• Nutrition Research and Practice
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• v.7 no.3
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• pp.185-191
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• 2013

In previous studies, we found that the consumption of legumes decreased bone turnover in ovariectomized rats. The purpose of the present study is to determine whether the protective effects on bone mineral density (BMD) and the microarchitecture of a diet containing legumes are comparable. In addition, we aim to determine their protective actions in bones by studying bone specific gene expression. Forty-two Sprague-Dawley rats are being divided into six groups during the 12 week study: 1) rats that underwent sham operations (Sham), 2) ovariectomized rats fed an AIN-93M diet (OVX), 3) ovariectomized rats fed an AIN-93M diet with soybeans (OVX-S), 4) ovariectomized rats fed an AIN-93M diet with mung beans (OVX-M), 5) ovariectomized rats fed an AIN-93M diet with cowpeas (OVX-C), and 6) ovariectomized rats fed an AIN-93M diet with azuki beans (OVX-A). Consumption of legumes significantly increased BMD of the spine and femur and bone volume of the femur compared to the OVX. Serum calcium and phosphate ratio, osteocalcin, expression of osteoprotegerin (OPG), and the receptor activator of nuclear factor ${\kappa}B$ ligand (RANKL) ratio increased significantly, while urinary excretion of calcium and deoxypyridinoline and expression of TNF-${\alpha}$ and IL-6 were significantly reduced in OVX rats fed legumes, compared to OVX rats that were not fed legumes. This study demonstrates that consumption of legumes has a beneficial effect on bone through modulation of OPG and RANKL expression in ovariectomized rats and that legume consumption can help compensate for an estrogen-deficiency by preventing bone loss induced by ovarian hormone deficiency.

• Journal of Nutrition and Health
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• v.51 no.5
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• pp.379-385
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• 2018

Purpose: Zinc (Zn) is an essential trace element for bone mineralization and osteoblast function. We examined the effects of Zn deficiency on osteoblast differentiation and mineralization in MC3T3-E1 cells. Methods: Osteoblastic MC3T3-E1 cells were cultured at concentration of 1 to $15{\mu}M$ $ZnCl_2$ (Zn- or Zn+) for 5, 15 and 25 days up to the calcification period. Extracellular matrix mineralization was detected by staining Ca and P deposits using Alizarin Red and von Kossa stain respectively, and alkaline phosphatase (ALP) activity was detected by ALP staining and colorimetric method. Results: Extracellular matrix mineralization was decreased in Zn deficiency over 5, 15, and 25 days. Similarly, staining of ALP activity as the sign of an osteoblast differentiation, was also decreased by Zn deficiency over the same period. Interestingly, the gene expression of bone-related markers (ALP, PTHR; parathyroid hormone receptor, OPN; osteopontin, OC; osteocalcin and COLI; collagen type I), and bone-specific transcription factor Runx2 were downregulated by Zn deficiency for 5 or 15 days, however, this was restored at 25 days. Conclusion: Our data suggests that Zn deficiency inhibits osteoblast differentiation by retarding bone marker gene expression and also inhibits bone mineralization by decreasing Ca/P deposition as well as ALP activity.

• Reproductive and Developmental Biology
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• v.32 no.1
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• pp.21-25
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• 2008

Bone marrow mesenchymal stem cells (BMMSCs) have the capacity for self-renewal and differentiation into a variety of cell types. They represent an attractive source of cells for gene and cell therapy. The purpose of this study is to direct the specific expression of the DsRed reporter gene in $Sca-1^+$ BMMSCs differentiated into a cardiomyogenic lineage. We constructed the prMLC-2v-DsRed vector expressing DsRed under the control of the 309 tp fragment of the rat MLC-2v 5'-flanking region. The specific expression of the DsRed reporter gene under the transcriptional control of the 309 bp fragment of the rat MLC-2v promoter was tested in 5-azacytidine healed-$Sca-1^+$ BMMSCs over 2 weeks after the prMLC-2v-DsRed transfection. The prMLC-2v-DsRed was specifically expressed in the $Sca-1^+$ BMMSCs with cardiomyogenic lineage differentiation and it demonstrates that the 309 bp sequences of the rat MLC-2v 5'-flanking region is sufficient to confer cardiac specific expression on a DsRed reporter gene. The cardiac-specific promoter-driven reporter vector provides an important tool for the study of stem cell differentiation and cell replacement therapy in ischemic cardiomyopathy.

