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Sample records for osteoblastic cell differentiation

  1. 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...

  2. 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.

  3. Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

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    Yun-Yun Ma

    Full Text Available Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification.

  4. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

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    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

  5. 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

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

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    Zunich Samantha M

    2012-07-01

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

  7. Differentiation of bovine spermatogonial stem cells into osteoblasts.

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    Qasemi-Panahi, Babak; Tajik, Parviz; Movahedin, Mansoureh; Moghaddam, Gholamali; Barzgar, Younes; Heidari-Vala, Hamed

    2011-07-01

    Spermatogonial Stem Cell (SSC) technologies provide multiple opportunities for research in the field of biotechnology and regenerative medicine. The therapeutic use of Embryonic Stem Cells (ESCs) is restricted due to severe ethical and immunological concerns. Therefore, we need a new pluripotent cell type. Despite well-known role of germ cells in the gametogenesis, some facts apparently show their multipotentiality. In the present study, bovine SSCs were co-cultured with Sertoli cell for 7 days. Sertoli cells and SSCs were identified by Vimentin and Oct-4 immunocytochemical staining method, respectively. In order to differentiate SSCs into osteoblasts, we used consecutive inducer media without separation of the colonies. We characterized osteoblasts using Alizarin red staining.

  8. 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.

  9. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

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    Yeh, Lee-Chuan C.; Ford, Jeffery J. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); Lee, John C. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States); Adamo, Martin L., E-mail: adamo@biochem.uthscsa.edu [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States)

    2014-07-18

    Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.

  10. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

    International Nuclear Information System (INIS)

    Yeh, Lee-Chuan C.; Ford, Jeffery J.; Lee, John C.; Adamo, Martin L.

    2014-01-01

    Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects

  11. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells.

    Science.gov (United States)

    Yeh, Lee-Chuan C; Ford, Jeffery J; Lee, John C; Adamo, Martin L

    2014-07-18

    Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. MEK5 suppresses osteoblastic differentiation

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    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.

  13. Cuscuta chinensis extract promotes osteoblast differentiation and mineralization in human osteoblast-like MG-63 cells.

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    Yang, Hyun Mo; Shin, Hyun-Kyung; Kang, Young-Hee; Kim, Jin-Kyung

    2009-02-01

    The aim of the present study was to investigate whether the aqueous extract of To-Sa-Za (TSZ-AE), the seed of Cuscuta chinensis Lam., which is a traditional medicinal herb commonly used in Korea and other oriental countries, could induce osteogenic activity in human osteoblast-like MG-63 cells. TSZ-AE treatment mildly promoted the proliferation of MG-63 cells at doses of 500 and 1,000 microg/mL in the 24-hour culture period. Dose-dependent increases in alkaline phosphatase (ALP) activity and collagen synthesis were shown at 48 and 72 hours of incubation. The release of bone morphogenetic protein (BMP)-2 but not osteocalcin in the MG-63 cells was induced by TSZ-AE at 72 hours (100-1,000 microg/mL). In addition, TSZ-AE markedly increased mRNA expression of ALP, collagen, and BMP-2 in the MG-63 cells in a dose-dependent manner. Mineralization in the culture of MG-63 cells was significantly induced at 500 and 1,000 microg/mL TSZ-AE treatment. In conclusion, this study shows that TSZ-AE enhanced ALP activity, collagen synthesis, BMP-2 expression, and mineralization in MG-63 cells. These results strongly suggest that C. chinensis can play an important role in osteoblastic bone formation and may possibly lead to the development of bone-forming drugs.

  14. Osteogenic differentiation of immature osteoblasts: Interplay of cell culture media and supplements.

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    Brauer, A; Pohlemann, T; Metzger, W

    2016-01-01

    Differentiation of immature osteoblasts to mature osteoblasts in vitro initially was induced by supplementing the medium with β-gylcerophosphate and dexamethasone. Later, ascorbic acid, vitamin D3, vitamin K3 and TGFβ1 were used in varying concentrations as supplements to generate a mature osteoblast phenotype. We tested the effects of several combinations of cell culture media, seeding protocols and osteogenic supplements on osteogenic differentiation of human primary osteoblasts. Osteogenic differentiation was analyzed by staining alkaline phosphatase (ALP) with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) and by von Kossa staining of deposited calcium phosphate. The combinations of culture media and supplements significantly influenced osteogenic differentiation, but the seeding protocol did not. Staining of ALP and calcium phosphate could be achieved only if our own mix of osteogenic supplements was used in combination with Dulbecco's modified Eagle medium or if a commercial mix of osteogenic supplements was used in combination with osteoblast growth medium. Especially for von Kossa, we observed great variations in the staining intensity. Because osteogenic differentiation is a complex process, the origin of the osteoblasts, cell culture media and osteogenic supplements should be established by preliminary experiments to achieve optimal differentiation. Staining of ALP or deposited calcium phosphate should be supplemented with qRT-PCR studies to learn more about the influence of specific supplements on osteogenic markers.

  15. Morphology and Differentiation of MG63 Osteoblast Cells on Saliva Contaminated Implant Surfaces

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    Neda Shams

    2015-11-01

    Full Text Available Objectives: Osteoblasts are the most important cells in the osseointegration process. Despite years of study on dental Implants, limited studies have discussed the effect of saliva on the adhesion process of osteoblasts to implant surfaces. The aim of this in vitro study was to evaluate the effect of saliva on morphology and differentiation of osteoblasts attached to implant surfaces.Materials and Methods: Twelve Axiom dental implants were divided into two groups. Implants of the case group were placed in containers, containing saliva, for 40 minutes. Then, all the implants were separately stored in a medium containing MG63 human osteoblasts for a week. Cell morphology and differentiation were assessed using a scanning electron microscope and their alkaline phosphatase (ALP activity was determined. The t-test was used to compare the two groups.Results: Scanning electron microscopic observation of osteoblasts revealed round or square cells with fewer and shorter cellular processes in saliva contaminated samples, whereas elongated, fusiform and well-defined cell processes were seen in the control group. ALP level was significantly lower in case compared to control group (P<0.05.Conclusion: Saliva contamination alters osteoblast morphology and differentiation and may subsequently interfere with successful osseointegration. Thus, saliva contamination of bone and implant must be prevented or minimized.

  16. Skeletal (stromal) stem cells: an update on intracellular signaling pathways controlling osteoblast differentiation.

    Science.gov (United States)

    Abdallah, Basem M; Jafari, Abbas; Zaher, Walid; Qiu, Weimin; Kassem, Moustapha

    2015-01-01

    Skeletal (marrow stromal) stem cells (BMSCs) are a group of multipotent cells that reside in the bone marrow stroma and can differentiate into osteoblasts, chondrocytes and adipocytes. Studying signaling pathways that regulate BMSC differentiation into osteoblastic cells is a strategy for identifying druggable targets for enhancing bone formation. This review will discuss the functions and the molecular mechanisms of action on osteoblast differentiation and bone formation; of a number of recently identified regulatory molecules: the non-canonical Notch signaling molecule Delta-like 1/preadipocyte factor 1 (Dlk1/Pref-1), the Wnt co-receptor Lrp5 and intracellular kinases. This article is part of a Special Issue entitled: Stem Cells and Bone. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Curcumin induces osteoblast differentiation through mild-endoplasmic reticulum stress-mediated such as BMP2 on osteoblast cells.

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    Son, Hyo-Eun; Kim, Eun-Jung; Jang, Won-Gu

    2018-01-15

    Curcumin (diferuloylmethane or [1E,6E]-1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6heptadiene-3,5-dione) is a phenolic natural product derived from the rhizomes of the turmeric plant, Curcuma longa. It is reported to have various biological actions such as anti-oxidative, anti-inflammatory, and anti-cancer effects. However, the molecular mechanism of osteoblast differentiation by curcumin has not yet been reported. The cytotoxicity of curcumin was identified using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression of osteogenic markers and endoplasmic reticulum (ER) stress markers in C3H1-T1/2 cells were measured using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity in C3H10T1/2 cells. Transcriptional activity was detected using a luciferase reporter assay. Curcumin increased the expression of genes such as distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OC), which subsequently induced osteoblast differentiation in C3H10T1/2 cells. In addition, ALP activity and mineralization was found to be increased by curcumin treatment. Curcumin also induced mild ER stress similar to bone morphogenetic protein 2 (BMP2) function in osteoblast cells. Next, we confirmed that curcumin increased mild ER stress and osteoblast differentiation similar to BMP2 in C3H10T1/2 mesenchymal stem cells. Transient transfection studies also showed that curcumin increased ATF6-Luc activity, while decreasing the activities of CREBH-Luc and SMILE-Luc. In addition, similar to BMP2, curcumin induced the phosphorylation of Smad 1/5/9. Overall, these results demonstrate that curcumin-induced mild ER stress increases osteoblast differentiation via ATF6 expression in C3H10T1/2 cells. Copyright © 2017. Published by Elsevier Inc.

  18. Tissue transglutaminase (TG2 activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line

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    Xiaoxue Yin

    2012-08-01

    Full Text Available Tissue transglutaminase (type II, TG2 has long been postulated to directly promote skeletal matrix calcification and play an important role in ossification. However, limited information is available on the expression, function and modulating mechanism of TG2 during osteoblast differentiation and mineralization. To address these issues, we cultured the well-established human osteosarcoma cell line SAOS-2 with osteo-inductive conditioned medium and set up three time points (culture days 4, 7, and 14 to represent different stages of SAOS-2 differentiation. Osteoblast markers, mineralization, as well as TG2 expression and activity, were then assayed in each stage. Furthermore, we inhibited TG activity with cystamine and then checked SAOS-2 differentiation and mineralization in each stage. The results showed that during the progression of osteoblast differentiation SAOS-2 cells presented significantly high levels of osteocalcin (OC mRNA, bone morphogenetic protein-2 (BMP-2 and collagen I, significantly high alkaline phosphatase (ALP activity, and the increased formation of calcified matrix. With the same tendency, TG2 expression and activity were up-regulated. Furthermore, inhibition of TG activity resulted in a significant decrease of OC, collagen I, and BMP-2 mRNA and of ALP activity and mineralization. This study demonstrated that TG2 is involved in osteoblast differentiation and may play a role in the initiation and regulation of the mineralization processes. Moreover, the modulating effects of TG2 on osteoblasts may be related to BMP-2.

  19. Osteoblast Differentiation and Bone Formation Gene Expression in Strontium-inducing Bone Marrow Mesenchymal Stem Cell

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    SILA-ASNA, MONNIPHA; BUNYARATVEJ, AHNOND; Maeda, Sakan; Kitaguchi, Hiromichi; BUNYARATAVEJ, NARONG

    2007-01-01

    Osteoblastic differentiation from human mesenchymal stem cell (hMSCs) is animportant step of bone formation. We studied the in vitro induction of hMSCs byusing strontium ranelate, a natural trace amount in water, food and human skeleton.The mRNA synthesis of various osteoblast specific genes was assessed by means ofreverse transcription polymerase chain reaction (RT-PCR). In the hMSCs culture,strontium ranelate could enhance the induction of hMSCs to differentiate intoosteoblasts. Cbfa1 gene ...

  20. Human mesenchymal stem cell osteoblast differentiation, ECM deposition, and biomineralization on PAH/PAA polyelectrolyte multilayers.

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    Pattabhi, Sudhakara Rao; Lehaf, Ali M; Schlenoff, Joseph B; Keller, Thomas C S

    2015-05-01

    Polyelectrolyte multilayer (PEMU) coatings built layer by layer with alternating pairs of polyelectrolytes can be tuned to improve cell interactions with surfaces and may be useful as biocompatible coatings to improve fixation between implants and tissues. Here, we show that human mesenchymal stromal cells (hMSCs) induced with bone differentiation medium (BDM) to become osteoblasts biomineralize crosslinked PEMUs built with the polycation poly(allylamine hydrochloride) (PAH) and the polyanion poly(acrylic acid) (PAA). Degrees of hMSC osteoblast differentiation and surface biomineralization on the smooth PAH-terminated PEMUs (PAH-PEMUs) and microstructured PAA-terminated PEMUs (PAA-PEMUs) reflect differences in cell-deposited extracellular matrix (ECM). BDM-induced hMSCs expressed higher levels of the early osteoblast differentiation marker alkaline phosphatase and collagen 1 (COL1) sooner on PAA-PEMUs than on PAH-PEMUs. Cells on both types of PEMUs proceeded to express the later stage osteoblast differentiation marker bone sialoprotein (BSP), but the BDM-induced cells organized a more amorphous Collagen I and denser BSP localization on PAA-PEMUs than on PAH-PEMUs. These ECM properties correlated with greater biomineralization on the PAA-PEMUs than on PAH-PEMUs. Together, these results confirm the suitability of PAH/PAA PEMUs as a substrate for hMSC osteogenesis and highlight the importance of substrate effects on ECM organization and BSP presentation on biomineralization. © 2014 Wiley Periodicals, Inc.

  1. Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells

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    Green, A.C. [St Vincent' s Institute, Fitzroy, Victoria 3065 (Australia); Department of Medicine at St. Vincent' s Hospital, The University of Melbourne, Victoria 3065 (Australia); Kocovski, P.; Jovic, T.; Walia, M.K. [St Vincent' s Institute, Fitzroy, Victoria 3065 (Australia); Chandraratna, R.A.S. [IO Therapeutics, Inc., Santa Ana, CA 92705 (United States); Martin, T.J.; Baker, E.K. [St Vincent' s Institute, Fitzroy, Victoria 3065 (Australia); Department of Medicine at St. Vincent' s Hospital, The University of Melbourne, Victoria 3065 (Australia); Purton, L.E., E-mail: lpurton@svi.edu.au [St Vincent' s Institute, Fitzroy, Victoria 3065 (Australia); Department of Medicine at St. Vincent' s Hospital, The University of Melbourne, Victoria 3065 (Australia)

    2017-01-01

    Low and high serum retinol levels are associated with increased fracture risk and poor bone health. We recently showed retinoic acid receptors (RARs) are negative regulators of osteoclastogenesis. Here we show RARs are also negative regulators of osteoblast and adipocyte differentiation. The pan-RAR agonist, all-trans retinoic acid (ATRA), directly inhibited differentiation and mineralisation of early osteoprogenitors and impaired the differentiation of more mature osteoblast populations. In contrast, the pan-RAR antagonist, IRX4310, accelerated differentiation of early osteoprogenitors. These effects predominantly occurred via RARγ and were further enhanced by an RARα agonist or antagonist, respectively. RAR agonists similarly impaired adipogenesis in osteogenic cultures. RAR agonist treatment resulted in significant upregulation of the Wnt antagonist, Sfrp4. This accompanied reduced nuclear and cytosolic β-catenin protein and reduced expression of the Wnt target gene Axin2, suggesting impaired Wnt/β-catenin signalling. To determine the effect of RAR inhibition in post-natal mice, IRX4310 was administered to male mice for 10 days and bones were assessed by µCT. No change to trabecular bone volume was observed, however, radial bone growth was impaired. These studies show RARs directly influence osteoblast and adipocyte formation from mesenchymal cells, and inhibition of RAR signalling in vivo impairs radial bone growth in post-natal mice. - Graphical abstract: Schematic shows RAR ligand regulation of osteoblast differentiation in vitro. RARγ antagonists±RARα antagonists promote osteoblast differentiation. RARγ and RARα agonists alone or in combination block osteoblast differentiation, which correlates with upregulation of Sfrp4, and downregulation of nuclear and cytosolic β-catenin and reduced expression of the Wnt target gene Axin2. Red arrows indicate effects of RAR agonists on mediators of Wnt signalling.

  2. Porous hydroxyapatite and biphasic calcium phosphate ceramics promote ectopic osteoblast differentiation from mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Lingli; Fan Hongsong; Zhang Xingdong [National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan 610064 (China); Hanagata, Nobutaka; Ikoma, Toshiyuki [Biomaterials Center, National Institute for Materials Science, Tsukuba, Ibaraki 305-0047 (Japan); Maeda, Megumi; Minowa, Takashi, E-mail: HANAGATA.Nobutaka@nims.go.j [Nanotechnology Innovation Center, National Institute for Materials Science, Tsukuba, Ibaraki 305-0047 (Japan)

    2009-04-15

    Because calcium phosphate (Ca-P) ceramics have been used as bone substitutes, it is necessary to investigate what effects the ceramics have on osteoblast maturation. We prepared three types of Ca-P ceramics with different Ca-P ratios, i.e. hydroxyapatite (HA), beta-tricalcium phosphate ({beta}-TCP), and biphasic calcium phosphate (BCP) ceramics with dense-smooth and porous structures. Comprehensive gene expression microarray analysis of mouse osteoblast-like cells cultured on these ceramics revealed that porous Ca-P ceramics considerably affected the gene expression profiles, having a higher potential for osteoblast maturation. In the in vivo study that followed, porous Ca-P ceramics were implanted into rat skeletal muscle. Sixteen weeks after the implantation, more alkaline-phosphatase-positive cells were observed in the pores of hydroxyapatite and BCP, and the expression of the osteocalcin gene (an osteoblast-specific marker) in tissue grown in pores was also higher in hydroxyapatite and BCP than in {beta}-TCP. In the pores of any Ca-P ceramics, 16 weeks after the implantation, we detected the expressions of marker genes of the early differentiation stage of chondrocytes and the complete differentiation stage of adipocytes, which originate from mesenchymal stem cells, as well as osteoblasts. These marker gene expressions were not observed in the muscle tissue surrounding the implanted Ca-P ceramics. These observations indicate that porous hydroxyapatite and BCP had a greater potential for promoting the differentiation of mesenchymal stem cells into osteoblasts than {beta}-TCP.

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

    Science.gov (United States)

    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.

  4. 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...

  5. Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche

    Directory of Open Access Journals (Sweden)

    LM McNamara

    2012-01-01

    Full Text Available Mesenchymal stem cells (MSCs within their native environment of the stem cell niche in bone receive biochemical stimuli from surrounding cells. These stimuli likely influence how MSCs differentiate to become bone precursors. The ability of MSCs to undergo osteogenic differentiation is well established in vitro;however, the role of the natural cues from bone’s regulatory cells, osteocytes and osteoblasts in regulating the osteogenic differentiation of MSCs in vivo are unclear. In this study we delineate the role of biochemical signalling from osteocytes and osteoblasts, using conditioned media and co-culture experiments, to understand how they direct osteogenic differentiation of MSCs. Furthermore, the synergistic relationship between osteocytes and osteoblasts is examined by transwell co-culturing of MSCs with both simultaneously. Osteogenic differentiation of MSCs was quantified by monitoring alkaline phosphatase (ALP activity, calcium deposition and cell number. Intracellular ALP was found to peak earlier and there was greater calcium deposition when MSCs were co-cultured with osteocytes rather than osteoblasts, suggesting that osteocytes are more influential than osteoblasts in stimulating osteogenesis in MSCs. Osteoblasts initially stimulated an increase in the number of MSCs, but ultimately regulated MSC differentiation down the same pathway. Our novel co-culture system confirmed a synergistic relationship between osteocytes and osteoblasts in producing biochemical signals to stimulate the osteogenic differentiation of MSCs. This study provides important insights into the mechanisms at work within the native stem cell niche to stimulate osteogenic differentiation and outlines a possible role for the use of co-culture or conditioned media methodologies for tissue engineering applications.

  6. Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane

    International Nuclear Information System (INIS)

    Rodríguez-Fuentes, Nayeli; Rodríguez-Hernández, Ana G.; Enríquez-Jiménez, Juana; Alcántara-Quintana, Luz E.; Fuentes-Mera, Lizeth; Piña-Barba, María C.; Zepeda-Rodríguez, Armando

    2013-01-01

    Highlights: •Nukbone showed to be a good scaffold for adhesion, proliferation and differentiation of stem cells. •Nukbone induced osteoblastic differentiation of human mesenchymal stem cells. •Results showed that Nukbone offer an excellent option for bone tissue regeneration due to properties. -- Abstract: Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects

  7. Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane

    Energy Technology Data Exchange (ETDEWEB)

    Rodríguez-Fuentes, Nayeli; Rodríguez-Hernández, Ana G. [Depto. Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Mexico City 04510 (Mexico); Enríquez-Jiménez, Juana [Depto. Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), México City 14000 (Mexico); Alcántara-Quintana, Luz E. [Subd. de Investigación, Centro Nacional de la Transfusión Sanguínea, Secretaria de Salud, Mexico City 07370 (Mexico); Fuentes-Mera, Lizeth [Depto. Biología Molecular e Histocompatibilidad, Hospital General “Dr. Manuel Gea González”, México City 4800 (Mexico); Piña-Barba, María C. [Depto. Materiales Metálicos y Cerámicos, Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México (UNAM), México City 04510 (Mexico); Zepeda-Rodríguez, Armando [Depto. Biología Celular y Tisular, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), México City 04510 (Mexico); and others

    2013-05-10

    Highlights: •Nukbone showed to be a good scaffold for adhesion, proliferation and differentiation of stem cells. •Nukbone induced osteoblastic differentiation of human mesenchymal stem cells. •Results showed that Nukbone offer an excellent option for bone tissue regeneration due to properties. -- Abstract: Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.

  8. [Impact of different degree pulpitis on cell proliferation and osteoblastic differentiation of dental pulp stem cell in Beagle immature premolars].

    Science.gov (United States)

    Ling, L; Zhao, Y M; Ge, L H

    2016-10-18

    To compare the proliferation and osteoblastic differentiation of dental pulp stem cell (DPSC) isolated from normal and inflamed pulps of different degrees in Beagle immature premolars, and provide evidence for the use of inflammatory DPSC (IDPSC). This study evaluated 14 Beagle's young premolars (21 roots). In the experiment group, irreversible pulpitis was induced by pulp exposure and the inflamed pulps were extracted 2 weeks and 6 weeks after the pulp chamber opening.For the control group, normal pulps were extracted immediately after the exposure. HE staining and real-time PCR were performed to confirm the inflammation. The cells were isolated from the inflamed and normal pulps (IDPSC and DPSC). Cell proliferation and osteoblastic differentiation potentials of the two cells were compared. Inflammation cells infiltration was observed in the inflamed pulps by HE staining. The expression of inflammatory factor was much higher in the 6 week inflamed pulp. IDPSC had higher potential of cell proliferation and osteoblastic differentiation potentials. Furthermore, the osteoblastic differentiation potentials of IDPSC from 2 week inflamed pulp were higher than those from 6 week inflamed pulp. The potential of cell proliferation and osteoblastic differentiation of DPSC was enhanced at early stage of irreversible pulpitis, and reduced at late stage in Beagle immature premolars.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2017-12-01

    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.

  11. Substrate Stiffness Controls Osteoblastic and Chondrocytic Differentiation of Mesenchymal Stem Cells without Exogenous Stimuli.

    Directory of Open Access Journals (Sweden)

    Rene Olivares-Navarrete

    Full Text Available Stem cell fate has been linked to the mechanical properties of their underlying substrate, affecting mechanoreceptors and ultimately leading to downstream biological response. Studies have used polymers to mimic the stiffness of extracellular matrix as well as of individual tissues and shown mesenchymal stem cells (MSCs could be directed along specific lineages. In this study, we examined the role of stiffness in MSC differentiation to two closely related cell phenotypes: osteoblast and chondrocyte. We prepared four methyl acrylate/methyl methacrylate (MA/MMA polymer surfaces with elastic moduli ranging from 0.1 MPa to 310 MPa by altering monomer concentration. MSCs were cultured in media without exogenous growth factors and their biological responses were compared to committed chondrocytes and osteoblasts. Both chondrogenic and osteogenic markers were elevated when MSCs were grown on substrates with stiffness <10 MPa. Like chondrocytes, MSCs on lower stiffness substrates showed elevated expression of ACAN, SOX9, and COL2 and proteoglycan content; COMP was elevated in MSCs but reduced in chondrocytes. Substrate stiffness altered levels of RUNX2 mRNA, alkaline phosphatase specific activity, osteocalcin, and osteoprotegerin in osteoblasts, decreasing levels on the least stiff substrate. Expression of integrin subunits α1, α2, α5, αv, β1, and β3 changed in a stiffness- and cell type-dependent manner. Silencing of integrin subunit beta 1 (ITGB1 in MSCs abolished both osteoblastic and chondrogenic differentiation in response to substrate stiffness. Our results suggest that substrate stiffness is an important mediator of osteoblastic and chondrogenic differentiation, and integrin β1 plays a pivotal role in this process.

  12. Behaviour of moderately differentiated osteoblast-like cells cultured in contact with bioactive glasses

    Directory of Open Access Journals (Sweden)

    Hattar S.

    2002-12-01

    Full Text Available Bioactive glasses have been shown to stimulate osteogenesis both in vivo and in vitro. However, the molecular mechanisms underlying this process are still poorly understood. In this study, we have investigated the behaviour of osteoblast-like cells (MG63, cultured in the presence of bioglass particles. Three types of granules were used: 45S5registered bioactive glass, 45S5registered granules preincubated in tris buffer and 60S non-reactive glass, used as control. Phase contrast microscopy permitted step-by-step visualization of cell cultures in contact with the particles. Ultrastructural observations of undecalcified sections revealed direct contacts of the cells and an electron-dense layer located at the periphery of the material. Protein synthesis was evaluated biochemically and showed a gradual increase throughout the culture time in the three types of cultures. Alkaline phosphatase was detected in situ, in clusters of packed cells either in contact with the material or in the background cell layer. Semi-quantitative RT-PCR analysis of the main osteoblastic markers showed that gene expression was maintained in all three cultures. The fact that osteocalcin was not detected, supports the fact that the MG63 cell line is composed of less differentiated osteogenic cells rather than mature osteoblasts. We also demonstrated for the first time in this cell line, the expression of Msx-2, Dlx-3 and Dlx-7 homeogenes, known to regulate in vivo foetal skeletogenesis as well as adult skeletal regeneration. However, no significant differences could be recognised in the expression pattern of bone markers between the three types of cultures. Yet these preliminary results indicate that bioactive glasses provided a suitable environment for the growth and proliferation of osteoblasts in vitro, since no drastic changes in phenotype expression of pre-osteoblasts was noted.

  13. Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells.

    Science.gov (United States)

    Yamamoto, Mioko; Kawashima, Nobuyuki; Takashino, Nami; Koizumi, Yu; Takimoto, Koyo; Suzuki, Noriyuki; Saito, Masahiro; Suda, Hideaki

    2014-03-01

    Three-dimensional (3D) spheroid culture is a method for creating 3D aggregations of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into odontoblasts. We cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture. Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signalling-related proteins were detected immunohistochemically. Odonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs (podonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Differentiation of Bovine Spermatogonial Stem Cells into Osteoblasts

    OpenAIRE

    Qasemi-Panahi, Babak; Tajik, Parviz; Movahedin, Mansoureh; Moghaddam, Gholamali; Barzgar, Younes; Heidari-Vala, Hamed

    2011-01-01

    Spermatogonial Stem Cell (SSC) technologies provide multiple opportunities for research in the field of biotechnology and regenerative medicine. The therapeutic use of Embryonic Stem Cells (ESCs) is restricted due to severe ethical and immunological concerns. Therefore, we need a new pluripotent cell type. Despite well-known role of germ cells in the gametogenesis, some facts apparently show their multipotentiality. In the present study, bovine SSCs were co-cultured with Sertoli cell for 7 da...

  15. Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Shi, Kaikai; Frary, Charles

    2015-01-01

    Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes, but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study, we demonstrated that stabilizing actin filaments by inhibiting gene...... expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs, enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro, as well as heterotopic bone formation in vivo. Similarly, treating hMSC with Phalloidin, which is known to stabilize...... polymerized actin filaments, increased hMSCs viability and OB differentiation. Conversely, Cytocholasin D, an inhibitor of actin polymerization, reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level, preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1...

  16. Secreted Clusterin protein inhibits osteoblast differentiation of bone marrow mesenchymal stem cells by suppressing ERK1/2 signaling pathway.

    Science.gov (United States)

    Abdallah, Basem M; Alzahrani, Abdullah M; Kassem, Moustapha

    2018-05-01

    Secreted Clusterin (sCLU, also known as Apolipoprotein J) is an anti-apoptotic glycoprotein involved in the regulation of cell proliferation, lipid transport, extracellular tissue remodeling and apoptosis. sCLU is expressed and secreted by mouse bone marrow-derived skeletal (stromal or mesenchymal) stem cells (mBMSCs), but its functional role in MSC biology is not known. In this study, we demonstrated that Clusterin mRNA expression and protein secretion in conditioned medium increased during adipocyte differentiation and decreased during osteoblast differentiation of mBMSCs. Treatment of mBMSC cultures with recombinant sCLU protein increased cell proliferation and exerted an inhibitory effect on the osteoblast differentiation while stimulated adipocyte differentiation in a dose-dependent manner. siRNA-mediated silencing of Clu expression in mBMSCs reduced adipocyte differentiation and stimulated osteoblast differentiation of mBMSCs. Furthermore, the inhibitory effect of sCLU on the osteoblast differentiation of mBMSCs was mediated by the suppression of extracellular signal-regulated kinase (ERK1/2) phosphorylation. In conclusion, we identified sCLU as a regulator of mBMSCs lineage commitment to osteoblasts versus adipocytes through a mechanism mediated by ERK1/2 signaling. Inhibiting sCLU is a possible therapeutic approach for enhancing osteoblast differentiation and consequently bone formation. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation.

    Science.gov (United States)

    Kaneto, Carla Martins; Pereira Lima, Patrícia S; Prata, Karen Lima; Dos Santos, Jane Lima; de Pina Neto, João Monteiro; Panepucci, Rodrigo Alexandre; Noushmehr, Houtan; Covas, Dimas Tadeu; de Paula, Francisco José Alburquerque; Silva, Wilson Araújo

    2017-06-01

    Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells were isolated for obtaining mesenchymal cells that had been expanded until osteogenic differentiation was induced. RNA was harvested at seven time points during the osteogenic differentiation period (D0, D+1, D+2, D+7, D+12, D+17 and D+21). Gene expression analysis was performed by the microarray technique and identified several differentially expressed genes. Some important genes for osteoblast differentiation had lower expression in OI patients, suggesting a smaller commitment of these patient's MSCs with the osteogenic lineage. Other genes also had their differential expression confirmed by RT-qPCR. An increase in the expression of genes related to adipocytes was observed, suggesting an increase of adipogenic differentiation at the expense osteogenic differentiation. Copyright © 2017. Published by Elsevier Masson SAS.

  18. Resveratrol augments the canonical Wnt signaling pathway in promoting osteoblastic differentiation of multipotent mesenchymal cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Haibin; Shang, Linshan; Li, Xi; Zhang, Xiyu; Gao, Guimin; Guo, Chenhong; Chen, Bingxi; Liu, Qiji [Key Laboratory of Experimental Teratology, MOE, Institute of Molecular Medicine and Genetics, Shandong University, 44 Wen Hua Xi Lu, Jinan, Shandong 250012 (China); Gong, Yaoqin, E-mail: yxg8@sdu.edu.cn [Key Laboratory of Experimental Teratology, MOE, Institute of Molecular Medicine and Genetics, Shandong University, 44 Wen Hua Xi Lu, Jinan, Shandong 250012 (China); Shao, Changshun, E-mail: shao@biology.rutgers.edu [Key Laboratory of Experimental Teratology, MOE, Institute of Molecular Medicine and Genetics, Shandong University, 44 Wen Hua Xi Lu, Jinan, Shandong 250012 (China); Department of Genetics, Rutgers University, Piscataway, NJ 08854 (United States)

    2009-10-15

    Resveratrol has been shown to possess many health-benefiting effects, including the promotion of bone formation. In this report we investigated the mechanism by which resveratrol promotes osteoblastic differentiation from pluripotent mesenchymal cells. Since Wnt signaling is well documented to induce osteoblastogenesis and bone formation, we characterized the factors involved in Wnt signaling in response to resveratrol treatment. Resveratrol treatment of mesenchymal cells led to an increase in stabilization and nuclear accumulation of {beta}-catenin dose-dependently and time-dependently. As a consequence of the increased nuclear accumulation of {beta}-catenin, the ability to activate transcription of {beta}-catenin-TCF/LEF target genes that are required for osteoblastic differentiation was upregulated. However, resveratrol did not affect the initial step of the Wnt signaling pathway, as resveratrol was as effective in upregulating the activity of {beta}-catenin in cells in which Lrp5 was knocked down as in control cells. In addition, while conditioned medium enriched in Wnt signaling antagonist Dkk1 was able to inhibit Wnt3a-induced {beta}-catenin upregulation, this inhibitory effect can be abolished in resveratrol-treated cells. Furthermore, we showed that the level of glycogen synthase kinase 3{beta} (GSK-3{beta}), which phosphorylates and destabilizes {beta}-catenin, was reduced in response to resveratrol treatment. The phosphorylation of GSK-3{beta} requires extracellular signal-regulated kinase (ERK)1/2. Together, our data indicate that resveratrol promotes osteoblastogenesis and bone formation by augmenting Wnt signaling.

  19. Resveratrol augments the canonical Wnt signaling pathway in promoting osteoblastic differentiation of multipotent mesenchymal cells

    International Nuclear Information System (INIS)

    Zhou, Haibin; Shang, Linshan; Li, Xi; Zhang, Xiyu; Gao, Guimin; Guo, Chenhong; Chen, Bingxi; Liu, Qiji; Gong, Yaoqin; Shao, Changshun

    2009-01-01

    Resveratrol has been shown to possess many health-benefiting effects, including the promotion of bone formation. In this report we investigated the mechanism by which resveratrol promotes osteoblastic differentiation from pluripotent mesenchymal cells. Since Wnt signaling is well documented to induce osteoblastogenesis and bone formation, we characterized the factors involved in Wnt signaling in response to resveratrol treatment. Resveratrol treatment of mesenchymal cells led to an increase in stabilization and nuclear accumulation of β-catenin dose-dependently and time-dependently. As a consequence of the increased nuclear accumulation of β-catenin, the ability to activate transcription of β-catenin-TCF/LEF target genes that are required for osteoblastic differentiation was upregulated. However, resveratrol did not affect the initial step of the Wnt signaling pathway, as resveratrol was as effective in upregulating the activity of β-catenin in cells in which Lrp5 was knocked down as in control cells. In addition, while conditioned medium enriched in Wnt signaling antagonist Dkk1 was able to inhibit Wnt3a-induced β-catenin upregulation, this inhibitory effect can be abolished in resveratrol-treated cells. Furthermore, we showed that the level of glycogen synthase kinase 3β (GSK-3β), which phosphorylates and destabilizes β-catenin, was reduced in response to resveratrol treatment. The phosphorylation of GSK-3β requires extracellular signal-regulated kinase (ERK)1/2. Together, our data indicate that resveratrol promotes osteoblastogenesis and bone formation by augmenting Wnt signaling.

  20. Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins during ex vivo Osteoblast Differentiation of Human Stromal Stem Cells

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Chen, Li; Nielsen, Maria Overbeck

    2012-01-01

    , is not fully established. To address these questions, we quantified the temporal dynamics of the human stromal (mesenchymal, skeletal) stem cell (hMSC) secretome during ex vivo OB differentiation using stable isotope labeling by amino acids in cell culture (SILAC). In addition, we employed pulsed SILAC...... the identification of novel factors produced by hMSC with potential role in OB differentiation. Our study demonstrates that the secretome of osteoblastic cells is more complex than previously reported and supports the emerging evidence that osteoblastic cells secrete proteins with endocrine functions and regulate...... regulators of OB differentiation. Furthermore, we studied the biological effects of one of these proteins, the hormone stanniocalcin 2 (STC2) and demonstrated its autocrine effects in enhancing osteoblastic differentiation of hMSC. In conclusion, combining complete and pulsed SILAC labeling facilitated...

  1. Hydroxyapatite and Calcified Elastin Induce Osteoblast-like Differentiation in Rat Aortic Smooth Muscle Cells

    Science.gov (United States)

    Lei, Yang; Sinha, Aditi; Nosoudi, Nasim; Grover, Ankit; Vyavahare, Naren

    2014-01-01

    Vascular calcification can be categorized into two different types. Intimal calcification related to atherosclerosis and elastin-specific medial arterial calcification (MAC). Osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) has been shown in both types; however, how this relates to initiation of vascular calcification is unclear. We hypothesize that the initial deposition of hydroxyapatite-like mineral in MAC occurs on degraded elastin first and that causes osteogenic transformation of VSMCs. To test this, rat aortic smooth muscle cells (RASMCs) were cultured on hydroxyapatite crystals and calcified aortic elastin. Using RT-PCR and specific protein assays, we demonstrate that RASMCs lose their smooth muscle lineage markers like alpha smooth muscle actin (SMA) and myosin heavy chain (MHC) and undergo chondrogenic/osteogenic transformation. This is indicated by an increase in the expression of typical chondrogenic proteins such as aggrecan, collagen type II alpha 1(Col2a1) and bone proteins such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, when calcified conditions are removed, cells return to their original phenotype. Our data supports the hypothesis that elastin degradation and calcification precedes VSMCs' osteoblast-like differentiation. PMID:24447384

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  3. The effects of 6-gingerol on proliferation, differentiation, and maturation of osteoblast-like MG-63 cells

    Energy Technology Data Exchange (ETDEWEB)

    Fan, J.Z.; Yang, X.; Bi, Z.G. [Department of Orthopedic Surgery, First Affiliated Hospital, Harbin Medicine University, Harbin (China)

    2015-04-28

    We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α). Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation.

  4. Culture conditions for equine bone marrow mesenchymal stem cells and expression of key transcription factors during their differentiation into osteoblasts

    Science.gov (United States)

    2013-01-01

    Background The use of equine bone marrow mesenchymal stem cells (BMSC) is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine: 1) if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2) the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum (FBS) or autologous horse serum (HS), and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 (BMP2), dexamethasone (DEX), or combination of the two. Alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-related transcription factor2 (Runx2), osterix (Osx), and T-box3 (Tbx3). Data were analyzed by ANOVA. Results Relative to control, FBS and HS increased cell number (133 ± 5 and 116 ± 5%, respectively; P  0.8). Runt-related transcription factor2 expression increased 3-fold (P equine BMSC into osteoblasts. In addition, expression of Runx2 and osterix increased and expression of Tbx3 is reduced during differentiation. PMID:24169030

  5. Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins During Ex Vivo Osteoblast Differentiation of Human Stromal Stem Cells*

    Science.gov (United States)

    Kristensen, Lars P.; Chen, Li; Nielsen, Maria Overbeck; Qanie, Diyako W.; Kratchmarova, Irina; Kassem, Moustapha; Andersen, Jens S.

    2012-01-01

    It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process, is not fully established. To address these questions, we quantified the temporal dynamics of the human stromal (mesenchymal, skeletal) stem cell (hMSC) secretome during ex vivo OB differentiation using stable isotope labeling by amino acids in cell culture (SILAC). In addition, we employed pulsed SILAC labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited more than 2-fold up or down-regulation. The pulsed SILAC method revealed a strong correlation between the fraction of isotope labeling and the subset of proteins known to be secreted and involved in OB differentiation. We verified SILAC data using qRT-PCR analysis of 9 identified potential novel regulators of OB differentiation. Furthermore, we studied the biological effects of one of these proteins, the hormone stanniocalcin 2 (STC2) and demonstrated its autocrine effects in enhancing osteoblastic differentiation of hMSC. In conclusion, combining complete and pulsed SILAC labeling facilitated the identification of novel factors produced by hMSC with potential role in OB differentiation. Our study demonstrates that the secretome of osteoblastic cells is more complex than previously reported and supports the emerging evidence that osteoblastic cells secrete proteins with endocrine functions and regulate cellular processes beyond bone formation. PMID:22801418

  6. The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

    International Nuclear Information System (INIS)

    Id Boufker, Hichame; Lagneaux, Laurence; Najar, Mehdi; Piccart, Martine; Ghanem, Ghanem; Body, Jean-Jacques; Journé, Fabrice

    2010-01-01

    The proto-oncogene Src is an important non-receptor protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and differentiation. It negatively regulates osteoblast activity, and, as such, its inhibition is a potential means to prevent bone loss. Dasatinib is a new dual Src/Bcr-Abl tyrosine kinase inhibitor initially developed for the treatment of chronic myeloid leukemia. It has also shown promising results in preclinical studies in various solid tumors. However, its effects on the differentiation of human osteoblasts have never been examined. We evaluated the effects of dasatinib on bone marrow-derived mesenchymal stromal cells (MSC) differentiation into osteoblasts, in the presence or absence of a mixture of dexamethasone, ascorbic acid and β-glycerophosphate (DAG) for up to 21 days. The differentiation kinetics was assessed by evaluating mineralization of the extracellular matrix, alkaline phosphatase (ALP) activity, and expression of osteoblastic markers (receptor activator of nuclear factor kappa B ligand [RANKL], bone sialoprotein [BSP], osteopontin [OPN]). Dasatinib significantly increased the activity of ALP and the level of calcium deposition in MSC cultured with DAG after, respectively, 7 and 14 days; it upregulated the expression of BSP and OPN genes independently of DAG; and it markedly downregulated the expression of RANKL gene and protein (decrease in RANKL/OPG ratio), the key factor that stimulates osteoclast differentiation and activity. Our results suggest a dual role for dasatinib in both (i) stimulating osteoblast differentiation leading to a direct increase in bone formation, and (ii) downregulating RANKL synthesis by osteoblasts leading to an indirect inhibition of osteoclastogenesis. Thus, dasatinib is a potentially interesting candidate drug for the treatment of osteolysis through its dual effect on bone metabolism

  7. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation

    Energy Technology Data Exchange (ETDEWEB)

    Godoy-Gallardo, Maria, E-mail: maria.godoy.gallardo@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Guillem-Marti, Jordi, E-mail: jordi.guillem.marti@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Sevilla, Pablo, E-mail: psevilla@euss.es [Department of Mechanics, Escola Universitària Salesiana de Sarrià (EUSS), C/ Passeig de Sant Bosco, 42, 08017 Barcelona (Spain); Manero, José M., E-mail: jose.maria.manero@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Gil, Francisco J., E-mail: francesc.xavier.gil@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); and others

    2016-02-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria–cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. - Highlights: • TESPSA silane induces osteoblast differentiation. • TESPSA reduces bacterial adhesion and biofilm formation. • TESPSA is a promising anchoring platform of biomolecules onto titanium.

  8. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation

    International Nuclear Information System (INIS)

    Godoy-Gallardo, Maria; Guillem-Marti, Jordi; Sevilla, Pablo; Manero, José M.; Gil, Francisco J.

    2016-01-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria–cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. - Highlights: • TESPSA silane induces osteoblast differentiation. • TESPSA reduces bacterial adhesion and biofilm formation. • TESPSA is a promising anchoring platform of biomolecules onto titanium.

  9. Using quantitative proteomics methods for studying the secreteome of human mesenchymal stem cells during osteoblast differentiation

    DEFF Research Database (Denmark)

    Kristensen, Lars Peter

      Betydningen af skelettet som et endokrint organ er et voksende felt indenfor knogle biologi. Der er imidlertid begræset information tilgængelig vedrørende de faktorer der seceneres af osteoblaster under deres differentiering og tidligere rapporterede kandidater er primært baseret på indirekte m...

  10. Lactate induces osteoblast differentiation by stabilization of HIF1α.

    Science.gov (United States)

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

    2017-09-05

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

  11. Retroviral-mediated gene therapy for the differentiation of primary cells into a mineralizing osteoblastic phenotype.

    Science.gov (United States)

    Phillips, Jennifer E; García, Andrés J

    2008-01-01

    Bone tissue engineering has emerged as a promising strategy for the repair of critical-sized skeletal fractures. However, the clinical application of this approach has been limited by the availability of a robust mineralizing cell source. Non-osteogenic cells, such as skin fibroblasts, are an attractive cell-source alternative because they are easy to harvest from autologous donor skin biopsies and display a high capacity for in vitro expansion. We have recently demonstrated that retroviral gene delivery of the osteoblastic transcription factor Runx2/Cbfa1 promotes osteogenic differentiation in primary dermal fibroblasts cultured in monolayer. Notably, sustained expression of Runx2 was not sufficient to promote functional osteogenesis in these cells, and co-treatment with the steroid hormone dexamethasone was required to induce deposition of biologically-equivalent matrix mineralization. On the basis of these results, we then investigated the osteogenic capacity of these genetically engineered fibroblasts when seeded on polymeric scaffolds in vitro and in vivo. These experiments demonstrated that Runx2-expressing fibroblasts seeded on collagen scaffolds produce significant levels of matrix mineralization after 28 days in vivo implantation in a subcutaneous, heterotopic site. Overall, these results offer evidence that transcription factor-based gene therapy may be a powerful strategy for the conversion of a non-osteogenic cellular phenotype into a mineralizing cell source for bone repair applications. This concept may also be applied to control functional differentiation in a broad range of cell types and tissue engineering applications. The chapter below outlines detailed methods for the isolation and ex vivo genetic modification of primary dermal fibroblasts using retroviral-mediated delivery of the Runx2 transgene in both monolayer culture and three-dimensional scaffolds.

  12. Resveratrol inhibits myeloma cell growth, prevents osteoclast formation, and promotes osteoblast differentiation

    DEFF Research Database (Denmark)

    Boissy, Patrice; Andersen, Thomas L; Abdallah, Basem M

    2005-01-01

    , a challenge for treating multiple myeloma is discovering drugs targeting not only myeloma cells but also osteoclasts and osteoblasts. Because resveratrol (trans-3,4',5-trihydroxystilbene) is reported to display antitumor activities on a variety of human cancer cells, we investigated the effects...... of this natural compound on myeloma and bone cells. We found that resveratrol reduces dose-dependently the growth of myeloma cell lines (RPMI 8226 and OPM-2) by a mechanism involving cell apoptosis. In cultures of human primary monocytes, resveratrol inhibits dose-dependently receptor activator of nuclear factor......RNA and cell surface protein levels and a decrease of NFATc1 stimulation and NF-kappaB nuclear translocation, whereas the gene expression of c-fms, CD14, and CD11a is up-regulated. Finally, resveratrol promotes dose-dependently the expression of osteoblast markers like osteocalcin and osteopontin in human bone...

  13. 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

  14. Culture & differentiation of mesenchymal stem cell into osteoblast on degradable biomedical composite scaffold: In vitro study

    Directory of Open Access Journals (Sweden)

    Krishan G Jain

    2015-01-01

    Full Text Available Background & objectives: There is a significant bone tissue loss in patients from diseases and traumatic injury. The current autograft transplantation gold standard treatment has drawbacks, namely donor site morbidity and limited supply. The field of tissue engineering has emerged with a goal to provide alternative sources for transplantations to bridge this gap between the need and lack of bone graft. The aim of this study was to prepare biocomposite scaffolds based on chitosan (CHT, polycaprolactone (PCL and hydroxyapatite (HAP by freeze drying method and to assess the role of scaffolds in spatial organization, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSCs in vitro, in order to achieve bone graft substitutes with improved physical-chemical and biological properties. Methods: Pure chitosan (100CHT and composites (40CHT/HAP, 30CHT/HAP/PCL and 25CHT/HAP/PCL scaffolds containing 40, 30, 25 parts per hundred resin (phr filler, respectively in acetic acid were freeze dried and the porous foams were studied for physicochemical and in vitro biological properties. Results: Scanning electron microscope (SEM images of the scaffolds showed porous microstructure (20-300 μm with uniform pore distribution in all compositions. Materials were tested under compressive load in wet condition (using phosphate buffered saline at pH 7.4. The in vitro studies showed that all the scaffold compositions supported mesenchymal stem cell attachment, proliferation and differentiation as visible from SEM images, [3-(4,5-dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide] (MTT assay, alkaline phosphatase (ALP assay and quantitative reverse transcription (qRT-PCR. Interpretation & conclusions: Scaffold composition 25CHT/HAP/PCL showed better biomechanical and osteoinductive properties as evident by mechanical test and alkaline phosphatase activity and osteoblast specific gene expression studies. This study suggests that this novel

  15. Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells

    Science.gov (United States)

    Ali, Dalia; Hamam, Rimi; Alfayez, Musaed; Kassem, Moustapha; Aldahmash, Abdullah

    2016-01-01

    The epigenetic mechanisms promoting lineage-specific commitment of human skeletal (mesenchymal or stromal) stem cells (hMSCs) into adipocytes or osteoblasts are still not fully understood. Herein, we performed an epigenetic library functional screen and identified several novel compounds, including abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell proliferation and differentiation that were targeted by abexinostat. Concordantly, ChIP-quantitative polymerase chain reaction revealed marked increase in H3K9Ac epigenetic mark on the promoter region of AdipoQ, FABP4, PPARγ, KLF15, CEBPA, SP7, and ALPL in abexinostat-treated hMSCs. Pharmacological inhibition of focal adhesion kinase (PF-573228) or insulin-like growth factor-1R/insulin receptor (NVP-AEW51) signaling exhibited significant inhibition of abexinostat-mediated adipocytic differentiation, whereas inhibition of WNT (XAV939) or transforming growth factor-β (SB505124) signaling abrogated abexinostat-mediated osteogenic differentiation of hMSCs. Our findings provide insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways governing adipocyte and osteoblast differentiation. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies and tissue engineering. Significance This unbiased epigenetic library functional screen identified several novel compounds, including abexinostat, that promoted adipocytic and osteoblastic differentiation of human skeletal (mesenchymal or stromal) stem cells (hMSCs). These data provide new insight into the understanding of the relationship between the epigenetic effect of histone deacetylase

  16. Barium titanate nanoparticles and hypergravity stimulation improve differentiation of mesenchymal stem cells into osteoblasts

    Directory of Open Access Journals (Sweden)

    Rocca A

    2015-01-01

    Full Text Available Antonella Rocca,1,2 Attilio Marino,1,2 Veronica Rocca,3 Stefania Moscato,4 Giuseppe de Vito,5,6 Vincenzo Piazza,5 Barbara Mazzolai,1 Virgilio Mattoli,1 Thu Jennifer Ngo-Anh,7 Gianni Ciofani1 1Istituto Italiano di Tecnologia, Center for Micro-BioRobotics @SSSA, Pontedera, Italy, 2Scuola Superiore Sant’Anna, The BioRobotics Institute, Pontedera, Italy, 3Università di Pisa, Dipartimento di Ingegneria dell’Informazione, Pisa, Italy, 4Università di Pisa, Dipartimento di Medicina Clinica e Sperimentale, Pisa, Italy, 5Istituto Italiano di Tecnologia, Center for Nanotechnology Innovation @NEST, Pisa, Italy, 6Scuola Normale Superiore, NEST, Pisa, Italy, 7Directorate of Human Spaceflight and Operations, European Space Agency, Noordwijk, the Netherlands Background: Enhancement of the osteogenic potential of mesenchymal stem cells (MSCs is highly desirable in the field of bone regeneration. This paper proposes a new approach for the improvement of osteogenesis combining hypergravity with osteoinductive nanoparticles (NPs.Materials and methods: In this study, we aimed to investigate the combined effects of hypergravity and barium titanate NPs (BTNPs on the osteogenic differentiation of rat MSCs, and the hypergravity effects on NP internalization. To obtain the hypergravity condition, we used a large-diameter centrifuge in the presence of a BTNP-doped culture medium. We analyzed cell morphology and NP internalization with immunofluorescent staining and coherent anti-Stokes Raman scattering, respectively. Moreover, cell differentiation was evaluated both at the gene level with quantitative real-time reverse-transcription polymerase chain reaction and at the protein level with Western blotting.Results: Following a 20 g treatment, we found alterations in cytoskeleton conformation, cellular shape and morphology, as well as a significant increment of expression of osteoblastic markers both at the gene and protein levels, jointly pointing to a substantial

  17. Irisin Enhances Osteoblast Differentiation In Vitro

    Directory of Open Access Journals (Sweden)

    Graziana Colaianni

    2014-01-01

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

  18. Differentiation and cytokine synthesis of human alveolar osteoblasts compared to osteoblast-like cells (MG63) in response to titanium surfaces.

