• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.4
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• pp.243-249
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• 2010

Tissue engineered bone (TEB) can replace an autogenous bone graft requiring an secondary operation site as well as avoid complications like inflammation or infection from xenogenic or synthetic bone graft. Adult mesenchymal stem cells (MSC) for TEB are considered to have various ranges of differentiation capacity or multipotency by the donor site and age. This study examined the effect of age on proliferation capacity, differentiation capacity and bone morphogenetic protein-2 (BMP-2) responsiveness of human bone marrow stromal cells (hBMSC) according to the age. In addition, to evaluate the effect on enhancement for osteoblast differentiation, the hBMSC were treated with Trichostatin A (TSA) and 5-Azacitidine (5-AZC) which was HDAC inhibitors and methyltransferase inhibitors respectively affecting chromatin remodeling temporarily and reversibly. The young and old group of hBMSC obtained from the iliac crest from total 9 healthy patients, showed similar proliferation capacity. Cell surface markers such as CD34, CD45, CD90 and CD105 showed uniform expression regardless of age. However, the young group showed more prominent transdifferentiation capacity with adipogenic differentiation. The osteoblast differentiation capacity or BMP responsiveness was low and similar between young and old group. TSA and 5-AZC showed potential for enhancing the BMP effect on osteoblast differentiation by increasing the expression level of osteogenic master gene, such as DLX5, ALP. More study will be needed to determine the positive effect of the reversible function of HDAC inhibitors or methyltransferase inhibitors on enhancing the low osteoblast differentiation capacity of hBMSC.

• Journal of the Korean Society of Food Science and Nutrition
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• v.34 no.6
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• pp.743-749
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• 2005

Estrogen is known to play an important role in maintaining bone mass, since the concentration of serum estrogen decrease after menopause and the estrogen deficiency results in bone loss. Phytoestrogens are plant compounds with estrogen-like biological activity, In this study, to investigate the bioactivities of phytoestrogen, which act on bone metabolism, we examined the effect of selected food-borne phytoestrogens (genistein, daidzein and resveratrol) on osteoblast proliferation and IGF-I production using MC3T3-El cells, a mouse calvaria osteoblast-like cell line. Cells were cultured in a serum free medium for 48 hr in the presence of genistein $(10^{-5}\;M)$, daidzein $(10^{-5}\;M)$ and resveratrol $(10^{-5}\;M)$. The effects of genistein, daidzein and resveratrol on the cell proliferation and growth were evaluated by total cell numbers, MTS assay and cell migration assay. Their effect was compared with the $17\beta-estradiol$. Genistein, daidzein and resveratrol exhibited stimulatory effects on the growth of MC3T3-El cells, and the most pronounced effect was shown with daidzein. In addition, these phytoestrogen increased alkaline phosphatase activity of MC3T3-El cells. These effects were similar to that of $17\beta-estradiol$ effects. Moreover, treatment with genistein, daidzein and resveratrol increased production of insulin like growth factor-I (IGF-I) in conditioned media, indicating that the growth promoting effects of these phytoestrogen were related to the changes in production of IGF-I by MC3T3-El cells. These results show that genistein, daidzein and resveratrol have a stimulatory effect on osteoblast function, and that these findings in a cell model may prove relevant to protecting against the loss of bone mass and the development of osteoporosis in human subjects.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.37 no.3
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• pp.214-224
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• 2011

Objective: This study examined the potential of the in vitro osteogenesis of microtopographically modified surfaces, RBM (resorbable blasting media) surfaces, which generate hydroxyapatite grit-blasting. Methods: RBM surfaces were modified hydroxyapatite grit-blasting to produce microtopographically modified surfaces and the surface morphology, roughness or elements were examined. To investigate the potential of the in vitro osteogenesis, the osteoblastic cell adhesion, proliferation, and differentiation were examined using the human osteoblast-like cell line, MG-63 cells. Osteoblastic cell proliferation was examined as a function of time. In addition, osteoblastic cell differentiation was verified using four different methods of an ALP activity assay, a mineralization assay using alizarin red-s staining, and gene expression of osteoblastic differentiation marker using RT-PCR or ELISA. Results: Osteoblastic cell adhesion, proliferation and ALP activity was elevated on the RBM surfaces compared to the machined group. The cells exhibited a high level of gene expression of the osteoblastic differentiation makers (osteonectin, type I collagen, Runx-2, osterix). imilar data was represented in the ELISA produced similar results in that the RBM surface increased the level of osteocalcin, osteopontin, TGF-beta1 and PGE2 secretion, which was known to stimulate the osteogenesis. Moreover, alizarin red-s staining revealed significantly more mineralized nodules on the RBM surfaces than the machined discs. Conclusion: RBM surfaces modified with hydroxyapatite grit-blasting stimulate the in vitro osteogenesis of MG-63 cells and may accelerate bone formation and increase bone-implant contact.

