• Journal of Periodontal and Implant Science
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• v.48 no.6
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• pp.383-394
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• 2018

Purpose: The purpose of this study was to evaluate the optimal diabetes duration for bone regeneration experiments in an alloxan monohydrate (ALX)-induced diabetic rabbit calvarial defect model by evaluating the association between diabetes duration and bone healing capacity. Methods: Twenty-four New Zealand white rabbits were used. Twenty-two rabbits were injected with 100 mg/kg of ALX to induce experimental diabetes. These rabbits were divided into 4 groups, including a control group and groups with diabetes durations of 1 week (group 1), 2 weeks (group 2), and 4 weeks (group 3). Calvarial defects were created at 1, 2, and 4 weeks after ALX injection and in the control rabbits. Cone-beam computed tomography (CBCT) scanning was performed on the day of surgery and at 2 and 4 weeks after surgery. The rabbits were sacrificed 4 weeks after surgery, followed by histological and immunofluorescence analysis. Results: The diabetic state of all diabetic rabbits was well-maintained throughout the experiment. Reconstructed 3-dimensional CBCT imaging showed more rapid and prominent bone regeneration in the control group than in the experimental groups. Histological staining showed notable bone regeneration in the control group, in contrast to scarce bone formation in the experimental groups. The appearance and immunoreactivity of receptor activator of nuclear factor-kappa B and osteoprotegerin did not show notable differences among the groups. Conclusion: ALX administration at 100 mg/kg successfully induced experimental diabetes in rabbits. The effect of diabetes on bone healing was evident when the interval between diabetes induction and the intervention was ${\geq}1$ week.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.37
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• pp.16.1-16.7
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• 2015

Background: This study aimed to investigate new bone formation using recombinant human bone morphogenetic protein 2 (rhBMP-2) and locally applied bisphosphonate in rat calvarial defects. Methods: Thirty-six rats were studied. Two circular 5 mm diameter bony defect were formed in the calvaria using a trephine bur. The bony defect were grafted with $Bio-Oss^{(R)}$ only (group 1, n = 9), $Bio-Oss^{(R)}$ wetted with rhBMP-2 (group 2, n = 9), $Bio-Oss^{(R)}$ wetted with rhBMP-2 and 1 mM alendronate (group 3, n = 9) and $Bio-Oss^{(R)}$ wetted with rhBMP-2 and 10 mM alendronate (group 4, n = 9). In each group, three animals were euthanized at 2, 4 and 8 weeks after surgery, respectively. The specimens were then analyzed by histology, histomorphometry and immunohistochemistry analysis. Results: There were significant decrease of bone formation area (p < 0.05) between group 4 and group 2, 3. Group 3 showed increase of new bone formation compared to group 2. In immunohistochemistry, collagen type I and osteoprotegerin (OPG) didn't show any difference. However, receptor activator of nuclear factor ${\kappa}B$ ligand (RANKL) decreased with time dependent except group 4. Conclusion: Low concentration bisphosphonate and rhBMP-2 have synergic effect on bone regeneration and this is result from the decreased activity of RANKL of osteoblast.

• The Korea Journal of Herbology
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• v.39 no.3
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• pp.85-95
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• 2024

Objectives : We proposed the hypothesis that administering Trigonella foenum-graecum seed extract (TSE) could alleviate menopausal symptoms and osteoporosis resulting from estrogen deficiency. Methods : Ovariectomized (OVX) rats were administered TSE at doses of 300 or 600 mg/kg body weight for 8 weeks, followed by measurement of serum lipid profile and serum bone markers using ELISA kits. Additionally, analysis of related genes in the femur and uterus was performed using Western blot and real-time PCR. Additionally, micro-CT analysis was performed to investigate the protective effect of TSE against bone loss due to oophorectomy. Results : The administration of TSE led to significant reductions in triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, and glucose levels in the serum of OVX rats. Furthermore, TSE increased estradiol levels in the serum and notably improved the levels of biochemical markers associated with bone metabolism. Additionally, TSE exerted significant regulatory effects on the mRNA levels of alkaline phosphatase (ALP) and receptor activator of nuclear factor kappa-B ligand (RANKL)-genes closely associated with bone metabolism in the femur. TSE also demonstrated pronounced effects on uterine tissue, with improvements observed in gene expression related to estrogen receptors. Conclusion : Our findings confirm the efficacy of TSE in ameliorating menopause symptoms by modulating elements associated with both bone and lipid metabolism in the serum, uterine tissue, and femur of OVX rats. The present findings suggest that TSE may offer potential therapeutic effects for symptoms related to menopause and osteoporosis in females.