    Science.gov (United States)

    Rausch-fan, Xiaohui; Qu, Zhe; Wieland, Marco; Matejka, Michael; Schedle, Andreas

    2008-01-01

    The aim of this study was to investigate the influence of different implant surface topographies and chemistries on the expression of differentiation/proliferation markers on MG63 cells and primary human alveolar osteoblasts. Hydrophobic acid-etched (A) and hydrophobic coarse-grit-blasted, acid-etched (SLA) surfaces and hydrophilic acid-etched (modA) and hydrophilic coarse-grit-blasted (modSLA) surfaces were produced. Thereby, modA and modSLA surfaces were rinsed under nitrogen protection and stored in a sealed glass tube containing isotonic NaCl solution at pH 4-6. Tissue culture plates without specimens served as controls. The behavior of MG63 cells and primary human alveolar osteoblasts (AOB) grown on all surfaces was compared through determination of alkaline phosphatase (ALP) activity, cell proliferation ((3)H-thymidin incorporation, MTT colorimetric assay) and expression of osteocalcin (OC), osteoprotegerin (OPG), transforming growth factor-beta1 (TGF-beta(1)) and vascular endothelial growth factor (VEGF), detected with commercial available test kits. Proliferation of MG63 and primary cells was highest on controls, followed by A surfaces, modA and SLA surfaces being almost on the same level and lowest on modSLA surfaces. modSLA surfaces exhibited highest ALP and OC production, followed by SLA, modA and A surfaces. Proliferation and OC production were comparable for MG63 cells and AOB. OPG, TGF-beta(1) and VEGF produced on primary cells showed a slightly different rank order on different surfaces compared to MG63 cells. modSLA still showed the highest production of OPG, TGF-beta(1) and VEGF, but was followed by modA, SLA and A. Statistical significance was checked by ANOVA (pmodA surfaces showed enhanced expression of OPG, TGF-beta(1) and VEGF on MG63 cells compared to primary human alveolar osteoblasts. Overall, the lowest proliferation rates and the highest expressions of differentiation markers and growth factor productions were observed on modSLA.

  19. UV-activated 7-dehydrocholesterol-coated titanium implants promote differentiation of human umbilical cord mesenchymal stem cells into osteoblasts.

    Science.gov (United States)

    Satué, María; Ramis, Joana M; Monjo, Marta

    2016-01-01

    Vitamin D metabolites are essential for bone regeneration and mineral homeostasis. The vitamin D precursor 7-dehydrocholesterol can be used after UV irradiation to locally produce active vitamin D by osteoblastic cells. Furthermore, UV-irradiated 7-dehydrocholesterol is a biocompatible coating for titanium implants with positive effects on osteoblast differentiation. In this study, we examined the impact of titanium implants surfaces coated with UV-irradiated 7-dehydrocholesterol on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. First, the synthesis of cholecalciferol (D3) was achieved through the incubation of the UV-activated 7-dehydrocholesterol coating for 48 h at 23℃. Further, we investigated in vitro the biocompatibility of this coating in human umbilical cord mesenchymal stem cells and its potential to enhance their differentiation towards the osteogenic lineage. Human umbilical cord mesenchymal stem cells cultured onto UV-irradiated 7-dehydrocholesterol-coated titanium implants surfaces, combined with osteogenic supplements, upregulated the gene expression of several osteogenic markers and showed higher alkaline phosphatase activity and calcein blue staining, suggesting increased mineralization. Thus, our results show that the use of UV irradiation on 7-dehydrocholesterol -treated titanium implants surfaces generates a bioactive coating that promotes the osteogenic differentiation of human umbilical cord mesenchymal stem cells, with regenerative potential for improving osseointegration in titanium-based bone anchored implants. © The Author(s) 2015.

  20. Accelerated differentiation of osteoblast cells on polycaprolactone scaffolds driven by a combined effect of protein coating and plasma modification

    Energy Technology Data Exchange (ETDEWEB)

    Yildirim, Eda D; Gueceri, Selcuk; Sun, Wei [Department of Mechanical Engineering and Mechanics, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104 (United States); Besunder, Robyn; Allen, Fred [Drexel University, School of Biomedical Engineering Science and Health System, 3141 Chestnut Street, Philadelphia, PA 19104 (United States); Pappas, Daphne, E-mail: edy22@drexel.ed [Army Research Laboratory, Aberdeen Proving Ground, MD 21005 (United States)

    2010-03-15

    A combined effect of protein coating and plasma modification on the quality of the osteoblast-scaffold interaction was investigated. Three-dimensional polycaprolactone (PCL) scaffolds were manufactured by the precision extrusion deposition (PED) system. The structural, physical, chemical and biological cues were introduced to the surface through providing 3D structure, coating with adhesive protein fibronectin and modifying the surface with oxygen-based plasma. The changes in the surface properties of PCL after those modifications were examined by contact angle goniometry, surface energy calculation, surface chemistry analysis (XPS) and surface topography measurements (AFM). The effects of modification techniques on osteoblast short-term and long-term functions were examined by cell adhesion, proliferation assays and differentiation markers, namely alkaline phosphatase activity (ALP) and osteocalcin secretion. The results suggested that the physical and chemical cues introduced by plasma modification might be sufficient for improved cell adhesion, but for accelerated osteoblast differentiation the synergetic effects of structural, physical, chemical and biological cues should be introduced to the PCL surface.

  1. Differentiation of Bone Marrow Mesenchymal Stem Cells in Osteoblasts and Adipocytes and its Role in Treatment of Osteoporosis.

    Science.gov (United States)

    Wang, Cheng; Meng, Haoye; Wang, Xin; Zhao, Chenyang; Peng, Jing; Wang, Yu

    2016-01-21

    Osteoporosis is a systemic metabolic bone disorder characterized by a decrease in bone mass and degradation of the bone microstructure, leaving bones that are fragile and prone to fracture. Most osteoporosis treatments improve symptoms, but to date there is no quick and effective therapy. Bone marrow mesenchymal stem cells (BMMSCs) have pluripotent potential. In adults, BMMSCs differentiate mainly into osteoblasts and adipocytes in the skeleton. However, if this differentiation is unbalanced, it may lead to a decrease in bone mass. If the number of adipocyte cells increases and that of osteoblast cells decreases, osteoporosis can result. A variety of hormones and cytokines play an important role in the regulation of BMMSCs bidirectional differentiation. Therefore, a greater understanding of the regulation mechanism of BMMSC differentiation may provide new methods to prevent and treat osteoporosis. In addition, autologous, allogeneic BMMSCs or genetically modified BMMSC transplantation can effectively increase bone mass and density, increase bone mechanical strength, correct the imbalance in bone metabolism, and increase bone formation, and is expected to provide a new strategy and method for the treatment of osteoporosis.

  2. Platelet-poor plasma stimulates the proliferation but inhibits the differentiation of rat osteoblastic cells in vitro.

    Science.gov (United States)

    Hamdan, Ahmad Abdel-Salam; Loty, Sabine; Isaac, Juliane; Bouchard, Philippe; Berdal, Ariane; Sautier, Jean-Michel

    2009-06-01

    Recent studies have shown that the use of platelet preparations in bone and implant surgery might stimulate bone formation. However, the biological mechanisms are not well understood. Moreover, few studies have attempted to evaluate the effect of platelet-poor plasma (PPP), which is a product of the platelet-rich plasma preparation process. Thus, this study investigated the behavior of osteoblasts isolated from fetal rat calvaria cultivated in the presence of homologous PPP. PPP was obtained by centrifugation of the rat mother's blood and used in replacement of fetal calf serum, which is classically used in primary culture procedures. Proliferation was measured by an MTT assay at 24, 48, and 72 h. Real-time PCR was performed to study the expression of Runx2, Dlx5, and osteocalcin (OC) on days 0 (4 h), 1, 3, 7, and 12. Alkaline phosphatase (ALP) biochemical activity was evaluated on days 0 (4 h), 1, 3, 7, and 12. Observations by phase-contrast microscopy showed that osteoblasts were able to differentiate until the mineralization of the matrix in the presence of PPP. PPP enhanced the proliferation significantly compared with the control group (Pexpressed by cells in the experimental group at lower levels compared with the control group. Biochemical assay of ALP showed a lower activity in the experimental group compared with the control group (P<0.001). These results suggest that, in the presence of homologous PPP, rat osteoblastic cells are able to maintain their phenotype, with a higher rate of proliferation. However, PPP seems to inhibit osteoblastic differentiation.

  3. Addition of Wollastonite Fibers to Calcium Phosphate Cement Increases Cell Viability and Stimulates Differentiation of Osteoblast-Like Cells

    Directory of Open Access Journals (Sweden)

    Juliana Almeida Domingues

    2017-01-01

    Full Text Available Calcium phosphate cement (CPC that is based on α-tricalcium phosphate (α-TCP is considered desirable for bone tissue engineering because of its relatively rapid degradation properties. However, such cement is relatively weak, restricting its use to areas of low mechanical stress. Wollastonite fibers (WF have been used to improve the mechanical strength of biomaterials. However, the biological properties of WF remain poorly understood. Here, we tested the response of osteoblast-like cells to being cultured on CPC reinforced with 5% of WF (CPC-WF. We found that both types of cement studied achieved an ion balance for calcium and phosphate after 3 days of immersion in culture medium and this allowed subsequent long-term cell culture. CPC-WF increased cell viability and stimulated cell differentiation, compared to nonreinforced CPC. We hypothesize that late silicon release by CPC-WF induces increased cell proliferation and differentiation. Based on our findings, we propose that CPC-WF is a promising material for bone tissue engineering applications.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Tridax procumbens flavonoids promote osteoblast differentiation and bone formation

    Directory of Open Access Journals (Sweden)

    Md. Abdullah Al Mamun

    Full Text Available BACKGROUND: Tridaxprocumbens flavonoids (TPFs are well known for their medicinal properties among local natives. Besides traditionally used for dropsy, anemia, arthritis, gout, asthma, ulcer, piles, and urinary problems, it is also used in treating gastric problems, body pain, and rheumatic pains of joints. TPFs have been reported to increase osteogenic functioning in mesenchymal stem cells. Our previous study showed that TPFs were significantly suppressed the RANKL-induced differentiation of osteoclasts and bone resorption. However, the effects of TPFs to promote osteoblasts differentiation and bone formation remain unclear. TPFs were isolated from Tridax procumbens and investigated for their effects on osteoblasts differentiation and bone formation by using primary mouse calvarial osteoblasts RESULTS: TPFs promoted osteoblast differentiation in a dose-dependent manner demonstrated by up-regulation of alkaline phosphatase and osteocalcin. TPFs also upregulated osteoblast differentiation related genes, including osteocalcin, osterix, and Runx2 in primary osteoblasts. TPFs treated primary osteoblast cells showed significant upregulation of bone morphogenetic proteins (BMPs including Bmp-2, Bmp-4, and Bmp-7. Addition of noggin, a BMP specific-antagonist, inhibited TPFs induced upregulation of the osteocalcin, osterix, and Runx2 CONCLUSION: Our findings point towards the induction of osteoblast differentiation by TPFs and suggested that TPFs could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis

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

    Science.gov (United States)

    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

  7. Transgelin is a TGFβ-inducible gene that regulates osteoblastic and adipogenic differentiation of human skeletal stem cells through actin cytoskeleston organization

    DEFF Research Database (Denmark)

    Elsafadi, E; Manikandan, M; Dawud, R. A.

    2016-01-01

    Regenerative medicine is a novel approach for treating conditions in which enhanced bone regeneration is required. We identified transgelin (TAGLN), a transforming growth factor beta (TGFβ)-inducible gene, as an upregulated gene during in vitro osteoblastic and adipocytic differentiation of human......MSC by regulating cytoskeleton organization. Targeting TAGLN is a plausible approach to enrich for committed hMSC cells needed for regenerative medicine application....... bone marrow-derived stromal (skeletal) stem cells (hMSC). siRNA-mediated gene silencing of TAGLN impaired lineage differentiation into osteoblasts and adipocytes but enhanced cell proliferation. Additional functional studies revealed that TAGLN deficiency impaired hMSC cell motility and in vitro...... transwell cell migration. On the other hand, TAGLN overexpression reduced hMSC cell proliferation, but enhanced cell migration, osteoblastic and adipocytic differentiation, and in vivo bone formation. In addition, deficiency or overexpression of TAGLN in hMSC was associated with significant changes...

  8. Appearance of cell-adhesion factor in osteoblast proliferation and differentiation of apatite coating titanium by blast coating method.

    Science.gov (United States)

    Umeda, Hirotsugu; Mano, Takamitsu; Harada, Koji; Tarannum, Ferdous; Ueyama, Yoshiya

    2017-08-01

    We have already reported that the apatite coating of titanium by the blast coating (BC) method could show a higher rate of bone contact from the early stages in vivo, when compared to the pure titanium (Ti) and the apatite coating of titanium by the flame spraying (FS) method. However, the detailed mechanism by which BC resulted in satisfactory bone contact is still unknown. In the present study, we investigated the importance of various factors including cell adhesion factor in osteoblast proliferation and differentiation that could affect the osteoconductivity of the BC disks. Cell proliferation assay revealed that Saos-2 could grow fastest on BC disks, and that a spectrophotometric method using a LabAssay TM ALP kit showed that ALP activity was increased in cells on BC disks compared to Ti disks and FS disks. In addition, higher expression of E-cadherin and Fibronectin was observed in cells on BC disks than Ti disks and FS disks by relative qPCR as well as Western blotting. These results suggested that the expression of cell-adhesion factors, proliferation and differentiation of osteoblast might be enhanced on BC disks, which might result higher osteoconductivity.

  9. Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

    Science.gov (United States)

    Leyva-Leyva, Margarita; López-Díaz, Annia; Barrera, Lourdes; Camacho-Morales, Alberto; Hernandez-Aguilar, Felipe; Carrillo-Casas, Erika M; Arriaga-Pizano, Lourdes; Calderón-Pérez, Jaime; García-Álvarez, Jorge; Orozco-Hoyuela, Gabriel; Piña-Barba, Cristina; Rojas-Martínez, Augusto; Romero-Díaz, Víktor; Lara-Arias, Jorge; Rivera-Bolaños, Nancy; López-Camarillo, César; Moncada-Saucedo, Nidia; Galván-De los Santos, Alejandra; Meza-Urzúa, Fátima; Villarreal-Gómez, Luis; Fuentes-Mera, Lizeth

    2015-11-01

    Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated β1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in

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

    NARCIS (Netherlands)

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

    2005-01-01

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

  11. Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

    Science.gov (United States)

    Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

    2014-01-01

    -forming activity of osteoclast-like cells cultured on dentin slices. These results suggest that arctigenin induces a dominant negative species of NFATc1, which inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

  12. Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

    Directory of Open Access Journals (Sweden)

    Teruhito Yamashita

    pit-forming activity of osteoclast-like cells cultured on dentin slices. These results suggest that arctigenin induces a dominant negative species of NFATc1, which inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

  13. Arctigenin Inhibits Osteoclast Differentiation and Function by Suppressing Both Calcineurin-Dependent and Osteoblastic Cell-Dependent NFATc1 Pathways

    Science.gov (United States)

    Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

    2014-01-01

    -forming activity of osteoclast-like cells cultured on dentin slices. These results suggest that arctigenin induces a dominant negative species of NFATc1, which inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways. PMID:24465763

  14. Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3{beta}

    Energy Technology Data Exchange (ETDEWEB)

    Zeadin, Melec G.; Butcher, Martin K.; Shaughnessy, Stephen G. [Department of Medicine, McMaster University, Hamilton, ON (Canada); Thrombosis and Atherosclerosis Research Institute, Hamilton, ON (Canada); Werstuck, Geoff H., E-mail: Geoff.Werstuck@taari.ca [Department of Medicine, McMaster University, Hamilton, ON (Canada); Thrombosis and Atherosclerosis Research Institute, Hamilton, ON (Canada)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Leptin promotes osteoblast differentiation of primary smooth muscle cells. Black-Right-Pointing-Pointer Leptin regulates the expression of genes involved in osteoblast differentiation. Black-Right-Pointing-Pointer Constitutively active GSK-3{beta} attenuates leptin-induced osteoblast differentiation. Black-Right-Pointing-Pointer This suggests that leptin signals through GSK-3{beta} to promote osteoblast differentiation. -- Abstract: In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 {mu}g/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3{beta} on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of {beta}-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3{beta} (Ad-GSK-3{beta} S9A) resulted in a >2-fold increase in GSK-3{beta} activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3{beta} activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-01-01

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

  16. Apc bridges Wnt/{beta}-catenin and BMP signaling during osteoblast differentiation of KS483 cells

    Energy Technology Data Exchange (ETDEWEB)

    Miclea, Razvan L., E-mail: R.L.Miclea@lumc.nl [Department of Pediatrics, Leiden University Medical Centre (LUMC), Leiden (Netherlands); Horst, Geertje van der, E-mail: G.van_der_Horst@lumc.nl [Department of Urology, LUMC, Leiden (Netherlands); Robanus-Maandag, Els C., E-mail: E.C.Robanus@lumc.nl [Department of Human Genetics, LUMC, Leiden (Netherlands); Loewik, Clemens W.G.M., E-mail: C.W.G.M.Lowik@lumc.nl [Department of Endocrinology and Metabolic Diseases, LUMC, Leiden (Netherlands); Oostdijk, Wilma, E-mail: W.Oostdijk@lumc.nl [Department of Pediatrics, Leiden University Medical Centre (LUMC), Leiden (Netherlands); Wit, Jan M., E-mail: J.M.Wit@lumc.nl [Department of Pediatrics, Leiden University Medical Centre (LUMC), Leiden (Netherlands); Karperien, Marcel, E-mail: H.B.J.Karperien@tnw.utwente.nl [MIRA Institute for Biomedical Technology and Technical Medicine, Department of Tissue Regeneration, University of Twente, Zuidhorst Room ZH 144, Drienerlolaan 5, 7522 NB Enschede (Netherlands)

    2011-06-10

    The canonical Wnt signaling pathway influences the differentiation of mesenchymal cell lineages in a quantitative and qualitative fashion depending on the dose of {beta}-catenin signaling. Adenomatous polyposis coli (Apc) is the critical intracellular regulator of {beta}-catenin turnover. To better understand the molecular mechanisms underlying the role of Apc in regulating the differentiation capacity of skeletal progenitor cells, we have knocked down Apc in the murine mesenchymal stem cell-like KS483 cells by stable expression of Apc-specific small interfering RNA. In routine culture, KSFrt-Apc{sub si} cells displayed a mesenchymal-like spindle shape morphology, exhibited markedly decreased proliferation and increased apoptosis. Apc knockdown resulted in upregulation of the Wnt/{beta}-catenin and the BMP/Smad signaling pathways, but osteogenic differentiation was completely inhibited. This effect could be rescued by adding high concentrations of BMP-7 to the differentiation medium. Furthermore, KSFrt-Apc{sub si} cells showed no potential to differentiate into chondrocytes or adipocytes. These results demonstrate that Apc is essential for the proliferation, survival and differentiation of KS483 cells. Apc knockdown blocks the osteogenic differentiation of skeletal progenitor cells, a process that can be overruled by high BMP signaling.

  17. Micro-/Nano- sized hydroxyapatite directs differentiation of rat bone marrow derived mesenchymal stem cells towards an osteoblast lineage

    Science.gov (United States)

    Huang, Yan; Zhou, Gang; Zheng, Lisha; Liu, Haifeng; Niu, Xufeng; Fan, Yubo

    2012-03-01

    Regenerative medicine consisting of cells and materials provides a new way for the repair and regeneration of tissues and organs. Nano-biomaterials are highlighted due to their advantageous features compared with conventional micro-materials. The aim of this study is to investigate the effects of micro-/nano- sized hydroxyapatite (μ/n-HA) on the osteogenic differentiation of rat bone marrow derived mesenchymal stem cells (rBMSCs). μ/n-HA were prepared by a microwave synthesizer and precipitation method, respectively. Different sizes of μ/n-HA were characterized by IR, XRD, SEM, TEM and co-cultured with rBMSCs. It was shown that rBMSCs expressed higher levels of osteoblast-related markers by n-HA than μ-HA stimulation. The size of HA is an important factor for affecting the osteogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.

  18. Stepwise Differentiation of Pluripotent Stem Cells into Osteoblasts Using Four Small Molecules under Serum-free and Feeder-free Conditions

    Directory of Open Access Journals (Sweden)

    Kosuke Kanke

    2014-06-01

    Full Text Available Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenylpyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide [TH] under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2, a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

  19. 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.

  20. Transcription factor ZNF25 is associated with osteoblast differentiation of human skeletal stem cells

    DEFF Research Database (Denmark)

    Twine, Natalie A.; Harkness, Linda; Kassem, Moustapha

    2016-01-01

    containing G protein-coupled receptor 5 and RAN-binding protein 3-like. We also observed enrichment in extracellular matrix organization, skeletal system development and regulation of ossification in the entire upregulated set of genes. Consistent with its function as a transcription factor during osteoblast...

  1. Osteoblastic differentiation and stress response of human mesenchymal stem cells exposed to alternating current electric fields

    Directory of Open Access Journals (Sweden)

    Kaplan David L

    2011-01-01

    Full Text Available Abstract Background Electric fields are integral to many biological events, from maintaining cellular homeostasis to embryonic development to healing. The application of electric fields offers substantial therapeutic potential, while optimal dosing regimens and the underlying mechanisms responsible for the positive clinical impact are poorly understood. Methods The purpose of this study was to track the differentiation profile and stress response of human bone marrow derived mesenchymal stem cells (hMSCs undergoing osteogenic differentiation during exposure to a 20 mV/cm, 60 kHz electric field. Morphological and biochemical changes were imaged using endogenous two-photon excited fluorescence (TPEF and quantitatively assessed through eccentricity calculations and extraction of the redox ratio from NADH, FAD and lipofuscin contributions. Real time reverse transcriptase-polymerase chain reactions (RT-PCR were used to track osteogenic differentiation markers, namely alkaline phosphatase (ALP and collagen type 1 (col1, and stress response markers, such as heat shock protein 27 (hsp27 and heat shock protein 70 (hsp70. Comparisons of collagen deposition between the stimulated hMSCs and controls were examined through second harmonic generation (SHG imaging. Results Quantitative differences in cell morphology, as described through an eccentricity ratio, were found on days 2 and days 5 (p Conclusions Electrical stimulation is a useful tool to improve hMSC osteogenic differentiation, while heat shock proteins may reveal underlying mechanisms, and optical non-invasive imaging may be used to monitor the induced morphological and biochemical changes.

  2. Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures

    DEFF Research Database (Denmark)

    Justesen, J; Dokkedahl, Karin Stenderup; Eriksen, E F

    2002-01-01

    Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP) is the ...

  3. Separate developmental programs for HLA-A and -B cell surface expression during differentiation from embryonic stem cells to lymphocytes, adipocytes and osteoblasts.

    Directory of Open Access Journals (Sweden)

    Hardee J Sabir

    Full Text Available A major problem of allogeneic stem cell therapy is immunologically mediated graft rejection. HLA class I A, B, and Cw antigens are crucial factors, but little is known of their respective expression on stem cells and their progenies. We have recently shown that locus-specific expression (HLA-A, but not -B is seen on some multipotent stem cells, and this raises the question how this is in other stem cells and how it changes during differentiation. In this study, we have used flow cytometry to investigate the cell surface expression of HLA-A and -B on human embryonic stem cells (hESC, human hematopoietic stem cells (hHSC, human mesenchymal stem cells (hMSC and their fully-differentiated progenies such as lymphocytes, adipocytes and osteoblasts. hESC showed extremely low levels of HLA-A and no -B. In contrast, multipotent hMSC and hHSC generally expressed higher levels of HLA-A and clearly HLA-B though at lower levels. IFNγ induced HLA-A to very high levels on both hESC and hMSC and HLA-B on hMSC. Even on hESC, a low expression of HLA-B was achieved. Differentiation of hMSC to osteoblasts downregulated HLA-A expression (P = 0.017. Interestingly HLA class I on T lymphocytes differed between different compartments. Mature bone marrow CD4(+ and CD8(+ T cells expressed similar HLA-A and -B levels as hHSC, while in the peripheral blood they expressed significantly more HLA-B7 (P = 0.0007 and P = 0.004 for CD4(+ and CD8(+ T cells, respectively. Thus different HLA loci are differentially regulated during differentiation of stem cells.

  4. 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.

  5. 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

  6. Fibroblast growth factor 2 inhibits up-regulation of bone morphogenic proteins and their receptors during osteoblastic differentiation of human mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Biver, Emmanuel, E-mail: ebiver@yahoo.fr [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France); Department of Rheumatology, Lille University Hospital, Roger Salengro Hospital, 59037 Lille cedex (France); Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14 (Switzerland); Soubrier, Anne-Sophie [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France); Department of Rheumatology, Lille University Hospital, Roger Salengro Hospital, 59037 Lille cedex (France); Thouverey, Cyril [Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14 (Switzerland); Cortet, Bernard [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France); Department of Rheumatology, Lille University Hospital, Roger Salengro Hospital, 59037 Lille cedex (France); Broux, Odile [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France); Caverzasio, Joseph [Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14 (Switzerland); Hardouin, Pierre [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer FGF modulates BMPs pathway in HMSCs by down-regulating BMP/BMPR expression. Black-Right-Pointing-Pointer This effect is mediated by ERK and JNK MAPKs pathways. Black-Right-Pointing-Pointer Crosstalk between FGF and BMPs must be taken into account in skeletal bioengineering. Black-Right-Pointing-Pointer It must also be considered in the use of recombinant BMPs in orthopedic and spine surgeries. -- Abstract: Understanding the interactions between growth factors and bone morphogenic proteins (BMPs) signaling remains a crucial issue to optimize the use of human mesenchymal stem cells (HMSCs) and BMPs in therapeutic perspectives and bone tissue engineering. BMPs are potent inducers of osteoblastic differentiation. They exert their actions via BMP receptors (BMPR), including BMPR1A, BMPR1B and BMPR2. Fibroblast growth factor 2 (FGF2) is expressed by cells of the osteoblastic lineage, increases their proliferation and is secreted during the healing process of fractures or in surgery bone sites. We hypothesized that FGF2 might influence HMSC osteoblastic differentiation by modulating expressions of BMPs and their receptors. BMP2, BMP4, BMPR1A and mainly BMPR1B expressions were up-regulated during this differentiation. FGF2 inhibited HMSCs osteoblastic differentiation and the up-regulation of BMPs and BMPR. This effect was prevented by inhibiting the ERK or JNK mitogen-activated protein kinases which are known to be activated by FGF2. These data provide a mechanism explaining the inhibitory effect of FGF2 on osteoblastic differentiation of HMSCs. These crosstalks between growth and osteogenic factors should be considered in the use of recombinant BMPs in therapeutic purpose of fracture repair or skeletal bioengineering.

  7. Osteoblastic differentiating potential of dental pulp stem cells in vitro cultured on a chemically modified microrough titanium surface.

    Science.gov (United States)

    DE Colli, Marianna; Radunovic, Milena; Zizzari, Vincenzo L; DI Giacomo, Viviana; DI Nisio, Chiara; Piattelli, Adriano; Calvo Guirado, José L; Zavan, Barbara; Cataldi, Amelia; Zara, Susi

    2018-03-30

    Titanium surface modification is critical for dental implant success. Our aim was to determine surfaces influence on dental pulp stem cells (DPSCs) viability and differentiation. Implants were divided into sandblasted/acid-etched (control) and sandblasted/acid-etched coated with calcium and magnesium ions (CaMg), supplied as composite (test). Proliferation was evaluated by MTT, differentiation checking osteoblastic gene expression, PGE2 secretion and matrix formation, inflammation by Interleukin 6 (IL-6) detection. MTT and IL-6 do not modify on test. A PGE2 increase on test is recorded. BMP2 is higher on test at early experimental points, Osterix and RUNX2 augment later. Alizarin-red S reveals higher matrix production on test. These results suggest that test surface is more osteoinductive, representing a start point for in vivo studies aiming at the construction of more biocompatible dental implants, whose integration and clinical performance are improved and some undesired effects, such as implant stability loss and further surgical procedures, are reduced.

  8. 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...

  9. Adhesion and differentiation of Saos-2 osteoblast-like cells on chromium-doped diamond-like carbon coatings.

    Science.gov (United States)

    Filova, Elena; Vandrovcova, Marta; Jelinek, Miroslav; Zemek, Josef; Houdkova, Jana; Jan Remsa; Kocourek, Tomas; Stankova, Lubica; Bacakova, Lucie

    2017-01-01

    Diamond-like carbon (DLC) thin films are promising for use in coating orthopaedic, dental and cardiovascular implants. The problem of DLC layers lies in their weak layer adhesion to metal implants. Chromium is used as a dopant for improving the adhesion of DLC films. Cr-DLC layers were prepared by a hybrid technology, using a combination of pulsed laser deposition (PLD) from a graphite target and magnetron sputtering. Depending on the deposition conditions, the concentration of Cr in the DLC layers moved from zero to 10.0 at.%. The effect of DLC layers with 0.0, 0.9, 1.8, 7.3, 7.7 and 10.0 at.% Cr content on the adhesion and osteogenic differentiation of human osteoblast-like Saos-2 cells was assessed in vitro. The DLC samples that contained 7.7 and 10.0 at.% of Cr supported cell spreading on day 1 after seeding. On day three after seeding, the most apparent vinculin-containing focal adhesion plaques were also found on samples with higher concentrations of chromium. On the other hand, the expression of type I collagen and alkaline phosphatase at the mRNA and protein level was the highest on Cr-DLC samples with a lower concentration of Cr (0-1.8 at.%). We can conclude that higher concentrations of chromium supported cell adhesion; however DLC and DLC doped with a lower concentration of chromium supported osteogenic cell differentiation.

  10. Angiogenic CXC chemokine expression during differentiation of human mesenchymal stem cells towards the osteoblastic lineage.

    Science.gov (United States)

    Bischoff, D S; Zhu, J H; Makhijani, N S; Kumar, A; Yamaguchi, D T

    2008-02-15

    The potential role of ELR(+) CXC chemokines in early events in bone repair was studied using human mesenchymal stem cells (hMSCs). Inflammation, which occurs in the initial phase of tissue healing in general, is critical to bone repair. Release of cytokines from infiltrating immune cells and injured bone can lead to recruitment of MSCs to the region of repair. CXC chemokines bearing the Glu-Leu-Arg (ELR) motif are also released by inflammatory cells and serve as angiogenic factors stimulating chemotaxis and proliferation of endothelial cells. hMSCs, induced to differentiate with osteogenic medium (OGM) containing ascorbate, beta-glycerophosphate (beta-GP), and dexamethasone (DEX), showed an increase in mRNA and protein secretion of the ELR(+) CXC chemokines CXCL8 and CXCL1. CXCL8 mRNA half-life studies reveal an increase in mRNA stability upon OGM stimulation. Increased expression and secretion is a result of DEX in OGM and is dose-dependent. Inhibition of the glucocorticoid receptor with mifepristone only partially inhibits DEX-stimulated CXCL8 expression indicating both glucocorticoid receptor dependent and independent pathways. Treatment with signal transduction inhibitors demonstrate that this expression is due to activation of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways and is mediated through the G(alphai)-coupled receptors. Angiogenesis assays demonstrate that OGM-stimulated conditioned media containing secreted CXCL8 and CXCL1 can induce angiogenesis of human microvascular endothelial cells in an in vitro Matrigel assay. Copyright 2007 Wiley-Liss, Inc.

  11. Patients With High Bone Mass Phenotype Exhibit Enhanced Osteoblast Differentiation and Inhibition of Adipogenesis of Human Mesenchymal Stem Cells

    DEFF Research Database (Denmark)

    Qiu, Weimin; Andersen, Tom; Bollerslev, Jens

    2007-01-01

    in iliac crest bone biopsies from patients with the HBM phenotype and controls. We also used retrovirus-mediated gene transduction to establish three different human mesenchymal stem cell (hMSC) strains stably expressing wildtype LRP5 (hMSC-LRP5WT), LRP5T244 (hMSC-LRP5T244, inactivation mutation leading...... to osteoporosis), or LRP5T253 (hMSC-LRP5T253, activation mutation leading to high bone mass). We characterized Wnt signaling activation using a dual luciferase assay, cell proliferation, lineage biomarkers using real-time PCR, and in vivo bone formation. Results: In bone biopsies, we found increased trabecular...... mineralized bone when implanted subcutaneously with hydroxyapatite/tricalcium phosphate in SCID/NOD mice. Conclusions: LRP5 mutations and the level of Wnt signaling determine differentiation fate of hMSCs into osteoblasts or adipocytes. Activation of Wnt signaling can thus provide a novel approach to increase...

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

    Science.gov (United States)

    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

  13. Time-lapse Raman imaging of osteoblast differentiation

    Science.gov (United States)

    Hashimoto, Aya; Yamaguchi, Yoshinori; Chiu, Liang-Da; Morimoto, Chiaki; Fujita, Katsumasa; Takedachi, Masahide; Kawata, Satoshi; Murakami, Shinya; Tamiya, Eiichi

    2015-07-01

    Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.

  14. Expression of cell adhesion and differentiation related genes in MC3T3 osteoblasts plated on titanium alloys: role of surface properties

    International Nuclear Information System (INIS)

    Sista, Subhash; Wen, Cuie; Hodgson, Peter D.; Pande, Gopal

    2013-01-01

    It is important to understand the cellular and molecular events that take place at the cell–material interface of implants used for bone repair. An understanding of the mechanisms involved in the initial stages of osteoblast interactions with the surface of the implant material is fundamental in deciding the fate of the cells that come in contact with it. In this study, we compared the relative gene expression of markers that are known to be associated with cell adhesion and differentiation in MC3T3 osteoblast cells, at various time points after plating the cells on surfaces of titanium (Ti) and its two alloys, titanium–zirconium (TiZr) and titanium–niobium (TiNb) by using Quantitative Real Time Polymerase Chain Reaction (RT-PCR). Our analysis indicated that expression of adhesion supporting genes was higher on TiZr surface as compared to Ti and TiNb. The behavior of these genes is possibly driven by a higher surface energy of TiZr. However no significant difference in the expression of differentiation related genes could be seen between the two alloys, although on both substrates it was higher as compared to unalloyed Ti. We propose that substrate composition of the alloys can influence the adhesion and differentiation related gene expression and that Ti alloys are better substrates for inducing osteogenesis as compared to unalloyed Ti. - Highlights: ► Methodology for comparing gene expression in osteoblasts plated on Ti, TiZr or TiNb ► Alloys with higher surface energy (TiZr) induce cell adhesion genes more efficiently ► Alloyed Ti is superior to unalloyed Ti to induce osteoblast differentiation genes

  15. 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.

  16. Biglycan deficiency increases osteoclast differentiation and activity due to defective osteoblasts

    DEFF Research Database (Denmark)

    Bi, Yanming; Nielsen, Karina L; Kilts, Tina M

    2006-01-01

    to be independent of the differential production of soluble RANKL and OPG and, instead, due to a decrease in osteoblast maturation accompanied by increase in osteoblastic proliferation. In addition to the imbalance between differentiation and proliferation, there was a differential decrease in secretory leukocyte......Bone mass is maintained by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. Although osteoblasts and osteoclasts have different developmental origins, it is generally believed that the differentiation, function, and survival of osteoclasts are regulated...... by osteogenic cells. We have previously shown that the extracellular matrix protein, biglycan (Bgn), plays an important role in the differentiation of osteoblast precursors. In this paper, we showed that Bgn is involved in regulating osteoclast differentiation through its effect on osteoblasts...

  17. 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

  18. Platelet-rich plasma stimulates osteoblastic differentiation in the presence of BMPs

    International Nuclear Information System (INIS)

    Tomoyasu, Akihiro; Higashio, Kanji; Kanomata, Kazuhiro; Goto, Masaaki; Kodaira, Kunihiko; Serizawa, Hiroko; Suda, Tatsuo; Nakamura, Atsushi; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu

    2007-01-01

    Platelet-rich plasma (PRP) is clinically used as an autologous blood product to stimulate bone formation in vivo. In the present study, we examined the effects of PRP on proliferation and osteoblast differentiation in vitro in the presence of bone morphogenetic proteins (BMPs). PRP and its soluble fraction stimulated osteoblastic differentiation of myoblasts and osteoblastic cells in the presence of BMP-2, BMP-4, BMP-6 or BMP-7. The soluble PRP fraction stimulated osteoblastic differentiation in 3D cultures using scaffolds made of collagen or hydroxyapatite. Moreover, heparin-binding fractions obtained from serum also stimulated osteoblastic differentiation in the presence of BMP-4. These results suggested that platelets contain not only growth factors for proliferation but also novel potentiator(s) for BMP-dependent osteoblastic differentiation

  19. Aryl Hydrocarbon Receptor Antagonists Mitigate the Effects of Dioxin on Critical Cellular Functions in Differentiating Human Osteoblast-Like Cells

    Directory of Open Access Journals (Sweden)

    Chawon Yun

    2018-01-01

    Full Text Available The inhibition of bone healing in humans is a well-established effect associated with cigarette smoking, but the underlying mechanisms are still unclear. Recent work using animal cell lines have implicated the aryl hydrocarbon receptor (AhR as a mediator of the anti-osteogenic effects of cigarette smoke, but the complexity of cigarette smoke mixtures makes understanding the mechanisms of action a major challenge. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin is a high-affinity AhR ligand that is frequently used to investigate biological processes impacted by AhR activation. Since there are dozens of AhR ligands present in cigarette smoke, we utilized dioxin as a prototype ligand to activate the receptor and explore its effects on pro-osteogenic biomarkers and other factors critical to osteogenesis using a human osteoblast-like cell line. We also explored the capacity for AhR antagonists to protect against dioxin action in this context. We found dioxin to inhibit osteogenic differentiation, whereas co-treatment with various AhR antagonists protected against dioxin action. Dioxin also negatively impacted cell adhesion with a corresponding reduction in the expression of integrin and cadherin proteins, which are known to be involved in this process. Similarly, the dioxin-mediated inhibition of cell migration correlated with reduced expression of the chemokine receptor CXCR4 and its ligand, CXCL12, and co-treatment with antagonists restored migratory capacity. Our results suggest that AhR activation may play a role in the bone regenerative response in humans exposed to AhR activators, such as those present in cigarette smoke. Given the similarity of our results using a human cell line to previous work done in murine cells, animal models may yield data relevant to the human setting. In addition, the AhR may represent a potential therapeutic target for orthopedic patients who smoke cigarettes, or those who are exposed to secondhand smoke or other

  20. MSM enhances GH signaling via the Jak2/STAT5b pathway in osteoblast-like cells and osteoblast differentiation through the activation of STAT5b in MSCs.

    Directory of Open Access Journals (Sweden)

    Youn Hee Joung

    Full Text Available Methylsulfonylmethane (MSM is a naturally occurring sulfur compound with well-known anti-oxidant properties and anti-inflammatory activities. But, its effects on bone are unknown. Growth hormone (GH is regulator of bone growth and bone metabolism. GH activates several signaling pathways such as the Janus kinase (Jak/signal transducers and activators of transcription (STAT pathway, thereby regulating expression of genes including insulin-like growth factor (IGF-1. GH exerts effects both directly and via IGF-1, which signals by activating the IGF-1 receptor (IGF-1R. In this study, we investigated the effects of MSM on the GH signaling via the Jak/STAT pathway in osteoblasts and the differentiation of primary bone marrow mesenchymal stem cells (MSCs. MSM was not toxic to osteoblastic cells and MSCs. MSM increased the expression of GH-related proteins including IGF-1R, p-IGF-1R, STAT5b, p-STAT5b, and Jak2 in osteoblastic cells and MSCs. MSM increased IGF-1R and GHR mRNA expression in osteoblastic cells. The expression of MSM-induced IGF-1R and GHR was inhibited by AG490, a Jak2 kinase inhibitor. MSM induced binding of STAT5 to the IGF-1R and increased IGF-1 and IGF-1R promoter activities. Analysis of cell extracts by immunoprecipitation and Western blot showed that MSM enhanced GH-induced activation of Jak2/STAT5b. We found that MSM and GH, separately or in combination, activated GH signaling via the Jak2/STAT5b pathway in UMR-106 cells. Using siRNA analysis, we found that STAT5b plays an essential role in GH signaling activation in C3H10T1/2 cells. Osteogenic marker genes (ALP, ON, OCN, BSP, OSX, and Runx2 were activated by MSM, and siRNA-mediated STAT5b knockdown inhibited MSM-induced expression of osteogenic markers. Furthermore, MSM increased ALP activity and the mineralization of MSCs. Taken together, these results indicated that MSM can promote osteogenic differentiation of MSCs through activation of STAT5b.

  1. Palmitic Acid Induces Osteoblastic Differentiation in Vascular Smooth Muscle Cells through ACSL3 and NF-κB, Novel Targets of Eicosapentaenoic Acid

    Science.gov (United States)

    Kageyama, Aiko; Matsui, Hiroki; Ohta, Masahiko; Sambuichi, Keisuke; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Yokoyama, Tomoyuki; Kurabayashi, Masahiko

    2013-01-01

    Free fatty acids (FFAs), elevated in metabolic syndrome and diabetes, play a crucial role in the development of atherosclerotic cardiovascular disease, and eicosapentaenoic acid (EPA) counteracts many aspects of FFA-induced vascular pathology. Although vascular calcification is invariably associated with atherosclerosis, the mechanisms involved are not completely elucidated. In this study, we tested the hypothesis that EPA prevents the osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC) induced by palmitic acid (PA), the most abundant long-chain saturated fatty acid in plasma. PA increased and EPA abolished the expression of the genes for bone-related proteins, including bone morphogenetic protein (BMP)-2, Msx2 and osteopontin in human aortic smooth muscle cells (HASMC). Among the long-chain acyl-CoA synthetase (ACSL) subfamily, ACSL3 expression was predominant in HASMC, and PA robustly increased and EPA efficiently inhibited ACSL3 expression. Importantly, PA-induced osteoblastic differentiation was mediated, at least in part, by ACSL3 activation because acyl-CoA synthetase (ACS) inhibitor or siRNA targeted to ACSL3 completely prevented the PA induction of both BMP-2 and Msx2. Conversely, adenovirus-mediated ACSL3 overexpression enhanced PA-induced BMP-2 and Msx2 expression. In addition, EPA, ACSL3 siRNA and ACS inhibitor attenuated calcium deposition and caspase activation induced by PA. Notably, PA induced activation of NF-κB, and NF-κB inhibitor prevented PA-induction of osteoblastic gene expression and calcium deposition. Immunohistochemistry revealed the prominent expression of ACSL3 in VSMC and macrophages in human non-calcifying and calcifying atherosclerotic plaques from the carotid arteries. These results identify ACSL3 and NF-κB as mediators of PA-induced osteoblastic differentiation and calcium deposition in VSMC and suggest that EPA prevents vascular calcification by inhibiting such a new molecular pathway elicited

  2. N-cadherin-mediated interaction with multiple myeloma cells inhibits osteoblast differentiation

    NARCIS (Netherlands)

    Groen, Richard W. J.; de Rooij, Martin F. M.; Kocemba, Kinga A.; Reijmers, Rogier M.; de Haan-Kramer, Anneke; Overdijk, Marije B.; Aalders, Linda; Rozemuller, Henk; Martens, Anton C. M.; Bergsagel, P. Leif; Kersten, Marie José; Pals, Steven T.; Spaargaren, Marcel

    2011-01-01

    Multiple myeloma is a hematologic malignancy characterized by a clonal expansion of malignant plasma cells in the bone marrow, which is accompanied by the development of osteolytic lesions and/or diffuse osteopenia. The intricate bi-directional interaction with the bone marrow microenvironment plays

  3. N-cadherin-mediated interaction with multiple myeloma cells inhibits osteoblast differentiation

    NARCIS (Netherlands)

    Groen, R.W.J.; de Rooij, M.F.M.; Kocemba, K.A.; Reijmers, R.M.; de Haan-Kramer, A.; Overdijk, M.B.; Aalders, L.; Rozemuller, H.; Martens, A.C.M.; Bergsagel, P.L.; Kersten, M.J.; Pals, S.T.; Spaargaren, M.

    2011-01-01

    Background Multiple myeloma is a hematologic malignancy characterized by a clonal expansion of malignant plasma cells in the bone marrow, which is accompanied by the development of osteolytic lesions and/or diffuse osteopenia. The intricate bi-directional interaction with the bone marrow

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

    Directory of Open Access Journals (Sweden)

    Rohaya Megat Abdul Wahab

    2017-06-01

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

  5. Separate Developmental Programs for HLA-A and -B Cell Surface Expression during Differentiation from Embryonic Stem Cells to Lymphocytes, Adipocytes and Osteoblasts

    DEFF Research Database (Denmark)

    Sabir, Hardee J; Nehlin, Jan O; Qanie, Diyako

    2013-01-01

    -A, but not -B) is seen on some multipotent stem cells, and this raises the question how this is in other stem cells and how it changes during differentiation. In this study, we have used flow cytometry to investigate the cell surface expression of HLA-A and -B on human embryonic stem cells (hESC), human...... hematopoietic stem cells (hHSC), human mesenchymal stem cells (hMSC) and their fully-differentiated progenies such as lymphocytes, adipocytes and osteoblasts. hESC showed extremely low levels of HLA-A and no -B. In contrast, multipotent hMSC and hHSC generally expressed higher levels of HLA-A and clearly HLA......A major problem of allogeneic stem cell therapy is immunologically mediated graft rejection. HLA class I A, B, and Cw antigens are crucial factors, but little is known of their respective expression on stem cells and their progenies. We have recently shown that locus-specific expression (HLA...

