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Sample records for murine mc3t3-e1 osteoblastic

  1. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    International Nuclear Information System (INIS)

    Kao, C T; Chen, C C; Cheong, U-I; Liu, S L; Huang, T H

    2014-01-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5 J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics. (paper)

  2. Cuscuta chinensis seeds water extraction protecting murine osteoblastic MC3T3-E1 cells against tertiary butyl hydroperoxide induced injury.

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    Gao, Jian-mei; Li, Ran; Zhang, Lei; Jia, Li-long; Ying, Xi-xiang; Dou, De-qiang; Li, Jian-chun; Li, Hai-bo

    2013-07-09

    Cuscuta chinensis (C. chinensis) is a well-known traditional Chinese herb that has been used to treat heart disease, diabetes, liver injury, cancer, and aging. Murine osteoblastic MC3T3-E1 cells were treated with various concentrations of C. chinensis water extraction at different time intervals. The antioxidant effect of C. chinensis on MC3T3-E1 cells was evaluated using MTT and TUNEL assays. The effect of C. chinensis on cell cycle was analyzed by flow cytometry with propidium iodide. Lipid peroxidation was measured by the HPLC method. The cellular redox status was determined from the reduced glutathione to oxidized glutathione ratio (GSH/GSSG) and the enzymes involved in glutathione metabolism, including glutathione reductase (GR), Glutathione S-transferase (GST), and Glucose-6-phosphate dehydrogenase (G6PD). The changes in relative mitochondrial transmembrane potential (ΔΨm) in the MC3T3-E1 cells were analyzed with rhodamine 123 staining. Western blot analysis was used to evaluate the levels of cytochrome c (cyto c), Bax, Bcl-2, caspase 3, Sirt3, and IDH2 expressions. The C. chinensis water extraction protects tertiary butyl hydroperoxide (TBHP)-treated MC3T3-E1 cells from death in a dose-dependent manner. C. chinensis treatment significantly inhibited the reactive oxygen species (ROS) generation, malondialdehyde (MDA) production, and increased the activity of superoxide dismutase (SOD), GR, GST, and G6PD. The release of cyto c from mitochondria was reduced by C. chinensis, which increased the expression of antiapoptotic IDH2, Sirt3, and Bcl-2 and decreased the expression of Bax, cyto c, and caspase 3. C. chinensis modulated the oxidative stress-induced apoptosis in MC3T3-E1 cells, probably due to its antioxidant activity and functioning via mitochondria-dependent pathways. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  3. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

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    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

  4. Effect of acetaminophen on osteoblastic differentiation and migration of MC3T3-E1 cells.

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    Nakatsu, Yoshihiro; Nakagawa, Fumio; Higashi, Sen; Ohsumi, Tomoko; Shiiba, Shunji; Watanabe, Seiji; Takeuchi, Hiroshi

    2018-02-01

    N-acetyl-p-aminophenol (APAP, acetaminophen, paracetamol) is a widely used analgesic/antipyretic with weak inhibitory effects on cyclooxygenase (COX) compared to non-steroidal anti-inflammatory drugs (NSAIDs). The mechanism of action of APAP is mediated by its metabolite that activates transient receptor potential channels, including transient receptor potential vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or the cannabinoid receptor type 1 (CB1). However, the exact molecular mechanism and target underlying the cellular actions of APAP remain unclear. Therefore, we investigated the effect of APAP on osteoblastic differentiation and cell migration, with a particular focus on TRP channels and CB1. Effects of APAP on osteoblastic differentiation and cell migration of MC3T3-E1, a mouse pre-osteoblast cell line, were assessed by the increase in alkaline phosphatase (ALP) activity, and both wound-healing and transwell-migration assays, respectively. APAP dose-dependently inhibited osteoblastic differentiation, which was well correlated with the effects on COX activity compared with other NSAIDs. In contrast, cell migration was promoted by APAP, and this effect was not correlated with COX inhibition. None of the agonists or antagonists of TRP channels and the CB receptor affected the APAP-induced cell migration, while the effect of APAP on cell migration was abolished by down-regulating TRPV4 gene expression. APAP inhibited osteoblastic differentiation via COX inactivation while it promoted cell migration independently of previously known targets such as COX, TRPV1, TRPA1 channels, and CB receptors, but through the mechanism involving TRPV4. APAP may have still unidentified molecular targets that modify cellular functions. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.

  5. Ginsenoside Re Promotes Osteoblast Differentiation in Mouse Osteoblast Precursor MC3T3-E1 Cells and a Zebrafish Model

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    Hye-Min Kim

    2016-12-01

    Full Text Available Bone homeostasis is tightly regulated to balance bone formation and bone resorption. Many anabolic drugs are used as bone-targeted therapeutic agents for the promotion of osteoblast-mediated bone formation or inhibition of osteoclast-mediated bone resorption. Previous studies showed that ginsenoside Re has the effect of the suppression of osteoclast differentiation in mouse bone-marrow derived macrophages and zebrafish. Herein, we investigated whether ginsenoside Re affects osteoblast differentiation and mineralization in in vitro and in vivo models. Mouse osteoblast precursor MC3T3-E1 cells were used to investigate cell viability, alkaline phosphatase (ALP activity, and mineralization. In addition, we examined osteoblastic signaling pathways. Ginsenoside Re affected ALP activity without cytotoxicity, and we also observed the stimulation of osteoblast differentiation through the activation of osteoblast markers including runt-related transcription factor 2, type 1 collagen, ALP, and osteocalcin in MC3T3-E1 cells. Moreover, Alizarin red S staining indicated that ginsenoside Re increased osteoblast mineralization in MC3T3-E1 cells and zebrafish scales compared to controls. These results suggest that ginsenoside Re promotes osteoblast differentiation as well as inhibits osteoclast differentiation, and it could be a potential therapeutic agent for bone diseases.

  6. Functional expression of 5-HT{sub 2A} receptor in osteoblastic MC3T3-E1 cells

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    Hirai, Takao; Kaneshige, Kota; Kurosaki, Teruko [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan); Nishio, Hiroaki, E-mail: nishio@fupharm.fukuyama-u.ac.jp [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan)

    2010-05-28

    In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT{sub 2} receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT{sub 2A} and 5-HT{sub 2C} receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT{sub 2A} receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT{sub 2A} receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.

  7. Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells

    International Nuclear Information System (INIS)

    Kimira, Yoshifumi; Ogura, Kana; Taniuchi, Yuri; Kataoka, Aya; Inoue, Naoki; Sugihara, Fumihito; Nakatani, Sachie; Shimizu, Jun; Wada, Masahiro; Mano, Hiroshi

    2014-01-01

    Highlights: • Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization. • Pro-Hyp significantly increased alkaline phosphatase activity. • Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. - Abstract: Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression

  8. Protective effect of Edaravone against hypoxia-induced cytotoxicity in osteoblasts MC3T3-E1 cells.

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    Cao, Bo; Chai, Chunxiang; Zhao, Sishun

    2015-12-01

    Edaravone is a newly developed clinical medicine for the treatment of acute cerebral infarction. Reduced blood supply to bones (hypoxia) has been involved in the pathological development of osteoporosis. In this study, we investigated the effect of Edaravone and its latent mechanism on hypoxia-induced cell toxicity in MC3T3-E1 cells. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were determined by the fluorescence dyes 2',7'-dichlorofluorescein diacetate (DCFH-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA), respectively. mRNA and proteins were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Edaravone significantly restored the hypoxia-induced reduction of MC3T3-E1 cell viability and inhibited lactate dehydrogenase release. In addition, we found that Edaravone inhibits the generation of ROS and NO. Hoechst staining results indicated that the nuclear condensation characteristic of apoptosis was increased in MC3T3-E1 cells after hypoxia exposure, which was significantly suppressed by Edaravone treatment. Mechanistically, we found that Edaravone markedly reduced the expression of cleaved caspase-3 and blunted the release of cytochrome c. These findings strongly suggested that Edaravone suppresses hypoxia-induced cytotoxicity in MC3T3-E1 cells. The pleiotropic effects of Edaravone on hypoxia exposure in osteoblasts suggest potential antiosteoporosis mechanisms of Edaravone. © 2015 International Union of Biochemistry and Molecular Biology.

  9. Linarin isolated from Buddleja officinalis prevents hydrogen peroxide-induced dysfunction in osteoblastic MC3T3-E1 cells.

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    Kim, Young Ho; Lee, Young Soon; Choi, Eun Mi

    2011-01-01

    The flowers and leaves buds of Buddleja officinalis MAXIM (Buddlejaceae) are used to treat eye troubles, hernia, gonorrhea and liver troubles in Asia. To elucidate the protective effects of linarin isolated from B. officinalis on the response of osteoblast to oxidative stress, osteoblastic MC3T3-E1 cells were pre-incubated with linarin for 1h before treatment with 0.3mM H(2)O(2) for 48h, and markers of osteoblast function and oxidative damage were examined. Linarin significantly (P<0.05) increased cell survival, alkaline phosphatase (ALP) activity, collagen content, calcium deposition, and osteocalcin secretion and decreased the production of receptor activator of nuclear factor-kB ligand (RANKL), protein carbonyl (PCO), and malondialdehyde (MDA) of osteoblastic MC3T3-E1 cells in the presence of hydrogen peroxide. These results demonstrate that linarin can protect osteoblasts against hydrogen peroxide-induced osteoblastic dysfunction and may exert anti-resorptive actions, at least in part, via the reduction of RANKL and oxidative damage. 2011 Elsevier Inc. All rights reserved.

  10. Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells

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    Yamauchi Mika

    2007-11-01

    Full Text Available Abstract Background Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Results Adiponectin receptor type 1 (AdipoR1 mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR, in the cells. AdipoR1 small interfering RNA (siRNA transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01–0.5 mM in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01–1.0 μg/ml also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively. Conclusion Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

  11. The effects of low dose X-irradiation on osteoblastic MC3T3-E1 cells in vitro

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    Xu Wei

    2012-06-01

    Full Text Available Abstract Background It has been indicated that moderate or high dose of X-irradiation could delay fracture union and cause osteoradionecrosis, in part, mediated by its effect on proliferation and differentiation of osteoblasts. However, whether low dose irradiation (LDI has similar roles on osteoblasts is still unknown. In this study, we investigated whether and to what extent LDI could affect the proliferation, differentiation and mineralization of osteoblasts in vitro. Methods The MC3T3-E1 cells were exposed to single dose of X-irradiation with 0, 0.1, 0.5, 1.0 Gy respectively. Cell proliferation, apoptosis, alkaline phosphatase (ALP activity, and mineralization was evaluated by methylthiazoletetrazolium (MTT and bromodeoxyuridine (BrdU assay, flow cytometry, ALP viability kit and von Kossa staining, respectively. Osteocalcin (OCN and core-binding factor α1 (Cbfα1 expressions were measured by real time-PCR and western blot, respectively. Results The proliferation of the cells exposed to 2.0 Gy was significantly lower than those exposed to ≤1.0 Gy (p  Conclusions LDI have different effects on proliferation and differentiation of osteoblasts from those of high dose of X-irradiation, which might suggest that LDI could lead to promotion of frature healing through enhancing the differentiation and mineralization of osteoblasts.

  12. Traf2 interacts with Smad4 and regulates BMP signaling pathway in MC3T3-E1 osteoblasts

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    Shimada, Koichi; Ikeda, Kyoko; Ito, Koichi

    2009-01-01

    Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate in the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from bone marrow, to indentify the proteins interacting with Smad4. cDNA clones for Tumor necrosis factor (TNF) receptor-associated factor 2 (Traf2) were identified, and the interaction between the endogenous proteins was confirmed in the mouse osteoblast cell line MC3T3-E1. To investigate the function of Traf2, we silenced it with siRNA. The level of BMP-2 protein in the medium, the expression levels of the Bmp2 gene and BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Sp7, and the phosphorylated-Smad1 protein level were increased in cells transfected with Traf2 siRNA. The nuclear accumulation of Smad1 increased with TNF-α stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-α-stimulated nuclear accumulation of Smad1 may be dependent on Traf2. Thus, the interaction between Traf2 and Smad4 may play a role in the cross-talk between TNF-α and BMP signaling pathways.

  13. miR-195 inhibited abnormal activation of osteoblast differentiation in MC3T3-E1 cells via targeting RAF-1.

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    Chao, Chen; Li, Feng; Tan, Zhiping; Zhang, Weizhi; Yang, Yifeng; Luo, Cheng

    2018-01-15

    Recent reports have demonstrated that RAF-1 L613V (a mutant of RAF-1) mutant mice show bone deformities similar to Noonan syndrome. It has been suggested that RAF-1 L613V might abnormally activate osteoblast differentiation of MC3T3-E1 cells. To demonstrate that RAF-1 is associated with bone deformity and that RAF-1 L613V dependent bone deformity could be inhibited by microRNA-195 (miR-195), we first investigated the amplifying influence of wild-type RAF-1 (WT) or RAF-1 L613V (L613V) on the viability and differentiation of MC3T3-E1 cells induced by bone morphogenetic protein-2 (BMP-2) via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Subsequently, we investigated the blocking effect and its mechanism of miR-195 for abnormal activation of osteoblast differentiation of MC3T3-E1 cells via targeting RAF-1. RAF-1, especially RAF-1 L613V , abnormally activates osteoblast differentiation of MC3T3-E1 cells induced by BMP-2. Meanwhile, miR-195 could inhibit the cell viability and differentiation of MC3T3-E1 cells. Transfection of miR-195 largely suppressed the L613V-induced viability and osteoblast differentiation of MC3T3-E1 cells and attenuated the accelerative effect of L613V on runt-related transcription factor-2 (Runx2), Osterix (OSX), alkaline phosphatase (ALP), osteocalcin (OCN), and distal-less homeobox 5 (DLX5) osteogenic gene expressions. In addition, miR-195 decreased the expression of RAF-1 mRNA and protein by directly targeting the 3'-untranslated regions (3'-UTR) of RAF-1 mRNA in MC3T3-E1 cells. Our findings indicated that miR-195 inhibited WT and L613V RAF-1 induced hyperactive osteoblast differentiation in MC3T3-E1 cells by targeting RAF-1. miR-195 might be a novel therapeutic agent for the treatment of L613V-induced bone deformity in Noonan syndrome. Copyright © 2017. Published by

  14. Osteoblast-like MC3T3-E1 Cells Prefer Glycolysis for ATP Production but Adipocyte-like 3T3-L1 Cells Prefer Oxidative Phosphorylation.

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    Guntur, Anyonya R; Gerencser, Akos A; Le, Phuong T; DeMambro, Victoria E; Bornstein, Sheila A; Mookerjee, Shona A; Maridas, David E; Clemmons, David E; Brand, Martin D; Rosen, Clifford J

    2018-06-01

    Mesenchymal stromal cells (MSCs) are early progenitors that can differentiate into osteoblasts, chondrocytes, and adipocytes. We hypothesized that osteoblasts and adipocytes utilize distinct bioenergetic pathways during MSC differentiation. To test this hypothesis, we compared the bioenergetic profiles of preosteoblast MC3T3-E1 cells and calvarial osteoblasts with preadipocyte 3T3L1 cells, before and after differentiation. Differentiated MC3T3-E1 osteoblasts met adenosine triphosphate (ATP) demand mainly by glycolysis with minimal reserve glycolytic capacity, whereas nondifferentiated cells generated ATP through oxidative phosphorylation. A marked Crabtree effect (acute suppression of respiration by addition of glucose, observed in both MC3T3-E1 and calvarial osteoblasts) and smaller mitochondrial membrane potential in the differentiated osteoblasts, particularly those incubated at high glucose concentrations, indicated a suppression of oxidative phosphorylation compared with nondifferentiated osteoblasts. In contrast, both nondifferentiated and differentiated 3T3-L1 adipocytes met ATP demand primarily by oxidative phosphorylation despite a large unused reserve glycolytic capacity. In sum, we show that nondifferentiated precursor cells prefer to use oxidative phosphorylation to generate ATP; when they differentiate to osteoblasts, they gain a strong preference for glycolytic ATP generation, but when they differentiate to adipocytes, they retain the strong preference for oxidative phosphorylation. Unique metabolic programming in mesenchymal progenitor cells may influence cell fate and ultimately determine the degree of bone formation and/or the development of marrow adiposity. © 2018 American Society for Bone and Mineral Research. © 2018 American Society for Bone and Mineral Research.

  15. Protection by the flavonoids quercetin and luteolin against peroxide- or menadione-induced oxidative stress in MC3T3-E1 osteoblast cells.

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    Fatokun, Amos A; Tome, Mercedes; Smith, Robert A; Darlington, L Gail; Stone, Trevor W

    2015-01-01

    Potential protective effects of the flavonoids quercetin and luteolin have been examined against the oxidative stress of MC3T3-E1 osteoblast-like cells. Although hydrogen peroxide and menadione reduced cell viability, the toxicity was prevented by desferrioxamine or catalase but not superoxide dismutase, suggesting the involvement of hydrogen peroxide in both cases. Quercetin and luteolin reduced the oxidative damage, especially that caused by hydrogen peroxide. When cultures were pre-incubated with quercetin or luteolin, protection was reduced or lost. Protection was also reduced when a 24 h pre-incubation with the flavonoids was followed by exposure to menadione alone. Pretreating cultures with luteolin impaired protection by quercetin, whereas quercetin pretreatment did not affect protection by luteolin. It is concluded that quercetin and luteolin suppress oxidative damage to MC3T3-E1 cells, especially caused by peroxide. The reduction in protection by pretreatment implies a down-regulation of part of the toxic transduction pathway.

  16. The role of non-thermal atmospheric pressure biocompatible plasma in the differentiation of osteoblastic precursor cells, MC3T3-E1.

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    Han, Ihn; Choi, Eun Ha

    2017-05-30

    Non-thermal atmospheric pressure plasma is ionized matter, composed of highly reactive species that include positive ions, negative ions, free radicals, neutral atoms, and molecules. Recent reports have suggested that non-thermal biocompatible plasma (NBP) can selectively kill a variety of cancer cells, and promote stem cell differentiation. However as of yet, the regulation of proliferation and differentiation potential of NBP has been poorly understood.Here, we investigated the effects of NBP on the osteogenic differentiation of precursor cell lines of osteoblasts, MC3T3 E1 and SaOS-2. For in vitro osteogenic differentiation, precursor cell lines were treated with NBP, and cultured with osteogenic induction medium. After 10 days of treatment, the NBP was shown to be effective in osteogenic differentiation in MC3T3 E1 cells by von Kossa and Alizarin Red S staining assay. Real-time PCR was then performed to investigate the expression of osteogenic specific genes, Runx2, OCN, COL1, ALP and osterix in MC3T3 E1 cells after treatment with NBP for 4 days. Furthermore, analysis of the protein expression showed that NBP treatment significantly reduced PI3K/AKT signaling and MAPK family signaling. However, p38 controlled phosphorylation of transcription factor forkhead box O1 (FoxO1) that related to cell differentiation with increased phosphorylated p38. These results suggest that non-thermal atmospheric pressure plasma can induce osteogenic differentiation, and enhance bone formation.

  17. Effects of irradiation on TGF-β1 mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line

    International Nuclear Information System (INIS)

    Song, Ju Seop; Kim, Kyoung A; Koh, Kwang Joon

    2008-01-01

    To investigate the effects of irradiation on transforming growth factor β1 (TGF-β 1 ) mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line. Cells were cultured in alpha-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum and antibiotics. When the cells reached the level of 70-80% confluence, culture media were changed with α-MEM supplemented with 10% FBS, 5 mM β-glycerol phosphate, and 50 μg/mL ascorbic acid. Thereafter the cells were irradiated with a single dose of 2, 4, 6, 8 Gy at a dose rate of 1.5 Gy/min. The expression pattern of TGF-β 1 mRNA, calcium content and calcific nodule formation were examined on day 3, 7, 14, 21, 28, respectively, after the irradiation. The amount of TGF-β 1 mRNA expression decreased significantly on day 7 after irradiation of 4, 6, 8 Gy. It also decreased on day 14 after irradiation of 6, 8 Gy, and decreased on day 21 after irradiation of 8 Gy. The amount of calcium deposition decreased significantly on day 7 after irradiation of 4, 8 Gy (P 1 mRNA expression that was associated with proliferation and the production of extracellular matrix in MC3T3-E1 osteoblastic cell line

  18. The role of kaempferol-induced autophagy on differentiation and mineralization of osteoblastic MC3T3-E1 cells.

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    Kim, In-Ryoung; Kim, Seong-Eon; Baek, Hyun-Su; Kim, Bok-Joo; Kim, Chul-Hoon; Chung, In-Kyo; Park, Bong-Soo; Shin, Sang-Hun

    2016-08-31

    Kaempferol, a kind of flavonol, has been reported to possess various osteogenic biological activities, such as inhibiting bone resorption of osteoclasts and promoting the differentiation and mineralization of preosteoblasts. However, the precise cellular mechanism of action of kaempferol in osteogenesis is elusive. Autophagy is a major intracellular degradation system, which plays an important role in cell growth, survival, differentiation and homeostasis in mammals. Recent studies showed that autophagy appeared to be involved in the degradation of osteoclasts, osteoblasts and osteocytes, potentially pointing to a new pathogenic mechanism of bone homeostasis and bone marrow disease. The potential correlation between autophagy, osteogenesis and flavonoids is unclear. The present study verified that kaempferol promoted osteogenic differentiation and mineralization and that it elevated osteogenic gene expression based on alkaline phosphatase (ALP) activity, alizarin red staining and quantitative PCR. And then we found that kaempferol induced autophagy by acridine orange (AO) and monodansylcadaverine (MDC) staining and autophagy-related protein expression. The correlation between kaempferol-induced autophagy and the osteogenic process was confirmed by the autophagy inhibitor 3-methyladenine (3-MA). Kaempferol promoted the proliferation, differentiation and mineralization of osteoblasts at a concentration of 10 μM. Kaempferol showed cytotoxic properties at concentrations above 50 μM. Concentrations above 10 μM decreased ALP activity, whereas those up to 10 μM increased ALP activity. Kaempferol at concentrations up to 10 μM also increased the expression of the osteoblast- activated factors RUNX-2, osterix, BMP-2 and collagen I according to RT-PCR analyses. 10 μM or less, the higher of the concentration and over time, kaempferol promoted the activity of osteoblasts. Kaempferol induced autophagy. It also increased the expression of the autophagy-related factors

  19. Knockdown of Indian hedgehog protein induces an inhibition of cell growth and differentiation in osteoblast MC3T3-E1 cells

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    Deng, Ang; Zhang, Hongqi; Hu, Minyu; Liu, Shaohua; Gao, Qile; Wang, Yuxiang; Guo, Chaofeng

    2017-01-01

    Indian hedgehog protein (Ihh) is evolutionarily conserved and serves important roles in controlling the differentiation of progenitor cells into osteoblasts. Ihh null mutant mice exhibit a failure of osteoblast development in endochondral bone. Although studies have demonstrated that Ihh signaling is a potent local factor that regulates osteoblast differentiation, the specific transcription factors that determine osteoblast differentiation remain unclear. Further studies are required to determine the precise mechanism through which Ihh regulates osteoblast differentiation. In the present study, Ihh was knocked down in osteoblast MC3T3-E1 cells using short hairpin RNA, to investigate the function of Ihh in osteoblast proliferation and differentiation and to examine the potential mechanism through which Ihh induces osteoblast apoptosis and cell cycle arrest. It was observed that the knockdown of Ihh induced a marked inhibition of cell growth and increased the apoptosis rate compared with the negative control osteoblasts. Downregulation of Ihh resulted in a cell cycle arrest at the G1 to S phase boundary in osteoblasts. In addition, the knockdown of Ihh decreased the alkaline phosphatase activity and mineral deposition of osteoblasts. The inhibitory roles of Ihh downregulation in osteoblast growth and differentiation may be associated with the transforming growth factor-β/mothers against decapentaplegic homolog and tumor necrosis factor receptor superfamily member 11B/tumor necrosis factor ligand superfamily member 11 signaling pathways. Manipulating either Ihh expression or its signaling components may be of benefit for the treatment of skeletal diseases. PMID:28990069

  20. Effects of Apatite Cement Containing Atelocollagen on Attachment to and Proliferation and Differentiation of MC3T3-E1 Osteoblastic Cells

    Directory of Open Access Journals (Sweden)

    Masaaki Takechi

    2016-04-01

    Full Text Available To improve the osteoconductivity of apatite cement (AC for reconstruction of bone defects after oral maxillofacial surgery, we previously fabricated AC containing atelocollagen (AC(ate. In the present study, we examined the initial attachment, proliferation and differentiation of mouse osteoblastic cells (MC3T3-E1 cells on the surface of conventional AC (c-AC, AC(ate and a plastic cell dish. The number of osteoblastic cells showing initial attachment to AC(ate was greater than those attached to c-AC and similar to the number attached to the plastic cell wells. We also found that osteoblastic cells were well spread and increased their number on AC(ate in comparison with c-AC and the wells without specimens, while the amount of procollagen type I carboxy-terminal peptide (PIPC produced in osteoblastic cells after three days on AC(ate was greater as compared to the others. There was no significant difference in regard to alkaline phosphatase (ALP activity and osteocalcin production by osteoblastic cells among the three surface types after three and six days. However, after 12 days, ALP activity and the produced osteocalcin were greater with AC(ate. In conclusion, AC(ate may be a useful material with high osteoconductivity for reconstruction of bone defects after oral maxillofacial surgery.

  1. Layer-by-layer assembly of peptide based bioorganic–inorganic hybrid scaffolds and their interactions with osteoblastic MC3T3-E1 cells

    International Nuclear Information System (INIS)

    Romanelli, Steven M.; Fath, Karl R.; Phekoo, Aruna P.; Knoll, Grant A.; Banerjee, Ipsita A.

    2015-01-01

    In this work we have developed a new family of biocomposite scaffolds for bone tissue regeneration by utilizing self-assembled fluorenylmethyloxycarbonyl protected Valyl-cetylamide (FVC) nanoassemblies as templates. To tailor the assemblies for enhanced osteoblast attachment and proliferation, we incorporated (a) Type I collagen, (b) a hydroxyapatite binding peptide sequence (EDPHNEVDGDK) derived from dentin sialophosphoprotein and (c) the osteoinductive bone morphogenetic protein-4 (BMP-4) to the templates by layer-by-layer assembly. The assemblies were then incubated with hydroxyapatite nanocrystals blended with varying mass percentages of TiO 2 nanoparticles and coated with alginate to form three dimensional scaffolds for potential applications in bone tissue regeneration. The morphology was examined by TEM and SEM and the binding interactions were probed by FITR spectroscopy. The scaffolds were found to be non-cytotoxic, adhered to mouse preosteoblast MC3T3-E1 cells and promoted osteogenic differentiation as indicated by the results obtained by alkaline phosphatase assay. Furthermore, they were found to be biodegradable and possessed inherent antibacterial capability. Thus, we have developed a new family of tissue-engineered biocomposite scaffolds with potential applications in bone regeneration. - Highlights: • Fmoc-val-cetylamide assemblies were used as templates. • Collagen, a short dentin sialophosphoprotein derived sequence and BMP-4 were incorporated. • Hydroxyapatite–TiO 2 nanocomposite blends and alginate were incorporated. • The 3D scaffold biocomposites adhered to preosteoblasts and promoted osteoblast differentiation. • The biocomposites also displayed antimicrobial activity

  2. Layer-by-layer assembly of peptide based bioorganic–inorganic hybrid scaffolds and their interactions with osteoblastic MC3T3-E1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Romanelli, Steven M. [Fordham University Department of Chemistry, 441 East Fordham Road, Bronx, NY 10458 (United States); Fath, Karl R. [The City University of New York, Queens College, Department of Biology, 65-30 Kissena Blvd, Flushing, NY 11367 (United States); The Graduate Center, The City University of New York, 365 Fifth Avenue, NY 10016 (United States); Phekoo, Aruna P. [The City University of New York, Queens College, Department of Biology, 65-30 Kissena Blvd, Flushing, NY 11367 (United States); Knoll, Grant A. [Fordham University Department of Chemistry, 441 East Fordham Road, Bronx, NY 10458 (United States); Banerjee, Ipsita A., E-mail: banerjee@fordham.edu [Fordham University Department of Chemistry, 441 East Fordham Road, Bronx, NY 10458 (United States)

    2015-06-01

    In this work we have developed a new family of biocomposite scaffolds for bone tissue regeneration by utilizing self-assembled fluorenylmethyloxycarbonyl protected Valyl-cetylamide (FVC) nanoassemblies as templates. To tailor the assemblies for enhanced osteoblast attachment and proliferation, we incorporated (a) Type I collagen, (b) a hydroxyapatite binding peptide sequence (EDPHNEVDGDK) derived from dentin sialophosphoprotein and (c) the osteoinductive bone morphogenetic protein-4 (BMP-4) to the templates by layer-by-layer assembly. The assemblies were then incubated with hydroxyapatite nanocrystals blended with varying mass percentages of TiO{sub 2} nanoparticles and coated with alginate to form three dimensional scaffolds for potential applications in bone tissue regeneration. The morphology was examined by TEM and SEM and the binding interactions were probed by FITR spectroscopy. The scaffolds were found to be non-cytotoxic, adhered to mouse preosteoblast MC3T3-E1 cells and promoted osteogenic differentiation as indicated by the results obtained by alkaline phosphatase assay. Furthermore, they were found to be biodegradable and possessed inherent antibacterial capability. Thus, we have developed a new family of tissue-engineered biocomposite scaffolds with potential applications in bone regeneration. - Highlights: • Fmoc-val-cetylamide assemblies were used as templates. • Collagen, a short dentin sialophosphoprotein derived sequence and BMP-4 were incorporated. • Hydroxyapatite–TiO{sub 2} nanocomposite blends and alginate were incorporated. • The 3D scaffold biocomposites adhered to preosteoblasts and promoted osteoblast differentiation. • The biocomposites also displayed antimicrobial activity.

  3. Direct effects of casein phosphopeptides on growth and differentiation of in vitro cultured osteoblastic cells (MC3T3-E1).

    Science.gov (United States)

    Tulipano, Giovanni; Bulgari, Omar; Chessa, Stefania; Nardone, Alessandro; Cocchi, Daniela; Caroli, Anna

    2010-02-25

    Casein phosphopeptides (CPPs) obtained by enzymatic hydrolysis in vitro of caseins, have been shown to enhance calcium solubility and to increase the calcification of embryonic rat bones in their diaphyseal area. Little is known about the direct effects of CPPs on cultured osteoblastic cells. Calcium in the microenvironment surrounding bone cells is not only important for the mineralization of the extracellular matrix, but it is believed to provide preosteblasts with a signal that modulates their proliferation and differentiation. The aim of the present study was to investigate the direct effects of four selected casein phosphopeptides on osteoblastic cell (MC3T3-E1 cells) viability and differentiation. The selected peptides have been obtained by chemical synthesis and differed in the number of phosphorylated sites and in the amino acid spacing out two phosphorylated sites, in order to further characterize the relationship between structure and function. The results obtained in this work demonstrated that CPPs may directly affect osteoblast-like cell growth, calcium uptake and ultimately calcium deposition in the extracellular matrix. The effects exerted by distinct CPPs on osteogenesis in vitro can be either stimulatory or inhibitory. Differential short amino acid sequences in their molecules, like the -SpEE- and the -SpTSpEE-motifs, are likely the molecular determinants for their biological activities on osteoblastic cells. Moreover, two genetic variants of CPPs showing one amino acid change in their sequence may profoundly differ in their biological activities. Finally, our data may also suggest important clues about the role of intrinsic phosphorylated peptides derived from endogenous phosphorylated proteins in bone metabolism, apart from extrinsic CPPs. Copyright 2009 Elsevier B.V. All rights reserved.

  4. Short-term administration of small molecule phenamil induced a protracted osteogenic effect on osteoblast-like MC3T3-E1 cells.

    Science.gov (United States)

    Lo, Kevin W-H; Kan, Ho Man; Laurencin, Cato T

    2016-06-01

    Sustained administration (21-day treatment) of the small molecule phenamil has been proposed as an alternative osteogenic factor when used in conjunction with a biodegradable scaffold for in vitro osteogenesis. While promising, the major issue associated with small molecules is non-specific cytotoxicity. The aim of this study was to minimize the side-effects from small-molecule drugs by reducing the frequency of administration. Toward this goal, we investigated whether a shorter phenamil treatment is sufficient to induce in vitro osteogenesis. We compared the effects of short-term (12 h) and continuous treatments of phenamil on osteoblastic differentiation and mineralization. Alkaline phosphatase (ALP) and osteopontin (OPN) activity were used as markers for osteoblastic differentiation. Measurement of the calcium content of the extracellular matrix was used as the hallmark for in vitro bone formation after 21 days of culture. Our findings revealed that both short and continuous phenamil treatment triggers osteoblastic differentiation and mineralization of MC3T3-E1 cells on a biodegradable polymeric scaffold composed of polylactic-co-glycolic acid (PLAGA) at the same time points. In addition, in order to fabricate a phenamil-loaded PLAGA scaffold, the small molecule phenamil was physically absorbed onto the surface of scaffolds and the bioactivity of the loaded scaffolds was evaluated. Furthermore, biochemical analysis indicated that short phenamil treatment of cells was accompanied by upregulation in protein expression of integrin α5, p125(FAK) and phosphorylation of CREB. These effects may contribute to the downstream signalling cascade necessary for osteogenesis, and such responses may account for our observed protracted osteogenic differentiation in vitro. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  5. The corrosion and biological behaviour of titanium alloys in the presence of human lymphoid cells and MC3T3-E1 osteoblasts

    International Nuclear Information System (INIS)

    Zhang Yumei; Zhao Yimin; Chai Feng; Hildebrand, Hartmut F; Hornez, Jean-Christophe; Li, Chang Liang; Traisnel, Michel

    2009-01-01

    Corrosion behaviour of biomedical alloys is generally determined in mineral electrolytes: unbuffered NaCl 0.9% (pH 7.4) or artificial saliva (pH 6.8). The assays with exclusive utilization of these electrolytes are of low relevance for the biological condition, to which the alloys will be exposed once implanted in the human organism. As an approach to the biological situation regarding the interaction of proteins, electrolytes and metals, we added the RPMI cell culture medium containing foetal calf serum as a biological electrolyte (pH 7.0). The analysis of corrosion behaviour was also performed in the presence of human lymphoid cells (CEM). The rest potential (E r ) and the global polarization were determined on cp-Ti, micro-arc oxidized cp-Ti (MAO-Ti), four different Ti-alloys (Ti6Al4V, Ti12Zr, Ti(AlMoZr), Ti(NbTaZr)) and 316L stainless steel. The 316L exhibited an appropriate E r and a good passive current density (I p ), but a high corrosion potential (E c ) and a very low breakdown potential (E b ) in all electrolytes. All Ti-alloys exhibited a much better electrochemical behaviour: better E r and E c and very high E b . No significant differences of the above parameters existed between the Ti-alloys, except for Zr-containing alloys that showed better corrosion behaviour. A remarkable difference, however, was stated with respect to the electrolytes. NaCl 0.9% induced strong variations between the Ti-alloys. More homogeneous results were obtained with artificial saliva and RPMI medium, which induced a favourable E c and an increased I p . The presence of cells further decreased these values. The unbuffered NaCl solution seems to be less appropriate for the analysis of corrosion of metals. Additional in vitro biological assessments with CEM cell suspensions and MC3T3-E1 osteoblasts confirmed the advantages of the Ti(AlMoZr) and Ti(NbTaZr) alloys with an improved cell proliferation and vitality rate.

  6. The corrosion and biological behaviour of titanium alloys in the presence of human lymphoid cells and MC3T3-E1 osteoblasts.

    Science.gov (United States)

    Zhang, Yu Mei; Chai, Feng; Hornez, Jean-Christophe; Li, Chang Liang; Zhao, Yi Min; Traisnel, Michel; Hildebrand, Hartmut F

    2009-02-01

    Corrosion behaviour of biomedical alloys is generally determined in mineral electrolytes: unbuffered NaCl 0.9% (pH 7.4) or artificial saliva (pH 6.8). The assays with exclusive utilization of these electrolytes are of low relevance for the biological condition, to which the alloys will be exposed once implanted in the human organism. As an approach to the biological situation regarding the interaction of proteins, electrolytes and metals, we added the RPMI cell culture medium containing foetal calf serum as a biological electrolyte (pH 7.0). The analysis of corrosion behaviour was also performed in the presence of human lymphoid cells (CEM). The rest potential (Er) and the global polarization were determined on cp-Ti, micro-arc oxidized cp-Ti (MAO-Ti), four different Ti-alloys (Ti6Al4V, Ti12Zr, Ti(AlMoZr), Ti(NbTaZr)) and 316L stainless steel. The 316L exhibited an appropriate Er and a good passive current density (Ip), but a high corrosion potential (Ec) and a very low breakdown potential (Eb) in all electrolytes. All Ti-alloys exhibited a much better electrochemical behaviour: better Er and Ec and very high Eb. No significant differences of the above parameters existed between the Ti-alloys, except for Zr-containing alloys that showed better corrosion behaviour. A remarkable difference, however, was stated with respect to the electrolytes. NaCl 0.9% induced strong variations between the Ti-alloys. More homogeneous results were obtained with artificial saliva and RPMI medium, which induced a favourable Ec and an increased Ip. The presence of cells further decreased these values. The unbuffered NaCl solution seems to be less appropriate for the analysis of corrosion of metals. Additional in vitro biological assessments with CEM cell suspensions and MC3T3-E1 osteoblasts confirmed the advantages of the Ti(AlMoZr) and Ti(NbTaZr) alloys with an improved cell proliferation and vitality rate.

  7. Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture.

    Science.gov (United States)

    Juran, C. M.; Blaber, E. A.; Almeida, E. A. C.

    2016-01-01

    Cell and animal studies conducted onboard the International Space Station and formerly on Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. However the intercellular communicative mechanisms associated with the regulation of bone synthesis and bone resorption cells are still largely unknown. Connexin-43 (CX43), a gap junction protein, is hypothesized to play a significant role in osteoblast and osteocyte signaling. The purpose of this investigation was to evaluate within a novel three-dimensional microenvironment how the osteocyte-osteoblast gap-junction expression changes when cultures are exposed to exaggerated mechanical load. MLO-Y4 osteocyte-like cells were cultured on a 3D-Biotek polystyrene insert and placed in direct contact with an MC3T3-E1 pre-osteoblast co-cultured monolayer and exposed to 48 h of mechanical stimulation (pulsatile fluid flow (PFF) or monolayer cyclic stretch (MCS)) then evaluated for viability, proliferation, metabolism, and CX43 expression. Mono-cultured MLO-Y4 and MC3T3-E1 control experiments were conducted under PFF and MCS stimulation to observe how strain application stimuli (PFF cell membrane shear or MCS cell focal adhesion/attachment loading) initiates different signaling pathways or downstream regulatory controls. TotalLive cell count, viability and metabolic reduction (Trypan Blue, LIVEDead and Alamar Blue analysis respectively) indicate that mechanical activation of MC3T3-E1 cells inhibits proliferation while maintaining an average 1.04E4 reductioncell metabolic rate, *p0.05 n4. MLO-Y4s in monolayer culture increase in number when exposed to MCS loading but the percent of live cells within the population is low (46.3 total count, *p0.05 n4), these results may indicate an apoptotic signaling cascade. PFF stimulation of the three-dimensional co-cultures elicits a universal increase in CX43 in MLO-Y4 and MC3T3-E1 cells, illustrated by

  8. CSN1S2 protein of goat milk inhibits the decrease of viability and increases the proliferation of MC3T3E1 pre-osteoblast cell in methyl glyoxal exposure

    Directory of Open Access Journals (Sweden)

    Choirunil Chotimah

    2015-03-01

    Full Text Available Objective: To investigate whether the CNS1S2 protein of goat milk is able to inhibit the toxicity of methyl glyoxal (MG towards MC3T3E1 pre-osteoblast cells. Methods: At confluency, pre-osteoblast cells were divided into five groups which included control (untreated, pre-osteoblast cells exposed to 5 µmol/L MG, pre-osteoblast cells exposed to MG in the presence of CSN1S2 protein at doses of 0.025, 0.050, and 0.100 mg/L, respectively. Analysis of reactive oxygen species was done with 2,7-dichlorodihydrofluorescein diacetate fluorochrome. The proliferation and viability of MC3T3E1 cells were measured by trypan blue staining. Malondialdehyde analysis was done colorimetrically. Results: Cell's viabilities were significantly lower in MG+0.050 mg/L CSN1S2 protein of goat milk compared to MG group (P<0.05. MG+0.100 mg/L CSN1S2 protein of goat milk significantly increased the cells viability compared to MG group (P<0.05. The levels of proliferation were significantly higher in MG+0.100 mg/L CSN1S2 protein of goat milk compared to control group and all treatment groups, respectively (P<0.05. Conclusions: High dose of CSN1S2 protein of goat milk (0.100 mg/L in high MG environment inhibits the decrease of viability due to the increases of the proliferation of MC3T3E1 preosteoblast cell.

  9. Inhibition of microRNA-214-5p promotes cell survival and extracellular matrix formation by targeting collagen type IV alpha 1 in osteoblastic MC3T3-E1 cells.

    Science.gov (United States)

    Li, Q S; Meng, F Y; Zhao, Y H; Jin, C L; Tian, J; Yi, X J

    2017-08-01

    This study aimed to investigate the functional effects of microRNA (miR)-214-5p on osteoblastic cells, which might provide a potential role of miR-214-5p in bone fracture healing. Blood samples were obtained from patients with hand fracture or intra-articular calcaneal fracture and from healthy controls (HCs). Expression of miR-214-5p was monitored by qRT-PCR at day 7, 14 and 21 post-surgery. Mouse osteoblastic MC3T3-E1 cells were transfected with antisense oligonucleotides (ASO)-miR-214-5p, collagen type IV alpha 1 (COL4A1) vector or their controls; thereafter, cell viability, apoptotic rate, and the expression of collagen type I alpha 1 (COL1A1), type II collagen (COL-II), and type X collagen (COL-X) were determined. Luciferase reporter assay, qRT-PCR, and Western blot were performed to ascertain whether COL4A1 was a target of miR-214-5p. Plasma miR-214-5p was highly expressed in patients with bone fracture compared with HCs after fracture (p extracellular matrix (ECM) formation of osteoblastic MC3T3-E1 cells by targeting COL4A1. Cite this article: Q. S. Li, F. Y. Meng, Y. H. Zhao, C. L. Jin, J. Tian, X. J. Yi. Inhibition of microRNA-214-5p promotes cell survival and extracellular matrix formation by targeting collagen type IV alpha 1 in osteoblastic MC3T3-E1 cells. Bone Joint Res 2017;6:464-471. DOI: 10.1302/2046-3758.68.BJR-2016-0208.R2. © 2017 Yi et al.

  10. Effects of 2-deoxy-D-glucose and quercetin on the expression of osteonectin and osteopontin during the differentiation of irradiated MC3T3-E1 osteoblastic cells

    International Nuclear Information System (INIS)

    Yu, Su Kyung; Koh, Kwang Joon; Kim, Kyoung A

    2008-01-01

    To characterize the effects of 2-deoxy-D-glucose (2-DG) and quercetin (QCT) on gene expression of osteonectin (ON) and osteopontin (OP) in irradiated MC3T3-E1 cells. When MC3T3-E1 osteoblastic cells had reached 70-80% confluence, cultures were transferred to a differentiating medium supplemented with 5 mM 2-DG or 10 μM QCT and then irradiated with 2, 4, 6, and 8 Gy. At various times after irradiation, the cells were analyzed for the expression of bone mineralization genes such as ON and OP. The mRNA expression of both ON and OP was increased according to the culture time in the differentiation medium, and the increase of the genes peaked at 14 days after the differentiation induction. In the case of OP, the increase of mRNA expression was maintained to 28 days after the differentiation, while the mRNA level of ON was reduced to the basal level at the same time. Irradiation adding 2-DG showed a significant peak value in the expression pattern of ON at 4 Gy 7 days after irradiation. Irradiation adding QCT increased the mRNA expression of ON and OP in a dose-dependant manner, but irradiation adding 2-DG did not show any differences between the control and experiments 14 days after irradiation. Irradiation adding QCT increased significantly the expression patterns of ON 21 days after irradiation. The results showed that QCT acted as a radiosensitizer in the gene expression of ON and OP during differentiation of the late stage of irradiated MC3T3-E1 osteoblastic cells in vitro.

  11. Knockdown of Indian hedgehog protein induces an inhibition of cell growth and differentiation in osteoblast MC3T3-E1 cells

    OpenAIRE

    Deng, Ang; Zhang, Hongqi; Hu, Minyu; Liu, Shaohua; Gao, Qile; Wang, Yuxiang; Guo, Chaofeng

    2017-01-01

    Indian hedgehog protein (Ihh) is evolutionarily conserved and serves important roles in controlling the differentiation of progenitor cells into osteoblasts. Ihh null mutant mice exhibit a failure of osteoblast development in endochondral bone. Although studies have demonstrated that Ihh signaling is a potent local factor that regulates osteoblast differentiation, the specific transcription factors that determine osteoblast differentiation remain unclear. Further studies are required to deter...

  12. PRELP (proline/arginine-rich end leucine-rich repeat protein) promotes osteoblastic differentiation of preosteoblastic MC3T3-E1 cells by regulating the β-catenin pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Haiying; Cui, Yazhou; Luan, Jing [School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Ji' nan, Shandong (China); Key Laboratory for Rare Disease Research of Shandong Province, Key Laboratory for Biotech Drugs of the Ministry of Health, Shandong Medical Biotechnological Center, Shandong Academy of Medical Sciences, Ji' nan, Shandong (China); Zhang, Xiumei [School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Ji' nan, Shandong (China); Li, Chengzhi; Zhou, Xiaoyan; Shi, Liang [School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Ji' nan, Shandong (China); Key Laboratory for Rare Disease Research of Shandong Province, Key Laboratory for Biotech Drugs of the Ministry of Health, Shandong Medical Biotechnological Center, Shandong Academy of Medical Sciences, Ji' nan, Shandong (China); Wang, Huaxin [Shandong University of Traditional Chinese Medicine, Ji' an, Shandong (China); Han, Jinxiang, E-mail: jxhan9888@aliyun.com [School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Ji' nan, Shandong (China); Key Laboratory for Rare Disease Research of Shandong Province, Key Laboratory for Biotech Drugs of the Ministry of Health, Shandong Medical Biotechnological Center, Shandong Academy of Medical Sciences, Ji' nan, Shandong (China)

    2016-02-12

    Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that β-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit the level of connexin43, a key regulator of osteoblast differentiation by affecting β-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating β-catenin/connexin43 pathway and osteoblast differentiation.

  13. The Effect of Exogenous Zinc Concentration on the Responsiveness of MC3T3-E1 Pre-Osteoblasts to Surface Microtopography: Part II (Differentiation

    Directory of Open Access Journals (Sweden)

    Kathryn Dorst

    2014-02-01

    Full Text Available Osseointegration of bone implants is a vital part of the recovery process. Numerous studies have shown that micropatterned geometries can promote cell-substrate associations and strengthen the bond between tissue and the implanted material. As demonstrated previously, exogenous zinc levels can influence the responsiveness of pre-osteoblasts to micropatterns and modify their migratory behavior. In this study, we sought to determine the effect of exogenous zinc on differentiation of osteoblasts cultured on micropatterned vs. planar substrates. Levels of activated metalloproteinase-2 (MMP-2 and transforming growth factor-beta 1 (TGF-β1, as well as early stage differentiation marker alkaline phosphatase, were altered with the addition of zinc. These results suggest that exogenous zinc concentration and micropatterning may interdependently modulate osteoblast differentiation.

  14. Divalent Metal Ions Induced Osteogenic Differentiation of MC3T3E1

    Science.gov (United States)

    Wang, Guoshou; Su, Wenta; Chen, Pohung; Huang, Teyang

    2017-12-01

    Biomaterial scaffolds blended with biochemical signal molecules with adequate osteoinductive and osteoconductive properties have attracted significant interest in bone tissue engineering regeneration. The divalent metal ions can gradually release from the scaffold into the culture medium and then induced osteoblastic differentiation of MC3T3E1. These MC3T3E1 cells expressed high activity of alkaline phosphatase, bone-related gene expression of collagen type I, Runx2, osteopontin, osteocalcin, and significantly enhanced deposited minerals on scaffold after 21 days of culture. This experiment provided a useful inducer for osteogenic differentiation in bone repair.

  15. Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing, E-mail: allenylq@hotmail.com; Liao, Er-Yuan, E-mail: eyliao@21cn.com

    2013-11-01

    Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1

  16. Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

    International Nuclear Information System (INIS)

    Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing; Liao, Er-Yuan

    2013-01-01

    Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1

  17. Calcification of MC3T3-E1 cells on titanium and zirconium.

    Science.gov (United States)

    Umezawa, Takayuki; Chen, Peng; Tsutsumi, Yusuke; Doi, Hisashi; Ashida, Maki; Suzuki, Shoichi; Moriyama, Keiji; Hanawa, Takao

    2015-01-01

    To confirm similarity of hard tissue compatibility between titanium and zirconium, calcification of MC3T3-E1 cells on titanium and zirconium was evaluated in this study. Mirror-polished titanium (Ti) and zirconium (Zr) disks and zirconium-sputter deposited titanium (Zr/Ti) were employed in this study. The surface of specimens were characterized using scanning electron microscopy and X-ray diffraction. Then, the cellular proliferation, differentiation and calcification of MC3T3-E1 cells on specimens were investigated. The surface of Zr/Ti was much smoother and cleaner than those of Ti and Zr. The proliferation of the cell was the same among three specimens, while the differentiation and calcification on Zr/Ti were faster than those on Ti and Zr. Therefore, Ti and Zr showed the identical hard tissue compatibility according to the evaluation with MC3T3-E1 cells. Sputter deposition may improve cytocompatibility.

  18. ClC-3 Promotes Osteogenic Differentiation in MC3T3-E1 Cell After Dynamic Compression.

    Science.gov (United States)

    Wang, Dawei; Wang, Hao; Gao, Feng; Wang, Kun; Dong, Fusheng

    2017-06-01

    ClC-3 chloride channel has been proved to have a relationship with the expression of osteogenic markers during osteogenesis, persistent static compression can upregulate the expression of ClC-3 and regulate osteodifferentiation in osteoblasts. However, there was no study about the relationship between the expression of ClC-3 and osteodifferentiation after dynamic compression. In this study, we applied dynamic compression on MC3T3-E1 cells to detect the expression of ClC-3, runt-related transcription factor 2 (Runx2), bone morphogenic protein-2 (BMP-2), osteopontin (OPN), nuclear-associated antigen Ki67 (Ki67), and proliferating cell nuclear antigen (PCNA) in biopress system, then we investigated the expression of these genes after dynamic compression with Chlorotoxin (specific ClC-3 chloride channel inhibitor) added. Under transmission electron microscopy, there were more cell surface protrusions, rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, abundant glycogen, and lysosomes scattered in the cytoplasm in MC3T3-E1 cells after dynamic compression. The nucleolus was more obvious. We found that ClC-3 was significantly up-regulated after dynamic compression. The compressive force also up-regulated Runx2, BMP-2, and OPN after dynamic compression for 2, 4 and 8 h. The proliferation gene Ki67 and PCNA did not show significantly change after dynamic compression for 8 h. Chlorotoxin did not change the expression of ClC-3 but reduced the expression of Runx2, BMP-2, and OPN after dynamic compression compared with the group without Cltx added. The data from the current study suggested that ClC-3 may promotes osteogenic differentiation in MC3T3-E1 cell after dynamic compression. J. Cell. Biochem. 118: 1606-1613, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. Effect of injection molded micro-structured polystyrene surfaces on proliferation of MC3T3-E1 cells

    Directory of Open Access Journals (Sweden)

    G. Lucchetta

    2015-04-01

    Full Text Available In this work, osteoinductive micro-pillared polystyrene surfaces were mass-produced for bone replacement applications, by means of the micro injection molding process. Firstly, the molding process parameters were optimized with a two-level, three-factor central composite face-centered plan to increase the quality of polystyrene micro pillars replication and to maximize the pillars height uniformity over the molded part. Secondly, osteoblastic MC3T3-E1 cells adhesion and proliferation on the replicated substrates were assessed as a function of micro topography parameters, such as pillars diameter, aspect ratio and spacing. Cell morphology and proliferation were evaluated through MTS test after 1, 3 and 7 days from seeding. The experimental results showed that cells adhesion and proliferation is more positively promoted on micro-pillared surfaces compared to flat surfaces, but no correlations were observed between cell proliferation and pillar diameter and spacing.

  20. Graphene Oxide Hybridized nHAC/PLGA Scaffolds Facilitate the Proliferation of MC3T3-E1 Cells

    Science.gov (United States)

    Liang, Chunyong; Luo, Yongchao; Yang, Guodong; Xia, Dan; Liu, Lei; Zhang, Xiaomin; Wang, Hongshui

    2018-01-01

    Biodegradable porous biomaterial scaffolds play a critical role in bone regeneration. In this study, the porous nano-hydroxyapatite/collagen/poly(lactic-co-glycolic acid)/graphene oxide (nHAC/PLGA/GO) composite scaffolds containing different amount of GO were fabricated by freeze-drying method. The results show that the synthesized scaffolds possess a three-dimensional porous structure. GO slightly improves the hydrophilicity of the scaffolds and reinforces their mechanical strength. Young's modulus of the 1.5 wt% GO incorporated scaffold is greatly increased compared to the control sample. The in vitro experiments show that the nHAC/PLGA/GO (1.5 wt%) scaffolds significantly cell adhesion and proliferation of osteoblast cells (MC3T3-E1). This present study indicates that the nHAC/PLGA/GO scaffolds have excellent cytocompatibility and bone regeneration ability, thus it has high potential to be used as scaffolds in the field of bone tissue engineering.

  1. [Effects of sintered bone modified with surface mineralization/P24 peptide composite biomaterial on the adhesion, proliferation and osteodifferentiation of MC3T3-E1 cells].

    Science.gov (United States)

    Li, Jingfeng; Zheng, Qixin; Guo, Xiaodong; Chen, Liaobin

    2014-10-01

    In the present research, the effects of sintered bone modified with surface mineralization/P24 peptide composite biomaterials on the adhesion, proliferation and osteodifferentiation of MC3T3-E1 cells were investigated. The experiments were divided into three groups due to biomaterials used: Group A (composite materials of sintered bone modified with surface mineralization and P24, a peptide of bone morphogenetic protein-2); Group B (sintered bone modified with surface mineralization) and Group C (sintered bone only). The three groups were observed by scanning electron microscopy (SEM) before the experiments, respectively. Then MC3T3-E1 cells were cultured on the surfaces of the three kinds of material, respectively. The cell adhesion rate was assessed by precipitation method. The proliferative ability of MC3T3-E1 cells were measured with MTT assay. And the ALP staining and measurement of alkaline phosphatase (ALP) activity were performed to assess the differentiation of cells into osteoblasts. The SEM results showed that the materials in the three groups retained the natural pore structure and the pore sizes were in the range between 200-850 μm. The adhesive ratio measurements and MTT assay suggested that adhesion and proliferation of MC3T3-E1 cells in Group A were much higher than those in Group B and Group C (P bone modified with surface mineralization/P24 composite material was confirmed to improve the adhesion rate and proliferation and osteodifferentiation of MC3T3-E1 cells, and maintained their morphology.

  2. The in vitro effects of macrophages on the osteogenic capabilities of MC3T3-E1 cells encapsulated in a biomimetic poly(ethylene glycol) hydrogel.

    Science.gov (United States)

    Saleh, Leila S; Carles-Carner, Maria; Bryant, Stephanie J

    2018-04-15

    Poly(ethylene glycol) PEG-based hydrogels are promising for cell encapsulation and tissue engineering, but are known to elicit a foreign body response (FBR) in vivo. The goal of this study was to investigate the impact of the FBR, and specifically the presence of inflammatory macrophages, on encapsulated cells and their ability to synthesize new extracellular matrix. This study employed an in vitro co-culture system with murine macrophages and MC3T3-E1 pre-osteoblasts encapsulated in a bone-mimetic hydrogel, which were cultured in transwell inserts, and exposed to an inflammatory stimulant, lipopolysaccharide (LPS). The co-culture was compared to mono-cultures of the cell-laden hydrogels alone and with LPS over 28 days. Two macrophage cell sources, RAW 264.7 and primary derived, were investigated. The presence of LPS-stimulated primary macrophages led to significant changes in the cell-laden hydrogel by a 5.3-fold increase in percent apoptotic osteoblasts at day 28, 4.2-fold decrease in alkaline phosphatase activity at day 10, and 7-fold decrease in collagen deposition. The presence of LPS-stimulated RAW macrophages led to significant changes in the cell-laden hydrogel by 5-fold decrease in alkaline phosphatase activity at day 10 and 4-fold decrease in collagen deposition. Mineralization, as measured by von Kossa stain or quantified by calcium content, was not sensitive to macrophages or LPS. Elevated interleukin-6 and tumor necrosis factor-α secretion were detected in mono-cultures with LPS and co-cultures. Overall, primary macrophages had a more severe inhibitory effect on osteoblast differentiation than the macrophage cell line, with greater apoptosis and collagen I reduction. In summary, this study highlights the detrimental effects of macrophages on encapsulated cells for bone tissue engineering. Poly(ethylene glycol) (PEG)-based hydrogels are promising for cell encapsulation and tissue engineering, but are known to elicit a foreign body response (FBR) in

  3. Bone morphogenetic protein-2 (BMP-2 and transforming growth factor-β1 (TGF-β1 alter connexin 43 phosphorylation in MC3T3-E1 Cells

    Directory of Open Access Journals (Sweden)

    Rudkin George H

    2001-07-01

    Full Text Available Abstract Background Bone morphogenetic proteins (BMPs and transforming growth factor-βs (TGF-βs are important regulators of bone repair and regeneration. BMP-2 and TGF-β1 have been shown to inhibit gap junctional intercellular communication (GJIC in MC3T3-E1 cells. Connexin 43 (Cx43 has been shown to mediate GJIC in osteoblasts and it is the predominant gap junctional protein expressed in these murine osteoblast-like cells. We examined the expression, phosphorylation, and subcellular localization of Cx43 after treatment with BMP-2 or TGF-β1 to investigate a possible mechanism for the inhibition of GJIC. Results Northern blot analysis revealed no detectable change in the expression of Cx43 mRNA. Western blot analysis demonstrated no significant change in the expression of total Cx43 protein. However, significantly higher ratios of unphosphorylated vs. phosphorylated forms of Cx43 were detected after BMP-2 or TGF-β1 treatment. Immunofluorescence and cell protein fractionation revealed no detectable change in the localization of Cx43 between the cytosol and plasma membrane. Conclusions BMP-2 and TGF-β1 do not alter expression of Cx43 at the mRNA or protein level. BMP-2 and TGF-β1 may inhibit GJIC by decreasing the phosphorylated form of Cx43 in MC3T3-E1 cells.

  4. In vitro behavior of MC3T3-E1 preosteoblast with different annealing temperature titania nanotubes.

    Science.gov (United States)

    Yu, W Q; Zhang, Y L; Jiang, X Q; Zhang, F Q

    2010-10-01

    Titanium oxide nanotube layers by anodization have excellent potential for dental implants because of good bone cell promotion. It is necessary to evaluate osteoblast behavior on different annealing temperature titania nanotubes for actual implant designs.  Scanning Electron Microscopy, X-Ray polycrystalline Diffractometer (XRD), X-ray photoelectron Spectroscope, and Atomic Force Microscopy (AFM) were used to characterize the different annealing temperature titania nanotubes. Confocal laser scanning microscopy, MTT, and Alizarin Red-S staining were used to evaluate the MC3T3-E1 preosteoblast behavior on different annealing temperature nanotubes.  The tubular morphology was constant when annealed at 450°C and 550°C, but collapsed when annealed at 650°C. XRD exhibited the crystal form of nanotubes after formation (amorphous), after annealing at 450°C (anatase), and after annealing at 550°C (anatase/rutile). Annealing led to the complete loss of fluorine on nanotubes at 550°C. Average surface roughness of different annealing temperature nanotubes showed no difference by AFM analysis. The proliferation and mineralization of preostoblasts cultured on anatase or anatase/rutile nanotube layers were shown to be significantly higher than smooth, amorphous nanotube layers.  Annealing can change the crystal form and composition of nanotubes. The nanotubes after annealing can promote osteoblast proliferation and mineralization in vitro. © 2010 John Wiley & Sons A/S.

  5. Genes Responsive to Low-Intensity Pulsed Ultrasound in MC3T3-E1 Preosteoblast Cells

    Directory of Open Access Journals (Sweden)

    Yoshiaki Tabuchi

    2013-11-01

    Full Text Available Although low-intensity pulsed ultrasound (LIPUS has been shown to enhance bone fracture healing, the underlying mechanism of LIPUS remains to be fully elucidated. Here, to better understand the molecular mechanism underlying cellular responses to LIPUS, we investigated gene expression profiles in mouse MC3T3-E1 preosteoblast cells exposed to LIPUS using high-density oligonucleotide microarrays and computational gene expression analysis tools. Although treatment of the cells with a single 20-min LIPUS (1.5 MHz, 30 mW/cm2 did not affect the cell growth or alkaline phosphatase activity, the treatment significantly increased the mRNA level of Bglap. Microarray analysis demonstrated that 38 genes were upregulated and 37 genes were downregulated by 1.5-fold or more in the cells at 24-h post-treatment. Ingenuity pathway analysis demonstrated that the gene network U (up contained many upregulated genes that were mainly associated with bone morphology in the category of biological functions of skeletal and muscular system development and function. Moreover, the biological function of the gene network D (down, which contained downregulated genes, was associated with gene expression, the cell cycle and connective tissue development and function. These results should help to further clarify the molecular basis of the mechanisms of the LIPUS response in osteoblast cells.

  6. Osteogenic differentiation of MC3T3-E1 cells on poly(L-lactide)/Fe{sub 3}O{sub 4} nanofibers with static magnetic field exposure

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Qing [State Key Laboratory of Organic–inorganic Composites, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 (China); Shi, Yuzhou; Shan, Dingying; Jia, Wenkai; Duan, Shun [Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 (China); Deng, Xuliang [Department of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081 (China); Yang, Xiaoping, E-mail: yangxp@mail.buct.edu.cn [State Key Laboratory of Organic–inorganic Composites, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 (China)

    2015-10-01

    Proliferation and differentiation of bone-related cells are modulated by many factors such as scaffold design, growth factor, dynamic culture system, and physical simulation. Nanofibrous structure and moderate-intensity (1 mT–1 T) static magnetic field (SMF) have been identified as capable of stimulating proliferation and differentiation of osteoblasts. Herein, magnetic nanofibers were prepared by electrospinning mixture solutions of poly(L-lactide) (PLLA) and ferromagnetic Fe{sub 3}O{sub 4} nanoparticles (NPs). The PLLA/Fe{sub 3}O{sub 4} composite nanofibers demonstrated homogeneous dispersion of Fe{sub 3}O{sub 4} NPs, and their magnetism depended on the contents of Fe{sub 3}O{sub 4} NPs. SMF of 100 mT was applied in the culture of MC3T3-E1 osteoblasts on pure PLLA and PLLA/Fe{sub 3}O{sub 4} composite nanofibers for the purpose of studying the effect of SMF on osteogenic differentiation of osteoblastic cells on magnetic nanofibrous scaffolds. On non-magnetic PLLA nanofibers, the application of external SMF could enhance the proliferation and osteogenic differentiation of MC3T3-E1 cells. In comparison with pure PLLA nanofibers, the incorporation of Fe{sub 3}O{sub 4} NPs could also promote the proliferation and osteogenic differentiation of MC3T3-E1 cells in the absence or presence of external SMF. The marriage of magnetic nanofibers and external SMF was found most effective in accelerating every aspect of biological behaviors of MC3T3-E1 osteoblasts. The findings demonstrated that the magnetic feature of substrate and microenvironment were applicable ways in regulating osteogenesis in bone tissue engineering. - Highlights: • Magnetic nanofibers containing well-dispersed Fe{sub 3}O{sub 4} nanoparticles were produced. • Static magnetic field (SMF) was applied to perform the culture of osteoblasts. • Osteogenic differentiation was enhanced on magnetic substrate with exposure to SMF.

  7. MC3T3-E1 cell response to stainless steel 316L with different surface treatments

    International Nuclear Information System (INIS)

    Zhang, Hongyu; Han, Jianmin; Sun, Yulong; Huang, Yongling; Zhou, Ming

    2015-01-01

    In the present study, stainless steel 316L samples with polishing, aluminum oxide blasting, and hydroxyapatite (HA) coating were prepared and characterized through a scanning electron microscope (SEM), optical interferometer (surface roughness, Sq), contact angle, surface composition and phase composition analyses. Osteoblast-like MC3T3-E1 cell adhesion on the samples was investigated by cell morphology using a SEM (4 h, 1 d, 3 d, 7 d), and cell proliferation was assessed by MTT method at 1 d, 3 d, and 7 d. In addition, adsorption of bovine serum albumin on the samples was evaluated at 1 h. The polished sample was smooth (Sq: 1.8 nm), and the blasted and HA coated samples were much rougher (Sq: 3.2 μm and 7.8 μm). Within 1 d of incubation, the HA coated samples showed the best cell morphology (e.g., flattened shape and complete spread), but there was no significant difference after 3 d and 7 d of incubation for all the samples. The absorbance value for the HA coated samples was the highest after 1 d and 3 d of incubation, indicating better cell viability. However, it reduced to the lowest value at 7 d. Protein adsorption on the HA coated samples was the highest at 1 h. The results indicate that rough stainless steel surface improves cell adhesion and morphology, and HA coating contributes to superior cell adhesion, but inhibits cell proliferation. - Highlights: • Rough stainless steel surface improves cell adhesion and proliferation. • HA coating results in superior cell morphology and cell attachment. • HA coating inhibits osteoblast cell proliferation after 7 d of incubation

  8. MC3T3-E1 cell response to stainless steel 316L with different surface treatments

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hongyu [State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua University, Beijing 100084 (China); Han, Jianmin, E-mail: siyanghan@163.com [Dental Materials Laboratory, National Engineering Laboratory for Digital and Material Technology of Stomatology, Peking University School and Hospital of Stomatology, Beijing 100081 (China); Sun, Yulong [State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua University, Beijing 100084 (China); Huang, Yongling [Jinghang Biomedicine Engineering Division, Beijing Institute of Aeronautical Material, Beijing 100095 (China); Zhou, Ming [State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua University, Beijing 100084 (China)

    2015-11-01

    In the present study, stainless steel 316L samples with polishing, aluminum oxide blasting, and hydroxyapatite (HA) coating were prepared and characterized through a scanning electron microscope (SEM), optical interferometer (surface roughness, Sq), contact angle, surface composition and phase composition analyses. Osteoblast-like MC3T3-E1 cell adhesion on the samples was investigated by cell morphology using a SEM (4 h, 1 d, 3 d, 7 d), and cell proliferation was assessed by MTT method at 1 d, 3 d, and 7 d. In addition, adsorption of bovine serum albumin on the samples was evaluated at 1 h. The polished sample was smooth (Sq: 1.8 nm), and the blasted and HA coated samples were much rougher (Sq: 3.2 μm and 7.8 μm). Within 1 d of incubation, the HA coated samples showed the best cell morphology (e.g., flattened shape and complete spread), but there was no significant difference after 3 d and 7 d of incubation for all the samples. The absorbance value for the HA coated samples was the highest after 1 d and 3 d of incubation, indicating better cell viability. However, it reduced to the lowest value at 7 d. Protein adsorption on the HA coated samples was the highest at 1 h. The results indicate that rough stainless steel surface improves cell adhesion and morphology, and HA coating contributes to superior cell adhesion, but inhibits cell proliferation. - Highlights: • Rough stainless steel surface improves cell adhesion and proliferation. • HA coating results in superior cell morphology and cell attachment. • HA coating inhibits osteoblast cell proliferation after 7 d of incubation.

  9. Behaviors of MC3T3-E1 cells on carbonated apatite films, with a characteristic network structure, fabricated on a titanium plate by aqueous spray coating

    International Nuclear Information System (INIS)

    Mochizuki, Chihiro; Hara, Hiroki; Oya, Kei; Aoki, Shun; Hayakawa, Tohru; Fujie, Hiromichi; Sato, Mitsunobu

    2014-01-01

    Four carbonated apatite films having average thicknesses of 1.3–0.11 μm, proportions of network sizes above 10 μm of 41–68%, and average border heights of the characteristic network structure of 0.98–0.29 μm were fabricated on a titanium plate by aqueous spray coating. These carbonated apatite films after heat treatment showed good mineralization ability in Hanks' balanced salt solution. Assessment of initial cell attachment and calcination on these films and on the Ti plate using osteoblastic MC3T3-E1 indicated that the carbonated apatite film heat treated at 600 °C, whose film thickness, proportion of network sizes above 10 μm, and border height were 0.11 μm, 61%, and 0.31 μm, respectively, was most preferred by osteoblastic cells. Field emission scanning electron microscopic observation of the cells attached to the films showed that the wide network and low border height of the network structure on the carbonated apatite film play an important role in the development of the filopodia of the osteoblastic cells. - Highlights: • Osteoblastic MC3T3-E1 behaviors on aqueous spray coating-derived carbonated apatite (CA) films • The network size of CA films is important. • CA films having a low network border height are better for cell proliferation

  10. Behaviors of MC3T3-E1 cells on carbonated apatite films, with a characteristic network structure, fabricated on a titanium plate by aqueous spray coating

    Energy Technology Data Exchange (ETDEWEB)

    Mochizuki, Chihiro; Hara, Hiroki [Division of Liberal Arts, Kogakuin University, 2665-1 Nakano, Hachioji, Tokyo 192-0015 (Japan); Oya, Kei [Research Institute for Science and Technology, Kogakuin University, 2665-1 Nakano, Hachioji, Tokyo 192-0015 (Japan); School of Engineering, Tokai University, 4-1-1 Kitakanane, Hiratsuka, Kanagawa 259-1292 (Japan); Aoki, Shun [Faculty of System Design, Tokyo Metropolitan University, 6-6 Asahigaoka, Hino, Tokyo 191-0065 (Japan); Hayakawa, Tohru [Department of Dental Engineering, Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Yokohama City, Kanagawa 230-8501 (Japan); Fujie, Hiromichi [Faculty of System Design, Tokyo Metropolitan University, 6-6 Asahigaoka, Hino, Tokyo 191-0065 (Japan); Sato, Mitsunobu, E-mail: lccsato@cc.kogakuin.ac.jp [Division of Liberal Arts, Kogakuin University, 2665-1 Nakano, Hachioji, Tokyo 192-0015 (Japan)

    2014-06-01

    Four carbonated apatite films having average thicknesses of 1.3–0.11 μm, proportions of network sizes above 10 μm of 41–68%, and average border heights of the characteristic network structure of 0.98–0.29 μm were fabricated on a titanium plate by aqueous spray coating. These carbonated apatite films after heat treatment showed good mineralization ability in Hanks' balanced salt solution. Assessment of initial cell attachment and calcination on these films and on the Ti plate using osteoblastic MC3T3-E1 indicated that the carbonated apatite film heat treated at 600 °C, whose film thickness, proportion of network sizes above 10 μm, and border height were 0.11 μm, 61%, and 0.31 μm, respectively, was most preferred by osteoblastic cells. Field emission scanning electron microscopic observation of the cells attached to the films showed that the wide network and low border height of the network structure on the carbonated apatite film play an important role in the development of the filopodia of the osteoblastic cells. - Highlights: • Osteoblastic MC3T3-E1 behaviors on aqueous spray coating-derived carbonated apatite (CA) films • The network size of CA films is important. • CA films having a low network border height are better for cell proliferation.

