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Sample records for human osteoblast cells

  1. 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

  2. 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.

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

    Science.gov (United States)

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

    2009-02-01

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

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

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    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.

  5. 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

  6. 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.

  7. 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.

  8. 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)

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

    Science.gov (United States)

    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.

  10. 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

  11. 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.

  12. 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...

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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...

  1. 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.

  2. 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...

  3. 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.

  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. 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.

  6. Effects of orbital spaceflight on human osteoblastic cell physiology and gene expression

    Science.gov (United States)

    Harris, S. A.; Zhang, M.; Kidder, L. S.; Evans, G. L.; Spelsberg, T. C.; Turner, R. T.

    2000-01-01

    During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation. It is not clear, however, whether the force imparted by gravity has direct effects on bone cells. To examine the response of bone forming cells to weightlessness, human fetal osteoblastic (hFOB) cells were cultured during the 17 day STS-80 space shuttle mission. Fractions of conditioned media were collected during flight and shortly after landing for analyses of glucose utilization and accumulation of type I collagen and prostaglandin E(2) (PGE(2)). Total cellular RNA was isolated from flight and ground control cultures after landing. Measurement of glucose levels in conditioned media indicated that glucose utilization occurred at a similar rate in flight and ground control cultures. Furthermore, the levels of type I collagen and PGE(2) accumulation in the flight and control conditioned media were indistinguishable. The steady-state levels of osteonectin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) were not significantly changed following spaceflight. Gene-specific reductions in mRNA levels for cytokines and skeletal growth factors were detected in the flight cultures using RNase protection assays. Steady-state mRNA levels for interleukin (IL)-1alpha and IL-6 were decreased 8 h following the flight and returned to control levels at 24 h postflight. Also, transforming growth factor (TGF)-beta(2) and TGF-beta(1) message levels were modestly reduced at 8 h and 24 h postflight, although the change was not statistically significant at 8 h. These data suggest that spaceflight did not significantly affect hFOB cell proliferation, expression of type I collagen, or PGE(2) production, further suggesting that the removal of osteoblastic cells from the context of the bone tissue results in a reduced ability to respond to weightlessness. However, spaceflight followed by return to earth significantly impacted the expression of cytokines and skeletal growth factors

  7. 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

  8. 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

  9. Effects of Curcumin on the Proliferation and Mineralization of Human Osteoblast-Like Cells: Implications of Nitric Oxide

    Directory of Open Access Journals (Sweden)

    Juan D. Pedrera-Zamorano

    2012-11-01

    Full Text Available Curcumin (diferuloylmethane is found in the rhizomes of the turmeric plant (Curcuma longa L. and has been used for centuries as a dietary spice and as a traditional Indian medicine used to treat different conditions. At the cellular level, curcumin modulates important molecular targets: transcription factors, enzymes, cell cycle proteins, cytokines, receptors and cell surface adhesion molecules. Because many of the curcumin targets mentioned above participate in the regulation of bone remodeling, curcumin may affect the skeletal system. Nitric oxide (NO is a gaseous molecule generated from L-arginine during the catalization of nitric oxide synthase (NOS, and it plays crucial roles in catalization and in the nervous, cardiovascular and immune systems. Human osteoblasts have been shown to express NOS isoforms, and the exact mechanism(s by which NO regulates bone formation remain unclear. Curcumin has been widely described to inhibit inducible nitric oxide synthase expression and nitric oxide production, at least in part via direct interference in NF-κB activation. In the present study, after exposure of human osteoblast-like cells (MG-63, we have observed that curcumin abrogated inducible NOS expression and decreased NO levels, inhibiting also cell prolifieration. This effect was prevented by the NO donor sodium nitroprusside. Under osteogenic conditions, curcumin also decreased the level of mineralization. Our results indicate that NO plays a role in the osteoblastic profile of MG-63 cells.

  10. Influence of the fetal bovine serum proteins on the growth of human osteoblast cells on graphene

    Czech Academy of Sciences Publication Activity Database

    Kalbáčová, M.; Brož, A.; Kalbáč, Martin

    100A, č. 11 (2012), s. 3001-3007 ISSN 1549-3296 R&D Projects: GA AV ČR IAA400400911; GA AV ČR KAN200100801; GA ČR GAP204/10/1677; GA ČR(CZ) GAP208/12/1062; GA MŠk ME09060 Institutional support: RVO:61388955 Keywords : human osteoblast * graphene * fetal bovine serum Subject RIV: CG - Electrochemistry Impact factor: 2.834, year: 2012

  11. Interaction of human osteoblast-like Saos-2 cells with stainless steel coated by silicalite-1 films

    Czech Academy of Sciences Publication Activity Database

    Jirka, Ivan; Vandrovcová, Marta; Plšek, Jan; Bouša, Milan; Brabec, Libor; Dragounová, Helena; Bačáková, Lucie

    2017-01-01

    Roč. 76, JUL 2017 (2017), s. 775-781 ISSN 0928-4931 R&D Projects: GA ČR(CZ) GA16-02681S Institutional support: RVO:61388955 ; RVO:67985823 Keywords : silicalite-1 film * biocompatibility * human osteoblast-like Saos-2 cells Subject RIV: CF - Physical ; Theoretical Chemistry; EI - Biotechnology ; Bionics (FGU-C) OBOR OECD: Physical chemistry; Biomaterials (as related to medical implants, devices, sensors) (FGU-C)

  12. 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

  13. 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

  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.

  15. Impact of silk fibroin-based scaffold structures on human osteoblast MG63 cell attachment and proliferation

    Directory of Open Access Journals (Sweden)

    Varkey A

    2015-10-01

    Full Text Available Aneesia Varkey,1,2 Elakkiya Venugopal,2 Ponjanani Sugumaran,2 Gopinathan Janarthanan,1 Mamatha M Pillai,2 Selvakumar Rajendran,2 Amitava Bhattacharyya1 1Advanced Textile and Polymer Research Laboratory, 2Tissue Engineering Laboratory, PSG Institute of Advanced Studies, Coimbatore, Tamil Nadu, India Abstract: The present study was carried out to investigate the impact of various types of silk fibroin (SF scaffolds on human osteoblast-like cell (MG63 attachment and proliferation. SF was isolated from Bombyx mori silk worm cocoons after degumming. Protein concentration in the degummed SF solution was estimated using Bradford method. Aqueous SF solution was used to fabricate three different types of scaffolds, viz, electrospun nanofiber mat, sponge, and porous film. The structures of the prepared scaffolds were characterized using optical micro­scopy and field emission scanning electron microscopy. The changes in the secondary structure of the proteins and the thermal behavior of the scaffolds were determined by Fourier transform infrared spectroscopy and thermo-gravimetric analysis, respectively. The biodegradation rate of scaffolds was determined by incubating the scaffolds in simulated body fluid for 4 weeks. MG63 cells were seeded on the scaffolds and their attachment and proliferation onto the scaffolds were studied. The MTT assay was carried out to deduce the toxicity of the developed scaffolds. All the scaffolds were found to be biocompatible. The amount of collagen produced by the osteoblast-like cells growing on different scaffolds was estimated. Keywords: silk fibroin scaffold, electrospun nanofiber, porous film, sponge, osteoblast

  16. 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.

  17. 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...

  18. 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...

  19. Procalcitonin NH2-terminal cleavage peptide has no mitogenic effect on normal human osteoblast-like cells

    International Nuclear Information System (INIS)

    Hassager, C.; Bonde, S.K.; Anderson, M.A.; Rink, H.; Spelsberg, T.C.; Riggs, B.L.

    1991-01-01

    The NH2-terminal cleavage peptide of procalcitonin (N-proCT) recently was reported to be a bone cell mitogen. The authors have investigated the effect of N-proCT on the proliferation of normal human cells that have the phenotype of mature osteoblasts (hOB cells). N-proCT treatment for 24, 48, or 96 h in concentrations from 1 nM to 1 microM did not significantly increase [3H]thymidine uptake (means ranged from -19% to 38% of control, no significant differences) in hOB cells (6-10 cell strains per experiment) plated at four different densities. However, the hOB cells responded significantly to treatment with transforming growth factor β (3 ng/ml), bovine insulin (300 micrograms/ml), or 30% fetal calf serum, which were included in all experiments as positive controls. The [3H]thymidine uptake data were confirmed in a direct cell count experiment tested at 96 h. Thus they data do not support the hypothesis that N-proCT is a potent mitogen for normal human osteoblasts

  20. 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

  1. 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.

  2. 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.

  3. 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...

  4. Titanium dioxide nanoparticles enhance production of superoxide anion and alter the antioxidant system in human osteoblast cells

    Directory of Open Access Journals (Sweden)

    Niska K

    2015-02-01

    Full Text Available Karolina Niska,1 Katarzyna Pyszka,1 Cecylia Tukaj,2 Michal Wozniak,1 Marek Witold Radomski,3–5 Iwona Inkielewicz-Stepniak1 1Department of Medical Chemistry, 2Department of Electron Microscopy, Medical University of Gdansk, Gdansk, Poland; 3School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, The University of Dublin Trinity College, Dublin, Ireland; 4Kardio-Med Silesia, 5Silesian Medical University, Zabrze, Poland Abstract: Titanium dioxide (TiO2 nanoparticles (NPs are manufactured worldwide for a variety of engineering and bioengineering applications. TiO2NPs are frequently used as a material for orthopedic implants. However, to the best of our knowledge, the biocompatibility of TiO2NPs and their effects on osteoblast cells, which are responsible for the growth and remodeling of the human skeleton, have not been thoroughly investigated. In the research reported here, we studied the effects of exposing hFOB 1.19 human osteoblast cells to TiO2NPs (5–15 nm for 24 and 48 hours. Cell viability, alkaline phosphatase (ALP activity, cellular uptake of NPs, cell morphology, superoxide anion (O2•- generation, superoxide dismutase (SOD activity and protein level, sirtuin 3 (SIR3 protein level, correlation between manganese (Mn SOD and SIR, total antioxidant capacity, and malondialdehyde were measured following exposure of hFOB 1.19 cells to TiO2NPs. Exposure of hFOB 1.19 cells to TiO2NPs resulted in: (1 cellular uptake of NPs; (2 increased cytotoxicity and cell death in a time- and concentration-dependent manner; (3 ultrastructure changes; (4 decreased SOD and ALP activity; (5 decreased protein levels of SOD1, SOD2, and SIR3; (6 decreased total antioxidant capacity; (7 increased O2•- generation; and (8 enhanced lipid peroxidation (malondialdehyde level. The linear relationship between the protein level of MnSOD and SIR3 and between O2•- content and SIR3 protein level was observed. Importantly, the cytotoxic

  5. Titanium dioxide nanoparticles enhance production of superoxide anion and alter the antioxidant system in human osteoblast cells

    Science.gov (United States)

    Niska, Karolina; Pyszka, Katarzyna; Tukaj, Cecylia; Wozniak, Michal; Radomski, Marek Witold; Inkielewicz-Stepniak, Iwona

    2015-01-01

    Titanium dioxide (TiO2) nanoparticles (NPs) are manufactured worldwide for a variety of engineering and bioengineering applications. TiO2NPs are frequently used as a material for orthopedic implants. However, to the best of our knowledge, the biocompatibility of TiO2NPs and their effects on osteoblast cells, which are responsible for the growth and remodeling of the human skeleton, have not been thoroughly investigated. In the research reported here, we studied the effects of exposing hFOB 1.19 human osteoblast cells to TiO2NPs (5–15 nm) for 24 and 48 hours. Cell viability, alkaline phosphatase (ALP) activity, cellular uptake of NPs, cell morphology, superoxide anion (O2•−2) generation, superoxide dismutase (SOD) activity and protein level, sirtuin 3 (SIR3) protein level, correlation between manganese (Mn) SOD and SIR, total antioxidant capacity, and malondialdehyde were measured following exposure of hFOB 1.19 cells to TiO2NPs. Exposure of hFOB 1.19 cells to TiO2NPs resulted in: (1) cellular uptake of NPs; (2) increased cytotoxicity and cell death in a time- and concentration-dependent manner; (3) ultrastructure changes; (4) decreased SOD and ALP activity; (5) decreased protein levels of SOD1, SOD2, and SIR3; (6) decreased total antioxidant capacity; (7) increased O2•− generation; and (8) enhanced lipid peroxidation (malondialdehyde level). The linear relationship between the protein level of MnSOD and SIR3 and between O2•− content and SIR3 protein level was observed. Importantly, the cytotoxic effects of TiO2NPs were attenuated by the pretreatment of hFOB 1.19 cells with SOD, indicating the significant role of O2•− in the cell damage and death observed. Thus, decreased expression of SOD leading to increased oxidizing stress may underlie the nanotoxic effects of TiO2NPs on human osteoblasts. PMID:25709434

  6. Micromachined Si channel width and tortuosity on human osteoblast cell attachment and proliferation

    International Nuclear Information System (INIS)

    Leber, Christopher; Choi, Hongsoo; Bose, Susmita; Bandyopadhyay, Amit

    2010-01-01

    In this study, influence of coating chemistry, channel width and tortuosity of various two-dimensional micro-channels were explored on micromachined Si using osteoblast precursor cells line 1 (OPC1). The rationale for our study is to delineate the influence of different porosity parameters on bone cell attachment and proliferation in vitro. Channel widths of 100, 200, 300, 400, and 600 μm; channel bends of 0, 1, and 2 right angles; and gold and silicon dioxide coatings on single-crystal Si were studied. Experiments were conducted with channel tops under glass covered and uncovered conditions keeping the channel depth at 220 μm. Independent samples were evaluated using SEM imaging and MTT assay to measure bone cell morphology and quantity. Images were taken of micro-channels and exterior chambers at 50x, 500x, 1000x, and 5000x magnifications. Channel and chamber cell densities were scored as follows: bare (score = 0), scattered (1), limited (2), abundant (3), and overflowing (4). Samples were then scored and statistically analyzed for major differences. In general, OPC1 cells proliferated at least 5% or better based on cell numbers under uncovered conditions than glass covered. Channel widths of 100 μm largely prohibited cell proliferation and diffusion by narrow path inhibition with the lowest average score of 1.17. Among channel bends of 0, 1, and 2 right angles, an increase in micro-channel tortuosity from 0-2 bends amplified OPC1 cell growth upwards of ∼ 6.6%. A one-way ANOVA showed significant differences in cell quantity for alternating channel tortuosity at a significance level of p < 0.05. No preference was found for gold or silicon dioxide coatings on Si for bone cell proliferation.

  7. 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.

  8. 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.

  9. 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

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

    Directory of Open Access Journals (Sweden)

    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

  11. 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...

  12. 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...

  13. In vitro biological evaluation of beta-TCP/HDPE--A novel orthopedic composite: a survey using human osteoblast and fibroblast bone cells.

    Science.gov (United States)

    Homaeigohar, S Sh; Shokrgozar, M A; Khavandi, A; Sadi, A Yari

    2008-02-01

    Beta-tricalcium phosphate reinforced high density polyethylene (beta-TCP/HDPE) was prepared to simulate bone composition and to study its capacity to act as bone tissue. This material was produced by replacing the mineral component and collagen soft tissue of the bone with beta-TCP and HDPE, respectively. The biocompatibility of the composite samples with different volume fractions of TCP (20, 30 and 40 vol %) was examined in vitro using two osteoblast cell lines G-292 and Saos-2, and also a type of fibroblast cell isolated from bone tissue, namely human bone fibroblast (HBF) by proliferation, and cell adhesion assays. Cell-material interaction with the surface of the composite samples was examined by scanning electron microscopy (SEM). The effect of beta-TCP/HDPE on the behavior of osteoblast and fibroblast cells was compared with those of composite and negative control samples; polyethylene (PE) and tissue culture polystyrene (TPS), respectively. In general, the results showed that the composite samples containing beta-TCP as reinforcement supported a higher rate of proliferation by various bone cells after 3, 7, and 14 days of incubation compared to the composite control sample. Furthermore, more osteoblast cells were attached to the surface of the composite samples when compared to the composite control samples after the above incubation periods (p HDPE composites are biocompatible, nontoxic, and act to stimulate proliferation and adhesion of the cells, whether osteoblast or fibroblast. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.

  14. 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...

  15. 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 ...

  16. Establishing quiescence in human bone marrow stem cells leads to enhanced osteoblast marker expression

    DEFF Research Database (Denmark)

    Harkness, Linda; Rumman, Mohammad; Kassem, Moustapha

    Human bone marrow stromal (skeletal) stem cells (hBMSC) are cells that retain a multi-lineage differentiation potential and are thus increasingly being investigated for use in clinical applications. In vivo BMSC, which comprise approximately 0.1% of the bone marrow compartment, are thought to mai...

  17. Different Motile Behaviors of Human Hematopoietic Stem versus Progenitor Cells at the Osteoblastic Niche

    Directory of Open Access Journals (Sweden)

    Katie Foster

    2015-11-01

    Full Text Available Despite advances in our understanding of interactions between mouse hematopoietic stem cells (HSCs and their niche, little is known about communication between human HSCs and the microenvironment. Using a xenotransplantation model and intravital imaging, we demonstrate that human HSCs display distinct motile behaviors to their hematopoietic progenitor cell (HPC counterparts, and the same pattern can be found between mouse HSCs and HPCs. HSCs become significantly less motile after transplantation, while progenitor cells remain motile. We show that human HSCs take longer to find their niche than previously expected and suggest that the niche be defined as the position where HSCs stop moving. Intravital imaging is the only technique to determine where in the bone marrow stem cells stop moving, and future analyses should focus on the environment surrounding the HSC at this point.

  18. Human alveolar bone cell proliferation, expression of osteoblastic phenotype, and matrix mineralization on porous titanium produced by powder metallurgy.

    Science.gov (United States)

    Rosa, Adalberto Luiz; Crippa, Grasiele Edilaine; de Oliveira, Paulo Tambasco; Taba, Mario; Lefebvre, Louis-Philippe; Beloti, Marcio Mateus

    2009-05-01

    This study aimed at investigating the influence of the porous titanium (Ti) structure on the osteogenic cell behaviour. Porous Ti discs were fabricated by the powder metallurgy process with the pore size typically between 50 and 400 microm and a porosity of 60%. Osteogenic cells obtained from human alveolar bone were cultured until subconfluence and subcultured on dense Ti (control) and porous Ti for periods of up to 17 days. Cultures grown on porous Ti exhibited increased cell proliferation and total protein content, and lower levels of alkaline phosphatase (ALP) activity than on dense Ti. In general, gene expression of osteoblastic markers-runt-related transcription factor 2, collagen type I, alkaline phosphatase, bone morphogenetic protein-7, and osteocalcin was lower at day 7 and higher at day 17 in cultures grown on porous Ti compared with dense Ti, a finding consistent with the enhanced growth rate for such cultures. The amount of mineralized matrix was greater on porous Ti compared with the dense one. These results indicate that the porous Ti is an appropriate substrate for osteogenic cell adhesion, proliferation, and production of a mineralized matrix. Because of the three-dimensional environment it provides, porous Ti should be considered an advantageous substrate for promoting desirable implant surface-bone interactions.

  19. 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.

  20. 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.

  1. Phenolic content of Sicilian virgin olive oils and their effect on MG-63 human osteoblastic cell proliferation

    Directory of Open Access Journals (Sweden)

    García-Martínez, O.

    2014-09-01

    Full Text Available The aim of this study was, first, to investigate the influence of olive variety and elevation of orchards on the phenolic compound content of Sicilian virgin olive oils (VOOs and, second, to investigate the effects of VOO phenolic extracts on osteoblast cell growth using the human MG-63 osteosarcoma cell line. Olive oil phenolic content and its effect on human osteosarcoma cell proliferation varied according to the type of cultivar and the grove altitude. This variation was also observed within the same type of cultivar. This observation demonstrates that the cultivar and the grove location can significantly affect the chemical composition and bioactivity of virgin olive oil. Although this study supports the hypothesis that virgin olive oil phenolic fractions exert a beneficial effect on bone health, further studies assessing the in vivo accessibility of virgin olive oil phenolic compounds to osteoblast cells should be carried out.El objetivo del presente trabajo es investigar la influencia de la variedad y la altitud del cultivo en el contenido fenólico de aceites de oliva virgen Sicilianos. Asimismo, se ha investigado el efecto de los extractos fenólicos de los aceites en el crecimiento de osteoblastos usando la línea celular de osteosarcoma humano MG-63. El contenido fenólico y el efecto de los extractos analizados en la proliferación de la línea celular osteoblástica muestra una variabilidad consistente de acuerdo con el tipo y la altitud del cultivo. Estos datos demuestran que estas características pueden afectar significativamente la composición química y los efectos en salud del aceite de oliva virgen. Los resultados de este trabajo soportan la hipótesis de que las fracciones fenólicas de los aceites de oliva vírgenes ejercen un efecto beneficioso en la salud ósea. Asimismo, se deben realizar más estudios que establezcan la accesibilidad in vivo de los compuestos fenólicos del aceite de oliva virgen a las células osteoblásticas.

  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. Transformation of human osteoblast cells to the tumorigenic phenotype by depleted uranium-uranyl chloride.

    OpenAIRE

    Miller, A C; Blakely, W F; Livengood, D; Whittaker, T; Xu, J; Ejnik, J W; Hamilton, M M; Parlette, E; John, T S; Gerstenberg, H M; Hsu, H

    1998-01-01

    Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Although the health effects of occupational uranium exposure are well known, limited data exist regarding the long-term health effects of internalized DU in humans. We established an in vitro cellular model to study DU exposure. Microdosimetric assessment, determined using a Monte Carlo computer simulation based on measured intracellular and extracellular uranium levels, showed that few (0.0014%) cell nuclei...

  4. In vitro analysis of low-level laser irradiation on human osteoblast-like cells proliferation

    Science.gov (United States)

    Bloise, Nora; Saino, Enrica; Bragheri, Francesca; Minzioni, Paolo; Cristiani, Ilaria; Imbriani, Marcello; Visai, Livia

    2011-07-01

    The objective of this study was to examine the in vitro effect of a single or a multiple doses of low-level laser irradiation (LLLI) on proliferation of the human osteosarcoma cell line, SAOS-2. SAOS-2 cells were divided in five groups and exposed to LLLI (659 nm diode laser; 11 mW power output): group I as a control (dark), group II exposed to a single laser dose of 1 J/cm2, group III irradiated with a single dose of 3 J/cm2, and group IV and V exposed for three consecutive days to 1 or 3 J/cm², respectively. Cellular proliferation was assessed daily up to 7 days of culturing. The obtained results showed an increase in proliferative capacity of SAOS-2 cells during the first 96 h of culturing time in once-irradiated cells, as compared to control cells. Furthermore, a significantly higher proliferation in the group IV and V was detected if compared to a single dose or to control group after 96 h and 7 days. In conclusion, the effect of the single dose on cell proliferation was transitory and repeated irradiations were necessary to observe a strong enhancement of SAOS-2 growth. As a future perspective, we would like to determine the potential of LLLI as a new approach for promoting bone regeneration onto biomaterials.

  5. 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...

  6. Trojan-Like Internalization of Anatase Titanium Dioxide Nanoparticles by Human Osteoblast Cells.

    Science.gov (United States)

    Ribeiro, A R; Gemini-Piperni, S; Travassos, R; Lemgruber, L; Silva, R C; Rossi, A L; Farina, M; Anselme, K; Shokuhfar, T; Shahbazian-Yassar, R; Borojevic, R; Rocha, L A; Werckmann, J; Granjeiro, J M

    2016-03-29

    Dentistry and orthopedics are undergoing a revolution in order to provide more reliable, comfortable and long-lasting implants to patients. Titanium (Ti) and titanium alloys have been used in dental implants and total hip arthroplasty due to their excellent biocompatibility. However, Ti-based implants in human body suffer surface degradation (corrosion and wear) resulting in the release of metallic ions and solid wear debris (mainly titanium dioxide) leading to peri-implant inflammatory reactions. Unfortunately, our current understanding of the biological interactions with titanium dioxide nanoparticles is still very limited. Taking this into consideration, this study focuses on the internalization of titanium dioxide nanoparticles on primary bone cells, exploring the events occurring at the nano-bio interface. For the first time, we report the selective binding of calcium (Ca), phosphorous (P) and proteins from cell culture medium to anatase nanoparticles that are extremely important for nanoparticle internalization and bone cells survival. In the intricate biological environment, anatase nanoparticles form bio-complexes (mixture of proteins and ions) which act as a kind of 'Trojan-horse' internalization by cells. Furthermore, anatase nanoparticles-induced modifications on cell behavior (viability and internalization) could be understand in detail. The results presented in this report can inspire new strategies for the use of titanium dioxide nanoparticles in several regeneration therapies.

  7. 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.

  8. The upregulation of receptor activator NF-kappaB ligand expression by interleukin-1alpha and Porphyromonas endodontalis in human osteoblastic cells.

    Science.gov (United States)

    Chen, S-C; Huang, F-M; Lee, S-S; Li, M-Z; Chang, Y-C

    2009-04-01

    To investigate the receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL) in osteoblastic cells stimulated with inflammatory mediators. The expression of RANKL in human osteoblastic cell line U2OS stimulated by pro-inflammatory cytokine interleukin (IL)-1alpha and black-pigmented bacteria Porphyromonas endodontalis was investigated by Western blot and enzyme-linked immunosorbent assay (ELISA). The significance of the results obtained from control and treated groups was statistically analysed by the paired Student's t-test. IL-1alpha was found to upregulate RANKL production in U2OS cells (P endodontalis also increased RANKL expression in U2OS cells after 4-h incubation period demonstrated by Western blot and ELISA (P endodontalis may be involved in developing apical periodontitis through the stimulation of RANKL production.

  9. 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

  10. 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.

  11. Effects on growth of human osteoblast-like cells of three nonsteroidal anti-inflammatory drugs: metamizole, dexketoprofen, and ketorolac.