• YAKHAK HOEJI
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• v.58 no.5
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• pp.307-313
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• 2014

Rumex crispus (curled dock), which is a perennial wild plant, has long been used as a laxative, astringent, and medicine to treat blood and skin diseases. We recently reported that the roots of R. crispus are an effective nutraceutical for bone. This study prepared ethanol extracts of the leaves and roots of R. crispus, and analyzed the major constituents using liquid chromatography and mass spectrometry. In addition, their effects on the proliferation and differentiation of human osteoblast-like MG-63 cells, such as cell viability, alkaline phosphatase (ALP) activity, collagen content, and mineralization, were compared. The chromatograms of the chemical constituents of the two extracts exhibited quite different profiles: quercetin and quercitrin were identified as major peaks in the leaf extract, whereas cinnamtannin B1 and procyanidin isomers were the major peaks for the root extract. Neither extract was cytotoxic at concentrations of < $25{\mu}g/ml$. ALP activity and collagen synthesis-which are markers of the early stage of osteogenesis-in MG-63 cells were significantly increased upon the addition of the root extract compared with the addition of the leaf extract. In contrast, the leaf extract had a more stimulatory effect on mineralization-which is marker of the late stage of osteogenesis-in MG-63 cells than did the root extract. In conclusion, extracts of both leaves and roots of R. crispus stimulated the bone-forming activity of osteoblasts; in particular, the root extract was more effective in the early stage of osteoblast differentiation, while the leaf extract was more effective in the late stage. This difference in anabolic activity may be due to differences in the constituents of the leaves and roots. The leaves and roots of R. crispus appear to complement each other as stimulators of bone formation.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.4
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• pp.347-352
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• 2014

Most known osteoporosis medicines are effective for bone resorption, and so there is an increasing demand for medicines that stimulate bone formation. Watercress (N. officinale R. Br.) is widely used as a salad green and herbal remedy. This study analyzed a watercress extract using ultra-performance liquid chromatography/mass spectrometry, and identified a rutin as one of its major constituents. Osteogenic-related assays were used to compare the effects of watercress containing rutin (WCR) and rutin alone on the proliferation and differentiation of human osteoblast-like MG-63 cells. The reported data are expressed as percentages relative to the control value (medium alone; assigned as 100%). WCR increased cell proliferation to $125.0{\pm}4.0%$ ($mean{\pm}SD$), as assessed using a cell viability assay, and increased the activity of alkaline phosphatase, an early differentiation marker, to $222.3{\pm}33.8%$. In addition, WCR increased the expression of collagen type I, another early differentiation marker, to $149.2{\pm}2.8%$, and increased the degree of mineralization, a marker of the late process of differentiation, to $122.9{\pm}3.9%$. Rutin alone also increased the activity of ALP (to $154.4{\pm}12.2%$), the expression of collagen type I (to $126.6{\pm}6.2%$), and the degree of mineralization (to $112.3{\pm}5.0%$). Daidzein, which is reported to stimulate bone formation, was used as a positive control; the effects of WCR on proliferation and differentiation were significantly greater than those of daidzein. These results indicate that WCR and rutin can both induce bone formation via the differentiation of MG-63 cells. This is the first study demonstrating the effectiveness of either WCR or rutin as an osteoblast stimulant.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.34 no.4
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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.

• Archives of Pharmacal Research
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• v.26 no.12
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• pp.1029-1035
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• 2003

Osteoporosis is recognized as one of the major hormonal deficiency diseases, especially in menopausal women and the elderly. When estrogen is reduced in the body, local factors such as IL-1 $\beta$ and IL-6, which are known to be related with bone resorption, are increased and promote osteoclastogenesis, which is responsible for bone resorption. In the present study, we investigated whether glucosidic isoflavones (Isocal, PIII) extracted from Sophorae fructus affect the proliferation of osteoblasts and prevent osteoclastogenesis in vitro by attenuating upstream cytokines such as IL-1$\beta$ and IL-6 in a human osteoblastic cell line (MG-63) and in a primary osteoblastic culture from SD rat femurs. Interestingly, IL-1$\beta$ and IL-6 mRNA were significantly suppressed in osteoblast-like cells treated with 17$\beta$-estradiol (E2) and PIII when compared to positive control (SDB), and this suppression was more effective at $10^{-8}$% than at the highest concentration of $10^{-4}$%. In addition, these were confirmed in protein levels using ELISA assay. In the cell line, the cells showed that E2 was the most effective in osteoblastic proliferation over the whole range of concentration ($10^{-4}%-10^{-12}$%), even though PIII also showed the second greatest effectiveness at $10^{-8}$%. Nitric oxide (NO) was significantly (p<0.05) upregulated in PIII and E2 over the concentration range $10^{-6}% to 10^{-8}$% when compared to SDB, without showing any dose dependency. In bone marrow primary culture, we found by TRAP assay that PIII effectively suppressed osteoclastogenesis next to E2 in comparison with SDB and culture media (control). In conclusion, these results suggest that local bone-resorbing cytokines can be regulated by PIII at lower concentrations and that, therefore, PIII may preferentially induce anti-osteoporosis response by attenuating osteoclastic differentiation and by upregulating NO.

