• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.5
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• pp.341-345
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• 2010

Introduction: Skeletal homeostasis is normally maintained by the stability between bone formation by osteoblasts and bone resorption by osteoclasts. However, the correlation between the inflammatory reaction and osteoblastic differentiation of cultured osteoprogenitor cells has not been fully investigated. This study examined the effects of inflammatory cytokines on the osteoblastic differentiation of cultured human periosteal-derived cells. Materials and Methods: Periosteal-derived cells were obtained from the mandibular periosteum and introduced into the cell culture. After passage 3, the periosteal-derived cells were further cultured in an osteogenic induction Dulbecco's modified Eagle's medium (DMEM) medium containing dexamethasone, ascorbic acid, and $\beta$-glycerophosphate. In this culture medium, tumor necrosis factor (TNF)-$\alpha$ with different concentrations (0.1, 1, and 10 ng/mL) or interleukin (IL)-$1{\beta}$ with different concentrations (0.01, 0.1, and 1 ng/mL) were added. Results: Both TNF-$\alpha$ and IL-$1{\beta}$ stimulated alkaline phosphatase (ALP) expression in the periosteal-derived cells. TNF-$\alpha$ and IL-$1{\beta}$ increased the level of ALP expression in a dose-dependent manner. Both TNF-$\alpha$ and IL-$1{\beta}$ also increased the level of alizarin red S staining in a dose-dependent manner during osteoblastic differentiation of cultured human periosteal-derived cells. Conclusion: These results suggest that inflammatory cytokines TNF-$\alpha$ and IL-$1{\beta}$ can stimulate the osteoblastic activity of cultured human periosteal-derived cells.

• Journal of Periodontal and Implant Science
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• v.31 no.1
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• pp.179-193
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• 2001

previous studies have demonstrated an increase in bone mass and density with use of bisphosphonate in osteoporosis. This agent acts as an inhibitor of osteoclastic activity and results in increase of net osteoblastic activity. The purpose of the present study was to examine the effect of the bisphosphonate on osteoblastic activity of the human periodontal ligament cells in vitro. Periodontal ligament cells were primarily obtained from extracted healthy third molars. Cells of 4th to 6th passage were cultured in Dulbecco's modified Eagle's medium containing alendronate sodium or etidronate disodium at the concentration of $10^{-12}{\sim}10^{-6}mol/L$ in 5% $Co_2$ incubator at $37^{\circ}C$. Cell count and MTT assay for cellular activity were done at 2 to 7 days of culture. Alkaline phosphatase activity at 4 to 7 days of culture and formation of mineralized nodules at 28 days of culture with addition of $50{\mu}g/m{\ell}$ ascorbic acid, 10 nM${\beta}-glycerophosphate$, $10^{-7}M$ dexamethasone were evaluated. 1. Alendronate sodium Compared to the control, the proliferation of periodontal ligament cells was generally increased and the cellular activity was maintained at 2 days of culture and generally decreased at 7 days of culture. Alkaline phosphatase activity of periodontal ligament cells was inceased and the formation of mineralized nodules by periodontal ligament cells was enhanced compared to the control. 2. Etidronate disodium The proliferation of periodontal ligament cells was increased at 2 days of culture and decreased or maintained at 7 days of culture. Compared to the control, the cellular activity of periodontal ligament cells was generally decreased. Alkaline phosphatase activity of peridontal ligament cells was increased and the formation of mineralized nodules by periodontal ligament cells was enhanced compared to the control. These results suggest that alendronate sodium and etidronate disodium may have a potential effect on osteoblastic lineage of periodontal ligament cells, distinct from their inhibitory action on osteoclasts and could contribute to enhance periodontal regeneration and alveolar bone regeneration.

