• Imaging Science in Dentistry
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• v.38 no.3
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• pp.125-132
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• 2008

Purpose : To investigate the effects of irradiation on transforming growth factor ${\beta}_1$ (TGF-${\beta}_1$) mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line. Materials and Methods : Cells were cultured in alpha-minimum essential medium ($\alpha$-MEM) supplemented with 10% fetal bovine serum and antibiotics. When the cells reached the level of 70-80% confluence, culture media were changed with $\alpha$-MEM supplemented with 10% FBS, 5 mM $\beta$-glycerol phosphate, and $50\;{\mu}g/mL$ ascorbic acid. Thereafter the cells were irradiated with a single dose of 2, 4, 6, 8 Gy at a dose rate of 1.5 Gy/min. The expression pattern of TGF-${\beta}_1$ mRNA, calcium content and calcific nodule formation were examined on day 3, 7, 14, 21, 28, respectively, after the irradiation. Results : The amount of TGF-${\beta}_1$ mRNA expression decreased significantly on day 7 after irradiation of 4, 6, 8 Gy. It also decreased on day 14 after irradiation of 6, 8 Gy. and decreased on day 21 after irradiation of 8 Gy. The amount of calcium deposition decreased significantly on day 7 after irradiation of 4, 8 Gy (P < 0.01) and showed a decreased tendency on day 14, 21 after irradiation of 4, 6, 8 Gy. The number of calcific nodules was decreased on day 7 after irradiation of 4, 8 Gy. Conclusion: Irradiation with a single dose of 4, 6, 8 Gy influences negatively the bone formation at the molecular level by affecting the TGF-${\beta}_1$ mRNA expression that was associated with proliferation and the production of extracellular matrix in MC3T3-E1 osteoblastic cell line.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.4
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• pp.247-265
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• 2008

Objectives: Many types of liver diseases can damage regenerative potential of mature hepatocytes, hepatic progenitor cells or oval cells. In such cases, a stem cell-based therapy can be an alternative therapeutic option. We examined whether human amnion-derived mesenchymal stem cells (HAM) and human umbilical cord-derived stem cells (HUC) could differentiate into hepatocyte-like cells as therapeutic cells for the liver diseases. Methods: HAM and HUC were isolated from the amnion and umbilical cord of the volunteers after a caesarean section with informed consent. In order to differentiate these cells into hepatocyte-like cells, cells were cultivated in hepatogenic medium using culture plates coated with fibronectin. Effects of hepatocyte growth factor, L-ascorbic acid 2-phosphate, insulin premixture fibroblast growth gactor 4, dimethylsulfoxide, oncostatin M and/or dexamethasone were examined on the hepatic differentiation. After differentiation, the cells were analyzed by RT-PCR, immunocytochemistry, immunoblotting, albumin ELISA, urea assay and periodic acid-schiffs staining. Results: Initial fibroblast-like appearance of HAM and HUC changed to a round shape during culture in the hepatogenic medium. However, in all hepatogenic conditions examined, HUC secreted more amounts of albumin or urea into medium than HAM. Expression of some of hepatocyte-specific genes increased and expression of new genes were observed in HUC following cultivation in hepatogenic medium. Results of immunocytochemistry and immunoblotting analyses demonstrated that HUC secreted albumin into the culture medium. PAS staining further demonstrated that HUC could store glycogen inside of the cells. Conclusions: Both HUC and HAM could differentiate into albumin-secreting, hepatocyte-like cells. Under the same hepatogenic conditions examined, HUC more efficiently differentiated into hepatocyte-like cells compared with the HAM. The results suggest that HUC and HAM could be used as sources of stem cells for the cell-based therapeutics such as in liver diseases.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.4
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• pp.279-288
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• 2007

