• Journal of Periodontal and Implant Science
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• v.35 no.3
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• pp.675-685
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• 2005

Bone resorption involves sequential stages of osteoclast precursor migration and differentiation of osteoclast precursors into multinucleated osteoclasts. Stromal cell derived factor (SDF)-1 is a chemotactic factor for osteoclast precursor migration. Matrix metalloproteinase (MMP)-9 is involved in migration of osteoclast precursors and activation of $interleukin(IL)-1{\beta}$. Alveolar bone destruction is a characteristic feature of periodontal disease. Treponema lecithinolyticum is a oral spirochete isolated from the periodontal lesions. The effect of lipopolysaccharide(LPS) from T. lecithinolyticum on expression of SDF-1 and MMP-9 was examined in cocultures of bone marrow cells and osteblasts derived from mouse calvariae. T. lecithinolyticum LPS increased expression of MMP-9 in the coculture. Polymyxin B, an inhibitor of LPS, abolished the increase of MMP-9 mRNA expression by LPS. LPS did not increase the expression of SDF-1, $IL-1{\beta}$ and tumor necrosis $factor(TNF)-{\alpha}$ mRNA in cocultures. Prostaglandin $E_2(PGE_2)$ up-regulated the expression of MMP-9 and NS398, an inhibitor of $PGE_2$ synthesis, down-regulated the induction of MMP-9 expression by T. lecitbinolyticm LPS. These results suggest that T. lecitbinolyticm LPS increases MMP-9 expression in bone cells via $PGE_2$ and that the induction of MMP-9 expression by T. lecitbinolyticm LPS is involved in alveolar bone destruction of periodontitis patients by the increase of osteoclast precursor migration and the activation of bone resorption-inducing cytokine.

• Nutritional Sciences
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• v.8 no.4
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• pp.242-249
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• 2005

It is well established that zinc plays an important role in bone metabolism and mineralization. The role of zinc in bone formation is well documented in animal models, but not much reported in cell models. In the present study, we evaluated zinc deficiency effects on osteoblastic cell proliferation, alkaline phosphatase activity and expression, and extracellular matrix bone nodule formation and bone-related gene expression in osteoblastic MC3T3-E1 cells. To deplete cellular zinc, chelexed-FBS and interpermeable zinc chelator TPEN were used. MC3T3-E1 cells were cultured in zinc concentration-dependent (0-15 ${\mu}M\;ZnCl_2$) and time-dependent (0-20 days) manners. MC3T3-E1 cell proliferation by MTT assay was increased as medium zinc level increased (p<0.05). Cellular Ca level and alkaline phosphatase activity were increased as medium zinc level increased (p<0.05). Alkaline phosphatase expression, a marker of commitment to the osteoblast lineage, measured by alkaline phosphatase staining was increased as medium zinc level increased. Extracellular calcium deposits measured by von Kossa staining for nodule formation also appeared higher in Zn+(15 ${\mu}M\;ZnCl_2$) than in Zn-(0 ${\mu}M\;ZnCl_2$). Bone formation marker genes, alkaline phosphatase and osteocalcin, were also expressed higher in Zn+ than in Zn-. The current work supports the beneficial effect of zinc on bone mineralization and bone-related gene expression. The results also promote further study as to the molecular mechanism of zinc deficiency for bone formation and thus facilitate to design preventive strategies for zinc-deficient bone diseases.

• 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.

• Journal of Periodontal and Implant Science
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• v.34 no.4
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• pp.759-769
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• 2004

