• Journal of Korean Dental Science
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• 제11권2호
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• pp.62-70
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• 2018

Purpose: Bone marrow has long been a source of primary cells. This study was performed to evaluate the effects of age and sex on the cellular viability and expression of stem cell markers of mRNA and on the protein expression of bone marrow stem cells (BMSCs) derived from healthy donors. Materials and Methods: Stem cells were isolated from human bone marrow and plated in culture plates. The shape of the BMSCs was observed under inverted microscope. Quantitative cellular viability was evaluated using a Cell-Counting Kit-8 assay. The expression of stem cell surface markers was tested and a series of quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot was performed to evaluate the expression in each group. Result: The shapes of the cells at 20s, 30s, and 50s were similar to each other. No significant changes in cellular viability were noted among different age groups or sex groups. The BMSCs expressed CD44, CD73, and CD90 surface markers but did not express CD14 and CD34. There were no noticeable differences in CD surface markers among the different age groups. The expressions of CD surface markers were similar between men and women. No significant differences in the secretion of vascular endothelial growth factors (VEGFs) were noted at Day 3 between different age groups. qRT-PCR regarding the expression showed differences between the age groups. However, Western blot analysis showed a decrease in expression but did not reach statistical significance (P>0.05). Conclusion: This study clearly showed no significant differences in shape, cell viability, expression of stem cell surface markers, or secretion of human VEGF among different age groups. However, western blot analysis showed a tendency of age-related decrease which did not reach statistical significance. Collectively, autologous or allogeneic BMSCs should be meticulously applied to obtain optimal results regarding age and sex.

• IMMUNE NETWORK
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• 제10권1호
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• pp.15-25
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• 2010

Background: Rat mast cells were regarded as a good model for mast cell function in immune response. Methods: Rat bone marrow mast cells (BMMC) were prepared both by recombinant rat IL-3 (rrIL-3) and by recombinant mouse stem cell factor (rmSCF), and investigated for both proliferation and differentiation in time course. Rat BMMC was induced by culture of rat bone marrow cells (BMCs) in the presence of both rrIL-3 (5 ng/ml) and rmSCF (5 ng/ml). Culture media were changed 2 times per week with the cell number condition of $5{\times}10^4/ml$ in 6 well plate. Proliferation was analyzed by cell number and cell counting kit-8 (CCK-8) and differentiation was by rat mast cell protease (RMCP) II and histamine. Results: Cell proliferation rates reached a maximum at 8 or 11 days of culture and decreased thereafter. However, both RMCP II production and histamine synthesis peaked after 11 days of culture. By real time RT-PCR, the level of histidine decarboxylase mRNA was more than 500 times higher on culture day 11 than on culture day 5. By transmission electron microscopy, the cells were heterogeneous in size and contained cytoplasmic granules. Using gated flow cytometry, we showed that cultured BMCs expressed high levels of $Fc{\varepsilon}RI$ and the mast cell antigen, ganglioside, on culture day 11. Conclusion: These results indicate that rat BMMCs were generated by culturing BMCs in the presence of rrII-3 and rmSCF and that the BMMCs have the characteristics of mucosal mast cells.

• Nutritional Sciences
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• 제8권4호
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• pp.242-249
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• 2005

It is well established that zinc plays an important role in bone metabolism and mineralization. The role of zinc in bone formation is well documented in animal models, but not much reported in cell models. In the present study, we evaluated zinc deficiency effects on osteoblastic cell proliferation, alkaline phosphatase activity and expression, and extracellular matrix bone nodule formation and bone-related gene expression in osteoblastic MC3T3-E1 cells. To deplete cellular zinc, chelexed-FBS and interpermeable zinc chelator TPEN were used. MC3T3-E1 cells were cultured in zinc concentration-dependent (0-15 ${\mu}M\;ZnCl_2$) and time-dependent (0-20 days) manners. MC3T3-E1 cell proliferation by MTT assay was increased as medium zinc level increased (p<0.05). Cellular Ca level and alkaline phosphatase activity were increased as medium zinc level increased (p<0.05). Alkaline phosphatase expression, a marker of commitment to the osteoblast lineage, measured by alkaline phosphatase staining was increased as medium zinc level increased. Extracellular calcium deposits measured by von Kossa staining for nodule formation also appeared higher in Zn+(15 ${\mu}M\;ZnCl_2$) than in Zn-(0 ${\mu}M\;ZnCl_2$). Bone formation marker genes, alkaline phosphatase and osteocalcin, were also expressed higher in Zn+ than in Zn-. The current work supports the beneficial effect of zinc on bone mineralization and bone-related gene expression. The results also promote further study as to the molecular mechanism of zinc deficiency for bone formation and thus facilitate to design preventive strategies for zinc-deficient bone diseases.

