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Sample records for differentiating mc3t3-e1 cells

  1. 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.

  2. 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.

  3. 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

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

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    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.

  5. 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.

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

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    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.

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

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    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.

  8. 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

  9. 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.

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

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    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

  11. 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

  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

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

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    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.

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

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    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.

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

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    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.

  16. 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

  17. 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

  18. Divalent Metal Ions Induced Osteogenic Differentiation of MC3T3E1

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    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.

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

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    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.

  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. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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)

  6. 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...

  7. 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.

  8. 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

  9. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. 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.

  12. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

  15. [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.

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

    Directory of Open Access Journals (Sweden)

    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.

  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. 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.

  19. 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. 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.

  3. 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.

  4. 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

  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. 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

  7. 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.

  8. 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. [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.

  6. 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.

  7. [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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

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

    International Nuclear Information System (INIS)

    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.

  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. 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.

  4. 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

  5. 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.

  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. 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

  8. 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.

  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. 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.

  11. 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...

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. The promising application of graphene oxide as coating materials in orthopedic implants: preparation, characterization and cell behavior

    International Nuclear Information System (INIS)

    Zhao, Changhong; Lu, Xiuzhen; Liu, Johan; Zanden, Carl

    2015-01-01

    To investigate the potential application of graphene oxide (GO) in bone repair, this study is focused on the preparation, characterization and cell behavior of graphene oxide coatings on quartz substrata. GO coatings were prepared on the substrata using a modified dip-coating procedure. Atomic force microscopy (AFM), scanning electron microscopy (SEM) and Raman spectroscopy results demonstrated that the as-prepared coatings in this study were homogeneous and had an average thickness of ∼67 nm. The rapid formation of a hydroxyapatite (HA) layer in the simulated body fluid (SBF) on GO coated substrata at day 14, as proved by SEM and x-ray diffraction (XRD), strongly indicated the bioactivity of coated substrata. In addition, MC3T3-E1 cells were cultured on the coated substrata to evaluate cellular activities. Compared with the non-coated substrata and tissue culture plates, no significant difference was observed on the coated substrata in terms of cytotoxicity, viability, proliferation and apoptosis. However, interestingly, higher levels of alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion were observed on the coated substrata, indicating that GO coatings enhanced cell differentiation compared with non-coated substrata and tissue culture plates. This study suggests that GO coatings had excellent biocompatibility and more importantly promoted MC3T3-E1 cell differentiation and might be a good candidate as a coating material for orthopedic implants. (paper)

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. Preparation and Evaluation of Dexamethasone (DEX/Growth and Differentiation Factor-5 (GDF-5 Surface-Modified Titanium Using β-Cyclodextrin-Conjugated Heparin (CD-Hep for Enhanced Osteogenic Activity In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Dae Hyeok Yang

    2017-08-01

    Full Text Available The most ideal implant models in the dental and orthopedic fields to minimize the failure rate of implantation involve the improvement of osseointegration with host bone. Therefore, a focus of this study is the preparation of surface-modified titanium (Ti samples of disc and screw types using dexamethasone (DEX and/or growth and differentiation factor-5 (GDF-5, as well as the evaluation of their efficacies on bone formation in vitro and in vivo. X-ray photoelectron spectroscopy (XPS, scanning electron microscopy (SEM and contact angle measurement were used to evaluate the surface chemical composition, surface morphology and wettability, respectively. The results showed that implant surfaces were successfully modified with DEX and/or GDF-5, and had rough surfaces along with hydrophilicity. DEX, GDF-5 or DEX/GDF-5 on the surface-modified samples were rapidly released within one day and released for 28 days in a sustained manner. The proliferation and bone formation of MC3T3-E1 cells cultured on pristine and surface-modified implants in vitro were examined by cell counting kit-8 (CCK-8 assay, as well as the measurements of alkaline phosphatase (ALP activity and calcium deposition, respectively. MC3T3-E1 cells cultured on DEX/GDF-5–Ti showed noticeable ALP activity and calcium deposition in vitro. Active bone formation and strong osseointegration occurred at the interface between DEX/GDF-5–Ti and host bone, as evaluated by micro computed-tomography (micro CT analysis. Surface modification using DEX/GDF-5 could be a good method for advanced implants for orthopaedic and dental applications.

  9. Exogenous nitric oxide (NO) generated by NO-plasma treatment modulates osteoprogenitor cells early differentiation

    International Nuclear Information System (INIS)

    Elsaadany, Mostafa; Subramanian, Gayathri; Ayan, Halim; Yildirim-Ayan, Eda

    2015-01-01

    In this study, we investigated whether nitric oxide (NO) generated using a non-thermal plasma system can mediate osteoblastic differentiation of osteoprogenitor cells without creating toxicity. Our objective was to create an NO delivery mechanism using NO-dielectric barrier discharge (DBD) plasma that can generate and transport NO with controlled concentration to the area of interest to regulate osteoprogenitor cell activity. We built a non-thermal atmospheric pressure DBD plasma nozzle system based on our previously published design and similar designs in the literature. The electrical and spectral analyses demonstrated that N 2 dissociated into NO under typical DBD voltage–current characteristics. We treated osteoprogenitor cells (MC3T3-E1) using NO-plasma treatment system. Our results demonstrated that we could control NO concentration within cell culture media and could introduce NO into the intracellular space using NO-plasma treatment with various treatment times. We confirmed that NO-plasma treatment maintained cell viability and did not create any toxicity even with prolonged treatment durations. Finally, we demonstrated that NO-plasma treatment induced early osteogenic differentiation in the absence of pro-osteogenic growth factors/proteins. These findings suggest that through the NO-plasma treatment system we are able to generate and transport tissue-specific amounts of NO to an area of interest to mediate osteoprogenitor cell activity without subsequent toxicity. This opens up the possibility to develop DBD plasma-assisted tissue-specific NO delivery strategies for therapeutic intervention in the prevention and treatment of bone diseases. (paper)

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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}.

  16. 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.

  17. 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.

  18. 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.

  19. A coupled diffusion-fluid pressure model to predict cell density distribution for cells encapsulated in a porous hydrogel scaffold under mechanical loading.

    Science.gov (United States)

    Zhao, Feihu; Vaughan, Ted J; Mc Garrigle, Myles J; McNamara, Laoise M

    2017-10-01

    Tissue formation within tissue engineering (TE) scaffolds is preceded by growth of the cells throughout the scaffold volume and attachment of cells to the scaffold substrate. It is known that mechanical stimulation, in the form of fluid perfusion or mechanical strain, enhances cell differentiation and overall tissue formation. However, due to the complex multi-physics environment of cells within TE scaffolds, cell transport under mechanical stimulation is not fully understood. Therefore, in this study, we have developed a coupled multiphysics model to predict cell density distribution in a TE scaffold. In this model, cell transport is modelled as a thermal conduction process, which is driven by the pore fluid pressure under applied loading. As a case study, the model is investigated to predict the cell density patterns of pre-osteoblasts MC3T3-e1 cells under a range of different loading regimes, to obtain an understanding of desirable mechanical stimulation that will enhance cell density distribution within TE scaffolds. The results of this study have demonstrated that fluid perfusion can result in a higher cell density in the scaffold region closed to the outlet, while cell density distribution under mechanical compression was similar with static condition. More importantly, the study provides a novel computational approach to predict cell distribution in TE scaffolds under mechanical loading. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. 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.

  1. Physicochemical properties of newly developed bioactive glass cement and its effects on various cells.

    Science.gov (United States)

    Washio, Ayako; Nakagawa, Aika; Nishihara, Tatsuji; Maeda, Hidefumi; Kitamura, Chiaki

    2015-02-01

    Biomaterials used in dental treatments are expected to have favorable properties such as biocompatibility and an ability to induce tissue formation in dental pulp and periapical tissue, as well as sealing to block external stimuli. Bioactive glasses have been applied in bone engineering, but rarely applied in the field of dentistry. In the present study, bioactive glass cement for dental treatment was developed, and then its physicochemical properties and effects on cell responses were analyzed. To clarify the physicochemical attributes of the cement, field emission scanning electron microscopy, X-ray diffraction, and pH measurement were carried out. Cell attachment, morphology, and viability to the cement were also examined to clarify the effects of the cement on odontoblast-like cells (KN-3 cells), osteoblastic cells (MC3T3-E1 cells), human periodontal ligament stem/progenitor cells and neuro-differentiative cells (PC-12 cells). Hydroxyapatite-like precipitation was formed on the surface of the hardened cement and the pH level changed from pH10 to pH9, then stabilized in simulate body fluid. The cement had no cytotxic effects on these cells, and particulary induced process elongation of PC-12 cells. Our results suggest that the newly developed bioactive glass cement have capability of the application in dental procedures as bioactive cement. © 2014 Wiley Periodicals, Inc.

  2. Plasma-Sprayed Titanium Patterns for Enhancing Early Cell Responses

    Science.gov (United States)

    Shi, Yunqi; Xie, Youtao; Pan, Houhua; Zheng, Xuebin; Huang, Liping; Ji, Fang; Li, Kai

    2016-06-01

    Titanium coating has been widely used as a biocompatible metal in biomedical applications. However, the early cell responses and long-term fixation of titanium implants are not satisfied. To obviate these defects, in this paper, micro-post arrays with various widths (150-1000 μm) and intervals (100-300 μm) were fabricated on the titanium substrate by template-assisted plasma spraying technology. In vitro cell culture experiments showed that MC3T3-E1 cells exhibited significantly higher osteogenic differentiation as well as slightly improved adhesion and proliferation on the micro-patterned coatings compared with the traditional one. The cell number on the pattern with 1000 µm width reached 130% after 6 days of incubation, and the expressions of osteopontin (OPN) as well as osteocalcin (OC) were doubled. No obvious difference was found in cell adhesion on various size patterns. The present micro-patterned coatings proposed a new modification method for the traditional plasma spraying technology to enhance the early cell responses and convenience for the bone in-growth.

  3. Electrically Reversible Redox-Switchable Polydopamine Films for Regulating Cell Behavior

    International Nuclear Information System (INIS)

    Tan, Guoxin; Liu, Yan; Wu, Yuxuan; Ouyang, Kongyou; Zhou, Lei; Yu, Peng; Liao, Jinwen; Ning, Chengyun

    2017-01-01

    Highlights: • The phenolic/quinone groups on polydopamine can redox-switchable reversible under electrical stimulation. • The quinone groups on PDA (oxidized PDA) enhanced cell spreading and proliferation. • The phenolic groups on PDA (reduced PDA) induced cell differentiation. - Abstract: Switchable surfaces that respond to external stimuli are important for regulating cell behavior. The results herein suggest that the redox process of polydopamine (PDA) is a switching reaction between oxidized polydopamine and reduced polydopamine, involving an interconversion of coupled two-proton (2H + ) and two-electron (2e − ) processes. The redox-switchable reversible surface potential arising from the potential-tunable redox reaction of the phenolic and quinone groups on PDA on titanium induced both cell adhesion and spreading. In vitro experiments demonstrated that the quinone groups on PDA greatly enhanced pre-osteoblasts MC3T3-E1 cell spreading and proliferation. Phenolic groups enhanced the induction of differentiation. The proposed methodology may allow further investigation of switchable surfaces for biological and medical applications.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Modified salicylanilide and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivatives as novel inhibitors of osteoclast differentiation and bone resorption.

    Science.gov (United States)

    Chen, Chun-Liang; Liu, Fei-Lan; Lee, Chia-Chung; Chen, Tsung-Chih; Ahmed Ali, Ahmed Atef; Sytwu, Huey-Kang; Chang, Deh-Ming; Huang, Hsu-Shan

    2014-10-09

    Inhibition of osteoclast formation is a potential strategy to prevent inflammatory bone resorption and to treat bone diseases. In the present work, the purpose was to discover modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivatives as potential antiosteoclastogenic agents. Their inhibitory effects on RANKL-induced osteoclastogenesis from RAW264.7 cells were evaluated by TRAP stain assay. The most potent compounds, 1d and 5d, suppressed RANKL-induced osteoclast formation and TRAP activity dose-dependently. The cytotoxicity assay on RAW264.7 cells suggested that the inhibition of osteoclastic bone resorption by these compounds did not result from their cytotoxicity. Moreover, both compounds downregulated RANKL-induced NF-κB and NFATc1 in the nucleus, suppressed the expression of osteoclastogenesis-related marker genes during osteoclastogenesis, and prevented osteoclastic bone resorption but did not impair osteoblast differentiation in MC3T3-E1. Therefore, these modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-diones could be potential lead compounds for the development of a new class of antiresorptive agents.

  9. Influences of doping mesoporous magnesium silicate on water absorption, drug release, degradability, apatite-mineralization and primary cells responses to calcium sulfate based bone cements

    International Nuclear Information System (INIS)

    Gu, Zhengrong; Wang, Sicheng; Weng, Weizong; Chen, Xiao; Cao, Liehu; Wei, Jie; Shin, Jung-Woog; Su, Jiacan

    2017-01-01

    In this study, composite cements containing mesoporous magnesium silicate (m-MS) and calcium sulfate (CS) were fabricated. The results revealed that the setting time of the m-MS/CS composite cements (m-MSC) slightly prolonged with the increase of m-MS content while the compressive strength suffered a little loss. The doping of m-MS improved the water absorption, drug release (vancomycin) and degradability of the m-MSC in Tris-HCl solution (pH = 7.4). In addition, addition of m-MS facilitated the apatite-mineralization of m-MSC in simulated body fluid (SBF), indicating good bioactivity. For cell cultural experiments, the results revealed that the m-MSC promoted the cells adhesion and proliferation, and improved the alkaline phosphatase (ALP) activity of MC3T3-E1 cells, revealing good cytocompatibility. It could be suggested that the m-MSC might be promising cements biomaterials for bone tissue regeneration. - Highlights: • The mesoporous magnesium silicate and calcium sulfate composite was fabricated. • The composite possessed good water absorption and drug release of vancomycin. • The bioactive composite could enhance the in vivo apatite formation in SBF. • The composite promoted cell adhesion, proliferation and osteogenic differentiation.

  10. Influences of doping mesoporous magnesium silicate on water absorption, drug release, degradability, apatite-mineralization and primary cells responses to calcium sulfate based bone cements

    Energy Technology Data Exchange (ETDEWEB)

    Gu, Zhengrong [Department of Trauma Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); The Department of Orthopaedics, Jing' an District Centre Hospital of Shanghai (Huashan Hospital Fudan University Jing' An Branch), 200040 (China); Wang, Sicheng [Department of Trauma Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Department of Orthopaedics, Zhongye Hospital, Shanghai 200941 (China); Weng, Weizong; Chen, Xiao; Cao, Liehu [Department of Trauma Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Wei, Jie [Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Shin, Jung-Woog [Department of Biomedical Engineering, Inje University, Gimhae, 621749 (Korea, Republic of); Su, Jiacan, E-mail: jiacansu@sina.com [Department of Trauma Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China)

    2017-06-01

    In this study, composite cements containing mesoporous magnesium silicate (m-MS) and calcium sulfate (CS) were fabricated. The results revealed that the setting time of the m-MS/CS composite cements (m-MSC) slightly prolonged with the increase of m-MS content while the compressive strength suffered a little loss. The doping of m-MS improved the water absorption, drug release (vancomycin) and degradability of the m-MSC in Tris-HCl solution (pH = 7.4). In addition, addition of m-MS facilitated the apatite-mineralization of m-MSC in simulated body fluid (SBF), indicating good bioactivity. For cell cultural experiments, the results revealed that the m-MSC promoted the cells adhesion and proliferation, and improved the alkaline phosphatase (ALP) activity of MC3T3-E1 cells, revealing good cytocompatibility. It could be suggested that the m-MSC might be promising cements biomaterials for bone tissue regeneration. - Highlights: • The mesoporous magnesium silicate and calcium sulfate composite was fabricated. • The composite possessed good water absorption and drug release of vancomycin. • The bioactive composite could enhance the in vivo apatite formation in SBF. • The composite promoted cell adhesion, proliferation and osteogenic differentiation.

  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. 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

  15. 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

  16. 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.

  17. 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...

  18. 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

  19. Effect of plasma surface functionalization on preosteoblast cells spreading and adhesion on a biomimetic hydroxyapatite layer formed on a titanium surface

    International Nuclear Information System (INIS)

    Myung, Sung Woon; Ko, Yeong Mu; Kim, Byung Hoon

    2013-01-01

    This study examined the plasma surface modification of biomimetic hydroxyapatite (HAp) formed on a titanium (Ti) surface as well as its influence on the behavior of preosteoblast cells. Ti substrates pre-treated with a plasma-polymerized thin film rich in carboxyl groups were subjected to a biomimetic process in a simulated body fluid solution to synthesize the HAp. The HAp layer grown on Ti substrate was then coated with two types of plasma polymerized acrylic acid and allyl amine thin film. The different types of Ti substrates were characterized by attenuated total reflection Fourier transform infrared spectroscopy, energy dispersive spectroscopy and X-ray diffraction. HAp with a Ca/P ratio from 1.25 to 1.38 was obtained on the Ti substrate and hydrophilic carboxyl (-COOH) and amine (-NH 2 ) functional groups were introduced to its surface. Scanning electron microscopy was used to observe the surface of the HAp coatings and the morphology of MC3T3-E1 cells. These results showed that the -COOH-modified HAp surfaces promoted the cell spreading synergistically by changing the surface morphology and chemical state.-NH 2 modified HAp had the lowest cell spreading and proliferation compared to HAp and -COOH-modified HAp. These results correspond to fluorescein analysis, which showed many more cell spreading of COOH/HAp/Ti surface compared to HAp and NH 2 modified HAp. A MTT assay was used to evaluate cell proliferation. The results showed that the proliferation of MC3T3-E1 cells increased in the order of COOH/HAp/Ti > HAp/Ti > NH 2 /Ti > Ti, corresponding to the effect of cell spreading for 6 days. The change in morphology and the chemical surface properties of the biomaterial via plasma polymerization can affect the behavior of MC3T3-E1 cells.

  20. Effect of plasma surface functionalization on preosteoblast cells spreading and adhesion on a biomimetic hydroxyapatite layer formed on a titanium surface

    Energy Technology Data Exchange (ETDEWEB)

    Myung, Sung Woon; Ko, Yeong Mu; Kim, Byung Hoon, E-mail: kim5055@chosun.ac.kr

    2013-12-15

    This study examined the plasma surface modification of biomimetic hydroxyapatite (HAp) formed on a titanium (Ti) surface as well as its influence on the behavior of preosteoblast cells. Ti substrates pre-treated with a plasma-polymerized thin film rich in carboxyl groups were subjected to a biomimetic process in a simulated body fluid solution to synthesize the HAp. The HAp layer grown on Ti substrate was then coated with two types of plasma polymerized acrylic acid and allyl amine thin film. The different types of Ti substrates were characterized by attenuated total reflection Fourier transform infrared spectroscopy, energy dispersive spectroscopy and X-ray diffraction. HAp with a Ca/P ratio from 1.25 to 1.38 was obtained on the Ti substrate and hydrophilic carboxyl (-COOH) and amine (-NH{sub 2}) functional groups were introduced to its surface. Scanning electron microscopy was used to observe the surface of the HAp coatings and the morphology of MC3T3-E1 cells. These results showed that the -COOH-modified HAp surfaces promoted the cell spreading synergistically by changing the surface morphology and chemical state.-NH{sub 2} modified HAp had the lowest cell spreading and proliferation compared to HAp and -COOH-modified HAp. These results correspond to fluorescein analysis, which showed many more cell spreading of COOH/HAp/Ti surface compared to HAp and NH{sub 2} modified HAp. A MTT assay was used to evaluate cell proliferation. The results showed that the proliferation of MC3T3-E1 cells increased in the order of COOH/HAp/Ti > HAp/Ti > NH{sub 2}/Ti > Ti, corresponding to the effect of cell spreading for 6 days. The change in morphology and the chemical surface properties of the biomaterial via plasma polymerization can affect the behavior of MC3T3-E1 cells.

  1. 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.

  2. Methionine restriction alters bone morphology and affects osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    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

  3. 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.

  4. 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

  5. Cell cytoskeletal changes effected by static compressive stress lead to changes in the contractile properties of tissue regenerative collagen membranes

    Directory of Open Access Journals (Sweden)

    K Gellynck

    2013-06-01

    Full Text Available Static compressive stress can influence the matrix, which subsequently affects cell behaviour and the cell’s ability to further transform the matrix. This study aimed to assess response to static compressive stress at different stages of osteoblast differentiation and assess the cell cytoskeleton’s role as a conduit of matrix-derived stimuli. Mouse bone marrow mesenchymal stem cells (MSCs (D1 ORL UVA, osteoblastic cells (MC3T3-E1 and post-osteoblast/pre-osteocyte-like cells (MLO-A5 were seeded in hydrated and compressed collagen gels. Contraction was quantified macroscopically, and cell morphology, survival, differentiation and mineralisation assessed using confocal microscopy, alamarBlue® assay, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR and histological stains, respectively. Confocal microscopy demonstrated cell shape changes and favourable microfilament organisation with static compressive stress of the collagen matrix; furthermore, cell survival was greater compared to the hydrated gels. The stage of osteoblast differentiation determined the degree of matrix contraction, with MSCs demonstrating the greatest amount. Introduction of microfilament disrupting inhibitors confirmed that pre-stress and tensegrity forces were under the influence of gel density, and there was increased survival and differentiation of the cells within the compressed collagen compared to the hydrated collagen. There was also relative stiffening and differentiation with time of the compressed cell-seeded collagen, allowing for greater manipulation. In conclusion, the combined collagen chemistry and increased density of the microenvironment can promote upregulation of osteogenic genes and mineralisation; MSCs can facilitate matrix contraction to form an engineered membrane with the potential to serve as a ‘pseudo-periosteum’ in the regeneration of bone defects.

  6. 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.

  7. 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

  8. Cellular attachment and differentiation on titania nanotubes exposed to air- or nitrogen-based non-thermal atmospheric pressure plasma.

    Directory of Open Access Journals (Sweden)

    Hye Yeon Seo

    Full Text Available The surface topography and chemistry of titanium implants are important factors for successful osseointegration. However, chemical modification of an implant surface using currently available methods often results in the disruption of topographical features and the loss of beneficial effects during the shelf life of the implant. Therefore, the aim of this study was to apply the recently highlighted portable non-thermal atmospheric pressure plasma jet (NTAPPJ, elicited from one of two different gas sources (nitrogen and air, to TiO2 nanotube surfaces to further improve their osteogenic properties while preserving the topographical morphology. The surface treatment was performed before implantation to avoid age-related decay. The surface chemistry and morphology of the TiO2 nanotube surfaces before and after the NTAPPJ treatment were determined using a field-emission scanning electron microscope, a surface profiler, a contact angle goniometer, and an X-ray photoelectron spectroscope. The MC3T3-E1 cell viability, attachment and morphology were confirmed using calcein AM and ethidium homodimer-1 staining, and analysis of gene expression using rat mesenchymal stem cells was performed using a real-time reverse-transcription polymerase chain reaction. The results indicated that both portable nitrogen- and air-based NTAPPJ could be used on TiO2 nanotube surfaces easily and without topographical disruption. NTAPPJ resulted in a significant increase in the hydrophilicity of the surfaces as well as changes in the surface chemistry, which consequently increased the cell viability, attachment and differentiation compared with the control samples. The nitrogen-based NTAPPJ treatment group exhibited a higher osteogenic gene expression level than the air-based NTAPPJ treatment group due to the lower atomic percentage of carbon on the surface that resulted from treatment. It was concluded that NTAPPJ treatment of TiO2 nanotube surfaces results in an increase in

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. Production of Composite Scaffold Containing Silk Fibroin, Chitosan, and Gelatin for 3D Cell Culture and Bone Tissue Regeneration.