    Science.gov (United States)

    De Luna-Bertos, Elvira; Ramos-Torrecillas, Javier; Manzano-Moreno, Francisco Javier; García-Martínez, Olga; Ruiz, Concepción

    2015-01-01

    Some nonsteroidal anti-inflammatory drugs (NSAIDs) have adverse effects on bone tissue. The objective of this study was to determine the effect of different doses of dexketoprofen, ketorolac, and metamizole on growth of the osteoblast MG63 cell line. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide spectrophotometry results showed that MG63 cell growth was significantly inhibited after 24 hr of culture with doses of 10, 20, 100, or 1,000 µM of each NSAID and with doses of 0.1, 1, or 5 µM of dexketoprofen and ketorolac but not metamizole. Cell-cycle studies revealed that dexketoprofen and ketorolac treatments significantly arrested the cell cycle in phase G0/G1, increasing the percentage of cells in this phase. Apoptosis/necrosis studies showed significant changes versus control cells, with an increased percentage of cells in apoptosis after treatment with 10, 100, or 1,000 µM of metamizole and after treatment with 1, 10, 100, or 1,000 µM of dexketoprofen or ketorolac. In conclusion, treatment of osteoblast-like cells with high doses of the NSAIDs tested increased not only the percentage of cells in apoptosis but also the percentage of necrotic cells. © The Author(s) 2014.

  12. TiO2 nanoparticles disrupt cell adhesion and the architecture of cytoskeletal networks of human osteoblast-like cells in a size dependent manner.

    Science.gov (United States)

    Ibrahim, Mohamed; Schoelermann, Julia; Mustafa, Kamal; Cimpan, Mihaela R

    2018-04-30

    Human exposure to titanium dioxide nanoparticles (nano-TiO 2 ) is increasing. An internal source of nano-TiO 2 is represented by titanium-based orthopedic and dental implants can release nanoparticles (NPs) upon abrasion. Little is known about how the size of NPs influences their interaction with cytoskeletal protein networks and the functional/homeostatic consequences that might follow at the implant-bone interface with regard to osteoblasts. We investigated the effects of size of anatase nano-TiO 2 on SaOS-2 human osteoblast-like cells exposed to clinically relevant concentrations (0.05, 0.5, 5 mg/L) of 5 and 40 nm spherical nano-TiO 2 . Cell viability and proliferation, adhesion, spread and migration were assessed, as well as the orientation of actin and microtubule cytoskeletal networks. The phosphorylation of focal adhesion kinase (p-FAK Y397 ) and the expression of vinculin in response to nano-TiO 2 were also assessed. Treatment with nano-TiO 2 disrupted the actin and microtubule cytoskeletal networks leading to morphological modifications of SaOS-2 cells. The phosphorylation of p-FAK Y397 and the expression of vinculin were also modified depending on the particle size, which affected cell adhesion. Consequently, the cell migration was significantly impaired in the 5 nm-exposed cells compared to unexposed cells. The present work shows that the orientation of cytoskeletal networks and the focal adhesion proteins and subsequently the adhesion, spread and migration of SaOS-2 cells were affected by the selected nano-TiO 2 in a size dependent manner. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

  13. The angiogenic behaviors of human umbilical vein endothelial cells (HUVEC) in co-culture with osteoblast-like cells (MG-63) on different titanium surfaces.

    Science.gov (United States)

    Shi, Bin; Andrukhov, Oleh; Berner, Simon; Schedle, Andreas; Rausch-Fan, Xiaohui

    2014-08-01

    Interaction between osteogenesis and angiogenesis plays an important role in implant osseointegration. In the present study we investigated the influence of titanium surface properties on the angiogenic behaviors of endothelial cells grown in direct contact co-culture with osteoblasts. Human umbilical vein endothelial cells (HUVECs) and osteoblast-like cells (MG-63 cells) were grown in direct co-culture on the following titanium surfaces: acid-etched (A), hydrophilic A (modA), coarse-gritblasted and acid-etched (SLA) and hydrophilic SLA (SLActive). Cell proliferation was evaluated by cell counting combined with flow cytometry. The expression of von Willebrand Factor (vWF), thrombomodulin (TM), endothelial cell protein C receptor (EPCR), E-Selectin, as well as vascular endothelial growth factor (VEGF) receptors Flt-1 and KDR in HUVECs and VEGF in MG-63 were measured by qPCR. The dynamic behavior of endothelial cells was recorded by time-lapse microscopy. Proliferation of HUVECs was highest on A, followed by SLA, modA and SLActive surfaces. The expression of vWF, TM, EPCR, E-Selectin and Flt-1 in HUVECs was significantly higher on A than on all other surfaces. The expression of KDR in HUVECs grown on A surface was below detection limit. VEGF expression in MG-63 cells was significantly higher on SLActive vs SLA and modA vs A surfaces. Time-lapse microscopy revealed that HUVECs moved quickest and formed cell clusters earlier on A surface, followed by SLA, modA and SLActive surface. In co-culture conditions, proliferation and expression of angiogenesis associated genes in HUVECs are promoted by smooth hydrophobic Ti surface, which is in contrast to previous mono-culture studies. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  14. 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.

  15. Zoledronic acid at subtoxic dose extends osteoblastic stage span of primary human osteoblasts.

    Science.gov (United States)

    Zara, Susi; De Colli, Marianna; di Giacomo, Viviana; Zizzari, Vincenzo Luca; Di Nisio, Chiara; Di Tore, Umberto; Salini, Vincenzo; Gallorini, Marialucia; Tetè, Stefano; Cataldi, Amelia

    2015-04-01

    This study aimed to check the effect of zoledronic acid (ZA) at subtoxic dose on human osteoblasts (HOs) in terms of cell viability, apoptosis occurrence, and differentiation induction. ZA belongs to the family of bisphosphonates (BPs), largely used in the clinical practice for the treatment of bone diseases, often associated with jaw osteonecrosis onset. Their pharmacological action consists in the direct block of the osteoclast-mediated bone resorption along with indirect action on osteoblasts. HOs were treated choosing the highest limit concentration (10(-5) M) which does not induce toxic effects. Live/dead staining, flow cytometry, mitochondrial membrane potential assay, osteocalcin western blotting, gp38 RT-PCR, collagen type I, PGE2, and IL-6 ELISA assays were performed. Similar viability level between control and ZA-treated samples is found along with no significant increase of apoptotic and necrotic cells in ZA-treated sample. To establish if an early apoptotic pathway was triggered, Bax expression and mitochondrial membrane potential were evaluated finding a higher protein expression in control sample and a good integrity of mitochondrial membrane in both experimental points. Type I collagen secretion and alkaline phosphatase (ALP) activity appear increased in ZA-treated sample, osteocalcin expression level is reduced in ZA-treated cells, whereas no modifications of gp38 mRNA level are evidenced. No statistical differences are identified in PGE2 secretion level whereas IL-6 secretion is lower in ZA-treated HOs with respect to control ones. These results highlight that ZA, delaying the osteoblastic differentiation process versus the osteocytic lineage, strengthens its pharmacological activity enhancing bone density. The knowledge of ZA effects on osteoblasts at subtoxic dose allows to improve therapeutic protocols in order to strengthen drug pharmacological activity through a combined action on both osteoclastic and osteoblastic cells.

  16. Effects of tunicamycin, mannosamine, and other inhibitors of glycoprotein processing on skeletal alkaline phosphatase in human osteoblast-like cells.

    Science.gov (United States)

    Farley, J R; Magnusson, P

    2005-01-01

    Skeletal alkaline phosphatase (sALP) is a glycoprotein- approximately 20% carbohydrate by weight, with five presumptive sites for N-linked glycosylation, as well as a carboxy-terminal site for attachment of the glycolipid structure (glycosylphosphatidylinositol, GPI), which anchors sALP to the outer surface of osteoblasts. The current studies were intended to characterize the effects of inhibiting glycosylation and glycosyl-processing on the synthesis, plasma membrane attachment, cellular-extracellular distribution, and reaction kinetics of sALP in human osteosarcoma (SaOS-2) cells. sALP synthesis, glycosylation, and GPI-anchor attachment were assessed as total protein synthesis/immunospecific sALP synthesis, sialic acid content (i.e., wheat germ agglutinin precipitation), and insolubility (i.e., temperature-dependent phase-separation), respectively. sALP reaction kinetics were characterized by analysis of dose-dependent initial velocity data, with a phosphoryl substrate. The results of these studies revealed that the inhibition of either N-linked glycosylation or oligosaccharide synthesis for GPI-anchor addition could affect the synthesis and the distribution of sALP, but not the kinetics of the phosphatase reaction. Tunicamycin-which blocks N-linked glycosylation by inhibiting core oligosaccharide synthesis-decreased cell layer protein and the total amount of sALP in the cells, while increasing the relative level of sALP in the cell-conditioned culture medium (CM, i.e., the amount of sALP released). These effects were attributed to dose- and time-dependent decreases in sALP synthesis and N-linked glycosylation, and an increase in apoptotic cell death (P sALP specific activity, in the cells and in the CM; and (3) increases in the percentages of both anchorless and wheat germ agglutinin (WGA)-soluble sALP in the medium, but not in the cells (P sALP to the outside of the plasma membrane surface. Neither mannosammine nor tunicamycin had any effect on the reaction

  17. The human umbilical cord blood: a potential source for osteoblast progenitor cells

    DEFF Research Database (Denmark)

    Kjeldsen, Cecilia Rosada; Melsvik, Dorte; Ebbesen, Peter

    2003-01-01

    -density mononuclear cells isolated from hUCB formed few adherent colonies with fibroblast-like morphology after a few days in culture. At confluence, the polyclonal cell populations were characterized. Using FACS analysis and immunocytochemistry, the cells were found to express HLA-ABC, CD9, vimentin, the b subunit...... visualized by Alizarin red staining. In the presence of normal horse serum and dexamethasone (10(-7) M), the cells formed foci of adipocytes. When the cells were implanted mixed with hydroxyapatite/tricalcium phosphate powder in the subcutis of immunocompromised mice for 8 weeks, they formed osteogenic...... tissue and a myelosupportive microenviroment that enclosed hematopoietic cells and adipocytes. Our results demonstrate the presence of circulating stem cells with osteogenic and adipogenic differentiation potential in hUCB and may encourage the use of hUCB as a potential source for stem cells...

  18. Effect of water-soluble P-chitosan and S-chitosan on human primary osteoblasts and giant cell tumor of bone stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Tang, T; Zhang, G; PY Lau, Carol; Zheng, L Z; Xie, X H; Wang, X L; Patrick, Y; Qin, L; Kumta, Shekhar M [Department of Orthopaedics and Traumatology, Chinese University of Hong Kong (Hong Kong); Wang, X H; He, K, E-mail: kumta@cuhk.edu.hk [Department of Mechanical Engineering, Institute of Bio-manufacturing Engineering, Tsinghua University, Beijing (China)

    2011-02-15

    Water-soluble phosphorylated chitosan (P-chitosan) and disodium (1 {yields} 4)-2-deoxy-2-sulfoamino-{beta}-D-glucopyranuronan (S-chitosan) are two chemically modified chitosans. In this study, we found that P-chitosan significantly promotes cell proliferation of both human primary osteoblasts (OBs) and the OB like stromal cell component of the giant cell tumor of bone (GCTB) cells at the concentration from 125 to 1000 {mu}g ml{sup -1} at all time points of 1, 3, 5 and 7 days after treatment. Further investigation of the osteogenic effect of the P-chitosan suggested that it regulates the levels of osteoclastogenic factors, receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression. An interesting finding is that S-chitosan at lower concentration (100 {mu}g ml{sup -1}) stimulates cell proliferation while a higher dose (1000 {mu}g ml{sup -1}) of S-chitosan inhibits it. The inhibitory effect of S-chitosan on human primary GCT stromal cells was greater than that of OBs (p < 0.05). Taken together, our findings elucidated the osteogenic effect of P-chitosan and the varying effects of S-chitosan on the proliferation of human primary OBs and GCT stromal cells and provided us the rationale for the construction of novel bone repair biomaterials with the dual properties of bone induction and bone tumor inhibition.

  19. 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-...

  20. 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

  1. Manipulation of Human Primary Endothelial Cell and Osteoblast Coculture Ratios to Augment Vasculogenesis and Mineralization

    Science.gov (United States)

    2014-06-01

    REFERENCES 1. Lieberman JFG, ed. Bone Regeneration and Repair: Biology and Clinical Applications. Totowa, NJ: Humana Press; 2005. 2. Lee K-B, Taghavi...2006;12:2521 2531. 22. Sieminski A, Padera R, Blunk T, et al. Systemic delivery of human growth hormone using genetically modified tissue-engineered

  2. Human osteoblast-like MG 63 cells on polysulfone modified with carbon nanotubes or carbon nanohorns

    Czech Academy of Sciences Publication Activity Database

    Staňková, Ľubica; Fraczek-Szczypta, A.; Blazewicz, M.; Filová, Elena; Blazewicz, S.; Lisá, Věra; Bačáková, Lucie

    2014-01-01

    Roč. 67, Feb 2014 (2014), s. 578-591 ISSN 0008-6223 R&D Projects: GA ČR(CZ) GAP108/12/1168 Institutional support: RVO:67985823 Keywords : carbon * nanotubes * cells Subject RIV: EI - Biotechnology ; Bionics Impact factor: 6.196, year: 2014

  3. Influence of surface roughness of carbon materials on human osteoblast-like cell growth

    Czech Academy of Sciences Publication Activity Database

    Starý, V.; Douděrová, M.; Bačáková, Lucie

    2014-01-01

    Roč. 102, č. 6 (2014), s. 1859-1879 ISSN 1549-3296 R&D Projects: GA ČR(CZ) GAP108/10/1858; GA ČR(CZ) GAP108/12/1168; GA AV ČR(CZ) KAN101120701 Institutional support: RVO:67985823 Keywords : carbon materials * roughness * biocompatibility * MG63 cells Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.369, year: 2014

  4. 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.

  5. 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

  6. 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

  7. Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells

    Directory of Open Access Journals (Sweden)

    Puri Christina

    2007-03-01

    Full Text Available Abstract Background Podoplanin is a membrane mucin that, among a series of tissues, is expressed on late osteoblasts and osteocytes. Since recent findings have focussed on podoplanin's potential role as a tumour progression factor, we aimed at identifying regulatory elements conferring PDPN promoter activity. Here, we characterized the molecular mechanism controlling basal PDPN transcription in human osteoblast-like MG63 versus Saos-2 cells. Results We cloned and sequenced 2056 nucleotides from the 5'-flanking region of the PDPN gene and a computational search revealed that the TATA and CAAT box-lacking promoter possesses features of a growth-related gene, such as a GC-rich 5' region and the presence of multiple putative Sp1, AP-4 and NF-1 sites. Reporter gene assays demonstrated a functional promoter in MG63 cells exhibiting 30-fold more activity than in Saos-2 cells. In vitro DNase I footprinting revealed eight protected regions flanked by DNaseI hypersensitive sites within the region bp -728 to -39 present in MG63, but not in Saos-2 cells. Among these regions, mutation and supershift electrophoretic mobility shift assays (EMSA identified four Sp1/Sp3 binding sites and two binding sites for yet unknown transcription factors. Deletion studies demonstrated the functional importance of two Sp1/Sp3 sites for PDPN promoter activity. Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells. In SL2 cells, Sp3 functioned as a repressor, while Sp1 and Sp3 acted positively synergistic. Weak PDPN promoter activity of Saos-2 cells correlated with low Sp1/Sp3 nuclear levels, which was confirmed by Sp1/Sp3 chromatin immunoprecipitations in vivo. Moreover, methylation-sensitive Southern blot analyses and bisulfite sequencing detected strong methylation of CpG sites upstream of bp -464 in MG63 cells, but hypomethylation of these sites in Saos-2 cells. Concomitantly

  8. 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.

  9. Three-dimensional, bioactive, biodegradable, polymer-bioactive glass composite scaffolds with improved mechanical properties support collagen synthesis and mineralization of human osteoblast-like cells in vitro.

    Science.gov (United States)

    Lu, Helen H; El-Amin, Saadiq F; Scott, Kimberli D; Laurencin, Cato T

    2003-03-01

    In the past decade, tissue engineering-based bone grafting has emerged as a viable alternative to biological and synthetic grafts. The biomaterial component is a critical determinant of the ultimate success of the tissue-engineered graft. Because no single existing material possesses all the necessary properties required in an ideal bone graft, our approach has been to develop a three dimensional (3-D), porous composite of polylactide-co-glycolide (PLAGA) and 45S5 bioactive glass (BG) that is biodegradable, bioactive, and suitable as a scaffold for bone tissue engineering (PLAGA-BG composite). The objectives of this study were to examine the mechanical properties of a PLAGA-BG matrix, to evaluate the response of human osteoblast-like cells to the PLAGA-BG composite, and to evaluate the ability of the composite to form a surface calcium phosphate layer in vitro. Structural and mechanical properties of PLAGA-BG were measured, and the formation of a surface calcium phosphate layer was evaluated by surface analysis methods. The growth and differentiation of human osteoblast-like cells on PLAGA-BG were also examined. A hypothesis was that the combination of PLAGA with BG would result in a biocompatible and bioactive composite, capable of supporting osteoblast adhesion, growth and differentiation, with mechanical properties superior to PLAGA alone. The addition of bioactive glass granules to the PLAGA matrix resulted in a structure with higher compressive modulus than PLAGA alone. Moreover, the PLAGA-BA composite was found to be a bioactive material, as it formed surface calcium phosphate deposits in a simulated body fluid (SBF), and in the presence of cells and serum proteins. The composite supported osteoblast-like morphology, stained positively for alkaline phosphatase, and supported higher levels of Type I collagen synthesis than tissue culture polystyrene controls. We have successfully developed a degradable, porous, polymer bioactive glass composite possessing

  10. Anti-citrullinated protein antibodies promote apoptosis of mature human Saos-2 osteoblasts via cell-surface binding to citrullinated heat shock protein 60.

    Science.gov (United States)

    Lu, Ming-Chi; Yu, Chia-Li; Yu, Hui-Chun; Huang, Hsien-Bin; Koo, Malcolm; Lai, Ning-Sheng

    2016-01-01

    We hypothesized that anti-citrullinated protein antibodies (ACPAs) react with osteoblast surface citrullinated proteins and affect cell function, leading to joint damage in patients with rheumatoid arthritis (RA). First, we purified ACPAs by cyclic citrullinated peptide (CCP)-conjugated affinity column chromatography. The cognate antigens of ACPAs on Saos-2 cells, a sarcoma osteogenic cell line generated from human osteoblasts, were probed by ACPAs, and the reactive bands were analyzed using proteomic analyses. We found that ACPAs bind to Saos-2 cell membrane, and several protein candidates, including HSP60, were identified. We then cloned and purified recombinant heat shock protein 60 (HSP60) and citrullinated HSP60 (citHSP60) and investigated the effect of ACPAs on Saos-2 cell. We confirmed that HSP60 obtained from Saos-2 cell membrane were citrullinated and reacted with ACPAs, which induces Saos-2 cells apoptosis via binding to surface-expressed citHSP60 through Toll-like receptor 4 signaling. ACPAs promoted interleukin (IL)-6 and IL-8 expression in Saos-2 cells. Finally, sera from patients with RA and healthy controls were examined for their titers of anti-HSP60 and anti-citHSP60 antibodies using an enzyme-linked immunosorbent assay. The radiographic change in patients with RA was evaluated using the Genant-modified Sharp scoring system. Patients with RA showed higher sera titers of anti-citHSP60, but not anti-HSP60, antibodies when compared with controls. In addition, the anti-citHSP60 level was positively associated with increased joint damage in patients with RA. In conclusion, Saos-2 cell apoptosis was mediated by ACPAs via binding to cell surface-expressed citHSP60 and the titer of anti-citHSP60 in patients with RA positively associated with joint damage. Copyright © 2015 Elsevier GmbH. All rights reserved.

  11. Attachment and proliferation of human osteoblast-like cells (MG-63) on laser-ablated titanium implant material

    International Nuclear Information System (INIS)

    Györgyey, Ágnes; Ungvári, Krisztina; Kecskeméti, Gabriella; Kopniczky, Judit; Hopp, Béla; Oszkó, Albert; Pelsöczi, István; Rakonczay, Zoltán; Nagy, Katalin; Turzó, Kinga

    2013-01-01

    Demand is increasing for shortening the long (3–6 months) osseointegration period to rehabilitate patients' damaged chewing apparatus in as short a time as possible. For dental implants, as for biomaterials in general, the bio- and osseointegration processes can be controlled at molecular and cellular levels by modification of the implant surface. One of the most promising of such surface modifications is laser ablation, as demonstrated by our previous results [46]. Commercially pure (CP4) sand-blasted, acid-etched titanium disks (Denti® System Ltd., Hungary) were irradiated with a KrF excimer laser (248 nm, fluence 0.4 J/cm 2 , FWHM 18 ns, 2000 pulses), or with a Nd:YAG laser (532 nm, 1.3 J/cm 2 , 10 ns, 200 pulses) then examined by SEM, AFM, and XPS. In vitro attachment (24 h) and proliferation (72 h) of MG-63 osteoblast cells were investigated via dimethylthiazol-diphenyl tetrazolium bromide (MTT), alamarBlue (AB) assays alkaline phosphatase quantification (ALP) and SEM. SEM and AFM revealed significant changes in morphology and roughness. XPS confirmed the presence of TiO 2 on each sample; after Nd:YAG treatment a reduced state of Ti (Ti 3+ ) was also observed. MTT, AB and ALP measurements detected an increase in the number of cells between the 24- and 72 hour observations; however, laser treatment did not affect cell attachment and proliferation significantly. - Highlights: • CP4 titanium implant surfaces were modified with Nd:YAG and KrF excimer laser. • SEM and AFM revealed significant changes in morphology and roughness. • XPS confirmed the presence of TiO 2 on each sample; after Nd:YAG treatment a reduced state of Ti (Ti 3+ ) was found. • Cell proliferation experiments detected an increased number of MG-63 cells between the 24 h and 72 h observations. • Laser treatments neither disturbed, nor enhanced MG-63 cell attachment and proliferation significantly

  12. Characteristics of minerals in vesicles produced by human osteoblasts hFOB 1.19 and osteosarcoma Saos-2 cells stimulated for mineralization.

    Science.gov (United States)

    Strzelecka-Kiliszek, Agnieszka; Bozycki, Lukasz; Mebarek, Saida; Buchet, Rene; Pikula, Slawomir

    2017-06-01

    Bone cells control initial steps of mineralization by producing extracellular matrix (ECM) proteins and releasing vesicles that trigger apatite nucleation. Using transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) we compared the quality of minerals in vesicles produced by two distinct human cell lines: fetal osteoblastic hFOB 1.19 and osteosarcoma Saos-2. Both cell lines, subjected to osteogenic medium with ascorbic acid (AA) and β-glycerophosphate (β-GP), undergo the entire osteoblastic differentiation program from proliferation to mineralization, produce the ECM and spontaneously release vesicles. We observed that Saos-2 cells mineralized better than hFOB 1.19, as probed by Alizarin Red-S (AR-S) staining, tissue nonspecific alkaline phosphatase (TNAP) activity and by analyzing the composition of minerals in vesicles. Vesicles released from Saos-2 cells contained and were surrounded by more minerals than vesicles released from hFOB 1.19. In addition, there were more F and Cl substituted apatites in vesicles from hFOB 1.19 than in those from Saos-2 cells as determined by ion ratios. Saos-2 and h-FOB 1.19 cells revealed distinct mineralization profiles, indicating that the process of mineralization may proceed differently in various types of cells. Our findings suggest that TNAP activity is correlated with the relative proportions of mineral-filled vesicles and mineral-surrounded vesicles. The origin of vesicles and their properties predetermine the onset of mineralization at the cellular level. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Surface modifications of dental ceramic implants with different glass solder matrices: in vitro analyses with human primary osteoblasts and epithelial cells.

    Science.gov (United States)

    Markhoff, Jana; Mick, Enrico; Mitrovic, Aurica; Pasold, Juliane; Wegner, Katharina; Bader, Rainer

    2014-01-01

    Ceramic materials show excellent esthetic behavior, along with an absence of hypersensitivity, making them a possible alternative implant material in dental surgery. However, their surface properties enable only limited osseointegration compared to titanium implants. Within this study, a novel surface coating technique for enhanced osseointegration was investigated biologically and mechanically. Specimens of tetragonal zirconia polycrystal (TZP) and aluminum toughened zirconia (ATZ) were modified with glass solder matrices in two configurations which mainly consisted of SiO2, Al2O3, K2O, and Na2O. The influence on human osteoblastic and epithelial cell viability was examined by means of a WST-1 assay as well as live/dead staining. A C1CP-ELISA was carried out to verify procollagen type I production. Uncoated/sandblasted ceramic specimens and sandblasted titanium surfaces were investigated as a reference. Furthermore, mechanical investigations of bilaterally coated pellets were conducted with respect to surface roughness and adhesive strength of the different coatings. These tests could demonstrate a mechanically stable implant coating with glass solder matrices. The coated ceramic specimens show enhanced osteoblastic and partly epithelial viability and matrix production compared to the titanium control. Hence, the new glass solder matrix coating could improve bone cell growth as a prerequisite for enhanced osseointegration of ceramic implants.