• Preventive Nutrition and Food Science
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• 제16권4호
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• pp.291-298
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• 2011

Yam extracts (Dioscorea batatas) have been reported to possess a variety of functions. However, studies on its osteogenic properties are limited. In this study, we investigated the effect of ethanol and water extracts on osteoblast proliferation and bone matrix protein synthesis, type I collagen and alkaline phosphatase (ALP), using osteoblastic MC3T3-E1 cell model. MC3T3-E1 cells were cultured with yam ethanol and water extracts (0~30 mg/L) within 39 days of osteoblast differentiation period. Cell proliferation was measured by MTT assay. Bone matrix proteins were assessed by the accumulation of type I collagen and ALP activity by staining the cell layers for matrix staining. Also, the secreted (media) matrix protein concentration (type I collagen) and enzyme activity (ALP) were measured colorimetrically. Yam ethanol and water extracts stimulated cell proliferation within the range of 15~30 mg/L at 15 day treatment. The accumulation of type I collagen in the extracellular matrix, as well as secreted collagen in the media, increased with increasing doses of yam ethanol (3~15 mg/L) and water (3~30 mg/L) extracts. ALP activity was not affected by yam ethanol extracts. Our results demonstrated that yam extracts stimulated osteoblast proliferation and enhanced the accumulation of the collagenous bone matrix protein type I collagen in the extracellular matrix. These results suggest that yam extracts may be a potential activator for bone formation by increasing osteoblast proliferation and increasing bone matrix protein type I collagen. Before confirming the osteogenic action of yam, further studies for clarifying how and whereby yam extracts can stimulate this ostegenesis action are required.

• 대한치과마취과학회지
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• 제14권2호
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• pp.107-114
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• 2014

Background: Angiogenesis has been recognized an essential precondition for osteogenesis. Because reduction and disruption of the blood supply to tissue cause tissue hypoxia, pathological bone loss affected by hypoxia often can occur in various clinical conditions. The effects of propofol on the process of osteogenesis have received little direct attention. Therefore, we investigated the effect of propofol on the growth and function of osteoblasts under hypoxic condition. Methods: After propofol (3, 30, $300{\mu}M$) preconditioning for 2 hours, hFOB 1.19 human osteoblast cells were cultured under 1 % oxygen tension for 48 hours. Using real time PCR and western blot analysis, we analyzed the expression of, BMP-2, TGF-${\beta}1$, type I collagen, osteocalcin, HIF-1s and Akt. Cell viability was also determined by MTT assay. Results: Propofol preconditioning on hypoxic-cultured osteoblast promoted the expressions of BMP-2, TGF-${\beta}1$, type I collagen and osteocalcin and induced hypoxia-mediated HIF-1 activation and the expression of Akt protein. Propofol with $300{\mu}M$ significant decreased cell viability compared to control. Conclusions: Clinically relevant concentrations of propofol are not cytotoxic to hypoxic osteoblasts in vitro. Propofol preconditioning on hypoxic-cultured osteoblast stimulates proliferation and differentiation of osteoblast through induced expression of BMP-2, TGF-${\beta}1$, type I collagen and osteocalcin. Propofol might promote angiogenesis and bone regeneration under hypoxic condition.