    Science.gov (United States)

    Li, Jianqing; Wang, Qiuke; Gu, Yebo; Zhu, Yu; Chen, Liang; Chen, Yunfeng

    2017-11-08

    BACKGROUND Bone tissue engineering, a powerful tool to treat bone defects, is highly dependent on use of scaffolds. Both silk fibroin (SF) and chitosan (Cs) are biocompatible and actively studied for reconstruction of tissue engineering. Gelatin (Gel) is also widely applied in the biomedical field due to its low antigenicity and physicochemical stability. MATERIAL AND METHODS In this study, 4 different types of scaffolds were constructed - SF, SF/Cs, SF/Gel, and SF/Cs/Gel - and we compared their physical and chemical properties as well as biological characterization of these scaffolds to determine the most suitable scaffold for use in bone regeneration. First, these scaffolds were produced via chemical cross-linking method and freeze-drying technique. Next, the characterization of internal structure was studied using scanning electron microscopy and the porosity was evaluated by liquid displacement method. Then, we compared physicochemical properties such as water absorption rate and degradation property. Finally, MC3T3-E1 cells were inoculated on the scaffolds to study the biocompatibility and osteogenesis of the three-dimensional (3D) scaffolds in vitro. RESULTS The composite scaffold formed by all 3 components was the best for use in bone regeneration. CONCLUSIONS We conclude that the best scaffold among the 4 studied for MC3T3-E1 cells is our SF/Cs/Gel scaffold, suggesting a new choice for bone regeneration that can be used to treat bone defects or fractures in clinical practice.

  15. 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

  16. 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.

  17. Surface potential-governed cellular osteogenic differentiation on ferroelectric polyvinylidene fluoride trifluoroethylene films.

    Science.gov (United States)

    Tang, Bolin; Zhang, Bo; Zhuang, Junjun; Wang, Qi; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

    2018-05-02

    Surface potential of biomaterials can dramatically influence cellular osteogenic differentiation. In this work, a wide range of surface potential on ferroelectric polyvinylidene fluoride trifluoroethylene (P(VDF-TrFE)) films was designed to get insight into the interfacial interaction of cell-charged surface. The P(VDF-TrFE) films poled by contact electric poling at various electric fields obtained well stabilized surface potential, with wide range from -3 to 915 mV. The osteogenic differentiation level of cells cultured on the films was strongly dependent on surface potential and reached the optimum at 391 mV in this system. Binding specificity assay indicated that surface potential could effectively govern the binding state of the adsorbed fibronectin (FN) with integrin. Molecular dynamic (MD) simulation further revealed that surface potential brought a significant difference in the relative distance between RGD and synergy PHSRN sites of adsorbed FN, resulting in a distinct integrin-FN binding state. These results suggest that the full binding of integrin α5β1 with both RGD and PHSRN sites of FN possesses a strong ability to activate osteogenic signaling pathway. This work sheds light on the underlying mechanism of osteogenic differentiation behavior on charged material surfaces, and also provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. The ferroelectric P(VDF-TrFE) films with steady and a wide range of surface potential were designed to understand underlying mechanism of cell-charged surface interaction. The results showed that the charged surface well favored upregulation of osteogenic differentiation of MC3T3-E1 cells, and more importantly, a highest level occurred on the film with a moderate surface potential. Experiments and molecular dynamics simulation demonstrated that the surface potential could govern fibronectin conformation and then the integrin-fibronectin binding. We propose that a full binding

  18. 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)

  19. 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)

  20. Spatial and temporal changes in the morphology of preosteoblastic cells seeded on microstructured tantalum surfaces

    DEFF Research Database (Denmark)

    Justesen, Jørn; Lorentzen, M.; Andersen, L. K.

    2009-01-01

    It has been widely reported that surface morphology on the micrometer scale affects cell function as well as cell shape. In this study, we have systematically compared the influence of 13 topographically micropatterned tantalum surfaces on the temporal development of morphology, including spreading......, and length of preosteoblastic cells (MC3T3-E1). Cells were examined after 0.5, 1, 4, and 24 h on different Ta microstructures with vertical dimensions (heights) of 0.25 and 1.6 mu m. Cell morphologies depended upon the underlying Surface topography, and the length and spreading of cells varied as a function...... to depend on the distance between the pillars with one specific pillar Structure exhibiting a decreased spreading combined with a radical change in morphology of the cells. Interestingly, this morphology on the particular pillar structure was associated with a markedly different distribution of the actio...

  1. 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

  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. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  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. Local cell metrics: a novel method for analysis of cell-cell interactions

    Directory of Open Access Journals (Sweden)

    Chen Chien-Chiang

    2009-10-01

    Full Text Available Abstract Background 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. Results 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. Conclusion 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

  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. Criticality in cell differentiation

    Indian Academy of Sciences (India)

    Indrani Bose

    2017-11-09

    Nov 9, 2017 ... Differentiation is mostly based on binary decisions with the progenitor cells ..... accounts for the dominant part of the remaining variation ... significant loss in information. ..... making in vitro: emerging concepts and novel tools.

  3. 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...

  4. 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.

  5. Enhanced attachment and growth of periodontal cells on glycine-arginine-glycine-aspartic modified chitosan membranes

    Directory of Open Access Journals (Sweden)

    Hsiao-Pei Tu

    2016-01-01

    Full Text Available Background: Chitosan, a polymeric carbohydrate derived from the exoskeleton of arthropod, has been suggested to be an excellent biomaterial for improving wound healing, especially for bones. To improve the periodontal cell attachment and growth, the cell adhesive peptide glycine-arginine-glycine-aspartic acid (Gly-Arg-Gly-Asp, GRGD grafted chitosan membrane was introduced in this study. Materials and Methods: Two types of commercial chitosan, three types of primary cultured cells, and two established cell lines were used. Human gingival and periodontal fibroblasts (hGF and hPDL, human root derived cell (hRDC, and rat calvaria bone cell (rCalB were cultured on the GRGD-fixed by ultraviolet light photochemical method on the chitosan membrane. With (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium assay and propidium iodine (PI staining, the cell adhesion and growth on GRGD-grafted chitosan were examined. Basal mRNA expressions of the receptors for GRGD, integrin αv (ITG αv and ITG β3, in the human gingival fibroblast cell line and mouse osteoblast cell line (MC3T3-E1 were examined with real-time polymerase chain reaction. Results: Because the cell adhesion/growth patterns on two chitosan membranes were similar, the GRGD modification was performed on one membrane (Primex only. For periodontal cells (hGFs, hPDLs, and hRDCs, the number of attached cells were increased on the membrane with the high concentration of GRGD than those on the membrane unmodified or modified with low concentration GRGD. For rCalBs cells, a different pattern was noted: GRGD modification did not enhance the calvaria cells attachment or growth. Moreover, mRNA expressions of ITG αv and β3 in AG09319 cells were significantly higher than those in MC3T3-E1 cells. Conclusions: With the limitation of this study, we suggested that GRGD-modified chitosan, especially at high concentration, could enhance the growth of various periodontal

  6. 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.

  7. 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.

  8. 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.

  9. 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....

  10. 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)

  11. 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.

  12. 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.

  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. 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

  15. Effects of RGD immobilization on light-induced cell sheet detachment from TiO{sub 2} nanodots films

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Kui; Wang, Tiantian [School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, Zhejiang University, Hangzhou 310027 (China); Yu, Mengliu [The Affiliated Stomatologic Hospital, Zhejiang University, Hangzhou 310003 (China); The First Affiliated Hospital of Medical College, Zhejiang University, Hangzhou, 310003 (China); Wan, Hongping [School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, Zhejiang University, Hangzhou 310027 (China); Lin, Jun [The First Affiliated Hospital of Medical College, Zhejiang University, Hangzhou, 310003 (China); Weng, Wenjian, E-mail: wengwj@zju.edu.cn [School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, Zhejiang University, Hangzhou 310027 (China); The Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050 (China); Wang, Huiming, E-mail: hmwang1960@hotmail.com [The Affiliated Stomatologic Hospital, Zhejiang University, Hangzhou 310003 (China); The First Affiliated Hospital of Medical College, Zhejiang University, Hangzhou, 310003 (China)

    2016-06-01

    Light-induced cell detachment is reported to be a safe and effective cell sheet harvest method. In the present study, the effects of arginine–glycine–aspartic acid (RGD) immobilization on cell growth, cell sheet construction and cell harvest through light illumination are investigated. RGD was first immobilized on TiO{sub 2} nanodots films through simple physical adsorption, and then mouse pre-osteoblastic MC3T3-E1 cells were seeded on the films. It was found that RGD immobilization promoted cell adhesion and proliferation. It was also observed that cells cultured on RGD immobilized films showed relatively high level of pan-cadherin. Cells harvested with ultraviolet illumination (365 nm) showed good viability on both RGD immobilized and unmodified TiO{sub 2} nanodot films. Single cell detachment assay showed that cells detached more quickly on RGD immobilized TiO{sub 2} nanodot films. That could be ascribed to the RGD release after UV365 illumination. The current study demonstrated that RGD immobilization could effectively improve both the cellular responses and light-induced cell harvest. - Highlights: • RGD immobilization on TiO{sub 2} nanodots film favors light-induced cell sheet detachment. • Physically adsorbed RGD detaches from the film through ultraviolet illumination. • RGD detachment promotes cells and cell sheets detachment.

  16. Micro/Nano Multilayered Scaffolds of PLGA and Collagen by Alternately Electrospinning for Bone Tissue Engineering

    Science.gov (United States)

    Kwak, Sanghwa; Haider, Adnan; Gupta, Kailash Chandra; Kim, Sukyoung; Kang, Inn-Kyu

    2016-07-01

    The dual extrusion electrospinning technique was used to fabricate multilayered 3D scaffolds by stacking microfibrous meshes of poly(lactic acid-co-glycolic acid) (PLGA) in alternate fashion to micro/nano mixed fibrous meshes of PLGA and collagen. To fabricate the multilayered scaffold, 35 wt% solution of PLGA in THF-DMF binary solvent (3:1) and 5 wt% solution of collagen in hexafluoroisopropanol (HFIP) with and without hydroxyapatite nanorods (nHA) were used. The dual and individual electrospinning of PLGA and collagen were carried out at flow rates of 1.0 and 0.5 mL/h, respectively, at an applied voltage of 20 kV. The density of collagen fibers in multilayered scaffolds has controlled the adhesion, proliferation, and osteogenic differentiation of MC3T3-E1 cells. The homogeneous dispersion of glutamic acid-modified hydroxyapatite nanorods (nHA-GA) in collagen solution has improved the osteogenic properties of fabricated multilayered scaffolds. The fabricated multilayered scaffolds were characterized using FT-IR, X-ray photoelectron spectroscopy, and transmission electron microscopy (TEM). The scanning electron microscopy (FE-SEM) was used to evaluate the adhesion and spreads of MC3T3-E1 cells on multilayered scaffolds. The activity of MC3T3-E1 cells on the multilayered scaffolds was evaluated by applying MTT, alkaline phosphatase, Alizarin Red, von Kossa, and cytoskeleton F-actin assaying protocols. The micro/nano fibrous PLGA-Col-HA scaffolds were found to be highly bioactive in comparison to pristine microfibrous PLGA and micro/nano mixed fibrous PLGA and Col scaffolds.

  17. 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...

  18. Theoretical modeling of mechanical homeostasis of a mammalian cell under gravity-directed vector.

    Science.gov (United States)

    Zhou, Lüwen; Zhang, Chen; Zhang, Fan; Lü, Shouqin; Sun, Shujin; Lü, Dongyuan; Long, Mian

    2018-02-01

    Translocation of dense nucleus along gravity vector initiates mechanical remodeling of a eukaryotic cell. In our previous experiments, we quantified the impact of gravity vector on cell remodeling by placing an MC3T3-E1 cell onto upward (U)-, downward (D)-, or edge-on (E)- orientated substrate. Our experimental data demonstrate that orientation dependence of nucleus longitudinal translocation is positively correlated with cytoskeletal (CSK) remodeling of their expressions and structures and also is associated with rearrangement of focal adhesion complex (FAC). However, the underlying mechanism how CSK network and FACs are reorganized in a mammalian cell remains unclear. In this paper, we developed a theoretical biomechanical model to integrate the mechanosensing of nucleus translocation with CSK remodeling and FAC reorganization induced by a gravity vector. The cell was simplified as a nucleated tensegrity structure in the model. The cell and CSK filaments were considered to be symmetrical. All elements of CSK filaments and cytomembrane that support the nucleus were simplified as springs. FACs were simplified as an adhesion cluster of parallel bonds with shared force. Our model proposed that gravity vector-directed translocation of the cell nucleus is mechanically balanced by CSK remodeling and FAC reorganization induced by a gravitational force. Under gravity, dense nucleus tends to translocate and exert additional compressive or stretching force on the cytoskeleton. Finally, changes of the tension force acting on talin by microfilament alter the size of FACs. Results from our model are in qualitative agreement with those from experiments.

  19. 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.

  20. 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)

  1. 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.

  2. Membrane-Dependent Bystander Effect Contributes to Amplification of the Response to Alpha-Particle Irradiation in Targeted and Nontargeted Cells

    International Nuclear Information System (INIS)

    Hanot, Maite; Hoarau, Jim; Carriere, Marie; Angulo, Jaime F.; Khodja, Hicham

    2009-01-01

    Purpose: Free radicals are believed to play an active role in the bystander response. This study investigated their origin as well as their temporal and spatial impacts in the bystander effect. Methods and Materials: We employed a precise alpha-particle microbeam to target a small fraction of subconfluent osteoblastic cells (MC3T3-E1). γH2AX-53BP1 foci, oxidative metabolism changes, and micronuclei induction in targeted and bystander cells were assessed. Results: Cellular membranes and mitochondria were identified as two distinct reactive oxygen species producers. The global oxidative stress observed after irradiation was significantly attenuated after cells were treated with filipin, evidence for the primal role of membrane in the bystander effect. To determine the membrane's impact at a cellular level, micronuclei yield was measured when various fractions of the cell population were individually targeted while the dose per cell remained constant. Induction of micronuclei increased in bystander cells as well as in targeted cells and was attenuated by filipin treatment, demonstrating a role for bystander signals between irradiated cells in an autocrine/paracrine manner. Conclusions: A complex interaction of direct irradiation and bystander signals leads to a membrane-dependent amplification of cell responses that could influence therapeutic outcomes in tissues exposed to low doses or to environmental exposure.

  3. Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering.

    Science.gov (United States)

    Lee, Sang Jin; Lee, Donghyun; Yoon, Taek Rim; Kim, Hyung Keun; Jo, Ha Hyeon; Park, Ji Sun; Lee, Jun Hee; Kim, Wan Doo; Kwon, Il Keun; Park, Su A

    2016-08-01

    For tissue engineering, a bio-porous scaffold which is applied to bone-tissue regeneration should provide the hydrophilicity for cell attachment as well as provide for the capability to bind a bioactive molecule such as a growth factor in order to improve cell differentiation. In this work, we prepared a three-dimensional (3D) printed polycaprolactone scaffold (PCLS) grafted with recombinant human bone morphogenic protein-2 (rhBMP2) attached via polydopamine (DOPA) chemistry. The DOPA coated PCL scaffold was characterized by contact angle, water uptake, and X-ray photoelectron spectroscopy (XPS) in order to certify that the surface was successfully coated with DOPA. In order to test the loading and release of rhBMP2, we examined the release rate for 28days. For the In vitro cell study, pre-osteoblast MC3T3-E1 cells were seeded onto PCL scaffolds (PCLSs), DOPA coated PCL scaffold (PCLSD), and scaffolds with varying concentrations of rhBMP2 grafted onto the PCLSD 100 and PCLSD 500 (100 and 500ng/ml loaded), respectively. These scaffolds were evaluated by cell proliferation, alkaline phosphatase activity, and real time polymerase chain reaction with immunochemistry in order to verify their osteogenic activity. Through these studies, we demonstrated that our fabricated scaffolds were well coated with DOPA as well as grafted with rhBMP2 at a quantity of 22.7±5ng when treatment with 100ng/ml rhBMP2 and 153.3±2.4ng when treated with 500ng/ml rhBMP2. This grafting enables rhBMP2 to be released in a sustained pattern. In the in vitro results, the cell proliferation and an osteoconductivity of PCLSD 500 groups was greater than any other group. All of these results suggest that our manufactured 3D printed porous scaffold would be a useful construct for application to the bone tissue engineering field. Tissue-engineered scaffolds are not only extremely complex and cumbersome, but also use organic solvents which can negatively influence cellular function. Thus, a rapid

  4. 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.

  5. Fibronectin adsorption and cell response on electroactive poly(vinylidene fluoride) films

    International Nuclear Information System (INIS)

    Ribeiro, C; Panadero, J A; Sencadas, V; Lanceros-Méndez, S; Tamaño, M N; Moratal, D; Salmerón-Sánchez, M; Gómez Ribelles, J L

    2012-01-01

    Due to the large potential of electroactive materials in novel tissue engineering strategies, the aim of this work is to determine if the crystalline phase and/or the surface electrical charge of electroactive poly(vinylidene fluoride), PVDF, have influence on the biological response in monolayer cell culture. Non-polar α-PVDF and electroactive β-PVDF were prepared. The β-PVDF films were poled by corona discharge to show negative or positive electrical surface charge density. It has been concluded that hydrophilicity of the PVDF substrates depends significantly on crystalline phase and polarity. Furthermore, by means of atomic force microscopy and an enzyme-linked immunosorbent assay test, it has been shown that positive or negative poling strongly influences the behavior of β-PVDF supports with respect to fibronectin (FN) adsorption, varying the exhibition of adhesion ligands of adsorbed FN. Culture of MC3T3-E1 pre-osteoeblasts proved that cell proliferation depends on surface polarity as well. These results open the viability of cell culture stimulation by mechanical deformation of a piezoelectric substrate that results in varying electrical charge densities on the substrate surface. (paper)

  6. Mitochondria in aging cell differentiation

    Czech Academy of Sciences Publication Activity Database

    Palková, Zdena; Váchová, Libuše

    2016-01-01

    Roč. 8, č. 7 (2016), s. 1287-1288 ISSN 1945-4589 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : mitochondria * cell differentiation * retrograde signaling Subject RIV: EE - Microbiology, Virology Impact factor: 4.867, year: 2016

  7. Cell-Adhesive Bioinspired and Catechol-Based Multilayer Freestanding Membranes for Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Maria P. Sousa

    2017-10-01

    Full Text Available Mussels are marine organisms that have been mimicked due to their exceptional adhesive properties to all kind of surfaces, including rocks, under wet conditions. The proteins present on the mussel’s foot contain 3,4-dihydroxy-l-alanine (DOPA, an amino acid from the catechol family that has been reported by their adhesive character. Therefore, we synthesized a mussel-inspired conjugated polymer, modifying the backbone of hyaluronic acid with dopamine by carbodiimide chemistry. Ultraviolet–visible (UV–Vis spectroscopy and nuclear magnetic resonance (NMR techniques confirmed the success of this modification. Different techniques have been reported to produce two-dimensional (2D or three-dimensional (3D systems capable to support cells and tissue regeneration; among others, multilayer systems allow the construction of hierarchical structures from nano- to macroscales. In this study, the layer-by-layer (LbL technique was used to produce freestanding multilayer membranes made uniquely of chitosan and dopamine-modified hyaluronic acid (HA-DN. The electrostatic interactions were found to be the main forces involved in the film construction. The surface morphology, chemistry, and mechanical properties of the freestanding membranes were characterized, confirming the enhancement of the adhesive properties in the presence of HA-DN. The MC3T3-E1 cell line was cultured on the surface of the membranes, demonstrating the potential of these freestanding multilayer systems to be used for bone 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. Regulators of Tfh cell differentiation

    Directory of Open Access Journals (Sweden)

    Gajendra Motiram Jogdand

    2016-11-01

    Full Text Available The follicular helper T (Tfh cells help is critical for activation of B cells, antibody class switching and germinal center formation. The Tfh cells are characterized by the expression of CXCR5, ICOS, PD-1, Bcl-6, and IL-21. They are involved in clearing infections and are adversely linked with autoimmune diseases and also have a role in viral replication as well as clearance. Tfh cells are generated from naïve CD4 T cells with sequential steps involving cytokine signaling (IL-21, IL-6, IL-12, activin A, migration and positioning in the germinal center by CXCR5, surface receptors (ICOS/ICOSL, SAP/SLAM as well as transcription factor (Bcl-6, c-Maf, STAT3 signaling and repressor miR155. On the other hand Tfh generation is negatively regulated at specific steps of Tfh generation by specific cytokine (IL-2, IL-7, surface receptor (PD-1, CTLA-4, transcription factors Blimp-1, STAT5, T-bet, KLF-2 signaling and repressor miR 146a. Interestingly, miR 17-92 and FOXO1 acts as a positive as well as a negative regulator of Tfh differentiation depending on the time of expression and disease specificity. Tfh cells are also generated from the conversion of other effector T cells as exemplified by Th1 cells converting into Tfh during viral infection. The mechanistic details of effector T cells conversion into Tfh are yet to be clear. To manipulate Tfh cells for therapeutic implication and or for effective vaccination strategies, it is important to know positive and negative regulators of Tfh generation. Hence, in this review we have highlighted and interlinked molecular signaling from cytokines, surface receptors, transcription factors, ubiquitin Ligase and miRNA as positive and negative regulators for Tfh differentiation.

  10. Effects of Neuropeptides and Mechanical Loading on Bone Cell Resorption in Vitro

    Directory of Open Access Journals (Sweden)

    Yeong-Min Yoo

    2014-04-01

    Full Text Available Neuropeptides such as vasoactive intestinal peptide (VIP and calcitonin gene-related peptide (CGRP are present in nerve fibers of bone tissues and have been suggested to potentially regulate bone remodeling. Oscillatory fluid flow (OFF-induced shear stress is a potent signal in mechanotransduction that is capable of regulating both anabolic and catabolic bone remodeling. However, the interaction between neuropeptides and mechanical induction in bone remodeling is poorly understood. In this study, we attempted to quantify the effects of combined neuropeptides and mechanical stimuli on mRNA and protein expression related to bone resorption. Neuropeptides (VIP or CGRP and/or OFF-induced shear stress were applied to MC3T3-E1 pre-osteoblastic cells and changes in receptor activator of nuclear factor kappa B (NF-κB ligand (RANKL and osteoprotegerin (OPG mRNA and protein levels were quantified. Neuropeptides and OFF-induced shear stress similarly decreased RANKL and increased OPG levels compared to control. Changes were not further enhanced with combined neuropeptides and OFF-induced shear stress. These results suggest that neuropeptides CGRP and VIP have an important role in suppressing bone resorptive activities through RANKL/OPG pathway, similar to mechanical loading.

  11. 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.

  12. A novel akermanite/poly (lactic-co-glycolic acid) porous composite scaffold fabricated via a solvent casting-particulate leaching method improved by solvent self-proliferating process.

    Science.gov (United States)

    Deng, Yao; Zhang, Mengjiao; Chen, Xianchun; Pu, Ximing; Liao, Xiaoming; Huang, Zhongbing; Yin, Guangfu

    2017-08-01

    Desirable scaffolds for tissue engineering should be biodegradable carriers to supply suitable microenvironments mimicked the extracellular matrices for desired cellular interactions and to provide supports for the formation of new tissues. In this work, a kind of slightly soluble bioactive ceramic akermanite (AKT) powders were aboratively selected and introduced in the PLGA matrix, a novel l-lactide modified AKT/poly (lactic- co -glycolic acid) (m-AKT/PLGA) composite scaffold was fabricated via a solvent casting-particulate leaching method improved by solvent self-proliferating process. The effects of m-AKT contents on properties of composite scaffolds and on MC3T3-E1 cellular behaviors in vitro have been primarily investigated. The fabricated scaffolds exhibited three-dimensional porous networks, in which homogenously distributed cavities in size of 300-400 μm were interconnected by some smaller holes in a size of 100-200 μm. Meanwhile, the mechanical structure of scaffolds was reinforced by the introduction of m-AKT. Moreover, alkaline ionic products released by m-AKT could neutralize the acidic degradation products of PLGA, and the apatite-mineralization ability of scaffolds could be largely improved. More valuably, significant promotions on adhesion, proliferation, and differentiation of MC3T3-E1 have been observed, which implied the calcium, magnesium and especially silidous ions released sustainably from composite scaffolds could regulate the behaviors of osteogenesis-related cells.

