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Sample records for mg63 osteoblast-like cells

  1. 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.

  2. 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.

  3. 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.

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

    Science.gov (United States)

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

    2018-04-01

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

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

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    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.

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

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    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.

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

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    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.

  8. In vitro investigation of anodization and CaP deposited titanium surface using MG63 osteoblast-like cells

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    Lee, J.M. [Department of Prosthodontics and Dental Research Institute, School of Dentistry, Seoul National University, 28 Yeongeon-dong, Jongno-gu, Seoul 110-749 (Korea, Republic of); Lee, J.I. [Department of Oral Pathology and Dental Research Institute, School of Dentistry, Seoul National University, Seoul (Korea, Republic of); Lim, Y.J., E-mail: limdds@snu.ac.kr [Department of Prosthodontics and Dental Research Institute, School of Dentistry, Seoul National University, 28 Yeongeon-dong, Jongno-gu, Seoul 110-749 (Korea, Republic of)

    2010-03-01

    The aim of the present study was to investigate surface characteristics in four different titanium surfaces (AN: anodized at 270 V; AN-CaP: anodic oxidation and CaP deposited; SLA: sandblasted and acid etched; MA: machined) and to evaluate biological behaviors such as cell adhesion, cell proliferation, cytoskeletal organization, and osteogenic protein expression of MG63 osteoblast-like cells at the early stage. Surface analysis was performed using scanning electron microscopy, thin-film X-ray diffractometry, and a confocal laser scanning microscope. In order to evaluate cellular responses, MG63 osteoblast-like cells were used. The cell viability was evaluated by MTT assay. Immunofluorescent analyses of actin, type I collagen, osteonectin and osteocalcin were performed. The anodized and CaP deposited specimen showed homogeneously distributed CaP particles around micropores and exhibited anatase type oxides, titanium, and HA crystalline structures. This experiment suggests that CaP particles on the anodic oxidation surface affect cellular attachment and spreading. When designing an in vitro biological study for CaP coated titanium, it must be taken into account that preincubation in medium prior to cell seeding and the cell culture medium may affect the CaP coatings. All these observations illustrate the importance of the experimental conditions and the physicochemical parameters of the CaP coating. It is considered that further evaluations such as long-term in vitro cellular assays and in vivo experiments should be necessary to figure out the effect of CaP deposition to biological responses.

  9. In vitro investigation of anodization and CaP deposited titanium surface using MG63 osteoblast-like cells

    International Nuclear Information System (INIS)

    Lee, J.M.; Lee, J.I.; Lim, Y.J.

    2010-01-01

    The aim of the present study was to investigate surface characteristics in four different titanium surfaces (AN: anodized at 270 V; AN-CaP: anodic oxidation and CaP deposited; SLA: sandblasted and acid etched; MA: machined) and to evaluate biological behaviors such as cell adhesion, cell proliferation, cytoskeletal organization, and osteogenic protein expression of MG63 osteoblast-like cells at the early stage. Surface analysis was performed using scanning electron microscopy, thin-film X-ray diffractometry, and a confocal laser scanning microscope. In order to evaluate cellular responses, MG63 osteoblast-like cells were used. The cell viability was evaluated by MTT assay. Immunofluorescent analyses of actin, type I collagen, osteonectin and osteocalcin were performed. The anodized and CaP deposited specimen showed homogeneously distributed CaP particles around micropores and exhibited anatase type oxides, titanium, and HA crystalline structures. This experiment suggests that CaP particles on the anodic oxidation surface affect cellular attachment and spreading. When designing an in vitro biological study for CaP coated titanium, it must be taken into account that preincubation in medium prior to cell seeding and the cell culture medium may affect the CaP coatings. All these observations illustrate the importance of the experimental conditions and the physicochemical parameters of the CaP coating. It is considered that further evaluations such as long-term in vitro cellular assays and in vivo experiments should be necessary to figure out the effect of CaP deposition to biological responses.

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

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    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.

  11. 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.

  12. 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

  13. 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

  14. 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

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

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    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.

  16. 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.

  17. 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

  18. 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.