  6. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

    International Nuclear Information System (INIS)

    Kawano, Michinao; Ariyoshi, Wataru; Iwanaga, Kenjiro; Okinaga, Toshinori; Habu, Manabu; Yoshioka, Izumi; Tominaga, Kazuhiro; Nishihara, Tatsuji

    2011-01-01

    Research highlights: → In this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. → MG63 cells were incubated with BMP-2 and HA for various time periods. → Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. → HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation. -- Abstract: Objectives: Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. Materials and methods: Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR. Results: BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and nuclear translocation

  7. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

    Energy Technology Data Exchange (ETDEWEB)

    Kawano, Michinao [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Ariyoshi, Wataru [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Iwanaga, Kenjiro [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Okinaga, Toshinori [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Habu, Manabu [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Yoshioka, Izumi [Division of Oral and Maxillofacial Surgery, Department of Medicine of Sensory and Motor Organs, University of Miyazaki, Kiyotake, Miyazaki 889-1692 (Japan); Tominaga, Kazuhiro [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Nishihara, Tatsuji, E-mail: tatsujin@kyu-dent.ac.jp [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, Kitakyushu 803-8580 (Japan)

    2011-02-25

    Research highlights: {yields} In this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. {yields} MG63 cells were incubated with BMP-2 and HA for various time periods. {yields} Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. {yields} HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation. -- Abstract: Objectives: Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. Materials and methods: Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR. Results: BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and

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

    Science.gov (United States)

    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.

  9. Enhanced Growth and Osteogenic Differentiation of Human Osteoblast-Like Cells on Boron-Doped Nanocrystalline Diamond Thin Films

    Czech Academy of Sciences Publication Activity Database

    Grausová, Ľubica; Kromka, Alexander; Burdíková, Zuzana; Eckhardt, Adam; Rezek, Bohuslav; Vacík, Jiří; Haenen, K.; Lisá, Věra; Bačáková, Lucie

    2011-01-01

    Roč. 6, č. 6 (2011), e20943 E-ISSN 1932-6203 R&D Projects: GA AV ČR(CZ) KAN400480701; GA AV ČR(CZ) IAAX00100902; GA ČR(CZ) GAP108/11/0794 Grant - others:GA AV ČR(CZ) KAN400100701 Program:KA Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z10480505; CEZ:AV0Z10100521 Keywords : osteoblast-like cells * boron * NCD films Subject RIV: EI - Biotechnology ; Bionics Impact factor: 4.092, year: 2011

  10. Heterotypic contact reveals a COX-2-mediated suppression of osteoblast differentiation by endothelial cells: A negative modulatory role for prostanoids in VEGF-mediated cell: cell communication?

    International Nuclear Information System (INIS)

    Clarkin, Claire E.; Garonna, Elena; Pitsillides, Andrew A.; Wheeler-Jones, Caroline P.D.

    2008-01-01

    In bone, angiogenesis must be initiated appropriately, but limited once remodelling or repair is complete. Our recent findings have supported a role for prostaglandins (PG), known modulators of osteoblast (OB) and endothelial cell (EC) behaviour, in facilitating VEGF-mediated paracrine communication from OBs to 'remotely located' ECs, but the mechanism(s) regulating OB:EC crosstalk when these cells are closely opposed are undefined. In this study we have examined: (i) the effects of exogenous PGE 2 on VEGF-driven events in ECs, and (ii) the role of endogenous COX-2-derived prostanoids in mediating communication between intimately opposed OBs and ECs in direct contact. Exposure of ECs to PGE 2 increased ERK1/2 phosphorylation, COX-2 induction, 6-keto-PGF 1α release and EC proliferation. In contrast, PGE 2 attenuated VEGF 165 -induced VEGFR2/Flk1 phosphorylation, ERK1/2 activation and proliferation of ECs, suggesting that exogenous PGE 2 restricts the actions of VEGF. However, the COX-2-selective inhibitor, NS398, also attenuated VEGF-induced proliferation, implying a distinct role for endogenous COX-2 activity in regulating EC behaviour. To examine the effect of OB:EC proximity and the role of COX-2 products further, we used a confrontational co-culture model. These studies showed that COX-2 blockade with NS398 enhanced EC-dependent increases in OB differentiation, that this effect was reversed by exogenous PGH 2 (immediate COX-2 product), and that exogenous VEGF did not influence EC-dependent OB differentiation under these conditions. Our findings indicate that locally produced prostanoids may serve distinct roles depending on OB:EC proximity and negatively modulate VEGF-mediated changes in EC behaviour when these cells are closely opposed to control angiogenesis during bone (re)modelling

  11. Notch Inhibits Osteoblast Differentiation and Causes Osteopenia

    Science.gov (United States)

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

    2008-01-01

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

  12. Osteoblasts Protect AML Cells from SDF-1-Induced Apoptosis

    Science.gov (United States)

    Kremer, Kimberly N.; Dudakovic, Amel; McGee-Lawrence, Meghan E.; Philips, Rachael L.; Hess, Allan D.; Smith, B. Douglas; van Wijnen, Andre J.; Karp, Judith E.; Kaufmann, Scott H.; Westendorf, Jennifer J.; Hedin, Karen E.

    2014-01-01

    The bone marrow provides a protective environment for acute myeloid leukemia (AML) cells that often allows leukemic stem cells to survive standard chemotherapeutic regimens. Targeting these leukemic stem cells within the bone marrow is critical for preventing relapse. We recently demonstrated that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis in AML cell lines and in patient samples expressing high levels of its receptor, CXCR4. Here we show that a subset of osteoblast lineage cells within the bone marrow can protect AML cells from undergoing apoptosis in response to the SDF-1 naturally present in that location. In co-culture systems, osteoblasts at various stages of differentiation protected AML cell lines and patient isolates from SDF-1-induced apoptosis. The differentiation of the osteoblast cell lines, MC3T3 and W-20-17, mediated this protection via a cell contact-independent mechanism. In contrast, bone marrow-derived mesenchymal cells, the precursors of osteoblasts, induced apoptosis in AML cells via a CXCR4-dependent mechanism and failed to protect AML cells from exogenously added SDF-1. These results indicate that osteoblasts in the process of differentiation potently inhibit the SDF-1-driven apoptotic pathway of CXCR4-expressing AML cells residing in the bone marrow. Drugs targeting this protective mechanism could potentially provide a new approach to treating AML by enhancing the SDF-1-induced apoptosis of AML cells residing within the bone marrow microenvironment. PMID:24851270

  13. 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

  14. ET-1 Promotes Differentiation of Periodontal Ligament Stem Cells into Osteoblasts through ETR, MAPK, and Wnt/β-Catenin Signaling Pathways under Inflammatory Microenvironment

    Science.gov (United States)

    Liang, Li; Zhou, Wei; Yang, Nan; Yu, Jifeng; Liu, Hongchen

    2016-01-01

    Periodontitis is a kind of chronic inflammatory disease that affects the tooth-supporting tissues. ET-1 is related to periodontitis and involved in the regulation of cytokines, but the mechanisms remain unclear. The aim of this study is to investigate how ET-1 affects proinflammatory cytokine expression and differentiation in human periodontal ligament stem cells (PDLSCs). PDLSCs were isolated from the periodontal ligament tissues of periodontitis patients and then treated with ET-1 (1, 10, or 100 nM) for 12 h, 24 h, or 72 h. The osteogenic potential of PDLSCs was tested using ALP staining. TNF-α, IL-1β, and IL-6 levels were evaluated by ELISA and western blot. Runx2, OCN, and COL1 mRNA and western levels were detected by RT-PCR and western blot, respectively. To examine the signaling pathways and molecular mechanisms involved in ET-1-mediated cytokine expression and osteogenic differentiation, ETR pathway, MAPKs pathway, Wnt/β-catenin pathway, and Wnt/Ca2+ pathway were detected by RT-PCR and western blot, respectively. ET-1 promoted differentiation of PDLSCs into osteoblasts by increasing secretion of TNF-α, IL-1β, and IL-6 in a dose- and time-dependent manner. ET-1 also increased expression of Runx2, OCN, and COL1. ET-1 promotes differentiation of PDLSCs into osteoblasts through ETR, MAPK, and Wnt/β-catenin signaling pathways under inflammatory microenvironment. PMID:26884650

  15. ET-1 Promotes Differentiation of Periodontal Ligament Stem Cells into Osteoblasts through ETR, MAPK, and Wnt/β-Catenin Signaling Pathways under Inflammatory Microenvironment

    Directory of Open Access Journals (Sweden)

    Li Liang

    2016-01-01

    Full Text Available Periodontitis is a kind of chronic inflammatory disease that affects the tooth-supporting tissues. ET-1 is related to periodontitis and involved in the regulation of cytokines, but the mechanisms remain unclear. The aim of this study is to investigate how ET-1 affects proinflammatory cytokine expression and differentiation in human periodontal ligament stem cells (PDLSCs. PDLSCs were isolated from the periodontal ligament tissues of periodontitis patients and then treated with ET-1 (1, 10, or 100 nM for 12 h, 24 h, or 72 h. The osteogenic potential of PDLSCs was tested using ALP staining. TNF-α, IL-1β, and IL-6 levels were evaluated by ELISA and western blot. Runx2, OCN, and COL1 mRNA and western levels were detected by RT-PCR and western blot, respectively. To examine the signaling pathways and molecular mechanisms involved in ET-1-mediated cytokine expression and osteogenic differentiation, ETR pathway, MAPKs pathway, Wnt/β-catenin pathway, and Wnt/Ca2+ pathway were detected by RT-PCR and western blot, respectively. ET-1 promoted differentiation of PDLSCs into osteoblasts by increasing secretion of TNF-α, IL-1β, and IL-6 in a dose- and time-dependent manner. ET-1 also increased expression of Runx2, OCN, and COL1. ET-1 promotes differentiation of PDLSCs into osteoblasts through ETR, MAPK, and Wnt/β-catenin signaling pathways under inflammatory microenvironment.

  16. Chondrocytic Atf4 regulates osteoblast differentiation and function via Ihh.

    Science.gov (United States)

    Wang, Weiguang; Lian, Na; Ma, Yun; Li, Lingzhen; Gallant, Richard C; Elefteriou, Florent; Yang, Xiangli

    2012-02-01

    Atf4 is a leucine zipper-containing transcription factor that activates osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation of skeletal development and bone formation is poorly understood. Investigations of the Atf4(-/-);Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteogenesis during development and bone remodeling postnatally. Atf4 overexpression in chondrocytes of the Atf4(-/-);Col2a1-Atf4 double mutants corrects the reduction in stature and limb in Atf4(-/-) embryos and rectifies the decrease in Ihh expression, Hh signaling, proliferation and accelerated hypertrophy that characterize the Atf4(-/-) developing growth plate cartilages. Unexpectedly, this genetic manipulation also restores the expression of osteoblastic marker genes, namely Ocn and bone sialoprotein, in Atf4(-/-) developing bones. In Atf4(-/-);Col2a1-Atf4 adult mice, all the defective bone parameters found in Atf4(-/-) mice, including bone volume, trabecular number and thickness, and bone formation rate, are rescued. In addition, the conditioned media of ex vivo cultures from wild-type or Atf4(-/-);Col2a1-Atf4, but not Atf4(-/-) cartilage, corrects the differentiation defects of Atf4(-/-) bone marrow stromal cells and Ihh-blocking antibody eliminates this effect. Together, these data indicate that Atf4 in chondrocytes is required for normal Ihh expression and for its paracrine effect on osteoblast differentiation. Therefore, the cell-autonomous role of Atf4 in chondrocytes dominates the role of Atf4 in osteoblasts during development for the control of early osteogenesis and skeletal growth.

  17. Adsorption of Amorphous Silica Nanoparticles onto Hydroxyapatite Surfaces Differentially Alters Surfaces Properties and Adhesion of Human Osteoblast Cells.

    Directory of Open Access Journals (Sweden)

    Priya Kalia

    Full Text Available Silicon (Si is suggested to be an important/essential nutrient for bone and connective tissue health. Silicon-substituted hydroxyapatite (Si-HA has silicate ions incorporated into its lattice structure and was developed to improve attachment to bone and increase new bone formation. Here we investigated the direct adsorption of silicate species onto an HA coated surface as a cost effective method of incorporating silicon on to HA surfaces for improved implant osseointegration, and determined changes in surface characteristics and osteoblast cell adhesion. Plasma-sprayed HA-coated stainless steel discs were incubated in silica dispersions of different concentrations (0-42 mM Si, at neutral pH for 12 h. Adsorbed Si was confirmed by XPS analysis and quantified by ICP-OES analysis following release from the HA surface. Changes in surface characteristics were determined by AFM and measurement of surface wettability. Osteoblast cell adhesion was determined by vinculin plaque staining. Maximum Si adsorption to the HA coated disc occurred after incubation in the 6 mM silica dispersion and decreased progressively with higher silica concentrations, while no adsorption was observed with dispersions below 6 mM Si. Comparison of the Si dispersions that produced the highest and lowest Si adsorption to the HA surface, by TEM-based analysis, revealed an abundance of small amorphous nanosilica species (NSP of ~1.5 nm in diameter in the 6 mM Si dispersion, with much fewer and larger NSP in the 42 mM Si dispersions. 29Si-NMR confirmed that the NSPs in the 6 mM silica dispersion were polymeric and similar in composition to the larger NSPs in the 42 mM Si dispersion, suggesting that the latter were aggregates of the former. Amorphous NSP adsorbed from the 6 mM dispersion on to a HA-coated disc surface increased the surface's water contact angle by 53°, whereas that adsorbed from the 42 mM dispersion decreased the contact angle by 18°, indicating increased and

  18. Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-β Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation

    Directory of Open Access Journals (Sweden)

    Nagarajan Selvamurugan

    2017-01-01

    Full Text Available Pulsed electromagnetic fields (PEMFs have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs’ cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF-β signaling pathway and microRNA 21 (miR21 were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF-β signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-β signaling pathway, was found to be miR21-5p’s putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF-β signaling pathway and stimulation of expression of miR21-5p in hBMSCs.

  19. Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation

    DEFF Research Database (Denmark)

    Foster, Leonard J; Zeemann, Patricia A; Li, Chen

    2005-01-01

    in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane...

  20. Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation

    International Nuclear Information System (INIS)

    Vezeridis, Peter S.; Semeins, Cornelis M.; Chen Qian; Klein-Nulend, Jenneke

    2006-01-01

    Osteocytes are thought to orchestrate bone remodeling, but it is unclear exactly how osteocytes influence neighboring bone cells. Here, we tested whether osteocytes, osteoblasts, and periosteal fibroblasts subjected to pulsating fluid flow (PFF) produce soluble factors that modulate the proliferation and differentiation of cultured osteoblasts and periosteal fibroblasts. We found that osteocyte PFF conditioned medium (CM) inhibited bone cell proliferation, and osteocytes produced the strongest inhibition of proliferation compared to osteoblasts and periosteal fibroblasts. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the inhibitory effects of osteocyte PFF CM, suggesting that a change in NO release is at least partially responsible for the inhibitory effects of osteocyte PFF CM. Furthermore, osteocyte PFF CM stimulated osteoblast differentiation measured as increased alkaline phosphatase activity, and L-NAME decreased the stimulatory effects of osteocyte PFF CM on osteoblast differentiation. We conclude that osteocytes subjected to PFF inhibit proliferation but stimulate differentiation of osteoblasts in vitro via soluble factors and that the release of these soluble factors was at least partially dependent on the activation of a NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast with respect to the production of soluble signaling molecules affecting osteoblast proliferation and differentiation

  1. Hedgehog signaling in tumor cells facilitates osteoblast-enhanced osteolytic metastases.

    Directory of Open Access Journals (Sweden)

    Shamik Das

    Full Text Available The remodeling process in bone yields numerous cytokines and chemokines that mediate crosstalk between osteoblasts and osteoclasts and also serve to attract and support metastatic tumor cells. The metastatic tumor cells disturb the equilibrium in bone that manifests as skeletal complications. The Hedgehog (Hh pathway plays an important role in skeletogenesis. We hypothesized that the Hh pathway mediates an interaction between tumor cells and osteoblasts and influences osteoblast differentiation in response to tumor cells. We have determined that breast tumor cells have an activated Hh pathway characterized by upregulation of the ligand, IHH and transcription factor GLI1. Breast cancer cells interact with osteoblasts and cause an enhanced differentiation of pre-osteoblasts to osteoblasts that express increased levels of the osteoclastogenesis factors, RANKL and PTHrP. There is sustained expression of osteoclast-promoting factors, RANKL and PTHrP, even after the osteoblast differentiation ceases and apoptosis sets in. Moreover, tumor cells that are deficient in Hh signaling are compromised in their ability to induce osteoblast differentiation and consequently are inefficient in causing osteolysis. The stimulation of osteoblast differentiation sets the stage for osteoclast differentiation and overall promotes osteolysis. Thus, in the process of developing newer therapeutic strategies against breast cancer metastasis to bone it would worthwhile to keep in mind the role of the Hh pathway in osteoblast differentiation in an otherwise predominant osteolytic phenomenon.

  2. Myeloma cells suppress osteoblasts through sclerostin secretion

    Energy Technology Data Exchange (ETDEWEB)

    Colucci, S; Brunetti, G; Oranger, A [Department of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Mori, G [Department of Biomedical Science, University of Foggia, Foggia (Italy); Sardone, F [Department of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Specchia, G; Rinaldi, E; Curci, P; Liso, V [Department of Emergency and Organ Transplantation, Hematology Section, Bari University Medical School, Bari (Italy); Passeri, G [Department of Internal Medicine and Biomedical Sciences, Center for Metabolic Bone Diseases, University of Parma, Parma (Italy); Zallone, A [Department of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Rizzi, R [Department of Emergency and Organ Transplantation, Hematology Section, Bari University Medical School, Bari (Italy); Grano, M [Department of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy)

    2011-06-01

    Wingless-type (Wnt) signaling through the secretion of Wnt inhibitors Dickkopf1, soluble frizzled-related protein-2 and -3 has a key role in the decreased osteoblast (OB) activity associated with multiple myeloma (MM) bone disease. We provide evidence that another Wnt antagonist, sclerostin, an osteocyte-expressed negative regulator of bone formation, is expressed by myeloma cells, that is, human myeloma cell lines (HMCLs) and plasma cells (CD138+ cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. We demonstrated that BM stromal cells (BMSCs), differentiated into OBs and co-cultured with HMCLs showed, compared with BMSCs alone, reduced expression of major osteoblastic-specific proteins, decreased mineralized nodule formation and attenuated the expression of members of the activator protein 1 transcription factor family (Fra-1, Fra-2 and Jun-D). Moreover, in the same co-culture system, the addition of neutralizing anti-sclerostin antibodies restored OB functions by inducing nuclear accumulation of β-catenin. We further demonstrated that the upregulation of receptor activator of nuclear factor κ-B ligand and the downregulation of osteoprotegerin in OBs were also sclerostin mediated. Our data indicated that sclerostin secretion by myeloma cells contribute to the suppression of bone formation in the osteolytic bone disease associated to MM.

  3. 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

  4. VEGF-C and TGF-β reciprocally regulate mesenchymal stem cell commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes.

    Science.gov (United States)

    Igarashi, Yasuyuki; Chosa, Naoyuki; Sawada, Shunsuke; Kondo, Hisatomo; Yaegashi, Takashi; Ishisaki, Akira

    2016-04-01

    The direction of mesenchymal stem cell (MSC) differentiation is regulated by stimulation with various growth factors and cytokines. We recently established MSC lines, [transforming growth factor-β (TGF-β)-responsive SG‑2 cells, bone morphogenetic protein (BMP)-responsive SG‑3 cells, and TGF-β/BMP-non-responsive SG‑5 cells], derived from the bone marrow of green fluorescent protein-transgenic mice. In this study, to compare gene expression profiles in these MSC lines, we used DNA microarray analysis to characterize the specific gene expression profiles observed in the TGF-β-responsive SG‑2 cells. Among the genes that were highly expressed in the SG‑2 cells, we focused on vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3), the gene product of FMS-like tyrosine kinase 4 (Flt4). We found that VEGF-C, a specific ligand of VEGFR3, significantly induced the cell proliferative activity, migratory ability (as shown by Transwell migration assay), as well as the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the SG‑2 cells. Additionally, VEGF-C significantly increased the expression of prospero homeobox 1 (Prox1) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), which are lymphatic endothelial cell markers, and decreased the expression of osteogenic differentiation marker genes in these cells. By contrast, TGF-β significantly increased the expression of early-phase osteogenic differentiation marker genes in the SG‑2 cells and markedly decreased the expression of lymphatic endothelial cell markers. The findings of our study strongly suggest the following: i) that VEGF-C promotes the proliferative activity and migratory ability of MSCs; and ii) VEGF-C and TGF-β reciprocally regulate MSC commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes, respectively. Our findings provide new insight into the molecular mechanisms underlying the regenerative ability of MSCs.

  5. Cell-penetrating superoxide dismutase attenuates oxidative stress-induced senescence by regulating the p53-p21Cip1 pathway and restores osteoblastic differentiation in human dental pulp stem cells

    Directory of Open Access Journals (Sweden)

    Park YJ

    2012-09-01

    Full Text Available Yoon Jung Choi,1,* Jue Yeon Lee,2,* Chong Pyoung Chung,2 Yoon Jeong Park,1,21Craniomaxillofacial Reconstructive Sciences, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea; 2Research Institute, Nano Intelligent Biomedical Engineering, Seoul, Republic of Korea*These authors contributed equally to this workBackground: Human dental pulp stem cells (DPSCs have potential applications in tissue regeneration because of their convenient cell harvesting procedures and multipotent capacity. However, the tissue regenerative potential of DPSCs is known to be negatively regulated by aging in long-term culture and under oxidative stress. With an aim of reducing cellular senescence and oxidative stress in DPSCs, an intracellular delivery system for superoxide dismutase 1 (SOD1 was developed. We conjugated SOD1 with a cell-penetrating peptide known as low-molecular weight protamine (LMWP, and investigated the effect of LMWP-SOD1 conjugates on hydrogen peroxide-induced cellular senescence and osteoblastic differentiation.Results: LMWP-SOD1 significantly attenuated enlarged and flattened cell morphology and increased senescence-associated β-galactosidase activity. Under the same conditions, LMWP-SOD1 abolished activation of the cell cycle regulator proteins, p53 and p21Cip1, induced by hydrogen peroxide. In addition, LMWP-SOD1 reversed the inhibition of osteoblastic differentiation and downregulation of osteogenic gene markers induced by hydrogen peroxide. However, LMWP-SOD1 could not reverse the decrease in odontogenesis caused by hydrogen peroxide.Conclusion: Overall, cell-penetrating LMWP-SOD1 conjugates are effective for attenuation of cellular senescence and reversal of osteoblastic differentiation of DPSCs caused by oxidative stress inhibition. This result suggests potential application in the field of antiaging and tissue engineering to overcome the limitations of senescent stem cells.Keywords: superoxide

  6. Co-Culture with Human Osteoblasts and Exposure to Extremely Low Frequency Pulsed Electromagnetic Fields Improve Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Sabrina Ehnert

    2018-03-01

    Full Text Available Human adipose-derived mesenchymal stem cells (Ad-MSCs have been proposed as suitable option for cell-based therapies to support bone regeneration. In the bone environment, Ad-MSCs will receive stimuli from resident cells that may favor their osteogenic differentiation. There is recent evidence that this process can be further improved by extremely low frequency pulsed electromagnetic fields (ELF-PEMFs. Thus, the project aimed at (i investigating whether co-culture conditions of human osteoblasts (OBs and Ad-MSCs have an impact on their proliferation and osteogenic differentiation; (ii whether this effect can be further improved by repetitive exposure to two specific ELF-PEMFs (16 and 26 Hz; (iii and the effect of these ELF-PEMFs on human osteoclasts (OCs. Osteogenic differentiation was improved by co-culturing OBs and Ad-MSCs when compared to the individual mono-cultures. An OB to Ad-MSC ratio of 3:1 had best effects on total protein content, alkaline phosphatase (AP activity, and matrix mineralization. Osteogenic differentiation was further improved by both ELF-PEMFs investigated. Interestingly, only repetitive exposure to 26 Hz ELF-PEMF increased Trap5B activity in OCs. Considering this result, a treatment with gradually increasing frequency might be of interest, as the lower frequency (16 Hz could enhance bone formation, while the higher frequency (26 Hz could enhance bone remodeling.

  7. Culture on fibrin matrices maintains the colony-forming capacity and osteoblastic differentiation of mesenchymal stem cells

    International Nuclear Information System (INIS)

    Colley, Helen; McArthur, Sally L; Stolzing, Alexandra; Scutt, Andy

    2012-01-01

    Mesenchymal stem cells (MSC) are multipotent cells capable of differentiating into a number of mesenchymal tissues including bone, cartilage, and tendon. Low numbers in vivo means exponential growth is needed in culture to enable therapeutic applications. MSC can expand rapidly in culture but usually lose their extensive capacity for differentiation that makes them therapeutically attractive. To try and maintain their capacity for differentiation and expansion in vitro, we cultured MSC on fibrin gels of different concentrations to create more physiological growth conditions for the cells. The cells were then re-plated onto tissue culture plastic and analysed. The cells that had been pre-cultured for seven days on fibrin, proliferated and maintained their differential potential to the osteogenic lineage better than tissue culture plastic expanded MSC. A concentration relationship between colony number and fibrin concentration was seen with decreasing numbers as fibrin concentration increased. These data support the concept that substrate signals significantly influence MSC growth and differentiation and that growth on a fibrin matrix could be used to maintain a stem cell phenotype during MSC expansion. (paper)

  8. 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.

  9. 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}.

  10. BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

    Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

  11. Osteoblastic differentiation of stem cells from human exfoliated deciduous teeth induced by thermosensitive hydrogels with strontium phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Su, Wen-Ta, E-mail: f10549@ntut.edu.tw [Department of Chemical Engineering and Biotechnology National Taipei University of Technology, Taipei, Taiwan (China); Chou, Wei-Ling [Department of Chemical Engineering and Biotechnology National Taipei University of Technology, Taipei, Taiwan (China); Chou, Chih-Ming [Department of Biochemistry, Taipei Medical University, Taipei, Taiwan (China)

    2015-07-01

    Stem cells from human exfoliated deciduous teeth (SHEDs) are a novel source of multi-potential stem cells for tissue engineering because of their potential to differentiate into multiple cell lineages. Strontium exhibits an important function in bone remodeling because it can simulate bone formation and decrease bone resorption. Hydrogels can mimic the natural cellular environment. The association of hydrogels with cell viability is determined using biological tests, including rheological experiments. In this study, osteogenic differentiation was investigated through SHED encapsulation in hydrogels containing strontium phosphate. Results of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and proliferating cell nuclear antigen (PCNA) immunofluorescence staining indicated that the cells grew well and SHEDs proliferated in the hydrogels. Strontium-loaded chitosan-based hydrogels induced the biomineralization and high expression of alkaline phosphatase. Moreover, the expression levels of bone-related genes, including type-I collagen, Runx2, osteopontin (OP), and osteonectin (ON), were up-regulated during the osteogenic differentiation of SHEDs. This study demonstrated that strontium can be an effective inducer of osteogenesis for SHEDs. Elucidating the function of bioceramics (such as strontium) is useful in designing and developing strategies for bone tissue engineering. - Highlights: • SHEDs have been considered as alternative sources of adult stem cells in tissue engineering. • Strontium phosphate can enhance the osteogenic differentiation of SHEDs. • Hydrogels can mimic the natural cellular environment. • Bioceramics (such as strontium) is useful in designing and developing strategies for bone tissue engineering.

  12. Ihh/Gli2 signaling promotes osteoblast differentiation by regulating Runx2 expression and function.

    Science.gov (United States)

    Shimoyama, Atsuko; Wada, Masahiro; Ikeda, Fumiyo; Hata, Kenji; Matsubara, Takuma; Nifuji, Akira; Noda, Masaki; Amano, Katsuhiko; Yamaguchi, Akira; Nishimura, Riko; Yoneda, Toshiyuki

    2007-07-01

    Genetic and cell biological studies have indicated that Indian hedgehog (Ihh) plays an important role in bone development and osteoblast differentiation. However, the molecular mechanism by which Ihh regulates osteoblast differentiation is complex and remains to be fully elucidated. In this study, we investigated the role of Ihh signaling in osteoblast differentiation using mesenchymal cells and primary osteoblasts. We observed that Ihh stimulated alkaline phosphatase (ALP) activity, osteocalcin expression, and calcification. Overexpression of Gli2- but not Gli3-induced ALP, osteocalcin expression, and calcification of these cells. In contrast, dominant-negative Gli2 markedly inhibited Ihh-dependent osteoblast differentiation. Ihh treatment or Gli2 overexpression also up-regulated the expression of Runx2, an essential transcription factor for osteoblastogenesis, and enhanced the transcriptional activity and osteogenic action of Runx2. Coimmunoprecipitation analysis demonstrated a physical interaction between Gli2 and Runx2. Moreover, Ihh or Gli2 overexpression failed to increase ALP activity in Runx2-deficient mesenchymal cells. Collectively, these results suggest that Ihh regulates osteoblast differentiation of mesenchymal cells through up-regulation of the expression and function of Runx2 by Gli2.

  13. Circadian rhythm of mechanically mediated differentiation of osteoblasts

    Science.gov (United States)

    Roberts, W. E.; Mozsary, P. G.; Klingler, E.

    1984-01-01

    The differential of osteoblasts in response to orthodontic pressure in the periodontal ligament of the maxillary-first-molar periodontal ligaments of 12-h-light/dark-entrained 7-wk-old male Simonsen outbred rats is measured by (H-3)-Thymidine nuclear-volume morphometry (Roberts et al., 1983) at hourly intervals throughout the circadian cycle. The results are presented in graphs and discussed. Preosteoblast large nuclei (D-cells) are found to synthesize DNA mainly in light and to divide in the following dark period, while small-nucleus osteoprogenitors (A-cells) synthesize in darkness and divide in light. These findings are seen as consistent with a model in which the sequence of proliferation and differentiation requires at least 60 h (five 12-h periods) and the shift from A to D cells lasts about 19 h.

  14. Adiponectin stimulates human osteoblasts proliferation and differentiation via the MAPK signaling pathway

    International Nuclear Information System (INIS)

    Luo Xianghang; Guo Lijuan; Yuan Lingqing; Xie Hui; Zhou Houde; Wu Xianping; Liao Eryuan

    2005-01-01

    Adipocytes can highly and specifically express adiponectin, and the adiponectin receptor (AdipoR) has been detected in bone-forming cells. The present study was undertaken to investigate the action of adiponectin on osteoblast proliferation and differentiation. AdipoR1 protein was detected in human osteoblasts. Adiponectin promoted osteoblast proliferation and resulted in a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, osteocalcin and type I collagen production, and an increase in mineralized matrix. Suppression of AdipoR1 with small-interfering RNA (siRNA) abolished the adiponectin-induced cell proliferation and ALP expression. Adiponectin induces activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal Kinase (JNK), but not ERK1/2 in osteoblasts, and these effects were blocked by suppression of AdipoR1 with siRNA. Furthermore, pretreatment of osteoblasts with the JNK inhibitor SP600125 abolished the adiponectin-induced cell proliferation. p38 inhibitor SB203580 blocked the adiponectin-induced ALP activity. These data indicate that adiponectin induces human osteoblast proliferation and differentiation, and the proliferation response is mediated by the AdipoR/JNK pathway, while the differentiation response is mediated via the AdipoR/p38 pathway. These findings suggest that osteoblasts are the direct targets of adiponectin

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-06-22

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Lamblin Anne-Francoise

    2007-10-01

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

  18. Nanoceramics on osteoblast proliferation and differentiation in bone tissue engineering.

    Science.gov (United States)

    Sethu, Sai Nievethitha; Namashivayam, Subhapradha; Devendran, Saravanan; Nagarajan, Selvamurugan; Tsai, Wei-Bor; Narashiman, Srinivasan; Ramachandran, Murugesan; Ambigapathi, Moorthi

    2017-05-01

    Bone, a highly dynamic connective tissue, consist of a bioorganic phase comprising osteogenic cells and proteins which lies over an inorganic phase predominantly made of CaPO 4 (biological apatite). Injury to bone can be due to mechanical, metabolic or inflammatory agents also owing pathological conditions like fractures, osteomyelitis, osteolysis or cysts may arise in enameloid, chondroid, cementum, or chondroid bone which forms the intermediate tissues of the body. Bone tissue engineering (BTE) applies bioactive scaffolds, host cells and osteogenic signals for restoring damaged or diseased tissues. Various bioceramics used in BTE can be bioactive (like glass ceramics and hydroxyapatite bioactive glass), bioresorbable (like tricalcium phosphates) or bioinert (like zirconia and alumina). Limiting the size of these materials to nano-scale has resulted in a higher surface area to volume ratio thereby improving multi-functionality, solubility, surface catalytic activity, high heat and electrical conductivity. Nanoceramics have been found to induce osteoconduction, osteointegration, osteogenesis and osteoinduction. The present review aims at summarizing the interactions of nanoceramics and osteoblast/stem cells for promoting the proliferation and differentiation of the osteoblast cells by nanoceramics as superior bone substitutes in bone tissue engineering applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Effect of various concentrations of Ti in hydrocarbon plasma polymer films on the adhesion, proliferation and differentiation of human osteoblast-like MG-63 cells

    Science.gov (United States)

    Vandrovcova, Marta; Grinevich, Andrey; Drabik, Martin; Kylian, Ondrej; Hanus, Jan; Stankova, Lubica; Lisa, Vera; Choukourov, Andrei; Slavinska, Danka; Biederman, Hynek; Bacakova, Lucie

    2015-12-01

    Hydrocarbon polymer films (ppCH) enriched with various concentrations of titanium were deposited on microscopic glass slides by magnetron sputtering from a Ti target. The maximum concentration of Ti (about 20 at.%) was achieved in a pure argon atmosphere. The concentration of Ti decreased rapidly after n-hexane vapors were introduced into the plasma discharge, and reached zero values at n-hexane flow of 0.66 sccm. The decrease in Ti concentration was associated with decreasing oxygen and titanium carbide concentration in the films, decreasing wettability (the water drop contact angle increased from 20° to 91°) and decreasing root-mean-square roughness (from 3.3 nm to 1.0 nm). The human osteoblast-like MG-63 cells cultured on pure ppCH films and on films with 20 at.% of Ti showed relatively high concentrations of ICAM-1, a marker of cell immune activation. Lower concentrations of Ti (mainly 5 at.%) improved cell adhesion and osteogenic differentiation, as revealed by higher concentrations of talin, vinculin and osteocalcin. Higher Ti concentrations (15 at.%) supported cell growth, as indicated by the highest final cell population densities on day 7 after seeding. Thus, enrichment of ppCH films with appropriate concentrations of Ti makes these films more suitable for potential coatings of bone implants.

  20. Induction of a program gene expression during osteoblast differentiation with strontium ranelate

    International Nuclear Information System (INIS)

    Zhu Lingling; Zaidi, Samir; Peng Yuanzhen; Zhou Hang; Moonga, Baljit S.; Blesius, Alexia; Dupin-Roger, Isabelle; Zaidi, Mone; Sun Li

    2007-01-01

    Strontium ranelate, a new agent for the treatment of osteoporosis, has been shown stimulate bone formation in various experimental models. This study examines the effect of strontium ranelate on gene expression in osteoblasts, as well as the formation of mineralized (von Kossa-positive) colony-forming unit-osteoblasts (CFU-obs). Bone marrow-derived stromal cells cultured for 21 days under differentiating conditions, when exposed to strontium ranelate, displayed a significant time- and concentration-dependent increase in the expression of the master gene, Runx2, as well as bone sialoprotein (BSP), but interestingly without effects on osteocalcin. This was associated with a significant increase in the formation of CFU-obs at day 21 of culture. In U-33 pre-osteoblastic cells, strontium ranelate significantly enhanced the expression of Runx2 and osteocalcin, but not BSP. Late, more mature osteoblastic OB-6 cells showed significant elevations in BSP and osteocalcin, but with only minimal effects on Runx2. In conclusion, strontium ranelate stimulates osteoblast differentiation, but the induction of the program of gene expression appears to be cell type-specific. The increased osteoblastic differentiation is the likely basis underlying the therapeutic bone-forming actions of strontium ranelate

  1. Disruption of kif3a results in defective osteoblastic differentiation in dental mesenchymal stem/precursor cells via the Wnt signaling pathway.

    Science.gov (United States)

    Jiang, Sicong; Chen, Guoqing; Feng, Lian; Jiang, Zongting; Yu, Mei; Bao, Jinku; Tian, Weidong

    2016-09-01

    The anterograde intraflagellar transport motor protein, kif3a, regulates the integrity of primary cilia and various cellular functions, however, the role of kif3a in dental mesenchymal stem/precursor cell differentiation remains to be fully elucidated. In the present study, the expression of kif3a was knocked down in human dental follicle cells (hDFCs) and human dental pulp cells (hDPCs) using short hairpin RNA. The results of subsequent immunofluorescence revealed that knocking down kif3a resulted in the loss of primary cilia, which led to impairment of substantial mineralization and expression of the differentiation‑associated markers, including alkaline phosphatase, Runt‑related transcription factor 2, dentin matrix protein 1 and dentin sialophosphoprotein in the hDFCs and hDPCs. The results of reverse transcription‑quantitative polymerase chain reaction and western blot analyses showed that the expression levels of Wnt3a‑mediated active β‑catenin and lymphoid enhancer‑binding factor 1 were attenuated, whereas the expression of phosphorylated glycogen synthase kinase 3β was enhanced, in the kif3a‑knockdown cells. In addition, exogenous Wnt3a partially rescued osteoblastic differentiation in the hDFCs and hDPCs. These results demonstrated that inhibition of kif3a in the hDFCs and hDPCs disrupted primary cilia formation and/or function, and indicated that kif3a is important in the differentiation of hDFCs and hDPCs through the Wnt pathway. These findings not only enhance current understanding of tooth development and diseases of tooth mineralization, but also indicate possible strategies to regulate mineralization during tooth repair and regeneration.

  2. Osteoblast Differentiation and Bone Matrix Formation In Vivo and In Vitro.

    Science.gov (United States)

    Blair, Harry C; Larrouture, Quitterie C; Li, Yanan; Lin, Hang; Beer-Stoltz, Donna; Liu, Li; Tuan, Rocky S; Robinson, Lisa J; Schlesinger, Paul H; Nelson, Deborah J

    2017-06-01

    We review the characteristics of osteoblast differentiation and bone matrix synthesis. Bone in air breathing vertebrates is a specialized tissue that developmentally replaces simpler solid tissues, usually cartilage. Bone is a living organ bounded by a layer of osteoblasts that, because of transport and compartmentalization requirements, produce bone matrix exclusively as an organized tight epithelium. With matrix growth, osteoblasts are reorganized and incorporated into the matrix as living cells, osteocytes, which communicate with each other and surface epithelium by cell processes within canaliculi in the matrix. The osteoblasts secrete the organic matrix, which are dense collagen layers that alternate parallel and orthogonal to the axis of stress loading. Into this matrix is deposited extremely dense hydroxyapatite-based mineral driven by both active and passive transport and pH control. As the matrix matures, hydroxyapatite microcrystals are organized into a sophisticated composite in the collagen layer by nucleation in the protein lattice. Recent studies on differentiating osteoblast precursors revealed a sophisticated proton export network driving mineralization, a gene expression program organized with the compartmentalization of the osteoblast epithelium that produces the mature bone matrix composite, despite varying serum calcium and phosphate. Key issues not well defined include how new osteoblasts are incorporated in the epithelial layer, replacing those incorporated in the accumulating matrix. Development of bone in vitro is the subject of numerous projects using various matrices and mesenchymal stem cell-derived preparations in bioreactors. These preparations reflect the structure of bone to variable extents, and include cells at many different stages of differentiation. Major challenges are production of bone matrix approaching the in vivo density and support for trabecular bone formation. In vitro differentiation is limited by the organization and

  3. MiR-29-mediated elastin down-regulation contributes to inorganic phosphorus-induced osteoblastic differentiation in vascular smooth muscle cells.

    Science.gov (United States)

    Sudo, Ryo; Sato, Fumiaki; Azechi, Takuya; Wachi, Hiroshi

    2015-12-01

    Vascular calcification increases the risk of cardiovascular mortality. We previously reported that expression of elastin decreases with progression of inorganic phosphorus (Pi)-induced vascular smooth muscle cell (VSMC) calcification. However, the regulatory mechanisms of elastin mRNA expression during vascular calcification remain unclear. MicroRNA-29 family members (miR-29a, b and c) are reported to mediate elastin mRNA expression. Therefore, we aimed to determine the effect of miR-29 on elastin expression and Pi-induced vascular calcification. Calcification of human VSMCs was induced by Pi and evaluated measuring calcium deposition. Pi stimulation promoted Ca deposition and suppressed elastin expression in VSMCs. Knockdown of elastin expression by shRNA also promoted Pi-induced VSMC calcification. Elastin pre-mRNA measurements indicated that Pi stimulation suppressed elastin expression without changing transcriptional activity. Conversely, Pi stimulation increased miR-29a and miR-29b expression. Inhibition of miR-29 recovered elastin expression and suppressed calcification in Pi-treated VSMCs. Furthermore, over-expression of miR-29b promoted Pi-induced VSMC calcification. RT-qPCR analysis showed knockdown of elastin expression in VSMCs induced expression of osteoblast-related genes, similar to Pi stimulation, and recovery of elastin expression by miR-29 inhibition reduced their expression. Our study shows that miR-29-mediated suppression of elastin expression in VSMCs plays a pivotal role in osteoblastic differentiation leading to vascular calcification. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  4. Adhesion and differentiation of Saos-2 osteoblast-like cells on chromium-doped diamond-like carbon coatings

    Czech Academy of Sciences Publication Activity Database

    Filová, Elena; Vandrovcová, Marta; Jelínek, Miroslav; Zemek, Josef; Houdková, Jana; Remsa, Jan; Kocourek, Tomáš; Staňková, Ľubica; Bačáková, Lucie

    2017-01-01

    Roč. 28, č. 1 (2017), č. článku 17. ISSN 0957-4530 R&D Projects: GA ČR(CZ) GA15-05864S; GA ČR(CZ) GA14-04790S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 ; RVO:68378271 Keywords : osteocalcin * osteogenic differentiation * hexavalent chromium * focal adhesion contact * cell spreading area Subject RIV: EI - Biotechnology ; Bionics OBOR OECD: Biomaterials (as related to medical implants, devices, sensors) Impact factor: 2.325, year: 2016

  5. A proteome study of secreted prostatic factors affecting osteoblastic activity: galectin-1 is involved in differentiation of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Andersen, H; Jensen, Ole N; Moiseeva, Elena P

    2003-01-01

    Prostate cancer cells metastasize to bone causing a predominantly osteosclerotic response. It has been shown that cells from the human prostate cancer cell line PC3 secrete factors that influence the behavior of osteoblast-like cells. Some of these factors with mitogenic activity have been found...... to be proteins with molecular weights between 20 and 30 kDa, but the identity of the osteoblastic mitogenic factor or factors produced by prostate cancer cells is still unknown. Therefore, the aim of this study was to characterize the protein profile of conditioned medium (CM) from PC3 cells in the molecular......BMS) cells. Furthermore, we tested whether adhesion of PC3 cells to plastic, laminin, fibronectin, and collagen type I was influenced by lactose, which inhibits galectin-1. Galectin-1 (1000 ng/ml) inhibited the proliferation of hBMS cells up to 70 +/- 12% (treated/control) of control in contrast to PC3 CM...

  6. 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.

  7. 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.

  8. Water soluble bioactives of nacre mediate antioxidant activity and osteoblast differentiation.

    Directory of Open Access Journals (Sweden)

    Ratna Chaturvedi

    Full Text Available The water soluble matrix of nacre is a proven osteoinductive material. In spite of the differences in the biomolecular compositions of nacre obtained from multiple species of oysters, the common biochemical properties of those principles substantiate their biological activity. However, the mechanism by which nacre stimulates bone differentiation remains largely unknown. Since the positive impact of antioxidants on bone metabolism is well acknowledged, in this study we investigated the antioxidant potential of a water soluble matrix (WSM obtained from the nacre of the marine oyster Pinctada fucata, which could regulate its osteoblast differentiation activity. Enhanced levels of ALP activity observed in pre-osteoblast cells upon treatment with WSM, suggested the induction of bone differentiation events. Furthermore, bone nodule formation and up-regulation of bone differentiation marker transcripts, i.e. collagen type-1 and osteocalcin by WSM confirmed its ability to induce differentiation of the pre-osteoblasts into mature osteoblasts. Remarkably, same WSM fraction upon pre-treatment lowered the H2O2 and UV-B induced oxidative damages in keratinocytes, thus indicating the antioxidant potential of WSM. This was further confirmed from the in vitro scavenging of ABTS and DPPH free radicals and inhibition of lipid peroxidation by WSM. Together, these results indicate that WSM poses both antioxidant potential and osteoblast differentiation property. Thus, bioactivities associated with nacre holds potential in the development of therapeutics for bone regeneration and against oxidative stress induced damages in cells.

  9. Effect of various concentrations of Ti in hydrocarbon plasma polymer films on the adhesion, proliferation and differentiation of human osteoblast-like MG-63 cells

    Energy Technology Data Exchange (ETDEWEB)

    Vandrovcova, Marta, E-mail: marta.vandrovcova@fgu.cas.cz [Department of Biomaterials and Tissue Engineering, Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4 (Czech Republic); Grinevich, Andrey; Drabik, Martin; Kylian, Ondrej; Hanus, Jan [Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holesovickach 2, 182 00 Prague 8 (Czech Republic); Stankova, Lubica; Lisa, Vera [Department of Biomaterials and Tissue Engineering, Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4 (Czech Republic); Choukourov, Andrei; Slavinska, Danka; Biederman, Hynek [Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holesovickach 2, 182 00 Prague 8 (Czech Republic); Bacakova, Lucie [Department of Biomaterials and Tissue Engineering, Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4 (Czech Republic)

    2015-12-01

    Graphical abstract: - Highlights: • Hydrocarbon plasma polymer films with Ti in concentration of 0–20 at.% were prepared. • The Ti concentration was positively correlated with the material surface wettability. • The optimum Ti concentrations for the MG-63 cells behavior were identified. • The Ti concentration also influenced the cell immune activation. - Abstract: Hydrocarbon polymer films (ppCH) enriched with various concentrations of titanium were deposited on microscopic glass slides by magnetron sputtering from a Ti target. The maximum concentration of Ti (about 20 at.%) was achieved in a pure argon atmosphere. The concentration of Ti decreased rapidly after n-hexane vapors were introduced into the plasma discharge, and reached zero values at n-hexane flow of 0.66 sccm. The decrease in Ti concentration was associated with decreasing oxygen and titanium carbide concentration in the films, decreasing wettability (the water drop contact angle increased from 20° to 91°) and decreasing root-mean-square roughness (from 3.3 nm to 1.0 nm). The human osteoblast-like MG-63 cells cultured on pure ppCH films and on films with 20 at.% of Ti showed relatively high concentrations of ICAM-1, a marker of cell immune activation. Lower concentrations of Ti (mainly 5 at.%) improved cell adhesion and osteogenic differentiation, as revealed by higher concentrations of talin, vinculin and osteocalcin. Higher Ti concentrations (15 at.%) supported cell growth, as indicated by the highest final cell population densities on day 7 after seeding. Thus, enrichment of ppCH films with appropriate concentrations of Ti makes these films more suitable for potential coatings of bone implants.

  10. 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.

  11. ent-Kaurane diterpenoids from Croton tonkinensis stimulate osteoblast differentiation

    DEFF Research Database (Denmark)

    Dao, Trong-Tuan; Lee, Kwang-Youl; Jeong, Hyung-Min

    2011-01-01

    Four new ent-kaurane diterpenoids (1-4) were isolated from the leaves of Croton tonkinensis by bioactivity-guided fractionation using an in vitro osteoblast differentiation assay. Their structures were identified as ent-11β-acetoxykaur-16-en-18-ol (1), ent-11α-hydroxy-18-acetoxykaur-16-ene (2), e...

  12. Pulsed electromagnetic fields promote the proliferation and differentiation of osteoblasts by reinforcing intracellular calcium transients.

    Science.gov (United States)

    Tong, Jie; Sun, Lijun; Zhu, Bin; Fan, Yun; Ma, Xingfeng; Yu, Liyin; Zhang, Jianbao

    2017-10-01

    Pulsed electromagnetic fields (PEMF) can be used to treat bone-related diseases, but the underlying mechanism remains unclear, especially the process by which PEMFs initiate biological effects. In this study, we demonstrated the effects of PEMF on proliferation and differentiation of osteoblasts using the model of calcium transients induced by high extracellular calcium. Our results showed that PEMF can increase both the percentage of responding cells and amplitude of intracellular calcium transients induced by high extracellular calcium stimulation. Compared with corresponding extracellular calcium levels, PEMF stimulation increased proliferation and differentiation of osteoblasts and related gene expressions, such as insulin-like growth factor 1 (IGF-1), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), which can be completely abolished by BAPTA-AM. Moreover, PEMF did not affect proliferation and differentiation of osteoblasts if no intracellular calcium transient was present in osteoblasts during PEMF exposure. Our results revealed that PEMF affects osteoblast proliferation and differentiation through enhanced intracellular calcium transients, which provided a cue to treat bone-related diseases with PEMF. Bioelectromagnetics. 38:541-549, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  13. Induction of osteoblast differentiation by selective activation of kinase-mediated actions of the estrogen receptor.