  13. Plasma Treated High-Density Polyethylene (HDPE Medpor Implant Immobilized with rhBMP-2 for Improving the Bone Regeneration

    Directory of Open Access Journals (Sweden)

    Jin-Su Lim

    2014-01-01

    Full Text Available We investigate the bone generation capacity of recombinant human bone morphogenetic protein-2 (rhBMP-2 immobilized Medpor surface through acrylic acid plasma-polymerization. Plasma-polymerization was carried out at a 20 W at an acrylic acid flow rate of 7 sccm for 5 min. The plasma-polymerized Medpor surface showed hydrophilic properties and possessed a high density of carboxyl groups. The rhBMP-2 was immobilized with covalently attached carboxyl groups using 1-ethyl-3-(3-dimethylaminopropyl carbodiimide and N-hydroxysuccinimide. Carboxyl groups and rhBMP-2 immobilization on the Medpor surface were identified by Fourier transform infrared spectroscopy. The activity of Medpor with rhBMP-2 immobilized was examined using an alkaline phosphatase assay on MC3T3-E1 cultured Medpor. These results showed that the rhBMP-2 immobilized Medpor increased the level of MC3T3-E1 cell differentiation. These results demonstrated that plasma surface modification has the potential to immobilize rhBMP-2 on polymer implant such as Medpor and can be used for the binding of bioactive nanomolecules in bone tissue engineering.

  14. [Effect of Ca(OH)2 on the cytotoxicity of lipopolysaccharide extracted from Porphyromonas endodontalis in vitro].

    Science.gov (United States)

    Guo, Jia-jie; Qiu, Li-hong; Yu, Ya-qiong; Xu, Li-ya; Fan, Yun-qian; Zhong, Ming

    2014-02-01

    To detect the degradation of Ca(OH)2 on lipopolysaccharide (LPS) extracted from Porphyromonas endodontalis (P.e) in vitro and estimate the influence of P.e LPS pretreated with Ca(OH)2 on the proliferation of MC3T3-E1 cells. The effect of Ca(OH)2 on MC3T3-E1 cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay. Then P.e LPS was treated with Ca(OH)2 for 30 mins or 60 mins at 37 degrees centigrade in vitro and the activity of P.e LPS was evaluated by Chromogenic End-point Tachypleus Amebocyte Lysate (CE TAL) test. Finally, MC3T3-E1 cells were exposed to P.e LPS pretreated with 15% Ca(OH)2 for 1, 3 and 5 d, and the cell proliferation was measured using the MTT assay comparing with the P.e LPS control group. SPSS 13.0 software package was used for statistical analysis. Compared with the negative control, exposing cells to 5%, 10% and 15% Ca(OH)2 had greatly promoted MC3T3-E1 cell proliferation. P.e LPS treated with 10% and 15% Ca(OH)2 both presented the best results by CE TAL and significant difference compared with P.e LPS control group. When 10 μg/mL P.e LPS was pretreated with 15% Ca(OH)2, no inhibition of MC3T3-E1 cell proliferation was noted. Ca(OH)2 detoxifies P.e LPS in vitro, mitigates the impact of P.e LPS on MC3T3-E1 cell proliferation. Supported by Science and Technology Projects of Liaoning Province (2011225020).

  15. Nuclear Mechanics and Stem Cell Differentiation.

    Science.gov (United States)

    Mao, Xinjian; Gavara, Nuria; Song, Guanbin

    2015-12-01

    Stem cells are characterized by their self-renewal and multi-lineage differentiation potential. Stem cell differentiation is a prerequisite for the application of stem cells in regenerative medicine and clinical therapy. In addition to chemical stimulation, mechanical cues play a significant role in regulating stem cell differentiation. The integrity of mechanical sensors is necessary for the ability of cells to respond to mechanical signals. The nucleus, the largest and stiffest cellular organelle, interacts with the cytoskeleton as a key mediator of cell mechanics. Nuclear mechanics are involved in the complicated interactions of lamins, chromatin and nucleoskeleton-related proteins. Thus, stem cell differentiation is intimately associated with nuclear mechanics due to its indispensable role in mechanotransduction and mechanical response. This paper reviews several main contributions of nuclear mechanics, highlights the hallmarks of the nuclear mechanics of stem cells, and provides insight into the relationship between nuclear mechanics and stem cell differentiation, which may guide clinical applications in the future.

  16. Can bone marrow differentiate into renal cells?

    Science.gov (United States)

    Imai, Enyu; Ito, Takahito

    2002-10-01

    A considerable plasticity of adult stem cells has been confirmed in a wide variety of tissues. In particular, the pluripotency of bone marrow-derived stem cells may influence the regeneration of injured tissues and may provide novel avenues in regenerative medicine. Bone marrow contains at least hematopoietic and mesenchymal stem cells, and both can differentiate into a wide range of differentiated cells. Side population (SP) cells, which are originally defined in bone marrow cells by high efflux of DNA-binding dye, seem to be a new class of multipotent stem cells. Irrespective of the approach used to obtain stem cells, the fates of marrow-derived cells following bone marrow transplantation can be traced by labeling donor cells with green fluorescence protein or by identifying donor Y chromosome in female recipients. So far, bone marrow-derived cells have been reported to differentiate into renal cells, including mesangial cells, endothelial cells, podocytes, and tubular cells in the kidney, although controversy exists. Further studies are required to address this issue. Cell therapy will be promising when we learn to control stem cells such as bone marrow-derived stem cells, embryonic stem cells, and resident stem cells in the kidney. Identification of factors that support stem cells or promote their differentiation should provide a relevant step towards cell therapy.

  17. 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.

  18. Transcriptome changes during intestinal cell differentiation

    DEFF Research Database (Denmark)

    Tadjali, Mehrdad; Seidelin, Jakob B; Olsen, Jørgen Lillelund

    2002-01-01

    The expression of 18149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change in the transc...

  19. 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

  20. Cancer stem cells and differentiation therapy.

    Science.gov (United States)

    Jin, Xiong; Jin, Xun; Kim, Hyunggee

    2017-10-01

    Cancer stem cells can generate tumors from only a small number of cells, whereas differentiated cancer cells cannot. The prominent feature of cancer stem cells is its ability to self-renew and differentiate into multiple types of cancer cells. Cancer stem cells have several distinct tumorigenic abilities, including stem cell signal transduction, tumorigenicity, metastasis, and resistance to anticancer drugs, which are regulated by genetic or epigenetic changes. Like normal adult stem cells involved in various developmental processes and tissue homeostasis, cancer stem cells maintain their self-renewal capacity by activating multiple stem cell signaling pathways and inhibiting differentiation signaling pathways during cancer initiation and progression. Recently, many studies have focused on targeting cancer stem cells to eradicate malignancies by regulating stem cell signaling pathways, and products of some of these strategies are in preclinical and clinical trials. In this review, we describe the crucial features of cancer stem cells related to tumor relapse and drug resistance, as well as the new therapeutic strategy to target cancer stem cells named "differentiation therapy."

  1. DNA repair in murine embryonic stem cells and differentiated cells

    International Nuclear Information System (INIS)

    Tichy, Elisia D.; Stambrook, Peter J.

    2008-01-01

    Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells

  2. A device for real-time live-cell microscopy during dynamic dual-modal mechanostimulation

    Science.gov (United States)

    Lorusso, D.; Nikolov, H. N.; Chmiel, T.; Beach, R. J.; Sims, S. M.; Dixon, S. J.; Holdsworth, D. W.

    2017-03-01

    Mechanotransduction - the process by which cells sense and respond to mechanical stimuli - is essential for several physiological processes including skeletal homeostasis. Mammalian cells are thought to be sensitive to different modes of mechanical stimuli, including vibration and fluid shear. To better understand the mechanisms underlying the early stages of mechanotransduction, we describe the development of devices for mechanostimulation (by vibration and fluid shear) of live cells that can be integrated with real-time optical microscopy. The integrated system can deliver up to 3 Pa of fluid shear simultaneous with high-frequency sinusoidal vibrations up to 1 g. Stimuli can be applied simultaneously or independently to cells during real-time microscopic imaging. A custom microfluidic chamber was prepared from polydimethylsiloxane on a glass-bottom cell culture dish. Fluid flow was applied with a syringe pump to induce shear stress. This device is compatible with a custom-designed motion control vibration system. A voice coil actuates the system that is suspended on linear air bushings. Accelerations produced by the system were monitored with an on-board accelerometer. Displacement was validated optically using particle tracking digital high-speed imaging (1200 frames per second). During operation at nominally 45 Hz and 0.3 g, displacements were observed to be within 3.56% of the expected value. MC3T3-E1 osteoblast like cells were seeded into the microfluidic device and loaded with the calcium sensitive fluorescent probe fura-2, then mounted onto the dual-modal mechanostimulation platform. Cells were then imaged and monitored for fluorescence emission. In summary, we have developed a system to deliver physiologically relevant vibrations and fluid shear to live cells during real-time imaging and photometry. Monitoring the behavior of live cells loaded with appropriate fluorescent probes will enable characterization of the signals activated during the initial

  3. Transcriptome changes during intestinal cell differentiation

    DEFF Research Database (Denmark)

    Tadjali, Mehrdad; Seidelin, Jakob B; Olsen, Jørgen

    2002-01-01

    The expression of 18149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change in the transc......The expression of 18149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change...... cells by performing reverse transcriptase-polymerase chain reaction on RNA extracted from laser dissected intestinal crypt and villi. In a screen of eight transcripts one - SART3 - was identified as a marker for human colonic crypts....

  4. Chromatin in embryonic stem cell neuronal differentiation.

    Science.gov (United States)

    Meshorer, E

    2007-03-01

    Chromatin, the basic regulatory unit of the eukaryotic genetic material, is controlled by epigenetic mechanisms including histone modifications, histone variants, DNA methylation and chromatin remodeling. Cellular differentiation involves large changes in gene expression concomitant with alterations in genome organization and chromatin structure. Such changes are particularly evident in self-renewing pluripotent embryonic stem cells, which begin, in terms of cell fate, as a tabula rasa, and through the process of differentiation, acquire distinct identities. Here I describe the changes in chromatin that accompany neuronal differentiation, particularly of embryonic stem cells, and discuss how chromatin serves as the master regulator of cellular destiny.

  5. Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells

    NARCIS (Netherlands)

    Guadix, Juan Antonio; Orlova, Valeria V.; Giacomelli, Elisa; Bellin, Milena; Ribeiro, Marcelo C.; Mummery, Christine L.; Pérez-Pomares, José M.; Passier, Robert

    2017-01-01

    Human pluripotent stem cells (hPSCs) are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced) to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA)

  6. 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

  7. Control of differentiation of melanoma cells

    International Nuclear Information System (INIS)

    Eguchi, Goro

    1980-01-01

    To develop the method to induce the appearance of differentiation in amelanotic melanoma, experimental control of differentiation in B-16 melanoma cells of mice was discussed. Human melanoma cells and yellow melanin pigment cells useful for a fundamental study of radiotherapy for cancer were cultured and were differentiated into some lines. Melanotic B-16 cells and amelanotic B-16 cells were irradiated with thermal neutron (neutron: 2.7 x 10 12 , γ-dose: 32.3 rad) after they were cultured in culture solution containing 10 γ/ml of 10 B-dopa for 13 hours. A fine structure 5 hours after the irradiation in one of 5 experimental cases showed aggregated disintegration of melanin pigment particles, markedly deformed and fragmentized nucleus, and structural changes in cell membrane. (Tsunoda, M.)

  8. 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

  9. Nucleotide excision repair in differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Wees, Caroline van der [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Jansen, Jacob [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Vrieling, Harry [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Laarse, Arnoud van der [Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Zeeland, Albert van [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Mullenders, Leon [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands)]. E-mail: l.mullenders@lumc.nl

    2007-01-03

    Nucleotide excision repair (NER) is the principal pathway for the removal of a wide range of DNA helix-distorting lesions and operates via two NER subpathways, i.e. global genome repair (GGR) and transcription-coupled repair (TCR). Although detailed information is available on expression and efficiency of NER in established mammalian cell lines, little is known about the expression of NER pathways in (terminally) differentiated cells. The majority of studies in differentiated cells have focused on repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4-photoproducts (6-4PP) because of the high frequency of photolesions at low level of toxicity and availability of sensitive technologies to determine photolesions in defined regions of the genome. The picture that emerges from these studies is blurred and rather complex. Fibroblasts and terminally differentiated myocytes of the rat heart display equally efficient GGR of 6-4PP but poor repair of CPD due to the absence of p48 expression. This repair phenotype is clearly different from human terminal differentiated neurons. Furthermore, both cell types were found to carry out TCR of CPD, thus mimicking the repair phenotype of established rodent cell lines. In contrast, in intact rat spermatogenic cells repair was very inefficient at the genome overall level and in transcriptionally active genes indicating that GGR and TCR are non-functional. Also, non-differentiated mouse embryonic stem (ES) cells exhibit low levels of NER after UV irradiation. However, the mechanisms that lead to low NER activity are clearly different: in differentiated spermatogenic cells differences in chromatin compaction and sequestering of NER proteins may underlie the lack of NER activity in pre-meiotic cells, whereas in non-differentiated ES cells NER is impaired by a strong apoptotic response.

  10. 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.

  11. Improving the osteointegration of Ti6Al4V by zeolite MFI coating

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yong [Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Jiao, Yilai [Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, Liaoning 110016 (China); Li, Xiaokang [Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Guo, Zheng, E-mail: guozheng@fmmu.edu.cn [Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China)

    2015-05-01

    Osteointegration is crucial for success in orthopedic implantation. In recent decades, there have been numerous studies aiming to modify titanium alloys, which are the most widely used materials in orthopedics. Zeolites are solid aluminosilicates whose application in the biomedical field has recently been explored. To this end, MFI zeolites have been developed as titanium alloy coatings and tested in vitro. Nevertheless, the effect of the MFI coating of biomaterials in vivo has not yet been addressed. The aim of the present work is to evaluate the effects of MFI-coated Ti6Al4V implants in vitro and in vivo. After surface modification, the surface was investigated using field emission scanning electron microscopy (FE-SEM) and energy dispersive spectroscopy (EDS). No difference was observed regarding the proliferation of MC3T3-E1 cells on the Ti6Al4V (Ti) and MFI-coated Ti6Al4V (M−Ti) (p > 0.05). However, the attachment of MC3T3-E1 cells was found to be better in the M−Ti group. Additionally, ALP staining and activity assays and quantitative real-time RT-PCR indicated that MC3T3-E1 cells grown on the M−Ti displayed high levels of osteogenic differentiation markers. Moreover, Van-Gieson staining of histological sections demonstrated that the MFI coating on Ti6Al4V scaffolds significantly enhanced osteointegration and promoted bone regeneration after implantation in rabbit femoral condylar defects at 4 and 12 weeks. Therefore, this study provides a method for modifying Ti6Al4V to achieve improved osteointegration and osteogenesis. - Highlights: • Osteointegration is a crucial factor for orthopedic implants. • We coated MFI zeolite on Ti6Al4V substrates and investigated the effects in vitro and in vivo. • The MFI coating displayed good biocompatibility and promoted osteogenic differentiation in vitro. • The MFI coating promoted osteointegration and osteogenesis peri-implant in vivo.

  12. 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...

  13. Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Juan Antonio Guadix

    2017-12-01

    Full Text Available Summary: Human pluripotent stem cells (hPSCs are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA and bone morphogenetic protein 4 (BMP4 promoted expression of the mesodermal marker PDGFRα, upregulated characteristic (proepicardial progenitor cell genes, and downregulated transcription of myocardial genes. We confirmed the (proepicardial-like properties of these cells using in vitro co-culture assays and in ovo grafting of hPSC-epicardial cells into chick embryos. Our data show that RA + BMP4-treated hPSCs differentiate into (proepicardial-like cells displaying functional properties (adhesion and spreading over the myocardium of their in vivo counterpart. The results extend evidence that hPSCs are an excellent model to study (proepicardial differentiation into cardiovascular cells in human development and evaluate their potential for cardiac regeneration. : The authors have shown that hPSCs can be instructed in vitro to differentiate into a specific cardiac embryonic progenitor cell population called the proepicardium. Proepicardial cells are required for normal formation of the heart during development and might contribute to the development of cell-based therapies for heart repair. Keywords: human pluripotent stem cells, proepicardium, progenitor cells, cardiovascular, differentiation

  14. Nanotopographical Control of Stem Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Laura E. McNamara

    2010-01-01

    Full Text Available Stem cells have the capacity to differentiate into various lineages, and the ability to reliably direct stem cell fate determination would have tremendous potential for basic research and clinical therapy. Nanotopography provides a useful tool for guiding differentiation, as the features are more durable than surface chemistry and can be modified in size and shape to suit the desired application. In this paper, nanotopography is examined as a means to guide differentiation, and its application is described in the context of different subsets of stem cells, with a particular focus on skeletal (mesenchymal stem cells. To address the mechanistic basis underlying the topographical effects on stem cells, the likely contributions of indirect (biochemical signal-mediated and direct (force-mediated mechanotransduction are discussed. Data from proteomic research is also outlined in relation to topography-mediated fate determination, as this approach provides insight into the global molecular changes at the level of the functional effectors.

  15. Biophysical regulation of stem cell differentiation.

    Science.gov (United States)

    Govey, Peter M; Loiselle, Alayna E; Donahue, Henry J

    2013-06-01

    Bone adaptation to its mechanical environment, from embryonic through adult life, is thought to be the product of increased osteoblastic differentiation from mesenchymal stem cells. In parallel with tissue-scale loading, these heterogeneous populations of multipotent stem cells are subject to a variety of biophysical cues within their native microenvironments. Bone marrow-derived mesenchymal stem cells-the most broadly studied source of osteoblastic progenitors-undergo osteoblastic differentiation in vitro in response to biophysical signals, including hydrostatic pressure, fluid flow and accompanying shear stress, substrate strain and stiffness, substrate topography, and electromagnetic fields. Furthermore, stem cells may be subject to indirect regulation by mechano-sensing osteocytes positioned to more readily detect these same loading-induced signals within the bone matrix. Such paracrine and juxtacrine regulation of differentiation by osteocytes occurs in vitro. Further studies are needed to confirm both direct and indirect mechanisms of biophysical regulation within the in vivo stem cell niche.

  16. The genetic network controlling plasma cell differentiation.

    Science.gov (United States)

    Nutt, Stephen L; Taubenheim, Nadine; Hasbold, Jhagvaral; Corcoran, Lynn M; Hodgkin, Philip D

    2011-10-01

    Upon activation by antigen, mature B cells undergo immunoglobulin class switch recombination and differentiate into antibody-secreting plasma cells, the endpoint of the B cell developmental lineage. Careful quantitation of these processes, which are stochastic, independent and strongly linked to the division history of the cell, has revealed that populations of B cells behave in a highly predictable manner. Considerable progress has also been made in the last few years in understanding the gene regulatory network that controls the B cell to plasma cell transition. The mutually exclusive transcriptomes of B cells and plasma cells are maintained by the antagonistic influences of two groups of transcription factors, those that maintain the B cell program, including Pax5, Bach2 and Bcl6, and those that promote and facilitate plasma cell differentiation, notably Irf4, Blimp1 and Xbp1. In this review, we discuss progress in the definition of both the transcriptional and cellular events occurring during late B cell differentiation, as integrating these two approaches is crucial to defining a regulatory network that faithfully reflects the stochastic features and complexity of the humoral immune response. 2011 Elsevier Ltd. All rights reserved.

  17. Thrombopoietin inhibits murine mast cell differentiation

    Science.gov (United States)

    Martelli, Fabrizio; Ghinassi, Barbara; Lorenzini, Rodolfo; Vannucchi, Alessandro M; Rana, Rosa Alba; Nishikawa, Mitsuo; Partamian, Sandra; Migliaccio, Giovanni; Migliaccio, Anna Rita

    2009-01-01

    We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin, or addition of this growth factor to bone marrow-derived mast cell cultures, severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target anti-apoptotic gene Bcl2. PMID:18276801

  18. 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.

  19. Demineralized dentin matrix composite collagen material for bone tissue regeneration.

    Science.gov (United States)

    Li, Jianan; Yang, Juan; Zhong, Xiaozhong; He, Fengrong; Wu, Xiongwen; Shen, Guanxin

    2013-01-01

    Demineralized dentin matrix (DDM) had been successfully used in clinics as bone repair biomaterial for many years. However, particle morphology of DDM limited it further applications. In this study, DDM and collagen were prepared to DDM composite collagen material. The surface morphology of the material was studied by scanning electron microscope (SEM). MC3T3-E1 cells responses in vitro and tissue responses in vivo by implantation of DDM composite collagen material in bone defect of rabbits were also investigated. SEM analysis showed that DDM composite collagen material evenly distributed and formed a porous scaffold. Cell culture and animal models results indicated that DDM composite collagen material was biocompatible and could support cell proliferation and differentiation. Histological evaluation showed that DDM composite collagen material exhibited good biocompatibility, biodegradability and osteoconductivity with host bone in vivo. The results suggested that DDM composite collagen material might have a significant clinical advantage and potential to be applied in bone and orthopedic surgery.

  20. Nano-hydroxyapatite reinforced polyhydroxybutyrate composites: A comprehensive study on the structural and in vitro biological properties

    Energy Technology Data Exchange (ETDEWEB)

    Sadat-Shojai, Mehdi, E-mail: MSadatShojai@gmail.com [Department of Biomaterials, Iran Polymer and Petrochemical Institute, P.O. Box 14965/115, Tehran (Iran, Islamic Republic of); Khorasani, Mohammad-Taghi [Department of Biomaterials, Iran Polymer and Petrochemical Institute, P.O. Box 14965/115, Tehran (Iran, Islamic Republic of); Jamshidi, Ahmad [Department of Novel Drug Delivery Systems, Iran Polymer and Petrochemical Institute, P.O. Box 14965/115, Tehran (Iran, Islamic Republic of); Irani, Shiva [Department of Biology, Science and Research Branch, Islamic Azad University, Tehran (Iran, Islamic Republic of)

    2013-07-01

    Nanocomposites based on polyhydroxybutyrate (PHB) and hydroxyapatite (HAp) have recently been proposed for application in bone repair and regeneration, but very limited studies have investigated the effect of HAp on the rheological and thermal behavior of PHB. More important, the efficiency of a biomaterial depends greatly on its ability to interact with cells, but little is known about this interaction for this kind of nanocomposite. Hence, this paper dealt with some of the characteristics of solution-casted PHB/HAp nanocomposite films, and tried to explore the effect of HAp nanoparticles on cellular responses. The results showed that both rheological and thermal properties can be tailored by incorporating appropriate amounts of nanoparticles. In vitro studies showed a significant increase in proliferation and differentiation of MC3T3-E1 on nanocomposites compared to the neat polymer. Surface examination indicated that topography and chemistry of surface are important factors influencing cellular processes; while no cell differentiation was found on the neat polymer, nanocomposite with 15 wt.% filler content exhibited a pronounced differentiation resulting from high surface roughness and large amount of exposed HAp. These results suggest that HAp particles play a much more important role in determining the biological performance of PHB than has previously been supposed. Highlights: • Preosteoblastic MC3T3-E1 cells exhibit an elongated shape in the presence of HAp. • The as-prepared nanoparticles acted as nucleating agent during PHB crystallization. • HAp efficiently induces cell proliferation and differentiation on PHB surface. • HAp nanoparticles significantly affect both the surface chemistry and topography. • A method was provided to simultaneously measure biomineralization and bioactivity.

