• BMB Reports
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• v.45 no.10
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• pp.571-576
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• 2012

Radiotherapy is considered to cause detrimental effects on bone tissue eventually increasing bone loss and fracture risk. However, there is a great controversy on the real effects of irradiation itself on osteoblasts, and the mechanisms by which irradiation affects osteoblast differentiation and mineralization are not completely understood. We explored how X-ray radiation influences differentiation and bone-specific gene expression in mouse calvarial osteoblasts. Irradiation at 2 Gy not only increased differentiation and mineralization of the cells, but also upregulated the expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin at early stages of differentiation. However, irradiation at higher doses (>2 Gy) did not stimulate osteoblast differentiation, rather it suppressed DNA synthesis by the cells without a toxic effect. Additional experiments suggested that transforming growth factor-beta 1 and runt-transcription factor 2 play important roles in irradiation- stimulated bone differentiation by acting as upstream regulators of bone-specific markers.

• BMB Reports
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• v.55 no.12
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• pp.627-632
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• 2022

Dickkopf-1 (DKK1) is a secreted protein that acts as an antagonist of the canonical WNT/β-catenin pathway, which regulates osteoblast differentiation. However, the role of DKK1 on osteoblast differentiation has not yet been fully clarified. Here, we investigate the functional role of DKK1 on osteoblast differentiation. Primary osteoprogenitor cells were isolated from human spinal bone tissues. To examine the role of DKK1 in osteoblast differentiation, we manipulated the expression of DKK1, and the cells were differentiated into mature osteoblasts. DKK1 overexpression in osteoprogenitor cells promoted matrix mineralization of osteoblast differentiation but did not promote matrix maturation. DKK1 increased Ca⁺ influx and activation of the Ca⁺/calmodulin-dependent protein kinase II Alpha (CAMK2A)-cAMP response element-binding protein 1 (CREB1) and increased translocation of p-CREB1 into the nucleus. In contrast, stable DKK1 knockdown in human osteosarcoma cell line SaOS2 exhibited reduced nuclear translocation of p-CREB1 and matrix mineralization. Overall, we suggest that manipulating DKK1 regulates the matrix mineralization of osteoblasts by Ca⁺-CAMK2A-CREB1, and DKK1 is a crucial gene for bone mineralization of osteoblasts.

• International Journal of Stem Cells
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• v.16 no.3
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• pp.342-355
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• 2023

Background and Objectives: Osteoblasts are derived from bone marrow mesenchymal stem cells (BMMSCs) and play important role in bone remodeling. While our previous studies have investigated the cell subtypes and heterogeneity in osteoblasts and BMMSCs separately, cell-to-cell communications between osteoblasts and BMMSCs in vivo in humans have not been characterized. The aim of this study was to investigate the cellular communication between human primary osteoblasts and bone marrow mesenchymal stem cells. Methods and Results: To investigate the cell-to-cell communications between osteoblasts and BMMSCs and identify new cell subtypes, we performed a systematic integration analysis with our single-cell RNA sequencing (scRNA-seq) transcriptomes data from BMMSCs and osteoblasts. We successfully identified a novel preosteoblasts subtype which highly expressed ATF3, CCL2, CXCL2 and IRF1. Biological functional annotations of the transcriptomes suggested that the novel preosteoblasts subtype may inhibit osteoblasts differentiation, maintain cells to a less differentiated status and recruit osteoclasts. Ligand-receptor interaction analysis showed strong interaction between mature osteoblasts and BMMSCs. Meanwhile, we found FZD1 was highly expressed in BMMSCs of osteogenic differentiation direction. WIF1 and SFRP4, which were highly expressed in mature osteoblasts were reported to inhibit osteogenic differentiation. We speculated that WIF1 and sFRP4 expressed in mature osteoblasts inhibited the binding of FZD1 to Wnt ligand in BMMSCs, thereby further inhibiting osteogenic differentiation of BMMSCs. Conclusions: Our study provided a more systematic and comprehensive understanding of the heterogeneity of osteogenic cells. At the single cell level, this study provided insights into the cell-to-cell communications between BMMSCs and osteoblasts and mature osteoblasts may mediate negative feedback regulation of osteogenesis process.