• Journal of Periodontal and Implant Science
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• v.32 no.1
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• pp.1-12
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• 2002

The periodontal ligament(PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. PDL-specific protein;PDLs22 had been previously identified as a novel protein isolated from cultured human PDL fibroblasts using subtraction hybridization between human gingival fibroblasts and PDL fibroblasts. The aim of this study was to examine the expression pattern and tissue localization of PDLs22 protein in embryonic and various postnatal stages of developing mouse using immunohistochemical staining. Embryos (E18) and postnatal (P1, P4, P5, P15, P18) were decapitated and the heads were fixed overnight in a freshly prepared solution of 4% paraformaldehyde. Some specimens were decalcified for $2{\sim}4$ weeks in a solution containing 10% of the disodium salt of ethylenediamine-tetraacetic acid (EDTA). Next, tissues were dehydrated, embedded in paraffin and sectioned serially at $6{\mu}m$ in thickness. Polyclonal antiserum raised against PDLs22 peptides, ISNKYLVKRQSRD, were made. The localization of PDLs22 in tissues was detected by polyclonal antibody against PDLs22 by means of immunohistochemical staining. The results were as follows; 1. Expression of PDLs22 protein was not detected in the tooth germ of bud and cap stage. 2. At the late bell stage and root formation stage, strong expression of PDLs22 protein was observed in developing tooth follicle, osteoblast-like cells, and subodontoblastic cells in the tooth pulp, but not in gingival fibroblasts, ameloblasts and odontoblasts of tooth germ 3. In erupted tooth, PDLs22 protein was intensely expressed in PDL and osteoblast-like cells of alveolar bone, but not in gingival fibroblasts, mature osteocytes and adjacent salivary glands. 4. In the developing alveolar bone and mid-palatal suture, expression of PDLs22 protein was seen in undifferentiated mesenchymal cells and osteoblast-like cells of developing mid-palatal suture, but not in mature osteocytes and chondrocytes. These results suggest that PDLs22 protein may play an important role in the differentiation of undifferentiated mesenchymal cells in the bone marrow and PDL cells, which can differentiate into multiple cell types including osteoblasts, cementoblasts, and PDL fibroblasts. However, more researches should be performed to gain a better understanding of the exact function of PDLs22 protein which related to the PDL cell differentiation.

• The korean journal of orthodontics
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• v.30 no.2 s.79
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• pp.197-204
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• 2000

[$K^+$]-selective ion channels were studied in excised inside-out membrane patches from human osteoblast-like cells (G292). Three classes of $K^+$channels were present and could be distinguished on the basis of conductance. Conductances were $270\pm27\;pS,\;113\pm12\;pS,\;48\pm8\;pS$ according to their approximate conductances in symmetrical 140 mM KCl saline at holding potential of -80 mV It was found that the small conductance (48 pS) $K^+$channel activation was dependent on membrane voltage. In current-voltage relationship, small conductance $K^+$channel showed outward rectification, and it was activated by the positive potential inside the membrane. In recordings, single channel currents were activayed by a negative pressure outside the membrane. The membrane pressure increased $P_{open}$ of the $K^+$ channel in a pressure-dependent manner. In the excised-patch clamp recordings, G292 osteoblast-like cells have been shown to contain three types of $K^+$ channels. Only the small conductance (48 pS) $K^+$channel is sensitive to the membrane stretch. These findings suggest that a hyperpolarizing current, mediated in part by this channel, may be associated with early events during the mechanical loading of the osteoblast. In G292 osteoblast-like cells, $K^+$channel is sensitive to membrane tension, and may represent a unique adaptation of the bone cell membrane to mechanical stress.

• Journal of Acupuncture Research
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• v.29 no.6
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• pp.11-21
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• 2012

Objectives : BHH10 is traditional medicine herb used for enhancing body resistance against various diseases. The aim of this study was to identify BHH10 extract induces osteogenic activity in human osteoblast-like MC3T3-E1 cells. Methods : MC3T3-E1, pre-osteoblast cell line, were treated with BHH10 of various concentrations($0.1{\mu}g/mL$, $1{\mu}g/mL$, $10{\mu}g/mL$). And then, the effect of BHH10 on osteoblast differentiation was examined by alkaline phosphatase(ALP) activity, von Kossa staining and RT-PCR for osteoblast differentiation markers such as osteocalcin(OCN), osteopontin(OPN). Results : BHH10 had dose-dependent effect on the viability of osteoblastic cells, and dose-dependently increased alkaline phosphatase(ALP) activity. BHH10 markedly increased mRNA expression for OCN, OPN in MC3T3-E1 cells. Also, BHH10 significantly induced mineralization in the culture of MC3T3-E1 cells. Conclusions : In conclusion, these results propose that BHH10 can play an important role in osteoblastic bone formation, osteogenesis, and may possibly lead to the development of bone-forming drugs.