  1. Nano-hydroxyapatite reinforced polyhydroxybutyrate composites: A comprehensive study on the structural and in vitro biological properties

    International Nuclear Information System (INIS)

    Sadat-Shojai, Mehdi; Khorasani, Mohammad-Taghi; Jamshidi, Ahmad; Irani, Shiva

    2013-01-01

    Nanocomposites based on polyhydroxybutyrate (PHB) and hydroxyapatite (HAp) have recently been proposed for application in bone repair and regeneration, but very limited studies have investigated the effect of HAp on the rheological and thermal behavior of PHB. More important, the efficiency of a biomaterial depends greatly on its ability to interact with cells, but little is known about this interaction for this kind of nanocomposite. Hence, this paper dealt with some of the characteristics of solution-casted PHB/HAp nanocomposite films, and tried to explore the effect of HAp nanoparticles on cellular responses. The results showed that both rheological and thermal properties can be tailored by incorporating appropriate amounts of nanoparticles. In vitro studies showed a significant increase in proliferation and differentiation of MC3T3-E1 on nanocomposites compared to the neat polymer. Surface examination indicated that topography and chemistry of surface are important factors influencing cellular processes; while no cell differentiation was found on the neat polymer, nanocomposite with 15 wt.% filler content exhibited a pronounced differentiation resulting from high surface roughness and large amount of exposed HAp. These results suggest that HAp particles play a much more important role in determining the biological performance of PHB than has previously been supposed. Highlights: • Preosteoblastic MC3T3-E1 cells exhibit an elongated shape in the presence of HAp. • The as-prepared nanoparticles acted as nucleating agent during PHB crystallization. • HAp efficiently induces cell proliferation and differentiation on PHB surface. • HAp nanoparticles significantly affect both the surface chemistry and topography. • A method was provided to simultaneously measure biomineralization and bioactivity

  2. Design and Characterization of a Sensorized Microfluidic Cell-Culture System with Electro-Thermal Micro-Pumps and Sensors for Cell Adhesion, Oxygen, and pH on a Glass Chip

    Directory of Open Access Journals (Sweden)

    Sebastian M. Bonk

    2015-07-01

    Full Text Available We combined a multi-sensor glass-chip with a microfluidic channel grid for the characterization of cellular behavior. The grid was imprinted in poly-dimethyl-siloxane. Mouse-embryonal/fetal calvaria fibroblasts (MC3T3-E1 were used as a model system. Thin-film platinum (Pt sensors for respiration (amperometric oxygen electrode, acidification (potentiometric pH electrodes and cell adhesion (interdigitated-electrodes structures, IDES allowed us to monitor cell-physiological parameters as well as the cell-spreading behavior. Two on-chip electro-thermal micro-pumps (ETμPs permitted the induction of medium flow in the system, e.g., for medium mixing and drug delivery. The glass-wafer technology ensured the microscopic observability of the on-chip cell culture. Connecting Pt structures were passivated by a 1.2 μm layer of silicon nitride (Si3N4. Thin Si3N4 layers (20 nm or 60 nm were used as the sensitive material of the pH electrodes. These electrodes showed a linear behavior in the pH range from 4 to 9, with a sensitivity of up to 39 mV per pH step. The oxygen sensors were circular Pt electrodes with a sensor area of 78.5 μm2. Their sensitivity was 100 pA per 1% oxygen increase in the range from 0% to 21% oxygen (air saturated. Two different IDES geometries with 30- and 50-μm finger spacings showed comparable sensitivities in detecting the proliferation rate of MC3T3 cells. These cells were cultured for 11 days in vitro to test the biocompatibility, microfluidics and electric sensors of our system under standard laboratory conditions.

  3. The Antigen Presenting Cells Instruct Plasma Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Wei eXu

    2014-01-01

    Full Text Available The professional antigen presenting cells (APCs, including many subsets of dendritic cells and macrophages, not only mediate prompt but nonspecific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells, which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only signal 1 (the antigen, but also signal 2 to directly instruct the differentiation process of plasma cells in a T cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

  4. Tracking plasma cell differentiation and survival.

    Science.gov (United States)

    Roth, Katrin; Oehme, Laura; Zehentmeier, Sandra; Zhang, Yang; Niesner, Raluca; Hauser, Anja E

    2014-01-01

    Plasma cells play a crucial role for the humoral immune response as they represent the body's factories for antibody production. The differentiation from a B cell into a plasma cell is controlled by a complex transcriptional network and happens within secondary lymphoid organs. Based on their lifetime, two types of antibody secreting cells can be distinguished: Short-lived plasma cells are located in extrafollicular sites of secondary lymphoid organs such as lymph node medullary cords and the splenic red pulp. A fraction of plasmablasts migrate from secondary lymphoid organs to the bone marrow where they can become long-lived plasma cells. Bone marrow plasma cells reside in special microanatomical environments termed survival niches, which provide factors promoting their longevity. Reticular stromal cells producing the chemokine CXCL12, which is known to attract plasmablasts to the bone marrow but also to promote plasma cell survival, play a crucial role in the maintenance of these niches. In addition, hematopoietic cells are contributing to the niches by providing other soluble survival factors. Here, we review the current knowledge on the factors involved in plasma cell differentiation, their localization and migration. We also give an overview on what is known regarding the maintenance of long lived plasma cells in survival niches of the bone marrow. © 2013 International Society for Advancement of Cytometry.

  5. 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.

  6. Bioprinting and Differentiation of Stem Cells

    Directory of Open Access Journals (Sweden)

    Scott A. Irvine

    2016-09-01

    Full Text Available The 3D bioprinting of stem cells directly into scaffolds offers great potential for the development of regenerative therapies; in particular for the fabrication of organ and tissue substitutes. For this to be achieved; the lineage fate of bioprinted stem cell must be controllable. Bioprinting can be neutral; allowing culture conditions to trigger differentiation or alternatively; the technique can be designed to be stimulatory. Such factors as the particular bioprinting technique; bioink polymers; polymer cross-linking mechanism; bioink additives; and mechanical properties are considered. In addition; it is discussed that the stimulation of stem cell differentiation by bioprinting may lead to the remodeling and modification of the scaffold over time matching the concept of 4D bioprinting. The ability to tune bioprinting properties as an approach to fabricate stem cell bearing scaffolds and to also harness the benefits of the cells multipotency is of considerable relevance to the field of biomaterials and bioengineering.

  7. Hydroxyapatite electrodeposition on anodized titanium nanotubes for orthopedic applications

    Energy Technology Data Exchange (ETDEWEB)

    Parcharoen, Yardnapar [Department of Biological Engineering, Faculty of Engineering, King Mongkut' s University of Technology Thonburi, Bangkok (Thailand); Kajitvichyanukul, Puangrat [Center of Excellence on Environmental Research and Innovation, Faculty of Engineering, Naresuan University, Phitsanulok (Thailand); Sirivisoot, Sirinrath [Department of Biological Engineering, Faculty of Engineering, King Mongkut' s University of Technology Thonburi, Bangkok (Thailand); Termsuksawad, Preecha, E-mail: preecha.ter@kmutt.ac.th [Division of Materials Technology, School of Energy, Environment and Materials, King Mongkut' s University of Technology Thonburi, 126 Pracha Uthit Rd., Bang Mod, ThungKhru, Bangkok 10140 (Thailand)

    2014-08-30

    Highlights: • We found that different anodization time of titanium significantly effects on nanotube length which further impacts adhesion strength of hydroxyapatite coating layers. • Adhesion strength of Hydroxyapatite (HA) coated on titanium dioxide nanotubes is better than that of HA coated on titanium plate. • Hydroxyapatite coated on titanium dioxide nanotubes showed higher cell density and better spreading of MC3T3-E1 cells (bone-forming cells) than that coated on titanium plate surface. - Abstract: Nanotubes modification for orthopedic implants has shown interesting biological performances (such as improving cell adhesion, cell differentiation, and enhancing osseointegration). The purpose of this study is to investigate effect of titanium dioxide (TiO{sub 2}) nanotube feature on performance of hydroxyapatite-coated titanium (Ti) bone implants. TiO{sub 2} nanotubes were prepared by anodization using ammonium fluoride electrolyte (NH{sub 4}F) with and without modifiers (PEG400 and Glycerol) at various potential forms, and times. After anodization, the nanotubes were subsequently annealed. TiO{sub 2} nanotubes were characterized by scanning electron microscope and X-ray diffractometer. The amorphous to anatase transformation due to annealing was observed. Smooth and highly organized TiO{sub 2} nanotubes were found when high viscous electrolyte, NH{sub 4}F in glycerol, was used. Negative voltage (−4 V) during anodization was confirmed to increase nanotube thickness. Length of the TiO{sub 2} nanotubes was significantly increased by times. The TiO{sub 2} nanotube was electrodeposited with hydroxyapatite (HA) and its adhesion was estimated by adhesive tape test. The result showed that nanotubes with the tube length of 560 nm showed excellent adhesion. The coated HA were tested for biological test by live/dead cell straining. HA coated on TiO{sub 2} nanotubes showed higher cells density, higher live cells, and more spreading of MC3T3-E1 cells than that

  8. The antigen presenting cells instruct plasma cell differentiation.

    Science.gov (United States)

    Xu, Wei; Banchereau, Jacques

    2014-01-06

    The professional antigen presenting cells (APCs), including many subsets of dendritic cells and macrophages, not only mediate prompt but non-specific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells (PCs), which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only "signal 1" (the antigen), but also "signal 2" to directly instruct the differentiation process of PCs in a T-cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching, and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

  9. Enhanced antiadhesive properties of chitosan/hyaluronic acid polyelectrolyte multilayers driven by thermal annealing: Low adherence for mammalian cells and selective decrease in adhesion for Gram-positive bacteria.

    Science.gov (United States)

    Muzzio, Nicolás E; Pasquale, Miguel A; Diamanti, Eleftheria; Gregurec, Danijela; Moro, Marta Martinez; Azzaroni, Omar; Moya, Sergio E

    2017-11-01

    The development of antifouling coatings with restricted cell and bacteria adherence is fundamental for many biomedical applications. A strategy for the fabrication of antifouling coatings based on the layer-by-layer assembly and thermal annealing is presented. Polyelectrolyte multilayers (PEMs) assembled from chitosan and hyaluronic acid were thermally annealed in an oven at 37°C for 72h. The effect of annealing on the PEM properties and topography was studied by atomic force microscopy, ζ-potential, circular dichroism and contact angle measurements. Cell adherence on PEMs before and after annealing was evaluated by measuring the cell spreading area and aspect ratio for the A549 epithelial, BHK kidney fibroblast, C2C12 myoblast and MC-3T3-E1 osteoblast cell lines. Chitosan/hyaluronic acid PEMs show a low cell adherence that decreases with the thermal annealing, as observed from the reduction in the average cell spreading area and more rounded cell morphology. The adhesion of S. aureus (Gram-positive) and E. coli (Gram-negative) bacteria strains was quantified by optical microscopy, counting the number of colony-forming units and measuring the light scattering of bacteria suspension after detachment from the PEM surface. A 20% decrease in bacteria adhesion was selectively observed in the S. aureus strain after annealing. The changes in mammalian cell and bacteria adhesion correlate with the changes in topography of the chitosan/hyaluronic PEMs from a rough fibrillar 3D structure to a smoother and planar surface after thermal annealing. Copyright © 2017. Published by Elsevier B.V.

  10. Schwann cells promote neuronal differentiation of bone marrow ...

    African Journals Online (AJOL)

    Administrator

    2011-04-25

    Apr 25, 2011 ... Bone marrow stromal cells (BMSCs), a type of multipotent stem cell, can differentiate into various types ... induced to differentiate into neuron-like cells when they are ... axonal regeneration and functional reconstruction do not.

  11. 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.

  12. 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

  13. Recombinant human IGF-1 produced by transgenic plant cell suspension culture enhances new bone formation in calvarial defects.

    Science.gov (United States)

    Poudel, Sher Bahadur; Bhattarai, Govinda; Kook, Sung-Ho; Shin, Yun-Ji; Kwon, Tae-Ho; Lee, Seung-Youp; Lee, Jeong-Chae

    2017-10-01

    Transgenic plant cell suspension culture systems have been utilized extensively as convenient and efficient expression systems for the production of recombinant human growth factors. We produced insulin-like growth factor-1 using a plant suspension culture system (p-IGF-1) and explored its effect on new bone formation in calvarial defects. We also compared the bone regenerating potential of p-IGF-1 with commercial IGF-1 derived from Escherichia coli (e-IGF-1). Male C57BL/6 mice underwent calvarial defect surgery, and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 13μg of p-IGF-1 (p-IGF-1 group) or e-IGF-1 (e-IGF-1 group). The sham group did not receive any treatment with ACS or IGFs after surgery. Live μCT and histological analyses showed critical-sized bone defects in the sham group, whereas greater bone formation was observed in the p-IGF-1 and e-IGF-1 groups than the ACS group both 5 and 10weeks after surgery. Bone mineral density, bone volume, and bone surface values were also higher in the IGF groups than in the ACS group. Local delivery of p-IGF-1 or e-IGF-1 more greatly enhanced the expression of osteoblast-specific markers, but inhibited osteoclast formation, in newly formed bone compared with ACS control group. Specifically, p-IGF-1 treatment induced higher expression of alkaline phosphatase, osteocalcin, and osteopontin in the defect site than did e-IGF-1. Furthermore, treatment with p-IGF-1, but not e-IGF-1, increased mineralization of MC3T3-E1 cells, with the attendant upregulation of osteogenic marker genes. Collectively, our findings suggest the potential of p-IGF-1 in promoting the processes required for bone regeneration. Copyright © 2017. Published by Elsevier Ltd.

  14. 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.

  15. Biomimetic synthesis and biocompatibility evaluation of carbonated apatites template-mediated by heparin

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Yi [Department of Oral and Maxillofacial Surgery, Laboratory of Interdisciplinary Studies, School and Hospital of Stomatology, Peking University, Beijing 100081 (China); Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China); Sun, Yuhua [Department of Oral and Maxillofacial Surgery, Laboratory of Interdisciplinary Studies, School and Hospital of Stomatology, Peking University, Beijing 100081 (China); Chen, Xiaofang [Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China); Zhu, Peizhi, E-mail: pzzhu@umich.edu [Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China); Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055 (United States); Wei, Shicheng, E-mail: sc-wei@pku.edu.cn [Department of Oral and Maxillofacial Surgery, Laboratory of Interdisciplinary Studies, School and Hospital of Stomatology, Peking University, Beijing 100081 (China); Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China)

    2013-07-01

    Biomimetic synthesis of carbonated apatites with good biocompatibility is a promising strategy for the broadening application of apatites for bone tissue engineering. Most researchers were interested in collagen or gelatin-based templates for synthesis of apatite minerals. Inspired by recent findings about the important role of polysaccharides in bone biomineralization, here we reported that heparin, a mucopolysaccharide, was used to synthesize carbonated apatites in vitro. The results indicated that the Ca/P ratio, carbon content, crystallinity and morphology of the apatites varied depending on the heparin concentration and the initial pH value. The morphology of apatite changed from flake-shaped to needle-shaped, and the degree of crystallinity decreased with the increasing of heparin concentration. Biocompatibility of the apatites was tested by proliferation and alkaline phosphatase activity of MC3T3-E1 cells. The results suggested that carbonated apatites synthesized in the presence of heparin were more favorable to the proliferation and differentiation of MC3T3-E1 cells compared with traditional method. In summary, the heparin concentration and the initial pH value play a key role in the chemical constitution and morphology, as well as biological properties of apatites. These biocompatible nano-apatite crystals hold great potential to be applied as bioactive materials for bone tissue engineering. - Highlights: • Heparin was used as a template to synthesize needle-shaped nano-apatite. • Changing the pH value and concentration led to different properties of apatite. • Apatite prepared by heparin was more favorable to the osteogenic differentiation. • Possible synthesis mechanism of apatite templated by heparin was described.

  16. Differential white cell count by centrifugal microfluidics.

    Energy Technology Data Exchange (ETDEWEB)

    Sommer, Gregory Jon; Tentori, Augusto M.; Schaff, Ulrich Y.

    2010-07-01

    We present a method for counting white blood cells that is uniquely compatible with centrifugation based microfluidics. Blood is deposited on top of one or more layers of density media within a microfluidic disk. Spinning the disk causes the cell populations within whole blood to settle through the media, reaching an equilibrium based on the density of each cell type. Separation and fluorescence measurement of cell types stained with a DNA dye is demonstrated using this technique. The integrated signal from bands of fluorescent microspheres is shown to be proportional to their initial concentration in suspension. Among the current generation of medical diagnostics are devices based on the principle of centrifuging a CD sized disk functionalized with microfluidics. These portable 'lab on a disk' devices are capable of conducting multiple assays directly from a blood sample, embodied by platforms developed by Gyros, Samsung, and Abaxis. [1,2] However, no centrifugal platform to date includes a differential white blood cell count, which is an important metric complimentary to diagnostic assays. Measuring the differential white blood cell count (the relative fraction of granulocytes, lymphocytes, and monocytes) is a standard medical diagnostic technique useful for identifying sepsis, leukemia, AIDS, radiation exposure, and a host of other conditions that affect the immune system. Several methods exist for measuring the relative white blood cell count including flow cytometry, electrical impedance, and visual identification from a stained drop of blood under a microscope. However, none of these methods is easily incorporated into a centrifugal microfluidic diagnostic platform.

  17. Fabrication and in vitro biocompatibility of biomorphic PLGA/nHA composite scaffolds for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Qian, Junmin, E-mail: jmqian@mail.xjtu.edu.cn; Xu, Weijun; Yong, Xueqing; Jin, Xinxia; Zhang, Wei

    2014-03-01

    In this study, biomorphic poly(DL-lactic-co-glycolic acid)/nano-hydroxyapatite (PLGA/nHA) composite scaffolds were successfully prepared using cane as a template. The porous morphology, phase, compression characteristics and in vitro biocompatibility of the PLGA/nHA composite scaffolds and biomorphic PLGA scaffolds as control were investigated. The results showed that the biomorphic scaffolds preserved the original honeycomb-like architecture of cane and exhibited a bimodal porous structure. The average channel diameter and micropore size of the PLGA/nHA composite scaffolds were 164 ± 52 μm and 13 ± 8 μm, respectively, with a porosity of 89.3 ± 1.4%. The incorporation of nHA into PLGA decreased the degree of crystallinity of PLGA, and significantly improved the compressive modulus of biomorphic scaffolds. The in vitro biocompatibility evaluation with MC3T3-E1 cells demonstrated that the biomorphic PLGA/nHA composite scaffolds could better support cell attachment, proliferation and differentiation than the biomorphic PLGA scaffolds. The localization depth of MC3T3-E1 cells within the channels of the biomorphic PLGA/nHA composite scaffolds could reach approximately 400 μm. The results suggested that the biomorphic PLGA/nHA composite scaffolds were promising candidates for bone tissue engineering. - Highlights: • Novel biomimetic PLGA/nHA composite scaffolds were successfully prepared. • nHA addition improved elastic modulus of PLGA scaffold and decreased its crystallinity. • PLGA/nHA composite scaffolds had better biocompatibility than PLGA scaffolds. • Biomorphic PLGA/nHA composite scaffold had great potential in bone tissue engineering.

  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. Dual-Functionalized Graphene Oxide Based siRNA Delivery System for Implant Surface Biomodification with Enhanced Osteogenesis.

    Science.gov (United States)

    Zhang, Li; Zhou, Qing; Song, Wen; Wu, Kaimin; Zhang, Yumei; Zhao, Yimin

    2017-10-11

    Surface functionalization by small interfering RNA (siRNA) is a novel strategy for improved implant osseointegration. A gene delivery system with safety and high transfection activity is a crucial factor for an siRNA-functionalized implant to exert its biological function. To this end, polyethylene glycol (PEG) and polyethylenimine (PEI) dual-functionalized graphene oxide (GO; nGO-PEG-PEI) may present a promising siRNA vector. In this study, nanosized nGO-PEG-PEI was prepared and optimized for siRNA delivery. Titania nanotubes (NTs) fabricated by anodic oxidation were biomodified with nGO-PEG-PEI/siRNA by cathodic electrodeposition, designated as NT-GPP/siRNA. NT-GPP/siRNA possessed benign cytocompatibility, as evaluated by cell adhesion and proliferation. Cellular uptake and knockdown efficiency of the NT-GPP/siRNA were assessed by MC3T3-E1 cells, which exhibited high siRNA delivery efficiency and sustained target gene silencing. Casein kinase-2 interacting protein-1 (Ckip-1) is a negative regulator of bone formation. siRNA-targeting Ckip-1 (siCkip-1) was introduced to the implant, and a series of in vitro and in vivo experiments were carried out to evaluate the osteogenic capacity of NT-GPP/siCkip-1. NT-GPP/siCkip-1 dramatically improved the in vitro osteogenic differentiation of MC3T3-E1 cells in terms of improved osteogenesis-related gene expression, and increased alkaline phosphatase (ALP) production, collagen secretion, and extracellular matrix (ECM) mineralization. Moreover, NT-GPP/siCkip-1 led to apparently enhanced in vivo osseointegration, as indicated by histological staining and EDX line scanning. Collectively, these findings suggest that NT-GPP/siRNA represents a practicable and promising approach for implant functionalization, showing clinical potential for dental and orthopedic applications.

  20. Fabrication and in vitro biocompatibility of biomorphic PLGA/nHA composite scaffolds for bone tissue engineering

    International Nuclear Information System (INIS)

    Qian, Junmin; Xu, Weijun; Yong, Xueqing; Jin, Xinxia; Zhang, Wei

    2014-01-01

    In this study, biomorphic poly(DL-lactic-co-glycolic acid)/nano-hydroxyapatite (PLGA/nHA) composite scaffolds were successfully prepared using cane as a template. The porous morphology, phase, compression characteristics and in vitro biocompatibility of the PLGA/nHA composite scaffolds and biomorphic PLGA scaffolds as control were investigated. The results showed that the biomorphic scaffolds preserved the original honeycomb-like architecture of cane and exhibited a bimodal porous structure. The average channel diameter and micropore size of the PLGA/nHA composite scaffolds were 164 ± 52 μm and 13 ± 8 μm, respectively, with a porosity of 89.3 ± 1.4%. The incorporation of nHA into PLGA decreased the degree of crystallinity of PLGA, and significantly improved the compressive modulus of biomorphic scaffolds. The in vitro biocompatibility evaluation with MC3T3-E1 cells demonstrated that the biomorphic PLGA/nHA composite scaffolds could better support cell attachment, proliferation and differentiation than the biomorphic PLGA scaffolds. The localization depth of MC3T3-E1 cells within the channels of the biomorphic PLGA/nHA composite scaffolds could reach approximately 400 μm. The results suggested that the biomorphic PLGA/nHA composite scaffolds were promising candidates for bone tissue engineering. - Highlights: • Novel biomimetic PLGA/nHA composite scaffolds were successfully prepared. • nHA addition improved elastic modulus of PLGA scaffold and decreased its crystallinity. • PLGA/nHA composite scaffolds had better biocompatibility than PLGA scaffolds. • Biomorphic PLGA/nHA composite scaffold had great potential in bone tissue engineering

  1. Manipulating the cell differentiation through lentiviral vectors.

    Science.gov (United States)

    Coppola, Valeria; Galli, Cesare; Musumeci, Maria; Bonci, Désirée

    2010-01-01

    The manipulation of cell differentiation is important to create new sources for the treatment of degenerative diseases or solve cell depletion after aggressive therapy against cancer. In this chapter, the use of a tissue-specific promoter lentiviral vector to obtain a myocardial pure lineage from murine embryonic stem cells (mES) is described in detail. Since the cardiac isoform of troponin I gene product is not expressed in skeletal or other muscle types, short mouse cardiac troponin proximal promoter is used to drive reporter genes. Cells are infected simultaneously with two lentiviral vectors, the first expressing EGFP to monitor the transduction efficiency, and the other expressing a puromycin resistance gene to select the specific cells of interest. This technical approach describes a method to obtain a pure cardiomyocyte population and can be applied to other lineages of interest.