• Journal of Physiology & Pathology in Korean Medicine
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• v.35 no.5
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• pp.169-176
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• 2021

This study was conducted to suggest the Cudrania tricuspidata leaf and Achyranthes japonica Nakai Complex (CAC) possibility of use as a functional natural material for improving bone disease. Cudrania tricuspidata leaf and Achyranthes japonica Nakai were mixed in the same amount, extracted with hot water, and then powdered and used in the study. After, the cytotoxicity of CAC for osteoblasts (MG63 cell), osteoclasts (differentiated RAW264.7 cell), and macrophages (RAW264.7 cell) were evaluated by MTT assay, and ALP assay and TRAP assay were performed to confirm the differentiation capacity of osteoblasts and osteoclasts, respectively. In addition, the anti-inflammatory effect in macrophages was evaluated by ELISA, qRT-PCR, and western blot assay. CAC did not proliferated osteoblasts and osteoclasts, but increased ALP activity against osteoblasts differentiation and decreased TRAP activity against osteoclasts differentiation. CAC did not proliferated macrophages but decreased nitric oxide production. Also, decreased NOS2, IL1B, IL6, PTGS2, and TNFA gene expression, and JNK and p38 protein phosphorylation in a concentration-dependent manner, but ERK protein phosphorylation was not changed. As a result, CAC increased the differentiation and activation of osteoblasts, inhibited the differentiation and activation of osteoclasts, and regulated the expression of inflammatory cytokines in macrophages. Therefore, it is thought that CAC can be used as a functional natural material that prevents bone disease and has an anti-inflammatory effect.

• Molecules and Cells
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• v.24 no.2
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• pp.177-184
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• 2007

Approximately 200 individual skeletal elements, which differ in shape and size, are the building blocks of the vertebrate skeleton. Various features of the individual skeletal elements, such as their location, shape, growth and differentiation rate, are being determined during embryonic development. A few skeletal elements, such as the lateral halves of the clavicle and parts of the skull are formed by a process called intramembranous ossification, whereby mesenchymal cells differentiate directly into osteoblasts, while the majority of skeletal elements are formed via endochondral ossification. The latter process starts with the formation of a cartilaginous template, which eventually is being replaced by bone. This requires co-regulation of differentiation of the cell-types specific for cartilage and bone, chondrocytes and osteoblasts, respectively. In recent years it has been demonstrated that Wnt family members and their respective intracellular pathways, such as non-canonical and the canonical $Wnt/{\beta}$-catenin pathway, play important and diverse roles during different steps of vertebrate skeletal development. Based on the recent discoveries modulation of the canonical Wnt-signaling pathway could be an interesting approach to direct stem cells into certain skeletal lineages.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.28 no.2
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• pp.114-125
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• 2002

This study was designed to evaluate the influence of fibroblasts or connective tissue from mouse oral mucosa on differentiation of neonatal mouse calvaria-derived osteoblasts and mineralization of bone nodules. Primary cell cultures from mouse calvarial osteoblasts and 2-4 passaged fibroblasts from oral mucosa were co-cultured in monolayer cultures, devided into 6 experimental group according to cell density or cell confluency. Osteoblasts were also co-cultured with fibroblasts in $Transwell^{(R)}$ culture plate with different co-cultured period according to osteoblast differentiation. The alkaline phosphatase activity were measured in monolayer cultures and cultures using $Transwell^{(R)}$. The mineralized bone nodules were presented by Von Kossa staining and density of mineralized nodules was measured by image analysis. The connective tissues with or without osteoblast seeding were cultured and examined histologically by Von Kossa and Trichrome Goldner staining. The results were as follows; 1. Prolonged maturation of matrix and delayed mineralization of bone nodules were resulted in monolayer cultures. 2. Co-culture of fibroblast with osteoblast using $Transwell^{(R)}$ during osteoblast proliferation stage stimulated proliferation of osteoblasts and increased alkaline phosphatase activity and mineralization of bone nodules. 3. Co-culture of fibroblast with osteoblast using $Transwell^{(R)}$ during matrix mineralization stage decreased and delayed mineralization of bone nodules. 4. In vitro cultured connective tissue with osteoblast seeding resulted in proliferation of osteoblasts and matrix formation with mineralization.