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

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    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.

  20. 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.

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

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

    Roč. 324, Jan 1 (2015), s. 99-105 ISSN 0169-4332 R&D Projects: GA ČR(CZ) GAP107/12/1025 Grant - others:GA ČR(CZ) GA13-09853S Institutional support: RVO:67985823 Keywords : nanoroughness * titanium * cells adhesion Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.150, year: 2015

  2. On the role of Nb-related sites of an oxidized β-TiNb alloy surface in its interaction with osteoblast-like MG-63 cells

    Energy Technology Data Exchange (ETDEWEB)

    Jirka, Ivan, E-mail: Ivan.Jirka@jh-inst.cas.cz [J. Heyrovský Institute of Physical Chemistry, Acad. Sci. CR, v.v.i. Dolejškova 3, 182 23 Prague 8 (Czech Republic); Vandrovcová, Marta [Institute of Physiology, Acad. Sci. CR, v.v.i., Vídeňská 1083, Prague 4 (Czech Republic); Frank, Otakar [J. Heyrovský Institute of Physical Chemistry, Acad. Sci. CR, v.v.i. Dolejškova 3, 182 23 Prague 8 (Czech Republic); Tolde, Zdeněk [Faculty of Mechanical Engineering, Czech Technical University in Prague, Institute of Materials Engineering, Karlovo nám. 13, Prague 2 (Czech Republic); Plšek, Jan [J. Heyrovský Institute of Physical Chemistry, Acad. Sci. CR, v.v.i. Dolejškova 3, 182 23 Prague 8 (Czech Republic); Luxbacher, Thomas [Anton Paar GmbH, Anton Paar Str. 20, 8054 Graz (Austria); Bačáková, Lucie [Institute of Physiology, Acad. Sci. CR, v.v.i., Vídeňská 1083, Prague 4 (Czech Republic); Starý, Vladimír [Faculty of Mechanical Engineering, Czech Technical University in Prague, Institute of Materials Engineering, Karlovo nám. 13, Prague 2 (Czech Republic)

    2013-04-01

    β-Stabilized titanium (Ti) alloys containing non-toxic elements, particularly niobium (Nb), are promising materials for the construction of bone implants. Their biocompatibility can be further increased by oxidation of their surface. Therefore, in this study, the adhesion, growth and viability of human osteoblast-like MG 63 cells in cultures on oxidized surfaces of a β-TiNb alloy were investigated and compared with the cell behavior on thermally oxidized Ti, i.e. a metal commonly used for constructing bone implants. Four experimental groups of samples were prepared: Ti or TiNb samples annealed to 600 °C for 60 min in a stream of dry air, and Ti and TiNb samples treated in Piranha solution prior to annealing. We found that on all TiNb-based samples, the cell population densities on days 1, 3 and 7 after seeding were higher than on the corresponding Ti-based samples. As revealed by XPS and Raman spectroscopy, and also by isoelectric point measurements, these results can be attributed to the presence of T-Nb{sub 2}O{sub 5} oxide phase in the surface of the alloy sample, which decreased its negative zeta (ζ)-potential in comparison with zeta (ζ)-potential of the Ti sample at physiological pH. This effect was tentatively explained by the presence of positively charged defects acting as Lewis sites of the surface Nb{sub 2}O{sub 5} phase. Piranha treatment slightly decreases the biocompatibility of the samples, which for the alloy samples may be explained by a decrease in the number of defective sites with this treatment. Thus, the presence of Nb and thermal oxidation of β-stabilized Ti alloys play a significant role in the increased biocompatibility of TiNb alloys. - Highlights: ► T-Nb{sub 2}O{sub 5} and rutile are the main components of the oxidized β-TiNb alloy surface. ► Negative value of ζ potential is reduced by presence of Nb in the alloy surface. ► Less negative ζ potential is beneficial for interaction of the alloy with cells. ► The β-TiNb alloy

  3. 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.

  4. Bone regeneration: in vitro evaluation of the behaviour of osteoblast-like MG63 cells placed in contact with polylactic-co-glycolic acid, deproteinized bovine bone and demineralized freeze-dried bone allograft.