  2. Cell differentiation: therapeutical challenges in diabetes.

    Science.gov (United States)

    Roche, Enrique; Vicente-Salar, Nestor; Arribas, Maribel; Paredes, Beatriz

    2012-01-01

    Stem cells, derived from either embryonic or adult tissues, are considered to be potential sources of insulin-secreting cells to be transplanted into type 1 and advanced stages of type 2 diabetic patients. Many laboratories have considered this possibility, resulting in a large amount of published protocols, with a wide degree of complexity among them. Our group was the first to report that it was possible to obtain insulin-secreting cells from mouse embryonic stem cells, proving the feasibility of this new challenge. The same observation was immediately reported using human embryonic stem cells. However, the resulting cell product was not properly characterised, affecting the reproducibility of the protocol by other groups. A more elaborated protocol was developed by Lumelsky and co-workers, demonstrating that neuroectodermal cells could be an alternative source for insulin-producing cells. However, the resulting cells of this protocol produced low amounts of the hormone. This aimed other groups to perform key changes in order to improve the insulin content of the resulting cells. Recently, Baetge's group has published a new protocol based on the knowledge accumulated in pancreatic development. In this protocol, human embryonic stem cells were differentiated into islet-like structures through a five step protocol, emulating the key steps during embryonic development of the endocrine pancreas. The final cell product, however, seemed to be in an immature state, thus further improvement is required. Despite this drawback, the protocol represents the culmination of work performed by different groups and offers new research challenges for the investigators in this exciting field. Concerning adult stem cells, the possibility of identifying pancreatic precursors or of reprogramming extrapancreatic derived cells are key possibilities that may circumvent the problems that appear when using embryonic stem cells, such as immune rejection and tumour formation.

  3. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Studies have shown that embryonic stem (ES) cells can be successfully differentiated into liver cells, which offer the potential unlimited cell source for a variety of end-stage liver disease. In our study, in order to induce mouse ES cells to differentiate into hepatocyte-like cells under chemically defined conditions, ES cells ...

  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. 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

  6. 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.

  7. The epigenomics of embryonic stem cell differentiation.

    Science.gov (United States)

    Kraushaar, Daniel C; Zhao, Keji

    2013-01-01

    Embryonic stem cells (ESCs) possess an open and highly dynamic chromatin landscape, which underlies their plasticity and ultimately maintains ESC pluripotency. The ESC epigenome must not only maintain the transcription of pluripotency-associated genes but must also, through gene priming, facilitate rapid and cell type-specific activation of developmental genes upon lineage commitment. Trans-generational inheritance ensures that the ESC chromatin state is stably transmitted from one generation to the next; yet at the same time, epigenetic marks are highly dynamic, reversible and responsive to extracellular cues. Once committed to differentiation, the ESC epigenome is remodeled and resolves into a more compact chromatin state. A thorough understanding of the role of chromatin modifiers in ESC fate and differentiation will be important if they are to be used for therapeutic purposes. Recent technical advances, particularly in next-generation sequencing technologies, have provided a genome-scale view of epigenetic marks and chromatin modifiers. More affordable and faster sequencing platforms have led to a comprehensive characterization of the ESC epigenome and epigenomes of differentiated cell types. In this review, we summarize and discuss the recent progress that has highlighted the central role of histone modifications, histone variants, DNA methylation and chromatin modifiers in ESC pluripotency and ESC fate. We provide a detailed and comprehensive discussion of genome-wide studies that are pertinent to our understanding of mammalian development.

  8. Proteomic cornerstones of hematopoietic stem cell differentiation

    DEFF Research Database (Denmark)

    Klimmeck, Daniel; Hansson, Jenny; Raffel, Simon

    2012-01-01

    Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors...... which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem....../progenitor cells (HSPCs, Lin(neg)Sca-1(+)c-Kit(+)) or myeloid committed precursors (Lin(neg)Sca-1(-)c-Kit(+)). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5,000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical...

  9. Directional differentiation of chicken embryonic stem cells into ...

    African Journals Online (AJOL)

    Jane

    2011-08-01

    Aug 1, 2011 ... In this study, the differentiation potential of chicken ES cells was investigated ... Key words: Chicken embryonic stem cells, in vitro, directional differentiation, .... synthesized by using the Revert Aid first strand cDNA synthesis kit.

  10. Regulatory T Cells in Skin Facilitate Epithelial Stem Cell Differentiation.

    Science.gov (United States)

    Ali, Niwa; Zirak, Bahar; Rodriguez, Robert Sanchez; Pauli, Mariela L; Truong, Hong-An; Lai, Kevin; Ahn, Richard; Corbin, Kaitlin; Lowe, Margaret M; Scharschmidt, Tiffany C; Taravati, Keyon; Tan, Madeleine R; Ricardo-Gonzalez, Roberto R; Nosbaum, Audrey; Bertolini, Marta; Liao, Wilson; Nestle, Frank O; Paus, Ralf; Cotsarelis, George; Abbas, Abul K; Rosenblum, Michael D

    2017-06-01

    The maintenance of tissue homeostasis is critically dependent on the function of tissue-resident immune cells and the differentiation capacity of tissue-resident stem cells (SCs). How immune cells influence the function of SCs is largely unknown. Regulatory T cells (Tregs) in skin preferentially localize to hair follicles (HFs), which house a major subset of skin SCs (HFSCs). Here, we mechanistically dissect the role of Tregs in HF and HFSC biology. Lineage-specific cell depletion revealed that Tregs promote HF regeneration by augmenting HFSC proliferation and differentiation. Transcriptional and phenotypic profiling of T regs and HFSCs revealed that skin-resident Tregs preferentially express high levels of the Notch ligand family member, Jagged 1 (Jag1). Expression of Jag1 on Tregs facilitated HFSC function and efficient HF regeneration. Taken together, our work demonstrates that Tregs in skin play a major role in HF biology by promoting the function of HFSCs. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Neuroendocrine differentiation of prostate cancer cells

    Czech Academy of Sciences Publication Activity Database

    Souček, Karel; Pernicová, Zuzana; Lincová, Eva; Staršíchová, Andrea; Kozubík, Alois

    2008-01-01

    Roč. 102, č. 5 (2008), s. 393 ISSN 0009-2770. [Mezioborové setkání mladých biologů, biochemiků a chemiků. Konference Sigma-Aldrich /8./. 10.06.2008-13.06.2008, Devět skal - Žďárské vrchy] R&D Projects: GA ČR(CZ) GA204/07/0834; GA ČR(CZ) GA310/07/0961 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : neuroendocrine differentiation * prostate cancer * neuroendocrine-like cells Subject RIV: BO - Biophysics

  12. Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

    Science.gov (United States)

    Sung, Li-Ying; Gao, Shaorong; Shen, Hongmei; Yu, Hui; Song, Yifang; Smith, Sadie L; Chang, Ching-Chien; Inoue, Kimiko; Kuo, Lynn; Lian, Jin; Li, Ao; Tian, X Cindy; Tuck, David P; Weissman, Sherman M; Yang, Xiangzhong; Cheng, Tao

    2006-11-01

    Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

  13. Probing stem cell differentiation using atomic force microscopy

    International Nuclear Information System (INIS)

    Liang, Xiaobin; Shi, Xuetao; Ostrovidov, Serge; Wu, Hongkai; Nakajima, Ken

    2016-01-01

    Graphical abstract: - Highlights: • Atomic force microscopy (AFM) was developed to probe stem cell differentiation. • The mechanical properties of stem cells and their ECMs can be used to clearly distinguish specific stem cell-differentiated lineages. • AFM is a facile and useful tool for monitoring stem cell differentiation in a non-invasive manner. - Abstract: A real-time method using atomic force microscopy (AFM) was developed to probe stem cell differentiation by measuring the mechanical properties of cells and the extracellular matrix (ECM). The mechanical properties of stem cells and their ECMs can be used to clearly distinguish specific stem cell-differentiated lineages. It is clear that AFM is a facile and useful tool for monitoring the differentiation of stem cells in a non-invasive manner.

  14. Probing stem cell differentiation using atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Xiaobin [Graduate School of Science and Engineering, Tokyo Institute of Technology, Ookayama 2-12-1, Meguro-ku, Tokyo 152-8550 (Japan); Shi, Xuetao, E-mail: mrshixuetao@gmail.com [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); Ostrovidov, Serge [WPI-Advanced Institute for Materials Research, Tohoku University, Sendai (Japan); Wu, Hongkai, E-mail: chhkwu@ust.hk [Department of Chemistry & Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Nakajima, Ken [Graduate School of Science and Engineering, Tokyo Institute of Technology, Ookayama 2-12-1, Meguro-ku, Tokyo 152-8550 (Japan)

    2016-03-15

    Graphical abstract: - Highlights: • Atomic force microscopy (AFM) was developed to probe stem cell differentiation. • The mechanical properties of stem cells and their ECMs can be used to clearly distinguish specific stem cell-differentiated lineages. • AFM is a facile and useful tool for monitoring stem cell differentiation in a non-invasive manner. - Abstract: A real-time method using atomic force microscopy (AFM) was developed to probe stem cell differentiation by measuring the mechanical properties of cells and the extracellular matrix (ECM). The mechanical properties of stem cells and their ECMs can be used to clearly distinguish specific stem cell-differentiated lineages. It is clear that AFM is a facile and useful tool for monitoring the differentiation of stem cells in a non-invasive manner.

  15. Unique B cell differentiation profile in tolerant kidney transplant patients.

    Science.gov (United States)

    Chesneau, M; Pallier, A; Braza, F; Lacombe, G; Le Gallou, S; Baron, D; Giral, M; Danger, R; Guerif, P; Aubert-Wastiaux, H; Néel, A; Michel, L; Laplaud, D-A; Degauque, N; Soulillou, J-P; Tarte, K; Brouard, S

    2014-01-01

    Operationally tolerant patients (TOL) display a higher number of blood B cells and transcriptional B cell signature. As they rarely develop an allo-immune response, they could display an abnormal B cell differentiation. We used an in vitro culture system to explore T-dependent differentiation of B cells into plasma cells. B cell phenotype, apoptosis, proliferation, cytokine, immunoglobulin production and markers of differentiation were followed in blood of these patients. Tolerant recipients show a higher frequency of CD20(+) CD24(hi) CD38(hi) transitional and CD20(+) CD38(lo) CD24(lo) naïve B cells compared to patients with stable graft function, correlating with a decreased frequency of CD20(-) CD38(+) CD138(+) differentiated plasma cells, suggestive of abnormal B cell differentiation. B cells from TOL proliferate normally but produce more IL-10. In addition, B cells from tolerant recipients exhibit a defective expression of factors of the end step of differentiation into plasma cells and show a higher propensity for cell death apoptosis compared to patients with stable graft function. This in vitro profile is consistent with down-regulation of B cell differentiation genes and anti-apoptotic B cell genes in these patients in vivo. These data suggest that a balance between B cells producing IL-10 and a deficiency in plasma cells may encourage an environment favorable to the tolerance maintenance. © Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.

  16. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... hepatocyte transplantation therapy and toxicity screening in drug discovery. Key words: Embryonic stem cells, hepatic-like cells, in vitro differentiation, sodium butyrate, ... from embryonic stem (ES) cell or induced pluripotent.

  17. [CONDITIONS OF SYNOVIAL MESENCHYMAL STEM CELLS DIFFERENTIATING INTO FIBROCARTILAGE CELLS].

    Science.gov (United States)

    Fu, Peiliang; Cong, Ruijun; Chen, Song; Zhang, Lei; Ding, Zheru; Zhou, Qi; Li, Lintao; Xu, Zhenyu; Wu, Yuli; Wu, Haishan

    2015-01-01

    To explore the conditions of synovial derived mesenchymal stem cells (SMSCs) differentiating into the fibrocartilage cells by using the orthogonal experiment. The synovium was harvested from 5 adult New Zealand white rabbits, and SMSCs were separated by adherence method. The flow cytometry and multi-directional differentiation method were used to identify the SMSCs. The conditions were found from the preliminary experiment and literature review. The missing test was carried out to screen the conditions and then 12 conditions were used for the orthogonal experiment, including transforming growth factor β1 (TGF-β1), bone morphogenic protein 2 (BMP-2), dexamethasone (DEX), proline, ascorbic acid (ASA), pyruvic acid, insulin + transferrin + selenious acid pre-mixed solution (ITS), bovin serum albumin (BSA), basic fibroblast growth factor (bFGF), intermittent hydraulic pressure (IHP), bone morphogenic protein 7 (BMP-7), and insulin-like growth factor (IGF). The L60 (212) orthogonal experiment was designed using the SPSS 18.0 with 2 level conditions and the cells were induced to differentiate on the small intestinal submucosa (SIS)-3D scaffold. The CD151+/CD44+ cells were detected with the flow cytometry and then the differentiation rate was recorded. The immumohistochemical staining, cellular morphology, toluidine blue staining, and semi-quantitative RT-PCR examination for the gene expressions of sex determining region Y (SRY)-box 9 gene (Sox9), aggrecan gene (AGN), collagen type I gene (Col I), collagen type II gene (Col II), collagen type IX gene (Col IX) were used for result confirmation. The differentiation rate was calculated as the product of CD151/CD44+ cells and cells with Col I high expression. The grow curve was detected with the DNA abundance using the PicoGreen Assay. The visual observation and the variances analysis among the variable were used to evaluate the result of the orthogonal experiment, 1 level interaction was considered. The q-test and the

  18. Nanomaterials modulate stem cell differentiation: biological interaction and underlying mechanisms.

    Science.gov (United States)

    Wei, Min; Li, Song; Le, Weidong

    2017-10-25

    Stem cells are unspecialized cells that have the potential for self-renewal and differentiation into more specialized cell types. The chemical and physical properties of surrounding microenvironment contribute to the growth and differentiation of stem cells and consequently play crucial roles in the regulation of stem cells' fate. Nanomaterials hold great promise in biological and biomedical fields owing to their unique properties, such as controllable particle size, facile synthesis, large surface-to-volume ratio, tunable surface chemistry, and biocompatibility. Over the recent years, accumulating evidence has shown that nanomaterials can facilitate stem cell proliferation and differentiation, and great effort is undertaken to explore their possible modulating manners and mechanisms on stem cell differentiation. In present review, we summarize recent progress in the regulating potential of various nanomaterials on stem cell differentiation and discuss the possible cell uptake, biological interaction and underlying mechanisms.

  19. Transplantation and differentiation of donor cells in the cloned pigs

    International Nuclear Information System (INIS)

    Shimada, Arata; Tomii, Ryo; Kano, Koichiro; Nagashima, Hiroshi

    2006-01-01

    The application of nuclear transfer technology is an interesting approach to investigate stem and progenitor cell transplantation therapy. If stem cells are used as a nuclear donor, donor cells can engraft into cloned animals without histocompatible problems. However, it is still uncertain whether donor cells can engraft to cloned animal and differentiate in vivo. To address this problem, we transplanted donor cells to dermal tissues of cloned pigs developed by using preadipocytes as donor cells. Preadipocytes are adipocytic progenitor which can differentiate to mature adipocytes in vitro. We showed that the donor preadipocytes were successfully transplanted into the cloned pigs without immune rejection and they differentiated into mature adipocytes in vivo 3 weeks after transplantation. In contrast, allogenic control preadipocytes, which can differentiate in vitro, did not differentiate in vivo. These results indicate that donor progenitor cells can differentiate in cloned animal

  20. Oligodendrocyte differentiation and implantation : new insights for remyelinating cell therapy

    NARCIS (Netherlands)

    Sher, Falak; Balasubramaniyan, Veerakumar; Boddeke, Erik; Copray, Sjef

    2008-01-01

    Purpose of review Recent research on oligodendrocyte development has yielded new insights on the involvement of morphogens and differentiation factors in oligodendrogenesis. This knowledge has improved strategies to control neural stem cell-derived oligodendrocyte differentiation and functional

  1. Downregulation of rRNA transcription triggers cell differentiation.

    Directory of Open Access Journals (Sweden)

    Yuki Hayashi

    Full Text Available Responding to various stimuli is indispensable for the maintenance of homeostasis. The downregulation of ribosomal RNA (rRNA transcription is one of the mechanisms involved in the response to stimuli by various cellular processes, such as cell cycle arrest and apoptosis. Cell differentiation is caused by intra- and extracellular stimuli and is associated with the downregulation of rRNA transcription as well as reduced cell growth. The downregulation of rRNA transcription during differentiation is considered to contribute to reduced cell growth. However, the downregulation of rRNA transcription can induce various cellular processes; therefore, it may positively regulate cell differentiation. To test this possibility, we specifically downregulated rRNA transcription using actinomycin D or a siRNA for Pol I-specific transcription factor IA (TIF-IA in HL-60 and THP-1 cells, both of which have differentiation potential. The inhibition of rRNA transcription induced cell differentiation in both cell lines, which was demonstrated by the expression of the common differentiation marker CD11b. Furthermore, TIF-IA knockdown in an ex vivo culture of mouse hematopoietic stem cells increased the percentage of myeloid cells and reduced the percentage of immature cells. We also evaluated whether differentiation was induced via the inhibition of cell cycle progression because rRNA transcription is tightly coupled to cell growth. We found that cell cycle arrest without affecting rRNA transcription did not induce differentiation. To the best of our knowledge, our results demonstrate the first time that the downregulation of rRNA levels could be a trigger for the induction of differentiation in mammalian cells. Furthermore, this phenomenon was not simply a reflection of cell cycle arrest. Our results provide a novel insight into the relationship between rRNA transcription and cell differentiation.

  2. Human invariant NKT cell subsets differentially promote differentiation, antibody production, and T cell stimulation by B cells in vitro.

    OpenAIRE

    O'REILLY, VINCENT

    2013-01-01

    PUBLISHED Invariant NK T (iNKT) cells can provide help for B cell activation and Ab production. Because B cells are also capable of cytokine production, Ag presentation, and T cell activation, we hypothesized that iNKT cells will also influence these activities. Furthermore, subsets of iNKT cells based on CD4 and CD8 expression that have distinct functional activities may differentially affect B cell functions. We investigated the effects of coculturing expanded human CD4(+), CD8α(+), and ...

  3. Directional differentiation of chicken embryonic stem cells into ...

    African Journals Online (AJOL)

    Chicken embryonic stem (ES) cells are useful for producing transgenic chickens and preserving genetic material in avian species. In this study, the differentiation potential of chicken ES cells was investigated in vitro. Chicken ES cells were differentiated into osteoblasts cultured for 15 to 21 days in the induction media ...

  4. Primitive human hematopoietic cells give rise to differentially specified daughter cells upon their initial cell division.

    NARCIS (Netherlands)

    Giebel, B.; Zhang, T.; Beckmann, J.; Spanholtz, J.; Wernet, P.; Ho, A.; Punzel, M.

    2006-01-01

    It is often predicted that stem cells divide asymmetrically, creating a daughter cell that maintains the stem-cell capacity, and 1 daughter cell committed to differentiation. While asymmetric stem-cell divisions have been proven to occur in model organisms (eg, in Drosophila), it remains illusive

  5. Symbiotic Cell Differentiation and Cooperative Growth in Multicellular Aggregates.

    Directory of Open Access Journals (Sweden)

    Jumpei F Yamagishi

    2016-10-01

    Full Text Available As cells grow and divide under a given environment, they become crowded and resources are limited, as seen in bacterial biofilms and multicellular aggregates. These cells often show strong interactions through exchanging chemicals, as evident in quorum sensing, to achieve mutualism and division of labor. Here, to achieve stable division of labor, three characteristics are required. First, isogenous cells differentiate into several types. Second, this aggregate of distinct cell types shows better growth than that of isolated cells without interaction and differentiation, by achieving division of labor. Third, this cell aggregate is robust with respect to the number distribution of differentiated cell types. Indeed, theoretical studies have thus far considered how such cooperation is achieved when the ability of cell differentiation is presumed. Here, we address how cells acquire the ability of cell differentiation and division of labor simultaneously, which is also connected with the robustness of a cell society. For this purpose, we developed a dynamical-systems model of cells consisting of chemical components with intracellular catalytic reaction dynamics. The reactions convert external nutrients into internal components for cellular growth, and the divided cells interact through chemical diffusion. We found that cells sharing an identical catalytic network spontaneously differentiate via induction from cell-cell interactions, and then achieve division of labor, enabling a higher growth rate than that in the unicellular case. This symbiotic differentiation emerged for a class of reaction networks under the condition of nutrient limitation and strong cell-cell interactions. Then, robustness in the cell type distribution was achieved, while instability of collective growth could emerge even among the cooperative cells when the internal reserves of products were dominant. The present mechanism is simple and general as a natural consequence of

  6. Non-genetic heterogeneity, criticality and cell differentiation.

    Science.gov (United States)

    Pal, Mainak; Ghosh, Sayantari; Bose, Indrani

    2014-11-27

    The different cell types in a living organism acquire their identity through the process of cell differentiation in which multipotent progenitor cells differentiate into distinct cell types. Experimental evidence and analysis of large-scale microarray data establish the key role played by a two-gene motif in cell differentiation in a number of cell systems. The two genes express transcription factors which repress each other's expression and autoactivate their own production. A number of theoretical models have recently been proposed based on the two-gene motif to provide a physical understanding of how cell differentiation occurs. In this paper, we study a simple model of cell differentiation which assumes no cooperativity in the regulation of gene expression by the transcription factors. The latter repress each other's activity directly through DNA binding and indirectly through the formation of heterodimers. We specifically investigate how deterministic processes combined with stochasticity contribute in bringing about cell differentiation. The deterministic dynamics of our model give rise to a supercritical pitchfork bifurcation from an undifferentiated stable steady state to two differentiated stable steady states. The stochastic dynamics of our model are studied using the approaches based on the Langevin equations and the linear noise approximation. The simulation results provide a new physical understanding of recent experimental observations. We further propose experimental measurements of quantities like the variance and the lag-1 autocorrelation function in protein fluctuations as the early signatures of an approaching bifurcation point in the cell differentiation process.

  7. Correlation between membrane fluidity cellular development and stem cell differentiation

    KAUST Repository

    Noutsi, Bakiza Kamal

    2016-01-01

    Cell membranes are made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as neuronal differentiation, cell membranes undergo dramatic structural

  8. Membrane glycoproteins of differentiating skeletal muscle cells

    International Nuclear Information System (INIS)

    Miller, K.R.; Remy, C.N.; Smith, P.B.

    1987-01-01

    The composition of N-linked glycoprotein oligosaccharides was studied in myoblasts and myotubes of the C2 muscle cell line. Oligosaccharides were radioactively labelled for 15 hr with [ 3 H] mannose and plasma membranes isolated. Ten glycopeptides were detected by SDS-PAGE and fluorography. The extent of labelling was 4-6 fold greater in myoblasts vs myotubes. A glycopeptide of Mr > 100,000 was found exclusively in myoblast membranes. Lectin chromatography revealed that the proportion of tri-, tetranntenary, biantennary and high mannose chains was similar throughout differentiation. The high mannose chain fraction was devoid of hybrid chains. The major high mannose chain contained nine mannose residues. The higher level of glycopeptide labelling in myoblasts vs myotubes corresponded to a 5-fold greater rate of protein synthesis. Pulse-chase experiments were used to follow the synthesis of the Dol-oligosaccharides. Myoblasts and myotubes labelled equivalently the glucosylated tetradecasaccharide but myoblasts labelled the smaller intermediates 3-4 greater than myotubes. Myoblasts also exhibited a 2-3 fold higher Dol-P dependent glycosyl transferase activity for chain elongation and Dol-sugar synthesis. Together these results show that the degree of protein synthesis and level of Dol-P are contributing factors in the higher capacity of myoblasts to produce N-glycoproteins compared to myotubes

  9. Differential marker expression by cultures rich in mesenchymal stem cells

    Science.gov (United States)

    2013-01-01

    Background Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. Results Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers – CD24, CD108 and CD40. Conclusion We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers. PMID:24304471

  10. Schwann cells promote neuronal differentiation of bone marrow ...

    African Journals Online (AJOL)

    It has been suggested that the BMSCs have the capacity to differentiate into neurons under specific experimental conditions, using chemical factors. In this study, we showed that BMSCs can be induced to differentiate into neuron-like cells when they are co-cultured with Schwann cells by Brdu pulse label technology.

  11. Isolation and Osteogenic Differentiation of Rat Periosteum-derived Cells

    OpenAIRE

    Declercq, Heidi Andrea; De Ridder, Leo Isabelle; Cornelissen, Maria Jozefa

    2005-01-01

    Selection of appropriate cultures having an osteogenic potential is a necessity if cell/biomaterial interactions are studied in long-term cultures. Osteoblastic cells derived from rat long bones or calvaria have the disadvantage of being in an advanced differentiation stage which results in terminal differentiation within 21 days. In this regard, less differentiated periosteum-derived osteoprogenitors could be more suitable.

