• Journal of Ginseng Research
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• v.39 no.1
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• pp.46-53
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• 2015

Background: Glucocorticoids (GCs) are commonly used in many chemotherapeutic protocols and play an important role in the normal regulation of bone remodeling. However, the prolonged use of GCs results in osteoporosis, which is partially due to apoptosis of osteoblasts and osteocytes. In this study, effects of Korean Red Ginseng (KRG) on GC-treated murine osteoblastic MC3T3-E1 cells and a GC-induced osteoporosis mouse model were investigated. Methods: MC3T3-E1 cells were exposed to dexamethasone (Dex) with or without KRG and cell viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Realtime polymerase chain reaction was performed to evaluate the apoptotic gene expression; osteogenic gene expression and alkaline phosphatase (ALP) activity were also measured. Western blotting was performed to evaluate the mitogen-activated protein kinase (MAPK) proteins. A GC-induced osteoporosis animal model was used for in vivo study. Results and conclusion: The MTT assay revealed that Korean Red Ginseng (KRG) prevents loss of cell viability caused by Dex-induced apoptosis in MC3T3E1 cells. Real-time polymerase chain reaction data showed that groups treated with both Dex and KRG exhibited lower mRNA levels of caspase-3 and -9, whereas the mRNA levels of Bcl2, IAPs, and XIAP increased. Moreover, groups treated with both Dex and KRG demonstrated increased mRNA levels of ALP, RUNX2, and bone morphogenic proteins as well as increased ALP activity in MC3T3-E1 cells, compared to cells treated with Dex only. In addition, KRG increased protein kinase B (AKT) phosphorylation and decreased c-Jun N-terminal kinase (JNK) phosphorylation. Moreover, microcomputed tomography analysis of the femurs showed that GC implantation caused trabecular bone loss. However, a significant reduction of bone loss was observed in the KRG-treated group. These results suggest that the molecular mechanism of KRG in the GC-induced apoptosis may lead to the development of therapeutic strategies to prevent and/or delay osteoporosis.

• Korean Journal of Clinical Laboratory Science
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• v.51 no.3
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• pp.379-385
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• 2019

Osteoporosis is a disease that increases the risk of fractures by inducing a decrease in bone strength by the changes in hormones and a decrease in minerals. Recent reports have indicated that the long-term administration of Adefovir dipivoxil (ADV), which is used as a treatment for the hepatitis virus and AIDS, may have osteoporotic side effects. On the other hand, there are few studies on the cytopathic correlation of these causes. In this study, the biological relevance of ADV was evaluated using osteoblast hFOB1.19 and vascular endothelial cell HUVEC. First, the cells were treated with ADV at different concentrations, and DAPI and crystal violet staining were performed for morphological analysis of each cell and nucleus. A CCK-8 assay, real-time PCR, alkaline phosphatase (ALP) staining, and activity was performed to evaluate the drug effects on cell proliferation, gene expression, and osteoblast differentiation. As a result, ADV induced cell hypertrophy in hFOB1.19 cells and HUVEC cells. Furthermore, ADV not only inhibited cell proliferation and TGF-${\beta}$ expression but was also involved in osteoblast differentiation. Overall, these results provide basic data to help better understand the mechanism of ADV-induced osteoporosis and its clinical implications.

• Journal of Periodontal and Implant Science
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• v.51 no.3
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• pp.213-223
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• 2021