    Science.gov (United States)

    Pappalardo, S; Mastrangelo, F; Reale Marroccia, D; Cappello, V; Ciampoli, C; Carlino, V; Tanteri, L; Costanzo, M; Sinatra, F; Tetè, S

    2008-01-01

    Insufficient bone density of the alveolar crests, caused by loss of the dental elements, sometimes impedes the primary stability of an integrated bone implant. The techniques of bone regeneration allow to obtain a sufficient quantity of alveolar bone to permit the implant rehabilitation of the edentulous crests. Today several grafting materials are available and they have different characteristics, according to their structure, which influence the different behaviour of the grafting materials to the bone and the implant surface. The aim of this study is to evaluate the interaction between a human osteosarcoma MG63 cell line and three different biomaterials: polylactic-co-glycolic acid (PLAGA), deproteinized bovine bone and demineralised freeze-dried bone allograft (DFDBA). From this study a different behaviour emerges of the osteoblast-like MG63 cells in relation to the sublayer on which these cells were placed in culture. The results of the study, in fact, demonstrate that the most osteoconductive material of the three analysed is the DFDBA, followed by DPBB. On the contrary, the PLGA, because of its roughness, does not seem to represent a valid support for cell growth, and does not encourage any morphologic modification in tumor cells. Furthermore, deproteinized bovine bone shows a differentiating effect which could lead to hypothesise an osteoconductive capacity of this biomaterial. Further studies should be carried out with the aim of explaining the results obtained.

  5. Polishing and coating carbon fiber-reinforced carbon composites with a carbon-titanium layer enhances adhesion and growth of osteoblast-like MG63 cells and vascular smooth muscle cells in vitro

    Czech Academy of Sciences Publication Activity Database

    Bačáková, Lucie; Starý, V.; Kofroňová, Olga; Lisá, Věra

    2001-01-01

    Roč. 54, č. 4 (2001), s. 567-578 ISSN 0021-9304 R&D Projects: GA ČR GA106/99/0626 Institutional research plan: CEZ:AV0Z5011922 Keywords : surface roughness * carbon particles * bone-derived cells Subject RIV: JI - Composite Materials Impact factor: 2.105, year: 2001

  6. 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.

  7. 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

  8. Cloning the Gravity and Shear Stress Related Genes from MG-63 Cells by Subtracting Hybridization

    Science.gov (United States)

    Zhang, Shu; Dai, Zhong-quan; Wang, Bing; Cao, Xin-sheng; Li, Ying-hui; Sun, Xi-qing

    2008-06-01

    Background The purpose of the present study was to clone the gravity and shear stress related genes from osteoblast-like human osteosarcoma MG-63 cells by subtractive hybridization. Method MG-63 cells were divided into two groups (1G group and simulated microgravity group). After cultured for 60 h in two different gravitational environments, two groups of MG-63 cells were treated with 1.5Pa fluid shear stress (FSS) for 60 min, respectively. The total RNA in cells was isolated. The gravity and shear stress related genes were cloned by subtractive hybridization. Result 200 clones were gained. 30 positive clones were selected using PCR method based on the primers of vector and sequenced. The obtained sequences were analyzed by blast. changes of 17 sequences were confirmed by RT-PCR and these genes are related to cell proliferation, cell differentiation, protein synthesis, signal transduction and apoptosis. 5 unknown genes related to gravity and shear stress were found. Conclusion In this part of our study, our result indicates that simulated microgravity may change the activities of MG-63 cells by inducing the functional alterations of specific genes.

  9. 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.

  10. Biological effects of polymers modified with carbon nanotubes on human osteoblast-like MG 63 cells

    Czech Academy of Sciences Publication Activity Database

    Grausová, Ľubica; Bačáková, Lucie; Fraczek, A.; Blazewicz, S.; Blazewicz, M.; Pepka, M.