  12. Demethylating agent, 5-azacytidine, reverses differentiation of embryonic stem cells

    International Nuclear Information System (INIS)

    Tsuji-Takayama, Kazue; Inoue, Toshiya; Ijiri, Yoshihiro; Otani, Takeshi; Motoda, Ryuichi; Nakamura, Shuji; Orita, Kunzo

    2004-01-01

    The de novo methylation activity is essential for embryonic development as well as embryonic stem (ES) cell differentiation, where the intensive and extensive DNA methylation was detected. In this study, we investigated the effects of a demethylating agent, 5-azacytidine (5-AzaC), on differentiated ES cells in order to study the possibility of reversing the differentiation process. We first induced differentiation of ES cells by forming embryoid bodies, and then the cells were treated with 5-AzaC. The cells showed some undifferentiated features such as stem cell-like morphology with unclear cell-to-cell boundary and proliferative responsiveness to LIF. Moreover, 5-AzaC increased the expressions of ES specific markers, SSEA-1, and alkaline phosphatase activity as well as ES specific genes, Oct4, Nanog, and Sox2. We also found that 5-AzaC demethylated the promoter region of H19 gene, a typical methylated gene during embryonic differentiation. These results indicate that 5-AzaC reverses differentiation state of ES cells through its DNA demethylating activity to differentiation related genes

  13. Hepatic differentiation potential of commercially available human mesenchymal stem cells.

    Science.gov (United States)

    Ong, Shin-Yeu; Dai, Hui; Leong, Kam W

    2006-12-01

    The ready availability and low immunogenicity of commercially available mesenchymal stem cells (MSC) render them a potential cell source for the development of therapeutic products. With cell source a major bottleneck in hepatic tissue engineering, we investigated whether commercially available human MSC (hMSC) can transdifferentiate into the hepatic lineage. Based on previous studies that find rapid gain of hepatic genes in bone marrow-derived stem cells cocultured with liver tissue, we used a similar approach to drive hepatic differentiation by coculturing the hMSC with rat livers treated or untreated with gadolinium chloride (GdCl(3)). After a 24-hour coculture period with liver tissue injured by GdCl(3) in a Transwell configuration, approximately 34% of the cells differentiated into albumin-expressing cells. Cocultured cells were subsequently maintained with growth factors to complete the hepatic differentiation. Cocultured cells expressed more hepatic gene markers, and had higher metabolic functions and P450 activity than cells that were only differentiated with growth factors. In conclusion, commercially available hMSC do show hepatic differentiation potential, and a liver microenvironment in culture can provide potent cues to accelerate and deepen the differentiation. The ability to generate hepatocyte-like cells from a commercially available cell source would find interesting applications in liver tissue engineering.

  14. Two pore channel 2 differentially modulates neural differentiation of mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Zhe-Hao Zhang

    Full Text Available Nicotinic acid adenine dinucleotide phosphate (NAADP is an endogenous Ca(2+ mobilizing nucleotide presented in various species. NAADP mobilizes Ca(2+ from acidic organelles through two pore channel 2 (TPC2 in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.

  15. NOV/CCN3 impairs muscle cell commitment and differentiation

    International Nuclear Information System (INIS)

    Calhabeu, Frederico; Lafont, Jerome; Le Dreau, Gwenvael; Laurent, Maryvonne; Kazazian, Chantal; Schaeffer, Laurent; Martinerie, Cecile; Dubois, Catherine

    2006-01-01

    NOV (nephroblastoma overexpressed) is a member of a family of proteins which encodes secreted matrix-associated proteins. NOV is expressed during development in dermomyotome and limb buds, but its functions are still poorly defined. In order to understand the role of NOV in myogenic differentiation, C2C12 cells overexpressing NOV (C2-NOV) were generated. These cells failed to engage into myogenic differentiation, whereas they retained the ability to differentiate into osteoblasts. In differentiating conditions, C2-NOV cells remained proliferative, failed to express differentiation markers and lost their ability to form myotubes. Inhibition of differentiation by NOV was also observed with human primary muscle cells. Further examination of C2-NOV cells revealed a strong downregulation of the myogenic determination genes MyoD and Myf5 and of IGF-II expression. MyoD forced expression in C2-NOV was sufficient to restore differentiation and IGF-II induction whereas 10 -6 M insulin treatment had no effects. NOV therefore acts upstream of MyoD and does not affect IGF-II induction and signaling. HES1, a target of Notch, previously proposed to mediate NOV action, was not implicated in the inhibition of differentiation. We propose that NOV is a specific cell fate regulator in the myogenic lineage, acting negatively on key myogenic genes thus controlling the transition from progenitor cells to myoblasts

  16. Diclofenac and triamcinolone acetonide impair tenocytic differentiation and promote adipocytic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Fredriksson, Maritha; Li, Yan; Stålman, Anders; Haldosén, Lars-Arne; Felländer-Tsai, Li

    2013-09-02

    Tendinopathies are often empirically treated with oral/topical nonsteroidal anti-inflammatory medications and corticosteroid injections despite their unclear effects on tendon regeneration. Recent studies indicate that tendon progenitors exhibit stem cell-like properties, i.e., differentiation to osteoblasts, adipocytes, and chondrocytes, in addition to tenocytes. Our present study aims at understanding the effects of triamcinolone acetonide and diclofenac on tenocytic differentiation of mesenchymal stem cells. The murine fibroblast C3H10T1/2 cell line was induced to tenocytic differentiation by growth differentiation factor-7. Cell proliferation and differentiation with the exposure of different concentrations of triamcinolone acetonide and diclofenac were measured by WST-1 assay and real-time polymerase chain reaction analysis, respectively. Cell proliferation was decreased in a concentration-dependent manner when exposed to triamcinolone acetonide and diclofenac. In addition to tenocytic differentiation, adipocyte formation was observed, both at gene expression and microscopic level, when the cells were exposed to triamcinolone acetonide or high concentrations of diclofenac. Our results indicate that triamcinolone acetonide and diclofenac might alter mesenchymal stem cell differentiation in a nonfavorable way regarding tendon regeneration; therefore, these medications should be used with more caution clinically.

  17. Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons

    Directory of Open Access Journals (Sweden)

    Vitor Fortuna

    2015-06-01

    Full Text Available The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs develop in close proximity to the dorsal aorta (DA and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA differentiation of SN precursors temporally coincides with vascular mural cell (VMC recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation.

  18. Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons.

    Science.gov (United States)

    Fortuna, Vitor; Pardanaud, Luc; Brunet, Isabelle; Ola, Roxana; Ristori, Emma; Santoro, Massimo M; Nicoli, Stefania; Eichmann, Anne

    2015-06-23

    The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Early stage differentiation of thallus cells of Porphyra haitanensis (Rhodophyta)

    Science.gov (United States)

    Wang, Sujuan; Sun, Yunlong; Lu, Anming; Wang, Guangyuan

    1987-09-01

    The early stage differentiation of thallus cells of Porphyra haitanensis T. J. Chang et B. F. Zheng was studied. Protoplasts or single cells were isolated from the blades using enzyme mixture comprising 2% sea snail gut enzyme and 1% cellulase. The isolated protoplasts or single cells were incubated in the MES medium. The cell differentiations were examined under the microscope at intervals after incubation. Four types of cell differentiation, namely, normal, abnormal, carposporangial and spermatorangial, and rhizoidal types, were observed. Since normal cell differentiations occur mostly in small thalli 50 mm in length and middle portions of big thalli 200 mm in length, it is essential to select tissues from these two kinds of thalli essential for commercial production.

  20. Differentiation of stem cells upon deprivation of exogenous FGF2

    DEFF Research Database (Denmark)

    Kjartansdóttir, Kristín Rós; Gabrielsen, Anette; Reda, Ahmed

    2012-01-01

    Establishing a model for in vitro differentiation of human embryonic stem cells (hESCs) towards the germ cell lineage could be used to identify molecular mechanisms behind germ cell differentiation that may help in understanding human infertility. Here, we evaluate whether a lack of exogenous...... fibroblast growth factor 2 (FGF2) is supporting spontaneous differentiation of hESCs cultured on human foreskin fibroblast (hFF) monolayers towards germ cell lineage. Additionally to depriving the hESCs of exogenous FGF2, cells were stimulated with all-trans retinoic acid (ATRA). To get a more comprehensive...... impression on effects of removal of FGF2 and stimulation with ATRA, we combined the results of three cell lines for each experimental setting. When combining gene expression profiles of three cell lines for 96 genes, only 6 genes showed a significant up-regulation in all cell lines, when no FGF2 was added...

  1. Epigenetic control of CD8+ T cell differentiation.

    Science.gov (United States)

    Henning, Amanda N; Roychoudhuri, Rahul; Restifo, Nicholas P

    2018-05-01

    Upon stimulation, small numbers of naive CD8 + T cells proliferate and differentiate into a variety of memory and effector cell types. CD8 + T cells can persist for years and kill tumour cells and virally infected cells. The functional and phenotypic changes that occur during CD8 + T cell differentiation are well characterized, but the epigenetic states that underlie these changes are incompletely understood. Here, we review the epigenetic processes that direct CD8 + T cell differentiation and function. We focus on epigenetic modification of DNA and associated histones at genes and their regulatory elements. We also describe structural changes in chromatin organization that affect gene expression. Finally, we examine the translational potential of epigenetic interventions to improve CD8 + T cell function in individuals with chronic infections and cancer.

  2. In vitro germ cell differentiation from cynomolgus monkey embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kaori Yamauchi

    Full Text Available BACKGROUND: Mouse embryonic stem (ES cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. METHODS AND FINDINGS: To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis. VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. CONCLUSION: VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the

  3. Id2 reinforces TH1 cell differentiation and inhibits E2A to repress TFH cell differentiation

    Science.gov (United States)

    Shaw, Laura A.; Bélanger, Simon; Omilusik, Kyla D.; Cho, Sunglim; Scott-Browne, James P.; Nance, J. Philip; Goulding, John; Lasorella, Anna; Lu, Li-Fan; Crotty, Shane; Goldrath, Ananda W.

    2016-01-01

    Differentiation of T helper (TH) effector subsets is critical for host protection. E protein transcription factors and Id proteins are important arbiters of T cell development, but their role in differentiation of TH1 and TFH cells is not well understood. TH1 cells showed robust Id2 expression compared to TFH cells, and RNAi depletion of Id2 increased TFH cell frequencies. Further, TH1 cell differentiation was blocked by Id2 deficiency, leading to E protein-dependent accumulation of effector cells with mixed characteristics during viral infection and severely impaired generation of TH1 cells following Toxoplasma gondii infection. The TFH-defining transcriptional repressor Bcl6 bound the Id2 locus, providing a mechanism for the bimodal Id2 expression and reciprocal development of TH1 and TFH cell fates. PMID:27213691

  4. Hypercholesterolemia Induces Differentiation of Regulatory T Cells in the Liver.

    Science.gov (United States)

    Mailer, Reiner K W; Gisterå, Anton; Polyzos, Konstantinos A; Ketelhuth, Daniel F J; Hansson, Göran K

    2017-05-26

    The liver is the central organ that responds to dietary cholesterol intake and facilitates the release and clearance of lipoprotein particles. Persistent hypercholesterolemia leads to immune responses against lipoprotein particles that drive atherosclerosis. However, the effect of hypercholesterolemia on hepatic T-cell differentiation remains unknown. To investigate hepatic T-cell subsets upon hypercholesterolemia. We observed that hypercholesterolemia elevated the intrahepatic regulatory T (Treg) cell population and increased the expression of transforming growth factor-β1 in the liver. Adoptive transfer experiments revealed that intrahepatically differentiated Treg cells relocated to the inflamed aorta in atherosclerosis-prone low-density lipoprotein receptor deficient ( Ldlr -/- ) mice. Moreover, hypercholesterolemia induced the differentiation of intrahepatic, but not intrasplenic, Th17 cells in wild-type mice, whereas the disrupted liver homeostasis in hypercholesterolemic Ldlr -/- mice led to intrahepatic Th1 cell differentiation and CD11b + CD11c + leukocyte accumulation. Our results elucidate a new mechanism that controls intrahepatic T-cell differentiation during atherosclerosis development and indicates that intrahepatically differentiated T cells contribute to the CD4 + T-cell pool in the atherosclerotic aorta. © 2017 American Heart Association, Inc.

  5. [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

  6. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.

    Science.gov (United States)

    Chen, Li; Hu, Huimin; Qiu, Weimin; Shi, Kaikai; Kassem, Moustapha

    2018-05-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Identification of transcript regulatory patterns in cell differentiation.

    Science.gov (United States)

    Gusnanto, Arief; Gosling, John Paul; Pope, Christopher

    2017-10-15

    Studying transcript regulatory patterns in cell differentiation is critical in understanding its complex nature of the formation and function of different cell types. This is done usually by measuring gene expression at different stages of the cell differentiation. However, if the gene expression data available are only from the mature cells, we have some challenges in identifying transcript regulatory patterns that govern the cell differentiation. We propose to exploit the information of the lineage of cell differentiation in terms of correlation structure between cell types. We assume that two different cell types that are close in the lineage will exhibit many common genes that are co-expressed relative to those that are far in the lineage. Current analysis methods tend to ignore this correlation by testing for differential expression assuming some sort of independence between cell types. We employ a Bayesian approach to estimate the posterior distribution of the mean of expression in each cell type, by taking into account the cell formation path in the lineage. This enables us to infer genes that are specific in each cell type, indicating the genes are involved in directing the cell differentiation to that particular cell type. We illustrate the method using gene expression data from a study of haematopoiesis. R codes to perform the analysis are available in http://www1.maths.leeds.ac.uk/∼arief/R/CellDiff/. a.gusnanto@leeds.ac.uk. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  8. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...... at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...

  9. Lactobacilli Differentially Activate Natural Killer Cells

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Christensen, Hanne Risager; Frøkiær, Hanne

    bacteria on regulatory functions of NK-cells. Here, we have investigated how human gut flora-derived non-pathogenic lactobacilli affect NK cells in vitro, by measuring proliferation and IFN-gamma production of human peripheral blood NK cells upon bacterial stimulation. CD3-CD56+ NK cells were isolated from...... having engulfed bacteria, stimulated the growth of the NK cells. In contrast, a Lactobacillus paracasei strain caused the NK cells to proliferate only in the presence of monocytes. These results demonstrate that various lactobacilli have the capacity to activate NK cells in vitro, in a monocyte dependent...

  10. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Hu, Huimin; Qiu, Weimin

    2018-01-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined...... the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte...

  11. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Ozcan

    2011-01-01

    in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...... at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...

  12. Differential TCR signals for T helper cell programming.

    Science.gov (United States)

    Morel, Penelope A

    2018-05-02

    Upon encounter with their cognate antigen naïve CD4 T cells become activated and are induced to differentiate into several possible T helper (Th) cell subsets. This differentiation depends on a number of factors including antigen presenting cells, cytokines and costimulatory molecules. The strength of the T cell receptor (TCR) signal, related to the affinity of TCR for antigen and antigen dose, has emerged as a dominant factor in determining Th cell fate. Recent studies have revealed that TCR signals of high or low strength do not simply induce quantitatively different signals in the T cells, but rather qualitatively distinct pathways can be induced based on TCR signal strength. This review examines the recent literature in this area and highlights important new developments in our understanding of Th cell differentiation and TCR signal strength. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  13. Conceptual Challenges of the Systemic Approach in Understanding Cell Differentiation.

    Science.gov (United States)

    Paldi, Andras

    2018-01-01

    The cells of a multicellular organism are derived from a single zygote and genetically identical. Yet, they are phenotypically very different. This difference is the result of a process commonly called cell differentiation. How the phenotypic diversity emerges during ontogenesis or regeneration is a central and intensely studied but still unresolved issue in biology. Cell biology is facing conceptual challenges that are frequently confused with methodological difficulties. How to define a cell type? What stability or change means in the context of cell differentiation and how to deal with the ubiquitous molecular variations seen in the living cells? What are the driving forces of the change? We propose to reframe the problem of cell differentiation in a systemic way by incorporating different theoretical approaches. The new conceptual framework is able to capture the insights made at different levels of cellular organization and considered previously as contradictory. It also provides a formal strategy for further experimental studies.

  14. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yoon Jung [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Lee, Jue Yeon [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Lee, Seung Jin [Department of Industrial Pharmacy, College of Pharmacy, Ewha Womans University, Seoul (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Chung, Chong-Pyoung [Department of Periodontology, School of Dentistry, Seoul National University, Seoul (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Park, Yoon Jeong, E-mail: parkyj@snu.ac.kr [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Doxazocin directly up-regulated bone metabolism at a low dose. Black-Right-Pointing-Pointer Doxazocin induced osteoblastic stem cell differentiation without affecting cell proliferation. Black-Right-Pointing-Pointer This osteogenic stem cell differentiation is mediated by ERK-signal dependent pathway. -- Abstract: Recent researches have indicated a role for antihypertensive drugs including alpha- or beta-blockers in the prevention of bone loss. Some epidemiological studies reported the protective effects of those agents on fracture risk. However, there is limited information on the association with those agents especially at the mechanism of action. In the present study, we investigated the effects of doxazosin, an alpha-blocker that is clinically used for the treatment of benign prostatic hyperplasia (BPH) along with antihypertensive medication, on the osteogenic stem cell differentiation. We found that doxazosin increased osteogenic differentiation of human mesenchymal stem cells, detected by Alizarin red S staining and calcein. Doxazosin not only induced expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin, it also resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK1/2), a MAP kinase involved in osteoblastic differentiation. Treatment with U0126, a MAP kinase inhibitor, significantly blocked doxazosin-induced osteoblastic differentiation. Unrelated to activation of osteogenic differentiation by doxazosin, we found that there were no significant changes in adipogenic differentiation or in the expression of adipose-specific genes, including peroxisome proliferator-activated receptor {gamma}, aP2, or LPL. In this report, we suggest that doxazosin has the ability to increase osteogenic cell differentiation via ERK1/2 activation in osteogenic differentiation of adult stem cells, which supports the protective effects of antihypertensive drug on fracture risk and

  15. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

    International Nuclear Information System (INIS)

    Choi, Yoon Jung; Lee, Jue Yeon; Lee, Seung Jin; Chung, Chong-Pyoung; Park, Yoon Jeong

    2011-01-01

    Highlights: ► Doxazocin directly up-regulated bone metabolism at a low dose. ► Doxazocin induced osteoblastic stem cell differentiation without affecting cell proliferation. ► This osteogenic stem cell differentiation is mediated by ERK-signal dependent pathway. -- Abstract: Recent researches have indicated a role for antihypertensive drugs including alpha- or beta-blockers in the prevention of bone loss. Some epidemiological studies reported the protective effects of those agents on fracture risk. However, there is limited information on the association with those agents especially at the mechanism of action. In the present study, we investigated the effects of doxazosin, an alpha-blocker that is clinically used for the treatment of benign prostatic hyperplasia (BPH) along with antihypertensive medication, on the osteogenic stem cell differentiation. We found that doxazosin increased osteogenic differentiation of human mesenchymal stem cells, detected by Alizarin red S staining and calcein. Doxazosin not only induced expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin, it also resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK1/2), a MAP kinase involved in osteoblastic differentiation. Treatment with U0126, a MAP kinase inhibitor, significantly blocked doxazosin-induced osteoblastic differentiation. Unrelated to activation of osteogenic differentiation by doxazosin, we found that there were no significant changes in adipogenic differentiation or in the expression of adipose-specific genes, including peroxisome proliferator-activated receptor γ, aP2, or LPL. In this report, we suggest that doxazosin has the ability to increase osteogenic cell differentiation via ERK1/2 activation in osteogenic differentiation of adult stem cells, which supports the protective effects of antihypertensive drug on fracture risk and according to our data doxazosin might be useful for application in the field of bone

  16. Division of Labor in Biofilms: the Ecology of Cell Differentiation.

    Science.gov (United States)

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    2015-04-01

    The dense aggregation of cells on a surface, as seen in biofilms, inevitably results in both environmental and cellular heterogeneity. For example, nutrient gradients can trigger cells to differentiate into various phenotypic states. Not only do cells adapt physiologically to the local environmental conditions, but they also differentiate into cell types that interact with each other. This allows for task differentiation and, hence, the division of labor. In this article, we focus on cell differentiation and the division of labor in three bacterial species: Myxococcus xanthus, Bacillus subtilis, and Pseudomonas aeruginosa. During biofilm formation each of these species differentiates into distinct cell types, in some cases leading to cooperative interactions. The division of labor and the cooperative interactions between cell types are assumed to yield an emergent ecological benefit. Yet in most cases the ecological benefits have yet to be elucidated. A notable exception is M. xanthus, in which cell differentiation within fruiting bodies facilitates the dispersal of spores. We argue that the ecological benefits of the division of labor might best be understood when we consider the dynamic nature of both biofilm formation and degradation.

  17. Molecular biological features of male germ cell differentiation

    Science.gov (United States)

    HIROSE, MIKA; TOKUHIRO, KEIZO; TAINAKA, HITOSHI; MIYAGAWA, YASUSHI; TSUJIMURA, AKIRA; OKUYAMA, AKIHIKO; NISHIMUNE, YOSHITAKE

    2007-01-01

    Somatic cell differentiation is required throughout the life of a multicellular organism to maintain homeostasis. In contrast, germ cells have only one specific function; to preserve the species by conveying the parental genes to the next generation. Recent studies of the development and molecular biology of the male germ cell have identified many genes, or isoforms, that are specifically expressed in the male germ cell. In the present review, we consider the unique features of male germ cell differentiation. (Reprod Med Biol 2007; 6: 1–9) PMID:29699260

  18. Differentiation of Dental Pulp Stem Cells into Neuron-Like Cells in Serum-Free Medium

    Directory of Open Access Journals (Sweden)

    Shahrul Hisham Zainal Ariffin

    2013-01-01

    Full Text Available Dental pulp tissue contains dental pulp stem cells (DPSCs. Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146+, Cd166+, and Cd31− in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

  19. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Hu, Huimin; Qiu, Weimin

    2018-01-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined...... differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte...... differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating h...

  20. Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation.

    Science.gov (United States)

    Martin, E C; Qureshi, A T; Llamas, C B; Burow, M E; King, A G; Lee, O C; Dasa, V; Freitas, M A; Forsberg, J A; Elster, E A; Davis, T A; Gimble, J M

    2018-02-07

    Stromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.

  1. Differentiation ability of rat postnatal dental pulp cells in vitro.

    NARCIS (Netherlands)

    Zhang, W.; Walboomers, X.F.; Wolke, J.G.C.; Bian, Z.; Fan, M.W.; Jansen, J.A.

    2005-01-01

    The current rapid progression in stem cell research has enhanced our knowledge of dental tissue regeneration. In this study, rat dental pulp cells were isolated and their differentiation ability was evaluated. First, dental pulp cells were obtained from maxillary incisors of male Wistar rats.

  2. Division of labor in biofilms : The ecology of cell differentiation

    NARCIS (Netherlands)

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    The dense aggregation of cells on a surface, as seen in biofilms, inevitably results in both environmental and cellular heterogeneity. For example, nutrient gradients can trigger cells to differentiate into various phenotypic states. Not only do cells adapt physiologically to the local environmental

  3. Pathways in pluripotency and differentiation of embryonic cells

    NARCIS (Netherlands)

    du Puy, L.

    2010-01-01

    Pluripotency - the potential to differentiate into derivatives of the three embryonic germ layers endoderm, ectoderm and mesoderm - is the main characteristic of embryonic stem (ES) cells. ES cells are derived from the inner cell mass (ICM) of a pre-implantation blastocyst and can self-renew

  4. Changes in total and differential white cell counts, total lymphocyte ...

    African Journals Online (AJOL)

    Background: Published reports on the possible changes in the various immune cell populations, especially the total lymphocyte and CD4 cell counts, during the menstrual cycle in Nigerian female subjects are relatively scarce. Aim: To determine possible changes in the total and differential white blood cell [WBC] counts, ...