Periodontal therapy has dealt primarily with attempts at arresting progression of disease, however, more recent techniques have focused on regenerating the periodontal ligament having the capacity to regenerate the periodontium. The effect of chitosan, a carbohydrate biopolymer extracted from chitin, on periodontal ligament regeneration is of particular interest. The purpose of this study was to evaluate the effect of chitosan on the expression of extracellular matrix proteins in primary rat calvarial cells in Vitro. In the control group, cells was cultured with BGjb media. In the experimental groups, cells were cultured with chitosan in concentration of 0.01, 0.1, 1.0 and 2.0 mg/ml. Then each group was characterized by examining alkaline phosphatase activity at 3 and 7 days, and the ability to produce mineralized nodules of rat calvarial cells at 14 and 21 days. Synthesis of type I collagen (COL-I), osteocalcin (OCN), bone sialoprotein (BSP) was evaluated by RT-PCR at 14 days. The results were as follows: 1. Alkaline phosphatase activity was significantly higher in the concentration of chitosan 0.01mg/ml, 0.1mg/ml and 1.0mg/ml compared to control (p<0.05). 2. The percentage of mineralized bone nodule was more in the concentration of chitosan 0.1mg/ml and 1.0mg/ml than the control. 3. At 14 day culture, the expression of OCN was increased by chitosan in concentration of 1.0 mg/ml and 2.0 mg/ml. These results suggested that chitosan in concentration of 0.1 and 1,0 mg/ml stimulate the extracellular matrix of primary rat calvarial cells and may facilitate the formation of bone.

• 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.

• International Journal of Oral Biology
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• v.46 no.1
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• pp.51-59
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• 2021

Periodontal disease is an inflammatory disease that affects the destruction of the bone supporting the tooth and connective tissues surrounding it. Periodontal ligament fibroblasts (PDLFs) induce overexpression of matrix metalloproteinase (MMP) involved in periodontal disease's inflammatory destruction. Osteoclasts take part in physiological bone remodeling, but they are also involved in bone destruction in many kinds of bone diseases, including osteoporosis and periodontal disease. This study examined the effect of baicalin on proteolytic enzymes' production and secretion of inflammatory cytokines in PDLFs and RAW 264.7 cells under the lipopolysaccharide (LPS)-induced inflammatory conditions. Baicalin inhibited the expression of the protein, MMP-1 and MMP-2, without affecting PDLFs' cell viability, suggesting its possibility because of the inhibition of phosphorylation activation of mitogen-activated protein kinase's p38, and the signal transduction process of nuclear factor κB (NFκB)-related protein. Also, baicalin reduced the expression of MMP-8 and MMP-9 in RAW 264.7 cells. This reduction is thought to be due to the inhibition of the signal transduction process of NFκB-related proteins affected by inhibiting p65RelA phosphorylation. Also, baicalin inhibited the secretion of nitric oxide and interleukin-6 induced by LPS in RAW 264.7 cells. These results suggest that baicalin inhibits connective tissue destruction in periodontal disease. The inhibition of periodontal tissue destruction may be a therapeutic strategy for treating inflammatory periodontal-diseased patients.

• Journal of Periodontal and Implant Science
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• v.30 no.2
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• pp.347-360
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• 2000

Bone remodeling results from the combined process of bone resorption and new bone formation which is regulated in part by some of the polypeptide growth factors such as platelet derived growth factor(PDGF), which has been known to be an important local regulator of bone cell activity and participate in normal bone remodeling. This process includes strictly regulated gene expression of several bone matrix proteins such as type I collagen and osteopontin, a 44 kDa phosphorylated glycoprotein, which has important roles in bone formation. The purpose of this study is to evaluate the effecs of PDGF-BB on the mRNA expression of bone matrix protein, type I collagen and osteopontin, in MC3T3- E1 cell culture. Cells were seeded at $5{\times}10^5$ cells in 10 ml of minimum essential medium alpha(${\alpha}-MEM$) containig 10% fetal bovine serum, 10 mM beta glycerophosphate. 0.1, 1, 10 ng/ml PDGF-BB were added to the cells for the day 3, 7, 14, 21, 28 and cultured for 24 hours. Type I collagen cDNA, Hf677, and osteopontin cDNA were used as probes for northern blot analysis. Total cellular RNA was purified at indicated day and northern blot analysis was performed. The results were as follows : Type I collagen mRNA expressions were higher at the day 3 and 7, and lower in the day 14, 21 in the control groups. In the experimental groups, mRNA expressions were increased when 0.1 ng/ml PDGF-BB were added on the day 3, 7, 21, and decreased in dose-dependent manner on the day 14, decreased at all added dose on the day 28. Osteopontin mRNA expressions were highest in the day 21 groups and lowest in the day 14 groups in the control groups. Interesting results were shown in the day 14 and 21 groups. We found that osteopontin mRNA level was increased in dose dependent manner in the day 14 groups, and decreased dose dependent manner in the day 21 groups. In conclusion, PDGF-BB may have various control effects on type I mRNA expression in the growth and differentiation process of MC3T3-E1 cells and may have contrary regulatory effects on osteopontin mRNA expression. For examples, when the baseline level of osteopontin mRNA was low, as in the day 14, PDGF-BB up-regulated osteopontin mRNA expression in dose dependent manner, and when the baseline level was high as in the day 21, PDGF-BB down-regulated dose dependent manner. Thus, it may be useful for clinical application in periodontal regeneration procedure if further study were performed.

