• Journal of Periodontal and Implant Science
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• 제35권3호
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• pp.747-760
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• 2005

Periodontal therapy has dealt primarily with attempts at arresting progression of disease. however, more recent techniques have focused on regenerating the periodontal ligament having the capacity to regenerate the periodontium. Recombinant human bone morphogenetic protein-7(rhBMP-7) can differentiate the osteoprogenitor cells and induce bone formation. The purpose of this study was to evaluate the effect of BMP-7 on rat periodontal ligament cells differentiation, in vitro. In the control group, cells was cultured with DMEM media. In the experimental groups, cells were cultured with rhBMP-7 in concentration of 10, 25, 50 and 100 ng/ml. Each group was characterized by examining alkaline phosphatase activity at 3 and 5 days of culture and the ability to produce mineralized nodules of rat calvarial cells at 14 days of culture. Synthesis of type I collagen(COL-I), osteocalcin(OCN), and bone sialoprotein(BSP) was evaluated by RT-PCR at 7 days of culture. Activation of Smad proteins and p38 MAP kinase was determined by western blot analysis of the cell lysates. Alkaline phosphatase activity was significantly increased in the concentration of BMP-7 50 ng/ml and 100 ng/ml compared to the control(p<0.05). The mineralized bone nodule formation was greater with addition of 50 ng/ml and 100 ng/ml BMP-7 than the control(p<0.01). In 7 days' culture, the expressions of COL-I, BSP, and OCN was increased by BMP-7 in concentration of 10 $ng/ml{\sim}100$ ng/ml. In western blot analysis, BMP-7 treated culture cells expressed Smad 1,5,8 in dose-dependent manner, whereas BMP-7 did not activate phosphorylated form of p38 MAP kinase. These result suggested that BMP-7 stimulate rat periodontal ligament cells to differentiate toward osteoblast phenotype and increase bone matrix production by activation of BMP-Smad pathway.

• Journal of Korean Neurosurgical Society
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• 제60권4호
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• pp.397-403
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• 2017

Objective : Cranioplasty using a cryopreserved skull flap is a wide spread practice. The most well-known complications of cranioplasty are postoperative surgical infections and bone flap resorption. In order to find biological evidence of cryopreserved cranioplasty, we investigated microorganism contamination of cryopreserved skulls and cultured osteoblasts from cryopreserved skulls. Methods : Cryopreserved skull flaps of expired patients stored in a bone bank were used. Cryopreserved skulls were packaged in a plastic bag and wrapped with cotton cloth twice. After being crushed by a hammer, cancellous bone between the inner and outer table was obtained. The cancellous bone chips were thawed in a water bath of $30^{\circ}C$ rapidly. After this, osteoblast culture and general microorganism culture were executed. Osteoblast cultures were done for 3 weeks. Microorganism cultures were done for 72 hours. Results : A total of 47 cryopreserved skull flaps obtained from craniectomy was enrolled. Of the sample, 11 people were women, and the average age of patients was 55.8 years. Twenty four people had traumatic brain injuries, and 23 people had vascular diseases. Among the patients with traumatic brain injuries, two had fracture compound comminuted depressed. The duration of cryopreservation was, on average, 83.2 months (9 to 161 months). No cultured osteoblast was observed. No microorganisms were cultured. Conclusion : In this study, neither microorganisms nor osteoblasts were cultured. The biological validity of cryopreserved skulls cranioplasty was considered low. However, the usage of cryopreserved skulls for cranioplasty is worthy of further investigation in the aspect of cost-effectiveness and risk-benefit of post-cranioplasty infection.