  11. Low-intensity pulsed ultrasound (LIPUS) stimulates mineralization of MC3T3-E1 cells through calcium and phosphate uptake.

    Science.gov (United States)

    Tassinary, João Alberto Fioravante; Lunardelli, Adroaldo; Basso, Bruno de Souza; Dias, Henrique Bregolin; Catarina, Anderson Velasque; Stülp, Simone; Haute, Gabriela Viegas; Martha, Bianca Andrade; Melo, Denizar Alberto da Silva; Nunes, Fernanda Bordignon; Donadio, Márcio Vinícius Fagundes; Oliveira, Jarbas Rodrigues de

    2018-03-01

    The present study aimed to evaluate the effect of low-intensity pulsed ultrasound (LIPUS) on pre-osteoblast mineralization using in vitro bioassays. Pre-osteoblastic MC3T3-E1 cells were exposed to LIPUS at 1 MHz frequency, 0.2 W/cm 2 intensity and 20% duty cycle for 30 min. The analyses were carried out up to 336 h (14 days) after exposure. The concentration of collagen, phosphate, alkaline phosphatase, calcium and transforming growth factor beta 1 (TGF-β1) in cell supernatant and the presence of calcium deposits in the cells were analyzed. Our results showed that LIPUS promotes mineralized nodules formation. Collagen, phosphate, and calcium levels were decreased in cell supernatant at 192 h after LIPUS exposure. However, alkaline phosphatase and TGF-β1 concentrations remained unchanged. Therapeutic pulsed ultrasound is capable of stimulating differentiation and mineralization of pre-osteoblastic MC3T3-E1 cells by calcium and phosphate uptake with consequent hydroxyapatite formation. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Enhancement of growth and osteogenic differentiation of MC3T3-E1 cells via facile surface functionalization of polylactide membrane with chitooligosaccharide based on polydopamine adhesive coating

    International Nuclear Information System (INIS)

    Li, Huihua; Luo, Chuang; Luo, Binghong; Wen, Wei; Wang, Xiaoying; Ding, Shan; Zhou, Changren

    2016-01-01

    Graphical abstract: - Highlights: • COS was conveniently immobilized on PDLLA membrane based on PDOPA adhesive layer. • The hydrophilicity of PDLLA membrane was improved by modified with PDOPA and COS. • COS-functionalized PDLLA membrane is favorable to cell adhesion and proliferation. • COS-coated PDLLA membrane notably promote osteogenic differentiation of MC3T3-E1. - Abstract: To develop a chitooligosaccharide(COS)-functionalized poly(D,L-lactide) (PDLLA) membrane to enhance growth and osteogenic differentiation of MC3T3-E1 cells, firstly a thin polydopamine (PDOPA) layer was adhered to the PDLLA membrane via the self-polymerization and strong adhesion behavior of dopamine. Subsequently, COS was immobilized covalently on the resultant PDLLA/PDOPA composite membrane by coupling with PDOPA active coating. The successful immobilization of the PDOPA and COS was confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Scanning electronic microscopy (SEM) and atomic force microscopy (AFM) results indicated that the surface topography and roughness of the membranes were changed, and the root mean square increased from 0.613 nm to 6.96 and 7.12 nm, respectively after coating PDOPA and COS. Water contact angle and surface energy measurements revealed that the membrane hydrophilicity was remarkably improved by surface modification. In vitro cells culture results revealed that the PDOPA- and COS-functionalized surfaces showed a significant increase in MC3T3-E1 cells adhesion, proliferation, osteogenic differentiation and alkaline phosphate activity compared to the pristine PDLLA substrate. Furthermore the COS-functionalized PDLLA membrane was more effectively at enhancing osteoblast activity than the PDOPA-functionalized PDLLA membrane.

  13. Sirtuin1 promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor γ in MC3T3-E1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Qu, Bo [Department of Orthopaedics, Chengdu Military General Hospital, Chengdu 610083 (China); Ma, Yuan [Department of Neurosurgery, Chengdu Military General Hospital, Chengdu 610083 (China); Yan, Ming [Department of Orthopaedics, Xijing Hospital of The Fourth Military Medical University, Xi’an 710032 (China); Gong, Kai; Liang, Feng; Deng, Shaolin; Jiang, Kai; Ma, Zehui [Department of Orthopaedics, Chengdu Military General Hospital, Chengdu 610083 (China); Pan, Xianming, E-mail: xianmingpanxj@163.com [Department of Orthopaedics, Chengdu Military General Hospital, Chengdu 610083 (China)

    2016-09-09

    Osteoporosis is a skeletal disorder characterized by bone loss, resulting in architectural deterioration of the skeleton, decreased bone strength and an increased risk of fragility fractures. Strengthening osteogenesis is an effective way to relieve osteoporosis. Sirtuin1 (Sirt1) is a nicotinamide adenine dinucleotide (NAD{sup +})-dependent deacetylase, which is reported to be involved in improving osteogenesis. Sirt1 targets peroxisome proliferator-activated receptor γ (PPARγ) in the regulation of adipose tissues; however, the molecular mechanism of Sirt1 in osteogenic differentiation is still unknown. PPARγ tends to induce more adipogenic differentiation rather than osteogenic differentiation. Hence, we hypothesized that Sirt1 facilitates osteogenic differentiation through downregulation of PPARγ signaling. Mouse pre-osteoblastic MC3T3-E1 cells were cultured under osteogenic medium. Sirt1 was overexpressed through plasmid transfection. The results showed that high expression of Sirt1 was associated with increased osteogenic differentiation, as indicated by quantitative PCR and Western blot analysis of osteogenic markers, and Von Kossa staining. Sirt1 overexpression also directly and negatively regulated the expression of PPARγ and its downstream molecules. Use of the PPARγ agonist Rosiglitazone, reversed the effects of Sirt1 on osteogenic differentiation. Using constructed luciferase plasmids, we demonstrated a role of Sirt1 in inhibiting PPARγ–induced activity and expression of adipocyte–specific genes, including acetyl-coenzyme A carboxylase (Acc) and fatty acid binding protein 4 (Fabp4). The interaction between Sirt1 and PPARγ was further confirmed using co-immunoprecipitation analysis. Together, these results reveal a novel mechanism for Sirt1 in osteogenic differentiation through downregulation of PPARγ activity. These findings suggest that the Sirt1–PPARγ pathway may represent a potential target for enhancement of osteogenesis and treatment

  14. Sirtuin1 promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor γ in MC3T3-E1 cells

    International Nuclear Information System (INIS)

    Qu, Bo; Ma, Yuan; Yan, Ming; Gong, Kai; Liang, Feng; Deng, Shaolin; Jiang, Kai; Ma, Zehui; Pan, Xianming

    2016-01-01

    Osteoporosis is a skeletal disorder characterized by bone loss, resulting in architectural deterioration of the skeleton, decreased bone strength and an increased risk of fragility fractures. Strengthening osteogenesis is an effective way to relieve osteoporosis. Sirtuin1 (Sirt1) is a nicotinamide adenine dinucleotide (NAD"+)-dependent deacetylase, which is reported to be involved in improving osteogenesis. Sirt1 targets peroxisome proliferator-activated receptor γ (PPARγ) in the regulation of adipose tissues; however, the molecular mechanism of Sirt1 in osteogenic differentiation is still unknown. PPARγ tends to induce more adipogenic differentiation rather than osteogenic differentiation. Hence, we hypothesized that Sirt1 facilitates osteogenic differentiation through downregulation of PPARγ signaling. Mouse pre-osteoblastic MC3T3-E1 cells were cultured under osteogenic medium. Sirt1 was overexpressed through plasmid transfection. The results showed that high expression of Sirt1 was associated with increased osteogenic differentiation, as indicated by quantitative PCR and Western blot analysis of osteogenic markers, and Von Kossa staining. Sirt1 overexpression also directly and negatively regulated the expression of PPARγ and its downstream molecules. Use of the PPARγ agonist Rosiglitazone, reversed the effects of Sirt1 on osteogenic differentiation. Using constructed luciferase plasmids, we demonstrated a role of Sirt1 in inhibiting PPARγ–induced activity and expression of adipocyte–specific genes, including acetyl-coenzyme A carboxylase (Acc) and fatty acid binding protein 4 (Fabp4). The interaction between Sirt1 and PPARγ was further confirmed using co-immunoprecipitation analysis. Together, these results reveal a novel mechanism for Sirt1 in osteogenic differentiation through downregulation of PPARγ activity. These findings suggest that the Sirt1–PPARγ pathway may represent a potential target for enhancement of osteogenesis and treatment of

  15. Multifunctional chitosan/polyvinyl pyrrolidone/45S5 Bioglass® scaffolds for MC3T3-E1 cell stimulation and drug release

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Qingqing [Institute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology & Optometry, Wenzhou Medical University, 270 Xueyuan Xi Road, Wenzhou, Zhejiang 325027 (China); Li, Wei [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, Erlangen 91058 (Germany); Yu, Shanshan; Ma, Liwei [Institute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology & Optometry, Wenzhou Medical University, 270 Xueyuan Xi Road, Wenzhou, Zhejiang 325027 (China); Jin, Dayong [Institute for Biomedical Materials and Devices, Faculty of Science, University of Technology Sydney, NSW 2007 (Australia); Advanced Cytometry Labs, ARC Center of Excellence for Nanoscale BioPhotonics, Macquarie University, Sydney, NSW 2109 (Australia); Boccaccini, Aldo R., E-mail: Aldo.Boccaccini@ww.uni-erlangen.de [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, Erlangen 91058 (Germany); Liu, Yong, E-mail: yongliu1980@hotmail.com [Institute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology & Optometry, Wenzhou Medical University, 270 Xueyuan Xi Road, Wenzhou, Zhejiang 325027 (China); Advanced Cytometry Labs, ARC Center of Excellence for Nanoscale BioPhotonics, Macquarie University, Sydney, NSW 2109 (Australia)

    2015-11-01

    Novel chitosan–polyvinyl pyrrolidone/45S5 Bioglass® (CS-PVP/BG) scaffolds were prepared via foam replication and chemical cross-linking techniques. The pristine BG, CS-PVP coated BG and genipin cross-linked CS-PVP/BG (G-CS-PVP/BG) scaffolds were synthesized and characterized in terms of chemical composition, physical structure and morphology respectively. Resistance to enzymatic degradation of the scaffold is improved significantly with the use of genipin cross-linked CS-PVP. The bio-effects of scaffolds on MC3T3-E1 osteoblast-like cells were evaluated by studying cell viability, adhesion and proliferation. The CCK-8 assay shows that cell viability on the resulting G-CS-PVP/BG scaffold is improved obviously after cross-linking of genipin. Cell skeleton images exhibit that well-stretched F-actin bundles are obtained on the G-CS-PVP/BG scaffold. SEM results present significant improvement on the cell adhesion and proliferation for cells cultured on the G-CS-PVP/BG scaffold. The drug release performance on the as-synthesized scaffold was studied in a phosphate buffered saline (PBS) solution. Vancomycin is found to be released in burst fashion within 24 h from the pristine BG scaffold, however, the release period from the G-CS-PVP/BG scaffold is enhanced to 7 days, indicating improved drug release properties of the G-CS-PVP/BG scaffold. Our results suggest that the G-CS-PVP/BG scaffolds possess promising physicochemical properties, sustained drug release capability and good biocompatibility for MC3T3-E1 cells' proliferation and adhesion, suggesting their potential applications in areas such as MC3T3-E1 cell stimulation and bone tissue engineering. - Highlights: • Novel genipi–chitosan–polyvinyl pyrrolidone/45S5 Bioglass® scaffolds are prepared. • Resistance to enzymatic degradation of the scaffold is improved significantly. • The resulting scaffold shows enhanced MC3T3-E1 cell adhesion and proliferation. • Release of antibiotic vancomycin from the

  16. Multifunctional chitosan/polyvinyl pyrrolidone/45S5 Bioglass® scaffolds for MC3T3-E1 cell stimulation and drug release

    International Nuclear Information System (INIS)

    Yao, Qingqing; Li, Wei; Yu, Shanshan; Ma, Liwei; Jin, Dayong; Boccaccini, Aldo R.; Liu, Yong

    2015-01-01

    Novel chitosan–polyvinyl pyrrolidone/45S5 Bioglass® (CS-PVP/BG) scaffolds were prepared via foam replication and chemical cross-linking techniques. The pristine BG, CS-PVP coated BG and genipin cross-linked CS-PVP/BG (G-CS-PVP/BG) scaffolds were synthesized and characterized in terms of chemical composition, physical structure and morphology respectively. Resistance to enzymatic degradation of the scaffold is improved significantly with the use of genipin cross-linked CS-PVP. The bio-effects of scaffolds on MC3T3-E1 osteoblast-like cells were evaluated by studying cell viability, adhesion and proliferation. The CCK-8 assay shows that cell viability on the resulting G-CS-PVP/BG scaffold is improved obviously after cross-linking of genipin. Cell skeleton images exhibit that well-stretched F-actin bundles are obtained on the G-CS-PVP/BG scaffold. SEM results present significant improvement on the cell adhesion and proliferation for cells cultured on the G-CS-PVP/BG scaffold. The drug release performance on the as-synthesized scaffold was studied in a phosphate buffered saline (PBS) solution. Vancomycin is found to be released in burst fashion within 24 h from the pristine BG scaffold, however, the release period from the G-CS-PVP/BG scaffold is enhanced to 7 days, indicating improved drug release properties of the G-CS-PVP/BG scaffold. Our results suggest that the G-CS-PVP/BG scaffolds possess promising physicochemical properties, sustained drug release capability and good biocompatibility for MC3T3-E1 cells' proliferation and adhesion, suggesting their potential applications in areas such as MC3T3-E1 cell stimulation and bone tissue engineering. - Highlights: • Novel genipi–chitosan–polyvinyl pyrrolidone/45S5 Bioglass® scaffolds are prepared. • Resistance to enzymatic degradation of the scaffold is improved significantly. • The resulting scaffold shows enhanced MC3T3-E1 cell adhesion and proliferation. • Release of antibiotic vancomycin from the

  17. Riboflavin and photoproducts in MC3T3-E1 differentiation

    NARCIS (Netherlands)

    Chaves Neto, Antonio Hernandes; Yano, Claudia Lumy; Paredes-Gamero, Edgar Julian; Machado, Daisy; Justo, Giselle Zenker; Peppelenbosch, Maikel P.; Ferreira, Carmen Verissima

    2010-01-01

    Photoderivatives of riboflavin can modulate the proliferation and survival of cancer cells. In this work, we examined the influence of riboflavin and photoderivatives on osteoblast differentiation induced by ascorbic acid and beta-glycerophosphate. These compounds decreased the osteoblast

  18. Poly(acrylic acid-regulated Synthesis of Rod-Like Calcium Carbonate Nanoparticles for Inducing the Osteogenic Differentiation of MC3T3-E1 Cells

    Directory of Open Access Journals (Sweden)

    Wei Yang

    2016-05-01

    Full Text Available Calcium carbonate, especially with nanostructure, has been considered as a good candidate material for bone regeneration due to its excellent biodegradability and osteoconductivity. In this study, rod-like calcium carbonate nanoparticles (Rod-CC NPs with desired water dispersibility were achieved with the regulation of poly (acrylic acid. Characterization results revealed that the Rod-CC NPs had an average length of 240 nm, a width of 90 nm with an average aspect ratio of 2.60 and a negative ζ-potential of −22.25 ± 0.35 mV. The degradation study illustrated the nanoparticles degraded 23% at pH 7.4 and 45% at pH 5.6 in phosphate-buffered saline (PBS solution within three months. When cultured with MC3T3-E1 cells, the Rod-CC NPs exhibited a positive effect on the proliferation of osteoblast cells. Alkaline phosphatase (ALP activity assays together with the osteocalcin (OCN and bone sialoprotein (BSP expression observations demonstrated the nanoparticles could induce the differentiation of MC3T3-E1 cells. Our study developed well-dispersed rod-like calcium carbonate nanoparticles which have great potential to be used in bone regeneration.

  19. Enhancing osteogenic differentiation of MC3T3-E1 cells by immobilizing RGD onto liquid crystal substrate

    International Nuclear Information System (INIS)

    Wu, Shaopeng; Yang, Xiaohui; Li, Wenqiang; Du, Lin; Zeng, Rong; Tu, Mei

    2017-01-01

    To understand the effects of GRGDF modification on MC3T3-E1 cell behavior, we cultured these cells onto a biomimetic liquid crystalline matrix modified with GRGDF peptide (OPC-GA-RGD). Successful immobilization of GRGDF on the liquid crystalline surface was verified by fluorescent labeling, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). OPC-GA-RGDs retained its liquid crystalline feature after surface modification. The RGD-immobilized OPC substrate was hardly beneficial to initial cell adhesion but could support long-term cell survival. The enhancement in cell proliferation did not correlate with RGD density. The lower GRGDF density immobilized on the liquid crystalline OPC matrix (OPC-GA-RGD3) promoted cell adhesion, proliferation, ALP expression level and mineralization, suggesting that both the viscoelasticity-based mechanical stimuli and receptor/ligand-based biochemical cue synergistically modulate MC3T3-E1 cell behavior. - Highlight: • A novel type of GRGDF-immobilized liquid crystalline matrices was fabricated and served as a substrate for the in vitro culture of MC3T3-E1 cells. • The lower RGD density might provide a better condition for initial cell adhesion and proliferation, up-regulation of ALP expression levels, and mineralization. • The intrinsic liquid crystalline feature of OPC matrix, instead of RGD efficiency, promoted initial cell adhesion. • Properties of the liquid crystalline OPC matrix together with the stable receptor-ligand binging synergistically modulated MC3T3-E1 cell behavior.

  20. Enhancing osteogenic differentiation of MC3T3-E1 cells by immobilizing RGD onto liquid crystal substrate

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Shaopeng; Yang, Xiaohui; Li, Wenqiang; Du, Lin; Zeng, Rong; Tu, Mei, E-mail: tumei@jnu.edu.cn

    2017-02-01

    To understand the effects of GRGDF modification on MC3T3-E1 cell behavior, we cultured these cells onto a biomimetic liquid crystalline matrix modified with GRGDF peptide (OPC-GA-RGD). Successful immobilization of GRGDF on the liquid crystalline surface was verified by fluorescent labeling, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). OPC-GA-RGDs retained its liquid crystalline feature after surface modification. The RGD-immobilized OPC substrate was hardly beneficial to initial cell adhesion but could support long-term cell survival. The enhancement in cell proliferation did not correlate with RGD density. The lower GRGDF density immobilized on the liquid crystalline OPC matrix (OPC-GA-RGD3) promoted cell adhesion, proliferation, ALP expression level and mineralization, suggesting that both the viscoelasticity-based mechanical stimuli and receptor/ligand-based biochemical cue synergistically modulate MC3T3-E1 cell behavior. - Highlight: • A novel type of GRGDF-immobilized liquid crystalline matrices was fabricated and served as a substrate for the in vitro culture of MC3T3-E1 cells. • The lower RGD density might provide a better condition for initial cell adhesion and proliferation, up-regulation of ALP expression levels, and mineralization. • The intrinsic liquid crystalline feature of OPC matrix, instead of RGD efficiency, promoted initial cell adhesion. • Properties of the liquid crystalline OPC matrix together with the stable receptor-ligand binging synergistically modulated MC3T3-E1 cell behavior.

  1. Synthesis of polypyrrole nanowires with positive effect on MC3T3-E1 cell functions through electrical stimulation

    Energy Technology Data Exchange (ETDEWEB)

    He, Yuan; Wang, Shihui [Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005 (China); Mu, Jing, E-mail: mujing@picb.ac.cn [Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005 (China); Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai 200031 (China); Dai, Lingfeng [Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005 (China); Zhang, Zhong [Department of Stomatology, The Affiliated Zhongshan Hospital of Xiamen University, Xiamen 361005 (China); Sun, Yanan; Shi, Wei [Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005 (China); Ge, Dongtao, E-mail: gedt@xmu.edu.cn [Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005 (China)

    2017-02-01

    Conducting polymer polypyrrole (PPy) possesses good biocompatibility and conductivity and has been used as functional coatings in bone tissue regeneration. In this study, a cholic acid doped PPy nanowires (PPy NWs) coating was electrochemically polymerized on the surface of titanium (Ti). The porous intertwined PPy NWs coating exhibited excellent electrical conductivity and electrochemical activity, better hydrophilicity and higher surface energy. In vitro cell experiments demonstrated that the PPy NWs coating together with a 10 μA substrate-mediate electrical stimulation (ES) was capable to positive regulate the functions of MC3T3-E1 such as cell adhesion, proliferation and differentiation. Further long-term functions of cell tests including alkaline phosphatase (ALP) activity, bone-carboxyglutamic acid-containing protein (BGP) and calcium deposition were all thoroughly increased. These confirmed that the combination of PPy NWs and ES could accelerate MC3T3-E1 cells mature and osteogenesis. Hence, the PPy NWs coating was an electro bioactive coating and may have potential applications in the treatment of bone damage repairing and regeneration with ES. - Highlights: • The porous PPy nanowire coating was electrochemically polymerized on Ti. • PPy NWs had good electrical conductivity, electrochemical stability and hydrophilicity. • Pure PPy NWs with ES could obviously promote the growth and cell functions of MC3T3-E1.

  2. Synthesis of polypyrrole nanowires with positive effect on MC3T3-E1 cell functions through electrical stimulation

    International Nuclear Information System (INIS)

    He, Yuan; Wang, Shihui; Mu, Jing; Dai, Lingfeng; Zhang, Zhong; Sun, Yanan; Shi, Wei; Ge, Dongtao

    2017-01-01

    Conducting polymer polypyrrole (PPy) possesses good biocompatibility and conductivity and has been used as functional coatings in bone tissue regeneration. In this study, a cholic acid doped PPy nanowires (PPy NWs) coating was electrochemically polymerized on the surface of titanium (Ti). The porous intertwined PPy NWs coating exhibited excellent electrical conductivity and electrochemical activity, better hydrophilicity and higher surface energy. In vitro cell experiments demonstrated that the PPy NWs coating together with a 10 μA substrate-mediate electrical stimulation (ES) was capable to positive regulate the functions of MC3T3-E1 such as cell adhesion, proliferation and differentiation. Further long-term functions of cell tests including alkaline phosphatase (ALP) activity, bone-carboxyglutamic acid-containing protein (BGP) and calcium deposition were all thoroughly increased. These confirmed that the combination of PPy NWs and ES could accelerate MC3T3-E1 cells mature and osteogenesis. Hence, the PPy NWs coating was an electro bioactive coating and may have potential applications in the treatment of bone damage repairing and regeneration with ES. - Highlights: • The porous PPy nanowire coating was electrochemically polymerized on Ti. • PPy NWs had good electrical conductivity, electrochemical stability and hydrophilicity. • Pure PPy NWs with ES could obviously promote the growth and cell functions of MC3T3-E1.

  3. Nano-hydroxyapatite particles induce apoptosis on MC3T3-E1 cells and tissue cells in SD rats

    Science.gov (United States)

    Wang, Liting; Zhou, Gang; Liu, Haifeng; Niu, Xufeng; Han, Jingyun; Zheng, Lisha; Fan, Yubo

    2012-04-01

    While the advantages of nanomaterials are being increasingly recognized, their potential toxicity is drawing more and more attention and concern. In this study, we explore the toxicity mechanism of 20-30 nm rod-shaped hydroxyapatite (HA) nanoparticles in vitro and in vivo. The nanoparticles were prepared by precipitation and characterized by IR, XRD and TEM. Concentrations of 0 μg mL-1, 10 μg mL-1, 100 μg mL-1, 1 mg mL-1, and 10 mg mL-1 were applied to the MC3T3-E1 cells for viability (MTT-test). Based on the characteristic differences of the two methods of cell death, the morphological features of the MC3T3-E1 cell line co-cultured with nano-hydroxyapatite (n-HA) (10 mg mL-1) for 24 h were also observed by TEM. Furthermore, important serum biochemical markers and histopathological examinations were used to evaluate the potential toxicological effect of n-HA on the major organs of SD rats injected intraperitoneally with n-HA (33.3 mg kg-1 body weight). In the results, we found cell growth inhibition and apoptosis in MC3T3-E1 cells co-cultured with n-HA. Moreover, apoptosis but not necrosis was illustrated in liver and renal tissue by using histopathology slices and serum biochemical markers. It suggests that apoptosis may be the possible mechanism of n-HA toxicity and provides a better understanding of the biocompatibility of nanomaterials applied in human bone repair.

  4. Effect of hierarchical pore structure on ALP expression of MC3T3-E1 cells on bioglass films.

    Science.gov (United States)

    Yu, Cuixia; Zhuang, Junjun; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

    2017-08-01

    Hierarchical porous bioglass films on the tantalum were designed to enhance osteointegration of metallic implants. The films were prepared by a sol-gel method using P123 as the mesopore template and polystyrene microsphere as the nanopore template. The films with 5.4nm mesopores and 100nm nanopores (MBG-100) elicited an obviously elongated morphology of the cultured MC3T3-E1 cells, as a result, a higher alkaline phosphatase level was expressed. It is suggested that the nanopores play an important role in regulating cellular behavior by initial protein adsorption through nanopore curvatures. The mesopores were proven very effective for loading rhBMP-2, and the rhBMP-2 loaded on MBG-100 films showed a better function of enhancing osteogenic differentiation, which is attributed to that the nanopore structure could expedite rhBMP-2 release and provide a microenvironment for intensifying the interaction of rhBMP-2 with the cells. Hence, the cell osteogenic differentiation can be enhanced by hierarchical porous bioglass films through both the porous structure and rhBMP-2 induction. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. [Effect of NF-κB on the expression of interleukin-6 induced by lipopolysaccharides of Porphyromonas endodontalis in MC3T3-E1 cells].

    Science.gov (United States)

    Yu, Ya-qiong; Guo, Jia-jie; Qiu, Li-hong; Lv, You; Jia, Ge; Guo, Yan

    2013-08-01

    To investigate the effect of NF-κB signaling on the expression of interleukin-6(IL-6) induced by lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) in MC3T3-El cells. MC3T3-E1 cells were pretreated with BAY-117082 for 1 h, and then were treated with 10 mg/L P.e-LPS for different times. The translocation of NF-κB was observed by immunofluorescence. The expression of IL-6 was detected by reverse transcription polymerse chain reaction (RT-PCR) and enzyme-linked immuno sorbent assay (ELISA). Statistical analysis was performed using multi-way ANOVA and Dunnett's t test with SPSS 13.0 software package. The staining of NF-κB was mostly in cytoplasm in untreated cells. Rapid translocation of NF-κB into nucleus was observed in the cells stimulated for 30 min and mostly relocalization of NF-κB from nucleus to cytoplasm was observed after 60 min. Pretreatment with 10 μmol/L BAY-117082 for 1h significantly inhibited P.e-LPS-induced translocation of NF-κB .The mRNA and proteins of IL-6 decreased significantly after pretreatment with 10 μmol/L BAY-117082 and the expression of IL-6 proteins was reduced from (774.983±6.585) ng/L to (377.384±14.620) ng/L (P<0.01). The group of treatment with BAY-117082 alone had no significant difference from the blank control group. P.e-LPS can induce translocation of NF-κB in mouse osteoblast MC3T3-El, and P.e-LPS may induce the expression of IL-6 in mouse osteoblast through the signaling of NF-κB.

  6. Oscillatory fluid flow elicits changes in morphology, cytoskeleton and integrin-associated molecules in MLO-Y4 cells, but not in MC3T3-E1 cells

    Directory of Open Access Journals (Sweden)

    Huiyun Xu

    2012-01-01

    Full Text Available Interstitial fluid flow stress is one of the most important mechanical stimulations of bone cells under physiological conditions. Osteocytes and osteoblasts act as primary mechanosensors within bones, and in vitro are able to respond to fluid shear stress, both morphologically and functionally. However, there is little information about the response of integrin-associated molecules using both osteoblasts and osteocytes. In this study, we investigated the changes in response to 2 hours of oscillatory fluid flow stress in the MLO-Y4 osteocyte-like cell line and the MC3T3-E1 osteoblast-like cell line. MLO-Y4 cells exhibited a significant increase in the expression of integrin-associated molecules, including OPN, CD44, vinculin and integrin avp3. However, there was no or limited increase observed in MC3T3-E1 osteoblast-like cells. Cell area and fiber stress formation were also markedly promoted by fluid flow only in MLO-Y4 cells. But the numbers of processes per cell remain unaffected in both cell lines.

  7. [The effects of platelet-rich fibrin extract on MC3T3-E1 cells cultured on the titanium discs].

    Science.gov (United States)

    Zhang, X J; Xu, S; Meng, W Y; Xiao, H J; Dong, K; Liu, Z H

    2017-01-09

    Objective: To evaluate the effect of platelet-rich fibrin extract (PRFe) on the adhesion, proliferation and differentiation of MC3T3-E1 cells cultured on the titanium discs. Methods: Samples were divided into experimental group (P) and control group (D). Group P used the α-minimal essential medium (α-MEM) containing PRFe (0.5%), while group D used only the α-MEM. Cell adhesion and cytoskeleton were observed using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). Methyl thiazolyl tetrazolium (MTT) assay to detect the number of the osteoblasts at 1, 3, 5, 7 d; the activity of alkaline phosphatase (ALP) to detect the differentiation of osteoblast at 1, 3, 5, 7 d; the level of osteogenetic biomarkers core-binding factorα1 (cbfα1) and osteocalcin (OCN) were quantified by quantitative real-time PCR (qRT-PCR) at 3 and 7 d. Results: SEM and LSCM showed that the adhesion and filaments of group P were higher than those of group D at each time point. MTT assay showed that the absorbance were significantly increased in group P (1 d: 0.299±0.002, 3 d: 0.517±0.004, 5 d: 0.810±0.002, 7 d: 1.203±0.011) compared with group D (1 d: 0.198±0.003, 3 d: 0.399±0.002, 5 d: 0.588±0.002, 7 d: 0.897±0.005) at each time points ( P< 0.05). Furthermore, the ALP activity of group P (1 d: 0.162±0.004, 3 d: 0.289±0.001, 5 d: 0.491±0.006, 7 d: 0.647±0.005) was significantly higher than that of group D (1 d: 0.121±0.003, 3 d: 0.191± 0.006, 5 d: 0.252±0.004, 7 d: 0.365±0.012), ( P< 0.05). Moreover, the qRT-PCR showed that the Cbfα1 and OCN gene expression in group P (Cfbα1, 3 d: 1.50±0.04, 7 d: 1.94±0.06; OCN, 3 d: 3.37±0.17, 7 d: 3.92± 0.04) were significantly higher than that in group D(Cfbα1, 3 d: 1, 7 d: 1.18±0.13; OCN, 3 d: 1, 7 d: 2.34± 0.09) ( P< 0.05). Conclusions: PRFe promoted the adhension, proliferation and differentiation of MC3T3-E1 cells on the titanium discs.

  8. Icaritin induces MC3T3-E1 subclone14 cell differentiation through estrogen receptor-mediated ERK1/2 and p38 signaling activation.

    Science.gov (United States)

    Wu, Zhidi; Ou, Ling; Wang, Chaopeng; Yang, Li; Wang, Panpan; Liu, Hengrui; Xiong, Yingquan; Sun, Kehuan; Zhang, Ronghua; Zhu, Xiaofeng

    2017-10-01

    Icaritin (ICT), a hydrolytic product of icariin from the genus Epimedium, has many indicated pharmacological and biological activities. Several studies have shown that ICT has potential osteoprotective effects, including stimulation of osteoblast differentiation and inhibition of osteoclast differentiation. However, the molecular mechanism for this anabolic action of ICT remains largely unknown. Here, we found that ICT could enhance MC3T3-E1 subclone 14 preosteoblastic cell differentiation associated with increased mRNA levels and protein expression of the differentiation markers alkaline phosphatase (ALP), type 1 collagen (COL1), osteocalcin (OC), osteoponin (OPN) and runt-related transcription factor 2 (RUNX2), and improved mineralization, confirmed by bone nodule formation and collagen synthesis. To characterize the underlying mechanisms, we examined the effect of ICT on estrogen receptor (ER) and mitogen-activated protein kinase (MAPK) signaling. ICT treatment induced p38 kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) activation, but it demonstrated at the same time point no effect on activation of c-Jun N-terminal kinase (JNK). ER antagonist ICI182780, p38 antagonist SB203580 and ERK1/2 antagonist PD98059 markedly inhibited the ICT-induced the mRNA expression of ALP, COL1, OC and OPN. ICI182780 attenuated the ICT-induced phosphorylation of p38 and ERK1/2. These observations indicate a potential mechanism of osteogenic effects of ICT involving the ERK1/2 and p38 pathway activation through the ER. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  9. 7-ketocholesterol induces apoptosis of MC3T3-E1 cells associated with reactive oxygen species generation, endoplasmic reticulum stress and caspase-3/7 dependent pathway

    Directory of Open Access Journals (Sweden)

    Yuta Sato

    2017-03-01

    Full Text Available Type 2 diabetes mellitus (T2DM is associated with an increased risk of bone fractures without reduction of bone mineral density. The cholesterol oxide 7-ketocholesterol (7KCHO has been implicated in numerous diseases such as atherosclerosis, Alzheimer's disease, Parkinson's disease, cancer, age-related macular degeneration and T2DM. In the present study, 7KCHO decreased the viability of MC3T3-E1 cells, increased reactive oxygen species (ROS production and apoptotic rate, and upregulated the caspase-3/7 pathway. Furthermore, these effects of 7KCHO were abolished by pre-incubation of the cells with N-acetylcysteine (NAC, an ROS inhibitor. Also, 7KCHO enhanced the mRNA expression of two endoplasmic reticulum (ER stress markers; CHOP and GRP78, in MC3T3-E1 cells. Pre-incubation of the cells with NAC suppressed the 7KCHO-induced upregulation of CHOP, but not GRP78. In conclusion, we demonstrated that 7KCHO induced apoptosis of MC3T3-E1 cells associated with ROS generation, ER stress, and caspase-3/7 activity, and the effects of 7KCHO were abolished by the ROS inhibitor NAC. These findings may provide new insight into the relationship between oxysterol and pathophysiology of osteoporosis seen in T2DM.

  10. Titania nanotube delivery fetal bovine serum for enhancing MC3T3-E1 activity and osteogenic gene expression

    International Nuclear Information System (INIS)

    Peng, Jing; Zhang, Xinming; Li, Zhaoyang; Liu, Yunde; Yang, Xianjin

    2015-01-01

    Titania nanotube (TNT) delivery of fetal bovine serum (FBS) was conducted on titanium (Ti) to enhance bone tissue repair. Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) showed FBS increased the tube wall thickness and decreased the tube diameter. Attenuated total reflectance Fourier transform infrared further confirmed that FBS completely covered the TNT and changed the surface composition. Water contact angle tests showed TNT/FBS possessed hydrophilic properties. Compared to original Ti, the TNT/FBS group had more attached osteoblasts after 2 h and enhanced filopodia growth at 0.5 h. Significantly, more osteoblasts were also observed on TNT/FBS after 7 d culturing. FBS was released steadily from TNT; about 70% of FBS had been released at 3 d and 90% at 5 d, as shown by the bicinchoninic acid method. TNT/FBS also enhanced subsequent osteoblast differentiation and gene expression; the quantum real-time polymerase chain reaction test showed that TNT/FBS up-regulated alkaline phosphatase and osteocalcin gene expression at 7 d and 14 d. Therefore, TNT/FBS delivered sustained in situ nutrition and enhanced osteoblast activity and osteogenic gene expression. - Highlights: • Fetal Bovine Serum (FBS) was filled in titania nanotube (TNT) structures. • FBS provided sustained-release in situ nutrition for surface osteoblast growth. • TNT/FBS enhanced osteoblast activity and osteogenic gene expression

  11. Titania nanotube delivery fetal bovine serum for enhancing MC3T3-E1 activity and osteogenic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Jing, E-mail: pengjingtd@163.com [Airport College, Civil Aviation University of China, Tianjin 300300 (China); Zhang, Xinming, E-mail: xinmingmail@163.com [Tianjin Product Quality Inspection Technology Research Institute, Tianjin 300384 (China); School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Li, Zhaoyang [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Liu, Yunde [School of Medical Laboratory, Tianjin Medical University, Tianjin 300203 (China); Yang, Xianjin [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China)

    2015-11-01

    Titania nanotube (TNT) delivery of fetal bovine serum (FBS) was conducted on titanium (Ti) to enhance bone tissue repair. Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) showed FBS increased the tube wall thickness and decreased the tube diameter. Attenuated total reflectance Fourier transform infrared further confirmed that FBS completely covered the TNT and changed the surface composition. Water contact angle tests showed TNT/FBS possessed hydrophilic properties. Compared to original Ti, the TNT/FBS group had more attached osteoblasts after 2 h and enhanced filopodia growth at 0.5 h. Significantly, more osteoblasts were also observed on TNT/FBS after 7 d culturing. FBS was released steadily from TNT; about 70% of FBS had been released at 3 d and 90% at 5 d, as shown by the bicinchoninic acid method. TNT/FBS also enhanced subsequent osteoblast differentiation and gene expression; the quantum real-time polymerase chain reaction test showed that TNT/FBS up-regulated alkaline phosphatase and osteocalcin gene expression at 7 d and 14 d. Therefore, TNT/FBS delivered sustained in situ nutrition and enhanced osteoblast activity and osteogenic gene expression. - Highlights: • Fetal Bovine Serum (FBS) was filled in titania nanotube (TNT) structures. • FBS provided sustained-release in situ nutrition for surface osteoblast growth. • TNT/FBS enhanced osteoblast activity and osteogenic gene expression.

  12. Ghrelin protects against depleted uranium-induced apoptosis of MC3T3-E1 cells through oxidative stress-mediated p38-mitogen-activated protein kinase pathway

    Energy Technology Data Exchange (ETDEWEB)

    Hao, Yuhui; Liu, Cong; Huang, Jiawei; Gu, Ying; Li, Hong; Yang, Zhangyou; Liu, Jing [State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, No. 30 Gaotanyan Street, Shapingba District, Chongqing 400038 (China); Wang, Weidong, E-mail: wwdwyl@sina.com [Department of Radiation Oncology, Shanghai Jiao Tong University Affiliated Sixth People Hospital, Shanghai 200233 (China); Li, Rong, E-mail: yuhui_hao@126.com [State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, No. 30 Gaotanyan Street, Shapingba District, Chongqing 400038 (China)

    2016-01-01

    Depleted uranium (DU) mainly accumulates in the bone over the long term. Osteoblast cells are responsible for the formation of bone, and they are sensitive to DU damage. However, studies investigating methods of reducing DU damage in osteoblasts are rarely reported. Ghrelin is a stomach hormone that stimulates growth hormones released from the hypothalamic–pituitary axis, and it is believed to play an important physiological role in bone metabolism. This study evaluates the impact of ghrelin on DU-induced apoptosis of the osteoblast MC3T3-E1 and investigates its underlying mechanisms. The results show that ghrelin relieved the intracellular oxidative stress induced by DU, eliminated reactive oxygen species (ROS) and reduced lipid peroxidation by increasing intracellular GSH levels; in addition, ghrelin effectively suppressed apoptosis, enhanced mitochondrial membrane potential, and inhibited cytochrome c release and caspase-3 activation after DU exposure. Moreover, ghrelin significantly reduced the expression of DU-induced phosphorylated p38-mitogen-activated protein kinase (MAPK). A specific inhibitor (SB203580) or specific siRNA of p38-MAPK could significantly suppress DU-induced apoptosis and related signals, whereas ROS production was not affected. In addition, ghrelin receptor inhibition could reduce the anti-apoptosis effect of ghrelin on DU and reverse the effect of ghrelin on intracellular ROS and p38-MAPK after DU exposure. These results suggest that ghrelin can suppress DU-induced apoptosis of MC3T3-E1 cells, reduce DU-induced oxidative stress by interacting with its receptor, and inhibit downstream p38-MAPK activation, thereby suppressing the mitochondrial-dependent apoptosis pathway. - Highlights: • Ghrelin suppressed DU-induced apoptosis of MC3T3-E1 cells. • Ghrelin inhibited DU-induced oxidative stress and further p38-MAPK activation. • Ghrelin further suppressed mitochondrial-dependent apoptosis pathway. • The anti-oxidation effect of

  13. Effect of surface topography and bioactive properties on early adhesion and growth behavior of mouse preosteoblast MC3T3-E1 cells.

    Science.gov (United States)

    Li, Na; Chen, Gang; Liu, Jue; Xia, Yang; Chen, Hanbang; Tang, Hui; Zhang, Feimin; Gu, Ning

    2014-10-08

    The effects of bioactive properties and surface topography of biomaterials on the adhesion and spreading properties of mouse preosteoblast MC3T3-E1 cells was investigated by preparation of different surfaces. Poly lactic-co-glycolic acid (PLGA) electrospun fibers (ES) were produced as a porous rough surface. In our study, coverslips were used as a substrate for the immobilization of 3,4-dihydroxyphenylalanine (DOPA) and collagen type I (COL I) in the preparation of bioactive surfaces. In addition, COL I was immobilized onto porous electrospun fibers surfaces (E-COL) to investigate the combined effects of bioactive molecules and topography. Untreated coverslips were used as controls. Early adhesion and growth behavior of MC3T3-E1 cells cultured on the different surfaces were studied at 6, 12, and 24 h. Evaluation of cell adhesion and morphological changes showed that the all the surfaces were favorable for promoting the adhesion and spreading of cells. CCK-8 assays and flow cytometry revealed that both topography and bioactive properties were favorable for cell growth. Analysis of β1, α1, α2, α5, α10 and α11 integrin expression levels by immunofluorescence, real-time RT-PCR, and Western blot and indicated that surface topography plays an important role in the early stage of cell adhesion. However, the influence of topography and bioactive properties of surfaces on integrins is variable. Compared with any of the topographic or bioactive properties in isolation, the combined effect of both types of properties provided an advantage for the growth and spreading of MC3T3-E1 cells. This study provides a new insight into the functions and effects of topographic and bioactive modifications of surfaces at the interface between cells and biomaterials for tissue engineering.

  14. MC3T3-E1 cell response of amorphous phase/TiO{sub 2} nanocrystal composite coating prepared by microarc oxidation on titanium

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Rui [Department of Materials Science and Engineering, Harbin Institute of Technology, Harbin 150001 (China); Wei, Daqing, E-mail: daqingwei@hit.edu.cn [Department of Materials Science and Engineering, Harbin Institute of Technology, Harbin 150001 (China); Yang, Haoyue; Feng, Wei [Department of Materials Science and Engineering, Harbin Institute of Technology, Harbin 150001 (China); Cheng, Su [Department of Mechanical Engineering, School of Architecture and Civil Engineering, Harbin University of Science and Technology, Harbin 150001 (China); Li, Baoqiang; Wang, Yaming; Jia, Dechang; Zhou, Yu [Department of Materials Science and Engineering, Harbin Institute of Technology, Harbin 150001 (China)

    2014-06-01

    Bioactive amorphous phase/TiO{sub 2} nanocrystal (APTN) composite coatings were fabricated by microarc oxidation (MAO) on Ti. The APTN coatings are composed of much amorphous phase with Si, Na, Ca, Ti and O elements and a few TiO{sub 2} nanocrystals. With increasing applied voltage, the micropore density of the APTN coating decreases and the micropore size of the APTN coating increases. The results indicate that less MC3T3-E1 cells attach on the APTN coatings as compared to Ti. However, the APTN coatings greatly enhance the cell proliferation ability and the activity of alkaline phosphatase. The amorphous phase and the concentrations of the released Ca and Si from the APTN coatings during cell culture have significant effects on the cell response. - Highlights: • Amorphous phase/TiO2 nanocrystal (APTN) composite coatings were fabricated. • The MC3T3-E1 cell response of the APTN coatings was evaluated. • The APTN coatings greatly enhanced the cell proliferation ability.

  15. MC3T3-E1 proliferation and differentiation on biphasic mixtures of Mg substituted β-tricalcium phosphate and amorphous calcium phosphate

    International Nuclear Information System (INIS)

    Singh, Satish S.; Roy, Abhijit; Lee, Boeun E.; Banerjee, Ipsita; Kumta, Prashant N.

    2014-01-01

    A low temperature aqueous approach was used to synthesize nanocrystalline, high surface area Mg 2+ substituted β-tricalcium phosphate (β-TCMP) to assess its potential use as a synthetic bone graft substitute. X-ray diffraction indicated that β-TCMP was the predominant crystalline phase formed. However, thermal analysis revealed the presence of a secondary amorphous phase which increased with increasing Mg 2+ concentration. Further analysis by Rietveld refinement indicated that the level of ionic substitution of Ca 2+ by Mg 2+ was significantly lower than the amount of Mg 2+ measured using elemental analysis, confirming the formation of a Mg 2+ rich secondary amorphous phase. MC3T3-E1 proliferation on substrates prepared using β-TCMP was assessed using the MTT assay. In comparison to commercially available β-TCP, increased proliferation was observed on samples prepared with 50% Mg, despite elevated Mg 2+ and PO 4 3− concentrations in culture media. Alkaline phosphatase (ALP) activity and qRT-PCR were used to study the effect of varying Mg 2+ substitution on osteogenic differentiation. Cells cultured on β-TCMP substrates prepared with increased Mg 2+ concentrations expressed significantly increased levels of ALP activity and osteogenic genes such as, osteocalcin, collagen-1, and Runx2, in comparison to those cultured on commercially available β-TCP

  16. Influence of MC3T3-E1 preosteoblast culture on the corrosion of a T6-treated AZ91 alloy

    Science.gov (United States)

    Brooks, Emily K.; Tobias, Menachem E.; Yang, Shuying; Bone, Lawrence B.; Ehrensberger, Mark T.

    2015-01-01

    This study investigated the corrosion of artificially aged T6 heat-treated Mg-9%Al-1%Zn (AZ91) for biomedical applications. Corrosion tests and surface analysis were completed both with and without a monolayer of mouse preosteoblast MC3T3-E1 cells cultured on the sample. Electrochemical impedance spectroscopy (EIS) and inductively coupled plasma mass spectroscopy (ICPMS) were used to explore the corrosion processes after either 3 or 21 days of AZ91 incubation in cell culture medium (CCM). The EIS showed both the inner layer resistance (Rin) and outer layer resistance (Rout) were lower for samples without cells cultured on the surface at 3 days (Rin = 2.64 e4 Ω/cm2, Rout = 140 Ω/cm2) compared to 21 days (Rin = 3.60 e4 Ω/cm2, Rout = 287 Ω/cm2) due to precipitation of magnesium and calcium phosphates over time. Samples with preosteoblasts cultured on the surface had a slower initial corrosion (3 day, Rin = 1.88 e5 Ω/cm2, Rout = 1060 Ω/cm2) which was observed to increase over time (21 day, Rin = 2.99 e4 Ω/cm2, Rout = 287 Ω/cm2). Changes in the corrosion processes were thought to be related to changes in the coverage provided by the cell layer. Our results reveal that the presence of cells and biological processes are able to significantly influence the corrosion rate of AZ91. PMID:25715925

  17. Structure, MC3T3-E1 cell response, and osseointegration of macroporous titanium implants covered by a bioactive microarc oxidation coating with microporous structure.

    Science.gov (United States)

    Zhou, Rui; Wei, Daqing; Cheng, Su; Feng, Wei; Du, Qing; Yang, Haoyue; Li, Baoqiang; Wang, Yaming; Jia, Dechang; Zhou, Yu

    2014-04-09

    Macroporous Ti with macropores of 50-400 μm size is prepared by sintering Ti microbeads with different diameters of 100, 200, 400, and 600 μm. Bioactive microarc oxidation (MAO) coatings with micropores of 2-5 μm size are prepared on the macroporous Ti. The MAO coatings are composed of a few TiO2 nanocrystals and lots of amorphous phases with Si, Ca, Ti, Na, and O elements. Compared to compact Ti, the MC3T3-E1 cell attachment is prolonged on macroporous Ti without and with MAO coatings; however, the cell proliferation number increases. These results are contributed to the effects of the space structure of macroporous Ti and the surface chemical feature and element dissolution of the MAO coatings during the cell culture. Macroporous Ti both without and with MAO coatings does not cause any adverse effects in vivo. The new bone grows well into the macropores and micropores of macroporous Ti with MAO coatings, showing good mechanical properties in vivo compared to Ti, MAO-treated Ti, and macroporous Ti because of its excellent osseointegration. Moreover, the MAO coatings not only show a high interface bonding strength with new bones but also connect well with macroporous Ti. Furthermore, the pushing out force for macroporous Ti with MAO coatings increases significantly with increasing microbead diameter.

  18. From 2D to 3D: The morphology, proliferation and differentiation of MC3T3-E1 on silk fibroin/chitosan matrices.

    Science.gov (United States)

    Li, Da-Wei; He, Feng-Li; He, Jin; Deng, Xudong; Liu, Ya-Li; Liu, Yang-Yang; Ye, Ya-Jing; Yin, Da-Chuan

    2017-12-15

    It has been widely accepted that cell culture in two-dimensional (2D) conditions may not be able to represent growth in three-dimensional (3D) conditions. Systematic comparisons between 2D and 3D cell cultures are needed to appropriately use the existing 2D results. In this work, we conducted a comparative study between 2D and 3D cell cultures of MC3T3-E1 using the same type of material (a mixture of silk fibroin (SF) and chitosan (CS)). Our results showed 3D SF/CS scaffold exhibited different effects on cell culture compared with the 2D cases. 1) The cells grown in 3D scaffold showed multiple morphologies. 2) The proliferation of cells in 3D scaffold was long-term and sustainable. 3) Cell differentiation occurred throughout the entire 3D scaffold. The results showed that cell culture in 3D SF/CS scaffold exhibited different features than 2D cases and 3D SF/CS scaffold could be a promising material for 3D cell culture. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Influence of MC3T3-E1 preosteoblast culture on the corrosion of a T6-treated AZ91 alloy.

    Science.gov (United States)

    Brooks, Emily K; Tobias, Menachem E; Yang, Shuying; Bone, Lawrence B; Ehrensberger, Mark T

    2016-02-01

    This study investigated the corrosion of artificially aged T6 heat-treated Mg-9%Al-1%Zn (AZ91) for biomedical applications. Corrosion tests and surface analysis were completed both with and without a monolayer of mouse preosteoblast MC3T3-E1 cells cultured on the sample. Electrochemical impedance spectroscopy (EIS) and inductively coupled plasma mass spectroscopy (ICPMS) were used to explore the corrosion processes after either 3 or 21 days of AZ91 incubation in cell culture medium (CCM). The EIS showed both the inner layer resistance (Rin ) and outer layer resistance (Rout ) were lower for samples without cells cultured on the surface at 3 days (Rin  = 2.64 e4 Ω/cm(2) , Rout  = 140 Ω/cm(2) ) compared to 21 days (Rin  = 3.60 e4 Ω/cm(2) , Rout  = 287 Ω/cm(2) ) due to precipitation of magnesium and calcium phosphates over time. Samples with preosteoblasts cultured on the surface had a slower initial corrosion (3 day, Rin  = 1.88 e5 Ω/cm(2) , Rout  = 1060 Ω/cm(2) ) which was observed to increase over time (21 day, Rin  = 2.99 e4 Ω/cm(2) , Rout  = 287 Ω/cm(2) ). Changes in the corrosion processes were thought to be related to changes in the coverage provided by the cell layer. Our results reveal that the presence of cells and biological processes are able to significantly influence the corrosion rate of AZ91. © 2015 Wiley Periodicals, Inc.

  20. Magnetically actuated mechanical stimuli on Fe3O4/mineralized collagen coatings to enhance osteogenic differentiation of the MC3T3-E1 cells.

    Science.gov (United States)

    Zhuang, Junjun; Lin, Suya; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

    2018-04-15

    Mechanical stimuli at the bone-implant interface are considered to activate the mechanotransduction pathway of the cell to improve the initial osseointegration establishment and to guarantee clinical success of the implant. However, control of the mechanical stimuli at the bone-implant interface still remains a challenge. In this study, we have designed a strategy of a magnetically responsive coating on which the mechanical stimuli is controlled because of coating deformation under static magnetic field (SMF). The iron oxide nanoparticle/mineralized collagen (IOP-MC) coatings were electrochemically codeposited on titanium substrates in different quantities of IOPs and distributions; the resulting coatings were verified to possess swelling behavior with flexibility same as that of hydrogel. The relative quantity of IOP to collagen and the IOP distribution in the coatings were demonstrated to play a critical role in mediating cell behavior. The cells present on the outer layer of the distributed IOP-MC (O-IOP-MC) coating with a mass ratio of 0.67 revealed the most distinct osteogenic differentiation activity being promoted, which could be attributed to the maximized mechanical stimuli with exposure to SMF. Furthermore, the enhanced osteogenic differentiation of the stimulated MC3T3-E1 cells originated from magnetically actuated mechanotransduction signaling pathway, embodying the upregulated expression of osteogenic-related and mechanotransduction-related genes. This work therefore provides a promising strategy for implementing mechanical stimuli to activate mechanotransduction on the bone-implant interface and thus to promote osseointegration. The magnetically actuated coating is designed to produce mechanical stimuli to cells for promoting osteogenic differentiation based on the coating deformation. Iron oxide nanoparticles (IOPs) were incorporated into the mineralized collagen coatings (MC) forming the composite coatings (IOP-MC) with spatially distributed IOPs

  1. Fluid shear stress suppresses TNF-α-induced apoptosis in MC3T3-E1 cells: Involvement of ERK5-AKT-FoxO3a-Bim/FasL signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Bin, Geng; Bo, Zhang; Jing, Wang; Jin, Jiang; Xiaoyi, Tan; Cong, Chen; Liping, An; Jinglin, Ma; Cuifang, Wang; Yonggang, Chen [The Second Hospital of Lanzhou University, #82 Cuiyingmen, Lanzhou, 730000 Gansu (China); Orthopaedics Key Laboratory of Gansu Province, Lanzhou, 730000 Gansu (China); Yayi, Xia, E-mail: xiayayildey@163.com [The Second Hospital of Lanzhou University, #82 Cuiyingmen, Lanzhou, 730000 Gansu (China); Orthopaedics Key Laboratory of Gansu Province, Lanzhou, 730000 Gansu (China)

    2016-05-01

    TNF-α is known to induce osteoblasts apoptosis, whereas mechanical stimulation has been shown to enhance osteoblast survival. In the present study, we found that mechanical stimulation in the form of fluid shear stress (FSS) suppresses TNF-α induced apoptosis in MC3T3-E1 cells. Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family that has been implicated in cell survival. We also demonstrated that FSS imposed by flow chamber in vitro leads to a markedly activation of ERK5, which was shown to be protective against TNF-α-induced apoptosis, whereas the transfection of siRNA against ERK5 (ERK5-siRNA) reversed the FSS-medicated anti-apoptotic effects. An initial FSS-mediated activation of ERK5 that phosphorylates AKT to increase its activity, and a following forkhead box O 3a (FoxO3a) was phosphorylated by activated AKT. Phosphorylated FoxO3a is sequestered in the cytoplasm, and prevents it from translocating to nucleus where it can increase the expression of FasL and Bim. The inhibition of AKT-FoxO3a signalings by a PI3K (PI3-kinase)/AKT inhibitor (LY294002) or the transfection of ERK5-siRNA led to the nuclear translocation of non-phosphorylated FoxO3a, and increased the protein expression of FasL and Bim. In addition, the activation of caspase-3 by TNF-α was significantly inhibited by aforementioned FSS-medicated mechanisms. In brief, the activation of ERK5-AKT-FoxO3a signaling pathways by FSS resulted in a decreased expression of FasL and Bim and an inhibition of caspase-3 activation, which exerts a protective effect that prevents osteoblasts from apoptosis. - Highlights: • Fluid shear stress inhibits osteoblast apoptosis induced by TNF-α. • Inhibition of ERK5 activity by transfection of ERK5 siRNA blocks FSS-mediated anti-apoptotic effect in osteoblast. • Activated ERK5-AKT-FoxO3a-Bim/FasL signaling pathways by FSS is required to protect osteoblast from apoptosis.

  2. Proliferation and differentiation of osteoblast-like MC3T3-E1 cells ons biomimetically and electrolytically deposited calcium phosphate coatings

    NARCIS (Netherlands)

    Wang, J.; de Boer, Jan; de Groot, K.

    2009-01-01

    Biomimetic and electrolytic deposition are versatile methods to prepare calcium phosphate coatings. In this article, we compared the effects of biomimetically deposited octacalcium phosphate and carbonate apatite coatings as well as electrolytically deposited carbonate apatite coating on the

  3. Effect of copper-doped silicate 13–93 bioactive glass scaffolds on the response of MC3T3-E1 cells in vitro and on bone regeneration and angiogenesis in rat calvarial defects in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yinan; Xiao, Wei [Department of Materials Science and Engineering, Missouri University of Science and Technology, Rolla, MO 65409 (United States); Bal, B. Sonny [Department of Orthopaedic Surgery, University of Missouri, Columbia, MO 65212 (United States); Rahaman, Mohamed N., E-mail: rahaman@mst.edu [Department of Materials Science and Engineering, Missouri University of Science and Technology, Rolla, MO 65409 (United States)

    2016-10-01

    The release of inorganic ions from biomaterials could provide an alternative approach to the use of growth factors for improving tissue healing. In the present study, the release of copper (Cu) ions from bioactive silicate (13–93) glass scaffolds on the response of cells in vitro and on bone regeneration and angiogenesis in vivo was studied. Scaffolds doped with varying concentrations of Cu (0–2.0 wt.% CuO) were created with a grid-like microstructure by robotic deposition. When immersed in simulated body fluid in vitro, the Cu-doped scaffolds released Cu ions into the medium in a dose-dependent manner and converted partially to hydroxyapatite. The proliferation and alkaline phosphatase activity of pre-osteoblastic MC3T3-E1 cells cultured on the scaffolds were not affected by 0.4 and 0.8 wt.% CuO in the glass but they were significantly reduced by 2.0 wt.% CuO. The percent new bone that infiltrated the scaffolds implanted for 6 weeks in rat calvarial defects (46 ± 8%) was not significantly affected by 0.4 or 0.8 wt.% CuO in the glass whereas it was significantly inhibited (0.8 ± 0.7%) in the scaffolds doped with 2.0 wt.% CuO. The area of new blood vessels in the fibrous tissue that infiltrated the scaffolds increased with CuO content of the glass and was significantly higher for the scaffolds doped with 2.0 wt.% CuO. Loading the scaffolds with bone morphogenetic protein-2 (1 μg/defect) significantly enhanced bone infiltration and reduced fibrous tissue in the scaffolds. These results showed that doping the 13–93 glass scaffolds with up to 0.8 wt.% CuO did not affect their biocompatibility whereas 2.0 wt.% CuO was toxic to cells and detrimental to bone regeneration. - Highlights: • First study to evaluate Cu ion release from silicate (13-93) bioactive glass scaffolds on osteogenesis in vivo • Released Cu ions influenced bone regeneration in a dose dependent manner • Lower concentrations of Cu ions had little effect on bone regeneration • Cu ion

  4. Effect of copper-doped silicate 13–93 bioactive glass scaffolds on the response of MC3T3-E1 cells in vitro and on bone regeneration and angiogenesis in rat calvarial defects in vivo

    International Nuclear Information System (INIS)

    Lin, Yinan; Xiao, Wei; Bal, B. Sonny; Rahaman, Mohamed N.

    2016-01-01

    The release of inorganic ions from biomaterials could provide an alternative approach to the use of growth factors for improving tissue healing. In the present study, the release of copper (Cu) ions from bioactive silicate (13–93) glass scaffolds on the response of cells in vitro and on bone regeneration and angiogenesis in vivo was studied. Scaffolds doped with varying concentrations of Cu (0–2.0 wt.% CuO) were created with a grid-like microstructure by robotic deposition. When immersed in simulated body fluid in vitro, the Cu-doped scaffolds released Cu ions into the medium in a dose-dependent manner and converted partially to hydroxyapatite. The proliferation and alkaline phosphatase activity of pre-osteoblastic MC3T3-E1 cells cultured on the scaffolds were not affected by 0.4 and 0.8 wt.% CuO in the glass but they were significantly reduced by 2.0 wt.% CuO. The percent new bone that infiltrated the scaffolds implanted for 6 weeks in rat calvarial defects (46 ± 8%) was not significantly affected by 0.4 or 0.8 wt.% CuO in the glass whereas it was significantly inhibited (0.8 ± 0.7%) in the scaffolds doped with 2.0 wt.% CuO. The area of new blood vessels in the fibrous tissue that infiltrated the scaffolds increased with CuO content of the glass and was significantly higher for the scaffolds doped with 2.0 wt.% CuO. Loading the scaffolds with bone morphogenetic protein-2 (1 μg/defect) significantly enhanced bone infiltration and reduced fibrous tissue in the scaffolds. These results showed that doping the 13–93 glass scaffolds with up to 0.8 wt.% CuO did not affect their biocompatibility whereas 2.0 wt.% CuO was toxic to cells and detrimental to bone regeneration. - Highlights: • First study to evaluate Cu ion release from silicate (13-93) bioactive glass scaffolds on osteogenesis in vivo • Released Cu ions influenced bone regeneration in a dose dependent manner • Lower concentrations of Cu ions had little effect on bone regeneration • Cu ion

  5. An axial distribution of seeding, proliferation, and osteogenic differentiation of MC3T3-E1 cells and rat bone marrow-derived mesenchymal stem cells across a 3D Thai silk fibroin/gelatin/hydroxyapatite scaffold in a perfusion bioreactor

    Energy Technology Data Exchange (ETDEWEB)

    Sinlapabodin, Salita; Amornsudthiwat, Phakdee; Damrongsakkul, Siriporn; Kanokpanont, Sorada, E-mail: sorada.k@chula.ac.th

    2016-01-01

    In cell culture, a perfusion bioreactor provides effective transportation of nutrients, oxygen, and waste removal to and from the core of the scaffold. In addition, it provides mechanical stimuli for enhancing osteogenic differentiation. In this study, we used an axial distribution of cell numbers, alkaline phosphatase (ALP) enzyme activity, and calcium content across 4 cross-sections of 10 mm thick scaffold, made of Thai silk fibroin (SF)/gelatin (G)/hydroxyapatite (HA), as a tool to evaluate the suitable perfusion flow rate. These evaluations cover all cellular developmental phases starting from seeding, to proliferation, and later osteogenic differentiation. Mouse pre-osteoblastic MC3T3-E1 cell lines were used as a cell model during seeding and proliferation. The bioreactor seeded scaffold provided more uniform cell distribution across the scaffold compared to centrifugal and agitation seeding, while the overall number of adhered cells from bioreactor seeding was slightly lower than agitation seeding. The dynamic culture using 1 ml/min perfusion flow rate (initial shear stress of 0.1 dyn/cm{sup 2}) enabled statistically higher MC3T3-E1 proliferation, ALP activity, and calcium deposition than those observed in the static-culturing condition. However, the perfusion flow rate of 1 ml/min seemed not to be enough for enhancing ALP expression across all sections of the scaffold. Rat bone marrow derived stromal cells (rMSC) were used in the detachment test and osteogenic differentiation. It was found that perfusion flow rate of 5 ml/min caused statistically higher cell detachment than that of 1 and 3 ml/min. The perfusion flow rate of 3 ml/min gave the highest rMSC osteogenic differentiation on a SF/G/HA scaffold than other flow rates, as observed from the significantly highest number of ALP enzyme activity and the calcium content without any significant cell growth. In addition, all of these parameters were evenly distributed across all scaffold sections. - Highlights

  6. The effects of BIG-3 on osteoblast differentiation are not dependent upon endogenously produced BMPs

    International Nuclear Information System (INIS)

    Gori, Francesca; Demay, Marie B.

    2005-01-01

    BMPs play an important role in both intramembranous and endochondral ossification. BIG-3, BMP-2-induced gene 3 kb, encodes a WD-40 repeat protein that accelerates the program of osteoblastic differentiation in vitro. To examine the potential interactions between BIG-3 and the BMP-2 pathway during osteoblastic differentiation, MC3T3-E1 cells stably transfected with BIG-3 (MC3T3E1-BIG-3), or with the empty vector (MC3T3E1-EV), were treated with noggin. Noggin treatment of pooled MC3T3E1-EV clones inhibited the differentiation-dependent increase in AP activity observed in the untreated MC3T3E1-EV clones but did not affect the increase in AP activity in the MC3T3E1-BIG-3 clones. Noggin treatment decreased the expression of Runx2 and type I collagen mRNAs and impaired mineralized matrix formation in MC3T3E1-EV clones but not in MC3T3E1-BIG-3 clones. To determine whether the actions of BIG-3 on osteoblast differentiation converged upon the BMP pathway or involved an alternate signaling pathway, Smad1 phosphorylation was examined. Basal phosphorylation of Smad1 was not altered in the MC3T3E1-BIG-3 clones. However, these clones did not exhibit the noggin-dependent decrease in phosphoSmad1 observed in the MC3T3E1-EV clones, nor did it decrease nuclear localization of phosphoSmad1. These observations suggest that BIG-3 accelerates osteoblast differentiation in MC3T3-E1 cells by inducing phosphorylation and nuclear translocation of Smad1 independently of endogenously produced BMPs

  7. Activating AMP-activated protein kinase by an α1 selective activator compound 13 attenuates dexamethasone-induced osteoblast cell death

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Shiguang [Department of Intensive Care Unit, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Mao, Li [Department of Endocrinology, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Ji, Feng, E-mail: huaiaifengjidr@163.com [Department of Orthopedics, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Wang, Shouguo; Xie, Yue; Fei, Haodong [Department of Orthopedics, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Wang, Xiao-dong, E-mail: xiaodongwangsz@163.com [The Center of Diagnosis and Treatment for Children' s Bone Diseases, The Children' s Hospital Affiliated to Soochow University, Suzhou (China)

    2016-03-18

    Excessive glucocorticoid (GC) usage may lead to non-traumatic femoral head osteonecrosis. Dexamethasone (Dex) exerts cytotoxic effect to cultured osteoblasts. Here, we investigated the potential activity of Compound 13 (C13), a novel α1 selective AMP-activated protein kinase (AMPK) activator, against the process. Our data revealed that C13 pretreatment significantly attenuated Dex-induced apoptosis and necrosis in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. AMPK activation mediated C13′ cytoprotective effect in osteoblasts. The AMPK inhibitor Compound C, shRNA-mediated knockdown of AMPKα1, or dominant negative mutation of AMPKα1 (T172A) almost abolished C13-induced AMPK activation and its pro-survival effect in osteoblasts. On the other hand, forced AMPK activation by adding AMPK activator A-769662 or exogenous expression a constitutively-active (ca) AMPKα1 (T172D) mimicked C13's actions and inhibited Dex-induced osteoblast cell death. Meanwhile, A-769662 or ca-AMPKα1 almost nullified C13's activity in osteoblast. Further studies showed that C13 activated AMPK-dependent nicotinamide adenine dinucleotide phosphate (NADPH) pathway to inhibit Dex-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary murine osteoblasts. Such effects by C13 were almost reversed by Compound C or AMPKα1 depletion/mutation. Together, these results suggest that C13 alleviates Dex-induced osteoblast cell death via activating AMPK signaling pathway. - Highlights: • Compound 13 (C13) attenuates dexamethasone (Dex)-induced osteoblast cell death. • C13-induced cytoprotective effect against Dex in osteoblasts requires AMPK activation. • Forced AMPK activation protects osteoblasts from Dex, nullifying C13's activities. • C13 increases NADPH activity and inhibits Dex-induced oxidative stress in osteoblasts.

  8. Activating AMP-activated protein kinase by an α1 selective activator compound 13 attenuates dexamethasone-induced osteoblast cell death

    International Nuclear Information System (INIS)

    Guo, Shiguang; Mao, Li; Ji, Feng; Wang, Shouguo; Xie, Yue; Fei, Haodong; Wang, Xiao-dong

    2016-01-01

    Excessive glucocorticoid (GC) usage may lead to non-traumatic femoral head osteonecrosis. Dexamethasone (Dex) exerts cytotoxic effect to cultured osteoblasts. Here, we investigated the potential activity of Compound 13 (C13), a novel α1 selective AMP-activated protein kinase (AMPK) activator, against the process. Our data revealed that C13 pretreatment significantly attenuated Dex-induced apoptosis and necrosis in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. AMPK activation mediated C13′ cytoprotective effect in osteoblasts. The AMPK inhibitor Compound C, shRNA-mediated knockdown of AMPKα1, or dominant negative mutation of AMPKα1 (T172A) almost abolished C13-induced AMPK activation and its pro-survival effect in osteoblasts. On the other hand, forced AMPK activation by adding AMPK activator A-769662 or exogenous expression a constitutively-active (ca) AMPKα1 (T172D) mimicked C13's actions and inhibited Dex-induced osteoblast cell death. Meanwhile, A-769662 or ca-AMPKα1 almost nullified C13's activity in osteoblast. Further studies showed that C13 activated AMPK-dependent nicotinamide adenine dinucleotide phosphate (NADPH) pathway to inhibit Dex-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary murine osteoblasts. Such effects by C13 were almost reversed by Compound C or AMPKα1 depletion/mutation. Together, these results suggest that C13 alleviates Dex-induced osteoblast cell death via activating AMPK signaling pathway. - Highlights: • Compound 13 (C13) attenuates dexamethasone (Dex)-induced osteoblast cell death. • C13-induced cytoprotective effect against Dex in osteoblasts requires AMPK activation. • Forced AMPK activation protects osteoblasts from Dex, nullifying C13's activities. • C13 increases NADPH activity and inhibits Dex-induced oxidative stress in osteoblasts.

  9. [Inhibitory effects of Porphyromonas endodontalis lipopolysaccharides on proliferation and differentiation of mouse osteoblast].

    Science.gov (United States)

    Li, Ren; Qiu, Li-hong; Yang, Di; Xue, Ming; Zhong, Ming

    2011-03-01

    To observe the effect of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on osteoblast cell proliferation and the activity of alkaline phosphatase (ALP) and interleukin (IL)-6 secretion and to investigate the role of Pe-LPS in osteoblast proliferation and differentiation. MC3T3-E1 cells were treated with different concentrations of Pe-LPS (10, 25, 50 mg/L) respectively. The relative growth rate (RGR) was detected by methyl thiazolyl tetrazolium (MTT) at different time point (12, 24, 48, 72 h). MC3T3-E1 cells were also stimulated with 10, 25 or 50 mg/L Pe-LPS for 6, 12, 24 and 48 h. The activity of ALP was detected by enzyme kinetics assay and the secretion of IL-6 was detected by enzyme-linked immunosorbent assay (ELISA). After the stimulation with 25 or 50 mg/L Pe-LPS for 72 h, the RGR of MC3T3-E1 cells descend to 87.46% and 71.12%. The ALP activities of MC3T3-E1 cells were inhibited obviously (P < 0.05) after the stimulation of different concentrations (10, 25, 50 mg/L) Pe-LPS for more than 24 hours. ELISA result showed that IL-6 increased to 32.21 ng/L treated with the 25 mg/L Pe-LPS for 6 h, 25 mg/L Pe-LPS gradually increased the expression of IL-6 from the ELISA results. Pe-LPS can induce the secretion of IL-6 in MC3T3-E1 and decrease the ALP activities of MC3T3-E1, the differentiation of osteoblasts was inhibited. with the long-time toxicity action of Pe-LPS, the proliferation rate of MC3T3-E1 also markedly decreased.