• Current Optics and Photonics
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• v.1 no.4
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• pp.412-420
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• 2017

Multinucleated bone resorptive osteoclasts differentiate from bone marrow-derived monocyte/macrophage precursor cells. During osteoclast differentiation, mononuclear pre-osteoclasts change their morphology and biochemical characteristics. In this study, Raman spectroscopy with multivariate techniques such as Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) were used to extract biochemical information related to various cellular events during osteoclastogenesis. This technique allowed for label-free and noninvasive monitoring of differentiating cells, and clearly discriminated four different time points during osteoclast differentiation. The Raman band intensity showed significant time-dependent changes that increased up to day 4. The results of Raman spectroscopy agreed with results from atomic force microscopy (AFM) and tartrate-resistant acid phosphatase (TRAP) staining, a conventional biological assay. Under AFM, normal spindle-like mononuclear pre-osteoclasts became round and smaller at day 2 after treatment with a receptor activator of nuclear $factor-{\kappa}B$ ligand and they formed multinucleated giant cells at day 4. Thus, Raman spectroscopy, in combination with PCA-LDA, may be useful for noninvasive label-free quality assessment of cell status during osteoclast differentiation, enabling more efficient optimization of the bioprocesses.

• The Journal of Korean Obstetrics and Gynecology
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• v.27 no.3
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• pp.12-27
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• 2014

Objectives: This study was performed to evaluate the effect of Guibi-tang water extract (GB) on osteoporosis. Methods: We examined the effect of GB on osteoclast differentiation using murine pre-osteoclastic RAW 264.7 cells treated with receptor activator of nuclear factor kappa-B ligand (RANKL). The effect of GB on osteoclast was measured by counting TRAP (+) multinucleated cells and measuring TRAP activity. The mRNA expressions of osteoclastogenesis-related genes (Cathepsin K, MMP-9, TRAP, NFATc1, MITF, TNF-${\alpha}$, IL-6, COX-2) were measured by real-time PCR. We examined the effect of GB on osteoblast proliferation, ALP activity, bone matrix protein synthesis and collagen synthesis using murine calvarial cell. Results: GB decreased the number of TRAP (+) multinucleated cells and inhibited TRAP activity in RANKL-stimulated RAW 264.7 cell. GB decreased the expression of genes related osteoclastogenesis such as Cathepsin K, MMP-9, TRAP, NFATc1, MITF, COX-2 in RANKL-stimulated RAW 264.7 cell. But GB did not decrease the expression of iNOS and increased the expression of TNF-${\alpha}$, IL-6 in RANKL-stimulated RAW 264.7 cell. These genes (iNOS, TNF-${\alpha}$, IL-6) are thought to be related with the inflammatory bone destruction. GB increased cell proliferation of rat calvarial cell and also increased ALP activity in rat calvarial cell. GB did not increase bone matrix protein synthesis but increased collagen synthesis in rat calvarial cell. Conclusions: This study suggests that GB may be effective in treating osteoporosis by inhibiting osteoclast differentiation and its related gene expression and by increasing osteoblast proliferation.

• Korean Journal of Community Nutrition
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• v.16 no.6
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• pp.762-770
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• 2011

