• 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.

• 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 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.

• Preventive Nutrition and Food Science
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• v.19 no.3
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• pp.194-203
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• 2014

Yam (Dioscorea batatas) is widely consumed as functional food for health promotion mainly in East Asia countries. We assessed whether yam root (tuber) or bark (peel) extracts stimulated the activity of osteoblasts for osteogenesis. MC3T3-E1 cells (mouse osteoblasts) were treated with yam root extracts (water or methanol) (study I) or bark extracts (water or hexane) (study II) within $0{\sim}10{\mu}g/mL$ during the periods of osteoblast proliferation (5~10 day), matrix maturation (11~15 day) and mineralization (16~20 day) as appropriate. In study I, both yam root water and methanol extracts increased cell proliferation as concentration-dependent manner. Cellular collagen synthesis and alkaline phosphatase (ALP) activity, both the indicators of bone matrix protein and inorganic phosphate production for calcification respectively, were also increased by yam root water and methanol extract. Osteoblast calcification as cell matrix Ca and P accumulation was also increased by the addition of yam root extracts. In study II, yam bark extracts (water and hexane) increased osteoblast proliferation and differentiation, as collagen synthesis and ALP activity and osteoblast matrix Ca and P deposition. The study results suggested that both yam root and bark extracts stimulate osteogenic function in osteoblasts by stimulating bone matrix maturation by increasing collagen synthesis, ALP activity, and matrix mineralization.

• Journal of Life Science
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• v.26 no.3
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• pp.331-337
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• 2016

Although fibroblast growth factor 23 (FGF23) is exclusively produced in osteoblasts and osteocytes, its main target is the kidney, where it decreases phosphate reabsorption by suppressing Na-phosphate cotransporters. Independently of its action on phosphate homeostasis, FGF23 also inhibits bone formation in vivo. In a calvarial osteoblastic cell model, FGF23 was shown to negatively affect extracellular matrix mineralization. This study investigated whether FGF23 had similar effects on osteoblast maturation, including differentiation and mineralization of bone marrow-derived mesenchymal stem cells (MSCs). D1 MSCs were cultured in an osteogenic medium containing β-glycerophosphate, ascorbic acid, and dexamethazone. Osteoblastic differentiation was evaluated by alkaline phosphatase (Alp) staining, and matrix mineralization was evaluated by alizarin red staining and calcium deposition. The expression of differentiation-stimulating genes Runx2, Alp, and osteocalcin and mineralization-inhibiting genes Enpp1 and Ank was analyzed using semiquantitative RT-PCR. Supraphysiological doses of FGF23 did not stimulate proliferation or osteoblastic differentiation of MSCs. Matrix mineralization 1, 2, and 3 weeks after the FGF23 treatment did not vary between control and FGF23 groups, although time-dependent enhancement of mineralization was obvious. Calcium deposition was also unchanged after the FGF23 treatment. mRNA expression levels of differentiation- and mineralization-related genes were also similar between the groups. Despite these negative findings, FGF23 signaling through FGF receptors seemed to function normally, with phosphorylation of the Erk protein more evident in the FGF23 group than in controls. These findings suggest that unlike calvarial osteoblasts, FGF23 is not likely to affect osteoblastic differentiation and mineralization of MSCs.

• Journal of Periodontal and Implant Science
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• v.41 no.4
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• pp.167-175
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• 2011

Purpose: Periodontal ligament (PDL) cell differentiation into osteoblasts is important in bone formation. Bone formation is a complex biological process and involves several tightly regulated gene expression patterns of bone-related proteins. The expression patterns of bone related proteins are regulated in a temporal manner both in vivo and in vitro. The aim of this study was to observe the gene expression profile in PDL cell proliferation, differentiation, and mineralization in vitro. Methods: PDL cells were grown until confluence, which were then designated as day 0, and nodule formation was induced by the addition of 50 ${\mu}g$/mL ascorbic acid, 10 mM ${\beta}$-glycerophosphate, and 100 nM dexamethasone to the medium. The dishes were stained with Alizarin Red S on days 1, 7, 14, and 21. Real-time polymerase chain reaction was performed for the detection of various genes on days 0, 1, 7, 14, and 21. Results: On day 0 with a confluent monolayer, in the active proliferative stage, c-myc gene expression was observed at its maximal level. On day 7 with a multilayer, alkaline phosphatase, bone morphogenetic protein (BMP)-2, and BMP-4 gene expression had increased and this was followed by maximal expression of osteocalcin on day 14 with the initiation of nodule mineralization. In relationship to apoptosis, c-fos gene expression peaked on day 21 and was characterized by the post-mineralization stage. Here, various genes were regulated in a temporal manner during PDL fibroblast proliferation, extracellular matrix maturation, and mineralization. The gene expression pattern was similar. Conclusions: We can speculate that the gene expression pattern occurs during PDL cell proliferation, differentiation, and mineralization. On the basis of these results, it might be possible to understand the various factors that influence PDL cell proliferation, extracellular matrix maturation, and mineralization with regard to gene expression patterns.