  10. Bidirectional communication between sensory neurons and osteoblasts in an in vitro coculture system.

    Science.gov (United States)

    Kodama, Daisuke; Hirai, Takao; Kondo, Hisataka; Hamamura, Kazunori; Togari, Akifumi

    2017-02-01

    Recent studies have revealed that the sensory nervous system is involved in bone metabolism. However, the mechanism of communication between neurons and osteoblasts is yet to be elucidated. In this study, we investigated the signaling pathways between sensory neurons of the dorsal root ganglion (DRG) and the osteoblast-like MC3T3-E1 cells using an in vitro coculture system. Our findings indicate that signal transduction from DRG-derived neurons to MC3T3-E1 cells is suppressed by antagonists of the AMPA receptor and the NK 1 receptor. Conversely, signal transduction from MC3T3-E1 cells to DRG-derived neurons is suppressed by a P2X 7 receptor antagonist. Our results suggest that these cells communicate with each other by exocytosis of glutamate, substance P in the efferent signal, and ATP in the afferent signal. © 2017 Federation of European Biochemical Societies.

  11. Effect of fibronectin adsorption on osteoblastic cellular responses to hydroxyapatite and alumina

    Energy Technology Data Exchange (ETDEWEB)

    Kawashita, Masakazu, E-mail: m-kawa@ecei.tohoku.ac.jp [Graduate School of Biomedical Engineering, Tohoku University, Sendai 980-8579 (Japan); Hasegawa, Maki [Graduate School of Biomedical Engineering, Tohoku University, Sendai 980-8579 (Japan); Kudo, Tada-aki; Kanetaka, Hiroyasu [Graduate School of Dentistry, Tohoku University, Sendai 980-8575 (Japan); Miyazaki, Toshiki [Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196 (Japan); Hashimoto, Masami [Japan Fine Ceramics Center, Nagoya 456-8587 (Japan)

    2016-12-01

    Initial cellular responses following implantation are important for inducing osteoconduction. We investigated cell adhesion, spreading, proliferation and differentiation of mouse MC3T3-E1 osteoblastic cells on untreated or fibronectin (Fn)-coated discs of hydroxyapatite (HAp) or alpha-type alumina (α-Al{sub 2}O{sub 3}). Fn coating significantly enhanced adhesion and spreading of MC3T3-E1 cells on HAp, but did not affect MC3T3-E1 cell proliferation and differentiation on HAp or α-Al{sub 2}O{sub 3}. Fn-coated HAp likely does not stimulate pre-osteoblast cells to initiate the process of osteoconduction; however, Fn adsorption might affect the response of inflammatory cells to the implanted material or, in conjunction with other serum proteins, stimulate pre-osteoblast cell proliferation and differentiation. Further studies on the effect of serum proteins in cell culture and the efficacy of Fn-coated HAp and α-Al{sub 2}O{sub 3}in vivo are warranted. - Highlights: • We studied osteoblast-like MC3T3-E1 cell responses on fibronectin (Fn)-coated discs (HAp/α-Al{sub 2}O{sub 3}). • Fn adsorption enhanced adhesion and spreading of MC3T3-E1 cells on HAp but not on α-Al{sub 2}O{sub 3}. • Fn adsorption hardly affected proliferation and differentiation of MC3T3-E1 cells on HAp and α-Al{sub 2}O{sub 3}. • Fn adsorption might stimulate osteoconduction on HAp along with other serum proteins.

  12. Effect of fibronectin adsorption on osteoblastic cellular responses to hydroxyapatite and alumina

    International Nuclear Information System (INIS)

    Kawashita, Masakazu; Hasegawa, Maki; Kudo, Tada-aki; Kanetaka, Hiroyasu; Miyazaki, Toshiki; Hashimoto, Masami

    2016-01-01

    Initial cellular responses following implantation are important for inducing osteoconduction. We investigated cell adhesion, spreading, proliferation and differentiation of mouse MC3T3-E1 osteoblastic cells on untreated or fibronectin (Fn)-coated discs of hydroxyapatite (HAp) or alpha-type alumina (α-Al 2 O 3 ). Fn coating significantly enhanced adhesion and spreading of MC3T3-E1 cells on HAp, but did not affect MC3T3-E1 cell proliferation and differentiation on HAp or α-Al 2 O 3 . Fn-coated HAp likely does not stimulate pre-osteoblast cells to initiate the process of osteoconduction; however, Fn adsorption might affect the response of inflammatory cells to the implanted material or, in conjunction with other serum proteins, stimulate pre-osteoblast cell proliferation and differentiation. Further studies on the effect of serum proteins in cell culture and the efficacy of Fn-coated HAp and α-Al 2 O 3 in vivo are warranted. - Highlights: • We studied osteoblast-like MC3T3-E1 cell responses on fibronectin (Fn)-coated discs (HAp/α-Al 2 O 3 ). • Fn adsorption enhanced adhesion and spreading of MC3T3-E1 cells on HAp but not on α-Al 2 O 3 . • Fn adsorption hardly affected proliferation and differentiation of MC3T3-E1 cells on HAp and α-Al 2 O 3 . • Fn adsorption might stimulate osteoconduction on HAp along with other serum proteins.

  13. Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress

    Directory of Open Access Journals (Sweden)

    Barrio Daniel A

    2001-08-01

    Full Text Available Abstract Background The tissue accumulation of protein-bound advanced glycation endproducts (AGE may be involved in the etiology of diabetic chronic complications, including osteopenia. The aim of this study was to investigate the effect of an AGE-modified type I collagen substratum on the adhesion, spreading, proliferation and differentiation of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 osteoblastic cells. We also studied the role of reactive oxygen species (ROS and nitric oxide synthase (NOS expression on these AGE-collagen mediated effects. Results AGE-collagen decreased the adhesion of UMR106 cells, but had no effect on the attachment of MC3T3E1 cells. In the UMR106 cell line, AGE-collagen also inhibited cellular proliferation, spreading and alkaline phosphatase (ALP activity. In preosteoblastic MC3T3E1 cells (24-hour culture, proliferation and spreading were significantly increased by AGE-collagen. After one week of culture (differentiated MC3T3E1 osteoblasts AGE-collagen inhibited ALP activity, but had no effect on cell number. In mineralizing MC3T3E1 cells (3-week culture AGE-collagen induced a decrease in the number of surviving cells and of extracellular nodules of mineralization, without modifying their ALP activity. Intracellular ROS production, measured after a 48-hour culture, was decreased by AGE-collagen in MC3T3E1 cells, but was increased by AGE-collagen in UMR106 cells. After a 24-hour culture, AGE-collagen increased the expression of endothelial and inducible NOS, in both osteoblastic cell lines. Conclusions These results suggest that the accumulation of AGE on bone extracellular matrix could regulate the proliferation and differentiation of osteoblastic cells. These effects appear to depend on the stage of osteoblastic development, and possibly involve the modulation of NOS expression and intracellular ROS pathways.

  14. Induction of DNA-strand breaks after X- irradiation in murine bone cells of various differentiation capacities

    Science.gov (United States)

    Lau, P.; Hellweg, C. E.; Kirchner, S.; Arenz, A.; Baumstark-Khan, C.; Horneck, G.

    Bone loss resulting from long-duration space flight is a well known medical risk for space travellers, as a weakened skeleton is more susceptible to bone fractures. In addition to weightlessness the astronaut is also exposed to cosmic ionizing radiation. In order to elucidate changes in bone cell metabolism by ionizing radiation, a ground-based bone cell model has been developed. This model consists of a bunch of immortalized murine osteocyte, osteoblast and pre-osteoblast cell lines representing discrete stages of differentiation: The osteocyte cell line MLO-Y4 (obtained from L. Bonewald, Kansas City, USA), the osteoblast cell line OCT-1 (obtained from D. Chen, San Antonio, USA), and the subclones 4 and 24 of the osteoblast cell line MC3T3-E1 (obtained from ATCC, Manassas, Virginia, USA). Regarding their growth properties, MLO-Y4 cells show the highest growth velocity with a doubling time of 15.8 h. The osteoblast cell line OCT-1 has a doubling time of 27.3 h. The respective values for MC3T3-E1 subclone 24 and S4 are 90.5 h and 51.6 h. To investigate the stage of differentiation, the expression of alkaline phosphatase, of osteocalcin and of E11 was examined. Survival after X-ray exposure was determined using the colony forming ability test. The resulting dose-effect relationships revealed significant differences. The parameter D0 of the survival curves ranges between 1.8 Gy for OCT-1, 1.9 Gy for MLO-Y4, 2.0 Gy for subclone 24 and 2,3 Gy for subclone 4. The quantitative acquisition of DNA-strand breaks was performed by Fluorescent Analysis of DNA-Unwinding (FADU). The results can be correlated with the corresponding survival curve. In conclusion, the cell lines with higher differentiation levels are less sensitive to radiation when compared to the lower differentiated osteoblast cell lines.

  15. Affecting osteoblastic responses with in vivo engineered potato pectin fragments

    NARCIS (Netherlands)

    Kokkonen, H.; Verhoef, R.P.; Kauppinen, K.; Muhonen, V.; Jorgensen, B.; Damager, I.; Schols, H.A.; Morra, M.; Ulvskov, P.; Tuukkanen, J.

    2012-01-01

    Pectins, complex plant-derived polysaccharides, are novel candidates for biomaterial nanocoatings. Pectic rhamnogalacturonan-I regions (RG-I) can be enzymatically treated to so-called modified hairy regions (MHR). We surveyed the growth and differentiation of murine preosteoblastic MC3T3-E1 cells on

  16. Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

    International Nuclear Information System (INIS)

    Kihara, Tasuku; Ichikawa, Saki; Yonezawa, Takayuki; Lee, Ji-Won; Akihisa, Toshihiro; Woo, Je Tae; Michi, Yasuyuki; Amagasa, Teruo; Yamaguchi, Akira

    2011-01-01

    Research highlights: → Acerogenin A stimulated osteoblast differentiation in osteogenic cells. → Acerogenin A-induced osteoblast differentiation was inhibited by noggin. → Acerogenin A increased Bmp-2, Bmp-4 and Bmp-7 mRNA expression in MC3T3-E1 cells. → Acerogenin A is a candidate agent for stimulating bone formation. -- Abstract: We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3 days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.

  17. Plant-derived pectin nanocoatings to prevent inflammatory cellular response of osteoblasts following Porphyromonas gingivalis infection

    Directory of Open Access Journals (Sweden)

    Meresta A

    2017-01-01

    Full Text Available Anna Meresta,1 Justyna Folkert,1 Timo Gaber,2 Korneliusz Miksch,1 Frank Buttgereit,2 Jacqueline Detert,2 Nicole Pischon,3,* Katarzyna Gurzawska3,4,* 1Environmental Biotechnology Department, Faculty of Power and Environmental, Silesian University of Technology, Gliwice, Poland; 2Department of Rheumatology and Clinical Immunology, 3Department of Periodontology, Charité University Medicine, Berlin, Germany; 4Oral Surgery Department, The School of Dentistry, University of Birmingham, Birmingham, UK *These authors contributed equally to this work Background: Bioengineered plant-derived Rhamnogalacturonan-Is (RG-Is from pectins are potential candidates for surface nanocoating of medical devices. It has recently been reported that RG-I nanocoatings may prevent bacterial infection and improve the biocompatibility of implants. The aim of the study was to evaluate in vitro impact of bioengineered RG-I nanocoatings on osteogenic capacity and proinflammatory cytokine response of murine osteoblasts following Porphyromonas gingivalis infection.Methods: Murine MC3T3-E1 osteoblasts and isolated primary calvarial osteoblasts from C57BL/6J (B6J osteoblasts mice were infected with P. gingivalis and incubated on tissue culture polystyrene plates with or without nanocoatings of unmodified RG-Is isolated from potato pulps (PU or dearabinanated RG-Is (PA. To investigate a behavior of infected osteoblasts cultured on RG-Is cell morphology, proliferation, metabolic activity, mineralization and osteogenic and pro-inflammatory gene expression were examined.Results: Following P. gingivalis infection, PA, but not PU, significantly promoted MC3T3-E1 and BJ6 osteoblasts proliferation, metabolic activity, and calcium deposition. Moreover, Il-1b, Il-6, TNF-α, and Rankl gene expressions were downregulated in cells cultured on PU and to a higher extent on PA as compared to the corresponding control, whereas Runx, Alpl, Col1a1, and Bglap gene expressions were upregulated vice

  18. SIRT3/SOD2 maintains osteoblast differentiation and bone formation by regulating mitochondrial stress

    OpenAIRE

    Gao, Jing; Feng, Zhihui; Wang, Xueqiang; Zeng, Mengqi; Liu, Jing; Han, Shujun; Xu, Jie; Chen, Lei; Cao, Ke; Long, Jiangang; Li, Zongfang; Shen, Weili; Liu, Jiankang

    2017-01-01

    Recent studies have revealed robust metabolic changes during cell differentiation. Mitochondria, the organelles where many vital metabolic reactions occur, may play an important role. Here, we report the involvement of SIRT3-regulated mitochondrial stress in osteoblast differentiation and bone formation. In both the osteoblast cell line MC3T3-E1 and primary calvarial osteoblasts, robust mitochondrial biogenesis and supercomplex formation were observed during differentiation, accompanied by in...

  19. Use of green fluorescent fusion protein to track activation of the transcription factor osterix during early osteoblast differentiation

    International Nuclear Information System (INIS)

    Tai Guangping; Christodoulou, Ioannis; Bishop, Anne E.; Polak, Julia M.

    2005-01-01

    Osterix (Osx) is a transcription factor required for the differentiation of preosteoblasts into fully functioning osteoblasts. However, the pattern of Osx activation during preosteoblast differentiation and maturation has not been clearly defined. Our aim was to study Osx activation during these processes in osteoblasts differentiating from murine and human embryonic stem cells (ESC). To do this, we constructed an Osx-GFP fusion protein reporter system to track Osx translocation within the cells. The distribution of Osx-GFP at representative stages of differentiation was also investigated by screening primary osteoblasts, mesenchymal stem cells, synoviocytes, and pre-adipocytes. Our experiments revealed that Osx-GFP protein was detectable in the cytoplasm of cultured, differentiated ESC 4 days after plating of enzymatically dispersed embryoid bodies. Osterix-GFP protein became translocated into the nucleus on day 7 following transfer of differentiated ESC to osteogenic medium. After 14 days of differentiation, cells showing nuclear translocation of Osx-GFP formed rudimentary bone nodules that continued to increase in number over the following weeks (through day 21). We also found that Osx translocated into the nuclei of mesenchymal stem cells (C3H10T1/2) and pre-osteoblasts (MC3T3-E1) and showed partial activation in pre-adipocytes (MC3T3-L1). These data suggest that Osx activation occurs at a very early point in the differentiation of the mesenchymal-osteoblastic lineage

  20. Vitamin a is a negative regulator of osteoblast mineralization.

    Directory of Open Access Journals (Sweden)

    Thomas Lind

    Full Text Available An excessive intake of vitamin A has been associated with an increased risk of fractures in humans. In animals, a high vitamin A intake leads to a reduction of long bone diameter and spontaneous fractures. Studies in rodents indicate that the bone thinning is due to increased periosteal bone resorption and reduced radial growth. Whether the latter is a consequence of direct effects on bone or indirect effects on appetite and general growth is unknown. In this study we therefore used pair-feeding and dynamic histomorphometry to investigate the direct effect of a high intake of vitamin A on bone formation in rats. Although there were no differences in body weight or femur length compared to controls, there was an approximately halved bone formation and mineral apposition rate at the femur diaphysis of rats fed vitamin A. To try to clarify the mechanism(s behind this reduction, we treated primary human osteoblasts and a murine preosteoblastic cell line (MC3T3-E1 with the active metabolite of vitamin A; retinoic acid (RA, a retinoic acid receptor (RAR antagonist (AGN194310, and a Cyp26 inhibitor (R115866 which blocks endogenous RA catabolism. We found that RA, via RARs, suppressed in vitro mineralization. This was independent of a negative effect on osteoblast proliferation. Alkaline phosphatase and bone gamma carboxyglutamate protein (Bglap, Osteocalcin were drastically reduced in RA treated cells and RA also reduced the protein levels of Runx2 and Osterix, key transcription factors for progression to a mature osteoblast. Normal osteoblast differentiation involved up regulation of Cyp26b1, the major enzyme responsible for RA degradation, suggesting that a drop in RA signaling is required for osteogenesis analogous to what has been found for chondrogenesis. In addition, RA decreased Phex, an osteoblast/osteocyte protein necessary for mineralization. Taken together, our data indicate that vitamin A is a negative regulator of osteoblast mineralization.

  1. Low-intensity pulsed ultrasound regulates proliferation and differentiation of osteoblasts through osteocytes

    International Nuclear Information System (INIS)

    Li, Lei; Yang, Zheng; Zhang, Hai; Chen, Wenchuan; Chen, Mengshi; Zhu, Zhimin

    2012-01-01

    Highlights: ► CM from LIPUS-stimulated osteocytes inhibits proliferation of osteoblasts. ► CM from LIPUS-stimulated osteocytes enhances differentiation of osteoblasts. ► LIPUS stimulates MLO-Y4 cells to secrete PGE 2 and NO. -- Abstract: Low-intensity pulsed ultrasound (LIPUS) has been used as a safe and effective modality to enhance fracture healing. As the most abundant cells in bone, osteocytes orchestrate biological activities of effector cells via direct cell-to-cell contacts and by soluble factors. In this study, we have used the osteocytic MLO-Y4 cells to study the effects of conditioned medium from LIPUS-stimulated MLO-Y4 cells on proliferation and differentiation of osteoblastic MC3T3-E1 cells. Conditioned media from LIPUS-stimulated MLO-Y4 cells (LIPUS-Osteocyte-CM) were collected and added on MC3T3-E1 cell cultures. MC3T3-E1 cells cultured in LIPUS-Osteocyte-CM demonstrated a significant inhibition of proliferation and an increased alkaline phosphatase activity. The results of PGE 2 and NO assay showed that LIPUS could enhance PGE 2 and NO secretion from MLO-Y4 cells at all time points within 24 h after LIPUS stimulation. We conclude that LIPUS regulates proliferation and differentiation of osteoblasts through osteocytes in vitro. Increased secretion of PGE 2 from osteocytes may play a role in this effect.

  2. The Role of Osteoblast-Derived Cytokines in Bone Metastatic Breast Cancer

    Science.gov (United States)

    2009-01-01

    transform infrared microspectroscopy [34]. The MC3T3-E1 cultures, however, contained material of an unknown origin that represented dystrophic...x- ray densitometer (GE Medical Systems, Fitchburg, WI) and scanned via a Dual Energy X- ray Absorptiometry (DEXA) scan. Company-provided software...including osteoporosis, rheumatoid arthritis , and skeletal metastases, resulting in osteoclast activity in excess of bone deposition by osteoblasts with

  3. Role of a new member of IGFBP superfamily, IGFBP-rP10, in proliferation and differentiation of osteoblastic cells

    International Nuclear Information System (INIS)

    Shibata, Yasuaki; Tsukazaki, Tomoo; Hirata, Kazunari; Xin Cheng; Yamaguchi, Akira

    2004-01-01

    Bone regeneration is critically regulated by various molecules. To identify the new genes involved in bone regeneration, we performed microarray-based gene expression analysis using a mouse bone regeneration model. We identified a new member of the IGFBP superfamily, designated IGFBP-rP10, whose expression is up-regulated at the early phase of bone regeneration. IGFBP-rP10 consists of an IGFBP homologous domain followed by a Kazal-type protein inhibitor domain and an immunoglobulin G-like domain. A real-time-based RT-PCR analysis demonstrated that various tissues including bone expressed IGFBP-rP10 mRNA in various degrees, and confirmed an up-regulation at the early phase of bone regeneration. In situ hybridization revealed that osteoblastic cells expressed IGFPB-rP10 mRNA during bone regeneration. Bone morphogenetic protein-2 increased the expression level of IGFBP-rP10 mRNA in various cells including C3H10T1/2, MC3T3-E1, C2C12, and primary murine osteoblastic cells. The addition of recombinant mouse IGFBP-rP10 promoted the proliferation of these cells but failed to stimulate alkaline phosphatase activity. These results suggest that IGFBP-rP10 is involved in the proliferation of osteoblasts during bone formation and bone regeneration

  4. Establishment of 3D culture and induction of osteogenic differentiation of pre-osteoblasts using wet-collected aligned scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Huifen [Hubei-MOSTKLOS & KLOBM, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China); Chongqing Affiliated Hospital of Stomatology, Chongqing University of Medical Sciences, Chongqing 400015 (China); Zhong, Junwen [Wuhan National Laboratory for Optoelectronics, School of Optical and Electronic Information, Huazhong University of Science and Technology, Wuhan 430074 (China); Xu, Fei; Song, Fangfang; Yin, Miao; Wu, Yanru; Hu, Qiyi [Hubei-MOSTKLOS & KLOBM, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China); Wang, Jiawei, E-mail: wangjwei@hotmail.com [Hubei-MOSTKLOS & KLOBM, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China)

    2017-02-01

    Aligned fibrous scaffolds have attracted much interest in bone tissue engineering, because they are supposed to induce osteogenic differentiation. For the first time, aligned silk fibroin nanofibres were loosely packed using a novel wet-collection electrospinning method. Moreover, three-dimensional (3D) culture of MC3T3-E1 pre-osteoblasts was established on these fibrous scaffolds. Physicochemical properties of the scaffolds and the behaviour of MC3T3-E1 pre-osteoblasts on the scaffolds were analysed and compared with scaffolds obtained using traditional method. Ethanol bath improved the uniformity and alignment of the fibres and increased the thickness and porosity of the scaffolds. Structures of the fibres were well maintained after immediate crosslinking in ethanol. Cells on the wet-collected scaffolds exhibited more ordered arrangement and elongated morphology as well as faster and deeper infiltration. The ordered infiltration resulted in the establishment of the 3D culture of cells, promoting proliferation and osteogenic differentiation of the pre-osteoblasts. Thus, the wet-collected aligned scaffolds with improved topographical and physicochemical properties presents significant potential application in bone regeneration. - Highlights: • Aligned silk fibroin nanofibres were loosely packed using a novel wet-collection electrospinning method. • Structural properties of the aligned nanofibres were improved. • Three-dimensional culture of MC3T3-E1 pre-osteoblasts was established. • The arrangement, morphology, infiltration, proliferation and osteogenic differentiation of cells were enhanced.

  5. NELL-1 increases pre-osteoblast mineralization using both phosphate transporter Pit1 and Pit2

    Energy Technology Data Exchange (ETDEWEB)

    Cowan, Catherine M. [Department of Bioengineering, University of California, Los Angeles, 420 Westwood Plaza,7523 Boelter Hall, Los Angeles, CA 90095 (United States); Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095 (United States); Zhang, Xinli; James, Aaron W.; Mari Kim, T.; Sun, Nichole [Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095 (United States); Wu, Benjamin [Department of Bioengineering, University of California, Los Angeles, 420 Westwood Plaza,7523 Boelter Hall, Los Angeles, CA 90095 (United States); Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095 (United States); Ting, Kang [Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095 (United States); Soo, Chia, E-mail: bsoo@ucla.edu [UCLA and Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic, Hospital Research Center, University of California, Los Angeles, 2641 Charles E. Young Dr. South, Los Angeles, CA 90095 (United States)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer NELL-1 accelerates extracellular matrix mineralization in MC3T3-E1 pre-osteoblasts. Black-Right-Pointing-Pointer NELL-1 significantly increases intracellular inorganic phosphate levels. Black-Right-Pointing-Pointer NELL-1 positively regulates osteogenesis but not proliferation in MC3T3-E1 cells. Black-Right-Pointing-Pointer NELL-1 regulates inorganic phosphate transporter activity. -- Abstract: NELL-1 is a potent osteoinductive molecule that enhances bone formation in multiple animal models through currently unidentified pathways. In the present manuscript, we hypothesized that NELL-1 may regulate osteogenic differentiation accompanied by alteration of inorganic phosphate (Pi) entry into the osteoblast via sodium dependent phosphate (NaPi) transporters. To determine this, MC3T3-E1 pre-osteoblasts were cultured in the presence of recombinant human (rh)NELL-1 or rhBMP-2. Analysis was performed for intracellular Pi levels through malachite green staining, Pit-1 and Pit-2 expression, and forced upregulation of Pit-1 and Pit-2. Results showed rhNELL-1 to increase MC3T3-E1 matrix mineralization and Pi influx associated with activation of both Pit-1 and Pit-2 channels, with significantly increased Pit-2 production. In contrast, Pi transport elicited by rhBMP-2 showed to be associated with increased Pit-1 production only. Next, neutralizing antibodies against Pit-1 and Pit-2 completely abrogated the Pi influx effect of rhNELL-1, suggesting rhNELL-1 is dependent on both transporters. These results identify one potential mechanism of action for rhNELL-1 induced osteogenesis and highlight a fundamental difference between NELL-1 and BMP-2 signaling.

  6. NELL-1 increases pre-osteoblast mineralization using both phosphate transporter Pit1 and Pit2

    International Nuclear Information System (INIS)

    Cowan, Catherine M.; Zhang, Xinli; James, Aaron W.; Mari Kim, T.; Sun, Nichole; Wu, Benjamin; Ting, Kang; Soo, Chia

    2012-01-01

    Highlights: ► NELL-1 accelerates extracellular matrix mineralization in MC3T3-E1 pre-osteoblasts. ► NELL-1 significantly increases intracellular inorganic phosphate levels. ► NELL-1 positively regulates osteogenesis but not proliferation in MC3T3-E1 cells. ► NELL-1 regulates inorganic phosphate transporter activity. -- Abstract: NELL-1 is a potent osteoinductive molecule that enhances bone formation in multiple animal models through currently unidentified pathways. In the present manuscript, we hypothesized that NELL-1 may regulate osteogenic differentiation accompanied by alteration of inorganic phosphate (Pi) entry into the osteoblast via sodium dependent phosphate (NaPi) transporters. To determine this, MC3T3-E1 pre-osteoblasts were cultured in the presence of recombinant human (rh)NELL-1 or rhBMP-2. Analysis was performed for intracellular Pi levels through malachite green staining, Pit-1 and Pit-2 expression, and forced upregulation of Pit-1 and Pit-2. Results showed rhNELL-1 to increase MC3T3-E1 matrix mineralization and Pi influx associated with activation of both Pit-1 and Pit-2 channels, with significantly increased Pit-2 production. In contrast, Pi transport elicited by rhBMP-2 showed to be associated with increased Pit-1 production only. Next, neutralizing antibodies against Pit-1 and Pit-2 completely abrogated the Pi influx effect of rhNELL-1, suggesting rhNELL-1 is dependent on both transporters. These results identify one potential mechanism of action for rhNELL-1 induced osteogenesis and highlight a fundamental difference between NELL-1 and BMP-2 signaling.

  7. Mechanical unloading reduces microtubule actin crosslinking factor 1 expression to inhibit β-catenin signaling and osteoblast proliferation.

    Science.gov (United States)

    Yin, Chong; Zhang, Yan; Hu, Lifang; Tian, Ye; Chen, Zhihao; Li, Dijie; Zhao, Fan; Su, Peihong; Ma, Xiaoli; Zhang, Ge; Miao, Zhiping; Wang, Liping; Qian, Airong; Xian, Cory J

    2018-07-01

    Mechanical unloading was considered a major threat to bone homeostasis, and has been shown to decrease osteoblast proliferation although the underlying mechanism is unclear. Microtubule actin crosslinking factor 1 (MACF1) is a cytoskeletal protein that regulates cellular processes and Wnt/β-catenin pathway, an essential signaling pathway for osteoblasts. However, the relationship between MACF1 expression and mechanical unloading, and the function and the associated mechanisms of MACF1 in regulating osteoblast proliferation are unclear. This study investigated effects of mechanical unloading on MACF1 expression levels in cultured MC3T3-E1 osteoblastic cells and in femurs of mice with hind limb unloading; and it also examined the role and potential action mechanisms of MACF1 in osteoblast proliferation in MACF1-knockdown, overexpressed or control MC3T3-E1 cells treated with or without the mechanical unloading condition. Results showed that the mechanical unloading condition inhibited osteoblast proliferation and MACF1 expression in MC3T3-E1 osteoblastic cells and mouse femurs. MACF1 knockdown decreased osteoblast proliferation, while MACF1 overexpression increased it. The inhibitory effect of mechanical unloading on osteoblast proliferation also changed with MACF1 expression levels. Furthermore, MACF1 was found to enhance β-catenin expression and activity, and mechanical unloading decreased β-catenin expression through MACF1. Moreover, β-catenin was found an important regulator of osteoblast proliferation, as its preservation by treatment with its agonist lithium attenuated the inhibitory effects of MACF1-knockdown or mechanical unloading on osteoblast proliferation. Taken together, mechanical unloading decreases MACF1 expression, and MACF1 up-regulates osteoblast proliferation through enhancing β-catenin signaling. This study has thus provided a mechanism for mechanical unloading-induced inhibited osteoblast proliferation. © 2017 Wiley Periodicals, Inc.

  8. Nicotine induces cell proliferation in association with cyclin D1 up-regulation and inhibits cell differentiation in association with p53 regulation in a murine pre-osteoblastic cell line

    International Nuclear Information System (INIS)

    Sato, Tsuyoshi; Abe, Takahiro; Nakamoto, Norimichi; Tomaru, Yasuhisa; Koshikiya, Noboru; Nojima, Junya; Kokabu, Shoichiro; Sakata, Yasuaki; Kobayashi, Akio; Yoda, Tetsuya

    2008-01-01

    Recent studies have suggested that nicotine critically affects bone metabolism. Many studies have examined the effects of nicotine on proliferation and differentiation, but the underlying molecular mechanisms remain unclear. We examined cell cycle regulators involved in the proliferation and differentiation of MC3T3-E1 cells. Nicotine induced cell proliferation in association with p53 down-regulation and cyclin D1 up-regulation. In differentiated cells, nicotine reduced alkaline phosphatase activity and mineralized nodule formation in dose-dependent manners. Furthermore, p53 expression was sustained in nicotine-treated cells during differentiation. These findings indicate that nicotine promotes the cell cycle and inhibits differentiation in association with p53 regulation in pre-osteoblastic cells

  9. A genomic characterization of the influence of silver nanoparticles on bone differentiation in MC3T3-E1 cells.

    Science.gov (United States)

    Qing, Tao; Mahmood, Meena; Zheng, Yuanting; Biris, Alexandru S; Shi, Leming; Casciano, Daniel A

    2018-02-01

    Silver nanoparticles (AgNPs) have been widely used in a variety of biomedical applications. Previous studies demonstrated that AgNPs significantly enhanced bone cell mineralization and differentiation in MC3T3-1 cells, a model in vitro system, when compared to several other NPs. This increased bone deposition was evaluated by phenotypic measurements and assessment of the expression of miRNAs associated with regulation of bone morphogenic proteins. In the present study, we used RNA-seq technology, a more direct measurement of gene expression, to investigate further the mechanisms of bone differentiation induced by AgNP treatment. Key factors associated with the osteoclast pathway were significantly increased in response to AgNP exposure including Bmp4, Bmp6 and Fosl1. In addition, genes of metabolism and toxicity pathways were significantly regulated as well. Although this study suggests the potential for AgNPs to influence bone morphogenesis in injury or disease applications, further investigation into the efficacy and safety of AgNPs in bone regeneration is warranted. Copyright © 2017 John Wiley & Sons, Ltd.

  10. Synthetic Peptides Analogue to Enamel Proteins Promote Osteogenic Differentiation of MC3T3-E1 and Mesenchymal Stem Cells

    Czech Academy of Sciences Publication Activity Database

    Rubert, M.; Ramis, J. M.; Vondrášek, Jiří; Gaya, A.; Lyngstadaas, S. P.; Monjo, M.

    2011-01-01

    Roč. 1, č. 2 (2011), s. 198-209 ISSN 2157-9083 Grant - others:GA ČR(CZ) GAP302/10/0427 Institutional research plan: CEZ:AV0Z40550506 Keywords : proline-rich regions * synthetic peptides * bone formation * mineralization * In Vitro Subject RIV: EI - Biotechnology ; Bionics

  11. Monosodium urate monohydrate crystals inhibit osteoblast viability and function: implications for development of bone erosion in gout.

    Science.gov (United States)

    Chhana, Ashika; Callon, Karen E; Pool, Bregina; Naot, Dorit; Watson, Maureen; Gamble, Greg D; McQueen, Fiona M; Cornish, Jillian; Dalbeth, Nicola

    2011-09-01

    Bone erosion is a common manifestation of chronic tophaceous gout. To investigate the effects of monosodium urate monohydrate (MSU) crystals on osteoblast viability and function. The MTT assay and flow cytometry were used to assess osteoblast cell viability in the MC3T3-E1 and ST2 osteoblast-like cell lines, and primary rat and primary human osteoblasts cultured with MSU crystals. Quantitative real-time PCR and von Kossa stained mineralised bone formation assays were used to assess the effects of MSU crystals on osteoblast differentiation using MC3T3-E1 cells. The numbers of osteoblasts and bone lining cells were quantified in bone samples from patients with gout. MSU crystals rapidly reduced viability in all cell types in a dose-dependent manner. The inhibitory effect on cell viability was independent of crystal phagocytosis and was not influenced by differing crystal length or addition of serum. Long-term culture of MC3T3-E1 cells with MSU crystals showed a reduction in mineralisation and decreased mRNA expression of genes related to osteoblast differentiation such as Runx2, Sp7 (osterix), Ibsp (bone sialoprotein), and Bglap (osteocalcin). Fewer osteoblast and lining cells were present on bone directly adjacent to gouty tophus than bone unaffected by tophus in patients with gout. MSU crystals have profound inhibitory effects on osteoblast viability and differentiation. These data suggest that bone erosion in gout occurs at the tophus-bone interface through alteration of physiological bone turnover, with both excessive osteoclast formation, and reduced osteoblast differentiation from mesenchymal stem cells.

  12. The aryl hydrocarbon receptor suppresses osteoblast proliferation and differentiation through the activation of the ERK signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Haitao; Du, Yuxuan; Zhang, Xulong; Sun, Ying; Li, Shentao; Dou, Yunpeng [Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069 (China); Li, Zhanguo [Department of Rheumatology and Immunology, Clinical Immunology Center, Peking University People' s Hospital, No. 11 Xizhimen South Street, Beijing 100044 (China); Yuan, Huihui, E-mail: huihui_yuan@163.com [Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069 (China); Zhao, Wenming, E-mail: zhao-wenming@163.com [Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069 (China)

    2014-11-01

    Ahr activation is known to be associated with synovitis and exacerbated rheumatoid arthritis (RA), but its contributions to bone loss have not been completely elucidated. Osteoblast proliferation and differentiation are abnormal at the erosion site in RA. Here, we reported that the expression of Ahr was increased in the hind paws' bone upon collagen-induced arthritis (CIA) in mice, and the levels of Ahr were negatively correlated with bone mineral density (BMD). In addition, immunofluorescent staining showed that the high expression of Ahr was mainly localized in osteoblasts from the CIA mice compared to normal controls. Moreover, the luciferase intensity of Ahr in the nucleus increased by 12.5% in CIA osteoblasts compared to that in normal controls. In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activation of the Ahr inhibited pre-osteoblast MC3T3-E1 cellular proliferation and differentiation in a dose-dependent manner. Interestingly, the levels of alkaline phosphatase (ALP) mRNA expression in the osteoblasts of CIA mice were reduced compared to normal controls. In contrast, decreased ALP expression by activated Ahr was completely reversed after pretreatment with an Ahr inhibitor (CH-223191) in MC3T3-E1 cell lines and primary osteoblasts on day 5. Our data further showed that activation of Ahr promoted the phosphorylation of ERK after 5 days. Moreover, Ahr-dependent activation of the ERK signaling pathway decreased the levels of proliferation cells and inhibited ALP activity in MC3T3-E1 cells. These results demonstrated that the high expression of Ahr may suppress osteoblast proliferation and differentiation through activation of the ERK signaling pathway, further enabling bone erosion in CIA mice. - Highlights: • The upregulation of Ahr was localized in osteoblasts of CIA mice. • The overexpression of Ahr suppressed osteoblast development. • The Ahr activated ERK signaling pathway to exacerbate bone erosion.

  13. [Effects of Porphyromonas endodontalis lipopolysaccharides on the expression of matrix metalloproteinase-9 in mouse osteoblasts].

    Science.gov (United States)

    Li, X L; Yu, Y Q; Qiu, L H; Yang, D; Wang, X M; Yu, J T

    2017-08-09

    Objective: To evaluate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of matrix metalloproteinase-9 (MMP-9) mRNA and protein as well as enzyme activity in MC3T3-E1 cells and the role of nuclear factor-κB (NF-κB) in the process, so as to investigate the expression of MMP-9 dependent signaling pathways in mouse osteoblasts induced by Pe LPS. Methods: The experiment was conducted in 3 sessions: MC3T3-E1 cells were treated with various concentrations of Pe LPS (0-20 mg/L) and 10 mg/L Pe LPS for different time intervals (0-48 h). The expression of MMP-9 mRNA and protein were detected by real-time reverse transcription-PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), while the enzyme activity was detected by gelatin zymography method. The expression of MMP-9 mRNA was also detected in 10 mg/L Pe LPS treated MC3T3-El cells after pretreated with specific NF-κB inhibitor BAY 11-7082 for l h. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. Results: The levels of MMP-9 mRNA and protein increased significantly after the treatment with various concentrations of Pe LPS (0-20 mg/L), which indicated that Pe LPS induced osteoblasts to express MMP-9 in dose dependent manners. The expression of MMP-9 protein increased from (5 395±362) ng/L (blank control group) to (12 684±375) ng/L (20 mg/L group). Maximal induction of MMP-9 mRNA expression was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 24 h. The expression of MMP-9 mRNA in the 20 mg/L group was about 7 times than that in the blank control group. After 24 h, the expression of MMP-9 mRNA decreased. Maximal expression of MMP-9 protein was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 48 h ([35 055±2 346] ng/L) showing the highest enzyme activity. The mRNA of MMP-9 decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. Conclusions: Pe LPS might

  14. Proliferation of mouse fibroblast-like and osteoblast-like cells on pure titanium films manufactured by electron beam melting.

    Science.gov (United States)

    Kawase, Mayu; Hayashi, Tatsuhide; Asakura, Masaki; Tomino, Masafumi; Mieki, Akimichi; Kawai, Tatsushi

    2016-10-01

    The physical characteristics and biological compatibility of surfaces produced by electron beam melting (EBM) are not well known. In particular, there are not many reports on biocompatibility qualities. In this study, pure Ti films were manufactured using EBM. While it is reported that moderately hydrophilic biomaterial surfaces display improved cell growth and biocompatibility, contact angle measurements on the EBM-produced pure Ti films showed slight hydrophobicity. Nonetheless, we found the cell count of both fibroblast-like cells (L929) and osteoblast-like cells (MC3T3-E1) increased on pure Ti films, especially the MC3T3-E1, which increased more than that of the control. In addition, the morphology of L929 and MC3T3-E1 was polygonal and spindle-shaped and the cytoskeleton was well developed in the pure Ti surface groups. Upon staining with Alizarin red S, a slight calcium deposition was observed and this level gradually rose to a remarkable level. These results indicate that pure Ti films manufactured by EBM have good biocompatibility and could be widely applied as biomedical materials in the near future. © 2016 International Federation for Cell Biology.

  15. [Effect of lipopolysaccharides from Porphyromonas endodontalis on the expression of macrophage colony stimulating factor in mouse osteoblasts].

    Science.gov (United States)

    Yu, Yaqiong; Qiu, Lihong; Guo, Jiajie; Qu, Liu; Xu, Liya; Zhong, Ming

    2014-09-01

    To investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of macrophage colony stimulating factor (M-CSF) mRNA and protein in MC3T3-E1 cells and the role of nucler factor-κB (NF-κB) in the process. MC3T3-E1 cells were treated with different concentrations of Pe-LPS (0-50 mg/L) and 10 mg/L Pe-LPS for different hours (0-24 h). The expression of M-CSF mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsordent assay (ELISA). The cells untreated by Pe-LPS served as control. The expression of M- CSF mRNA and protein was also detected in 10 mg/L Pe- LPS treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor. The groups were divided as follows, control group, BAY group (10 µmol/L BAY 11-7082 treated alone MC3T3-E1 cells), Pe-LPS group (10 mg/L Pe-LPS stimulated MC3T3-E1 cells for 6 h), BAY combine with Pe-LPS group (10 µmol/L BAY 11-7082 pretreated cells for 1 h and 10 mg/L of Pe-LPS stimulated MC3T3-E1 cells for 6 h). The level of M- CSF mRNA and protein increased significantly after treatment with different concentrations of Pe-LPS (0-50 mg/L), which indicated that Pe-LPS induced osteoblasts to express M-CSF mRNA and protein in dose dependent manners. The expression of M-CSF protein increased from (35 ± 2) ng/L (control group) to (170 ± 8) ng/L (50 mg/L group). Maximal induction of M-CSF mRNA expression was found in the MC3T3- E1 cells treated with 10 mg/L Pe-LPS for 6 h. After 6 h, the expression of M-CSF mRNA decreased gradually. The expression of M-CSF protein also increased with the treatment of 10 mg/L Pe-LPS for 10 h [(122 ± 4) ng/L]. After 10 h, the expression of M-CSF protein decreased gradually. The mRNA and proteins of M-CSF decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. There was no significant difference between BAY group and the control. Pe-LPS may induce

  16. [Effect of lipopolysaccharides from Porphyromonas endodontalis on the expression of interleukin-34 in mouse osteoblasts].

    Science.gov (United States)

    Yu, Ya-Qiong; Guo, Jia-Jie; Qiu, Li-Hong; Li, Xiao-Lin; Yang, Di; Guo, Yan

    2017-02-01

    To investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (P.e) on the expression of interleukin-34 (IL-34) mRNA in MC3T3-E1 cells and the role of p38MAPK, ERK1/2, NF-κB and SIRT1 in the process. MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 20 mg/L P.e-LPS for different time (0-24 h). The expression of IL-34 mRNA was detected by real-time reverse transcription-polymerase chain reaction (real time RT-PCR). MC3T3-E1 cells were pretreated with inhibitor of NF-κB(BAY 11-7082),inhibitor of p38MAPK (SB203580), inhibitor of ERK1/2 (PD98059), agonist of sirtuin1 (SIRT1) [resveratrol (RES)] and inhibitor of SIRT1 (EX-527) for 1 h, and then were treated with 20 mg/L P.e-LPS. The expression of IL-34 mRNA was detected by real time RT-PCR. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. The level of IL-34 mRNA increased significantly after treatment with different concentrations of P.e-LPS(0-50 mg/L),which indicated that P.e-LPS induced osteoblasts to express IL-34 mRNA in a dose-dependent manner. Maximal induction of IL-34 mRNA expression was observed in MC3T3-E1 cells treated with 20 mg/L P.e-LPS for 24 h.At 48 h, the expression of IL-34 mRNA decreased gradually. The mRNA of IL-34 decreased significantly after pretreatment with 10 μmol/L BAY-117082, SB203580 and PD98059 for 1 h. P.e-LPS-induced IL-34 upregulation was attenuated by pretreatment with RES, but increased by EX-527. These results suggest that P.e-LPS may mediate IL-34 mRNA expression in MC3T3-E1 cells. This process is dependent, at least in part, on p38MAPK, ERK1/2, NF-κB and SIRT1 signaling pathways.

  17. Low-intensity pulsed ultrasound regulates proliferation and differentiation of osteoblasts through osteocytes

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lei, E-mail: geraldleelei@163.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Yang, Zheng [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Zhang, Hai [Department of Restorative Dentistry, School of Dentistry, University of Washington, Seattle, WA (United States); Chen, Wenchuan [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Chen, Mengshi [Department of Biomechanics, Sichuan University, Chengdu (China); Zhu, Zhimin, E-mail: hxzhimin@163.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer CM from LIPUS-stimulated osteocytes inhibits proliferation of osteoblasts. Black-Right-Pointing-Pointer CM from LIPUS-stimulated osteocytes enhances differentiation of osteoblasts. Black-Right-Pointing-Pointer LIPUS stimulates MLO-Y4 cells to secrete PGE{sub 2} and NO. -- Abstract: Low-intensity pulsed ultrasound (LIPUS) has been used as a safe and effective modality to enhance fracture healing. As the most abundant cells in bone, osteocytes orchestrate biological activities of effector cells via direct cell-to-cell contacts and by soluble factors. In this study, we have used the osteocytic MLO-Y4 cells to study the effects of conditioned medium from LIPUS-stimulated MLO-Y4 cells on proliferation and differentiation of osteoblastic MC3T3-E1 cells. Conditioned media from LIPUS-stimulated MLO-Y4 cells (LIPUS-Osteocyte-CM) were collected and added on MC3T3-E1 cell cultures. MC3T3-E1 cells cultured in LIPUS-Osteocyte-CM demonstrated a significant inhibition of proliferation and an increased alkaline phosphatase activity. The results of PGE{sub 2} and NO assay showed that LIPUS could enhance PGE{sub 2} and NO secretion from MLO-Y4 cells at all time points within 24 h after LIPUS stimulation. We conclude that LIPUS regulates proliferation and differentiation of osteoblasts through osteocytes in vitro. Increased secretion of PGE{sub 2} from osteocytes may play a role in this effect.

  18. The Role of Moderate Static Magnetic Fields on Biomineralization of Osteoblasts on Sulfonated Polystryene Films

    Energy Technology Data Exchange (ETDEWEB)

    X Ba; M Hadjiargyrou; E DiMasi; Y Meng; M Simon; Z Tan; M Rafailovich

    2011-12-31

    We have investigated the effects of moderate static magnetic fields (SMFs) on murine MC3T3-E1 osteoblasts, and found that they enhance proliferations and promote differentiation. The increase in proliferation rates in response to SMFs was greater in cultures grown on partially sulfonated polytstyrene (SPS, degree of sulfonation: 33%) than in cultures grown on tissue culture plastic. We have previously shown that when the degree of sulfonation exceeded a critical value (12%) [1], spontaneous fibrillogenesis occured which allowed for direct observation of the ECM fibrillar organization under the influence of external fields. We found that the ECM produced in cultures grown on the SPS in the presence of the SMFs assembled into a lattice with larger dimensions than the ECM of the cultures grown in the absence of SMFs. During the early stages of the biomineralization process (day 7), the SMF exposed cultures also templated mineral deposition more rapidly than the control cultures. The rapid response is attributed to orientation of diamagnetic ECM proteins already present in the serum, which could then initiate further cellular signaling. SMFs also influenced late stage osteoblast differentiation as measured by the increased rate of osteocalcin secretion and gene expression beginning 15 days after SFM exposure. This correlated with a large increase in mineral deposition, and in cell modulus. GIXD and EDXS analysis confirmed early deposition of crystalline hydroxyapatite. Previous studies on the effects of moderate SMF had focused on cellular gene and protein expression, but did not consider the organization of the ECM fibers. Our ability to form these fibers has allowed us explore this additional effect and highlight its significance in the initiation of the biomineralization process.

  19. Prostate cancer cells induce osteoblastic differentiation via semaphorin 3A.

    Science.gov (United States)

    Liu, Fuzhou; Shen, Weiwei; Qiu, Hao; Hu, Xu; Zhang, Chao; Chu, Tongwei

    2015-03-01

    Prostate cancer metastasis to bone is the second most commonly diagnosed malignant disease among men worldwide. Such metastatic disease is characterized by the presence of osteoblastic bone lesions, and is associated with high rates of mortality. However, the various mechanisms involved in prostate cancer-induced osteoblastic differentiation have not been fully explored. Semaphorin 3A (Sema 3A) is a newly identified regulator of bone metabolism which stimulates differentiation of pre-osteoblastic cells under physiological conditions. We investigated in this study whether prostate cancer cells can mediate osteoblastic activity through Sema 3A. We cultured osteoprogenitor MC3T3-E1 cells in prostate cancer-conditioned medium, and analyzed levels of Sema 3A protein in diverse prostate cancer cell lines to identify cell lines in which Sema 3A production showed a positive correlation with osteo-stimulation. C4-2 cells were stably transfected with Sema 3A short hairpin RNA to further determine whether Sema 3A contributes to the ability of C4-2 cells to induce osteoblastic differentiation. Down-regulation of Sema 3A expression decreased indicators of C4-2 CM-induced osteoblastic differentiation, including alkaline phosphatase production and mineralization. Additionally, silencing or neutralizing Sema 3A in C4-2 cells resulted in diminished β-catenin expression in osteogenitor MC3T3-E1 cells. Our results suggest that prostate cancer-induced osteoblastic differentiation is at least partially mediated by Sema 3A, and may be regulated by the β-catenin signalling pathway. Sema 3A may represent a novel target for treatment of prostate cancer-induced osteoblastic lesions. © 2014 Wiley Periodicals, Inc.

  20. Matrix metalloproteinases (MMPs) safeguard osteoblasts from apoptosis during transdifferentiation into osteocytes

    DEFF Research Database (Denmark)

    Karsdal, M A; Levin Andersen, Thomas; Bonewald, L

    2004-01-01

    of osteoblasts forced to transdifferentiate into osteocytes in 3D type I collagen gels were inhibited by more than 50% when exposed to 10 microM GM6001 and to Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), a natural MT1-MMP inhibitor. This shows the importance of MMPs in safeguarding osteoblasts from......Osteoblasts undergo apoptosis or differentiate into either osteocytes or bone-lining cells after termination of bone matrix synthesis. In this study, we investigated the role of matrix metalloproteinases (MMPs) in differentiation of osteoblasts, bone formation, transdifferentiation into osteocytes......, and osteocyte apoptosis. This was accomplished by using calvarial sections from the MT1-MMP-deficient mouse and by culture of the mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts. We found that a synthetic matrix metalloprotease inhibitor, GM6001, strongly inhibited bone formation...

  1. [Effect of lipopolysaccharides extracted from Porphyromonas endodontalis on the expression of CD14 in osteoblast].

    Science.gov (United States)

    Jia, Ge; Qiu, Li-hong; Li, Ren; Yang, Di; Guo, Yan

    2010-10-01

    To investigate the effect of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of CD14 in osteoblast. Under the condition with or without serum, MC3T3-E1 cells were stimulated with 10 μg/mL P.e-LPS for 24 hours, then the expression of CD14 was measured using flow cytometry; the membrane CD14 mRNA was detected at different time point (0, 1, 3, 6, 12, 24 h) by RT-PCR. Statistical analysis was performed using two-sample t test, one-way ANOVA and Dunnett t test with SPSS13.0 software package. Flow cytometry showed that CD14 protein increased after the stimulation of P.e-LPS for 24 h, while the non-serum group demonstrated more increase; the membrane CD14 mRNA level was up-regulated by 10 μg/mL P.e-LPS at 1 hour, and reached the maximum at 3 h and declined after 6 h. P.e-LPS can induce the expression of CD14 in osteoblast MC3T3-E1, which indicates that P.e-LPS may play an important role in osteoblast through CD14.

  2. Affecting osteoblastic responses with in vivo engineered potato pectin fragments

    DEFF Research Database (Denmark)

    Kokkonen, Hanna; Verhoef, Renè; Kauppinen, Kyösti

    2012-01-01

    Pectins, complex plant-derived polysaccharides, are novel candidates for biomaterial nanocoatings. Pectic rhamnogalacturonan-I regions (RG-I) can be enzymatically treated to so-called modified hairy regions (MHR). We surveyed the growth and differentiation of murine preosteoblastic MC3T3-E1 cells...... subtler. However, tetracycline-stained calcium-containing mineral was detected merely only on uncoated TCPS. These results indicate the possibility to affect bone cell growth with in vivo-modified pectin fragments, consecutively providing information on the significance of certain monosaccharides...... on the biocompatibility of these polysaccharides. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012....

  3. Calcite as a bone substitute. Comparison with hydroxyapatite and tricalcium phosphate with regard to the osteoblastic activity

    Energy Technology Data Exchange (ETDEWEB)

    Monchau, F., E-mail: Francine.monchau@univ-artois.fr [Laboratoire Genie Civil et geo-Environnement (EA 4515, Universite Lille Nord de France), Equipe Biomateriaux Artois (Universite d' Artois), IUT/GMP, 1230, rue de l' Universite, BP 819, 62408 Bethune cedex (France); Hivart, Ph.; Genestie, B. [Laboratoire Genie Civil et geo-Environnement (EA 4515, Universite Lille Nord de France), Equipe Biomateriaux Artois (Universite d' Artois), IUT/GMP, 1230, rue de l' Universite, BP 819, 62408 Bethune cedex (France); Chai, F. [Laboratoire Medicaments et Biomateriaux a Liberation Controlee (INSERM U 1008, Universite Lille Nord de France), Groupe de Recherche sur les Biomateriaux (Universite Lille-2), Faculte de Medecine, 1, place de Verdun, 59045 Lille cedex (France); and others

    2013-01-01

    Close to the bone mineral phase, the calcic bioceramics, such as hydroxyapatite (HA) and {beta}-tricalcium phosphate ({beta}-TCP), are commonly used as substitutes or filling materials in bone surgery. Besides, calcium carbonate (CaCO{sub 3}) is also used for their excellent biocompatibility and bioactivity. However, the problem with the animal-origin aragonite demands the new technique to synthesize pure calcite capable of forming 3D bone implant. This study aims to manufacture and evaluate a highly-pure synthetic crystalline calcite with good cytocompatibility regarding to the osteoblasts, comparing to that of HA and {beta}-TCP. After the manufacture of macroporous bioceramic scaffolds with the identical internal architecture, their cytocompatibility is studied through MC3T3-E1 osteoblasts with the tests of cell viability, proliferation, vitality, etc. The results confirmed that the studied process is able to form a macroporous material with a controlled internal architecture, and this synthesized calcite is non-cytotoxic and facilitate the cell proliferation. Indeed requiring further improvement, the studied calcite is definitely an interesting alternative not only to coralline aragonite but also to calcium phosphate ceramics, particularly in bone sites with the large bone remodelling. Highlights: Black-Right-Pointing-Pointer Macroporous calcite manufacturing with controlled architecture as bone substitute Black-Right-Pointing-Pointer Cytotoxicity: adaptation of the colony-forming method with the target cells: MC3T3-E1 osteoblasts Black-Right-Pointing-Pointer Study of osteoblast proliferation and activity on calcite, HA and TCP.

  4. Adenovirus-mediated siRNA targeting TNF-α and overexpression of bone morphogenetic protein-2 promotes early osteoblast differentiation on a cell model of Ti particle-induced inflammatory response in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Guo, H.H.; Yu, C.C.; Sun, S.X. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedic Surgery, Yinchuan (China); Ma, X.J. [Ningxia Medical Autonomous Region of the First People' s Hospital, Department of Orthopedic Surgery, Yinchuan (China); Yang, X.C.; Sun, K.N.; Jin, Q.H. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedic Surgery, Yinchuan (China)

    2013-10-02

    Wear particles are phagocytosed by macrophages and other inflammatory cells, resulting in cellular activation and release of proinflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty failure. During this pathological process, tumor necrosis factor-alpha (TNF-α) plays an important role in wear-particle-induced osteolysis. In this study, recombination adenovirus (Ad) vectors carrying both target genes [TNF-α small interfering RNA (TNF-α-siRNA) and bone morphogenetic protein 2 (BMP-2)] were synthesized and transfected into RAW264.7 macrophages and pro-osteoblastic MC3T3-E1 cells, respectively. The target gene BMP-2, expressed on pro-osteoblastic MC3T3-E1 cells and silenced by the TNF-α gene on cells, was treated with titanium (Ti) particles that were assessed by real-time PCR and Western blot. We showed that recombinant adenovirus (Ad-siTNFα-BMP-2) can induce osteoblast differentiation when treated with conditioned medium (CM) containing RAW264.7 macrophages challenged with a combination of Ti particles and Ad-siTNFα-BMP-2 (Ti-ad CM) assessed by alkaline phosphatase activity. The receptor activator of nuclear factor-κB ligand was downregulated in pro-osteoblastic MC3T3-E1 cells treated with Ti-ad CM in comparison with conditioned medium of RAW264.7 macrophages challenged with Ti particles (Ti CM). We suggest that Ad-siTNFα-BMP-2 induced osteoblast differentiation and inhibited osteoclastogenesis on a cell model of a Ti particle-induced inflammatory response, which may provide a novel approach for the treatment of periprosthetic osteolysis.

  5. The effect of fluoride surface modification of ceramic TiO{sub 2} on the surface properties and biological response of osteoblastic cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Tiainen, H; Knychala, J; Lyngstadaas, S P; Haugen, H J [Department of Biomaterials, Institute for Clinical Dentistry, University of Oslo, PO Box 1109 Blindern, NO-0317 Oslo (Norway); Monjo, M [Department of Fundamental Biology and Health Sciences, Research Institute on Health Sciences (IUNICS), University of the Balearic Islands, Cra. de Valldemossa, km 7.5, 07122 Palma de Mallorca (Spain); Nilsen, O [Department of Chemistry, University of Oslo, PO Box 1033 Blindern, NO-0315 Oslo (Norway); Ellingsen, J E, E-mail: h.j.haugen@odont.uio.no [Oral Research Laboratory, Institute for Clinical Dentistry, University of Oslo, PO Box 1109 Blindern, NO-0317 Oslo (Norway)

    2011-08-15

    This study investigates the effect of fluoride surface modification on the surface properties of polycrystalline ceramic TiO{sub 2} and the biological response of murine osteoblast cells to fluoride-modified TiO{sub 2} in vitro. Fluoride concentrations up to 9 at.% were detected and the fluoride was found to bind to the surface in a ligand exchange reaction between surface hydroxyl groups and the fluoride anions from the HF. No significant changes in the surface topography were detected. In vitro experiments were performed in order to evaluate the biological response of the MC3T3-E1 cells to the fluoride-modified ceramic TiO{sub 2} surfaces. No difference in the lactate dehydrogenase (LDH) activity was seen in comparison to unmodified samples, apart from the highest fluoride concentration ({approx}9 at.%) which was found to be more toxic to the cells. Real-time PCR analysis showed no conclusive evidence for the fluoride-induced promotion of osteoblast differentiation as no significant increase in the collagen-1, osteocalcin, or BMP-2 mRNA levels was detected on the fluoride-modified ceramic TiO{sub 2} surfaces apart from one group, which showed an elevated osteocalcin level and higher number of cells. Since the observed grain boundary corrosion is also anticipated to reduce the mechanical properties of ceramic TiO{sub 2}, this surface modification method may not be an ideal method for improving the osteogenic response of ceramic TiO{sub 2} scaffolds.

  6. [Effect of lipopolysaccharides extracted from Porphyromonas endodontalis on the expression of p38 and ERK1/2 in osteoblast].

    Science.gov (United States)

    Lv, You; Jia, Ge; Qiu, Li-hong; Bao, Mu-rong; Yu, Ya-qiong; Guo, Yan

    2012-08-01

    To investigate the effect of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of p38 and ERK1/2 in osteoblast. MC3T3-E1 cells were stimulated with 10 μg/mL P.e-LPS for 0,5,15,30,60,180 min. The phosphorylation of p38 and ERK1/2 was measured by Western blot. Statistical analysis was performed using one- way ANOVA and Dunnett t test with SPSS11.0 software package. 10 μg/mL LPS could significantly activate p38 MAPK. The peak of phosphorylated p38 was detected at 5 to 30 min(P<0.01) and returned to baseline within 60 min; the level of phosphorylated ERK1/2 increased after the stimulation of LPS for 5 min and reached maximum at 15 min (P<0.01) and declined after 30 min. P.e-LPS can induce the expression of p38 and ERK1/2 in osteoblast MC3T3-E1, which indicates that P.e-LPS may play an important role in osteoblast through p38 and ERK1/2.

  7. Wnt/β-catenin signaling activates nephronectin expression in osteoblasts

    International Nuclear Information System (INIS)

    Ikehata, Mikiko; Yamada, Atsushi; Morimura, Naoko; Itose, Masakatsu; Suzawa, Tetsuo; Shirota, Tatsuo; Chikazu, Daichi; Kamijo, Ryutaro

    2017-01-01

    Nephronectin (Npnt), an extracellular matrix protein, is considered to play critical roles as an adhesion molecule in the development and functions of various organs and tissues, such as the kidneys and bone. In the present study, we found that Wnt3a strongly enhanced Npnt mRNA expression in osteoblast-like MC3T3-E1 cells, while it also induced an increase in Npnt gene expression in both time- and dose-dependent manners via the Wnt/β-catenin signaling pathway. These results suggest novel mechanisms for Wnt3a-induced osteoblast proliferation and cell survival via Npnt gene expression. - Highlights: • Wnt3a enhances nephronectin gene expression. • Nephronectin gene induction by Wnt3a is occurred by time- and dose-dependent manner. • Expression of nephronectin is regulated via β-catenin activation.

  8. MEK5 suppresses osteoblastic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Kaneshiro, Shoichi [Department of Orthopaedic Surgery, Japan Community Health Care Organization Osaka Hospital, 4-2-78 Fukushima, Fukushima Ward, Osaka City, Osaka 553-0003 (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Otsuki, Dai; Yoshida, Kiyoshi; Yoshikawa, Hideki [Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Higuchi, Chikahisa, E-mail: c-higuchi@umin.ac.jp [Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2015-07-31

    Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and is activated by its upstream kinase, MAPK kinase 5 (MEK5), which is a member of the MEK family. Although the role of MEK5 has been investigated in several fields, little is known about its role in osteoblastic differentiation. In this study, we have demonstrated the role of MEK5 in osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. We found that treatment with BIX02189, an inhibitor of MEK5, increased alkaline phosphatase (ALP) activity and the gene expression of ALP, osteocalcin (OCN) and osterix, as well as it enhanced the calcification of the extracellular matrix. Moreover, osteoblastic cell proliferation decreased at a concentration of greater than 0.5 μM. In addition, knockdown of MEK5 using siRNA induced an increase in ALP activity and in the gene expression of ALP, OCN, and osterix. In contrast, overexpression of wild-type MEK5 decreased ALP activity and attenuated osteoblastic differentiation markers including ALP, OCN and osterix, but promoted cell proliferation. In summary, our results indicated that MEK5 suppressed the osteoblastic differentiation, but promoted osteoblastic cell proliferation. These results implied that MEK5 may play a pivotal role in cell signaling to modulate the differentiation and proliferation of osteoblasts. Thus, inhibition of MEK5 signaling in osteoblasts may be of potential use in the treatment of osteoporosis. - Highlights: • MEK5 inhibitor BIX02189 suppresses proliferation of osteoblasts. • MEK5 knockdown and MEK5 inhibitor promote differentiation of osteoblasts. • MEK5 overexpression inhibits differentiation of osteoblasts.

  9. α1B-Adrenergic receptor signaling controls circadian expression of Tnfrsf11b by regulating clock genes in osteoblasts

    Directory of Open Access Journals (Sweden)

    Takao Hirai

    2015-11-01

    Full Text Available Circadian clocks are endogenous and biological oscillations that occur with a period of <24 h. In mammals, the central circadian pacemaker is localized in the suprachiasmatic nucleus (SCN and is linked to peripheral tissues through neural and hormonal signals. In the present study, we investigated the physiological function of the molecular clock on bone remodeling. The results of loss-of-function and gain-of-function experiments both indicated that the rhythmic expression of Tnfrsf11b, which encodes osteoprotegerin (OPG, was regulated by Bmal1 in MC3T3-E1 cells. We also showed that REV-ERBα negatively regulated Tnfrsf11b as well as Bmal1 in MC3T3-E1 cells. We systematically investigated the relationship between the sympathetic nervous system and the circadian clock in osteoblasts. The administration of phenylephrine, a nonspecific α1-adrenergic receptor (AR agonist, stimulated the expression of Tnfrsf11b, whereas the genetic ablation of α1B-AR signaling led to the alteration of Tnfrsf11b expression concomitant with Bmal1 and Per2 in bone. Thus, this study demonstrated that the circadian regulation of Tnfrsf11b was regulated by the clock genes encoding REV-ERBα (Nr1d1 and Bmal1 (Bmal1, also known as Arntl, which are components of the core loop of the circadian clock in osteoblasts.

  10. Influence of radiation on initial attachment of osteoblast-like cells on titanium plate

    International Nuclear Information System (INIS)

    Kakuta, Saburo; Hamazaki, Miki; Mitsumoto, Kazuyo; Itabashi, Yuto; Fujimori, Shinya; Miyazaki, Takashi; Nagumo, Masao

    1996-01-01

    Radiotherapy is a useful and convenient therapy for oral cancer. However, there are many side effects such as stomatitis and radionecrosis of jaws. Radionecrosis may cause loosing or infection of biomaterials used for reconstruction of jaws. In this experiment, in vitro investigation was performed to clarify the influence of radiation on initial attachment of osteoblast-like cells to the titanium plate. UMR-106 and MC3T3-E1 cells were used as osteoblast-like cells. Cell attachment was evaluated by alkaline phosphatase activity and staining attached cells with crystal violet. The results revealed that initial attachment of osteoblast-like cells to the titanium plate was dose-dependently decreased by radiation and that radiosensitivity of each cell was different respectively. Furthermore, the participation of active oxygen was suggested because of partial recovery of cell attachment by addition of superoxide dismutase and/or an antioxidant such as ascorbic acid. (author)

  11. Modified titanium surface with gelatin nano gold composite increases osteoblast cell biocompatibility

    International Nuclear Information System (INIS)

    Lee, Young-Hee; Bhattarai, Govinda; Aryal, Santosh; Lee, Nan-Hee; Lee, Min-Ho; Kim, Tae-Gun; Jhee, Eun-Chung; Kim, Hak-Yong; Yi, Ho-Keun

    2010-01-01

    This study examined the gelatin nano gold (GnG) composite for surface modification of titanium in addition to insure biocompatibility on dental implants or biomaterials. The GnG composite was constructed by gelatin and hydrogen tetrachloroaurate in presence of reducing agent, sodium borohydrate (NabH 4 ). The GnG composite was confirmed by UV-VIS spectroscopy and transmission electron microscopy (TEM). A dipping method was used to modify the titanium surface by GnG composite. Surface was characterized by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX). The MC-3T3 E1 cell viability was assessed by trypan blue and the expression of proteins to biocompatibility were analyzed by Western blotting. The GnG composite showed well dispersed character, the strong absorption at 530 nm, roughness, regular crystal and clear C, Na, Cl, P, and Au signals onto titanium. Further, this composite allowed MC-3T3 E1 growth and viability compared to gelatin and pure titanium. It induced ERK activation and the expression of cell adherent molecules, FAK and SPARC, and growth factor, VEGF. However, GnG decreased the level of SAPK/JNK. This shows that GnG composite coated titanium surfaces have a good biocompatibility for osteoblast growth and attachment than in intact by simple and versatile dipping method. Furthermore, it offers good communication between cell and implant surfaces by regulating cell signaling and adherent molecules, which are useful to enhance the biocompatibility of titanium surfaces.

  12. Effect of nanometer scale surface roughness of titanium for osteoblast function

    Directory of Open Access Journals (Sweden)

    Satoshi Migita

    2017-02-01

    Full Text Available Surface roughness is an important property for metallic materials used in medical implants or other devices. The present study investigated the effects of surface roughness on cellular function, namely cell attachment, proliferation, and differentiation potential. Titanium (Ti discs, with a hundred nanometer- or nanometer-scale surface roughness (rough and smooth Ti surface, respectively were prepared by polishing with silicon carbide paper. MC3T3-E1 mouse osteoblast-like cells were cultured on the discs, and their attachment, spreading area, proliferation, and calcification were analyzed. Cells cultured on rough Ti discs showed reduced attachment, proliferation, and calcification ability suggesting that the surface inhibited osteoblast function. The findings can provide a basis for improving the biocompatibility of medical devices.

  13. SATB2 participates in regulation of menadione-induced apoptotic insults to osteoblasts.

    Science.gov (United States)

    Wei, Jyh-Ding; Lin, Yi-Ling; Tsai, Cheng-Hsiu; Shieh, Hui-Shan; Lin, Pei-I; Ho, Wei-Pin; Chen, Ruei-Ming

    2012-07-01

    Special AT-rich sequence binding protein 2 (SATB2), a nuclear matrix attachment region-binding protein, can regulate embryonic development, cell differentiation, and cell survival. Previous studies showed that SATB2 is involved in osteoblast differentiation and skeletal development. In this study, we evaluated the role of SATB2 in oxidative stress-induced apoptotic insults to human osteoblast-like MG63 cells and mouse MC3T3-E1 cells. Exposure of MG63 cells to menadione increased intracellular reactive oxygen species levels in a concentration- and time-dependent manner. Simultaneously, menadione-induced oxidative stress triggered cell shrinkage and decreased cell viability. In addition, treatment of MG63 cells with menadione time-dependently decreased the mitochondrial membrane potential but enhanced caspase-3 activity. As a result, menadione-induced DNA fragmentation and cell apoptosis. As to the mechanism, exposure of MG63 cells to menadione amplified SATB2 messenger (m)RNA and protein expression in a time-dependent manner. Knockdown of translation of SATB2 mRNA using RNA interference led to chromatin disruption and nuclear damage. When MG63 cells and MC3T3-E1 cells were treated with SATB2 small interfering RNA, menadione-induced cell apoptosis was increased. We conclude that menadione causes oxidative stress in human osteoblasts and induces cellular apoptosis via a mitochondrion-caspase protease pathway. In addition, SATB2 may play a crucial role in protecting against oxidative stress-induced osteoblast apoptosis. Copyright © 2012 Orthopaedic Research Society.

  14. [Scanning electron microscopy observation of the growth of osteoblasts on Ti-24Nb-4Zr-8Sn modified by micro-arc oxidation and alkali-heat treatment and implant-bone interface].

    Science.gov (United States)

    Han, Xue; Liu, Hong-Chen; Wang, Dong-Sheng; Li, Shu-Jun; Yang, Rui

    2011-01-01

    To observe the efficacy of micro-arc oxidation and alkali-heat treatment (MAH) on Ti-24Nb-4Zr-8Sn (Ti2448). Disks (diameter of 14.5 mm, thickness of 1 mm) and cylinders (diameter of 3 mm, height of 10 mm) were fabricated from Ti2448 alloy. Samples were divided into three groups: polished (Ti2448), micro-arc oxidation(MAO-Ti2448), micro-arc oxidation and alkali-heat treatment (MAH-Ti2448). MC3T3-E1 osteoblastic cells were cultured on the disks and cell morphology was observed with scanning electron microscopy (SEM) aftre 3 days. The cylinder samples were implanted in the tibia of dogs and implant-bone interface was observed with SEM after 3 months. A rough and porous structure was shown in both MAO and MAH group. The MC3T3-E1 cells on the MAH-Ti2448 discs spread fully in intimate contact with the underlying coarse surface through active cytoskeletal extentions. Osseointegration was formed in the implant-bone interface in MAH samples. MAH treatment can provide a more advantageous Ti2448 surface to osteoblastic cells than MAO treatment does, and the former can improve the implant-bone integration.

  15. Kidney tubular-cell secretion of osteoblast growth factor is increased by kaempferol: a scientific basis for "the kidney controlling the bone" theory of Chinese medicine.

    Science.gov (United States)

    Long, Mian; Li, Shun-xiang; Xiao, Jiang-feng; Wang, Jian; Lozanoff, Scott; Zhang, Zhi-guang; Luft, Benjamin J; Johnson, Francis

    2014-09-01

    To study, at the cytological level, the basic concept of Chinese medicine that "the Kidney (Shen) controls the bone". Kaempferol was isolated form Rhizoma Drynariae (Gu Sui Bu, GSB) and at several concentrations was incubated with opossum kidney (OK) cells, osteoblasts (MC3T3 E1) and human fibroblasts (HF) at cell concentrations of 2×10(4)/mL. Opossum kidney cell-conditioned culture media with kaempferol at 70 nmol/L (70kaeOKM) and without kaempferol (0OKM) were used to stimulate MC3T3 E1 and HF proliferation. The bone morphological protein receptors I and II (BMPR I and II) in OK cells were identified by immune-fluorescence staining and Western blot analysis. Kaempferol was found to increase OK cell growth (Pkaempferol increases kidney cell secretion of OGF. Neither of these media had any significant effect on HF growth. Kaempferol also was found to increase the level of the BMPR II in OK cells. This lends strong support to the original idea that the Kidney has a significant influence over bone-formation, as suggested by some long-standing Chinese medical beliefs, kaempferol may also serve to stimulate kidney repair and indirectly stimulate bone formation.