    2006-01-01

    Roč. 9, č. 58-60 (2006), s. 14-16 ISSN 1429-7248 R&D Projects: GA ČR(CZ) GA204/06/0225 Institutional research plan: CEZ:AV0Z50110509 Keywords : bone tissue engineering * carbon nanoparticles * synthetic polymers Subject RIV: EI - Biotechnology ; Bionics

  11. 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.

  12. 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)

  13. Nanocrystalline diamond: In vitro biocompatibility assessment by MG63 and human bone marrow cells cultures.

    Science.gov (United States)

    Amaral, M; Dias, A G; Gomes, P S; Lopes, M A; Silva, R F; Santos, J D; Fernandes, M H

    2008-10-01

    Nanocrystalline diamond (NCD) has a great potential for prosthetic implants coating. Nevertheless, its biocompatibility still has to be better understood. To do so, we employed several materials characterization techniques (SEM, AFM, micro-Raman spectroscopy) and cell culture assays using MG63 osteoblast-like and human bone marrow cells. Biochemical routines (MTT assays, Lowry's method, ALP activity) supported by SEM and confocal microscopy characterization were carried out. We used silicon nitride (Si3N4) substrates for NCD coatings based on a previous demonstration of the superior adhesion and tribological performance of these NCD coated ceramics. Results demonstrate an improved human osteoblast proliferation and the stimulation of differentiated markers, like ALP activity and matrix mineralization, compared with standard polystyrene tissue culture plates. The nanometric featuring of NCD, associated to its chemical affinity are key points for bone regeneration purposes.

  14. Aberrant tropoelastin secretion in MG-63 human osteosarcoma cells

    International Nuclear Information System (INIS)

    Curtiss, S.W.

    1989-01-01

    The secretion of newly synthesized tropoelastin, the soluble precursor of the extracellular matrix protein elastin, is not well understood. MG-63 human osteosarcoma cells were found by immunoblot analysis to synthesize 62 kD and 64 kD tropoelastins. Media from 63 cells labelled for five hours with [ 3 H]-valine contain no detectable tropoelastin, unlike media from other tropoelastin-synthesizing cells. Immunoblots of conditioned media and 1Ox-concentrated conditioned media left on the cells for six days also show an absence of tropoelastin from the cell media. No insoluble elastin is associated with the cell layer, as determined by amino acid analysis and electron microscopy of 18-21 day cell cultures. The absence of tropoelastin from the cell medium and elastin from the extracellular matrix indicates that MG63 cells do not secrete tropoelastin as expected, but accumulate it intracellularly. This accumulation is transient: immunoblots and immunofluorescence microscopy show that cells three days after passage have the highest steady-state levels of tropoelastin per cell, that day 8 cells contain lower but still significant amounts of tropoelastin, and that by day 22 tropoelastin is no longer present in the cell cultures. Cell density is a critical factor in the observed pattern of tropoelastin expression. Cells seeded at ten fold their usual initial density have high tropoelastin levels at one day after passage, sooner than cells seeded normally. Tropoelastin also disappears from high density-seeded cells more quickly and is no longer detectable at day 10. Lysosome-like vesicles containing membranous structures appear by immunoelectron microscopy to be the primary site of intracellular tropoelastin localization

  15. 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.

  16. 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

  17. 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)

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. The influence of chemical structure of aliphatic polyesters on adhesion and growth of osteoblast-like MG63 cells

    Czech Academy of Sciences Publication Activity Database

    Pamula, E.; Bačáková, Lucie; Buczynska, J.; Filová, Elena; Nosková, Lenka; Dobrzynski, P.; Bero, M.

    2004-01-01

    Roč. 7, č. 37 (2004), s. 14-17 ISSN 1429-7248. [Konferencja Naukowa Biomaterialy w medycynie i weterynarii /14./. Rytro, 10.10.2004-13.10.2004] R&D Projects: GA MŠk OC 527.130 Grant - others:Polish Committee for Scientific Research(PL) PBZ-KBN-082/T08/2002 Institutional research plan: CEZ:AV0Z5011922 Keywords : tissue engineering * degradable copolymers * physicochemical surface properties Subject RIV: EI - Biotechnology ; Bionics

  4. 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.

  5. 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

  6. 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.