  14. Surface Modifications of Dental Ceramic Implants with Different Glass Solder Matrices: In Vitro Analyses with Human Primary Osteoblasts and Epithelial Cells

    Science.gov (United States)

    Mick, Enrico

    2014-01-01

    Ceramic materials show excellent esthetic behavior, along with an absence of hypersensitivity, making them a possible alternative implant material in dental surgery. However, their surface properties enable only limited osseointegration compared to titanium implants. Within this study, a novel surface coating technique for enhanced osseointegration was investigated biologically and mechanically. Specimens of tetragonal zirconia polycrystal (TZP) and aluminum toughened zirconia (ATZ) were modified with glass solder matrices in two configurations which mainly consisted of SiO2, Al2O3, K2O, and Na2O. The influence on human osteoblastic and epithelial cell viability was examined by means of a WST-1 assay as well as live/dead staining. A C1CP-ELISA was carried out to verify procollagen type I production. Uncoated/sandblasted ceramic specimens and sandblasted titanium surfaces were investigated as a reference. Furthermore, mechanical investigations of bilaterally coated pellets were conducted with respect to surface roughness and adhesive strength of the different coatings. These tests could demonstrate a mechanically stable implant coating with glass solder matrices. The coated ceramic specimens show enhanced osteoblastic and partly epithelial viability and matrix production compared to the titanium control. Hence, the new glass solder matrix coating could improve bone cell growth as a prerequisite for enhanced osseointegration of ceramic implants. PMID:25295270

  15. Surface Modifications of Dental Ceramic Implants with Different Glass Solder Matrices: In Vitro Analyses with Human Primary Osteoblasts and Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Jana Markhoff

    2014-01-01

    Full Text Available Ceramic materials show excellent esthetic behavior, along with an absence of hypersensitivity, making them a possible alternative implant material in dental surgery. However, their surface properties enable only limited osseointegration compared to titanium implants. Within this study, a novel surface coating technique for enhanced osseointegration was investigated biologically and mechanically. Specimens of tetragonal zirconia polycrystal (TZP and aluminum toughened zirconia (ATZ were modified with glass solder matrices in two configurations which mainly consisted of SiO2, Al2O3, K2O, and Na2O. The influence on human osteoblastic and epithelial cell viability was examined by means of a WST-1 assay as well as live/dead staining. A C1CP-ELISA was carried out to verify procollagen type I production. Uncoated/sandblasted ceramic specimens and sandblasted titanium surfaces were investigated as a reference. Furthermore, mechanical investigations of bilaterally coated pellets were conducted with respect to surface roughness and adhesive strength of the different coatings. These tests could demonstrate a mechanically stable implant coating with glass solder matrices. The coated ceramic specimens show enhanced osteoblastic and partly epithelial viability and matrix production compared to the titanium control. Hence, the new glass solder matrix coating could improve bone cell growth as a prerequisite for enhanced osseointegration of ceramic implants.

  16. Cytokine expression in human osteoblasts after antiseptic treatment: a comparative study between polyhexanide and chlorhexidine.

    Science.gov (United States)

    Röhner, Eric; Hoff, Paula; Gaber, Timo; Lang, Annemarie; Vörös, Pauline; Buttgereit, Frank; Perka, Carsten; Windisch, Christoph; Matziolis, Georg

    2015-02-01

    Chlorhexidine and polyhexanide are frequently used antiseptics in clinical practice and have a broad antimicrobial range. Both antiseptics are helpful medical agents for septic wound treatment with a high potential for defeating joint infections. Their effect on human osteoblasts has, so far, not been sufficiently evaluated. The aim of this study was to investigate the activating potential of polyhexanide and chlorhexidine on inflammatory cytokines/chemokines in human osteoblasts in vitro. Human osteoblasts were isolated and cultivated in vitro and then treated separately with 0.1% and 2% chlorhexidine and 0.04% polyhexanide as commonly applied concentrations in clinical practice. Detection of cell structure and cell morphology was performed by light microscopic inspection. Cytokine and chemokine secretion was determined by using a multiplex suspension array. Cell shrinking, defective cell membrane, and the loss of cell adhesion indicated cell damage of human osteoblasts after treatment with both antiseptics was evaluated by using light microscopy. Polyhexanide, but not chlorhexidine, caused human osteoblasts to secrete various interleukins (1β, 6, and 7), interferon γ, tumor necrosis factor α, vascular endothelial growth factor, eotaxin, fibroblast growth factor basic, and granulocyte macrophage colony-stimulating factor as quantified by multiplex suspension array. Both antiseptics induced morphological cell damage at an optimum exposure between 1 and 10 min. But only polyhexanide mediated a pronounced secretion of inflammatory cytokines and chemokines in human osteoblasts. Therefore, we recommend a preferred usage of chlorhexidine in septic surgery to avoid the induction of an inflammatory reaction.

  17. Anti-inflammatory drugs suppress proliferation and induce apoptosis through altering expressions of cell cycle regulators and pro-apoptotic factors in cultured human osteoblasts

    International Nuclear Information System (INIS)

    Chang, J.-K.; Li, C.-J.; Liao, H.-J.; Wang, C.-K.; Wang, G.-J.; Ho, M.-L.

    2009-01-01

    It has been reported that anti-inflammatory drugs (AIDs) inhibited bone repair in animal studies, and suppressed proliferation and induced cell death in rat osteoblast cultures. In this study, we further investigated the molecular mechanisms of AID effects on proliferation and cell death in human osteoblasts (hOBs). We examined the effects of dexamethasone (10 -7 and 10 -6 M), non-selective non-steroidal anti-inflammatory drugs (NSAIDs): indomethacin, ketorolac, piroxicam and diclofenac (10 -5 and 10 -4 M), and COX-2 inhibitor: celecoxib (10 -6 and 10 -5 M) on proliferation, cytotoxicity, cell death, and mRNA and protein levels of cell cycle and apoptosis-related regulators in hOBs. All the tested AIDs significantly inhibited proliferation and arrested cell cycle at G0/G1 phase in hOBs. Celecoxib and dexamethasone, but not non-selective NSAIDs, were found to have cytotoxic effects on hOB, and further demonstrated to induce apoptosis and necrosis (at higher concentration) in hOBs. We further found that indomethacin, celecoxib and dexamethasone increased the mRNA and protein expressions of p27 kip1 and decreased those of cyclin D2 and p-cdk2 in hOBs. Bak expression was increased by celecoxib and dexamethasone, while Bcl-XL level was declined only by dexamethasone. Furthermore, the replenishment of PGE1, PGE2 or PGF2α did not reverse the effects of AIDs on proliferation and expressions of p27 kip1 and cyclin D2 in hOBs. We conclude that the changes in expressions of regulators of cell cycle (p27 kip1 and cyclin D2) and/or apoptosis (Bak and Bcl-XL) by AIDs may contribute to AIDs caused proliferation suppression and apoptosis in hOBs. This effect might not relate to the blockage of prostaglandin synthesis by AIDs

  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. Attachment and proliferation of human osteoblast-like cells (MG-63) on laser-ablated titanium implant material

    Energy Technology Data Exchange (ETDEWEB)

    Györgyey, Ágnes; Ungvári, Krisztina [Department of Oral Biology and Experimental Dental Research, Faculty of Dentistry, University of Szeged, H-6720 Szeged (Hungary); Kecskeméti, Gabriella; Kopniczky, Judit [Department of Optics and Quantum Electronics, Faculty of Science and Informatics, University of Szeged, H-6720 Szeged (Hungary); Hopp, Béla [Research Group on Laser Physics, Hungarian Academy of Sciences and University of Szeged, H-6720 Szeged (Hungary); Oszkó, Albert [Department of Physical Chemistry and Materials Science, Faculty of Science and Informatics, University of Szeged, H-6720 Szeged (Hungary); Pelsöczi, István; Rakonczay, Zoltán [Department of Oral Biology and Experimental Dental Research, Faculty of Dentistry, University of Szeged, H-6720 Szeged (Hungary); Nagy, Katalin [Department of Oral Surgery, Faculty of Dentistry, University of Szeged, H-6720 Szeged (Hungary); Turzó, Kinga, E-mail: kturzo@yahoo.com [Department of Oral Biology and Experimental Dental Research, Faculty of Dentistry, University of Szeged, H-6720 Szeged (Hungary)

    2013-10-15

    Demand is increasing for shortening the long (3–6 months) osseointegration period to rehabilitate patients' damaged chewing apparatus in as short a time as possible. For dental implants, as for biomaterials in general, the bio- and osseointegration processes can be controlled at molecular and cellular levels by modification of the implant surface. One of the most promising of such surface modifications is laser ablation, as demonstrated by our previous results [46]. Commercially pure (CP4) sand-blasted, acid-etched titanium disks (Denti® System Ltd., Hungary) were irradiated with a KrF excimer laser (248 nm, fluence 0.4 J/cm{sup 2}, FWHM 18 ns, 2000 pulses), or with a Nd:YAG laser (532 nm, 1.3 J/cm{sup 2}, 10 ns, 200 pulses) then examined by SEM, AFM, and XPS. In vitro attachment (24 h) and proliferation (72 h) of MG-63 osteoblast cells were investigated via dimethylthiazol-diphenyl tetrazolium bromide (MTT), alamarBlue (AB) assays alkaline phosphatase quantification (ALP) and SEM. SEM and AFM revealed significant changes in morphology and roughness. XPS confirmed the presence of TiO{sub 2} on each sample; after Nd:YAG treatment a reduced state of Ti (Ti{sup 3+}) was also observed. MTT, AB and ALP measurements detected an increase in the number of cells between the 24- and 72 hour observations; however, laser treatment did not affect cell attachment and proliferation significantly. - Highlights: • CP4 titanium implant surfaces were modified with Nd:YAG and KrF excimer laser. • SEM and AFM revealed significant changes in morphology and roughness. • XPS confirmed the presence of TiO{sub 2} on each sample; after Nd:YAG treatment a reduced state of Ti (Ti{sup 3+}) was found. • Cell proliferation experiments detected an increased number of MG-63 cells between the 24 h and 72 h observations. • Laser treatments neither disturbed, nor enhanced MG-63 cell attachment and proliferation significantly.

  20. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

    International Nuclear Information System (INIS)

    Muthukumaran, Padmalosini; Lim, Chwee Teck; Lee, Taeyong

    2012-01-01

    Highlights: ► Estradiol induced stiffness changes of osteoblasts were quantified using AFM. ► Estradiol causes significant decrease in the stiffness of osteoblasts. ► Decreased stiffness was caused by decreased density of f-actin network. ► Stiffness changes were not associated with mineralized matrix of osteoblasts. ► Estradiol increases inherent alkaline phosphatase activity of osteoblasts. -- Abstract: Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E ∗ ) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with β-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E ∗ . The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E ∗ of osteoblasts significantly decreased by 43–46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness changes of osteoblasts were not associated with changes in the synthesized mineralized matrix of the cells. Thus, a decrease in osteoblast stiffness with estrogen treatment was

  1. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

    Energy Technology Data Exchange (ETDEWEB)

    Muthukumaran, Padmalosini [Department of Bioengineering, National University of Singapore (Singapore); Lim, Chwee Teck [Department of Bioengineering, National University of Singapore (Singapore); Department of Mechanical Engineering, National University of Singapore (Singapore); Mechanobiology Institute, National University of Singapore (Singapore); Singapore-MIT Alliance for Research and Technology (SMART), National University of Singapore (Singapore); Lee, Taeyong, E-mail: bielt@nus.edu.sg [Department of Bioengineering, National University of Singapore (Singapore)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Estradiol induced stiffness changes of osteoblasts were quantified using AFM. Black-Right-Pointing-Pointer Estradiol causes significant decrease in the stiffness of osteoblasts. Black-Right-Pointing-Pointer Decreased stiffness was caused by decreased density of f-actin network. Black-Right-Pointing-Pointer Stiffness changes were not associated with mineralized matrix of osteoblasts. Black-Right-Pointing-Pointer Estradiol increases inherent alkaline phosphatase activity of osteoblasts. -- Abstract: Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E{sup Asterisk-Operator }) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with {beta}-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E{sup Asterisk-Operator }. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E{sup Asterisk-Operator} of osteoblasts significantly decreased by 43-46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness

  2. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    International Nuclear Information System (INIS)

    Ozeki, Nobuaki; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kinoshita, Katsue; Hase, Naoko; Nakata, Kazuhiko; Kondo, Ayami; Mogi, Makio; Nakamura, Hiroshi

    2014-01-01

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7 + hSMSC)-derived osteoblast-like (α7 + hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7 + hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7 + hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7 + hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form via ADAM-28

  3. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kinoshita, Katsue; Hase, Naoko; Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya, Aichi 464-8650 (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya, Aichi 464-8650 (Japan); Nakamura, Hiroshi [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan)

    2014-04-15

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7{sup +}hSMSC)-derived osteoblast-like (α7{sup +}hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7{sup +}hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7{sup +}hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7{sup +}hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form via

  4. 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

  5. Strontium-Substituted Bioceramics Particles: A New Way to Modulate MCP-1 and Gro-α Production by Human Primary Osteoblastic Cells

    Directory of Open Access Journals (Sweden)

    Julien Braux

    2016-12-01

    Full Text Available Background: To avoid morbidity and limited availability associated with autografts, synthetic calcium phosphate (CaP ceramics were extensively developed and used as bone filling materials. Controlling their induced-inflammatory response nevertheless remained a major concern. Strontium-containing CaP ceramics were recently demonstrated for impacting cytokines’ secretion pattern of human primary monocytes. The present study focuses on the ability of strontium-containing CaP to control the human primary bone cell production of two major inflammatory and pro-osteoclastogenic mediators, namely MCP-1 and Gro-α, in response to ceramics particles. Methods: This in vitro study was performed using human primary osteoblasts in which their response to ceramics was evaluated by PCR arrays, antibody arrays were used for screening and real-time PCR and ELISA for more focused analyses. Results: Study of mRNA and protein expression highlights that human primary bone cells are able to produce these inflammatory mediators and reveal that the adjunction of CaP in the culture medium leads to their enhanced production. Importantly, the current work determines the down-regulating effect of strontium-substituted CaP on MCP-1 and Gro-α production. Conclusion: Our findings point out a new capability of strontium to modulate human primary bone cells’ communication with the immune system.

  6. Influence of Different Three-Dimensional Open Porous Titanium Scaffold Designs on Human Osteoblasts Behavior in Static and Dynamic Cell Investigations

    Directory of Open Access Journals (Sweden)

    Jana Markhoff

    2015-08-01

    Full Text Available In the treatment of osseous defects micro-structured three-dimensional materials for bone replacement serve as leading structure for cell migration, proliferation and bone formation. The scaffold design and culture conditions are crucial for the limited diffusion distance of nutrients and oxygen. In static culture, decreased cell activity and irregular distribution occur within the scaffold. Dynamic conditions entail physical stimulation and constant medium perfusion imitating physiological nutrient supply and metabolite disposal. Therefore, we investigated the influence of different scaffold configurations and cultivation methods on human osteoblasts. Cells were seeded on three-dimensional porous Ti-6Al-4V scaffolds manufactured with selective laser melting (SLM or electron beam melting (EBM varying in porosity, pore size and basic structure (cubic, diagonal, pyramidal and cultured under static and dynamic conditions. Cell viability, migration and matrix production were examined via mitochondrial activity assay, fluorescence staining and ELISA. All scaffolds showed an increasing cell activity and matrix production under static conditions over time. Expectations about the dynamic culture were only partially fulfilled, since it enabled proliferation alike the static one and enhanced cell migration. Overall, the SLM manufactured scaffold with the highest porosity, small pore size and pyramidal basic structure proved to be the most suitable structure for cell proliferation and migration.

  7. Influence of Different Three-Dimensional Open Porous Titanium Scaffold Designs on Human Osteoblasts Behavior in Static and Dynamic Cell Investigations.

    Science.gov (United States)

    Markhoff, Jana; Wieding, Jan; Weissmann, Volker; Pasold, Juliane; Jonitz-Heincke, Anika; Bader, Rainer

    2015-08-24

    In the treatment of osseous defects micro-structured three-dimensional materials for bone replacement serve as leading structure for cell migration, proliferation and bone formation. The scaffold design and culture conditions are crucial for the limited diffusion distance of nutrients and oxygen. In static culture, decreased cell activity and irregular distribution occur within the scaffold. Dynamic conditions entail physical stimulation and constant medium perfusion imitating physiological nutrient supply and metabolite disposal. Therefore, we investigated the influence of different scaffold configurations and cultivation methods on human osteoblasts. Cells were seeded on three-dimensional porous Ti-6Al-4V scaffolds manufactured with selective laser melting (SLM) or electron beam melting (EBM) varying in porosity, pore size and basic structure (cubic, diagonal, pyramidal) and cultured under static and dynamic conditions. Cell viability, migration and matrix production were examined via mitochondrial activity assay, fluorescence staining and ELISA. All scaffolds showed an increasing cell activity and matrix production under static conditions over time. Expectations about the dynamic culture were only partially fulfilled, since it enabled proliferation alike the static one and enhanced cell migration. Overall, the SLM manufactured scaffold with the highest porosity, small pore size and pyramidal basic structure proved to be the most suitable structure for cell proliferation and migration.

  8. Direct conversion of human fibroblasts into functional osteoblasts by defined factors.

    Science.gov (United States)

    Yamamoto, Kenta; Kishida, Tsunao; Sato, Yoshiki; Nishioka, Keisuke; Ejima, Akika; Fujiwara, Hiroyoshi; Kubo, Toshikazu; Yamamoto, Toshiro; Kanamura, Narisato; Mazda, Osam

    2015-05-12

    Osteoblasts produce calcified bone matrix and contribute to bone formation and remodeling. In this study, we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some defined factors and culturing in osteogenic medium. Osteoblast-specific transcription factors, Runt-related transcription factor 2 (Runx2), and Osterix, in combination with Octamer-binding transcription factor 3/4 (Oct4) and L-Myc (RXOL) transduction, converted ∼ 80% of the fibroblasts into osteocalcin-producing cells. The directly converted osteoblasts (dOBs) induced by RXOL displayed a similar gene expression profile as normal human osteoblasts and contributed to bone repair after transplantation into immunodeficient mice at artificial bone defect lesions. The dOBs expressed endogenous Runx2 and Osterix, and did not require continuous expression of the exogenous genes to maintain their phenotype. Another combination, Oct4 plus L-Myc (OL), also induced fibroblasts to produce bone matrix, but the OL-transduced cells did not express Osterix and exhibited a more distant gene expression profile to osteoblasts compared with RXOL-transduced cells. These findings strongly suggest successful direct reprogramming of fibroblasts into functional osteoblasts by RXOL, a technology that may provide bone regeneration therapy against bone disorders.

  9. 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

  10. 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.

  11. Generation of Directly Converted Human Osteoblasts That Are Free of Exogenous Gene and Xenogenic Protein.

    Science.gov (United States)

    Yamamoto, Kenta; Sato, Yoshiki; Honjo, Kenichi; Ichioka, Hiroaki; Oseko, Fumishige; Sowa, Yoshihiro; Yamamoto, Toshiro; Kanamura, Narisato; Kishida, Tsunao; Mazda, Osam

    2016-11-01

    Generation of osteoblasts from human somatic cells may be applicable in an effective transplantation therapy against bone diseases. Recently we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some transcription factor genes via retroviral vectors. However, retroviral vector-mediated transduction may potentially cause tumor formation from the infected cells, thus a non-viral gene transfection method may be more preferable for preparation of osteoblasts to be used for transplantation therapy. Here, we constructed a plasmid vector encoding Oct4, Osterix, and L-Myc that were an appropriate combination of transcription factors for this purpose. Osteoblast-like phenotypes including high alkaline phosphatase (ALP) activity, bone matrix production and osteoblast-specific gene expression were induced in normal human fibroblasts that were transfected with the plasmid followed by culturing in osteogenic medium. The plasmid-driven directly converted osteoblasts (p-dOBs) were obtained even in the absence of a xenogenic protein. The plasmid vector sequence had fallen out of the p-dOBs. The cells formed deposition of calcified bodies in situ after transplantation into mice. These results strongly suggest that p-dOBs can be put into practical use for a novel cell-based therapy against bone diseases. J. Cell. Biochem. 117: 2538-2545, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. Treatment with 1,25-dihydroxyvitamin D3 reduces impairment of human osteoblast functions during cellular aging in culture

    DEFF Research Database (Denmark)

    Kveiborg, M.; Rattan, Suresh; Eriksen, E.F.

    2001-01-01

    is due to impaired responsiveness to calcitriol known to be important for the regulation of biological activities of the osteoblasts. Thus, we examined changes in vitamin D receptor (VDR) system and the osteoblastic responses to calcitriol treatment during in vitro osteoblast aging. We found no change...... in the amount of VDR at either steady state mRNA level or protein level with increasing in vitro osteoblast age and examination of VDR localization, nuclear translocation and DNA binding activity revealed no in vitro age-related changes. Furthermore, calcitriol (10(-8)M) treatment of early-passage osteoblastic......Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D(3) (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long...

  13. Comparison of low-intensity pulsed ultrasound and pulsed electromagnetic field treatments on OPG and RANKL expression in human osteoblast-like cells

    NARCIS (Netherlands)

    Borsje, Manon A.; Ren, Yijin; de Haan-Visser, H. Willy; Kuijer, Roel

    OBJECTIVE: To compare two clinically applied treatments to stimulate bone healing-low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF)-for their effects on RANKL and OPG expression in osteoblast-like cells in vitro. MATERIALS AND METHODS: LIPUS or PEMF was applied to

  14. Electrical activity of ferroelectric biomaterials and its effects on the adhesion, growth and enzymatic activity of human osteoblast-like cells

    Czech Academy of Sciences Publication Activity Database

    Vaněk, Přemysl; Kolská, Z.; Luxbacher, T.; García, J.A.L.; Lehocký, M.; Vandrovcová, Marta; Bačáková, Lucie; Petzelt, Jan

    2016-01-01

    Roč. 49, č. 17 (2016), 1-12, č. článku 175403. ISSN 0022-3727 R&D Projects: GA ČR(CZ) GA15-01558S Institutional support: RVO:68378271 ; RVO:67985823 Keywords : biomaterials * ferroelectric * zeta potential * osteoblast-like cells Subject RIV: BO - Biophysics Impact factor: 2.588, year: 2016

  15. 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...

  16. Adhesion and Growth of Human Osteoblast-Like Cell in Cultures on Nanocomposite Carbon-Based Materials

    Czech Academy of Sciences Publication Activity Database

    Bačáková, Lucie; Grausová, Ľubica; Vacík, Jiří; Lavrentiev, Vasyl; Blazewicz, S.; Fraczek, A.; Kromka, Alexander; Haenen, K.

    2011-01-01

    Roč. 3, č. 1 (2011), s. 99-109 ISSN 1941-4900 R&D Projects: GA AV ČR(CZ) IAAX00100902; GA MŠk(CZ) 2B06173; GA AV ČR(CZ) KAN400480701 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z10480505; CEZ:AV0Z10100521 Keywords : nanoscale surface roughness * electrical conductivity * osteoblasts * bone tissue engineering Subject RIV: EI - Biotechnology ; Bionics Impact factor: 0.528, year: 2011

  17. 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

  18. 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.

  19. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-16

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

  20. 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

  1. 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.

  2. Electrical activity of ferroelectric biomaterials and its effects on the adhesion, growth and enzymatic activity of human osteoblast-like cells

    Science.gov (United States)

    Vaněk, P.; Kolská, Z.; Luxbacher, T.; García, J. A. L.; Lehocký, M.; Vandrovcová, M.; Bačáková, L.; Petzelt, J.

    2016-05-01

    Ferroelectrics have been, among others, studied as electroactive implant materials. Previous investigations have indicated that such implants induce improved bone formation. If a ferroelectric is immersed in a liquid, an electric double layer and a diffusion layer are formed at the interface. This is decisive for protein adsorption and bioactive behaviour, particularly for the adhesion and growth of cells. The charge distribution can be characterized, in a simplified way, by the zeta potential. We measured the zeta potential in dependence on the surface polarity on poled ferroelectric single crystalline LiNbO3 plates. Both our results and recent results of colloidal probe microscopy indicate that the charge distribution at the surface can be influenced by the surface polarity of ferroelectrics under certain ‘ideal’ conditions (low ionic strength, non-contaminated surface, very low roughness). However, suggested ferroelectric coatings on the surface of implants are far from ideal: they are rough, polycrystalline, and the body fluid is complex and has high ionic strength. In real cases, it can therefore be expected that there is rather low influence of the sign of the surface polarity on the electric diffusion layer and thus on the specific adsorption of proteins. This is supported by our results from studies of the adhesion, growth and the activity of alkaline phosphatase of human osteoblast-like Saos-2 cells on ferroelectric LiNbO3 plates in vitro.

  3. Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone.

    Science.gov (United States)

    El-Amin, Saadiq F; Attawia, Mohamed; Lu, Helen H; Shah, Asist K; Chang, Richard; Hickok, Noreen J; Tuan, Rocky S; Laurencin, Cato T

    2002-01-01

    The use of biodegradable polymers in the field of orthopaedic surgery has gained increased popularity, as surgical pins and screws, and as potential biological scaffolds for repairing cartilage and bone defects. One such group of polymers that has gained considerable attention are the polyesters, poly(lactide-co-glycolide) (PLAGA) and polylactic acid (PLA), because of their minimal tissue inflammatory response, favorable biocompatibility and degradation characteristics. The objective of this study was to evaluate human osteoblastic cell adherence and growth on PLAGA and PLA scaffolds by examining integrin receptor (alpha2, alpha3, alpha4, alpha5, alpha6 and beta1) expression. Primary human osteoblastic cells isolated from trabecular bone adhered efficiently to both PLAGA and PLA, with the rate of adherence on PLAGA comparable to that of control tissue culture polystyrene (TCPS), and significantly higher than on PLA polymers at 3, 6 and 12 h. Human osteoblastic phenotypic expression, alkaline phosphatase (ALP) activity was positive on both degradable matrices, whereas osteocalcin levels were significantly higher on cells grown on PLAGA than on PLA composites. Interestingly, the integrin subunits, alpha2, alpha3, alpha4, alpha5, alpha6 and beta1 were all expressed at higher levels by osteoblasts cultured on PLAGA than those on PLA as analyzed by westerns blots and by flow cytometry. Among the integrins, alpha2, beta5 and beta1 showed the greatest difference in levels between the two surfaces. Thus, both PLA and PLAGA support osteoblastic adhesion and its accompanying engagement of integrin receptor and expression of osteocalcin and ALP. However PLAGA consistently appeared to be a better substrate for osteoblastic cells based on these parameters. This study is one of the first to investigate the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable polymers PLAGA and PLA, and to examine the expression of their key

  4. Effect of blood component coatings of enosseal implants on proliferation and synthetic activity of human osteoblasts and cytokine production of peripheral blood mononuclear cells

    Czech Academy of Sciences Publication Activity Database

    Himlová, L.; Kubies, Dana; Hulejová, H.; Bartová, J.; Riedel, Tomáš; Štikarová, J.; Suttnar, J.; Pešáková, V.