  16. Sulforaphane reverses glucocorticoid-induced apoptosis in osteoblastic cells through regulation of the Nrf2 pathway

    Directory of Open Access Journals (Sweden)

    Lin H

    2014-07-01

    Full Text Available Hao Lin,1,* Bo Wei,1,* Guangsheng Li,1 Jinchang Zheng,1 Jiecong Sun,1 Jiaqi Chu,2 Rong Zeng,1 Yanru Niu21Department of Spinal Surgery, Affiliated Hospital of Guangdong Medical College, Zhanjiang, People’s Republic of China; 2Laboratory Institute of Minimally Invasive Orthopedic Surgery, Affiliated Hospital of Guangdong Medical College, Zhanjiang, People’s Republic of China *These authors contributed equally to this work Abstract: Apoptosis of osteoblasts triggered by high-dose glucocorticoids (GCs has been identified as a major cause of osteoporosis. However, the underlying molecular mechanisms accounting for this action remain elusive, which has impeded the prevention and cure of this side effect. Sulforaphane (SFP is a naturally occurring isothiocyanate that has huge health benefits for humans. In this study, by using osteoblastic MC3T3-E1 cells as a model, we demonstrate the protective effects of SFP against dexamethasone (Dex-induced apoptosis and elucidate the underlying molecular mechanisms. The results show that SFP could effectively inhibit the Dex-induced growth inhibition and release of lactate dehydrogenase in MC3T3-E1 cells. Treatment with Dex induced caspase-dependent apoptosis in MC3T3-E1 cells, as evidenced by an increase in the Sub-G1 phase, chromatin condensation, and deoxyribonucleic acid fragmentation, which were significantly suppressed by coincubation with SFP. Mitochondria-mediated apoptosis pathway contributed importantly to Dex-induced apoptosis, as revealed by the activation of caspase-3/-9 and subsequent cleavage of poly adenosine diphosphate ribose polymerase, which was also effectively blocked by SFP. Moreover, treatments of Dex strongly induced overproduction of reactive oxygen species and inhibited the expression of nuclear factor erythroid 2-related factor 2 (Nrf2 and the downstream effectors HO1 and NQO1. However, cotreatment with SFP effectively reversed this action of Dex. Furthermore, silencing of Nrf2 by

  17. Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors

    Directory of Open Access Journals (Sweden)

    Lamblin Anne-Francoise

    2007-10-01

    Full Text Available Abstract Background Osteoblast differentiation requires the coordinated stepwise expression of multiple genes. Histone deacetylase inhibitors (HDIs accelerate the osteoblast differentiation process by blocking the activity of histone deacetylases (HDACs, which alter gene expression by modifying chromatin structure. We previously demonstrated that HDIs and HDAC3 shRNAs accelerate matrix mineralization and the expression of osteoblast maturation genes (e.g. alkaline phosphatase, osteocalcin. Identifying other genes that are differentially regulated by HDIs might identify new pathways that contribute to osteoblast differentiation. Results To identify other osteoblast genes that are altered early by HDIs, we incubated MC3T3-E1 preosteoblasts with HDIs (trichostatin A, MS-275, or valproic acid for 18 hours in osteogenic conditions. The promotion of osteoblast differentiation by HDIs in this experiment was confirmed by osteogenic assays. Gene expression profiles relative to vehicle-treated cells were assessed by microarray analysis with Affymetrix GeneChip 430 2.0 arrays. The regulation of several genes by HDIs in MC3T3-E1 cells and primary osteoblasts was verified by quantitative real-time PCR. Nine genes were differentially regulated by at least two-fold after exposure to each of the three HDIs and six were verified by PCR in osteoblasts. Four of the verified genes (solute carrier family 9 isoform 3 regulator 1 (Slc9a3r1, sorbitol dehydrogenase 1, a kinase anchor protein, and glutathione S-transferase alpha 4 were induced. Two genes (proteasome subunit, beta type 10 and adaptor-related protein complex AP-4 sigma 1 were suppressed. We also identified eight growth factors and growth factor receptor genes that are significantly altered by each of the HDIs, including Frizzled related proteins 1 and 4, which modulate the Wnt signaling pathway. Conclusion This study identifies osteoblast genes that are regulated early by HDIs and indicates pathways that

  18. A combination of CO2 laser and plasma surface modification of poly(etheretherketone) to enhance osteoblast response

    International Nuclear Information System (INIS)

    Zheng, Yanyan; Xiong, Chengdong; Wang, Zhecun; Li, Xiaoyu; Zhang, Lifang

    2015-01-01

    Highlights: • COOH and microgrooves containing micropores or microcraters structure were constructed on PEEK surface by a combination of CO 2 laser and plasma treatment. • The mechanical properties of PEEK are maintained after single or dual surface treatment. • Pre-osteoblast cells (MC3T3-E1) adhesion, spreading and proliferation were improved remarkably on dual treated PEEK surface. • Cell pseudopodia protrude into the micropores or microcraters, in favor of forming firmer bone-implant integration. - Abstract: Poly(etheretherketone) (PEEK) is a rigid semicrystalline polymer that combines excellent mechanical properties, broad chemical resistance and bone-like stiffness and is widely used in biomedical fields. However, the bio-inert surface of PEEK tends to hinder its biomedical applications when direct osteointegration between the implants and the host tissue is desired. In this work, we demonstrate a dual modification method, which combines the laser and plasma surface treatment to combine advantages of both chemical states and microstructures for osteoblasts responses. While the plasma treatment introduces surface carboxyl groups (−COOH) onto PEEK surface, the laser treatment constructs microstructures over the PEEK surface. Our results indicated that −COOH as well as microgrooves containing micropores or microcraters structure are constructed on PEEK surface and plasma treatment has no apparent effect on the morphology of microstructures produced by laser micromachining. Unexpectedly, the superior mechanical properties of PEEK were maintained irrespective of the treatment used. Compared to native PEEK and single treated PEEK, dual modified PEEK is more favorable for pre-osteoblasts (MC3T3-E1) adhesion, spreading and proliferation. Moreover, cell pseudopodia protrude into the micropores or microcraters, in favor of forming firmer bone-implant integration. Our study illustrates enhanced osteoblasts responses to dual treated PEEK surface, which gives

  19. A combination of CO{sub 2} laser and plasma surface modification of poly(etheretherketone) to enhance osteoblast response

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yanyan [Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xiong, Chengdong [Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041 (China); Wang, Zhecun [Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Xiaoyu [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Zhang, Lifang, E-mail: zhanglfcioc@163.com [Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041 (China)

    2015-07-30

    Highlights: • COOH and microgrooves containing micropores or microcraters structure were constructed on PEEK surface by a combination of CO{sub 2} laser and plasma treatment. • The mechanical properties of PEEK are maintained after single or dual surface treatment. • Pre-osteoblast cells (MC3T3-E1) adhesion, spreading and proliferation were improved remarkably on dual treated PEEK surface. • Cell pseudopodia protrude into the micropores or microcraters, in favor of forming firmer bone-implant integration. - Abstract: Poly(etheretherketone) (PEEK) is a rigid semicrystalline polymer that combines excellent mechanical properties, broad chemical resistance and bone-like stiffness and is widely used in biomedical fields. However, the bio-inert surface of PEEK tends to hinder its biomedical applications when direct osteointegration between the implants and the host tissue is desired. In this work, we demonstrate a dual modification method, which combines the laser and plasma surface treatment to combine advantages of both chemical states and microstructures for osteoblasts responses. While the plasma treatment introduces surface carboxyl groups (−COOH) onto PEEK surface, the laser treatment constructs microstructures over the PEEK surface. Our results indicated that −COOH as well as microgrooves containing micropores or microcraters structure are constructed on PEEK surface and plasma treatment has no apparent effect on the morphology of microstructures produced by laser micromachining. Unexpectedly, the superior mechanical properties of PEEK were maintained irrespective of the treatment used. Compared to native PEEK and single treated PEEK, dual modified PEEK is more favorable for pre-osteoblasts (MC3T3-E1) adhesion, spreading and proliferation. Moreover, cell pseudopodia protrude into the micropores or microcraters, in favor of forming firmer bone-implant integration. Our study illustrates enhanced osteoblasts responses to dual treated PEEK surface, which

  20. Stimulatory effect of undecylenic acid on mouse osteoblast differentiation.

    Science.gov (United States)

    Kim, Myung Hee; Shim, Ki Shuk; Lee, Su-Ui; Kim, Young Sup; Min, Yong Ki; Kim, Seong Hwan

    2010-04-01

    Natural compounds with bone-forming (or anabolic) activity have been recently focused on in bone research. The present study investigated the effect of undecylenic acid (UA) on osteoblast differentiation in mouse osteoblastic MC3T3-E1 subclone 4 cells and primary mouse calvarial cells. Low concentrations of UA (up to 5 microM) exhibited no cytotoxicity and significantly increased the expression and activity of alkaline phosphatase (early differentiation marker of osteoblast) and calcium deposition with the induction of expression of the osteocalcin gene in both cells. Interestingly, at low concentration of UA, the induction of NF-kappaB p65 translocation into nucleus and the up-regulation of AP-1 and NFATc1 transcript levels were also observed, suggesting that the stimulatory effect of UA on osteoblast differentiation could be mediated through the activation of transcription factors. Additionally, although the patterns of UA-induced activation of MAP kinases (JNK and p38) were not completely consistent with the increase of both ALP activity and calcium deposition by UA, MAP kinases might be partially involved in the biological function of UA during the early and late stages of osteoblast differentiation. Copyright (c) 2009 John Wiley & Sons, Ltd.

  1. Effects of high-frequency near-infrared diode laser irradiation on the proliferation and migration of mouse calvarial osteoblasts.

    Science.gov (United States)

    Kunimatsu, Ryo; Gunji, Hidemi; Tsuka, Yuji; Yoshimi, Yuki; Awada, Tetsuya; Sumi, Keisuke; Nakajima, Kengo; Kimura, Aya; Hiraki, Tomoka; Abe, Takaharu; Naoto, Hirose; Yanoshita, Makoto; Tanimoto, Kotaro

    2018-01-04

    Laser irradiation activates a range of cellular processes and can promote tissue repair. Here, we examined the effects of high-frequency near-infrared (NIR) diode laser irradiation on the proliferation and migration of mouse calvarial osteoblastic cells (MC3T3-E1). MC3T3-E1 cells were cultured and exposed to high-frequency (30 kHz) 910-nm diode laser irradiation at a dose of 0, 1.42, 2.85, 5.7, or 17.1 J/cm 2 . Cell proliferation was evaluated with BrdU and ATP concentration assays. Cell migration was analyzed by quantitative assessment of wound healing using the Incucyt ® ZOOM system. In addition, phosphorylation of mitogen-activated protein kinase (MAPK) family members including p38 mitogen-activated protein kinase (p38), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK), and extracellular signal-regulated protein kinase (ERK)1/2) after laser irradiation was examined with western blotting. Compared to the control, cell proliferation was significantly increased by laser irradiation at a dose of 2.85, 5.7, or 17.1 J/cm 2 . Laser irradiation at a dose of 2.85 J/cm 2 induced MC3T3-E1 cells to migrate more rapidly than non-irradiated control cells. Irradiation with the high-frequency 910-nm diode laser at a dose of 2.85 J/cm 2 induced phosphorylation of MAPK/ERK1/2 15 and 30 min later. However, phosphorylation of p38 MAPK and SAPK/JNK was not changed by NIR diode laser irradiation at a dose of 2.85 J/cm 2 . Irradiation with a high-frequency NIR diode laser increased cell division and migration of MT3T3-E1 cells, possibly via MAPK/ERK signaling. These observations may be important for enhancing proliferation and migration of osteoblasts to improve regeneration of bone tissues.

  2. Murine osteoblastic and osteoclastic differentiation on strontium releasing hydroxyapatite forming cements

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Satish S., E-mail: sss42@pitt.edu [Department of Chemical & Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Roy, Abhijit, E-mail: abr20@pitt.edu [Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Lee, Boeun, E-mail: bol11@pitt.edu [Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Parekh, Shrey, E-mail: smp116@pitt.edu [Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Kumta, Prashant N., E-mail: pkumta@pitt.edu [Department of Chemical & Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Center for Craniofacial Regeneration, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Department of Mechanical Engineering and Materials Science, University of Pittsburgh, PA 15261 (United States); Center for Complex Engineered Multifunctional Materials, University of Pittsburgh, PA 15261 (United States)

    2016-06-01

    Ionic substitutions in hydroxyapatite (HA) scaffolds and self-setting cements containing Sr{sup 2+} ions incorporated are particularly of interest in bone regeneration. To date, the approach widely used to incorporate Sr{sup 2+} ions into HA cements has been the addition of Sr{sup 2+} containing salts, such as SrCO{sub 3}, SrCl{sub 2} ∙ 6H{sub 2}O, or SrHPO{sub 4}. However, this approach is dependent upon the relative solubility of Sr{sup 2+} containing salts with respect to calcium phosphate (CaP) precursors. Therefore, in the current study Sr{sup 2+} substituted dicalcium phosphate dihydrate (DCPD) was first synthesized and directly reacted with tetracalcium phosphate (TTCP) to form Sr{sup 2+} substituted HA forming cements. Rietveld refinement indicated that after one week of aging in phosphate buffered saline, cements prepared with and without Sr{sup 2+} were composed of 75% HA and 25% unreacted TTCP by weight. Cements prepared with 10% Sr{sup 2+} DCPD exhibited increased compressive strengths in comparison to unsubstituted cements. Increased MC3T3-E1 proliferation and differentiation were also observed on the cements prepared with increasing Sr{sup 2+} content. It was concluded that both the scaffold microstructure and Sr{sup 2+} ion release supported osteogenic differentiation. With respect to osteoclastic differentiation, no statistically significant differences in TRAP activity or cell morphology were observed. This suggests that the amount of Sr{sup 2+} released may have been too low to influence osteoclast formation in comparison to unsubstituted cements. The results obtained herein demonstrate that the use of Sr{sup 2+} substituted DCPD precursors rather than individually separate Sr{sup 2+} containing salts may be a useful approach to prepare Sr{sup 2+} containing HA cements. - Highlights: • Sr{sup 2+} containing HA cement was prepared by reacting TTCP with Sr{sup 2+} substituted DCPD. • Cements with increased Sr{sup 2+} supported increased

  3. Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-α

    International Nuclear Information System (INIS)

    Tsukasaki, Masayuki; Yamada, Atsushi; Suzuki, Dai; Aizawa, Ryo; Miyazono, Agasa; Miyamoto, Yoichi; Suzawa, Tetsuo; Takami, Masamichi; Yoshimura, Kentaro; Morimura, Naoko; Yamamoto, Matsuo; Kamijo, Ryutaro

    2011-01-01

    Highlights: → TNF-α inhibits POEM gene expression. → Inhibition of POEM gene expression is caused by NF-κB activation by TNF-α. → Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-α. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-α (TNF-α), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-κB) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-α in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-α-induced inhibition of osteoblast differentiation. These results suggest that TNF-α inhibits POEM expression through the NF-κB signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-α.

  4. Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress

    International Nuclear Information System (INIS)

    Tang, C.-H.; Chiu, Y.-C.; Huang, C.-F.; Chen, Y.-W.; Chen, P.-C.

    2009-01-01

    Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromal cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.

  5. Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Tsukasaki, Masayuki [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Suzuki, Dai [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Aizawa, Ryo [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Department of Periodontology, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta, Tokyo 145-8515 (Japan); Miyazono, Agasa [Department of Periodontology, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta, Tokyo 145-8515 (Japan); Miyamoto, Yoichi; Suzawa, Tetsuo; Takami, Masamichi; Yoshimura, Kentaro [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Morimura, Naoko [Laboratory for Comparative Neurogenesis, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Yamamoto, Matsuo [Department of Periodontology, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta, Tokyo 145-8515 (Japan); Kamijo, Ryutaro [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan)

    2011-07-15

    Highlights: {yields} TNF-{alpha} inhibits POEM gene expression. {yields} Inhibition of POEM gene expression is caused by NF-{kappa}B activation by TNF-{alpha}. {yields} Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-{alpha}. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-{alpha} (TNF-{alpha}), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-{alpha}-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-{kappa}B) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-{alpha} in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-{alpha}-induced inhibition of osteoblast differentiation. These results suggest that TNF-{alpha} inhibits POEM expression through the NF-{kappa}B signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-{alpha}.

  6. Limonene protects osteoblasts against methylglyoxal-derived adduct formation by regulating glyoxalase, oxidative stress, and mitochondrial function.

    Science.gov (United States)

    Suh, Kwang Sik; Chon, Suk; Choi, Eun Mi

    2017-12-25

    Methylglyoxal (MG) is a potent protein glycating agent and an important precursor of advanced glycation end products, which are involved in the pathogenesis of diabetic osteopathy. In this study, we investigated the effects of limonene on MG-induced damage in osteoblastic MC3T3-E1 cells. Pretreating cells with limonene prevented MG-induced protein adduct formation, tumor necrosis factor alpha and interleukin-6 release, mitochondrial superoxide production, and cardiolipin peroxidation. In addition, limonene increased glyoxalase I activity, and glutathione and heme oxygenase-1 levels in the presence of MG. Pretreatment with limonene prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation and adenosine triphosphate loss, and reduced the levels of adenosine monophosphate-activated protein kinase, peroxisome proliferator activated receptor γ coactivator 1α, and nitric oxide. These results demonstrate that limonene may prevent the development of diabetic osteopathy. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Heat Shock Protein 70 Negatively Regulates TGF-β-Stimulated VEGF Synthesis via p38 MAP Kinase in Osteoblasts

    Directory of Open Access Journals (Sweden)

    Go Sakai

    2017-11-01

    Full Text Available Background/Aims: We previously demonstrated that transforming growth factor-β (TGF-β stimulates the synthesis of vascular endothelial growth factor (VEGF through the activation of p38 mitogen-activated protein (MAP kinase in osteoblast-like MC3T3-E1 cells. Heat shock protein70 (HSP70 is a ubiquitously expressed molecular chaperone. In the present study, we investigated the involvement of HSP70 in the TGF-β-stimulated VEGF synthesis and the underlying mechanism in these cells. Methods: Culture MC3T3-E1 cells were stimulated by TGF-β. Released VEGF was measured using an ELISA assay. VEGF mRNA level was quantified by RT-PCR. Phosphorylation of each protein kinase was analyzed by Western blotting. Results: VER-155008 and YM-08, both of HSP70 inhibitors, significantly amplified the TGF-β-stimulated VEGF release. In addition, the expression level of VEGF mRNA induced by TGF-β was enhanced by VER-155008. These inhibitors markedly strengthened the TGF-β-induced phosphorylation of p38 MAP kinase. The TGF-β-induced phosphorylation of p38 MAP kinase was amplified in HSP70-knockdown cells. SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by these inhibitors of the TGF-β-induced VEGF release. Conclusion: These results strongly suggest that HSP70 acts as a negative regulator in the TGF-β-stimulated VEGF synthesis in osteoblasts, and that the inhibitory effect of HSP70 is exerted at a point upstream of p38 MAP kinase.

  8. [Effects of lipopolysaccharides from various Porphyromonas on the expression of CD14 and TLRs in mouse osteoblast].

    Science.gov (United States)

    Jia, Ge; Xue, Ming; Li, Ren; Lv, You; Qiu, Li-hong

    2011-12-01

    To observe the effect of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) and Porphyromonas gingivals(P.g) on the expression of CD14 and TLRs in osteoblast. MC3T3-E1 cells were stimulated with 10μg/mL P.e-LPS and P.g-LPS. The change of CD14,TLR2 and TLR4 mRNA was observed at different time point (0,1,3,6,12,24h) using RT-PCR,and the expression of CD14,TLR2 and TLR4 protein was measured by flow cytometry at 24-hour. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS11.0 software package. MC3T3-E1 cells were stimulated with 10μg/mL P.e-LPS for 1h,the expression of CD14 and TLR4 mRNA increased significantly. There was no increase of TLR2 mRNA with stimulation of P.e-LPS. The CD14,TLR2 and TLR4 mRNA expression increased significantly after stimulation with 10μg/mL P.g-LPS. Flow cytometry showed that CD14 and TLR4 protein increased significantly after stimulation with 10μg/mL P.e-LPS. CD14,TLR2 and TLR4 protein increased significantly after treatment with 10μg/mL P.g-LPS. CD14,TLR4 receptors are involved in P.e-LPS effect and CD14,TLR2 and TLR4 receptors are involved in P.g-LPS effect in mouse osteoblast.

  9. Effect of heparin and alendronate coating on titanium surfaces on inhibition of osteoclast and enhancement of osteoblast function

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Ho-Jin; Yun, Young-Pil [Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Han, Choong-Wan; Kim, Min Sung [Department of Oral and Maxillofacial Radiology, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Institute of Oral Biology, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kim, Sung Eun; Bae, Min Soo [Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kim, Gyu-Tae; Choi, Yong-Suk; Hwang, Eui-Hwan [Department of Oral and Maxillofacial Radiology, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Institute of Oral Biology, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Lee, Joon Woo [Department of Technology Commercialization Information, Korea Institute of Science and Technology Information (KISTI), 66, Hoegi-ro, Dongdaemun-gu, Seoul 130-741 (Korea, Republic of); Lee, Jin-Moo; Lee, Chang-Hoon [Department of Oriental Gynecology, College of Oriental Medicine, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kim, Duck-Su [Department of Conservative Dentistry, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kwon, Il Keun, E-mail: kwoni@khu.ac.kr [Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Institute of Oral Biology, Kyung Hee University, Seoul 130-701 (Korea, Republic of)

    2011-09-23

    Highlights: {yields} We examine bone metabolism of engineered alendronate attached to Ti surfaces. {yields} Alendronate-immobilized Ti enhances activation of osteoblast differentiation. {yields} Alendronate-immobilized Ti inhibits osteoclast differentiation. {yields} Alendronate-immobilized Ti may be a bioactive implant with dual functions. -- Abstract: The failure of orthopedic and dental implants has been attributed mainly to loosening of the implant from host bone, which may be due to weak bonding of the implant material to bone tissue. Titanium (Ti) is used in the field of orthopedic and dental implants because of its excellent biocompatibility and outstanding mechanical properties. Therefore, in the field of materials science and tissue engineering, there has been extensive research to immobilize bioactive molecules on the surface of implant materials in order to provide the implants with improved adhesion to the host bone tissue. In this study, chemically active functional groups were introduced on the surface of Ti by a grafting reaction with heparin and then the Ti was functionalized by immobilizing alendronate onto the heparin-grafted surface. In the MC3T3-E1 cell osteogenic differentiation study, the alendronate-immobilized Ti substrates significantly enhanced alkaline phosphatase activity (ALP) and calcium content. Additionally, nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells was inhibited with the alendronate-immobilized Ti as confirmed by TRAP analysis. Real time PCR analysis showed that mRNA expressions of osteocalcin and osteopontin, which are markers for osteogenesis, were upregulated in MC3T3-E1 cells cultured on alendronate-immobilized Ti. The mRNA expressions of TRAP and Cathepsin K, markers for osteoclastogenesis, in RAW264.7 cells cultured on alendronate-immobilized Ti were down-regulated. Our study suggests that alendronate-immobilized Ti may be a bioactive implant with dual functions to enhance

  10. The role of surface charge on the uptake and biocompatibility of hydroxyapatite nanoparticles with osteoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen Liang; Mccrate, Joseph M; Li Hao [Department of Mechanical and Aerospace Engineering, University of Missouri, Columbia, MO 65211 (United States); Lee, James C-M, E-mail: liha@missouri.edu [Department of Biological Engineering, University of Missouri, Columbia, MO 65211 (United States)

    2011-03-11

    The objective of this study is to evaluate the effect of hydroxyapatite (HAP) nanoparticles with different surface charges on the cellular uptake behavior and in vitro cell viability and proliferation of MC3T3-E1 cell lines (osteoblast). The nanoparticles' surface charge was varied by surface modification with two carboxylic acids: 12-aminododecanoic acid (positive) and dodecanedioic acid (negative). The untreated HAP nanoparticles and dodecanoic acid modified HAP nanoparticles (neutral) were used as the control. X-ray diffraction (XRD) revealed that surface modifications by the three carboxylic acids did not change the crystal structure of HAP nanoparticles; Fourier transform infrared spectroscopy (FT-IR) confirmed the adsorption and binding of the carboxylic acids on the HAP nanoparticles' surfaces; and zeta potential measurement confirmed that the chemicals successfully modified the surface charge of HAP nanoparticles in water based solution. Transmission electron microscopy (TEM) images showed that positively charged, negatively charged and untreated HAP nanoparticles, with similar size and shape, all penetrated into the cells and cells had more uptake of HAP nanoparticles with positive charge compared to those with negative charge, which might be attributed to the attractive or repulsive interaction between the negatively charged cell membrane and positively/negatively charged HAP nanoparticles. The neutral HAP nanoparticles could not penetrate the cell membrane due to their larger size. MTT assay and LDH assay results indicated that as compared with the polystyrene control, greater cell viability and cell proliferation were measured on MC3T3-E1 cells treated with the three kinds of HAP nanoparticles (neutral, positive, and untreated), among which positively charged HAP nanoparticles showed the strongest improvement for cell viability and cell proliferation. In summary, the surface charge of HAP nanoparticles can be modified to influence the cellular

  11. The role of surface charge on the uptake and biocompatibility of hydroxyapatite nanoparticles with osteoblast cells

    International Nuclear Information System (INIS)

    Chen Liang; Mccrate, Joseph M; Li Hao; Lee, James C-M

    2011-01-01

    The objective of this study is to evaluate the effect of hydroxyapatite (HAP) nanoparticles with different surface charges on the cellular uptake behavior and in vitro cell viability and proliferation of MC3T3-E1 cell lines (osteoblast). The nanoparticles' surface charge was varied by surface modification with two carboxylic acids: 12-aminododecanoic acid (positive) and dodecanedioic acid (negative). The untreated HAP nanoparticles and dodecanoic acid modified HAP nanoparticles (neutral) were used as the control. X-ray diffraction (XRD) revealed that surface modifications by the three carboxylic acids did not change the crystal structure of HAP nanoparticles; Fourier transform infrared spectroscopy (FT-IR) confirmed the adsorption and binding of the carboxylic acids on the HAP nanoparticles' surfaces; and zeta potential measurement confirmed that the chemicals successfully modified the surface charge of HAP nanoparticles in water based solution. Transmission electron microscopy (TEM) images showed that positively charged, negatively charged and untreated HAP nanoparticles, with similar size and shape, all penetrated into the cells and cells had more uptake of HAP nanoparticles with positive charge compared to those with negative charge, which might be attributed to the attractive or repulsive interaction between the negatively charged cell membrane and positively/negatively charged HAP nanoparticles. The neutral HAP nanoparticles could not penetrate the cell membrane due to their larger size. MTT assay and LDH assay results indicated that as compared with the polystyrene control, greater cell viability and cell proliferation were measured on MC3T3-E1 cells treated with the three kinds of HAP nanoparticles (neutral, positive, and untreated), among which positively charged HAP nanoparticles showed the strongest improvement for cell viability and cell proliferation. In summary, the surface charge of HAP nanoparticles can be modified to influence the cellular uptake of

  12. Effects of different magnitudes of mechanical strain on Osteoblasts in vitro

    International Nuclear Information System (INIS)

    Tang Lin; Lin Zhu; Li Yongming

    2006-01-01

    In addition to systemic and local factors, mechanical strain plays a crucial role in bone remodeling during growth, development, and fracture healing, and especially in orthodontic tooth movement. Although many papers have been published on the effects of mechanical stress on osteoblasts or osteoblastic cells, little is known about the effects of different magnitudes of mechanical strain on such cells. In the present study, we investigated how different magnitudes of cyclic tensile strain affected osteoblasts. MC3T3-E1 osteoblastic cells were subjected to 0%, 6%, 12% or 18% elongation for 24 h using a Flexercell Strain Unit, and then the mRNA and protein expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were examined. The results showed that cyclic tensile strain induced a magnitude-dependent increase (0%, 6%, 12%, and 18%) in OPG synthesis and a concomitant decrease in RANKL mRNA expression and sRANKL release from the osteoblasts. Furthermore, the induction of OPG mRNA expression by stretching was inhibited by indomethacin or genistein, and the stretch-induced reduction of RANKL mRNA was inhibited by PD098059. These results indicate that different magnitudes of cyclic tensile strain influence the biological behavior of osteoblasts, which profoundly affects bone remodeling

  13. Surface modification of biphasic calcium phosphate scaffolds by non-thermal atmospheric pressure nitrogen and air plasma treatment for improving osteoblast attachment and proliferation

    International Nuclear Information System (INIS)

    Choi, Yu-Ri; Kwon, Jae-Sung; Song, Doo-Hoon; Choi, Eun Ha; Lee, Yong-Keun; Kim, Kyoung-Nam; Kim, Kwang-Mahn

    2013-01-01

    Surface modifications induced by non-thermal plasma have been used extensively in biomedical applications. The attachment and proliferation of osteoblast cells are important in bone tissue engineering using scaffolds. Hence the effect of non-thermal plasma on hydroxyapatite/β-tri-calcium phosphate (HA/β-TCP) scaffolds in terms of improving osteoblast attachment and proliferation was investigated. Experimental groups were treated with non-thermal plasma for 10 min and 20 min and a control group was not treated with non-thermal plasma. For surface chemistry analysis, X-ray photoelectron spectroscopy (XPS) analysis was carried out. The hydrophilicity was determined from contact angle measurement on the surface. Atomic force microscopy analysis (AFM) was used to test the change in surface roughness and cell attachment and proliferation were evaluated using MC3T3-E1 osteoblast cells. XPS spectra revealed a decreased amount of carbon on the surface of the plasma-treated sample. The contact angle was also decreased following plasma treatment, indicating improved hydrophilicity of plasma-treated surfaces compared to the untreated disc. A significant increase in MC3T3E-1 cell attachment and proliferation was noted on plasma-treated samples as compared to untreated specimens. The results suggest that non-thermal atmospheric pressure nitrogen and air plasma treatments provide beneficial surface characteristics on HA/β-TCP scaffolds. - Highlights: ► Non-thermal plasma increased OH- and decreased C on biphasic scaffold. ► Non-thermal plasma had no effect on surface roughness. ► Non-thermal plasma resulted in hydrophilic surface. ► Non-thermal plasma resulted in better cell attachment and proliferation. ► Non-thermal plasma treatment on biphasic scaffold is useful for tissue engineering

  14. Effects of imatinib and nilotinib on the whole transcriptome of cultured murine osteoblasts.

    Science.gov (United States)

    Kirschner, Gyöngyi; Balla, Bernadett; Horváth, Péter; Kövesdi, Andrea; Lakatos, Gergely; Takács, István; Nagy, Zsolt; Tóbiás, Bálint; Árvai, Kristóf; Kósa, János Pál; Lakatos, Péter

    2016-09-01

    Numerous clinical observations have confirmed that breakpoint cluster region-abelson fusion oncoprotein tyrosine kinase inhibitors used in leukemia treatment alter bone physiology in a complex manner. The aim of the present study was to analyze the whole transcriptome of cultured murine osteoblasts and determine the changes following treatment with imatinib and nilotinib using Sequencing by Oligonucleotide Ligation and Detection next generation RNA sequencing. This study also aimed to identify candidate signaling pathways and network regulators by multivariate Ingenuity Pathway Analysis. Based on the right-tailed Fisher's exact test, significantly altered pathways including upstream regulators were defined for each drug. The correlation between these pathways and bone metabolism was also examined. The preliminary results suggest the two drugs have different mechanisms of action on osteoblasts, and imatinib was shown to have a greater effect on gene expression. Data also indicated the potential role of a number of genes and signaling cascades that may contribute to identifying novel targets for the treatment of metabolic bone diseases.

  15. Celecoxib inhibits osteoblast maturation by suppressing the expression of Wnt target genes

    Directory of Open Access Journals (Sweden)

    Akihiro Nagano

    2017-01-01

    Full Text Available Non-steroidal anti-inflammatory drugs (NSAIDs have been shown to impair bone healing. We previously reported that in colon cancer cells, celecoxib, a COX-2-selective NSAID, inhibited the canonical Wnt/β-catenin signaling pathway. Since this pathway also plays an important role in osteoblast growth and differentiation, we examined the effect of celecoxib on maturation of osteoblast-like cell line MC3T3-E1. Celecoxib induced degradation of transcription factor 7-like 2, a key transcription factor of the canonical Wnt pathway. Subsequently, we analyzed the effect of celecoxib on two osteoblast differentiation markers; runt-related transcription factor 2 (RUNX2 and alkaline phosphatase (ALP, both of which are the products of the canonical Wnt pathway target genes. Celecoxib inhibited the expression of both RUNX2 and ALP by suppressing their promoter activity. Consistent with these observations, celecoxib also strongly inhibited osteoblast-mediated mineralization. These results suggest that celecoxib inhibits osteoblast maturation by suppressing Wnt target genes, and this could be the mechanism that NSAIDs inhibit bone formation and fracture healing.

  16. Differences in responses to X-ray exposure between osteoclast and osteoblast cells

    International Nuclear Information System (INIS)

    Zhang, Jian; Wang, Ziyang; Wu, Anqing; Nie, Jing; Pei, Hailong; Hu, Wentao; Wang, Bing; Shang, Peng; Li, Bingyan; Zhou, Guangming

    2017-01-01

    Radiation-induced bone loss is a potential health concern for cancer patients undergoing radiotherapy. Enhanced bone resorption by osteoclasts and decreased bone formation by osteoblasts were thought to be the main reasons. In this study, we showed that both pre-differentiating and differentiating osteoclasts were relatively sensitive to X-rays compared with osteoblasts. X-rays decreased cell viability to a greater degree in RAW264.7 cells and in differentiating cells than than in osteoblastic MC3T3-E1 cells. X-rays at up to 8 Gy had little effects on osteoblast mineralization. In contrast, X-rays at 1 Gy induced enhanced osteoclastogenesis by enhanced cell fusion, but had no effects on bone resorption. A higher dose of X-rays at 8 Gy, however, had an inhibitory effect on bone resorption. In addition, actin ring formation was disrupted by 8 Gy of X-rays and reorganized into clusters. An increased activity of Caspase 3 was found after X-ray exposure. Actin disorganization and increased apoptosis may be the potential effects of X-rays at high doses, by inhibiting osteoclast differentiation. Taken together, our data indicate high radiosensitivity of osteoclasts. X-ray irradiation at relatively low doses can activate osteoclastogenesis, but not osteogenic differentiation. The radiosensitive osteoclasts are the potentially responsive cells for X-ray-induced bone loss.

  17. Bioenergetics during calvarial osteoblast differentiation reflect strain differences in bone mass.

    Science.gov (United States)

    Guntur, Anyonya R; Le, Phuong T; Farber, Charles R; Rosen, Clifford J

    2014-05-01

    Osteoblastogenesis is the process by which mesenchymal stem cells differentiate into osteoblasts that synthesize collagen and mineralize matrix. The pace and magnitude of this process are determined by multiple genetic and environmental factors. Two inbred strains of mice, C3H/HeJ and C57BL/6J, exhibit differences in peak bone mass and bone formation. Although all the heritable factors that differ between these strains have not been elucidated, a recent F1 hybrid expression panel (C3H × B6) revealed major genotypic differences in osteoblastic genes related to cellular respiration and oxidative phosphorylation. Thus, we hypothesized that the metabolic rate of energy utilization by osteoblasts differed by strain and would ultimately contribute to differences in bone formation. In order to study the bioenergetic profile of osteoblasts, we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) first in a preosteoblastic cell line MC3T3-E1C4 and subsequently in primary calvarial osteoblasts from C3H and B6 mice at days 7, 14, and 21 of differentiation. During osteoblast differentiation in media containing ascorbic acid and β-glycerophosphate, all 3 cell types increased their oxygen consumption and extracellular acidification rates compared with the same cells grown in regular media. These increases are sustained throughout differentiation. Importantly, C3H calvarial osteoblasts had greater oxygen consumption rates than B6 consistent with their in vivo phenotype of higher bone formation. Interestingly, osteoblasts utilized both oxidative phosphorylation and glycolysis during the differentiation process although mature osteoblasts were more dependent on glycolysis at the 21-day time point than oxidative phosphorylation. Thus, determinants of oxygen consumption reflect strain differences in bone mass and provide the first evidence that during collagen synthesis osteoblasts use both glycolysis and oxidative phosphorylation to synthesize and

  18. Improving the Osteoblast Cell Adhesion on Electron Beam Controlled TiO2 Nanotubes

    Directory of Open Access Journals (Sweden)

    Sung Wook Yoon

    2014-01-01

    Full Text Available Here we investigate the osteogenesis and synostosis processes on the surface-modified TiO2 nanotubes via electron beam irradiation. The TiO2 nanotubes studied were synthesized by anodization process under different anodizing voltage. For the anodization voltage of 15, 20, and 25 V, TiO2 nanotubes with diameters of 59, 82, and 105 nm and length of 115, 276, and 310 nm were obtained, respectively. MC3T3-E1 osteoblast cell line was incubated on the TiO2 nanotubes to monitor the change in the cell adhesion before and after the electron beam irradiation. We observe that the electron beam irradiation affects the number of surviving osteoblast cells as well as the cultivation time. In particular, the high adhesion rate of 155% was obtained when the osteoblast cells were cultivated for 2 hours on the TiO2 nanotube, anodized under 20 V, and irradiated with 5,000 kGy of electron beam.

  19. In vitro response of pre-osteoblastic cells to laser microgrooved PEEK

    International Nuclear Information System (INIS)

    Cordero, D; López-Álvarez, M; Rodríguez-Valencia, C; Serra, J; Chiussi, S; González, P

    2013-01-01

    Polyetheretherketone (PEEK) is currently being used in implants as an alternative to titanium, due to its mechanical properties, cytocompatibility and inertness. Several studies have demonstrated that certain patterning on the implants promotes the oriented cell growth of osteoblasts, favouring the formation of bone tissue. This patterning improves the implant's osteointegration in the bone and its mechanical stability. Therefore, the objective of this work is to micro-structure PEEK by laser radiation and to carry out an exhaustive study of the orientation of pre-osteoblast cells that grow on this material. Parallel microgrooves were obtained using an ArF excimer laser coupled with a mask projection unit with distances of 25, 50, 75 and 100 µm between grooves. The cell growth on these PEEK surfaces was studied, in order to compare the effect of different distances between grooves on the biological response of MC3T3-E1 pre-osteoblastic cells. Preferential cell orientation was observed for all studied distances, which was more pronounced in the 25 and 50 µm ones. (paper)

  20. Enhanced osteoblast responses to poly ether ether ketone surface modified by water plasma immersion ion implantation.

    Science.gov (United States)

    Wang, Heying; Lu, Tao; Meng, Fanhao; Zhu, Hongqin; Liu, Xuanyong

    2014-05-01

    Poly ether ether ketone (PEEK) offers a set of characteristics superior for human implants; however, its application is limited by the bio-inert surface property. In this work, PEEK surface was modified using single step plasma immersion ion implantation (PIII) treatment with a gas mixture of water vapor as a plasma resource and argon as an ionization assistant. Field emission scanning electron microscopy, atomic force microscopy and X-ray photoelectron spectroscopy were used to investigate the microstructure and composition of the modified PEEK surface. The water contact angle and zeta-potential of the surfaces were also measured. Osteoblast precursor cells MC3T3-E1 and rat bone mesenchymal stem cells were cultured on the PEEK samples to evaluate their cytocompatibility. The obtained results show that the hydroxyl groups as well as a "ravined structure" are constructed on water PIII modified PEEK. Compared with pristine PEEK, the water PIII treated PEEK is more favorable for osteoblast adhesion, spreading and proliferation, besides, early osteogenic differentiation indicated by the alkaline phosphatase activity is also up-regulated. Our study illustrates enhanced osteoblast responses to the PEEK surface modified by water PIII, which gives positive information in terms of future biomedical applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Understanding improved osteoblast behavior on select nanoporous anodic alumina

    Science.gov (United States)

    Ni, Siyu; Li, Changyan; Ni, Shirong; Chen, Ting; Webster, Thomas J

    2014-01-01

    The aim of this study was to prepare different sized porous anodic alumina (PAA) and examine preosteoblast (MC3T3-E1) attachment and proliferation on such nanoporous surfaces. In this study, PAA with tunable pore sizes (25 nm, 50 nm, and 75 nm) were fabricated by a two-step anodizing procedure in oxalic acid. The surface morphology and elemental composition of PAA were characterized by field emission scanning electron microscopy and X-ray photoelectron spectroscopy analysis. The nanopore arrays on all of the PAA samples were highly regular. X-ray photoelectron spectroscopy analysis suggested that the chemistry of PAA and flat aluminum surfaces were similar. However, contact angles were significantly greater on all of the PAA compared to flat aluminum substrates, which consequently altered protein adsorption profiles. The attachment and proliferation of preosteoblasts were determined for up to 7 days in culture using field emission scanning electron microscopy and a Cell Counting Kit-8. Results showed that nanoporous surfaces did not enhance initial preosteoblast attachment, whereas preosteoblast proliferation dramatically increased when the PAA pore size was either 50 nm or 75 nm compared to all other samples (Paluminum by modifying surface nano-roughness alone (and not changing chemistry) through an anodization process to improve osteoblast density, and, thus, should be further studied as a bioactive interface for orthopedic applications. PMID:25045263

  2. Receptor for advanced glycation end products inhibits proliferation in osteoblast through suppression of Wnt, PI3K and ERK signaling

    International Nuclear Information System (INIS)

    Li, Guofeng; Xu, Jingren; Li, Zengchun

    2012-01-01

    Highlights: ► RAGE overexpression suppresses cell proliferation in MC3T3-E1 cells. ► RAGE overexpression decreases Wnt/β-catenin signaling. ► RAGE overexpression decreases ERK and PI3K signaling. ► Inhibition of Wnt signaling abolishes PI3K signaling restored by RAGE blockade. ► Inhibition of Wnt signaling abolishes ERK signaling restored by RAGE blockade. -- Abstract: Expression of receptor for advanced glycation end products (RAGE) plays a crucial role in bone metabolism. However, the role of RAGE in the control of osteoblast proliferation is not yet evaluated. In the present study, we demonstrate that RAGE overexpression inhibits osteoblast proliferation in vitro. The negative regulation of RAGE on cell proliferation results from suppression of Wnt, PI3K and ERK signaling, and is restored by RAGE neutralizing antibody. Prevention of Wnt signaling using Sfrp1 or DKK1 rescues RAGE-decreased PI3K and ERK signaling and cell proliferation, indicating that the altered cell growth in RAGE overexpressing cells is in part secondary to alterations in Wnt signaling. Consistently, RAGE overexpression inhibits the expression of Wnt targets cyclin D1 and c-myc, which is partially reversed by RAGE blockade. Overall, these results suggest that RAGE inhibits osteoblast proliferation via suppression of Wnt, PI3K and ERK signaling, which provides novel mechanisms by which RAGE regulates osteoblast growth.

  3. Receptor for advanced glycation end products inhibits proliferation in osteoblast through suppression of Wnt, PI3K and ERK signaling

    Energy Technology Data Exchange (ETDEWEB)

    Li, Guofeng [Department of Emergency Surgery, East Hospital, Tongji University School of Medicine, Shanghai 200120 (China); Xu, Jingren [Department of Traditional Chinese Orthopaedics, East Hospital, Tongji University School of Medicine, Shanghai 200120 (China); Li, Zengchun, E-mail: lizc.2007@yahoo.com.cn [Department of Emergency Surgery, East Hospital, Tongji University School of Medicine, Shanghai 200120 (China)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer RAGE overexpression suppresses cell proliferation in MC3T3-E1 cells. Black-Right-Pointing-Pointer RAGE overexpression decreases Wnt/{beta}-catenin signaling. Black-Right-Pointing-Pointer RAGE overexpression decreases ERK and PI3K signaling. Black-Right-Pointing-Pointer Inhibition of Wnt signaling abolishes PI3K signaling restored by RAGE blockade. Black-Right-Pointing-Pointer Inhibition of Wnt signaling abolishes ERK signaling restored by RAGE blockade. -- Abstract: Expression of receptor for advanced glycation end products (RAGE) plays a crucial role in bone metabolism. However, the role of RAGE in the control of osteoblast proliferation is not yet evaluated. In the present study, we demonstrate that RAGE overexpression inhibits osteoblast proliferation in vitro. The negative regulation of RAGE on cell proliferation results from suppression of Wnt, PI3K and ERK signaling, and is restored by RAGE neutralizing antibody. Prevention of Wnt signaling using Sfrp1 or DKK1 rescues RAGE-decreased PI3K and ERK signaling and cell proliferation, indicating that the altered cell growth in RAGE overexpressing cells is in part secondary to alterations in Wnt signaling. Consistently, RAGE overexpression inhibits the expression of Wnt targets cyclin D1 and c-myc, which is partially reversed by RAGE blockade. Overall, these results suggest that RAGE inhibits osteoblast proliferation via suppression of Wnt, PI3K and ERK signaling, which provides novel mechanisms by which RAGE regulates osteoblast growth.

  4. Porphyromonas endodontalis lipopolysaccharides induce RANKL by mouse osteoblast in a way different from that of Escherichia coli lipopolysaccharide.

    Science.gov (United States)

    Tang, Yin; Sun, Feifei; Li, Xiaoting; Zhou, Yuan; Yin, Shihai; Zhou, Xuedong

    2011-12-01

    Porphyromonas endodontalis lipopolysaccharide (LPS) has been shown to have a high positive rate in infected root canals and symptomatic apical periodontitis. It may play an integral role as a potent stimulator of inflammatory cytokines involved in apical lesions. The receptor activator of nuclear factor-κB ligand (RANKL) has been proven to be the key regulator of bone remodeling. This study investigated P. endodontalis LPS-induced RANKL production and LPS signaling in mouse osteoblasts. LPS-induced RANKL production in mouse osteoblast MC3T3-E1 cells was measured by Western blot and real-time polymerase chain reaction, and the Toll-like receptors (TLRs) were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. Both of the anti-TLR2 and anti-TLR4 antibodies significantly (P endodontalis LPS; only anti-TLR2 antibody had a significant (P endodontalis LPS-infected osteoblasts (P endodontalis LPS has the ability to promote the expression of RANKL in mouse osteoblasts, and this induction was mainly through the TLR2/4-JNK signaling pathway, a situation quite different from that of typical bacterial endotoxin (E. coli LPS). Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  5. Recql4 haploinsufficiency in mice leads to defects in osteoblast progenitors: Implications for low bone mass phenotype

    International Nuclear Information System (INIS)

    Yang Jieping; Murthy, Sreemala; Winata, Therry; Werner, Sean; Abe, Masumi; Prahalad, Agasanur K.; Hock, Janet M.

    2006-01-01

    The cellular and molecular mechanisms that underlie skeletal abnormalities in defective Recql4-related syndromes are poorly understood. Our objective in this study was to explore the function of Recql4 in osteoblast biology both in vitro and in vivo. Immunohistochemistry on adult mouse bone showed Recql4 protein localization in active osteoblasts around growth plate, but not in fully differentiated osteocytes. Consistent with this finding, Recql4 gene expression was high in proliferating mouse osteoblastic MC3T3.E1 cells and decreased as cells progressively lost their proliferation activity during differentiation. Recql4 overexpression in osteoblastic cells exhibited higher proliferation activity, while its depletion impeded cell growth. In addition, bone marrow stromal cells from male Recql4+/- mice had fewer progenitor cells, including osteoprogenitors, indicated by reduced total fibroblast colony forming units (CFU-f) and alkaline phosphatase-positive CFU-f colonies concomitant with reduced bone mass. These findings provide evidence that Recql4 functions as a regulatory protein during osteoprogenitor proliferation, a critical cellular event during skeleton development

  6. Silver oxide-containing hydroxyapatite coating supports osteoblast function and enhances implant anchorage strength in rat femur.

    Science.gov (United States)

    Eto, Shuichi; Miyamoto, Hiroshi; Shobuike, Takeo; Noda, Iwao; Akiyama, Takayuki; Tsukamoto, Masatsugu; Ueno, Masaya; Someya, Shinsuke; Kawano, Shunsuke; Sonohata, Motoki; Mawatari, Masaaki

    2015-09-01

    Antibacterial silver with hydroxyapatite (Ag-HA) is a promising coating material for imparting antibacterial properties to implants. We previously reported that 3% (w/w) silver with HA (3% Ag-HA) has both antibacterial activity and osteoconductivity. In this study, we investigated the effects of Ag-HA on the in vitro osteoblast function and the in vivo anchorage strength and osteoconductivity of implants. Production of the osteoblast marker alkaline phosphatase, but not cytotoxicity, was observed in cells of the osteoblast cell line MC3T3-E1 cultured on the 3% Ag-HA-coated surface. These results were similar to those observed with silver-free HA coating. In contrast, a significant high level of cytotoxicity was observed when the cells were cultured on a 50% Ag-HA-coated surface. The anchorage strength of implants inserted into the femur of Sprague-Dawley (SD) rats was enhanced by coating the implants with 3% Ag-HA. On the 3% Ag-HA-coated surface, both metaphyseal and diaphyseal areas were largely covered with new bone and had adequate osteoconductivity. These results suggest that 3% Ag-HA, like conventional HA, promotes osteogenesis by supporting osteoblast viability and function and thereby contributes to sufficient anchorage strength of implants. Application of 3% Ag-HA, which combines the osteoconductivity of HA and the antibacterial activity of silver, to prosthetic joints will help prevent postoperative infections. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  7. A trans-acting enhancer modulates estrogen-mediated transcription of reporter genes in osteoblasts.

    Science.gov (United States)

    Sasaki-Iwaoka, H; Maruyama, K; Endoh, H; Komori, T; Kato, S; Kawashima, H

    1999-02-01

    The presence of bone-specific estrogen agonists and discovery of the osteoblast-specific transcription factor (TF), Cbfa1, together with the discovery of synergism between a TF Pit-1 and estrogen receptor alpha (ERalpha) on rat prolactin gene, led to investigation of Cbfa1 in the modulation of osteoblast-specific actions of estrogen. Reverse transcribed-polymerase chain reaction demonstrated expression of Cbfa1 in the osteoblastic cell lines, MG63, ROS17/2.8, and MC3T3E1, but not in nonosteoblastic cell lines, MCF7, C3H10T1/2, and HeLa. An ER expression vector and a series of luciferase (Luc) reporter plasmids harboring the Cbfa1 binding site OSE2 (the osteoblast-specific cis element in the osteocalcin promoter) and palindromic estrogen response elements (EREs) were cotransfected into both osteoblastic and nonosteoblastic cells. OSE2 worked as a cis- acting element in osteoblastic cells but not nonosteoblastic cells, whereas EREs were cis- acting in all cell lines. Synergistic transactivation was observed in osteoblastic cells only when both ERE and OSE2 were placed in juxtaposition to the promoter. Forced expression of Cbfa1 in C3H10T1/2 cells also induced synergism. Tamoxifen, a partial agonist/antagonist of estrogen, acted as an osteoblast-specific agonist in cells transfected with a promoter containing ERE and acted synergistically with a promoter containing the ERE-OSE2 enhancer combination. These results support the idea that bone-specific TFs modulate the actions of estrogen in a tissue-specific manner.

  8. Identification of Rorβ targets in cultured osteoblasts and in human bone

    Energy Technology Data Exchange (ETDEWEB)

    Roforth, Matthew M., E-mail: roforth.matthew@mayo.edu; Khosla, Sundeep, E-mail: khosla.sundeep@mayo.edu; Monroe, David G., E-mail: monroe.david@mayo.edu

    2013-11-01

    Highlights: •We examine the gene expression patterns controlled by Rorβ in osteoblasts. •Genes involved in extracellular matrix regulation and proliferation are affected. •Rorβ mRNA levels increase in aged, human bone biopsies. •Rorβ may affect osteoblast activity by modulation of these pathways. -- Abstract: Control of osteoblastic bone formation involves the cumulative action of numerous transcription factors, including both activating and repressive functions that are important during specific stages of differentiation. The nuclear receptor retinoic acid receptor-related orphan receptor β (Rorβ) has been recently shown to suppress the osteogenic phenotype in cultured osteoblasts, and is highly upregulated in bone marrow-derived osteogenic precursors isolated from aged osteoporotic mice, suggesting Rorβ is an important regulator of osteoblast function. However the specific gene expression patterns elicited by Rorβ are unknown. Using microarray analysis, we identified 281 genes regulated by Rorβ in an MC3T3-E1 mouse osteoblast cell model (MC3T3-Rorβ-GFP). Pathway analysis revealed alterations in genes involved in MAPK signaling, genes involved in extracellular matrix (ECM) regulation, and cytokine-receptor interactions. Whereas the identified Rorβ-regulated ECM genes normally decline during osteoblastic differentiation, they were highly upregulated in this non-mineralizing MC3T3-Rorβ-GFP model system, suggesting that Rorβ may exert its anti-osteogenic effects through ECM disruption. Consistent with these in vitro findings, the expression of both RORβ and a subset of RORβ-regulated genes were increased in bone biopsies from postmenopausal women (73 ± 7 years old) compared to premenopausal women (30 ± 5 years old), suggesting a role for RORβ in human age-related bone loss. Collectively, these data demonstrate that Rorβ regulates known osteogenic pathways, and may represent a novel therapeutic target for age-associated bone loss.

  9. Osteoclasts but not osteoblasts are affected by a calcified surface treated with zoledronic acid in vitro

    International Nuclear Information System (INIS)

    Schindeler, Aaron; Little, David G.

    2005-01-01

    Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption. Recent interest has centered on the effects of bisphosphonates on osteoblasts. Chronic dosing of osteoblasts with solubilized bisphosphonates has been reported to enhance osteogenesis and mineralization in vitro. However, this methodology poorly reflects the in vivo situation, where free bisphosphonate becomes rapidly bound to mineralized bone surfaces. To establish a more clinically relevant cell culture model, we cultured bone cells on calcium phosphate coated quartz discs pre-treated with the potent nitrogen-containing bisphosphonate, zoledronic acid (ZA). Binding studies utilizing [ 14 C]-labeled ZA confirmed that the bisphosphonate bound in a concentration-dependent manner over the 1-50 μM dose range. When grown on ZA-treated discs, the viability of bone-marrow derived osteoclasts was greatly reduced, while the viability and mineralization of the osteoblastic MC3T3-E1 cell line were largely unaffected. This suggests that only bone resorbing cells are affected by bound bisphosphonate. However, this system does not account for transient exposure to unbound bisphosphonate in the hours following a clinical dosing. To model this event, we transiently treated osteoblasts with ZA in the absence of a calcified surface. Osteoblasts proved highly resistant to all transitory treatment regimes, even when utilizing ZA concentrations that prevented mineralization and/or induced cell death when dosed chronically. This study represents a pharmacologically more relevant approach to modeling bisphosphonate treatment on cultured bone cells and implies that bisphosphonate therapies may not directly affect osteoblasts at bone surfaces

  10. HSP90 inhibitors potentiate PGF2α-induced IL-6 synthesis via p38 MAP kinase in osteoblasts.

    Directory of Open Access Journals (Sweden)

    Kazuhiko Fujita

    Full Text Available Heat shock protein 90 (HSP90 that is ubiquitously expressed in various tissues, is recognized to be a major molecular chaperone. We have previously reported that prostaglandin F2α (PGF2α, a potent bone remodeling mediator, stimulates the synthesis of interleukin-6 (IL-6 through p44/p42 mitogen-activated protein (MAP kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that Rho-kinase acts at a point upstream of p38 MAP kinase. In the present study, we investigated the involvement of HSP90 in the PGF2α-stimulated IL-6 synthesis and the underlying mechanism in MC3T3-E1 cells. Geldanamycin, an inhibitor of HSP90, significantly amplified both the PGF2α-stimulated IL-6 release and the mRNA expression levels. In addition, other HSP90 inhibitors, 17-allylamino-17demethoxy-geldanamycin (17-AAG and 17-dimethylamino-ethylamino-17-demethoxy-geldanamycin (17-DMAG and onalespib, enhanced the PGF2α-stimulated IL-6 release. Geldanamycin, 17-AAG and onalespib markedly strengthened the PGF2α-induced phosphorylation of p38 MAP kinase. Geldanamycin and 17-AAG did not affect the PGF2α-induced phosphorylation of p44/p42 MAP kinase and myosin phosphatase targeting subunit (MYPT-1, a substrate of Rho-kinase, and the protein levels of RhoA and Rho-kinase. In addition, HSP90-siRNA enhanced the PGF2α-induced phosphorylation of p38 MAP kinase. Furthermore, SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by geldanamycin, 17-AAG or 17-DMAG of the PGF2α-stimulated IL-6 release. Our results strongly suggest that HSP90 negatively regulates the PGF2α-stimulated IL-6 synthesis in osteoblasts, and that the effect of HSP90 is exerted through regulating p38 MAP kinase activation.

  11. The roles of P2Y2 purinergic receptors in osteoblasts and mechanotransduction.

    Directory of Open Access Journals (Sweden)

    Yanghui Xing

    Full Text Available We previously demonstrated, using osteoblastic MC3T3-E1 cells, that P2Y2 purinergic receptors are involved in osteoblast mechanotransduction. In this study, our objective was to further investigate, using a knockout mouse model, the roles of P2Y2 receptors in bone mechanobiology. We first examined bone structure with micro-CT and measured bone mechanical properties with three point bending experiments in both wild type mice and P2Y2 knockout mice. We found that bones from P2Y2 knockout mice have significantly decreased bone volume, bone thickness, bone stiffness and bone ultimate breaking force at 17 week old age. In order to elucidate the mechanisms by which P2Y2 receptors contribute to bone biology, we examined differentiation and mineralization of bone marrow cells from wild type and P2Y2 knockout mice. We found that P2Y2 receptor deficiency reduces the differentiation and mineralization of bone marrow cells. Next, we compared the response of primary osteoblasts, from both wild type and P2Y2 knockout mice, to ATP and mechanical stimulation (oscillatory fluid flow, and found that osteoblasts from wild type mice have a stronger response, in terms of ERK1/2 phosphorylation, to both ATP and fluid flow, relative to P2Y2 knockout mice. However, we did not detect any difference in ATP release in response to fluid flow between wild type and P2Y2 knock out osteoblasts. Our findings suggest that P2Y2 receptors play important roles in bone marrow cell differentiation and mineralization as well as in bone cell mechanotransduction, leading to an osteopenic phenotype in P2Y2 knockout mice.

  12. Osteoblast interaction with DLC-coated Si substrates.

    Science.gov (United States)

    Chai, Feng; Mathis, Nicolas; Blanchemain, Nicolas; Meunier, Cathy; Hildebrand, Hartmut F

    2008-09-01

    Diamond-like carbon (DLC) coating is a convenient means of modifying material surfaces that are sensitive to wear, such as titanium and silica substrates. This work aims to evaluate the osteoblast-like cells' response to DLC-coated Si (Si-DLC), which was treated under different conditions. DLC and deuterated DLC films were deposited by plasma-enhanced chemical vapor deposition to obtain a 200-nm-thick layer on all the samples. Three types of precursor gas were applied for deposition: pure methane (CH(4)), pure deuterated methane (CD(4)) and their half/half mixture. All surface treatments were performed under two different self-bias voltages (V(sb)): -400 and -600V. The modified surfaces were characterized by X-ray photoelectron spectroscopy, Raman spectroscopy, Rutherford backscattering spectroscopy, elastic recoil detection analysis, X-ray reflectometry and the sessile-drop method. MC3T3-E1 osteoblasts were cultured on the Si-DLC wafers for 3 and 6 days. Biological tests to measure cell proliferation, cell vitality, cell morphology and cell adhesion were performed. All DLC coatings produced a slightly more hydrophobic state than non-treated Si. Certain types of amorphous DLC coating, such as the surface treated under the V(sb) of -600V in pure methane (600CH(4)) or in pure deuterated methane (600CD(4)), offered a significantly higher cell proliferation rate to Si substrate. Scanning electron microscopy observations confirmed that the optimal cell adhesion behavior, among all the treated surfaces, occurred on the surface of the 600CH(4) and 600CD(4) groups, which showed increased amounts of filopodia and microvilli to enhance cell-environment exchange. In conclusion, DLC coating on Si could produce better surface stability and improved cellular responses.

  13. Dioxinodehydroeckol Enhances the Differentiation of Osteoblasts by Regulating the Expression of Phospho-Smad1/5/8

    Directory of Open Access Journals (Sweden)

    Byul-Nim Ahn

    2016-09-01

    Full Text Available Lack of bone formation-related health problems are a major problem for the aging population in the modern world. As a part of the ongoing trend of developing natural substances that attenuate osteoporotic bone loss conditions, dioxinodehydroeckol (DHE from edible brown alga Ecklonia cava was tested for its effects on osteoblastogenic differentiation in MC3T3-E1 pre-osteoblasts. DHE was observed to successfully enhance osteoblast differentiation, as indicated by elevated cell proliferation, alkaline phosphatase activity, intracellular cell mineralization, along with raised levels of osteoblastogenesis indicators at the concentration of 20 μM. Results suggested a possible intervening of DHE on the bone morphogenetic protein (BMP signaling pathway, according to elevated protein levels of BMP-2, collagen-I, and Smads. In addition, the presence of DHE was also able to raise the phosphorylated extracellular signal–regulated kinase (ERK and c-Jun N-terminal kinase (JNK levels which are also activated by the BMP signaling pathway. In conclusion, DHE is suggested to be a potential bioactive compound against bone loss that could enhance osteoblastogenesis with a suggested BMP pathway interaction.

  14. Purinergic signaling is required for fluid shear stress-induced NF-{kappa}B translocation in osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Genetos, Damian C., E-mail: dgenetos@ucdavis.edu [Department of Anatomy, Cell Biology, and Physiology, School of Veterinary Medicine, University of California, Davis, CA (United States); Karin, Norman J. [Cell Biology and Biochemistry, Pacific Northwest National Laboratory, Richland, WA (United States); Geist, Derik J. [Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN (United States); Donahue, Henry J. [Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, Pennsylvania State College of Medicine, Hershey, PA (United States); Duncan, Randall L. [Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN (United States)

    2011-04-01

    Fluid shear stress regulates gene expression in osteoblasts, in part by activation of the transcription factor NF-{kappa}B. We examined whether this process was under the control of purinoceptor activation. MC3T3-E1 osteoblasts under static conditions expressed the NF-{kappa}B inhibitory protein I{kappa}B{alpha} and exhibited cytosolic localization of NF-{kappa}B. Under fluid shear stress, I{kappa}B{alpha} levels decreased, and concomitant nuclear localization of NF-{kappa}B was observed. Cells exposed to fluid shear stress in ATP-depleted medium exhibited no significant reduction in I{kappa}B{alpha}, and NF-{kappa}B remained within the cytosol. Similar results were found using oxidized ATP or Brilliant Blue G, P2X{sub 7} receptor antagonists, indicating that the P2X{sub 7} receptor is responsible for fluid shear-stress-induced I{kappa}B{alpha} degradation and nuclear accumulation of NF-{kappa}B. Pharmacologic blockage of the P2Y6 receptor also prevented shear-induced I{kappa}B{alpha} degradation. These phenomena involved neither ERK1/2 signaling nor autocrine activation by P2X{sub 7}-generated lysophosphatidic acid. Our results suggest that fluid shear stress regulates NF-{kappa}B activity through the P2Y{sub 6} and P2X{sub 7} receptor.

  15. Large gradient high magnetic fields affect osteoblast ultrastructure and function by disrupting collagen I or fibronectin/αβ1 integrin.

    Directory of Open Access Journals (Sweden)

    Ai-Rong Qian

    Full Text Available The superconducting magnet generates a field and field gradient product that can levitate diamagnetic materials. In this study a specially designed superconducting magnet with a large gradient high magnetic field (LG-HMF, which can provide three apparent gravity levels (μ-g, 1-g, and 2-g, was used to simulate a space-like gravity environment. The effects of LG-HMF on the ultrastructure and function of osteoblast-like cells (MG-63 and MC3T3-E1 and the underlying mechanism were investigated by transmission electromicroscopy (TEM, MTT, and cell western (ICW assays. Under LG-HMF significant morphologic changes in osteoblast-like cells occurred, including expansion of endoplasmic reticulum and mitochondria, an increased number of lysosomes, distorted microvilli, and aggregates of actin filaments. Compared to controls, cell viability and alkaline phosphatase (ALP secretion were significantly increased, and collagen I (col I, fibronectin (FN, vinculin, integrin α3, αv, and β1 expression were changed under LG-HMF conditions. In conclusion, LG-HMF affects osteoblast ultrastructure, cell viability, and ALP secretion, and the changes caused by LG-HMF may be related to disrupting col I or FN/αβ1 integrin.

  16. Osthole Enhances Osteogenesis in Osteoblasts by Elevating Transcription Factor Osterix via cAMP/CREB Signaling In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Zhong-Rong Zhang

    2017-06-01

    Full Text Available Anabolic anti-osteoporotic agents are desirable for treatment and prevention of osteoporosis and fragility fractures. Osthole is a coumarin derivative extracted from the medicinal herbs Cnidium monnieri (L. Cusson and Angelica pubescens Maxim.f. Osthole has been reported with osteogenic and anti-osteoporotic properties, whereas the underlying mechanism of its benefit still remains unclear. The objective of the present study was to investigate the osteopromotive action of osthole on mouse osteoblastic MC3T3-E1 cells and on mouse femoral fracture repair, and to explore the interaction between osthole-induced osteopromotive effect and cyclic adenosine monophosphate (cAMP elevating effect. Osthole treatment promoted osteogenesis in osteoblasts by enhancing alkaline phosphatase (ALP activity and mineralization. Oral gavage of osthole enhanced fracture repair and increased bone strength. Mechanistic study showed osthole triggered the cAMP/CREB pathway through the elevation of the intracellular cAMP level and activation of the phosphorylation of the cAMP response element-binding protein (CREB. Blockage of cAMP/CREB downstream signals with protein kinase A (PKA inhibitor KT5720 partially suppressed osthole-mediated osteogenesis by inhibiting the elevation of transcription factor, osterix. In conclusion, osthole shows osteopromotive effect on osteoblasts in vitro and in vivo. Osthole-mediated osteogenesis is related to activation of the cAMP/CREB signaling pathway and downstream osterix expression.

  17. Fabrication of dopamine-modified hyaluronic acid/chitosan multilayers on titanium alloy by layer-by-layer self-assembly for promoting osteoblast growth

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xinming, E-mail: xmzhang@tju.edu.cn [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Li, Zhaoyang, E-mail: zyli@tju.edu.cn [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072 (China); Yuan, Xubo [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Cui, Zhenduo; Yang, Xianjin [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072 (China)

    2013-11-01

    The bare inert surface of titanium (Ti) alloy typically causes early failures in implants. Layer-by-layer self-assembly is one of the simple methods for fabricating bioactive multilayer coatings on titanium implants. In this study, a dopamine-modified hyaluronic acid/chitosan (DHA/CHI) bioactive multilayer was built on the surface of Ti–24Nb–2Zr (TNZ) alloy. Zeta potential oscillated between −2 and 17 mV for DHA- and CHI-ending layers during the assembly process, respectively. The DHA/CHI multilayer considerably decreased the contact angle and dramatically improved the wettability of TNZ alloy. Atomic force microscopy results revealed a rough surface on the original TNZ alloy, while the surface became smoother and more homogeneous after the deposition of approximately 5 bilayers (TNZ/(DHA/CHI){sub 5}). X-ray photoelectron spectroscopy analysis indicated that the TNZ/(DHA/CHI){sub 5} sample was completely covered by polyelectrolytes. Pre-osteoblast MC3T3-E1 cells were cultured on the original TNZ alloy and TNZ/(DHA/CHI){sub 5} to evaluate the effects of DHA/CHI multilayer on osteoblast proliferation in vitro. The proliferation of osteoblasts on TNZ/(DHA/CHI){sub 5} was significantly higher than that on the original TNZ alloy. The results of this study indicate that the proposed technique improves the biocompatibility of TNZ alloy and can serve as a potential modification method in orthopedic applications.

  18. Fabrication of dopamine-modified hyaluronic acid/chitosan multilayers on titanium alloy by layer-by-layer self-assembly for promoting osteoblast growth

    International Nuclear Information System (INIS)

    Zhang, Xinming; Li, Zhaoyang; Yuan, Xubo; Cui, Zhenduo; Yang, Xianjin

    2013-01-01

    The bare inert surface of titanium (Ti) alloy typically causes early failures in implants. Layer-by-layer self-assembly is one of the simple methods for fabricating bioactive multilayer coatings on titanium implants. In this study, a dopamine-modified hyaluronic acid/chitosan (DHA/CHI) bioactive multilayer was built on the surface of Ti–24Nb–2Zr (TNZ) alloy. Zeta potential oscillated between −2 and 17 mV for DHA- and CHI-ending layers during the assembly process, respectively. The DHA/CHI multilayer considerably decreased the contact angle and dramatically improved the wettability of TNZ alloy. Atomic force microscopy results revealed a rough surface on the original TNZ alloy, while the surface became smoother and more homogeneous after the deposition of approximately 5 bilayers (TNZ/(DHA/CHI) 5 ). X-ray photoelectron spectroscopy analysis indicated that the TNZ/(DHA/CHI) 5 sample was completely covered by polyelectrolytes. Pre-osteoblast MC3T3-E1 cells were cultured on the original TNZ alloy and TNZ/(DHA/CHI) 5 to evaluate the effects of DHA/CHI multilayer on osteoblast proliferation in vitro. The proliferation of osteoblasts on TNZ/(DHA/CHI) 5 was significantly higher than that on the original TNZ alloy. The results of this study indicate that the proposed technique improves the biocompatibility of TNZ alloy and can serve as a potential modification method in orthopedic applications.

  19. Osteomacs interact with megakaryocytes and osteoblasts to regulate murine hematopoietic stem cell function.

    Science.gov (United States)

    Mohamad, Safa F; Xu, Linlin; Ghosh, Joydeep; Childress, Paul J; Abeysekera, Irushi; Himes, Evan R; Wu, Hao; Alvarez, Marta B; Davis, Korbin M; Aguilar-Perez, Alexandra; Hong, Jung Min; Bruzzaniti, Angela; Kacena, Melissa A; Srour, Edward F

    2017-12-12

    Networking between hematopoietic stem cells (HSCs) and cells of the hematopoietic niche is critical for stem cell function and maintenance of the stem cell pool. We characterized calvariae-resident osteomacs (OMs) and their interaction with megakaryocytes to sustain HSC function and identified distinguishing properties between OMs and bone marrow (BM)-derived macrophages. OMs, identified as CD45 + F4/80 + cells, were easily detectable (3%-5%) in neonatal calvarial cells. Coculture of neonatal calvarial cells with megakaryocytes for 7 days increased OM three- to sixfold, demonstrating that megakaryocytes regulate OM proliferation. OMs were required for the hematopoiesis-enhancing activity of osteoblasts, and this activity was augmented by megakaryocytes. Serial transplantation demonstrated that HSC repopulating potential was best maintained by in vitro cultures containing osteoblasts, OMs, and megakaryocytes. With or without megakaryocytes, BM-derived macrophages were unable to functionally substitute for neonatal calvarial cell-associated OMs. In addition, OMs differentiated into multinucleated, tartrate resistant acid phosphatase-positive osteoclasts capable of bone resorption. Nine-color flow cytometric analysis revealed that although BM-derived macrophages and OMs share many cell surface phenotypic similarities (CD45, F4/80, CD68, CD11b, Mac2, and Gr-1), only a subgroup of OMs coexpressed M-CSFR and CD166, thus providing a unique profile for OMs. CD169 was expressed by both OMs and BM-derived macrophages and therefore was not a distinguishing marker between these 2 cell types. These results demonstrate that OMs support HSC function and illustrate that megakaryocytes significantly augment the synergistic activity of osteoblasts and OMs. Furthermore, this report establishes for the first time that the crosstalk between OMs, osteoblasts, and megakaryocytes is a novel network supporting HSC function.