  7. Correlation between E-cadherin-regulated cell adhesion and human osteosarcoma MG-63 cell anoikis.

    Science.gov (United States)

    Lin, Ding-Sheng; Cai, Le-Yi; Ding, Jian; Gao, Wei-Yang

    2014-01-01

    The aim of this study was to investigate the relationship between cell adhesion and anoikis evasion among human osteosarcoma cells (MG-63), and to further study the molecular mechanisms. Human osteosarcoma cells (MG-63) were assessed for apoptosis, and caspase-3, E-cadherin and β-catenin expression in EDTA and control non-EDTA groups. MG-63 cells were predominantly aggregated when in suspension, and the suspended cells were more dispersed in the EDTA group. Following culture in suspension for 24 h, 48 h, or 72 h, the rates of apoptosis were 34.88%±3.64%, 59.3%±7.22% and 78.5%±5.21% in the experimental group and 7.34%±2.13%, 14.7%±3.69%, and 21.4%±3.60% in the control group, respectively. Caspase-3 expression progressively increased and E-cadherin and β-catenin were decreased in the experimental group, whereas there was no change in the control group. MG-63 cells could avoid anoikis through cell adhesion, and E-cadherin might play a role in this process.

  8. 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...

  9. 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

  10. [Experimental research in vitro of TK/GCV system for osteosarcoma MG-63 cell damage].

    Science.gov (United States)

    Zhang, Hua-Dong; Lu, Zhi; Feng, Yi; Liu, Xiao-Li; Hou, Hui-Ming

    2014-03-01

    To study the killing effects of the liposome-mediated thymidine kinase (TK)/ganciclovir (GCV) system on MG-63 osteosarcoma (OS) cells and its bystander effects. Liposome-mediated TK gene transfected into MG-63 OS cells, the efficiency of transfection was analyzed by flow cytometry and observed under inverted fluorescence microscope. Non-transfected osteosarcoma MG-63 cells were divided into three groups,in the experimental group 1 transfected TK/GCV cells cultured in solutiona liquid mixture by supernatant by 1/10,1/7,1/5,1/2 ratio to original broth; in the experimental group 2 transfected cells cultured in solutiona liquid mixture of supernatant filtered through 0.22 microm filter by 1/10,1/7, 1/5, 1/2 ratio to original broth, in control group the transfection cells cultured in original culture solution. Cell growth inhibition rate and osteosarcoma cell sensitivity to TK/GCV system were measured by MTT assay in each group. The TK gene was transfected into MG-63 OS cells successfully by liposome-mediated, flow cytometry instrument detection TK gene transfection cell transfection efficiency can reach 75.5%. Six days later the MTT assay showed that in the experimental group 1 inhibition rate of all concentration ratio of the mixed culture fluid were statistically significant as compared with the control group (P culture medium was not statistically significant as compared with the control group (P > 0.05). TK gene transfected MG-63 cells increased with the the GCV concentration,the cell apoptosis rate increased. The experiment demonstrated that the MG-63 OS cells are sensitive to the liposome-mediated TK/GCV system and bystander effects are significant.

  11. Girdin/GIV is upregulated by cyclic tension, propagates mechanical signal transduction, and is required for the cellular proliferation and migration of MG-63 cells

    International Nuclear Information System (INIS)

    Hu, Jiang-Tian; Li, Yan; Yu, Bing; Gao, Guo-Jie; Zhou, Ting; Li, Song

    2015-01-01

    To explore how Girdin/GIV is regulated by cyclic tension and propagates downstream signals to affect cell proliferation and migration. Human osteoblast-like MG-63 cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h, produced by a four-point bending system. Cyclic tension force upregulated Girdin and Akt expression and phosphorylation in cultured MG-63 cells. Girdin and Akt each promoted the phosphorylation of the other under stimulated tension. In vitro MTT and transwell assays showed that Girdin and Akt are required for cell proliferation and migration during cellular quiescence. Moreover, STAT3 was determined to be essential for Girdin expression under stimulated tension force in the physiological condition, as well as for osteoblast proliferation and migration during quiescence. These findings suggest that the STAT3/Girdin/Akt pathway activates in osteoblasts in response to mechanical stimulation and may play a significant role in triggering osteoblast proliferation and migration during orthodontic treatment. - Highlights: • Tension force upregulates Girdin and Akt expression and phosphorylation. • Girdin and Akt promotes the phosphorylation of each other under tension stimulation. • Girdin and Akt are required for MG-63 cell proliferation and migration. • STAT3 is essential for Girdin expression after application of the tension forces