    2016-01-01

    Roč. 2016, č. 2016 (2016), 8769347_1-8769347_15 ISSN 0962-9351 R&D Projects: GA MZd(CZ) NT13297; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61389013 Keywords : peripheral blood mononuclear cells * cytokine * osteoblasts Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.232, year: 2016

  5. 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.

  6. 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...

  7. 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

  8. 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

  9. Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells

    NARCIS (Netherlands)

    J. Morhayim (Jess); J. van de Peppel (Jeroen); E. Braakman (Eric); Rombouts, E.W.J.C. (Elwin W. J. C.); M.N.D. Ter Borg (Mariëtte N. D.); A. Dudakovic (Amel); H. Chiba (Hideki); B.C.J. van der Eerden (Bram); M.H.G.P. Raaijmakers (Marc H.G.P.); A.J. van Wijnen (André); J.J. Cornelissen (Jan); J.P.T.M. van Leeuwen (Hans)

    2016-01-01

    textabstractOsteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2009-02-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Strong influence of hierarchically structured diamond nano-topography on adhesion of human osteoblasts and mesenchymal cells

    Czech Academy of Sciences Publication Activity Database

    Brož, A.; Barešová, V.; Kromka, Alexander; Rezek, Bohuslav; Kalbáčová, M.

    2009-01-01

    Roč. 206, č. 9 (2009), s. 2038-2041 ISSN 1862-6300 R&D Projects: GA AV ČR KAN400100701 Institutional research plan: CEZ:AV0Z10100521 Keywords : diamond morphology * cells adhesion * cells behaviour Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 1.228, year: 2009

  14. Effect of different surface nanoroughness of titanium dioxide films on the growth of human osteoblast-like MG63 cells

    Czech Academy of Sciences Publication Activity Database

    Vandrovcová, Marta; Hanuš, J.; Drábik, M.; Kylián, O.; Biederman, H.; Lisá, Věra; Bačáková, Lucie

    100A, č. 4 (2012), s. 1016-1032 ISSN 1549-3296 R&D Projects: GA AV ČR(CZ) KAN101120701 Institutional research plan: CEZ:AV0Z50110509 Keywords : titanium dioxide * nanoscale surface roughness * MG63 cells Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.834, year: 2012

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-11-01

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

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

    OpenAIRE

    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 ...

  19. 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

  20. 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...

  1. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. 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.

  4. A comparison of the cytotoxicity and proinflammatory cytokine production of EndoSequence root repair material and ProRoot mineral trioxide aggregate in human osteoblast cell culture using reverse-transcriptase polymerase chain reaction.

    Science.gov (United States)

    Ciasca, Maria; Aminoshariae, Anita; Jin, Ge; Montagnese, Thomas; Mickel, Andre

    2012-04-01

    The purpose of this study was to compare the cytotoxicity and cytokine expression profiles of EndoSequence Root Repair Material (ERRM; Brasseler, Savannah, GA) putty, ERRM flowable, and ProRoot mineral trioxide aggregate (MTA; Dentsply Tulsa Dental, Johnson City, TN) using osteoblast cells (MG-63). Four millimeters in diameter of each material was placed in the center of a 6-well culture plate, and a 2-mL suspension (10(5) cells/mL) of human osteoblasts was seeded in each well. Photomicrograph images were used to evaluate cytotoxicity as evidenced by the lack of osteoblast cell growth in relation to the materials with AH-26 (Dentsply Tulsa Dental) as the positive control. In addition, reverse-transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the expression of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α). Cytokine expression of MG-63 cells upon lipopolysaccharide treatment was used as controls. RT-PCR results were normalized by the expression of the housekeeping gene β-actin and were used to measure cytokine expression. Statistical analysis was performed using analysis of variance. Results showed that ERRM putty and MTA exhibited minimal levels of cytotoxicity; however, ERRM was slightly more cytotoxic although not statistically significant. The expression of IL-1β, IL-6, and IL-8 was detected in all samples with minimal TNF-α expression. We concluded that ERRM and MTA showed similar cytotoxicity and cytokine expressions. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  5. 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

  6. Human immunodeficiency virus envelope protein Gp120 induces proliferation but not apoptosis in osteoblasts at physiologic concentrations.

    Directory of Open Access Journals (Sweden)

    Nathan W Cummins

    Full Text Available Patients with HIV infection have decreased numbers of osteoblasts, decreased bone mineral density and increased risk of fracture compared to uninfected patients; however, the molecular mechanisms behind these associations remain unclear. We questioned whether Gp120, a component of the envelope protein of HIV capable of inducing apoptosis in many cell types, is able to induce cell death in bone-forming osteoblasts. We show that treatment of immortalized osteoblast-like cells and primary human osteoblasts with exogenous Gp120 in vitro at physiologic concentrations does not result in apoptosis. Instead, in the osteoblast-like U2OS cell line, cells expressing CXCR4, a receptor for Gp120, had increased proliferation when treated with Gp120 compared to control (P<0.05, which was inhibited by pretreatment with a CXCR4 inhibitor and a G-protein inhibitor. This suggests that Gp120 is not an inducer of apoptosis in human osteoblasts and likely does not directly contribute to osteoporosis in infected patients by this mechanism.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Differentiation of bovine spermatogonial stem cells into osteoblasts.

    Science.gov (United States)

    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.

  9. Effect of thorium, cerium and lanthanium metals on the radiosensitivity of human osteoblasts

    International Nuclear Information System (INIS)

    Iwahara, Lucas Kiyoshi da Fonseca

    2016-01-01

    This work analyzed the effects of Th, Ce and La combinations on the human osteoblast proliferation. Due to the osteotropic potential of actinides and lanthanides, a human osteoblast cell line was used to evaluate the effects of metals on cell radiosensitivity using cell proliferation and total proteins as indicators. Assays were performed using cultures exposed to metal alone and in combination and to ionising radiation. It was not observed effects on proliferation for cultures exposed to the metals alone. Concerning the influence of the three elements on the radiosensitivity, it was seen that all three metals were able to interfere on this indicator, in a concentration dependent manner. Evaluating cultures exposed to binary mixtures (Th-Ce and Th-La) and a ternary mixture (Th-Ce-La), it was verified that there were chemical interactions between the metals, for the combinations tested. The results showed very strong antagonism on the inhibition of cell proliferation in cultures exposed to Th-La and Th- Ce-La combinations. Regarding the osteoblasts exposed to mixtures and to radiation it was seen an antagonistic effect on the cell proliferation in all tested combinations, and the Th-Ce combination with a higher degree. These results show that metal mixtures containing thorium, in association with ionising radiation, induced different effects on cell proliferation, regarding the exposure to the metals alone, suggesting the possibility that the combinations interfere on osteoblast radiosensitivity expressing the increase of the occupational hazard among workers involved with monazite sands. The results also indicate that the analysis of the effects of metal mixtures on human cells is a more realistic risk assessment in comparison with the analysis of risk for single elements. The work displays the need to development of risk assessment models that include the study of mixtures obtained in the work environment for the evaluation of cytotoxic and radiotoxic effects in

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

    Science.gov (United States)

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

    2017-12-04

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

  11. 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. Detachment strength of human osteoblasts cultured on hydroxyapatite with various surface roughness. Contribution of integrin subunits.

    Science.gov (United States)

    Kokkinos, Petros A; Koutsoukos, Petros G; Deligianni, Despina D

    2012-06-01

    Hydroxyapatite (HA) has been widely used as a bone substitute in dental, maxillofacial and orthopaedic surgery and as osteoconductive bone substitute or precoating of pedicle screws and cages in spine surgery. The aim of the present study was to investigate the osteoblastic adhesion strength on HA substrata with different surface topography and biochemistry (pre-adsorption of fibronectin) after blocking of specific integrin subunits with monoclonal antibodies. Stoichiometric HA was prepared by precipitation followed by ageing and characterized by SEM, EDX, powder XRD, Raman spectroscopy, TGA, and specific surface area analysis. Human bone marrow derived osteoblasts were cultured on HA disc-shaped substrata which were sintered and polished resulting in two surface roughness grades. For attachment evaluation, cells were incubated with monoclonal antibodies and seeded for 2 h on the substrata. Cell detachment strength was determined using a rotating disc device. Cell detachment strength was surface roughness, fibronectin preadsorption and intergin subunit sensitive.

  13. Transcriptional Regulation of Frizzled-1 in Human Osteoblasts by Sp1.

    Directory of Open Access Journals (Sweden)

    Shibing Yu

    Full Text Available The wingless pathway has a powerful influence on bone metabolism and is a therapeutic target in skeletal disorders. Wingless signaling is mediated in part through the Frizzled (FZD receptor family. FZD transcriptional regulation is poorly understood. Herein we tested the hypothesis that Sp1 plays an important role in the transcriptional regulation of FZD1 expression in osteoblasts and osteoblast mineralization. To test this hypothesis, we conducted FZD1 promoter assays in Saos2 cells with and without Sp1 overexpression. We found that Sp1 significantly up-regulates FZD1 promoter activity in Saos2 cells. Chromatin immunoprecipitation (ChIP and electrophoretic mobility shift (EMSA assays identified a novel and functional Sp1 binding site at -44 to -40 from the translation start site in the FZD1 promoter. The Sp1-dependent activation of the FZD1 promoter was abolished by mithramycin A (MMA, an antibiotic affecting both Sp1 binding and Sp1 protein levels in Saos2 cells. Similarly, down-regulation of Sp1 in hFOB cells resulted in less FZD1 expression and lower alkaline phosphatase activity. Moreover, over-expression of Sp1 increased FZD1 expression and Saos2 cell mineralization while MMA decreased Sp1 and FZD1 expression and Saos2 cell mineralization. Knockdown of FZD1 prior to Sp1 overexpression partially abolished Sp1 stimulation of osteoblast differentiation markers. Taken together, our results suggest that Sp1 plays a role in human osteoblast differentiation and mineralization, which is at least partially mediated by Sp1-dependent transactivation of FZD1.

  14. 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.

  15. 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

  16. 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

  17. Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells

    DEFF Research Database (Denmark)

    Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen

    2010-01-01

    -alpha downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-alpha expression was only seen in hOBs and mesenchymal stem cells......Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co......-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR...

  18. 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.

  19. 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...

  20. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

    International Nuclear Information System (INIS)

    Mizoshiri, N.; Kishida, T.; Yamamoto, K.; Shirai, T.; Terauchi, R.; Tsuchida, S.; Mori, Y.; Ejima, A.; Sato, Y.; Arai, Y.; Fujiwara, H.; Yamamoto, T.; Kanamura, N.; Mazda, O.; Kubo, T.

    2015-01-01

    Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.

  1. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Mizoshiri, N. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kishida, T. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, K. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Shirai, T.; Terauchi, R.; Tsuchida, S. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mori, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Ejima, A. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Sato, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Arai, Y.; Fujiwara, H. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, T.; Kanamura, N. [Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, O., E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kubo, T. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2015-11-27

    Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.

  2. 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

  3. 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

  4. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. 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

  6. 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

  7. The influence of pore size on colonization of poly(L-lactide-glycolide) scaffolds with human osteoblast-like MG 63 cells in vitro

    Czech Academy of Sciences Publication Activity Database

    Pamula, E.; Bačáková, Lucie; Filová, Elena; Buczynska, J.; Dobrzynski, P.; Nosková, Lenka; Grausová, Ľubica

    2008-01-01

    Roč. 19, č. 1 (2008), s. 425-435 ISSN 0957-4530 R&D Projects: GA ČR(CZ) GA106/06/1576; GA MŠk 1P05OC012 Grant - others:Polish Budget Fund for Scientific Research(PL) 3T08D 019 28 Institutional research plan: CEZ:AV0Z50110509 Keywords : bone tissue engineering * degradable scaffolds * osteoblasts Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.508, year: 2008

  8. Interaction of Human Osteoblast-Like Saos-2 and MG-63 Cells with Thermally Oxidized Surfaces of a Titanium-Niobium Alloy

    Czech Academy of Sciences Publication Activity Database

    Vandrovcová, Marta; Jirka, Ivan; Novotná, Katarína; Lisá, Věra; Frank, Otakar; Kolská, Z.; Starý, V.; Bačáková, Lucie

    2014-01-01

    Roč. 9, č. 6 (2014), e100475 E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GAP108/10/1858; GA ČR(CZ) GAP107/12/1025; GA MPO FR-TI3/088 Institutional support: RVO:67985823 ; RVO:61388955 Keywords : thermally oxidized surface * titanium-niobium * TiO2 * osteoblast * macrophage Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.234, year: 2014

  9. Therapeutic touch affects DNA synthesis and mineralization of human osteoblasts in culture.

    Science.gov (United States)

    Jhaveri, Ankur; Walsh, Stephen J; Wang, Yatzen; McCarthy, MaryBeth; Gronowicz, Gloria

    2008-11-01

    Complementary and alternative medicine (CAM) techniques are commonly used in hospitals and private medical facilities; however, the effectiveness of many of these practices has not been thoroughly studied in a scientific manner. Developed by Dr. Dolores Krieger and Dora Kunz, Therapeutic Touch is one of these CAM practices and is a highly disciplined five-step process by which a practitioner can generate energy through their hands to promote healing. There are numerous clinical studies on the effects of TT but few in vitro studies. Our purpose was to determine if Therapeutic Touch had any effect on osteoblast proliferation, differentiation, and mineralization in vitro. TT was performed twice a week for 10 min each on human osteoblasts (HOBs) and on an osteosarcoma-derived cell line, SaOs-2. No significant differences were found in DNA synthesis, assayed by [(3)H]-thymidine incorporation at 1 or 2 weeks for SaOs-2 or 1 week for HOBs. However, after four TT treatments in 2 weeks, TT significantly (p = 0.03) increased HOB DNA synthesis compared to controls. Immunocytochemistry for Proliferating Cell Nuclear Antigen (PCNA) confirmed these data. At 2 weeks in differentiation medium, TT significantly increased mineralization in HOBs (p = 0.016) and decreased mineralization in SaOs-2 (p = 0.0007), compared to controls. Additionally, Northern blot analysis indicated a TT-induced increase in mRNA expression for Type I collagen, bone sialoprotein, and alkaline phosphatase in HOBs and a decrease of these bone markers in SaOs-2 cells. In conclusion, Therapeutic Touch appears to increase human osteoblast DNA synthesis, differentiation and mineralization, and decrease differentiation and mineralization in a human osteosarcoma-derived cell line. (c) 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-01-01

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

  11. 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.

  12. 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.

  13. 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)

  14. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering.

    Science.gov (United States)

    El-Amin, S F; Lu, H H; Khan, Y; Burems, J; Mitchell, J; Tuan, R S; Laurencin, C T

    2003-03-01

    The nature of the extracellular matrix (ECM) is crucial in regulating cell functions via cell-matrix interactions, cytoskeletal organization, and integrin-mediated signaling. In bone, the ECM is composed of proteins such as collagen (CO), fibronectin (FN), laminin (LM), vitronectin (VN), osteopontin (OP) and osteonectin (ON). For bone tissue engineering, the ECM should also be considered in terms of its function in mediating cell adhesion to biomaterials. This study examined ECM production, cytoskeletal organization, and adhesion of primary human osteoblastic cells on biodegradable matrices applicable for tissue engineering, namely polylactic-co-glycolic acid 50:50 (PLAGA) and polylactic acid (PLA). We hypothesized that the osteocompatible, biodegradable polymer surfaces promote the production of bone-specific ECM proteins in a manner dependent on polymer composition. We first examined whether the PLAGA and PLA matrices could support human osteoblastic cell growth by measuring cell adhesion at 3, 6 and 12h post-plating. Adhesion on PLAGA was consistently higher than on PLA throughout the duration of the experiment, and comparable to tissue culture polystyrene (TCPS). ECM components, including CO, FN, LM, ON, OP and VN, produced on the surface of the polymers were quantified by ELISA and localized by immunofluorescence staining. All of these proteins were present at significantly higher levels on PLAGA compared to PLA or TCPS surfaces. On PLAGA, OP and ON were the most abundant ECM components, followed by CO, FN, VN and LN. Immunofluorescence revealed an extracellular distribution for CO and FN, whereas OP and ON were found both intracellularly as well as extracellularly on the polymer. In addition, the actin cytoskeletal network was more extensive in osteoblasts cultured on PLAGA than on PLA or TCPS. In summary, we found that osteoblasts plated on PLAGA adhered better to the substrate, produced higher levels of ECM molecules, and showed greater cytoskeletal

  15. Combinatorial MAPLE gradient thin film assemblies signalling to human osteoblasts

    International Nuclear Information System (INIS)

    Axente, Emanuel; Sima, Felix; Elena Sima, Livia; Serban, Natalia; Ristoscu, Carmen; Mihailescu, Ion N; Erginer, Merve; Toksoy Oner, Ebru; Eroglu, Mehmet S; Petrescu, Stefana M

    2014-01-01

    There is increased interest in smart bioactive materials to control tissue regeneration for the engineering of cell instructive scaffolds. We introduced combinatorial matrix-assisted pulsed laser evaporation (C-MAPLE) as a new method for the fabrication of organic thin films with a compositional gradient. Synchronized C-MAPLE of levan and oxidized levan was employed to assemble a two-compound biopolymer film structure. The gradient of the film composition was validated by fluorescence microscopy. In this study, we investigated the cell response induced by the compositional gradient using imaging of early osteoblast attachment and analysis of signalling phosphoprotein expression. Cells attached along the gradient in direct proportion to oxidized levan concentration. During this process distinct areas of the binary gradient have been shown to modulate the osteoblasts’ extracellular signal-regulated kinase signalling with different propensity. The proposed fabrication method results in the preparation of a new bioactive material, which could control the cell signalling response. This approach can be extended to screen new bioactive interfaces for tissue regeneration. (papers)

  16. Biocompatibility and Inflammatory Potential of Titanium Alloys Cultivated with Human Osteoblasts, Fibroblasts and Macrophages

    Science.gov (United States)

    Markhoff, Jana; Krogull, Martin; Schulze, Christian; Rotsch, Christian; Hunger, Sandra; Bader, Rainer

    2017-01-01

    The biomaterials used to maintain or replace functions in the human body consist mainly of metals, ceramics or polymers. In orthopedic surgery, metallic materials, especially titanium and its alloys, are the most common, due to their excellent mechanical properties, corrosion resistance, and biocompatibility. Aside from the established Ti6Al4V alloy, shape memory materials such as nickel-titanium (NiTi) have risen in importance, but are also discussed because of the adverse effects of nickel ions. These might be reduced by specific surface modifications. In the present in vitro study, the osteoblastic cell line MG-63 as well as primary human osteoblasts, fibroblasts, and macrophages were cultured on titanium alloys (forged Ti6Al4V, additive manufactured Ti6Al4V, NiTi, and Diamond-Like-Carbon (DLC)-coated NiTi) to verify their specific biocompatibility and inflammatory potential. Additive manufactured Ti6Al4V and NiTi revealed the highest levels of metabolic cell activity. DLC-coated NiTi appeared as a suitable surface for cell growth, showing the highest collagen production. None of the implant materials caused a strong inflammatory response. In general, no distinct cell-specific response could be observed for the materials and surface coating used. In summary, all tested titanium alloys seem to be biologically appropriate for application in orthopedic surgery. PMID:28772412

  17. Biocompatibility and Inflammatory Potential of Titanium Alloys Cultivated with Human Osteoblasts, Fibroblasts and Macrophages

    Directory of Open Access Journals (Sweden)

    Jana Markhoff

    2017-01-01

    Full Text Available The biomaterials used to maintain or replace functions in the human body consist mainly of metals, ceramics or polymers. In orthopedic surgery, metallic materials, especially titanium and its alloys, are the most common, due to their excellent mechanical properties, corrosion resistance, and biocompatibility. Aside from the established Ti6Al4V alloy, shape memory materials such as nickel-titanium (NiTi have risen in importance, but are also discussed because of the adverse effects of nickel ions. These might be reduced by specific surface modifications. In the present in vitro study, the osteoblastic cell line MG-63 as well as primary human osteoblasts, fibroblasts, and macrophages were cultured on titanium alloys (forged Ti6Al4V, additive manufactured Ti6Al4V, NiTi, and Diamond-Like-Carbon (DLC-coated NiTi to verify their specific biocompatibility and inflammatory potential. Additive manufactured Ti6Al4V and NiTi revealed the highest levels of metabolic cell activity. DLC-coated NiTi appeared as a suitable surface for cell growth, showing the highest collagen production. None of the implant materials caused a strong inflammatory response. In general, no distinct cell-specific response could be observed for the materials and surface coating used. In summary, all tested titanium alloys seem to be biologically appropriate for application in orthopedic surgery.

  18. Specific proliferation rates of human osteoblasts on calcium phosphate surfaces with variable concentrations of α-TCP

    International Nuclear Information System (INIS)

    Santos, Euler A. dos; Farina, Marcos; Soares, Gloria A.

    2007-01-01

    Ideally, ceramics used in the repair of bone defects need to be resorbed and replaced by newly formed bone in vivo. Tricalcium phosphate (TCP) has been widely used in association with hydroxyapatite (HA) due to its higher resorption kinetics when compared with HA alone. The aim of our study was to quantitatively investigate the effect of α-tricalcium phosphate (α-TCP) on human osteoblasts' adhesion and proliferation. Ceramic samples with variable concentrations of α-TCP and HA were produced by the calcination of calcium-deficient and stoichiometric HA. Human osteoblasts were cultured on the materials in three distinct experiments with different concentrations of cells. Numerical evaluation of cellular growth along time in culture was performed for each condition. The quantity of cells seeded onto the ceramics seems to influence the osteoblast behavior once proliferation was lower when more cells were seeded onto the samples. However, a smaller content of α-TCP in relation to that of HA did not significantly modify the specific proliferation rates of the osteoblasts. Only after a long time in culture, the increasing of the α-TCP content seems to change the cells' behavior

  19. 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...

  20. Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

    Science.gov (United States)

    Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

    2010-09-01

    Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point

  1. The Impact of Metal Ion Exposure on the Cellular Behavior of Human Osteoblasts and PBMCs: In Vitro Analyses of Osteolytic Processes

    Directory of Open Access Journals (Sweden)

    Anika Jonitz-Heincke

    2017-07-01

    Full Text Available Osteolysis in the periprosthetic tissue can be caused by metallic wear particles and ions that can originate from implant surface corrosion. These products influence cellular behavior and stimulate the expression of proinflammatory cytokines. The purpose of this study was to evaluate the impact of CoCr29Mo6 ions on cell survival, differentiation, and cytokine expression in human osteoblasts and peripheral blood mononuclear cells (PBMCs. Thus, we exposed cells with a mixture of 200 µg/L ion solution and determined cell viability and apoptosis/necrosis. Gene expression analyses of osteoblastic and osteoclastic differentiation markers as well as pro-osteolytic mediators (IL-6, IL-8, TNF-α, MCP-1, MMP1, TIMP1 were performed. These markers were also investigated in mixed cultures of adherent and non-adherent PBMCs as well as in co-cultures of human osteoblasts and PBMCs. The ion solution induced necrosis in osteoblasts and PBMCs in single cultures. All examined mediators were highly expressed in the co-culture of osteoblasts and PBMCs whereas in the single cell cultures only IL-6, IL-8, and MMP1 were found to be stimulated. While the applied concentration of the CoCr29Mo6 ion solutions had only marginal effects on human osteoblasts and PBMCs alone, the co-culture may provide a comprehensive model to study osteolytic processes in response to Co and Cr ions.

  2. Evaluation of the effect of three monazite constituents on the radiosensitivity of human osteoblasts

    International Nuclear Information System (INIS)

    Iwahara, Lucas Kiyoshi da Fonseca; Oliveira, Monica Stuck de; Alencar, Marcus Alexandre Vallim de

    2017-01-01

    Thorium has gained notoriety in recent years, as a potential source of nuclear energy, substituting uranium in power plants. Monazite is an important font of thorium, as well of uranium and rare earths elements. Professionals involved in the extraction and manipulation of this mineral are occupationally exposed to aerosols containing metals and to ionizing radiation. This paper analyzed the effects of thorium, cerium and lanthanum on cell radiosensitivity. As an osteotropic substance, thorium is mostly deposited in bone tissue and may interfere in cellular radiosensitivity. A human osteoblast cell line was used to evaluate the effects of thorium, cerium and lanthanum on cell radiosensitivity, using proliferation as indicator. Assays were performed using cell cultures exposed to metals and to ionizing radiation. As a result, metals in combination with ionizing radiation induced changes on cell proliferation, in a concentration-dependent manner, in comparison with the exposure to metals alone. That suggests the possibility of combination interfering with radiosensitivity of osteoblasts, indicating an enhancement in occupational risk for workers that manipulate monazite byproducts and are subject to radiation in the environment. Thus, the development of risk assessment models that include the evaluation of metal-radiation mixtures and their cytotoxic and radiotoxic effects on tissues and organs must be highlighted. (author)

  3. Mechanisms underlying the attachment and spreading of human osteoblasts: from transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces.

    Science.gov (United States)

    Brun, Paola; Scorzeto, Michele; Vassanelli, Stefano; Castagliuolo, Ignazio; Palù, Giorgio; Ghezzo, Francesca; Messina, Grazia M L; Iucci, Giovanna; Battaglia, Valentina; Sivolella, Stefano; Bagno, Andrea; Polzonetti, Giovanni; Marletta, Giovanni; Dettin, Monica

    2013-04-01

    The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  4. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts.

    Science.gov (United States)

    Mizoshiri, N; Kishida, T; Yamamoto, K; Shirai, T; Terauchi, R; Tsuchida, S; Mori, Y; Ejima, A; Sato, Y; Arai, Y; Fujiwara, H; Yamamoto, T; Kanamura, N; Mazda, O; Kubo, T

    2015-11-27

    Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

    International Nuclear Information System (INIS)

    Ma, W.H.; Liu, Y.J.; Wang, W.; Zhang, Y.Z.