    Science.gov (United States)

    Kousteni, Stavroula; Almeida, Maria; Han, Li; Bellido, Teresita; Jilka, Robert L; Manolagas, Stavros C

    2007-02-01

    Estrogens control gene transcription by cis or trans interactions of the estrogen receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-estren-3alpha,17beta-diol (estren) or an estradiol-dendrimer conjugate, each a synthetic compound that stimulates kinase-mediated ER actions 1,000 to 10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17beta-estradiol (E(2)) suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E(2), stimulated Wnt/beta-catenin-mediated transcription in T-cell factor-lacZ transgenic mice. Moreover, E(2) stimulated BMP signaling in mice in which ERalpha lacks DNA binding activity and classical estrogen response element-mediated transcription (ERalpha(NERKI/-)) but not in wild-type controls. This evidence reveals for the first time the existence of a large signalosome in which inputs from the ER, kinases, bone morphogenetic proteins, and Wnt signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.

  14. Enhancement of osteoblastic differentiation in alginate gel beads with bioactive octacalcium phosphate particles

    International Nuclear Information System (INIS)

    Endo, Kosei; Anada, Takahisa; Suzuki, Osamu; Yamada, Masumi; Seki, Minoru; Sasaki, Keiichi

    2015-01-01

    The present study investigated whether alginate (Alg) hydrogel microbeads have a role in maintaining mouse bone marrow stromal ST-2 cells and release the cells after being stimulated by synthetic octacalcium phosphate (OCP), which is a mineral crystal capable of stimulating osteoblastic differentiation during a conversion process to hydroxyapatite (HA). The ST-2 cell suspension in the alginate solution, which contained various concentrations of OCP granules with diameters less than 53 μm, was extruded drop-wise into a stirred gelation solution containing BaCl 2 using an encapsulator with nitrogen gas stream. The Alg-microbeads (Alg/OCP · ST-2 microbeads) that were generated, which had a diameter of approximately 400 μm, were incubated for up to 14 d and then assessed for osteoblastic differentiation. Alg-microbeads with cells were also incubated to identify the possible conversion from OCP to HA. Osteoblast differentiation markers in ST-2 cells, alkaline phosphatase (ALP) and collagen type I, were up-regulated in the presence of higher amounts of OCP. X-ray diffraction analysis and Fourier transform infrared spectroscopy confirmed that the OCP tended to convert to HA over time, suggesting that the OCP in Alg-microbeads interacts three-dimensionally with ST-2 cells and stimulates its osteoblastic differentiation. The release of ST-2 cells from the microbeads was also estimated. ST-2 cells were identified outside of the microbeads, although the cell number tended to decrease with increasing OCP. These results suggest that Alg/OCP microbeads could be used as a vehicle to activate osteoblastic cells and deliver them to sites where bone regeneration is needed. (paper)

  15. Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells

    DEFF Research Database (Denmark)

    Ali, D.; Hamam, R.; Alfayez, M.

    2016-01-01

    proliferation and differentiation that were targeted by abexinostat. Concordantly, ChIP-quantitative polymerase chain reaction revealed marked increase in H3K9Ac epigenetic mark on the promoter region of AdipoQ, FABP4, PPARγ, KLF15, CEBPA, SP7, and ALPL in abexinostat-treated hMSCs. Pharmacological inhibition...

  16. 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.

  17. Secreted Clusterin protein inhibits osteoblast differentiation of bone marrow mesenchymal stem cells by suppressing ERK1/2 signaling pathway

    DEFF Research Database (Denmark)

    Abdallah, Basem; Alzahrani, Abdullah M; Kassem, Moustapha

    2018-01-01

    Secreted Clusterin (sCLU, also known as Apolipoprotein J) is an anti-apoptotic glycoprotein involved in the regulation of cell proliferation, lipid transport, extracellular tissue remodeling and apoptosis. sCLU is expressed and secreted by mouse bone marrow-derived skeletal (stromal or mesenchyma...

  18. Arctigenin Inhibits Osteoclast Differentiation and Function by Suppressing Both Calcineurin-Dependent and Osteoblastic Cell-Dependent NFATc1 Pathways

    OpenAIRE

    Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

    2014-01-01

    Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblasti...

  19. The Effects of Orbital Spaceflight on Human Osteoblastic Cell Physiology and Gene Expression

    Science.gov (United States)

    Turner, R. T.

    1999-01-01

    The purpose of the proposed study is to establish whether changes in gravitational loading have a direct effect on osteoblasts to regulate TGF-6 expression. The effects of spaceflight and reloading on TGF-B MRNA and peptide levels will be studied in a newly developed line of immortalized human fetal osteoblasts (HFOB) transfected with an SV-40 temperature dependent mutant to generate proliferating, undifferentiated hFOB cells at 33-34 C and a non-proliferating, differentiated HFOB cells at 37-39'C. Unlike previous cell culture models, HFOB cells have unlimited proliferative capacity yet can be precisely regulated to differentiate into mature cells which express mature osteoblast function. If isolated osteoblasts respond to changes in mechanical loading in a manner similar to their response in animals, the cell system could provide a powerful model to investigate the signal transduction pathway for gravitational loading.

  20. 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

  1. Wnt3a induces the expression of acetylcholinesterase during osteoblast differentiation via the Runx2 transcription factor.

    Science.gov (United States)

    Xu, Miranda L; Bi, Cathy W C; Liu, Etta Y L; Dong, Tina T X; Tsim, Karl W K

    2017-07-28

    Acetylcholinesterase (AChE) hydrolyzes acetylcholine to terminate cholinergic transmission in neurons. Apart from this AChE activity, emerging evidence suggests that AChE could also function in other, non-neuronal cells. For instance, in bone, AChE exists as a proline-rich membrane anchor (PRiMA)-linked globular form in osteoblasts, in which it is proposed to play a noncholinergic role in differentiation. However, this hypothesis is untested. Here, we found that in cultured rat osteoblasts, AChE expression was increased in parallel with osteoblastic differentiation. Because several lines of evidence indicate that AChE activity in osteoblast could be triggered by Wnt/β-catenin signaling, we added recombinant human Wnt3a to cultured osteoblasts and found that this addition induced expression of the ACHE gene and protein product. This Wnt3a-induced AChE expression was blocked by the Wnt-signaling inhibitor Dickkopf protein-1 (DKK-1). We hypothesized that the Runt-related transcription factor 2 (Runx2), a downstream transcription factor in Wnt/β-catenin signaling, is involved in AChE regulation in osteoblasts, confirmed by the identification of a Runx2-binding site in the ACHE gene promoter, further corroborated by ChIP. Of note, Runx2 overexpression in osteoblasts induced AChE expression and activity of the ACHE promoter tagged with the luciferase gene. Moreover, deletion of the Runx2-binding site in the ACHE promoter reduced its activity during osteoblastic differentiation, and addition of 5-azacytidine and trichostatin A to differentiating osteoblasts affected AChE expression, suggesting epigenetic regulation of the ACHE gene. We conclude that AChE plays a role in osteoblastic differentiation and is regulated by both Wnt3a and Runx2. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Proliferation and osteoblastic differentiation of hMSCs on cellulose-based hydrogels.

    Science.gov (United States)

    Raucci, Maria Grazia; Alvarez-Perez, Marco Antonio; Demitri, Christian; Sannino, Alessandro; Ambrosio, Luigi

    2012-01-01

    The aim of this project was to study the proliferation and differentiation of human Mesenchymal Stem Cells (hMSCs) onto a cellulose-based hydrogel for bone tissue engineering. Modified-cellulose hydrogel was prepared via double esterification crosslinking using citric acid. The response of human Mesenchymal Stem Cells (hMSCs) in terms of cell proliferation and differentiation into osteoblastic phenotype was evaluated by using Alamar blue assay and Alkaline phosphatase activity. The results showed that CMCNa and CMCNa_CA have no negative effect on hMSC, adhesion and proliferation. Moreover, the increase of the ALP expression for CMCNa_CA confirms the ability of the hydrogels to support the osteoblastic differentiation. The cellulose-based hydrogels have a potential application as filler in bone tissue regeneration.

  3. Lactoferrin promote primary rat osteoblast proliferation and differentiation via up-regulation of insulin-like growth factor-1 expression.

    Science.gov (United States)

    Hou, Jian-ming; Wu, Man; Lin, Qing-ming; Lin, Fan; Xue, Ying; Lan, Xu-hua; Chen, En-yu; Wang, Mei-li; Yang, Hai-yan; Wang, Feng-xiong

    2014-08-01

    The aim of this study was to explore the effect of lactoferrin (LF) in primary fetal rat osteoblasts proliferation and differentiation and investigate the underlying molecular mechanisms. Primary rat osteoblasts were obtained from the calvarias of neonatal rats. Osteoblasts were treated with LF (0.1-1000 μg/mL), or OSI-906 [a selective inhibitor of insulin-like growth factor 1 (IGF-1) receptor and insulin receptor]. The IGF-1 was then knocked down by small hairpin RNA (shRNA) technology and then was treated with recombinant human IGF-1 or LF. Cell proliferation and differentiation were measured by MTT assay and alkaline phosphatase (ALP) assay, respectively. The expression of IGF-1 and IGF binding protein 2 (IGFBP2) mRNA were analyzed using real-time PCR. LF promotes the proliferation and differentiation of osteoblasts in a certain range (1-100 μg/mL) in time- and dose-dependent manner. The mRNA level of IGF-1 was significantly increased, while the expression of IGFBP2 was suppressed by LF treatment. Knockdown of IGF-1 by shRNA in primary rat osteoblast dramatically decreased the abilities of proliferation and differentiation of osteoblasts and blocked the proliferation and differentiation effect of LF in osteoblasts. OSI906 (5 μM) blocked the mitogenic and differentiation of LF in osteoblasts. Proliferation and differentiation of primary rat osteoblasts in response to LF are mediated in part by stimulating of IGF-1 gene expression and alterations in the gene expression of IGFBP2.

  4. Expression of osterix Is Regulated by FGF and Wnt/β-Catenin Signalling during Osteoblast Differentiation.

    Directory of Open Access Journals (Sweden)

    Katharina Felber

    Full Text Available Osteoblast differentiation from mesenchymal cells is regulated by multiple signalling pathways. Here we have analysed the roles of Fibroblast Growth Factor (FGF and canonical Wingless-type MMTV integration site (Wnt/β-Catenin signalling pathways on zebrafish osteogenesis. We have used transgenic and chemical interference approaches to manipulate these pathways and have found that both pathways are required for osteoblast differentiation in vivo. Our analysis of bone markers suggests that these pathways act at the same stage of differentiation to initiate expression of the osteoblast master regulatory gene osterix (osx. We use two independent approaches that suggest that osx is a direct target of these pathways. Firstly, we manipulate signalling and show that osx gene expression responds with similar kinetics to that of known transcriptional targets of the FGF and Wnt pathways. Secondly, we have performed ChIP with transcription factors for both pathways and our data suggest that a genomic region in the first intron of osx mediates transcriptional activation. Based upon these data, we propose that FGF and Wnt/β-Catenin pathways act in part by directing transcription of osx to promote osteoblast differentiation at sites of bone formation.

  5. 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.

  6. LIF inhibits osteoblast differentiation at least in part by regulation of HAS2 and its product hyaluronan.

    Science.gov (United States)

    Falconi, Dominic; Aubin, Jane E

    2007-08-01

    LIF arrests osteogenesis in fetal rat calvaria cells in a differentiation stage-specific manner. Differential display identified HAS2 as a LIF-induced gene and its product, HA, modulated osteoblast differentiation similarly to LIF. Our data suggest that LIF arrests osteoblast differentiation by altering HA content of the extracellular matrix. Leukemia inhibitory factor (LIF) elicits both anabolic and catabolic effects on bone. We previously showed in the fetal rat calvaria (RC) cell system that LIF inhibits osteoblast differentiation at the late osteoprogenitor/early osteoblast stage. To uncover potential molecular mediators of this inhibitory activity, we used a positive-negative genome-wide differential display screen to identify LIF-induced changes in the developing osteoblast transcriptome. Although LIF signaling is active throughout the RC cell proliferation-differentiation sequence, only a relatively small number of genes, in several different functional clusters, are modulated by LIF specifically during the LIF-sensitive inhibitory time window. Based on their known and predicted functions, most of the LIF-regulated genes identified are plausible candidates to be involved in the LIF-induced arrest of osteoprogenitor differentiation. To test this hypothesis, we further analyzed the function of one of the genes identified, hyaluronan synthase 2 (HAS2), in the LIF-induced inhibition. Synthesis of hyaluronan (HA), the product of HAS enzymatic activity, was stimulated by LIF and mimicked the HAS2 expression profile, with highest expression in early/proliferative and late/maturing cultures and lowest levels in intermediate/late osteoprogenitor-early osteoblast cultures. Exogenously added high molecular weight HA, the product of HAS2, dose-dependently inhibited osteoblast differentiation, with pulse-treatment effective in the same differentiation stage-specific inhibitory window as seen with LIF. In addition, however, pulse treatment with HA in early cultures

  7. 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

  8. Lithium doped calcium phosphate cement maintains physical mechanical properties and promotes osteoblast proliferation and differentiation.

    Science.gov (United States)

    Li, Li; Wang, Renchong; Li, Baichuan; Liang, Wei; Pan, Haobo; Cui, Xu; Tang, Jingli; Li, Bing

    2017-07-01

    Calcium phosphate cement (CPC) has been widely used in bone tissue repairing due to its physical mechanical properties and biocompatibility. Addition of trace element to CPC has shown promising evidence to improve the physical properties and biological activities of CPC. Lithium (Li) has effect on osteoblast proliferation and differentiation. In this study, we incorporated Li to CPC and examined the physical properties of Li/CPC and its effect on osteoblast proliferation and differentiation. We found that Li doped CPC maintained similar setting time, pore size distribution, compressive strength, composition, and morphology as CPC without Li. Additionally, Li doped CPC improved osteoblast proliferation and differentiation significantly compared to CPC without Li. To our knowledge, our results, for the first time, show that Li doped CPC has beneficial effect on osteoblast in cell culture while keeps the excellent physical-mechanical properties of CPC. This study will lead to potential application of Li doped CPC in bone tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 944-952, 2017. © 2016 Wiley Periodicals, Inc.

  9. 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.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  11. Adipose derived mesenchymal stem cells – Their osteogenicity and osteoblast in vitro mineralization on titanium granule carriers

    DEFF Research Database (Denmark)

    Dahl, Morten; Syberg, Susanne; Jørgensen, Niklas Rye

    2013-01-01

    Adipose derived mesenchymal stem cells (ADMSCs) may be osteogenic, may generate neoangiogenisis and may be progenitors for differentiated osteoblast mineralization. Titanium granules may be suitable as carriers for these cells. The aim was to demonstrate the osteogenic potential of ADMSCs...

  12. 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.

  13. Electrical Polarization of Titanium Surfaces for the Enhancement of Osteoblast Differentiation

    Science.gov (United States)

    Gittens, Rolando A.; Olivares-Navarrete, Rene; Rettew, Robert; Butera, Robert J.; Alamgir, Faisal M.; Boyan, Barbara D.; Schwartz, Zvi

    2014-01-01

    Electrical stimulation has been used clinically to promote bone regeneration in cases of fractures with delayed union or nonunion, with several in vitro and in vivo reports suggesting its beneficial effects on bone formation. However, the use of electrical stimulation of titanium (Ti) implants to enhance osseointegration is less understood, in part because of the few in vitro models that attempt to represent the in vivo environment. In this article, the design of a new in vitro system that allows direct electrical stimulation of osteoblasts through their Ti substrates without the flow of exogenous currents through the media is presented, and the effect of applied electrical polarization on osteoblast differentiation and local factor production was evaluated. A custom-made polycarbonate tissue culture plate was designed to allow electrical connections directly underneath Ti disks placed inside the wells, which were supplied with electrical polarization ranging from 100 to 500 mV to stimulate MG63 osteoblasts. Our results show that electrical polarization applied directly through Ti substrates on which the cells are growing in the absence of applied electrical currents may increase osteoblast differentiation and local factor production in a voltage-dependent manner. PMID:23996899

  14. Production and action of transforming growth factor-beta in human osteoblast cultures: dependence on cell differentiation and modulation by calcitriol

    DEFF Research Database (Denmark)

    Kassem, M; Kveiborg, Marie; Eriksen, E F

    2000-01-01

    Transforming growth factor beta (TGF-beta) plays an important role in skeletal remodelling. However, few studies have examined its effects on cultured human osteoblasts. Our aim is to characterise the biological effects of TGF-beta1 on human osteoblasts and to examine the interaction between TGF-...

  15. Osteocalcin, a marker of differentiated function during calcification of cultured chick osteoblasts

    International Nuclear Information System (INIS)

    Lian, J.; Chipman, S.; Glowacki, J.; Gerstenfeld, L.

    1986-01-01

    The expression of differentiated function was examined in cultured osteoblasts isolated from 17-day embryonic chicken calvarie. Cell cultures grown in the absence (control) or presence of 10 mM β-Glycerol Phosphate (βGP) (stimulus for calcification) were analyzed at 6-day intervals over a 30-day period for total mineral, alkaline phosphatase (AP) activity, osteocalcin levels and collagen. AP was first detected in both cultures between days 6 and 9 when cells became crowded. Control cultures maintained high levels of enzyme activity (30-50 fold) while β GPO 4 culture activity declined after day 18 when extensive mineralization occurred. Osteocalcin, the vitamin K-dependent, bone-specific, calcium-binding protein showed a similar pattern of induction as AP with at 50-100-fold increase in both cultures. Collagen accumulated through out the 30-day experimental period for both β GPO 4 and control cultures while collagen synthesis ( 3 H-proline pulse) peaked at day 15 in culture. These results suggest that with time in culture, osteoblast differentiation may be occurring. The increased mineralization of β GPO 4 cultures appeared to down regulate the enzyme activity of AP in comparison to control culture, while osteocalcin synthesis was enhanced. In conclusion, the chick osteoblast system offers a model to study bone cell differentiation, protein synthesis and matrix calcification

  16. 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

  17. Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

    Science.gov (United States)

    Ma, Ming-San; Kannan, Vishnu; de Vries, Anneriek E; Czepiel, Marcin; Wesseling, Evelyn M; Balasubramaniyan, Veerakumar; Kuijer, Roel; Vissink, Arjan; Copray, Sjef C V M; Raghoebar, Gerry M

    2017-01-01

    New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

  18. Identification of differentiation-stage specific markers that define the ex vivo osteoblastic phenotype

    DEFF Research Database (Denmark)

    Twine, Natalie A; Chen, Li; Pang, Chi N

    2014-01-01

    The phenotype of osteoblastic (OB) cells in culture is currently defined using a limited number of markers of low sensitivity and specificity. For the clinical use of human skeletal (stromal, mesenchymal) stem cells (hMSC) in therapy, there is also a need to identify a set of gene markers...... clustering and Pearson's correlation generated 4 groups of genes: early stage differentiation genes (peak expression: 0-24h, n=28) which were enriched for extracellular matrix organisation, e.g. COL1A1, LOX, and SERPINH1; middle stage differentiating genes (peak expression days: 3 and 6, n=20) which were...

  19. Osteoblast cell response to surface-modified carbon nanotubes

    International Nuclear Information System (INIS)

    Zhang Faming; Weidmann, Arne; Nebe, J. Barbara; Burkel, Eberhard

    2012-01-01

    In order to investigate the interaction of cells with modified multi-walled carbon nanotubes (MWCNTs) for their potential biomedical applications, the MWCNTs were chemically modified with carboxylic acid groups (–COOH), polyvinyl alcohol (PVA) polymer and biomimetic apatite on their surfaces. Additionally, human osteoblast MG-63 cells were cultured in the presence of the surface-modified MWCNTs. The metabolic activities of osteoblastic cells, cell proliferation properties, as well as cell morphology were studied. The surface modification of MWCNTs with biomimetic apatite exhibited a significant increase in the cell viability of osteoblasts, up to 67.23%. In the proliferation phases, there were many more cells in the biomimetic apatite-modified MWCNT samples than in the MWCNTs–COOH. There were no obvious changes in cell morphology in osteoblastic MG-63 cells cultured in the presence of these chemically-modified MWCNTs. The surface modification of MWCNTs with apatite achieves an effective enhancement of their biocompatibility.

  20. Notch Signaling in Prostate Cancer Cells Promotes Osteoblastic Metastasis

    Science.gov (United States)

    2017-06-01

    information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this...function and number while inducing osteoblast proliferation. Our results suggest that Notch signaling from cancer cells promotes osteoblastic...Participants and other collaborating organizations: I initiated collaboration with Dr. Evan Keller at University of Michigan to interrogate PCa bone

  1. The role of surface microtopography in the modulation of osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    JS Hayes

    2010-07-01

    Full Text Available The osteoinductive and conductive capabilities of commercially pure titanium and its alloys is well documented, as is their ability to provide long-term stability for permanent implantable devices. Fracture fixation in paediatric and trauma patients generally requires transient fixation after which the implant becomes redundant and requires removal. Removal can be complicated due to excessive bony over-growth which is encouraged by the standard micro-rough implant surface. We have shown in vivo that removal related morbidity can be significantly reduced with surface polishing, a technique which reduces the micro-roughness of clinically available materials. However, tissue integration at the bone-implant interface requires activation of key regulatory pathways which influences osteoblastic differentiation and maturation therefore we do not believe this effect to be purely mechanical. To elucidate potential mechanisms by which surface polishing exerts its effect on bone regeneration this study assessed in vitro the effect of surface polishing commercially pure titanium on cell growth, morphology and on the regulation of core binding factor 1, osterix, collagen I, alkaline phosphatase, bone sialoprotein and osteocalcin for primary rat calvarial osteoblasts. Results indicate that polishing differentially influences osteoblast differentiation in a surface dependent manner and that these changes are potentially linked to surface dependent morphology, but not to differences in cell proliferation.

  2. Effects of platelet rich plasma (PRP) on human gingival fibroblast, osteoblast and periodontal ligament cell behaviour.

    Science.gov (United States)

    Kobayashi, Eizaburo; Fujioka-Kobayashi, Masako; Sculean, Anton; Chappuis, Vivianne; Buser, Daniel; Schaller, Benoit; Dőri, Ferenc; Miron, Richard J

    2017-06-02

    The use of platelet rich plasma (PRP, GLO) has been used as an adjunct to various regenerative dental procedures. The aim of the present study was to characterize the influence of PRP on human gingival fibroblasts, periodontal ligament (PDL) cells and osteoblast cell behavior in vitro. Human gingival fibroblasts, PDL cells and osteoblasts were cultured with conditioned media from PRP and investigated for cell migration, proliferation and collagen1 (COL1) immunostaining. Furthermore, gingival fibroblasts were tested for genes encoding TGF-β, PDGF and COL1a whereas PDL cells and osteoblasts were additionally tested for alkaline phosphatase (ALP) activity, alizarin red staining and mRNA levels of osteoblast differentiation markers including Runx2, COL1a2, ALP and osteocalcin (OCN). It was first found that PRP significantly increased cell migration of all cells up to 4 fold. Furthermore, PRP increased cell proliferation at 3 and 5 days of gingival fibroblasts, and at 3 days for PDL cells, whereas no effect was observed on osteoblasts. Gingival fibroblasts cultured with PRP increased TGF-β, PDGF-B and COL1 mRNA levels at 7 days and further increased over 3-fold COL1 staining at 14 days. PDL cells cultured with PRP increased Runx2 mRNA levels but significantly down-regulated OCN mRNA levels at 3 days. No differences in COL1 staining or ALP staining were observed in PDL cells. Furthermore, PRP decreased mineralization of PDL cells at 14 days post seeding as assessed by alizarin red staining. In osteoblasts, PRP increased COL1 staining at 14 days, increased COL1 and ALP at 3 days, as well as increased ALP staining at 14 days. No significant differences were observed for alizarin red staining of osteoblasts following culture with PRP. The results demonstrate that PRP promoted gingival fibroblast migration, proliferation and mRNA expression of pro-wound healing molecules. While PRP induced PDL cells and osteoblast migration and proliferation, it tended to have

  3. 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.

  4. The predominant mechanism of intercellular calcium wave propagation changes during long-term culture of human osteoblast-like cells

    DEFF Research Database (Denmark)

    Henriksen, Zanne; Hiken, Jeffrey F; Steinberg, Thomas H

    2006-01-01

    cells still responded to addition of ATP, but P2Y desensitization did not inhibit ICW propagation. Our data indicate that the relative role of P2Y-mediated and gap junction-mediated ICW changes during osteoblast differentiation in vitro. In less differentiated cells, P2Y-mediated ICW predominate......Intercellular calcium waves (ICW) are calcium transients that spread from cell to cell in response to different stimuli. We previously demonstrated that human osteoblast-like cells in culture propagate ICW in response to mechanical stimulation by two mechanisms. One mechanism involves autocrine...... activation of P2Y receptors, and the other requires gap junctional communication. In the current work we ask whether long-term culture of osteoblast-like cells affects the propagation of ICW by these two mechanisms. Human osteoblast-like cells were isolated from bone marrow. Mechanically induced ICW were...

  5. Dual Effect of Chrysanthemum indicum Extract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and Resorption In Vitro

    Directory of Open Access Journals (Sweden)

    Jong Min Baek

    2014-01-01

    Full Text Available The risk of bone-related diseases increases due to the imbalance between bone resorption and bone formation by osteoclasts and osteoblasts, respectively. The goal in the development of antiosteoporotic treatments is an agent that will improve bone through simultaneous osteoblast stimulation and osteoclast inhibition without undesirable side effects. To achieve this goal, numerous studies have been performed to identify novel approaches using natural oriental herbs to treat bone metabolic diseases. In the present study, we investigated the effect of Chrysanthemum indicum extract (CIE on the differentiation of osteoclastic and osteoblastic cells. CIE inhibited the formation of TRAP-positive mature osteoclasts and of filamentous-actin rings and disrupted the bone-resorbing activity of mature osteoclasts in a dose-dependent manner. CIE strongly inhibited Akt, GSK3β, and IκB phosphorylation in RANKL-stimulated bone marrow macrophages and did not show any effects on MAP kinases, including p38, ERK, and JNK. Interestingly, CIE also enhanced primary osteoblast differentiation via upregulation of the expression of alkaline phosphatase and the level of extracellular calcium concentrations during the early and terminal stages of differentiation, respectively. Our results revealed that CIE could have a potential therapeutic role in bone-related disorders through its dual effects on osteoclast and osteoblast differentiation.

  6. Telomerase activity promotes osteoblast differentiation by modulating IGF-signaling pathway

    DEFF Research Database (Denmark)

    Saeed, Hamid; Qiu, Weimin; Li, Chen

    2015-01-01

    -regulation of several components of insulin-like growth factor (IGF) signaling. Specifically, a significant increase in IGF-induced AKT phosphorylation and alkaline phosphatase (ALP) activity were observed in hMSC-TERT. Enhanced ALP activity was reduced in presence of IGF1 receptor inhibitor: picropodophyllin....... In addition, telomerase deficiency caused significant reduction in IGF signaling proteins in osteoblastic cells cultured from telomerase deficient mice (Terc (-/-)). The low bone mass exhibited by Terc (-/-) mice was associated with significant reduction in serum levels of IGF1 and IGFBP3 as well as reduced...... skeletal mRNA expression of Igf1, Igf2, Igf2r, Igfbp5 and Igfbp6. IGF1-induced osteoblast differentiation was also impaired in Terc (-/-) MSC. In conclusion, our data demonstrate that impaired IGF/AKT signaling contributes to the observed decreased bone mass and bone formation exhibited by telomerase...

  7. Bone conditioned media (BCM) improves osteoblast adhesion and differentiation on collagen barrier membranes.

    Science.gov (United States)

    Fujioka-Kobayashi, Masako; Caballé-Serrano, Jordi; Bosshardt, Dieter D; Gruber, Reinhard; Buser, Daniel; Miron, Richard J

    2016-07-04

    The use of autogenous bone chips during guided bone regeneration procedures has remained the gold standard for bone grafting due to its excellent combination of osteoconduction, osteoinduction and osteogenesis. Recent protocols established by our group have characterized specific growth factors and cytokines released from autogenous bone that have the potential to be harvested and isolated into bone conditioned media (BCM). Due to the advantageous osteo-promotive properties of BCM, the aims of the present study was to pre-coat collagen barrier membranes with BCM and investigate its effect on osteoblast adhesion, proliferation and differentiation for possible future clinical use. Scanning electron microscopy (SEM) was first used to qualitative assess BCM protein accumulation on the surface of collagen membranes. Thereafter, undifferentiated mouse ST2 stromal bone marrow cells were seeded onto BioGide porcine derived collagen barrier membranes (control) or barrier membranes pre-coated with BCM (test group). Control and BCM samples were compared for cell adhesion at 8 h, cell proliferation at 1, 3 and 5 days and real-time PCR at 5 days for osteoblast differentiation markers including Runx2, alkaline phosphatase (ALP), osteocalcin (OCN) and bone sialoprotein (BSP). Mineralization was further assessed with alizarin red staining at 14 days post seeding. SEM images demonstrated evidence of accumulated proteins found on the surface of collagen membranes following coating with BCM. Analysis of total cell numbers revealed that the additional pre-coating with BCM markedly increased cell attachment over 4 fold when compared to cells seeded on barrier membranes alone. No significant difference could be observed for cell proliferation at all time points. BCM significantly increased mRNA levels of osteoblast differentiation markers including ALP, OCN and BSP at 5 days post seeding. Furthermore, barrier membranes pre-coated with BCM demonstrated a 5-fold increase in alizarin

  8. Osteoblast Differentiation Decreases Hypergravity-Stimulated Release of PGE(sub 2)

    Science.gov (United States)

    Searby, Nancy D.; Steele, Charles R.; Globus, Ruth K.

    2002-01-01

    We determined if progressive differentiation of osteoblasts influences their sensitivity to gravitational loading. Osteoblasts were cultured for 4 days (confluent monolayer), 6 days (prenodules), 9 days (nodules) and 19 days (mineralized nodules), then centrifuged at 10 times gravity (g) or 50-g for 3 hours using the NASA Ames 1-ft. Diameter Centrifuge. Stationary controls were placed in an adjacent incubator. Following centrifugation, conditioned media were collected and analyzed for PGE, by ELISA. Microtubules were fluorescently labeled and analyzed by confocal microscopy to determine microtubule network morphology and height. Centrifugation at 10-g reduced microtubule network height by 15% on d4 and 10% on d6, with variable changes in more mature cultures. No major changes in microtubule morphology were observed. PGE(sub 2) release by d4 cultures increased in a dose-dependent fashion (3-fold at 10-g and 6-fold at 50-g relative to controls). D6 cultures produced a 5-fold increase for both 10-g and 50-g. PGE(sub 2) increased only 1.5-fold by d9, and by d19, PGE(sub 2) was not delectable in either the control or hypergravity-stimulated cells. Thus, as osteoblasts differentiate in culture, responsiveness of the microtubule cytoskeleton and the PGE(sub 2) pathway to hypergravity declines.

  9. 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.

  10. 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

  11. 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

  12. Effects of extracellular magnesium extract on the proliferation and differentiation of human osteoblasts and osteoclasts in coculture.

    Science.gov (United States)

    Wu, Lili; Feyerabend, Frank; Schilling, Arndt F; Willumeit-Römer, Regine; Luthringer, Bérengère J C

    2015-11-01

    Coculture of osteoblasts and osteoclasts is a subject of interest in the understanding of how magnesium (Mg)-based implants influence the bone metabolism and remodeling upon degradation. Human telomerase reverse transcriptase (hTERT) transduced mesenchymal stem cells (SCP-1) were first differentiated into osteoblasts with osteogenic supplements and then further cocultured with peripheral blood mononucleated cells (PBMC) without the addition of osteoclastogenesis promoting factors. Concomitantly, the cultures were exposed to variable Mg extract dilutions (0, 30×, 10×, 5×, 3×, 2× and 1×). Phenotype characterization documented that while 2× dilution of Mg extract was extremely toxic to osteoclast monoculture, monocytes in coculture with osteoblasts exhibited a greater tolerance to higher Mg extract concentration. The dense growth of osteoblasts in cultures with 1× dilution of Mg extract suggested that high concentration of Mg extract promoted osteoblast proliferation/differentiation behavior. The results of intracellular alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities as well as protein and gene expressions of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoclast-associated receptor (OSCAR) revealed significantly enhanced formation of osteoblasts whereas decreased osteoclastogenesis in the cultures with high concentrations of Mg extract (2× and 1× dilutions). In conclusion, while an increased osteoinductivity has been demonstrated, the impact of potentially decreased osteoclastogenesis around the Mg-based implants should be also taken into account. Cocultures containing both bone-forming osteoblasts and bone-resorbing osteoclasts should be preferentially performed for in vitro cytocompatibility assessment of Mg-based implants as they more closely mimic the in vivo environment. An attractive human osteoblasts and osteoclasts cocultivation regime was

  13. Responses of human normal osteoblast cells and osteoblast-like cell line, MG-63 cells, to pulse electromagnetic field (PEMF

    Directory of Open Access Journals (Sweden)

    Suttatip Kamolmatyakul

    2008-01-01

    Full Text Available The objective of this in vitro study is to investigate the effect of pulsed electromagnetic field (PEMF on cellular proliferation and osteocalcin production of osteoblast-like cell line, MG-63 cells, and human normal osteoblast cells (NHOC obtained from surgical bone specimens. The cells were placed in 24-well culture plates in the amount of 3x104 cell/wells with 2 ml αMEM media supplemented with 10% FBS. The experimental plates were placed between a pair of Helmoltz coils powered by a pulse generator (PEMF, 50 Hz, 1.5 mV/cm in the upper compartment of a dual incubator (Forma. The control plates were placed in the lower compartment of the incubator without Helmotz coils. After three days, the cell proliferation was measured by the method modified from Mossman (J. Immunol Methods 1983; 65: 55-63. Other sets of plates were used for osteocalcin production assessment. Media from these sets were collected after 6 days and assessed for osteocalcin production using ELISA kits. The data were analyzed using a one-way analysis of variance (ANOVA. The results showed that MG-63 cells from the experimental group proliferated significantly more than those from the control group (20% increase, p<0.05. No significant difference in osteocalcin production was detected between the two groups. On the other hand, NHOC from the experimental group produced larger amount of osteocalcin (25% increase, p<0.05 and proliferated significantly more than those from the control group (100% increase, p<0.05. In conclusion, PEMF effect on osteoblasts might depend on their cell type of origin. For osteoblast-like cell line, MG-63 cells, PEMF increased proliferation rate but not osteocalcin production of the cells. However, PEMF stimulation effect on human normal osteoblast cells was most likely associated with enhancement of both osteocalcin production and cell proliferation.

  14. Effects of interfacial micromotions on vitality and differentiation of human osteoblasts.

    Science.gov (United States)

    Ziebart, J; Fan, S; Schulze, C; Kämmerer, P W; Bader, R; Jonitz-Heincke, A

    2018-02-01

    Enhanced micromotions between the implant and surrounding bone can impair osseointegration, resulting in fibrous encapsulation and aseptic loosening of the implant. Since the effect of micromotions on human bone cells is sparsely investigated, an in vitro system, which allows application of micromotions on bone cells and subsequent investigation of bone cell activity, was developed. Micromotions ranging from 25 µm to 100 µm were applied as sine or triangle signal with 1 Hz frequency to human osteoblasts seeded on collagen scaffolds. Micromotions were applied for six hours per day over three days. During the micromotions, a static pressure of 527 Pa was exerted on the cells by Ti6Al4V cylinders. Osteoblasts loaded with Ti6Al4V cylinders and unloaded osteoblasts without micromotions served as controls. Subsequently, cell viability, expression of the osteogenic markers collagen type I, alkaline phosphatase, and osteocalcin, as well as gene expression of osteoprotegerin, receptor activator of NF-κB ligand, matrix metalloproteinase-1, and tissue inhibitor of metalloproteinase-1, were investigated. Live and dead cell numbers were higher after 25 µm sine and 50 µm triangle micromotions compared with loaded controls. Collagen type I synthesis was downregulated in respective samples. The metabolic activity and osteocalcin expression level were higher in samples treated with 25 µm micromotions compared with the loaded controls. Furthermore, static loading and micromotions decreased the osteoprotegerin/receptor activator of NF-κB ligand ratio. Our system enables investigation of the behaviour of bone cells at the bone-implant interface under shear stress induced by micromotions. We could demonstrate that micromotions applied under static pressure conditions have a significant impact on the activity of osteoblasts seeded on collagen scaffolds. In future studies, higher mechanical stress will be applied and different implant surface structures will be

  15. [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.

  16. Inhibition of osteoclastogenesis by osteoblast-like cells genetically engineered to produce interleukin-10.

    Science.gov (United States)

    Fujioka, Kazuki; Kishida, Tsunao; Ejima, Akika; Yamamoto, Kenta; Fujii, Wataru; Murakami, Ken; Seno, Takahiro; Yamamoto, Aihiro; Kohno, Masataka; Oda, Ryo; Yamamoto, Toshiro; Fujiwara, Hiroyoshi; Kawahito, Yutaka; Mazda, Osam

    2015-01-16

    Bone destruction at inflamed joints is an important complication associated with rheumatoid arthritis (RA). Interleukin-10 (IL-10) may suppress not only inflammation but also induction of osteoclasts that play key roles in the bone destruction. If IL-10-producing osteoblast-like cells are induced from patient somatic cells and transplanted back into the destructive bone lesion, such therapy may promote bone remodeling by the cooperative effects of IL-10 and osteoblasts. We transduced mouse fibroblasts with genes for IL-10 and Runx2 that is a crucial transcription factor for osteoblast differentiation. The IL-10-producing induced osteoblast-like cells (IL-10-iOBs) strongly expressed osteoblast-specific genes and massively produced bone matrix that were mineralized by calcium phosphate in vitro and in vivo. Culture supernatant of IL-10-iOBs significantly suppressed induction of osteoclast from RANKL-stimulated Raw264.7 cells as well as LPS-induced production of inflammatory cytokine by macrophages. The IL-10-iOBs may be applicable to novel cell-based therapy against bone destruction associated with RA. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Effect of various concentrations of Ti in hydrocarbon plasma polymer films on the adhesion, proliferation and differentiation of human osteoblast-like MG-63 cells

    Czech Academy of Sciences Publication Activity Database

    Vandrovcová, Marta; Grinevich, A.; Drábik, M.; Kylián, O.; Hanuš, J.; Staňková, Ľubica; Lisá, Věra; Choukourov, A.; Slavínská, D.; Biederman, H.; Bačáková, Lucie

    2015-01-01

    Roč. 357, part A (2015), s. 459-472 ISSN 0169-4332 R&D Projects: GA ČR(CZ) GAP108/12/1168; GA ČR(CZ) GA14-04790S Institutional support: RVO:67985823 Keywords : metal carbon composite films * surface wettability * nanoscale roughness * osteoblasts * bone implants Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.150, year: 2015

  18. Mechanisms of palmitate-induced cell death in human osteoblasts

    Science.gov (United States)

    Gunaratnam, Krishanthi; Vidal, Christopher; Boadle, Ross; Thekkedam, Chris; Duque, Gustavo

    2013-01-01

    Summary Lipotoxicity is an overload of lipids in non-adipose tissues that affects function and induces cell death. Lipotoxicity has been demonstrated in bone cells in vitro using osteoblasts and adipocytes in coculture. In this condition, lipotoxicity was induced by high levels of saturated fatty acids (mostly palmitate) secreted by cultured adipocytes acting in a paracrine manner. In the present study, we aimed to identify the underlying mechanisms of lipotoxicity in human osteoblasts. Palmitate induced autophagy in cultured osteoblasts, which was preceded by the activation of autophagosomes that surround palmitate droplets. Palmitate also induced apoptosis though the activation of the Fas/Jun kinase (JNK) apoptotic pathway. In addition, osteoblasts could be protected from lipotoxicity by inhibiting autophagy with the phosphoinositide kinase inhibitor 3-methyladenine or by inhibiting apoptosis with the JNK inhibitor SP600125. In summary, we have identified two major molecular mechanisms of lipotoxicity in osteoblasts and in doing so we have identified a new potential therapeutic approach to prevent osteoblast dysfunction and death, which are common features of age-related bone loss and osteoporosis. PMID:24285710

  19. Smurf1 Inhibits Osteoblast Differentiation, Bone Formation, and Glucose Homeostasis through Serine 148

    Directory of Open Access Journals (Sweden)

    Junko Shimazu

    2016-04-01

    Full Text Available The E3 ubiquitin ligase Smurf1 targets the master regulator of osteoblast differentiation, Runx2, for degradation, yet the function of Smurf1, if any, during osteoblast differentiation in vivo is ill defined. Here, we show that Smurf1 prevents osteoblast differentiation by decreasing Runx2 accumulation in osteoblasts. Remarkably, mice harboring a substitution mutation at serine 148 (S148 in Smurf1 that prevents its phosphorylation by AMPK (Smurf1ki/ki display a premature osteoblast differentiation phenotype that is equally severe as that of Smurf1−/− mice, as well as a high bone mass, and are also hyperinsulinemic and hypoglycemic. Consistent with the fact that Smurf1 targets the insulin receptor for degradation, there is, in Smurf1ki/ki mice, an increase in insulin signaling in osteoblasts that triggers a rise in the circulating levels of osteocalcin, a hormone that favors insulin secretion. These results identify Smurf1 as a determinant of osteoblast differentiation during the development of bone formation and glucose homeostasis post-natally and demonstrate the necessity of S148 for these functions.

  20. 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.

  1. FLUOXETINE INHIBITS OSTEOBLAST DIFFERENTIATION & MINERALIZATION IN FRACTURE HEALING

    Science.gov (United States)

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

    2016-01-01

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

  2. Bioactive treatment promotes osteoblast differentiation on titanium materials fabricated by selective laser melting technology.

    Science.gov (United States)

    Tsukanaka, Masako; Fujibayashi, Shunsuke; Takemoto, Mitsuru; Matsushita, Tomiharu; Kokubo, Tadashi; Nakamura, Takashi; Sasaki, Kiyoyuki; Matsuda, Shuichi

    2016-01-01

    Selective laser melting (SLM) technology is useful for the fabrication of porous titanium implants with complex shapes and structures. The materials fabricated by SLM characteristically have a very rough surface (average surface roughness, Ra=24.58 µm). In this study, we evaluated morphologically and biochemically the specific effects of this very rough surface and the additional effects of a bioactive treatment on osteoblast proliferation and differentiation. Flat-rolled titanium materials (Ra=1.02 µm) were used as the controls. On the treated materials fabricated by SLM, we observed enhanced osteoblast differentiation compared with the flat-rolled materials and the untreated materials fabricated by SLM. No significant differences were observed between the flat-rolled materials and the untreated materials fabricated by SLM in their effects on osteoblast differentiation. We concluded that the very rough surface fabricated by SLM had to undergo a bioactive treatment to obtain a positive effect on osteoblast differentiation.

  3. Effects of fluoridation of porcine hydroxyapatite on osteoblastic activity of human MG63 cells

    International Nuclear Information System (INIS)

    Li, Zhipeng; Huang, Baoxin; Mai, Sui; Wu, Xiayi; Zhang, Hanqing; Qiao, Wei; Luo, Xin; Chen, Zhuofan

    2015-01-01

    Biological hydroxyapatite, derived from animal bones, is the most widely used bone substitute in orthopedic and dental treatments. Fluorine is the trace element involved in bone remodeling and has been confirmed to promote osteogenesis when administered at the appropriate dose. To take advantage of this knowledge, fluorinated porcine hydroxyapatite (FPHA) incorporating increasing levels of fluoride was derived from cancellous porcine bone through straightforward chemical and thermal treatments. Physiochemical characteristics, including crystalline phases, functional groups and dissolution behavior, were investigated on this novel FPHA. Human osteoblast-like MG63 cells were cultured on the FPHA to examine cell attachment, cytoskeleton, proliferation and osteoblastic differentiation for in vitro cellular evaluation. Results suggest that fluoride ions released from the FPHA play a significant role in stimulating osteoblastic activity in vitro, and appropriate level of fluoridation (1.5 to 3.1 atomic percents of fluorine) for the FPHA could be selected with high potential for use as a bone substitute. (paper)

  4. Periostin inhibits mechanical stretch-induced apoptosis in osteoblast-like MG-63 cells.

    Science.gov (United States)

    Yu, Kai-Wen; Yao, Chung-Chen; Jeng, Jiiang-Huei; Shieh, Hao-Ying; Chen, Yi-Jane

    2018-04-01

    Appropriate mechanical stress plays an important role in regulating the proliferation and differentiation of osteoblasts, whereas high-level mechanical stress may be harmful and compromise cell survival. Periostin, a matricellular protein, is essential in maintaining functional integrity of bone and collagen-rich connective tissue in response to mechanical stress. This study investigated whether or not high-level mechanical stretch induces cell apoptosis and the regulatory role of periostin in mechanical stretch-induced apoptosis in osteoblastic cells. Osteoblast-like MG-63 cells were seeded onto Bio-Flex I culture plates and subjected to cyclic mechanical stretching (15% elongation, 0.1 Hz) in a Flexercell tension plus system-5000. The same process was applied to cells pre-treated with exogenous human recombinant periostin before mechanical stretching. We used a chromatin condensation and membrane permeability dead cell apoptosis kit to evaluate the stretch-induced cell responses. Expression of caspase-3 and cPARP was examined by immunofluorescent stain and flow cytometry. The expression of periostin in MG-63 cells is involved in the TGF-β signaling pathway. High-level cyclic mechanical stretch induced apoptotic responses in MG-63 osteoblastic cells. The percentages of apoptotic cells and cells expressing cPARP protein increased in the groups of cells subjected to mechanical stretch, but these responses were absent in the presence of exogenous periostin. Our study revealed that high-level mechanical stretch induces apoptotic cell death, and that periostin plays a protective role against mechanical stretch-induced apoptosis in osteoblastic cells. Copyright © 2017. Published by Elsevier B.V.

  5. Slight changes in the mechanical stimulation affects osteoblast- and osteoclast-like cells in co-culture.

    Science.gov (United States)

    Kadow-Romacker, Anke; Duda, Georg N; Bormann, Nicole; Schmidmaier, Gerhard; Wildemann, Britt

    2013-12-01

    Osteoblast- and osteoclast-like cells are responsible for coordinated bone maintenance, illustrated by a balanced formation and resorption. Both parameters appear to be influenced by mechanical constrains acting on each of these cell types individually. We hypothesized that the interactions between both cell types are also influenced by mechanical stimulation. Co-cultures of osteoblast- and osteoclast-like cells were stimulated with 1,100 µstrain, 0.1 or 0.3 Hz for 1-5 min/day over 5 days. Two different setups depending on the differentiation of the osteoclast-like cells were used: i) differentiation assay for the fusion of pre-osteoclasts to osteoclasts, ii) resorption assay to determine the activity level of osteoclast-like cells. In the differentiation assay (co-culture of osteoblasts with unfused osteoclast precursor cells) the mechanical stimulation resulted in a significant decrease of collagen-1 and osteocalcin produced by osteoblast-like cells. Significantly more TRAP-iso5b was measured after stimulation for 3 min with 0.1 Hz, indicating enhanced osteoclastogenesis. In the resorption assay (co-culture of osteoblasts with fused osteoclasts) the stimulation for 3 min with 0.3 Hz significantly increased the resorption activity of osteoclasts measured by the pit formation and the collagen resorption. The same mechanical stimulation resulted in an increased collagen-1 production by the osteoblast-like cells. The ratio of RANKL/OPG was not different between the groups. These findings demonstrate that already small changes in duration or frequency of mechanical stimulation had significant consequences for the behavior of osteoblast- and osteoclast-like cells in co-culture, which partially depend on the differentiation status of the osteoclast-like cells.

  6. [Low-frequency pulsed electromagnetic fields promotes rat osteoblast differentiation in vitro through cAMP/PKA signal pathway].

    Science.gov (United States)

    Fang, Qing-Qing; Li, Zhi-Zhong; Zhou, Jian; Shi, Wen-Gui; Yan, Juan-Li; Xie, Yan-Fang; Chen, Ke-Ming

    2016-11-20

    To study whether low-frequency pulsed electromagnetic fields promotes the differentiation of cultured rat osteoblasts through the cAMP/PKA signal pathway. Rat calvarial osteoblasts isolated by enzyme digestion were exposed to 50 Hz 0.6 mT low-frequency pulsed electromagnetic field for varying lengths of time, and the concentration of cAMP and levels of phosphorylated PKA in the cells were assayed. In cells treated with DDA to inhibit the activity of adenylate cyclase, the changes of ALP activity and transcription of osteogenic gene were detected after exposure to low-frequency pulsed electromagnetic field. The changes of osteogenic gene transcription and protein expression were tested in the osteoblasts pretreated with KT5720 in response to low-frequency pulsed electromagnetic field exposure. The intracellular cAMP concentration in the cells increased significantly at 20 min during exposure to low-frequency pulsed electromagnetic field, began to decrease at 40 min during the exposure, and increased again after a 2-h exposure; the same pattern of variation was also observed in p-PKA level. Application of DDA and KT5720 pretreatment both suppressed the increase in ALP activity and osteogenic gene transcription induced by electromagnetic field exposure. Low- frequency pulsed electromagnetic field exposure improves the differentiation of cultured rat osteoblasts by activating cAMP/PKA signal pathway.