• Journal of Life Science
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• v.20 no.4
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• pp.607-613
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• 2010

Osteoporosis is a disease involving a decrease in bone mineral density and an increased risk of fractures. The MC3T3-E1 pre-osteoblastic cell line is a well-accepted model of osteogenesis in vitro. Pine needles have long been used as a traditional health-promoting medicinal food in Korea. In this study, MTT assay, the alkaline phosphatase (ALP) activity and collagen synthesis of osteoblast cells were investigated to determine the effects of pine needle extracts on cell proliferation and differentiation. Pine needle extracts were prepared using hexane, ethanol and water. The effects of the pine needle extracts were examined by comparing the results with those of commercial agents, such as proanthocyanidin. The MC3T3-E1 cells exposed to proanthocyanidin showed increased proliferation in a concentration-dependent manner. The cells exposed to the hexane extract showed a similar increase in proliferation to that observed with proanthocyanidin. The hexane extract showed the highest ALP activity. Moreover, a supplement of pine needle extracts induced collagen synthesis in MC3T3-E1 cells. The pine needle extract produced the highest level of collagen synthesis at concentrations of $10{\sim}50\;{\mu}g/ml$. These results indicate that pine needle extracts have an anabolic effect on bone by promoting osteoblastic differentiation, and may be used in the treatment of common metabolic bone diseases.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.3
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• pp.197-205
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• 2007

Angiogenesis is a essential part for bone formation and bone fracture healing. Vascular endothelial growth factor (VEGF), one of the most important molecules among many angiogenic factors, is a specific mitogen for vascular endothelial cells. VEGF-mediated angiogenesis is required for bone formation and repair. However, the effect of VEGF on osteoblastic cells during osteogenesis is still controversial. In recent days, substantial progress have been made toward developing tissue-engineered alternatives to autologous bone grafting for maxillofacial bony defects. Periosteum has received considerable interest as a better source of adult stem cells. Periosteum has the advantage of easy harvest and contains various cell types and progenitor cells that are able to differentiate into a several mesenchymal lineages, including bone. Several studies have reported the bone formation potential of periosteal cells, however, the correlation between VEGF signaling and cultured human periosteal cell-derived osteogenesis has not been fully investigated yet. The purpose of this study was to examine the correlation between VEGF signaling and cultured human periosteal-derived cells osteogenesis. Periosteal tissues of $5\;{\times}\;20\;mm$ were obtained from mandible during surgical extraction of lower impacted third molar from 3 patients. Periosteal-derived cells were introduced into the cell culture and were subcultured once they reached confluence. After passage 3, the periosteal-derived cells were further cultured for 42 days in an osteogenic inductive culture medium containing dexamethasone, ascorbic acid, and ${\beta}-glycerophosphate$. We evaluated the alkaline phosphatase (ALP) activity, the expression of Runx2 and VEGF, alizarin red S staining, and the quantification of osteocalcin and VEGF secretion in the periosteal-derived cells. The ALP activity increased rapidly up to day 14, followed by decrease in activity to day 35. Runx2 was expressed strongly at day 7, followed by decreased expression at day 14, and its expression was not observed thereafter. Both VEGF 165 and VEGF 121 were expressed strongly at day 35 and 42 of culture, particularly during the later stages of differentiation. Alizarin red S-positive nodules were first observed on day 14 and then increased in number during the entire culture period. Osteocalcin and VEGF were first detected in the culture medium on day 14, and their levels increased thereafter in a time-dependent manner. These results suggest that VEGF secretion from cultured human periosteal-derived cells increases along with mineralization process of the extracellular matrix. The level of VEGF secretion from periosteal-derived cells might depend on the extent of osteoblastic differentiation.

• Journal of Oral Medicine and Pain
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• v.26 no.1
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• pp.39-50
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• 2001

