed in the sutural mesenchyme, osteogenic fronts and mildly expressed in the dura mater during the embryonic stage. Dlx5 mRNA was intensely expressed osteogenic fronts and the periostem of parietal bones. To further examine the upstream signaling molecules of transcription factor Msx2 and Dlx5, we have done in vitro experiments in E15.5 mouse calvarial explants. Interestingly, implantation of BMP2-, BMP4-soaked beads onto the osteogenic fronts after 48 hours organ culture induced etopic expressions of Msx2 and Dlx5 genes. On the other hand, overexpression of $TGF{\beta}1$, GDF-6, -7, FGF-2, -4 and Shh did not induce the expression of Msx2 and Dlx5. Taken together. these data indicate that transcription factor Msx2 and Dlx5 play critical roles in the calvarial bone and suture development, and that BMP siganling is involved in the osteogenesis of calvarial bones and the maintenance of cranial sutures through regulating these two transcriotpn factors. Furthermore, different expression patterns between Msx2 and Dlx5 suggest their specific functions in the osteoblast differentiation.

• Journal of Life Science
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• v.24 no.3
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• pp.297-303
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• 2014

The effects of Eisenia bicyclis extracts on osteoblast differentiation and osteoclast formation were investigated. The proliferation of MC3T3-E1 osteoblastic cells was tested in an MTT assay. Treatment with E. bicyclis ethanol extract increased cell proliferation by approximately 128% at a concentration of 10 ${\mu}g/ml$. The ALP activities in the MC3T3-E1 cells was 179% higher when the E. bicyclis ethanol extract was processed at a concentration of 50 ${\mu}g/ml$. The proliferation of RAW 264.7 osteoclastic cells decreased significantly in response to treatment with the E. bicyclis extracts. Moreover, the proliferation of the RAW 264.7 osteoclastic cells treated with E. bicyclis hot water extract decreased by nearly 80%. In addition, the E. bicyclis extract reduced the number of tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cells from osteoclastic RAW 264.7 cells. These results indicate that E. bicyclis extracts have an anabolic effect on bone through the promotion of osteoclast differentiation and suggest that the extracts could be used in the treatment of common metabolic bone diseases.

• Journal of the Korean Society of Food Science and Nutrition
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• v.34 no.7
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• pp.929-936
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• 2005

The purpose of this study was to examine the ability of alkaline phosphatase (ALP) synthesis of MC3T3-E1 cells when above edible sources, Solidago virga-aurea var. gigantea Miq. root (SVR) extracts, were supplimented. MC3T3-E1 cells were cultured with $\alpha-MEM$(vehicle control), dexamethasone and genestein (positive control), and SVR extracts for 27 days. The effects of SVR MeOH extracts and its fractions on cell proliferation were measured by MTT assay. At 10, 100${\mu}g/mL$ of SVR methanol extract treated, that were elevated of cell proliferation to 140 and $120\%$ via vehicle control, respectively. And then ALP synthesis was measured by spectrophotometer for enzyme activity and by naphthol AS-BI staining for morphometry at 3, 9, 18, and 27th day. As the results, every extracts and fractions were promoted ALP activity by time course at 1, 10, 100${\mu}g/mL$, except n-hexane and chloroform fractions. Remarkably, the MeOH extracts were increased ALP activity more than 4.4 times compared with vehicle control, 2.2 times via positive control at 27th day (p<0.05). The SVR MeOH extracts treated cells, especially at a concentration of 10${\mu}g/mL$, showed remarkably higher than vehicle-treated control cells of mineralization which were checked by Alizarin red staining. These results indicate that SVR methanol extract have an induction ability of proliferation and differentiation on osteoblast.