  7. Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yinxiang Wang

    2018-01-01

    Full Text Available Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc, enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.

  8. Biocompatibility of chitosan-coated iron oxide nanoparticles with osteoblast cells

    Directory of Open Access Journals (Sweden)

    Shi S

    2012-10-01

    Full Text Available Si-Feng Shi,1 Jing-Fu Jia,2 Xiao-Kui Guo,3 Ya-Ping Zhao,2 De-Sheng Chen,1 Yong-Yuan Guo,1 Tao Cheng,1 Xian-Long Zhang11Department of Orthopedic Surgery, Shanghai Sixth People’s Hospital, School of Medicine, 2School of Chemistry and Chemical Technology, 3Department of Medical Microbiology and Parasitology, School of Medicine, Shanghai Jiao Tong University Shanghai, ChinaBackground: Bone disorders (including osteoporosis, loosening of a prosthesis, and bone infections are of great concern to the medical community and are difficult to cure. Therapies are available to treat such diseases, but all have drawbacks and are not specifically targeted to the site of disease. Chitosan is widely used in the biomedical community, including for orthopedic applications. The aim of the present study was to coat chitosan onto iron oxide nanoparticles and to determine its effect on the proliferation and differentiation of osteoblasts.Methods: Nanoparticles were characterized using transmission electron microscopy, dynamic light scattering, x-ray diffraction, zeta potential, and vibrating sample magnetometry. Uptake of nanoparticles by osteoblasts was studied by transmission electron microscopy and Prussian blue staining. Viability and proliferation of osteoblasts were measured in the presence of uncoated iron oxide magnetic nanoparticles or those coated with chitosan. Lactate dehydrogenase, alkaline phosphatase, total protein synthesis, and extracellular calcium deposition was studied in the presence of the nanoparticles.Results: Chitosan-coated iron oxide nanoparticles enhanced osteoblast proliferation, decreased cell membrane damage, and promoted cell differentiation, as indicated by an increase in alkaline phosphatase and extracellular calcium deposition. Chitosan-coated iron oxide nanoparticles showed good compatibility with osteoblasts.Conclusion: Further research is necessary to optimize magnetic nanoparticles for the treatment of bone disease

  9. Sr-substituted bone cements direct mesenchymal stem cells, osteoblasts and osteoclasts fate.

    Directory of Open Access Journals (Sweden)

    Monica Montesi

    Full Text Available Strontium-substituted apatitic bone cements enriched with sodium alginate were developed as a potential modulator of bone cells fate. The biological impact of the bone cement were investigated in vitro through the study of the effect of the nanostructured apatitic composition and the doping of strontium on mesenchymal stem cells, pre-osteoblasts and osteoclasts behaviours. Up to 14 days of culture the bone cells viability, proliferation, morphology and gene expression profiles were evaluated. The results showed that different concentrations of strontium were able to evoke a cell-specific response, in fact an inductive effect on mesenchymal stem cells differentiation and pre-osteoblasts proliferation and an inhibitory effect on osteoclasts activity were observed. Moreover, the apatitic structure of the cements provided a biomimetic environment suitable for bone cells growth. Therefore, the combination of biological features of this bone cement makes it as promising biomaterials for tissue regeneration.

  10. 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

  11. Osteoblastic cells trigger gate currents on nanocrystalline diamond transistor

    Czech Academy of Sciences Publication Activity Database

    Ižák, Tibor; Krátká, Marie; Kromka, Alexander; Rezek, Bohuslav

    2015-01-01

    Roč. 129, May (2015), 95-99 ISSN 0927-7765 R&D Projects: GA ČR GAP108/12/0996 Grant - others:AVČR(CZ) M100101209 Institutional support: RVO:68378271 Keywords : field-effect transistors * nanocrystalline diamond * osteoblastic cells * leakage currents Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 3.902, year: 2015

  12. Identification of differentiation-stage specific molecular markers for the osteoblastic phenotype

    DEFF Research Database (Denmark)

    Twine, Natalie; Chen, Li; Wilkins, Marc

    to age-matched control (n=4). Using RNA-seq and cluster analysis, we identified a set of stage-specific molecular markers that define the progression of OB phenotype during ex vivo culture of hMSC, predict in vivo bone formation capacity of hMSC and can be employed to study the mechanisms of impaired......The phenotype of osteoblastic (OB) cells in culture is currently defined using a limited number of markers of low sensitivity and specificity which belong mostly to extracellular matrix proteins. Also, for clinical use of human skeletal (mesenchymal) stem cells (hMSC) in bone regeneration......, there is a need to identify predictive markers for in vivo bone forming capacity. Thus, we employed Illumina RNA sequencing (RNASeq) to examine changes in gene expression across 8 time points between 0-12 days of ex vivo OB differentiation of hMSC. We identified a subset of expressed genes as potentially...

  13. Hypoxia inhibits the growth, differentiation and bone-forming capacity of rat osteoblasts

    International Nuclear Information System (INIS)

    Utting, J.C.; Robins, S.P.; Brandao-Burch, A.; Orriss, I.R.; Behar, J.; Arnett, T.R.

    2006-01-01

    We investigated the effect of hypoxia on rat osteoblast function in long-term primary cultures. Reduction of pO 2 from 20% to 5% and 2% decreased formation of mineralized bone nodules 1.7-fold and 11-fold, respectively. When pO 2 was reduced further to 0.2%, bone nodule formation was almost abolished. The inhibitory effect of hypoxia on bone formation was partly due to decreased osteoblast proliferation, as measured by 3 H-thymidine incorporation. Hypoxia also sharply reduced osteoblast alkaline phosphatase (ALP) activity and expression of mRNAs for ALP and osteocalcin, suggesting inhibition of differentiation to the osteogenic phenotype. Hypoxia did not increase the apoptosis of osteoblasts but induced a reversible state of quiescence. Transmission electron microscopy revealed that collagen fibrils deposited by osteoblasts cultured in 2% O 2 were less organized and much less abundant than in 20% O 2 cultures. Furthermore, collagen produced by hypoxic osteoblasts contained a lower percentage of hydroxylysine residues and exhibited an increased sensitivity to pepsin degradation. These data demonstrate the absolute oxygen requirement of osteoblasts for successful bone formation and emphasize the importance of the vasculature in maintaining bone health. We recently showed that hypoxia also acts in a reciprocal manner as a powerful stimulator of osteoclast formation. Considered together, our results help to explain the bone loss that occurs at the sites of fracture, tumors, inflammation and infection, and in individuals with vascular disease or anemia

  14. Cooperative effects in differentiation and proliferation between PDGF-BB and matrix derived synthetic peptides in human osteoblasts

    Directory of Open Access Journals (Sweden)

    Vordemvenne Thomas

    2011-11-01

    Full Text Available Abstract Background Enhancing osteogenic capabilities of bone matrix for the treatment of fractures and segmental defects using growth factors is an active area of research. Recently, synthetic peptides like AC- 100, TP508 or p-15 corresponding to biologically active sequences of matrix proteins have been proven to stimulate bone formation. The platelet-derived growth factor (PDGF BB has been identified as an important paracrine factor in early bone healing. We hypothesized that the combined use of PDGF-BB with synthetic peptides could result in an increase in proliferation and calcification of osteoblast-like cells. Methods Osteoblast-like cell cultures were treated with PDGF and synthetic peptides, singly and as combinations, and compared to non-treated control cell cultures. The cultures were evaluated at days 2, 5, and 10 in terms of cell proliferation, calcification and gene expression of alkaline phosphate, collagen I and osteocalcin. Results Experimental findings revealed that the addition of PDGF, p-15 and TP508 and combinations of PDGF/AC-100, PDGF/p-15 and PDGF/TP508 resulted in an increase in proliferating osteoblasts, especially in the first 5 days of cultivation. Proliferation did not significantly differ between single factors and factor combinations (p > 0.05. The onset of calcification in osteoblasts occurred earlier and was more distinct compared to the corresponding control or PDGF stimulation alone. Significant difference was found for the combined use of PDGF/p-15 and PDGF/AC-100 (p Conclusions Our findings indicate that PDGF exhibits cooperative effects with synthetic peptides in differentiation and proliferation. These cooperative effects cause a significant early calcification of osteoblast-like cells (p

  15. 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.

  16. 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

  17. Surface modification of TiO{sub 2} nanotubes with osteogenic growth peptide to enhance osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Min, E-mail: minlai@jsnu.edu.cn [School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116 (China); Jin, Ziyang [School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116 (China); Su, Zhiguo [Department of Pharmacy, The Affiliated hospital of Qingdao University, Qingdao, Shandong 266555 (China)

    2017-04-01

    To investigate the influence of surface-biofunctionalized substrates on osteoblast behavior, a layer of aligned TiO{sub 2} nanotubes with a diameter of around 70 nm was fabricated on titanium surface by anodization, and then osteogenic growth peptide (OGP) was conjugated onto TiO{sub 2} nanotubes through the intermediate layer of polydopamine. The morphology, composition and wettability of different surfaces were characterized by field-emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements, respectively. The effects of OGP-modified TiO{sub 2} nanotube substrates on the morphology, proliferation and differentiation of osteoblasts were examined in vitro. Immunofluorescence staining revealed that the OGP-functionalized TiO{sub 2} nanotubes were favorable for cell spreading. However, there was no significant difference in cell proliferation observed among the different groups. Cells grown onto OGP-functionalized TiO{sub 2} nanotubes showed significantly higher (p < 0.05 or p < 0.01) levels of alkaline phosphatase (ALP) and mineralization after 4, 7 and 14 days of culture, respectively. Cells grown on OGP-functionalized TiO{sub 2} nanotubes had significantly higher (p < 0.05 or p < 0.01) expression of osteogenic-related genes including runt related transcription factor 2 (Runx2), ALP, collagen type I (Col I), osteopontin (OPN) and osteocalcin (OC) after 14 days of culture. These data suggest that surface functionalization of TiO{sub 2} nanotubes with OGP was beneficial for cell spreading and differentiation. This study provides a novel platform for the development and fabrication of titanium-based implants that enhance the propensity for osseointegration between the native tissue and implant interface. - Highlights: • The OGP functionalized TiO{sub 2} nanotube substrates were successfully fabricated through a direct and effective method. • The OGP functionalized substrates

  18. 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.

  19. 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.

  20. Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance.

    Science.gov (United States)

    Ando, Kazunori; Shibata, Eri; Hans, Stefan; Brand, Michael; Kawakami, Atsushi

    2017-12-04

    Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Gold nanoparticles stimulate differentiation and mineralization of primary osteoblasts through the ERK/MAPK signaling pathway

    International Nuclear Information System (INIS)

    Zhang, Dawei; Liu, Dandan; Zhang, Jinchao; Fong, Chichun; Yang, Mengsu

    2014-01-01

    Gold nanoparticles (AuNPs) have shown great promise for a variety of applications, including chemistry, biology, and medicine. Recently, AuNPs have found promising applications in cartilage and bone repair. However, to realize the above promised applications, more work needs to be carried out to clarify the interactions between biological systems and AuNPs. In the present study, primary osteoblasts were used to evaluate the biocompatibility of 20-nm and 40-nm AuNPs, including morphology, proliferation, differentiation, gene and protein expression, and the underlying mechanisms. The results demonstrated that AuNPs were taken up by osteoblasts and aggregated in perinuclear compartment and vescular structures, but no morphological changes were observed. AuNPs could significantly promote the proliferation of osteoblasts, enhance the ALP activities, and increase the number of bone nodules and calcium content in vitro. In addition, the expression of BMP-2, Runx-2, OCN and Col-1 was remarkably up-regulated in the presence of AuNPs. It is noteworthy that 20-nm AuNPs are more potent than 40-nm AuNPs in regulating osteoblast activities. Besides, AuNPs increased the level of ERK phosphorylation/total ERK, suggesting the activation of ERK/MAPK pathway is involved in above activities. In conclusion, AuNPs exhibited great biocompatibility with osteoblasts, and have tremendous potential to be used as drug and/or gene delivery carrier for bone and tissue engineering in the future. - Highlights: • AuNPs aggregated in perinuclear compartment and vescular structures of osteoblasts. • AuNPs up-regulated the expression of Runx-2, BMP-2, OCN and Col I of osteoblasts. • AuNPs enhanced osteoblast differentiation by activating the ERK/MAPK pathway. • The size of nanoparticles may be important to exhibit their biological effects. • AuNPs have tremendous potential in bone and tissue engineering in future

  2. Tanshinol Attenuates the Deleterious Effects of Oxidative Stress on Osteoblastic Differentiation via Wnt/FoxO3a Signaling

    Directory of Open Access Journals (Sweden)

    Yajun Yang

    2013-01-01

    Full Text Available There is now increasing evidence which suggests a pivotal role for oxidative stress in the development and progression of osteoporosis. We confirm herein the protective effects of natural antioxidant Tanshinol against oxidative stress in osteoblastic differentiation and the underlying mechanism. Our results show that hydrogen peroxide (H2O2 leads to accumulation of reactive oxygen species (ROS, decrease in cell viability, cell cycle arrest and apoptosis in a caspase-3-dependent manner, and inhibition of osteoblastic differentiation. Tanshinol reverses these deleterious consequence triggered by oxidative stress. Moreover, under the condition of oxidative stress, Tanshinol suppresses the activation of FoxO3a transcription factor and expressions of its target genes Gadd45a and catalase (CAT and simultaneously counteracts the inhibition of Wnt signalling and expressions of target genes Axin2, alkaline phosphatase (ALP, and Osteoprotegerin (OPG. The findings are further consolidated using FoxO3a siRNA interference and overexpression of Tcf4. The results illustrate that Tanshinol attenuates oxidative stress via down-regulation of FoxO3a signaling, and rescues the decrease of osteoblastic differentiation through upregulation of Wnt signal under oxidative stress. The present findings suggest that the beneficial effects of Tanshinol may be adopted as a novel therapeutic approach in recently recognized conditions of niche targeting osteoporosis.

  3. Mechanisms in endocrinology: micro-RNAs: targets for enhancing osteoblast differentiation and bone formation.

    Science.gov (United States)

    Taipaleenmäki, Hanna; Bjerre Hokland, Lea; Chen, Li; Kauppinen, Sakari; Kassem, Moustapha

    2012-03-01

    Osteoblast differentiation and bone formation (osteogenesis) are regulated by transcriptional and post-transcriptional mechanisms. Recently, a novel class of regulatory factors termed micro-RNAs (miRNAs) has been identified as playing an important role in the regulation of many aspects of osteoblast biology including proliferation, differentiation, metabolism and apoptosis. Also, preliminary data from animal disease models suggest that targeting miRNAs in bone can be a novel approach to increase bone mass. This review highlights the current knowledge of miRNA biology and their role in bone formation and discusses their potential use in future therapeutic applications for metabolic bone diseases.

  4. UV-killed Staphylococcus aureus enhances adhesion and differentiation of osteoblasts on bone-associated biomaterials.

    Science.gov (United States)

    Somayaji, Shankari N; Huet, Yvette M; Gruber, Helen E; Hudson, Michael C

    2010-11-01

    Titanium alloys (Ti) are the preferred material for orthopedic applications. However, very often, these metallic implants loosen over a long period and mandate revision surgery. For implant success, osteoblasts must adhere to the implant surface and deposit a mineralized extracellular matrix (ECM). Here, we utilized UV-killed Staphylococcus aureus as a novel osteoconductive coating for Ti surfaces. S. aureus expresses surface adhesins capable of binding to bone and biomaterials directly. Furthermore, interaction of S. aureus with osteoblasts activates growth factor-related pathways that potentiate osteogenesis. Although UV-killed S. aureus cells retain their bone-adhesive ability, they do not stimulate significant immune modulator expression. All of the abovementioned properties were utilized for a novel implant coating so as to promote osteoblast recruitment and subsequent cell functions on the bone-implant interface. In this study, osteoblast adhesion, proliferation, and mineralized ECM synthesis were measured on Ti surfaces coated with fibronectin with and without UV-killed bacteria. Osteoblast adhesion was enhanced on Ti alloy surfaces coated with bacteria compared to uncoated surfaces, while cell proliferation was sustained comparably on both surfaces. Osteoblast markers such as collagen, osteocalcin, alkaline phosphatase activity, and mineralized nodule formation were increased on Ti alloy coated with bacteria compared to uncoated surfaces.

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

    Science.gov (United States)

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

    2017-12-12

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

  6. Fabrication of nanofibrous scaffold using a PLA and hagfish thread keratin composite; its effect on cell adherence, growth, and osteoblast differentiation

    International Nuclear Information System (INIS)

    Kim, Beom-Su; Lee, Jun; Park, Ko Eun; Park, Won Ho

    2013-01-01

    Electrospinning is a useful method for the production of nanofibrous scaffolds in the field of tissue engineering. Keratin has been used as a biomaterial for electrospinning and can be used in a variety of biomedical applications because it is a natural protein, giving it the ability to improve cell affinity of scaffolds. In this study, keratin was extracted from hagfish slime thread (H-keratin) and blended with polylactic acid (PLA) polymer solution to construct a nanofibrous scaffold. Wool keratin (W-keratin) was used as a control for the comparison of morphological, physical, and biological properties. The results of Fourier transform infrared spectroscopy showed the presence of both W-keratin and H-keratin in the electrospun PLA/keratin. Observations with a scanning electron microscope revealed that PLA, PLA/W-keratin, and PLA/H-keratin had similar average diameters (∼800 nm). Cell attachment experiments showed that MG-63 cells adhered more rapidly and spread better onto PLA/H-keratin than onto the pure PLA or PLA/W-keratin. Cell proliferation assay, DNA content, live/dead, and alkaline phosphatase activity assays showed that PLA/H-keratin scaffolds could accelerate the viability, proliferation, and osteogenesis of MG-63 cells relative to pure PLA or PLA/W-keratin nanofibrous scaffolds. These findings suggest that H-keratin can improve cellular attraction and has great potential to be used as a biomaterial in bone tissue engineering. (paper)

  7. Activity of the rat osteocalcin basal promoter in osteoblastic cells is dependent upon homeodomain and CP1 binding motifs.

    Science.gov (United States)

    Towler, D A; Bennett, C D; Rodan, G A

    1994-05-01

    A detailed analysis of the transcriptional machinery responsible for osteoblast-specific gene expression should provide tools useful for understanding osteoblast commitment and differentiation. We have defined three cis-elements important for basal activity of the rat osteocalcin (OC) promoter, located at about -200 to -180, -170 to -138, and -121 to -64 relative to the transcription initiation site. A motif (TCTGATTGTGT) present in the region between -200 and -170 that binds a multisubunit CP1/NFY/CBF-like CAAT factor complex contributes significantly to high level basal activity and presumably functions as the CAAT box for the rat OC promoter. We show that the region -121 to 32 is sufficient to confer osteoblastic cell type specificity in transient transfection assays of cultured cell lines using luciferase as a reporter. The basal promoter is active in rodent osteoblastic cell lines, but not in rodent fibroblastic or muscle cell lines. Although the rat OC box (-100 to -74) contains a CAAT motif, we could not detect CP1-like CAAT factor binding to this region. In fact, we demonstrate that a Msx-1 (Hox 7.1) homeodomain binding motif (ACTAATTG; bottom strand) in the 3'-end of the rat OC box is necessary for high level activity of the rat OC basal promoter in osteoblastic cells. A nuclear factor that recognizes this motif appears to be present in osteoblastic ROS 17/2.8 cells, which produce OC, but not in fibroblastic ROS 25/1 cells, which fail to express OC. This ROS 17/2.8 nuclear factor also recognizes the A/T-rich DNA cognates of the homeodomain-containing POU family of transcription factors. Taken together, these data suggest that a ubiquitous CP1-like CAAT factor and a cell type-restricted homeodomain containing (Msx or POU family) transcription factor interact with the proximal rat OC promoter to direct appropriate basal OC transcription in osteoblastic cells.

  8. Effects of nano-emulsion preparations of tocopherols and tocotrienols on oxidative stress and osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Song Liang-Song

    2017-01-01

    Full Text Available Tocopherols and tocotrienols are two groups of compounds in the vitamin E family, of which the tocopherols are widely used as antioxidant dietary supplements. Recent studies have shown mixed observations for tocopherol functions in bone homeostasis. We have evaluated the potency of suspension- and nano-emulsion formulation-based delivery of different vitamin E family members in lipopolysaccharide (LPS-induced oxidative stress and osteoblast differentiation. Our results showed the both tocopherols and tocotrienols could reduce oxidative stress as evaluated by the levels of reactive oxygen species (ROS. Their effects were enhanced when applied in the nano-emulsion mode of delivery due to increased bioavailability. In addition, our results showed that tocotrienols increased osteoblast differentiation, while tocopherols showed reduced osteoblast differentiation, which may be due to their differential effects on SMAD and p65 signaling. Together, these findings indicate that tocotrienols delivered through nano-emulsion exhibit superior antioxidant properties and osteoblast differentiation, and could serve as a better alternative to tocopherol-based vitamin E supplements.

  9. The flavonoid fisetin promotes osteoblasts differentiation through Runx2 transcriptional activity.

    Science.gov (United States)

    Léotoing, Laurent; Davicco, Marie-Jeanne; Lebecque, Patrice; Wittrant, Yohann; Coxam, Véronique

    2014-06-01

    Flavonoids represent a group of polyphenolic compounds commonly found in daily nutrition with proven health benefits. Among this group, the flavonol fisetin has been previously shown to protect bone by repressing osteoclast differentiation. In the present study, we investigated the role of fisetin in regulating osteoblasts physiology. In vivo mice treated with LPSs exhibited osteoporosis features associated with a dramatic repression of osteoblast marker expression. In this model, inhibition of osteocalcin and type I collagen alpha 1 transcription was partially countered by a daily consumption of fisetin. Interestingly, in vitro, fisetin promoted both osteoblast alkaline phosphatase activity and mineralization process. To decipher how fisetin may exert its positive effect on osteoblastogenesis, we analyzed its ability to control the runt-related transcription factor 2 (Runx2), a key organizer in developing and maturing osteoblasts. While fisetin did not impact Runx2 mRNA and protein levels, it upregulated its transcriptional activity. Actually, fisetin stimulated the luciferase activity of a reporter plasmid driven by the osteocalcin gene promoter that contains Runx2 binding sites and promoted the mRNA expression of osteocalcin and type I collagen alpha 1 targets. Bone sparing properties of fisetin also rely on its positive influence on osteoblast differentiation and activity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. 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.

  11. Lecithin blended polyamide-6 high aspect ratio nanofiber scaffolds via electrospinning for human osteoblast cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Nirmala, R. [Bio-nano System Engineering, College of Engineering, Chonbuk National University, Jeonju, 561 756 (Korea, Republic of); Park, Hye-Min [Department of Pharmacology and Toxicology, College of Veterinary Medicine, Chonbuk National University, Jeonju 561 756 (Korea, Republic of); Navamathavan, R. [School of Advanced Materials Engineering, Chonbuk National University, Jeonju 561 756 (Korea, Republic of); Kang, Hyung-Sub [Department of Pharmacology and Toxicology, College of Veterinary Medicine, Chonbuk National University, Jeonju 561 756 (Korea, Republic of); El-Newehy, Mohamed H. [Petrochemical Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh 11451 (Saudi Arabia); Kim, Hak Yong, E-mail: khy@jbnu.ac.kr [Petrochemical Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh 11451 (Saudi Arabia); Center for Healthcare Technology and Development, Chonbuk National University, Jeonju, 561 756 (Korea, Republic of)

    2011-03-12

    In this study, we focused on the preparation and characterization of lecithin blended polyamide-6 nanofibers via an electrospinning process for human osteoblastic (HOB) cell culture applications. The morphological, structural characterizations and thermal properties of polyamide-6/lecithin nanofibers were determined by using scanning electron microscopy (SEM), field-emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA). SEM images revealed that the nanofibers were well-oriented with good incorporation of lecithin. FT-IR results indicated the presence of amino groups of lecithin in the blended nanofibers. TGA analysis revealed that the onset degradation temperature decreased with increasing lecithin content in the blended nanofibers. The morphological features of cells attached on polyamide-6/lecithin nanofibers were confirmed by SEM. The adhesion, viability and proliferation properties of osteoblast cells on the polyamide-6/lecithin blended nanofibers were analyzed by in vitro cell compatibility test. This study demonstrated the non-cytotoxic behavior of electrospun polyamide-6/lecithin nanofibers for the osteoblast cell culture.

  12. Lecithin blended polyamide-6 high aspect ratio nanofiber scaffolds via electrospinning for human osteoblast cell culture

    International Nuclear Information System (INIS)

    Nirmala, R.; Park, Hye-Min; Navamathavan, R.; Kang, Hyung-Sub; El-Newehy, Mohamed H.; Kim, Hak Yong

    2011-01-01

    In this study, we focused on the preparation and characterization of lecithin blended polyamide-6 nanofibers via an electrospinning process for human osteoblastic (HOB) cell culture applications. The morphological, structural characterizations and thermal properties of polyamide-6/lecithin nanofibers were determined by using scanning electron microscopy (SEM), field-emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA). SEM images revealed that the nanofibers were well-oriented with good incorporation of lecithin. FT-IR results indicated the presence of amino groups of lecithin in the blended nanofibers. TGA analysis revealed that the onset degradation temperature decreased with increasing lecithin content in the blended nanofibers. The morphological features of cells attached on polyamide-6/lecithin nanofibers were confirmed by SEM. The adhesion, viability and proliferation properties of osteoblast cells on the polyamide-6/lecithin blended nanofibers were analyzed by in vitro cell compatibility test. This study demonstrated the non-cytotoxic behavior of electrospun polyamide-6/lecithin nanofibers for the osteoblast cell culture.

  13. Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells

    Science.gov (United States)

    Yang, Wuchen; Guo, Dayong; Harris, Marie A.; Cui, Yong; Gluhak-Heinrich, Jelica; Wu, Junjie; Chen, Xiao-Dong; Skinner, Charles; Nyman, Jeffry S.; Edwards, James R.; Mundy, Gregory R.; Lichtler, Alex; Kream, Barbara E.; Rowe, David W.; Kalajzic, Ivo; David, Val; Quarles, Darryl L.; Villareal, Demetri; Scott, Greg; Ray, Manas; Liu, S.; Martin, James F.; Mishina, Yuji; Harris, Stephen E.

    2013-01-01

    Summary We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKOob) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKOob mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKOob osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA+ MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells. PMID:23843612

  14. The controlled release of simvastatin from TiO{sub 2} nanotubes to promote osteoblast differentiation and inhibit osteoclast resorption

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Min, E-mail: minlai@jsnu.edu.cn [School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116 (China); Jin, Ziyang; Yang, Xinyi; Wang, Huaying [School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116 (China); Xu, Kui [Biomedical Engineering Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211 (China)

    2017-02-28

    Highlights: • The TiO{sub 2} nanotube substrates filled with simvastatin were successfully coated using chitosan/gelatin multilayers. • The bio-functionalized substrates display controlled release of simvastatin in a sustained manner. • The bio-functionalized substrates have great potential for improving osteoblast differentiation. • The bio-functionalized substrates effectively inhibit osteoclast differentiation. - Abstract: The aim of this study was to fabricate a novel drug-releasing bioactive platform that has excellent potential for improving osteoblast differentiation and inhibiting osteoclast resorption. TiO{sub 2} nanotubes (TNTs) with an outer diameter of around 70 nm were prepared by an anodization method. TNTs were filled with simvastatin (SV) and then coated using chitosan/gelatin multilayers (TNT-SV-LBL). The successful fabrication of TNT-SV-LBL substrates was confirmed by field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurement, respectively. The in vitro release behavior of simvastatin from TNT-SV-LBL substrates showed a sustained release as compared to the uncoated group. Osteoblasts adhering to TNT-SV-LBL substrates attached well and displayed significantly higher (p < 0.01) cell viability compared with the other substrates. More importantly, osteoblasts grown on TNT-SV-LBL substrates displayed a statistically significant (p < 0.01 or p < 0.05) increase in protein production levels of alkaline phosphatase (ALP), osteocalcin (OC) and mRNA expression of runt related transcription factor 2 (Runx2), ALP, collagen type I (Col I), osteopontin (OPN), OC and osteoprotegerin (OPG) compared to the other groups after 4, 7 and 14 days of culture, respectively. Additionally, multinuclear osteoclastic differentiation of RAW264.7 cells grown on TNT-SV-LBL substrates was inhibited as confirmed by tartrate-resistant acid phosphatase (TRAP) analysis. These

  15. Receptors and effects of gut hormones in three osteoblastic cell lines

    Directory of Open Access Journals (Sweden)

    Wilson Peter JM

    2011-07-01

    Full Text Available Abstract Background In recent years the interest on the relationship of gut hormones to bone processes has increased and represents one of the most interesting aspects in skeletal research. The proportion of bone mass to soft tissue is a relationship that seems to be controlled by delicate and subtle regulations that imply "cross-talks" between the nutrient intake and tissues like fat. Thus, recognition of the mechanisms that integrate a gastrointestinal-fat-bone axis and its application to several aspects of human health is vital for improving treatments related to bone diseases. This work analysed the effects of gut hormones in cell cultures of three osteoblastic cell lines which represent different stages in osteoblastic development. Also, this is the first time that there is a report on the direct effects of glucagon-like peptide 2, and obestatin on osteoblast-like cells. Methods mRNA expression levels of five gut hormone receptors (glucose-dependent insulinotropic peptide [GIP], glucagon-like peptide 1 [GLP-1], glucagon-like peptide 2 [GLP-2], ghrelin [GHR] and obestatin [OB] were analysed in three osteoblastic cell lines (Saos-2, TE-85 and MG-63 showing different stages of osteoblast development using reverse transcription and real time polymerase chain reaction. The responses to the gut peptides were studied using assays for cell viability, and biochemical bone markers: alkaline phosphatase (ALP, procollagen type 1 amino-terminal propeptides (P1NP, and osteocalcin production. Results The gut hormone receptor mRNA displayed the highest levels for GIP in Saos-2 and the lowest levels in MG-63, whereas GHR and GPR39 (the putative obestatin receptor expression was higher in TE-85 and MG-63 and lower in Saos-2. GLP-1 and GLP-2 were expressed only in MG-63 and TE-85. Treatment of gut hormones to cell lines showed differential responses: higher levels in cell viability in Saos-2 after GIP, in TE-85 and MG-63 after GLP-1, GLP-2, ghrelin and

  16. Effect of Irradiation on Apoptosis, Cell Cycle Arrest and Calcified Nodule Formation of Rat Calvarial Osteoblast

    International Nuclear Information System (INIS)

    Lee, Young Mi; Choi, Hang Moon; Heo, Min Suk; Lee, Sam Sun; Choi, Soon Chul; Park, Tae Won

    2000-01-01

    The study was aimed to detect the induction of apoptosis, cell cycle arrest and calcified nodule formation after irradiation on primarily cultured osteoblasts. Using rat calvarial osteoblasts, the effects of irradiation on apoptosis, cell cycle arrest, and calcified nodule formation were studied. The single irradiation of 10, 20 Gy was done with 5.38 Gy/min dose rate using the 137 Cs cell irradiator at 4th and 14th day of culture. Apoptosis induction and cell cycle arrest were assayed by the flow cytometry at 1, 2, 3, and 4 days after irradiation. The formation of calcified nodules was observed by alizarin red staining at 1, 3, 10, 14 days after irradiation at 4th day of culture, and at 1, 4, 5 days after irradiation at 14th day of culture. Apoptosis was not induced by 10 or 20 Gy independent of irradiation and culture period. Irradiation did not induced G1 arrest in post-irradiated osteoblasts. After irradiation at 4th-day of culture, G2 arrest was induced but it was not statistically significant after irradiation at 14th-day of culture. In the case of irradiated cells at 4th day of culture, calcified nodules were not formed and at 14th-day of culture after irradiation, calcified nodule formation did not affected. Taken together, these results suggest that irradiation at the dose of 10-20 Gy would not affect apoptosis induction of osteoblasts. Cell cycle and calcified nodule formation were influenced by the level of differentiation of osteblasts.

  17. The role of osteoblast cells in the pathogenesis of unicameral bone cysts.

    Science.gov (United States)

    Aarvold, Alexander; Smith, James O; Tayton, Edward R; Edwards, Caroline J; Fowler, Darren J; Gent, Edward D; Oreffo, Richard O C

    2012-08-01

    The pathogenesis of unicameral bone cysts (UBCs) remains largely unknown. Osteoclasts have been implicated, but the role of osteoblastic cells has, to date, not been explored. This study investigated the pathophysiology of UBCs by examining the interactions between the cyst fluid and human bone marrow stromal cells (hBMSCs) and the effect of the fluid on osteogenesis. Fluid was aspirated from two UBCs and analysed for protein, electrolyte and cytokine levels. Graded concentrations of the fluid were used as culture media for hBMSCs to determine the effects of the fluid on hBMSC proliferation and osteogenic differentiation. The fibrocellular lining was analysed histologically and by electron microscopy. Alkaline phosphatase (ALP) staining of hBMSCs that were cultured in cyst fluid demonstrated increased cell proliferation and osteogenic differentiation compared to basal media controls. Biochemical analysis of these hBMSCs compared to basal controls confirmed a marked increase in DNA content (as a marker of proliferation) and ALP activity (as a marker of osteogenic differentiation) which was highly significant (p < 0.001). Osteoclasts were demonstrated in abundance in the cyst lining. The cyst fluid cytokine profile revealed levels of the pro-osteoclast cytokines IL-6, MIP-1α and MCP-1 that were 19×, 31× and 35× greater than those in reference serum. Cyst fluid promoted osteoblastic growth and differentiation. Despite appearing paradoxical that the cyst fluid promoted osteogenesis, osteoblastic cells are required for osteoclastogenesis through RANKL signalling. Three key cytokines in this pathway (IL-6, MIP-1α, MCP-1) were highly elevated in cyst fluid. These findings may hold the key to the pathogenesis of UBCs, with implications for treatment methods.

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

    Science.gov (United States)

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

    2015-11-01

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

  19. 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

  20. Myostatin inhibits osteoblastic differentiation by suppressing osteocyte-derived exosomal microRNA-218: A novel mechanism in muscle-bone communication.

    Science.gov (United States)

    Qin, Yiwen; Peng, Yuanzhen; Zhao, Wei; Pan, Jianping; Ksiezak-Reding, Hanna; Cardozo, Christopher; Wu, Yingjie; Divieti Pajevic, Paola; Bonewald, Lynda F; Bauman, William A; Qin, Weiping

    2017-06-30

    Muscle and bone are closely associated in both anatomy and function, but the mechanisms that coordinate their synergistic action remain poorly defined. Myostatin, a myokine secreted by muscles, has been shown to inhibit muscle growth, and the disruption of the myostatin gene has been reported to cause muscle hypertrophy and increase bone mass. Extracellular vesicle-exosomes that carry microRNA (miRNA), mRNA, and proteins are known to perform an important role in cell-cell communication. We hypothesized that myostatin may play a crucial role in muscle-bone interactions and may promote direct effects on osteocytes and on osteocyte-derived exosomal miRNAs, thereby indirectly influencing the function of other bone cells. We report herein that myostatin promotes expression of several bone regulators such as sclerostin (SOST), DKK1, and RANKL in cultured osteocytic (Ocy454) cells, concomitant with the suppression of miR-218 in both parent Ocy454 cells and derived exosomes. Exosomes produced by Ocy454 cells that had been pretreated with myostatin could be taken up by osteoblastic MC3T3 cells, resulting in a marked reduction of Runx2, a key regulator of osteoblastic differentiation, and in decreased osteoblastic differentiation via the down-regulation of the Wnt signaling pathway. Importantly, the inhibitory effect of myostatin-modified osteocytic exosomes on osteoblast differentiation is completely reversed by expression of exogenous miR-218, through a mechanism involving miR-218-mediated inhibition of SOST. Together, our findings indicate that myostatin directly influences osteocyte function and thereby inhibits osteoblastic differentiation, at least in part, through the suppression of osteocyte-derived exosomal miR-218, suggesting a novel mechanism in muscle-bone communication. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Lung cancer-derived Dickkopf1 is associated with bone metastasis and the mechanism involves the inhibition of osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Tianqing; Teng, Jiajun; Jiang, Liyan; Zhong, Hua; Han, Baohui, E-mail: baohuihan1@163.com

    2014-01-17

    Highlights: •DKK1 level was associated with NSCLC bone metastases. •Lung tumor cells derived DKK1 inhibited osteoblast differentiation. •Lung tumor cells derived DKK1 modulates β-catenin and RUNX2. -- Abstract: Wnt/β-catenin signaling and Dickkopf1 (DKK1) play important roles in the progression of lung cancer, which preferably metastasizes to skeleton. But the role of them in bone dissemination is poorly understood. This study aims to define the role of DKK1 in lung cancer bone metastases and investigate the underlying mechanism. Our results demonstrated that DKK1 over-expression was a frequent event in non-small-cell lung cancer (NSCLC) blood samples, and serous DKK1 level was much higher in bone metastatic NSCLC compared to non-bone metastatic NSCLC. We also found that conditioned medium from DKK1 over-expressing lung cancer cells inhibited the differentiation of osteoblast, determined by alkaline phosphatase activity and osteocalcin secretion, whereas the conditioned medium from DKK1 silencing lung cancer cells exhibited the opposite effects. Mechanistically, DKK1 reduced the level of β-catenin and RUNX2, as well as inhibiting the nuclear translocation of β-catenin. Taken together, these results suggested that lung cancer-produced DKK1 may be an important mechanistic link between NSCLC and bone metastases, and targeting DKK1 may be an effective method to treat bone metastase of NSCLC.

  2. Lung cancer-derived Dickkopf1 is associated with bone metastasis and the mechanism involves the inhibition of osteoblast differentiation

    International Nuclear Information System (INIS)

    Chu, Tianqing; Teng, Jiajun; Jiang, Liyan; Zhong, Hua; Han, Baohui

    2014-01-01

    Highlights: •DKK1 level was associated with NSCLC bone metastases. •Lung tumor cells derived DKK1 inhibited osteoblast differentiation. •Lung tumor cells derived DKK1 modulates β-catenin and RUNX2. -- Abstract: Wnt/β-catenin signaling and Dickkopf1 (DKK1) play important roles in the progression of lung cancer, which preferably metastasizes to skeleton. But the role of them in bone dissemination is poorly understood. This study aims to define the role of DKK1 in lung cancer bone metastases and investigate the underlying mechanism. Our results demonstrated that DKK1 over-expression was a frequent event in non-small-cell lung cancer (NSCLC) blood samples, and serous DKK1 level was much higher in bone metastatic NSCLC compared to non-bone metastatic NSCLC. We also found that conditioned medium from DKK1 over-expressing lung cancer cells inhibited the differentiation of osteoblast, determined by alkaline phosphatase activity and osteocalcin secretion, whereas the conditioned medium from DKK1 silencing lung cancer cells exhibited the opposite effects. Mechanistically, DKK1 reduced the level of β-catenin and RUNX2, as well as inhibiting the nuclear translocation of β-catenin. Taken together, these results suggested that lung cancer-produced DKK1 may be an important mechanistic link between NSCLC and bone metastases, and targeting DKK1 may be an effective method to treat bone metastase of NSCLC

  3. Surface modification of parylene-N films for the culture of osteoblast-like cells (MG-63)

    Energy Technology Data Exchange (ETDEWEB)

    Liaqat, Usman [Graduate Program of Nano Science and Technology, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Ko, Hyuk [Department of Materials Science and Engineering, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Suh, Hwal [Graduate Program of Nano Science and Technology, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Department of Medical Engineering, College of Medicine, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul, 120-749 (Korea, Republic of); Lee, Misu [Division of Life Sciences, College of Life Science and Bioengineering, Incheon National University, Incheon 406-772 (Korea, Republic of); Pyun, Jae-Chul, E-mail: jcpyun@yonsei.ac.kr [Department of Materials Science and Engineering, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of)

    2016-08-15

    Highlights: • Osteoblast-like cells (MG-63) was cultured on differently modified surfaces of parylene films. • Proliferation of MG-63 was observed to be far increased on UV-treated parylene-N film. • The influences of UV-treatment were found out on cell viability, proliferation rate and cell cycle. • The influence was estimated to be negligible on the protein synthesis, cell differentiation. • The UV-treated parylene-N was demonstrated to be effectively used for the culture of MG-63. - Abstract: The influence of microenvironments on the culture of osteoblast-like cells (MG-63) has been investigated using parylene films with different surfaces, such as parylene-N film, UV-modified parylene-N film, functional parylene film with amine groups (parylene-A), and UV-modified parylene-A film. In this work, parylene-N film was found to induce dramatic changes in cell adhesion and cell viability before and after UV-treatment with respect to the culture of osteoblast-like cells (MG-63). The influences of such a chemical environment on cell culture were investigated in relation to the cell proliferation (viability and proliferation rate) and the cell physiology (cell cycle, protein synthesis, and differentiation) of cells grown on parylene-N film, UV-modified parylene-N film, parylene-A film, and UV-modified parylene-A film in comparison with cells grown on a polystyrene surface.

  4. Thin films of single-walled carbon nanotubes promote human osteoblastic cells (Saos-2) proliferation in low serum concentrations

    International Nuclear Information System (INIS)

    Akasaka, Tsukasa; Yokoyama, Atsuro; Matsuoka, Makoto; Hashimoto, Takeshi; Watari, Fumio

    2010-01-01

    One strategy used for the regeneration of bone is the development of cell culture substrates and scaffolds that can control osteoblast proliferation and differentiation. In recent investigations, carbon nanotubes (CNTs) have been utilized as scaffolds for osteoblastic cell cultures; however, there are only a few reports describing the proliferation of osteoblastic cells on thin CNT films; in particular, the effects of serum concentration on cell proliferation have not been studied. In the present study, we prepared culture dishes with homogeneous thin or thick films of non-modified CNTs and examined the effect of serum concentrations on human osteoblastic cells (Saos-2) proliferation in these culture dishes. We demonstrated that the ratio of cell proliferation was strongly affected by the concentration of serum. Interestingly, single-walled carbon nanotube (SWNT) thin films were found to be the most effective substrate for the proliferation of Saos-2 cells in low concentrations of serum. Thus, thin SWNT films may be used as an effective biomaterial for the culture of Saos-2 cells in low serum concentrations.

  5. Synergistic effects of tributyltin and 2,3,7,8-tetrachlorodibenzo-p-dioxin on differentiating osteoblasts and osteoclasts

    International Nuclear Information System (INIS)

    Koskela, Antti; Viluksela, Matti; Keinänen, Meeri; Tuukkanen, Juha; Korkalainen, Merja

    2012-01-01

    The purpose of this study was to examine the effects of the persistent and accumulative environmental pollutants tributyltin (TBT) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) individually and in combination on differentiating bone cells. TBT and TCDD are chemically distinct compounds with different mechanisms of toxicity, but they typically have the same sources of exposure and both have been shown to affect bone development at low exposure levels. Bone marrow stem cells were isolated from femurs and tibias of C57BL/6 J mice, differentiated in culture into osteoblasts or osteoclasts and exposed to 0.1–10 nM TBT, 0.01–1 nM TCDD or 10 nM TBT + 1 nM TCDD. In osteoblasts, the combined exposure to TBT and TCDD significantly decreased the mRNA expression of alkaline phosphatase and osteocalcin more than TBT or TCDD alone. PCR array showed different gene expression profiles for TBT and TCDD individually, and the combination evoked several additional alterations in gene expression. Expression of aryl hydrocarbon receptor repressor (AHRR) was increased by TCDD as expected, but simultaneous exposure to TBT prevented the increase thus potentially strengthening AHR-mediated effects of TCDD. The number of osteoclasts was reduced by TCDD alone and in combination with TBT, but TBT alone had no effect. However, the total area of resorbed bone was remarkably lower after combined exposure than after TBT or TCDD alone. In conclusion, very low concentrations of TBT and TCDD have synergistic deleterious effects on bone formation and additive effects on bone resorption. -- Highlights: ► Combined exposure to TCDD and TBT evoked a unique gene expression profile. ► Osteoblast differentiation was synergistically disturbed after combined exposure. ► Bone resorbing activity was additively decreased after combined exposure.

  6. Synergistic effects of tributyltin and 2,3,7,8-tetrachlorodibenzo-p-dioxin on differentiating osteoblasts and osteoclasts

    Energy Technology Data Exchange (ETDEWEB)

    Koskela, Antti, E-mail: antti.koskela@oulu.fi [University of Oulu, Department of Anatomy and Cell Biology, Oulu (Finland); Viluksela, Matti [National Institute for Health and Welfare, Department of Environmental Health, Kuopio (Finland); Keinänen, Meeri; Tuukkanen, Juha [University of Oulu, Department of Anatomy and Cell Biology, Oulu (Finland); Korkalainen, Merja [National Institute for Health and Welfare, Department of Environmental Health, Kuopio (Finland)

    2012-09-01

    The purpose of this study was to examine the effects of the persistent and accumulative environmental pollutants tributyltin (TBT) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) individually and in combination on differentiating bone cells. TBT and TCDD are chemically distinct compounds with different mechanisms of toxicity, but they typically have the same sources of exposure and both have been shown to affect bone development at low exposure levels. Bone marrow stem cells were isolated from femurs and tibias of C57BL/6 J mice, differentiated in culture into osteoblasts or osteoclasts and exposed to 0.1–10 nM TBT, 0.01–1 nM TCDD or 10 nM TBT + 1 nM TCDD. In osteoblasts, the combined exposure to TBT and TCDD significantly decreased the mRNA expression of alkaline phosphatase and osteocalcin more than TBT or TCDD alone. PCR array showed different gene expression profiles for TBT and TCDD individually, and the combination evoked several additional alterations in gene expression. Expression of aryl hydrocarbon receptor repressor (AHRR) was increased by TCDD as expected, but simultaneous exposure to TBT prevented the increase thus potentially strengthening AHR-mediated effects of TCDD. The number of osteoclasts was reduced by TCDD alone and in combination with TBT, but TBT alone had no effect. However, the total area of resorbed bone was remarkably lower after combined exposure than after TBT or TCDD alone. In conclusion, very low concentrations of TBT and TCDD have synergistic deleterious effects on bone formation and additive effects on bone resorption. -- Highlights: ► Combined exposure to TCDD and TBT evoked a unique gene expression profile. ► Osteoblast differentiation was synergistically disturbed after combined exposure. ► Bone resorbing activity was additively decreased after combined exposure.

  7. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Claire [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France); Lafosse, Jean-Michel [CHU Toulouse, Hopital Rangueil, Service d' orthopedie et Traumatologie, Toulouse F-31000 (France); Malavaud, Bernard [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France); CHU Toulouse, Hopital Rangueil, Service d' Urologie et de Transplantation Renale, Toulouse F-31000 (France); Cuvillier, Olivier, E-mail: olivier.cuvillier@ipbs.fr [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France)

    2010-01-01

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

  8. Response of MG63 osteoblast-like cells onto polycarbonate membrane surfaces with different micropore sizes.

    Science.gov (United States)

    Lee, Sang Jin; Choi, Jin San; Park, Ki Suk; Khang, Gilson; Lee, Young Moo; Lee, Hai Bang

    2004-08-01

    Response of different types of cells on materials is important for the applications of tissue engineering and regenerative medicine. It is recognized that the behavior of the cell adhesion, proliferation, and differentiation on materials depends largely on surface characteristics such as wettability, chemistry, charge, rigidity, and roughness. In this study, we examined the behavior of MG63 osteoblast-like cells cultured on a polycarbonate (PC) membrane surfaces with different micropore sizes (0.2-8.0 microm in diameter). Cell adhesion and proliferation to the PC membrane surfaces were determined by cell counting and MTT assay. The effect of surface micropore on the MG63 cells was evaluated by cell morphology, protein content, and alkaline phosphatase (ALP) specific activity. It seems that the cell adhesion and proliferation were progressively inhibited as the PC membranes had micropores with increasing size, probably due to surface discontinuities produced by track-etched pores. Increasing micropore size of the PC membrane results in improved protein synthesis and ALP specific activity in isolated cells. There was a statistically significant difference (Pmicropore sizes. The MG63 cells also maintained their phenotype under conditions that support a round cell shape. RT-PCR analysis further confirmed the osteogenic phenotype of the MG63 cells onto the PC membranes with different micropore sizes. In results, as micropore size is getting larger, cell number is reduced and cell differentiation and matrix production is increased. This study demonstrated that the surface topography plays an important role for phenotypic expression of the MG63 osteoblast-like cells.

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

    Directory of Open Access Journals (Sweden)

    Michael S Stalvey

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

  10. Toxicity of uranium and lead on osteoblastic bone cells

    International Nuclear Information System (INIS)

    Milgram, S.; Thiebault, C.; Carriere, M.; Gouget, B.; Malaval, L.