Purpose: The atmospheric pressure plasma jet (APPJ) has been introduced as an effective disinfection method for titanium surfaces due to their massive radical generation at low temperatures. Helium (He) has been widely applied as a discharge gas in APPJ due to its bactericidal effects and was proven to be effective in our previous study. This study aimed to evaluate the safety and effects of He-APPJ application at both the cell and tissue levels. Methods: Cellular-level responses were examined using human gingival fibroblasts and osteoblasts (MC3T3-E1 cells). He-APPJ was administered to the cells in the experimental group, while the control group received only He-gas treatment. Immediate cell responses and recovery after He-APPJ treatment were examined in both cell groups. The effect of He-APPJ on osteogenic differentiation was evaluated via an alkaline phosphatase activity assay. In vivo, He-APPJ treatment was administered to rat calvarial bone and the adjacent periosteum, and samples were harvested for histological examination. Results: He-APPJ treatment for 5 minutes induced irreversible effects in both human gingival fibroblasts and osteoblasts in vitro. Immediate cell detachment of human gingival fibroblasts and osteoblasts was shown regardless of treatment time. However, the detached areas in the groups treated for 1 or 3 minutes were completely repopulated within 7 days. Alkaline phosphatase activity was not influenced by 1 or 3 minutes of plasma treatment, but was significantly lower in the 5 minute-treated group (P=0.002). In vivo, He-APPJ treatment was administered to rat calvaria and periosteum for 1 or 3 minutes. No pathogenic changes occurred at 7 days after He-APPJ treatment in the He-APPJ-treated group compared to the control group (He gas only). Conclusions: Direct He-APPJ treatment for up to 3 minutes showed no harmful effects at either the cell or tissue level.

• 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.

• Nutrition Research and Practice
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• v.11 no.3
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• pp.190-197
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• 2017

BACKGROUND/OBJECTIVES: Gallus gallus domesticus (GD) is a natural mutant breed of chicken in Korea with an atypical characterization of melanin in its tissue. This study investigated the effects of melanin extracts of GD on osteoblast differentiation and inhibition of osteoclast formation. MATERIALS/METHODS: The effects of the melanin extract of GD on human osteoblast MG-63 cell differentiation were examined by evaluating cell viability, osteoblast differentiation, and expression of osteoblast-specific transcription factors such as bone morphogenetic protein 2 (BMP-2), small mothers against decapentaplegic homologs 5 (SMAD5), runt-related transcription factor 2 (RUNX2), osteocalcin and type 1 collagen (COL-1) by reverse transcription-polymerase chain reaction and western blotting analysis. We investigated the inhibitory effect of melanin on the osteoclasts formation through tartrate-resistant acid phosphatase (TRAP) activity and TRAP stains in Raw 264.7 cell. RESULTS: The melanin extract of GD was not cytotoxic to MG-63 cells at concentrations of $50-250{\mu}g/mL$. Alkaline phosphatase (ALP) activity and bone mineralization of melanin extract-treated cells increased in a dose-dependent manner from 50 to $250{\mu}g/mL$ and were 149% and 129% at $250{\mu}g/mL$ concentration, respectively (P < 0.05). The levels of BMP-2, osteocalcin, and COL-1 gene expression were significantly upregulated by 1.72-, 4.44-, and 2.12-fold in melanin-treated cells than in the control cells (P < 0.05). The levels of RUNX2 and SMAD5 proteins were higher in melanin-treated cells than in control vehicle-treated cells. The melanin extract attenuated the formation of receptor activator of nuclear factor kappa-B ligand-induced TRAP-positive multinucleated RAW 264.7 cells by 22%, and was 77% cytotoxic to RAW 264.7 macrophages at a concentration of $500{\mu}g/mL$. CONCLUSIONS: This study provides evidence that the melanin extract promoted osteoblast differentiation by activating BMP/SMADs/RUNX2 signaling and regulating transcription of osteogenic genes such as ALP, type I collagen, and osteocalcin. These results suggest that the effective osteoblastic differentiation induced by melanin extract from GD makes it potentially useful in maintaining bone health.