• Journal of the korean academy of Pediatric Dentistry
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• v.31 no.4
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• pp.651-658
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• 2004

Runx2 is a transcription factor in homologous with Drosophila runt gene and it is essential for bone formation during embryogenesis and a critical gene for osteoblast differentiation and osteoblast function. Runx2-haploinsufficency causes cleidocranial dysplasia (CCD). CCD is an autosomal-dominant inherited disorder characterized by hypoplastic clevicle and delayed ossification in fontanelles and wormian bones. Dental defects are possibly shown to CCD patients : multiple supernumerary teeth, irregular and compressed permanent tooth crowns, hypoplastic and hypomineralized defects in enamel and dentin, an excess of epithelial root remnants, the absence of cellular cementum, and abnormally shaped roots. In addition, delayed eruption of the secondary dentition is a constant finding. The aim of this study is to evaluate the role of Runx2 in the tooth development and eruption through analyzing the expression pattern of Runx2 by in situ hybridization during crown (late bell stage) and root formation of tooth, using postnatal day 1, 4, 7, 14 and 21 mice mandibular molar teeth. mRNA of Runx2-full length is expressed in dental follicle and surrounding tissue at postnatal day1 and 4. At postnatal day 7, it is expressed in ameloblasts of occlusal surface of enamel and bone area surrounding the tooth. In comparison with previous stage, at postnatal day 14, it is expressed in ameloblasts of proximal surface of enamel. At postnatal day 21 it's expression is observed only in bone area. mRNA of Runx2-typeII is not expressed. At postnatal day 1 and 7. At postnatal day 14 and 21, it's expression is observed in the bone area. In this study, we suggest that Runx2 have a relation of ameloblasts differentiation and an important role to tooth eruption made by dental follicle during intraosseous eruption stage. Also we can confirm that Runx2 has a role to bone formation.

• The Journal of Korean Academy of Prosthodontics
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• v.53 no.3
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• pp.198-206
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• 2015

Purpose: We aimed to investigate the effect of combined various microgrooves and thermal oxidation on the titanium (Ti) and to evaluate various in vitro responses of human periodontal ligament cells (PLCs). Materials and methods: Grade II titanium disks were fabricated. Microgrooves were applied on titanium discs to have $0/0{{\mu}m}$, $15/3.5{{\mu}m}$, $30/10{{\mu}m}$, and $60/10{{\mu}m}$ of respective width/depth by photolithography. Thermal oxidation was performed on the microgrooves of Ti substrata for 3 h at $700^{\circ}C$ in air. The experiments were divided into 3 groups: control group (ST), thermal oxidation group (ST/TO), and combined microgrooves and thermal oxidation group (Gr15-TO, Gr30-TO, Gr60-TO). Surface characterization was performed by field-emission scanning microscopy. Cell adhesion, osteoblastic differentiation, and mineralization were analyzed using the bromodeoxyurdine (BrdU), Alkaline phosphatase (ALP) activity, and extracellular calcium deposition assays, respectively. Statistical analysis was performed using the oneway analysis of variance and Pearson's bivariate correlation analysis (SPSS Version 17.0). Results: In general, the combined microgrooves and thermal oxidation group (Gr15-TO, Gr30-TO, Gr60-TO) showed significantly higher levels compared with the control (ST) or thermal oxidation (ST-TO) groups in the BrdU expression, ALP activity, and extracellular calcium deposition. Gr60-TO group induced highest levels of cell adhesion and osteoblastic differentiation. Conclusion: Within the limitation of this study, we conclude that the Ti surface treatment using combined microgrooves and thermal oxidation is highly effective in inducing the cell adhesion andosteoblastic differentiation. The propose surface is also expected to be effective in inducing rapid and strong osseointegration of Ti oral implants.

• Journal of the korean academy of Pediatric Dentistry
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• v.28 no.2
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• pp.207-218
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• 2001