    2015-01-01

    Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation

  6. Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

    Energy Technology Data Exchange (ETDEWEB)

    Ma, W.H.; Liu, Y.J.; Wang, W.; Zhang, Y.Z. [The Third Hospital of Hebei Medical University, The Provincial Key Laboratory for Orthopedic Biomechanics of Hebei, Shijiazhuang, Hebei Province (China)

    2015-02-13

    Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation.

  7. 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

  8. The impact of doped silicon quantum dots on human osteoblasts

    Czech Academy of Sciences Publication Activity Database

    Ostrovská, L.; Brož, Antonín; Fučíková, A.; Bělinová, T.; Sugimoto, H.; Kanno, T.; Fujii, M.; Valenta, J.; Kalbáčová, M.H.

    2016-01-01

    Roč. 6, č. 68 (2016), s. 63403-63413 ISSN 2046-2069 Institutional support: RVO:67985823 Keywords : silicon quantum dots * osteoblasts * cytotoxicity * photoluminiscence bioimaging Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.108, year: 2016

  9. 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

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

    Science.gov (United States)

    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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. Comparison of in vitro biocompatibility of NanoBone(®) and BioOss(®) for human osteoblasts.

    Science.gov (United States)

    Liu, Qin; Douglas, Timothy; Zamponi, Christiane; Becker, Stephan T; Sherry, Eugene; Sivananthan, Sureshan; Warnke, Frauke; Wiltfang, Jörg; Warnke, Patrick H

    2011-11-01

    Scaffolds for bone tissue engineering seeded with the patient's own cells might be used as a preferable method to repair bone defects in the future. With the emerging new technologies of nanostructure design, new synthetic biomaterials are appearing on the market. Such scaffolds must be tested in vitro for their biocompatibility before clinical application. However, the choice between a natural or a synthetic biomaterial might be challenging for the doctor and the patient. In this study, we compared the biocompatibility of a synthetic bone substitute, NanoBone(®) , to the widely used natural bovine bone replacement material BioOss(®) . The in vitro behaviour of human osteoblasts on both materials was investigated. Cell performance was determined using scanning electron microscopy (SEM), cell vitality staining and four biocompatibility tests (LDH, MTT, WST, BrdU). We found that both materials showed low cytotoxicity and good biocompatibility. The MTT proliferation test was superior for Nanobone(®) . Both scaffolds caused only little damage to human osteoblasts and justify their clinical application. However, NanoBone(®) was able to support and promote proliferation of human osteoblasts slightly better than BioOss(®) in our chosen test set-up. The results may guide doctors and patients when being challenged with the choice between a natural or a synthetic biomaterial. Further experiments are necessary to determine the comparison of biocompatibility in vivo. © 2011 John Wiley & Sons A/S.

  20. 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

  1. 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

  2. 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.

  3. Engineering a morphogenetically active hydrogel for bioprinting of bioartificial tissue derived from human osteoblast-like SaOS-2 cells.

    Science.gov (United States)

    Neufurth, Meik; Wang, Xiaohong; Schröder, Heinz C; Feng, Qingling; Diehl-Seifert, Bärbel; Ziebart, Thomas; Steffen, Renate; Wang, Shunfeng; Müller, Werner E G

    2014-10-01

    Sodium alginate hydrogel, stabilized with gelatin, is a suitable, biologically inert matrix that can be used for encapsulating and 3D bioprinting of bone-related SaOS-2 cells. However, the cells, embedded in this matrix, remain in a non-proliferating state. Here we show that addition of an overlay onto the bioprinted alginate/gelatine/SaOS-2 cell scaffold, consisting of agarose and the calcium salt of polyphosphate [polyP·Ca(2+)-complex], resulted in a marked increase in cell proliferation. In the presence of 100 μm polyP·Ca(2+)-complex, the cells proliferate with a generation time of approximately 47-55 h. In addition, the hardness of the alginate/gelatin hydrogel substantially increases in the presence of the polymer. The reduced Young's modulus for the alginate/gelatin hydrogel is approximately 13-14 kPa, and this value drops to approximately 0.5 kPa after incubation of the cell containing scaffolds for 5 d. In the presence of 100 μm polyP·Ca(2+)-complex, the reduced Young's modulus increases to about 22 kPa. The hardness of the polyP·Ca(2+)-complex containing hydrogel remains essentially constant if cells are absent in the matrix, but it drops to 3.2 kPa after a 5 d incubation period in the presence of SaOS-2 cells, indicating that polyP·Ca(2+)-complex becomes metabolized, degraded, by the cells. The alginate/gelatine-agarose system with polyP·Ca(2+)-complex cause a significant increase in the mineralization of the cells. SEM analyses revealed that the morphology of the mineral nodules formed on the surface of the cells embedded in the alginate/gelatin hydrogel do not significantly differ from the nodules on cells growing in monolayer cultures. The newly developed technique, using cells encapsulated into an alginate/gelatin hydrogel and a secondary layer containing the morphogenetically active, growth promoting polymer polyP·Ca(2+)-complex opens new possibilities for the application of 3D bioprinting in bone tissue engineering. Copyright

  4. 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.

  5. Ectopic bone formation in nude rats using human osteoblasts seeded poly(3)hydroxybutyrate embroidery and hydroxyapatite-collagen tapes constructs.

    Science.gov (United States)

    Mai, Ronald; Hagedorn, Manolo Gunnar; Gelinsky, Michael; Werner, Carsten; Turhani, Dritan; Späth, Heike; Gedrange, Tomas; Lauer, Günter

    2006-09-01

    The aim of this study was to evaluate the ectopic bone formation using tissue engineered cell-seeded constructs with two different scaffolds and primary human maxillary osteoblasts in nude rats over an implantation period of up to 96 days. Collagen I-coated Poly(3)hydroxybutyrate (PHB) embroidery and hydroxyapatite (HAP) collagen tapes were seeded with primary human maxillary osteoblasts (hOB) and implanted into athymic rnu/run rats. A total of 72 implants were placed into the back muscles of 18 rats. 24, 48 and 96 days after implantation, histological and histomorphometric analyses were made. The osteoblastic character of the cells was confirmed by immunocytochemistry and RT-PCR for osteocalcin. Histological analysis demonstrated that all cell-seeded constructs induced ectopic bone formation after 24, 48 and 96 days of implantation. There was more mineralized tissue in PHB constructs than in HAP-collagen tapes (at day 24; p embroidery or HAP-collagen tapes can induce ectopic bone formation. However, the amount of bone formed decreased with increasing length of implantation.

  6. Expression of LRP1 by human osteoblasts: a mechanism for the delivery of lipoproteins and vitamin K1 to bone

    DEFF Research Database (Denmark)

    Niemeier, Andreas; Kassem, Moustapha; Toedter, Klaus

    2005-01-01

    Accumulating clinical and experimental data show the importance of dietary lipids and lipophilic vitamins, such as vitamin K1, for bone formation. The molecular mechanism of how they enter the osteoblast is unknown. Here we describe the expression of the multifunctional LRP1 by human osteoblasts...... in vitro and in vivo. We provide evidence that LRP1 plays an important role in the uptake of postprandial lipoproteins and vitamin K1 by human osteoblasts....

  7. 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.

  8. 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.

  9. 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.

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

    International Nuclear Information System (INIS)

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

    1996-01-01

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

  11. Tantalum coating on TiO{sub 2} nanotubes induces superior rate of matrix mineralization and osteofunctionality in human osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Frandsen, Christine J.; Brammer, Karla S. [Materials Science and Engineering, University of California at San Diego, La Jolla, CA 92093 (United States); Noh, Kunbae [Corporate Research Institute, Cheil Industries, Inc., Gocheon-Dong, Uiwang-Si, Gyeonggi-Do, 437-711 (Korea, Republic of); Johnston, Gary [Materials Science and Engineering, University of California at San Diego, La Jolla, CA 92093 (United States); Jin, Sungho, E-mail: jin@ucsd.edu [Materials Science and Engineering, University of California at San Diego, La Jolla, CA 92093 (United States); Mechanical and Aerospace Engineering, University of California at San Diego, La Jolla, CA 92093 (United States)

    2014-04-01

    Nanostructured surface geometries have been the focus of a multitude of recent biomaterial research, and exciting findings have been published. However, only a few publications have directly compared nanostructures of various surface chemistries. The work herein directly compares the response of human osteoblast cells to surfaces of identical nanotube geometries with two well-known orthopedic biomaterials: titanium oxide (TiO{sub 2}) and tantalum (Ta). The results reveal that the Ta surface chemistry on the nanotube architecture enhances alkaline phosphatase activity, and promotes a ∼ 30% faster rate of matrix mineralization and bone-nodule formation when compared to results on bare TiO{sub 2} nanotubes. This study implies that unique combinations of surface chemistry and nanostructure may influence cell behavior due to distinctive physico-chemical properties. These findings are of paramount importance to the orthopedics field for understanding cell behavior in response to subtle alterations in nanostructure and surface chemistry, and will enable further insight into the complex manipulation of biomaterial surfaces. With increased focus in the field of orthopedic materials research on nanostructured surfaces, this study emphasizes the need for careful and systematic review of variations in surface chemistry in concurrence with nanotopographical changes. - Highlights: • A TiO{sub 2} nanotube surface structure was coated with tantalum. • Osteoblast cell response was compared between the tantalum coated and as-formed TiO{sub 2} nanotube surface. • We observed superior rates of bone matrix mineralization and osteoblast maturation on the tantalum coated nanotube surface.

  12. Tantalum coating on TiO2 nanotubes induces superior rate of matrix mineralization and osteofunctionality in human osteoblasts

    International Nuclear Information System (INIS)

    Frandsen, Christine J.; Brammer, Karla S.; Noh, Kunbae; Johnston, Gary; Jin, Sungho

    2014-01-01

    Nanostructured surface geometries have been the focus of a multitude of recent biomaterial research, and exciting findings have been published. However, only a few publications have directly compared nanostructures of various surface chemistries. The work herein directly compares the response of human osteoblast cells to surfaces of identical nanotube geometries with two well-known orthopedic biomaterials: titanium oxide (TiO 2 ) and tantalum (Ta). The results reveal that the Ta surface chemistry on the nanotube architecture enhances alkaline phosphatase activity, and promotes a ∼ 30% faster rate of matrix mineralization and bone-nodule formation when compared to results on bare TiO 2 nanotubes. This study implies that unique combinations of surface chemistry and nanostructure may influence cell behavior due to distinctive physico-chemical properties. These findings are of paramount importance to the orthopedics field for understanding cell behavior in response to subtle alterations in nanostructure and surface chemistry, and will enable further insight into the complex manipulation of biomaterial surfaces. With increased focus in the field of orthopedic materials research on nanostructured surfaces, this study emphasizes the need for careful and systematic review of variations in surface chemistry in concurrence with nanotopographical changes. - Highlights: • A TiO 2 nanotube surface structure was coated with tantalum. • Osteoblast cell response was compared between the tantalum coated and as-formed TiO 2 nanotube surface. • We observed superior rates of bone matrix mineralization and osteoblast maturation on the tantalum coated nanotube surface

  13. Lysophosphatidic acid-functionalised titanium as a superior surface for supporting human osteoblast (MG63 maturation

    Directory of Open Access Journals (Sweden)

    JP Mansell

    2012-05-01

    Full Text Available Covalent modifications of titanium with small molecules known to promote human osteoblast maturation are especially attractive in developing superior biomaterials. An important step in securing competent bone formation at implant sites is promoting the formation of mature osteoblasts, either from committed pre-osteoblasts or from their mesenchymal progenitors. To this end our research has focussed on identifying molecules that enhance human osteoblast formation and maturation and to develop ways of covalently attaching these molecules to implant surfaces so that they are more likely to withstand the rigors of the implantation process whilst still retaining their bioactivity. Herein we report the novel production of lipid-functionalised titanium using lysophosphatidic acid or a related compound, (3S 1-fluoro-3-hydroxy-4-butyl-1-phosphonate. Both lipids were especially effective at co-operating with calcitriol to promote human osteoblast maturation at these modified Ti surfaces in vitro. The novel findings presented offer enticing new developments towards the fabrication of next-generation implant devices with the potential to significantly enhance the osseointegration process and with it improvements in future prosthesis performance and longevity.

  14. Vitamin D activation of functionally distinct regulatory miRNAs in primary human osteoblasts.

    Science.gov (United States)

    Lisse, Thomas S; Chun, Rene F; Rieger, Sandra; Adams, John S; Hewison, Martin

    2013-06-01

    When bound to the vitamin D receptor (VDR), the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of osteoblast transcription. Less clear is the impact of 1,25D on posttranscriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOBs) identified human miRNAs that were differentially regulated by >1.5-fold following treatment with 1,25D (10 nM, 6 hours), which included miRNAs 637 and 1228. Quantitative reverse transcription PCR analyses showed that the host gene for miR-1228, low-density lipoprotein receptor-related protein 1 (LRP1), was coinduced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1-10 nM, 6 hours). By contrast, the endogenous host gene for miR-637, death-associated protein kinase 3 (DAPK3), was transcriptionally repressed by following treatment with 1,25D. Analysis of two potential targets for miR-637 and miR-1228 in HOB, type IV collagen (COL4A1) and bone morphogenic protein 2 kinase (BMP2K), respectively, showed that 1,25D-mediates suppression of these targets via distinct mechanisms. In the case of miR-637, suppression of COL4A1 appears to occur via decreased levels of COL4A1 mRNA. By contrast, suppression of BMP2K by miR-1228 appears to occur by inhibition of protein translation. In mature HOBs, small interfering RNA (siRNA) inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated downregulation of BMP2K protein expression. This was associated with suppression of prodifferentiation responses to 1,25D in HOB, as represented by parallel decrease in osteocalcin and alkaline phosphatase expression. These data show for the first time that the effects of 1,25D on human bone cells are not restricted to classical VDR-mediated transcriptional responses but also involve miRNA-directed posttranscriptional mechanisms. Copyright © 2013 American Society for Bone and

  15. Influence of the covalent immobilization of graphene oxide in poly(vinyl alcohol) on human osteoblast response.

    Science.gov (United States)

    Linares, Javier; Matesanz, María Concepción; Feito, María José; Salavagione, Horacio Javier; Martínez, Gerardo; Gómez-Fatou, Marián; Portolés, María Teresa

    2016-02-01

    The differences in the response of human Saos-2 osteoblasts to nanocomposites of poly(vinyl alcohol) (PVA) and 1.5wt.% graphene oxide (GO) prepared by covalent linking (PVA/GO-c) and simple blending (PVA/GO-m) have been evaluated through different biocompatibility parameters. The effects produced on osteoblasts by these two nanocomposites were analysed in parallel and compared with the direct action of GO and with the effect of PVA films without GO. The intracellular content of reactive oxygen species (ROS) and the levels of interleukin-6 (IL-6) were measured to evaluate oxidative stress induction and protective response, respectively. The results demonstrate that the combination of GO with PVA reduces both the proliferation delay and the internal cell complexity alterations induced by GO on human osteoblasts. Moreover, the covalent attachment of GO to the PVA chains increases both cell viability and IL-6 levels, reducing both apoptosis and intracellular ROS content when compared to simple blending of both materials. The use of this strategy to modulate the biointerface reduces the toxic effects of graphene while preserving the reinforcement characteristics for application in tissue engineering scaffolds, and has enormous interest for polymer/graphene biomaterials development. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Abdallah, Basem; Kassem, Moustapha

    2008-01-01

    Mesenchymal stem cells (MSC) are a group of clonogenic cells present among the bone marrow stroma and capable of multilineage differentiation into mesoderm-type cells such as osteoblasts, adipocytes and chondrocytes. Due to their ease of isolation and their differentiation potential, MSC are being...... introduced into clinical medicine in variety of applications and through different ways of administration. Here, we discuss approaches for isolation, characterization and directing differentiation of human mesenchymal stem cells (hMSC). An update of the current clinical use of the cells is also provided....

  17. Transforming growth factor-beta1 stimulates the production of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in human bone marrow stromal osteoblast progenitors

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Flyvbjerg, Allan; Eriksen, E F

    2001-01-01

    While transforming growth factor-beta1 (TGF-beta1) regulates proliferation and differentiation of human osteoblast precursor cells, the mechanisms underlying these effects are not known. Several hormones and locally acting growth factors regulate osteoblast functions through changes in the insulin......-like growth factors (IGFs) and IGF-binding proteins (IGFBPs). Thus, we studied the effects of TGF-beta1 on IGFs and IGFBPs in human marrow stromal (hMS) osteoblast precursor cells. TGF-beta1 increased the steady-state mRNA level of IGF-I up to 8.5+/-0.6-fold (P...

  18. 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.

  19. 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.

  20. The effect of a new direct Factor Xa inhibitor on human osteoblasts: an in-vitro study comparing the effect of rivaroxaban with enoxaparin

    LENUS (Irish Health Repository)

    Solayar, Gandhi N

    2011-10-28

    Abstract Background Current treatments for the prevention of thromboembolism include heparin and low-molecular weight heparins (LMWHs). A number of studies have suggested that long term administration of these drugs may adversely affect osteoblasts and therefore, bone metabolism. Xarelto™ (Rivaroxaban) is a new anti-thrombotic drug for the prevention of venous thromboembolism in adult patients undergoing elective hip and knee replacement surgery. The aim of this in vitro study was to investigate the possible effects of rivaroxaban on osteoblast viability, function and gene expression compared to enoxaparin, a commonly used LMWH. Methods Primary human osteoblast cultures were treated with varying concentrations of rivaroxaban (0.013, 0.13, 1.3 and 13 μg\\/ml) or enoxaparin (1, 10 and 100 μg\\/ml). The effect of each drug on osteoblast function was evaluated by measuring alkaline phosphatase activity. The MTS assay was used to assess the effect of drug treatments on cell proliferation. Changes in osteocalcin, Runx2 and BMP-2 messenger RNA (mRNA) expression following drug treatments were measured by real-time polymerase chain reaction (PCR). Results Rivaroxaban and enoxaparin treatment did not adversely affect osteoblast viability. However, both drugs caused a significant reduction in osteoblast function, as measured by alkaline phosphatase activity. This reduction in osteoblast function was associated with a reduction in the mRNA expression of the bone marker, osteocalcin, the transcription factor, Runx2, and the osteogenic factor, BMP-2. Conclusions These data show that rivaroxaban treatment may negatively affect bone through a reduction in osteoblast function.

  1. 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.

  2. 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.

  3. 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.

  4. Human prostatic cancer cells, PC3, elaborate mitogenic activity which selectively stimulates human bone cells

    International Nuclear Information System (INIS)

    Perkel, V.S.; Mohan, S.; Herring, S.J.; Baylink, D.J.; Linkhart, T.A.

    1990-01-01

    Prostatic cancer typically produces osteoblastic metastases which are not attended by marrow fibrosis. In the present study we sought to test the hypothesis that prostatic cancer cells produce factor(s) which act selectively on human osteoblasts. Such a paracrine mechanism would explain the observed increase in osteoblasts, unaccompanied by an increase in marrow fibroblasts. To test this hypothesis we investigated the mitogenic activity released by the human prostatic tumor cell line, PC3. PC3 cells have been reported previously to produce mitogenic activity for cells that was relatively specific for rat osteoblasts compared to rat fibroblasts. However, the effects of this activity on human cells has not been examined previously. PC3-conditioned medium (CM) (5-50 micrograms CM protein/ml) stimulated human osteoblast proliferation by 200-950% yet did not stimulate human fibroblast proliferation ([3H]thymidine incorporation). PC3 CM also increased cell numbers in human osteoblast but not fibroblast cell cultures. To determine whether the osteoblast-specific mitogenic activity could be attributed to known bone growth factors, specific assays for these growth factors were performed. PC3 CM contained 10 pg insulin-like growth factor (IGF) I, less than 2 pg IGF II, 54 pg basic fibroblast growth factor, and 16 pg transforming growth factor beta/microgram CM protein. None of these growth factors alone or in combination could account for the observed osteoblast-specific PC3 cell-derived mitogenic activity. Furthermore, when 5 micrograms/ml PC3 CM was tested in combination with maximally effective concentrations of either basic fibroblast growth factor, IGF I, IGF II, or transforming growth factor beta, it produced an additive effect suggesting that PC3 CM stimulates osteoblast proliferation by a mechanism independent of these bone mitogens

  5. Quercetin Protects Primary Human Osteoblasts Exposed to Cigarette Smoke through Activation of the Antioxidative Enzymes HO-1 and SOD-1

    Directory of Open Access Journals (Sweden)

    Karl F. Braun

    2011-01-01

    Full Text Available Smokers frequently suffer from impaired fracture healing often due to poor bone quality and stability. Cigarette smoking harms bone cells and their homeostasis by increased formation of reactive oxygen species (ROS. The aim of this study was to investigate whether Quercetin, a naturally occurring antioxidant, can protect osteoblasts from the toxic effects of smoking. Human osteoblasts exposed to cigarette smoke medium (CSM rapidly produced ROS and their viability decreased concentration- and time-dependently. Co-, pre- and postincubation with Quercetin dose-dependently improved their viability. Quercetin increased the expression of the anti-oxidative enzymes heme-oxygenase- (HO- 1 and superoxide-dismutase- (SOD- 1. Inhibiting HO-1 activity abolished the protective effect of Quercetin. Our results demonstrate that CSM damages human osteoblasts by accumulation of ROS. Quercetin can diminish this damage by scavenging the radicals and by upregulating the expression of HO-1 and SOD-1. Thus, a dietary supplementation with Quercetin could improve bone matter, stability and even fracture healing in smokers.

  6. 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.

  7. Derivation of Stromal (Skeletal, Mesenchymal) Stem-like cells from Human Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Mahmood, Amer; Harkness, Linda; Abdallah, Basem

    2012-01-01

    EBs using BMP2 (bone morphogenic protein 2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold......Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESC) is a pre-requisite for their use in clinical applications. However, there is no standard protocol for differentiating hESC into osteoblastic cells. The aim of this study was to identify the emergence of a human...... stromal (mesenchymal, skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESC in a feeder-free environment using serum replacement and as suspension aggregates (embryoid...

  8. Biocompatibility of the titanium-based implant surfaces: Effect of the calcium dihydrogen phosphate on osteoblast cells

    Directory of Open Access Journals (Sweden)

    Kaluđerović Milena R.

    2016-01-01

    Full Text Available The influence of the presence of calcium dihydrogen phosphate in acid media on titanium-based implant surfaces, Ticer, employed in clinics, and its white form (Ticer white, on osteoblast cells was investigated. Novel surfaces M1 and M2 were obtained by immersing Ticer and Ticer white surfaces in calcium dihydrogen phosphate solution at pH 3.5. The surfaces were characterized by SEM, EDS and X-ray diffraction. The results related to interaction of investigated surfaces and human osteoblast cells from indirect biocompatibility (MTT and SRB assays, proliferation (DAPI assay and mode of cell death (acridine orange/ethidium bromide (AO/EB double staining were found to be in good agreement, as well as findings from osteocalcin (OC and bone sialoprotein (BSP expression. Surfaces were obtained by employing anodic plasma-electrochemical oxidation with spark discharges without subsequent surface modifications were found to be more compatible. Soaking of Ticer and Ticer white in phosphate solution gave toxic materials (M1 and M2 which induced apoptosis and secondary necrosis in osteoblast cells.

  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. 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

  12. 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

  13. Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro

    Science.gov (United States)

    Elbaz, Alexandre; Wu, Xiying; Rivas, Daniel; Gimble, Jeffrey M; Duque, Gustavo

    2010-01-01

    Abstract Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell–cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase. PMID:19382912

  14. MiR-9 is overexpressed in spontaneous canine osteosarcoma and promotes a metastatic phenotype including invasion and migration in osteoblasts and osteosarcoma cell lines.

    Science.gov (United States)

    Fenger, Joelle M; Roberts, Ryan D; Iwenofu, O Hans; Bear, Misty D; Zhang, Xiaoli; Couto, Jason I; Modiano, Jaime F; Kisseberth, William C; London, Cheryl A

    2016-10-10

    MicroRNAs (miRNAs) regulate the expression of networks of genes and their dysregulation is well documented in human malignancies; however, limited information exists regarding the impact of miRNAs on the development and progression of osteosarcoma (OS). Canine OS exhibits clinical and molecular features that closely resemble the corresponding human disease and it is considered a well-established spontaneous animal model to study OS biology. The purpose of this study was to investigate miRNA dysregulation in canine OS. We evaluated miRNA expression in primary canine OS tumors and normal canine osteoblast cells using the nanoString nCounter system. Quantitative PCR was used to validate the nanoString findings and to assess miR-9 expression in canine OS tumors, OS cell lines, and normal osteoblasts. Canine osteoblasts and OS cell lines were stably transduced with pre-miR-9 or anti-miR-9 lentiviral constructs to determine the consequences of miR-9 on cell proliferation, apoptosis, invasion and migration. Proteomic and gene expression profiling of normal canine osteoblasts with enforced miR-9 expression was performed using 2D-DIGE/tandem mass spectrometry and RNA sequencing and changes in protein and mRNA expression were validated with Western blotting and quantitative PCR. OS cell lines were transduced with gelsolin (GSN) shRNAs to investigate the impact of GSN knockdown on OS cell invasion. We identified a unique miRNA signature associated with primary canine OS and identified miR-9 as being significantly overexpressed in canine OS tumors and cell lines compared to normal osteoblasts. Additionally, high miR-9 expression was demonstrated in tumor-specific tissue obtained from primary OS tumors. In normal osteoblasts and OS cell lines transduced with miR-9 lentivirus, enhanced invasion and migration were observed, but miR-9 did not affect cell proliferation or apoptosis. Proteomic and transcriptional profiling of normal canine osteoblasts overexpressing miR-9 identified

  15. CBFA1 and topoisomerase I mRNA levels decline during cellular aging of human trabecular osteoblasts

    DEFF Research Database (Denmark)

    Christiansen, Mette; Kveiborg, M.; Kassem, M.

    2000-01-01

    In order to understand the reasons for age-related impairment of the function of bone forming osteoblasts, we have examined the steady-state mRNA levels of the transcription factor CBFA1 and topoisomerase I during cellular aging of normal human trabecular osteoblasts, by the use of semiquantitati...

  16. 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.