  5. In vitro evaluation of three-dimensional single-walled carbon nanotube composites for bone tissue engineering.

    Science.gov (United States)

    Gupta, Ashim; Main, Benjamin J; Taylor, Brittany L; Gupta, Manu; Whitworth, Craig A; Cady, Craig; Freeman, Joseph W; El-Amin, Saadiq F

    2014-11-01

    The purpose of this study was to develop three-dimensional single-walled carbon nanotube composites (SWCNT/PLAGA) using 10-mg single-walled carbon nanotubes (SWCNT) for bone regeneration and to determine the mechanical strength of the composites, and to evaluate the interaction of MC3T3-E1 cells via cell adhesion, growth, survival, proliferation, and gene expression. PLAGA (polylactic-co-glycolic acid) and SWCNT/PLAGA microspheres and composites were fabricated, characterized, and mechanical testing was performed. MC3T3-E1 cells were seeded and cell adhesion/morphology, growth/survival, proliferation, and gene expression analysis were performed to evaluate biocompatibility. Imaging studies demonstrated microspheres with uniform shape and smooth surfaces, and uniform incorporation of SWCNT into PLAGA matrix. The microspheres bonded in a random packing manner while maintaining spacing, thus resembling trabeculae of cancellous bone. Addition of SWCNT led to greater compressive modulus and ultimate compressive strength. Imaging studies revealed that MC3T3-E1 cells adhered, grew/survived, and exhibited normal, nonstressed morphology on the composites. SWCNT/PLAGA composites exhibited higher cell proliferation rate and gene expression compared with PLAGA. These results demonstrate the potential of SWCNT/PLAGA composites for musculoskeletal regeneration, for bone tissue engineering, and are promising for orthopedic applications as they possess the combined effect of increased mechanical strength, cell proliferation, and gene expression. © 2014 Wiley Periodicals, Inc.

  6. Pituitary cell differentiation from stem cells and other cells: toward restorative therapy for hypopituitarism?

    Science.gov (United States)

    Willems, Christophe; Vankelecom, Hugo

    2014-01-01

    The pituitary gland, key regulator of our endocrine system, produces multiple hormones that steer essential physiological processes. Hence, deficient pituitary function (hypopituitarism) leads to severe disorders. Hypopituitarism can be caused by defective embryonic development, or by damage through tumor growth/resection and traumatic brain injury. Lifelong hormone replacement is needed but associated with significant side effects. It would be more desirable to restore pituitary tissue and function. Recently, we showed that the adult (mouse) pituitary holds regenerative capacity in which local stem cells are involved. Repair of deficient pituitary may therefore be achieved by activating these resident stem cells. Alternatively, pituitary dysfunction may be mended by cell (replacement) therapy. The hormonal cells to be transplanted could be obtained by (trans-)differentiating various kinds of stem cells or other cells. Here, we summarize the studies on pituitary cell regeneration and on (trans-)differentiation toward hormonal cells, and speculate on restorative therapies for pituitary deficiency.

  7. Differential radiosensitivity among B cell subpopulations

    International Nuclear Information System (INIS)

    Riggs, J.E.

    1988-01-01

    The selective radiosensitivity of sIgM >> sIgD marginal zone B cells is associated with the selective loss of B cell function. The simultaneous restoration of impaired function and recovery of these cells with time supports this premise. B cell recovery, delayed one week after irradiation, is in progress at two weeks, and virtually complete by three weeks. XID mice reveal similar recovery kinetics although there are fewer recovering cells and these bear reduced levels of Ia. This observation represents additional evidence that xid B cells are distinct from those of normal mice. The simultaneous loss, and concurrent recovery, of sIgM >> sIgD B cells and TI-2 responsiveness in irradiated mice suggests the existence of a unique B cell subpopulation possessing both phenotypes. Additional support for this hypothesis is provided by demonstrating that splenocytes, depleted of IgD + cells adoptively reconstitute this response in XID mice. The peritoneal B cell pool, which, compared to the spleen, consist of increased numbers of sIgM >> sIgD B cells, is shown to be a source of radiosensitive B cells that are TI-2 responsive. These observations represent additional evidence for an association between sIgM >> sIgD B cells and TI-2 responsiveness

  8. [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.

  9. Chemical strategies for pancreatic β cell differentiation, reprogramming, and regeneration.

    Science.gov (United States)

    Ma, Xiaojie; Zhu, Saiyong

    2017-04-01

    Generation of unlimited functional pancreatic β cells is critical for the study of pancreatic biology and treatment of diabetes mellitus. Recent advances have suggested several promising directions, including directed differentiation of pancreatic β cells from pluripotent stem cells, reprogramming of pancreatic β cells from other types of somatic cells, and stimulated proliferation and enhanced functions of existing pancreatic β cells. Small molecules are useful in generating unlimited numbers of functional pancreatic cells in vitro and could be further developed as drugs to stimulate endogenous pancreatic regeneration. Here, we provide an updated summary of recent major achievements in pancreatic β cell differentiation, reprogramming, proliferation, and function. These studies will eventually lead to significant advances in the field of pancreatic biology and regeneration. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Cell differentiation and matrix organization in engineered teeth.

    Science.gov (United States)

    Nait Lechguer, A; Couble, M L; Labert, N; Kuchler-Bopp, S; Keller, L; Magloire, H; Bleicher, F; Lesot, H

    2011-05-01

    Embryonic dental cells were used to check a series of criteria to be achieved for tooth engineering. Implantation of cultured cell-cell re-associations led to crown morphogenesis, epithelial histogenesis, organ vascularization, and root and periodontium development. The present work aimed to investigate the organization of predentin/dentin, enamel, and cementum which formed and mineralized after implantation. These implants were processed for histology, transmission electron microscopy, x-ray microanalysis, and electron diffraction. After two weeks of implantation, the re-associations showed gradients of differentiating odontoblasts. There were ciliated, polarized, and extended cell processes in predentin/dentin. Ameloblasts became functional. Enamel crystals showed a typical oriented arrangement in the inner and outer enamel. In the developing root, odontoblasts differentiated, cementogenesis occurred, and periodontal ligament fibroblasts interacted with the root surface and newly formed bone. The implantation of cultured dental cell re-associations allows for reproduction of complete functional differentiation at the cell, matrix, and mineral levels.

  11. Differentiation of Spermatogonia Stem Cells into Functional Mature Neurons Characterized with Differential Gene Expression.

    Science.gov (United States)

    Bojnordi, Maryam Nazm; Azizi, Hossein; Skutella, Thomas; Movahedin, Mansoureh; Pourabdolhossein, Fereshteh; Shojaei, Amir; Hamidabadi, Hatef Ghasemi

    2017-09-01

    Transplantation of embryonic stem cells (ESCs) is a promising therapeutic approach for the treatment of neurodegenerative diseases. However, ESCs are not usable clinically due to immunological and ethical limitations. The identification of an alternative safe cell source opens novel options via autologous transplantation in neuro-regeneration circumventing these problems. Here, we examined the neurogenic capacity of embryonic stem-like cells (ES-like cells) derived from the testis using neural growth factor inducers and utilized them to generate functional mature neurons. The neuronal differentiation of ES-like cells is induced in three stages. Stage 1 is related to embryoid body (EB) formation. To induce neuroprogenitor cells, EBs were cultured in the presence of retinoic acid, N 2 supplement and fibroblast growth factor followed by culturing in a neurobasal medium containing B 27 , N 2 supplements for additional 10 days, to allow the maturation and development of neuronal progenitor cells. The neurogenic differentiation was confirmed by immunostaining for markers of mature neurons. The differentiated neurons were positive for Tuj1 and Tau1. Real-time PCR dates indicated the expression of Nestin and Neuro D (neuroprogenitor markers) in induced cells at the second stage of the differentiation protocol. The differentiated mature neurons exhibited the specific neuron markers Map2 and β-tubulin. The functional maturity of neurons was confirmed by an electrophysiological analysis of passive and active neural membrane properties. These findings indicated a differentiation capacity of ES-like cells derived from the testis to functionally mature neurons, which proposes them as a novel cell source for neuroregenerative medicine.

  12. Electrospun bioactive mats enriched with Ca-polyphosphate/retinol nanospheres as potential wound dressing

    OpenAIRE

    Müller, Werner E.G.; Tolba, Emad; Dorweiler, Bernhard; Schröder, Heinz C.; Diehl-Seifert, Bärbel; Wang, Xiaohong

    2015-01-01

    Background While electrospun materials have been frequently used in tissue engineering no wound dressings exist that significantly improved wound healing effectively. Methods We succeeded to fabricate three-dimensional (3D) electrospun poly(D,l-lactide) (PLA) fiber mats into which nanospheres, formed from amorphous calcium polyphosphate (polyP) nanoparticles (NP) and encapsulated retinol (“retinol/aCa-polyP-NS” nanospheres [NS]), had been incorporated. Results Experiments with MC3T3-E1 cells ...

  13. 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.

  14. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    Directory of Open Access Journals (Sweden)

    Li Chen

    2018-05-01

    Full Text Available Human stromal stem cells (hMSCs differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs: Cofilin 1 (CFL1 and Destrin (DSTN or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4. In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1 which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Keywords: Actin cytoskeleton, Actin depolymerizing factors, Adipocyte differentiation, Human stromal stem cells

  15. Current perspectives in Set7 mediated stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Nazanin Karimnia

    2016-12-01

    Full Text Available Set7 is a key regulatory enzyme involved in the methylation of lysine residues of histone and non-histone proteins. This lysine methyltransferase is induced during stem cell differentiation and regulates lineage specific gene transcription and cell fate. In this article we discuss recent experimental evidence identifying regulatory targets under the control of Set7 as well as emerging evidence of regulation in stem cell differentiation. Furthermore, we discuss the function of non-coding RNAs regulated by Set7 implicated in cell plasticity.

  16. In vitro differentiation of primordial germ cells and oocyte-like cells from stem cells.

    Science.gov (United States)

    Costa, José J N; Souza, Glaucinete B; Soares, Maria A A; Ribeiro, Regislane P; van den Hurk, Robert; Silva, José R V

    2018-02-01

    Infertility is the result of failure due to an organic disorder of the reproductive organs, especially their gametes. Recently, much progress has been made on generating germ cells, including oocytes, from various types of stem cells. This review focuses on advances in female germ cell differentiation from different kinds of stem cells, with emphasis on embryonic stem cells, adult stem cells, and induced pluripotent stem cells. The advantages and disadvantages of the derivation of female germ cells from several types of stem cells are also highlighted, as well as the ability of stem cells to generate mature and functional female gametes. This review shows that stem cell therapies have opened new frontiers in medicine, especially in the reproductive area, with the possibility of regenerating fertility.

  17. Redox environment in stem and differentiated cells: A quantitative approach

    Directory of Open Access Journals (Sweden)

    O.G. Lyublinskaya

    2017-08-01

    Full Text Available Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H2DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed. Keywords: Embryonic stem cells, Differentiated cells, ROS, Redox status, H2DCFDA, HyPer, Flow cytometry, Quantitative redox biology

  18. Engineering kidney cells: reprogramming and directed differentiation to renal tissues.

    Science.gov (United States)

    Kaminski, Michael M; Tosic, Jelena; Pichler, Roman; Arnold, Sebastian J; Lienkamp, Soeren S

    2017-07-01

    Growing knowledge of how cell identity is determined at the molecular level has enabled the generation of diverse tissue types, including renal cells from pluripotent or somatic cells. Recently, several in vitro protocols involving either directed differentiation or transcription-factor-based reprogramming to kidney cells have been established. Embryonic stem cells or induced pluripotent stem cells can be guided towards a kidney fate by exposing them to combinations of growth factors or small molecules. Here, renal development is recapitulated in vitro resulting in kidney cells or organoids that show striking similarities to mammalian embryonic nephrons. In addition, culture conditions are also defined that allow the expansion of renal progenitor cells in vitro. Another route towards the generation of kidney cells is direct reprogramming. Key transcription factors are used to directly impose renal cell identity on somatic cells, thus circumventing the pluripotent stage. This complementary approach to stem-cell-based differentiation has been demonstrated to generate renal tubule cells and nephron progenitors. In-vitro-generated renal cells offer new opportunities for modelling inherited and acquired renal diseases on a patient-specific genetic background. These cells represent a potential source for developing novel models for kidney diseases, drug screening and nephrotoxicity testing and might represent the first steps towards kidney cell replacement therapies. In this review, we summarize current approaches for the generation of renal cells in vitro and discuss the advantages of each approach and their potential applications.

  19. Insulin redirects differentiation from cardiogenic mesoderm and endoderm to neuroectoderm in differentiating human embryonic stem cells.

    NARCIS (Netherlands)

    Freund, C.M.A.H.; Ward-van Oostwaard, D.; Monshouwer-Kloots, J.; van den Brink, S.; van Rooijen, M.A.; Xu, X.; Zweigerdt, R.; Mummery, C.L.; Passier, R.

    2008-01-01

    Human embryonic stem cells (hESC) can proliferate indefinitely while retaining the capacity to form derivatives of all three germ layers. We have reported previously that hESC differentiate into cardiomyocytes when cocultured with a visceral endoderm-like cell line (END-2). Insulin/insulin-like

  20. Cancer stem cell markers in patterning differentiation and in prognosis of oral squamous cell carcinoma.

    Science.gov (United States)

    Mohanta, Simple; Siddappa, Gangotri; Valiyaveedan, Sindhu Govindan; Dodda Thimmasandra Ramanjanappa, Ravindra; Das, Debashish; Pandian, Ramanan; Khora, Samanta Sekhar; Kuriakose, Moni Abraham; Suresh, Amritha

    2017-06-01

    Differentiation is a major histological parameter determining tumor aggressiveness and prognosis of the patient; cancer stem cells with their slow dividing and undifferentiated nature might be one of the factors determining the same. This study aims to correlate cancer stem cell markers (CD44 and CD147) with tumor differentiation and evaluate their subsequent effect on prognosis. Immunohistochemical analysis in treatment naïve oral cancer patients (n = 53) indicated that the expression of CD147 was associated with poorly differentiated squamous cell carcinoma and moderately differentiated squamous cell carcinoma (p squamous cell carcinoma and poorly differentiated squamous cell carcinoma patients were CD44 high /CD147 high as compared to only 10% of patients with well-differentiated squamous cell carcinoma. A three-way analysis indicated that differentiation correlated with recurrence and survival (p oral squamous cell carcinoma cell lines originating from different grades of oral cancer. Flowcytometry-based analysis indicated an increase in CD44 + /CD147 + cells in cell lines of poorly differentiated squamous cell carcinoma (94.35 ± 1.14%, p squamous cell carcinoma origin (93.49 ± 0.47%, p squamous cell carcinoma origin (23.12% ± 0.49%). Expression profiling indicated higher expression of cancer stem cell and epithelial-mesenchymal transition markers in SCC029B (poorly differentiated squamous cell carcinoma originated; p ≤ 0.001), which was further translated into increased spheroid formation, migration, and invasion (p squamous cell carcinoma origin. This study suggests that CD44 and CD147 together improve the prognostic efficacy of tumor differentiation; in vitro results further point out that these markers might be determinant of differentiation characteristics, imparting properties of increased self-renewal, migration, and invasion.

  1. Effect of coumarins on HL-60 cell differentiation.

    Science.gov (United States)

    Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

    2000-01-01

    Twenty-eight coumarins, including 7 furocoumarins, were examined for their activity of induction of terminal differentiation of human promyelocytic leukemia cells (HL-60) by nitro blue tetrazolium (NBT) reducing, nonspecific esterase, specific esterase and phagocytic activities. Esculetin, nordalbergin, 6,7-dihydroxy-4-methylcoumarin and imperatorin had strong activity among the coumarins examined. HL-60 cells treated with these coumarins differentiated into mature monocyte/macrophage. The structure-activity relationship established from the results revealed that 6,7-dihydroxy moiety had an important role in the induction of differentiation of HL-60.

  2. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito

    2005-01-01

    that compare cells grown in suspension to similar cells grown attached to one another as aggregates have suggested that it is adhesion to the extracellular matrix of the basal membrane that confers resistance to apoptosis and, hence, resistance to cytotoxins. The genes whose expression correlates with poor...... in cell adhesion and the cytoskeleton. If the proteins involved in tethering cells to the extracellular matrix are important in conferring drug resistance, it may be possible to improve chemotherapy by designing drugs that target these proteins....

  3. Influence of Porcine Intervertebral Disc Matrix on Stem Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Hans-Lothar Fuchsbauer

    2011-08-01

    Full Text Available For back disorders, cell therapy is one approach for a real regeneration of a degenerated nucleus pulposus. Human mesenchymal stem cells (hMSC could be differentiated into nucleus pulposus (NP-like cells and used for cell therapy. Therefore it is necessary to find a suitable biocompatible matrix, which supports differentiation. It could be shown that a differentiation of hMSC in a microbial transglutaminase cross-linked gelatin matrix is possible, but resulted in a more chondrocyte-like cell type. The addition of porcine NP extract to the gelatin matrix caused a differentiation closer to the desired NP cell phenotype. This concludes that a hydrogel containing NP extract without any other supplements could be suitable for differentiation of hMSCs into NP cells. The NP extract itself can be cross-linked by transglutaminase to build a hydrogel free of NP atypical substrates. As shown by side-specific biotinylation, the NP extract contains molecules with free glutamine and lysine residues available for the transglutaminase.

  4. Efficient differentiation of human embryonic stem cells to definitive endoderm.

    Science.gov (United States)

    D'Amour, Kevin A; Agulnick, Alan D; Eliazer, Susan; Kelly, Olivia G; Kroon, Evert; Baetge, Emmanuel E

    2005-12-01

    The potential of human embryonic stem (hES) cells to differentiate into cell types of a variety of organs has generated much excitement over the possible use of hES cells in therapeutic applications. Of great interest are organs derived from definitive endoderm, such as the pancreas. We have focused on directing hES cells to the definitive endoderm lineage as this step is a prerequisite for efficient differentiation to mature endoderm derivatives. Differentiation of hES cells in the presence of activin A and low serum produced cultures consisting of up to 80% definitive endoderm cells. This population was further enriched to near homogeneity using the cell-surface receptor CXCR4. The process of definitive endoderm formation in differentiating hES cell cultures includes an apparent epithelial-to-mesenchymal transition and a dynamic gene expression profile that are reminiscent of vertebrate gastrulation. These findings may facilitate the use of hES cells for therapeutic purposes and as in vitro models of development.

  5. Cell Fate and Differentiation of Bone Marrow Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Shoichiro Kokabu

    2016-01-01

    Full Text Available Osteoblasts and bone marrow adipocytes originate from bone marrow mesenchymal stem cells (BMMSCs and there appears to be a reciprocal relationship between adipogenesis and osteoblastogenesis. Alterations in the balance between adipogenesis and osteoblastogenesis in BMMSCs wherein adipogenesis is increased relative to osteoblastogenesis are associated with decreased bone quality and quantity. Several proteins have been reported to regulate this reciprocal relationship but the exact nature of the signals regulating the balance between osteoblast and adipocyte formation within the bone marrow space remains to be determined. In this review, we focus on the role of Transducin-Like Enhancer of Split 3 (TLE3, which was recently reported to regulate the balance between osteoblast and adipocyte formation from BMMSCs. We also discuss evidence implicating canonical Wnt signalling, which plays important roles in both adipogenesis and osteoblastogenesis, in regulating TLE3 expression. Currently, there is demand for new effective therapies that target the stimulation of osteoblast differentiation to enhance bone formation. We speculate that reducing TLE3 expression or activity in BMMSCs could be a useful approach towards increasing osteoblast numbers and reducing adipogenesis in the bone marrow environment.

  6. Histone h1 depletion impairs embryonic stem cell differentiation.

    Science.gov (United States)

    Zhang, Yunzhe; Cooke, Marissa; Panjwani, Shiraj; Cao, Kaixiang; Krauth, Beth; Ho, Po-Yi; Medrzycki, Magdalena; Berhe, Dawit T; Pan, Chenyi; McDevitt, Todd C; Fan, Yuhong

    2012-01-01

    Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.

  7. Proteomic analysis of osteogenic differentiation of dental follicle precursor cells

    DEFF Research Database (Denmark)

    Morsczeck, Christian; Petersen, Jørgen; Völlner, Florian

    2009-01-01

    of differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B....... The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic...... proteins, plastin 3 T-isoform, beta-actin, superoxide dismutases, and transgelin were found to be highly up-regulated, whereas cofilin-1, pro-alpha 1 collagen, destrin, prolyl 4-hydrolase and dihydrolipoamide dehydrogenase were found to be highly down-regulated. The group of up-regulated proteins...

  8. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

    Science.gov (United States)

    Wang, Zhongde

    2011-01-01

    Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

  9. Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

    Science.gov (United States)

    Miyatsuka, Takeshi; Li, Zhongmei; German, Michael S.

    2009-01-01

    OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. PMID:19478145

  10. Differentiation of breast cancer stem cells by knockdown of CD44: promising differentiation therapy

    Directory of Open Access Journals (Sweden)

    Pham Phuc V

    2011-12-01

    Full Text Available Abstract Background Breast cancer stem cells (BCSCs are the source of breast tumors. Compared with other cancer cells, cancer stem cells show high resistance to both chemotherapy and radiotherapy. Targeting of BCSCs is thus a potentially promising and effective strategy for breast cancer treatment. Differentiation therapy represents one type of cancer stem-cell-targeting therapy, aimed at attacking the stemness of cancer stem cells, thus reducing their chemo- and radioresistance. In a previous study, we showed that down-regulation of CD44 sensitized BCSCs to the anti-tumor agent doxorubicin. This study aimed to determine if CD44 knockdown caused BCSCs to differentiate into breast cancer non-stem cells (non-BCSCs. Methods We isolated a breast cancer cell population (CD44+CD24- cells from primary cultures of malignant breast tumors. These cells were sorted into four sub-populations based on their expression of CD44 and CD24 surface markers. CD44 knockdown in the BCSC population was achieved using small hairpin RNA lentivirus particles. The differentiated status of CD44 knock-down BCSCs was evaluated on the basis of changes in CD44+CD24- phenotype, tumorigenesis in NOD/SCID mice, and gene expression in relation to renewal status, metastasis, and cell cycle in comparison with BCSCs and non-BCSCs. Results Knockdown of CD44 caused BCSCs to differentiate into non-BCSCs with lower tumorigenic potential, and altered the cell cycle and expression profiles of some stem cell-related genes, making them more similar to those seen in non-BCSCs. Conclusions Knockdown of CD44 is an effective strategy for attacking the stemness of BCSCs, resulting in a loss of stemness and an increase in susceptibility to chemotherapy or radiation. The results of this study highlight a potential new strategy for breast cancer treatment through the targeting of BCSCs.

  11. Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells.

    Directory of Open Access Journals (Sweden)

    Opas Traitanon

    Full Text Available The direct effect of immunosuppressive drugs calcineurin inhibitor (Tacrolimus, TAC and mTOR inhibitor (Sirolimus, SRL on B cell activation, differentiation and proliferation is not well documented. Purified human B cells from healthy volunteers were stimulated through the B Cell Receptor with Anti-IgM + anti-CD40 + IL21 in the absence / presence of TAC or SRL. A variety of parameters of B cell activity including activation, differentiation, cytokine productions and proliferation were monitored by flow cytometry. SRL at clinically relevant concentrations (6 ng/ml profoundly inhibited CD19(+ B cell proliferation compared to controls whereas TAC at similar concentrations had a minimal effect. CD27(+ memory B cells were affected more by SRL than naïve CD27- B cells. SRL effectively blocked B cell differentiation into plasma cells (CD19(+CD138(+ and Blimp1(+/Pax5(low cells even at low dose (2 ng/ml, and totally eliminated them at 6 ng/ml. SRL decreased absolute B cell counts, but the residual responding cells acquired an activated phenotype (CD25(+/CD69(+ and increased the expression of HLA-DR. SRL-treated stimulated B cells on a per cell basis were able to enhance the proliferation of allogeneic CD4(+CD25(- T cells and induce a shift toward the Th1 phenotype. Thus, SRL and TAC have different effects on B lymphocytes. These data may provide insights into the clinical use of these two agents in recipients of solid organ transplants.