Osteoprotegerin (OPG) plays a core role in bone reformation by antagonizing the effect of receptor activator of nuclear factor ${\kappa}$-B ligand (RANKL), and mediates vascular calcification in cardiovascular disease patients. Thus, we aimed to examine the relationship between serum OPG levels and cardiovascular factors and inflammatory markers in metabolic syndrome patients (MS). This cross-sectional study included 96 men who visited the diet clinic between May and July 2011. Patients were classified into 2 groups based on NCEP-ATP guidelines: normal and with MS (n = 50 and 46, respectively). Physical measurements, biochemical assay were measured. Serum OPG and IL-6, diponectin and hs-CRP were assessed. MS were aged $50.02{\pm}10.85$ years, and normal patients $52.07{\pm}9.56$ years, with no significant differences. Significant differences were not observed in BMI between the 2 groups. Moreover, significant differences were not observed in serum OPG, however, the serum OPG level ($4.41{\pm}1.86pmol/L$) differed significantly between an overweight MS (BMI > 25) and normal patients. OPG was correlated to age (r = 0.410, p = 0.000), HDL-cholesterol (r = 0.209, p = 0.015), and log adiponectin (r = 0.175, p = 0.042). Multiple regression analyses using the enter method showed that age (${\beta}$ = 0.412, p = 0.000) and BMI (${\beta}$ = 0.265, p = 0.000) considerably affected OPG. In conclusion, out study showed that serum OPG levels are correlated with cardiovascular risk factors, such as BMI, HDL-cholesterol and adiponectin in MS and adiponectin, suggesting that serum OPG has potential as a cardiovascular disease indicator and predictor.

• Journal of Physiology & Pathology in Korean Medicine
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• v.26 no.3
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• pp.320-324
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• 2012

Osteoporosis is an important public health issue in postmenopausal women. It is a major public health concern and is widely believed that osteoporosis results from imbalance between bone resorption and bone formation. Recently natural products from plants have been extensively studied as therapeutic drugs to treat and prevent various diseases. Hoelen (scientific name, Poria cocos) is a mushroom that is used in traditional Chinese medicine. Hoelen exhibits anti-inflammatory activity and has a protective effect on tumor progression. However, the effect of hoelen in osteoclast differentiation remains unknown. Thus, we examined the effect of hoelen in receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL)-induced osteoclast differentiation. Hoelen significantly inhibited RANKL-induced osteoclast differentiation in bone marrow-derived macrophages (BMMs) in dose dependent manner without toxicity. Also, we showed that hoelen significantly inhibited the mRNA expression of tartrate-resistant acid phophatase (TRAP) and nuclear factor of activated T cells 1 (NFATc1) in BMMs treated with RANKL. In Particular, hoelen greatly inhibited the protein expression of NFATc1. Ectopic expression of NFATc1 partially reverses hoelen-mediated inhibition of osteoclast differentiation. Taken together, our results demonstrated that hoelen may be useful treatment option of bone-related disease such as osteoporosis, reumatoid arthritis, and periodontitis.

• The Korean Journal of Food And Nutrition
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• v.29 no.2
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• pp.178-185
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• 2016

This study was designed to develop and to qualify a coffee alternative beverage using a mixture of coffee beans and roasted black beans (Rhynchosia nulubilis). Therefore, the total isoflavone content (TIC), total phenol content (TPC), antioxidant activity, anti-inflammatory activity, NFATc1 (Nuclear factor of activated T-cells c1) expression in RANKL (receptor activator of nuclear factor kappa-B ligand)-stimulated RAW264.7 cells and sensory evaluation were measured for 5 different Cb (coffee bean)-RoS (roasted seomoktae) mixture extracts (Cb100RoS0, Cb75RoS25, Cb50RoS50, Cb25RoS75, and Cb0RoS100). Cb0RoS100 had the highest TIC ($516.83{\pm}36.61mg/100g$) and TPC ($18.11{\pm}1.77mg$ TAE/100 g) along with the highest antioxidant activity as measured by DPPH radical scavenging activity ($73.55{\pm}8.11%$) and ABTS radical scavenging activity ($63.27{\pm}7.27%$). Also, Cb0RoS100 showed the highest anti-inflammatory activity as measured by NO production ($13.57{\pm}2.21{\mu}M$) and PGE2 production ($3.25{\pm}0.21ng/mL$). The more the RoS ratio was increased in the mixtures of Cb-RoS, the more the NFATc1 protein expression was decreased in RANKL-stimulated RAW264.7 cells. In case of sensory evaluation, Cb50RoS50 had the highest scores for flavor, delicate flavor and overall quality, which were similar to those in Cb alone (Cb100RoS0). We suggest that the use of RoS replacement instead of Cb in/as a coffee alternative beverage may help to reduce the risk of caffeine-related bone loss and/or bone disease by effectively blocking NFATc1 expression in RANKL-stimulated RAW264.7 cells compared with Cb alone.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.1
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• pp.64-70
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• 2010