  17. 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...

  18. Vitamins D3 and K2 may partially counterbalance the detrimental effects of pentosidine in ex vivo human osteoblasts.

    Science.gov (United States)

    Sanguineti, R; Monacelli, F; Parodi, A; Furfaro, A L; Borghi, R; Pacini, D; Pronzato, M A; Odetti, P; Molfetta, L; Traverso, N

    2016-01-01

    Osteoporosis is a metabolic multifaceted disorder, characterized by insufficient bone strength. It has been recently shown that advanced glycation end products (AGEs) play a role in senile osteoporosis, through bone cell impairment and altered biomechanical properties. Pentosidine (PENT), a wellcharacterized AGE, is also considered a biomarker of bone fracture. Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D 3 , are prerequisites for optimal osteoblasts functioning. Vitamin K 2 is known to enhance in vitro and in vitro vitamin D-induced bone formation. The aim of the study was to assess the effects of Vitamins D 3 and K 2 and PENT on in vitro osteoblast activity, to convey a possible translational clinical message. Ex vivo human osteoblasts cultured, for 3 weeks, with vitamin D 3 and vitamin K 2 were exposed to PENT, a well-known advanced glycoxidation end product for the last 72 hours. Experiments with PENT alone were also carried out. Gene expression of specific markers of bone osteoblast maturation [alkaline phosphatase, ALP; collagen I, COL Iα1; and osteocalcin (bone-Gla-protein) BGP] was measured, together with the receptor activator of nuclear factor kappa-B ligand/osteoproteregin (RANKL/OPG) ratio to assess bone remodeling. Expression of RAGE, a well-characterized receptor of AGEs, was also assessed. PENT+vitamins slightly inhibited ALP secretion while not affecting gene expression, indicating hampered osteoblast functional activity. PENT+vitamins up-regulated collagen gene expression, while protein secretion was unchanged. Intracellular collagen levels were partially decreased, and a significant reduction in BGP gene expression and intracellular protein concentration were both reported after PENT exposure. The RANKL/OPG ratio was increased, favouring bone reabsorption. RAGE gene expression significantly decreased. These results were confirmed by a lower mineralization rate. We provided in vitro evidence that glycoxidation might

  19. 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

  20. 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.

  1. Gene expression analysis in human osteoblasts exposed to dexamethasone identifies altered developmental pathways as putative drivers of osteoporosis

    Directory of Open Access Journals (Sweden)

    Sadlier Denise M

    2007-02-01

    Full Text Available Abstract Background Osteoporosis, a disease of decreased bone mineral density represents a significant and growing burden in the western world. Aging population structure and therapeutic use of glucocorticoids have contributed in no small way to the increase in the incidence of this disease. Despite substantial investigative efforts over the last number of years the exact molecular mechanism underpinning the initiation and progression of osteoporosis remain to be elucidated. This has meant that no significant advances in therapeutic strategies have emerged, with joint replacement surgery being the mainstay of treatment. Methods In this study we have used an integrated genomics profiling and computational biology based strategy to identify the key osteoblast genes and gene clusters whose expression is altered in response to dexamethasone exposure. Primary human osteoblasts were exposed to dexamethasone in vitro and microarray based transcriptome profiling completed. Results These studies identified approximately 500 osteoblast genes whose expression was altered. Functional characterization of the transcriptome identified developmental networks as being reactivated with 106 development associated genes found to be differentially regulated. Pathway reconstruction revealed coordinate alteration of members of the WNT signaling pathway, including frizzled-2, frizzled-7, DKK1 and WNT5B, whose differential expression in this setting was confirmed by real time PCR. Conclusion The WNT pathway is a key regulator of skeletogenesis as well as differentiation of bone cells. Reactivation of this pathway may lead to altered osteoblast activity resulting in decreased bone mineral density, the pathological hallmark of osteoporosis. The data herein lend weight to the hypothesis that alterations in developmental pathways drive the initiation and progression of osteoporosis.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. Beneficial Effects of Concentrated Growth Factors and Resveratrol on Human Osteoblasts In Vitro Treated with Bisphosphonates

    Directory of Open Access Journals (Sweden)

    Elisa Borsani

    2018-01-01

    Full Text Available Bisphosphonates are primary pharmacological agents against osteoclast-mediated bone loss and widely used in the clinical practice for prevention and treatment of a variety of skeletal conditions, such as low bone density and osteogenesis imperfecta, and pathologies, such as osteoporosis, malignancies metastatic to bone, Paget disease of bone, multiple myeloma, and hypercalcemia of malignancy. However, long-term bisphosphonate treatment is associated with pathologic conditions including osteonecrosis of the jaw, named BRONJ, which impaired bone regeneration process. Clinical management of BRONJ is controversy and one recent approach is the use of platelet concentrates, such as Concentrated Growth Factors, alone or together with biomaterials or antioxidants molecules, such as resveratrol. The aim of the present study was to investigate the in vitro effects of Concentrated Growth Factors and/or resveratrol on the proliferation and differentiation of human osteoblasts, treated or not with bisphosphonates. Human osteoblasts were stimulated for 3 days in complete medium and for 21 days in mineralization medium. At the end of the experimental period, the in vitro effect on osteoblast proliferation and differentiation was evaluated using different techniques such as MTT, ELISA for the quantification/detection of osteoprotegerin and bone morphogenetic protein-2, immunohistochemistry for sirtuin 1 and collagen type I, and the Alizarin Red S staining for the rate of mineralization. Results obtained showed that Concentrated Growth Factors and/or resveratrol significantly increased osteoblast proliferation and differentiation and that the cotreatment with Concentrated Growth Factors and resveratrol had a protective role on osteoblasts treated with bisphosphonates. In conclusion, these data suggest that this approach could be promised in the clinical management of BRONJ.

  10. 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.

  11. [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.

  12. Osteoblast Cell Response on the Ti6Al4V Alloy Heat-Treated

    Directory of Open Access Journals (Sweden)

    Mercedes Paulina Chávez-Díaz

    2017-04-01

    Full Text Available In an effort to examine the effect of the microstructural changes of the Ti6Al4V alloy, two heat treatments were carried out below (Ti6Al4V800 and above (Ti6Al4V1050 its β-phase transformation temperature. After each treatment, globular and lamellar microstructures were obtained. Saos-2 pre-osteoblast human osteosarcoma cells were seeded onto Ti6Al4V alloy disks and immersed in cell culture for 7 days. Electrochemical assays in situ were performed using OCP and EIS measurements. Impedance data show a passive behavior for the three Ti6Al4V alloys; additionally, enhanced impedance values were recorded for Ti6Al4V800 and Ti6Al4V1050 alloys. This passive behavior in culture medium is mostly due to the formation of TiO2 during their sterilization. Biocompatibility and cell adhesion were characterized using the SEM technique; Ti6Al4V as received and Ti6Al4V800 alloys exhibited polygonal and elongated morphology, whereas Ti6Al4V1050 alloy displayed a spherical morphology. Ti and O elements were identified by EDX analysis due to the TiO2 and signals of C, N and O, related to the formation of organic compounds from extracellular matrix. These results suggest that cell adhesion is more likely to occur on TiO2 formed in discrete α-phase regions (hcp depending on its microstructure (grains.

  13. 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.

  14. 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

  15. 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.

  16. Investigation of integrin expression on the surface of osteoblast-like cells by atomic force microscopy

    International Nuclear Information System (INIS)

    Caneva Soumetz, Federico; Saenz, Jose F.; Pastorino, Laura; Ruggiero, Carmelina; Nosi, Daniele; Raiteri, Roberto

    2010-01-01

    The transforming growth factor β1 (TGF-β1) is a human cytokine which has been demonstrated to modulate cell surface integrin repertoire. In this work integrin expression in response to TGF-β1 stimulation has been investigated on the surface of human osteoblast-like cells. We used atomic force microscopy (AFM) and confocal laser scanning microscopy to assess integrin expression and to evaluate their distribution over the dorsal side of the plasma membrane. AFM probes have been covalently functionalised with monoclonal antibodies specific to the β1 integrin subunit. Force curves have been collected in order to obtain maps of the interaction between the immobilized antibody and the respective cell membrane receptors. Adhesion peaks have been automatically detected by means of an ad hoc developed data analysis software. The specificity of the detected interactions has been assessed by adding free antibody in the solution and monitoring the dramatic decrease in the recorded interactions. In addition, the effect of TGF-β1 treatment on both the fluorescence signal and the adhesion events has been tested. The level of expression of the β1 integrin subunit was enhanced by TGF-β1. As a further analysis, the adhesion force of the single living cells to the substrate was measured by laterally pushing the cell with the AFM tip and measuring the force necessary to displace it. The treatment with TGF-β1 resulted in a decrease of the cell/substrate adhesion force. Results obtained by AFM have been validated by confocal laser scanning microscopy thus demonstrating the high potential of the AFM technique for the investigation of cell surface receptors distribution and trafficking at the nanoscale.

  17. Investigation of integrin expression on the surface of osteoblast-like cells by atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Caneva Soumetz, Federico [Department of Communication, Computer and System Sciences, University of Genova, Via Opera Pia, 13-16145 Genova (Italy); Saenz, Jose F. [Biophysical and Electronic Engineering Department, University of Genova, Via All' Opera Pia 11a, 16145 Genova (Italy); Pastorino, Laura; Ruggiero, Carmelina [Department of Communication, Computer and System Sciences, University of Genova, Via Opera Pia, 13-16145 Genova (Italy); Nosi, Daniele [Department of Anatomy, Histology and Forensic Medicine, Bio-photonic Laboratory, University of Florence, viale Morgagni, 85 Firenze, CAP 50134 Florence (Italy); Raiteri, Roberto, E-mail: rr@unige.it [Biophysical and Electronic Engineering Department, University of Genova, Via All' Opera Pia 11a, 16145 Genova (Italy)

    2010-03-15

    The transforming growth factor {beta}1 (TGF-{beta}1) is a human cytokine which has been demonstrated to modulate cell surface integrin repertoire. In this work integrin expression in response to TGF-{beta}1 stimulation has been investigated on the surface of human osteoblast-like cells. We used atomic force microscopy (AFM) and confocal laser scanning microscopy to assess integrin expression and to evaluate their distribution over the dorsal side of the plasma membrane. AFM probes have been covalently functionalised with monoclonal antibodies specific to the {beta}1 integrin subunit. Force curves have been collected in order to obtain maps of the interaction between the immobilized antibody and the respective cell membrane receptors. Adhesion peaks have been automatically detected by means of an ad hoc developed data analysis software. The specificity of the detected interactions has been assessed by adding free antibody in the solution and monitoring the dramatic decrease in the recorded interactions. In addition, the effect of TGF-{beta}1 treatment on both the fluorescence signal and the adhesion events has been tested. The level of expression of the {beta}1 integrin subunit was enhanced by TGF-{beta}1. As a further analysis, the adhesion force of the single living cells to the substrate was measured by laterally pushing the cell with the AFM tip and measuring the force necessary to displace it. The treatment with TGF-{beta}1 resulted in a decrease of the cell/substrate adhesion force. Results obtained by AFM have been validated by confocal laser scanning microscopy thus demonstrating the high potential of the AFM technique for the investigation of cell surface receptors distribution and trafficking at the nanoscale.

  18. 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.

  19. Optimization of the Static Human Osteoblast/Osteoclast Co-culture System

    Directory of Open Access Journals (Sweden)

    James Jam Jolly

    2018-03-01

    Full Text Available Osteoblasts (OBs and osteoclasts (OCs are 2 major groups of bone cells. Their cell-to-cell interactions are important to ensure the continuity of the bone-remodeling process. Therefore, the present study was carried out to optimize an OB/OC co-culture system utilizing the human OB cell line hFOB 1.19 and OCs extracted from peripheral blood mononuclear cells (PBMNCs. It was a 2-step procedure, involving the optimization of the OB culture and the co-culture of the OBs with PBMNCs at an optimum ratio. Firstly, pre-OBs were cultured to 90% confluency and the time required for differentiation was determined. OB differentiation was determined using the van Gieson staining to detect the presence of collagen and Alizarin Red for calcium. Secondly, OBs and OCs were co-cultured at the ratios of 1 OC: 1 OB, 1 OC: 4 OBs, 2 OCs: 1 OB, and 1 OC: 2 OBs. Tartrate-resistant acid phosphatase (TRAP staining was used to detect the differentiation of the OCs. The results showed that collagen was present on day 1, whereas calcium was detected as early as day 3. Based on the result of TRAP staining, 1 OC: 2 OBs was taken as the most appropriate ratio. No macrophage colony-stimulating factor and receptor activator of the nuclear factor-κB ligand were added because they were provided by the OBs. In conclusion, these optimization processes are vital as they ensure the exact time point and ratio of the OB/OC co-culture in order to produce a reliable and reproducible co-culture system.

  20. 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...

  1. Biocompatibility evaluation in vitro. Part I: Morphology expression and proliferation of human and rat osteoblasts on the biomaterials

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The biocompatibility evaluation of calcium phosphate based biomaterials is performed by tissue culture in vitro model. Three kinds of bioceramic materials which are potential to deal with bone trauma and/or conduct tissue growth are recommodated. The biological research results show that human and animal osteoblast cells anchor the materials surface in two hours in culture. Confocal laser scanning microscopy (CLSM) demonstrated the normal cell distribution and proliferation on both of dense and porous biomaterials. Hydroxyapatite and tricalcium phosphate stimulate cell proliferation. However, DNA and protein synthesis were considerably limited and the apoptosis phenomenon would be present on the hydroxyapatite (HA) materials by adding Al, Mg elements. Several important methods of biocompatibility evaluation of implant materials are described and the related biological molecular techniques such as tissue culture, cell transfection, cellular DNA stain, and Lowry assay are involved in the present research.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. 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.

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Lin H

    2014-07-01

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

  6. Interaction of CO2 laser-modified nylon with osteoblast cells in relation to wettability

    International Nuclear Information System (INIS)

    Waugh, D.G.; Lawrence, J.; Morgan, D.J.; Thomas, C.L.

    2009-01-01

    It has been amply demonstrated previously that CO 2 lasers hold the ability to surface modify various polymers. In addition, it has been observed that these surface enhancements can augment the biomimetic nature of the laser irradiated materials. This research has employed a CO 2 laser marker to produce trench and hatch topographical patterns with peak heights of around 1 μm on the surface of nylon 6,6. The patterns generated have been analysed using white light interferometry, optical microscopy and X-ray photoelectron spectroscopy was employed to determine the surface oxygen content. Contact angle measurements were used to characterize each sample in terms of wettability. Generally, it was seen that as a result of laser processing the contact angle, surface roughness and surface oxygen content increased whilst the apparent polar and total surface energies decreased. The increase in contact angle and reduction in surface energy components was found to be on account of a mixed intermediate state wetting regime owing to the change in roughness due to the induced topographical patterns. To determine the biomimetic nature of the modified and as-received control samples each one was seeded with 2 x 10 4 cells/ml normal human osteoblast cells and observed after periods of 24 h and 4 days using optical microscopy and SEM to determine mean cell cover densities and variations in cell morphology. In addition, a haemocytometer was used to show that the cell count for the laser patterned samples had increased by up to a factor of 1.5 compared to the as-received control sample after 4 days of incubation. Significantly, it was determined that all laser-induced patterns gave rise to better cell response in comparison to the as-received control sample studied due to increased preferential cell growth on those surfaces with increased surface roughness.

  7. 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

  8. 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.

  9. Carbon Nanotubes-Hydroxyapatite Nanocomposites for an Improved Osteoblast Cell Response

    Directory of Open Access Journals (Sweden)

    Sabrina Constanda

    2016-01-01

    Full Text Available An alternative and simple coprecipitation method was developed to obtain carbon nanotube-hydroxyapatite (CNTs:HAp based nanocomposites. The incorporation of CNTs (in a concentration of 5% and 10% of total weight of the nanocomposite and their impact on both structural and biological properties were studied by using a set of standard complementary biological, microscopic, and spectroscopic techniques. The characteristic peaks of carbon structure in CNTs were not observed in the CNTs-HAp composites by X-ray diffraction analysis. Moreover, FTIR and Raman spectroscopies confirmed the presence of HAp as the main phase of the synthesized CNTs:HAp nanocomposites. The addition of CNTs considerably affected the nanocomposite morphology by increasing the average crystallite size from 18.7 nm (for raw HAp to 28.6 nm (for CNTs:HAp-10, confirming their proper incorporation. The biocompatibility evaluation of CNTs:HAp-5 and CNTs:HAp-10 nanocomposites included the assessment of several parameters, such as cell viability, antioxidant response, and lipid peroxidation, on human G-292 osteoblast cell line. Our findings revealed good biocompatibility properties for CNTs:HAp nanocomposites prepared by the coprecipitation method supporting their potential uses in orthopedics and prosthetics.

  10. 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.

  11. 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

  12. 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

  13. Early cell adhesion events differ between osteoporotic and non-osteoporotic osteoblasts.

    Science.gov (United States)

    Perinpanayagam, H; Zaharias, R; Stanford, C; Brand, R; Keller, J; Schneider, G

    2001-11-01

    In osteoporosis, the regenerative capacity of bone is compromised, which may involve altered osteoblast (OB) activity. This could be attributed to an inappropriate synthesis and assembly of an extracellular matrix (ECM), altered cell adhesion to the ECM, or be due to inappropriate downstream activation of adhesion-mediated signaling cascades through proteins such as focal adhesion kinase (FAK). The purpose of our study was to compare early adhesion-mediated events using previously described and characterized clinically derived OBs obtained from human patients undergoing major joint arthroplasty for osteoporosis or osteoarthritis. The presence or absence of osteoporosis was established with a radiographic index. Using light microscopy and crystal violet staining, we show that OB cells derived from sites of osteoporosis do not attach and spread as well as non-osteoporotic (OP) OB cells. OP cells initially have a more rounded morphology, and show significantly less (P attachment to serum-coated tissue culture plastic over a 24 h time period. Immunofluorescent labeling after 24 h of attachment showed that OP OB focal adhesions (FAs) and stress fibers were less defined, and that the OP cells were smaller and had a more motile phenotype. When normalized protein lysates were Western blotted for phosphotyrosine (PY) a band corresponding to pp125FAK was identified. FAK tyrosine phosphorylation was evident at 6 h in both the OP and non-OP OBs, but decreased or was absent through 24 h in OP OBs. These results suggest early adhesion-mediated events, such as cell adhesion, attachment, and FAK signaling via PY may be altered in OP OBs.

  14. In Vitro Proliferation and Anti-Apoptosis of the Papain-Generated Casein and Soy Protein Hydrolysates towards Osteoblastic Cells (hFOB1.19).

    Science.gov (United States)

    Pan, Xiao-Wen; Zhao, Xin-Huai

    2015-06-17

    Casein and soy protein were digested by papain to three degrees of hydrolysis (DH) 7.3%-13.3%, to obtain respective six casein and soy protein hydrolysates, aiming to clarify their in vitro proliferation and anti-apoptosis towards a human osteoblastic cell line (hFOB1.19 cells). Six casein and soy protein hydrolysates at five levels (0.01-0.2 mg/mL) mostly showed proliferation as positive 17β-estradiol did, because they conferred the osteoblasts with cell viability of 100%-114% and 104%-123%, respectively. The hydrolysates of higher DH values had stronger proliferation. Casein and soy protein hydrolysates of the highest DH values altered cell cycle progression, and enhanced cell proportion of S-phase from 50.5% to 56.5% and 60.5%. The two also antagonized etoposide- and NaF-induced osteoblast apoptosis. In apoptotic prevention, apoptotic cells were decreased from 31.6% to 22.6% and 15.6% (etoposide treatment), or from 19.5% to 17.7% and 12.4% (NaF treatment), respectively. In apoptotic reversal, soy protein hydrolysate decreased apoptotic cells from 13.3% to 11.7% (etoposide treatment), or from 14.5% to 11.0% (NaF treatment), but casein hydrolysate showed no reversal effect. It is concluded that the hydrolysates of two kinds had estradiol-like action on the osteoblasts, and soy protein hydrolysates had stronger proliferation and anti-apoptosis on the osteoblasts than casein hydrolysates.

  15. 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)

  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. Particles induced surface nanoroughness of titanium surface and its influence on adhesion of osteoblast-like MG-63 cells

    Science.gov (United States)

    Solař, P.; Kylián, O.; Marek, A.; Vandrovcová, M.; Bačáková, L.; Hanuš, J.; Vyskočil, J.; Slavínská, D.; Biederman, H.

    2015-01-01

    Titanium is one of the most common materials employed for production of implants, which is due to its good biocompatibility. However, the colonization of titanium surface by osteoblast cells may be influenced by its roughness and therefore precise control of roughness of titanium surface as well as identification of its optimal value for growth of cells is of high importance. In this study the nanorough titanium surfaces were prepared on polished disks of TiAlV by two step method of deposition. In the first step TiAlV were coated by nanoparticles generated by gas aggregation sources. Such prepared films of nanoparticles were subsequently covered with a titanium overlayer. Different values of surface roughness in the range 1-100 nm were achieved by variation of the size and number of the nanoparticles. Such prepared surfaces were subsequently used for investigation of influence of roughness of titanium surfaces on the adhesion of human osteoblast-like MG-63 cells. It was found out that 7 days after seeding the highest number of adhering cells was observed for samples with root-mean-square roughness of 30 nm.

  18. 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.

  19. Omentin-1 Stimulates Human Osteoblast Proliferation through PI3K/Akt Signal Pathway

    Directory of Open Access Journals (Sweden)

    Shan-Shan Wu

    2013-01-01

    Full Text Available It has been presumed that adipokines deriving from adipose tissue may play important roles in bone metabolism. Omentin-1, a novel adipokine, which is selectively expressed in visceral adipose tissue, has been reported to stimulate proliferation and inhibit differentiation of mouse osteoblast. However, little information refers to the effect of omentin-1 on human osteoblast (hOB proliferation. The current study examined the potential effects of omentin-1 on proliferation in hOB and the signal pathway involved. Omentin-1 promoted hOB proliferation in a dose-dependent manner as determined by [3H]thymidine incorporation. Western blot analysis revealed that omentin-1 induced activation of Akt (phosphatidylinositol-3 kinase downstream effector and such effect was impeded by transfection of hOB with Akt-siRNA. Furthermore, LY294002 (a selective PI3K inhibitor and HIMO (a selective Akt inhibitor abolished the omentin-1-induced hOB proliferation. These findings indicate that omentin-1 induces hOB proliferation via the PI3K/Akt signaling pathway and suggest that osteoblast is a direct target of omentin-1.

  20. 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.

  1. Low proliferation and high apoptosis of osteoblastic cells on hydrophobic surface are associated with defective Ras signaling

    International Nuclear Information System (INIS)

    Chang, Eun-Ju; Kim, Hong-Hee; Huh, Jung-Eun; Kim, In-Ae; Seung Ko, Jea; Chung, Chong-Pyoung; Kim, Hyun-Man

    2005-01-01

    The hydrophobic (HPB) nature of most polymeric biomaterials has been a major obstacle in using those materials in vivo due to low compatibility with cells. However, there is little knowledge of the molecular detail to explain how surface hydrophobicity affects cell responses. In this study, we compared the proliferation and apoptosis of human osteoblastic MG63 cells adhered to hydrophilic (HPL) and hydrophobic surfaces. On the hydrophobic surface, less formation of focal contacts and actin stress fibers, a delay in cell cycle progression, and an increase in apoptosis were observed. By using fibroblast growth factor 1 (FGF1) as a model growth factor, we also investigated intracellular signaling pathways on hydrophilic and hydrophobic surfaces. The activation of Ras, Akt, and ERK by FGF1 was impaired in MG63 cells on the hydrophobic surface. The overexpression of constitutively active form of Ras and Akt rescued those cells from apoptosis and recovered cell cycle progression. Furthermore, their overexpression also restored the actin cytoskeletal organization on the hydrophobic surface. Finally, the proliferative, antiapoptotic, and cytoskeletal effects of constitutively active Ras in MG63 cells on the hydrophobic surface were blocked by wortmannin and PD98059 that inhibit Akt and ERK activation, respectively. Therefore, our results suggest that the activation of Ras and its downstream molecules Akt and ERK to an appropriate level is one of crucial elements in the determination of osteoblast cell responses. The Ras pathway may represent a cell biological target that should be considered for successful surface modification of biomaterials to induce adequate cell responses in the bone tissue

  2. Human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Zaher, Walid; Al-Nbaheen, May

    2012-01-01

    Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self......-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number...

  3. 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.

  4. 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...

  5. 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.

  6. 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.