• International Journal of Oral Biology
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• 제33권4호
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• pp.179-186
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• 2008

Several lines of evidence suggest that osteocytes play a critical role in bone remodeling. Both healthy and apoptotic osteocytes can send signals to other bone surface cells such as osteoblasts, osteoclasts, osteoclast precursors, and bone lining cells through canalicular networks. Osteocytes responding to mechanical strain may also send signals to other cells. To determine the role for osteocytes an mechanical strain in bone remodeling, we examined the effects of fluid flow shear stress on osteoclast precursor cell and osteoblast proliferation and recruitment induced by osteocytes. In addition, the effects of fluid flow shear stress on osteocyte M-CSF, RANKL, and OPG mRNA expression were also examined. MLO-Y4 cells were used as an in vitro model for osteocytes, RAW 264.7 cells and MOCP-5 cells as osteoclast precursors, and 2T3 cells as osteoblasts. MLO-Y4 cells conditioned medium (Y4-CM) was collected after 24h culture. For fluid flow experiments, MLO-Y4 cells were exposed to 2h of pulsatile fluid flow (PFF) at 2, 4, 8, $16{\pm}0.6\;dynes/cm^2$ using the Flexcell $Streamer^{TM}$ system. For proliferation assays, MOCP-5, RAW 264.7, and 2T3 cells were cultured with control media or 10-100% Y4 CM. Cells were cultured for 3d, and then cells were counted. RAW 264.7 and 2T3 cell migration was assayed using transwells with control media or 10-100% Y4-CM. M-CSF, RANKL and OPG in MLO-Y4 mRNA expression was determined by semiquantitative RT-PCR. Y4-CM increased osteoclast precursor proliferation and migration, but decreased 2T3 cell proliferation and migration. CM from MLO-Y4 cells exposed to PFF caused decreased RAW 267.4 cell proliferation and migration and 2T3 migration compared to control Y4-CM. However, Y4-CM from cells exposed to PFF had no effect on 2T3 osteoblastic cell proliferation. PFF decreased RNAKL mRNA and increased OPG mRNA in MLO-Y4 cells compared to control(without PFF). PFF had no effect on M-CSF mRNA expression in MLO-Y4 cells. These results suggest that osteocytes can regulate bone remodeling by communication with osteoclast precursors and osteoblasts and that osteocytes can communicate mechanical signals to other cells.

• Corrosion Science and Technology
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• 제8권2호
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• pp.62-67
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• 2009

Ti and Ti-alloys have good biocompatibility, appropriate mechanical properties and excellent corrosion resistance. However, the widely used Ti-6Al-4V is found to release toxic ions (Al and V) into the body, leading to undesirable long-term effects. Ti-6Al-4V has much higher elastic modulus (100 GPa) than cortical bone (20 GPa). Therefore, titanium alloys with low elastic modulus have been developed as biomaterials to minimize stress shielding. The electrochemical behavior of surface-modified and MC3T3-E1 cell-cultured Ti-30(Nb,Ta) alloys with low elastic modulus have been investigated using various electrochemical methods. Surfaces of test samples were treated as follows: $0.3{\mu}m$ polished; $25{\mu}m$, $50{\mu}m$ and $125{\mu}m$ sandblasted. Specimen surfaces were cultured with MC3T3-E1 cells for 2 days. Average surface roughness ($R_a$) and morphology of specimens were determined using a surface profilometer, OM, and FE-SEM. Corrosion behavior was investigated using a potentiostat(EG&G PARSTAT 2273), and electrochemical impedance spectroscopy was performed (10 mHz to 100 kHz) in 0.9% NaCl solution at $36.5{\pm}1^{\circ}C$. The microstructures of the Ti-30(Ta,Nb) alloys had a needle-like appearance. The $R_a$ of polished Ti-30Ta and Ti-30Nb alloys was lower than that of the sandblasted Ti alloy. Cultured cells displayed round shapes. For polished alloy samples, cells were well-cultured on all surfaces compared to sandblasted alloy samples. In sandblasted and cell-cultured Ti-30(Nb,Ta) alloy, the pitting potential decreased and passive current density increased as $R_a$ increased. Anodic polarization curves of cell-cultured Ti alloys showed unstable behavior in the passive region compared to non-cell-cultured alloys. From impedance tests of sandblasted and cell-cultured alloys, the polarization resistance decreased as $R_a$ increased, whereas, $R_a$ for cell-cultured Ti alloys increased compared to non-cell-cultured Ti alloys.