  12. The role of purinergic receptors in stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Constanze Kaebisch

    2015-01-01

    Full Text Available A major challenge modern society has to face is the increasing need for tissue regeneration due to degenerative diseases or tumors, but also accidents or warlike conflicts. There is great hope that stem cell-based therapies might improve current treatments of cardiovascular diseases, osteochondral defects or nerve injury due to the unique properties of stem cells such as their self-renewal and differentiation potential. Since embryonic stem cells raise severe ethical concerns and are prone to teratoma formation, adult stem cells are still in the focus of research. Emphasis is placed on cellular signaling within these cells and in between them for a better understanding of the complex processes regulating stem cell fate. One of the oldest signaling systems is based on nucleotides as ligands for purinergic receptors playing an important role in a huge variety of cellular processes such as proliferation, migration and differentiation. Besides their natural ligands, several artificial agonists and antagonists have been identified for P1 and P2 receptors and are already used as drugs. This review outlines purinergic receptor expression and signaling in stem cells metabolism. We will briefly describe current findings in embryonic and induced pluripotent stem cells as well as in cancer-, hematopoietic-, and neural crest-derived stem cells. The major focus will be placed on recent findings of purinergic signaling in mesenchymal stem cells addressed in in vitro and in vivo studies, since stem cell fate might be manipulated by this system guiding differentiation towards the desired lineage in the future.

  13. Clonal analysis of stem cells in differentiation and disease.

    Science.gov (United States)

    Colom, Bartomeu; Jones, Philip H

    2016-12-01

    Tracking the fate of individual cells and their progeny by clonal analysis has redefined the concept of stem cells and their role in health and disease. The maintenance of cell turnover in adult tissues is achieved by the collective action of populations of stem cells with an equal likelihood of self-renewal or differentiation. Following injury stem cells exhibit striking plasticity, switching from homeostatic behavior in order to repair damaged tissues. The effects of disease states on stem cells are also being uncovered, with new insights into how somatic mutations trigger clonal expansion in early neoplasia. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  14. Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells.

    Science.gov (United States)

    Vieira Torquato, Heron F; Ribeiro-Filho, Antonio C; Buri, Marcus V; Araújo Júnior, Roberto T; Pimenta, Renata; de Oliveira, José Salvador R; Filho, Valdir C; Macho, Antonio; Paredes-Gamero, Edgar J; de Oliveira Martins, Domingos T

    2017-04-01

    Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin + ) and leukemia stem cell population (CD34 + CD38 - Lin -/low ). Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G 0 /G 1 (7μM) and G 2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Dexamethasone Suppresses Oxysterol-Induced Differentiation of Monocytic Cells

    Directory of Open Access Journals (Sweden)

    Yonghae Son

    2016-01-01

    Full Text Available Oxysterol like 27-hydroxycholesterol (27OHChol has been reported to induce differentiation of monocytic cells into a mature dendritic cell phenotype. We examined whether dexamethasone (Dx affects 27OHChol-induced differentiation using THP-1 cells. Treatment of monocytic cells with Dx resulted in almost complete inhibition of transcription and surface expression of CD80, CD83, and CD88 induced by 27OHChol. Elevated surface levels of MHC class I and II molecules induced by 27OHChol were reduced to basal levels by treatment with Dx. A decreased endocytosis ability caused by 27OHChol was recovered by Dx. We also examined effects of Dx on expression of CD molecules involved in atherosclerosis. Increased levels of surface protein and transcription of CD105, CD137, and CD166 by treatment with 27OHChol were significantly inhibited by cotreatment with Dx. These results indicate that Dx inhibits 27OHChol-induced differentiation of monocytic cells into a mature dendritic cell phenotype and expression of CD molecules whose levels are associated with atherosclerosis. In addition, we examined phosphorylation of AKT induced by 27OHChol and effect of Dx, where cotreatment with Dx inhibited the phosphorylation of AKT. The current study reports that Dx regulates oxysterol-mediated dendritic cell differentiation of monocytic cells.

  16. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during

  17. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells

    NARCIS (Netherlands)

    Fernandes, H.; Mentink, A.; Bank, R.; Stoop, R.; Blitterswijk, C. van; Boer, J. de

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  18. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  19. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, H.A.M.; Mentink-Leusink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  20. Somatic mutation and cell differentiation in neoplastic transformation

    International Nuclear Information System (INIS)

    Huberman, E.; Collart, F.R.

    1987-01-01

    In brief, the authors suggest that tumor formation may result from continuous expression of growth facilitating genes that, as a result of irreversible changes during the initiation step, are placed under the control of genes expressed during normal differentiation. Thus, to understand carcinogenesis, we must decipher the processes that lead to the acquisition of a mature phenotype in both normal and tumor cells and characterize the growth dependency of tumor cells to inducers of cell differentiation. Furthermore, the growth of a variety of tumors may be controlled through the use of inducers of maturation that activate genes located beyond the gene that is altered during tumor initiation. 22 refs., 3 figs

  1. Differential migration and proliferation of geometrical ensembles of cell clusters

    International Nuclear Information System (INIS)

    Kumar, Girish; Chen, Bo; Co, Carlos C.; Ho, Chia-Chi

    2011-01-01

    Differential cell migration and growth drives the organization of specific tissue forms and plays a critical role in embryonic development, tissue morphogenesis, and tumor invasion. Localized gradients of soluble factors and extracellular matrix have been shown to modulate cell migration and proliferation. Here we show that in addition to these factors, initial tissue geometry can feedback to generate differential proliferation, cell polarity, and migration patterns. We apply layer by layer polyelectrolyte assembly to confine multicellular organization and subsequently release cells to demonstrate the spatial patterns of cell migration and growth. The cell shapes, spreading areas, and cell-cell contacts are influenced strongly by the confining geometry. Cells within geometric ensembles are morphologically polarized. Symmetry breaking was observed for cells on the circular pattern and cells migrate toward the corners and in the direction parallel to the longest dimension of the geometric shapes. This migration pattern is disrupted when actomyosin based tension was inhibited. Cells near the edge or corner of geometric shapes proliferate while cells within do not. Regions of higher rate of cell migration corresponded to regions of concentrated growth. These findings demonstrate that multicellular organization can result in spatial patterns of migration and proliferation.

  2. Directed neuronal differentiation of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Noggle Scott A

    2003-10-01

    Full Text Available Abstract Background We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs to neural precursors and neurons. HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII. Results A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7–10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons. Conclusion This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4+ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.

  3. Osteogenic differentiation of human dental papilla mesenchymal cells

    International Nuclear Information System (INIS)

    Ikeda, Etsuko; Hirose, Motohiro; Kotobuki, Noriko; Shimaoka, Hideki; Tadokoro, Mika; Maeda, Masahiko; Hayashi, Yoshiko; Kirita, Tadaaki; Ohgushi, Hajime

    2006-01-01

    We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of β-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate that mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering

  4. Transplantation Dose Alters the Differentiation Program of Hematopoietic Stem Cells.

    Science.gov (United States)

    Brewer, Casey; Chu, Elizabeth; Chin, Mike; Lu, Rong

    2016-05-24

    Hematopoietic stem cell (HSC) transplantation is the most prevalent stem cell therapy, but it remains a risky procedure. To improve this treatment, it is important to understand how transplanted stem cells rebuild the blood and immune systems and how this process is impacted by transplantation variables such as the HSC dose. Here, we find that, in the long term following transplantation, 70%-80% of donor-HSC-derived clones do not produce all measured blood cell types. High HSC doses lead to more clones that exhibit balanced lymphocyte production, whereas low doses produce more T-cell-specialized clones. High HSC doses also produce significantly higher proportions of early-differentiating clones compared to low doses. These complex differentiation behaviors uncover the clonal-level regeneration dynamics of hematopoietic regeneration and suggest that transplantation dose can be exploited to improve stem cell therapy. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  5. Effects of ionizing radiation on differentiation of murine bone marrow cells into mast cells

    International Nuclear Information System (INIS)

    Murakami, Sho; Yoshino, Hironori; Ishikawa, Junya; Yamaguchi, Masaru; Tsujiguchi, Takakiyo; Nishiyama, Ayaka; Yokoyama, Kouki; Kashiwakura, Ikuo

    2015-01-01

    Mast cells, immune effector cells produced from bone marrow cells, play a major role in immunoglobulin E–mediated allergic responses. Ionizing radiation affects the functions of mast cells, which are involved in radiation-induced tissue damage. However, whether ionizing radiation affects the differential induction of mast cells is unknown. Here we investigated whether bone marrow cells of X-irradiated mice differentiated into mast cells. To induce mast cells, bone marrow cells from X-irradiated and unirradiated mice were cultured in the presence of cytokines required for mast cell induction. Although irradiation at 0.5 Gy and 2 Gy decreased the number of bone marrow cells 1 day post-irradiation, the cultured bone marrow cells of X-irradiated and unirradiated mice both expressed mast cell–related cell-surface antigens. However, the percentage of mast cells in the irradiated group was lower than in the unirradiated group. Similar decreases in the percentage of mast cells induced in the presence of X-irradiation were observed 10 days post irradiation, although the number of bone marrow cells in irradiated mice had recovered by this time. Analysis of mast cell function showed that degranulation of mast cells after immunoglobulin E–mediated allergen recognition was significantly higher in the X-irradiated group compared with in the unirradiated group. In conclusion, bone marrow cells of X-irradiated mice differentiated into mast cells, but ionizing radiation affected the differentiation efficiency and function of mast cells. (author)

  6. Glutathione, cell proliferation and differentiation | Ashtiani | African ...

    African Journals Online (AJOL)

    All organisms require an equivalent source for living. Reduced glutathione is the most abundant thiol containing protein in mammalian cells and organs. Glutathione was discovered by Hopkins in 1924 who published his findings in JBC. It is a three peptide containing glutamic acid, cystein and glycin and is found in reduced ...

  7. Effect of cell density on adipogenic differentiation of mesenchymal stem cells

    International Nuclear Information System (INIS)

    Lu, Hongxu; Guo, Likun; Wozniak, Michal J.; Kawazoe, Naoki; Tateishi, Tetsuya; Zhang, Xingdong; Chen, Guoping

    2009-01-01

    The effect of cell density on the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) was investigated by using a patterning technique to induce the formation of a cell density gradient on a micropatterned surface. The adipogenic differentiation of MSCs at a density gradient from 5 x 10 3 to 3 x 10 4 cells/cm 2 was examined. Lipid vacuoles were observed at all cell densities after 1-3 weeks of culture in adipogenic differentiation medium although the lipid vacuoles were scarce at the low cell density and abundant at the high cell density. Real-time RT-PCR analysis showed that adipogenesis marker genes encoding peroxisome proliferator-activated receptor γ2 (PPARγ2), lipoprotein lipase (LPL), and fatty acid binding protein-4 (FABP4) were detected in the MSCs cultured at all cell densities. The results suggest that there was no apparent effect of cell density on the adipogenic differentiation of human MSCs.

  8. Characterisation of insulin-producing cells differentiated from tonsil derived mesenchymal stem cells.

    Science.gov (United States)

    Kim, So-Yeon; Kim, Ye-Ryung; Park, Woo-Jae; Kim, Han Su; Jung, Sung-Chul; Woo, So-Youn; Jo, Inho; Ryu, Kyung-Ha; Park, Joo-Won

    2015-01-01

    Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on β-mercaptoethanol and insulin-transferin-selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin-transferin-selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  9. The Role of Lymphatic Niches in T Cell Differentiation

    Science.gov (United States)

    Capece, Tara; Kim, Minsoo

    2016-01-01

    Long-term immunity to many viral and bacterial pathogens requires CD8+ memory T cell development, and the induction of long-lasting CD8+ memory T cells from a naïve, undifferentiated state is a major goal of vaccine design. Formation of the memory CD8+ T cell compartment is highly dependent on the early activation cues received by naïve CD8+ T cells during primary infection. This review aims to highlight the cellularity of various niches within the lymph node and emphasize recent evidence suggesting that distinct types of T cell activation and differentiation occur within different immune contexts in lymphoid organs. PMID:27306645

  10. Parathyroid Hormone Activates Phospholipase C (PLC)-Independent Protein Kinase C Signaling Pathway via Protein Kinase A (PKA)-Dependent Mechanism: A New Defined Signaling Route Would Induce Alternative Consideration to Previous Conceptions.

    Science.gov (United States)

    Tong, Guojun; Meng, Yue; Hao, Song; Hu, Shaoyu; He, Youhua; Yan, Wenjuan; Yang, Dehong

    2017-04-20

    BACKGROUND Parathyroid hormone (PTH) is an effective anti-osteoporosis agent, after binding to its receptor PTHR1, several signaling pathways, including cAMP/protein kinase A (PKA) and phospholipase C (PLC)/protein kinase C (PKC), are initiated through G proteins; with the cAMP/PKA pathway as the major pathway. Earlier studies have reported that PTHR1 might also activate PKC via a PLC-independent mechanism, but this pathway remains unclear. MATERIAL AND METHODS In HEK293 cells, cAMP accumulation was measured with ELISA and PKC was measured with fluorescence resonance energy transfer (FRET) analysis using CKAR plasmid. In MC3T3-E1 cells, real-time PCR was performed to examine gene expressions. Then assays for cell apoptosis, cell differentiation, alkaline phosphatase activity, and mineralization were performed. RESULTS The FRET analysis found that PTH(1-34), [G1,R19]PTH(1-34) (GR(1-34), and [G1,R19]PTH(1-28) (GR(1-28) were all activated by PKC. The PKC activation ability of GR(1-28) was blocked by cAMP inhibitor (Rp-cAMP) and rescued with the addition of active PKA-α and PKA-β. The PKC activation ability of GR(1-34) was partially inhibited by Rp-cAMP. In MC3T3-E1 cells, gene expressions of ALP, CITED1, NR4a2, and OSX that was regulated by GR(1-28) were significantly changed by the pan-PKC inhibitor Go6983. After pretreatment with Rp-cAMP, the gene expressions of ALP, CITED1, and OPG were differentially regulated by GR(1-28) or GR(1-34), and the difference was blunted by Go6983. PTH(1-34), GR(1-28), and GR(1-34) significantly decreased early apoptosis and augmented osteoblastic differentiation in accordance with the activities of PKA and PKC. CONCLUSIONS PLC-independent PKC activation induced by PTH could be divided into two potential mechanisms: one was PKA-dependent and associated with PTH(1-28); the other was PKA-independent and associated with PTH(29-34). We also found that PTH could activate PLC-independent PKC via PKA-dependent mechanisms.

  11. Mechanisms of dealing with DNA damage in terminally differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Fortini, P. [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy); Dogliotti, E., E-mail: eugenia.dogliotti@iss.it [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy)

    2010-03-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

  12. Chronology of endocrine differentiation and beta-cell neogenesis.

    Science.gov (United States)

    Miyatsuka, Takeshi

    2016-01-01

    Diabetes is a chronic and incurable disease, which results from absolute or relative insulin insufficiency. Therefore, pancreatic beta cells, which are the only type of cell that expresses insulin, is considered to be a potential target for the cure of diabetes. Although the findings regarding beta-cell neogenesis during pancreas development have been exploited to induce insulin-producing cells from non-beta cells, there are still many hurdles towards generating fully functional beta cells that can produce high levels of insulin and respond to physiological signals. To overcome these problems, a solid understanding of pancreas development and beta-cell formation is required, and several mouse models have been developed to reveal the unique features of each endocrine cell type at distinct developmental time points. Here I review our understanding of pancreas development and endocrine differentiation focusing on recent progresses in improving temporal cell labeling in vivo.

  13. Mechanisms of dealing with DNA damage in terminally differentiated cells

    International Nuclear Information System (INIS)

    Fortini, P.; Dogliotti, E.

    2010-01-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

  14. Effect of Ultrasonic Vibration on Proliferation and Differentiation of Cells

    Directory of Open Access Journals (Sweden)

    Haruka Hino

    2016-12-01

    Full Text Available The effect of mechanical stimulation of vibration on proliferation and differentiation of cells has been studied in vitro. To apply the vibration on the cells, a piezoelectric element was attached on the outside surface of the bottom of the culture plate of six wells. The piezoelectric element was vibrated by sinusoidally alternating voltage at 1.0 MHz generated by a function generator. Five kinds of cells were used in the experiment: C2C12 (mouse myoblast cell, L929 (fibroblast connective tissue of mouse, Hepa1-6 (mouse hepatoma cell, HUVEC (human umbilical vein endothelial cell, and Neuro-2a (mouse neural crest-derived cell line. After the incubation for 24 hours, cells were exposed to the ultrasonic vibration intermittently for three days: for thirty minutes per day. At the end of the experiment, the number of cells was counted by colorimetric method with a microplate photometer. In the case of Neuro-2a, the total length of the neurite was calculated at the microscopic image. The experimental study shows following results. Cells are exfoliated by the strong vibration. Proliferation and differentiation of cells are accelerated with mild vibration. The optimum intensity of vibration depends on the kind of cells.

  15. Induction of differentiation of murine embryonal carcinoma cells by ouabain

    International Nuclear Information System (INIS)

    Zimmerman, B.T.

    1986-01-01

    Embryonal carcinoma (EC) cells can be induced to differentiate by ouabain at concentrations which inhibit Na + , K + -ATPase activity as measured by inhibition of 86 Rb + uptake. Since the pharmacologic action of ouabain is thought to be specific, the authors investigated the role of Na + , K + -ATPase inhibition and specific metabolic consequences of this inhibition in the induction of EC differentiation, and explored whether this might be a common mode of action for a variety of structurally diverse inducers. The Na + , K + -ATPase maintains ionic gradients in cells. However, results of studies utilizing specific ionophores, channel blockers, and media deficient in specific components failed to demonstrate a consistent role for ion flux or concentration in the differentiation process. The Na + , K + -ATPase is a major consumer of ATP. They therefore examined the effect of Na + , K + -ATPase inhibition on the adenylate energy charge as measured by high performance liquid chromatography of adenylate nucleotides. Ouabain was found to significantly decrease the energy charge in sensitive cells suggesting a role for suppression of ATP turnover is triggering differentiation. However, direct inhibition of glycolysis also induced differentiation without decreasing the energy charge, suggesting that reduction of the energy charge is not a common mechanism for induction of differentiation of EC

  16. Regulation of T cell differentiation and function by EZH2

    Directory of Open Access Journals (Sweden)

    THEODOROS KARANTANOS

    2016-05-01

    Full Text Available The enhancer of zeste homologue 2 (EZH2, one of the polycomb group (PcG proteins, is the catalytic subunit of Polycomb-repressive complex 2 (PRC2 and induces the trimethylation of the histone H3 lysine 27 (H3K27me3 promoting epigenetic gene silencing. EZH2 contains a SET domain promoting the methyltransferase activity while the three other protein components of PRC2, namely EED, SUZ12 and RpAp46/48 induce compaction of the chromatin permitting EZH2 enzymatic activity. Numerous studies highlight the role of this evolutionary conserved protein as a master regulator of differentiation in humans involved in the repression of the homeotic (Hox gene and the inactivation of X-chromosome. Through its effects in the epigenetic regulation of critical genes, EZH2 has been strongly linked to cell cycle progression, stem cell pluripotency and cancer biology. Most recently, EZH2 has been associated with hematopoietic stem cell proliferation and differentiation, thymopoiesis and lymphopoiesis. Several studies have evaluated the role of EZH2 in the regulation of T cell differentiation and plasticity as well as its implications in the development of autoimmune diseases and graft versus host disease (GvHD. In this review we will briefly summarize the current knowledge regarding the role of EZH2 in the regulation of T cell differentiation, effector function and homing in the tumor microenvironment and we will discuss possible therapeutic targeting of EZH2 in order to alter T cell immune functions.

  17. Pericytes Stimulate Oligodendrocyte Progenitor Cell Differentiation during CNS Remyelination

    Directory of Open Access Journals (Sweden)

    Alerie Guzman De La Fuente

    2017-08-01

    Full Text Available The role of the neurovascular niche in CNS myelin regeneration is incompletely understood. Here, we show that, upon demyelination, CNS-resident pericytes (PCs proliferate, and parenchymal non-vessel-associated PC-like cells (PLCs rapidly develop. During remyelination, mature oligodendrocytes were found in close proximity to PCs. In Pdgfbret/ret mice, which have reduced PC numbers, oligodendrocyte progenitor cell (OPC differentiation was delayed, although remyelination proceeded to completion. PC-conditioned medium accelerated and enhanced OPC differentiation in vitro and increased the rate of remyelination in an ex vivo cerebellar slice model of demyelination. We identified Lama2 as a PC-derived factor that promotes OPC differentiation. Thus, the functional role of PCs is not restricted to vascular homeostasis but includes the modulation of adult CNS progenitor cells involved in regeneration.

  18. Expression of assayable residual stem cell damage in erythroid differentiation

    International Nuclear Information System (INIS)

    Huebner, G.E.; Miller, M.E.; Cronkite, E.P.

    1985-01-01

    In rodents, residual damage is inducible in hematopoietic stem cells by exposure to ionizing radiation or alkylating agents. This damage can b e assayed in mice by transferring bone marrow into lethally irradiated syngeneic recipients and subsequently measuring the incremental increase of-( 125 I)iodo-2'-deoxyuridine incorporation in spleens. In this study, bone marrow from mice treated 3 weeks previously with Methylnitrosourea (50 mg/kg) or 450 rad was injected into recipients in order to determine possible residual effects of treatment of erythroid cell differentiation following stem cell seeding. Such effects were detected by a reduced amount of 59 Fe incorporation into spleens, thus indicatin g transfer of residual stem cell damage to differentiating cells. (orig.)

  19. Lineage-specific interface proteins match up the cell cycle and differentiation in embryo stem cells

    DEFF Research Database (Denmark)

    Re, Angela; Workman, Christopher; Waldron, Levi

    2014-01-01

    The shortage of molecular information on cell cycle changes along embryonic stem cell (ESC) differentiation prompts an in silico approach, which may provide a novel way to identify candidate genes or mechanisms acting in coordinating the two programs. We analyzed germ layer specific gene expression...... changes during the cell cycle and ESC differentiation by combining four human cell cycle transcriptome profiles with thirteen in vitro human ESC differentiation studies. To detect cross-talk mechanisms we then integrated the transcriptome data that displayed differential regulation with protein...... interaction data. A new class of non-transcriptionally regulated genes was identified, encoding proteins which interact systematically with proteins corresponding to genes regulated during the cell cycle or cell differentiation, and which therefore can be seen as interface proteins coordinating the two...

  20. Generation of male differentiated germ cells from various types of stem cells.

    Science.gov (United States)

    Hou, Jingmei; Yang, Shi; Yang, Hao; Liu, Yang; Liu, Yun; Hai, Yanan; Chen, Zheng; Guo, Ying; Gong, Yuehua; Gao, Wei-Qiang; Li, Zheng; He, Zuping

    2014-06-01

    Infertility is a major and largely incurable disease caused by disruption and loss of germ cells. It affects 10-15% of couples, and male factor accounts for half of the cases. To obtain human male germ cells 'especially functional spermatids' is essential for treating male infertility. Currently, much progress has been made on generating male germ cells, including spermatogonia, spermatocytes, and spermatids, from various types of stem cells. These germ cells can also be used in investigation of the pathology of male infertility. In this review, we focused on advances on obtaining male differentiated germ cells from different kinds of stem cells, with an emphasis on the embryonic stem (ES) cells, the induced pluripotent stem (iPS) cells, and spermatogonial stem cells (SSCs). We illustrated the generation of male differentiated germ cells from ES cells, iPS cells and SSCs, and we summarized the phenotype for these stem cells, spermatocytes and spermatids. Moreover, we address the differentiation potentials of ES cells, iPS cells and SSCs. We also highlight the advantages, disadvantages and concerns on derivation of the differentiated male germ cells from several types of stem cells. The ability of generating mature and functional male gametes from stem cells could enable us to understand the precise etiology of male infertility and offer an invaluable source of autologous male gametes for treating