  7. Osteoblasts Interaction with PLGA Membranes Functionalized with Titanium Film Nanolayer by PECVD. In vitro Assessment of Surface Influence on Cell Adhesion during Initial Cell to Material Interaction

    Science.gov (United States)

    Terriza, Antonia; Vilches-Pérez, José I.; González-Caballero, Juan L.; de la Orden, Emilio; Yubero, Francisco; Barranco, Angel; Gonzalez-Elipe, Agustín R.; Vilches, José; Salido, Mercedes

    2014-01-01

    New biomaterials for Guided Bone Regeneration (GBR), both resorbable and non-resorbable, are being developed to stimulate bone tissue formation. Thus, the in vitro study of cell behavior towards material surface properties turns a prerequisite to assess both biocompatibility and bioactivity of any material intended to be used for clinical purposes. For this purpose, we have developed in vitro studies on normal human osteoblasts (HOB®) HOB® osteoblasts grown on a resorbable Poly (lactide-co-glycolide) (PLGA) membrane foil functionalized by a very thin film (around 15 nm) of TiO2 (i.e., TiO2/PLGA membranes), designed to be used as barrier membrane. To avoid any alteration of the membranes, the titanium films were deposited at room temperature in one step by plasma enhanced chemical vapour deposition. Characterization of the functionalized membranes proved that the thin titanium layer completely covers the PLGA foils that remains practically unmodified in their interior after the deposition process and stands the standard sterilization protocols. Both morphological changes and cytoskeletal reorganization, together with the focal adhesion development observed in HOB osteoblasts, significantly related to TiO2 treated PLGA in which the Ti deposition method described has revealed to be a valuable tool to increase bioactivity of PLGA membranes, by combining cell nanotopography cues with the incorporation of bioactive factors. PMID:28788538

  8. Osteoblasts Interaction with PLGA Membranes Functionalized with Titanium Film Nanolayer by PECVD. In vitro Assessment of Surface Influence on Cell Adhesion during Initial Cell to Material Interaction

    Directory of Open Access Journals (Sweden)

    Antonia Terriza

    2014-03-01

    Full Text Available New biomaterials for Guided Bone Regeneration (GBR, both resorbable and non-resorbable, are being developed to stimulate bone tissue formation. Thus, the in vitro study of cell behavior towards material surface properties turns a prerequisite to assess both biocompatibility and bioactivity of any material intended to be used for clinical purposes. For this purpose, we have developed in vitro studies on normal human osteoblasts (HOB® HOB® osteoblasts grown on a resorbable Poly (lactide-co-glycolide (PLGA membrane foil functionalized by a very thin film (around 15 nm of TiO2 (i.e., TiO2/PLGA membranes, designed to be used as barrier membrane. To avoid any alteration of the membranes, the titanium films were deposited at room temperature in one step by plasma enhanced chemical vapour deposition. Characterization of the functionalized membranes proved that the thin titanium layer completely covers the PLGA foils that remains practically unmodified in their interior after the deposition process and stands the standard sterilization protocols. Both morphological changes and cytoskeletal reorganization, together with the focal adhesion development observed in HOB osteoblasts, significantly related to TiO2 treated PLGA in which the Ti deposition method described has revealed to be a valuable tool to increase bioactivity of PLGA membranes, by combining cell nanotopography cues with the incorporation of bioactive factors.

  9. 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.

  10. 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.

  11. The antiarrhythmic peptide analog rotigaptide (ZP123) stimulates gap junction intercellular communication in human osteoblasts and prevents decrease in femoral trabecular bone strength in ovariectomized rats

    DEFF Research Database (Denmark)

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

    2005-01-01

    Gap junctions play an important role in bone development and function, but the lack of pharmacological tools has hampered the gap junction research. The antiarrhythmic peptides stimulate gap junction communication between cardiomyocytes, but effects in noncardiac tissue are unknown. The purpose...... of this study was to examine whether antiarrhythmic peptides, which are small peptides increasing gap junctional conductivity, show specific binding to osteoblasts and investigate the effect of the stable analog rotigaptide (ZP123) on gap junctional intercellular communication in vitro and on bone mass...... and strength in vivo. Cell coupling and calcium signaling were assessed in vitro on human, primary, osteoblastic cells. In vivo effects of rotigaptide on bone strength and density were determined 4 wk after ovariectomy in rats treated with either vehicle, sc injection twice daily (300 nmol per kilogram body...

  12. 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.

  13. The influence of bisphosphonates on human osteoblast migration and integrin aVb3/tenascin C gene expression in vitro

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    Said Yekta Sareh

    2011-02-01

    Full Text Available Abstract Background Bisphosphonates are therapeutics of bone diseases, such as Paget's disease, multiple myeloma or osteoclastic metastases. As a severe side effect the bisphosphonate induced osteonecrosis of the jaw (BONJ often requires surgical treatment and is accompanied with a disturbed wound healing. Therefore, the influence on adhesion and migration of human osteoblasts (hOB after bisphosphonate therapy has been investigated by morphologic as well as gene expression methods. Methods By a scratch wound experiment, which measures the reduction of defined cell layer gap, the morphology and migration ability of hOB was evaluated. A test group of hOB, which was stimulated by zoledronate 5 × 10-5M, and a control group of unstimulated hOB were applied. Furthermore the gene expression of integrin aVb3 and tenascin C was quantified by Real-Time rtPCR at 5data points over an experimental period of 14 days. The bisphosphonates zoledronate, ibandronate and clodronate have been compared with an unstimulated hOB control. Results After initially identical migration and adhesion characteristics, zoledronate inhibited hOB migration after 50 h of stimulation. The integrinavb3 and tenascin C gene expression was effected by bisphosphonates in a cell line dependent manner with decreased, respectively inconsistent gene expression levels over time. The non-nitrogen containing bisphosphonates clodronate led to decreased gene expression levels. Conclusion Bisphosphonates seem to inhibit hOB adhesion and migration. The integrin aVb3 and tenascin C gene expression seem to be dependent on the cell line. BONJ could be enhanced by an inhibition of osteoblast adhesion and migration. The gene expression results, however, suggest a cell line dependent effect of bisphosphonates, which could explain the interindividual differences of BONJ incidences.

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

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    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. 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.

  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. 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.

  18. 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.

  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. 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.

  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. 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. MiR-9 is overexpressed in spontaneous canine osteosarcoma and promotes a metastatic phenotype including invasion and migration in osteoblasts and osteosarcoma cell lines

    International Nuclear Information System (INIS)

    Fenger, Joelle M.; Roberts, Ryan D.; Iwenofu, O. Hans; Bear, Misty D.; Zhang, Xiaoli; Couto, Jason I.; Modiano, Jaime F.; Kisseberth, William C.; London, Cheryl A.

    2016-01-01

    MicroRNAs (miRNAs) regulate the expression of networks of genes and their dysregulation is well documented in human malignancies; however, limited information exists regarding the impact of miRNAs on the development and progression of osteosarcoma (OS). Canine OS exhibits clinical and molecular features that closely resemble the corresponding human disease and it is considered a well-established spontaneous animal model to study OS biology. The purpose of this study was to investigate miRNA dysregulation in canine OS. We evaluated miRNA expression in primary canine OS tumors and normal canine osteoblast cells using the nanoString nCounter system. Quantitative PCR was used to validate the nanoString findings and to assess miR-9 expression in canine OS tumors, OS cell lines, and normal osteoblasts. Canine osteoblasts and OS cell lines were stably transduced with pre-miR-9 or anti-miR-9 lentiviral constructs to determine the consequences of miR-9 on cell proliferation, apoptosis, invasion and migration. Proteomic and gene expression profiling of normal canine osteoblasts with enforced miR-9 expression was performed using 2D-DIGE/tandem mass spectrometry and RNA sequencing and changes in protein and mRNA expression were validated with Western blotting and quantitative PCR. OS cell lines were transduced with gelsolin (GSN) shRNAs to investigate the impact of GSN knockdown on OS cell invasion. We identified a unique miRNA signature associated with primary canine OS and identified miR-9 as being significantly overexpressed in canine OS tumors and cell lines compared to normal osteoblasts. Additionally, high miR-9 expression was demonstrated in tumor-specific tissue obtained from primary OS tumors. In normal osteoblasts and OS cell lines transduced with miR-9 lentivirus, enhanced invasion and migration were observed, but miR-9 did not affect cell proliferation or apoptosis. Proteomic and transcriptional profiling of normal canine osteoblasts overexpressing miR-9 identified

  5. 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

  6. 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.

  7. 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.

  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. 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

  11. 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.

  12. 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.

  13. Improving effects of chitosan nanofiber scaffolds on osteoblast proliferation and maturation

    Science.gov (United States)

    Ho, Ming-Hua; Liao, Mei-Hsiu; Lin, Yi-Ling; Lai, Chien-Hao; Lin, Pei-I; Chen, Ruei-Ming

    2014-01-01

    Osteoblast maturation plays a key role in regulating osteogenesis. Electrospun nanofibrous products were reported to possess a high surface area and porosity. In this study, we developed chitosan nanofibers and examined the effects of nanofibrous scaffolds on osteoblast maturation and the possible mechanisms. Macro- and micro observations of the chitosan nanofibers revealed that these nanoproducts had a flat surface and well-distributed fibers with nanoscale diameters. Mouse osteoblasts were able to attach onto the chitosan nanofiber scaffolds, and the scaffolds degraded in a time-dependent manner. Analysis by scanning electron microscopy further showed mouse osteoblasts adhered onto the scaffolds along the nanofibers, and cell–cell communication was also detected. Mouse osteoblasts grew much better on chitosan nanofiber scaffolds than on chitosan films. In addition, human osteoblasts were able to adhere and grow on the chitosan nanofiber scaffolds. Interestingly, culturing human osteoblasts on chitosan nanofiber scaffolds time-dependently increased DNA replication and cell proliferation. In parallel, administration of human osteoblasts onto chitosan nanofibers significantly induced osteopontin, osteocalcin, and alkaline phosphatase (ALP) messenger (m)RNA expression. As to the mechanism, chitosan nanofibers triggered runt-related transcription factor 2 mRNA and protein syntheses. Consequently, results of ALP-, alizarin red-, and von Kossa-staining analyses showed that chitosan nanofibers improved osteoblast mineralization. Taken together, results of this study demonstrate that chitosan nanofibers can stimulate osteoblast proliferation and maturation via runt-related transcription factor 2-mediated regulation of osteoblast-associated osteopontin, osteocalcin, and ALP gene expression. PMID:25246786

  14. 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.

  15. 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.

  16. Differences in otosclerotic and normal human stapedial osteoblast properties are normalized by alendronate in vitro.

    Science.gov (United States)

    Gronowicz, Gloria; Richardson, Yvonne L; Flynn, John; Kveton, John; Eisen, Marc; Leonard, Gerald; Aronow, Michael; Rodner, Craig; Parham, Kourosh

    2014-10-01

    Identify and compare phenotypic properties of osteoblasts from patients with otosclerosis (OSO), normal bones (HOB), and normal stapes (NSO) to determine a possible cause for OSO hypermineralization and assess any effects of the bisphosphonate, alendronate. OSO (n = 11), NSO (n = 4), and HOB (n = 13) cultures were assayed for proliferation, adhesion, mineralization, and gene expression with and without 10(-10)M-10(-8)M alendronate. Academic hospital. Cultures were matched for age, sex, and passage number. Cell attachment and proliferation + alendronate were determined by Coulter counting cells and assaying tritiated thymidine uptake, respectively. At 7, 14, and 21 days of culture + alendronate, calcium content and gene expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were determined. OSO had significantly more cells adhere but less proliferation than NSO or HOB. Calcification was significantly increased in OSO compared to HOB and NSO. NSO and HOB had similar cell adhesion and proliferation rates. A dose-dependent effect of alendronate on OSO adhesion, proliferation, and mineralization was found, resulting in levels equal to NSO and HOB. All cultures expressed osteoblast-specific genes such as RUNX2, alkaline phosphatase, type I collagen, and osteocalcin. However, osteopontin was dramatically reduced, 9.4-fold at 14 days, in OSO compared to NSO. Receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG), important in bone resorption, was elevated in OSO with decreased levels of OPG levels. Alendronate had little effect on gene expression in HOB but in OSO increased osteopontin levels and decreased RANKL/OPG. OSO cultures displayed properties of hypermineralization due to decreased osteopontin (OPN) and also had increased RANKL/OPG, which were normalized by alendronate. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2014.

  17. 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.

  18. 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).

  19. 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

  20. 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...

  1. 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.

  2. Cytotoxic and phenotypic effects of uranium and lead on osteoblastic cells are highly dependent on metal speciation

    International Nuclear Information System (INIS)

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

    2008-01-01

    Bone is one of the main retention organs for uranium (U) and lead (Pb). The clinical effects of U or Pb poisoning are well known: acute and chronic intoxications impair bone formation. However, only few studies dealt with the cellular and molecular mechanisms of their toxicity. The purpose of this study was to investigate acute cytotoxicity of U and Pb and their phenotypic effects on rat and human osteoblasts, the cells responsible for bone formation. The most likely species of the toxicants in contact with cells after blood contamination were selected for cell exposure. Results showed that the cytotoxic effect of U and Pb is highly dependent on their speciation. Thus, Pb was cytotoxic when left free in the exposure medium or when complexed with carbonate, cystein or citrate, but not when complexed with albumin or phosphate, under an insoluble form. U was cytotoxic whatever its speciation, but differences in sensitivity were observed as a function of speciation. Population growth recovery could be obtained after exposure to low doses of U or Pb, except for some U-carbonate complexes which had irreversible effects whatever the dose. The activation of two markers of bone formation and mineralization, osteocalcin and bone sialoprotein (BSP), was observed after exposure to non-toxic doses or non-toxic species of U or Pb while their inhibition was observed after toxic exposure to both metals. This work provides new elements to better understand the complex mechanisms of U and Pb toxicity to osteoblasts. Our results also illustrate the importance of a strictly controlled speciation of the metals in toxicological studies

  3. 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.

  4. The Cooperative Effect of Genistein and Protein Hydrolysates on the Proliferation and Survival of Osteoblastic Cells (hFOB 1.19

    Directory of Open Access Journals (Sweden)

    Shuo Wang

    2016-11-01

    Full Text Available Chum salmon skin gelatin, de-isoflavoned soy protein, and casein were hydrolyzed at two degrees of hydrolysis. Genistein, the prepared hydrolysates, and genistein-hydrolysate combinations were assessed for their proliferative and anti-apoptotic effects on human osteoblasts (hFOB 1.19 to clarify potential cooperative effects between genistein and these hydrolysates in these two activities. Genistein at 2.5 μg/L demonstrated the highest proliferative activity, while the higher dose of genistein inhibited cell growth. All hydrolysates promoted osteoblast proliferation by increasing cell viability to 102.9%–131.1%. Regarding etoposide- or NaF-induced osteoblast apoptosis, these hydrolysates at 0.05 g/L showed both preventive and therapeutic effects against apoptosis. In the mode of apoptotic prevention, the hydrolysates decreased apoptotic cells from 32.9% to 15.2%–23.7% (etoposide treatment or from 23.6% to 14.3%–19.6% (NaF treatment. In the mode of apoptotic rescue, the hydrolysates lessened the extent of apoptotic cells from 15.9% to 13.0%–15.3% (etoposide treatment or from 13.3% to 10.9%–12.7% (NaF treatment. Gelatin hydrolysates showed the highest activities among all hydrolysates in all cases. All investigated combinations (especially the genistein-gelatin hydrolysate combination had stronger proliferation, apoptotic prevention, and rescue than genistein itself or their counterpart hydrolysates alone, suggesting that genistein cooperated with these hydrolysates, rendering greater activities in osteoblast proliferation and anti-apoptosis.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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...

  15. 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.

  16. Effects of titanium surface anodization with CaP incorporation on human osteoblastic response

    Science.gov (United States)

    OLIVEIRA, Natássia Cristina Martins; MOURA, Camilla Christian Gomes; ZANETTA-BARBOSA, Darceny; MENDONÇA, Daniela Baccelli Silveira; MENDONÇA, Gustavo; DECHICHI, Paula

    2015-01-01

    In this study we investigated whether anodization with calcium phosphate (CaP) incorporation (Vulcano®) enhances growth factors secretion, osteoblast-specific gene expression, and cell viability, when compared to acid etched surfaces (Porous®) and machined surfaces (Screw®) after 3 and 7 days. Results showed significant cell viability for Porous and Vulcano at day 7, when compared with Screw (p=0.005). At the same time point, significant differences regarding runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and bone sialoprotein (BSP) expression were found for all surfaces (p0.05). Although no significant correlation was found for growth factors secretion and Runx2 expression, a significant positive correlation between this gene and ALP/BSP expression showed that their strong association is independent on the type of surface. The incorporation of CaP affected the biological parameters evaluated similar to surfaces just acid etched. The results presented here support the observations that roughness also may play an important role in determining cell response. PMID:23498218

  17. 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)

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. Influence of sterilization methods on cell behavior and functionality of osteoblasts cultured on TiO2 nanotubes

    International Nuclear Information System (INIS)

    Oh, Seunghan; Brammer, Karla S.; Moon, Kyung-Suk; Bae, Ji-Myung; Jin, Sungho

    2011-01-01

    We investigated the adhesion, proliferation and osteogenic functionality of osteoblasts cultured on titanium dioxide (TiO 2 ) nanotubes in response to different sterilization methods (dry autoclaving vs. wet autoclaving). We prepared various sizes (30-100 nm diameter) of TiO 2 nanotubes on titanium substrates by anodization, sterilized nanotubes by different conditions, and seeded osteoblast cells onto the nanotube surfaces with two different cell seeding densities (10,000 vs. 50,000 cells/well in 12-culture well). The result of this study indicates that the adhesion, proliferation and alkaline phosphatase activity of osteoblasts cultured on only the larger 70 and 100 nm TiO 2 nanotube arrays were dramatically changed by the different sterilization conditions at a low cell seeding density. However, with a higher cell seeding density (50,000 cells/well in 12-cell culture well), the results revealed no significant difference among altered nanotube geometry, 30-100 nm diameters, nor sterilization methods. Next, it was revealed that the nanofeatures of proteins adhered on nanotubular TiO 2 morphology are altered by the sterilization method. It was determined that this protein adhesion effect, in combination with the cell density of osteoblasts seeded onto such TiO 2 nanotube surfaces, has profound effects on cell behavior. This study clearly shows that these are some of the important in vitro culture factors that need to be taken into consideration, as well as TiO 2 nanotube diameters which play an important role in the improvement of cell behavior and functionality.

  8. Vertically aligned carbon nanotubes as cytocompatible material for enhanced adhesion and proliferation of osteoblast-like cells.

    Science.gov (United States)

    Giannona, Suna; Firkowska, Izabela; Rojas-Chapana, José; Giersig, Michael

    2007-01-01

    In this study, we describe the spatial organization of CAL-72 osteoblast-like cells on arrays of vertically aligned multi-walled carbon nanotubes (VACNTs). It was observed that, unlike cell growth on non-patterned surfaces, the cell attachment and spreading process on VACNTs was significantly enhanced. Additionally, since carbon nanotubes are known to possess resilient mechanical properties and are chemically stable, the effect of periodic arrays of VACNTs on CAL-72 osteoblast-like cells was also studied. The periodicity and alignment of VACNTs considerably influenced growth, shape and orientation of the cells by steering toward the nanopattern. This situation is of great interest for the potential application of VACNTs in bone bioenginnering. This data provides evidence that CAL-72 osteoblast-like cells can sense physical features at the nanoscale. These results give a fascinating insight into the ways in which cell growth can be influenced by man-made nanostructures and could provide a framework for achieving controlled cell guidance with controlled organization and special physical properties.

  9. 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

  10. 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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-05-28

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

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  13. Response of Human Osteoblast to n-HA/PEEK—Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite

    Science.gov (United States)

    Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

    2016-03-01

    Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.

  14. Response of Human Osteoblast to n-HA/PEEK—Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite

    Science.gov (United States)

    Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

    2016-01-01

    Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery. PMID:26956660

  15. Chitosan–Cellulose Multifunctional Hydrogel Beads: Design, Characterization and Evaluation of Cytocompatibility with Breast Adenocarcinoma and Osteoblast Cells

    Science.gov (United States)

    Trivedi, Poonam; Saloranta-Simell, Tiina; Gradišnik, Lidija; Prabhakar, Neeraj; Smått, Jan-Henrik; Mohan, Tamilselvan; Gericke, Martin; Heinze, Thomas

    2018-01-01

    Cytocompatible polysaccharide-based functional scaffolds are potential extracellular matrix candidates for soft and hard tissue engineering. This paper describes a facile approach to design cytocompatible, non-toxic, and multifunctional chitosan-cellulose based hydrogel beads utilising polysaccharide dissolution in sodium hydroxide-urea-water solvent system and coagulation under three different acidic conditions, namely 2 M acetic acid, 2 M hydrochloric acid, and 2 M sulfuric acid. The effect of coagulating medium on the final chemical composition of the hydrogel beads is investigated by spectroscopic techniques (ATR–FTIR, Raman, NMR), and elemental analysis. The beads coagulated in 2 M acetic acid displayed an unchanged chitosan composition with free amino groups, while the beads coagulated in 2 M hydrochloric and sulfuric acid showed protonation of amino groups and ionic interaction with the counterions. The ultrastructural morphological study of lyophilized beads showed that increased chitosan content enhanced the porosity of the hydrogel beads. Furthermore, cytocompatibility evaluation of the hydrogel beads with human breast adenocarcinoma cells (soft tissue) showed that the beads coagulated in 2 M acetic acid are the most suitable for this type of cells in comparison to other coagulating systems. The acetic acid fabricated hydrogel beads also support osteoblast growth and adhesion over 192 h. Thus, in future, these hydrogel beads can be tested in the in vitro studies related to breast cancer and for bone regeneration. PMID:29315214

  16. Chitosan-Cellulose Multifunctional Hydrogel Beads: Design, Characterization and Evaluation of Cytocompatibility with Breast Adenocarcinoma and Osteoblast Cells.

    Science.gov (United States)

    Trivedi, Poonam; Saloranta-Simell, Tiina; Maver, Uroš; Gradišnik, Lidija; Prabhakar, Neeraj; Smått, Jan-Henrik; Mohan, Tamilselvan; Gericke, Martin; Heinze, Thomas; Fardim, Pedro

    2018-01-09

    Cytocompatible polysaccharide-based functional scaffolds are potential extracellular matrix candidates for soft and hard tissue engineering. This paper describes a facile approach to design cytocompatible, non-toxic, and multifunctional chitosan-cellulose based hydrogel beads utilising polysaccharide dissolution in sodium hydroxide-urea-water solvent system and coagulation under three different acidic conditions, namely 2 M acetic acid, 2 M hydrochloric acid, and 2 M sulfuric acid. The effect of coagulating medium on the final chemical composition of the hydrogel beads is investigated by spectroscopic techniques (ATR-FTIR, Raman, NMR), and elemental analysis. The beads coagulated in 2 M acetic acid displayed an unchanged chitosan composition with free amino groups, while the beads coagulated in 2 M hydrochloric and sulfuric acid showed protonation of amino groups and ionic interaction with the counterions. The ultrastructural morphological study of lyophilized beads showed that increased chitosan content enhanced the porosity of the hydrogel beads. Furthermore, cytocompatibility evaluation of the hydrogel beads with human breast adenocarcinoma cells (soft tissue) showed that the beads coagulated in 2 M acetic acid are the most suitable for this type of cells in comparison to other coagulating systems. The acetic acid fabricated hydrogel beads also support osteoblast growth and adhesion over 192 h. Thus, in future, these hydrogel beads can be tested in the in vitro studies related to breast cancer and for bone regeneration.

  17. New Parameters to Quantitatively Express the Invasiveness of Bacterial Strains from Implant-Related Orthopaedic Infections into Osteoblast Cells

    Directory of Open Access Journals (Sweden)

    Davide Campoccia

    2018-04-01

    Full Text Available Complete eradication of bacterial infections is often a challenging task, especially in presence of prosthetic devices. Invasion of non-phagocytic host cells appears to be a critical mechanism of microbial persistence in host tissues. Hidden within host cells, bacteria elude host defences and antibiotic treatments that are intracellularly inactive. The intracellular invasiveness of bacteria is generally measured by conventional gentamicin protection assays. The efficiency of invasion, however, markedly differs across bacterial species and adjustments to the titre of the microbial inocula used in the assays are often needed to enumerate intracellular bacteria. Such changes affect the standardisation of the method and hamper a direct comparison of bacteria on a same scale. This study aims at investigating the precise relation between inoculum, in terms of multiplicity of infection (MOI, and internalised bacteria. The investigation included nine Staphylococcus aureus, seven Staphylococcus epidermidis, five Staphylococcus lugdunensis and two Enterococcus faecalis clinical strains, which are co-cultured with MG63 human osteoblasts. Unprecedented insights are offered on the relations existing between MOI, number of internalised bacteria and per cent of internalised bacteria. New parameters are identified that are of potential use for qualifying the efficiency of internalization and compare the behaviour of bacterial strains.

  18. New Parameters to Quantitatively Express the Invasiveness of Bacterial Strains from Implant-Related Orthopaedic Infections into Osteoblast Cells.

    Science.gov (United States)

    Campoccia, Davide; Montanaro, Lucio; Ravaioli, Stefano; Cangini, Ilaria; Testoni, Francesca; Visai, Livia; Arciola, Carla Renata

    2018-04-03

    Complete eradication of bacterial infections is often a challenging task, especially in presence of prosthetic devices. Invasion of non-phagocytic host cells appears to be a critical mechanism of microbial persistence in host tissues. Hidden within host cells, bacteria elude host defences and antibiotic treatments that are intracellularly inactive. The intracellular invasiveness of bacteria is generally measured by conventional gentamicin protection assays. The efficiency of invasion, however, markedly differs across bacterial species and adjustments to the titre of the microbial inocula used in the assays are often needed to enumerate intracellular bacteria. Such changes affect the standardisation of the method and hamper a direct comparison of bacteria on a same scale. This study aims at investigating the precise relation between inoculum, in terms of multiplicity of infection (MOI), and internalised bacteria. The investigation included nine Staphylococcus aureus , seven Staphylococcus epidermidis , five Staphylococcus lugdunensis and two Enterococcus faecalis clinical strains, which are co-cultured with MG63 human osteoblasts. Unprecedented insights are offered on the relations existing between MOI, number of internalised bacteria and per cent of internalised bacteria. New parameters are identified that are of potential use for qualifying the efficiency of internalization and compare the behaviour of bacterial strains.

  19. HIV-1 protein induced modulation of primary human osteoblast differentiation and function via a Wnt/β-catenin-dependent mechanism.

    LENUS (Irish Health Repository)

    Butler, Joseph S

    2013-02-01

    HIV infection is associated with metabolic bone disease resulting in bone demineralization and reduced bone mass. The molecular mechanisms driving this disease process have yet to be elucidated. Wnt\\/β-catenin signaling plays a key role in bone development and remodeling. We attempted to determine the effects of the HIV-1 protein, gp120, on Wnt\\/β-catenin signaling at an intracellular and transcriptional level in primary human osteoblasts (HOBs). This work, inclusive of experimental controls, was part of a greater project assessing the effects of a variety of different agents on Wnt\\/β-catenin signaling (BMC Musculoskelet Disord 2010;11:210).We examined the phenotypic effects of silencing and overexpressing the Wnt antagonist, Dickkopf-1 (Dkk1) in HOBs treated with gp120. HOBs exposed to gp120 displayed a significant reduction in alkaline phosphatase activity (ALP) activity and cell proliferation and increased cellular apoptosis over a 48 h time course. Immunocytochemistry demonstrated a significant reduction in intracytosolic and intranuclear β-catenin in response to HIV-1 protein exposure. These changes were associated with a reduction of TCF\\/LEF-mediated transcription, the transcriptional outcome of canonical Wnt β-catenin signaling. Silencing Dkk1 expression in HOBs exposed to gp120 resulted in increased ALP activity and cell proliferation, and decreased cellular apoptosis relative to scrambled control. Dkk1 overexpression exacerbated the inhibitory effect of gp120 on HOB function, with decreases in ALP activity and cell proliferation and increased cellular apoptosis relative to vector control. Wnt\\/β-catenin signaling plays a key regulatory role in HIV-associated bone loss, with Dkk1, aputative central mediator in this degenerative process. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 218-226, 2013.