• Journal of Periodontal and Implant Science
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• 제35권4호
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• pp.1097-1108
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• 2005

The present study was to determine the influence of micro-macro biphasic calcium phosphate(MBCP) on proliferation and differentiation of human marrow-derived mesenchymal stem cells. Primary stem cells were cultured from bone marrow and 3-4 passaged cells were used. This study tested the proliferative effects by cell counting. Collagen sythensis, alkaline phosphatase activity, expression of osteocalcin and bone sialoprotein by Western blot analysis were evaluated. The cellular proliferation of ASC was not influenced by MBCP. Collagen synthesis of ASC cultured on MBCP significantly increased at 5th and 7th days(p<0.05). The ALP activity in ASC cultured on MBCP significantly increased at 5th and 7th days(p<0.05). The expression of OC and BSP incresaed in ASC cultured on MBCP. These results suggest that MBCP may stimulates the osteoblastic activity of ASC.

• Journal of Periodontal and Implant Science
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• 제32권4호
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• pp.865-873
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• 2002

The primary cause of tooth loss after 30 years of age is periodontal disease. Destruction of alveolar bone by periodontal disease is done by bone resorbing activity of osteoclasts. Understanding differentiation and activation mechanism of osteoclasts is essential for controling periodontal disease. The purpose of this study is to identify the possible effects of Vitamin D and cytokines affecting osteoclasts and its precursor cells. Four to six week-old mice were killed and humerus, radius, tibia and femur were removed aseptically and washed two times with Hank's solution containing penicillin-streptomycin and then soft tissue were removed. Bone marrow cells were collected by 22 gauge needle. Cells were cultured in Hank's solution containing 1 mg/ml type II collagenase, 0.05% trypsin, 41mM EDTA. Supernatant solution was removed 5 times after 15 minutes of digestion with above mentioned enzyme solution, and remained bone particles were maintained in alpha-MEM for 15 minutes and $4^{\circ}C$ temperature. Bone particles were agitated for 1 minute and supernatant solution containing osteoclast precursor cells were filtrated with cell stainer. These separated osteoclast precursor cells were dispensed with 100-mm culture dish by $1{\times}10^7$ cells unit and cultured in ${\alpha}$- MEM containing 20 ng/ml recombinant human M-CSF, 30 ng/ml recombinant human soluble osteoclast differentiation factor and 10% fetal calf serum for 2 and 7 days. Total RNA of osteoclast precursor cells were extracted using RNeasy kit. One ${\mu}g$ of total RNA was reverse transcribed in $42^{\circ}C$ for 30 minutes using SuperScriptII reverse transcriptase. Expression of transcribed receptors of each hormone and cytokine were traced with 1 ${\mu}l$ of cDNA solution by PCR amplification. Vitamin D receptor WAS found in cells cultured for 7 days. TNF-${\alpha}$ receptor was found in cells cultured for 2 days and amount of receptors were increased by 7 days. IL-1 type I receptor was not found in cells cultured 2 and 7 days. But, IL-1 receptor type II was found in cells cultured for 2 days. TGF-${\alpha},{\beta}$type I receptor was found in cells cultured 2 and 7 days, and amount of receptors were increased by 7 days of culture. These results implies Vitamin D and cytokines can affect osteoclasts directly, and affecting period in differentiation cycle of osteoclasts is different by Vitamin D and cytokines.

• Animal cells and systems
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• 제4권3호
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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.