• Journal of Periodontal and Implant Science
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• v.47 no.5
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• pp.273-291
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• 2017

Purpose: Although static magnetic fields (SMFs) have been used in dental prostheses and osseointegrated implants, their biological effects on osteoblastic and cementoblastic differentiation in cells involved in periodontal regeneration remain unknown. This study was undertaken to investigate the effects of SMFs (15 mT) on the osteoblastic and cementoblastic differentiation of human osteoblasts, periodontal ligament cells (PDLCs), and cementoblasts, and to explore the possible mechanisms underlying these effects. Methods: Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, mineralized nodule formation based on Alizarin red staining, calcium content, and the expression of marker mRNAs assessed by reverse transcription polymerase chain reaction (RT-PCR). Signaling pathways were analyzed by western blotting and immunocytochemistry. Results: The activities of the early marker ALP and the late markers matrix mineralization and calcium content, as well as osteoblast- and cementoblast-specific gene expression in osteoblasts, PDLCs, and cementoblasts were enhanced. SMFs upregulated the expression of Wnt proteins, and increased the phosphorylation of glycogen synthase $kinase-3{\beta}$ ($GSK-3{\beta}$) and total ${\beta}-catenin$ protein expression. Furthermore, p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) pathways were activated. Conclusions: SMF treatment enhanced osteoblastic and/or cementoblastic differentiation in osteoblasts, cementoblasts, and PDLCs. These findings provide a molecular basis for the beneficial osteogenic and/or cementogenic effect of SMFs, which could have potential in stimulating bone or cementum formation during bone regeneration and in patients with periodontal disease.

• BMB Reports
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• v.46 no.11
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• pp.527-532
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• 2013

Gangliosides are complex glycosphingolipids that are the major component of cytoplasmic cell membranes, and play a role in the control of biological processes. Human mesenchymal stem cells (hMSCs) have received considerable attention as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. In this study, we focus on various functional roles of gangliosides in the differentiation of hMSCs into osteoblasts or neuronal cells. A relationship between gangliosides and epidermal growth factor receptor (EGFR) activation during osteoblastic differentiation of hMSCs was observed, and the gangliosides may play a major role in the regulation of the differentiation. The roles of gangliosides in osteoblast differentiation are dependent on the origin of hMSCs. The reduction of ganglioside biosynthesis inhibited the neuronal differentiation of hMSCs during an early stage of the differentiation process, and the ganglioside expression can be used as a marker for the identification of neuronal differentiation from hMSCs.

• Korean Journal of Pharmacognosy
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• v.40 no.3
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• pp.190-195
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• 2009

Osteoblasts play an important role in bone metabolism by bone formation. Natural medicines having a stimulatory activity on osteoblast proliferation and differentiation can improve bone diseases such as osteoporosis. The methanol extracts of 159 herbal medicines were screened for the stimulatory activity on osteoblast proliferation by MTT assay and differentiation in the presence of ascorbic acid and $\beta$-glycerophosphate. Among the tested extracts, Alpiniae Semen, Amomi Semen, Anemarrhenae Rhizoma, Bambusae Folium, Cannabis Semen, Dalbergiae odoriferae Lignum, and Luffae Fructus Retinervus showed relatively strong stimulatory activity on osteoblast proliferation, whereas Amomi Semen showed strong stimulatory activity on osteoblast differentiation.

• BMB Reports
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• v.52 no.7
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• pp.469-474
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• 2019

Kruppel-like factor 2 (KLF2) has been implicated in the regulation of cell proliferation, differentiation, and survival in a variety of cells. Recently, it has been reported that KLF2 regulates the p65-mediated transactivation of $NF-{\kappa}B$. Although the $NF-{\kappa}B$ pathway plays an important role in the differentiation of osteoclasts and osteoblasts, the role of KLF2 in these bone cells has not yet been fully elucidated. In this study, we demonstrated that KLF2 regulates osteoclast and osteoblast differentiation. The overexpression of KLF2 in osteoclast precursor cells inhibited osteoclast differentiation by downregulating c-Fos, NFATc1, and TRAP expression, while KLF2 overexpression in osteoblasts enhanced osteoblast differentiation and function by upregulating Runx2, ALP, and BSP expression. Conversely, the downregulation of KLF2 with KLF2-specific siRNA increased osteoclast differentiation and inhibited osteoblast differentiation. Moreover, the overexpression of interferon regulatory protein 2-binding protein 2 (IRF2BP2), a regulator of KLF2, suppressed osteoclast differentiation and enhanced osteoblast differentiation and function. These effects were reversed by downregulating KLF2. Collectively, our data provide new insights and evidence to suggest that the IRF2BP2/KLF2 axis mediates osteoclast and osteoblast differentiation, thereby affecting bone homeostasis.