  20. Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radioablative conditioning.

    Science.gov (United States)

    Olson, Timothy S; Caselli, Anna; Otsuru, Satoru; Hofmann, Ted J; Williams, Richard; Paolucci, Paolo; Dominici, Massimo; Horwitz, Edwin M

    2013-06-27

    Successful hematopoietic stem cell (HSC) transplantation requires donor HSC engraftment within specialized bone marrow microenvironments known as HSC niches. We have previously reported a profound remodeling of the endosteal osteoblastic HSC niche after total body irradiation (TBI), defined as relocalization of surviving megakaryocytes to the niche site and marked expansion of endosteal osteoblasts. We now demonstrate that host megakaryocytes function critically in expansion of the endosteal niche after preparative radioablation and in the engraftment of donor HSC. We show that TBI-induced migration of megakaryocytes to the endosteal niche depends on thrombopoietin signaling through the c-MPL receptor on megakaryocytes, as well as CD41 integrin-mediated adhesion. Moreover, niche osteoblast proliferation post-TBI required megakaryocyte-secreted platelet-derived growth factor-BB. Furthermore, blockade of c-MPL-dependent megakaryocyte migration and function after TBI resulted in a significant decrease in donor HSC engraftment in primary and competitive secondary transplantation assays. Finally, we administered thrombopoietin to mice beginning 5 days before marrow radioablation and ending 24 hours before transplant to enhance megakaryocyte function post-TBI, and found that this strategy significantly enhanced donor HSC engraftment, providing a rationale for improving hematopoietic recovery and perhaps overall outcome after clinical HSC transplantation.

  1. Mechanisms involved in regulation of osteoclastic differentiation by mechanical stress-loaded osteoblasts

    International Nuclear Information System (INIS)

    Kaneuji, Takeshi; Ariyoshi, Wataru; Okinaga, Toshinori; Toshinaga, Akihiro; Takahashi, Tetsu; Nishihara, Tatsuji

    2011-01-01

    Highlights: → Effect of compressive force on osteoblasts were examined. → Compressive force induced OPG expression and suppressed osteoclastogenesis. → This enhancement of OPG is dependent on Wnt/Ca2+ signal pathway. -- Abstract: Mechanical stress is known to be important for regulation of bone turnover, though the detailed mechanisms are not fully understood. In the present study, we examined the effect of mechanical stress on osteoblasts using a novel compression model. Mouse osteoblastic MC3T3-E1 cells were embedded in three-dimensional (3D) gels and cultured with continuous compressive force (0-10.0 g/cm 2 ) for 48 h, and the conditioned medium were collected. RAW264.7 cells were then incubated with the conditioned medium for various times in the presence of receptor activator of nuclear factor-κB ligand (RANKL). Conditioned medium was found to inhibit the differentiation of RAW264.7 cells into osteoclasts induced by RANKL via down-regulation of the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of IκBα, and nuclear translocation of p50 and p65. Interestingly, the conditioned medium also had a high level of binding activity to RANKL and blocked the binding of RANK to RANKL. Furthermore, the binding activity of conditioned medium to RANKL was reduced when the 3D gel was supplemented with KN-93, an inhibitor of non-canonical Wnt/Ca 2+ pathway. In addition, expression level of osteoprotegerin (OPG) mRNA was increased in time- and force-dependent manners, and remarkably suppressed by KN-93. These results indicate that osteoblastic cells subjected to mechanical stress produce OPG, which binds to RANKL. Furthermore, this binding activity strongly inhibited osteoclastogenesis through suppression of TRAF6 and the nuclear factor-kappa B (NF-κB) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt/Ca 2+ pathway.

  2. Does Translational Symmetry Matter on the Micro Scale? Fibroblastic and Osteoblastic Interactions with the Topographically Distinct Poly(ε-caprolactone)/Hydroxyapatite Thin Films

    Science.gov (United States)

    2015-01-01

    Material composition and topography of the cell-contacting material interface are important considerations in the design of biomaterials at the nano and micro scales. This study is one of the first to have assessed the osteoblastic response to micropatterned polymer–ceramic composite surfaces. In particular, the effect of topographic variations of composite poly(ε-caprolactone)/hydroxyapatite (PCL/HAp) films on viability, proliferation, migration and osteogenesis of fibroblastic and osteoblastic MC3T3-E1 cells was evaluated. To that end, three different micropatterned PCL/HAp films were compared: flat and textured, the latter of which included films comprising periodically arranged and randomly distributed oval topographic features 10 μm in diameter, 20 μm in separation and 10 μm in height, comparable to the dimensions of MC3T3-E1 cells. PCL/HAp films were fabricated by the combination of a bottom-up, soft chemical synthesis of the ceramic, nanoparticulate phase and a top-down, photolithographic technique for imprinting fine, microscale features on them. X-ray diffraction analysis indicated an isotropic orientation of both the polymeric chains and HAp crystallites in the composite samples. Biocompatibility tests indicated no significant decrease in their viability when grown on PCL/HAp films. Fibroblast proliferation and migration onto PCL/HAp films proceeded slower than on the control borosilicate glass, with the flat composite film fostering more cell migration activity than the films containing topographic features. The gene expression of seven analyzed osteogenic markers, including procollagen type I, osteocalcin, osteopontin, alkaline phosphatase, and the transcription factors Runx2 and TGFβ-1, was, however, consistently upregulated in cells grown on PCL/HAp films comprising periodically ordered topographic features, suggesting that the higher levels of symmetry of the topographic ordering impose a moderate mechanochemical stress on the adherent cells

  3. The interplay between nanostructured carbon-grafted chitosan scaffolds and protein adsorption on the cellular response of osteoblasts: structure-function property relationship.

    Science.gov (United States)

    Depan, D; Misra, R D K

    2013-04-01

    The rapid adsorption of proteins occurs during the early stages of biomedical device implantation into physiological systems. In this regard, the adsorption of proteins is a strong function of the nature of a biomedical device, which ultimately governs the biological functions. The objective of this study was to elucidate the interplay between nanostructured carbon-modified (graphene oxide and single-walled carbon nanohorn) chitosan scaffolds and consequent protein adsorption and biological function (osteoblast function). We compare and contrast the footprint of protein adsorption on unmodified chitosan and nanostructured carbon-modified chitosan. A comparative analysis of cell-substrate interactions using an osteoblast cell line (MC3T3-E1) implied that biological functions were significantly enhanced in the presence of nanostructured carbon, compared with unmodified chitosan. The difference in their respective behaviors is related to the degree and topography of protein adsorption on the scaffolds. Furthermore, there was a synergistic effect of nanostructured carbon and protein adsorption in terms of favorably modulating biological functions, including cell attachment, proliferation and viability, with the effect being greater on nanostructured carbon-modified scaffolds. The study also underscores that protein adsorption is favored in nanostructured carbon-modified scaffolds such that bioactivity and biological function are promoted. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  4. Methionine restriction alters bone morphology and affects osteoblast differentiation

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    Amadou Ouattara

    2016-12-01

    Full Text Available Methionine restriction (MR extends the lifespan of a wide variety of species, including rodents, drosophila, nematodes, and yeasts. MR has also been demonstrated to affect the overall growth of mice and rats. The objective of this study was to evaluate the effect of MR on bone structure in young and aged male and female C57BL/6J mice. This study indicated that MR affected the growth rates of males and young females, but not aged females. MR reduced volumetric bone mass density (vBMD and bone mineral content (BMC, while bone microarchitecture parameters were decreased in males and young females, but not in aged females compared to control-fed (CF mice. However, when adjusted for bodyweight, the effect of MR in reducing vBMD, BMC and microarchitecture measurements was either attenuated or reversed suggesting that the smaller bones in MR mice is appropriate for its body size. In addition, CF and MR mice had similar intrinsic strength properties as measured by nanoindentation. Plasma biomarkers suggested that the low bone mass in MR mice could be due to increased collagen degradation, which may be influenced by leptin, IGF-1, adiponectin and FGF21 hormone levels. Mouse preosteoblast cell line cultured under low sulfur amino acid growth media attenuated gene expression levels of Col1al, Runx2, Bglap, Alpl and Spp1 suggesting delayed collagen formation and bone differentiation. Collectively, our studies revealed that MR altered bone morphology which could be mediated by delays in osteoblast differentiation. Keywords: Methionine restriction, Aged mice, Micro-computed tomography, Nanoindentation, MC3T3-E1 subclone 4

  5. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    International Nuclear Information System (INIS)

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha; Jamal, Shazia; Levi, Edi; Rishi, Arun K.; Datta, Nabanita S.

    2013-01-01

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  6. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Jamal, Shazia [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Levi, Edi [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Rishi, Arun K. [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); VA Medical Center, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Datta, Nabanita S., E-mail: ndatta@med.wayne.edu [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States)

    2013-07-12

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  7. Overexpression of osteoprotegerin promotes preosteoblast differentiation to mature osteoblasts

    NARCIS (Netherlands)

    Yu, Hongyou; de Vos, Paul; Ren, Yijin

    OBJECTIVE: The hypothesis of the present study is that overexpression of osteoprotegerin (OPG) promotes preosteoblast maturation. MATERIALS AND METHODS: The preosteoblast cell line MC3T3-E1 was transfected with OPG overexpression. OPG expression was confirmed by enzyme-linked immunosorbent assay

  8. Pomegranate Peel Extract Prevents Bone Loss in a Preclinical Model of Osteoporosis and Stimulates Osteoblastic Differentiation in Vitro

    Directory of Open Access Journals (Sweden)

    Mélanie Spilmont

    2015-11-01

    Full Text Available The nutritional benefits of pomegranate have attracted great scientific interest. The pomegranate, including the pomegranate peel, has been used worldwide for many years as a fruit with medicinal activity, mostly antioxidant properties. Among chronic diseases, osteoporosis, which is associated with bone remodelling impairment leading to progressive bone loss, could eventually benefit from antioxidant compounds because of the involvement of oxidative stress in the pathogenesis of osteopenia. In this study, with in vivo and ex vivo experiments, we investigated whether the consumption of pomegranate peel extract (PGPE could limit the process of osteopenia. We demonstrated that in ovariectomized (OVX C57BL/6J mice, PGPE consumption was able to significantly prevent the decrease in bone mineral density (−31.9%; p < 0.001 vs. OVX mice and bone microarchitecture impairment. Moreover, the exposure of RAW264.7 cells to serum harvested from mice that had been given a PGPE-enriched diet elicited reduced osteoclast differentiation and bone resorption, as shown by the inhibition of the major osteoclast markers. In addition, PGPE appeared to substantially stimulate osteoblastic MC3T3-E1 alkaline phosphatase (ALP activity at day 7, mineralization at day 21 and the transcription level of osteogenic markers. PGPE may be effective in preventing the bone loss associated with ovariectomy in mice, and offers a promising alternative for the nutritional management of this disease.

  9. Pomegranate Peel Extract Prevents Bone Loss in a Preclinical Model of Osteoporosis and Stimulates Osteoblastic Differentiation in Vitro.

    Science.gov (United States)

    Spilmont, Mélanie; Léotoing, Laurent; Davicco, Marie-Jeanne; Lebecque, Patrice; Miot-Noirault, Elisabeth; Pilet, Paul; Rios, Laurent; Wittrant, Yohann; Coxam, Véronique

    2015-11-11

    The nutritional benefits of pomegranate have attracted great scientific interest. The pomegranate, including the pomegranate peel, has been used worldwide for many years as a fruit with medicinal activity, mostly antioxidant properties. Among chronic diseases, osteoporosis, which is associated with bone remodelling impairment leading to progressive bone loss, could eventually benefit from antioxidant compounds because of the involvement of oxidative stress in the pathogenesis of osteopenia. In this study, with in vivo and ex vivo experiments, we investigated whether the consumption of pomegranate peel extract (PGPE) could limit the process of osteopenia. We demonstrated that in ovariectomized (OVX) C57BL/6J mice, PGPE consumption was able to significantly prevent the decrease in bone mineral density (-31.9%; p < 0.001 vs. OVX mice) and bone microarchitecture impairment. Moreover, the exposure of RAW264.7 cells to serum harvested from mice that had been given a PGPE-enriched diet elicited reduced osteoclast differentiation and bone resorption, as shown by the inhibition of the major osteoclast markers. In addition, PGPE appeared to substantially stimulate osteoblastic MC3T3-E1 alkaline phosphatase (ALP) activity at day 7, mineralization at day 21 and the transcription level of osteogenic markers. PGPE may be effective in preventing the bone loss associated with ovariectomy in mice, and offers a promising alternative for the nutritional management of this disease.

  10. ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Xinhua [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China); Wang, Xiaoyuan [Department of Nephrology, Xi An Honghui Hospital, Xi an (China); Hu, Xiongke; Chen, Yong; Zeng, Kefeng [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China); Zhang, Hongqi, E-mail: zhq9699@126.com [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China)

    2015-07-01

    Although 17β-estradial (E2) is known to stimulate bone formation, the underlying mechanisms are not fully understood. Recent studies have implicated the Wnt/β-catenin pathway as a major signaling cascade in bone biology. The interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. In this study, E2 significantly increased the expression of β-catenin by inducing phosphorylations of GSK3β at serine 9. ERβ siRNAs were transfected into MC3T3-E1 cells and revealed that ERβ involved E2-induced osteoblasts proliferation and differentiation via Wnt/β-catenin signaling. The osteoblast differentiation genes (BGP, ALP and OPN) and proliferation related gene (cyclin D1) expression were significantly induced by E2-mediated ERβ. Furthermore immunofluorescence and immunoprecipitation analysis demonstrated that E2 induced the accumulation of β-catenin protein in the nucleus which leads to interaction with T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors. Taken together, these findings suggest that E2 promotes osteoblastic proliferation and differentiation by inducing proliferation-related and differentiation-related gene expression via ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. Our findings provide novel insights into the mechanisms of action of E2 in osteoblastogenesis. - Highlights: • 17β-estradial (E2) promotes GSK3-β phosphorylation. • E2 activates the Wnt/β-catenin signaling pathway. • The Wnt/β-catenin signaling pathway interacts with estrogen signaling pathways. • E2-mediated ER induced osteoblast differentiation and proliferation related genes expression.

  11. ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways

    International Nuclear Information System (INIS)

    Yin, Xinhua; Wang, Xiaoyuan; Hu, Xiongke; Chen, Yong; Zeng, Kefeng; Zhang, Hongqi

    2015-01-01

    Although 17β-estradial (E2) is known to stimulate bone formation, the underlying mechanisms are not fully understood. Recent studies have implicated the Wnt/β-catenin pathway as a major signaling cascade in bone biology. The interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. In this study, E2 significantly increased the expression of β-catenin by inducing phosphorylations of GSK3β at serine 9. ERβ siRNAs were transfected into MC3T3-E1 cells and revealed that ERβ involved E2-induced osteoblasts proliferation and differentiation via Wnt/β-catenin signaling. The osteoblast differentiation genes (BGP, ALP and OPN) and proliferation related gene (cyclin D1) expression were significantly induced by E2-mediated ERβ. Furthermore immunofluorescence and immunoprecipitation analysis demonstrated that E2 induced the accumulation of β-catenin protein in the nucleus which leads to interaction with T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors. Taken together, these findings suggest that E2 promotes osteoblastic proliferation and differentiation by inducing proliferation-related and differentiation-related gene expression via ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. Our findings provide novel insights into the mechanisms of action of E2 in osteoblastogenesis. - Highlights: • 17β-estradial (E2) promotes GSK3-β phosphorylation. • E2 activates the Wnt/β-catenin signaling pathway. • The Wnt/β-catenin signaling pathway interacts with estrogen signaling pathways. • E2-mediated ER induced osteoblast differentiation and proliferation related genes expression

  12. In vitro evaluation of caseinophosphopeptides from different genetic variants on bone mineralization

    Directory of Open Access Journals (Sweden)

    Giovanni Tulipano

    2010-01-01

    Full Text Available Casein phosphopeptides (CPPs have been shown to enhance calcium solubility and to increase the calcification by in vitro analyses. The aim of our study was to investigate the effects of four selected casein peptides, which differ in the number of phosphorylated serines, on osteoblast mineralization in vitro. The chosen peptides, related to different casein genetic variants, were obtained by chemical synthesis and tested on murine osteoblast cell line (MC3T3-E1. Our results suggest that the distinct peptides in protein hydrolysates may differentially affect calcium deposition in the extracellular matrix and that the genetic variation within the considered peptides is involved in their differential effect.

  13. Mechanical Stimulation and IGF-1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT-mTOR Pathway.

    Science.gov (United States)

    Bakker, Astrid D; Gakes, Tom; Hogervorst, Jolanda M A; de Wit, Gerard M J; Klein-Nulend, Jenneke; Jaspers, Richard T

    2016-06-01

    Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts. © 2015 Wiley Periodicals, Inc.

  14. TRIB3 mediates the expression of Wnt5a and activation of nuclear factor-κB in Porphyromonas endodontalis lipopolysaccharide-treated osteoblasts.

    Science.gov (United States)

    Yu, Y; Qiu, L; Guo, J; Yang, D; Qu, L; Yu, J; Zhan, F; Xue, M; Zhong, M

    2015-08-01

    Porphyromonas endodontalis lipopolysaccharide (LPS) is considered to be correlated with the progression of bone resorption in periodontal and periapical diseases. Wnt5a has recently been implicated in inflammatory processes, but its role is unclear as a P. endodontalis LPS-induced mediator in osteoblasts. Tribbles homolog 3 (TRIB3) encodes a pseudokinase and has been linked to inflammation in certain situations. Here, we found that P. endodontalis LPS induced Wnt5a expression in a dose- and time-dependent manner and it also upregulated translocation, phosphorylation and transcriptional activity of nuclear factor-κB (NF-κB) in MC3T3-E1 cells. Bay 11-7082 blocked the translocation of NF-κB and Wnt5a expression induced by P. endodontalis LPS. Chromatin immunoprecipitation assay further established that induction of Wnt5a by P. endodontalis LPS was mediated through the NF-κB p65 subunit. Additionally, P. endodontalis LPS increased expression of TRIB3 in osteoblasts after 10 h simulated time. Overexpression of TRIB3 enhanced NF-κB phosphorylation and Wnt5a induction, whereas knockdown of TRIB3 inhibited NF-κB phosphorylation and Wnt5a expression in P. endodontalis LPS-stimulated osteoblasts. These results suggest that P. endodontalis LPS has the ability to promote the expression of Wnt5a in mouse osteoblasts, and this induction is mainly mediated by NF-κB pathway. TRIB3 seems to modulate the sustained expression of Wnt5a in osteoblasts stimulated by P. endodontalis LPS, as well as regulating NF-κB phosphorylation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Selective Estrogen Receptor Modulator (SERM)-like Activities of Diarylheptanoid, a Phytoestrogen from Curcuma comosa, in Breast Cancer Cells, Pre-osteoblast Cells, and Rat Uterine Tissues.

    Science.gov (United States)

    Thongon, Natthakan; Boonmuen, Nittaya; Suksen, Kanoknetr; Wichit, Patsorn; Chairoungdua, Arthit; Tuchinda, Patoomratana; Suksamrarn, Apichart; Winuthayanon, Wipawee; Piyachaturawat, Pawinee

    2017-05-03

    Diarylheptanoids from Curcuma comosa, of the Zingiberaceae family, exhibit diverse estrogenic activities. In this study we investigated the estrogenic activity of a major hydroxyl diarylheptanoid, 7-(3,4 -dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene (compound 092) isolated from C. comosa. The compound elicited different transcriptional activities of estrogen agonist at low concentrations (0.1-1 μM) and antagonist at high concentrations (10-50 μM) using luciferase reporter gene assay in HEK-293T cells. In human breast cancer (MCF-7) cells, compound 092 showed an anti-estrogenic activity by down-regulating ERα-signaling and suppressing estrogen-responsive genes, whereas it attenuated the uterotrophic effect of estrogen in immature ovariectomized rats. Of note, compound 092 promoted mouse pre-osteoblastic (MC3T3-E1) cell differentiation and the related bone markers, indicating its positive osteogenic effect. Our findings highlight a new, nonsteroidal, estrogen agonist/antagonist of catechol diarylheptanoid from C. comosa, which is scientific evidence supporting its potential as a dietary supplement to prevent bone loss with low risk of breast and uterine cancers in postmenopausal women.

  16. Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

    Science.gov (United States)

    Imamura, Katsuyuki; Maeda, Shingo; Kawamura, Ichiro; Matsuyama, Kanehiro; Shinohara, Naohiro; Yahiro, Yuhei; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

    2014-04-04

    Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

  17. Optimal Zn-Modified Ca–Si-Based Ceramic Nanocoating with Zn Ion Release for Osteoblast Promotion and Osteoclast Inhibition in Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Jiangming Yu

    2017-01-01

    Full Text Available We investigated the slow release of Zn ion (Zn2+ from nanocoatings and compared the in vitro response of osteoblasts (MC3T3-E1 and proosteoclasts (RAW 264.7 cultured on Ca2ZnSi2O7 nanocoated with different Zn/Ca molar ratios on a Ti-6Al-4V (i.e., Ti substrate to optimize cell behaviors and molecule levels. Significant morphology differences were observed among samples. By comparing with pure Ti and CaSiO3 nanocoating, the morphology of Ca2ZnSi2O7 ceramic nanocoatings was rough and contained small nanoparticles or aggregations. Slow Zn2+ release from nanocoatings was observed and Zn2+ concentration was regulated by varying the Zn/Ca ratios. The cell-response results showed Ca2ZnSi2O7 nanocoating at different Zn/Ca molar ratios for osteoblasts and osteoclasts. Compared to other nanocoatings and Ti, sample Zn/Ca (0.3 showed the highest cell viability and upregulated expression of the osteogenic differentiation genes ALP, COL-1, and OCN. Additionally, sample Zn/Ca (0.3 showed the greatest inhibition of RAW 264.7 cell growth and decreased the mRNA levels of osteoclast-related genes OAR, TRAP, and HYA1. Therefore, the optimal Zn-Ca ratio of 0.3 in Ca2ZnSi2O7 ceramic nanocoating on Ti had a dual osteoblast-promoting and osteoclast-inhibiting effect to dynamically balance osteoblasts/osteoclasts. These optimal Zn-Ca ratios are valuable for Ca2ZnSi2O7 ceramic nanocoating on Ti-coated implants for potential applications in bone tissue regeneration.

  18. Evaluation of low doses of gamma irradiation in the formation of mineralization nodules in osteoblasts culture

    Energy Technology Data Exchange (ETDEWEB)

    Targino, Bárbara; Pinto, Thais Lazarine; Silva, Evily Fernandes; Somessari, E.S.R.; Bellini, Maria Helena; Affonso, Regina [Instituto De Pesquisas Energéticas e Nucleares (IPEN/CNEN-SP), São Paulo, SP (Brazil)

    2017-07-01

    Full text: Introduction: Osteoblasts are specialized fibroblasts that secrete and mineralize the bone matrix. The mineralized extracellular matrix is mainly composed of type I collagen, osteocalcin, and the inorganic mineral hydroxylapatite1. The use of radiation as therapy in some cancers causes great bone loss. However, low dose radiation may have the opposite effect. Low dose X-irradiation on osteoblastic culture had effects on proliferation and differentiation with increase of mineralization nodules2. However, there is little information on the potential therapeutic efficacy of low-dose gamma-irradiation in the formation of mineralization nodules. Objective: To evaluate the effects of irradiation with 60Co γ-rays in low doses in the formation of mineralization nodules in culture of osteoblasts. Methods: MC3T3-E1 cells were bought by the Banco de Células do Rio de Janeiro, Brazil (MC3T3-E1 Subclone 14). The cells were cultured in α-MEM medium consisting of 10% FBS and without β-glycerophosphate and L-ascorbic acid (GIBCO, Custom Product, Catalog No. A1049001) (Zhao Y, Guan H, Liu S et al. Biol. Pharm. Bull. 2005, 28(8):1371-1376). Plating efficiency assays: cells were plated at a density of 100 cell/plate into 60 mm Petri dishes. After 14 days the places were stained with violet crystal and the colonies were counted. -glycerophosphate and 50 mg/ml ascorbic acid, and analyzed on days 7, 14 and 21. Osteoblast culture irradiation assay: cells were plated at a density of 1 x 105 cells/plate on 60 mm dishes and the next day were irradiated by 60Co source with 0 (as the control), 0.5, 1.0, 1.5 and 2.0 Gy using the GammaCell 220 - Irradiation Unit of Canadian-Atomic Energy Commission Ltd. (CTR-IPEN). On day 21 of culture, undifferentiated (without ascorbic acid and β-glycerophosphate), differentiating cells (0 Gy) and irradiated cells at different doses, the medium was removed, cells were washed with phosphate buffer saline, fixed with 70% ethyl alcohol and

  19. Evaluation of low doses of gamma irradiation in the formation of mineralization nodules in osteoblasts culture

    International Nuclear Information System (INIS)

    Targino, Bárbara; Pinto, Thais Lazarine; Silva, Evily Fernandes; Somessari, E.S.R.; Bellini, Maria Helena; Affonso, Regina

    2017-01-01

    Full text: Introduction: Osteoblasts are specialized fibroblasts that secrete and mineralize the bone matrix. The mineralized extracellular matrix is mainly composed of type I collagen, osteocalcin, and the inorganic mineral hydroxylapatite1. The use of radiation as therapy in some cancers causes great bone loss. However, low dose radiation may have the opposite effect. Low dose X-irradiation on osteoblastic culture had effects on proliferation and differentiation with increase of mineralization nodules2. However, there is little information on the potential therapeutic efficacy of low-dose gamma-irradiation in the formation of mineralization nodules. Objective: To evaluate the effects of irradiation with 60Co γ-rays in low doses in the formation of mineralization nodules in culture of osteoblasts. Methods: MC3T3-E1 cells were bought by the Banco de Células do Rio de Janeiro, Brazil (MC3T3-E1 Subclone 14). The cells were cultured in α-MEM medium consisting of 10% FBS and without β-glycerophosphate and L-ascorbic acid (GIBCO, Custom Product, Catalog No. A1049001) (Zhao Y, Guan H, Liu S et al. Biol. Pharm. Bull. 2005, 28(8):1371-1376). Plating efficiency assays: cells were plated at a density of 100 cell/plate into 60 mm Petri dishes. After 14 days the places were stained with violet crystal and the colonies were counted. -glycerophosphate and 50 mg/ml ascorbic acid, and analyzed on days 7, 14 and 21. Osteoblast culture irradiation assay: cells were plated at a density of 1 x 105 cells/plate on 60 mm dishes and the next day were irradiated by 60Co source with 0 (as the control), 0.5, 1.0, 1.5 and 2.0 Gy using the GammaCell 220 - Irradiation Unit of Canadian-Atomic Energy Commission Ltd. (CTR-IPEN). On day 21 of culture, undifferentiated (without ascorbic acid and β-glycerophosphate), differentiating cells (0 Gy) and irradiated cells at different doses, the medium was removed, cells were washed with phosphate buffer saline, fixed with 70% ethyl alcohol and

  20. High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

    OpenAIRE

    Kongsfelt, Iben Boutrup; Byskov, Kristina; Pedersen, Lasse Ebdrup; Pedersen, Lene

    2014-01-01

    The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expre...

  1. Down-Regulation by Resveratrol of Basic Fibroblast Growth Factor-Stimulated Osteoprotegerin Synthesis through Suppression of Akt in Osteoblasts

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    Gen Kuroyanagi

    2014-10-01

    Full Text Available It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2 on osteoprotegerin (OPG synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK, and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation.

  2. Innovative biodegradable poly(L-lactide/collagen/hydroxyapatite composite fibrous scaffolds promote osteoblastic proliferation and differentiation

    Directory of Open Access Journals (Sweden)

    Zhou GQ

    2017-10-01

    Full Text Available Guoqiang Zhou,1–3 Sudan Liu,1 Yanyan Ma,1 Wenshi Xu,1 Wei Meng,1 Xue Lin,1 Wenying Wang,1,3 Shuxiang Wang,1–3 Jinchao Zhang1–3 1College of Chemistry and Environmental Science, 2Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, 3Key Laboratory of Chemical Biology of Hebei Province, Hebei University, Baoding, Hebei, People’s Republic of China Abstract: The development of an artificial bone graft which can promote the regeneration of fractures or diseased bones is currently the most challenging aspect in bone tissue engineering. To achieve the purpose of promoting bone proliferation and differentiation, the artificial graft needs have a similar structure and composition of extracellular matrix. One-step electrospinning method of biocomposite nanofibers containing hydroxyapatite (HA nanoparticles and collagen (Coll were developed for potential application in bone tissue engineering. Nanocomposite scaffolds of poly(L-lactide (PLLA, PLLA/HA, PLLA/Coll, and PLLA/Coll/HA were fabricated by electrospinning. The morphology, diameter, elements, hydrophilicity, and biodegradability of the composite scaffolds have been investigated. The biocompatibility of different nanocomposite scaffolds was assessed using mouse osteoblasts MC3T3-E1 in vitro, and the proliferation, differentiation, and mineralization of cells on different nanofibrous scaffolds were investigated. The results showed that PLLA/Coll/HA nanofiber scaffolds enhanced cell adhesion, spreading, proliferation, differentiation, mineralization, and gene expression of osteogenic markers compared to other scaffolds. In addition, the nanofibrous scaffolds maintained a stable composition at the beginning of the degradation period and morphology wastage and weight loss were observed when incubated for up to 80 days in physiological simulated conditions. The PLLA/Coll/HA composite nanofibrous scaffolds could be a potential material for guided bone regeneration

  3. Anti-osteoporotic activity of harpagide by regulation of bone formation in osteoblast cell culture and ovariectomy-induced bone loss mouse models.

    Science.gov (United States)

    Chung, Hwa-Jin; Kyung Kim, Won; Joo Park, Hyen; Cho, Lan; Kim, Me-Riong; Kim, Min Jeong; Shin, Joon-Shik; Ho Lee, Jin; Ha, In-Hyuk; Kook Lee, Sang

    2016-02-17

    Harpagide, an iridoid glucoside, is a constituent of the root of Harpagophytum procumbens var. sublobatum (Engl.) Stapf, Devil's claw which has been used in patients with osteoarthritis (OA). In the present study, we investigated the anti-osteoporotic potential of harpagide and its underlying mechanism of action in in vitro cell culture and in vivo bone loss animal models. Harpagide was obtained from the alkalic hydrolysis of harpagoside, a major constituent of H. procumbens var. sublobatum Analysis of biomarkers for bone formation in osteoblastic MC3T3-E1 cells and bone resorption in osteoclast cells derived from mouse bone marrow cells was performed to evaluate the mechanism of action. The protective activity of harpagide against bone loss was also evaluated in ovariectomized (OVX) mouse model. Harpagide improved bone properties by stimulating the process of differentiation and maturation of osteoblast cells and suppressing the process of RANKL-induced differentiation of osteoclast cells. In OVX-induced bone loss mouse model, oral administration of harpagide significantly improved recovery of bone mineral density, trabecular bone volume, and trabecular number in the femur. Harpagide also prevented increase of trabecular separation and structure model index induced by OVX. Harpagide effectively inhibited the serum levels of biochemical markers of bone loss, including alkaline phosphatase, osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase. Taken together, the present study demonstrates that harpagide has a potential for prevention of bone loss in OVX mice by regulating the stimulation of osteoblast differentiation and the suppression of osteoclast formation. Therefore, these findings suggest that harpagide might serve as a bioactive compound derived from H. procumbens var. sublobatum for improvement of age-dependent bone destruction disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  4. Impact of isolation method on doubling time and the quality of chondrocyte and osteoblast differentiated from murine dental pulp stem cells

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    Rohaya Megat Abdul Wahab

    2017-06-01

    Full Text Available Background Stem cells are normally isolated from dental pulps using the enzymatic digestion or the outgrowth method. However, the effects of the isolation method on the quality of the isolated stem cells are not studied in detail in murine models. The aim of this study was to compare the matrices secreted by osteoblast and chondrocytes differentiated from dental pulp stem cells isolated through different means. Method DPSC from murine incisors were isolated through either the outgrowth (DPSC-OG or the enzymatic digestion (DPSC-ED method. Cells at passage 4 were used in this study. The cells were characterized through morphology and expression of cell surface markers. The cells’ doubling time when cultured using different seeding densities was calculated and analyzed using one-way ANOVA and Tukey’s multiple comparison post-test. The ability of cells to differentiate to chondrocyte and osteoblast was evaluated through staining and analysis on the matrices secreted. Results Gene expression analysis showed that DPSC-OG and DPSC-ED expressed dental pulp mesenchymal stem cell markers, but not hematopoietic stem cell markers. The least number of cells that could have been used to culture DPSC-OG and DPSC-ED with the shortest doubling time was 5 × 102 cells/cm2 (11.49 ± 2.16 h and 1 × 102 cells/cm2 (10.55 h ± 0.50, respectively. Chondrocytes differentiated from DPSC-ED produced  2 times more proteoglycan and at a faster rate than DPSC-OG. FTIR revealed that DPSC-ED differentiated into osteoblast also secreted matrix, which more resembled a calvaria. Discussion Isolation approaches might have influenced the cell populations obtained. This, in turn, resulted in cells with different proliferation and differentiation capability. While both DPSC-OG and DPSC-ED expressed mesenchymal stem cell markers, the percentage of cells carrying each marker might have differed between the two methods. Regardless, enzymatic digestion clearly yielded cells

  5. Mechanisms involved in regulation of osteoclastic differentiation by mechanical stress-loaded osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Kaneuji, Takeshi [Division of Oral and Maxillofacial Reconstructive Surgery, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Ariyoshi, Wataru; Okinaga, Toshinori; Toshinaga, Akihiro [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Takahashi, Tetsu [Division of Oral and Maxillofacial Reconstructive Surgery, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Nishihara, Tatsuji, E-mail: tatsujin@kyu-dent.ac.jp [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan)

    2011-04-29

    Highlights: {yields} Effect of compressive force on osteoblasts were examined. {yields} Compressive force induced OPG expression and suppressed osteoclastogenesis. {yields} This enhancement of OPG is dependent on Wnt/Ca2+ signal pathway. -- Abstract: Mechanical stress is known to be important for regulation of bone turnover, though the detailed mechanisms are not fully understood. In the present study, we examined the effect of mechanical stress on osteoblasts using a novel compression model. Mouse osteoblastic MC3T3-E1 cells were embedded in three-dimensional (3D) gels and cultured with continuous compressive force (0-10.0 g/cm{sup 2}) for 48 h, and the conditioned medium were collected. RAW264.7 cells were then incubated with the conditioned medium for various times in the presence of receptor activator of nuclear factor-{kappa}B ligand (RANKL). Conditioned medium was found to inhibit the differentiation of RAW264.7 cells into osteoclasts induced by RANKL via down-regulation of the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of I{kappa}B{alpha}, and nuclear translocation of p50 and p65. Interestingly, the conditioned medium also had a high level of binding activity to RANKL and blocked the binding of RANK to RANKL. Furthermore, the binding activity of conditioned medium to RANKL was reduced when the 3D gel was supplemented with KN-93, an inhibitor of non-canonical Wnt/Ca{sup 2+} pathway. In addition, expression level of osteoprotegerin (OPG) mRNA was increased in time- and force-dependent manners, and remarkably suppressed by KN-93. These results indicate that osteoblastic cells subjected to mechanical stress produce OPG, which binds to RANKL. Furthermore, this binding activity strongly inhibited osteoclastogenesis through suppression of TRAF6 and the nuclear factor-kappa B (NF-{kappa}B) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt

  6. Peroxisomes in Different Skeletal Cell Types during Intramembranous and Endochondral Ossification and Their Regulation during Osteoblast Differentiation by Distinct Peroxisome Proliferator-Activated Receptors.

    Directory of Open Access Journals (Sweden)

    Guofeng Qian

    Full Text Available Ossification defects leading to craniofacial dysmorphism or rhizomelia are typical phenotypes in patients and corresponding knockout mouse models with distinct peroxisomal disorders. Despite these obvious skeletal pathologies, to date no careful analysis exists on the distribution and function of peroxisomes in skeletal tissues and their alterations during ossification. Therefore, we analyzed the peroxisomal compartment in different cell types of mouse cartilage and bone as well as in primary cultures of calvarial osteoblasts. The peroxisome number and metabolism strongly increased in chondrocytes during endochondral ossification from the reserve to the hypertrophic zone, whereas in bone, metabolically active osteoblasts contained a higher numerical abundance of this organelle than osteocytes. The high abundance of peroxisomes in these skeletal cell types is reflected by high levels of Pex11β gene expression. During culture, calvarial pre-osteoblasts differentiated into secretory osteoblasts accompanied by peroxisome proliferation and increased levels of peroxisomal genes and proteins. Since many peroxisomal genes contain a PPAR-responsive element, we analyzed the gene expression of PPARɑ/ß/ɣ in calvarial osteoblasts and MC3T3-E1 cells, revealing higher levels for PPARß than for PPARɑ and PPARɣ. Treatment with different PPAR agonists and antagonists not only changed the peroxisomal compartment and associated gene expression, but also induced complex alterations of the gene expression patterns of the other PPAR family members. Studies in M3CT3-E1 cells showed that the PPARß agonist GW0742 activated the PPRE-mediated luciferase expression and up-regulated peroxisomal gene transcription (Pex11, Pex13, Pex14, Acox1 and Cat, whereas the PPARß antagonist GSK0660 led to repression of the PPRE and a decrease of the corresponding mRNA levels. In the same way, treatment of calvarial osteoblasts with GW0742 increased in peroxisome number and

  7. Understanding improved osteoblast behavior on select nanoporous anodic alumina

    Directory of Open Access Journals (Sweden)

    Ni S

    2014-07-01

    Full Text Available Siyu Ni,1 Changyan Li,1 Shirong Ni,2 Ting Chen,1 Thomas J Webster3,4 1College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, People’s Republic of China; 2Department of Pathophysiology, Wenzhou Medical University, Wenzhou, People’s Republic of China; 3Department of Chemical Engineering, College of Engineering, Northeastern University, Boston, MA, USA; 4Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia Abstract: The aim of this study was to prepare different sized porous anodic alumina (PAA and examine preosteoblast (MC3T3-E1 attachment and proliferation on such nanoporous surfaces. In this study, PAA with tunable pore sizes (25 nm, 50 nm, and 75 nm were fabricated by a two-step anodizing procedure in oxalic acid. The surface morphology and elemental composition of PAA were characterized by field emission scanning electron microscopy and X-ray photoelectron spectroscopy analysis. The nanopore arrays on all of the PAA samples were highly regular. X-ray photoelectron spectroscopy analysis suggested that the chemistry of PAA and flat aluminum surfaces were similar. However, contact angles were significantly greater on all of the PAA compared to flat aluminum substrates, which consequently altered protein adsorption profiles. The attachment and proliferation of preosteoblasts were determined for up to 7 days in culture using field emission scanning electron microscopy and a Cell Counting Kit-8. Results showed that nanoporous surfaces did not enhance initial preosteoblast attachment, whereas preosteoblast proliferation dramatically increased when the PAA pore size was either 50 nm or 75 nm compared to all other samples (P<0.05. Thus, this study showed that one can alter surface energy of aluminum by modifying surface nano-roughness alone (and not changing chemistry through an anodization process to improve osteoblast density, and, thus, should be

  8. Sirtuin 1 regulates matrix metalloproteinase-13 expression induced by Porphyromonas endodontalis lipopolysaccharide via targeting nuclear factor–κB in osteoblasts

    Science.gov (United States)

    Qu, Liu; Yu, Yaqiong; Qiu, Lihong; Yang, Di; Yan, Lu; Guo, Jiajie; Jahan, Rabita

    2017-01-01

    ABSTRACT Porphyromonas endodontalis lipopolysaccharide (P.e LPS) is an important initiating factor for periapical inflammation and bone destruction. Matrix metalloproteinase-13 (MMP-13) has been shown to participate in the formation and diffusion of periapical bone lesion in chronic apical periodontitis. Sirtuin 1 (SIRT1) is a key regulator of inflammation in mammalian cells which suppresses the release of inflammatory mediators. This study aimed to explore the role of SIRT1 in regulating MMP-13 expression induced by P.e LPS in osteoblasts. P.e LPS stimulated MMP-13 expression in MC3T3-E1 cells. Knockdown of SIRT1 reinforced the increase of MMP-13mRNA expression induced by P.e LPS. SIRT1 activator resveratrol significantly reduced the expression of MMP-13 and SIRT1 inhibitor EX-527 enhanced the expression of MMP-13. Moreover, SIRT1 activation with resveratrol inhibited acetylation of NF-κB p65 and NF-κB transcriptional activity, which were enhanced by P.e LPS. In addition, NF-κB p65 was involved in P.e LPS-induced MMP-13 expression via directly binding to the MMP-13 promoter. However, SIRT1 activation significantly interfered with this binding. These findings strongly suggest that P.e LPS induces MMP-13 expression in osteoblasts, and SIRT1 suppresses this expression of MMP-13 through targeting NF-κB p65. This provides new insights into understanding the actions of SIRT1 on anti-inflammatory and anti-bone resorption activity. PMID:28473882

  9. Sirtuin 1 regulates matrix metalloproteinase-13 expression induced by Porphyromonas endodontalis lipopolysaccharide via targeting nuclear factor-κB in osteoblasts.

    Science.gov (United States)

    Qu, Liu; Yu, Yaqiong; Qiu, Lihong; Yang, Di; Yan, Lu; Guo, Jiajie; Jahan, Rabita

    2017-01-01

    Porphyromonas endodontalis lipopolysaccharide (P.e LPS) is an important initiating factor for periapical inflammation and bone destruction. Matrix metalloproteinase-13 (MMP-13) has been shown to participate in the formation and diffusion of periapical bone lesion in chronic apical periodontitis. Sirtuin 1 (SIRT1) is a key regulator of inflammation in mammalian cells which suppresses the release of inflammatory mediators. This study aimed to explore the role of SIRT1 in regulating MMP-13 expression induced by P.e LPS in osteoblasts. P.e LPS stimulated MMP-13 expression in MC3T3-E1 cells. Knockdown of SIRT1 reinforced the increase of MMP-13mRNA expression induced by P.e LPS. SIRT1 activator resveratrol significantly reduced the expression of MMP-13 and SIRT1 inhibitor EX-527 enhanced the expression of MMP-13. Moreover, SIRT1 activation with resveratrol inhibited acetylation of NF-κB p65 and NF-κB transcriptional activity, which were enhanced by P.e LPS. In addition, NF-κB p65 was involved in P.e LPS-induced MMP-13 expression via directly binding to the MMP-13 promoter. However, SIRT1 activation significantly interfered with this binding. These findings strongly suggest that P.e LPS induces MMP-13 expression in osteoblasts, and SIRT1 suppresses this expression of MMP-13 through targeting NF-κB p65. This provides new insights into understanding the actions of SIRT1 on anti-inflammatory and anti-bone resorption activity.

  10. Influence of nanostructural environment and fluid flow on osteoblast-like cell behavior: a model for cell-mechanics studies.

    Science.gov (United States)

    Prodanov, L; Semeins, C M; van Loon, J J W A; te Riet, J; Jansen, J A; Klein-Nulend, J; Walboomers, X F

    2013-05-01

    Introducing nanoroughness on various biomaterials has been shown to profoundly effect cell-material interactions. Similarly, physical forces act on a diverse array of cells and tissues. Particularly in bone, the tissue experiences compressive or tensile forces resulting in fluid shear stress. The current study aimed to develop an experimental setup for bone cell behavior, combining a nanometrically grooved substrate (200 nm wide, 50 nm deep) mimicking the collagen fibrils of the extracellular matrix, with mechanical stimulation by pulsatile fluid flow (PFF). MC3T3-E1 osteoblast-like cells were assessed for morphology, expression of genes involved in cell attachment and osteoblastogenesis and nitric oxide (NO) release. The results showed that both nanotexture and PFF did affect cellular morphology. Cells aligned on nanotexture substrate in a direction parallel to the groove orientation. PFF at a magnitude of 0.7 Pa was sufficient to induce alignment of cells on a smooth surface in a direction perpendicular to the applied flow. When environmental cues texture and flow were interacting, PFF of 1.4 Pa applied parallel to the nanogrooves initiated significant cellular realignment. PFF increased NO synthesis 15-fold in cells attached to both smooth and nanotextured substrates. Increased collagen and alkaline phosphatase mRNA expression was observed on the nanotextured substrate, but not on the smooth substrate. Furthermore, vinculin and bone sialoprotein were up-regulated after 1 h of PFF stimulation. In conclusion, the data show that interstitial fluid forces and structural cues mimicking extracellular matrix contribute to the final bone cell morphology and behavior, which might have potential application in tissue engineering. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  11. Chitosan scaffold modified with D-(+) raffinose and enriched with thiol-modified gelatin for improved osteoblast adhesion

    International Nuclear Information System (INIS)

    Galli, C; Parisi, L; Smerieri, A; Lumetti, S; Manfredi, E; Macaluso, G M; Elviri, L; Bianchera, A; Bettini, R; Lagonegro, P

    2016-01-01

    The aim of the present study was to investigate whether chitosan-based scaffolds modified with D-(+) raffinose and enriched with thiol-modified gelatin could selectively improve osteoblast adhesion and proliferation. 2, 3 and 4.5% chitosan films were prepared. Chitosan suitability for tissue engineering was confirmed by protein adsorption assay. Scaffolds were incubated with a 2.5 mg ml −1 BSA solution and the decrease of protein content in the supernatants was measured by spectrophotometry. Chitosan films were then enriched with thiol-modified gelatin and their ability to bind BSA was also measured. Then, 2% chitosan discs with or without thiol-modified gelatin were used as culture substrates for MC3T3-E1 cells. After 72 h cells were stained with trypan blue or with calcein AM and propidium iodide for morphology, viability and proliferation assays. Moreover, cell viability was measured at 48, 72, 96 and 168 h to obtain a growth curve. Chitosan films efficiently bound and retained BSA proportionally to the concentration of chitosan discs. The amount of protein retained was higher on chitosan enriched with thiol-modified gelatin. Moreover, chitosan discs allowed the adhesion and the viability of cells, but inhibited their proliferation. The functionalization of chitosan with thiol-modified gelatin enhanced cell spreading and proliferation. Our data confirm that chitosan is a suitable material for tissue engineering. Moreover, our data show that the enrichment of chitosan with thiol-modified gelatin enhances its biological properties. (paper)

  12. Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts

    International Nuclear Information System (INIS)

    Wan, Qiaoqiao; Cho, Eunhye; Yokota, Hiroki; Na, Sungsoo

    2013-01-01

    Highlights: •Shear stress increased TCF/LEF activity and stimulated β-catenin nuclear localization. •Rac1, Cdc42, and RhoA displayed distinct dynamic activity patterns under flow. •Rac1 and Cdc42, but not RhoA, regulate shear stress-driven TCF/LEF activation. •Cytoskeleton did not significantly affect shear stress-induced TCF/LEF activation. -- Abstract: Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm 2 ) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate

  13. Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Qiaoqiao; Cho, Eunhye [Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 (United States); Yokota, Hiroki [Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 (United States); Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Na, Sungsoo, E-mail: sungna@iupui.edu [Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 (United States)

    2013-04-19

    Highlights: •Shear stress increased TCF/LEF activity and stimulated β-catenin nuclear localization. •Rac1, Cdc42, and RhoA displayed distinct dynamic activity patterns under flow. •Rac1 and Cdc42, but not RhoA, regulate shear stress-driven TCF/LEF activation. •Cytoskeleton did not significantly affect shear stress-induced TCF/LEF activation. -- Abstract: Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm{sup 2}) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate

  14. Low-level ultrahigh-frequency and ultrashort-pulse blue laser irradiation enhances osteoblast extracellular calcification by upregulating proliferation and differentiation via transient receptor potential vanilloid 1.

    Science.gov (United States)

    Mikami, Risako; Mizutani, Koji; Aoki, Akira; Tamura, Yukihiko; Aoki, Kazuhiro; Izumi, Yuichi

    2018-04-01

    Low-level laser irradiation (LLLI) exerts various biostimulative effects, including promotion of wound healing and bone formation; however, few studies have examined biostimulation using blue lasers. The purpose of this study was to investigate the effects of low-level ultrahigh-frequency (UHF) and ultrashort-pulse (USP) blue laser irradiation on osteoblasts. The MC3T3-E1 osteoblast cell line was used in this study. Following LLLI with a 405 nm newly developed UHF-USP blue laser (80 MHz, 100 fs), osteoblast proliferation, and alkaline phosphatase (ALP) activity were assessed. In addition, mRNA levels of the osteoblast differentiation markers, runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), and osteopontin (Opn) was evaluated, and extracellular calcification was quantified. To clarify the involvement of transient receptor potential (TRP) channels in LLLI-induced biostimulation, cells were treated prior to LLLI with capsazepine (CPZ), a selective inhibitor of TRP vanilloid 1 (TRPV1), and subsequent proliferation and ALP activity were measured. LLLI with the 405 nm UHF-USP blue laser significantly enhanced cell proliferation and ALP activity, compared with the non-irradiated control and LLLI using continuous-wave mode, without significant temperature elevation. LLLI promoted osteoblast proliferation in a dose-dependent manner up to 9.4 J/cm 2 and significantly accelerated cell proliferation in in vitro wound healing assay. ALP activity was significantly enhanced at doses up to 5.6 J/cm 2 , and expression of Osx and Alp mRNAs was significantly increased compared to that of the control on days 3 and 7 following LLLI at 5.6 J/cm 2 . The extent of extracellular calcification was also significantly higher as a result of LLLI 3 weeks after the treatment. Measurement of TRPV1 protein expression on 0, 3, and 7 days post-irradiation revealed no differences between the LLLI and control groups; however, promotion of cell

  15. Evaluation of epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes and concerns on osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Chenyu; Deng, Jia [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Xiang, Lin; Wu, Yingying [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Wei, Xiawei [State Key Laboratory of Biotherapy and Laboratory for Aging Research, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan 610041 (China); Qu, Yili [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Man, Yi, E-mail: manyi780203@126.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China)

    2016-10-01

    Collagen membranes have ideal biological and mechanical properties for supporting infiltration and proliferation of osteoblasts and play a vital role in guided bone regeneration (GBR). However, pure collagen can lead to inflammation, resulting in progressive bone resorption. Therefore, a method for regulating the level of inflammatory cytokines at surgical sites is paramount for the healing process. Epigallocatechin-3-gallate (EGCG) is a component extracted from green tea with numerous biological activities including an anti-inflammatory effect. Herein, we present a novel cross-linked collagen membrane containing different concentrations of EGCG (0.0064%, 0.064%, and 0.64%) to regulate the level of inflammatory factors secreted by pre-osteoblast cells; improve cell proliferation; and increase the tensile strength, wettability, and thermal stability of collagen membranes. Scanning electron microscope images show that the surfaces of collagen membranes became smoother and the collagen fiber diameters became larger with EGCG treatment. Measurement of the water contact angle demonstrated that introducing EGCG improved membrane wettability. Fourier transform infrared spectroscopy analyses indicated that the backbone of collagen was intact, and the thermal stability was significant improved in differential scanning calorimetry. The mechanical properties of 0.064% and 0.64% EGCG-treated collagen membranes were 1.5-fold greater than those of the control. The extent of cross-linking was significantly increased, as determined by a 2,4,6-trinitrobenzenesulfonic acid solution assay. The Cell Counting Kit-8 (CCK-8) and live/dead assays revealed that collagen membrane cross-linked by 0.0064% EGCG induced greater cell proliferation than pure collagen membranes. Additionally, real-time polymerase chain reaction and enzyme-linked immunosorbent assay results showed that EGCG significantly affected the production of inflammatory factors secreted by MC3T3-E1 cells. Taken together, our

  16. Hierarchical micro-nano structured Ti6Al4V surface topography via two-step etching process for enhanced hydrophilicity and osteoblastic responses.

    Science.gov (United States)

    Moon, Byeong-Seok; Kim, Sungwon; Kim, Hyoun-Ee; Jang, Tae-Sik

    2017-04-01

    Hierarchical micro-nano (HMN) surface structuring of dental implants is a fascinating strategy for achieving fast and mechanically stable fixation due to the synergetic effect of micro- and nano-scale surface roughness with surrounding tissues. However, the introduction of a well-defined nanostructure on a microstructure having complex surface geometry is still challenging. As a means of fabricating HMN surface on Ti6Al4V-ELI, target-ion induced plasma sputtering (TIPS) was used onto a sand-blasted, large-grit and acid-etched substrate. The HMN surface topography was simply controlled by adjusting the tantalum (Ta) target power of the TIPS technique, which is directly related to the Ta ion flux and the surface chemical composition of the substrate. Characterization using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and laser scanning microscopy (LSM) verified that well-defined nano-patterned surface structures with a depth of ~300 to 400nm and a width of ~60 to 70nm were uniformly distributed and followed the complex micron-sized surface geometry. In vitro cellular responses of pre-osteoblast cells (MC3T3-E1) were assessed by attachment and proliferation of cells on flat, nano-roughened, micro-roughened, and an HMN surface structure of Ti6Al4V-ELI. Moreover, an in vivo dog mandible defect model study was used to investigate the biological effect of the HMN surface structure compared with the micro-roughened surface. The results showed that the surface nanostructure significantly increased the cellular activities of flat and micro-roughened Ti, and the bone-to-implant contact area and new bone volume were significantly improved on the HMN surface structured Ti. These results support the idea that an HMN surface structure on Ti6Al4V-ELI alloy has great potential for enhancing the biological performance of dental implants. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. [Effects of lipopolysaccharides from Porphyromonas endodontalis on the expression of MIP-1α in mouse osteoblasts].

    Science.gov (United States)

    Yu, Ya-Qiong; Li, Xiao-Lin; Qiu, Li-Hong; Guo, Jia-Jie; Yang, Di; Guo, Yan

    2017-06-01

    To investigate the effects of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of macrophageinflammatoryprotein-1α (MIP-1α) mRNA and protein levels in MC3T3-E1 cells and the influence of curcumin in the process. MC3T3-E1 cells were treated with 20 mg/L P.e-LPS for different times (0-48 h). The expression of MIP-1α mRNA and protein was detected by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) and enzyme linked immunosorbent assay(ELISA). MC3T3-E1 cells were pretreated with inhibitor of (curcumin) for 1 h, and then treated with 20 mg/L P.e-LPS. The expression of MIP-1α was also detected by real-time RT-PCR and ELISA.Statistical analysis was performed using one-way ANOVA and Dunnett's t test with SPSS 13.0 software package. In the observation time (0-48 h), the impact of 20 P.e-LPS mg/L on induction of MIP-1α in MC3T3-El cells exhibited a time-dependent manner. The expression of MIP-1α mRNA and protein decreased significantly after pretreatment with 10 μmol/L curcumin for 1 h. The results suggest that P.e-LPS may mediate MIP-1α expression in MC3T3-E1 cells, and curcumin has a significant inhibitory effect on this process.

  18. Osteoblast interaction with laser cladded HA and SiO2-HA coatings on Ti-6Al-4V

    International Nuclear Information System (INIS)

    Yang Yuling; Serpersu, Kaan; He Wei; Paital, Sameer R.; Dahotre, Narendra B.

    2011-01-01

    In order to improve the bioactivity and biocompatibility of titanium endosseous implants, the morphology and composition of the surfaces were modified. Polished Ti-6Al-4V substrates were coated by a laser cladding process with different precursors: 100 wt.% HA and 25 wt.% SiO 2 -HA. X-ray diffraction of the laser processed samples showed the presence of CaTiO 3 , Ca 3 (PO 4 ) 2 , and Ca 2 SiO 4 phases within the coatings. From in vitro studies, it was observed that compared to the unmodified substrate all laser cladded samples presented improved cellular interactions and bioactivity. The samples processed with 25 wt.% SiO 2 -HA precursor showed a significantly higher HA precipitation after immersion in simulated body fluid than 100 wt.% HA precursor and titanium substrates. The in vitro biocompatibility of the laser cladded coatings and titanium substrate was investigated by culturing of mouse MC3T3-E1 pre-osteoblast cell line and analyzing the cell viability, cell proliferation, and cell morphology. A significantly higher cell attachment and proliferation rate were observed for both laser cladded 100 wt.% HA and 25 wt.% SiO 2 -HA samples. Compared to 100 wt.% HA sample, 25 wt.% SiO 2 -HA samples presented a slightly improved cellular interaction due to the addition of SiO 2 . The staining of the actin filaments showed that the laser cladded samples induced a normal cytoskeleton and well-developed focal adhesion contacts. Scanning electron microscopic image of the cell cultured samples revealed better cell attachment and spreading for 25 wt.% SiO 2 -HA and 100 wt.% HA coatings than titanium substrate. These results suggest that the laser cladding process improves the bioactivity and biocompatibility of titanium. The observed biological improvements are mainly due to the coating induced changes in surface chemistry and surface morphology. Highlights: → Laser cladding of Ti alloys with bioceramics creates new phases. → Laser cladded samples with SiO 2 -doped

  19. Osteoblast interaction with laser cladded HA and SiO{sub 2}-HA coatings on Ti-6Al-4V

    Energy Technology Data Exchange (ETDEWEB)

    Yang Yuling [Department of Physics, Northeastern University, Shenyang 110004 (China); Department of Materials Science and Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Serpersu, Kaan [Department of Materials Science and Engineering, University of Tennessee, Knoxville, TN 37996 (United States); He Wei, E-mail: whe5@utk.edu [Department of Materials Science and Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Department of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Paital, Sameer R. [Department of Materials Science and Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Dahotre, Narendra B. [Department of Materials Science and Engineering, University of North Texas, Denton, TX 76207 (United States)

    2011-12-01

    In order to improve the bioactivity and biocompatibility of titanium endosseous implants, the morphology and composition of the surfaces were modified. Polished Ti-6Al-4V substrates were coated by a laser cladding process with different precursors: 100 wt.% HA and 25 wt.% SiO{sub 2}-HA. X-ray diffraction of the laser processed samples showed the presence of CaTiO{sub 3}, Ca{sub 3}(PO{sub 4}){sub 2}, and Ca{sub 2}SiO{sub 4} phases within the coatings. From in vitro studies, it was observed that compared to the unmodified substrate all laser cladded samples presented improved cellular interactions and bioactivity. The samples processed with 25 wt.% SiO{sub 2}-HA precursor showed a significantly higher HA precipitation after immersion in simulated body fluid than 100 wt.% HA precursor and titanium substrates. The in vitro biocompatibility of the laser cladded coatings and titanium substrate was investigated by culturing of mouse MC3T3-E1 pre-osteoblast cell line and analyzing the cell viability, cell proliferation, and cell morphology. A significantly higher cell attachment and proliferation rate were observed for both laser cladded 100 wt.% HA and 25 wt.% SiO{sub 2}-HA samples. Compared to 100 wt.% HA sample, 25 wt.% SiO{sub 2}-HA samples presented a slightly improved cellular interaction due to the addition of SiO{sub 2}. The staining of the actin filaments showed that the laser cladded samples induced a normal cytoskeleton and well-developed focal adhesion contacts. Scanning electron microscopic image of the cell cultured samples revealed better cell attachment and spreading for 25 wt.% SiO{sub 2}-HA and 100 wt.% HA coatings than titanium substrate. These results suggest that the laser cladding process improves the bioactivity and biocompatibility of titanium. The observed biological improvements are mainly due to the coating induced changes in surface chemistry and surface morphology. Highlights: {yields} Laser cladding of Ti alloys with bioceramics creates new

  20. New cyclic peptides with osteoblastic proliferative activity from Dianthus superbus.

    Science.gov (United States)

    Tong, Yun; Luo, Jian-Guang; Wang, Rui; Wang, Xiao-Bing; Kong, Ling-Yi

    2012-03-01

    Two new cyclic peptides, dianthins G-H (1 and 2), together with the known dianthin E (3), were isolated from the traditional Chinese medicinal plant Dianthus superbus. The sequences of cyclic peptides 1 and 2 were elucidated as cyclo (-Gly(1)-Pro(2)-Leu(3)-Thr(4)-Leu(5)-Phe(6)-) and cyclo (-Gly(1)-Pro(2)-Val(3)-Thr(4)-Ile(5)-Phe(6)-), on the basis of ESI tandem mass fragmentation analysis, extensive 2D NMR methods and X-ray diffraction. The isolated three compounds all increase proliferation of MC3T3-E1 cells in vitro using MTT method. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Improved biocompatibility of poly (styrene-b-(ethylene-co-butylene)-b-styrene) elastomer by a surface graft polymerization of hyaluronic acid.

    Science.gov (United States)

    Li, Xiaomeng; Luan, Shifang; Shi, Hengchong; Yang, Huawei; Song, Lingjie; Jin, Jing; Yin, Jinghua; Stagnaro, Paola

    2013-02-01

    Hyaluronic acid (HA) is an important component of extracellular matrix (ECM) in many tissues, providing a hemocompatible and supportive environment for cell growth. In this study, glycidyl methacrylate-hyaluronic acid (GMHA) was first synthesized and verified by proton nuclear magnetic resonance ((1)H NMR) spectroscopy. GMHA was then grafted to the surface of biomedical elastomer poly (styrene-b-(ethylene-co-butylene)-b-styrene) (SEBS) via an UV-initiated polymerization, monitored by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). The further improvement of biocompatibility of the GMHA-modified SEBS films was assessed by platelet adhesion experiments and in vitro response of murine osteoblastic cell line MC-3T3-E1 with the virgin SEBS surface as the reference. It showed that the surface modification with HA strongly resisted platelet adhesion whereas improved cell-substrate interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Hierarchical micro-nano structured Ti6Al4V surface topography via two-step etching process for enhanced hydrophilicity and osteoblastic responses

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Byeong-Seok; Kim, Sungwon; Kim, Hyoun-Ee; Jang, Tae-Sik, E-mail: cgamja@snu.ac.kr

    2017-04-01

    Hierarchical micro-nano (HMN) surface structuring of dental implants is a fascinating strategy for achieving fast and mechanically stable fixation due to the synergetic effect of micro- and nano-scale surface roughness with surrounding tissues. However, the introduction of a well-defined nanostructure on a microstructure having complex surface geometry is still challenging. As a means of fabricating HMN surface on Ti6Al4V-ELI, target-ion induced plasma sputtering (TIPS) was used onto a sand-blasted, large-grit and acid-etched substrate. The HMN surface topography was simply controlled by adjusting the tantalum (Ta) target power of the TIPS technique, which is directly related to the Ta ion flux and the surface chemical composition of the substrate. Characterization using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and laser scanning microscopy (LSM) verified that well-defined nano-patterned surface structures with a depth of ~ 300 to 400 nm and a width of ~ 60 to 70 nm were uniformly distributed and followed the complex micron-sized surface geometry. In vitro cellular responses of pre-osteoblast cells (MC3T3-E1) were assessed by attachment and proliferation of cells on flat, nano-roughened, micro-roughened, and an HMN surface structure of Ti6Al4V-ELI. Moreover, an in vivo dog mandible defect model study was used to investigate the biological effect of the HMN surface structure compared with the micro-roughened surface. The results showed that the surface nanostructure significantly increased the cellular activities of flat and micro-roughened Ti, and the bone-to-implant contact area and new bone volume were significantly improved on the HMN surface structured Ti. These results support the idea that an HMN surface structure on Ti6Al4V-ELI alloy has great potential for enhancing the biological performance of dental implants. - Highlights: • A micro-nano-hierarchical (MNH) surface structure on Ti6Al4V-ELI was fabricated via TIPS

  3. Hierarchical micro-nano structured Ti6Al4V surface topography via two-step etching process for enhanced hydrophilicity and osteoblastic responses

    International Nuclear Information System (INIS)

    Moon, Byeong-Seok; Kim, Sungwon; Kim, Hyoun-Ee; Jang, Tae-Sik

    2017-01-01

    Hierarchical micro-nano (HMN) surface structuring of dental implants is a fascinating strategy for achieving fast and mechanically stable fixation due to the synergetic effect of micro- and nano-scale surface roughness with surrounding tissues. However, the introduction of a well-defined nanostructure on a microstructure having complex surface geometry is still challenging. As a means of fabricating HMN surface on Ti6Al4V-ELI, target-ion induced plasma sputtering (TIPS) was used onto a sand-blasted, large-grit and acid-etched substrate. The HMN surface topography was simply controlled by adjusting the tantalum (Ta) target power of the TIPS technique, which is directly related to the Ta ion flux and the surface chemical composition of the substrate. Characterization using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and laser scanning microscopy (LSM) verified that well-defined nano-patterned surface structures with a depth of ~ 300 to 400 nm and a width of ~ 60 to 70 nm were uniformly distributed and followed the complex micron-sized surface geometry. In vitro cellular responses of pre-osteoblast cells (MC3T3-E1) were assessed by attachment and proliferation of cells on flat, nano-roughened, micro-roughened, and an HMN surface structure of Ti6Al4V-ELI. Moreover, an in vivo dog mandible defect model study was used to investigate the biological effect of the HMN surface structure compared with the micro-roughened surface. The results showed that the surface nanostructure significantly increased the cellular activities of flat and micro-roughened Ti, and the bone-to-implant contact area and new bone volume were significantly improved on the HMN surface structured Ti. These results support the idea that an HMN surface structure on Ti6Al4V-ELI alloy has great potential for enhancing the biological performance of dental implants. - Highlights: • A micro-nano-hierarchical (MNH) surface structure on Ti6Al4V-ELI was fabricated via TIPS

  4. Immobilization of calcium and phosphate ions improves the osteoconductivity of titanium implants

    International Nuclear Information System (INIS)

    Sunarso; Toita, Riki; Tsuru, Kanji; Ishikawa, Kunio

    2016-01-01

    In this work, to elevate weak osteoconductivity of titanium (Ti) implant, we prepared a Ti implant having both calcium and phosphate ions on its surface. To modify calcium and phosphate ions onto Ti, phosphate ions were first immobilized by treating the Ti with a NaH 2 PO 4 solution, followed by CaCl 2 treatment to immobilize calcium ions, which created the calcium and phosphate ions-modified Ti (Ca-P-Ti). X-ray photoelectron spectroscopy and thin-layer X-ray diffraction measurement confirmed that both phosphate and calcium ions were co-immobilized onto the Ti surface on the molecular level. Three-hour after seeding MC3T3-E1 murine pre-osteoblast cells on substrates, cell number on Ca-P-Ti was much larger than that of Ti and phosphate-modified Ti (P-Ti), but was similar to that of calcium-modified Ti (Ca-Ti). Also, MC3T3-E1 cells on Ca-P-Ti expressed larger amount of vinculin, a focal adhesion protein, than those on other substrates, probably resulting in larger cell size as well as greater cell proliferation on Ca-P-Ti than those on other substrates. Alkaline phosphatase activity of cells on Ca-P-Ti was greater than those on Ti and P-Ti, but was almost comparable to that of Ca-Ti. Moreover, the largest amount of bone-like nodule formation was observed on Ca-P-Ti. These results provide evidence that calcium and phosphate ions-co-immobilization onto Ti increased the osteoconductivity of Ti by stimulating the responses of pre-osteoblast cells. This simple modification would be promising technique for bone tissue implant including dental and orthopedic implants. - Highlights: • Phosphate and calcium ions have been successfully co-immobilize on Ti surface. • Co-immobilization of Ca and phosphate ions (Ca-P-Ti) did not alter the original surface morphology. • Ca-P-Ti significantly improved initial MC3T3-E1 cell adhesion. • Ca-P-Ti demonstrated remarkable cell proliferation, differentiation and mineralization. • Overall, Ca-P-Ti would be a promising bone

  5. Involvement of integrins in the adhesion of osteoblastic cells to a type-I collagen matrix

    OpenAIRE

    Antonio Desmond McCarthy; Toshimasa Uemura; Susana Beatriz Etcheverry; Ana María Cortizo

    2006-01-01

    Se han desarrollado varios biomateriales con potencial aplicaci ón en la reconstrucción de tejidos. En este sentido, existe un creciente interés en el diseño de materiales de implante óseo con máxima biocompatibilidad y adecuada adhesividad celular. El objetivo de este trabajo fue estudiar cuáles receptores integrinas participan en la adhesión de osteoblastos a una matriz de colágeno tipo-I. Se analizaron dos líneas celulares osteoblásticas: UMR106, derivada de osteosarcoma de rata; y MC3T3E1...

  6. Templating Biomineralization: Surface Directed Protein Self-assembly and External Magnetic Field Stimulation of Osteoblasts

    Science.gov (United States)

    Ba, Xiaolan

    Biomineralization is a wide-spread phenomenon in the biological systems, which is the process of mineral formation by organisms through interaction between its organic contents and the inorganic minerals. The process is essential in a broad spectrum of biological phenomena ranging from bone and tooth formation to pathological mineralization under hypoxic conditions or cancerous formations. In this thesis I studied biomineralization at the earliest stages in order to obtain a better understanding of the fundamental principals involved. This knowledge is essential if we want to engineer devices which will increase bone regeneration or prevent unwanted mineral deposits. Extracellular matrix (ECM) proteins play an essential role during biomineralization in bone and engineered tissues. In this dissertation, I present an approach to mimic the ECM in vitro to probe the interactions of these proteins with calcium phosphate mineral and with each other. Early stage of mineralization is investigated by mechanical properties of the protein fibers using Scanning Probe Microscopy (SPM) and Shear Modulation Force Microscopy (SMFM). The development of mineral crystals on the protein matrices is also characterized by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Grazing Incidence X-ray Diffraction (GIXRD). The results demonstrate complementary actions of the two ECM proteins to collect cations and template calcium phosphate mineral, respectively. Magnets have been clinically used as an "induction source" in various bone or orthodontic treatments. However, the mechanism and effects of magnetic fields remain unclear. In this dissertation, I also undertake the present investigation to study the effects of 150 mT static magnetic fields (SMF) on ECM development and cell biomineralization using MC3T3-E1 osteobalst-like cells. Early stage of biomineralization is characterized by SPM, SMFM and confocal laser scanning microscopy (CSLM). Late stage of

  7. Effect of Modified Pectin Molecules on the Growth of Bone Cells

    NARCIS (Netherlands)

    Kokkonen, H.E.; Ilvesaro, J.M.; Morra, M.; Schols, H.A.; Tuukkanen, J.

    2007-01-01

    The aim of this study was to investigate molecular candidates for bone implant nanocoatings, which could improve biocompatibility of implant materials. Primary rat bone cells and murine preosteoblastic MC3T3-E1 cells were cultured on enzymatically modified hairy regions (MHR-A and MHR-B) of apple

  8. Protective effects of myricitrin against osteoporosis via reducing reactive oxygen species and bone-resorbing cytokines

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Qiang; Gao, Bo; Wang, Long; Hu, Ya-Qian; Lu, Wei-Guang; Yang, Liu; Luo, Zhuo-Jing; Liu, Jian, E-mail: liujianhq@sina.com

    2014-11-01

    Oxidative stress is a crucial pathogenic factor in the development of osteoporosis. Myricitrin, isolated from Myrica cerifera, is a potent antioxidant. We hypothesized that myricitrin possessed protective effects against osteoporosis by partially reducing reactive oxygen species (ROS) and bone-resorbing cytokines in osteoblastic MC3T3-E1 cells and human bone marrow stromal cells (hBMSCs). We investigated myricitrin on osteogenic differentiation under oxidative stress. Hydrogen peroxide (H{sub 2}O{sub 2}) was used to establish an oxidative cell injury model. Our results revealed that myricitrin significantly improved some osteogenic markers in these cells. Myricitrin decreased lipid production and reduced peroxisome proliferator-activated receptor gamma-2 (PPARγ2) expression in hBMSCs. Moreover, myricitrin reduced the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and IL-6 and partially suppressed ROS production. In vivo, we established a murine ovariectomized (OVX) osteoporosis model. Our results demonstrated that myricitrin supplementation reduced serum malondialdehyde (MDA) activity and increased reduced glutathione (GSH) activity. Importantly, it ameliorated the micro-architecture of trabecular bones in the 4th lumbar vertebrae (L4) and distal femur. Taken together, these results indicated that the protective effects of myricitrin against osteoporosis are linked to a reduction in ROS and bone-resorbing cytokines, suggesting that myricitrin may be useful in bone metabolism diseases, particularly osteoporosis. - Highlights: • Myricitrin protects MC3T3-E1 cells and hBMSCs from oxidative stress. • It is accompanied by a decrease in oxidative stress and bone-resorbing cytokines. • Myricitrin decreases serum reactive oxygen species to some degree. • Myricitrin partly reverses ovariectomy effects in vivo. • Myricitrin may represent a beneficial anti-osteoporosis treatment method.