  20. Toxicity of iron oxide nanoparticles against osteoblasts

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  1. Toxicity of iron oxide nanoparticles against osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-09-15

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

  2. In vitro study of biocompatibility of a graphene composite with gold nanoparticles and hydroxyapatite on human osteoblasts.

    Science.gov (United States)

    Crisan, Liana; Crisan, Bogdan; Soritau, Olga; Baciut, Mihaela; Biris, Alexandru Radu; Baciut, Grigore; Lucaciu, Ondine

    2015-10-01

    The purpose of this study was to evaluate the biocompatibility of some composites consisting of different proportions of graphene in combination with gold nanoparticles (AuNPs) and nanostructured hydroxyapatite (HA) on osteoblast viability, proliferation and differentiation. Au/HA@graphene composites synthesized by the catalytic chemical vapor deposition induction heating method with acetylene as the carbon source and over an Au/HA catalyst, were characterized by transmission electron microscopy, thermogravimetric analysis and Raman spectroscopy and showed that the few-layer graphene was grown over the Au/HA catalyst. The cytocompatibility study was performed using the fluorescein diacetate assay for assessment of the viability and proliferation of osteoblasts cultivated in the presence of HA, Au/HA and Au/HA@graphene composites as colloidal suspensions or as substrates. The most favorable composites for cell adhesion and proliferation were HA, Au/HA and Au/HA composites with 1.6% and 3.15% concentration of graphenes. Immunocytochemical staining performed after 19 days of osteoblasts cultivation on substrates showed that the graphene composites induced low expression of alkaline phosphatase compared to the control group and HA and Au/HA substrates. The presence of graphene in the substrate composition also induced an increased level of intracellular osteopontin and cytoskeleton reorganization (actin-F) depending on graphene concentration, suggesting cell activation, increased cellular adhesion and acquisition of a mechanosensorial osteocyte phenotype. Copyright © 2015 John Wiley & Sons, Ltd.

  3. 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.

  4. 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.

  5. MEK5 suppresses osteoblastic differentiation

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-07-31

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

  6. 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

  7. 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

  8. Antioxidant and bone repair properties of quercetin-functionalized hydroxyapatite: An in vitro osteoblast-osteoclast-endothelial cell co-culture study.

    Science.gov (United States)

    Forte, Lucia; Torricelli, Paola; Boanini, Elisa; Gazzano, Massimo; Rubini, Katia; Fini, Milena; Bigi, Adriana

    2016-03-01

    Quercetin (3,3',4',5,7-pentahydroxy-flavone) is a flavonoid known for its pharmacological activities, which include antioxidant and anti-inflammatory properties, as well as possible beneficial action on diseases involving bone loss. In this work, we explored the possibility to functionalize hydroxyapatite (HA) with quercetin in order to obtain new materials for bone repair through local administration of the flavonoid. HA was synthesized in presence of different concentrations of quercetin according to two different procedures: direct synthesis and phase transition from monetite. Direct synthesis lead to composite nanocrystals containing up to 3.1 wt% quercetin, which provokes a reduction of the crystals mean dimensions and of the length of the coherently scattering domains. Synthesis conditions provoke a partial oxidation of quercetin and, as a consequence, a significant reduction of its radical scavenging activity (RSA). On the other hand, synthesis through phase transition yields samples containing up to 1.3 wt% of quercetin incorporated into hydroxyapatite, with minor structural modifications, which exhibit relevant anti-oxidant activities, as testified by their high RSA levels, (slightly lower than that of pure quercetin). The biological response to these materials was tested using an innovative triculture model involving osteoblast, osteoclast and endothelial cells, in order to mimic bone microenvironment. The results show that the presence of quercetin in the composite materials enhances human osteoblast-like MG63 proliferation and differentiation, whereas it downregulates osteoclastogenesis of osteoclast precursors 2T-110, and supports proliferation and differentiation of human umbilical vein endothelial cells (HUVEC). The pharmacological activities of the flavonoid quercetin include anti-oxidant and antiinflammatory properties, as well as capability to prevent bone loss. In this paper, we demonstrate that it is possible to synthesize hydroxyapatite

  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. Enhancement of Osteoblastic-Like Cell Activity by Glow Discharge Plasma Surface Modified Hydroxyapatite/β-Tricalcium Phosphate Bone Substitute

    Directory of Open Access Journals (Sweden)

    Eisner Salamanca

    2017-11-01

    Full Text Available Glow discharge plasma (GDP treatments of biomaterials, such as hydroxyapatite/β-tricalcium phosphate (HA/β-TCP composites, produce surfaces with fewer contaminants and may facilitate cell attachment and enhance bone regeneration. Thus, in this study we used argon glow discharge plasma (Ar-GDP treatments to modify HA/β-TCP particle surfaces and investigated the physical and chemical properties of the resulting particles (HA/β-TCP + Ar-GDP. The HA/β-TCP particles were treated with GDP for 15 min in argon gas at room temperature under the following conditions: power: 80 W; frequency: 13.56 MHz; pressure: 100 mTorr. Scanning electron microscope (SEM observations showed similar rough surfaces of HA/β-TCP + Ar-GDP HA/β-TCP particles, and energy dispersive spectrometry analyses showed that HA/β-TCP surfaces had more contaminants than HA/β-TCP + Ar-GDP surfaces. Ca/P mole ratios in HA/β-TCP and HA/β-TCP + Ar-GDP were 1.34 and 1.58, respectively. Both biomaterials presented maximal intensities of X-ray diffraction patterns at 27° with 600 a.u. At 25° and 40°, HA/β-TCP + Ar-GDP and HA/β-TCP particles had peaks of 200 a.u., which are similar to XRD intensities of human bone. In subsequent comparisons, MG-63 cell viability and differentiation into osteoblast-like cells were assessed on HA/β-TCP and HA/β-TCP + Ar-GDP surfaces, and Ar-GDP treatments led to improved cell growth and alkaline phosphatase activities. The present data indicate that GDP surface treatment modified HA/β-TCP surfaces by eliminating contaminants, and the resulting graft material enhanced bone regeneration.

  11. Osteoblast adhesion, migration, and proliferation variations on chemically patterned nanocrystalline diamond films evaluated by live-cell imaging

    Czech Academy of Sciences Publication Activity Database

    Brož, Antonín; Ukraintsev, Egor; Kromka, Alexander; Rezek, Bohuslav; Kalbáčová, M.H.

    2017-01-01

    Roč. 105, č. 5 (2017), s. 1469-1478 ISSN 1549-3296 R&D Projects: GA ČR(CZ) GA14-04790S; GA MZd(CZ) NV15-32497A Institutional support: RVO:67985823 ; RVO:68378271 Keywords : live-cell imaging * osteoblasts * adhesion * proliferation * migration * patterned surface Subject RIV: EI - Biotechnology ; Bionics OBOR OECD: Biomaterials (as related to medical implants, devices, sensors) Impact factor: 3.076, year: 2016

  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. Effects of activated and nonactivated platelet-rich plasma on proliferation of human osteoblasts in vitro

    Czech Academy of Sciences Publication Activity Database

    Slapnička, J.; Fassmann, A.; Strašák, Luděk; Augustin, P.; Vaněk, J.

    2008-01-01

    Roč. 66, č. 2 (2008), s. 297-301 ISSN 0278-2391 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : platelet-rich plasma * osteoblast * proliferation Subject RIV: BO - Biophysics Impact factor: 1.241, year: 2008

  14. 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.

  15. SPILANTHES ACMELLA AND PHYSICAL EXERCISE INCREASED TESTOSTERONE LEVELS AND OSTEOBLAST CELLS IN GLUCOCORTICOID-INDUCED OSTEOPOROSIS MALE MICE

    Directory of Open Access Journals (Sweden)

    Hening Laswati

    2015-08-01

    Full Text Available Background: Glucocorticoid-induced osteoporosis is leading cause of secondary osteoporosis by decreasing formation activity and increasing resorption activity. Spilanthes acmella, is one of Indonesia medicinal plants that contain of polyphenol and flavonoids. Previously in vitro study showed that buthanol and water fraction from this plant have increased alkaline phosphatase that known as marker of bone formation. The objective of this study to analyze the effect of Spilanthes acmella  and physical exercise in increasing testosterone and  osteoblast cells of femoral’s trabecular glucocorticoid-induced osteoporosis male mice. Method: This study using a posttest control group design, 36 male healthy mice (5 months old  were randomizely devided into 6 groups, there are : 1.Healthy control group (without induction dexamethaxone, 2.Osteoporosis groups (induction with dexamethaxone without treatment, 3.Positive control receive suspension alendronat, 4.70% Ethanol extract of Spilanthes acmella group, 5.Combination group of 70% extract ethanol of Spilanthes acmella and exercise, and 6.Exercise group  (walking using mice treadmill 10m/minute, 5-12 minutes 3 times a week. All of the intervention were given for 4 weeks. The serum levels of testosterone were determined using  immunoserology (ELISA and osteoblast cells were determined histomorphometry by light microscopy.  All statistical test were carried out using SPSS 23 and statistical significance was  set at p<0.05 for all analysis. The testosterone levels  between group were compared using Mann-Whitney test and osteoblast cells between group were compared with multiple comparison. Results: It showed that the alendronate group, combination group and the exercise group increasing testosterone level (p<0.05 from that osteoporotic group. There were also increasing osteoblast cells (p<0.05 in the alendronate group and combination group. There was no correlation between testosterone level and

  16. 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.

  17. Modulation of insulin-like growth factor 1 levels in human osteoarthritic subchondral bone osteoblasts.

    Science.gov (United States)

    Massicotte, Frédéric; Fernandes, Julio Cesar; Martel-Pelletier, Johanne; Pelletier, Jean-Pierre; Lajeunesse, Daniel

    2006-03-01

    Human osteoarthritis (OA) is characterized by cartilage loss, bone sclerosis, osteophyte formation and inflammation of the synovial membrane. We previously reported that OA osteoblasts (Ob) show abnormal phenotypic characteristics possibly responsible for bone sclerosis and that two subgroups of OA patients can be identified by low or high endogenous production of prostaglandin E2 (PGE2) by OA Ob. Here, we determined that the elevated PGE2 levels in the high OA subgroup were linked with enhanced cyclooxygenase-2 (COX-2) protein levels compared to normal and low OA Ob. A linear relationship was observed between endogenous PGE2 levels and insulin-like growth factor 1 (IGF-1) levels in OA Ob. As parathyroid hormone (PTH) and PGE2 are known stimulators of IGF-1 production in Ob, we next evaluated their effect in OA Ob. Both subgroups increased their IGF-1 production similarly in response to PGE2, while the high OA subgroup showed a blunted response to PTH compared to the low OA group. Conversely, only the high OA group showed a significant inhibition of IGF-1 production when PGE2 synthesis was reduced with Naproxen, a non-steroidal antiinflammatory drug (NSAID) that inhibits cyclooxygenases (COX). The PGE2-dependent stimulation of IGF-1 synthesis was due in part to the cAMP/protein kinase A pathway since both the direct inhibition of this pathway with H-89 and the inhibition of EP2 or EP4 receptors, linked to cAMP production, reduced IGF-1 synthesis. The production of the most abundant IGF-1 binding proteins (IGFBPs) in bone tissue, IGFBP-3, -4, and -5, was lower in OA compared to normal Ob independently of the OA group. Under basal condition, OA Ob expressed similar IGF-1 mRNA to normal Ob; however, PGE2 stimulated IGF-1 mRNA expression more in OA than normal Ob. These data suggest that increased IGF-1 levels correlate with elevated endogenous PGE2 levels in OA Ob and that higher IGF-1 levels in OA Ob could be important for bone sclerosis in OA.

  18. 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

    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 osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit

  19. 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

    Full Text Available 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 osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA, a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the

  20. Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts.

    Science.gov (United States)

    Milovancev, Milan; Hilgart-Martiszus, Ian; McNamara, Michael J; Goodall, Cheri P; Seguin, Bernard; Bracha, Shay; Wickramasekara, Samanthi I

    2013-06-13

    Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data

  1. 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

    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 osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit

  2. 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.

  3. 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.

  4. 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.

  5. Surface modification of nano-silica on the ligament advanced reinforcement system for accelerated bone formation: primary human osteoblasts testing in vitro and animal testing in vivo.

    Science.gov (United States)

    Li, Mengmeng; Wang, Shiwen; Jiang, Jia; Sun, Jiashu; Li, Yuzhuo; Huang, Deyong; Long, Yun-Ze; Zheng, Wenfu; Chen, Shiyi; Jiang, Xingyu

    2015-05-07

    The Ligament Advanced Reinforcement System (LARS) has been considered as a promising graft for ligament reconstruction. To improve its biocompatibility and effectiveness on new bone formation, we modified the surface of a polyethylene terephthalate (PET) ligament with nanoscale silica using atom transfer radical polymerization (ATRP) and silica polymerization. The modified ligament is tested by both in vitro and in vivo experiments. Human osteoblast testing in vitro exhibits an ∼21% higher value in cell viability for silica-modified grafts compared with original grafts. Animal testing in vivo shows that there is new formed bone in the case of a nanoscale silica-coated ligament. These results demonstrate that our approach for nanoscale silica surface modification on LARS could be potentially applied for ligament reconstruction.

  6. 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.

  7. Dual Action of Lysophosphatidate-Functionalised Titanium: Interactions with Human (MG63 Osteoblasts and Methicillin Resistant Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Mette Elena Skindersoe

    Full Text Available Titanium (Ti is a widely used material for surgical implants; total joint replacements (TJRs, screws and plates for fixing bones and dental implants are forged from Ti. Whilst Ti integrates well into host tissue approximately 10% of TJRs will fail in the lifetime of the patient through a process known as aseptic loosening. These failures necessitate revision arthroplasties which are more complicated and costly than the initial procedure. Finding ways of enhancing early (osseointegration of TJRs is therefore highly desirable and continues to represent a research priority in current biomaterial design. One way of realising improvements in implant quality is to coat the Ti surface with small biological agents known to support human osteoblast formation and maturation at Ti surfaces. Lysophosphatidic acid (LPA and certain LPA analogues offer potential solutions as Ti coatings in reducing aseptic loosening. Herein we present evidence for the successful bio-functionalisation of Ti using LPA. This modified Ti surface heightened the maturation of human osteoblasts, as supported by increased expression of alkaline phosphatase. These functionalised surfaces also deterred the attachment and growth of Staphylococcus aureus, a bacterium often associated with implant failures through sepsis. Collectively we provide evidence for the fabrication of a dual-action Ti surface finish, a highly desirable feature towards the development of next-generation implantable devices.

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

    Science.gov (United States)

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

    2017-05-03

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

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

    Science.gov (United States)

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

    2013-05-01

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

  10. 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

  11. 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.

  12. The expression and role of serotonin receptor 5HTR2A in canine osteoblasts and an osteosarcoma cell line.

    Science.gov (United States)

    Bracha, Shay; Viall, Austin; Goodall, Cheri; Stang, Bernadette; Ruaux, Craig; Seguin, Bernard; Chappell, Patrick E

    2013-12-12

    The significance of the serotonergic system in bone physiology and, more specifically, the importance of the five hydroxytryptamine receptor 2A (5HTR2A) in normal osteoblast proliferation have been previously described; however the role of serotonin in osteosarcoma remains unclear. Particularly, the expression and function of 5HTR2A in canine osteosarcoma has not yet been studied, thus we sought to determine if this indoleamine modulates cellular proliferation in vitro. Using real time quantitative reverse transcription PCR and immunoblot analyses, we explored receptor expression and signaling differences between non-neoplastic canine osteoblasts (CnOb) and an osteosarcoma cell line (COS). To elucidate specific serotonergic signaling pathways triggered by 5HTR2A, we performed immunoblots for ERK and CREB. Finally, we compared cell viability and the induction of apoptosis in the presence 5HTR2A agonists and antagonists. 5HTR2A was overexpressed in the malignant cell line in comparison to normal cells. In CnOb cells, ERK phosphorylation (ERK-P) decreased in response to both serotonin and a specific 5HTR2A antagonist, ritanserin. In contrast, ERK-P abundance increased in COS cells following either treatment. While endogenous CREB was undetectable in CnOb, CREB was observed constitutively in COS, with expression and exhibited increased CREB phosphorylation following escalating concentrations of ritanserin. To determine the influence of 5HTR2A signaling on cell viability we challenged cells with ritanserin and serotonin. Our findings confirmed that serotonin treatment promoted cell viability in malignant cells but not in normal osteoblasts. Conversely, ritanserin reduced cell viability in both the normal and osteosarcoma cells. Further, ritanserin induced apoptosis in COS at the same concentrations associated with decreased cell viability. These findings confirm the existence of a functional 5HTR2A in a canine osteosarcoma cell line. Results indicate that intracellular

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

    NARCIS (Netherlands)

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

    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

  14. 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.

  15. Comparative Analysis of the Oxygen Supply and Viability of Human Osteoblasts in Three-Dimensional Titanium Scaffolds Produced by Laser-Beam or Electron-Beam Melting

    Directory of Open Access Journals (Sweden)

    Anika Jonitz-Heincke

    2013-11-01

    Full Text Available Synthetic materials for bone replacement must ensure a sufficient mechanical stability and an adequate cell proliferation within the structures. Hereby, titanium materials are suitable for producing patient-individual porous bone scaffolds by using generative techniques. In this in vitro study, the viability of human osteoblasts was investigated in porous 3D Ti6Al4V scaffolds, which were produced by electron-beam (EBM or laser-beam melting (LBM. For each examination, two cylindrical scaffolds (30 mm × 10 mm in size, 700 µm × 700 µm macropores were placed on each other and seeded with cells. The oxygen consumption and the acidification in the center of the structures were investigated by means of microsensors. Additionally, the synthesis of pro-collagen type 1 was analyzed. On the LBM titanium scaffolds, vital bone cells were detected in the center and in the periphery after 8 days of cultivation. In the EBM titanium constructs, however, vital cells were only visible in the center. During the cultivation period, the cells increasingly produced procollagen type 1 in both scaffolds. In comparison to the periphery, the oxygen content in the center of the scaffolds slightly decreased. Furthermore, a slight acidification of the medium was detectable. Compared to LBM, the EBM titanium scaffolds showed a less favorable behavior with regard to cell seeding.

  16. 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

  17. Estrogen and estrogen receptor alpha promotes malignancy and osteoblastic tumorigenesis in prostate cancer.

    Science.gov (United States)

    Mishra, Sweta; Tai, Qin; Gu, Xiang; Schmitz, James; Poullard, Ashley; Fajardo, Roberto J; Mahalingam, Devalingam; Chen, Xiaodong; Zhu, Xueqiong; Sun, Lu-Zhe

    2015-12-29

    The role of estrogen signaling in regulating prostate tumorigenesis is relatively underexplored. Although, an increasing body of evidence has linked estrogen receptor beta (ERß) to prostate cancer, the function of estrogen receptor alpha (ERα) in prostate cancer is not very well studied. We have discovered a novel role of ERα in the pathogenesis of prostate tumors. Here, we show that prostate cancer cells express ERα and estrogen induces oncogenic properties in prostate cancer cells through ERα. Importantly, ERα knockdown in the human prostate cancer PacMetUT1 cells as well as pharmacological inhibition of ERα with ICI 182,780 inhibited osteoblastic lesion formation and lung metastasis in vivo. Co-culture of pre-osteoblasts with cancer cells showed a significant induction of osteogenic markers in the pre-osteoblasts, which was attenuated by knockdown of ERα in cancer cells suggesting that estrogen/ERα signaling promotes crosstalk between cancer and osteoblastic progenitors to stimulate osteoblastic tumorigenesis. These results suggest that ERα expression in prostate cancer cells is essential for osteoblastic lesion formation and lung metastasis. Thus, inhibition of ERα signaling in prostate cancer cells may be a novel therapeutic strategy to inhibit the osteoblastic lesion development as well as lung metastasis in patients with advanced prostate cancer.

  18. Phenolic Modified Ceramic Coating on Biodegradable Mg Alloy: The Improved Corrosion Resistance and Osteoblast-Like Cell Activity

    Directory of Open Access Journals (Sweden)

    Hung-Pang Lee

    2017-06-01

    Full Text Available Magnesium alloys have great potential for developing orthopedic implants due to their biodegradability and mechanical properties, but the rapid corrosion rate of the currently-available alloys limits their clinical applications. To increase the corrosion resistance of the substrate, a protective ceramic coating is constructed by a micro-arc oxidation (MAO process on ZK60 magnesium alloy. The porous ceramic coating is mainly composed of magnesium oxide and magnesium silicate, and the results from cell cultures show it can stimulate osteoblastic cell growth and proliferation. Moreover, gallic acid, a phenolic compound, was successfully introduced onto the MAO coating by grafting on hydrated oxide and chelating with magnesium ions. The gallic acid and rough surface of MAO altered the cell attachment behavior, making it difficult for fibroblasts to adhere to the MAO coating. The viability tests showed that gallic acid could suppress fibroblast growth and stimulate osteoblastic cell proliferation. Overall, the porous MAO coating combined with gallic acid offered a novel strategy for increasing osteocompatibility.

  19. 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

  20. 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

  1. Porous titanium obtained by a new powder metallurgy technique: Preliminary results of human osteoblast adhesion on surface polished substrates.

    Science.gov (United States)

    Biasotto, M; Ricceri, R; Scuor, N; Schmid, C; Sandrucci, M A; Di Lenarda, R; Matteazzi, P

    2003-01-01

    This study concerns a novel powder metallurgy method for producing porous titanium (pTi) exhibiting high mechanical properties. The preparation procedure consisted of the following stages: first, the preparation of Ti and titanium hydride (TiH2) powder mixtures and their consolidation with a cold isostatic press, followed by a sintering of the green bodies performed with hot isostatic press (HIP) equipment. Thermal decomposition in controlled environment of the TiH2 phase results in the foam structure. The resulting porosity percolates with a volume fraction of approximately 20%. The final material exhibits interesting mechanical properties, comparable to those of full density titanium (between grade 2 and grade 3), with the advantage of a minor density. The samples produced were tested to verify their biological response by studying the effectiveness of osteoblast adhesion and growth. In this preliminary study, osteoblastic cell morphology was investigated and compared to that observed on fully dense commercially pure titanium (Ti-cp) (ASTM, grade 3). The preliminary results were promising regarding cellular adhesion and spreading. (Journal of Applied Biomaterials & Biomechanics 2003; 1: 172-7).

  2. 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...

  3. Collageneous matrix coatings on titanium implants modified with decorin and chondroitin sulfate: characterization and influence on osteoblastic cells.

    Science.gov (United States)

    Bierbaum, Susanne; Douglas, Timothy; Hanke, Thomas; Scharnweber, Dieter; Tippelt, Sonja; Monsees, Thomas K; Funk, Richard H W; Worch, Hartmut

    2006-06-01

    Studies in developmental and cell biology have established the fact that responses of cells are influenced to a large degree by morphology and composition of the extracellular matrix. Goal of this work is to use this basic principle to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM), utilizing the natural self-assembly potential of collagen in combination with further ECM components in close analogy to the situation in vivo. Aiming at load-bearing applications in bone contact, collagen type I in combination with the proteoglycan decorin and the glycosaminoglycan chondroitin sulfate (CS) was used; fibrillogenesis, fibril morphology, and adsorption of differently composed fibrils onto titanium were assessed. Both decorin and CS could be integrated into the fibrils during fibrillogenesis, the amount bound respectively desorbed depending on the ionic strength of fibrillogenesis buffer. Including decorin always resulted in a significant decrease of fibril diameter, CS in only a slight decrease or even increase, depending on the collagen preparation used. No significant changes in adsorption to titanium could be detected. Osteoblastic cells showed different reactions for cytoskeletal arrangement and osteopontin expression depending on the composition of the ECM, with CS enhancing the osteoblast phenotype.

  4. 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.

  5. A novel rotating electrochemically anodizing process to fabricate titanium oxide surface nanostructures enhancing the bioactivity of osteoblastic cells.

    Science.gov (United States)

    Chang, Chih-Hung; Lee, Hsin-Chun; Chen, Chia-Chun; Wu, Yi-Hau; Hsu, Yuan-Ming; Chang, Yin-Pen; Yang, Ta-I; Fang, Hsu-Wei

    2012-07-01

    Titanium oxide (TiO(2) ) surface layers with various surface nanostructures (nanotubes and nanowires) have been developed using an anodizing technique. The pore size and length of TiO(2) nanotubes can be tailored by changing the anodizing time and applied voltage. We developed a novel method to transform the upper part of the formed TiO(2) nanotubes into a nanowire-like structure by rotating the titanium anode during anodizing process. The transformation of nanotubes contributed to the preferential chemical dissolution of TiO(2) on the areas with intense interface tension stress. Furthermore, we further compared the effect of various TiO(2) surface nanostructures including flat, nanotubes, and nanowires on bioactive applications. The MG-63 osteoblastic cells cultured on the TiO(2) nanowires exhibited a polygonal shape with extending filopodia and showed highest levels of cell viability and alkaline phosphatase activity (ALP). The TiO(2) nanowire structure formed by our novel method can provide beneficial effects for MG-63 osteoblastic cells in attachment, proliferation, and secretion of ALP on the TiO(2) surface layer. Copyright © 2012 Wiley Periodicals, Inc.

  6. 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 b