• Development and Reproduction
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• v.17 no.1
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• pp.1-8
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• 2013

Osteoprotegerin (OPG) is a secreted glycoprotein that regulates bone resorption by inhibiting differentiation and activation of osteoclast, thereby potentially useful for the treatment of many bone diseases associated with increased bone loss. In this study, we designed a novel cDNA expression cassette by modifying the potent and mammary gland-specific goat ${\beta}$-casein/hGH hybrid gene construct and examined human OPG (hOPG) cDNA expression in transgenic mice. Six transgenic mice all successfully expressed hOPG in their milk at the level of 0.06-2,000 ${\mu}g/ml$. An estimated molecular weight of the milk hOPG was 55 kDa in SDS-PAGE, which is the same as a naturally glycosylated monomer. This hOPG expression was highly specific to the mammary glands of transgenic mice. hOPG mRNA was not detected in any organs analyzed except mammary gland. Functional integrity of milk hOPG was evaluated by TRAP (tartrate-resistant acid phosphatase) activity assay in bone marrow cell cultures. OPG ligand (OPG-L) treatment increased TRAP activity by two fold but it was completely abolished by co-treatment with transgenic milk containing hOPG. Taken together, our novel cDNA expression cassette could direct an efficient expression of biologically active hOPG, a potential candidate pharmaceutical for bone diseases, only in the mammary gland of transgenic mice.

• Journal of Nutrition and Health
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• v.51 no.1
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• pp.23-30
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• 2018

Purpose: Runx2 (runt-related transcription factor 2), a bone-specific transcription factor, is a key regulator of osteoblast differentiation and its expression is induced by the activation of BMP-2 signaling. This study examined whether zinc modulates BMP-2 signaling and therefore stimulates Runx2 and osteoblast differentiation gene expression. Methods: Two osteoblastic MC3T3-E1 cell lines (subclones 4 as a high osteoblast differentiation and subclone 24 as a low osteoblastic differentiation) were cultured in an osteogenic medium (OSM) as the normal control, Zn-($1{\mu}M$ Zn) or Zn+($15{\mu}M$ Zn) for 24 h. The genes and proteins for BMP-2 signaling (BMP-2, Smad-1/p-Smad-1), transcription factors (Runx2, osterix), and osteoblast differentiation marker proteins were assessed. Results: In both cell lines, BMP-2 mRAN and protein expression and extracellular BMP-2 secretion all decreased in Zn-. The expression of Smad-1 (downstream regulator of BMP-2 signaling) and p-Smad-1 (phosphorylated Smad-1) also downregulated in Zn-. Furthermore, the expression of the bone-specific transcription factors, Runx2 and osterix, decreased in Zn-, which might be due to the decreased BMP-2 expression and Smad-1 activation (p-Smad-1) by Zn-, because Runx2 and osterix both are downstream in BMP-2 signaling. Bone marker gene expression, such as alkaline phosphatase (ALP), collagen type I (COLI), osteocalcin, and osteopontin were also downregulated in Zn-. Conclusion: The results suggest that a zinc deficiency in osteoblasts suppresses the BMP-2 signaling pathway via the suppression of Smad-1 activation, and this suppressed BMP-2 signaling can cause poor osteoblast differentiation.

• Journal of Pharmaceutical Investigation
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• v.33 no.4
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• pp.281-286
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• 2003

To design gene deliver systems which can deliver higher amounts of genes into stem cells, we studied the expression of receptors involved in the receptor-mediated endocytosis of bone marrow stromal stem cells. Bone marrow was isolated from ICR mice, and bone marrow stromal stem cells were isolated based on their plastic adherence property. Several culture conditions were screened for effective and continuous culture of marrow stromal stem cells. MesenCult medium was finally used to cultivate marrow stromal stem cells in vitro. As candidate receptors, various chemokine receptors were studied. Both bone marrow cells ad marrow-derived stromal stem cells showed expression of CC chemokine receptors (CCR) and CXC chemokine receptors (CXCR). Marrow stromal stem cells showed higher expression of CCR5 ad CXCR4 chemokine receptors as compared to other types of chemokine receptors. Moreover, though the expression of chemokine receptors generally decreased in most chemokine receptors with the cultivaton of marrow stromal stem cells, CCR5 and CXCR4 chemokine receptors retained the higher level of receptor expressions over prolonged periods. These results suggest that the ligands exhibiting specific binding to CCR5 or CXCR4 might be used to modify gene delivery systems for increased levels of receptor-mediated gene delivery into stromal stem cells.