    2007-01-01

    Bone is one of the main retention organs affected by uranium (U) and lead (Pb). Intoxications have been documented to inhibit bone formation and impair bone modeling and remodeling. However, only few studies dealt with cellular and molecular mechanisms of their toxicity. The purpose of this study was to investigate the acute cytotoxicity of U and Pb and their phenotypic effects on ROS17/2.8 osteoblastic cells. The most likely forms of the toxics in contact with cells after blood contamination were selected for cell exposure. Results show that whatever their speciation, bone cells are always more sensitive to Pb than to U. Moreover, Pb is toxic when it is left free in the exposure medium or when it is complexed with bicarbonate, cysteine or citrate, but not with albumin or phosphate. U is more cytotoxic when it is complexed with transferrin than with bicarbonate. A direct correlation between toxicity and cellular accumulation could be observed. Beside, exposure of U or Pb to bone cells induces a speciation-dependant variation of RNA expression of two markers of bone formation and mineralization: osteocalcin (OCN) and bone sialoprotein (BSP). OCN and BSP-expression could be activated in sub-toxic condition, respectively, by Pb-albumin (1.6-fold) and U-bicarbonate (2.3-fold). In the meantime, U-transferrin and Pb-citrate lead to an inhibition of the two markers. This study shows a complex mechanism of toxicity of two heavy metals with a significant phenotypic impact on osteoblastic cells highly dependant on metal speciation which controls cell accumulation. (authors)

  11. Osteoblast-Prostate Cancer Cell Interaction in Prostate Cancer Bone Metastases

    National Research Council Canada - National Science Library

    Navone, Nora

    2001-01-01

    .... This suggests that prostate cancer cells interact with cells from the osteoblastic lineage. To understand the molecular bases of prostatic bone metastases, we established two prostate cancer cell lines, MDA PCa 2a and MDA PCa 2b (1...

  12. Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells

    DEFF Research Database (Denmark)

    Post, S; Abdallah, B M; Bentzon, J F

    2008-01-01

    differentiation into one particular lineage. However, this inverse relationship between bone and fat is not consistent and under certain in vivo conditions, bone and fat can change independently suggesting separate precursor cell populations. In order to test for this hypothesis, we extensively characterized two...... of mature adipocytes visualized by Oil Red O staining. On the other hand, mMSC2 and not mMSC1 differentiated to osteoblast lineage as demonstrated by up-regulation of osteoblastic makers (CBFA1/RUNX2, Osterix, alkaline phosphatase, bone sialoprotein and osteopontin) and formation of alizarin red stained...... that are committed to either osteoblast or adipocyte lineage. These cell populations may undergo independent changes during aging and in bone diseases and thus represent important targets for therapy....

  13. Biphasic Response to Luteolin in MG-63 Osteoblast-Like Cells under High Glucose‑Induced Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Naser Abbasi

    2016-03-01

    Full Text Available Background: Clinical evidence indicates the diabetes-induced impairment of osteogenesis caused by a decrease in osteoblast activity. Flavonoids can increase the differentiation and mineralization of osteoblasts in a high-glucose state. However, some flavonoids such as luteolin may have the potential to induce cytotoxicity in osteoblast-like cells. This study was performed to investigate whether a cytoprotective concentration range of luteolin could be separated from a cytotoxic concentration range in human MG-63 osteoblast-like cells in high-glucose condition. Methods: Cells were cultured in a normal- or high-glucose medium. Cell viability was determined with the MTT assay. The formation of intracellular reactive oxygen species (ROS was measured using probe 2’,7’ -dichlorofluorescein diacetate, and osteogenic differentiation was evaluated with an alkaline phosphatase bioassay. Results: ROS generation, reduction in alkaline phosphatase activity, and cell death induced by high glucose were inhibited by lower concentrations of luteolin (EC50, 1.29±0.23 µM. Oxidative stress mediated by high glucose was also overcome by N-acetyl-L-cysteine. At high concentrations, luteolin caused osteoblast cell death in normal- and high-glucose states (IC50, 34±2.33 and 27±2.42 µM, respectively, as represented by increased ROS and decreased alkaline phosphatase activity. Conclusion: Our results indicated that the cytoprotective action of luteolin in glucotoxic condition was manifested in much lower concentrations, by a factor of approximately 26 and 20, than was its cytotoxic activity, which occurred under normal or glucotoxic condition, respectively.

  14. 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 ...

  15. 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.

  16. Dual reporter transgene driven by 2.3Col1a1 promoter is active in differentiated osteoblasts

    Science.gov (United States)

    Marijanovic, Inga; Jiang, Xi; Kronenberg, Mark S.; Stover, Mary Louise; Erceg, Ivana; Lichtler, Alexander C.; Rowe, David W.

    2003-01-01

    AIM: As quantitative and spatial analyses of promoter reporter constructs are not easily performed in intact bone, we designed a reporter gene specific to bone, which could be analyzed both visually and quantitatively by using chloramphenicol acetyltransferase (CAT) and a cyan version of green fluorescent protein (GFPcyan), driven by a 2.3-kb fragment of the rat collagen promoter (Col2.3). METHODS: The construct Col2.3CATiresGFPcyan was used for generating transgenic mice. Quantitative measurement of promoter activity was performed by CAT analysis of different tissues derived from transgenic animals; localization was performed by visualized GFP in frozen bone sections. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed for CAT and GFP activity. RESULTS: In mice, CAT activity was detected in the calvaria, long bone, teeth, and tendon, whereas histology showed that GFP expression was limited to osteoblasts and osteocytes. In cell culture, increased activity of CAT correlated with increased differentiation, and GFP activity was restricted to mineralized nodules. CONCLUSION: The concept of a dual reporter allows a simultaneous visual and quantitative analysis of transgene activity in bone.

  17. Zirconium ions up-regulate the BMP/SMAD signaling pathway and promote the proliferation and differentiation of human osteoblasts.

    Directory of Open Access Journals (Sweden)

    Yongjuan Chen

    Full Text Available Zirconium (Zr is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2 or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV oxynitrate (ZrO(NO32 at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.

  18. Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

    Science.gov (United States)

    Chen, Yongjuan; Roohani-Esfahani, Seyed-Iman; Lu, ZuFu; Zreiqat, Hala; Dunstan, Colin R.

    2015-01-01

    Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV) oxynitrate (ZrO(NO3)2) at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling. PMID:25602473

  19. 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

  20. Collagen-lactoferrin fibrillar coatings enhance osteoblast proliferation and differentiation

    Czech Academy of Sciences Publication Activity Database

    Vandrovcová, Marta; Douglas, T.E.L.; Heinemann, S.; Scharnweber, D.; Dubruel, P.; Bačáková, Lucie

    2015-01-01

    Roč. 103, č. 2 (2015), s. 525-533 ISSN 1549-3296 R&D Projects: GA ČR(CZ) GBP108/12/G108 Institutional support: RVO:67985823 Keywords : lactoferin * collagen * bone cells Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.263, year: 2015

  1. Evaluation of the attachment, proliferation, and differentiation of osteoblast on a calcium carbonate coating on titanium surface

    Energy Technology Data Exchange (ETDEWEB)

    Liu Yi; Jiang Tao; Zhou Yi; Zhang Zhen; Wang Zhejun [Key Laboratory for Oral Biomedical Engineering, Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Tong Hua; Shen Xinyu [College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 (China); Wang Yining, E-mail: wang.yn@whu.edu.cn [Key Laboratory for Oral Biomedical Engineering, Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China)

    2011-07-20

    Titanium has been reported to have some limitations in dental and orthopaedic clinical application. This study described a coating process using a simple chemical method to prepare calcium carbonate coatings on smooth titanium (STi) and sandblasted and acid-etched titanium (SATi), and evaluated the biological response of the materials in vitro. The surfaces of STi, SATi, calcium carbonate coated STi (CC-STi) and calcium carbonate coated SATi (CC-SATi) were characterized for surface roughness, contact angles, surface morphology and surface chemistry. The morphology of MG63 cells cultured on the surfaces was observed by SEM and Immuno-fluorescence staining. Cell attachment/proliferation was assessed by MTT assay, and cell differentiation was evaluated by alkaline phosphatase (ALP) activity. MG63 was found to attach favorably to calcium carbonate crystals with longer cytoplasmic extensions on CC-STi and CC-SATi, resulting in lower cell proliferation but higher ALP activity when compared to STi and SATi respectively. Moreover, CC-SATi is more favorable than CC-STi in terms of biological response. In conclusion, the calcium carbonate coatings on titanium were supposed to improve the osteointegration process and stimulate osteoblast differentiation, especially in early stage. And this method could possibly be a feasible alternative option for future clinical application. Highlights: {yields} Calcium carbonate coatings were prepared on titanium substrates. {yields} The coating process is simple and cost-effective. {yields} Calcium carbonate coating could induce differentiation toward an osteoblastic phenotype. {yields} Calcium carbonate coating could enhance the osteointegration process especially in early stage.

  2. Interaction of osteoblast-like cells with serum and fibronectin: effects on cell motility and proliferation in vitro

    International Nuclear Information System (INIS)

    Zuk, A.

    1986-01-01

    Osteoblast migration and proliferation are believed to occur during bone remodelling, in particular after osteoclastic bone resorption and prior to osteoblastic bone formation. In order to study migration and proliferation in vitro, the model of Alessandri et al. (1983) was modified. The model entailed seeding osteoblast-like cells into wells cut in agar and quantifying migration and proliferation peripheral to the well. Cell morphology also was described. The data indicated that on growth surfaces enriched with varying concentrations of fetal calf serum (FSC), the quantification of migration and proliferation was related both to percent cell attachment and to FCS-concentration. Because few osteoblast-like cells incorporated ( 3 H-TdR), it was concluded that the appearance of cells peripheral to the well was due to migration, and not to proliferation. Cell morphology and myosin distribution and organization indicated that osteoblast-like cells at the periphery of the cell culture (i.e. leading edge) may have been directionally migrating whereas cells behind the leading edge may have been engaged in non-directional migration. The migration, proliferation, and morphology of osteoblast-like cells cultured on fibronectin (FN) enriched growth surfaces also was examined. The quantification of migration and proliferation was related to the FN-concentration applied to the growth surface. Because few osteoblast-like cells incorporated 3 H-TdR and cell morphology indicated migration, it was concluded that osteoblast-like cells on FN-enriched growth surfaces are specialized, in part, for migration

  3. 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.

  4. Cord blood-derived macrophage-lineage cells rapidly stimulate osteoblastic maturation in mesenchymal stem cells in a glycoprotein-130 dependent manner.

    Directory of Open Access Journals (Sweden)

    Tania J Fernandes

    Full Text Available In bone, depletion of osteoclasts reduces bone formation in vivo, as does osteal macrophage depletion. How osteoclasts and macrophages promote the action of bone forming osteoblasts is, however, unclear. Since recruitment and differentiation of multi-potential stromal cells/mesenchymal stem cells (MSC generates new active osteoblasts, we investigated whether human osteoclasts and macrophages (generated from cord blood-derived hematopoietic progenitors induce osteoblastic maturation in adipose tissue-derived MSC. When treated with an osteogenic stimulus (ascorbate, dexamethasone and β-glycerophosphate these MSC form matrix-mineralising, alkaline phosphatase-expressing osteoblastic cells. Cord blood-derived progenitors were treated with macrophage colony stimulating factor (M-CSF to form immature proliferating macrophages, or with M-CSF plus receptor activator of NFκB ligand (RANKL to form osteoclasts; culture medium was conditioned for 3 days by these cells to study their production of osteoblastic factors. Both osteoclast- and macrophage-conditioned medium (CM greatly enhanced MSC osteoblastic differentiation in both the presence and absence of osteogenic medium, evident by increased alkaline phosphatase levels within 4 days and increased mineralisation within 14 days. These CM effects were completely ablated by antibodies blocking gp130 or oncostatin M (OSM, and OSM was detectable in both CM. Recombinant OSM very potently stimulated osteoblastic maturation of these MSC and enhanced bone morphogenetic protein-2 (BMP-2 actions on MSC. To determine the influence of macrophage activation on this OSM-dependent activity, CM was collected from macrophage populations treated with M-CSF plus IL-4 (to induce alternative activation or with GM-CSF, IFNγ and LPS to cause classical activation. CM from IL-4 treated macrophages stimulated osteoblastic maturation in MSC, while CM from classically-activated macrophages did not. Thus, macrophage-lineage cells

  5. Potential of Osteoblastic Cells Derived from Bone Marrow and Adipose Tissue Associated with a Polymer/Ceramic Composite to Repair Bone Tissue.

    Science.gov (United States)

    Freitas, Gileade P; Lopes, Helena B; Almeida, Adriana L G; Abuna, Rodrigo P F; Gimenes, Rossano; Souza, Lucas E B; Covas, Dimas T; Beloti, Marcio M; Rosa, Adalberto L

    2017-09-01

    One of the tissue engineering strategies to promote bone regeneration is the association of cells and biomaterials. In this context, the aim of this study was to evaluate if cell source, either from bone marrow or adipose tissue, affects bone repair induced by osteoblastic cells associated with a membrane of poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT). Mesenchymal stem cells (MSC) were isolated from rat bone marrow and adipose tissue and characterized by detection of several surface markers. Also, both cell populations were cultured under osteogenic conditions and it was observed that MSC from bone marrow were more osteogenic than MSC from adipose tissue. The bone repair was evaluated in rat calvarial defects implanted with PVDF-TrFE/BT membrane and locally injected with (1) osteoblastic cells differentiated from MSC from bone marrow, (2) osteoblastic cells differentiated from MSC from adipose tissue or (3) phosphate-buffered saline. Luciferase-expressing osteoblastic cells derived from bone marrow and adipose tissue were detected in bone defects after cell injection during 25 days without difference in luciferin signal between cells from both sources. Corroborating the in vitro findings, osteoblastic cells from bone marrow combined with the PVDF-TrFE/BT membrane increased the bone formation, whereas osteoblastic cells from adipose tissue did not enhance the bone repair induced by the membrane itself. Based on these findings, it is possible to conclude that, by combining a membrane with cells in this rat model, cell source matters and that bone marrow could be a more suitable source of cells for therapies to engineer bone.

  6. Integrin αv in the mechanical response of osteoblast lineage cells

    Energy Technology Data Exchange (ETDEWEB)

    Kaneko, Keiko [Department of Bone and Joint Disease, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511 (Japan); Ito, Masako [Medical Work-Life-Balance Center, Nagasaki University Hospital, Nagasaki 852-8501 (Japan); Naoe, Yoshinori [Department of Mechanism of Aging, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511 (Japan); Lacy-Hulbert, Adam [Department of Pediatrics, Massachusetts General Hospital, Boston, MA 02114 (United States); Ikeda, Kyoji, E-mail: kikeda@ncgg.go.jp [Department of Bone and Joint Disease, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511 (Japan)

    2014-05-02

    Highlights: • Deletion of integrin αv in osteoblast lineage results in an impaired SOST response to loading in vivo. • c-Src–p130Cas–JNK–YAP/TAZ is activated via integrin αv on osteoblasts in response to FSS. • Deletion of integrin αv in osteoblasts results in impaired responses to mechanical stimulation. • Integrin αv is a key component of the mechanosensing machinery in bone. - Abstract: Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation with fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src–JNK–YAP/TAZ pathway in response to mechanical stimulation.

  7. Integrin αv in the mechanical response of osteoblast lineage cells

    International Nuclear Information System (INIS)

    Kaneko, Keiko; Ito, Masako; Naoe, Yoshinori; Lacy-Hulbert, Adam; Ikeda, Kyoji

    2014-01-01

    Highlights: • Deletion of integrin αv in osteoblast lineage results in an impaired SOST response to loading in vivo. • c-Src–p130Cas–JNK–YAP/TAZ is activated via integrin αv on osteoblasts in response to FSS. • Deletion of integrin αv in osteoblasts results in impaired responses to mechanical stimulation. • Integrin αv is a key component of the mechanosensing machinery in bone. - Abstract: Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation with fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src–JNK–YAP/TAZ pathway in response to mechanical stimulation

  8. A role for the retinoblastoma protein as a regulator of mouse osteoblast cell adhesion: implications for osteogenesis and osteosarcoma formation.

    Directory of Open Access Journals (Sweden)

    Bernadette Sosa-García

    2010-11-01

    Full Text Available The retinoblastoma protein (pRb is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sophie Thiolloy

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

  11. Therapeutic Doses of Nonsteroidal Anti-Inflammatory Drugs Inhibit Osteosarcoma MG-63 Osteoblast-Like Cells Maturation, Viability, and Biomineralization Potential

    Science.gov (United States)

    De Luna-Bertos, E.; Ramos-Torrecillas, J.; García-Martínez, O.; Guildford, A.; Santin, M.; Ruiz, C.

    2013-01-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used to reduce pain and inflammation. However, their effect on bone metabolisms is not well known, and results in the literature are contradictory. The present study focusses on the effect of dexketoprofen, ketorolac, metamizole, and acetylsalicylic acid, at therapeutic doses, on different biochemical and phenotypic pathways in human osteoblast-like cells. Osteoblasts (MG-63 cell line) were incubated in culture medium with 1–10 μM of dexketoprofen, ketorolac, metamizole, and acetylsalicylic acid. Flow cytometry was used to study antigenic profile and phagocytic activity. The osteoblastic differentiation was evaluated by mineralization and synthesis of collagen fibers by microscopy and alkaline phosphatase activity (ALP) by spectrophotometric assay. Short-term treatment with therapeutic doses of NSAIDs modulated differentiation, antigenic profile, and phagocyte activity of osteoblast-like cells. The treatment reduced ALP synthesis and matrix mineralization. However, nonsignificant differences were observed on collagen syntheses after treatments. The percentage of CD54 expression was increased with all treatments. CD80, CD86, and HLA-DR showed a decreased expression, which depended on NSAID and the dose applied. The treatments also decreased phagocyte activity in this cellular population. The results of this paper provide evidences that NSAIDs inhibit the osteoblast differentiation process thus reducing their ability to produce new bone mineralized extracellular matrix. PMID:24170983

  12. Therapeutic Doses of Nonsteroidal Anti-Inflammatory Drugs Inhibit Osteosarcoma MG-63 Osteoblast-Like Cells Maturation, Viability, and Biomineralization Potential

    Directory of Open Access Journals (Sweden)

    E. De Luna-Bertos

    2013-01-01

    Full Text Available Nonsteroidal anti-inflammatory drugs (NSAIDs are frequently used to reduce pain and inflammation. However, their effect on bone metabolisms is not well known, and results in the literature are contradictory. The present study focusses on the effect of dexketoprofen, ketorolac, metamizole, and acetylsalicylic acid, at therapeutic doses, on different biochemical and phenotypic pathways in human osteoblast-like cells. Osteoblasts (MG-63 cell line were incubated in culture medium with 1–10 μM of dexketoprofen, ketorolac, metamizole, and acetylsalicylic acid. Flow cytometry was used to study antigenic profile and phagocytic activity. The osteoblastic differentiation was evaluated by mineralization and synthesis of collagen fibers by microscopy and alkaline phosphatase activity (ALP by spectrophotometric assay. Short-term treatment with therapeutic doses of NSAIDs modulated differentiation, antigenic profile, and phagocyte activity of osteoblast-like cells. The treatment reduced ALP synthesis and matrix mineralization. However, nonsignificant differences were observed on collagen syntheses after treatments. The percentage of CD54 expression was increased with all treatments. CD80, CD86, and HLA-DR showed a decreased expression, which depended on NSAID and the dose applied. The treatments also decreased phagocyte activity in this cellular population. The results of this paper provide evidences that NSAIDs inhibit the osteoblast differentiation process thus reducing their ability to produce new bone mineralized extracellular matrix.

  13. Reconstruction of rat calvarial defects with human mesenchymal stem cells and osteoblast-like cells in poly-lactic-co-glycolic acid scaffolds

    Directory of Open Access Journals (Sweden)

    C Zong

    2010-09-01

    Full Text Available Human mesenchymal stem cells (hMSCs can be used for xenogenic transplantation due to their low immunogenicity, high proliferation rate, and multi-differentiation potentials. Therefore, hMSCs are an ideal seeding source for tissue engineering. The present study evaluates the reconstruction effects of hMSCs and osteoblast-like cells differentiated from hMSCs in poly-lactic-co-glycolic acid (PLGA scaffolds on the calvarial defect of rats. Two bilateral full-thickness defects (5mm in diameter were created in the calvarium of nonimmunosuppressed Sprague-Dawley rats. The defects were filled by PLGA scaffolds with hMSCs (hMSC Construct or with osteoblast-like cells differentiated from hMSCs (Osteoblast Construct. The defects without any graft (Blank Defect or filled with PLGA scaffold without any cells (Blank Scaffold were used as controls. Evaluation was performed using macroscopic view, histology and immunohistochemical analysis respectively at 10 and 20 weeks after transplantation. In addition, fluorescent carbocyanine CM-Dil was used to track the implanted cells in vivo during transplantation. The results showed that while both hMSC Construct and Osteoblast Construct led to an effective reconstruction of critical-size calvarial defects, the bone reconstruction potential of hMSC Construct was superior to that of Osteoblast Construct in non-autogenous applications. Our findings verify the feasibility of the use of xenogenic MSCs for tissue engineering and demonstrate that undifferentiated hMSCs are more suitable for bone reconstruction in xenotransplantation models.

  14. 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.

  15. Cellular lead toxicity and metabolism in primary and clonal osteoblastic bone cells

    International Nuclear Information System (INIS)

    Long, G.J.; Rosen, J.F.; Pounds, J.G.

    1990-01-01

    A knowledge of bone lead metabolism is critical for understanding the toxicological importance of bone lead, as a toxicant both to bone cells and to soft tissues of the body, as lead is mobilized from large reservoirs in hard tissues. To further understand the processes that mediate metabolism of lead in bone, it is necessary to determine lead metabolism at the cellular level. Experiments were conducted to determine the intracellular steady-state 210 Pb kinetics in cultures of primary and clonal osteoblastic bone cells. Osteoblastic bone cells obtained by sequential collagenase digestion of mouse calvaria or rat osteosarcoma (ROS 17/2.8) cells were labeled with 210 Pb as 5 microM lead acetate for 20 hr, and kinetic parameters were determined by measuring the efflux of 210 Pb from the cells over a 210 -min period. The intracellular metabolism of 210 Pb was characterized by three kinetic pools of 210 Pb in both cell types. Although the values of these parameters differed between the primary osteoblastic cells and ROS cells, the profile of 210 Pb was remarkably similar in both cell types. Both types exhibited one large, slowly exchanging pool (S3), indicative of mitochondrial lead. These data show that primary osteoblastic bone cells and ROS cells exhibit similar steady-state lead kinetics, and intracellular lead distribution. These data also establish a working model of lead kinetics in osteoblastic bone cells and now permit an integrated view of lead kinetics in bone

  16. Calcitonin, phosphate, and the osteocyte--osteoblast bone cell unit

    Energy Technology Data Exchange (ETDEWEB)

    Talmage, R.V.; Matthews, J.L.; Martin, J.H.; Kennedy, J.W. III; Davis, W.L.; Roycroft, J.H. Jr.

    1974-01-01

    In this report we have attempted to correlate the morphological and chemical changes that occur in the long bone (tibia) of rats with the hypocalcemia that is produced following calcitonin injection or release from its gland of origin. By varying the supply of phosphate available to the rat, it has been possible to demonstrate that changes produced by CT both in bone and in plasma calcium concentrations were dependent upon an adequate supply of this ion. It is, therefore, postulated that the hypocalcemia produced by calcitonin is secondary to the formation of a calcium phosphate complex in and around osteocytes and lining cells. It is suggested that this complex, which is normally prevented from transforming to apatite crystal by the presence of an inhibitor, reduces the availability of calcium for rapid transport to the ECF. The reduction in calcium flux from bone to ECF results in a rapid and transient hypocalcemia. Regardless of the status of this postulate, we have at least demonstrated that the osteocyte-osteoblast unit of compact bone reacts rapidly to calcitonin in a process requiring phosphate in a sequence of events which can be closely correlated to the hypocalcemic action of the hormone.

  17. Synergistic effects of tributyltin and 2,3,7,8-tetrachlorodibenzo-p-dioxin on differentiating osteoblasts and osteoclasts.

    Science.gov (United States)

    Koskela, Antti; Viluksela, Matti; Keinänen, Meeri; Tuukkanen, Juha; Korkalainen, Merja

    2012-09-01

    The purpose of this study was to examine the effects of the persistent and accumulative environmental pollutants tributyltin (TBT) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) individually and in combination on differentiating bone cells. TBT and TCDD are chemically distinct compounds with different mechanisms of toxicity, but they typically have the same sources of exposure and both have been shown to affect bone development at low exposure levels. Bone marrow stem cells were isolated from femurs and tibias of C57BL/6J mice, differentiated in culture into osteoblasts or osteoclasts and exposed to 0.1-10nM TBT, 0.01-1nM TCDD or 10nM TBT+ 1nM TCDD. In osteoblasts, the combined exposure to TBT and TCDD significantly decreased the mRNA expression of alkaline phosphatase and osteocalcin more than TBT or TCDD alone. PCR array showed different gene expression profiles for TBT and TCDD individually, and the combination evoked several additional alterations in gene expression. Expression of aryl hydrocarbon receptor repressor (AHRR) was increased by TCDD as expected, but simultaneous exposure to TBT prevented the increase thus potentially strengthening AHR-mediated effects of TCDD. The number of osteoclasts was reduced by TCDD alone and in combination with TBT, but TBT alone had no effect. However, the total area of resorbed bone was remarkably lower after combined exposure than after TBT or TCDD alone. In conclusion, very low concentrations of TBT and TCDD have synergistic deleterious effects on bone formation and additive effects on bone resorption. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. 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)

  19. 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

  20. Sr-containing hydroxyapatite: morphologies of HA crystals and bioactivity on osteoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Aina, Valentina [Department of Chemistry, Università degli Studi di Torino, Via P. Giuria 7, 10125 Torino (Italy); Centre of Excellence NIS (Nanostructured Interfaces and Surface) Università degli Studi di Torino (Italy); INSTM (Italian National Consortium for Materials Science and Technology), UdR Università di Torino (Italy); Bergandi, Loredana, E-mail: loredana.bergandi@unito.it [Department of Oncology, Università degli Studi di Torino, Via Santena 5/bis, 10126 Torino (Italy); Lusvardi, Gigliola; Malavasi, Gianluca [Department of Chemical and Geological Sciences, Università di Modena and Reggio Emilia, Via Campi 183, 41125 Modena (Italy); Imrie, Flora E.; Gibson, Iain R. [School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD (United Kingdom); Cerrato, Giuseppina [Department of Chemistry, Università degli Studi di Torino, Via P. Giuria 7, 10125 Torino (Italy); Centre of Excellence NIS (Nanostructured Interfaces and Surface) Università degli Studi di Torino (Italy); INSTM (Italian National Consortium for Materials Science and Technology), UdR Università di Torino (Italy); Ghigo, Dario [Department of Oncology, Università degli Studi di Torino, Via Santena 5/bis, 10126 Torino (Italy)

    2013-04-01

    A series of Sr-substituted hydroxyapatites (HA), of general formula Ca{sub (10−x)}Sr{sub x}(PO{sub 4}){sub 6}(OH){sub 2}, where x = 2 and 4, were synthesized by solid state methods and characterized extensively. The reactivity of these materials in cell culture medium was evaluated, and the behavior towards MG-63 osteoblast cells (in terms of cytotoxicity and proliferation assays) was studied. Future in vivo studies will give further insights into the behavior of the materials. A paper by Lagergren et al. (1975), concerning Sr-substituted HA prepared by a solid state method, reports that the presence of Sr in the apatite composition strongly influences the apatite diffraction patterns. Zeglinsky et al. (2012) investigated Sr-substituted HA by ab initio methods and Rietveld analyses and reported changes in the HA unit cell volume and shape due to the Sr addition. To further clarify the role played by the addition of Sr on the physico-chemical properties of these materials we prepared Sr-substituted HA compositions by a solid state method, using different reagents, thermal treatments and a multi-technique approach. Our results indicated that the introduction of Sr at the levels considered here does influence the structure of HA. There is also evidence of a decrease in the crystallinity degree of the materials upon Sr addition. The introduction of increasing amounts of Sr into the HA composition causes a decrease in the specific surface area and an enrichment of Sr-apatite phase at the surface of the samples. Bioactivity tests show that the presence of Sr causes changes in particle size and/or morphology during soaking in MEM solution; on the contrary the morphology of pure HA does not change after 14 days of reaction. The presence of Sr, as Sr-substituted HA and SrCl{sub 2,} in cultures of human MG-63 osteoblasts did not produce any cytotoxic effect. In fact, Sr-substituted HA increased the proliferation of osteoblast cells and enhanced cell differentiation: Sr in

  1. Sr-containing hydroxyapatite: morphologies of HA crystals and bioactivity on osteoblast cells

    International Nuclear Information System (INIS)

    Aina, Valentina; Bergandi, Loredana; Lusvardi, Gigliola; Malavasi, Gianluca; Imrie, Flora E.; Gibson, Iain R.; Cerrato, Giuseppina; Ghigo, Dario

    2013-01-01

    A series of Sr-substituted hydroxyapatites (HA), of general formula Ca (10−x) Sr x (PO 4 ) 6 (OH) 2 , where x = 2 and 4, were synthesized by solid state methods and characterized extensively. The reactivity of these materials in cell culture medium was evaluated, and the behavior towards MG-63 osteoblast cells (in terms of cytotoxicity and proliferation assays) was studied. Future in vivo studies will give further insights into the behavior of the materials. A paper by Lagergren et al. (1975), concerning Sr-substituted HA prepared by a solid state method, reports that the presence of Sr in the apatite composition strongly influences the apatite diffraction patterns. Zeglinsky et al. (2012) investigated Sr-substituted HA by ab initio methods and Rietveld analyses and reported changes in the HA unit cell volume and shape due to the Sr addition. To further clarify the role played by the addition of Sr on the physico-chemical properties of these materials we prepared Sr-substituted HA compositions by a solid state method, using different reagents, thermal treatments and a multi-technique approach. Our results indicated that the introduction of Sr at the levels considered here does influence the structure of HA. There is also evidence of a decrease in the crystallinity degree of the materials upon Sr addition. The introduction of increasing amounts of Sr into the HA composition causes a decrease in the specific surface area and an enrichment of Sr-apatite phase at the surface of the samples. Bioactivity tests show that the presence of Sr causes changes in particle size and/or morphology during soaking in MEM solution; on the contrary the morphology of pure HA does not change after 14 days of reaction. The presence of Sr, as Sr-substituted HA and SrCl 2, in cultures of human MG-63 osteoblasts did not produce any cytotoxic effect. In fact, Sr-substituted HA increased the proliferation of osteoblast cells and enhanced cell differentiation: Sr in HA has a positive effect

  2. 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.

  3. Comparative proteomic analysis of plasma membrane proteins between human osteosarcoma and normal osteoblastic cell lines

    International Nuclear Information System (INIS)

    Zhang, Zhiyu; Ma, Fang; Cai, Zhengdong; Zhang, Lijun; Hua, Yingqi; Jia, Xiaofang; Li, Jian; Hu, Shuo; Peng, Xia; Yang, Pengyuan; Sun, Mengxiong

    2010-01-01

    Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and adolescents. However, the knowledge in diagnostic modalities has progressed less. To identify new biomarkers for the early diagnosis of OS as well as for potential novel therapeutic candidates, we performed a sub-cellular comparative proteomic research. An osteosarcoma cell line (MG-63) and human osteoblastic cells (hFOB1.19) were used as our comparative model. Plasma membrane (PM) was obtained by aqueous two-phase partition. Proteins were analyzed through iTRAQ-based quantitative differential LC/MS/MS. The location and function of differential proteins were analyzed through GO database. Protein-protein interaction was examined through String software. One of differentially expressed proteins was verified by immunohistochemistry. 342 non-redundant proteins were identified, 68 of which were differentially expressed with 1.5-fold difference, with 25 up-regulated and 43 down-regulated. Among those differential proteins, 69% ware plasma membrane, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and interaction with each other. One protein--CD151 located in net nodes was verified to be over-expressed in osteosarcoma tissue by immunohistochemistry. It is the first time to use plasma membrane proteomics for studying the OS membrane proteins according to our knowledge. We generated preliminary but comprehensive data about membrane protein of osteosarcoma. Among these, CD151 was further validated in patient samples, and this small molecule membrane might be a new target for OS research. The plasma membrane proteins identified in this study may provide new insight into osteosarcoma biology and potential diagnostic and therapeutic biomarkers

  4. 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.

  5. A 3D printed nano bone matrix for characterization of breast cancer cell and osteoblast interactions

    Science.gov (United States)

    Zhu, Wei; Castro, Nathan J.; Cui, Haitao; Zhou, Xuan; Boualam, Benchaa; McGrane, Robert; Glazer, Robert I.; Zhang, Lijie Grace

    2016-08-01

    Bone metastasis is one of the most prevalent complications of late-stage breast cancer, in which the native bone matrix components, including osteoblasts, are intimately involved in tumor progression. The development of a successful in vitro model would greatly facilitate understanding the underlying mechanism of breast cancer bone invasion as well as provide a tool for effective discovery of novel therapeutic strategies. In the current study, we fabricated a series of in vitro bone matrices composed of a polyethylene glycol hydrogel and nanocrystalline hydroxyapatite of varying concentrations to mimic the native bone microenvironment for the investigation of breast cancer bone metastasis. A stereolithography-based three-dimensional (3D) printer was used to fabricate the bone matrices with precisely controlled architecture. The interaction between breast cancer cells and osteoblasts was investigated in the optimized bone matrix. Using a Transwell® system to separate the two cell lines, breast cancer cells inhibited osteoblast proliferation, while osteoblasts stimulated breast cancer cell growth, whereas, both cell lines increased IL-8 secretion. Breast cancer cells co-cultured with osteoblasts within the 3D bone matrix formed multi-cellular spheroids in comparison to two-dimensional monolayers. These findings validate the use of our 3D printed bone matrices as an in vitro metastasis model, and highlights their potential for investigating breast cancer bone metastasis.

  6. Osteoblastic cell response to spark plasma-sintered zirconia/titanium cermets.

    Science.gov (United States)

    Fernandez-Garcia, Elisa; Guillem-Marti, Jordi; Gutierrez-Gonzalez, Carlos F; Fernandez, Adolfo; Ginebra, Maria-Pau; Lopez-Esteban, Sonia

    2015-01-01

    Ceramic/metal composites, cermets, arise from the idea to combine the dissimilar properties in the pure materials. This work aims to study the biocompatibility of new micro-nanostructured 3 Y-TZP/Ti materials with 25, 50 and 75 vol.% Ti, which have been successfully obtained by spark slasma sintering technology, as well as to correlate their surface properties (roughness, wettability and chemical composition) with the osteoblastic cell response. All samples had isotropic and slightly waved microstructure, with sub-micrometric average roughness. Composites with 75 vol.% Ti had the highest surface hydrophilicity. Surface chemical composition of the cermets correlated well with the relative amounts used for their fabrication. A cell viability rate over 80% dismissed any cytotoxicity risk due to manufacturing. Cell adhesion and early differentiation were significantly enhanced on materials containing the nanostructured 3 Y-TZP phase. Proliferation and differentiation of SaOS-2 were significantly improved in their late-stage on the composite with 75 vol.% Ti that, from the osseointegration standpoint, is presented as an excellent biomaterial for bone replacement. Thus, spark plasma sintering is consolidated as a suitable technology for manufacturing nanostructured biomaterials with enhanced bioactivity. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  7. Pharmacological activation of aldehyde dehydrogenase 2 promotes osteoblast differentiation via bone morphogenetic protein-2 and induces bone anabolic effect

    Energy Technology Data Exchange (ETDEWEB)

    Mittal, Monika; Pal, Subhashis; China, Shyamsundar Pal; Porwal, Konica [Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, Lucknow 226031 (India); Dev, Kapil [Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Shrivastava, Richa [Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Raju, Kanumuri Siva Rama; Rashid, Mamunur [Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Trivedi, Arun Kumar; Sanyal, Sabyasachi [Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Wahajuddin, Muhammad [Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Bhaduria, Smrati [Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Maurya, Rakesh [Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Chattopadhyay, Naibedya, E-mail: n_chattopadhyay@cdri.res.in [Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, Lucknow 226031 (India)

    2017-02-01

    Aldehyde dehydrogenases (ALDHs) are a family of enzymes involved in detoxifying aldehydes. Previously, we reported that an ALDH inhibitor, disulfiram caused bone loss in rats and among ALDHs, osteoblast expressed only ALDH2. Loss-of-function mutation in ALDH2 gene is reported to cause bone loss in humans which suggested its importance in skeletal homeostasis. We thus studied whether activating ALDH2 by N-(1, 3-benzodioxol-5-ylmethyl)-2, 6-dichlorobenzamide (alda-1) had osteogenic effect. We found that alda-1 increased and acetaldehyde decreased the differentiation of rat primary osteoblasts and expressions of ALDH2 and bone morphogenetic protein-2 (BMP-2). Silencing ALDH2 in osteoblasts abolished the alda-1 effects. Further, alda-1 attenuated the acetaldehyde-induced lipid-peroxidation and oxidative stress. BMP-2 is essential for bone regeneration and alda-1 increased its expression in osteoblasts. We then showed that alda-1 (40 mg/kg dose) augmented bone regeneration at the fracture site with concomitant increase in BMP-2 protein compared with control. The osteogenic dose (40 mg/kg) of alda-1 attained a bone marrow concentration that was stimulatory for osteoblast differentiation, suggesting that the tissue concentration of alda-1 matched its pharmacologic effect. In addition, alda-1 promoted modeling-directed bone growth and peak bone mass achievement, and increased bone mass in adult rats which reiterated its osteogenic effect. In osteopenic ovariectomized (OVX) rats, alda-1 reversed trabecular osteopenia with attendant increase in serum osteogenic marker (procollagen type I N-terminal peptide) and decrease in oxidative stress. Alda-1 has no effect on liver and kidney function. We conclude that activating ALDH2 by alda-1 had an osteoanabolic effect involving increased osteoblastic BMP-2 production and decreased OVX-induced oxidative stress. - Highlights: • Alda-1 induced osteoblast differentiation that involved upregulation of ALDH2 and BMP-2 • Alda-1

  8. 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.

  9. Sodium nitroprusside induces autophagic cell death in glutathione-depleted osteoblasts.

    Science.gov (United States)

    Son, Min Jeong; Lee, Seong-Beom; Byun, Yu Jeong; Lee, Hwa Ok; Kim, Ho-Shik; Kwon, Oh-Joo; Jeong, Seong-Whan

    2010-01-01

    Previous studies reported that high levels of nitric oxide (NO) induce apoptotic cell death in osteoblasts. We examined molecular mechanisms of cytotoxic injury induced by sodium nitroprusside (SNP), a NO donor, in both glutathione (GSH)-depleted and control U2-OS osteoblasts. Cell viability was reduced by much lower effective concentrations of SNP in GSH-depleted cells compared to normal cells. The data suggest that the level of intracellular GSH is critical in SNP-induced cell death processes of osteoblasts. The level of oxidative stress due to SNP treatments doubled in GSH-depleted cells when measured with fluorochrome H2DCFDA. Pretreatment with the NO scavenger PTIO preserved the viability of cells treated with SNP. Viability of cells treated with SNP was recovered by pretreatment with Wortmannin, an autophagy inhibitor, but not by pretreatment with zVAD-fmk, a pan-specific caspase inhibitor. Large increases of LC3-II were shown by immunoblot analysis of the SNP-treated cells, and the increase was blocked by pretreatment with PTIO or Wortmannin; this implies that under GSH-depleted conditions SNP induces different molecular signaling that lead to autophagic cell death. The ultrastructural morphology of SNP-treated cells in transmission electron microscopy showed numerous autophagic vacuoles. These data suggest NO produces oxidative stress and cellular damage that culminate in autophagic cell death of GSH-depleted osteoblasts. Copyright 2010 Wiley Periodicals, Inc.

  10. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Henriksen, Z; Sørensen, O H

    2004-01-01

    . Osteoblast phenotypes were induced by either dexamethasone (Dex) or bone morphogenetic protein-2 (BMP-2). Bone marrow was obtained from biopsies at the posterior iliac spine. Cells were isolated by gradient centrifugation and grown to confluence. Cells were treated with 1 nM 1,25-dihydroxyvitamin D (vitamin...... activity was increased by Dex, but not by BMP-2 treatment. P1NP production was decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with vitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels......, but in combination with vitamin D, BMP-2 increased the osteocalcin production, while Dex treatment completely suppressed osteocalcin production. Further, PTH-induced cAMP production was greatly enhanced by Dex treatment, whereas BMP-2 did not affect cAMP production. Finally, in vitro mineralization was greatly...

  11. Human osteoblast cells: isolation, characterization, and growth on polymers for musculoskeletal tissue engineering.

    Science.gov (United States)

    El-Amin, Saadiq F; Botchwey, Edward; Tuli, Richard; Kofron, Michelle D; Mesfin, Addisu; Sethuraman, Swaminathan; Tuan, Rocky S; Laurencin, Cato T

    2006-03-01

    We performed a detailed examination of the isolation, characterization, and growth of human osteoblast cells derived from trabecular bone. We further examined the morphology, phenotypic gene expression, mineralization,and growth of these human osteoblasts on polyester polymers used for musculoskeletal tissue engineering. Polylactic-co-glycolic acid [PLAGA (85:15, 50:50, 75:25)], and poly-lactic acid (L-PLA, D,L-PLA) were examined. The osteoblastic expression of key phenotypic markers osteocalcin, alkaline phosphatase, collagen, and bone sialoprotein at 4 and 8 weeks was examined. Reverse transcription-polymerase chain reaction studies revealed that trabecular-derived osteoblasts were positive for all markers evaluated with higher levels expressed over long-term culture. These cells also revealed mineralization and maturation as evidenced by energy dispersive X-ray analysis and scanning electron microscopy. Growth studies on PLAGA at 50:50,75:25, and 85:15 ratios and PLA in the L and DL isoforms revealed that human osteoblasts actively grew, with significantly higher cell numbers attached to scaffolds composed of PLAGA 50:50 in the short term and PLAGA 85:15 in the long term compared with PLA (p < 0.05). We believe human cell adhesion among these polymeric materials may be dependent on differences in cellular integrin expression and extracellular matrix protein elaboration. (c) 2005 Wiley Periodicals, Inc.

  12. Hydroxyapatite coatings deposited by liquid precursor plasma spraying: controlled dense and porous microstructures and osteoblastic cell responses

    International Nuclear Information System (INIS)

    Huang Yi; Song Lei; Liu Xiaoguang; Xiao Yanfeng; Wu Yao; Chen Jiyong; Wu Fang; Gu Zhongwei

    2010-01-01

    Hydroxyapatite coatings were deposited on Ti-6Al-4V substrates by a novel plasma spraying process, the liquid precursor plasma spraying (LPPS) process. X-ray diffraction results showed that the coatings obtained by the LPPS process were mainly composed of hydroxyapatite. The LPPS process also showed excellent control on the coating microstructure, and both nearly fully dense and highly porous hydroxyapatite coatings were obtained by simply adjusting the solid content of the hydroxyapatite liquid precursor. Scanning electron microscope observations indicated that the porous hydroxyapatite coatings had pore size in the range of 10-200 μm and an average porosity of 48.26 ± 0.10%. The osteoblastic cell responses to the dense and porous hydroxyapatite coatings were evaluated with human osteoblastic cell MG-63, in respect of the cell morphology, proliferation and differentiation, with the hydroxyapatite coatings deposited by the atmospheric plasma spraying (APS) process as control. The cell experiment results indicated that the heat-treated LPPS coatings with a porous structure showed the best cell proliferation and differentiation among all the hydroxyapatite coatings. Our results suggest that the LPPS process is a promising plasma spraying technique for fabricating hydroxyapatite coatings with a controllable microstructure, which has great potential in bone repair and replacement applications.

  13. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting...... in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal...... of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx...

  14. Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells

    International Nuclear Information System (INIS)

    Komm, B.S.; Terpening, C.M.; Benz, D.J.; Graeme, K.A.; Gallegos, A.; Korc, M.; Greene, G.L.; O'Malley, B.W.; Haussler, M.R.

    1988-01-01

    High specific activity estradiol labeled with iodine-125 was used to detect approximately 200 saturable, high-affinity (dissociation constant approximately equal to 1.0 nM) nuclear binding sites in rat (ROS 17/2.8) and human (HOS TE85) clonal osteoblast-like osteosarcoma cells. Of the steroids tested, only testosterone exhibited significant cross-reactivity with estrogen binding. RNA blot analysis with a complementary DNA probe to the human estrogen receptor revealed putative receptor transcripts of 6 to 6.2 kilobases in both rat and human osteosarcoma cells. Type I procollagen and transforming growth factor-beta messenger RNA levels were enhanced in cultured human osteoblast-like cells treated with 1 nM estradiol. Thus, estrogen can act directly on osteoblasts by a receptor-mediated mechanism and thereby modulate the extracellular matrix and other proteins involved in the maintenance of skeletal mineralization and remodeling

  15. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

    DEFF Research Database (Denmark)

    Eskildsen, Tilde; Taipaleenmäki, Hanna; Stenvang, Jan

    2011-01-01

    Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators......-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore...

  16. Real-time observations of mechanical stimulus-induced enhancements of mechanical properties in osteoblast cells

    International Nuclear Information System (INIS)

    Zhang Xu; Liu Xiaoli; Sun Jialun; He Shuojie; Lee, Imshik; Pak, Hyuk Kyu

    2008-01-01

    Osteoblast, playing a key role in the pathophysiology of osteoporosis, is one of the mechanical stress sensitive cells. The effects of mechanical load-induced changes of mechanical properties in osteoblast cells were studied at real-time. Osteoblasts obtained from young Wister rats were exposed to mechanical loads in different frequencies and resting intervals generated by atomic force microscopy (AFM) probe tip and simultaneously measured the changes of the mechanical properties by AFM. The enhancement of the mechanical properties was observed and quantified by the increment of the apparent Young's modulus, E * . The observed mechanical property depended on the frequency of applied tapping loads. For the resting interval is 50 s, the mechanical load-induced enhancement of E * -values disappears. It seems that the enhanced mechanical property was recover able under no additional mechanical stimulus

  17. Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells.

    Science.gov (United States)

    Farley, J R; Stilt-Coffing, B

    2001-01-01

    Although quantitative measurement of skeletal alkaline phosphatase (sALP) activity in serum can provide an index of the rate of bone formation, the metabolic process that determines the release of sALP - from the surface of osteoblasts, into circulation-is unknown. The current studies were intended to examine the hypothesis that the release of sALP from human osteoblasts is a consequence of apoptotic cell death. We measured the release of sALP activity from human osteosarcoma (SaOS-2) cells and normal human bone cells, under basal conditions and in response to agents that increased apoptosis (TNF-a, okadiac acid) and agents that inhibit apoptosis (IGF-I, calpain, and caspase inhibitors). Apoptosis was determined by the presence of nucleosomes (histone-associated DNA) in the cytoplasm of the cells by using a commercial kit. The results of these studies showed that TNF-a and okadiac acid caused dose- and time-dependent increases in apoptosis in the SaOS-2 cells (r = 0.78 for doses of TNF-a and r = 0.93 for doses of okadiac acid, P sALP activity (e.g., r = 0.89 for TNF-a and r = 0.75 for okadiac acid, P sALP activity (P sALP activity (P sALP release. The associations between apoptosis and sALP release were not unique to osteosarcoma (i.e., SaOS-2) cells, but also seen with osteoblast-line cells derived from normal human bone. Together, these data demonstrate that the release of sALP activity from human osteoblast-line cells in vitro is associated with, and may be a consequence of, apoptotic cell death. These findings are consistent with the general hypothesis that the appearance of sALP activity in serum may reflect the turnover of osteoblast-line cells.

  18. Short communication: selective cytotoxicity of curcumin on osteosarcoma cells compared to healthy osteoblasts

    Directory of Open Access Journals (Sweden)

    Chang R

    2014-01-01

    Full Text Available Run Chang,1 Linlin Sun,1 Thomas J Webster1,21Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 2Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi ArabiaAbstract: Curcumin is a natural phenolic compound extracted from the plant Curcuma longa L. In previous studies, curcumin has been shown to have anticancer, antioxidant, and anti-inflammatory effects. In this study, the cytotoxicity of different concentrations (5, 10, 25, 50, 75, and 100 µM of curcumin dissolved in dimethyl sulfoxide was compared between MG-63 osteosarcoma and healthy human osteoblast cells. Consequently, the viability of osteosarcoma cells was less than 50% at a concentration of 10 µM compared to the control sample without curcumin, but healthy osteoblast cells had at least 80% viability throughout all the concentrations tested. The results demonstrated that MG-63 osteosarcoma cells were much more sensitive in terms of cytotoxicity to curcumin, while the healthy human osteoblasts exhibited a higher healthy viability after 24 hours of curcumin treatment. Therefore, this study showed that at the right concentrations (5 µM to 25 µM, curcumin, along with a proper nanoparticle drug delivery carrier, may selectively kill bone cancer cells over healthy bone cells.Keywords: curcumin, osteosarcoma, human osteoblast, viability, bone cancer

  19. Delta-like 1/fetal antigen 1(DLK1/FA1) inhibits BMP2 induced osteoblast differentiation through modulation of NFκB signaling pathway

    DEFF Research Database (Denmark)

    Qiu, Weimin; Abdallah, Basem; Kassem, Moustapha

    DLK1/FA1 (delta-like 1/fetal antigen-1) is a negative regulator of bone mass that acts to inhibit osteoblast differentiation and stimulate osteoclast differentiation. However, the molecular mechanisms underlying these effects are not known. Thus, we studied the effect of DLK1/FA1 on different...... osteogenic factors-induced osteoblast differentiation. We identified DLK1/FA1 as an inhibitor of BMP2-induced osteogenesis in mouse myoblast C2C12 cells. Stable overexpression of DLK1/FA1 in C2C12 cells or the addition of its soluble form protein FA1 significantly inhibited BMP2-induced osteogenesis...... as assessed by reduced Alp activity and osteogenic gene expression including Alp, Col1a1, Runx2 and Bglap. In addition, DLK1/FA1 inhibited BMP signaling as demonstrated by reduced gene expression of BMP-responsive genes: Junb and Id1, reduced BMP2 induced luciferase activity in C2C12 BMP luciferase reporter...

