• 약학회지
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• 제40권5호
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• pp.550-557
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• 1996

One of therapeutics in liver disease (morbus wilson) is D-penicillamin (D-pen: D-3-mercapto-valin). Especially the cross-linking of collagen molecules could be inhibited by D-pe n in extracellular space. In this study we investigated the antifibrotic effects of D-pen in rats that were induced the liver fibrosis by bile duct ligation and scission (BDL/S). Rats were treated for 4 weeks with D-pen after BDL/S operation or sham operation. The balance between fibrogenesis-marker (PNIIIP) and the fibrolysis-maker (PNIVP) were observed in sera by RIA (radioimmunoassay), and the parameter of collagen deposition in liver tissue (hydroxyproline: HYP) was measured by colorimetry. The weight of liver in BDL/S operated group was increased significantly in compared with sham operation group (15.2g${\pm}$1.1, vs 11.9g${\pm}$3.9: p<0.005, p<0.05). The rats group treated by D-pen showed the lower level of PNIIIP (6.7ng/ml${\pm}$1.5, vs 9.5ng/ml${\pm}$2.8) and the higher value of PIVCP (14.0ng/ml${\pm}$1.9, vs 7.9ng/ml${\pm}$1.5) in sera that compared to untreated rats. The content of HYP was decreased by 141% in BDL/S with D-pen treated group than that of it in BDL/S group. No correlation was revealed between collagen parameters in sera and HYP in liver tissue of BDL/S operated and D-pen treated rats. The group treated with D-pen showed the lower value of clinical biochemistry parameters (GOT: glutamate oxalacetate transaminase, Total-Bilirubin) in compared with only BDL/S operated rats, but the value of GPT (glutamate pyruvate transaminase) and Alkaline phosphatase in two BDL/S groups was nearly same. In the histological finding, we observed mild bile duct proliferation, weak inflammation and fibrosis in BDL/S with D-pen treated group, but BDL/S operated group showed the formation of septum (island of hepatocytes), massive bile duct proliferation. This result represents that the BDL/S operation induces liver fibrosis (cirrhosis) in 4 weeks, and D-pen inhibits the synthesis of collagen weakly and stimulates the degradation of collagen in the extracellular space. We conclude that the monitoring of PNIIIP, PIVCP in sera is useful parameter for screening of antifibrotic effect, and D-pen delay the liver fibrosis.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제39권
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• pp.7.1-7.9
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• 2017

Background: This study was to investigate the effect of biomechanical stimulation on osteoblast differentiation of human periosteal-derived stem cell using the newly developed bioreactor. Methods: Human periosteal-derived stem cells were harvested from the mandible during the extraction of an impacted third molar. Using the new bioreactor, 4% cyclic equibiaxial tension force (0.5 Hz) was applied for 2 and 8 h on the stem cells and cultured for 3, 7, and 14 days on the osteogenic medium. Biochemical changes of the osteoblasts after the biomechanical stimulation were investigated. No treatment group was referred to as control group. Results: Alkaline phosphatase (ALP) activity and ALP messenger RNA (mRNA) expression level were higher in the strain group than those in the control group. The osteocalcin and osteonectin mRNA expressions were higher in the strain group compared to those in the control group on days 7 and 14. The vascular endothelial growth factor (VEGF) mRNA expression was higher in the strain group in comparison to that in the control group. Concentration of alizarin red S corresponding to calcium content was higher in the strain group than in the control group. Conclusions: The study suggests that cyclic tension force could influence the osteoblast differentiation of periosteal-derived stem cells under optimal stimulation condition and the force could be applicable for tissue engineering.

• International Journal of Oral Biology
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• 제36권4호
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• pp.173-178
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• 2011