• Journal of Periodontal and Implant Science
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• 제32권3호
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• pp.489-500
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• 2002

The purpose of this study was to evaluate newly fabricated tricalcium phosphate(TCP)/chitosan microgranuls as bone substitutes. TCP/chitosan microgranules were fabricated by dropping TCP-chitosan suspension into the NaOH/ethanol solution. The size of microgranules could be controllable via airflow rate. PDGF-BB was loaded into the fabricated granules via freeze-drying methods(300 ng/20 mg). To evaluate cell proliferation, cultured osteoblasts cell lines(MC3T3-El) was dropped on the BioOss(R), chitosan microgranules, TCP/chitosan microgranules and cultured for 1, 7 , 14, and 28 days. Scanning electron microscopic observation was done after 7 days of culture and light microscopic examination was done after 28 days of culture. PDGF-BB release from the microgranules was tested. Rabbit calvarial defects(8 mm in diameter) were formed and chitosan, TCP/chitosan, PDGF-TCP/chitosan microgranules, and BioGran(R) were grafted to test the ability of new bone formation. At SEM view, the size of prepared microgranules was 250-1000 um and TCP powders were observed at the surface of TCP/chitosan microgranules. TCP powders gave roughness to the granules and this might help the attachment of osteoblasts. The pores formed between microgranules might be able to allow new bone ingrowth and vascularization. There were no significant differences in cell number among BioOss(R) and two microgranules at 28 day. Light and scanning electron microscopic examination showed that seeded osteoblastic cells were well attached to TCP/chitosan microgranules and proliferated in a multi-layer. PDGF-BB released from TCP/chitosan microgranules was at therapeutic concentration for at least 1 week. In rabbit calvarial defect models, PDGF-TCP/chitosan microgranules grafted sites showed thicker bone trabeculae pattern and faster bone maturation than others. These results suggested that the TCP/chitosan microgranules showed the potential as bone substitutes.

• Journal of Nutrition and Health
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• 제51권4호
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• pp.316-322
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• 2018

Purpose: The Glycyrrhiza uralensis species (Leguminosae) as a medicinal biocompound, and one of its root components, isoliquritigenin (ISL), which is a flavonoid, has been reported to have anti-tumor activity in vitro and in vivo. However, its function in bone formation has not been studied yet. In this study, we tested the effect of Glycyrrhiza uralensis (ErLR) and baked Glycyrrhiza uralensis (EdLR) extracts on osteoblast proliferation, alkaline phosphatase (ALP) activity, and bone-related gene expression in osteoblastic MC3T3-E1 cells. Methods: MC3T3-E1 cells were cultured in various levels of ErLR (0, 5, 10, 15, $20{\mu}g/mL$), EdLR (0, 5, 10, 15, $20{\mu}g/mL$), or ISL (0, 5, 10, 15, $20{\mu}M$) in time sequences (1, 5, and 20 days). Also, isoliquritigenin (ISL) was tested for comparison to those two biocompound extracts. Results: MTT assay results showed that all three compounds (ErLR, EdLR, and ISL) increased osteoblastic-cell proliferation in a concentration-dependent manner for one day. In addition, both ErLR and EdLR compounds elevated the osteoblast proliferation for 5 or 20 days. Extracellular ALP activity was also increased as ErLR, EdLR, and ISL concentration increased at 20 days, which implies the positive effect of Glycyrrhiza species on osteoblast mineralization. The bone-related marker mRNAs were upregulated in the ErLR-treated osteoblastic MC3T3-E1 cells for 20 days. Bone-specific transcription factor Runx2 gene expression was also elevated in the ErLR- and EdLR-treated osteoblastic MC3T3-E1 cells for 20 days. Conclusion: These results demonstrated that Glycyrrhiza uralensis extracts may be useful for preventing osteoporosis by increasing cell proliferation, ALP activity, and bone-marker gene expression in osteoblastic cells.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제32권6호
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• pp.524-529
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• 2006

For the repairing of bone defect, autogenous or allogenic bone grafting remains the standard. However, these methods have numerous disadvantages including limited amount, donor site morbidity and spread of diseases. Tissue engineering technique by culturing stem cells may allow for a smart solution for this problem. Adipose tissue contains mesenchymal stem cells that can be differentiate into bone, cartilage, fat or muscle by exposing them to specific growth conditions. In this study, the authors procured the stem cell from buccal fat pad and differentiate them into osteoblast and are to examine the bone induction capacity. Buccal fat-derived cells (BFDC) were obtained from human buccal fat pad and cultured. BFDC were analyzed for presence of stem cell by immunofluorescent staining against CD-34, CD-105 and STRO-1. After BFDC were differentiated in osteogenic medium for three passages, their ability to differentiate into osteogenic pathway were checked by alkaline phosphatase (ALP) staining, Alizarin red staining and RT-PCR for osteocalcin (OC) gene expression. Immunofluorescent and biochemical assays demonstrated that BFDC might be a distinguished stem cells and mineralization was accompanied by increased activity or expression of ALP and OC. And calcium phosphate deposition was also detected in their extracelluar matrix. The current study supports the presence of stem cells within the buccal fat pad and the potential implications for human bone tissue engineering for maxillofacial reconstruction.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제28권3호
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• pp.216-225
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• 2002