  20. Cell fusion in osteoclasts plays a critical role in controlling bone mass and osteoblastic activity

    International Nuclear Information System (INIS)

    Iwasaki, Ryotaro; Ninomiya, Ken; Miyamoto, Kana; Suzuki, Toru; Sato, Yuiko

    2008-01-01

    The balance between osteoclast and osteoblast activity is central for maintaining the integrity of bone homeostasis. Here we show that mice lacking dendritic cell specific transmembrane protein (DC-STAMP), an essential molecule for osteoclast cell-cell fusion, exhibited impaired bone resorption and upregulation of bone formation by osteoblasts, which do not express DC-STAMP, which led to increased bone mass. On the contrary, DC-STAMP over-expressing transgenic (DC-STAMP-Tg) mice under the control of an actin promoter showed significantly accelerated cell-cell fusion of osteoclasts and bone resorption, with decreased osteoblastic activity and bone mass. Bone resorption and formation are known to be regulated in a coupled manner, whereas DC-STAMP regulates bone homeostasis in an un-coupled manner. Thus our results indicate that inhibition of a single molecule provides both decreased osteoclast activity and increased bone formation by osteoblasts, thereby increasing bone mass in an un-coupled and a tissue specific manner.

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

    Science.gov (United States)

    Long, Fanxin

    2011-12-22

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

  2. Comparative of fibroblast and osteoblast cells adhesion on surface modified nanofibrous substrates based on polycaprolactone.

    Science.gov (United States)

    Sharifi, Fereshteh; Irani, Shiva; Zandi, Mojgan; Soleimani, Masoud; Atyabi, Seyed Mohammad

    2016-12-01

    One of the determinant factors for successful bioengineering is to achieve appropriate nano-topography and three-dimensional substrate. In this research, polycaprolactone (PCL) nano-fibrous mat with different roughness modified with O 2 plasma was fabricated via electrospinning. The purpose of this study was to evaluate the effect of plasma modification along with surface nano-topography of mats on the quality of human fibroblast (HDFs) and osteoblast cells (OSTs)-substrate interaction. Surface properties were studied using scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle, Fourier-transformation infrared spectroscopy. We evaluated mechanical properties of fabricated mats by tensile test. The viability and proliferation of HDFs and OSTs on the substrates were followed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT). Mineralization of the substrate was determined by alizarin red staining method and calcium content of OSTs was determined by calcium content kit. Cells morphology was studied by SEM analysis. The results revealed that the plasma-treated electrospun nano-fibrous substrate with higher roughness was an excellent designed substrate. A bioactive topography for stimulating proliferation of HDFs and OSTs is to accelerate the latter's differentiation time. Therefore, the PCL substrate with high density and major nano-topography were considered as a bio-functional and elegant bio-substrate for tissue regeneration applications.

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

    Directory of Open Access Journals (Sweden)

    Matthew M. Cook

    2013-09-01

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

  4. Comparative evaluation of different calcium phosphate-based bone graft granules - an in vitro study with osteoblast-like cells.

    Science.gov (United States)

    Bernhardt, Anne; Lode, Anja; Peters, Fabian; Gelinsky, Michael

    2013-04-01

    Granule-shaped calcium phosphate-based bone graft materials are often required for bone regeneration especially in implant dentistry. Two newly developed bone graft materials are Ceracell(®) , an open-celled highly porous bioceramic from β-tricalcium phosphate (β-TCP) under addition of bioglass and Osseolive(®) , an open porous glass ceramic with the general formula Ca2 KNa(PO4 )2 . The goal of this study was to characterize different modifications of the two bone graft materials in vitro in comparison to already established ceramic bone grafts Cerasorb M(®) , NanoBone(®) and BONIT Matrix(®) . Adhesion and proliferation of SaOS-2 osteoblast-like cells were evaluated quantitatively by determining DNA content and lactate dehydrogenase (LDH) activity and qualitatively by scanning electron microscopy (SEM). In addition, MTT cell-vitality staining was applied to confirm the attachment of viable cells to the different materials. Osteogenic differentiation was evaluated by measurement of alkaline phosphatase (ALP) activity as well as gene expression analysis of osteogenic markers using reverse transcriptase PCR. DNA content and LDH activity revealed good cell attachment and proliferation for Ceracell and Cerasorb M. When pre-incubated with cell-culture medium, also Osseolive showed good cell attachment and proliferation. Attachment and proliferation of osteoblast-like cells on NanoBone and BONIT Matrix was very low, even after pre-incubation with cell-culture medium. Specific ALP activity on Ceracell(®) , Osseolive (®) and Cerasorb M(®) increased with time and expression of bone-related genes ALP, osteonectin, osteopontin and bone sialoprotein II was demonstrated. Ceracell as well as Osseolive granules support proliferation and osteogenic differentiation in vitro and may be promising candidates for in vivo applications. © 2011 John Wiley & Sons A/S.

  5. Additively manufactured 3D porous Ti-6Al-4V constructs mimic trabecular bone structure and regulate osteoblast proliferation, differentiation and local factor production in a porosity and surface roughness dependent manner

    International Nuclear Information System (INIS)

    Cheng, Alice; Boyan, Barbara D; Humayun, Aiza; Cohen, David J; Schwartz, Zvi

    2014-01-01

    Additive manufacturing by laser sintering is able to produce high resolution metal constructs for orthopedic and dental implants. In this study, we used a human trabecular bone template to design and manufacture Ti-6Al-4V constructs with varying porosity via laser sintering. Characterization of constructs revealed interconnected porosities ranging from 15–70% with compressive moduli of 2579–3693 MPa. These constructs with macro porosity were further surface-treated to create a desirable multi-scale micro-/nano-roughness, which has been shown to enhance the osseointegration process. Osteoblasts (MG63 cells) exhibited high viability when grown on the constructs. Proliferation (DNA) and alkaline phosphatase specific activity, an early differentiation marker, decreased as porosity increased, while osteocalcin, a late differentiation marker, as well as osteoprotegerin, vascular endothelial growth factor and bone morphogenetic proteins 2 and 4 increased with increasing porosity. Three-dimensional (3D) constructs with the highest porosity and surface modification supported the greatest osteoblast differentiation and local factor production. These results indicate that additively manufactured 3D porous constructs mimicking human trabecular bone and produced with additional surface treatment can be customized for increased osteoblast response. Increased factors for osteoblast maturation and differentiation on high porosity constructs suggest the enhanced performance of these surfaces for increasing osseointegration in vivo. (paper)

  6. Additively manufactured 3D porous Ti-6Al-4V constructs mimic trabecular bone structure and regulate osteoblast proliferation, differentiation and local factor production in a porosity and surface roughness dependent manner.

    Science.gov (United States)

    Cheng, Alice; Humayun, Aiza; Cohen, David J; Boyan, Barbara D; Schwartz, Zvi

    2014-10-07

    Additive manufacturing by laser sintering is able to produce high resolution metal constructs for orthopedic and dental implants. In this study, we used a human trabecular bone template to design and manufacture Ti-6Al-4V constructs with varying porosity via laser sintering. Characterization of constructs revealed interconnected porosities ranging from 15-70% with compressive moduli of 2579-3693 MPa. These constructs with macro porosity were further surface-treated to create a desirable multi-scale micro-/nano-roughness, which has been shown to enhance the osseointegration process. Osteoblasts (MG63 cells) exhibited high viability when grown on the constructs. Proliferation (DNA) and alkaline phosphatase specific activity, an early differentiation marker, decreased as porosity increased, while osteocalcin, a late differentiation marker, as well as osteoprotegerin, vascular endothelial growth factor and bone morphogenetic proteins 2 and 4 increased with increasing porosity. Three-dimensional (3D) constructs with the highest porosity and surface modification supported the greatest osteoblast differentiation and local factor production. These results indicate that additively manufactured 3D porous constructs mimicking human trabecular bone and produced with additional surface treatment can be customized for increased osteoblast response. Increased factors for osteoblast maturation and differentiation on high porosity constructs suggest the enhanced performance of these surfaces for increasing osseointegration in vivo.

  7. 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.

  8. 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.

  9. Osteoblast recruitment routes in human cancellous bone remodeling

    DEFF Research Database (Denmark)

    Kristensen, Helene Bjørg; Andersen, Thomas Levin; Marcussen, Niels

    2014-01-01

    It is commonly proposed that bone forming osteoblasts recruited during bone remodeling originate from bone marrow perivascular cells, bone remodeling compartment canopy cells, or bone lining cells. However, an assessment of osteoblast recruitment during adult human cancellous bone remodeling...... is lacking. We addressed this question by quantifying cell densities, cell proliferation, osteoblast differentiation markers, and capillaries in human iliac crest biopsy specimens. We found that recruitment occurs on both reversal and bone-forming surfaces, as shown by the cell density and osterix levels...

  10. Nanoparticles prepared from the water extract of Gusuibu (Drynaria fortunei J. Sm. protects osteoblasts against insults and promotes cell maturation

    Directory of Open Access Journals (Sweden)

    Hsu C-K

    2011-07-01

    Full Text Available Chung-King Hsu1,2, Mei-Hsiu Liao3, Yu-Tyng Tai4, Shing-Hwa Liu5, Keng-Liang Ou6, Hsu-Wei Fang7, I-Jung Lee8, Ruei-Ming Chen2,31Institute of Materials Science and Engineering, National Taipei University of Technology, 2Cell Physiology and Molecular Image Research Center, Taipei Medical University-Wan Fang Medical Center, 3Graduate Institute of Medical Sciences, Taipei Medical University, 4Department of Anesthesiology, Taipei Medical University-Wan Fang Medical Center, 5Institute of Toxicology, College of Medicine, National Taiwan University, 6Graduate Institute of Biomedical Materials and Engineering, Taipei Medical University, 7Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, 8Division of Information and Herbarium, National Research Institute of Chinese Medicine, Taipei, TaiwanAbstract: Our previous study showed that Gusuibu (Drynaria fortunei J. Sm. can stimulate osteoblast maturation. This study was further designed to evaluate the effects of nanoparticles prepared from the water extract of Gusuibu (WEG on osteoblast survival and maturation. Primary osteoblasts were exposed to 1, 10, 100, and 1000 µg/mL nanoparticles of WEG (nWEG for 24, 48, and 72 hours did not affect morphologies, viability, or apoptosis of osteoblasts. In comparison, treatment of osteoblasts with 1000 µg/mL WEG for 72 hours decreased cell viability and induced DNA fragmentation and cell apoptosis. nWEG had better antioxidant bioactivity in protecting osteoblasts from oxidative and nitrosative stress-induced apoptosis than WEG. In addition, nWEG stimulated greater osteoblast maturation than did WEG. Therefore, this study shows that WEG nanoparticles are safer to primary osteoblasts than are normal-sized products, and may promote better bone healing by protecting osteoblasts from apoptotic insults, and by promoting osteogenic maturation.Keywords: Gusuibu, nanoparticles, cell protection, osteoblast maturation

  11. Enhanced adhesion of osteoblastic cells on polystyrene films by independent control of surface topography and wettability.

    Science.gov (United States)

    Yang, Seung Yun; Kim, Eung-Sam; Jeon, Gumhye; Choi, Kwan Yong; Kim, Jin Kon

    2013-04-01

    We independently controlled surface topography and wettability of polystyrene (PS) films by CF4 and oxygen plasma treatments, respectively, to evaluate the adhesion and proliferation of human fetal osteoblastic (hFOB) cells on the films. Among the CF4 plasma-treated PS films with the average surface roughness ranging from 0.9 to 70 nm, the highest adhesion of hFOB cells was observed on a PS film with roughness of ~11 nm. When this film was additionally treated by oxygen plasma to provide a hydrophilic surface with a contact angle less than 10°, the proliferation of bone-forming cell was further enhanced. Thus, the plasma-based independent modification of PS film into an optimum nanotexture for human osteoblast cells could be appplied to materials used in bone tissue engineering. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Effect of the gamma radiation and common antioxidants on some aspects of osteoblast differentiation during the formation of bone tissue in an in-vivo model

    International Nuclear Information System (INIS)

    Quinones O, M. G.

    2015-01-01

    Gamma radiation is the emission of energy through short electromagnetic waves to a higher level of frequency with respect to ultraviolet light. This type of energy in the medical application is used as a tool to kill cancer cells in humans, however, adverse damages to its exposure can produce secondary effects in the short and long term depending on the damage in cells and tissues nearby to the irradiation zone, the human body will present various injuries and conditions. In bone tissue, secondary effects that have been observed, is an alteration of the architecture and integrity of bone extracellular matrix of cortical and trabecular tissue, which causes loss of bone density. However, the reason that the bone tissue is affected is not clear, but is believed to be related to the formation of free radicals, which generate oxidative damage in biomolecules of the cells, damaging the tissue structure, organs and systems of the human body. The studies to identify the main reasons that will affect bone tissue as a result of radiotherapy have been carried out by models In-vitro and some In-vivo. In most studies in-vitro with cells with osteoblast phenotype, the results suggest alterations in proliferation and differentiation of these cells. However, the etiology and the role of these changes in disorders and bone injuries as adverse secondary effects of the radiotherapy are very poorly understood to date. In the present study an In-vivo model was used, that are ectopic bone plates which are developed by endochondral ossification, after having implanted demineralized bone particles at 16 days of development, at which time they are constituted by bone tissue. Ectopic bone plates were used with the aim of knowing as gamma radiation indirectly modifies to cellular level the osteoblast differentiation, cells that are involved in the formation and mineralization of bone extracellular matrix. One of the well known effects of gamma radiation is the generation of free radicals

  13. Biocompatibility evaluation of HDPE-UHMWPE reinforced β-TCP nanocomposites using highly purified human osteoblast cells.

    Science.gov (United States)

    Shokrgozar, M A; Farokhi, M; Rajaei, F; Bagheri, M H A; Azari, Sh; Ghasemi, I; Mottaghitalab, F; Azadmanesh, K; Radfar, J

    2010-12-15

    Biocompatibility of β-TCP/HDPE-UHMWPE nanocomposite as a new bone substitute material was evaluated by using highly purified human osteoblast cells. Human osteoblast cells were isolated from bone tissue and characterized by immunofluorescence Staining before and after purification using magnetic bead system. Moreover, proliferation, alkaline phosphatase production, cell attachment, calcium deposition, gene expression, and morphology of osteoblast cells on β-TCP/HDPE-UHMWPE nanocomposites were evaluated. The results have shown that the human osteoblast cells were successfully purified and were suitable for subsequent cell culturing process. The high proliferation rate of osteoblast cells on β-TCP/HDPE-UHMWPE nanocomposite confirmed the great biocompatibility of the scaffold. Expression of bone-specific genes was taken place after the cells were incubated in composite extract solutions. Furthermore, osteoblast cells were able to mineralize the matrix next to composite samples. Scanning electron microscopy demonstrated that cells had normal morphology on the scaffold. Thus, these results indicated that the nanosized β-TCP/HDPE-UHMWPE blend composites could be potential scaffold, which is used in bone tissue engineering. Copyright © 2010 Wiley Periodicals, Inc.

  14. 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)

  15. Targeting of Mesenchymal Stromal Cells by Cre-Recombinase Transgenes Commonly Used to Target Osteoblast Lineage Cells.

    Science.gov (United States)

    Zhang, Jingzhu; Link, Daniel C

    2016-11-01

    The targeting specificity of tissue-specific Cre-recombinase transgenes is a key to interpreting phenotypes associated with their use. The Ocn-Cre and Dmp1-Cre transgenes are widely used to target osteoblasts and osteocytes, respectively. Here, we used high-resolution microscopy of bone sections and flow cytometry to carefully define the targeting specificity of these transgenes. These transgenes were crossed with Cxcl12 gfp mice to identify Cxcl12-abundant reticular (CAR) cells, which are a perivascular mesenchymal stromal population implicated in hematopoietic stem/progenitor cell maintenance. We show that in addition to osteoblasts, Ocn-Cre targets a majority of CAR cells and arteriolar pericytes. Surprisingly, Dmp1-Cre also targets a subset of CAR cells, in which expression of osteoblast-lineage genes is enriched. Finally, we introduce a new tissue-specific Cre-recombinase, Tagln-Cre, which efficiently targets osteoblasts, a majority of CAR cells, and both venous sinusoidal and arteriolar pericytes. These data show that Ocn-Cre and Dmp1-Cre target broader stromal cell populations than previously appreciated and may aid in the design of future studies. Moreover, these data highlight the heterogeneity of mesenchymal stromal cells in the bone marrow and provide tools to interrogate this heterogeneity. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

  16. Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Briolay, A. [ICBMS, UMR CNRS 5246, University of Lyon 1, Bâtiment Raulin, 43 Bd du 11 novembre 1918, 69622 Villeurbanne Cedex (France); Lencel, P. [Physiopathology of Inflammatory Bone Diseases, EA4490, ULCO. Quai Masset, Bassin Napoléon BP120, 62327 Boulogne/Mer (France); Bessueille, L. [ICBMS, UMR CNRS 5246, University of Lyon 1, Bâtiment Raulin, 43 Bd du 11 novembre 1918, 69622 Villeurbanne Cedex (France); Caverzasio, J. [Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14 (Switzerland); Buchet, R. [ICBMS, UMR CNRS 5246, University of Lyon 1, Bâtiment Raulin, 43 Bd du 11 novembre 1918, 69622 Villeurbanne Cedex (France); Magne, D., E-mail: david.magne@univ-lyon1.fr [ICBMS, UMR CNRS 5246, University of Lyon 1, Bâtiment Raulin, 43 Bd du 11 novembre 1918, 69622 Villeurbanne Cedex (France)

    2013-01-18

    Highlights: ► Ankylosing spondylitis (AS) leads to bone fusions and ankylosis. ► TNF-α stimulates osteoblasts through growth factors in AS. ► We compare the involvement of canonical vs non-canonical Wnt signaling. ► Canonical Wnt signaling is not involved in TNF-α effects in differentiating hMSCs. ► TNF-α stimulates osteoblasts through Wnt5a autocrine secretion in hMSCs. -- Abstract: Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased

  17. Enhanced adhesion of osteoblastic cells on polystyrene films by independent control of surface topography and wettability

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Seung Yun [National Creative Research Center for Block Copolymer Self-Assembly, Departments of Environmental Science and Engineering and Chemical Engineering, Pohang University of Science and Technology, Pohang, 790-784 (Korea, Republic of); Kim, Eung-Sam [School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, 790-784 (Korea, Republic of); Jeon, Gumhye [National Creative Research Center for Block Copolymer Self-Assembly, Departments of Environmental Science and Engineering and Chemical Engineering, Pohang University of Science and Technology, Pohang, 790-784 (Korea, Republic of); Choi, Kwan Yong, E-mail: kchoi@postech.ac.kr [School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, 790-784 (Korea, Republic of); Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, 790-784 (Korea, Republic of); Kim, Jin Kon, E-mail: jkkim@postech.ac.kr [National Creative Research Center for Block Copolymer Self-Assembly, Departments of Environmental Science and Engineering and Chemical Engineering, Pohang University of Science and Technology, Pohang, 790-784 (Korea, Republic of)

    2013-04-01

    We independently controlled surface topography and wettability of polystyrene (PS) films by CF{sub 4} and oxygen plasma treatments, respectively, to evaluate the adhesion and proliferation of human fetal osteoblastic (hFOB) cells on the films. Among the CF{sub 4} plasma-treated PS films with the average surface roughness ranging from 0.9 to 70 nm, the highest adhesion of hFOB cells was observed on a PS film with roughness of ∼ 11 nm. When this film was additionally treated by oxygen plasma to provide a hydrophilic surface with a contact angle less than 10°, the proliferation of bone-forming cell was further enhanced. Thus, the plasma-based independent modification of PS film into an optimum nanotexture for human osteoblast cells could be appplied to materials used in bone tissue engineering. Highlights: ► New approach based on plasma treatment to independently control the surface topography and wettability ► The adhesion of human fetal osteoblast (hFOB) was enhanced on a surface with an average roughness of ∼ 11 nm. ► The adhesion and proliferation of hFOB was maximized when nanotextured surface became highly hydrophilic.

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

    Directory of Open Access Journals (Sweden)

    Sung Wook Yoon

    2014-01-01

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

  19. Enhanced adhesion of osteoblastic cells on polystyrene films by independent control of surface topography and wettability

    International Nuclear Information System (INIS)

    Yang, Seung Yun; Kim, Eung-Sam; Jeon, Gumhye; Choi, Kwan Yong; Kim, Jin Kon

    2013-01-01

    We independently controlled surface topography and wettability of polystyrene (PS) films by CF 4 and oxygen plasma treatments, respectively, to evaluate the adhesion and proliferation of human fetal osteoblastic (hFOB) cells on the films. Among the CF 4 plasma-treated PS films with the average surface roughness ranging from 0.9 to 70 nm, the highest adhesion of hFOB cells was observed on a PS film with roughness of ∼ 11 nm. When this film was additionally treated by oxygen plasma to provide a hydrophilic surface with a contact angle less than 10°, the proliferation of bone-forming cell was further enhanced. Thus, the plasma-based independent modification of PS film into an optimum nanotexture for human osteoblast cells could be appplied to materials used in bone tissue engineering. Highlights: ► New approach based on plasma treatment to independently control the surface topography and wettability ► The adhesion of human fetal osteoblast (hFOB) was enhanced on a surface with an average roughness of ∼ 11 nm. ► The adhesion and proliferation of hFOB was maximized when nanotextured surface became highly hydrophilic

  20. DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yong; Fang, Shi-ji [The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000 (China); Zhu, Li-juan [Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and Institute of Neuroscience, Soochow University, Suzhou, Jiangsu 215021 (China); Zhu, Lun-qing, 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, Jiangsu 215000 (China); Zhou, Xiao-zhong, E-mail: zhouxz@suda.edu.cn [The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000 (China)

    2014-10-24

    Highlights: • LDI increases ALP activity, promotes type I collagen (Col I)/Runx2 mRNA expression. • LDI induces DNA–PKcs activation, which is required for osteoblast differentiation. • Akt activation mediates LDI-induced ALP activity and Col I/Runx2 mRNA increase. • DNA–PKcs–SIN1 complexation mediates LDI-induced Akt Ser-473 phosphorylation. • DNA–PKcs–SIN1 complexation is important for osteoblast differentiation. - Abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1 Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.

  1. Comparative of fibroblast and osteoblast cells adhesion on surface modified nanofibrous substrates based on polycaprolactone

    OpenAIRE

    Sharifi, Fereshteh; Irani, Shiva; Zandi, Mojgan; Soleimani, Masoud; Atyabi, Seyed Mohammad

    2016-01-01

    One of the determinant factors for successful bioengineering is to achieve appropriate nano-topography and three-dimensional substrate. In this research, polycaprolactone (PCL) nano-fibrous mat with different roughness modified with O2 plasma was fabricated via electrospinning. The purpose of this study was to evaluate the effect of plasma modification along with surface nano-topography of mats on the quality of human fibroblast (HDFs) and osteoblast cells (OSTs)-substrate interaction. Surfac...

  2. Biocompatibility of Poly-ε-caprolactone-hydroxyapatite composite on mouse bone marrow-derived osteoblasts and endothelial cells

    Directory of Open Access Journals (Sweden)

    Wooley Paul H

    2009-02-01

    Full Text Available Abstract Background Tissue-engineered bone may be developed by seeding the cells capable of both osteogenesis and vascularization on biocompatible composite scaffolds. The current study investigated the performance of mice bone marrow-derived osteogenic cells and endothelial cells as seeded on hydroxyapatite (HA and poly-ε-caprolactone (PCL composite scaffolds. Methods Mononuclear cells were induced to osteoblasts and endothelial cells respectively, which were defined by the expression of osteocalcin, alkaline phosphatase (ALP, and deposits of calcium-containing crystal for osteoblasts, or by the expression of vascular endothelial growth factor receptor-2 (VEGFR-2 and von Willebrand factor (vWF, and the formation of a capillary network in Matrigel™ for endothelial cells. Both types of cell were seeded respectively on PCL-HA scaffolds at HA to PCL weight ratio of 1:1, 1:4, or 0:1 and were evaluated using scanning electron microscopy, ALP activity (of osteoblasts and nitric oxide production (of endothelial cells plus the assessment of cell viability. Results The results indicated that HA led to a positive stimulation of osteoblasts viability and ALP activity, while HA showed less influence on endothelial cells viability. An elevated nitric oxide production of endothelial cells was observed in HA-containing group. Conclusion Supplement of HA into PCL improved biocompatible for bone marrow-derived osteoblasts and endothelial cells. The PCL-HA composite integrating with two types of cells may provide a useful system for tissue-engineered bone grafts with vascularization.

  3. Dexmedetomidine attenuates H2O2-induced cell death in human osteoblasts.

    Science.gov (United States)

    Yoon, Ji-Young; Park, Jeong-Hoon; Kim, Eun-Jung; Park, Bong-Soo; Yoon, Ji-Uk; Shin, Sang-Wook; Kim, Do-Wan

    2016-12-01

    Reactive oxygen species play critical roles in homeostasis and cell signaling. Dexmedetomidine, a specific agonist of the α 2 -adrenoceptor, has been commonly used for sedation, and it has been reported to have a protective effect against oxidative stress. In this study, we investigated whether dexmedetomidine has a protective effect against H 2 O 2 -induced oxidative stress and the mechanism of H 2 O 2 -induced cell death in normal human fetal osteoblast (hFOB) cells. Cells were divided into three groups: control group-cells were incubated in normoxia without dexmedetomidine, hydrogen peroxide (H 2 O 2 ) group-cells were exposed to H 2 O 2 (200 µM) for 2 h, and Dex/H 2 O 2 group-cells were pretreated with dexmedetomidine (5 µM) for 2 h then exposed to H 2 O 2 (200 µM) for 2 h. Cell viability and apoptosis were evaluated. Osteoblast maturation was determined by assaying bone nodular mineralization. Expression levels of bone-related proteins were determined by western blot. Cell viability was significantly decreased in the H 2 O 2 group compared with the control group, and this effect was improved by dexmedetomidine. The Hoechst 33342 and Annexin-V FITC/PI staining revealed that dexmedetomidine effectively decreased H 2 O 2 -induced hFOB cell apoptosis. Dexmedetomidine enhanced the mineralization of hFOB cells when compared to the H 2 O 2 group. In western blot analysis, bone-related protein was increased in the Dex/H 2 O 2 group. We demonstrated the potential therapeutic value of dexmedetomidine in H 2 O 2 -induced oxidative stress by inhibiting apoptosis and enhancing osteoblast activity. Additionally, the current investigation could be evidence to support the antioxidant potential of dexmedetomidine in vitro.

  4. 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.

  5. Implantable Self-Powered Low-Level Laser Cure System for Mouse Embryonic Osteoblasts' Proliferation and Differentiation.

    Science.gov (United States)

    Tang, Wei; Tian, Jingjing; Zheng, Qiang; Yan, Lin; Wang, Jiangxue; Li, Zhou; Wang, Zhong Lin

    2015-08-25

    Bone remodeling or orthodontic treatment is usually a long-term process. It is highly desirable to speed up the process for effective medical treatment. In this work, a self-powered low-level laser cure system for osteogenesis is developed using the power generated by the triboelectric nanogenerator. It is found that the system significantly accelerated the mouse embryonic osteoblasts' proliferation and differentiation, which is essential for bone and tooth healing. The system is further demonstrated to be driven by a living creature's motions, such as human walking or a mouse's breathing, suggesting its practical use as a portable or implantable clinical cure for bone remodeling or orthodontic treatment.

  6. Activation of the PI3K/Akt pathway by oxidative stress mediates high glucose-induced increase of adipogenic differentiation in primary rat osteoblasts.

    Science.gov (United States)

    Zhang, Yu; Yang, Jian-Hong

    2013-11-01

    Diabetes mellitus is associated with increased risk of osteopenia and bone fracture that may be related to hyperglycemia. However, the mechanisms accounting for diabetic bone disorder are unclear. Here, we showed that high glucose significantly promoted the production of reactive oxygen species (ROS) in rat primary osteoblasts. Most importantly, we reported for the first time that ROS induced by high glucose increased alkaline phosphatase activity, inhibited type I collagen (collagen I) protein level and cell mineralization, as well as gene expression of osteogenic markers including runt-related transcription factor 2 (Runx2), collagen I, and osteocalcin, but promoted lipid droplet formation and gene expression of adipogenic markers including peroxisome proliferator-activated receptor gamma, adipocyte fatty acid binding protein (aP2), and adipsin, which were restored by pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger. Moreover, high glucose-induced oxidative stress activated PI3K/Akt pathway to inhibited osteogenic differentiation but stimulated adipogenic differentiation. In contrast, NAC and a PI3K inhibitor, LY-294002, reversed the down-regulation of osteogenic markers and the up-regulation of adipogenic markers as well as the activation of Akt under high glucose. These results indicated that oxidative stress played a key role in high glucose-induced increase of adipogenic differentiation, which contributed to the inhibition of osteogenic differentiation. This process was mediated by PI3K/Akt pathway in rat primary osteoblasts. Hence, suppression of oxidative stress could be a potential therapeutic approach for diabetic osteopenia. © 2013 Wiley Periodicals, Inc.

  7. Osteoblastic and Vascular Endothelial Niches, Their Control on Normal Hematopoietic Stem Cells, and Their Consequences on the Development of Leukemia

    Directory of Open Access Journals (Sweden)

    Bella S. Guerrouahen

    2011-01-01

    Full Text Available Stem cell self-renewal is regulated by intrinsic mechanisms and extrinsic signals mediated via specialized microenvironments called “niches.” The best-characterized stem cell is the hematopoietic stem cell (HSC. Self-renewal and differentiation ability of HSC are regulated by two major elements: endosteal and vascular regulatory elements. The osteoblastic niche localized at the inner surface of the bone cavity might serve as a reservoir for long-term HSC storage in a quiescent state. Whereas the vascular niche, which consists of sinusoidal endothelial cell lining blood vessel, provides an environment for short-term HSC proliferation and differentiation. Both niches act together to maintain hematopoietic homeostasis. In this paper, we provide some principles applying to the hematopoietic niches, which will be useful in the study and understanding of other stem cell niches. We will discuss altered microenvironment signaling leading to myeloid lineage disease. And finally, we will review some data on the development of acute myeloid leukemia from a subpopulation called leukemia-initiating cells (LIC, and we will discuss on the emerging evidences supporting the influence of the microenvironment on chemotherapy resistance.

  8. Extracellular Vesicles from Adipose-Derived Mesenchymal Stem Cells Downregulate Senescence Features in Osteoarthritic Osteoblasts

    Directory of Open Access Journals (Sweden)

    Miguel Tofiño-Vian

    2017-01-01

    Full Text Available Osteoarthritis (OA affects all articular tissues leading to pain and disability. The dysregulation of bone metabolism may contribute to the progression of this condition. Adipose-derived mesenchymal stem cells (ASC are attractive candidates in the search of novel strategies for OA treatment and exert anti-inflammatory and cytoprotective effects on cartilage. Chronic inflammation in OA is a relevant factor in the development of cellular senescence and joint degradation. In this study, we extend our previous observations of ASC paracrine effects to study the influence of conditioned medium and extracellular vesicles from ASC on senescence induced by inflammatory stress in OA osteoblasts. Our results in cells stimulated with interleukin- (IL- 1β indicate that conditioned medium, microvesicles, and exosomes from ASC downregulate senescence-associated β-galactosidase activity and the accumulation of γH2AX foci. In addition, they reduced the production of inflammatory mediators, with the highest effect on IL-6 and prostaglandin E2. The control of mitochondrial membrane alterations and oxidative stress may provide a mechanism for the protective effects of ASC in OA osteoblasts. We have also shown that microvesicles and exosomes mediate the paracrine effects of ASC. Our study suggests that correction of abnormal osteoblast metabolism by ASC products may contribute to their protective effects.

  9. Peen treatment on a titanium implant: effect of roughness, osteoblast cell functions, and bonding with bone cement

    Directory of Open Access Journals (Sweden)

    Khandaker M

    2016-02-01

    Full Text Available Morshed Khandaker,1,4 Shahram Riahinezhad,1 Fariha Sultana,1 Melville B Vaughan,2,4 Joshua Knight,2 Tracy L Morris3,4 1Department of Engineering & Physics, 2Department of Biology, 3Department of Mathematics and Statistics, 4Center for Interdisciplinary Biomedical Education and Research, University of Central Oklahoma, Edmond, OK, USA Abstract: Implant failure due to poor integration of the implant with the surrounding biomaterial is a common problem in various orthopedic and orthodontic surgeries. Implant fixation mostly depends upon the implant surface topography. Micron to nanosize circular-shaped groove architecture with adequate surface roughness can enhance the mechanical interlock and osseointegration of an implant with the host tissue and solve its poor fixation problem. Such groove architecture can be created on a titanium (Ti alloy implant by laser peening treatment. Laser peening produces deep, residual compressive stresses in the surfaces of metal parts, delivering increased fatigue life and damage tolerance. The scientific novelty of this study is the controlled deposition of circular-shaped rough spot groove using laser peening technique and understanding the effect of the treatment techniques for improving the implant surface properties. The hypothesis of this study was that implant surface grooves created by controlled laser peen treatment can improve the mechanical and biological responses of the implant with the adjoining biomaterial. The objective of this study was to measure how the controlled laser-peened groove architecture on Ti influences its osteoblast cell functions and bonding strength with bone cement. This study determined the surface roughness and morphology of the peen-treated Ti. In addition, this study compared the osteoblast cell functions (adhesion, proliferation, and differentiation between control and peen-treated Ti samples. Finally, this study measured the fracture strength between each kind of Ti samples

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

    OpenAIRE

    Fredriksson, Maritha; Li, Yan; St?lman, Anders; Haldos?n, Lars-Arne; Fell?nder-Tsai, Li

    2013-01-01

    Background 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 differentiat...

  11. Fabrication and evaluation of osteoblastic differentiation of human mesenchymal stem cells on novel CaO-SiO2-P2O5-B2O3 glass-ceramics.

    Science.gov (United States)

    Lee, Jae Hyup; Seo, Jun-Hyuk; Lee, Kyung Mee; Ryu, Hyun-Seung; Baek, Hae-Ri

    2013-07-01

    Apatite-wollastonite glass-ceramics have high mechanical strength, and CaO-SiO2 -B2 O3 glass-ceramics showed excellent bioactivity and high biodegradability. A new type of CaO-SiO2 -P2 O5 -B2 O3 system of bioactive glass-ceramics (BGS-7) was fabricated, and the effect and usefulness was evaluated via bioactivity using simulated body fluid and human mesenchymal stem cells (hMSCs). The purpose of this study was to compare BGS-7 and hydroxyapatite (HA) using hMSCs in order to evaluate the bioactivity of BGS-7 and its possibility as a bone graft extender. Alkaline phosphatase (ALP) staining, ALP activity, cell proliferation 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, Alizarin Red-S (AR-S) staining, calcium levels, the mRNA expression of ALP, osteocalcin, osteopontin, and runt-related transcription factor 2 (runx-2) using reverse-transcription polymerase chain reaction (RT-PCR) and the protein expression of osteocalcin and runx-2 using Western blot were measured by transplanting hMSC onto a tissue culture plate, HA, and BGS-7. The ALP staining and AR-S staining of BGS-7 was greater than that of HA and control. The ALP value of BGS-7 was significantly higher than that of HA and control. The MTS results showed that BGS-7 had a higher value than the groups transplanted onto HA and control on day 15. The calcium level was higher than the control in both HA and BGS-7, and was especially high in BGS-7. There were more mineral products on BGS-7 than on the HA when analyzed by scanning electron microscopy. The mRNA expression of ALP, osteopontin, osteocalcin, and runx-2 were higher on BGS-7 than on HA and the control when analyzed by RT-PCR. The relative gene expression of osteopontin and runx-2 were found to be higher on BGS-7 than on HA and the control by Western blot. Accordingly, it is predicted that BGS-7 would have high biocompatibility and good osteoconductivity, and presents a possibility as a new

  12. Wnt7b can replace Ihh to induce hypertrophic cartilage vascularization but not osteoblast differentiation during endochondral bone development.

    Science.gov (United States)

    Joeng, Kyu Sang; Long, Fanxin

    2014-01-01

    Indian hedgehog (Ihh) is an essential signal that regulates endochondral bone development. We have previously shown that Wnt7b promotes osteoblast differentiation during mouse embryogenesis, and that its expression in the perichondrium is dependent on Ihh signaling. To test the hypothesis that Wnt7b may mediate some aspects of Ihh function during endochondral bone development, we activated Wnt7b expression from the R26-Wnt7b allele with Col2-Cre in the Ihh(-/-) mouse. Artificial expression of Wnt7b rescued vascularization of the hypertrophic cartilage in the Ihh(-/-) mouse, but failed to restore orthotopic osteoblast differentiation in the perichondrium. Similarly, Wnt7b did not recover Ihh-dependent perichondral bone formation in the Ihh(-/-); Gli3(-/-) embryo. Interestingly, Wnt7b induced bone formation at the diaphyseal region of long bones in the absence of Ihh, possibly due to increased vascularization in the area. Thus, Ihh-dependent expression of Wnt7b in the perichondrium may contribute to vascularization of the hypertrophic cartilage during endochondral bone development.

  13. 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.

  14. Phenolic Compounds in Extra Virgin Olive Oil Stimulate Human Osteoblastic Cell Proliferation

    Science.gov (United States)

    García-Martínez, Olga; De Luna-Bertos, Elvira; Ramos-Torrecillas, Javier; Ruiz, Concepción; Milia, Egle; Lorenzo, María Luisa; Jimenez, Brigida; Sánchez-Ortiz, Araceli; Rivas, Ana

    2016-01-01

    In this study, we aimed to clarify the effects of phenolic compounds and extracts from different extra virgin olive oil (EVOO) varieties obtained from fruits of different ripening stages on osteoblast cells (MG-63) proliferation. Cell proliferation was increased by hydroxytyrosol, luteolin, apigenin, p-coumaric, caffeic, and ferulic acids by approximately 11–16%, as compared with controls that were treated with one vehicle alone, while (+)-pinoresinol, oleuropein, sinapic, vanillic acid and derivative (vanillin) did not affect cell proliferation. All phenolic extracts stimulated MG-63 cell growth, and they induced higher cell proliferation rates than individual compounds. The most effective EVOO phenolic extracts were those obtained from the Picual variety, as they significantly increased cell proliferation by 18–22%. Conversely, Arbequina phenolic extracts increased cell proliferation by 9–13%. A decline in osteoblast proliferation was observed in oils obtained from olive fruits collected at the end of the harvest period, as their total phenolic content decreases at this late stage. Further research on the signaling pathways of olive oil phenolic compounds involved in the processes and their metabolism should be carried out to develop new interventions and adjuvant therapies using EVOO for bone health (i.e.osteoporosis) in adulthood and the elderly. PMID:26930190

  15. Phenolic Compounds in Extra Virgin Olive Oil Stimulate Human Osteoblastic Cell Proliferation.

    Science.gov (United States)

    García-Martínez, Olga; De Luna-Bertos, Elvira; Ramos-Torrecillas, Javier; Ruiz, Concepción; Milia, Egle; Lorenzo, María Luisa; Jimenez, Brigida; Sánchez-Ortiz, Araceli; Rivas, Ana

    2016-01-01

    In this study, we aimed to clarify the effects of phenolic compounds and extracts from different extra virgin olive oil (EVOO) varieties obtained from fruits of different ripening stages on osteoblast cells (MG-63) proliferation. Cell proliferation was increased by hydroxytyrosol, luteolin, apigenin, p-coumaric, caffeic, and ferulic acids by approximately 11-16%, as compared with controls that were treated with one vehicle alone, while (+)-pinoresinol, oleuropein, sinapic, vanillic acid and derivative (vanillin) did not affect cell proliferation. All phenolic extracts stimulated MG-63 cell growth, and they induced higher cell proliferation rates than individual compounds. The most effective EVOO phenolic extracts were those obtained from the Picual variety, as they significantly increased cell proliferation by 18-22%. Conversely, Arbequina phenolic extracts increased cell proliferation by 9-13%. A decline in osteoblast proliferation was observed in oils obtained from olive fruits collected at the end of the harvest period, as their total phenolic content decreases at this late stage. Further research on the signaling pathways of olive oil phenolic compounds involved in the processes and their metabolism should be carried out to develop new interventions and adjuvant therapies using EVOO for bone health (i.e.osteoporosis) in adulthood and the elderly.

  16. The influence of surface chemistry and topography on the contact guidance of MG63 osteoblast cells.

    Science.gov (United States)

    Ismail, F S Magdon; Rohanizadeh, R; Atwa, S; Mason, R S; Ruys, A J; Martin, P J; Bendavid, A

    2007-05-01

    The purpose of the present study was to determine in vitro the effects of different surface topographies and chemistries of commercially pure titanium (cpTi) and diamond-like carbon (DLC) surfaces on osteoblast growth and attachment. Microgrooves (widths of 2, 4, 8 and 10 microm and a depth of 1.5-2 microm) were patterned onto silicon (Si) substrates using microlithography and reactive ion etching. The Si substrates were subsequently vapor coated with either cpTi or DLC coatings. All surfaces were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. Using the MG63 Osteoblast-Like cell line, we determined cell viability, adhesion, and morphology on different substrates over a 3 day culture period. The results showed cpTi surfaces to be significantly more hydrophilic than DLC for groove sizes larger than 2 microm. Cell contact guidance was observed for all grooved samples in comparison to the unpatterned controls. The cell viability tests indicated a significantly greater cell number for 8 and 10 microm grooves on cpTi surfaces compared to other groove sizes. The cell adhesion study showed that the smaller groove sizes, as well as the unpatterned control groups, displayed better cell adhesion to the substrate.

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

    Science.gov (United States)

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

    2013-06-27

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

  18. 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.

  19. Four-point bending protocols to study the effects of dynamic strain in osteoblastic cells in vitro.

    Science.gov (United States)

    Galea, Gabriel L; Price, Joanna S

    2015-01-01

    Strain engendered within bone tissue by mechanical loading of the skeleton is a major influence on the processes of bone modeling and remodeling and so a critical determinant of bone mass and architecture. The cells best placed to respond to strain in bone tissue are the resident osteocytes and osteoblasts. To address the mechanisms of strain-related responses in osteoblast-like cells, our group uses both in vivo and in vitro approaches, including a system of four-point bending of the substrate on which cells are cultured. A range of cell lines can be studied using this system but we routinely compare their responses to those in primary cultures of osteoblast-like cells derived from explants of mouse long bones. These cells show a range of well-characterized responses to physiological levels of strain, including increased proliferation, which in vivo is a feature of the osteogenic response.

  20. Zirconia coated titanium for implants and their interactions with osteoblast cells

    International Nuclear Information System (INIS)

    Kaluđerović, Milena R.; Schreckenbach, Joachim P.; Graf, Hans-Ludwig

    2014-01-01

    The anodic plasma-electrochemical oxidation in aqueous electrolytes of Zr(SO 4 ) 2 was used to prepare new zirconia/titania-based surfaces M1 (Ti, Zr and O: 7–10, 22–27 and 65–69 at.%) and M2 (Ti, Zr and O: 11–13, 20–23 and 64–69 at.%). The chemical composition and the microstructure of these coatings were characterized by surface and solid state techniques such as scanning electron microscopy, electron probe microanalysis, Raman spectroscopy and X-ray diffraction. These mixed oxides of ZrO 2 /TiO 2 surfaces consist up to 84% (m/m) of ZrO 2 and 16% (m/m) of TiO 2 . Monoclinic zirconia was detected as the dominant microcrystalline phase. In vitro studies were conducted on primary human osteoblast cells. MTT and DAPI assays were used for assessment on cell proliferation. Immunohistochemical analyses of morphology, cell cluster formation and expression of bone sialoprotein (BSP) and osteocalcin (OC) were performed. Novel surfaces M1 and M2 induced proliferation and expression of OC and BSP similarly to Ticer, used in clinical practice. Furthermore, the presence of zirconia on titanium surface has a higher beneficial effect on the osteoblast morphological changes and cell cluster formation. - Highlights: • Surfaces M1 and M2 (up to 84% (m/m) ZrO 2 and 16% (m/m) TiO 2 ) were prepared. • Novel materials promote proliferation of human osteoblasts similarly to Ticer. • Morphological changes and cell cluster formation are induced faster on M1 and M2. • Higher expression of OC and BSP is caused by M1 and M2. • M1 and M2 may influence the rate of bone formation

  1. Cellular reactions of osteoblast-like cells to a novel nanocomposite membrane for guided bone regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Meng Yao [State Key Laboratory of Oral Diseases, West China Stomatology Hospital, Sichuan University, Chengdu 610041 (China); Department of Orthodontics, West China Stomatology Hospital, Sichuan University, Chengdu 610041 (China); Liu Man [State Key Laboratory of Oral Diseases, West China Stomatology Hospital, Sichuan University, Chengdu 610041 (China); Stomatology Health Care Center, Shenzhen Maternity and Child Healthcare Hospital, Shenzhen 518048 (China); Wang Shaoan [State Key Laboratory of Oral Diseases, West China Stomatology Hospital, Sichuan University, Chengdu 610041 (China); Mo Anchun [State Key Laboratory of Oral Diseases, West China Stomatology Hospital, Sichuan University, Chengdu 610041 (China)], E-mail: moanchun@163.com; Huang, Cui [State Key Laboratory of Oral Diseases, West China Stomatology Hospital, Sichuan University, Chengdu 610041 (China); Zuo Yi; Li Jidong [Research Center for Nano-biomaterials, Sichuan University, Chengdu 610041 (China)

    2008-11-15

    This study investigated the bioactivity and biocompatibility of hydroxyapatite nanoparticles (n-HA)/Polyamide-66 (PA66) nanocomposite membrane and expanded-polytetrafluoroethylene (e-PTFE) membrane (as control) to MG63 osteoblast-like cells. The attachment and proliferation of the cells on the porous surface of nHA/PA66 membrane and the surface of e-PTFE membrane were evaluated by scanning electron microscope (SEM) observation and the MTT assay. The bioactivity of the cells on the surface of the two membranes was evaluated by testing cell viability and alkaline phosphatase (ALP) activities. The results suggested that the bioresponse of MG63 osteoblast-like cells on the porous surface of nHA/PA66 membrane was better than the bioresponse on the opposite surface of e-PTFE membrane. Because of a better cell attachment manner, there is a potential utilization of the guided bone regeneration (GBR) membrane to substitute nHA/PA66 membrane for e-PTFE membra0008.

  2. Cellular reactions of osteoblast-like cells to a novel nanocomposite membrane for guided bone regeneration

    International Nuclear Information System (INIS)

    Meng Yao; Liu Man; Wang Shaoan; Mo Anchun; Huang, Cui; Zuo Yi; Li Jidong

    2008-01-01

    This study investigated the bioactivity and biocompatibility of hydroxyapatite nanoparticles (n-HA)/Polyamide-66 (PA66) nanocomposite membrane and expanded-polytetrafluoroethylene (e-PTFE) membrane (as control) to MG63 osteoblast-like cells. The attachment and proliferation of the cells on the porous surface of nHA/PA66 membrane and the surface of e-PTFE membrane were evaluated by scanning electron microscope (SEM) observation and the MTT assay. The bioactivity of the cells on the surface of the two membranes was evaluated by testing cell viability and alkaline phosphatase (ALP) activities. The results suggested that the bioresponse of MG63 osteoblast-like cells on the porous surface of nHA/PA66 membrane was better than the bioresponse on the opposite surface of e-PTFE membrane. Because of a better cell attachment manner, there is a potential utilization of the guided bone regeneration (GBR) membrane to substitute nHA/PA66 membrane for e-PTFE membrane

  3. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

    International Nuclear Information System (INIS)

    Brady, Robert T.; O'Brien, Fergal J.; Hoey, David A.