• Archives of Plastic Surgery
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• 제34권2호
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• pp.156-162
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• 2007

Purpose: In the previous in vitro studies the bone marrow stromal cells(BSCs) have shown the superior effect for wound healing activity than fibroblasts, which includes cell proliferation, type I collagen synthesis, and the production of bFGF, VEGF and TGF-${\beta}$ in chronic wound healing. The aim of this study is to compare the effects of BSCs and fibroblasts on wound healing activity in vivo, especially on collagen synthesis. Methods: The fibroblasts and BSCs were harvested from patients and cultured. The cultured cells were infiltrated into the pores of polyethylene discs. These discs were divided into three groups according to the mixed cells. In groups I, II and III the discs were loaded with no cells, fibroblasts and BSCs, respectively. Twelve discs per group(total 36 discs) were made for this study. After creating 6 pockets in the back of each rats, each discs was implanted into each pockets. At three time intervals from 1 to 3 weeks, the implanted discs were harvested for the histological and quantitative analysis. The amount of collagen produced was evaluated using ELISA. Statistical comparisons were made using the Mann-Whitney U-test. Results: There was great difference in the collagen synthesis among the three groups by the 1st and 2nd weeks. The BSC group showed highest collagen level, followed by fibroblast group and no cell group(p<0.05). The 3rd week specimens also showed greater collagen amount in BSC and fibroblast groups compared to those of no cell group(p<0.05). However, there was little difference between BSC and fibroblast groups. Conclusion: This result demonstrates that BSC has superior effect on stimulating wound healing than fibroblast, which is currently used for wound healing.

• Journal of Periodontal and Implant Science
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• 제34권4호
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• pp.759-769
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• 2004

Periodontal therapy has dealt primarily with attempts at arresting progression of disease, however, more recent techniques have focused on regenerating the periodontal ligament having the capacity to regenerate the periodontium. The effect of chitosan, a carbohydrate biopolymer extracted from chitin, on periodontal ligament regeneration is of particular interest. The purpose of this study was to evaluate the effect of chitosan on the expression of extracellular matrix proteins in primary rat calvarial cells in Vitro. In the control group, cells was cultured with BGjb media. In the experimental groups, cells were cultured with chitosan in concentration of 0.01, 0.1, 1.0 and 2.0 mg/ml. Then each group was characterized by examining alkaline phosphatase activity at 3 and 7 days, and the ability to produce mineralized nodules of rat calvarial cells at 14 and 21 days. Synthesis of type I collagen (COL-I), osteocalcin (OCN), bone sialoprotein (BSP) was evaluated by RT-PCR at 14 days. The results were as follows: 1. Alkaline phosphatase activity was significantly higher in the concentration of chitosan 0.01mg/ml, 0.1mg/ml and 1.0mg/ml compared to control (p<0.05). 2. The percentage of mineralized bone nodule was more in the concentration of chitosan 0.1mg/ml and 1.0mg/ml than the control. 3. At 14 day culture, the expression of OCN was increased by chitosan in concentration of 1.0 mg/ml and 2.0 mg/ml. These results suggested that chitosan in concentration of 0.1 and 1,0 mg/ml stimulate the extracellular matrix of primary rat calvarial cells and may facilitate the formation of bone.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제27권2호
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• pp.103-110
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• 2001

This study was performed to observe the effect of ultrasound(1.0MHz, $0.75W/cm^2\;and\;1.0W/cm^2$) irradiation on cultured MC3T3-E1 cell, osteoblastic like cell with respect to the proliferation, protein synthesis, and alkaline phosphatase activity of the cells. The results were as follows: 1. The proliferation of MC3T3-E1 cells was increased on ultrasound irradiated group compared with control group. 2. The protein synthesis was not apparently increased on ultrasound irradiated group compared with control group. 3. The alkaline phosphatase activity level was not apparently increased on ultrasound irradiated group compared with control group. From the above results and other literatures, we could suggest that the ultrasound with the appropriate intensity and frequency may have important roles in stimulation of cell proliferation. Therefore the ultrasound may be used in the acceleration of the bone regeneration and bone fracture healing.