Hyaluronic acid (HA) is an almost essential component of extracellular matrices. Early in embryogenesis mesenchymal cells migrate, proliferate and differentiate, in part, because of the influence of HA. Since the features of embryogenesis are revisited during wound repair, including bone fracture repair, this study was initiated to evaluate whether HA has an effect on calcification and bone formation in an in vitro system of osteogenesis. Mouse calvaria Pre-osteoblast (MC3T3-E1) cells were cultured in ${\alpha}-MEM$ medium with microorganism-derivative hyaluronic acid that was produced by Strep. zooepidemicus which of molecular weight was 3 million units. The dosages were categorized in each 0.5, 1.0 and 2.0 mg/ml concentration experimental groups. After 2 and 4 days cultures in expeirmental and control groups, the tendency of cell proliferation, MTT assay, protein synthesis ability, collagen synthesis and alkaline phosphatase activity were analysed and bone nodule formation capacity were measured with Alizarin Red S stain after 29 days cultures. The cell proliferation was increased in time, especially the group of 0.5 and 1.0 mg/ml concentration of HA were showed prominent cell proliferation. After 2 and 4 days culture, experimental groups in general were greater cell activity in MTT assay. The protein synthesis was increased in all experimental groups compared to control group, especially most prominent in 1.0 mg/ml concentration group. The collagen synthesis capacity were increased in HA experimental groups, especially prominent in 1.0 mg/ml group and the activity of alkaline phosphatase were increased, especially also prominent in 1.0 mg/ml group, compared to control group. Above these, the activity of mouse carvarial pre-osteoblast cells was showed greater bone osteogenesis activity in all applied HA experimental group, especially group of 1.0 mg/ml concentration of HA.

• Restorative Dentistry and Endodontics
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• 제24권1호
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• pp.187-199
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• 1999

Bone remodeling is characterized by the continuing processes of osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Bone metabolism is tightly regulated at the local level by networks of hormones, cytokines, and other factors. In pathological conditions of bone remodeling, including osteoporosis and periodontal diseases, inflammatory cytokines and local mediators are responsible for enhancement of osteoclast resorption and inhibition of repair at the sites of bone resorption. TNF-${\alpha}$ is a pleiotropic hormone with actions on the differentiation, growth, and functional activities of normal and malignant cells from numerous tissues. TNF-${\alpha}$ has been proposed as a local mediator of the control of bone turnover in situations of chronic inflammation, and it has been assumed that the local source of TNF-${\alpha}$ is the monocyte in the adjacent bone marrow or the local circulation. TNF-${\alpha}$ is a potent inducer of bone resorption. TNF-${\alpha}$ is known to induce the activation of apoptotic signaling pathway, which leads to the apoptosis of bone cells. We demonstrated that treatment of murine osteoblastic MC3T3E1 cells with TNF-${\alpha}$ decreases proliferation as well as alkaline phosphatase (ALP) activity in a dose depenent manner. In addition, TNF-${\alpha}$ increases osteoclast-like cell formation in $1{\alpha}$, 25(OH)2D3 or PGE2-treated bone marrow cell culture. When cells were cultured in TNF-${\alpha}$ free ${\alpha}$-MEM, this inhibitory effect of ALP activity was reversible up to 10 ng/ml TNF-${\alpha}$, in contrast, at the 20 ng/ml TNF-${\alpha}$, irreversible. In this concentration, TNF-${\alpha}$ may induce apoptosis in MC3T3E1 cells. In this study, TNF-${\alpha}$ induces apoptosis resulting in chromosomal DNA fragmentation, preceded by JNK/SAPKs and caspase-3 activation. Our present results show that JNK/SAPKs and caspase-3 are activated by TNF-${\alpha}$, suggesting that the JNK/SAPKs and caspase-3 participate in the bone resorption, associated with apoptosis.