The purpose of this study was to investigate the direct inhibitory effect of calcium hydroxide materials on differentiation and activation of osteoclast. we used the osteoclast progenitor cells isolated from bone marrow cell of chick embryo tibia and four experimental materials [$Ca(OH)_2$ powder, Metapaste$^{(R)}$, Vitapex$^{(R)}$, Pulpdent$^{(R)}$] diluted at 0.1, 0.01, $0.05{\mu}g/ml$. There were measured both the number of differentiated osteoclast and the area of resorption lacunae. Also, we conducted MTT assay on U2OS osteoblast to examine of cytotoxic effect and obtained following result. 1. Considering the result of the inhibitory effects upon osteoclast differentiation, There were shown a significant difference increased in the following order: Metapaste$^{(R)}$, $Ca(OH)_2$ powder, Vitapex$^{(R)}$. But no significant difference was found in pulpdent group that the number of differentiated osteoclast was increased at all concentrations(p<0.05). 2. Among the three experimental groups, that is, $Ca(OH)_2$ powder, Metapaste$^{(R)}$, Vitapex$^{(R)}$ at $0.1{\mu}g/ml$ dilution that were statistically significant in reduction of the number of differentiated osteoclast. Vitapex group showed significant cytotoxic effect compared to control and another two groups exhibited no significant difference. Also, 0.2% DMSO group was shown statistically siginificant cytotoxicity (p<0.05). 3. Examining pattern and measured area of resorption lacunae in the control and the three experimental groups ,that is, $Ca(OH)_2$ powder, Metapaste$^{(R)}$, Vitapex$^{(R)}$ at $0.1{\mu}g/ml$ dilution, except $Ca(OH)_2$ powder group, statistically significant differences were found between experimental groups and control group. Also, DMSO group showed statistically significant decrease (p<0.05). From these results, we think that calcium hydroxide is responsible for suppression of hard tissue resorption by a direct inhibition of dfferentiation and activation of osteoclast.

• Journal of Dental Rehabilitation and Applied Science
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• v.25 no.4
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• pp.361-374
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• 2009

For successful osteogenesis around the implants, interaction between implant surface and surrounding tissue is important. Biomechanical bonding and biochemical bonding are considered to influence the response of adherent cells. But the focus has shifted surface chemistry. The purpose of this study is to evaluate the MC3T3-E1 osteoblast like cell responses of magnesium (Mg) ion implanted titanium surface produced using a plasma source ion implantation method. Commercially pure titanium disc was used as substrates. The discs were prepared to produce four different surface, A: Machine turned surface, B: Mg implanted surface, C: sandblasted surface, D: sandblasted and Mg implanted surface. MC3T3 El osteoblastic like cells were cultured on the disc specimens. Cell adhesion, proliferation, differentiation, and synthesis of extracellular matrix were evaluated. The cell adhesion morphology was evaluated by SEM. RT PCR assay was used for assessment of cell adhesion, proliferation and differentiation. ALP activity was measured for cell differentiation. The results of this study were as follows: 1. SEM showed that cell on Mg ion groups was more proliferative than that of non Mg ion groups. On the machine turned surface, cell showed some degree of contact guidance in aligning with the machining grooves. 2. In RT PCR analysis, osteonectin and c-fos mRNA were more expressed on sandblasted and Mg ion implanted group. 3. ALP activity was not significantly different among all groups. Within the limitations of this study, the following conclusions were drawn: It might indicate Mg ion implanted titanium surface induce better bone response than non Mg ion groups.

• Journal of Periodontal and Implant Science
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• v.33 no.3
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• pp.457-474
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• 2003