• Molecules and Cells
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• v.40 no.6
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• pp.386-392
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• 2017

Periodontal ligament stem cells (PDLSCs) are multipotent stem cells derived from periodontium and have mesenchymal stem cell (MSC)-like characteristics. Recently, the perivascular region was recognized as the developmental origin of MSCs, which suggests the in vivo angiogenic potential of PDLSCs. In this study, we investigated whether PDLSCs could be a potential source of perivascular cells, which could contribute to in vivo angiogenesis. PDLSCs exhibited typical MSC-like characteristics such as the expression pattern of surface markers (CD29, CD44, CD73, and CD105) and differentiation potentials (osteogenic and adipogenic differentiation). Moreover, PDLSCs expressed perivascular cell markers such as NG2, ${\alpha}-smooth$ muscle actin, platelet-derived growth factor receptor ${\beta}$, and CD146. We conducted an in vivo Matrigel plug assay to confirm the in vivo angiogenic potential of PDLSCs. We could not observe significant vessel-like structures with PDLSCs alone or human umbilical vein endothelial cells (HUVECs) alone at day 7 after injection. However, when PDLSCs and HUVECs were co-injected, there were vessel-like structures containing red blood cells in the lumens, which suggested that anastomosis occurred between newly formed vessels and host circulatory system. To block the $SDF-1{\alpha}$ and CXCR4 axis between PDLSCs and HUVECs, AMD3100, a CXCR4 antagonist, was added into the Matrigel plug. After day 3 and day 7 after injection, there were no significant vessel-like structures. In conclusion, we demonstrated the perivascular characteristics of PDLSCs and their contribution to in vivo angiogenesis, which might imply potential application of PDLSCs into the neovascularization of tissue engineering and vascular diseases.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.225-232
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• 2013

Satellite cells were derived from muscular tissue in postnatal pig. Satellite cell is an important to growth and development in animal tissues or organs. However, the progress underlying induced differentiation is not clear. The aim of this study was to evaluate the morphologic and the transcriptome changes in porcine satellite cell (PSC) treated with insulin, rosiglitazone, or dexamethasone respectively. PSC was obtained from postnatal muscle tissue. In study 1, for study the effect of insulin and FBS on the differentiated satellite cells, cells were cultured at absence or presence of insulin treated with FBS. Total RNA was extracted for determining the expression levels of myogenic PAX3, PAX7, Myf5, MyoD, and myogenin genes by real-time PCR. Myogenic genes decreased expression levels of mRNA in treated with insulin. In study 2, in order to clarify the relationship between rosiglitazone and lipid in differentiated satellite cells, we further examined the effect of FBS on lipid accumulation in the presence or absence of the rosiglitazone and lipid. Significant differences were observed between rosiglitazone and lipid by FBS. The mRNA of FABP4 and $PPAR{\gamma}$ increased in rosiglitazone treatment. In study 3, we examined the effect of dexamethasone on osteogenic differentiation in PSC. The mRNA was increased osteoblasotgenic ALP and ON genes treated with dexamethasone in 2% FBS. Dexamethasone induces osteoblastogenesis in differentiated PSC. Taken together, in differentiated PSCs, FABP4 and $PPAR{\gamma}$ increased to rosiglitazone. Whereas, no differences to FBS and lipid. These results were not comparable with previous reports. Our results suggest that adipogenic, myogenic, and osteoblastogenic could be isolated from porcine skeletal muscle, and identify culture conditions which optimize proliferation and differentiation formation of PSC.

• Polymer(Korea)
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• v.33 no.1
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• pp.26-32
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• 2009

The aim of this research was to prepare microparticulate systems based on poly (lactide-co-glycolide)(PLGA) for the local release of ipriflavone in order to reduce bone loss. We developed the IP loaded PLGA microspheres using relatively simple oil-in-water(O/W) solvent evaporation method. HPLC was used to perform the in vitro release test of IP and morphology of cell attached on the micro-spheres was investigated using SEM. Cytotoxicity was assayed by cell counting kit-8 (CCK-8) test. Osteogenic differential cells were analyzed by ALP activity. Through RT-PCR analysis, we observed osteocalcin, ALP, and Type I collagen mRNA expression. The release of IP in vitro was more prolonged over 42 days and IP/PLGA microspheres showed the improvement on the cell proliferation, ALP activity and RT-PCR comparing with control (only PLGA). This initial research will be used to direct future work involved in developing this composite injectable bone tissue engineering system.