• Archives of Craniofacial Surgery
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• v.24 no.6
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• pp.251-259
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• 2023

Background: Periosteum-mediated bone regeneration (PMBR) is a recognized method for mandibular reconstruction. Despite its unpredictable nature and the limited degree to which it is understood, it does not share the concerns of developmental changes to donor and recipient tissues that other treatment options do. The definitive role of the periosteum in bone regeneration in any mammal remains largely unexplored. The purpose of this study was to identify the genetic determinants of PMBR in mammals through a systematic review. Methods: Our search methodology was designed in accordance with the PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis) guidelines. We conducted a quality assessment of each publication, and evaluated the differences in gene expression between days 7 and 15. Results: A total of four studies satisfied the inclusion criteria. The subjects and tissues examined in these studies were Wistar rat calvaria in two studies, mini-pigs in one study, and calves and mice in one study. Three out of the four studies achieved the necessary quality score of ≥ 3. Gene expression analysis showed increased activity of genes responsible for angiogenesis, cytokine activities, and immune-inflammatory responses on day 7. Additionally, genes related to skeletal development and signaling pathways were upregulated on day 15. Conclusions: The results suggest that skeletal morphogenesis is regulated by genes associated with skeletal development, and the gene expression patterns of PMBR may be characterized by specific pathways.

• BMB Reports
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• v.43 no.8
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• pp.513-523
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• 2010

Cytokines that bind to and signal through the gp130 co-receptor subunit include interleukin (IL)-6, IL-11, oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and ciliary neutrophic factor (CNTF). Apart from contributing to inflammation, gp130 signalling cytokines also function in the maintenance of bone homeostasis. Expression of each of these cytokines and their ligand-specific receptors is observed in bone and joint cells, and bone-active hormones and inflammatory cytokines regulate their expression. gp130 signalling cytokines have been shown to regulate the differentiation and activity of osteoblasts, osteoclasts and chondrocytes. Furthermore, cytokine and receptor specific gene-knockout mouse models have identified distinct roles for each of these cytokines in regulating bone resorption, bone formation and bone growth. This review will discuss the current models of paracrine and endocrine actions of gp130-signalling cytokines in bone remodelling and growth, as well as their impact in pathologic bone remodelling evident in periodontal disease, rheumatoid arthritis, spondylarthropathies and osteoarthritis.

• Journal of Periodontal and Implant Science
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• v.42 no.3
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• pp.95-104
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• 2012

Purpose: The purpose of this study was to determine whether increasing the Ti6Al4V surface oxide negative charge through heat ($600^{\circ}C$) or radiofrequency plasma glow discharge (RFGD) pretreatment, with or without a subsequent coating with fibronectin, stimulated osteoblast gene marker expression in the MC3T3 osteoprogenitor cell line. Methods: Quantitative real-time polymerase chain reaction was used to measure changes over time in the mRNA levels for osteoblast gene markers, including alkaline phosphatase, bone sialoprotein, collagen type I (${\alpha}1$), osteocalcin, osteopontin and parathyroid hormone-related peptide (PTH-rP), and the osteoblast precursor genes Runx2 and osterix. Results: Osteoprogenitors began to differentiate earlier on disks that were pretreated with heat or RFGD. The pretreatments increased gene marker expression in the absence of a fibronectin coating. However, pretreatments increased osteoblast gene expression for fibronectin-coated disks more than uncoated disks, suggesting a surface oxide-mediated specific enhancement of fibronectin's bioactivity. Heat pretreatment had greater effects on the mRNA expression of genes for PTH-rP, alkaline phosphatase and osteocalcin while RFGD pretreatment had greater effects on osteopontin and bone sialoprotein gene expression. Conclusions: The results suggest that heat and RFGD pretreatments of the Ti6Al4V surface oxide stimulated osteoblast differentiation through an enhancement of (a) coated fibronectin's bioactivity and (b) the bioactivities of other serum or matrix proteins. The quantitative differences in the effects of the two pretreatments on osteoblast gene marker expression may have arisen from the unique physico-chemical characteristics of each resultant oxide surface. Therefore, engineering the Ti6Al4V surface oxide to become more negatively charged can be used to accelerate osteoblast differentiation through fibronectin-dependent and independent mechanisms.

• Journal of Nutrition and Health
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• v.51 no.4
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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.