• Korean Journal of Clinical Laboratory Science
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• v.37 no.1
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• pp.35-40
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• 2005

This paper reports the morphological nature of the remodelled interface process between implants and surrounding bone after 1, 4, 6, 8 and 12 weeks of implantation of smooth machined implants into rat tibias. After 4 weeks of implantation, histochemical analysis showed that the new bone was growing in direct contact with the implant. In the forming process, the activatived osteoblast cells migrated toward the interface and colonized the surface at the contact areas. This immature woven bone, rich in osteocyte lacunae, was deposited directly onto the implant surface. Osteoblast activity was found to continue ill 12 weeks of implantation The osteoblasts in lacunar areas developed numerous processes and synthesized bone matrix, after all, surrounded by secreting matrix. At the 12th week, the amount of newly formed bone matrix between bone and implant increased in mineralization. The mineralized mature bone contained well organized collagen fibers with characteristic banding pattern bone tissue formation around the implant.

• Journal of Acupuncture Research
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• v.22 no.2
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• pp.181-190
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• 2005

Background & Object : The differentiation of osteoblasts controlled by various growth factors and matrix proteins expression in bone. The aim of this study was to identify the Astragalus membranaceus that may induce the osteogenic activity in human osteoblast-like SaOS-2 cells. Methods : The osteogenic activity of Astragalus membranaceus were evaluated by WST-8 assay, ALP activity, RT-PCR analysis of VEGF, OCN, OPN, Col I mRNA, and ELISA or colorimetric analysis, and mineralization by Alizarin red staining in SaOS-2 cells. Results : Astragalus membranaceus had no effect on viability of osteoblastic cells, and dose dependently increased alkaline phosphatase (ALP) activity. Astragalus membranaceus markedly increased mRNA expression for vascular endothelial growth factor (VEGF), osteocalcin (OCN), osteopontin (OPN), and type I collagen (Col 1) in SaOS-2 cells. Extracellular accumulation of proteins such as VEGF, and Col I was increased in a dose-dependent manner. Also, Astragalus membranaceus significantly induced mineralization in the culture of SaOS-2 cells. Conclusion : This study showed that Astragalus membranaceus not affect on viability, but it enhanced ALP activity, VEGF, bone matrix proteins such as OCN, OPN and Col I, and mineralization in SaOS-2 cells. These results propose that Astragalus membranaceus plays an important role in osteoblastic bone formation, and possibly lead to the development of bone-forming drug.

• 대한치주과학회:학술대회논문집
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• 2002.11a
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• pp.48-49
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• 2002

• Journal of Periodontal and Implant Science
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• v.37 no.sup2
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• pp.447-463
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• 2007

Periodontal ligament (PDL) cells have been known as multipotential cells, and as playing an important rolesin periodontal regeneration. The PDL cells are composed of heterogeneous cell populations which have the capacity to differentiate into either cementoblasts or osteoblasts, depending on needs and conditions. Therefore, PDL cells have the capacity to produce mineralized nodules in vitro in mineralization medium which include ascorbic acid, ${\beta}$-glycerophosphate and dexamethasone. In spite of these well-known osteoblast like properties of PDL cells, very little is known about the molecules involved in the formation of the mineralized nodules in the PDL cells. In the present study, we analysed gene-expression profiles during the mineralization process of cultured PDL cells by means of a cDNA microarray consisting of 3063 genes. Nodules of mineralized matrix were strongly stained with alizarin red S on the PDL cells cultured in the media with mineralization supplements. Among 3,063 genes analyzed, 35 were up-regulated more than two-fold at one or more time points in cells that developed matrix mineralization nodules, and 38 were down-regulated to less than half their normal level of expression. In accord with the morphological change we observed, several genes related to calcium-related or mineral metabolism were induced in PDL cells during osteogenesis, such as IGF-II and IGFBP-2. Proteogycan 1, fibulin-5, keratin 5, ,${\beta}$-actin, ${\alpha}$-smooth muscle actin and capping protein, and cytoskeleton and extracellular matrix proteins were up-regulated during mineralization. Several genes encoding proteins related to apoptosis weredifferentially expressed in PDL cells cultured in the medium containing mineralization supplements. Dkk-I and Nip3, which are apoptosis-inducing agents, were up-regulated, and Btf and TAXlBP1, which have an anti-apoptosis activity, were down-regulated during mineralization. Also periostin and S100 calciumbinding protein A4 were down-regulated during mineralization.