In order to investigate the effect of Transforming growth factor ${\beta}1$(below TGF-${\beta}1$) and osteogenic protein-1(below Op-1) onto the myogenic differentiation, C2C12 satellite myoblastic cell line was cultured and treated with both growth factors. At first morphological changes with microscopical examination were examined, and isolated total RNA to analyse mRNA expression of bone marker proteins, muscle regulatory proteins, TGF-${\beta}$ receptor and their ligands by Northern blot analysis. And cellular proliferative inducibility of both growth factors was also tested to C2C12 cells. Incubating the cell with $5ng/m{\ell}$ of TGF-${\beta}1$ until 4 days almost inhibited multinucleated myotube formation expressing muscular regulatory proteins, and induced decreasing Id proteins. However, no osteoblastic phenotypes was induced by TGF-${\beta}1$ in C2C12 cells. The mRNA expression of TGF-${\beta}$ receptors with TGF-${\beta}1$ was conversed after 48 hours cultured. Type I TGF-${\beta}$ receptor was seemed to play a role in negative signalling for inhibition of myogenic differentiation. OP-1 dose dependently induced ALP activity, osteopontine production and bone sialoprotein production at concentrations above $100ng/m{\ell}$ and osteocalcin production at concentrations above $300ng/m{\ell}$. The concentration of OP-1 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubation with above $100ng/m{\ell}$ OP-1 suppressed the expression of mRNA for muscular egulatory proteins from 2 days after incubation. Expression of Id-1, 2, 3 mRNA were stimulated by OP-1 at concentration above $300ng/m{\ell}$. When C2C12 cells were treated with both growth factors, TGF-${\beta}1$ potentiated the inhibitory effect of OP-1 on myotube formation and expression of mRNA for myogenin at 12 days. And TGF-${\beta}1$ reduced osteocalcin and bone sialoprotein production induced by OP-1 at 12 days in C2C12 cells. Both growth factor had no mitogenic effect. These results indicate that OP-1 converts the differentiation pathway of C2C12 myoblasts into that of osteoblastic lineage cells and it's not heritable, but TGF-${\beta}1$ does not and has reversible inhibitory activity on the myogenic differentiation. TGF-${\beta}1$ and OP-1 play a role in myogenic differentiation via different mechanism between them.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.27 no.2
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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.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.6
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• pp.1821-1824
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• 2004

In order to investigate the effects of Radix Achyranthis Bidentatae (RAB) on the growth and differentiation of human osteoblast-like cells, we supplemented the culture medium of MG-63 cells with various concentrations of RAB water extracts. RAB extracts significantly stimulated cell growth, as confirmed by the colorimetric MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. RAB extracts also increased the alkaline phosphatase (ALP) activity, which is a osteoblast differentiation marker. These results suggest that RAB can stimulate osteoblastic activity and may represent new pharmacological tools for the treatment of osteoporosis.

• Proceedings of the Korean Institute of Surface Engineering Conference
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• 2012.05a
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• pp.255-255
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• 2012

The interactions between cells and nanotopographies are increasingly interested as nanotopographies that may be more efficient in promoting cell functions and, titanium nanotubes have attracted much attention. Titanium nanotube can be fabricated easily with precisely controlled diameters and lengths. Titanium nanotubes with the suitable tube dimensions have been observed to enhance bone cell functions, whereas, there is still some controversy. These nanotubes can also serve as carriers for drugs such as growth factors, antibacterial agents and other drugs and show promise in bone implant applications.

• International Journal of Oral Biology
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• v.32 no.4
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• pp.119-125
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• 2007

Dlx3 is a homeodomain protein and is known to play a role in development and differentiation of many tissues. Deletion of four base pairs in DLX3 (NT3198) is causally related to tricho-dento-osseous (TDO) syndrome (OMIM #190320), a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium. The molecular mechanisms that explain the phenotypic characteristics of TDO syndrome have not been clearly determined. In this study, we examined phenotypic characteristics of wild type DLX3(wtDlx3) and 4-BP DEL DLX3 (TDO mtDlx3) in C2C12 cells. To investigate how wtDlx3 and TDO mtDlx3 differentially regulate osteoblastic differentiation, reporter assays were performed by using luciferase reporters containing the promoters of alkaline phosphatase, bone sialoprotein or osteocalcin. Both wtDlx3 and TDO mtDlx3 enhanced significantly all the reporter activities but the effect of mtDlx3 was much weaker than that of wtDlx3. In spite of these differences in reporter activity, electrophoretic mobility shift assay showed that both wtDlx3 and TDO mtDlx3 formed similar amounts of DNA binding complexes with Dlx3 binding consensus sequence or with ALP promoter oligonucleotide bearing the Dlx3 binding core sequence. TDO mtDlx3 exhibits a longer half-life than wtDlx3 and it corresponds to PESTfind analysis result showing that potential PEST sequence was missed in carboxy terminal of TDO mtDlx3. In addition, co-immunoprecipitation demonstrated that TDO mtDlx3 binds to Msx2 more strongly than wtDlx3. Taken together, though TDO mtDlx3 acted as a weaker transcriptional activator than wtDlx3 in osteoblastic cells, there is possibility that during in vivo osteoblast differentiation TDO mtDlx3 may antagonize transcriptional repressor activity of Msx2 more effectively and for longer period than wtDlx3, resulting in enhancement of osteoblast differentiation.

• Journal of Periodontal and Implant Science
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• v.35 no.2
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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.