  9. Deferoxamine immobilized poly(D,L-lactide) membrane via polydopamine adhesive coating: The influence on mouse embryo osteoblast precursor cells and human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Huihua [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Luo, Binghong, E-mail: tluobh@jnu.edu.cn [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Wen, Wei [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Zhou, Changren, E-mail: tcrz9@jnu.edu.cn [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Tian, Lingling [Center for Nanofibers & Nanotechnology, Department of Mechanical Engineering, National University of Singapore, Singapore 117576 (Singapore); Ramakrishna, Seeram [Center for Nanofibers & Nanotechnology, Department of Mechanical Engineering, National University of Singapore, Singapore 117576 (Singapore); Guangdong-Hongkong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou 510632 (China)

    2017-01-01

    Osteogenesis and angiogenesis play the prominent role in the bone regeneration. In this study, deferoxamine (DFO), an induced agent for osteogenesis and angiogenesis, was modified onto the surface of poly(D,L-lactide) (PDLLA) membrane via a facile and convenient approach based on the self-polymerization of dopamine (DOPA). The surface composition, morphology, hydrophilicity and surface energy of the original and modified PDLLA membranes were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electronic microscopy (SEM), atomic force microscopy (AFM) and contact angle measurement. The surface roughness and hydrophilicity of the PDLLA membrane were obviously increased by introducing either the single polydopamine (PDOPA) or the dual layers of PDOPA and DFO. In vitro cells culture experiments indicated that both the PDLLA/PDOPA and PDLLA/PDOPA-DFO composite membranes were more beneficial to the attachment, proliferation and spreading of MC3T3-E1 cells and HUVECs compared to the original PDLLA membrane. The PDLLA/PDOPA-DFO membrane was supportive for the proliferation of both MC3T3-E1 cells and HUVECs, and especially for HUVECs. The results suggested that the as-prepared PDLLA/PDOPA-DFO composite can be expected to be used as a promising bone regenerative material with promoted angiogenesis. - Highlights: • DFO was conveniently immobilized on PDLLA membrane based on PDOPA adhesive layer. • Hydrophilicity of PDLLA membrane was improved by modification with PDOPA and DFO. • Modified membranes were more favorable to the growth of MC3T3-E1 cells and HUVECs. • DFO was supportive for the growth of two kinds of cells, especially for HUVECs.

  10. The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest.

    Science.gov (United States)

    Gascoyne, Duncan M; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E; Croucher, Peter I; Banham, Alison H

    2015-01-01

    Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology.

  11. Osteoblast CFTR inactivation reduces differentiation and osteoprotegerin expression in a mouse model of cystic fibrosis-related bone disease.

    Directory of Open Access Journals (Sweden)

    Michael S Stalvey

    Full Text Available Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF. CF-related bone disease (CFBD is characterized by uncoupled bone turnover--impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR, the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr-/- mouse model. In the murine calvarial organ culture assay, Cftr-/- calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+ littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr-/- compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl mRNA was detected, significantly less osteoprotegerin (Opg was expressed in Cftr-/- compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr-/- murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt

  12. Edaravone protects osteoblastic cells from dexamethasone through inhibiting oxidative stress and mPTP opening.

    Science.gov (United States)

    Sun, Wen-xiao; Zheng, Hai-ya; Lan, Jun

    2015-11-01

    Existing evidences have emphasized an important role of oxidative stress in dexamethasone (Dex)-induced osteoblastic cell damages. Here, we investigated the possible anti-Dex activity of edaravone in osteoblastic cells, and studied the underlying mechanisms. We showed that edaravone dose-dependently attenuated Dex-induced death and apoptosis of established human or murine osteoblastic cells. Further, Dex-mediated damages to primary murine osteoblasts were also alleviated by edaravone. In osteoblastic cells/osteoblasts, Dex induced significant oxidative stresses, tested by increased levels of reactive oxygen species and lipid peroxidation, which were remarkably inhibited by edaravone. Meanwhile, edaravone repressed Dex-induced mitochondrial permeability transition pore (mPTP) opening, or mitochondrial membrane potential reduction, in osteoblastic cells/osteoblasts. Significantly, edaravone-induced osteoblast-protective activity against Dex was alleviated with mPTP inhibition through cyclosporin A or cyclophilin-D siRNA. Together, we demonstrate that edaravone protects osteoblasts from Dex-induced damages probably through inhibiting oxidative stresses and following mPTP opening.

  13. Development of Flexible Capacitive Ultrasound Transducers and the Use of Ultrasound for Bone Repair

    Science.gov (United States)

    Wentzell, Scott A.

    Ultrasound is a widely applicable technique for therapy in the biomedical arena. However, conventional ultrasound transducers are not conducive for non-planar surfaces. Therefore, we developed flexible transducers capable of performing ultrasound and evaluated their use in biomedical applications. Flexible capacitive ultrasound transducers based on micrometer-thick dielectric tapes were developed and fabricated. These transducers were able to be made by hand at low-cost while still demonstrating good tolerances in center operating frequency. Intensities of up to 120 mW/cm2 were recorded and operation was dependent upon the applied AC and DC voltages along with the thickness of the dielectric insulation. These capacitive ultrasound transducers were used to stimulate MC3T3-E1 murine osteoblast cells to investigate the effects of low-frequency ultrasound on osteogenic gene expression and anabolic signaling pathways. After stimulation by 94.5 kHz continuous wave ultrasound for 20 minutes, significant increases in the activation of the Wnt signaling pathway and concentration of intracellular calcium were observed. Daily stimulation by ultrasound showed a trend of increased osteogenic gene expression across the phases of matrix deposition, maturation and calcification by osteoblasts. Finally, the heating of osteoblasts for stimulating osteoclastogenic responses was investigated. The application of increased temperatures of 42 and 47 degrees Celsius for 5 minutes showed significant increases in the RANKL/OPG ratio in media conditioned by osteoblasts. However, the altered RANKL/OPG ratio was not able to generate increases in osteoclastogenesis for RAW 264.7 murine macrophage cells culture in the condition media. This was possibly due to high overall osteoprotegerin expression, or unwanted inducement of M1 and M2 macrophage activation in the cell population. The overall work of this thesis demonstrates the development of novel capacitive transducers. These conformable

  14. Aluminum Silicate Nanotube Coating of Siloxane-Poly(lactic acid-Vaterite Composite Fibermats for Bone Regeneration

    Directory of Open Access Journals (Sweden)

    Shuji Yamazaki

    2012-01-01

    Full Text Available In our earlier work, a flexible fibermat consisting of a biodegradable composite with soluble silicate species, which has been reported to enhance bone formation, was prepared successfully using poly(L-lactic acid and siloxane-containing calcium carbonate particles by electrospinning. The fibermat showed enhanced bone formation in an in vivo test. In the present work, to improve the hydrophilicity of skeletal fibers in a fibermat, they were coated with nanotubular aluminum silicate crystals, which have a hydrophilic surface that has excellent affinity to body fluids and a high surface area advantageous for pronounced protein adsorption. The nanotubes were coated easily on the fiber surface using an electrophoretic method. In a conventional contact angle test, a drop of water rapidly penetrated into the nanotube-coated fibermat. The culture test using murine osteoblast-like cells (MC3T3-E1 showed that the cell attachment to the nanotube-coated fibermat at an early stage after seeding was enhanced in comparison with that to the noncoated one. This approach may provide a new method of improving the surface of polymer-based biomaterials.

  15. High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

    DEFF Research Database (Denmark)

    Kongsfelt, Iben Boutrup; Byskov, Kristina; Pedersen, Lasse Ebdrup

    2014-01-01

    overexpression led to faster cell spreading. The final total numbers of attached cells did, however, not differ between cultures of PiT1 overexpressing cells and control cells of neither cell type. We suggest that the PiT1-mediated fast adhesion potentials allow the cells to go faster out of G0/G1 and thereby......The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells......, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expression supports that cells utilize the PiT1 levels to control proliferation, with non-proliferating cells showing...

  16. Expression and Dynamics of Podoplanin in Cultured Osteoblasts with Mechanostress and Mineralization Stimulus.

    Science.gov (United States)

    Takenawa, Tomohiro; Kanai, Takenori; Kitamura, Tetsuya; Yoshimura, Yoshitaka; Sawa, Yoshihiko; Iida, Junichiro

    2018-02-27

    This study investigates the significance of the expression and dynamics of podoplanin in mechanostress and mineralization in cultured murine osteoblasts. Podoplanin increased in osteoblasts subjected to straining in non-mineralization medium, suggesting that the mechanostress alone is a podoplanin induction factor. In osteoblasts subjected to vertical elongation straining in the mineralization medium, the mRNA amounts of podoplanin, osteopontin, and osteocalcin were significantly larger than those in cells not subjected to straining, suggesting that mechanostress is the cause of a synergistic effect in the expression of these proteins. In osteoblasts in the mineralization medium, significant increases in osteocalcin mRNA occurred earlier in cells subjected to straining than in the cells not subjected to straining, suggesting that the mechanostress is a critical factor to enhance the expression of osteocalcin. Western blot and ELISA analysis showed increased podoplanin production in osteoblasts with longer durations of straining. There was significantly less mineralization product in osteoblasts with antibodies for podoplanin, osteopontin, and osteocalcin. There was also less osteopontin and osteocalcin produced in osteoblasts with anti-podoplanin. These findings suggest that mechanostress induces the production of podoplanin in osteoblasts and that podoplanin may play a role in mineralization in cooperation with bone-associated proteins.

  17. Characterization of bioactive RGD peptide immobilized onto poly(acrylic acid) thin films by plasma polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Hyun Suk; Ko, Yeong Mu; Shim, Jae Won [Department of Dental Materials, School of Dentistry, MRC Center, Chosun University, Gwangju (Korea, Republic of); Lim, Yun Kyong; Kook, Joong-Ki [Department of Oral Biochemistry, School of Dentistry, Chosun University, Gwangju (Korea, Republic of); Cho, Dong-Lyun [School of Applied Chemical Engineering and Center for Functional Nano Fine Chemicals, Chonnam National University, Gwangju (Korea, Republic of); Kim, Byung Hoon, E-mail: kim5055@chosun.ac.kr [Department of Dental Materials, School of Dentistry, MRC Center, Chosun University, Gwangju (Korea, Republic of)

    2010-11-01

    Plasma surface modification can be used to improve the surface properties of commercial pure Ti by creating functional groups to produce bioactive materials with different surface topography. In this study, a titanium surface was modified with acrylic acid (AA) using a plasma treatment and immobilized with bioactive arginine-glycine-aspartic acid (RGD) peptide, which may accelerate the tissue integration of bone implants. Both terminals containing the -NH{sub 2} of RGD peptide sequence and -COOH of poly(acrylic acid) (PAA) thin film were combined with a covalent bond in the presence of 1-ethyl-3-3-dimethylaminopropyl carbodiimide (EDC). The chemical structure and morphology of AA film and RGD immobilized surface were investigated by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), atomic force microscopy (AFM), and scanning electron microscopy (SEM). All chemical analysis showed full coverage of the Ti substrate with the PAA thin film containing COOH groups and the RGD peptide. The MC3T3-E1 cells were cultured on each specimen, and the cell alkaline phosphatase (ALP) activity were examined. The surface-immobilized RGD peptide has a significantly increased the ALP activity of MC3T3-E1 cells. These results suggest that the RGD peptide immobilization on the titanium surface has an effect on osteoblastic differentiation of MC3T3-E1 cells and potential use in osteo-conductive bone implants.

  18. Osteoblast role in osteoarthritis pathogenesis.

    Science.gov (United States)

    Maruotti, Nicola; Corrado, Addolorata; Cantatore, Francesco P

    2017-11-01

    Even if osteoarthritis pathogenesis is still poorly understood, numerous evidences suggest that osteoblasts dysregulation plays a key role in osteoarthritis pathogenesis. An abnormal expression of OPG and RANKL has been described in osteoarthritis osteoblasts, which is responsible for abnormal bone remodeling and decreased mineralization. Alterations in genes expression are involved in dysregulation of osteoblast function, bone remodeling, and mineralization, leading to osteoarthritis development. Moreover, osteoblasts produce numerous transcription factors, growth factors, and other proteic molecules which are involved in osteoarthritis pathogenesis. © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  19. Biological and Mechanical Effects of Micro-Nanostructured Titanium Surface on an Osteoblastic Cell Line In vitro and Osteointegration In vivo.

    Science.gov (United States)

    Hao, Jingzu; Li, Ying; Li, Baoe; Wang, Xiaolin; Li, Haipeng; Liu, Shimin; Liang, Chunyong; Wang, Hongshui

    2017-09-01

    Hybrid micro-nanostructure implant surface was produced on titanium (Ti) surface by acid etching and anodic oxidation to improve the biological and mechanical properties. The biological properties of the micro-nanostructure were investigated by simulated body fluid (SBF) soaking test and MC3T3-E1 cell co-culture experiment. The cell proliferation, spreading, and bone sialoprotein (BSP) gene expression were examined by MTT, SEM, and reverse transcription-polymerase chain reaction (RT-PCR), respectively. In addition, the mechanical properties were evaluated by instrumented nanoindentation test and friction-wear test. Furthermore, the effect of the micro-nanostructure surface on implant osteointegration was examined by in vivo experiment. The results showed that the formation of bone-like apatite was accelerated on the micro-nanostructured Ti surface after immersion in simulated body fluid, and the proliferation, spreading, and BSP gene expression of the MC3T3-E1 cells were also upregulated on the modified surface. The micro-nanostructured Ti surface displayed decreased friction coefficient, stiffness value, and Young's modulus which were much closer to those of the cortical bone, compared to the polished Ti surface. This suggested much better mechanical match to the surrounding bone tissue of the micro-nanostructured Ti surface. Furthermore, the in vivo animal experiment showed that after implantation in the rat femora, the micro-nanostructure surface displayed higher bonding strength between bone tissues and implant; hematoxylin and eosin (H&E) staining suggested that much compact osteoid tissue was observed at the interface of Micro-nano-Ti-bone than polished Ti-bone interface after implantation. Based on these results mentioned above, it was concluded that the improved biological and mechanical properties of the micro-nanostructure endowed Ti surface with good biocompatibility and better osteointegration, implying the enlarged application of the micro

  20. Effect of alginate hydrogel containing polyproline-rich peptides on osteoblast differentiation

    International Nuclear Information System (INIS)

    Rubert, M; Monjo, M; Ramis, J M; Lyngstadaas, S P

    2012-01-01

    Polyproline-rich synthetic peptides have previously been shown to induce bone formation and mineralization in vitro and to decrease bone resorption in vivo. Alginate hydrogel formulations containing these synthetic peptides (P2, P5, P6) or Emdogain® (EMD) were tested for surface coating of bone implants. In an aqueous environment, the alginate hydrogels disclosed a highly compact structure suitable for cell adhesion and proliferation. Lack of cytotoxicity of the alginate-gel coating containing peptides was tested in MC3T3-E1 cell cultures. In the present study, relative mRNA expression levels of integrin alpha 8 were induced by P5 compared to untreated alginate gel, and osteopontin mRNA levels were increased after 21 days of culture by treatment with synthetic peptides or EMD compared to control. Further, in agreement with previous results when the synthetic peptides were administered in the culture media, osteocalcin mRNA was significantly upregulated after long-term treatment with the formulated synthetic peptides compared to untreated and EMD alginate gel. These results indicate that the alginate gel is a suitable carrier for the delivery of synthetic peptides, and that the formulation is promising as biodegradable and biocompatible coating for bone implants. (paper)

  1. [Effect of CD-14 and toll like receptors on the expression of interleukin-6 induced by lipopolysaccharides of Porphyromonas endodontalis].

    Science.gov (United States)

    Jia, Ge; Qiu, Li-Hong; Li, Ren; Lü, You; Yu, Ya-Qiong; Zhong, Ming

    2011-09-01

    To evaluate the effect of cluster of differentiation 14 (CD-14) and Toll like receptors (TLR) on the expression of interleukin-6 (IL-6) mRNA induced by Porphyromonas endodontalis (Pe) lipopolysaccharides (LPS). MC3T3-E1 cells were treated with 10 mg/L Pe-LPS for different hours, and the cells uninvolved by anything as the blank group. The expression of IL-6 was detected by reverse transcription polymerse chain reaction (RT-PCR) and enzyme-liked immunosorbent assay (ELISA). The expression of CD-14, TLR-2 and TLR-4 mRNA was observed at different time point (0 - 24 h) by RT-PCR. The protein of CD-14, TLR-2 and TLR-4 was analyzed with a flow cytometer. MC3T3-E1 cells were pretreated with anti-CD-14, anti-TLR-2 and anti-TLR-4 antibody for 1 h, and then cells were stimulated with 10 mg/L Pe-LPS for 6 h. The expression of IL-6 mRNA was examined by RT-PCR. Statistical analysis was performed using one-way ANOVA Dunnett-t test with SPSS 11.0 software package. The IL-6 mRNA and proteins increased significantly after treatment with Pe-LPS. When MC3T3-E1 cells treated by Pe-LPS for 6 h, the expression of proteins soared from (11.696 ± 0.672) ng/L to (36.534 ± 0.574) ng/L (P < 0.01); In the control group, the CD-14 and TLR-4 mRNA are ambly-expression, and the ratios of CD-14 and TLR-4 positive cells were (39.038 ± 3.131)% and (11.438 ± 0.385)% respectively in MC3T3-E1. After treatment by Pe-LPS, the expression of CD-14 and TLR-4 mRNA increased significantly, and the ratios of CD-14 and TLR-4 positive cells markedly increased to (62.407 ± 1.800)% and (21.367 ± 2.271)%. TLR-2 expression did not change apparently after Pe-LPS treatment. The expression of IL-6 mRNA was partly inhibited by anti-CD-14 or anti-TLR-4 antibody, but not by TLR-2. Pe-LPS can induce the expression of IL-6 in osteoblast MC3T3-E1 through CD-14 and TLR-4, but not TLR-2.

  2. Cellular response of Murine Osteoblasts to Cryopreservation: the ...

    African Journals Online (AJOL)

    AJB SERVER

    2006-11-02

    Nov 2, 2006 ... growth (Porter, 2006), while on the other hand, cell adhesion precedes ... Mouse fibroblast cell line L929 fixed to glass and silicon were studied by ..... since viability immediately after thawing drops from ~80-. 90% for OB cells ...

  3. Biological responses of brushite-forming Zn- and ZnSr- substituted beta-tricalcium phosphate bone cements

    Directory of Open Access Journals (Sweden)

    S Pina

    2010-09-01

    Full Text Available The core aim of this study was to investigate zinc (Zn- and zinc and strontium (ZnSr-containing brushite-forming beta-tricalcium phosphate (TCP cements for their effects on proliferation and differentiation of osteoblastic-like cells (MC3T3-E1 cell line as well as for their in vivo behaviour in trabecular bone cylindrical defects in a pilot study. In vitro proliferation and maturation responses of MC3T3-E1 osteoblastic-like cells to bone cements were studied at the cellular and molecular levels. The Zn- and Sr-containing brushite cements were found to stimulate pre-osteoblastic proliferation and osteoblastic maturation. Indeed, MC3T3-E1 cells exposed to the powdered cements had increased proliferative rates and higher adhesiveness capacity, in comparison to control cells. Furthermore, they exhibited higher alkaline phosphatase (ALP activity and increased Type-I collagen secretion and fibre deposition into the extracellular matrix. Proliferative and collagen deposition properties were more evident for cells grown in cements doped with Sr. The in vivo osteoconductive propertiesof the ZnCPC and ZnSrCPC cements were also pursued. Histological and histomorphometric analyses were performed at 1 and 2 months after implantation, using carbonated apatite cement (Norian SRS® as control. There was no evidence of cement-induced adverse foreign body reactions, and furthermore ZnCPC and ZnSrCPC cements revealed better in vivo performance in comparison to the control apatite cement. Additionally, the presence of both zinc and strontium resulted in the highest rate of new bone formation. These novel results indicate that the investigated ZnCPC and ZnSrCPC cements are both biocompatible and osteoconductive, being good candidate materials to use as bone substitutes.

  4. Investigation of silk fibroin nanoparticle-decorated poly(L-lactic acid composite scaffolds for osteoblast growth and differentiation

    Directory of Open Access Journals (Sweden)

    Chen BQ

    2017-03-01

    Full Text Available Biao-Qi Chen,1 Ranjith Kumar Kankala,1,2 Ai-Zheng Chen,1,2 Ding-Zhu Yang,1 Xiao-Xia Cheng,1 Ni-Na Jiang,1,2 Kai Zhu,3,4 Shi-Bin Wang1,2 1Institute of Biomaterials and Tissue Engineering, 2Fujian Provincial Key Laboratory of Biochemical Technology, Huaqiao University, Xiamen, Fujian, 3Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, 4Shanghai Institute of Cardiovascular Disease, Shanghai, People’s Republic of China Abstract: Attempts to reflect the physiology of organs is quite an intricacy during the tissue engineering process. An ideal scaffold and its surface topography can address and manipulate the cell behavior during the regeneration of targeted tissue, affecting the cell growth and differentiation significantly. Herein, silk fibroin (SF nanoparticles were incorporated into poly(L-lactic acid (PLLA to prepare composite scaffolds via phase-inversion technique using supercritical carbon dioxide (SC-CO2. The SF nanoparticle core increased the surface roughness and hydrophilicity of the PLLA scaffolds, leading to a high affinity for albumin attachment. The in vitro cytotoxicity test of SF/PLLA scaffolds in L929 mouse fibroblast cells indicated good biocompatibility. Then, the in vitro interplay between mouse preosteoblast cell (MC3T3-E1 and various topological structures and biochemical cues were evaluated. The cell adhesion, proliferation, osteogenic differentiation and their relationship with the structures as well as SF content were explored. The SF/PLLA weight ratio (2:8 significantly affected the MC3T3-E1 cells by improving the expression of key players in the regulation of bone formation, ie, alkaline phosphatase (ALP, osteocalcin (OC and collagen 1 (COL-1. These results suggest not only the importance of surface topography and biochemical cues but also the potential of applying SF/PLLA composite scaffolds as biomaterials in bone tissue engineering. Keywords: super critical fluids, surface topography, bone

  5. Osteogenic Transcription Regulated by Exaggerated Stretch Loading via Convergent Wnt Signaling

    Science.gov (United States)

    Juran, Cassandra M.; Blaber, Elizabeth A.; Almeida, Eduardo A. C.

    2017-01-01

    Cell and animal studies conducted onboard the International Space Station and formerly the Shuttle flights have provided data illuminating the deleterious biological response of bone to mechanical unloading. Down regulation of proliferative mechanisms within stem cell populations of the osteogenic niche is a suggested mechanism for loss of bone mass. However the intercellular communicative cues from osteoblasts and osteocytes in managing stem cell proliferation and osteogenic differentiation are largely unknown. In this investigation, MLO-Y4 osteocyte-like and MC3T3-E1 osteoblast-like cells, are co-culture under dynamic tensile conditions and evaluated for phenotypic expression of biochemical signaling proteins influential in driving stem cell differentiation. MLO-Y4 and MC3T3-E1 were co-cultured on polyethersulfone membrane with a 0.45m porosity to permit soluble factor transfer and direct cell-cell gap junction signaling. Cyclic tensile stimulation was applied for 48 h at a frequency of 0.1Hz and strain of 0.1. Total Live cell counts indicate mechanical activation of MC3T3-E1s inhibits proliferation while MLO-Y4s increase in number. However, the percent of live MLO-Y4s within the population is low (46.3 total count, *p0.05, n4) suggesting a potential apoptotic signaling cascade. Immunofluorescence demonstrated that stimulation of co-cultures elicits increased gap junction communication. Previously reported PCR evaluation of osteogenic markers further corroborate that the co-cultured populations communicative networks play a role in translating mechanical signals to molecular messaging. These findings suggest that an osteocyte-osteoblast signaling feedback mechanism may regulate mechanotransduction of an apoptotic cascade within osteocytes and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed.

  6. Cytotoxicity and biocompatibility of Zirconia (Y-TZP posts with various dental cements

    Directory of Open Access Journals (Sweden)

    Hyeongsoon Shin

    2016-08-01

    Full Text Available Objectives Endodontically treated teeth with insufficient tooth structure are often restored with esthetic restorations. This study evaluated the cytotoxicity and biological effects of yttria partially stabilized zirconia (Y-TZP blocks in combination with several dental cements. Materials and Methods Pairs of zirconia cylinders with medium alone or cemented with three types of dental cement including RelyX U200 (3M ESPE, FujiCEM 2 (GC, and Panavia F 2.0 (Kuraray were incubated in medium for 14 days. The cytotoxicity of each supernatant was determined using 3-(4,5-dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide (MTT assays on L929 fibroblasts and MC3T3-E1 osteoblasts. The levels of interleukin-6 (IL-6 mRNA were evaluated by reverse transcription polymerase chain reaction (RT-PCR, and IL-6 protein was evaluated by enzyme-linked immunosorbent assays (ELISA. The data were analyzed using one-way ANOVA and Tukey post-hoc tests. A p < 0.05 was considered statistically significant. Results The MTT assays showed that MC3T3-E1 osteoblasts were more susceptible to dental cements than L929 fibroblasts. The resin based dental cements increased IL-6 expression in L929 cells, but reduced IL-6 expression in MC3T3-E1 cells. Conclusions Zirconia alone or blocks cemented with dental cement showed acceptable biocompatibilities. The results showed resin-modified glass-ionomer based cement less produced inflammatory cytokines than other self-adhesive resin-based cements. Furthermore, osteoblasts were more susceptible than fibroblasts to the biological effects of dental cement.

  7. Polyetheretherketone Hybrid Composites with Bioactive Nanohydroxyapatite and Multiwalled Carbon Nanotube Fillers

    Directory of Open Access Journals (Sweden)

    Chen Liu

    2016-12-01

    Full Text Available Polyetheretherketone (PEEK hybrid composites reinforced with inorganic nanohydroxyapatite (nHA and multiwalled carbon nanotube (MWNT were prepared by melt-compounding and injection molding processes. The additions of nHA and MWNT to PEEK were aimed to increase its elastic modulus, tensile strength, and biocompatibility, rendering the hybrids suitable for load-bearing implant applications. The structural behavior, mechanical property, wettability, osteoblastic cell adhesion, proliferation, differentiation, and mineralization of the PEEK/nHA-MWNT hybrids were studied. X-ray diffraction and SEM observation showed that both nHA and MWNT fillers are incorporated into the polymer matrix of PEEK-based hybrids. Tensile tests indicated that the elastic modulus of PEEK can be increased from 3.87 to 7.13 GPa by adding 15 vol % nHA and 1.88 vol % MWNT fillers. The tensile strength and elongation at break of the PEEK/(15% nHA-(1.88% MWNT hybrid were 64.48 MPa and 1.74%, respectively. Thus the tensile properties of this hybrid were superior to those of human cortical bones. Water contact angle measurements revealed that the PEEK/(15% nHA-(1.88% MWNT hybrid is hydrophilic due to the presence of nHA. Accordingly, hydrophilic PEEK/(15% nHA-(1.88% MWNT hybrid promoted the adhesion, proliferation, differentiation, and mineralization of murine MC3T3-E1 osteoblasts on its surface effectively on the basis of cell culture, fluorescence microscopy, MTT assay, WST-1 assay, alkaline phosphatase activity, and Alizarin red staining tests. Thus the PEEK/(15% nHA-(1.88% MWNT hybrid has the potential to be used for fabricating load-bearing bone implants.

  8. Osteoblastic cells: differentiation and trans-differentiation

    DEFF Research Database (Denmark)

    Kassem, Moustapha; Abdallah, Basem; Saeed, Hamid

    2008-01-01

    The osteoblast is the bone forming cell and is derived from mesenchymal stem cells (MSC) present among the bone marrow stroma. MSC are capable of multi-lineage differentiation into mesoderm-type cells such as osteoblasts and adipocytes. Understanding the mechanisms underlying osteoblast different...

  9. Engraftment Outcomes after HPC Co-Culture with Mesenchymal Stromal Cells and Osteoblasts

    Directory of Open Access Journals (Sweden)

    Matthew M. Cook

    2013-09-01

    Full Text Available Haematopoietic stem cell (HSC transplantation is an established cell-based therapy for a number of haematological diseases. To enhance this therapy, there is considerable interest in expanding HSCs in artificial niches prior to transplantation. This study compared murine HSC expansion supported through co-culture on monolayers of either undifferentiated mesenchymal stromal cells (MSCs or osteoblasts. Sorted Lineage− Sca-1+ c-kit+ (LSK haematopoietic stem/progenitor cells (HPC demonstrated proliferative capacity on both stromal monolayers with the greatest expansion of LSK shown in cultures supported by osteoblast monolayers. After transplantation, both types of bulk-expanded cultures were capable of engrafting and repopulating lethally irradiated primary and secondary murine recipients. LSKs co-cultured on MSCs showed comparable, but not superior, reconstitution ability to that of freshly isolated LSKs. Surprisingly, however, osteoblast co-cultured LSKs showed significantly poorer haematopoietic reconstitution compared to LSKs co-cultured on MSCs, likely due to a delay in short-term reconstitution. We demonstrated that stromal monolayers can be used to maintain, but not expand, functional HSCs without a need for additional haematopoietic growth factors. We also demonstrated that despite apparently superior in vitro performance, co-injection of bulk cultures of osteoblasts and LSKs in vivo was detrimental to recipient survival and should be avoided in translation to clinical practice.

  10. Evaluating the feasibility of utilizing the small molecule phenamil as a novel biofactor for bone regenerative engineering.

    Science.gov (United States)

    Lo, Kevin W-H; Ulery, Bret D; Kan, Ho Man; Ashe, Keshia M; Laurencin, Cato T

    2014-09-01

    Osteoblast cell adhesion and differentiation on biomaterials are important achievements necessary for implants to be useful in bone regenerative engineering. Recombinant bone morphogenetic proteins (BMPs) have been shown to be important for these processes; however, there are many challenges associated with the widespread use of these proteins. A recent report demonstrated that the small molecule phenamil, a diuretic derivative, was able to induce osteoblast differentiation and mineralization in vitro via the canonical BMP signalling cascade (Park et al., 2009). In this study, the feasibility of using phenamil as a novel biofactor in conjunction with a biodegradable poly(lactide-co-glycolide acid) (PLAGA) polymeric scaffold for engineering bone tissue was evaluated. The in vitro cellular behaviour of osteoblast-like MC3T3-E1 cells cultured on PLAGA scaffolds in the presence of phenamil at 10 μM were characterized with regard to initial cell adhesion, proliferation, alkaline phosphatase (ALP) activity and matrix mineralization. The results demonstrate that phenamil supported cell proliferation, promoted ALP activity and facilitated matrix mineralization of osteoblast-like MC3T3-E1 cells. Moreover, in this study, we found that phenamil promoted integrin-mediated cell adhesion on PLAGA scaffolds. It was also shown that phenamil encapsulated within porous, microsphere PLAGA scaffolds retained its osteogenic activity upon release. Based on these findings, the small molecule phenamil has the potential to serve as a novel biofactor for the repair and regeneration of bone tissues. Copyright © 2012 John Wiley & Sons, Ltd.

  11. Structure and functional regulation of the CD38 promoter

    International Nuclear Information System (INIS)

    Sun Li; Iqbal, Jameel; Zaidi, Samir; Zhu Linglng; Zhang Xuefeng; Peng Yuanzheng; Moonga, Baljit S.; Zaidi, Mone

    2006-01-01

    CD38 has multiple roles in biology, including T lymphocyte signaling, neutrophil migration, neurotransmission, cell proliferation, apoptosis, and bone remodeling. To study the transcriptional control of the CD38 gene, we cloned a putative 1.8 kb promoter fragment from a rabbit genomic DNA library. Primer extension analysis indicated two transcription start sites consistent with the absence of a TATA box. Sequence analysis revealed several AP-1, AP-4, myo-D, GATA, and SP-1 sequences. MC3T3.E1 (osteoblast) or RAW-C3 (osteoclast precursor macrophage) cells were then transfected with the CD38 promoter or its deletion fragments ligated to the luciferase reporter gene. Except for the shortest 41 bp fragment, all fragments showed significant luciferase activity. There was a marked stimulation of basal activity in the 93 bp fragment that contained a GC box and SP-1 site. Furthermore, there were significant differences in the activity of the fragments in MC3T3.E1 and RAW-C3 cells. Intracellular Ca 2+ elevations by ionomycin (10 μM) in MC3T3.E1 cells inhibited promoter activity, except in the short 41 bp. In contrast, cAMP elevation by exposure to forskolin (100 μM) inhibited activation of all fragments, except the 0.6 and 1.2 kb fragments. Finally, TNF-α stimulated promoter activity in RAW-C3 cells transfected with the 93 bp and 1.0 kb fragments, consistent with the stimulation of CD38 mRNA by TNF-α. Physiologically, therefore, modulation of the expression of the NAD + -sensing enzyme, CD38, by Ca 2+ , cAMP, and cytokines, such as TNF-α may contribute to coupling the intense metabolic activity of osteoclasts and osteoblasts to their respective bone-resorbing and bone-forming functions

  12. A role for PERK in the mechanism underlying fluoride-induced bone turnover

    International Nuclear Information System (INIS)

    Sun, Fei; Li, Xining; Yang, Chen; Lv, Peng; Li, Guangsheng; Xu, Hui

    2014-01-01

    While it has been well-documented that excessive fluoride exposure caused the skeletal disease and osteoblasts played a critical role in the advanced skeletal fluorosis, the underlying mechanism that mediated these effects remain poorly understood. The present study was undertaken to examine the effect of fluoride on bone of rats and MC3T3-E1 cells in vitro. Herein we found pathological features of high bone turnover in fluoride-treated rats, which was supported by an increase of osteogenic and osteoclastogenic genes expression in different stages of fluoride exposure. The skeletal toxicity of fluoride was accompanied by activation of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR). A novel finding of this study was that expression of PKR-like endoplasmic reticulum kinase (PERK) was the same trend with receptor activator for nuclear factor-κ B ligand (RANKL), and NF-E2 p45-related factor 2 (Nrf2) was the same trend with Runt-related transcription factor 2 (Runx2) in bones of rats exposed to varied fluoride condition. Based on these data, we hypothesized that up-regulation of PERK probably played a role in mediating bone turnover induced by fluoride. Action of fluoride on MC3T3-E1 cells differentiation was demonstrated through analysis of alkaline phosphatase (ALP) activity and mineralized nodules formation. Meantime, an increase of binding immunoglobulin protein (BiP) expression indicated the active ER stress in cells exposed to various dose of fluoride. Blocking PERK expression using siRNA showed the obvious decrease of osteogenic and osteoclastogenic factors expression in MC3T3-E1 cells exposed to certain dose of fluoride that could positively stimulate osteoblastic viability. In conclusion these findings underscore the importance of PERK in modulating fluoride induced bone formation and bone resorption. Understanding the link between PERK and bone turnover could probe into the mechanism underlying different bone lesion of

  13. Isoform-specific regulation of osteogenic factors by polypeptide N-Acetylgalactosaminyltransferases 1 and 4

    International Nuclear Information System (INIS)

    Tang, Juan; Zheng, Hanxi; Chen, Ling; Gao, Shangshang; Shi, Xiaorui; Liu, Jingjing; Xu, Lan

    2017-01-01

    The family of UDP-GalNAc polypeptide: N-Acetylgalactosaminlytransfersases (ppGalNAcTs) catalyzes the initial step of O-linked protein glycosylation. Mucin-type O-glycoproteins are abundant in the bone and may play an important role in osteogenesis. Herein, we examined the effects of ppGalNAc-T isoforms on osteogenesis of MC3T3-E1 pre-osteoblasts. We found that ppGalNAc-T1 and -T4 isoforms were highly expressed during osteogenesis of MC3T3-E1 and their knockdown by short hairpin RNA (shRNA) decreased osteoblast formation and bone mineralization. Knockdown of ppGalNAc-T1 or -T4 decreased mRNA and protein levels of bone sialoprotein (BSP). Knockdown of ppGalNAc-T1decreased mRNA levels of osteocalcin (OC), osteoprotegerin (OPG). Knockdown ofppGalNAc-T4 isoform decreased mRNA levels of OC, OPG and vitamin D receptor (VDR). While knockdown of T1 or T4 isoforms did not change the expression of osteopontin (OPN), COLLI, receptor activator for nuclear factor-κB ligand (RANKL) and transforming growth factor-β (TGF-β). Our results demonstrated that the ppGalNAc-T4 was highly expressed in MC3T3-E1 cells during osteogenesis for the first time. We also found that ppGalNAc-T1 and -T4 affected the expression of different osteogenic factors, suggesting distinct roles ppGalNAc-T isoformsplay in regulating osteogenesis in vitro. - Highlights: • ppGalNAc-T1 and T4 are highly expressed during MC3T3 cell osteogenesis. • Knockdown of ppGalNAc-T1 and -T4 decreases osteogenic differentiation and mineralization. • Expression of osteogenic factors are differentially affected by decreased ppGalNAc-T1 and -T4 expression.

  14. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Chieri [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp [Division of Pharmacotherapy, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

  15. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    International Nuclear Information System (INIS)

    Sato, Chieri; Iwasaki, Tsuyoshi; Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime

    2012-01-01

    Highlights: ► We investigated the role of S1P signaling for osteoblast differentiation. ► Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. ► S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. ► MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P receptor-mediated signaling plays a crucial role for osteoblast differentiation.

  16. The effect of porosity on cell ingrowth into accurately defined, laser-made, polylactide-based 3D scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Danilevicius, Paulius; Georgiadi, Leoni [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece); Pateman, Christopher J.; Claeyssens, Frederik [Kroto Research Institute, Department of Materials Science and Engineering, University of Sheffield, Broad Lane, Sheffield S3 7HQ (United Kingdom); Chatzinikolaidou, Maria, E-mail: mchatzin@materials.uoc.gr [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece); Department of Materials Science and Technology, University of Crete, PO Box 2208, 71303 Heraklion (Greece); Farsari, Maria, E-mail: mfarsari@iesl.forth.gr [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece)

    2015-05-01

    Highlights: • We studied the porosity of laser-made 3D scaffolds on MC3T3-E1 pre-osteoblastic cells. • We made polylactide 3D scaffolds with pores 25–110 μm. - Abstract: The aim of this study is to demonstrate the accuracy required for the investigation of the role of solid scaffolds’ porosity in cell proliferation. We therefore present a qualitative investigation into the effect of porosity on MC3T3-E1 pre-osteoblastic cell ingrowth of three-dimensional (3D) scaffolds fabricated by direct femtosecond laser writing. The material we used is a purpose made photosensitive pre-polymer based on polylactide. We designed and fabricated complex, geometry-controlled 3D scaffolds with pore sizes ranging from 25 to 110 μm, representing porosities 70%, 82%, 86%, and 90%. The 70% porosity scaffolds did not support cell growth initially and in the long term. For the other porosities, we found a strong adhesion of the pre-osteoblastic cells from the first hours after seeding and a remarkable proliferation increase after 3 weeks and up to 8 weeks. The 86% porosity scaffolds exhibited a higher efficiency compared to 82% and 90%. In addition, bulk material degradation studies showed that the employed, highly-acrylated polylactide is degradable. These findings support the potential use of the proposed material and the scaffold fabrication technique in bone tissue engineering.

  17. In vitro and in vivo characterization of anodised zirconium as a potential material for biomedical applications.

    Science.gov (United States)

    Katunar, Maria R; Gomez Sanchez, Andrea; Santos Coquillat, Ana; Civantos, Ana; Martinez Campos, Enrique; Ballarre, Josefina; Vico, Tamara; Baca, Matias; Ramos, Viviana; Cere, Silvia

    2017-06-01

    In vitro studies offer the insights for the understanding of the mechanisms at the tissue-implant interface that will provide an effective functioning in vivo. The good biocompatibility of zirconium makes a good candidate for biomedical applications and the attractive in vivo performance is mainly due to the presence of a protective oxide layer. The aim of this study is to evaluate by in vitro and in vivo approach, the influence of surface modification achieved by anodisation at 30 and 60V on zirconium implants on the first steps of the osseointegration process. In this study cell attachment, proliferation and morphology of mouse myoblast C2C12-GFP and in mouse osteoprogenitor MC3T3-E1 cells was evaluated. Also, together with the immune system response, osteoclast differentiation and morphology with RAW 264.7 murine cell line were analysed. It was found that anodisation treatment at 60V enhanced cell spreading and the osteoblastic and osteoclastic cells morphology, showing a strong dependence on the surface characteristics. In vivo tests were performed in a rat femur osteotomy model. Dynamical and static histological and histomorphometric analyses were developed 15 and 30days after surgery. Newly formed bone around Zr60V implants showed a continuous newly compact and homogeneous bone just 15 after surgery, as judged by the enhanced thickness and mineralization rate. The results indicate that anodising treatment at 60V could be an effective improvement in the osseointegration of zirconium by stimulating adhesion, proliferation, morphology, new bone thickness and bone mineral apposition, making zirconium an emerging candidate material for biomedical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. In vitro and in vivo characterization of anodised zirconium as a potential material for biomedical applications

    Energy Technology Data Exchange (ETDEWEB)

    Katunar, Maria R, E-mail: mkatunar@fi.mdp.edu.ar [INTEMA, Universidad Nacional de Mar del Plata-CONICET, Juan B. Justo, 4302, B7608FDQ, Mar del Plata (Argentina); Gomez Sanchez, Andrea [INTEMA, Universidad Nacional de Mar del Plata-CONICET, Juan B. Justo, 4302, B7608FDQ, Mar del Plata (Argentina); Santos Coquillat, Ana [Instituto de Estudios Biofuncionales, Universidad Complutense de Madrid, Madrid, España (Spain); Civantos, Ana [Instituto de Ciencia y Tecnología de Polímeros, CSIC, Madrid (Spain); Martinez Campos, Enrique [Instituto de Estudios Biofuncionales, Universidad Complutense de Madrid, Madrid, España (Spain); Ballarre, Josefina; Vico, Tamara [INTEMA, Universidad Nacional de Mar del Plata-CONICET, Juan B. Justo, 4302, B7608FDQ, Mar del Plata (Argentina); Baca, Matias [Traumatologia y Ortopedia, Hospital Interzonal General de Agudos “Oscar Alende”, Mar del Plata (Argentina); Ramos, Viviana [Instituto de Ciencia y Tecnología de Polímeros, CSIC, Madrid (Spain); Cere, Silvia [INTEMA, Universidad Nacional de Mar del Plata-CONICET, Juan B. Justo, 4302, B7608FDQ, Mar del Plata (Argentina)

    2017-06-01

    In vitro studies offer the insights for the understanding of the mechanisms at the tissue–implant interface that will provide an effective functioning in vivo. The good biocompatibility of zirconium makes a good candidate for biomedical applications and the attractive in vivo performance is mainly due to the presence of a protective oxide layer. The aim of this study is to evaluate by in vitro and in vivo approach, the influence of surface modification achieved by anodisation at 30 and 60 V on zirconium implants on the first steps of the osseointegration process. In this study cell attachment, proliferation and morphology of mouse myoblast C2C12-GFP and in mouse osteoprogenitor MC3T3-E1 cells was evaluated. Also, together with the immune system response, osteoclast differentiation and morphology with RAW 264.7 murine cell line were analysed. It was found that anodisation treatment at 60 V enhanced cell spreading and the osteoblastic and osteoclastic cells morphology, showing a strong dependence on the surface characteristics. In vivo tests were performed in a rat femur osteotomy model. Dynamical and static histological and histomorphometric analyses were developed 15 and 30 days after surgery. Newly formed bone around Zr60V implants showed a continuous newly compact and homogeneous bone just 15 after surgery, as judged by the enhanced thickness and mineralization rate. The results indicate that anodising treatment at 60 V could be an effective improvement in the osseointegration of zirconium by stimulating adhesion, proliferation, morphology, new bone thickness and bone mineral apposition, making zirconium an emerging candidate material for biomedical applications. - Highlights: • Surface modification by anodisation stimulates cell attachment and proliferation. • The anodising process on Zr as a substrate modification improves bone formation. • The mineral processes are accelerated in the Zr60V showing a faster cell response.

  19. In vitro and in vivo corrosion, cytocompatibility and mechanical properties of biodegradable Mg-Y-Ca-Zr alloys as implant materials.

    Science.gov (United States)

    Chou, Da-Tren; Hong, Daeho; Saha, Partha; Ferrero, Jordan; Lee, Boeun; Tan, Zongqing; Dong, Zhongyun; Kumta, Prashant N

    2013-11-01

    This study introduces a class of biodegradable Mg-Y-Ca-Zr alloys novel to biological applications and presents evaluations for orthopedic and craniofacial implant applications. Mg-Y-Ca-Zr alloys were processed using conventional melting and casting techniques. The effects of increasing Y content from 1 to 4 wt.% as well as the effects of T4 solution treatment were assessed. Basic material phase characterization was conducted using X-ray diffraction, optical microscopy and scanning electron microscopy. Compressive and tensile tests allowed for the comparison of mechanical properties of the as-cast and T4-treated Mg-Y-Ca-Zr alloys to pure Mg and as-drawn AZ31. Potentiodynamic polarization tests and mass loss immersion tests were used to evaluate the corrosion behavior of the alloys. In vitro cytocompatibility tests on MC3T3-E1 pre-osteoblast cells were also conducted. Finally, alloy pellets were implanted into murine subcutaneous tissue to observe in vivo corrosion as well as local host response through H&E staining. SEM/EDS analysis showed that secondary phase intermetallics rich in yttrium were observed along the grain boundaries, with the T4 solution treatment diffusing the secondary phases into the matrix while increasing the grain size. The alloys demonstrated marked improvement in mechanical properties over pure Mg. Increasing the Y content contributed to improved corrosion resistance, while solution-treated alloys resulted in lower strength and compressive strain compared to as-cast alloys. The Mg-Y-Ca-Zr alloys demonstrated excellent in vitro cytocompatibility and normal in vivo host response. The mechanical, corrosion and biological evaluations performed in this study demonstrated that Mg-Y-Ca-Zr alloys, especially with the 4 wt.% Y content, would perform well as orthopedic and craniofacial implant biomaterials. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  20. Post-genome wide association studies and functional analyses identify association of MPP7 gene variants with site-specific bone mineral density.

    Science.gov (United States)

    Xiao, Su-Mei; Kung, Annie Wai Chee; Gao, Yi; Lau, Kam-Shing; Ma, Alvin; Zhang, Zhen-Lin; Liu, Jian-Min; Xia, Wiebo; He, Jin-Wei; Zhao, Lin; Nie, Min; Fu, Wei-Zhen; Zhang, Min-Jia; Sun, Jing; Kwan, Johnny S H; Tso, Gloria Hoi Wan; Dai, Zhi-Jie; Cheung, Ching-Lung; Bow, Cora H; Leung, Anskar Yu Hung; Tan, Kathryn Choon Beng; Sham, Pak Chung

    2012-04-01

    Our previous genome-wide association study (GWAS) in a Hong Kong Southern Chinese population with extreme bone mineral density (BMD) scores revealed suggestive association with MPP7, which ranked second after JAG1 as a candidate gene for BMD. To follow-up this suggestive signal, we replicated the top single-nucleotide polymorphism rs4317882 of MPP7 in three additional independent Asian-descent samples (n= 2684). The association of rs4317882 reached the genome-wide significance in the meta-analysis of all available subjects (P(meta)= 4.58 × 10(-8), n= 4204). Site heterogeneity was observed, with a larger effect on spine than hip BMD. Further functional studies in a zebrafish model revealed that vertebral bone mass was lower in an mpp7 knock-down model compared with the wide-type (P= 9.64 × 10(-4), n= 21). In addition, MPP7 was found to have constitutive expression in human bone-derived cells during osteogenesis. Immunostaining of murine MC3T3-E1 cells revealed that the Mpp7 protein is localized in the plasma membrane and intracytoplasmic compartment of osteoblasts. In an assessment of the function of identified variants, an electrophoretic mobility shift assay demonstrated the binding of transcriptional factor GATA2 to the risk allele 'A' but not the 'G' allele of rs4317882. An mRNA expression study in human peripheral blood mononuclear cells confirmed that the low BMD-related allele 'A' of rs4317882 was associated with lower MPP7 expression (P= 9.07 × 10(-3), n= 135). Our data suggest a genetic and functional association of MPP7 with BMD variation.

  1. In vitro and in vivo characterization of anodised zirconium as a potential material for biomedical applications

    International Nuclear Information System (INIS)

    Katunar, Maria R; Gomez Sanchez, Andrea; Santos Coquillat, Ana; Civantos, Ana; Martinez Campos, Enrique; Ballarre, Josefina; Vico, Tamara; Baca, Matias; Ramos, Viviana; Cere, Silvia

    2017-01-01

    In vitro studies offer the insights for the understanding of the mechanisms at the tissue–implant interface that will provide an effective functioning in vivo. The good biocompatibility of zirconium makes a good candidate for biomedical applications and the attractive in vivo performance is mainly due to the presence of a protective oxide layer. The aim of this study is to evaluate by in vitro and in vivo approach, the influence of surface modification achieved by anodisation at 30 and 60 V on zirconium implants on the first steps of the osseointegration process. In this study cell attachment, proliferation and morphology of mouse myoblast C2C12-GFP and in mouse osteoprogenitor MC3T3-E1 cells was evaluated. Also, together with the immune system response, osteoclast differentiation and morphology with RAW 264.7 murine cell line were analysed. It was found that anodisation treatment at 60 V enhanced cell spreading and the osteoblastic and osteoclastic cells morphology, showing a strong dependence on the surface characteristics. In vivo tests were performed in a rat femur osteotomy model. Dynamical and static histological and histomorphometric analyses were developed 15 and 30 days after surgery. Newly formed bone around Zr60V implants showed a continuous newly compact and homogeneous bone just 15 after surgery, as judged by the enhanced thickness and mineralization rate. The results indicate that anodising treatment at 60 V could be an effective improvement in the osseointegration of zirconium by stimulating adhesion, proliferation, morphology, new bone thickness and bone mineral apposition, making zirconium an emerging candidate material for biomedical applications. - Highlights: • Surface modification by anodisation stimulates cell attachment and proliferation. • The anodising process on Zr as a substrate modification improves bone formation. • The mineral processes are accelerated in the Zr60V showing a faster cell response.

  2. Addition of MgO nanoparticles and plasma surface treatment of three-dimensional printed polycaprolactone/hydroxyapatite scaffolds for improving bone regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Roh, Hee-Sang; Lee, Chang-Min; Hwang, Young-Hyoun [Department of Dental Materials, School of Dentistry, Chosun University, Gwangju 61452 (Korea, Republic of); Kook, Min-Suk [Department of Oral & Maxillofacial Surgery, School of Dentistry, Chonnam National University, Gwangju 61186 (Korea, Republic of); Yang, Seong-Won [Department of Ophthalmology, College of Medicine, Chosun University, Gwangju 61452 (Korea, Republic of); Lee, Donghun [Department of Herbal Pharmacology, Kyung Hee University College of Korean Medicine, Seoul 130-701 (Korea, Republic of); Kim, Byung-Hoon, E-mail: kim5055@chosun.ac.kr [Department of Dental Materials, School of Dentistry, Chosun University, Gwangju 61452 (Korea, Republic of)

    2017-05-01

    Magnesium (Mg) plays an important role in the body in mediating cell-extracellular matrix interactions and controlling bone apatite structure and density. Hydroxyapatite (HAp) has been used for osteoconductive bone replacement because of its good compressive strength and biocompatibility. The object of this study is to investigate the effects of adding Magnesium oxide (MgO) nanoparticles to polycaprolactone (PCL)/HAp composites and treating PCL/HAp/MgO scaffolds with oxygen and nitrogen plasma. The 3D PCL/HAp/MgO scaffolds were fabricated using a 3D bioextruder. PCL was mixed with 1–15 wt% of MgO and HAp. The scaffolds were treated with oxygen and nitrogen plasma under anisotropic etching conditions to improve the bioactivity. The plasma-treated surfaces were analyzed by X-ray photoelectron spectroscopy, scanning electron microscopy, and atomic force microscopy. In addition, the proliferation and differentiation of pre-osteoblast (MC3T3-E1) cells were examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and alkaline phosphatase activity. Cell mineralization within the produced scaffolds was analyzed by the quantification of alizarin stainings. The addition of MgO/HAp nanoparticles and plasma treatment enhanced the adhesion, proliferation, and differentiation of MC3T3-E1 cells in the PCL scaffolds. Hence, changes in physical surface morphology and surface chemical properties of the 3D scaffold by plasma treatment can affect the behavior of MC3T3-E1 cells. - Highlights: • 3D-printed PCL/HAp/MgO showed good porosity and interconnectivity. • O{sub 2} and N{sub 2} plasma improved the surface roughness and hydrophilicity on scaffolds. • Addition of HAp/MgO nanoparticles enhanced the cell behavior of preosteoblast.

  3. Functionalization of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds via surface heparinization for bone tissue engineering.

    Science.gov (United States)

    Jiang, Tao; Khan, Yusuf; Nair, Lakshmi S; Abdel-Fattah, Wafa I; Laurencin, Cato T

    2010-06-01

    Scaffolds exhibiting biological recognition and specificity play an important role in tissue engineering and regenerative medicine. The bioactivity of scaffolds in turn influences, directs, or manipulates cellular responses. In this study, chitosan/poly(lactic acid-co-glycolic acid) (chitosan/PLAGA) sintered microsphere scaffolds were functionalized via heparin immobilization. Heparin was successfully immobilized on chitosan/PLAGA scaffolds with controllable loading efficiency. Mechanical testing showed that heparinization of chitosan/PLAGA scaffolds did not significantly alter the mechanical properties and porous structures. In addition, the heparinized chitosan/PLAGA scaffolds possessed a compressive modulus of 403.98 +/- 19.53 MPa and a compressive strength of 9.83 +/- 0.94 MPa, which are in the range of human trabecular bone. Furthermore, the heparinized chitosan/PLAGA scaffolds had an interconnected porous structure with a total pore volume of 30.93 +/- 0.90% and a median pore size of 172.33 +/- 5.89 mum. The effect of immobilized heparin on osteoblast-like MC3T3-E1 cell growth was investigated. MC3T3-E1 cells proliferated three dimensionally throughout the porous structure of the scaffolds. Heparinized chitosan/PLAGA scaffolds with low heparin loading (1.7 microg/scaffold) were shown to be capable of stimulating MC3T3-E1 cell proliferation by MTS assay and cell differentiation as evidenced by elevated osteocalcin expression when compared with nonheparinized chitosan/PLAGA scaffold and chitosan/PLAGA scaffold with high heparin loading (14.1 microg/scaffold). This study demonstrated the potential of functionalizing chitosan/PLAGA scaffolds via heparinization with improved cell functions for bone tissue engineering applications.

  4. Preparation, characterization, and in vitro cytotoxicity evaluation of a novel anti-tuberculosis reconstruction implant.

    Directory of Open Access Journals (Sweden)

    JunFeng Dong

    Full Text Available Reconstruction materials currently used in clinical for osteoarticular tuberculosis (TB are unsatisfactory due to a variety of reasons. Rifampicin (RFP is a well-known and highly effective first-line anti-tuberculosis (anti-TB drug. Poly-DL-lactide (PDLLA and nano-hydroxyapatite (nHA are two promising materials that have been used both for orthopedic reconstruction and as carriers for drug release. In this study we report the development of a novel anti-TB implant for osteoarticular TB reconstruction using a combination of RFP, PDLLA and nHA.RFP, PDLLA and nHA were used as starting materials to produce a novel anti-TB activity implant by the solvent evaporation method. After manufacture, the implant was characterized and its biodegradation and drug release profile were tested. The in vitro cytotoxicity of the implant was also evaluated in pre-osteoblast MC3T3-E1 cells using multiple methodologies.A RFP/PDLLA/nHA composite was successfully synthesized using the solvent evaporation method. The composite has a loose and porous structure with evenly distributed pores. The production process was steady and no chemical reaction occurred as proved by Fourier Transform Infrared Spectroscopy (FTIR and X-Ray Diffraction (XRD. Meanwhile, the composite blocks degraded and released drug for at least 12 weeks. Evaluation of in vitro cytotoxicity in MC3T3-E1 cells verified that the synthesized composite blocks did not affect cell growth and proliferation.It is feasible to manufacture a novel bioactive anti-TB RFP/PDLLA/nHA composite by the solvent evaporation method. The composite blocks showed appropriate properties such as degradation, drug release and biosafety to MC3T3-E1 cells. In conclusion, the novel composite blocks may have great potential for clinical applications in repairing bone defects caused by osteoarticular TB.

  5. Chemical modification of chitosan film via surface grafting of citric acid molecular to promote the biomineralization

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yang, E-mail: liuyang@cczu.edu.cn [Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou 213164 (China); Engineering Research Center of Nano-Geo Materials of Ministry of Education, China University of Geosciences, Wuhan 430074 (China); School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); Shen, Xin; Zhou, Huan [Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou 213164 (China); Wang, Yingjun [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); Deng, Linhong, E-mail: dlh@cczu.edu.cn [Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou 213164 (China)

    2016-05-01

    Graphical abstract: - Highlights: • Chitosan film was modified by surface grafting of citric acid. • The modified film has good hydrophilicity and moisture-retaining capacity. • The citric acid grafting treatment significantly promote the biomineralization. • MC3T3-E1 osteoblasts research confirms the biocompatibility of the film. - Abstract: We develop a novel chitosan–citric acid film (abbreviated as CS–CA) suitable for biomedical applications in this study. In this CS–CA film, the citric acid, which is a harmless organic acid has been extensively investigated as a modifying agent on carbohydrate polymers, was cross-linked by 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) onto the surface of chitosan (CS) film. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) confirms the graft copolymerization of the modified chitosan film (CS–CA). Surface wettability, moisturizing performance, the capacity of mineralization in vitro and biocompatibility of the films were characterized. After modification, this CS–CA film has good hydrophilicity. It is very evident that the citric acid grafting treatment significantly promotes the biomineralization of the chitosan based substrates. Cell experiments show that the MC3T3-E1 osteoblasts can adhere and proliferate well on the surface of CS–CA film. This CS–CA film, which can be prepared in large quantities and at low cost, should have potential application in bone tissue engineering.

  6. Long-term antibacterial characteristics and cytocompatibility of titania nanotubes loaded with Au nanoparticles without photocatalytic effects

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Guomin [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (China); Feng, Hongqing, E-mail: fenghongqing@binn.cas.cn [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (China); Beijing Institute of Nanoenergy and Nanosystems, Chinese Academy of Sciences, National Center for Nanoscience and Technology (NCNST), Beijing 100083 (China); Jin, Weihong; Gao, Ang; Peng, Xiang; Li, Wan; Wu, Hao [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (China); Li, Zhou [Beijing Institute of Nanoenergy and Nanosystems, Chinese Academy of Sciences, National Center for Nanoscience and Technology (NCNST), Beijing 100083 (China); Chu, Paul K., E-mail: paul.chu@cityu.edu.hk [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (China)

    2017-08-31

    Highlights: • Au@TiO{sub 2}-NT shows long-term antibacterial effects without photocatalytical effects. • An ROS-free antibacterial process is confirmed. • Au@TiO{sub 2}-NT shows good compatibility with osteoblasts and does not induce ROS production. - Abstract: Au nanoparticles (NPs) can endow titania nanotubes (Au@TiO{sub 2}-NT) with light-independent antibacterial properties which bode well for in vivo application because of the dark environment inside tissues. In this work, the long-term antibacterial bactericidal properties and cytocompatibility of Au@TiO{sub 2}-NT without photocatalytic effects are studied in details. The materials exhibit antibacterial effects against Staphylococcus aureus according to antibacterial tests carried out for a total time of 21 days, which are normally long enough for early stage tissue healing after surgery. In addition, adhesion and proliferation of MC3T3-E1 osteoblasts on Au@TiO{sub 2}-NT reveal cytocompatibility comparable to that of TiO{sub 2}-NT. No reactive oxygen species (ROS) are detected from either the bacteria or MC3T3-E1 cells cultured on the Au@TiO{sub 2}-NT surface. The absence of ROS, long-term antibacterial properties, and cytocompatibility make Au@TiO{sub 2}-NT promising biomaterials in orthopedic devices and implants.

  7. Tailored biodegradable triblock copolymer coatings obtained by MAPLE: a parametric study

    Science.gov (United States)

    Brajnicov, S.; Neacsu, P.; Moldovan, A.; Marascu, V.; Bonciu, A.; Ion, R.; Dinca, V.; Cimpean, A.; Dinescu, M.

    2017-11-01

    Biocompatible and biodegradable coatings with controllable and tailored chemical and physical characteristics (i.e. morphology and roughness) are of great interest in bone related research applications. Within this research direction, in this work, a series of novel biodegradable coatings based on triblock copolymers poly(lactide- co-caprolactone)-block-poly(ethylene-glycol)-block-poly(lactide- co-caprolactone) (PLCL-PEG-PLCL) were obtained by matrix-assisted pulsed laser evaporation (MAPLE) and their morphological characteristics and roughness were modulated by varying target composition and laser fluence. The coatings were used for preliminary in vitro testing with MC3T3-E1 pre-osteoblasts. It was found that for a specific range of fluences, the main functional groups in the MAPLE-deposited thin films, as determined by Fourier transform infrared spectroscopy, are similar to the molecular structures of the initial material. Depending on the deposition parameters, significant changes in morphologies, i.e. material accumulation in the form of droplets, wrinkles, or carpet-like structures were revealed by atomic force microscopy (AFM) and scanning electron microscopy. The optimized coating characteristics were further correlated to MC3T3-E1 pre-osteoblasts response. The ability to control the morphology and to maintain unaltered the chemistry of the deposited material through MAPLE is an important step in creating functional bio-interfaces in the field of biomedical research and tissue engineering.

  8. Graphene-containing PCL- coated Porous 13-93B3 Bioactive Glass Scaffolds for Bone Regeneration

    Science.gov (United States)

    Türk, Mert; Deliormanlı, Aylin M.

    2018-04-01

    Borate-based 13-93B3 bioactive glass scaffolds were coated with the graphene-containing poly-caprolactone (PCL) solution to prepare electrically conductive composites for biomedical applications. Results revealed that electrical conductivity of the scaffolds increased with increasing concentration of graphene nanoparticles. Significant difference was not observed in hydroxyapatite forming ability of the bare and the graphene-containing scaffolds immersed in simulated body fluid. In vitro cytotoxicity experiments (XTT tests) showed that pre-osteoblastic MC3T3-E1 cell viability percentages of the graphene- containing samples was higher than control group samples after 7 days of incubation. However, a decrease in cell viability rates was obtained after 14 days of incubation for samples coated with PCL containing graphene starting from 3 wt%. Additionally, results obtained in the live-dead assay were consistent with the results of XTT tests. A higher ALP activity was detected in cells cultured on the graphene-containing borate glass scaffolds than those on the bare PCL coated 13-93B3 scaffolds suggesting the presence of graphene nanopowders stimulated an early stage of osteoblastic differentiation. SEM analysis showed that MC3T3-E1 cells exhibited a flat appearance and spread out through the surface in all groups of scaffolds starting from 3 days of incubation.

  9. Mechanical stress activates Smad pathway through PKCδ to enhance interleukin-11 gene transcription in osteoblasts.

    Directory of Open Access Journals (Sweden)

    Shinsuke Kido

    Full Text Available BACKGROUND: Mechanical stress rapidly induces ΔFosB expression in osteoblasts, which binds to interleukin (IL-11 gene promoter to enhance IL-11 expression, and IL-11 enhances osteoblast differentiation. Because bone morphogenetic proteins (BMPs also stimulate IL-11 expression in osteoblasts, there is a possibility that BMP-Smad signaling is involved in the enhancement of osteoblast differentiation by mechanical stress. The present study was undertaken to clarify whether mechanical stress affects BMP-Smad signaling, and if so, to elucidate the role of Smad signaling in mechanical stress-induced enhancement of IL-11 gene transcription. METHODOLOGY/PRINCIPAL FINDINGS: Mechanical loading by fluid shear stress (FSS induced phosphorylation of BMP-specific receptor-regulated Smads (BR-Smads, Smad1/5, in murine primary osteoblasts (mPOBs. FSS rapidly phosphorylated Y311 of protein kinase C (PKCδ, and phosphorylated PKCδ interacted with BR-Smads to phosphorylate BR-Smads. Transfection of PKCδ siRNA or Y311F mutant PKCδ abrogated BR-Smads phosphorylation and suppressed IL-11 gene transcription enhanced by FSS. Activated BR-Smads bound to the Smad-binding element (SBE of IL-11 gene promoter and formed complex with ΔFosB/JunD heterodimer via binding to the C-terminal region of JunD. Site-directed mutagenesis in the SBE and the AP-1 site revealed that both SBE and AP-1 sites were required for full activation of IL-11 gene promoter by FSS. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that PKCδ-BR-Smads pathway plays an important role in the intracellular signaling in response to mechanical stress, and that a cross-talk between PKCδ-BR-Smads and ΔFosB/JunD pathways synergistically stimulates IL-11 gene transcription in response to mechanical stress.

  10. Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes.

    Directory of Open Access Journals (Sweden)

    Padma P Srinivasan

    Full Text Available Voltage-sensitive calcium channels (VSCC regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3-E1 and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degeneration.

  11. Constitutive β-catenin activation in osteoblasts impairs terminal osteoblast differentiation and bone quality

    Energy Technology Data Exchange (ETDEWEB)

    Bao, Quanwei; Chen, Sixu; Qin, Hao [State Key Laboratory of Trauma, Burn and Combined injury, Department of Trauma Surgery, Daping Hospital, Third Military Medical University, ChongQing 400042 (China); Feng, Jianquan [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A& M Health Science Center, Dallas, TX 75246 (United States); Liu, Huayu; Liu, Daocheng; Li, Ang; Shen, Yue; Zhong, Xiaozheng; Li, Junfeng [State Key Laboratory of Trauma, Burn and Combined injury, Department of Trauma Surgery, Daping Hospital, Third Military Medical University, ChongQing 400042 (China); Zong, Zhaowen, E-mail: zongzhaowen@sina.cn [State Key Laboratory of Trauma, Burn and Combined injury, Department of Trauma Surgery, Daping Hospital, Third Military Medical University, ChongQing 400042 (China)

    2017-01-01

    Accumulating evidence suggests that Wnt/β-catenin signaling plays a central role in controlling bone mass. We previously reported that constitutive activation of β-catenin (CA-β-catenin) in osteoblasts potentially has side effects on the bone growth and bone remodeling process, although it could increase bone mass. The present study aimed to observe the effects of osteoblastic CA-β-catenin on bone quality and to investigate possible mechanisms of these effects. It was found that CA-β-catenin mice exhibited lower mineralization levels and disorganized collagen in long bones as confirmed by von Kossa staining and sirius red staining, respectively. Also, bone strength decreased significantly in CA-β-catenin mice. Then the effect of CA-β-catenin on biological functions of osteoblasts were investigated and it was found that the expression levels of osteocalcin, a marker for the late differentiation of osteoblasts, decreased in CA-β-catenin mice, while the expression levels of osterix and alkaline phosphatase, two markers for the early differentiation of osteoblasts, increased in CA-β-catenin mice. Furthermore, higher proliferation rate were revealed in osteoblasts that were isolated from CA-β-catenin mice. The Real-time PCR and western blot examination found that the expression level of c-myc and cyclin D1, two G1 progression-related molecules, increased in osteoblasts that were isolated from the CA-β-catenin mice, and the expression levels of CDK14 and cyclin Y, two mitotic-related molecules that can accelerate cells entering into S and G2/M phases, increased in osteoblasts that were isolated from the CA-β-catenin mice. In summary, osteoblastic CA-β-catenin kept osteoblasts in high proliferative state and impaired the terminal osteoblast differentiation, and this led to changed bone structure and decreased bone strength. - Highlights: • Wnt/β-catenin signaling plays a central role in controlling bone mass. • CA-β-catenin has side effects on the bone

  12. Single walled carbon nanotube composites for bone tissue engineering.

    Science.gov (United States)

    Gupta, Ashim; Woods, Mia D; Illingworth, Kenneth David; Niemeier, Ryan; Schafer, Isaac; Cady, Craig; Filip, Peter; El-Amin, Saadiq F

    2013-09-01

    The purpose of this study was to develop single walled carbon nanotubes (SWCNT) and poly lactic-co-glycolic acid (PLAGA) composites for orthopedic applications and to evaluate the interaction of human stem cells (hBMSCs) and osteoblasts (MC3T3-E1 cells) via cell growth, proliferation, gene expression, extracellular matrix production and mineralization. PLAGA and SWCNT/PLAGA composites were fabricated with various amounts of SWCNT (5, 10, 20, 40, and 100 mg), characterized and degradation studies were performed. Cells were seeded and cell adhesion/morphology, growth/survival, proliferation and gene expression analysis were performed to evaluate biocompatibility. Imaging studies demonstrated uniform incorporation of SWCNT into the PLAGA matrix and addition of SWCNT did not affect the degradation rate. Imaging studies revealed that MC3T3-E1 and hBMSCs cells exhibited normal, non-stressed morphology on the composites and all were biocompatible. Composites with 10 mg SWCNT resulted in highest rate of cell proliferation (p PLAGA composites imparted beneficial cellular growth capabilities and gene expression, and mineralization abilities were well established. These results demonstrate the potential of SWCNT/PLAGA composites for musculoskeletal regeneration and bone tissue engineering (BTE) and are promising for orthopedic applications. Copyright © 2013 Orthopaedic Research Society.