Soybean is of particular interest as a food supplement of isoflavones for inhibiting bone resorption in postmenopausal woman. These beneficial effects of isoflavones are caused by functioning as partial agonists or antagonists of estrogen, of which anti-resorptive effect is mediated indirectly through paracrine factors produced by osteoblasts that act on osteoclasts. In this study, the indirect effect of soybean on osteoclastic differentiation of RAW264.7 cells were investigated. The conditioned medium was collected from MC3T3-E1 osbeoblasts treated with 0.001 mg/mL~0.1 mg/mL soybean extracts for 6 days, mixed in 1:1 ratio with osteoclast medium, and then added into RAW264.7 cells with receptor activator of nuclear factor kappa B ligand (RANKL), a differentiation inducer for 3 days. Of paracrine factors in the conditioned medium, the protein expression of osteoprotegerin (OPG) with soybean extract was specifically higher in a dose dependent manner than with $10^{-9}$ M~$10^{-6}$ M of estrogen, genistein or daidzein standards. In RAW264.7 cells, the conditioned medium with soybean inhibited RANKL induced osteoclastic differentiation as total number of multinucleated tartrateresistant alkaline phosphatase (TRAP)-positive osteoclasts and protein expression of MMP-9 were significantly decreased. Coupled with the low expression of estrogen receptor $\alpha$ and $\beta$ proteins in RANKL treated RAW264.7 cells, we demonstrate that the conditioned medium of soybean treated osteoblasts inhibits RANKL induced differentiation of osteoclasts with the selective expression of OPG in osteoblasts.

• Journal of Periodontal and Implant Science
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• v.42 no.6
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• pp.185-195
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• 2012

Purpose: (-)-epigallocatechin-3-gallate (EGCG) has been reported to exert anti-inflammatory and antibacterial effects in periodontitis. However, its exact mechanism of action has yet to be determined. The present in vitro study evaluated the anti-in-flammatory effects of EGCG on human periodontal ligament fibroblasts (hPDLFs) and human periodontal ligament stem cells (hPDLSCs) affected by bacterial lipopolysaccharide (LPS) extracted from Porphyromonas gingivalis. Methods: hPDLFs and hPDLSCs were extracted from healthy young adults and were treated with EGCG and/or P. gingivalis LPS. After 1, 3, 5, and 7 days from treatment, cytotoxic and proliferative effects were evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and bromodeoxyuridine assay, respectively. And then, the gene expressions of hPDLFs and hPDLSCs were observed for interleukin (IL)-$1{\beta}$, IL-6, tumor necrosis factor (TNF)-${\alpha}$, osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B ligand (RANKL), and RANKL/OPG using real-time polymerase chain reaction (PCR) at 0, 6, 24, and 48 hours after treatment. The experiments were performed with the following groups for hPDLFs and hPDLSCs; 1) No treat, 2) EGCG alone, 3) P. gingivalis LPS alone, 4) EGCG+P. gingivalis LPS. Results: The 20 ${\mu}M$ of EGCG and 20 ${\mu}g/mL$ of P. gingivalis LPS had the lowest cytotoxic effects, so those concentrations were used for further experiments. The proliferations of hPDLFs and hPDLSCs increased in all groups, though the 'EGCG alone' showed less increase. In real-time PCR, the hPDLFs and hPDLSCs of 'EGCG alone' showed similar gene expressions to those cells of 'no treat'. The gene expressions of 'P. gingivalis LPS alone' in both hPDLFs and hPDLSCs were highly increased at 6 hours for IL-$1{\beta}$, IL-6, TNF-${\alpha}$, RANKL, and RANKL/OPG, except the RANKL/OPG in hPDLSCs. However, those increased gene expressions were down-regulated in 'EGCG+P. gingivalis LPS' by the additional treatment of EGCG. Conclusions: Our results demonstrate that EGCG could exert an anti-inflammatory effect in hPDLFs and hPDLSCs against a major pathogen of periodontitis, P. gingivalis LPS.