Tumor necrosis factor alpha ($TNF{\alpha}$) is a multifunctional cytokine that is elevated in inflammatory diseases such as atherosclerosis, diabetes and rheumatoid arthritis. Recent evidence has suggested that ${\beta}2$ adrenergic receptor (${\beta}2AR$) activation in osteoblasts suppresses osteogenic activity. In the present study, we explored whether $TNF{\alpha}$ modulates ${\beta}AR$ expression in osteoblastic cells and whether this regulation is associated with the inhibition of osteoblast differentiation by $TNF{\alpha}$. In the experiments, we used C2C12 cells, MC3T3-E1 cells and primary cultured mouse bone marrow stromal cells. Among the three subtypes of ${\beta}AR$, ${\beta}2$ and ${\beta}3AR$ were found in our analysis to be upregulated by $TNF{\alpha}$. Moreover, isoproterenol-induced cAMP production was observed to be significantly enhanced in $TNF{\alpha}$-primed C2C12 cells, indicating that $TNF{\alpha}$ enhances ${\beta}2AR$ signaling in osteoblasts. $TNF{\alpha}$ was further found in C2C12 cells to suppress bone morphogenetic protein 2-induced alkaline phosphatase (ALP) activity and the expression of osteogenic marker genes including Runx2, ALP and osteocalcin. Propranolol, a ${\beta}2AR$ antagonist, attenuated this $TNF{\alpha}$ suppression of osteogenic differentiation. $TNF{\alpha}$ increased the expression of receptor activator of NF-${\kappa}B$ ligand (RANKL), an essential osteoclastogenic factor, in C2C12 cells which was again blocked by propranolol. In summary, our data show that $TNF{\alpha}$ increases ${\beta}2AR$ expression in osteoblasts and that a blockade of ${\beta}2AR$ attenuates the suppression of osteogenic differentiation and stimulation of RANKL expression by $TNF{\alpha}$. These findings imply that a crosstalk between $TNF{\alpha}$ and ${\beta}2AR$ signaling pathways might occur in osteoblasts to modulate their function.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제34권4호
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• pp.419-427
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• 2008

The present study aimed to investigate the osteogenic potentials of differentiated osteoblast-like cells (DOCs) induced from bone marrow-derived mesenchymal stem cells (MSCs) on ${\beta}-tricalcium$ phosphate (${\beta}-TCP$) with recombinant human bone morphogenetic protein (rhBMP-2) in vitro. Osteoblast differentiation was induced in confluent cultures by adding 100 nM dexamethasone, 10 mM ${\beta}$-glycerophosphate, 50 mM L-ascorbic acid. The Alizarin red S staining and reverse transcriptase-polymerase chain reaction (RT-PCR) were perfomed to examine the mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), receptor activator for nuclear factor ${\kappa}B$ ligand (RANKL), runt-related transcription factor 2 (RUNX2), collagen-Ⅰ (COL-Ⅰ). There were no significant differences in the osteogenic potentials of DOCs induced from MSCs on ${\beta}-TCP(+/-)$. According to the incubation period, there were significant increasing of Alizadin red S staining in the induction 3 weeks. The mRNA expression of ALP, RUNX2, and RANKL were higher in DOCs/${\beta}-TCP(-)$ than DOCs/${\beta}-TCP(+)$. According to rhBMP-2 concentrations, the mRNA expression of BSP was significantly increased in DOCs/${\beta}-TCP(+)$ compared to that of DOCs/${\beta}-TCP(-)$ on rhBMP 10 ng/ml. Our study presented the ${\beta}-TCP$ will have the possibility that calcium phosphate directly affect the osteoblastic differentiation of the bone marrowderived MSCs.

• 치위생과학회지
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• 제16권1호
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• pp.70-76
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• 2016

이번 연구의 목적은 하이드록시아파타이트를 이용하여 블라스팅 처리한 뒤 양극산화 방법을 이용하여 하이드록시아파타이트와 이산화티탄이 복합된 표면을 만들고, 이에 대해 세포부착양상을 관찰하고 ALP 활성도와 칼슘 침착량을 측정함으로써 세포 분화능을 평가하여 표면의 생체적합성을 평가하는 데 있다. 이를 위해 실험을 진행한 결과 다음과 같은 결과를 얻었다. 접촉각 분석 결과, 복합처리된 HA+100과 HA+MAO 150은 유의하게 낮은 접촉각을 나타내었다(p<0.05). 세포부착 관찰 결과 연마 처리한 SM 시편에서는 납작한 모양으로 세포가 붙어있는 모습을 관찰할 수 있었으나, 표면 처리된 실험군에서는 세포의 모양이 시편을 따라 좀 더 길게 뻗어 부착된 모습을 관찰할 수 있었다. ALP 활성도 측정 결과 HA+MAO 150과 HA+MAO 200은 1, 2, 3주 모든 기간에서 가장 높은 ALP 활성도를 나타내었다(p>0.05). 칼슘양 측정 결과 HA+MAO 150 과 HA+MAO 200은 1, 2, 3주 모든 기간에서 가장 많은 칼슘양을 나타내었다(p<0.05). 따라서 하이드록시아파타이트 이산화티탄이 복합 코팅된 표면은 높은 표면에너지를 가지며 우수한 세포활성도를 나타내어 치과용 임플란트 표면으로 유용하게 사용될 수 있을 것이라고 생각된다.