The ultimate objective of periodontal treatment is to get rid of an on-going periodontal disease and further regenerate the supporting tissue, which is already destroyed, functionally. Currently, the bone grafting operation using various kinds of bone grafting materials and the operation for induced regeneration of periodontal tissue using the blocking membrane are performed for regeneration of the destroyed periodontal tissue. However, there are respective limitations Galenical preparations, which are used for regeneration of periodontal of tissue, has less risk of rejective reaction or toxicity that may be incidental to degradation and their effect is sustainable. Thus, in case they are applicable to a clinic, they can he used economically. Chitosan has such compatibility, biological actions including antibacterial activity, acceleration of wound treatment, etc., and excellent mechanical characteristics, which has recently aroused more interest in it. Also, it has been reported that it promotes osteogenesis directly or indirectly by functioning as a matrix to promote migration and differentiation of a specific precussor cell (for example, osteoblast) and further inhibiting the function of such a cell as fibroblast to prevent osteogenesis. In this study, the pure chitosan solution, which was obtained by purifying chitosan, was used. However, since this chitosan is of a liquiform, it is difficult to sustain it in a defective region. It is, therefore, essential to use a carrier for delivering chitosan to, and sustaining it gradually in the defective region. In the calvarial defect model of the Sprague-Dawley rat, it is relatively easy to maintain a space. Therefore, in this study, the chitosan solution with which ACS was wetted was grafted onto the defective region, For an experimental model, a calvarial defect of rat m s selected, and a critical size of the defective region was a circular defect with a diameter of 8 mm. A group in which no treatment was conducted for the calvarial defect was set as a negative control group. Another group in which treatment was conducted with ACS only was set as a positive control group (ACS group). And another group in which treatment was conducted was conducted with by grafting the pure chitosan solution onto the defective region through ACS which was wetted with the chitosan solution was set an experimental group (Chitosan/ACS group). Chitosan was applied to the Sprague-Dawley rat's calvarial bone by applying ACS which was wetted with the chitosan solution, and each Sprague-Dawley rat was sacrificed respectively 2 weeks and 8 weeks after the operation for such application. Then, the treatment results were compared and observed histologically and his tometrically. Thereby, the following conclusions were obtained. 1. In the experimental group, a pattern was shown that from 2 weeks after the operation, vascular proliferation proceeded and osteogenesis proceeded through osteoblast infiltration, and at 8 week after the operation, ACS was almost absorbed, the amount of osteogensis was increased and many osteoid tissue layers were observed. 2. At 2 weeks after the operation, each amount of osteogenesis appeared to be 8.70.8 %, 13.62.3 % and 4.80.7 % respectively in the experimental group, the positive control group and the negative control group. Accordingly, it appeared to be higher in the Experimental group and the positive control group than in the negative control group, but there was no significant difference statistically (p<0.01). 3. At 8 weeks after the operation, each amount of osteogenesis appeared to be 62.26.1%, 17.42.5 % and 8.21.4 % respectively in the experimental group, the positive control group and the negative control group. Accordingly, it appeared to be substantially higher in the experimental group than in the positive control group and the negative control group, and there was a significant difference statistically (p<0.01). As a result of conducting the experiment, when ACS was used as a carrier for chitosan, chitosan showed effective osteogenesis in the perforated defective region of the Sprague-Dawley rat's calvarial bone.

• Journal of Periodontal and Implant Science
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• v.31 no.1
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• pp.1-23
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• 2001

Chitosan is biodegradable natural polymer that has been demonstrated its ability to improve wound healing, and calcium metaphosphate(CMP) is a unique class of phosphate minerals having a polymeric structure. In this study, chitosan/CMP and platelet derived growth factor(PDGF-BB) loaded chitosan/CMP sponges were developed, and the effect of the sponges on bone regeneration and their possibility as scaffolds for bone formation by three-dimensional osteoblast culture were examined. PDGF-BB loaded chitosan/CMP sponges were prepared by freeze-drying of a mixture of chitosan solution and CMP powder, and soaking in a PDGF-BB solution. Fabricated sponge retained its 3-dimensional porous structure with $100-200\;{\mu}m$ pores. The release kinetics of PDGF-BB loaded onto the sponge were measured in vitro with $^{125}I-labeled$ PDGF-BB. In order to examine their possibility as scaffolds for bone formation, fetal rat calvarial osteoblastic cells were isolated, cultured, and seeded into the sponges. The cell-sponge constructs were cultured for 28 days. Cell proliferation, alkaline phosphatase activity were measured at 1, 7, 14 and 28 days, and histologic examination was performed. In order to examine the effect on the healing of bone defect, the sponges were implanted into rat calvarial defects. Rats were sacrificed 2 and 4 weeks after implantation and histologic and histomorphometrical examination were performed. An effective therapeutic concentration of PDGF-BB following a high initial burst release was maintained throughout the examination period. PDGF-BB loaded chitosan/CMP sponges supported the proliferation of seeded osteoblastic cells as well as their differentiation as indicated by high alkaline phosphatase activities. Histologic findings indicated that seeded osteoblastic cells well attached to sponge matrices and proliferated in a multi-layer fashion. In the experiments of implantation in rat calvarial defects, histologic and histomorphometric examination revealed that chitosan/CMP sponge promoted osseous healing as compared to controls. PDGF-BB loaded chitosan/CMP sponge further echanced bone regeneration. These results suggested that PDGF-BB loaded chitosan/CMP sponge was a feasable scaffolding material to grow osteoblast in a three-dimentional structure for transplantation into a site for bone regeneration.