    2015-01-01

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting

  4. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Brady, Robert T. [Tissue Engineering Research Group, Dept. of Anatomy, Royal College of Surgeons in Ireland (Ireland); Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Advanced Materials and BioEngineering Research Centre (AMBER), Trinity College Dublin & Royal College of Surgeons in Ireland (Ireland); Dept. of Mechanical, Aeronautical and Biomedical Engineering, University of Limerick (Ireland); O' Brien, Fergal J. [Tissue Engineering Research Group, Dept. of Anatomy, Royal College of Surgeons in Ireland (Ireland); Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Advanced Materials and BioEngineering Research Centre (AMBER), Trinity College Dublin & Royal College of Surgeons in Ireland (Ireland); Hoey, David A., E-mail: david.hoey@ul.ie [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Dept. of Mechanical, Aeronautical and Biomedical Engineering, University of Limerick (Ireland); The Centre for Applied Biomedical Engineering Research, University of Limerick (Ireland); Materials & Surface Science Institute, University of Limerick (Ireland)

    2015-03-27

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting

  5. Comparison of the biological effects of exogenous and endogenous 1,25-dihydroxyvitamin D3 on the mature osteoblast cell line MLO-A5.

    Science.gov (United States)

    Yang, Dongqing; Anderson, Paul H; Turner, Andrew G; Morris, Howard A; Atkins, Gerald J

    2016-11-01

    Clinical and animal data indicate that serum 25-hydroxyvitamin D 3 (25D) exerts an anabolic effect on bone while serum 1α,25-dihydroxyvitamin D 3 (1,25D) stimulates bone mineral loss, although the mechanism responsible for these divergent actions is unknown. Biological effects of 25D on bone cells are dependent on the local conversion to 1,25D by the 25-hydroxyvitamin D-1α-hydroxylase enzyme, CYP27B1. Therefore, identification of possible differential activities of locally produced and exogenously supplied 1,25D in bone is likely to be informative for guiding optimal administration of vitamin D supplements for bone health. The mature osteoblastic cell line MLO-A5 expresses both the vitamin D receptor (Vdr) and Cyp27b1, and therefore is a suitable model for comparing the activities of 1,25D arising from these sources. Biologically, exogenous and endogenous sources of 1,25D have similar effects on proliferation, mineralisation and induction of a range of genes by MLO-A5 osteoblasts under osteogenic conditions although endogenous 1,25D levels are markedly lower than exogenous levels. Significant differences of pharmacokinetics and pharmacodynamics of 1,25D are evident between these two sources particularly in terms of modulating gene expression for Cyp24a1 and other genes largely expressed by embedded osteoblasts/osteocytes suggesting that endogenously synthesised 1,25D is more efficiently utilised by the differentiating osteoblast. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. 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...

  7. The In-Vitro Effects of Sea Cucumber (Stichopus sp1 Extract on Human Osteoblast Cell Line

    Directory of Open Access Journals (Sweden)

    A Shahrulazua

    2013-03-01

    Full Text Available Despite its claimed therapeutic effects, the action of sea cucumber (known as gamat in the Malay language on human osteoblast cells is still unknown. We performed in vitro studies utilising extract of Stichopus sp1 (gamat to elucidate its effects on cell viability and functional activity. We found an inverse relationship between gamat concentration and its effect on osteoblast cell viability (p<0.001. Only gamat concentration at 1mg/ml significantly promoted cell viability at day 3 of incubation. There was a trend towards increased osteoblast cell function in the presence of gamat at 5mg/ml and 10mg/ml but this observation was not consistent at different incubation periods.

  8. Plasma deposited composite coatings to control biological response of osteoblast-like MG-63 cells

    Science.gov (United States)

    Keremidarska, M.; Radeva, E.; Eleršič, K.; Iglič, A.; Pramatarova, L.; Krasteva, N.

    2014-12-01

    The successful osseointegration of a bone implant is greatly dependent on its ability to support cellular adhesion and functions. Deposition of thin composite coatings onto the implant surface is a promising approach to improve interactions with cells without compromising implant bulk properties. In this work, we have developed composite coatings, based on hexamethyldisiloxane (HMDS) and detonation nanodiamond (DND) particles and have studied adhesion, growth and function of osteoblast-like MG-63 cells. PPHMDS/DND composites are of interest for orthopedics because they combine superior mechanical properties and good biocompatibility of DND with high adherence of HMDS to different substrata including glass, metals and plastics. We have used two approaches of the implementation of DND particles into a polymer matrix: pre-mixture of both components followed by plasma polymerization and layer-by-layer deposition of HMDS and DND particles and found that the deposition approach affects significantly the surface properties of the resulting layers and cell behaviour. The composite, prepared by subsequent deposition of monomer and DND particles was hydrophilic, with a rougher surface and MG-63 cells demonstrated better spreading, growth and function compared to the other composite which was hydrophobic with a smooth surface similarly to unmodified polymer. Thus, by varying the deposition approach, different PPHMDS/DND composite coatings, enhancing or inhibiting osteoblast adhesion and functions, can be obtained. In addition, the effect of fibronectin pre-adsorption was studied and was found to increase greatly MG-63 cell spreading.

  9. Effect of Q-switched Laser Surface Texturing of Titanium on Osteoblast Cell Response

    Science.gov (United States)

    Voisey, K. T.; Scotchford, C. A.; Martin, L.; Gill, H. S.

    Titanium and its alloys are important biomedical materials. It is known that the surface texture of implanted medical devices affects cell response. Control of cell response has the potential to enhance fixation of implants into bone and, in other applications, to prevent undesired cell adhesion. The potential use of a 100W Q-switched YAG laser miller (DMG Lasertec 60 HSC) for texturing titanium is investigated. A series of regular features with dimensions of the order of tens of micrometers are generated in the surface of titanium samples and the cell response to these features is determined. Characterisation of the laser milled features reveals features with a lengthscale of a few microns superposed on the larger scale structures, this is attributed to resolidification of molten droplets generated and propelled over the surface by individual laser pulses. The laser textured samples are exposed to osteoblast cells and it is seen that cells do respond to the features in the laser textured surfaces.

  10. 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.

  11. 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.

  12. Intrinsically superparamagnetic Fe-hydroxyapatite nanoparticles positively influence osteoblast-like cell behaviour

    Science.gov (United States)

    2012-01-01

    Background Superparamagnetic nanoparticles (MNPs) have been progressively explored for their potential in biomedical applications and in particular as a contrast agent for diagnostic imaging, for magnetic drug delivery and more recently for tissue engineering applications. Considering the importance of having safe MNPs for such applications, and the essential role of iron in bone remodelling, this study developed and analysed novel biocompatible and bioreabsorbable superparamagnetic nanoparticles, that avoid the use of poorly tolerated magnetite based nanoparticles, for bone tissue engineering applications. Results MNPs were obtained by doping hydroxyapatite (HA) with Fe ions, by directly substituting Fe2+ and Fe3+ into the HA structure yielding superparamagnetic bioactive phase. In the current study, we have investigated the effects of increasing concentrations (2000 μg/ml; 1000 μg/ml; 500 μg/ml; 200 μg/ml) of FeHA MNPs in vitro using Saos-2 human osteoblast-like cells cultured for 1, 3 and 7 days with and without the exposure to a static magnetic field of 320 mT. Results demonstrated not only a comparable osteoblast viability and morphology, but increased in cell proliferation, when compared to a commercially available Ha nanoparticles, even with the highest dose used. Furthermore, FeHA MNPs exposure to the static magnetic field resulted in a significant increase in cell proliferation throughout the experimental period, and higher osteoblast activity. In vivo preliminary results demonstrated good biocompatibility of FeHA superparamagnetic material four weeks after implantation into a critical size lesion of the rabbit condyle. Conclusions The results of the current study suggest that these novel FeHA MNPs may be particularly relevant for strategies of bone tissue regeneration and open new perspectives for the application of a static magnetic field in a clinical setting of bone replacement, either for diagnostic imaging or magnetic drug delivery

  13. Mycobacterium leprae downregulates the expression of PHEX in Schwann cells and osteoblasts

    Directory of Open Access Journals (Sweden)

    Sandra R Boiça Silva

    2010-08-01

    Full Text Available Neuropathy and bone deformities, lifelong sequelae of leprosy that persist after treatment, result in significant impairment to patients and compromise their social rehabilitation. Phosphate-regulating gene with homologies to endopeptidase on the X chromosome (PHEX is a Zn-metalloendopeptidase, which is abundantly expressed in osteoblasts and many other cell types, such as Schwann cells, and has been implicated in phosphate metabolism and X-linked rickets. Here, we demonstrate that Mycobacterium leprae stimulation downregulates PHEX transcription and protein expression in a human schwannoma cell line (ST88-14 and human osteoblast lineage. Modulation of PHEX expression was observed to a lesser extent in cells stimulated with other species of mycobacteria, but was not observed in cultures treated with latex beads or with the facultative intracellular bacterium Salmonella typhimurium. Direct downregulation of PHEX by M. leprae could be involved in the bone resorption observed in leprosy patients. This is the first report to describe PHEX modulation by an infectious agent.

  14. Interaction of Protein Phosphatase 1δ with Nucleophosmin in Human Osteoblastic Cells

    International Nuclear Information System (INIS)

    Haneji, Tatsuji; Teramachi, Jumpei; Hirashima, Kanji; Kimura, Koji; Morimoto, Hiroyuki

    2012-01-01

    Protein phosphorylation and dephosphorylation has been recognized as an essential mechanism in the regulation of cellular metabolism and function in various tissues. Serine and threonine protein phosphatases (PP) are divided into four categories: PP1, PP2A, PP2B, and PP2C. At least four isoforms of PP1 catalytic subunit in rat, PP1α, PP1γ1, PP1γ2, and PP1δ, were isolated. In the present study, we examined the localization and expression of PP1δ in human osteoblastic Saos-2 cells. Anti-PP1δ antibody recognized a protein present in the nucleolar regions in Saos-2 cells. Cellular fractionation revealed that PP1δ is a 37 kDa protein localized in the nucleolus. Nucleophosmin is a nucleolar phosphoprotein and located mainly in the nucleolus. Staining pattern of nucleophosmin in Saos-2 cells was similar to that of PP1δ. PP1δ and nucleophosmin were specifically stained as dots in the nucleus. Dual fluorescence images revealed that PP1δ and nucleophosmin were localized in the same regions in the nucleolus. Similar distribution patterns of PP1δ and nucleophosmin were observed in osteoblastic MG63 cells. The interaction of PP1δ and nucleophosmin was also shown by immunoprecipitation and Western analysis. These results indicated that PP1δ associate with nucleophosmin directly in the nucleolus and suggested that nucleophosmin is one of the candidate substrate for PP1δ

  15. Romidepsin Promotes Osteogenic and Adipocytic Differentiation of Human Mesenchymal Stem Cells through Inhibition of Histondeacetylase Activity

    Directory of Open Access Journals (Sweden)

    Dalia Ali

    2018-01-01

    Full Text Available Bone marrow mesenchymal stem cells (BMSCs are adult multipotent stem cells that can differentiate into mesodermal lineage cells, including adipocytes and osteoblasts. However, the epigenetic mechanisms governing the lineage-specific commitment of BMSCs into adipocytes or osteoblasts are under investigation. Herein, we investigated the epigenetic effect of romidepsin, a small molecule dual inhibitor targeting HDAC1 and HDAC2 identified through an epigenetic library functional screen. BMSCs exposed to romidepsin (5 nM exhibited enhanced adipocytic and osteoblastic differentiation. Global gene expression and signaling pathway analyses of differentially expressed genes revealed a strong enrichment of genes involved in adipogenesis and osteogenesis in romidepsin-treated BMSCs during induction into adipocytes or osteoblasts, respectively. Pharmacological inhibition of FAK signaling during adipogenesis or inhibition of FAK or TGFβ signaling during osteogenesis diminished the biological effects of romidepsin on BMSCs. The results of chromatin immunoprecipitation combined with quantitative polymerase chain reaction indicated a significant increase in H3K9Ac epigenetic markers in the promoter regions of peroxisome proliferator-activated receptor gamma (PPARγ and KLF15 (related to adipogenesis or SP7 (Osterix and alkaline phosphatase (ALP (related to osteogenesis in romidepsin-treated BMSCs. Our data indicated that romidepsin is a novel in vitro modulator of adipocytic and osteoblastic differentiation of BMSCs.

  16. Absolute Configuration of Andrographolide and Its Proliferation of Osteoblast Cell Lines

    Science.gov (United States)

    Chantrapromma, S.; Boonnak, N.; Pitakpornpreecha, T.; Yordthong, T.; Chidan Kumar, C. S.; Fun, H. K.

    2018-05-01

    Andrographolide, C20H30O5, is a labdane diterpenoid which was isolated from the leave of Andrographis paniculata. Its crystal structure is determined by single crystal X-ray diffraction: monoclinic, sp. gr. P21, Z = 2. Absolute configuration is determined by the refinement of the Flack parameter to 0.21(19). In the crystal, molecules are linked by O-H···O hydrogen bonds and C-H···O interactions into two dimensional network parallel to the (001) plane. Its proliferation of osteoblast cell lines is reported.

  17. 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.

  18. Comparison of the effect of activated or non-activated PRP in various concentrations on osteoblast and fibroblast cell line proliferation.

    Science.gov (United States)

    Vahabi, Surena; Yadegari, Zahra; Mohammad-Rahimi, Hossein

    2017-09-01

    Platelet-rich plasma (PRP) contains growth factors which positively affect cell proliferation, cell differentiation, chemotaxis and intracellular matrix synthesis. All these processes are involved in wound healing and tissue regeneration; thus, PRP as a source of growth factors can be used in periodontal regenerative therapies. The purpose of the present study was to assess the effect of various concentrations of activated and non-activated PRP on proliferation of osteoblasts and fibroblasts in vitro. PRP was obtained from three healthy volunteers. 75, 50, 25, and 10% concentrations of f PRP were prepared by dilution in Dulbecco's modified Eagle's medium. In activated PRP groups, PRP concentrations were activated by adding calcium gluconate. Human gingival fibroblast (HGF) cell line and MG-63 (osteosarcoma) human osteoblast-like cell line were used in the study. The MTT proliferation assay was used to assess the effect of different types of PRP concentrates on proliferation of HGF and MG-63 cells, in 24, 48 and 72 h. After 24, 48, and 72 h, the proliferation rate of both cell lines was higher in the positive control group, except in 72 h in HGF cell lines, that 10% non-activated PRP group and 10 and 25% activated PRP groups has higher proliferation rate than the positive control group, which it was not significant. Proliferation rate in cells with 10% activated PRP was highest among samples containing PRP. The current study failed to show the significant effect of activated or non-activated PRP on proliferation of HGFs or MG-63 osteoblast-like cells. However, our results showed that activated PRP had a greater effect than non-activated PRP.

  19. Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction.

    Science.gov (United States)

    Godin, Lindsay M; Suzuki, Sakiko; Jacobs, Christopher R; Donahue, Henry J; Donahue, Seth W

    2007-11-01

    An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca (2+)](i)). This study analyzed the [Ca (2+)](i) wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca (2+)](i) wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca (2+)](i) wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca (2+)](i) wave area in the first bout it was likely to have a large [Ca (2+)](i) wave area in the second bout (r (2) = 0.933). These findings support the idea that individual bone cells have "calcium fingerprints" (i.e., a unique [Ca (2+)](i) wave profile that is reproducible for repeated exposure to a given stimulus).

  20. The Degradation Interface of Magnesium Based Alloys in Direct Contact with Human Primary Osteoblast Cells.

    Directory of Open Access Journals (Sweden)

    Nezha Ahmad Agha

    Full Text Available Magnesium alloys have been identified as a new generation material of orthopaedic implants. In vitro setups mimicking physiological conditions are promising for material / degradation analysis prior to in vivo studies however the direct influence of cell on the degradation mechanism has never been investigated. For the first time, the direct, active, influence of human primary osteoblasts on magnesium-based materials (pure magnesium, Mg-2Ag and Mg-10Gd alloys is studied for up to 14 days. Several parameters such as composition of the degradation interface (directly beneath the cells are analysed with a scanning electron microscope equipped with energy dispersive X-ray and focused ion beam. Furthermore, influence of the materials on cell metabolism is examined via different parameters like active mineralisation process. The results are highlighting the influences of the selected alloying element on the initial cells metabolic activity.

  1. The Degradation Interface of Magnesium Based Alloys in Direct Contact with Human Primary Osteoblast Cells.

    Science.gov (United States)

    Ahmad Agha, Nezha; Willumeit-Römer, Regine; Laipple, Daniel; Luthringer, Bérengère; Feyerabend, Frank

    2016-01-01

    Magnesium alloys have been identified as a new generation material of orthopaedic implants. In vitro setups mimicking physiological conditions are promising for material / degradation analysis prior to in vivo studies however the direct influence of cell on the degradation mechanism has never been investigated. For the first time, the direct, active, influence of human primary osteoblasts on magnesium-based materials (pure magnesium, Mg-2Ag and Mg-10Gd alloys) is studied for up to 14 days. Several parameters such as composition of the degradation interface (directly beneath the cells) are analysed with a scanning electron microscope equipped with energy dispersive X-ray and focused ion beam. Furthermore, influence of the materials on cell metabolism is examined via different parameters like active mineralisation process. The results are highlighting the influences of the selected alloying element on the initial cells metabolic activity.

  2. Carriers in mesenchymal stem cell osteoblast mineralization-State-of-the-art

    DEFF Research Database (Denmark)

    Dahl, Morten; Jørgensen, Niklas Rye; Hørberg, Mette

    2014-01-01

    PURPOSE: Tissue engineering is a new way to regenerate bone tissue, where osteogenic capable cells combine with an appropriate scaffolding material. Our aim was in a Medline Search to evaluate osteoblast mineralization in vitro and in vivo including gene expressing combining mesenchymal stem cells...... (MSCs) and five different carriers, titanium, collagen, calcium carbonate, calcium phosphate and polylactic acid-polyglycolic acid copolymer for purpose of a meta-or a descriptive analysis. MATERIALS AND METHODS: The search included the following MeSH words in different combinations-mesenchymal stem...... cells, alkaline phosphatase, bone regeneration, tissue engineering, drug carriers, tissue scaffolds, titanium, collagen, calcium carbonate, calcium phosphates and polylactic acid-polyglycolic acid copolymer. RESULTS: Two out of 80 articles included numerical values and as control, carriers and cells...

  3. Proton concentrations can be a major contributor to the modification of osteoclast and osteoblast differentiation, working independently of extracellular bicarbonate ions.

    Science.gov (United States)

    Kato, Kohtaro; Matsushita, Misao

    2014-01-01

    We established a system to separately analyze the role of protons and bicarbonate ions in vitro in which the pH of the medium was controlled by HEPES at various concentrations of sodium bicarbonate (NaHCO3) in the absence of carbon dioxide (CO2). Using this system, we demonstrated that acidosis promoted osteoclast formation independently of extracellular NaHCO3 in a short-term culture. Protons and bicarbonate ions acted on osteoclast differentiation with opposite effects, the former positively and the latter negatively. The HEPES-based system maintained pH in the absence of extracellular NaHCO3 without CO2. Therefore, we could demonstrate that osteoblast differentiation was promoted at higher pH in a long-term culture system without NaHCO3 in which ALP activity and nodule mineralization were enhanced. This finding indicates that protons negatively control osteoblast differentiation independently of extracellular bicarbonate ions. However, the difference in the concentration of NaHCO3 did not have any influence on nodule mineralization. The opposite effects of protons, the promotion of osteoclast formation and the inhibition of osteoblast differentiation, were suppressed in the presence of 5 mM N-acetyl cysteine, a reagent activating the scavenging of reactive oxygen species (ROS), implying that ROS act on both systems, the promotion of large osteoclast formation and the deterioration of osteoblast formation under acidosis.

  4. Different Expression and Localization of Phosphoinositide Specific Phospholipases C in Human Osteoblasts, Osteosarcoma Cell Lines, Ewing Sarcoma and Synovial Sarcoma

    Directory of Open Access Journals (Sweden)

    V.Vasco

    2017-06-01

    Full Text Available Background: Bone hardness and strength depends on mineralization, which involves a complex process in which calcium phosphate, produced by bone-forming cells, was shed around the fibrous matrix. This process is strictly regulated, and a number of signal transduction systems were interested in calcium metabolism, such as the phosphoinositide (PI pathway and related phospholipase C (PLC enzymes. Objectives: Our aim was to search for common patterns of expression in osteoblasts, as well as in ES and SS. Methods: We analysed the PLC enzymes in human osteoblasts and osteosarcoma cell lines MG-63 and SaOS-2. We compared the obtained results to the expression of PLCs in samples of patients affected with Ewing sarcoma (ES and synovial sarcoma (SS. Results: In osteoblasts, MG-63 cells and SaOS-2 significant differences were identified in the expression of PLC δ4 and PLC η subfamily isoforms. Differences were also identified regarding the expression of PLCs in ES and SS. Most ES and SS did not express PLCB1, which was expressed in most osteoblasts, MG-63 and SaOS-2 cells. Conversely, PLCB2, unexpressed in the cell lines, was expressed in some ES and SS. However, PLCH1 was expressed in SaOS-2 and inconstantly expressed in osteoblasts, while it was expressed in ES and unexpressed in SS. The most relevant difference observed in ES compared to SS regarded PLC ε and PLC η isoforms. Conclusion: MG-63 and SaOS-2 osteosarcoma cell lines might represent an inappropriate experimental model for studies about the analysis of signal transduction in osteoblasts

  5. Compositional effects on the formation of a calcium phosphate layer and the response of osteoblast-like cells on polymer-bioactive glass composites.

    Science.gov (United States)

    Lu, Helen H; Tang, Amy; Oh, Seong Cheol; Spalazzi, Jeffrey P; Dionisio, Kathie

    2005-11-01

    Biodegradable polymer-ceramic composites are attractive systems for bone tissue engineering applications. These composites have the combined advantages of the component phases, as well as the inherent ease in optimization where desired material properties can be tailored in a well-controlled manner. This study focuses on the optimization of a polylactide-co-glycolide (PLAGA) and 45S5 bioactive glass (BG) composite for bone tissue engineering. The first objective is to examine the effects of composition or overall BG content on the formation of a Ca-P layer on the PLAGA-BG composite. It is expected that with increasing BG content (0%, 10%, 25%, 50% by weight), the required incubation time in a simulated body fluid (SBF) for the composite to form a detectable surface Ca-P layer will decrease. Both the kinetics and the chemistry will be determined using SEM+EDAX, FTIR, and mu-CT methods. Solution phosphorous and calcium concentrations will also be measured. The second objective of the study is to determine the effects of BG content on the maturation of osteoblast-like cells on the PLAGA-BG composite. It is hypothesized that mineralization will increase with increasing BG content, and the composite will support the proliferation and differentiation of osteoblasts. Specifically, cell proliferation, alkaline phosphatase activity and mineralization will be monitored as a function of BG content (0%, 10%, 50% by weight) and culturing time. It was found that the kinetics of Ca-P layer formation and the resulting Ca-P chemistry were dependent on BG content. The response of human osteoblast-like cells to the PLAGA-BG composite was also a function of BG content. The 10% and 25% BG composite supported greater osteoblast growth and differentiation compared to the 50% BG group. The results of this study suggest that there is a threshold BG content which is optimal for osteoblast growth, and the interactions between PLAGA and BG may modulate the kinetics of Ca-P formation and the

  6. Substance P Promotes the Proliferation, but Inhibits Differentiation and Mineralization of Osteoblasts from Rats with Spinal Cord Injury via RANKL/OPG System.

    Directory of Open Access Journals (Sweden)

    Hai-Juan Liu

    Full Text Available Spinal cord injury (SCI causes a significant amount of bone loss, which results in osteoporosis (OP. The neuropeptide substance P (SP and SP receptors may play important roles in the pathogenesis of OP after SCI. To identify the roles of SP in the bone marrow mesenchymal stem cell derived osteoblasts (BMSC-OB in SCI rats, we investigated the expression of neurokinin-1 receptors (NK1R in BMSC-OB and the effects of SP on bone formation by development of BMSC-OB cultures. Sixty young male Sprague-Dawley rats were randomized into two groups: SHAM and SCI. The expression of NK1R protein in BMSC-OB was observed using immunohistochemistry and Western blot analysis. The dose- and time-dependent effects of SP on the proliferation, differentiation and mineralization of BMSC-OB and the expression of osteoblastic markers by in vitro experiments. The expression of NK1R in BMSC-OB was observed on plasma membranes and in cytoplasm. One week after osteogenic differentiation, the expression of NK1R was significantly increased after SCI at mRNA and protein levels. However, this difference was gradually attenuated at 2 or 3 weeks later. SP have the function to enhance cell proliferation, inhibite cell differentiation and mineralization at a proper concentration and incubation time, and this effect would be inhibited by adding SP or NK1R antagonist. The expression of RANKL/OPG was significantly increased in tibiae after SCI. Similarly, the RANKL/OPG expression in SCI rats was significantly increased when treating with 10-8 M SP. SP plays a very important role in the pathogenesis of OP after SCI. The direct effect of SP may lead to increased bone resorption through the RANKL/OPG axis after SCI. In addition, high expression of SP also results in the suppression of osteogenesis in SCI rats. Then, the balance between bone resorption and bone formation was broken and finally osteoporosis occurred.

  7. Differentiation potential of STRO-1+ dental pulp stem cells changes during cell passaging

    Directory of Open Access Journals (Sweden)

    Wang Ruoning

    2010-05-01

    Full Text Available Abstract Background Dental pulp stem cells (DPSCs can be driven into odontoblast, osteoblast, and chondrocyte lineages in different inductive media. However, the differentiation potential of naive DPSCs after serial passaging in the routine culture system has not been fully elucidated. Results DPSCs were isolated from human/rat dental pulps by the magnetic activated cell sorting based on STRO-1 expression, cultured and passaged in the conventional culture media. The biological features of STRO-1+ DPSCs at the 1st and 9th passages were investigated. During the long-term passage, the proliferation ability of human STRO-1+ DPSCs was downregulated as indicated by the growth kinetics. When compared with STRO-1+ DPSCs at the 1st passage (DPSC-P1, the expression of mature osteoblast-specific genes/proteins (alkaline phosphatase, bone sialoprotein, osterix, and osteopontin, odontoblast-specific gene/protein (dentin sialophosphoprotein and dentin sialoprotein, and chondrocyte-specific gene/protein (type II collagen was significantly upregulated in human STRO-1+ DPSCs at the 9th passage (DPSC-P9. Furthermore, human DPSC-P9 cells in the mineralization-inducing media presented higher levels of alkaline phosphatase at day 3 and day 7 respectively, and produced more mineralized matrix than DPSC-P9 cells at day 14. In vivo transplantation results showed that rat DPSC-P1 cell pellets developed into dentin, bone and cartilage structures respectively, while DPSC-P9 cells can only generate bone tissues. Conclusions These findings suggest that STRO-1+ DPSCs consist of several interrelated subpopulations which can spontaneously differentiate into odontoblasts, osteoblasts, and chondrocytes. The differentiation capacity of these DPSCs changes during cell passaging, and DPSCs at the 9th passage restrict their differentiation potential to the osteoblast lineage in vivo.

  8. Influence of high glucose and advanced glycation end-products (ages) levels in human osteoblast-like cells gene expression.

    Science.gov (United States)

    Miranda, Cristina; Giner, Mercè; Montoya, M José; Vázquez, M Angeles; Miranda, M José; Pérez-Cano, Ramón

    2016-08-31

    Type 2 diabetes mellitus (T2DM) is associated with an increased risk of osteoporotic fracture. Several factors have been identified as being potentially responsible for this risk, such as alterations in bone remodelling that may have been induced by changes in circulating glucose or/and by the presence of non-oxidative end products of glycosylation (AGEs). The aim of this study is to assess whether such variations generate a change in the gene expression related to the differentiation and osteoblast activity (OPG, RANKL, RUNX2, OSTERIX, and AGE receptor) in primary cultures of human osteoblast-like cells (hOB). We recruited 32 patients; 10 patients had osteoporotic hip fractures (OP group), 12 patients had osteoporotic hip fractures with T2DM (T2DM group), and 10 patients had hip osteoarthritis (OA group) with no osteoporotic fractures and no T2DM. The gene expression was analyzed in hOB cultures treated with physiological glucose concentration (4.5 mM) as control, high glucose (25 mM), and high glucose plus AGEs (2 μg/ml) for 24 h. The hOB cultures from patients with hip fractures presented slower proliferation. Additionally, the hOB cultures from the T2DM group were the most negatively affected with respect to RUNX2 and OSX gene expression when treated solely with high glucose or with high glucose plus AGEs. Moreover, high levels of glucose induced a major decrease in the RANKL/OPG ratio when comparing the OP and the T2DM groups to the OA group. Our data indicates an altered bone remodelling rate in the T2DM group, which may, at least partially, explain the reduced bone strength and increased incidence of non-traumatic fractures in diabetic patients.

  9. Mechanisms regulating osteoblast response to surface microtopography and vitamin D

    Science.gov (United States)

    Bell, Bryan Frederick, Jr.

    A comprehensive understanding of the interactions between orthopaedic and dental implant surfaces with the surrounding host tissue is essential in the design of advanced biomaterials that better promote bone growth and osseointegration of implants. Dental implants with roughened surfaces and high surface energy are well known to promote osteoblast differentiation in vitro and promote increased bone-to-implant contact in vivo. In addition, increased surface roughness increases osteoblasts response to the vitamin D metabolite 1alpha,25(OH)2D3. However, the exact mechanisms mediating cell response to surface properties and 1alpha,25(OH)2D3 are still being elucidated. The central aim of the thesis is to investigate whether integrin signaling in response to rough surface microtopography enhances osteoblast differentiation and responsiveness to 1alpha,25(OH)2D3. The hypothesis is that the integrin alpha5beta1 plays a role in osteoblast response to surface microtopography and that 1alpha,25(OH) 2D3 acts through VDR-independent pathways involving caveolae to synergistically enhance osteoblast response to surface roughness and 1alpha,25(OH) 2D3. To test this hypothesis the objectives of the studies performed in this thesis were: (1) to determine if alpha5beta 1 signaling is required for osteoblast response to surface microstructure; (2) to determine if increased responsiveness to 1alpha,25(OH)2D 3 requires the vitamin D receptor, (3) to determine if rough titanium surfaces functionalized with the peptides targeting integrins (RGD) and transmembrane proteoglycans (KRSR) will enhance both osteoblast proliferation and differentiation, and (4) to determine whether caveolae, which are associated with integrin and 1alpha,25(OH)2D3 signaling, are required for enhance osteogenic response to surface microstructure and 1alpha,25(OH)2D 3. The results demonstrate that integrins, VDR, and caveolae play important roles in mediating osteoblast response to surface properties and 1alpha,25

  10. Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice

    International Nuclear Information System (INIS)

    Otsuru, Satoru; Tamai, Katsuto; Yamazaki, Takehiko; Yoshikawa, Hideki; Kaneda, Yasufumi

    2007-01-01

    Recent studies have suggested the existence of osteoblastic cells in the circulation, but the origin and role of these cells in vivo are not clear. Here, we examined how these cells contribute to osteogenesis in a bone morphogenetic protein (BMP)-induced model of ectopic bone formation. Following lethal dose-irradiation and subsequent green fluorescent protein-transgenic bone marrow cell-transplantation (GFP-BMT) in mice, a BMP-2-containing collagen pellet was implanted into muscle. Three weeks later, a significant number of GFP-positive osteoblastic cells were present in the newly generated ectopic bone. Moreover, peripheral blood mononuclear cells (PBMNCs) from the BMP-2-implanted mouse were then shown to include osteoblast progenitor cells (OPCs) in culture. Passive transfer of the PBMNCs isolated from the BMP-2-implanted GFP-mouse to the BMP-2-implanted nude mouse led to GFP-positive osteoblast accumulation in the ectopic bone. These data provide new insight into the mechanism of ectopic bone formation involving bone marrow-derived OPCs in circulating blood

  11. 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

  12. 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

  13. Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche.

    Science.gov (United States)

    Zhang, Wenting; Gu, Yexin; Sun, Qiaoling; Siegel, David S; Tolias, Peter; Yang, Zheng; Lee, Woo Y; Zilberberg, Jenny

    2015-01-01

    We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC) using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1) optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2) replenishing OSB during culture as a practical means of prolonging MMC's viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses.

  14. Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche.

    Directory of Open Access Journals (Sweden)

    Wenting Zhang

    Full Text Available We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC using an osteoblast (OSB-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1 optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2 replenishing OSB during culture as a practical means of prolonging MMC's viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses.

  15. Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Jensen, Charlotte H; Gutierrez, Gloria

    2004-01-01

    Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. INTRODUCTION......: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. MATERIALS AND METHODS: As a model for hMSCs, we have...... was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. RESULTS: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high...

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

    Directory of Open Access Journals (Sweden)

    Jinhu Xiong

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

  17. In Vitro Evaluation the Influence of Glass-Ceramic Degradation Products on Osteoblast Cells.

    Directory of Open Access Journals (Sweden)

    Israa K. Sabree

    2016-03-01

    Full Text Available Regenerative medicine focuses on using biomaterials as three-dimensional (3D porous scaffolds, specifically designed to mimic the nature of host tissue and hence to promote cell growth and tissue regeneration. 3D bioactive glass-ceramic scaffolds are one of the most frequently studied types of scaffolds for bone tissue engineering because of their excellent bioactivity and potential for stimulating osteogenesis and angiogenesis. For such purposes, porous 3D 70%SiO2-30%CaO bioactive glass-ceramic scaffolds with three different pore sizes and identical porosity are used in present study to investigate In vitro, the effect of pore size on the degradation rate of scaffold which is achieved through examining changes in the composition of the immersion solution(SBF, simulated body fluid, and to investigate the action of released ions from the bioactive glass-ceramic scaffold during soaking process on osteoblast cells The results confirmed that all three scaffolds behaved in a similar manner and the ions release from the three scaffolds were of comparable concentration, which may be attributable to the identical porosity for all the scaffolds in addition to the using static immersion which delays ions diffusion. The pH of culture media increased from 7.6 to 8.2 after one day soaking. The optical microscopy images demonstrated that high ion concentration (Si, Ca, P in the culture medium could have a negative effect on the cells and induce cell death, while low concentration of ionic dissolution products induces osteoblast proliferation in dilute culture medium.

  18. LncRNA TUG1 sponges miR-204-5p to promote osteoblast differentiation through upregulating Runx2 in aortic valve calcification.

    Science.gov (United States)

    Yu, Cong; Li, Lifu; Xie, Fei; Guo, Shichao; Liu, Fayuan; Dong, Nianguo; Wang, Yongjun

    2018-01-01

    Emerging evidence indicates that long non-coding RNAs (lncRNAs) play a vital role in cardiovascular physiology and pathology. Although the lncRNA TUG1 is implicated in atherosclerosis, its function in calcific aortic valve disease (CAVD) remains unknown. In this study, we found that TUG1 was highly expressed in human aortic valves and primary valve interstitial cells (VICs). Moreover, TUG1 knockdown induced inhibition of osteoblast differentiation in CAVD both in vitro and in vivo. Mechanistically, silencing of TUG1 increased the expression of miR-204-5p and subsequently inhibited Runx2 expression at the post-transcriptional level. Importantly, TUG1 directly interacted with miR-204-5p and downregulation of miR-204-5p efficiently reversed the suppression of Runx2 induced by TUG1 short hairpin RNA (shRNA). Thus, TUG1 positively regulated the expression of Runx2, through sponging miR-204-5p, and promoted osteogenic differentiation in CAVD. All together, the evidence generated by our study elucidates the role of lncRNA TUG1 as a miRNA sponge in CAVD, and sheds new light on lncRNA-directed diagnostics and therapeutics in CAVD. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions please email: journals.permissions@oup.com.

  19. 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

  20. 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)

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

    Directory of Open Access Journals (Sweden)

    Chi Zhang

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

  2. Caffeine Induces Cell Death via Activation of Apoptotic Signal and Inactivation of Survival Signal in Human Osteoblasts

    Directory of Open Access Journals (Sweden)

    Wen-Hsiung Chan

    2008-05-01

    Full Text Available Caffeine consumption is a risk factor for osteoporosis, but the precise regulatory mechanisms are currently unknown. Here, we show that cell viability decreases in osteoblasts treated with caffeine in a dose-dependent manner. This cell death is attributed primarily to apoptosis and to a smaller extent, necrosis. Moreover, caffeine directly stimulates intracellular oxidative stress. Our data support caffeine-induced apoptosis in osteoblasts via a mitochondria-dependent pathway. The apoptotic biochemical changes were effectively prevented upon pretreatment with ROS scavengers, indicating that ROS plays a critical role as an upstream controller in the caffeine-induced apoptotic cascade. Additionally, p21-activated protein kinase 2 (PAK2 and c-Jun N-terminal kinase (JNK were activated in caffeine-treated osteoblasts. Experiments further found that PAK2 activity is required for caffeine-induced JNK activation and apoptosis. Importantly, our data also show that caffeine triggers cell death via inactivation of the survival signal, including the ERK- and Akt-mediated anti-apoptotic pathways. Finally, exposure of rats to dietary water containing 10~20 μM caffeine led to bone mineral density loss. These results demonstrate for the first time that caffeine triggers apoptosis in osteoblasts via activation of mitochondria-dependent cell death signaling and inactivation of the survival signal, and causes bone mineral density loss in vivo.

  3. The influence of the crystallinity of electrostatic spray deposition-derived coatings on osteoblast-like cell behavior, in vitro.

    NARCIS (Netherlands)

    Siebers, M.C.; Walboomers, X.F.; Leeuwenburgh, S.C.G.; Wolke, J.G.C.; Jansen, J.A.

    2006-01-01

    This article describes the influence of the crystallinity of carbonate apatite (CA) coatings on osteoblast-like cell behavior. Porous CA coatings were produced with electrostatic spray deposition (ESD), and subsequently, received heat treatments of 400, 500, or 700 degrees C to induce various

  4. Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells

    Science.gov (United States)

    Cook, T. F.; Burke, J. S.; Bergman, K. D.; Quinn, C. O.; Jeffrey, J. J.; Partridge, N. C.

    1994-01-01

    Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.

  5. 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.

  6. Cell-metal interactions: A comparison of natural uranium to other common metals in renal cells and bone osteoblasts

    International Nuclear Information System (INIS)

    Milgram, S.; Carriere, M.; Thiebault, C.; Berger, P.; Khodja, H.; Gouget, B.

    2007-01-01

    Uranium acute intoxication has been documented to induce nephrotoxicity. Kidneys are the main target organs after short term exposures to high concentrations of the toxic, while chronic exposures lead to its accumulation in the skeleton. In this paper, chemical toxicity of uranium is investigated for rat osteoblastic bone cells and compared to results previously obtained on renal cells. We show that bone cells are less sensitive to uranium than renal cells. The influence of the chemical form on U cytotoxicity is demonstrated. For both cell types, a comparison of uranium toxicity with other metals or metalloids toxicities (Mn, Ni, Co, Cu, Zn, Se and Cd) permits classification of Cd, Zn, Se IV and Cu as the most toxic and Ni, Se VI , Mn and U as the least toxic. Chemical toxicity of natural uranium proves to be far less than that of cadmium. To try to explain the differences in sensitivities observed between metals and different cell types, cellular accumulations in cell monolayers are quantified by inductively coupled plasma-mass spectroscopy (ICP-MS), function of time or function of dose: lethal doses which simulate acute intoxications and sub-lethal doses which are more realistic with regard to environmentally metals concentrations. In addition to being more resistant, bone cells accumulated much more uranium than did renal cells. Moreover, for both cell models, Mn, U-citrate and U-bicarbonate are strongly accumulated whereas Cu, Zn and Ni are weakly accumulated. On the other hand, a strong difference in Cd behaviour between the two cell types is shown: whereas Cd is very weakly accumulated in bone cells, it is very strongly accumulated in renal cells. Finally, elemental distribution of the toxics is determined on a cellular scale using nuclear microprobe analysis. For both renal and osteoblastic cells, uranium was accumulated in as intracellular precipitates similar to those observed previously by SEM/EDS

  7. Cell-metal interactions: A comparison of natural uranium to other common metals in renal cells and bone osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Milgram, S. [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France); Carriere, M. [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France); Thiebault, C. [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France); Berger, P. [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France); Khodja, H. [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France); Gouget, B. [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France)]. E-mail: barbara.gouget@cea.fr

    2007-07-15

    Uranium acute intoxication has been documented to induce nephrotoxicity. Kidneys are the main target organs after short term exposures to high concentrations of the toxic, while chronic exposures lead to its accumulation in the skeleton. In this paper, chemical toxicity of uranium is investigated for rat osteoblastic bone cells and compared to results previously obtained on renal cells. We show that bone cells are less sensitive to uranium than renal cells. The influence of the chemical form on U cytotoxicity is demonstrated. For both cell types, a comparison of uranium toxicity with other metals or metalloids toxicities (Mn, Ni, Co, Cu, Zn, Se and Cd) permits classification of Cd, Zn, Se{sup IV} and Cu as the most toxic and Ni, Se{sup VI}, Mn and U as the least toxic. Chemical toxicity of natural uranium proves to be far less than that of cadmium. To try to explain the differences in sensitivities observed between metals and different cell types, cellular accumulations in cell monolayers are quantified by inductively coupled plasma-mass spectroscopy (ICP-MS), function of time or function of dose: lethal doses which simulate acute intoxications and sub-lethal doses which are more realistic with regard to environmentally metals concentrations. In addition to being more resistant, bone cells accumulated much more uranium than did renal cells. Moreover, for both cell models, Mn, U-citrate and U-bicarbonate are strongly accumulated whereas Cu, Zn and Ni are weakly accumulated. On the other hand, a strong difference in Cd behaviour between the two cell types is shown: whereas Cd is very weakly accumulated in bone cells, it is very strongly accumulated in renal cells. Finally, elemental distribution of the toxics is determined on a cellular scale using nuclear microprobe analysis. For both renal and osteoblastic cells, uranium was accumulated in as intracellular precipitates similar to those observed previously by SEM/EDS.

  8. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    International Nuclear Information System (INIS)

    Wu, Li-An; Yuan, Guohua; Yang, Guobin; Ortiz-Gonzalez, Iris; Yang, Wuchen; Cui, Yong; MacDougall, Mary; Donly, Kevin J.; Harris, Stephen; Chen, Shuo

    2009-01-01

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  9. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Li-An [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi-an (China); Yuan, Guohua; Yang, Guobin [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Key Laboratory of Oral Biomedical Engineering Ministry of Education, Wuhan (China); Ortiz-Gonzalez, Iris [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Yang, Wuchen; Cui, Yong [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); MacDougall, Mary [Department of Oral/Maxillofacial Surgery, University of Alabama, Birmingham, AL (United States); Donly, Kevin J. [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Harris, Stephen [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); Chen, Shuo, E-mail: chens0@uthscsa.edu [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States)

    2009-08-14

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  10. Effects of space-relevant radiation on pre-osteoblasts

    International Nuclear Information System (INIS)

    Hu, Yueyuan

    2014-01-01

    Until now limited research has been conducted to address the mechanisms leading ionizing radiation exposure induced bone loss. This is relevant for cancer radiotherapy and human spaceflight. Exposure to radiation can result in elevated bone fracture risk in patients receiving cancer radiotherapy. In human spaceflight, astronauts are exposed to space radiation which is a very complex mixture consisting primarily of high-energy charged particles. Osteoblasts are of mesenchymal origin and responsible for creating and maintaining skeletal architecture; these cells produce extracellular matrix proteins and regulators of matrix mineralization during initial bone formation and later bone remodeling. The aim of this work was to investigate the effects of ionizing radiation on pre-osteoblasts including cellular survival, cell cycle regulation and differentiation modification. Experiments with the pre-osteoblast cell line OCT-1 and the mesenchymal stem cell line C3H10T1/2 showed that radiation cell killing depends on dose and linear energy transfer (LET) and is most effective at an LET of ∝150 keV/μm. High-LET radiation has a much more pronounced ability to induce cell cycle arrest in the G2/M phase. After both X-rays and heavy ions exposure, expression of the cell cycle regulator CDKN1A was significantly up-regulated in a dose-dependent manner. The findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression. Radiation exposure enhances osteoblastic differentiation and maturation, and mediates Runx2 and TGF-β1 expression during early differentiation of pre-osteoblasts. Osteogenic differentiation did not alter cellular radiosensitivity, DNA repair of radiation-induced damages and the effects of radiation on proliferation. Further experiments are needed to elucidate possible synergistic effects of microgravity and radiation on osteoblast differentiation. This may

  11. Effects of space-relevant radiation on pre-osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Yueyuan

    2014-02-12

    Until now limited research has been conducted to address the mechanisms leading ionizing radiation exposure induced bone loss. This is relevant for cancer radiotherapy and human spaceflight. Exposure to radiation can result in elevated bone fracture risk in patients receiving cancer radiotherapy. In human spaceflight, astronauts are exposed to space radiation which is a very complex mixture consisting primarily of high-energy charged particles. Osteoblasts are of mesenchymal origin and responsible for creating and maintaining skeletal architecture; these cells produce extracellular matrix proteins and regulators of matrix mineralization during initial bone formation and later bone remodeling. The aim of this work was to investigate the effects of ionizing radiation on pre-osteoblasts including cellular survival, cell cycle regulation and differentiation modification. Experiments with the pre-osteoblast cell line OCT-1 and the mesenchymal stem cell line C3H10T1/2 showed that radiation cell killing depends on dose and linear energy transfer (LET) and is most effective at an LET of ∝150 keV/μm. High-LET radiation has a much more pronounced ability to induce cell cycle arrest in the G2/M phase. After both X-rays and heavy ions exposure, expression of the cell cycle regulator CDKN1A was significantly up-regulated in a dose-dependent manner. The findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression. Radiation exposure enhances osteoblastic differentiation and maturation, and mediates Runx2 and TGF-β1 expression during early differentiation of pre-osteoblasts. Osteogenic differentiation did not alter cellular radiosensitivity, DNA repair of radiation-induced damages and the effects of radiation on proliferation. Further experiments are needed to elucidate possible synergistic effects of microgravity and radiation on osteoblast differentiation. This may

  12. Biological Effects of Osteoblast-Like Cells on Nanohydroxyapatite Particles at a Low Concentration Range

    Directory of Open Access Journals (Sweden)

    Xiaochen Liu

    2011-01-01

    Full Text Available The biological effects of osteoblast-like MG-63 cells on nanohydroxyapatite (n-HA at the low concentration range (5–25 g/mL for 5 days was investigated. The results showed the viability and actin cytoskeleton of the cells descended with the increase of the concentration of n-HA, and the actin cytoskeleton of cells was depolymerised and became more disordered. Apoptotic rate of cells (1.85%, 1.99%, and 2.29% increased with the increase of n-HA concentration (5, 15, and 25 g/mL and become significantly higher than the control. Total intracellular protein content decreased with n-HA concentration increase, showing significant difference between 25 g/mL and the control, and no significant change of ALP activity was observed at the 5th day. The results revealed that the cell growth was inhibited by n-HA in a concentration-dependent manner, and the obvious biological effects of MG-63 cells on n-HA existed at the low concentration range from 5 to 25 g/mL.

  13. Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells

    Science.gov (United States)

    Drury, Jeanie L.; Moussi, Joelle; Taylor-Pashow, Kathryn M. L.

    2016-01-01

    Monosodium titanates (MST) are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II). In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II) complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II) complexes for 24–72 h. A CellTiter-Blue® assay was employed to assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II) suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II) suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II) are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes. PMID:28044136

  14. Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells

    Directory of Open Access Journals (Sweden)

    Yen-Wei Chen

    2016-01-01

    Full Text Available Monosodium titanates (MST are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II. In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II complexes for 24–72 h. A CellTiter-Blue® assay was employed to assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes.

  15. Parathyroid hormone depresses cytosolic pH and DNA synthesis in osteoblast-like cells

    International Nuclear Information System (INIS)

    Reid, I.R.; Civitelli, R.; Avioli, L.V.; Hruska, K.A.

    1988-01-01

    It has recently become apparent that a number of hormones and growth factors modulate cytosolic pH (pH i ) and there is some evidence that this in turn may influence cell growth. The authors have examined the effects of parathyroid hormone (PTH) on both these parameters in an osteoblast-like cell line, UMR 106. Preliminary studies, using the pH-sensitive fluorescent probe 2',7'-bis(2-carboxyethyl)-5,(6)-carboxyfluorescein indicated that these cells regulate pH i by means of an amiloride-inhibitable Na + -H + exchanger. Rat PTH-(1-34) (rPTH) caused a progressive dose-related decrease in pH i with a half-maximal effect at 10 -11 M. The diacylglycerol analogue, phorbol 12-myristate 13-acetate, increased both pH i and [ 3 H]thymidine incorporation, and amiloride reduced both indexes. However, rPTH remained a potent inhibitor of [ 3 H]thymidine incorporation in the presence of amiloride, even though it did not affect pH i in these circumstances. It is concluded that PTH decreases pH i and growth in UMR 106 cells but that these changes can be dissociated. Depression of pH i may have other important effects on bone metabolism, such as reducing cell-cell communication, and may be associated with alkalinization of the bone fluid compartment

  16. 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

  17. 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

  18. Osteoblast</