  13. Three-dimensional bioactive glass implants fabricated by rapid prototyping based on CO(2) laser cladding.

    Science.gov (United States)

    Comesaña, R; Lusquiños, F; Del Val, J; López-Álvarez, M; Quintero, F; Riveiro, A; Boutinguiza, M; de Carlos, A; Jones, J R; Hill, R G; Pou, J

    2011-09-01

    Three-dimensional bioactive glass implants were produced by rapid prototyping based on laser cladding without using moulds. CO(2) laser radiation was employed to melt 45S5 and S520 bioactive glass particles and to deposit the material layer by layer following a desired geometry. Controlled thermal input and cooling rate by fine tuning of the processing parameters allowed the production of crack-free fully dense implants. Microstructural characterization revealed chemical composition stability, but crystallization during processing was extensive when 45S5 bioactive glass was used. Improved results were obtained using the S520 bioactive glass, which showed limited surface crystallization due to an expanded sintering window (the difference between the glass transition temperature and crystallization onset temperature). Ion release from the S520 implants in Tris buffer was similar to that of amorphous 45S5 bioactive glass prepared by casting in graphite moulds. Laser processed S520 scaffolds were not cytotoxic in vitro when osteoblast-like MC3T3-E1 cells were cultured with the dissolution products of the glasses; and the MC3T3-E1 cells attached and spread well when cultured on the surface of the materials. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  14. PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: In vitro cell culture studies

    International Nuclear Information System (INIS)

    Milovac, Dajana; Gamboa-Martínez, Tatiana C.; Ivankovic, Marica; Gallego Ferrer, Gloria; Ivankovic, Hrvoje

    2014-01-01

    In the present study, we examined the potential of using highly porous poly(ε-caprolactone) (PCL)-coated hydroxyapatite (HAp) scaffold derived from cuttlefish bone for bone tissue engineering applications. The cell culture studies were performed in vitro with preosteoblastic MC3T3-E1 cells in static culture conditions. Comparisons were made with uncoated HAp scaffold. The attachment and spreading of preosteoblasts on scaffolds were observed by Live/Dead staining Kit. The cells grown on the HAp/PCL composite scaffold exhibited greater spreading than cells grown on the HAp scaffold. DNA quantification and scanning electron microscopy (SEM) confirmed a good proliferation of cells on the scaffolds. DNA content on the HAp/PCL scaffold was significantly higher compared to porous HAp scaffolds. The amount of collagen synthesis was determined using a hydroxyproline assay. The osteoblastic differentiation of the cells was evaluated by determining alkaline phosphatase (ALP) activity and collagen type I secretion. Furthermore, cell spreading and cell proliferation within scaffolds were observed using a fluorescence microscope. - Highlights: • Hydroxyapatite/poly(ε-caprolactone) scaffold with interconnected pores was prepared • Cytotoxicity test showed that the scaffold was not cytotoxic towards MC3T3-E1 cells • The scaffold supported the attachment, proliferation and differentiation of cells • A 3D cell colonization was confirmed using the fluorescence microscopy • The scaffold might be a promising candidate for bone tissue engineering

  15. Alpha-particles microbeam irradiation: impact of reactive oxygen species in bystander effect

    International Nuclear Information System (INIS)

    Hanot, M.

    2008-11-01

    Ionizing radiation-induced bystander effects arise in bystander cells that receive signals from directly irradiated cells. To date, free radicals are believed to play an active role in the bystander response, but this is incompletely characterized. To mark temporal and spatial impacts of bystander effect, we employed a precise α-particle microbeam to target a small fraction of sub-confluent osteoblastic cell cultures (MC3T3-E1). We identified the cellular membrane and mitochondria like two distinct places generating reactive oxygen species. The global oxidative stress observed after irradiation was significantly attenuated after filipin treatment, evidencing the pivotal role of membrane in MC3T3-E1 cells bystander response. To determine impact of bystander effect at a cell level, cellular consequences of this membrane-dependant bystander effect were then investigated. A variable fraction of the cell population (10 to 100%) was individually targeted. In this case, mitotic death and micronuclei yield both increased in bystander cells as well as in targeted cells demonstrating a role of bystander signals between irradiated cells in an autocrine or paracrine manner. Our results indicate a complex interaction of direct irradiation and bystander signals that lead to a membrane-dependant amplification of cell responses. (author)

  16. FLUOXETINE INHIBITS OSTEOBLAST DIFFERENTIATION & MINERALIZATION IN FRACTURE HEALING

    Science.gov (United States)

    Bradaschia-Correa, Vivian; Josephson, Anne M; Mehta, Devan; Mizrahi, Matthew; Neibart, Shane S; Liu, Chao; Kennedy, Oran; Castillo, Alesha B; Egol, Kenneth A; Leucht, Philipp

    2016-01-01

    Chronic use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression has been linked to osteoporosis. In this study, we investigated the effect of chronic SSRI use on fracture healing in two murine models of bone regeneration. First, we performed a comprehensive analysis of endochondral bone healing in a femur fracture model. C57/BL6 mice treated with fluoxetine, the most commonly prescribed SSRI, developed a normal cartilaginous soft-callus at 14 days after fracture and demonstrated a significantly smaller and biomechanically weaker bony hard-callus at 28 days. In order to further dissect the mechanism that resulted in a smaller bony regenerate, we used an intramembranous model of bone healing and revealed that fluoxetine treatment resulted in a significantly smaller bony callus at 7 and 14 days postinjury. In order to test whether the smaller bony regenerate following fluoxetine treatment was caused by an inhibition of osteogenic differentiation and/or mineralization, we employed in vitro experiments, which established that fluoxetine treatment decreases osteogenic differentiation and mineralization and that this effect is serotonin-independent. Finally, in a translational approach, we tested whether cessation of the medication would result in restoration of the regenerative potential. However, histologic and µCT analysis revealed non-union formation in these animals with fibrous tissue interposition within the callus. In conclusion, fluoxetine exerts a direct, inhibitory effect on osteoblast differentiation and mineralization, shown in two disparate murine models of bone repair. Discontinuation of the drug did not result in restoration of the healing potential, but rather led to complete arrest of the repair process. Besides the well-established effect of SSRIs on bone homeostasis, our study provides strong evidence that fluoxetine use negatively impacts fracture healing. PMID:27869327

  17. Toxicity of iron oxide nanoparticles against osteoblasts

    International Nuclear Information System (INIS)

    Shi Sifeng; Jia Jingfu; Guo Xiaokui; Zhao Yaping; Liu Boyu; Chen Desheng; Guo Yongyuan; Zhang Xianlong

    2012-01-01

    Magnetic nanoparticles have been widely used for tissue repair, magnetic resonance imaging, immunoassays and drug delivery. They are very promising in orthopaedic applications and several magnetic nanoparticles have been exploited for the treatment of orthopaedic disease. Here, we conducted an in vitro study to examine the interaction of magnetic iron oxide nanoparticles with human osteoblasts to evaluate the dose-related toxicity of the nanoparticles on osteoblasts. A transmission electron microscope was used to visualise the internalised magnetic nanoparticles in osteoblasts. The CCK-8 results revealed increased cell viability (107.5 % vitality compared with the control group) when co-cultured at a low concentration (20 μg/mL) and decreased cell viability (59.5 % vitality in a concentration of 300 μg/mL and 25.9 % in 500 μg/mL) when co-cultured in high concentrations. The flow cytometric detection revealed similar results with 5.48 % of apoptosis in a concentration of 20 μg/mL, 23.40 % of apoptosis in a concentration of 300 μg/mL and 28.49 % in a concentration of 500 μg/mL. The disrupted cytoskeleton of osteoblasts was also revealed using a laser scanning confocal microscope. We concluded that use of a low concentration of magnetic iron oxide nanoparticles is important to avoid damage to osteoblasts.

  18. Toxicity of iron oxide nanoparticles against osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Shi Sifeng [Shanghai Jiao Tong University, Department of Orthopaedic Surgery, Shanghai Sixth People' s Hospital (China); Jia Jingfu [Shanghai Jiao Tong University, School of Chemistry and Chemical Technology (China); Guo Xiaokui [Shanghai Jiao Tong University School of Medicine, Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences (China); Zhao Yaping [Shanghai Jiao Tong University, School of Chemistry and Chemical Technology (China); Liu Boyu [Shanghai Jiao Tong University School of Medicine, Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences (China); Chen Desheng; Guo Yongyuan; Zhang Xianlong, E-mail: zhangxianlong20101@163.com [Shanghai Jiao Tong University, Department of Orthopaedic Surgery, Shanghai Sixth People' s Hospital (China)

    2012-09-15

    Magnetic nanoparticles have been widely used for tissue repair, magnetic resonance imaging, immunoassays and drug delivery. They are very promising in orthopaedic applications and several magnetic nanoparticles have been exploited for the treatment of orthopaedic disease. Here, we conducted an in vitro study to examine the interaction of magnetic iron oxide nanoparticles with human osteoblasts to evaluate the dose-related toxicity of the nanoparticles on osteoblasts. A transmission electron microscope was used to visualise the internalised magnetic nanoparticles in osteoblasts. The CCK-8 results revealed increased cell viability (107.5 % vitality compared with the control group) when co-cultured at a low concentration (20 {mu}g/mL) and decreased cell viability (59.5 % vitality in a concentration of 300 {mu}g/mL and 25.9 % in 500 {mu}g/mL) when co-cultured in high concentrations. The flow cytometric detection revealed similar results with 5.48 % of apoptosis in a concentration of 20 {mu}g/mL, 23.40 % of apoptosis in a concentration of 300 {mu}g/mL and 28.49 % in a concentration of 500 {mu}g/mL. The disrupted cytoskeleton of osteoblasts was also revealed using a laser scanning confocal microscope. We concluded that use of a low concentration of magnetic iron oxide nanoparticles is important to avoid damage to osteoblasts.

  19. Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation.

    NARCIS (Netherlands)

    Glass, D.A.; Bialek, P.; Ahn, J.D.; Starbuck, M.; Patel, M.S.; Clevers, J.C.; Taketo, M.M.; Long, F.; McMahon, A.P.; Lang, R.A.; Karsenty, G.

    2005-01-01

    Inactivation of beta-catenin in mesenchymal progenitors prevents osteoblast differentiation; inactivation of Lrp5, a gene encoding a likely Wnt coreceptor, results in low bone mass (osteopenia) by decreasing bone formation. These observations indicate that Wnt signaling controls osteoblast

  20. Osteokalzinexpression and regulation in hematologic malignancies and in cultured cells

    International Nuclear Information System (INIS)

    Wihlidal, P.

    2010-01-01

    Main issue of this work was to gain further insight into the association of haematopoiesis and osteopoiesis. A crucial cue for that is the fact that haematopoietic stem cells of haematopoietic diseases, which are characterised by c-KIT (CD117) expression, express the osteoblast marker osteocalcin. Thus, attention was focussed on the expression and regulation of osteocalcin, on one hand in blood and bone marrow samples of haematological diseases and on the other hand in leukaemic and osteosarcoma cell lines, i.e., by 1. investigating the expression of osteocalcin (OCN) splicing variants in haematological malignancies. We analysed bone marrow obtained from two patients with chronic myeloid leukaemia (CML), seven patients with other myeloproliferative diseases (MPD) and four patients with acute myeloid leukaemia (AML). RT-PCR analyses were performed in order to assess and quantify spliced (OCNs) and unspliced (OCNu) mRNA, the associated transcription factors (AML1 and AML3) as well as c-KIT, which is a marker for activated stem cells. Our data indicate that OCNs mRNA and OCN protein are expressed in c-KIT positive neoplastic stem cells in haematological malignancies. 2. It has been suggested that the tyrosine kinase inhibitor imatinib mesylate (IM), which has proven anti-proliferative effect, influences osteogenesis and bone turnover in treated patients. Thus, we aimed to quantify OCN mRNA, its splicing variants, the associated Runt-domain transcription factors AML1 and AML3, c-KIT and several metabolic genes to gain evidence about the differentiation state in the HL-60 leukaemia cell line as well as MG63 and U2OS osteosarcoma cells and murine primary osteoblasts MC3T3-E1. Our data indicate that IM induces inhibition of proliferation and synthesis of total OCN-mRNA in all cell lines, but a relative increase of OCNs-mRNA was observed in the human cell lines. On the other hand, differentiation-associated genes appeared to be stimulated. This may also indicate an

  1. Overexpression of α-catenin increases osteoblastic differentiation in mouse mesenchymal C3H10T1/2 cells

    International Nuclear Information System (INIS)

    Kim, Dohee; Yang, Jae-Yeon; Shin, Chan Soo

    2009-01-01

    α- and β-Catenin link cadherins to the actin-based cytoskeleton at adherens junctions and regulate cell-cell adhesion. Although roles of cadherins and canonical Wnt-/β-catenin-signaling in osteoblastic differentiation have been extensively studied, the role of α-catenin is not known. Murine embryonic mesenchymal stem cells, C3H10T1/2 cells, were transduced with retrovirus encoding α-catenin (MSCV-α-catenin-HA-GFP). In the presence of Wnt-3A conditioned medium or osteogenic medium (β-glycerol phosphate and ascorbic acid), cells overexpressing α-catenin showed enhanced osteoblastic differentiation as measured by alkaline phosphatase (ALP) staining and ALP activity assay compared to cells transduced with empty virus (MSCV-GFP). In addition, mRNA expression of osteocalcin and Runx2 was significantly increased compared to control. Cell aggregation assay revealed that α-catenin overexpression has significantly increased cell-cell aggregation. However, cellular β-catenin levels (total, cytoplasmic-nuclear ratio) and β-catenin-TCF/LEF transcriptional activity did not change by overexpression of α-catenin. Knock-down of α-catenin using siRNA decreased osteoblastic differentiation as measured by ALP assay. These results suggest that α-catenin overexpression increases osteoblastic differentiation by increasing cell-cell adhesion rather than Wnt-/β-catenin-signaling.

  2. Dysregulated gene expression in the primary osteoblasts and osteocytes isolated from hypophosphatemic Hyp mice.

    Directory of Open Access Journals (Sweden)

    Kazuaki Miyagawa

    Full Text Available Osteocytes express multiple genes involved in mineral metabolism including PHEX, FGF23, DMP1 and FAM20C. In Hyp mice, a murine model for X-linked hypophosphatemia (XLH, Phex deficiency results in the overproduction of FGF23 in osteocytes, which leads to hypophosphatemia and impaired vitamin D metabolism. In this study, to further clarify the abnormality in osteocytes of Hyp mice, we obtained detailed gene expression profiles in osteoblasts and osteocytes isolated from the long bones of 20-week-old Hyp mice and wild-type (WT control mice. The expression of Fgf23, Dmp1, and Fam20c was higher in osteocytic cells than in osteoblastic cells in both genotypes, and was up-regulated in Hyp cells. Interestingly, the up-regulation of these genes in Hyp bones began before birth. On the other hand, the expression of Slc20a1 encoding the sodium/phosphate (Na+/Pi co-transporter Pit1 was increased in osteoblasts and osteocytes from adult Hyp mice, but not in Hyp fetal bones. The direct effects of extracellular Pi and 1,25-dihydroxyvitamin D3 [1,25(OH2D3] on isolated osteoblastic and osteocytic cells were also investigated. Twenty-four-hour treatment with 10-8 M 1,25(OH2D3 increased the expression of Fgf23 in WT osteoblastic cells but not in osteocytic cells. Dmp1 expression in osteocytic cells was increased due to the 24-hour treatment with 10 mM Pi and was suppressed by 10-8 M 1,25(OH2D3 in WT osteocytic cells. We also found the up-regulation of the genes for FGF1, FGF2, their receptors, and Egr-1 which is a target of FGF signaling, in Hyp osteocytic cells, suggesting the activation of FGF/FGFR signaling. These results implicate the complex gene dysregulation in osteoblasts and osteocytes of Hyp mice, which might contribute to the pathogenesis.

  3. Osteoblast and osteocyte: games without frontiers.

    Science.gov (United States)

    Capulli, Mattia; Paone, Riccardo; Rucci, Nadia

    2014-11-01

    The portrait of osteoblasts and osteocytes has been subjected to a revision, since a large body of evidence is attributing these cells amazing roles both inside and outside the bone. The osteoblast, long confined to its bone building function, is actually a very eclectic cell, actively regulating osteoclast formation and function as well as hematopoietic stem cells homeostasis. It is also an endocrine cell, affecting energy metabolism, male fertility and cognition through the release of osteocalcin, a perfect definition-fitting hormone in its uncarboxylated state. As for the osteocytes, many evidence shows that they do not merely represent the final destination of the osteoblasts, but they are instead very active cells that, besides a mechanosensorial function, actively contribute to the bone remodelling by regulating bone formation and resorption. The regulation is exerted by the production of sclerostin (SOST), which in turn inhibits osteoblast differentiation by blocking Wnt/beta-catenin pathway. At the same time, osteocytes influence bone resorption both indirectly, by producing RANKL, which stimulates osteoclastogenesis, and directly by means of a local osteolysis, which is observed especially under pathological conditions. The great versatility of both these cells reflects the complexity of the bone tissue, which has not only a structural role, but influences and is influenced by different organs, taking part in homeostatic and adaptive responses affecting the whole organism. Copyright © 2014. Published by Elsevier Inc.

  4. Irisin Enhances Osteoblast Differentiation In Vitro

    Directory of Open Access Journals (Sweden)

    Graziana Colaianni

    2014-01-01

    Full Text Available It has been recently demonstrated that exercise activity increases the expression of the myokine Irisin in skeletal muscle, which is able to drive the transition of white to brown adipocytes, likely following a phenomenon of transdifferentiation. This new evidence supports the idea that muscle can be considered an endocrine organ, given its ability to target adipose tissue by promoting energy expenditure. In accordance with these new findings, we hypothesized that Irisin is directly involved in bone metabolism, demonstrating its ability to increase the differentiation of bone marrow stromal cells into mature osteoblasts. Firstly, we confirmed that myoblasts from mice subjected to 3 weeks of free wheel running increased Irisin expression compared to nonexercised state. The conditioned media (CM collected from myoblasts of exercised mice induced osteoblast differentiation in vitro to a greater extent than those of mice housed in resting conditions. Furthermore, the differentiated osteoblasts increased alkaline phosphatase and collagen I expression by an Irisin-dependent mechanism. Our results show, for the first time, that Irisin directly targets osteoblasts, enhancing their differentiation. This finding advances notable perspectives in future studies which could satisfy the ongoing research of exercise-mimetic therapies with anabolic action on the skeleton.

  5. Magnesium substitution in the structure of orthopedic nanoparticles: A comparison between amorphous magnesium phosphates, calcium magnesium phosphates, and hydroxyapatites

    International Nuclear Information System (INIS)

    Nabiyouni, Maryam; Ren, Yufu; Bhaduri, Sarit B.

    2015-01-01

    As biocompatible materials, magnesium phosphates have received a lot of attention for orthopedic applications. During the last decade multiple studies have shown advantages for magnesium phosphate such as lack of cytotoxicity, biocompatibility, strong mechanical properties, and high biodegradability. The present study investigates the role of Mg +2 and Ca +2 ions in the structure of magnesium phosphate and calcium phosphate nanoparticles. To directly compare the effect of Mg +2 and Ca +2 ions on structure of nanoparticles and their biological behavior, three groups of nanoparticles including amorphous magnesium phosphates (AMPs) which release Mg +2 , calcium magnesium phosphates (CMPs) which release Mg +2 and Ca +2 , and hydroxyapatites (HAs) which release Ca +2 were studied. SEM, TEM, XRD, and FTIR were used to evaluate the morphology, crystallinity, and chemical properties of the particles. AMP particles were homogeneous nanospheres, whereas CMPs were combinations of heterogeneous nanorods and nanospheres, and HAs which contained heterogeneous nanosphere particles. Cell compatibility was monitored in all groups to determine the cytotoxicity effect of particles on studied MC3T3-E1 preosteoblasts. AMPs showed significantly higher attachment rate than the HAs after 1 day and both AMPs and CMPs showed significantly higher proliferation rate when compared to HAs after 7 days. Gene expression level of osteoblastic markers ALP, COL I, OCN, OPN, RUNX2 were monitored and they were normalized to GAPDH housekeeping gene. Beta actin expression level was monitored as the second housekeeping gene to confirm the accuracy of results. In general, AMPs and CMPs showed higher expression level of osteoblastic genes after 7 days which can further confirm the stimulating role of Mg + 2 and Ca +2 ions in increasing the proliferation rate, differentiation, and mineralization of MC3T3-E1 preosteoblasts. - Highlights: • Role of Mg 2+ and Ca 2+ ions in proliferation, and differentiation

  6. Osteocytes, not Osteoblasts or Lining Cells, are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone.

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    Jinhu Xiong

    Full Text Available The cytokine receptor activator of nuclear factor kappa B ligand (RANKL, encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone.

  7. An osteoblast-derived proteinase controls tumor cell survival via TGF-beta activation in the bone microenvironment.

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    Thiolloy, Sophie; Edwards, James R; Fingleton, Barbara; Rifkin, Daniel B; Matrisian, Lynn M; Lynch, Conor C

    2012-01-01

    Breast to bone metastases frequently induce a "vicious cycle" in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment. To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays). Collectively, these studies identify a novel "mini-vicious cycle" between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases.

  8. An osteoblast-derived proteinase controls tumor cell survival via TGF-beta activation in the bone microenvironment.

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    Sophie Thiolloy

    Full Text Available Breast to bone metastases frequently induce a "vicious cycle" in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment.To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry. Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry. Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1 the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay; and 2 that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays.Collectively, these studies identify a novel "mini-vicious cycle" between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases.

  9. Osteoblast-specific transcription factor Osterix increases vitamin D receptor gene expression in osteoblasts.

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    Chi Zhang

    Full Text Available Osterix (Osx is an osteoblast-specific transcription factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR is a member of the nuclear hormone receptor superfamily that regulates target gene transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for transcriptional regulation by Osx in osteoblasts. For example, calvaria obtained from Osx-null embryos displayed dramatic reductions in VDR expression compared to wild-type calvaria. Stable overexpression of Osx stimulated VDR expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of VDR. In contrast, Osx levels remained unchanged in osteoblasts in VDR-null mice. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the VDR promoter in a dose-dependent manner. To define the region of the VDR promoter that was responsive to Osx, a series of VDR promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 120 bp of the VDR promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VDR promoter activation by Osx. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native VDR promoter in primary osteoblasts in vivo. Cumulatively, these data strongly support a direct regulatory role for Osx in VDR gene expression. They further provide new insight into potential mechanisms and pathways that Osx controls in osteoblasts and during the process of osteoblastic cell differentiation.

  10. Cells responding to surface structure of calcium phosphate ceramics for bone regeneration.

    Science.gov (United States)

    Zhang, Jingwei; Sun, Lanying; Luo, Xiaoman; Barbieri, Davide; de Bruijn, Joost D; van Blitterswijk, Clemens A; Moroni, Lorenzo; Yuan, Huipin

    2017-11-01

    Surface structure largely affects the inductive bone-forming potential of calcium phosphate (CaP) ceramics in ectopic sites and bone regeneration in critical-sized bone defects. Surface-dependent osteogenic differentiation of bone marrow stromal cells (BMSCs) partially explained the improved bone-forming ability of submicron surface structured CaP ceramics. In this study, we investigated the possible influence of surface structure on different bone-related cells, which may potentially participate in the process of improved bone formation in CaP ceramics. Besides BMSCs, the response of human brain vascular pericytes (HBVP), C2C12 (osteogenic inducible cells), MC3T3-E1 (osteogenic precursors), SV-HFO (pre-osteoblasts), MG63 (osteoblasts) and SAOS-2 (mature osteoblasts) to the surface structure was evaluated in terms of cell proliferation, osteogenic differentiation and gene expression. The cells were cultured on tricalcium phosphate (TCP) ceramics with either micron-scaled surface structure (TCP-B) or submicron-scaled surface structure (TCP-S) for up to 14 days, followed by DNA, alkaline phosphatase (ALP) and quantitative polymerase chain reaction gene assays. HBVP were not sensitive to surface structure with respect to cell proliferation and osteogenic differentiation, but had downregulated angiogenesis-related gene expression (i.e. vascular endothelial growth factor) on TCP-S. Without additional osteogenic inducing factors, submicron-scaled surface structure enhanced ALP activity and osteocalcin gene expression of human (h)BMSCs and C2C12 cells, favoured the proliferation of MC3T3-E1, MG63 and SAOS-2, and increased ALP activity of MC3T3-E1 and SV-HFO. The results herein indicate that cells with osteogenic potency (either osteogenic inducible cells or osteogenic cells) could be sensitive to surface structure and responded to osteoinductive submicron-structured CaP ceramics in cell proliferation, ALP production or osteogenic gene expression, which favour bone

  11. In vitro characterization of two different atmospheric plasma jet chemical functionalizations of titanium surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Mussano, F., E-mail: federico.mussano@unito.it [CIR Dental School, Department of Surgical Sciences UNITO, via Nizza 230, 10126, Turin (Italy); Genova, T. [CIR Dental School, Department of Surgical Sciences UNITO, via Nizza 230, 10126, Turin (Italy); Department of Life Sciences and Systems Biology, UNITO, via Accademia Albertina 13, 10123, Turin (Italy); Verga Falzacappa, E. [Department of Molecular Science and Nanosystems, UNIVE, Via Torino 155, 30170, Venezia (Italy); Nadir srl, Via Torino 155, 30170 Venezia (Italy); Scopece, P. [Nadir srl, Via Torino 155, 30170 Venezia (Italy); Munaron, L. [Department of Life Sciences and Systems Biology, UNITO, via Accademia Albertina 13, 10123, Turin (Italy); Centre for Nanostructured Interfaces and Surfaces (NIS) (Italy); Rivolo, P.; Mandracci, P. [Politecnico di Torino, Department of Applied Science and Technology, Materials and Microsoystems Laboratory (ChiLab), Corso Duca degli Abruzzi 24, 10129, Torino (Italy); Benedetti, A. [Department of Molecular Science and Nanosystems, UNIVE, Via Torino 155, 30170, Venezia (Italy); Carossa, S. [CIR Dental School, Department of Surgical Sciences UNITO, via Nizza 230, 10126, Turin (Italy); Patelli, A. [Department of Physics and Astronomy, UNIPD, via Marzolo 8, 35122 Padova (Italy)

    2017-07-01

    Highlights: • NH{sub 2}-Ti and COOH/R-Ti obtained via atmospheric plasma jet RF-APPJ portable equipment. • Higher quantity of adsorbed proteins and improved cell adhesion on treated surfaces. • More tapered and elongated cells on NH{sub 2}-Ti compared to COOH/R-Ti. • Higher osteocalcin expression on NH{sub 2}-Ti. - Abstract: Plasma surface activation and plasma polymers deposition are promising technologies capable to modulate biologically relevant surface features of biomaterials. The purpose of this study was to evaluate the biological effects of two different surface modifications, i.e. amine (NH{sub 2}-Ti) and carboxylic/esteric (COOH/R-Ti) functionalities obtained from 3-aminopropyltriethoxysilane (3-APTES) and methylmethacrylate (MMA) precursors, respectively, through an atmospheric plasma jet RF-APPJ portable equipment. The coatings were characterized by Scanning Electron Microscopy, FT-IR spectroscopy, XPS and surface energy calculations. Stability in water and after UV sterilization were also verified. The pre-osteoblastic murine cell line MC3T3-E1 was used to perform the in-vitro tests. The treated samples showed a higher quantity of adsorbed proteins and improved osteoblast cells adhesion on the surfaces compared to the pristine titanium, in particular the COOH/R-Ti led to a nearly two-fold improvement. Cell proliferation on coated samples was initially (at 24 h) lower than on titanium control, while, at 48 h, COOH/R-Ti reached the proliferation rate of pristine titanium. Cells grown on NH{sub 2}-Ti were more tapered and elongated in shape with lower areas than on COOH/R-Ti enriched surfaces. Finally, NH{sub 2}-Ti significantly enhanced osteocalcin production, starting from 14 days, while COOH/R-Ti had this effect only from 21 days. Notably, NH{sub 2}-Ti was more efficient than COOH/R-Ti at 21 days. The amine functionality elicited the most relevant osteogenic effect in terms of osteocalcin expression, thus establishing an interesting correlation

  12. Endothelial-to-Osteoblast Conversion Generates Osteoblastic Metastasis of Prostate Cancer.

    Science.gov (United States)

    Lin, Song-Chang; Lee, Yu-Chen; Yu, Guoyu; Cheng, Chien-Jui; Zhou, Xin; Chu, Khoi; Murshed, Monzur; Le, Nhat-Tu; Baseler, Laura; Abe, Jun-Ichi; Fujiwara, Keigi; deCrombrugghe, Benoit; Logothetis, Christopher J; Gallick, Gary E; Yu-Lee, Li-Yuan; Maity, Sankar N; Lin, Sue-Hwa

    2017-06-05

    Prostate cancer (PCa) bone metastasis is frequently associated with bone-forming lesions, but the source of the osteoblastic lesions remains unclear. We show that the tumor-induced bone derives partly from tumor-associated endothelial cells that have undergone endothelial-to-osteoblast (EC-to-OSB) conversion. The tumor-associated osteoblasts in PCa bone metastasis specimens and patient-derived xenografts (PDXs) were found to co-express endothelial marker Tie-2. BMP4, identified in PDX-conditioned medium, promoted EC-to-OSB conversion of 2H11 endothelial cells. BMP4 overexpression in non-osteogenic C4-2b PCa cells led to ectopic bone formation under subcutaneous implantation. Tumor-induced bone was reduced in trigenic mice (Tie2 cre /Osx f/f /SCID) with endothelial-specific deletion of osteoblast cell-fate determinant OSX compared with bigenic mice (Osx f/f /SCID). Thus, tumor-induced EC-to-OSB conversion is one mechanism that leads to osteoblastic bone metastasis of PCa. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Notch Inhibits Osteoblast Differentiation and Causes Osteopenia

    Science.gov (United States)

    Zanotti, Stefano; Smerdel-Ramoya, Anna; Stadmeyer, Lisa; Durant, Deena; Radtke, Freddy; Canalis, Ernesto

    2008-01-01

    Notch receptors are determinants of cell fate decisions. To define the role of Notch in the adult skeleton, we created transgenic mice overexpressing the Notch intracellular domain (NICD) under the control of the type I collagen promoter. First-generation transgenics were small and osteopenic. Bone histomorphometry revealed that NICD caused a decrease in bone volume, secondary to a reduction in trabecular number; osteoblast and osteoclast number were decreased. Low fertility of founder mice and lethality of young pups did not allow the complete establishment of transgenic lines. To characterize the effect of Notch overexpression in vitro, NICD was induced in osteoblasts and stromal cells from Rosanotch mice, in which a STOP cassette flanked by loxP sites is upstream of NICD, by transduction with an adenoviral vector expressing Cre recombinase (Cre) under the control of the cytomegalovirus (CMV) promoter (Ad-CMV-Cre). NICD impaired osteoblastogenesis and inhibited Wnt/β-catenin signaling. To determine the effects of notch1 deletion in vivo, mice in which notch1 was flanked by loxP sequences (notch1loxP/loxP) were mated with mice expressing Cre recombinase under the control of the osteocalcin promoter. Conditional null notch1 mice had no obvious skeletal phenotype, possibly because of rescue by notch2; however, 1-month-old females exhibited a modest increase in osteoclast surface and eroded surface. Osteoblasts from notch1loxP/loxP mice, transduced with Ad-CMV-Cre and transfected with Notch2 small interfering RNA, displayed increased alkaline phosphatase activity. In conclusion, Notch signaling in osteoblasts causes osteopenia and impairs osteo-blastogenesis by inhibiting the Wnt/β-catenin pathway. PMID:18420737

  14. Lactate induces osteoblast differentiation by stabilization of HIF1α.

    Science.gov (United States)

    Wu, Yu; Wang, Miaomiao; Feng, Haihua; Peng, Ying; Sun, Jieyun; Qu, Xiuxia; Li, Chunping

    2017-09-05

    Aerobic glycolysis is involved in osteoblast differentiation induced by Wnt signaling or PTH treatment. However, it is still unclear whether lactate, the end product of aerobic glycolysis, plays any role in osteoblast differentiation. Herein we report that in cultures of osteoblast-lineage cells, lactate promoted alkaline phosphatase-positive cell formation, increased the activity of alkaline phosphatase, and induced the expression of osteocalcin. This osteoblast differentiation-inducing effect of lactate can be inhibited by blocking its entry into cells with MCT1 siRNA or inhibitors, and by interfering with its metabolism by using specific siRNAs for LDHB and PDH. Moreover, lactate stabilized HIF1α expression and inhibited HIF1α activity, with BAY87-2243 lowering the osteoblast differentiation-inducing effect of lactate. Thus, these findings reveal an unrecognized role for aerobic glycolysis in osteoblast differentiation via its end product, lactate. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Osteoblast-secreted collagen upregulates paracrine Sonic hedgehog signaling by prostate cancer cells and enhances osteoblast differentiation

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    Zunich Samantha M

    2012-07-01

    Full Text Available Abstract Background Induction of osteoblast differentiation by paracrine Sonic hedgehog (Shh signaling may be a mechanism through which Shh-expressing prostate cancer cells initiate changes in the bone microenvironment and promote metastases. A hallmark of osteoblast differentiation is the formation of matrix whose predominant protein is type 1 collagen. We investigated the formation of a collagen matrix by osteoblasts cultured with prostate cancer cells, and its effects on interactions between prostate cancer cells and osteoblasts. Results In the presence of exogenous ascorbic acid (AA, a co-factor in collagen synthesis, mouse MC3T3 pre-osteoblasts in mixed cultures with human LNCaP prostate cancer cells or LNCaP cells modified to overexpress Shh (LNShh cells formed collagen matrix with distinct fibril ultrastructural characteristics. AA increased the activity of alkaline phosphatase and the expression of the alkaline phosphatase gene Akp2, markers of osteoblast differentiation, in MC3T3 pre-osteoblasts cultured with LNCaP or LNShh cells. However, the AA-stimulated increase in Akp2 expression in MC3T3 pre-osteoblasts cultured with LNShh cells far exceeded the levels observed in MC3T3 cells cultured with either LNCaP cells with AA or LNShh cells without AA. Therefore, AA and Shh exert a synergistic effect on osteoblast differentiation. We determined whether the effect of AA on LNShh cell-induced osteoblast differentiation was mediated by Shh signaling. AA increased the expression of Gli1 and Ptc1, target genes of the Shh pathway, in MC3T3 pre-osteoblasts cultured with LNShh cells to at least twice their levels without AA. The ability of AA to upregulate Shh signaling and enhance alkaline phosphatase activity was blocked in MC3T3 cells that expressed a dominant negative form of the transcription factor GLI1. The AA-stimulated increase in Shh signaling and Shh-induced osteoblast differentiation was also inhibited by the specific collagen synthesis

  16. Building strong bones: molecular regulation of the osteoblast lineage.

    Science.gov (United States)

    Long, Fanxin

    2011-12-22

    The past 15 years have witnessed tremendous progress in the molecular understanding of osteoblasts, the main bone-forming cells in the vertebrate skeleton. In particular, all of the major developmental signals (including WNT and Notch signalling), along with an increasing number of transcription factors (such as RUNX2 and osterix), have been shown to regulate the differentiation and/or function of osteoblasts. As evidence indicates that osteoblasts may also regulate the behaviour of other cell types, a clear understanding of the molecular identity and regulation of osteoblasts is important beyond the field of bone biology.

  17. Serotonin regulates osteoblast proliferation and function in vitro

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    Dai, S.Q.; Yu, L.P. [Department of Orthopedic Surgery, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Shi, X. [Department of Obstetrics and Gynecology, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Wu, H. [Emergency Department, The First Affiliated Hospital, Soochow University, Suzhou (China); Shao, P.; Yin, G.Y.; Wei, Y.Z. [Department of Orthopedic Surgery, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China)

    2014-08-01

    The monoamine serotonin (5-hydroxytryptamine, 5-HT), a well-known neurotransmitter, also has important functions outside the central nervous system. The objective of this study was to investigate the role of 5-HT in the proliferation, differentiation, and function of osteoblasts in vitro. We treated rat primary calvarial osteoblasts with various concentrations of 5-HT (1 nM to 10 µM) and assessed the rate of osteoblast proliferation, expression levels of osteoblast-specific proteins and genes, and the ability to form mineralized nodules. Next, we detected which 5-HT receptor subtypes were expressed in rat osteoblasts at different stages of osteoblast differentiation. We found that 5-HT could inhibit osteoblast proliferation, differentiation, and mineralization at low concentrations, but this inhibitory effect was mitigated at relatively high concentrations. Six of the 5-HT receptor subtypes (5-HT{sub 1A}, 5-HT{sub 1B}, 5-HT{sub 1D}, 5-HT{sub 2A}, 5-HT{sub 2B}, and 5-HT{sub 2C}) were found to exist in rat osteoblasts. Of these, 5-HT{sub 2A} and 5-HT{sub 1B} receptors had the highest expression levels, at both early and late stages of differentiation. Our results indicated that 5-HT can regulate osteoblast proliferation and function in vitro.

  18. Nanofibrous yet injectable polycaprolactone-collagen bone tissue scaffold with osteoprogenitor cells and controlled release of bone morphogenetic protein-2

    Energy Technology Data Exchange (ETDEWEB)

    Subramanian, Gayathri; Bialorucki, Callan [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Yildirim-Ayan, Eda, E-mail: eda.yildirimayan@utoledo.edu [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Department of Orthopaedic Surgery, University of Toledo Medical Center, Toledo, OH 43614 (United States)

    2015-06-01

    In this work, we developed a nanofibrous, yet injectable orthobiologic tissue scaffold that is capable of hosting osteoprogenitor cells and controlling kinetic release profile of the encapsulated pro-osteogenic factor without diminishing its bioactivity over 21 days. This innovative injectable scaffold was synthesized by incorporating electrospun and subsequently O{sub 2} plasma-functionalized polycaprolactone (PCL) nanofibers within the collagen type-I solution along with MC3T3-E1 cells (pre-osteoblasts) and bone morphogenetic protein-2 (BMP2). Through changing the PCL nanofiber concentration within the injectable scaffolds, we were able to tailor the mechanical strength, protein retention capacity, bioactivity preservation, and osteoinductive potential of the scaffolds. The nanofibrous internal structure of the scaffold allowed us to use a low dose of BMP2 (200 ng/ml) to achieve osteoblastic differentiation in in vitro culture. The osteogenesis capacity of the injectable scaffolds were evaluated though measuring MC3T3-E1 cell proliferation, ALP activity, matrix mineralization, and early- and late-osteoblast specific gene expression profiles over 21 days. The results demonstrated that the nanofibrous injectable scaffold provides not only an osteoinductive environment for osteoprogenitor cells to differentiate, but also a suitable biomechanical and biochemical environment to act as a reservoir for osteogenic factors with controlled release profile. - Highlights: • Injectable nanofibrous scaffold with osteoprogenitor cells and BMP2 was synthesized. • PCL nanofiber concentration within collagen scaffold affected the BMP2 retention and bioactivity. • Optimal PCL concentration was identified for mechanical stability, injectability, and osteogenic activity. • Scaffolds exhibited long-term osteoinductive capacity for bone repair and regeneration.

  19. Zoledronic acid at subtoxic dose extends osteoblastic stage span of primary human osteoblasts.

    Science.gov (United States)

    Zara, Susi; De Colli, Marianna; di Giacomo, Viviana; Zizzari, Vincenzo Luca; Di Nisio, Chiara; Di Tore, Umberto; Salini, Vincenzo; Gallorini, Marialucia; Tetè, Stefano; Cataldi, Amelia

    2015-04-01

    This study aimed to check the effect of zoledronic acid (ZA) at subtoxic dose on human osteoblasts (HOs) in terms of cell viability, apoptosis occurrence, and differentiation induction. ZA belongs to the family of bisphosphonates (BPs), largely used in the clinical practice for the treatment of bone diseases, often associated with jaw osteonecrosis onset. Their pharmacological action consists in the direct block of the osteoclast-mediated bone resorption along with indirect action on osteoblasts. HOs were treated choosing the highest limit concentration (10(-5) M) which does not induce toxic effects. Live/dead staining, flow cytometry, mitochondrial membrane potential assay, osteocalcin western blotting, gp38 RT-PCR, collagen type I, PGE2, and IL-6 ELISA assays were performed. Similar viability level between control and ZA-treated samples is found along with no significant increase of apoptotic and necrotic cells in ZA-treated sample. To establish if an early apoptotic pathway was triggered, Bax expression and mitochondrial membrane potential were evaluated finding a higher protein expression in control sample and a good integrity of mitochondrial membrane in both experimental points. Type I collagen secretion and alkaline phosphatase (ALP) activity appear increased in ZA-treated sample, osteocalcin expression level is reduced in ZA-treated cells, whereas no modifications of gp38 mRNA level are evidenced. No statistical differences are identified in PGE2 secretion level whereas IL-6 secretion is lower in ZA-treated HOs with respect to control ones. These results highlight that ZA, delaying the osteoblastic differentiation process versus the osteocytic lineage, strengthens its pharmacological activity enhancing bone density. The knowledge of ZA effects on osteoblasts at subtoxic dose allows to improve therapeutic protocols in order to strengthen drug pharmacological activity through a combined action on both osteoclastic and osteoblastic cells.

  20. Notch Signaling in Prostate Cancer Cells Promotes Osteoblastic Metastasis

    Science.gov (United States)

    2017-06-01

    information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this...function and number while inducing osteoblast proliferation. Our results suggest that Notch signaling from cancer cells promotes osteoblastic...Participants and other collaborating organizations: I initiated collaboration with Dr. Evan Keller at University of Michigan to interrogate PCa bone

  1. Tridax procumbens flavonoids promote osteoblast differentiation and bone formation

    Directory of Open Access Journals (Sweden)

    Md. Abdullah Al Mamun

    Full Text Available BACKGROUND: Tridaxprocumbens flavonoids (TPFs are well known for their medicinal properties among local natives. Besides traditionally used for dropsy, anemia, arthritis, gout, asthma, ulcer, piles, and urinary problems, it is also used in treating gastric problems, body pain, and rheumatic pains of joints. TPFs have been reported to increase osteogenic functioning in mesenchymal stem cells. Our previous study showed that TPFs were significantly suppressed the RANKL-induced differentiation of osteoclasts and bone resorption. However, the effects of TPFs to promote osteoblasts differentiation and bone formation remain unclear. TPFs were isolated from Tridax procumbens and investigated for their effects on osteoblasts differentiation and bone formation by using primary mouse calvarial osteoblasts RESULTS: TPFs promoted osteoblast differentiation in a dose-dependent manner demonstrated by up-regulation of alkaline phosphatase and osteocalcin. TPFs also upregulated osteoblast differentiation related genes, including osteocalcin, osterix, and Runx2 in primary osteoblasts. TPFs treated primary osteoblast cells showed significant upregulation of bone morphogenetic proteins (BMPs including Bmp-2, Bmp-4, and Bmp-7. Addition of noggin, a BMP specific-antagonist, inhibited TPFs induced upregulation of the osteocalcin, osterix, and Runx2 CONCLUSION: Our findings point towards the induction of osteoblast differentiation by TPFs and suggested that TPFs could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis

  2. Composite coating of 58S bioglass and hydroxyapatite on a poly (ethylene terepthalate) artificial ligament graft for the graft osseointegration in a bone tunnel

    Energy Technology Data Exchange (ETDEWEB)

    Li Hong; Wu Yang; Ge Yunsheng; Jiang Jia; Gao Kai [Department of Sports Medicine, Huashan Hospital, Shanghai (China); Zhang Pengyun; Wu Lingxiang [Shanghai Research Center of Biomedical Engineering, Shanghai (China); Chen Shiyi, E-mail: cshiyi@163.com [Department of Sports Medicine, Huashan Hospital, Shanghai (China)

    2011-09-01

    The purpose of this study was to determine the effect of the combination of hydroxyapatite (HA) and bioglass (BG) on polyethylene terephthalate (PET) artificial ligament graft osseointegration within the bone tunnel. The results of in vitro culturing of MC3T3-E1 mouse osteoblastic cells proved that this HA/BG composite coating can promote the cell compatibility of grafts. A rabbit extraarticular tendon-to-bone healing model was used to evaluate the effect of this composite coating on PET artificial ligaments in vivo. The final results demonstrated that HA/BG coating improved new bone formation at the graft-bone interface and increased the load-to-failure property of graft in bone tunnel compared to the control group at early time. The study has shown that HA/BG composite coating on the PET artificial ligament surface has a positive effect in the induction of artificial ligament osseointegration within the bone tunnel.

  3. Composite coating of 58S bioglass and hydroxyapatite on a poly (ethylene terepthalate) artificial ligament graft for the graft osseointegration in a bone tunnel

    International Nuclear Information System (INIS)

    Li Hong; Wu Yang; Ge Yunsheng; Jiang Jia; Gao Kai; Zhang Pengyun; Wu Lingxiang; Chen Shiyi

    2011-01-01

    The purpose of this study was to determine the effect of the combination of hydroxyapatite (HA) and bioglass (BG) on polyethylene terephthalate (PET) artificial ligament graft osseointegration within the bone tunnel. The results of in vitro culturing of MC3T3-E1 mouse osteoblastic cells proved that this HA/BG composite coating can promote the cell compatibility of grafts. A rabbit extraarticular tendon-to-bone healing model was used to evaluate the effect of this composite coating on PET artificial ligaments in vivo. The final results demonstrated that HA/BG coating improved new bone formation at the graft-bone interface and increased the load-to-failure property of graft in bone tunnel compared to the control group at early time. The study has shown that HA/BG composite coating on the PET artificial ligament surface has a positive effect in the induction of artificial ligament osseointegration within the bone tunnel.

  4. Comparison study of biomimetic strontium-doped calcium phosphate coatings by electrochemical deposition and air plasma spray: morphology, composition and bioactive performance.

    Science.gov (United States)

    Li, Ling; Lu, Xia; Meng, Yizhi; Weyant, Christopher M

    2012-10-01

    In this study, strontium-doped calcium phosphate coatings were deposited by electrochemical deposition and plasma spray under different process parameters to achieve various coating morphologies. The coating composition was investigated by energy dispersive X-ray spectroscopy and X-ray diffraction. The surface morphologies of the coatings were studied through scanning electron microscopy while the cytocompatibility and bioactivity of the strontium-doped calcium phosphate coatings were evaluated using bone cell culture using MC3T3-E1 osteoblast-like cells. The addition of strontium leads to enhanced proliferation suggesting the possible benefits of strontium incorporation in calcium phosphate coatings. The morphology and composition of deposited coatings showed a strong influence on the growth of cells.

  5. The in vitro biomineralization and cytocompatibility of polydopamine coated carbon nanotubes

    International Nuclear Information System (INIS)

    Yan Penghua; Wang Jinqing; Wang Lin; Liu Bin; Lei Ziqiang; Yang Shengrong

    2011-01-01

    In this work, polydopamine coated carbon nanotubes were firstly prepared by a simple and feasible route. Then, for comparison, the in vitro bioactivity and cytocompatibility of the carbon nanotubes and the polydopamine coated carbon nanotubes were assessed by immersion study in simulated body fluids and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide test using osteoblast cells (MC3T3-E1), respectively. As a result, it has been demonstrated that the introduction of polydopamine coating can greatly enhance the bioactivity and promote cell proliferation of the carbon nanotubes. The improvement of bioactive behavior is attributed to the good combination of catecholamines structure of the polydopamine and the structural advantages of carbon nanotubes as a framework material. It is anticipated that the polydopamine coated carbon nanotubes would find potential applications in bone tissue engineering and other biomedical areas.

  6. The in vitro biomineralization and cytocompatibility of polydopamine coated carbon nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Yan Penghua [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, 18 Tianshui Middle Road, Lanzhou 730000 (China); Key Laboratory of Polymer Materials of Gansu Province, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China); Wang Jinqing, E-mail: jqwang@licp.cas.cn [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, 18 Tianshui Middle Road, Lanzhou 730000 (China); Wang Lin; Liu Bin [School of Stomatology, Lanzhou University, Lanzhou 730000 (China); Lei Ziqiang [Key Laboratory of Polymer Materials of Gansu Province, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China); Yang Shengrong, E-mail: sryang@lzb.ac.cn [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, 18 Tianshui Middle Road, Lanzhou 730000 (China); Key Laboratory of Polymer Materials of Gansu Province, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China)

    2011-03-15

    In this work, polydopamine coated carbon nanotubes were firstly prepared by a simple and feasible route. Then, for comparison, the in vitro bioactivity and cytocompatibility of the carbon nanotubes and the polydopamine coated carbon nanotubes were assessed by immersion study in simulated body fluids and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide test using osteoblast cells (MC3T3-E1), respectively. As a result, it has been demonstrated that the introduction of polydopamine coating can greatly enhance the bioactivity and promote cell proliferation of the carbon nanotubes. The improvement of bioactive behavior is attributed to the good combination of catecholamines structure of the polydopamine and the structural advantages of carbon nanotubes as a framework material. It is anticipated that the polydopamine coated carbon nanotubes would find potential applications in bone tissue engineering and other biomedical areas.

  7. Intracortical osteoblastic osteosarcoma with oncogenic rickets

    International Nuclear Information System (INIS)

    Hasegawa, T.; Hirohashi, Setsuo; Shimoda, Tadakazu; Yokoyama, Ryohei; Beppu, Yasuo; Maeda, Shotaro

    1999-01-01

    Intracortical osteosarcoma is the rarest variant of osteosarcoma, occurring within, and usually confined to, the cortical bone. Oncogenic osteomalacia, or rickets, is an unusual clinicopathologic entity in which vitamin D-resistant osteomalacia, or rickets, occurs in association with some tumors of soft tissue or bone. We present a case of oncogenic rickets associated with intracortical osteosarcoma of the tibia in a 9-year-old boy, whose roentgenographic abnormalities of rickets disappeared and pertinent laboratory data except for serum alkaline phosphatase became normal after surgical resection of the tumor. Histologically, the tumor was an osteosarcoma with a prominent osteoblastic pattern. An unusual microscopic feature was the presence of matrix mineralization showing rounded calcified structures (calcified spherules). Benign osteoblastic tumors, such as osteoid osteoma and osteoblastoma, must be considered in the differential diagnosis because of the relatively low cellular atypia and mitotic activity of this tumor. The infiltrating pattern with destruction or engulfment of normal bone is a major clue to the correct diagnosis of intracortical osteosarcoma. The co-existing radiographic changes of rickets were due to the intracortical osteosarcoma. (orig.)

  8. Intracortical osteoblastic osteosarcoma with oncogenic rickets

    Energy Technology Data Exchange (ETDEWEB)

    Hasegawa, T.; Hirohashi, Setsuo [Pathology Division, National Cancer Center Research Institute, Tokyo (Japan); Shimoda, Tadakazu [Clinical Laboratory Division, National Cancer Center Hospital, Tokyo (Japan); Yokoyama, Ryohei; Beppu, Yasuo [Orthopedic Division, National Cancer Center Hospital, Tokyo (Japan); Maeda, Shotaro [Department of Pathology, Nippon Medical School Hospital, Tokyo (Japan)

    1999-01-01

    Intracortical osteosarcoma is the rarest variant of osteosarcoma, occurring within, and usually confined to, the cortical bone. Oncogenic osteomalacia, or rickets, is an unusual clinicopathologic entity in which vitamin D-resistant osteomalacia, or rickets, occurs in association with some tumors of soft tissue or bone. We present a case of oncogenic rickets associated with intracortical osteosarcoma of the tibia in a 9-year-old boy, whose roentgenographic abnormalities of rickets disappeared and pertinent laboratory data except for serum alkaline phosphatase became normal after surgical resection of the tumor. Histologically, the tumor was an osteosarcoma with a prominent osteoblastic pattern. An unusual microscopic feature was the presence of matrix mineralization showing rounded calcified structures (calcified spherules). Benign osteoblastic tumors, such as osteoid osteoma and osteoblastoma, must be considered in the differential diagnosis because of the relatively low cellular atypia and mitotic activity of this tumor. The infiltrating pattern with destruction or engulfment of normal bone is a major clue to the correct diagnosis of intracortical osteosarcoma. The co-existing radiographic changes of rickets were due to the intracortical osteosarcoma. (orig.) With 8 figs., 25 refs.

  9. Myeloma cells suppress osteoblasts through sclerostin secretion

    Energy Technology Data Exchange (ETDEWEB)

    Colucci, S; Brunetti, G; Oranger, A [Department of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Mori, G [Department of Biomedical Science, University of Foggia, Foggia (Italy); Sardone, F [Department of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Specchia, G; Rinaldi, E; Curci, P; Liso, V [Department of Emergency and Organ Transplantation, Hematology Section, Bari University Medical School, Bari (Italy); Passeri, G [Department of Internal Medicine and Biomedical Sciences, Center for Metabolic Bone Diseases, University of Parma, Parma (Italy); Zallone, A [Department of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Rizzi, R [Department of Emergency and Organ Transplantation, Hematology Section, Bari University Medical School, Bari (Italy); Grano, M [Department of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy)

    2011-06-01

    Wingless-type (Wnt) signaling through the secretion of Wnt inhibitors Dickkopf1, soluble frizzled-related protein-2 and -3 has a key role in the decreased osteoblast (OB) activity associated with multiple myeloma (MM) bone disease. We provide evidence that another Wnt antagonist, sclerostin, an osteocyte-expressed negative regulator of bone formation, is expressed by myeloma cells, that is, human myeloma cell lines (HMCLs) and plasma cells (CD138+ cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. We demonstrated that BM stromal cells (BMSCs), differentiated into OBs and co-cultured with HMCLs showed, compared with BMSCs alone, reduced expression of major osteoblastic-specific proteins, decreased mineralized nodule formation and attenuated the expression of members of the activator protein 1 transcription factor family (Fra-1, Fra-2 and Jun-D). Moreover, in the same co-culture system, the addition of neutralizing anti-sclerostin antibodies restored OB functions by inducing nuclear accumulation of β-catenin. We further demonstrated that the upregulation of receptor activator of nuclear factor κ-B ligand and the downregulation of osteoprotegerin in OBs were also sclerostin mediated. Our data indicated that sclerostin secretion by myeloma cells contribute to the suppression of bone formation in the osteolytic bone disease associated to MM.

  10. Osteoblastic Metastases Mimickers on Contrast Enhanced CT

    Directory of Open Access Journals (Sweden)

    Fahad Al-Lhedan

    2017-01-01

    Full Text Available Secondary osseous involvement in lymphoma is more common compared to primary bone lymphoma. The finding of osseous lesion can be incidentally discovered during the course of the disease. However, osseous metastases are infrequently silent. Detection of osseous metastases is crucial for accurate staging and optimal treatment planning of lymphoma. The aim of imaging is to identify the presence and extent of osseous disease and to assess for possible complications such as pathological fracture of the load-bearing bones and cord compression if the lesion is spinal. We are presenting two patients with treated lymphoma who were in complete remission. On routine follow-up contrast enhanced CT, there were new osteoblastic lesions in the spine worrisome for metastases. Additional studies were performed for further evaluation of both of them which did not demonstrate any corresponding suspicious osseous lesion. The patients have a prior history of chronic venous occlusive thrombosis that resulted in collaterals formation. Contrast enhancement of the vertebral body marrow secondary to collaterals formation and venous flow through the vertebral venous plexus can mimic the appearance of spinal osteoblastic metastases.

  11. Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radioablative conditioning

    OpenAIRE

    Olson, Timothy S.; Caselli, Anna; Otsuru, Satoru; Hofmann, Ted J.; Williams, Richard; Paolucci, Paolo; Dominici, Massimo; Horwitz, Edwin M.

    2013-01-01

    After radioablative conditioning, host megakaryocytes promote endosteal HSC niche expansion and donor stem cell engraftment.Thrombopoietin administration before radiation and bone marrow transplant enhances megakaryocyte promotion of HSC engraftment.

  12. Electrospun nanofibrous scaffolds of poly (L-lactic acid)-dicalcium silicate composite via ultrasonic-aging technique for bone regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Shengjie [Department of Orthopaedics, The First Affiliated Hospital of Soochow University, 188 Shizi St, Suzhou, Jiangsu 215006 (China); Sun, Junying, E-mail: wodaoshi@sohu.com [Department of Orthopaedics, The First Affiliated Hospital of Soochow University, 188 Shizi St, Suzhou, Jiangsu 215006 (China); Li, Yadong [College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, Jiangsu 215123 (China); Li, Jun [Department of Orthopaedics, The First Affiliated Hospital of Soochow University, 188 Shizi St, Suzhou, Jiangsu 215006 (China); Cui, Wenguo [Orthopedic Institute, Soochow University, 708 Renmin Rd, Suzhou, Jiangsu 215007 (China); Li, Bin, E-mail: binli@suda.edu.cn [Department of Orthopaedics, The First Affiliated Hospital of Soochow University, 188 Shizi St, Suzhou, Jiangsu 215006 (China)

    2014-02-01

    Polymeric nanofibrous composite scaffolds incorporating bioglass and bioceramics have been increasingly promising for bone tissue engineering. In the present study, electrospun poly (L-lactic acid) (PLLA) scaffolds containing dicalcium silicate (C{sub 2}S) nanoparticles (approximately 300 nm) were fabricated. Using a novel ultrasonic dispersion and aging method, uniform C{sub 2}S nanoparticles were prepared and they were homogenously distributed in the PLLA nanofibers upon electrospinning. In vitro, the PLLA-C{sub 2}S fibers induced the formation of HAp on the surface when immersed in simulated body fluid (SBF). During culture, the osteoblastic MC3T3-E1 cells adhered well on PLLA-C{sub 2}S scaffolds, as evidenced by the well-defined actin stress fibers and well-spreading morphology. Further, compared to pure PLLA scaffolds without C{sub 2}S, PLLA-C{sub 2}S scaffolds markedly promoted the proliferation of MC3T3-E1 cells as well as their osteogenic differentiation, which was characterized by the enhanced alkaline phosphatase (ALP) activity. Together, findings from this study clearly demonstrated that PLLA-C{sub 2}S composite scaffold may function as an ideal candidate for bone tissue engineering. - Highlights: • Dicalcium silicate (C{sub 2}S) nanoparticles were prepared via a sol–gel process. • C{sub 2}S nanoparticles were stabilized using ultrasonic-aging technique. • PLLA-C{sub 2}S composite nanofibers were fabricated through electrospinning technique. • C{sub 2}S nanoparticles could be homogenously distributed in nanofibers. • The composite scaffolds enhanced proliferation and differentiation of osteoblasts.

  13. In vitro evaluation of poly(ethylene glycol)-block-poly(ε-caprolactone) methyl ether copolymer coating effects on cells adhesion and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Rusen, Laurentiu [National Institute for Lasers, Plasma and Radiation Physics, Bucharest (Romania); Neacsu, Patricia; Cimpean, Anisoara [University of Bucharest, Department of Biochemistry and Molecular Biology, Bucharest (Romania); Valentin, Ion; Brajnicov, Simona; Dumitrescu, L.N. [National Institute for Lasers, Plasma and Radiation Physics, Bucharest (Romania); Banita, Janina [University of Bucharest, Faculty of Chemistry, Bucharest (Romania); IBAR, Institute of Biochemistry of the Romanian Academy, 296 Splaiul Independentei, RO-060031 Bucharest (Romania); Dinca, Valentina, E-mail: valentina.dinca@inflpr.ro [National Institute for Lasers, Plasma and Radiation Physics, Bucharest (Romania); Dinescu, Maria [National Institute for Lasers, Plasma and Radiation Physics, Bucharest (Romania)

    2016-06-30

    Understanding and controlling natural and synthetic biointerfaces is known to be the key to a wide variety of application within cell culture and tissue engineering field. As both material characteristics and methods are important in tailoring biointerfaces characteristics, in this work we explore the feasibility of using Matrix Assisted Pulsed Laser Evaporation technique for obtaining synthetic copolymeric biocoatings (i.e. poly(ethylene glycol)-block-poly(ε-caprolactone) methyl ether) for evaluating in vitro Vero and MC3T3-E1 pre-osteoblasts cell response. Characterization and evaluation of the coated substrates were carried out using different techniques. The Fourier transform infrared spectroscopy data demonstrated that the main functional groups in the MAPLE-deposited films remained intact. Atomic Force Microscopy images showed the coatings to be continuous, with the surface roughness depending on the deposition parameters. Moreover, the behaviour of the coatings in medium mimicking the pH and temperature of the human body was studied and corelated to degradation. Spectro-ellipsometry (SE) and AFM measurements revealed the degradation trend during immersion time by the changes in coating thickness and roughness. In vitro biocompatibility was studied by indirect contact tests on Vero cells in accordance with ISO 10993-5/2009. The results obtained in terms of cell morphology (phase contrast microscopy) and cytotoxicity (LDH and MTT assays) proved biocompatibility. Furthermore, direct contact assays on MC3T3-E1 pre-osteoblasts demonstrated the capacity of all analyzed specimens to support cell adhesion, normal cellular morphology and growth.

  14. PDMP, a ceramide analogue, acts as an inhibitor of mTORC1 by inducing its translocation from lysosome to endoplasmic reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Ode, Takashi [Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Research Fellow of the Japan Society for the Promotion of Science (JSPS), 5-3-1 Kojimachi, Chiyoda-ku, Tokyo 102-0083 (Japan); Podyma-Inoue, Katarzyna A.; Terasawa, Kazue [Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Inokuchi, Jin-ichi [Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558 (Japan); Kobayashi, Toshihide [Lipid Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); CNRS, UMR 7213, University of Strasbourg, 67401 Illkirch (France); Watabe, Tetsuro [Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Izumi, Yuichi [Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Hara-Yokoyama, Miki, E-mail: m.yokoyama.bch@tmd.ac.jp [Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan)

    2017-01-01

    Mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism, and cell differentiation. Recent studies have revealed that the recruitment of mTORC1 to lysosomes is essential for its activation. The ceramide analogue 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a well known glycosphingolipid synthesis inhibitor, also affects the structures and functions of various organelles, including lysosomes and endoplasmic reticulum (ER). We investigated whether PDMP regulates the mTORC1 activity through its effects on organellar behavior. PDMP induced the translocation of mTORC1 from late endosomes/lysosomes, leading to the dissociation of mTORC1 from its activator Rheb in MC3T3-E1 cells. Surprisingly, we found mTORC1 translocation to the ER upon PDMP treatment. This effect of PDMP was independent of its action as the inhibitor, since two stereoisomers of PDMP, with and without the inhibitor activity, showed essentially the same effect. We confirmed that PDMP inhibits the mTORC1 activity based on the decrease in the phosphorylation of ribosomal S6 kinase, a downstream target of mTORC1, and the increase in LC3 puncta, reflecting autophagosome formation. Furthermore, PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation and differentiation of MC3T3-E1 cells. Accordingly, the present results reveal a novel mechanism of PDMP, which inhibits the mTORC1 activity by inducing the translocation of mTOR from lysosomes to the ER. - Highlights: • The ceramide analogue, PDMP, suppressed the activation of mTORC1. • PDMP induced the translocation of mTOR from lysosomes to ER. • PDMP led to the dissociation of mTOR from its activator Rheb. • PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation.

  15. Covalent attachment of cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) to poly(etheretherketone) surface by tailored silanization layers technique

    International Nuclear Information System (INIS)

    Zheng, Yanyan; Xiong, Chengdong; Li, Xiaoyu; Zhang, Lifang

    2014-01-01

    Highlights: • The carbonyl groups on PEEK surface were effectively reduced to hydroxyl groups using sodium borohydride. • Silanization layers technique was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on hydroxylation-pretreated PEEK sheet surface by covalent chemical attachment. • XPS, surface profiler and water contact angle measurements proved the presence of GRGD on PEEK surface. • Osteoblast-like cells (MC3T3-E1) attachment and proliferation were improved effectively on GRGD-modified PEEK surface. - Abstract: Poly(etheretherketone) (PEEK) is a rigid semicrystalline polymer that combines excellent mechanical properties, broad chemical resistance and bone-like stiffness and is widely used in biomedical fields. However, PEEK is naturally bioinert, leading to limited biomedical applications, especially when a direct bone-implant osteointegration is desired. In this study, a three-step reaction procedure was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on the surface of PEEK sheet by covalent chemical attachment to favor cell adhesion and proliferation. First, hydroxylation-pretreated PEEK surfaces were silanized with 7-Oct-1-enyltrichlorosilane (OETS) in dry cyclohexane, resulting in a silanization layer with terminal ethenyl. Second, the terminal ethylenic double bonds of the silanization layer on PEEK surface were converted to carboxyl groups through acidic potassium manganate oxidation. Finally, GRGD was covalently attached by carbodiimide mediated condensation between the carboxyl on PEEK surface and amine presents in GRGD. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, surface profiler and water contact angle measurements were applied to characterize the modified surfaces. The effect of cells attachment and proliferation on each specimen was investigated. Pre-osteoblast cells (MC3T3-E1) attachment, spreading and proliferation

  16. Covalent attachment of cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) to poly(etheretherketone) surface by tailored silanization layers technique

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yanyan [Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xiong, Chengdong [Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041 (China); Li, Xiaoyu [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Zhang, Lifang, E-mail: zhanglfcioc@163.com [Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041 (China)

    2014-11-30

    Highlights: • The carbonyl groups on PEEK surface were effectively reduced to hydroxyl groups using sodium borohydride. • Silanization layers technique was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on hydroxylation-pretreated PEEK sheet surface by covalent chemical attachment. • XPS, surface profiler and water contact angle measurements proved the presence of GRGD on PEEK surface. • Osteoblast-like cells (MC3T3-E1) attachment and proliferation were improved effectively on GRGD-modified PEEK surface. - Abstract: Poly(etheretherketone) (PEEK) is a rigid semicrystalline polymer that combines excellent mechanical properties, broad chemical resistance and bone-like stiffness and is widely used in biomedical fields. However, PEEK is naturally bioinert, leading to limited biomedical applications, especially when a direct bone-implant osteointegration is desired. In this study, a three-step reaction procedure was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on the surface of PEEK sheet by covalent chemical attachment to favor cell adhesion and proliferation. First, hydroxylation-pretreated PEEK surfaces were silanized with 7-Oct-1-enyltrichlorosilane (OETS) in dry cyclohexane, resulting in a silanization layer with terminal ethenyl. Second, the terminal ethylenic double bonds of the silanization layer on PEEK surface were converted to carboxyl groups through acidic potassium manganate oxidation. Finally, GRGD was covalently attached by carbodiimide mediated condensation between the carboxyl on PEEK surface and amine presents in GRGD. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, surface profiler and water contact angle measurements were applied to characterize the modified surfaces. The effect of cells attachment and proliferation on each specimen was investigated. Pre-osteoblast cells (MC3T3-E1) attachment, spreading and proliferation

  17. In vitro evaluation of poly(ethylene glycol)-block-poly(ɛ-caprolactone) methyl ether copolymer coating effects on cells adhesion and proliferation

    Science.gov (United States)

    Rusen, Laurentiu; Neacsu, Patricia; Cimpean, Anisoara; Valentin, Ion; Brajnicov, Simona; Dumitrescu, L. N.; Banita, Janina; Dinca, Valentina; Dinescu, Maria

    2016-06-01

    Understanding and controlling natural and synthetic biointerfaces is known to be the key to a wide variety of application within cell culture and tissue engineering field. As both material characteristics and methods are important in tailoring biointerfaces characteristics, in this work we explore the feasibility of using Matrix Assisted Pulsed Laser Evaporation technique for obtaining synthetic copolymeric biocoatings (i.e. poly(ethylene glycol)-block-poly(ɛ-caprolactone) methyl ether) for evaluating in vitro Vero and MC3T3-E1 pre-osteoblasts cell response. Characterization and evaluation of the coated substrates were carried out using different techniques. The Fourier transform infrared spectroscopy data demonstrated that the main functional groups in the MAPLE-deposited films remained intact. Atomic Force Microscopy images showed the coatings to be continuous, with the surface roughness depending on the deposition parameters. Moreover, the behaviour of the coatings in medium mimicking the pH and temperature of the human body was studied and corelated to degradation. Spectro-ellipsometry (SE) and AFM measurements revealed the degradation trend during immersion time by the changes in coating thickness and roughness. In vitro biocompatibility was studied by indirect contact tests on Vero cells in accordance with ISO 10993-5/2009. The results obtained in terms of cell morphology (phase contrast microscopy) and cytotoxicity (LDH and MTT assays) proved biocompatibility. Furthermore, direct contact assays on MC3T3-E1 pre-osteoblasts demonstrated the capacity of all analyzed specimens to support cell adhesion, normal cellular morphology and growth.

  18. In Vitro and In Vivo Evaluation of Zinc-Modified Ca–Si-Based Ceramic Coating for Bone Implants

    Science.gov (United States)

    Zheng, Xuebin; He, Dannong; Ye, Xiaojian; Wang, Meiyan

    2013-01-01

    The host response to calcium silicate ceramic coatings is not always favorable because of their high dissolution rates, leading to high pH within the surrounding physiological environment. Recently, a zinc-incorporated calcium silicate-based ceramic Ca2ZnSi2O7 coating, developed on a Ti-6Al-4V substrate using plasma-spray technology, was found to exhibit improved chemical stability and biocompatibility. This study aimed to investigate and compare the in vitro response of osteoblastic MC3T3-E1 cells cultured on Ca2ZnSi2O7 coating, CaSiO3 coating, and uncoated Ti-6Al-4V titanium control at cellular and molecular level. Our results showed Ca2ZnSi2O7 coating enhanced MC3T3-E1 cell attachment, proliferation, and differentiation compared to CaSiO3 coating and control. In addition, Ca2ZnSi2O7 coating increased mRNA levels of osteoblast-related genes (alkaline phosphatase, procollagen α1(I), osteocalcin), insulin-like growth factor-I (IGF-I), and transforming growth factor-β1 (TGF-β1). The in vivo osteoconductive properties of Ca2ZnSi2O7 coating, compared to CaSiO3 coating and control, was investigated using a rabbit femur defect model. Histological and histomorphometrical analysis demonstrated new bone formation in direct contact with the Ca2ZnSi2O7 coating surface in absence of fibrous tissue and higher bone-implant contact rate (BIC) in the Ca2ZnSi2O7 coating group, indicating better biocompatibility and faster osseointegration than CaSiO3 coated and control implants. These results indicate Ca2ZnSi2O7 coated implants have applications in bone tissue regeneration, since they are biocompatible and able to osseointegrate with host bone. PMID:23483914

  19. In vitro and in vivo evaluation of zinc-modified ca-si-based ceramic coating for bone implants.

    Science.gov (United States)

    Yu, Jiangming; Li, Kai; Zheng, Xuebin; He, Dannong; Ye, Xiaojian; Wang, Meiyan

    2013-01-01

    The host response to calcium silicate ceramic coatings is not always favorable because of their high dissolution rates, leading to high pH within the surrounding physiological environment. Recently, a zinc-incorporated calcium silicate-based ceramic Ca2ZnSi2O7 coating, developed on a Ti-6Al-4V substrate using plasma-spray technology, was found to exhibit improved chemical stability and biocompatibility. This study aimed to investigate and compare the in vitro response of osteoblastic MC3T3-E1 cells cultured on Ca2ZnSi2O7 coating, CaSiO3 coating, and uncoated Ti-6Al-4V titanium control at cellular and molecular level. Our results showed Ca2ZnSi2O7 coating enhanced MC3T3-E1 cell attachment, proliferation, and differentiation compared to CaSiO3 coating and control. In addition, Ca2ZnSi2O7 coating increased mRNA levels of osteoblast-related genes (alkaline phosphatase, procollagen α1(I), osteocalcin), insulin-like growth factor-I (IGF-I), and transforming growth factor-β1 (TGF-β1). The in vivo osteoconductive properties of Ca2ZnSi2O7 coating, compared to CaSiO3 coating and control, was investigated using a rabbit femur defect model. Histological and histomorphometrical analysis demonstrated new bone formation in direct contact with the Ca2ZnSi2O7 coating surface in absence of fibrous tissue and higher bone-implant contact rate (BIC) in the Ca2ZnSi2O7 coating group, indicating better biocompatibility and faster osseointegration than CaSiO3 coated and control implants. These results indicate Ca2ZnSi2O7 coated implants have applications in bone tissue regeneration, since they are biocompatible and able to osseointegrate with host bone.

  20. [Development of a novel absorbable nanofiber chitosan-collagen membrane by electrospinning and the preliminary study on guided bone regeneration].