• Journal of Periodontal and Implant Science
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• 제30권1호
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• pp.39-52
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• 2000

Polypeptide growth factor belong to a class of potent biologic mediator which regulate cell differentiation, proliferation, migration and metabolism. 1,25-dihydroxyvitamin $D_3$ decrease cell proliferation, and stimulate alkaline phosphatase activity which express in osteoblast during cell differentiation period. IGF-I is known to stimulate cell proliferation and differentiation too. 1,25-dihydroxyvitamin $D_3$ is known to increase IGF-I binding sites and IGF binding protein which inhibite the effect of IGF. The purpose of this study is to evaluate potential role of IGF-I as mediator that control the action of 1,25-dihydroxyvitamin $D_3$. MC3T3-E1 cell were seeded $5{\times}10^5/ml$ at 100mm culture plate in ${\alpha}-MEM$ containing 10% fetal bovine serum. After 48 hour incubation period, medium were changed ${\alpha}-MEM$ containing 5% fetal bovine serum. After 24 hours, $10^{-9}M$ 1,25-dihydroxyvitamin $D_3$ added. Total mRNA was extracted at 0, 6, 24, 48, 72 hour. PRPCR method was programed for the detection of IGF-I mRNA. In the both groups of 1,25-dihydroxy vitamin $D_3$ treated and control, alternative splicing form of IGF-I, IGF-IA and IGF-IB were expressed. In the 1,25-dihydroxyvitamin $D_3$ treated group, IGF-I mRNA expression was matained until 24 hour, there after expression was decresed. MC3T3-E1 cell were seeded $2.5{\times}10^4/ml$ at 24well plate in ${\alpha}-MEM$ containing 10% fetal bovine serum. After 48 hour incubation period, medium were changed ${\alpha}-MEM$ containing 3% fetal bovine serum. After 24 hours, $10^{-9}M$ 1,25-dihydroxyvitamin $D_3$ and 10 ng/ml IGF-I were added separately or together. Cell were cultured for 1 and 3 days, $2{\mu}Ci/ml\;[^3H]$ -thymidine was added for the last 24h of culture of each days. ${[^3H]}$-thymidine incorporation in to DNA was measured and expressed counter per minute(CPM). DNA synthetic activity was significantly decreased by 1,25-dihydroxyvitamin $D_3$ both at 1 day and 3 day, and in the combination group of 1,25-dihydroxyvitamin $D_3$ and IGF-I, DNA synthetic activity was also decreased both at 1 day and 3 days. IGF-I did not affect the DNA synthetic activity compared to control group both at 1 day and 3 day. From the above results, 1,25-dihydroxyvitamin $D_3$ was potent inhibitor of cell proliferaton in MC3T3-E1 cells. It assumed that the effect of 1,25-dihydroxyvitamin $D_3$ on osteoblast proliferation may be mediated in part by decreased level of IGF-I.

• Journal of Periodontal and Implant Science
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• 제35권2호
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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.

• The Journal of Advanced Prosthodontics
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• 제7권6호
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• pp.496-505
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• 2015

PURPOSE. To determine the effect of fibronectin (FN)-conjugated, microgrooved titanium (Ti) on osteoblast differentiation and gene expression in human bone marrow-derived mesenchymal stem cells (MSCs). MATERIALS AND METHODS. Photolithography was used to fabricate the microgrooved Ti, and amine functionalization (silanization) was used to immobilize fibronectin on the titanium surfaces. Osteoblast differentiation and osteoblast marker gene expression were analyzed by means of alkaline phosphatase activity assay, extracellular calcium deposition assay, and quantitative real-time PCR. RESULTS. The conjugation of fibronectin on Ti significantly increased osteoblast differentiation in MSCs compared with non-conjugated Ti substrates. On the extracellular calcium deposition assays of MSCs at 21 days, an approximately two-fold increase in calcium concentration was observed on the etched 60-${\mu}m$-wide/10-${\mu}m$-deep microgrooved surface with fibronectin (E60/10FN) compared with the same surface without fibronectin (E60/10), and a more than four-fold increase in calcium concentration was observed on E60/10FN compared with the non-etched control (NE0) and etched control (E0) surfaces. Through a series of analyses to determine the expression of osteoblast marker genes, a significant increase in all the marker genes except type I collagen ${\alpha}1$ mRNA was seen with E60/10FN more than with any of the other groups, as compared with NE0. CONCLUSION. The FN-conjugated, microgrooved Ti substrate can provide an effective surface to promote osteoblast differentiation and osteoblast marker gene expression in MSCs.