• Journal of Periodontal and Implant Science
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• v.39 no.sup2
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• pp.279-286
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• 2009

Purpose: Gene therapy (ex vivo) has recently been used as a means of delivering bone morphogenetic proteins (BMPs) to sites of tissue regeneration. In the present study, we investigated the effect of co-transduction of adenoviruses expressing BMP-2 and BMP-7 on osteogenesisof C2C12 cells in vitro. Methods: A replication-defective human adenovirus 5 (Ad5) containing a cDNA for BMPs in the E1 region of the virus (Ad5BMP-2 and Ad5BMP-7) was constructed by in vivo homologous recombination. Functional activity of Ad5BMP-2 and Ad5BMP-7 were evaluated in mouse stromal cells (W20-17cells). C2C12 cells are transduced with various MOI (multiplicity of infection) of Ad5BMP-2 and Ad5BMP-7 to assess most effective and stable titer. Based on this result, C2C12 cells were transduced with Ad5BMP-2 and Ad5BMP-7 alone or by combination. BMPs expression, alkaline phosphatase (ALPase) activity, cell proliferation, and mineralization were assessed. Results: Ad5BMP-2 and Ad5BMP-7 are successfully transduced to W20-17 cells, and secreted BMPs stimulated cell differentiation. Also, C2C12 cells transduced with Ad5BMPs showed expression of BMPs and increased ALPaseactivity. In all groups, cell proliferation was observed over times. At 7days, cells co-transduced with Ad5BMP-2 and Ad5BMP-7 showed lower proliferation than the others. C2C12 cells co-transduced with Ad5BMP-2 and Ad5BMP-7 had greater ALPaseactivity than that would be predicted if effect of individual Ad5BMPs were additive. Little mineralized nodule formation was detected in cells transduced with individual Ad5BMPs. In contrast, Ad5BMP-2 and Ad5BMP-7 combination stimulated mineralization after culturing for 10 days in mineralizing medium. Conclusions: Present study demonstrated that adenoviruses expressing BMPs gene successfully produced BMPs protein and these BMPs stimulated cells to be differentiated into osteoblastic cells. In addition, the osteogenic activity of Ad5BMPs can be synergistically increased by co-transduction of cells with Ad5BMP-2 and Ad5BMP-7.

• The Journal of Korean Academy of Prosthodontics
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• v.46 no.6
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• pp.620-627
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• 2008

STATEMENT OF PROBLEM: A biochemical approach for surface modification has offered an alternative for physicochemical and morphological methods to obtain desirable bone-implant interfaces. PURPOSE: The purpose of the present study was to investigate cell responses to poly (D,L-lactide-co-glycolide) (PLGA)/$1{\alpha}$,25-(OH)$_2D_3$ coating with reference to cellular proliferation and differentiation in vitro. MATERIAL AND METHODS: 96 titanium discs were fabricated and divided into four groups. Group 1 was anodized under 300 V as control. Group 2, 3 and 4 were anodized then coated with 3 ml PLGA/$1{\alpha}$,25-(OH)$_2D_3$ solutions. Amount of the solutions were 2 ul, 20 ul and 200ul respectively. The osteoblast-like Human Osteogenic Sarcoma (HOS) cells were seeded and cultured for 1, 3 and 7 days. MTSbased cell proliferation assay and ALPase activity test were carried out. RESULTS: PLGA nanoparticles were observed as fine, smooth and round and HOS cells attached to the anodized surfaces through strand-like and sheet-like filopodia. After 3 days of culture, the dendritic filopodia were exaggerated and sheet-like cytoplasmic projections covered the coated titanium surfaces. After 3 days of culture, all of the groups showed increased cellular proliferation and the lowest proliferation rate was measured on group 2. Higher amount of incorporated $1{\alpha}$,25-(OH)$_2D_3$ (Group 3 and 4) improved cellular proliferation but the differences were not significant statistically (P > .05). But they increased the rate of ALP activities than the control group at day 3 (P < .05). CONCLUSION: Biodegradable PLGA nanoparticles incorporated with vitamin D metabolite positively affected proliferation and differentiation of cells on the anodized titanium surface.