In the present study, we focused on stem cells in the dental papilla of the tooth germ. The tooth germ, sometimes called the tooth bud, is the primordial structure from which a tooth is formed. The tooth germ consists of the enamel organ, the dental papilla, and the dental follicle. The dental papilla lies below a cellular aggregation of the enamel organ. Mesenchymal cells within the dental papilla are responsible for formation of dentin and pulp of a tooth. Tooth germ disappears as a tooth is formed, but that of a third molar stays in the jawbone of a human until the age of 10 to 16, because third molars grow slowly. Impacted third molar tooth germs from young adults are sometimes extracted for orthodontic treatment. In the present study, we evaluated the osteogenic activity and mineralization of cultured human dental papilla-derived cells. Dental papillas were harvested from mandible during surgical extraction of lower impacted third molar from 3 patients aged 13-15 years. After passage 3, the dental papilla-derived cells were trypsinized and subsequently suspended in the osteogenic induction DMEM medium supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM -glycerophosphate at a density of $1\;{\times}10^6\;cells/dish$ in a 100-mm culture dish. The dental papilla-derived cells were then cultured for 6 weeks and the medium was changes every 3 days during the incubation period. Dental papilla-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 7 of culture period, then decreased in intensity during the culture period. ALP mRNA level was largely elevated at 1 weeks and gradually decreased with culture time. Osteocalcin mRNA expression appeared at day 14 in culture, after that its expression continuously increased in a time-dependent manner up to day 28. The expression remained constant thereafter. Runx2 expression appeared at day 7 with no detection thereafter. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. Osteocalcin secretion was detectable in the culture medium from 1 week. The secretion of osteocalcin from dental papilla-derived cells into the medium greatly increased after 3 weeks although it showed a shallow increase by then. In conclusion, our study showed that cultured human dental papilla-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix.

• Journal of Nutrition and Health
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• v.41 no.8
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• pp.701-710
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• 2008

The wild simulated ginseng (WSG) has been effectively used in folk medicine as a remedy against hepatic disease, hypertension and arthritic disease. However, there is still lack of scientific proof about its antioxidant capability. The present study has been conducted to evaluate the protective role of the WSG ethanol extract in the CCl4-induced oxidative stress and resultant hepatic disfunction in ICR mice. The electron donating abilities and IC50 of WSG etnanol extract were 76.86 ${\pm}$ 1.06% and 33.3 ${\mu}g$/mL (that of ascobic acid was 16.5 ${\mu}g$/mL), respectively. Total antioxidant status of WSG extract was 2.13 ${\pm}$ 0.06 mmoL/mg, while the values of ascorbic acid and BHT were 3.63 ${\pm}$ 0.06 and 3.12 ${\pm}$ 0.02, respectively. ICR mice (aged 3weeks) were fed for 4 weeks on AIN-93M diet and had free access to food and water. The animals were divided into three groups: normal group (intraperitoneally (i.p) injected with PBS at 100 ${\mu}L$/mouse), group C; CCl4-induced and without any treatment. (i.p injected only PBS, 100 ${\mu}L$ /mice), group G; CCl4-induced and treated with WSG (i.p injected with 5 mg WSG extract per mouse, suspended in 100 ${\mu}L$ phosphate buffer). After the i.p. injection of WSG or PBS (5 times for 7weeks), all mice were administered CCl4 in olive oil at the last day of the experiment, except for normal group. The normal group was administered only olive oil. Determination of plasma triglyceride, total cholersterol, fasting glucose and GPT activity was performed using automatic blood analyzer. To evaluate the protective effect against the oxidative stress, DNA fragmentation and TBARS were determined in blood leucocytes and RBC and hepatocyte, respectively. Body and organs weights and food intake did not show significant differences among the groups. Blood total cholesterol of group G was similar to that of normal group, which was the lowest in group C. The fasting blood glucose level was the highest in normal group (205.20 ${\pm}$ 135.24), which were decreased in group C (134.2 ${\pm}$ 79.31) and group G (126.48 ${\pm}$ 77.05). TBARS values in a red blood cell and hepatic tisuue homogenate were lower in group G comparing to the group C. DNA% in tail, tail length (TL) and tail moment (TM) of blood leucoocytes showed the highest values in group C (20.11 ${\pm}$ 2.47, 17.36 ${\pm}$ 2.58, 94.11 ${\pm}$ 12.29) and they were significantly diminished in group G (9.63 ${\pm}$ 1.19, 7.04 ${\pm}$ 1.50, 38.64 ${\pm}$ 7.60). In conclusion, wild simulated ginseng might be a protective agent against the oxidative stress.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.28 no.6
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• pp.511-519
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• 2006