    Science.gov (United States)

    Gao, B; Li, X J; Lin, M; Li, Y Y; Dong, Y

    2018-02-09

    Objective: To evaluate the application effect of nanofiber chitosan-collagen membrane (NCM) on guided bone regeneration (GBR). Methods: The mixture of collagen, chitosan, polyethylene oxide was used to make up the NCM by electrospinning, then the NCM was crosslinked by glutaraldehyde vapor. The physical property of the NCM was measured by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). MC3T3-E1 osteoblasts were cultured on NCM to characterize the biocompatibility. The effectiveness of four groups [contrast group, Bio-gide membrane (BGM), compressed chitosan-collagen menbrane (CCM), NCM/CCM] on bone regeneration were evaluated in critical-sized defects (diameter = 5 mm) in SD rats. Results: When the mixed solution consists of 4.0% collagen, 1.0% chitosan and 3.5% polyethylene oxide, the NCM could be validly fabricated by electrospinning. After cross-linking by glutaraldehyde vapor, the tensile strength and the stability of NCM in damp was enhanced. No cytotoxicity of the NCM was detected on MC3T3-E1 osteoblasts. In vivo study showed that the new bone regeneration ratio of NCM/CCM group was [(43.10±1.49)%], and this was similar to that of the group of BGM [(41.36±2.60)%] ( P> 0.05), but higher than that of the CCM group [(33.10±1.41)%] and the contrast group [(7.22±2.46)%] ( P< 0.05). Conclusions: The NCM can promote new bone regeneration effectively in GBR procedure.

  1. Characterization and antibacterial performance of bioactive Ti–Zn–O coatings deposited on titanium implants

    International Nuclear Information System (INIS)

    Tsai, Ming-Tzu; Chang, Yin-Yu; Huang, Heng-Li; Hsu, Jui-Ting; Chen, Ya-Chi; Wu, Aaron Yu-Jen

    2013-01-01

    Titanium (Ti)-based materials have been used for dental and orthopedic implants because of their excellent biological compatibility, superior mechanical strength, and high corrosion resistance. The hypothesis of this present study was to manufacture the Zn-doped TiO 2 layer possessing the biocompatibility and antibacterial ability on the surface of Ti specimens. TiO 2 , ZnO, and Ti(Zn)O 2 coatings were deposited on polished pure Ti substrates using a cathodic arc deposition system. Murine osteoblasts (MC3T3-E1) and human Staphylococcus aureus (S. aureus) were cultured onto the surface with different deposited coatings, respectively. The biocompatibility was examined by cell viability and osteogenic gene expression. The antibacterial ability was determined by SYTO9 nucleic acid staining. A porous Zn-doped TiO 2 coating was successfully produced. The ZnO exhibited a fibrous structure with nanorods showing a hydrophobic feature (contact angle approximately 89°). These material properties affected the following biological performance. The antibacterial testing found no apparent difference between the uncoated Ti plate and the TiO 2 coating. However, significantly lower numbers of S. aureus were observed on ZnO and Ti(Zn)O 2 coatings compared to that on the uncoated Ti. The biocompatible testing exhibited that TiO 2 and Ti(Zn)O 2 coatings enhanced greater cell viability and proliferation than the uncoated Ti plate and ZnO coating. The osteogenic gene expression of Dlx-5 and osterix also improved for the TiO 2 and Ti(Zn)O 2 coatings. However, a significant inhibition of cell viability was found for the ZnO coating. These findings suggested that the composite Ti(Zn)O 2 coating with a lower content of Zn (7.6 ± 1.3 at.%) not only improved antibacterial activity, but also maintained the biocompatibility to bone cells. - Highlights: ► TiO 2 , Ti(Zn)O 2 and ZnO coatings were deposited by cathodic arc evaporation. ► Zn may incorporated with Ti to form Zn-doped TiO 2 .

  2. Characterization and antibacterial performance of bioactive Ti–Zn–O coatings deposited on titanium implants

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, Ming-Tzu [Department of Biomedical Engineering, Hungkuang University, Taichung 433, Taiwan (China); Chang, Yin-Yu, E-mail: yinyu@nfu.edu.tw [Department of Mechanical and Computer-Aided Engineering, National Formosa University, Yunlin 632, Taiwan (China); Huang, Heng-Li; Hsu, Jui-Ting [School of Dentistry, College of Medicine China Medical University, Taichung 404, Taiwan (China); Chen, Ya-Chi [Department of Materials Science and Engineering, Mingdao University, Changhua 523, Taiwan (China); Wu, Aaron Yu-Jen [Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung 833, Taiwan (China)

    2013-01-01

    Titanium (Ti)-based materials have been used for dental and orthopedic implants because of their excellent biological compatibility, superior mechanical strength, and high corrosion resistance. The hypothesis of this present study was to manufacture the Zn-doped TiO{sub 2} layer possessing the biocompatibility and antibacterial ability on the surface of Ti specimens. TiO{sub 2}, ZnO, and Ti(Zn)O{sub 2} coatings were deposited on polished pure Ti substrates using a cathodic arc deposition system. Murine osteoblasts (MC3T3-E1) and human Staphylococcus aureus (S. aureus) were cultured onto the surface with different deposited coatings, respectively. The biocompatibility was examined by cell viability and osteogenic gene expression. The antibacterial ability was determined by SYTO9 nucleic acid staining. A porous Zn-doped TiO{sub 2} coating was successfully produced. The ZnO exhibited a fibrous structure with nanorods showing a hydrophobic feature (contact angle approximately 89°). These material properties affected the following biological performance. The antibacterial testing found no apparent difference between the uncoated Ti plate and the TiO{sub 2} coating. However, significantly lower numbers of S. aureus were observed on ZnO and Ti(Zn)O{sub 2} coatings compared to that on the uncoated Ti. The biocompatible testing exhibited that TiO{sub 2} and Ti(Zn)O{sub 2} coatings enhanced greater cell viability and proliferation than the uncoated Ti plate and ZnO coating. The osteogenic gene expression of Dlx-5 and osterix also improved for the TiO{sub 2} and Ti(Zn)O{sub 2} coatings. However, a significant inhibition of cell viability was found for the ZnO coating. These findings suggested that the composite Ti(Zn)O{sub 2} coating with a lower content of Zn (7.6 ± 1.3 at.%) not only improved antibacterial activity, but also maintained the biocompatibility to bone cells. - Highlights: ► TiO{sub 2}, Ti(Zn)O{sub 2} and ZnO coatings were deposited by cathodic arc

  3. In vitro study of 3D PLGA/n-HAp/β-TCP composite scaffolds with etched oxygen plasma surface modification in bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Roh, Hee-Sang [Department of Dental Materials, School of Dentistry, Chosun University, 309 Pilmun-daero, Dong-gu, Gwangju 61452 (Korea, Republic of); Jung, Sang-Chul [Department of Environmental Engineering, Sunchon National University, 255 Jungang-ro, Sunchon 57922 (Korea, Republic of); Kook, Min-Suk [Department of Oral and Maxillofacial Surgery, School of Dentistry, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 61186 (Korea, Republic of); Kim, Byung-Hoon, E-mail: kim5055@chosun.ac.kr [Department of Dental Materials, School of Dentistry, Chosun University, 309 Pilmun-daero, Dong-gu, Gwangju 61452 (Korea, Republic of)

    2016-12-01

    Highlights: • PLGA and PLGA/n-HAp/β-TCP scaffolds were successfully fabricated by 3D printing. • Oxygen plasma etching increases the wettability and surface roughness. • Bioceramics and oxygen plasma etching and could be used to improve the cell affinity. - Abstract: Three-dimensional (3D) scaffolds have many advantageous properties for bone tissue engineering application, due to its controllable properties such as pore size, structural shape and interconnectivity. In this study, effects on oxygen plasma surface modification and adding of nano-hydroxyapatite (n-HAp) and β-tricalcium phosphate (β-TCP) on the 3D PLGA/n-HAp/β-TCP scaffolds for improving preosteoblast cell (MC3T3-E1) adhesion, proliferation and differentiation were investigated. The 3D PLGA/n-HAp/β-TCP scaffolds were fabricated by 3D Bio-Extruder equipment. The 3D scaffolds were prepared with 0°/90° architecture and pore size of approximately 300 μm. In addition 3D scaffolds surface were etched by oxygen plasma to enhance the hydrophilic property and surface roughness. After oxygen plasma treatment, the surface chemistry and morphology were investigated by Fourier transform infrared spectroscopy, scanning electron microscopy, and atomic force microscopy. And also hydrophilic property was measured by contact angle. The MC3T3-E1 cell proliferation and differentiation were investigated by MTT assay and ALP activity. In present work, the 3D PLGA/HAp/beta-TCP composite scaffold with suitable structure for the growth of osteoblast cells was successfully fabricated by 3D rapid prototyping technique. The surface hydrophilicity and roughness of 3D scaffold increased by oxygen plasma treatment had a positive effect on cell adhesion, proliferation, and differentiation. Furthermore, the differentiation of MC3T3-E1 cell was significantly enhanced by adding of n-HAp and β-TCP on 3D PLGA scaffold. As a result, combination of bioceramics and oxygen plasma treatment showed a synergistic effect on

  4. MiR-132 regulates osteogenic differentiation via downregulating Sirtuin1 in a peroxisome proliferator-activated receptor β/δ–dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Kai; Qu, Bo; Liao, Dongfa; Liu, Da; Wang, Cairu; Zhou, Jingsong; Pan, Xianming, E-mail: xianmingpanxj@163.com

    2016-09-09

    MicroRNAs (miRNAs) play significant roles in multiple diseases by regulating the expression of their target genes. Type 2 diabetes mellitus (T2DM) is a chronic endocrine and metabolic disease with complex mechanisms. T2DM can result in diabetic osteoporosis (DO), which is characterized by bone loss, decreased bone mineral density and increased bone fractures. The promotion of osteogenic differentiation of osteoblasts is an effective way to treat osteoporosis. In the present study, high glucose (HG) and free fatty acids (FFA) were employed to mimic T2DM in MC3T3-E1 cells. To induce osteogenic differentiation, MC3T3-E1 cells were cultured in osteogenic medium. The results showed that osteogenic differentiation was significantly suppressed by HG and FFA. We found that miR-132 expression was significantly upregulated and much higher in HG-FFA–induced cells than other selected miRNAs, indicating that miR-132 might play an important role in DO. Furthermore, overexpression of miR-132 markedly inhibited the expression of key markers of osteogenic differentiation and alkaline phosphatase (ALP) activity. Reciprocally, inhibition of miR-132 restored osteogenic differentiation, even under treatment with HG-FFA. We also showed that Sirtuin 1 (Sirt1) was one of the target genes of miR-132, whose expression was controlled by miR-132. Ectopic expression of Sirt1 reversed the decrease in osteogenic differentiation caused by miR-132 and HG-FFA. These results demonstrated the direct role of miR-132 in suppressing osteogenic differentiation through downregulating Sirt1. Moreover, we demonstrated that peroxisome proliferator-activated receptor β/δ (PPARβ/δ) was a downstream molecule of Sirt1, and its knockout by PPARβ/δ siRNA significantly abolished the promotive effects of Sirt1 on osteogenic differentiation, indicating that Sirt1 functioned in a PPARβ/δ–dependent manner. Taken together, we provide crucial evidence that miR-132 plays a key role in regulating osteogenic

  5. MiR-132 regulates osteogenic differentiation via downregulating Sirtuin1 in a peroxisome proliferator-activated receptor β/δ–dependent manner

    International Nuclear Information System (INIS)

    Gong, Kai; Qu, Bo; Liao, Dongfa; Liu, Da; Wang, Cairu; Zhou, Jingsong; Pan, Xianming

    2016-01-01

    MicroRNAs (miRNAs) play significant roles in multiple diseases by regulating the expression of their target genes. Type 2 diabetes mellitus (T2DM) is a chronic endocrine and metabolic disease with complex mechanisms. T2DM can result in diabetic osteoporosis (DO), which is characterized by bone loss, decreased bone mineral density and increased bone fractures. The promotion of osteogenic differentiation of osteoblasts is an effective way to treat osteoporosis. In the present study, high glucose (HG) and free fatty acids (FFA) were employed to mimic T2DM in MC3T3-E1 cells. To induce osteogenic differentiation, MC3T3-E1 cells were cultured in osteogenic medium. The results showed that osteogenic differentiation was significantly suppressed by HG and FFA. We found that miR-132 expression was significantly upregulated and much higher in HG-FFA–induced cells than other selected miRNAs, indicating that miR-132 might play an important role in DO. Furthermore, overexpression of miR-132 markedly inhibited the expression of key markers of osteogenic differentiation and alkaline phosphatase (ALP) activity. Reciprocally, inhibition of miR-132 restored osteogenic differentiation, even under treatment with HG-FFA. We also showed that Sirtuin 1 (Sirt1) was one of the target genes of miR-132, whose expression was controlled by miR-132. Ectopic expression of Sirt1 reversed the decrease in osteogenic differentiation caused by miR-132 and HG-FFA. These results demonstrated the direct role of miR-132 in suppressing osteogenic differentiation through downregulating Sirt1. Moreover, we demonstrated that peroxisome proliferator-activated receptor β/δ (PPARβ/δ) was a downstream molecule of Sirt1, and its knockout by PPARβ/δ siRNA significantly abolished the promotive effects of Sirt1 on osteogenic differentiation, indicating that Sirt1 functioned in a PPARβ/δ–dependent manner. Taken together, we provide crucial evidence that miR-132 plays a key role in regulating osteogenic

  6. Osteoblast recruitment routes in human cancellous bone remodeling

    DEFF Research Database (Denmark)

    Kristensen, Helene Bjørg; Andersen, Thomas Levin; Marcussen, Niels

    2014-01-01

    It is commonly proposed that bone forming osteoblasts recruited during bone remodeling originate from bone marrow perivascular cells, bone remodeling compartment canopy cells, or bone lining cells. However, an assessment of osteoblast recruitment during adult human cancellous bone remodeling...... is lacking. We addressed this question by quantifying cell densities, cell proliferation, osteoblast differentiation markers, and capillaries in human iliac crest biopsy specimens. We found that recruitment occurs on both reversal and bone-forming surfaces, as shown by the cell density and osterix levels...

  7. Local transplantation is an effective method for cell delivery in the osteogenesis imperfecta murine model.

    Science.gov (United States)

    Pauley, Penelope; Matthews, Brya G; Wang, Liping; Dyment, Nathaniel A; Matic, Igor; Rowe, David W; Kalajzic, Ivo

    2014-09-01

    Osteogenesis imperfecta is a serious genetic disorder that results from improper type I collagen production. We aimed to evaluate whether bone marrow stromal cells (BMSC) delivered locally into femurs were able to engraft, differentiate into osteoblasts, and contribute to formation of normal bone matrix in the osteogenesis imperfect murine (oim) model. Donor BMSCs from bone-specific reporter mice (Col2.3GFP) were expanded in vitro and transplanted into the femoral intramedullary cavity of oim mice. Engraftment was evaluated after four weeks. We detected differentiation of donor BMSCs into Col2.3GFP+ osteoblasts and osteocytes in cortical and trabecular bone of transplanted oim femurs. New bone formation was detected by deposition of dynamic label in the proximity to the Col2.3GFP+ osteoblasts, and new bone showed more organized collagen structure and expression of type I α2 collagen. Col2.3GFP cells were not found in the contralateral femur indicating that transplanted osteogenic cells did not disseminate by circulation. No osteogenic engraftment was observed following intravenous transplantation of BMSCs. BMSC cultures derived from transplanted femurs showed numerous Col2.3GFP+ colonies, indicating the presence of donor progenitor cells. Secondary transplantation of cells recovered from recipient femurs and expanded in vitro also showed Col2.3GFP+ osteoblasts and osteocytes confirming the persistence of donor stem/progenitor cells. We show that BMSCs delivered locally in oim femurs are able to engraft, differentiate into osteoblasts and osteocytes and maintain their progenitor potential in vivo. This suggests that local delivery is a promising approach for introduction of autologous MSC in which mutations have been corrected.

  8. Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

    International Nuclear Information System (INIS)

    Nemoto, Eiji; Ebe, Yukari; Kanaya, Sousuke; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi

    2012-01-01

    Highlights: ► Wnt5a is identified in osteoblasts in tibial growth plate and bone marrow. ► Osteoblastic differentiation is associated with increased expression of Wnt5a/Ror2. ► Wnt5a/Ror2 signaling is important for BMP-2-mediated osteoblastic differentiation. ► Wnt5a/Ror2 operates independently of BMP-Smad pathway. -- Abstract: Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.

  9. Measurement of oxygen consumption rate of osteoblasts from ...

    African Journals Online (AJOL)

    Jane

    2011-05-10

    May 10, 2011 ... E-mail: kedongsong@dlut.edu.cn. Tel: +86 411 ... the experiments, including inverted phase contrast microscope. (IX70-Olympus ... The pictures showed that the osteoblasts still had very high cellular viability. consumption of ...

  10. Transdifferentiation of adipocytes to osteoblasts: potential for orthopaedic treatment.

    Science.gov (United States)

    Lin, Daphne P L; Dass, Crispin R

    2018-03-01

    As both adipocytes and osteoblasts originate from the same pool of mesenchymal stem cells, increasing clinical evidence has emerged of the plasticity between the two lineages. For instance, the downregulation of osteoblast differentiation and upregulation of adipogenesis are common features of conditions such as multiple myeloma, obesity and drug-induced bone loss in diabetes mellitus. However, despite in-vitro and in-vivo observations of adipocyte transdifferentiation into osteoblasts, little is known of the underlying mechanisms. This review summarises the current knowledge of this particular transdifferentiation process whereby the Wnt/β-catenin signalling pathway and Runx2 overexpression have been postulated to play a critical role. Furthermore, due to the possibility of a novel therapy in the treatment of bone conditions, a number of agents with the potential to induce adipo-to-osteoblast transdifferentiation have been investigated such as all-trans retinoic acid, bone morphogenetic protein-9 and vascular endothelial growth factor. © 2018 Royal Pharmaceutical Society.

  11. Mechanisms regulating osteoblast response to surface microtopography and vitamin D

    Science.gov (United States)

    Bell, Bryan Frederick, Jr.

    A comprehensive understanding of the interactions between orthopaedic and dental implant surfaces with the surrounding host tissue is essential in the design of advanced biomaterials that better promote bone growth and osseointegration of implants. Dental implants with roughened surfaces and high surface energy are well known to promote osteoblast differentiation in vitro and promote increased bone-to-implant contact in vivo. In addition, increased surface roughness increases osteoblasts response to the vitamin D metabolite 1alpha,25(OH)2D3. However, the exact mechanisms mediating cell response to surface properties and 1alpha,25(OH)2D3 are still being elucidated. The central aim of the thesis is to investigate whether integrin signaling in response to rough surface microtopography enhances osteoblast differentiation and responsiveness to 1alpha,25(OH)2D3. The hypothesis is that the integrin alpha5beta1 plays a role in osteoblast response to surface microtopography and that 1alpha,25(OH) 2D3 acts through VDR-independent pathways involving caveolae to synergistically enhance osteoblast response to surface roughness and 1alpha,25(OH) 2D3. To test this hypothesis the objectives of the studies performed in this thesis were: (1) to determine if alpha5beta 1 signaling is required for osteoblast response to surface microstructure; (2) to determine if increased responsiveness to 1alpha,25(OH)2D 3 requires the vitamin D receptor, (3) to determine if rough titanium surfaces functionalized with the peptides targeting integrins (RGD) and transmembrane proteoglycans (KRSR) will enhance both osteoblast proliferation and differentiation, and (4) to determine whether caveolae, which are associated with integrin and 1alpha,25(OH)2D3 signaling, are required for enhance osteogenic response to surface microstructure and 1alpha,25(OH)2D 3. The results demonstrate that integrins, VDR, and caveolae play important roles in mediating osteoblast response to surface properties and 1alpha,25

  12. The impact of doped silicon quantum dots on human osteoblasts

    Czech Academy of Sciences Publication Activity Database

    Ostrovská, L.; Brož, Antonín; Fučíková, A.; Bělinová, T.; Sugimoto, H.; Kanno, T.; Fujii, M.; Valenta, J.; Kalbáčová, M.H.

    2016-01-01

    Roč. 6, č. 68 (2016), s. 63403-63413 ISSN 2046-2069 Institutional support: RVO:67985823 Keywords : silicon quantum dots * osteoblasts * cytotoxicity * photoluminiscence bioimaging Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.108, year: 2016

  13. Osteoblast cell response to surface-modified carbon nanotubes

    International Nuclear Information System (INIS)

    Zhang Faming; Weidmann, Arne; Nebe, J. Barbara; Burkel, Eberhard

    2012-01-01

    In order to investigate the interaction of cells with modified multi-walled carbon nanotubes (MWCNTs) for their potential biomedical applications, the MWCNTs were chemically modified with carboxylic acid groups (–COOH), polyvinyl alcohol (PVA) polymer and biomimetic apatite on their surfaces. Additionally, human osteoblast MG-63 cells were cultured in the presence of the surface-modified MWCNTs. The metabolic activities of osteoblastic cells, cell proliferation properties, as well as cell morphology were studied. The surface modification of MWCNTs with biomimetic apatite exhibited a significant increase in the cell viability of osteoblasts, up to 67.23%. In the proliferation phases, there were many more cells in the biomimetic apatite-modified MWCNT samples than in the MWCNTs–COOH. There were no obvious changes in cell morphology in osteoblastic MG-63 cells cultured in the presence of these chemically-modified MWCNTs. The surface modification of MWCNTs with apatite achieves an effective enhancement of their biocompatibility.

  14. Ultrastructural and metabolic changes in osteoblasts exposed to uranyl nitrate

    International Nuclear Information System (INIS)

    Tasat, D.R.; Orona, N.S.; Mandalunis, P.M.; Cabrini, R.L.; Ubios, A.M.

    2007-01-01

    Exposure to uranium is an occupational hazard to workers who continually handle uranium and an environmental risk to the population at large. Since the cellular and molecular pathways of uranium toxicity in osteoblast cells are still unknown, the aim of the present work was to evaluate the adverse effects of uranyl nitrate (UN) on osteoblasts both in vivo and in vitro. Herein we studied the osteoblastic ultrastructural changes induced by UN in vivo and analyzed cell proliferation, generation of reactive oxygen species (ROS), apoptosis, and alkaline phosphatase (APh) activity in osteoblasts exposed to various UN concentrations (0.1, 1, 10, and 100 μM) in vitro. Cell proliferation was quantified by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, ROS was determined using the nitro blue tetrazolium test, apoptosis was morphologically determined using Hoechst 3332 and APh activity was assayed spectrophotometrically. Electron microscopy revealed that the ultrastructure of active and inactive osteoblasts exposed to uranium presented cytoplasmic and nuclear alterations. In vitro, 1-100 μM UN failed to modify cell proliferation ratio and to induce apoptosis. ROS generation increased in a dose-dependent manner in all tested doses. APh activity was found to decrease in 1-100 μM UN-treated cells vs. controls. Our results show that UN modifies osteoblast cell metabolism by increasing ROS generation and reducing APh activity, suggesting that ROS may play a more complex role in cell physiology than simply causing oxidative damage. (orig.)

  15. The role of osteoblasts in peri-prosthetic osteolysis.

    LENUS (Irish Health Repository)

    O'Neill, S C

    2013-08-01

    Peri-prosthetic osteolysis and subsequent aseptic loosening is the most common reason for revising total hip replacements. Wear particles originating from the prosthetic components interact with multiple cell types in the peri-prosthetic region resulting in an inflammatory process that ultimately leads to peri-prosthetic bone loss. These cells include macrophages, osteoclasts, osteoblasts and fibroblasts. The majority of research in peri-prosthetic osteolysis has concentrated on the role played by osteoclasts and macrophages. The purpose of this review is to assess the role of the osteoblast in peri-prosthetic osteolysis. In peri-prosthetic osteolysis, wear particles may affect osteoblasts and contribute to the osteolytic process by two mechanisms. First, particles and metallic ions have been shown to inhibit the osteoblast in terms of its ability to secrete mineralised bone matrix, by reducing calcium deposition, alkaline phosphatase activity and its ability to proliferate. Secondly, particles and metallic ions have been shown to stimulate osteoblasts to produce pro inflammatory mediators in vitro. In vivo, these mediators have the potential to attract pro-inflammatory cells to the peri-prosthetic area and stimulate osteoclasts to absorb bone. Further research is needed to fully define the role of the osteoblast in peri-prosthetic osteolysis and to explore its potential role as a therapeutic target in this condition.

  16. Mechanisms of palmitate-induced cell death in human osteoblasts

    Science.gov (United States)

    Gunaratnam, Krishanthi; Vidal, Christopher; Boadle, Ross; Thekkedam, Chris; Duque, Gustavo

    2013-01-01

    Summary Lipotoxicity is an overload of lipids in non-adipose tissues that affects function and induces cell death. Lipotoxicity has been demonstrated in bone cells in vitro using osteoblasts and adipocytes in coculture. In this condition, lipotoxicity was induced by high levels of saturated fatty acids (mostly palmitate) secreted by cultured adipocytes acting in a paracrine manner. In the present study, we aimed to identify the underlying mechanisms of lipotoxicity in human osteoblasts. Palmitate induced autophagy in cultured osteoblasts, which was preceded by the activation of autophagosomes that surround palmitate droplets. Palmitate also induced apoptosis though the activation of the Fas/Jun kinase (JNK) apoptotic pathway. In addition, osteoblasts could be protected from lipotoxicity by inhibiting autophagy with the phosphoinositide kinase inhibitor 3-methyladenine or by inhibiting apoptosis with the JNK inhibitor SP600125. In summary, we have identified two major molecular mechanisms of lipotoxicity in osteoblasts and in doing so we have identified a new potential therapeutic approach to prevent osteoblast dysfunction and death, which are common features of age-related bone loss and osteoporosis. PMID:24285710

  17. Time-lapse Raman imaging of osteoblast differentiation

    Science.gov (United States)

    Hashimoto, Aya; Yamaguchi, Yoshinori; Chiu, Liang-Da; Morimoto, Chiaki; Fujita, Katsumasa; Takedachi, Masahide; Kawata, Satoshi; Murakami, Shinya; Tamiya, Eiichi

    2015-07-01

    Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.

  18. Nanoporous hydroxyapatite/sodium titanate bilayer on titanium implants for improved osteointegration.

    Science.gov (United States)

    Carradò, A; Perrin-Schmitt, F; Le, Q V; Giraudel, M; Fischer, C; Koenig, G; Jacomine, L; Behr, L; Chalom, A; Fiette, L; Morlet, A; Pourroy, G

    2017-03-01

    The aim of this study was to improve the strength and quality of the titanium-hydroxyapatite interface in order to prevent long-term failure of the implanted devices originating from coating delamination and to test it in an in-vivo model. Ti disks and dental commercial implants were etched in Kroll solution. Thermochemical treatments of the acid-etched titanium were combined with sol-gel hydroxyapatite (HA) coating processes to obtain a nanoporous hydroxyapatite/sodium titanate bilayer. The sodium titanate layer was created by incorporating sodium ions onto the Ti surface during a NaOH alkaline treatment and stabilized using a heat treatment. HA layer was added by dip-coating in a sol-gel solution. The bioactivity was assessed in vitro with murine MC3T3-E1 and human SaOs-2 cells. Functional and histopathological evaluations of the coated Ti implants were performed at 22, 34 and 60days of implantation in a dog lower mandible model. Nanoporous hydroxyapatite/sodium titanate bilayer on titanium implants was sensitive neither to crack propagation nor to layer delamination. The in vitro results on murine MC3T3-E1 and human SaOs-2 cells confirm the advantage of this coating regarding the capacity of cell growth and differentiation. Signs of progressive bone incorporation, such as cancellous bone formed in contact with the implant over the existing compact bone, were notable as early as day 22. Overall, osteoconduction and osteointegration mean scores were higher for test implants compared to the controls at 22 and 34 days. Nanoporous hydroxyapatite/sodium titanate bilayer improves the in-vivo osteoconduction and osteointegration. It prevents the delamination during the screwing and it could increase HA-coated dental implant stability without adhesive failures. The combination of thermochemical treatments with dip coating is a low-cost strategy. Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  19. Evolution of the osteoblast: skeletogenesis in gar and zebrafish

    Directory of Open Access Journals (Sweden)

    Eames B Frank

    2012-03-01

    Full Text Available Abstract Background Although the vertebrate skeleton arose in the sea 500 million years ago, our understanding of the molecular fingerprints of chondrocytes and osteoblasts may be biased because it is informed mainly by research on land animals. In fact, the molecular fingerprint of teleost osteoblasts differs in key ways from that of tetrapods, but we do not know the origin of these novel gene functions. They either arose as neofunctionalization events after the teleost genome duplication (TGD, or they represent preserved ancestral functions that pre-date the TGD. Here, we provide evolutionary perspective to the molecular fingerprints of skeletal cells and assess the role of genome duplication in generating novel gene functions. We compared the molecular fingerprints of skeletogenic cells in two ray-finned fish: zebrafish (Danio rerio--a teleost--and the spotted gar (Lepisosteus oculatus--a "living fossil" representative of a lineage that diverged from the teleost lineage prior to the TGD (i.e., the teleost sister group. We analyzed developing embryos for expression of the structural collagen genes col1a2, col2a1, col10a1, and col11a2 in well-formed cartilage and bone, and studied expression of skeletal regulators, including the transcription factor genes sox9 and runx2, during mesenchymal condensation. Results Results provided no evidence for the evolution of novel functions among gene duplicates in zebrafish compared to the gar outgroup, but our findings shed light on the evolution of the osteoblast. Zebrafish and gar chondrocytes both expressed col10a1 as they matured, but both species' osteoblasts also expressed col10a1, which tetrapod osteoblasts do not express. This novel finding, along with sox9 and col2a1 expression in developing osteoblasts of both zebrafish and gar, demonstrates that osteoblasts of both a teleost and a basally diverging ray-fin fish express components of the supposed chondrocyte molecular fingerprint. Conclusions Our

  20. Local cell metrics: a novel method for analysis of cell-cell interactions.

    Science.gov (United States)

    Su, Jing; Zapata, Pedro J; Chen, Chien-Chiang; Meredith, J Carson

    2009-10-23

    The regulation of many cell functions is inherently linked to cell-cell contact interactions. However, effects of contact interactions among adherent cells can be difficult to detect with global summary statistics due to the localized nature and noise inherent to cell-cell interactions. The lack of informatics approaches specific for detecting cell-cell interactions is a limitation in the analysis of large sets of cell image data, including traditional and combinatorial or high-throughput studies. Here we introduce a novel histogram-based data analysis strategy, termed local cell metrics (LCMs), which addresses this shortcoming. The new LCM method is demonstrated via a study of contact inhibition of proliferation of MC3T3-E1 osteoblasts. We describe how LCMs can be used to quantify the local environment of cells and how LCMs are decomposed mathematically into metrics specific to each cell type in a culture, e.g., differently-labelled cells in fluorescence imaging. Using this approach, a quantitative, probabilistic description of the contact inhibition effects in MC3T3-E1 cultures has been achieved. We also show how LCMs are related to the naïve Bayes model. Namely, LCMs are Bayes class-conditional probability functions, suggesting their use for data mining and classification. LCMs are successful in robust detection of cell contact inhibition in situations where conventional global statistics fail to do so. The noise due to the random features of cell behavior was suppressed significantly as a result of the focus on local distances, providing sensitive detection of cell-cell contact effects. The methodology can be extended to any quantifiable feature that can be obtained from imaging of cell cultures or tissue samples, including optical, fluorescent, and confocal microscopy. This approach may prove useful in interpreting culture and histological data in fields where cell-cell interactions play a critical role in determining cell fate, e.g., cancer, developmental

  1. Hydroxyapatite coating on damaged tooth surfaces by immersion

    International Nuclear Information System (INIS)

    Lim, Byoung-Ki; Ryu, Su-Chak; Sun, Fangfang; Koh, Kwangnak; Han, Dong-Wook; Lee, Jaebeom

    2009-01-01

    Hydroxyapatite (HAp) was coated on scratched areas of a human tooth and HAp disks by the immersion method in a HAp colloidal solution (≤20 μm of average diameter dispersed in DI water). The surface morphologies of the scratched area after immersion for 1-3 months were investigated showing that the damaged surfaces were remarkably recovered. Then, the mechanical property and chemical stability of the HAp coating layers on both specimens were determined via the Vickers hardness test and concentration measurement of extracted Ca 2+ ions, respectively, after strong acidic treatment. The cellular behavior of mouse calvaria-derived pre-osteoblastic cells (MC3T3-E1) was also examined on the HAp layers regenerated on micro-scratched HAp disks for the purpose of their potential applications on maxillofacial bone conservation and reconstruction for prosthetic dentistry, and artificial disk preparation of a vertebral column. The notable loss of Ca 2+ ions under a highly acidic condition was not observed in the layers coated by HAp adsorption, indicating that the coating surface was well adhered with the original surfaces of the respective specimen. Moreover, the HAp adsorption did not adversely affect the adhesion, growth and proliferation of MC3T3-E1 cells on the coated HAp layers for up to 21 days. These results suggest that the HAp coating on the scratched areas of the tooth would be effectively applicable for the development of long-term prevention of micro-cleavage and tooth health supporters to reduce discoloration and further maxillofacial and orthopedic applications.

  2. Repair of Cranial Bone Defects Using rhBMP2 and Submicron Particle of Biphasic Calcium Phosphate Ceramics with Through-Hole

    Directory of Open Access Journals (Sweden)

    Byung-Chul Jeong

    2015-01-01

    Full Text Available Recently a submicron particle of biphasic calcium phosphate ceramic (BCP with through-hole (donut-shaped BCP (d-BCP was developed for improving the osteoconductivity. This study was performed to examine the usefulness of d-BCP for the delivery of osteoinductive rhBMP2 and the effectiveness on cranial bone regeneration. The d-BCP was soaked in rhBMP2 solution and then freeze-dried. Scanning electron microscope (SEM, energy dispersive spectroscopy (EDS, and Raman spectroscopy analyses confirmed that rhBMP2 was well delivered onto the d-BCP surface and the through-hole. The bioactivity of the rhBMP2/d-BCP composite was validated in MC3T3-E1 cells as an in vitro model and in critical-sized cranial defects in C57BL/6 mice. When freeze-dried d-BCPs with rhBMP2 were placed in transwell inserts and suspended above MC3T3-E1, alkaline phosphatase activity and osteoblast-specific gene expression were increased compared to non-rhBMP2-containing d-BCPs. For evaluating in vivo effectiveness, freeze-dried d-BCPs with or without rhBMP2 were implanted into critical-sized cranial defects. Microcomputed tomography and histologic analysis showed that rhBMP2-containing d-BCPs significantly enhanced cranial bone regeneration compared to non-rhBMP2-containing control. These results suggest that a combination of d-BCP and rhBMP2 can accelerate bone regeneration, and this could be used to develop therapeutic strategies in hard tissue healing.

  3. Repair of Cranial Bone Defects Using rhBMP2 and Submicron Particle of Biphasic Calcium Phosphate Ceramics with Through-Hole.

    Science.gov (United States)

    Jeong, Byung-Chul; Choi, Hyuck; Hur, Sung-Woong; Kim, Jung-Woo; Oh, Sin-Hye; Kim, Hyun-Seung; Song, Soo-Chang; Lee, Keun-Bae; Park, Kwang-Bum; Koh, Jeong-Tae

    2015-01-01

    Recently a submicron particle of biphasic calcium phosphate ceramic (BCP) with through-hole (donut-shaped BCP (d-BCP)) was developed for improving the osteoconductivity. This study was performed to examine the usefulness of d-BCP for the delivery of osteoinductive rhBMP2 and the effectiveness on cranial bone regeneration. The d-BCP was soaked in rhBMP2 solution and then freeze-dried. Scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), and Raman spectroscopy analyses confirmed that rhBMP2 was well delivered onto the d-BCP surface and the through-hole. The bioactivity of the rhBMP2/d-BCP composite was validated in MC3T3-E1 cells as an in vitro model and in critical-sized cranial defects in C57BL/6 mice. When freeze-dried d-BCPs with rhBMP2 were placed in transwell inserts and suspended above MC3T3-E1, alkaline phosphatase activity and osteoblast-specific gene expression were increased compared to non-rhBMP2-containing d-BCPs. For evaluating in vivo effectiveness, freeze-dried d-BCPs with or without rhBMP2 were implanted into critical-sized cranial defects. Microcomputed tomography and histologic analysis showed that rhBMP2-containing d-BCPs significantly enhanced cranial bone regeneration compared to non-rhBMP2-containing control. These results suggest that a combination of d-BCP and rhBMP2 can accelerate bone regeneration, and this could be used to develop therapeutic strategies in hard tissue healing.

  4. Synthesis of bioactive and machinable miserite glass-ceramics for dental implant applications.

    Science.gov (United States)

    Saadaldin, Selma A; Dixon, S Jeffrey; Costa, Daniel O; Rizkalla, Amin S

    2013-06-01

    To synthesize and characterize machinable, bioactive glass-ceramics (GCs) suitable for dental implant applications. A glass in the SiO2-Al2O3-CaO-CaF2-K2O-B2O3-La2O3 system was synthesized by wet chemical methods, followed by calcination, melting and quenching. Crystallization kinetics were determined by differential thermal analysis (DTA). GC discs were produced by cold pressing of the glass powder and sintered using schedules determined by DTA. The crystalline phases and microstructure of GC samples were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. Dynamic Young's modulus (E), true hardness (Ho), fracture toughness (KIC) and brittleness index (BI) were evaluated. Bioactivity was studied by examining the formation of hydroxyapatite (HA) on the GC surfaces after soaking in simulated body fluid (SBF). Attachment and proliferation of MC3T3-E1 osteoblastic cells were assessed in vitro. Miserite [KCa5(Si2O7)(Si6O15)(OH)F] was the main crystalline phase of the GC with additional secondary phases. Microstructural studies revealed interlocking lath-like crystalline morphology. E, Ho, and KIC values for the GCs were 96±3 GPa, 5.27±0.26 GPa and 4.77±0.27 MPa m(0.5), respectively. The BI was found to be 1.11±0.05 μm(-0.5), indicating outstanding machinability. An HA surface layer was formed on the GC surfaces when soaked in SBF, indicating potential bioactivity. MC3T3-E1 cells exhibited attachment, spreading and proliferation on GC surfaces, demonstrating excellent biocompatibility. We present a novel approach for the synthesis of miserite GC with the physical and biological properties required for non-metallic dental implant applications. Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  5. Influence of engineered surface on cell directionality and motility

    International Nuclear Information System (INIS)

    Tang, Qing Yuan; Pang, Stella W; Tong, Wing Yin; Shi, Peng; Lam, Yun Wah; Shi, Jue

    2014-01-01

    Control of cell migration is important in numerous key biological processes, and is implicated in pathological conditions such as cancer metastasis and inflammatory diseases. Many previous studies indicated that cell migration could be guided by micropatterns fabricated on cell culture surfaces. In this study, we designed a polydimethylsiloxane cell culture substrate with gratings punctuated by corners and ends, and studied its effects on the behavior of MC3T3-E1 osteoblast cells. MC3T3-E1 cells elongated and aligned with the gratings, and the migration paths of the cells appeared to be guided by the grating pattern. Interestingly, more than 88% of the cells cultured on these patterns were observed to reverse their migration directions at least once during the 16 h examination period. Most of the reversal events occurred at the corners and the ends of the pattern, suggesting these localized topographical features induce an abrupt loss in directional persistence. Moreover, the cell speed was observed to increase temporarily right after each directional reversal. Focal adhesion complexes were more well-established in cells on the angular gratings than on flat surfaces, but the formation of filipodia appeared to be imbalanced at the corners and the ends, possibly leading to the loss of directional persistence. This study describes the first engineered cell culture surface that consistently induces changes in the directional persistence of adherent cells. This will provide an experimental model for the study of this phenomenon and a valuable platform to control the cell motility and directionality, which can be used for cell screening and selection. (paper)

  6. Cuscuta chinensis extract promotes osteoblast differentiation and mineralization in human osteoblast-like MG-63 cells.

    Science.gov (United States)

    Yang, Hyun Mo; Shin, Hyun-Kyung; Kang, Young-Hee; Kim, Jin-Kyung

    2009-02-01

    The aim of the present study was to investigate whether the aqueous extract of To-Sa-Za (TSZ-AE), the seed of Cuscuta chinensis Lam., which is a traditional medicinal herb commonly used in Korea and other oriental countries, could induce osteogenic activity in human osteoblast-like MG-63 cells. TSZ-AE treatment mildly promoted the proliferation of MG-63 cells at doses of 500 and 1,000 microg/mL in the 24-hour culture period. Dose-dependent increases in alkaline phosphatase (ALP) activity and collagen synthesis were shown at 48 and 72 hours of incubation. The release of bone morphogenetic protein (BMP)-2 but not osteocalcin in the MG-63 cells was induced by TSZ-AE at 72 hours (100-1,000 microg/mL). In addition, TSZ-AE markedly increased mRNA expression of ALP, collagen, and BMP-2 in the MG-63 cells in a dose-dependent manner. Mineralization in the culture of MG-63 cells was significantly induced at 500 and 1,000 microg/mL TSZ-AE treatment. In conclusion, this study shows that TSZ-AE enhanced ALP activity, collagen synthesis, BMP-2 expression, and mineralization in MG-63 cells. These results strongly suggest that C. chinensis can play an important role in osteoblastic bone formation and may possibly lead to the development of bone-forming drugs.

  7. Nanostructured magnesium has fewer detrimental effects on osteoblast function

    Directory of Open Access Journals (Sweden)

    Weng L

    2013-05-01

    Full Text Available Lucy Weng, Thomas J Webster School of Engineering and Department of Orthopedics, Brown University, Providence, RI, USA Abstract: Efforts have been made recently to implement nanoscale surface features on magnesium, a biodegradable metal, to increase bone formation. Compared with normal magnesium, nanostructured magnesium has unique characteristics, including increased grain boundary properties, surface to volume ratio, surface roughness, and surface energy, which may influence the initial adsorption of proteins known to promote the function of osteoblasts (bone-forming cells. Previous studies have shown that one way to increase nanosurface roughness on magnesium is to soak the metal in NaOH. However, it has not been determined if degradation of magnesium is altered by creating nanoscale features on its surface to influence osteoblast density. The aim of the present in vitro study was to determine the influence of degradation of nanostructured magnesium, created by soaking in NaOH, on osteoblast density. Our results showed a less detrimental effect of magnesium degradation on osteoblast density when magnesium was treated with NaOH to create nanoscale surface features. The detrimental degradation products of magnesium are of significant concern when considering use of magnesium as an orthopedic implant material, and this study identified a surface treatment, ie, soaking in NaOH to create nanoscale features for magnesium that can improve its use in numerous orthopedic applications. Keywords: nanostructured magnesium, degradation, detrimental effects, osteoblasts

  8. Osteoblasts Protect AML Cells from SDF-1-Induced Apoptosis

    Science.gov (United States)

    Kremer, Kimberly N.; Dudakovic, Amel; McGee-Lawrence, Meghan E.; Philips, Rachael L.; Hess, Allan D.; Smith, B. Douglas; van Wijnen, Andre J.; Karp, Judith E.; Kaufmann, Scott H.; Westendorf, Jennifer J.; Hedin, Karen E.

    2014-01-01

    The bone marrow provides a protective environment for acute myeloid leukemia (AML) cells that often allows leukemic stem cells to survive standard chemotherapeutic regimens. Targeting these leukemic stem cells within the bone marrow is critical for preventing relapse. We recently demonstrated that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis in AML cell lines and in patient samples expressing high levels of its receptor, CXCR4. Here we show that a subset of osteoblast lineage cells within the bone marrow can protect AML cells from undergoing apoptosis in response to the SDF-1 naturally present in that location. In co-culture systems, osteoblasts at various stages of differentiation protected AML cell lines and patient isolates from SDF-1-induced apoptosis. The differentiation of the osteoblast cell lines, MC3T3 and W-20-17, mediated this protection via a cell contact-independent mechanism. In contrast, bone marrow-derived mesenchymal cells, the precursors of osteoblasts, induced apoptosis in AML cells via a CXCR4-dependent mechanism and failed to protect AML cells from exogenously added SDF-1. These results indicate that osteoblasts in the process of differentiation potently inhibit the SDF-1-driven apoptotic pathway of CXCR4-expressing AML cells residing in the bone marrow. Drugs targeting this protective mechanism could potentially provide a new approach to treating AML by enhancing the SDF-1-induced apoptosis of AML cells residing within the bone marrow microenvironment. PMID:24851270

  9. Adhesion of osteoblasts to a nanorough titanium implant surface

    Directory of Open Access Journals (Sweden)

    Gongadze E

    2011-08-01

    Full Text Available Ekaterina Gongadze1, Doron Kabaso2, Sebastian Bauer3, Tomaž Slivnik2, Patrik Schmuki3, Ursula van Rienen1, Aleš Iglič21Institute of General Electrical Engineering, University of Rostock, Rostock, Germany; 2Laboratory of Biophysics, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia; 3Department of Materials Science, Friedrich-Alexander University of Erlangen-Nurenberg, Erlangen, GermanyAbstract: This work considers the adhesion of cells to a nanorough titanium implant surface with sharp edges. The basic assumption was that the attraction between the negatively charged titanium surface and a negatively charged osteoblast is mediated by charged proteins with a distinctive quadrupolar internal charge distribution. Similarly, cation-mediated attraction between fibronectin molecules and the titanium surface is expected to be more efficient for a high surface charge density, resulting in facilitated integrin mediated osteoblast adhesion. We suggest that osteoblasts are most strongly bound along the sharp convex edges or spikes of nanorough titanium surfaces where the magnitude of the negative surface charge density is the highest. It is therefore plausible that nanorough regions of titanium surfaces with sharp edges and spikes promote the adhesion of osteoblasts.Keywords: osteoblasts, nanostructures, adhesion, titanium implants, osteointegration

  10. Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance.

    Science.gov (United States)

    Ando, Kazunori; Shibata, Eri; Hans, Stefan; Brand, Michael; Kawakami, Atsushi

    2017-12-04

    Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

    International Nuclear Information System (INIS)

    Ecarot-Charrier, B.; Bouchard, F.; Delloye, C.

    1989-01-01

    Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with [35S] sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I

  12. Effects of space-relevant radiation on pre-osteoblasts

    International Nuclear Information System (INIS)

    Hu, Yueyuan

    2014-01-01

    Until now limited research has been conducted to address the mechanisms leading ionizing radiation exposure induced bone loss. This is relevant for cancer radiotherapy and human spaceflight. Exposure to radiation can result in elevated bone fracture risk in patients receiving cancer radiotherapy. In human spaceflight, astronauts are exposed to space radiation which is a very complex mixture consisting primarily of high-energy charged particles. Osteoblasts are of mesenchymal origin and responsible for creating and maintaining skeletal architecture; these cells produce extracellular matrix proteins and regulators of matrix mineralization during initial bone formation and later bone remodeling. The aim of this work was to investigate the effects of ionizing radiation on pre-osteoblasts including cellular survival, cell cycle regulation and differentiation modification. Experiments with the pre-osteoblast cell line OCT-1 and the mesenchymal stem cell line C3H10T1/2 showed that radiation cell killing depends on dose and linear energy transfer (LET) and is most effective at an LET of ∝150 keV/μm. High-LET radiation has a much more pronounced ability to induce cell cycle arrest in the G2/M phase. After both X-rays and heavy ions exposure, expression of the cell cycle regulator CDKN1A was significantly up-regulated in a dose-dependent manner. The findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression. Radiation exposure enhances osteoblastic differentiation and maturation, and mediates Runx2 and TGF-β1 expression during early differentiation of pre-osteoblasts. Osteogenic differentiation did not alter cellular radiosensitivity, DNA repair of radiation-induced damages and the effects of radiation on proliferation. Further experiments are needed to elucidate possible synergistic effects of microgravity and radiation on osteoblast differentiation. This may

  13. Identification and proteomic analysis of osteoblast-derived exosomes

    Energy Technology Data Exchange (ETDEWEB)

    Ge, Min; Ke, Ronghu; Cai, Tianyi; Yang, Junyi; Mu, Xiongzheng, E-mail: cranio@vip.163.com

    2015-11-06

    Exosomes are nanometer-sized vesicles with the function of intercellular communication, and they are released by various cell types. To reveal the knowledge about the exosomes from osteoblast, and explore the potential functions of osteogenesis, we isolated microvesicles from supernatants of mouse Mc3t3 by ultracentrifugation, characterized exosomes by electron microscopy and immunoblotting and presented the protein profile by proteomic analysis. The result demonstrated that microvesicles were between 30 and 100 nm in diameter, round shape with cup-like concavity and expressed exosomal marker tumor susceptibility gene (TSG) 101 and flotillin (Flot) 1. We identified a total number of 1069 proteins among which 786 proteins overlap with ExoCarta database. Gene Oncology analysis indicated that exosomes mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The Ingenuity Pathway Analysis showed pathways are mostly involved in exosome biogenesis, formation, uptake and osteogenesis. Among the pathways, eukaryotic initiation factor 2 pathways played an important role in osteogenesis. Our study identified osteoblast-derived exosomes, unveiled the content of them, presented potential osteogenesis-related proteins and pathways and provided a rich proteomics data resource that will be valuable for further studies of the functions of individual proteins in bone diseases. - Highlights: • We for the first time identified exosomes from mouse osteoblast. • Osteoblasts-derived exosomes contain osteoblast peculiar proteins. • Proteins from osteoblasts-derived exosomes are intently involved in EIF2 pathway. • EIF2α from the EIF2 pathway plays an important role in osteogenesis.

  14. Effects of space-relevant radiation on pre-osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Yueyuan

    2014-02-12

    Until now limited research has been conducted to address the mechanisms leading ionizing radiation exposure induced bone loss. This is relevant for cancer radiotherapy and human spaceflight. Exposure to radiation can result in elevated bone fracture risk in patients receiving cancer radiotherapy. In human spaceflight, astronauts are exposed to space radiation which is a very complex mixture consisting primarily of high-energy charged particles. Osteoblasts are of mesenchymal origin and responsible for creating and maintaining skeletal architecture; these cells produce extracellular matrix proteins and regulators of matrix mineralization during initial bone formation and later bone remodeling. The aim of this work was to investigate the effects of ionizing radiation on pre-osteoblasts including cellular survival, cell cycle regulation and differentiation modification. Experiments with the pre-osteoblast cell line OCT-1 and the mesenchymal stem cell line C3H10T1/2 showed that radiation cell killing depends on dose and linear energy transfer (LET) and is most effective at an LET of ∝150 keV/μm. High-LET radiation has a much more pronounced ability to induce cell cycle arrest in the G2/M phase. After both X-rays and heavy ions exposure, expression of the cell cycle regulator CDKN1A was significantly up-regulated in a dose-dependent manner. The findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression. Radiation exposure enhances osteoblastic differentiation and maturation, and mediates Runx2 and TGF-β1 expression during early differentiation of pre-osteoblasts. Osteogenic differentiation did not alter cellular radiosensitivity, DNA repair of radiation-induced damages and the effects of radiation on proliferation. Further experiments are needed to elucidate possible synergistic effects of microgravity and radiation on osteoblast differentiation. This may

  15. Fludarabine inhibits STAT1-mediated up-regulation of caspase-3 expression in dexamethasone-induced osteoblasts apoptosis and slows the progression of steroid-induced avascular necrosis of the femoral head in rats.

    Science.gov (United States)

    Feng, Zhenhua; Zheng, Wenhao; Tang, Qian; Cheng, Liang; Li, Hang; Ni, Wenfei; Pan, Xiaoyun

    2017-08-01

    Steroid-induced avascular necrosis of the femoral head (SANFH) is a major limitation of long-term or excessive clinical administration of glucocorticoids. Fludarabine, which is a compound used to treat various hematological malignancies, such as chronic lymphocytic leukemia, acts by down-regulating signal transducer and activator of transcription 1 (STAT1) by inhibiting STAT1 phosphorylation in both normal and cancer cells. This study assessed the effects of fludarabine in vitro (primary murine osteoblasts) and in vivo (rat SANFH model). In vitro, pretreatment with fludarabine significantly inhibited Dexamethasone (Dex)-induced apoptosis in osteoblasts, which was examined by TUNEL staining. Treatment with Dex caused a remarkable decrease in the expression of Bcl-2; an increase in cytochrome c release; activation of BAX, caspase-9, and caspase-3; and an obvious enhancement in STAT1 phosphorylation. However, treatment resulted in the up-regulation of caspase-3 expression. Enhanced P-STAT1 activity and up-regulation of caspase-3 expression were also observed in osteoblasts. In vivo, the subchondral trabeculae in fludarabine-treated rats exhibited less bone loss and a lower ratio of empty lacunae. Taken together, our results suggest that STAT1-mediated up-regulation of caspase-3 is involved in osteoblast apoptosis induced by Dex and indicates that fludarabine may serve as a potential agent for the treatment of SANFH.

  16. EFEK APLIKASI PADA HIDROKSIAPATIT TERHADAP PERLEKATAN SEL OSTEOBLAS

    Directory of Open Access Journals (Sweden)

    Siti Sunarintyas

    2011-12-01

    Full Text Available Hydroxyapatite (HA is often used as a human bone graft. Modification of HA surface is developed to increase osseointegration process which is influenced by cells attachment surrounded the tissue. Sericin has polar side groups which accelerate cells attachment. The aim of this research was to determine the effect of sericin application over HA surface on the attachment of osteoblast cells. The research used HA synthesized from gypsum (Kulon Progo, Yogyakarta. Sericin was extracted from silkworm cocoons of Bombyx mori. Sericin of 0.01, 0.05, 0.1% were applied over HA discs surface (10 mm in diameter, 3 mm thick. The discs were inserted into osteoblast cells culture of MC3T3E1 for attachment test. The data were analyzed by ANOVA and LSD. The result showed that the amount of osteoblast cells attached to HA (7.60±0.42 was significantly difference (p<0.05 with HA + sericin 0.01% (10.30±0.52, HA + sericin 0.5% (10.63±0.70, HA + sericin 0.1% (10.67±0.87. Variation of sericin concentrations applied over HA did not influence any significant difference on the amount of cells attachment (p>0.05. In conclusion, sericin application over HA surface increased the amount of osteoblast cells attachment. Concentrations of sericin application over HA (0.01, 0.05, 0.1% did not influence osteoblast cells attachment.

  17. EFEK APLIKASI PADA HIDROKSIAPATIT TERHADAP PERLEKATAN SEL OSTEOBLAS

    Directory of Open Access Journals (Sweden)

    Siti Sunarintyas

    2011-12-01

    Full Text Available Hydroxyapatite (HA is often used as a human bone graft. Modification of HA surface is developed to increase osseointegration process which is influenced by cells attachment surrounded the tissue. Sericin has polar side groups which accelerate cells attachment. The aim of this research was to determine the effect of sericin application over HA surface on the attachment of osteoblast cells. The research used HA synthesized from gypsum (Kulon Progo, Yogyakarta. Sericin was extracted from silkworm cocoons of Bombyx mori. Sericin of 0.01, 0.05, 0.1% were applied over HA discs surface (10 mm in diameter, 3 mm thick. The discs were inserted into osteoblast cells culture of MC3T3E1 for attachment test. The data were analyzed by ANOVA and LSD. The result showed that the amount of osteoblast cells attached to HA (7.60±0.42 was significantly difference (p0.05. In conclusion, sericin application over HA surface increased the amount of osteoblast cells attachment. Concentrations of sericin application over HA (0.01, 0.05, 0.1% did not influence osteoblast cells attachment.

  18. Nanostructured magnesium has fewer detrimental effects on osteoblast function.

    Science.gov (United States)

    Weng, Lucy; Webster, Thomas J

    2013-01-01

    Efforts have been made recently to implement nanoscale surface features on magnesium, a biodegradable metal, to increase bone formation. Compared with normal magnesium, nanostructured magnesium has unique characteristics, including increased grain boundary properties, surface to volume ratio, surface roughness, and surface energy, which may influence the initial adsorption of proteins known to promote the function of osteoblasts (bone-forming cells). Previous studies have shown that one way to increase nanosurface roughness on magnesium is to soak the metal in NaOH. However, it has not been determined if degradation of magnesium is altered by creating nanoscale features on its surface to influence osteoblast density. The aim of the present in vitro study was to determine the influence of degradation of nanostructured magnesium, created by soaking in NaOH, on osteoblast density. Our results showed a less detrimental effect of magnesium degradation on osteoblast density when magnesium was treated with NaOH to create nanoscale surface features. The detrimental degradation products of magnesium are of significant concern when considering use of magnesium as an orthopedic implant material, and this study identified a surface treatment, ie, soaking in NaOH to create nanoscale features for magnesium that can improve its use in numerous orthopedic applications.

  19. ent-Kaurane diterpenoids from Croton tonkinensis stimulate osteoblast differentiation

    DEFF Research Database (Denmark)

    Dao, Trong-Tuan; Lee, Kwang-Youl; Jeong, Hyung-Min

    2011-01-01

    Four new ent-kaurane diterpenoids (1-4) were isolated from the leaves of Croton tonkinensis by bioactivity-guided fractionation using an in vitro osteoblast differentiation assay. Their structures were identified as ent-11β-acetoxykaur-16-en-18-ol (1), ent-11α-hydroxy-18-acetoxykaur-16-ene (2), e...

  20. Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yinxiang Wang

    2018-01-01

    Full Text Available Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc, enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.

  1. Rotary culture enhances pre-osteoblast aggregation and mineralization.

    Science.gov (United States)

    Facer, S R; Zaharias, R S; Andracki, M E; Lafoon, J; Hunter, S K; Schneider, G B

    2005-06-01

    Three-dimensional environments have been shown to enhance cell aggregation and osteoblast differentiation. Thus, we hypothesized that three-dimensional (3D) growth environments would enhance the mineralization rate of human embryonic palatal mesenchymal (HEPM) pre-osteoblasts. The objective of this study was to investigate the potential use of rotary cell culture systems (RCCS) as a means to enhance the osteogenic potential of pre-osteoblast cells. HEPM cells were cultured in a RCCS to create 3D enviroments. Tissue culture plastic (2D) cultures served as our control. 3D environments promoted three-dimensional aggregate formations. Increased calcium and phosphorus deposition was significantly enhanced three- to 18-fold (P < 0.001) in 3D cultures as compared with 2D environments. 3D cultures mineralized in 1 wk as compared with the 2D cultures, which took 4 wks, a decrease in time of nearly 75%. In conclusion, our studies demonstrated that 3D environments enhanced osteoblast cell aggregation and mineralization.

  2. Nanostructured magnesium has fewer detrimental effects on osteoblast function

    Science.gov (United States)

    Weng, Lucy; Webster, Thomas J

    2013-01-01

    Efforts have been made recently to implement nanoscale surface features on magnesium, a biodegradable metal, to increase bone formation. Compared with normal magnesium, nanostructured magnesium has unique characteristics, including increased grain boundary properties, surface to volume ratio, surface roughness, and surface energy, which may influence the initial adsorption of proteins known to promote the function of osteoblasts (bone-forming cells). Previous studies have shown that one way to increase nanosurface roughness on magnesium is to soak the metal in NaOH. However, it has not been determined if degradation of magnesium is altered by creating nanoscale features on its surface to influence osteoblast density. The aim of the present in vitro study was to determine the influence of degradation of nanostructured magnesium, created by soaking in NaOH, on osteoblast density. Our results showed a less detrimental effect of magnesium degradation on osteoblast density when magnesium was treated with NaOH to create nanoscale surface features. The detrimental degradation products of magnesium are of significant concern when considering use of magnesium as an orthopedic implant material, and this study identified a surface treatment, ie, soaking in NaOH to create nanoscale features for magnesium that can improve its use in numerous orthopedic applications. PMID:23674891

  3. Measurement of oxygen consumption rate of osteoblasts from ...

    African Journals Online (AJOL)

    The cells were evaluated through live/dead assay, hematoxylin-eosin (HE) and alkaline phosphatase (ALP) staining. Moreover, Von-Kossa staining and Alizarin Red S staining were carried out for mineralized nodule formation. Following this, the oxygen consumption rates of osteoblasts in the earlier mentioned different ...

  4. Osteoblastic cells trigger gate currents on nanocrystalline diamond transistor

    Czech Academy of Sciences Publication Activity Database

    Ižák, Tibor; Krátká, Marie; Kromka, Alexander; Rezek, Bohuslav

    2015-01-01

    Roč. 129, May (2015), 95-99 ISSN 0927-7765 R&D Projects: GA ČR GAP108/12/0996 Grant - others:AVČR(CZ) M100101209 Institutional support: RVO:68378271 Keywords : field-effect transistors * nanocrystalline diamond * osteoblastic cells * leakage currents Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 3.902, year: 2015

  5. Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

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    Yun-Yun Ma

    Full Text Available Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification.

  6. Osteoblast growth behavior on porous-structure titanium surface

    International Nuclear Information System (INIS)

    Tian Yuan; Ding Siyang; Peng Hui; Lu Shanming; Wang Guoping; Xia Lu; Wang Peizhi

    2012-01-01

    Highlights: ► Micro-arc oxidation technology formed a porous feature on titanium surface. ► This porous surface accelerated adhesion, proliferation and differentiation compared with smooth surface. ► Osteogenesis-related proteins and genes were up regulated by this porous surface. ► It is anticipated that micro-arc oxidation surface could enhance osteoblastic activity and bone regeneration. - Abstract: A bioavailable surface generated by nano-technology could accelerate implant osteointegration, reduce healing time and enable implants to bear early loading. In this study, a nano-porous surface of titanium wafers was modified using micro-arc oxidation technique; surface of smooth titanium was used as control group. Surface characteristic was evaluated by investigating morphology, roughness and hydrophilicity of titanium wafers. In vitro studies, osteoblastic adhesion, proliferation and ALP activity, as well as gene and protein expressions relative to mineralization were assayed. Our results showed that a crater-liked nano-porous surface with greater roughness and better hydrophilicity were fabricated by micro-arc oxidation. It was further indicated that nano-porous surface could enhance adhesion, proliferation and ALP activity of osteoblasts compared with smooth surfaces. In addition, gene and protein expression of collagen-I, osteocalcin and osteopontin were also obviously increased. In summary, micro-arc oxidized techniques could form an irregular nano-porous morphology on implant surface which is favorable to improve osteoblastic function and prospected to be a potent modification of dental implant.

  7. Osteoblastic response to pectin nanocoating on titanium surfaces

    DEFF Research Database (Denmark)

    Gurzawska, Katarzyna; Svava, Rikke; Yihua, Yu

    2014-01-01

    with respect to surface properties and osteogenic response in osteoblastic cells. Nanocoatings on titanium surfaces were evaluated by scanning electron microscopy, contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy. The effect of coated RG-Is on cell adhesion, cell...

  8. Ecotropic murine leukemia virus-induced fusion of murine cells

    International Nuclear Information System (INIS)

    Pinter, A.; Chen, T.; Lowy, A.; Cortez, N.G.; Silagi, S.

    1986-01-01

    Extensive fusion occurs upon cocultivation of murine fibroblasts producing ecotropic murine leukemia viruses (MuLVs) with a large variety of murine cell lines in the presence of the polyene antibiotic amphotericin B, the active component of the antifungal agent Fungizone. The resulting polykaryocytes contain nuclei from both infected and uninfected cells, as evidenced by autoradiographic labeling experiments in which one or the other parent cell type was separately labeled with [ 3 H]thymidine and fused with an unlabeled parent. This cell fusion specifically requires the presence of an ecotropic MuLV-producing parent and is not observed for cells producing xenotropic, amphotropic, or dualtropic viruses. Mouse cells infected with nonecotropic viruses retain their sensitivity toward fusion, whereas infection with ecotropic viruses abrogates the fusion of these cells upon cocultivation with other ecotropic MuLV-producing cells. Nonmurine cells lacking the ecotropic gp70 receptor are not fused under similar conditions. Fusion is effectively inhibited by monospecific antisera to gp70, but not by antisera to p15(E), and studies with monoclonal antibodies identify distinct amino- and carboxy-terminal gp70 regions which play a role in the fusion reaction. The enhanced fusion which occurs in the presence of amphotericin B provides a rapid and sensitive assay for the expression of ecotropic MuLVs and should facilitate further mechanistic studies of MuLV-induced fusion of murine cells

  9. Chondrocytic Atf4 regulates osteoblast differentiation and function via Ihh.

    Science.gov (United States)

    Wang, Weiguang; Lian, Na; Ma, Yun; Li, Lingzhen; Gallant, Richard C; Elefteriou, Florent; Yang, Xiangli

    2012-02-01

    Atf4 is a leucine zipper-containing transcription factor that activates osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation of skeletal development and bone formation is poorly understood. Investigations of the Atf4(-/-);Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteogenesis during development and bone remodeling postnatally. Atf4 overexpression in chondrocytes of the Atf4(-/-);Col2a1-Atf4 double mutants corrects the reduction in stature and limb in Atf4(-/-) embryos and rectifies the decrease in Ihh expression, Hh signaling, proliferation and accelerated hypertrophy that characterize the Atf4(-/-) developing growth plate cartilages. Unexpectedly, this genetic manipulation also restores the expression of osteoblastic marker genes, namely Ocn and bone sialoprotein, in Atf4(-/-) developing bones. In Atf4(-/-);Col2a1-Atf4 adult mice, all the defective bone parameters found in Atf4(-/-) mice, including bone volume, trabecular number and thickness, and bone formation rate, are rescued. In addition, the conditioned media of ex vivo cultures from wild-type or Atf4(-/-);Col2a1-Atf4, but not Atf4(-/-) cartilage, corrects the differentiation defects of Atf4(-/-) bone marrow stromal cells and Ihh-blocking antibody eliminates this effect. Together, these data indicate that Atf4 in chondrocytes is required for normal Ihh expression and for its paracrine effect on osteoblast differentiation. Therefore, the cell-autonomous role of Atf4 in chondrocytes dominates the role of Atf4 in osteoblasts during development for the control of early osteogenesis and skeletal growth.

  10. Improving effects of chitosan nanofiber scaffolds on osteoblast proliferation and maturation

    Science.gov (United States)

    Ho, Ming-Hua; Liao, Mei-Hsiu; Lin, Yi-Ling; Lai, Chien-Hao; Lin, Pei-I; Chen, Ruei-Ming

    2014-01-01

    Osteoblast maturation plays a key role in regulating osteogenesis. Electrospun nanofibrous products were reported to possess a high surface area and porosity. In this study, we developed chitosan nanofibers and examined the effects of nanofibrous scaffolds on osteoblast maturation and the possible mechanisms. Macro- and micro observations of the chitosan nanofibers revealed that these nanoproducts had a flat surface and well-distributed fibers with nanoscale diameters. Mouse osteoblasts were able to attach onto the chitosan nanofiber scaffolds, and the scaffolds degraded in a time-dependent manner. Analysis by scanning electron microscopy further showed mouse osteoblasts adhered onto the scaffolds along the nanofibers, and cell–cell communication was also detected. Mouse osteoblasts grew much better on chitosan nanofiber scaffolds than on chitosan films. In addition, human osteoblasts were able to adhere and grow on the chitosan nanofiber scaffolds. Interestingly, culturing human osteoblasts on chitosan nanofiber scaffolds time-dependently increased DNA replication and cell proliferation. In parallel, administration of human osteoblasts onto chitosan nanofibers significantly induced osteopontin, osteocalcin, and alkaline phosphatase (ALP) messenger (m)RNA expression. As to the mechanism, chitosan nanofibers triggered runt-related transcription factor 2 mRNA and protein syntheses. Consequently, results of ALP-, alizarin red-, and von Kossa-staining analyses showed that chitosan nanofibers improved osteoblast mineralization. Taken together, results of this study demonstrate that chitosan nanofibers can stimulate osteoblast proliferation and maturation via runt-related transcription factor 2-mediated regulation of osteoblast-associated osteopontin, osteocalcin, and ALP gene expression. PMID:25246786

  11. Responses of human normal osteoblast cells and osteoblast-like cell line, MG-63 cells, to pulse electromagnetic field (PEMF

    Directory of Open Access Journals (Sweden)

    Suttatip Kamolmatyakul

    2008-01-01

    Full Text Available The objective of this in vitro study is to investigate the effect of pulsed electromagnetic field (PEMF on cellular proliferation and osteocalcin production of osteoblast-like cell line, MG-63 cells, and human normal osteoblast cells (NHOC obtained from surgical bone specimens. The cells were placed in 24-well culture plates in the amount of 3x104 cell/wells with 2 ml αMEM media supplemented with 10% FBS. The experimental plates were placed between a pair of Helmoltz coils powered by a pulse generator (PEMF, 50 Hz, 1.5 mV/cm in the upper compartment of a dual incubator (Forma. The control plates were placed in the lower compartment of the incubator without Helmotz coils. After three days, the cell proliferation was measured by the method modified from Mossman (J. Immunol Methods 1983; 65: 55-63. Other sets of plates were used for osteocalcin production assessment. Media from these sets were collected after 6 days and assessed for osteocalcin production using ELISA kits. The data were analyzed using a one-way analysis of variance (ANOVA. The results showed that MG-63 cells from the experimental group proliferated significantly more than those from the control group (20% increase, p<0.05. No significant difference in osteocalcin production was detected between the two groups. On the other hand, NHOC from the experimental group produced larger amount of osteocalcin (25% increase, p<0.05 and proliferated significantly more than those from the control group (100% increase, p<0.05. In conclusion, PEMF effect on osteoblasts might depend on their cell type of origin. For osteoblast-like cell line, MG-63 cells, PEMF increased proliferation rate but not osteocalcin production of the cells. However, PEMF stimulation effect on human normal osteoblast cells was most likely associated with enhancement of both osteocalcin production and cell proliferation.

  12. Effects of total flavonoids from Drynariae Rhizoma prevent bone loss in vivo and in vitro

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    Shuang-hong Song

    2016-12-01

    Full Text Available Estrogen deficiency is one of the major causes of osteoporosis in postmenopausal women. Drynariae Rhizoma is a widely used traditional Chinese medicine for the treatment of bone diseases. In this study, we investigated the therapeutic effects of the total Drynariae Rhizoma flavonoids (DRTF on estrogen deficiency-induced bone loss using an ovariectomized rat model and osteoblast-like MC3T3-E1 cells. Our results indicated that DRTF produced osteo-protective effects on the ovariectomized rats in terms of bone loss reduction, including decreased levels of bone turnover markers, enhanced biomechanical femur strength and trabecular bone microarchitecture deterioration prevention. In vitro experiments revealed that the actions of DRTF on regulating osteoblastic activities were mediated by the estrogen receptor (ER dependent pathway. Our data also demonstrated that DRTF inhibited osteoclastogenesis via up-regulating osteoprotegrin (OPG, as well as down-regulating receptor activator of NF–κB ligand (RANKL expression. In conclusion, this study indicated that DRTF treatment effectively suppressed bone mass loss in an ovariectomized rat model, and in vitro evidence suggested that the effects were exerted through actions on both osteoblasts and osteoclasts. Keywords: Osteoporosis, Osteoblast, Osteoclast, Ovariectomy, Drynariae Rhizoma

  13. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

    International Nuclear Information System (INIS)

    Kawano, Michinao; Ariyoshi, Wataru; Iwanaga, Kenjiro; Okinaga, Toshinori; Habu, Manabu; Yoshioka, Izumi; Tominaga, Kazuhiro; Nishihara, Tatsuji

    2011-01-01

    Research highlights: → In this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. → MG63 cells were incubated with BMP-2 and HA for various time periods. → Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. → HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation. -- Abstract: Objectives: Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. Materials and methods: Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR. Results: BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and nuclear translocation

  14. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

    Energy Technology Data Exchange (ETDEWEB)

    Kawano, Michinao [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Ariyoshi, Wataru [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Iwanaga, Kenjiro [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Okinaga, Toshinori [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Habu, Manabu [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Yoshioka, Izumi [Division of Oral and Maxillofacial Surgery, Department of Medicine of Sensory and Motor Organs, University of Miyazaki, Kiyotake, Miyazaki 889-1692 (Japan); Tominaga, Kazuhiro [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Nishihara, Tatsuji, E-mail: tatsujin@kyu-dent.ac.jp [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, Kitakyushu