• Journal of Periodontal and Implant Science
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• 제37권4호
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• pp.859-869
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• 2007

Naked DNA and standard vectors have been previously used for gene delivery. Among these, PEI can efficiently condense DNA and has high intrinsic endosomal activities. The aim of this study is to investigate whether the cationic polycation PEI could increase the transfection efficiency of BMP expressing DNA using a vector-loaded collagen sponge model. BMP-2/pcDNA3.1 plasmid was constructed by subcloning human BMP-2 cDNA into the pcDNA3.1 plasmid vector. PEI/DNA complexes were prepared by mixing PEI and BMP-2/pcDNA3.1 and the constructed complexes were loaded into the collagen sponges. In vitro studies, BMSCs were transfected with the PEI/BMP-2/pcDNA3.1 complexes from collgen sponge. The level of secreted BMP-2 and alkaline phosphatase activities of transfected BMSCs were significantly higher in PEI/BMP-2/pcDNA3.1 group than in BMP-2/pcDNA3.1 group (p<0.05). Transfected BMSCs were cultured and mineralization was observed only in cells treated with PEI/BMP-2/pcDNA3.1 complexes. In vivo studies, PEI/BMP-2/pcDNA3.1/collagen, BMP-2/pcDNA3.1/collagen and blank collagen were grafted in skeletal muscle of nude mice. Ectopic bone formation was shown in PEI/BMP-2/pcDNA3.1/collagen grafted mouse 4 weeks postimplantation, while not in BMP-2/pcDNA3.1 grafted tissue. This study suggests that PEI-condensed DNA encoding for BMP-2 is capable of inducing bone formation in ectopic site and might increase the transfection rate of BMP-2/pcDNA3.1. As a non-viral vector, PEI offers the potential in gene therapy for bone engineering.

• Archives of Pharmacal Research
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• 제28권7호
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• pp.791-798
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• 2005

The hepatoprotective activity of flavonol glycosides rich fraction (F-2), prepared from 70% alcohol extract of the aerial parts of V calcarata Desf., was evaluated in a rat model with a liver injury induced by daily oral administration of $CCl_4$ (100 mg/kg, b.w) for four weeks. Treatment of the animals with F-2 using a dose of (25 mg/kg, b.w) during the induction of hepatic damage by $CCl_4$ significantly reduced the indices of liver injuries. The hepatoprotective effects of F-2 significantly reduced the elevated levels of the following serum enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). The antioxidant activity of F-2 markedly ameliorated the antioxidant parameters including glutathione (GSH) content, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), plasma catalase (CAT) and packed erythrocytes glucose-6-phosphate dehydrogenase (G6PDH) to be comparable with normal control levels. In addition, it normalized liver malondialdehyde (MDA) levels and creatinine concentration. Chromatographic purification of F-2 resulted in the isolation of two flavonol glycosides that rarely occur in the plant kingdom, identified as quercetin-3,5-di-O-$\beta$-D-diglucoside (5) and kaempferol-3,5-di-O-$\beta$-D-diglucoside (4) in addition to the three known compounds identified as quercetin-3-O-$\alpha$-L-rhamnosyl- (${\rightarrow}6$)-$\beta$-D-glucoside [rutin, 3], quercetin-3-O-$\beta$-D-glucoside [isoquercitrin, 2] and kaempferol-3-O-$\beta$-D-glucoside [astragalin, 1]. These compounds were identified based on interpretation of their physical, chemical, and spectral data. Moreover, the spectrophotometric estimation of the flavonoids content revealed that the aerial parts of the plant contain an appreciable amount of flavonoids (0.89%) calculated as rutin. The data obtained from this study revealed that the flavonol glycosides of F-2 protect the rat liver from hepatic damage induced by $CCl_4$ through inhibition of lipid peroxidation caused by $CCl_4$ reactive free radicals.