Autogenous bone grafts have been considered the gold standard for maxillofacial bony defects. However, this procedure could entail a complicated surgical procedure as well as potential donor site morbidity. Possibly the best solution for bone-defect regeneration is a tissue engineering approach, i.e. the use of a combination of a suitable scaffold with osteogenic cells. A major source of osteogenic cells is the bone marrow. Bone marrow-derived mesenchymal stem cells are multipotent and have the ability to differentiate into osteoblastic, chondrocytic, and adipocytic lineage cells. However, the isolation of cells from bone marrow has someproblems when used in clinical setting. Bone marrow aspiration is sometimes potentially more invasive and painful procedure and carries of a risk of morbidity and infection. A minimally invasive, easily accessible alternative would be cells derived from periosteum. The periosteum also contains multipotent cells that have the potential to differentiate into osteoblasts and chondrocytes. In the present study, we evaluated the osteogenic activity and mineralization of cultured human periosteal-derived cells. Periosteal explants were harvested from mandibule during surgical extraction of lower impacted third molar. The periosteal cells were cultured in the osteogenic inductive medium consisting of DMEM supplemented with 10% fetal calf serum, 50g/ml L-ascorbic acid 2-phosphate, 10 nmol dexamethasone and 10 mM -glycerophosphate for 42 days. Periosteal-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 14 of culture period, then decreased in intensity during the culture period. ALP mRNA expression increased up to day 14 with a decrease thereafter. Osteocalcin mRNA expression appeared at day 7 in culture, after that its expression continuously increased in a time-dependent manner up to the entire duration of culture. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. In conclusion, our study showed that cultured human periosteal-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix. As the periosteal-derived cells, easily harvested from intraoral procedure such as surgical extraction of impacted third molar, has the excellent potential of osteogenic capacity, tissue-engineered bone using periosteal-derived cells could be the best choice in reconstruction of maxillofacial bony defects.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.3
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• pp.199-206
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• 2010

This study investigated the effects of strontium on osteoblastic phenotypes of cultured human periostealderived cells. Periosteal tissues were harvested from mandible during surgical extraction of lower impacted third molar. Periosteal-derived cells were introduced into cell culture. After passage 3, the periostealderived cells were further cultured for 28 days in an osteogenic induction DMEM medium supplemented with fetal bovine serum, ascorbic acid 2-phosphate, dexamethasone and at a density of $3{\times}10^4$ cells/well in a 6-well plate. In this culture medium, strontium at different concentrations (1, 5, 10, and 100 ${\mu}g$/mL) was added. The medium was changed every 3 days during the incubation period. We examined the cellular proliferation, histochemical detection and biochemical measurements of alkaline phosphatase (ALP), the RT-PCR analysis for ALP and osteocalcin, and von Kossa staining and calcium contents in the periostealderived cells. Cell proliferation was not associated with the addition of strontium in periosteal-derived cells. The ALP activity in the periosteal-derived cells was higher in 5, 10, and 100 ${\mu}g$/ml strontium-treated cells than in untreated cells at day 14 of culture. Among the strontium-treated cells, the ALP activity was appreciably higher in 100 ${\mu}g$/ml strontium-treated cells than in 5 and 10 ${\mu}g$/ml strontium-treated cells. The levels of ALP and osteocalcin mRNA in the periosteal-derived cells was also higher in strontium-treated cells than in untreated cells at day 14 of culture. Their levels were increased in a dose-dependent manner. Von Kossa-positive mineralization nodules were strongly observed in the 1 ${\mu}g$/ml strontium-treated cells at day 21 and 28 of culture. The calcium content in the periosteal-derived cells was also higher in 1 ${\mu}g$/ml strontium-treated cells at day 28 of culture. These results suggest that low concentration of strontium stimulates the osteoblastic phenotypes of more differentiated periosteal-derived cells, whereas high concentration of strontium stimulates the osteoblastic phenotypes of less differentiated periosteal-derived cells. The effects of strontium on osteoblastic phenotypes of periosteal-derived cells appear to be associated with differentiation-extent.