• Journal of Korean Dental Science
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• v.5 no.2
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• pp.77-87
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• 2012

Purpose: This study examined the scanning electron microscopic feature, protein marker expression and osteoinductive activity of demineralized dentin matrix (DDM) from human for nude mice. Materials and Methods: Twenty healthy nude mice, weighing about 20 g were used for study. DDM from Human was prepared and implanted into the dorsal portion of nude mouse. Before implantation, DDM was examined by scanning electron microscopy (SEM). Nude mice were sacrificed at 2 weeks, 4 weeks and 8 weeks after DDM grafting and evaluated histologically by H-E, MT staining. And also immunohistochemistry analysis (ostecalcin, osteopontin) was performed. Result: Dentinal tubules and collagen fibers were observed by SEM of dentin surface of DDM. The DDM induced bone and cartilage independently in soft tissues. And, the histological findings showed bone forming cells like osteoblasts, fibroblasts at 2, 4 and 8 weeks. On immunohistochemistry analysis, osteocalcin and osteopontin positive bone forming cells were observed. Conclusion: This results showed that the DDM from human has osteoinductive ability and is a good alternative to autogenous bone graft materials.

• Restorative Dentistry and Endodontics
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• v.39 no.3
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• pp.187-194
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• 2014

Objectives: The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. Materials and Methods: MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). Results: Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). Conclusions: These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.

• Nutrition Research and Practice
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• v.10 no.6
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• pp.569-574
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• 2016

BACKGROUND/OBJECTIVES: We investigated the anti-osteoarthritic effects of deer bone extract on the gene expressions of matrix metalloproteinases (MMPs) and collagen type II (COL2) in interleukin-$1{\beta}$-induced osteoarthritis (OA) chondrocytes. MATERIALS/METHODS: Primary rabbit chondrocytes were treated as follows: CON (PBS treatment), NC (IL-$1{\beta}$ treatment), PC (IL-$1{\beta}+100{\mu}g/mL$ glucosamine sulphate/chondroitin sulphate mixture), and DB (IL-$1{\beta}+100{\mu}g/mL$ deer bone extract). RESULTS: The results of the cell viability assay indicated that deer bone extract at doses ranging from 100 to $500{\mu}g/mL$ inhibits cell death in chondrocytes induced by IL-$1{\beta}$. Deer bone extract was able to significantly recover the mRNA expression of COL2 that was down-regulated by IL-$1{\beta}$ (NC: 0.79 vs. DB: 0.87, P < 0.05) and significantly decrease the mRNA expression of MMP-3 (NC: 2.24 vs. DB: 1.75) and -13 (NC: 1.28 vs. DB: 0.89) in OA chondrocytes (P < 0.05).CONCLUSIONS: We concluded that deer bone extract induces accumulation of COL2 through the down-regulation of MMPs in IL-$1{\beta}$-induced OA chondrocytes. Our results suggest that deer bone extract, which contains various components related to OA, including chondroitin sulphate, may possess anti-osteoarthritic properties and be of value in inhibiting the pathogenesis of OA.