  12. Girdin/GIV is upregulated by cyclic tension, propagates mechanical signal transduction, and is required for the cellular proliferation and migration of MG-63 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Jiang-Tian; Li, Yan; Yu, Bing; Gao, Guo-Jie; Zhou, Ting; Li, Song, E-mail: song_li59@126.com

    2015-08-21

    To explore how Girdin/GIV is regulated by cyclic tension and propagates downstream signals to affect cell proliferation and migration. Human osteoblast-like MG-63 cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h, produced by a four-point bending system. Cyclic tension force upregulated Girdin and Akt expression and phosphorylation in cultured MG-63 cells. Girdin and Akt each promoted the phosphorylation of the other under stimulated tension. In vitro MTT and transwell assays showed that Girdin and Akt are required for cell proliferation and migration during cellular quiescence. Moreover, STAT3 was determined to be essential for Girdin expression under stimulated tension force in the physiological condition, as well as for osteoblast proliferation and migration during quiescence. These findings suggest that the STAT3/Girdin/Akt pathway activates in osteoblasts in response to mechanical stimulation and may play a significant role in triggering osteoblast proliferation and migration during orthodontic treatment. - Highlights: • Tension force upregulates Girdin and Akt expression and phosphorylation. • Girdin and Akt promotes the phosphorylation of each other under tension stimulation. • Girdin and Akt are required for MG-63 cell proliferation and migration. • STAT3 is essential for Girdin expression after application of the tension forces.

  13. Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells

    Science.gov (United States)

    Drury, Jeanie L.; Moussi, Joelle; Taylor-Pashow, Kathryn M. L.

    2016-01-01

    Monosodium titanates (MST) are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II). In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II) complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II) complexes for 24–72 h. A CellTiter-Blue® assay was employed to assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II) suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II) suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II) are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes. PMID:28044136

  14. Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells

    Directory of Open Access Journals (Sweden)

    Yen-Wei Chen

    2016-01-01

    Full Text Available Monosodium titanates (MST are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II. In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II complexes for 24–72 h. A CellTiter-Blue® assay was employed to assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes.

  15. Parathyroid hormone depresses cytosolic pH and DNA synthesis in osteoblast-like cells

    International Nuclear Information System (INIS)

    Reid, I.R.; Civitelli, R.; Avioli, L.V.; Hruska, K.A.

    1988-01-01

    It has recently become apparent that a number of hormones and growth factors modulate cytosolic pH (pH i ) and there is some evidence that this in turn may influence cell growth. The authors have examined the effects of parathyroid hormone (PTH) on both these parameters in an osteoblast-like cell line, UMR 106. Preliminary studies, using the pH-sensitive fluorescent probe 2',7'-bis(2-carboxyethyl)-5,(6)-carboxyfluorescein indicated that these cells regulate pH i by means of an amiloride-inhibitable Na + -H + exchanger. Rat PTH-(1-34) (rPTH) caused a progressive dose-related decrease in pH i with a half-maximal effect at 10 -11 M. The diacylglycerol analogue, phorbol 12-myristate 13-acetate, increased both pH i and [ 3 H]thymidine incorporation, and amiloride reduced both indexes. However, rPTH remained a potent inhibitor of [ 3 H]thymidine incorporation in the presence of amiloride, even though it did not affect pH i in these circumstances. It is concluded that PTH decreases pH i and growth in UMR 106 cells but that these changes can be dissociated. Depression of pH i may have other important effects on bone metabolism, such as reducing cell-cell communication, and may be associated with alkalinization of the bone fluid compartment

  16. Rho A Regulates Epidermal Growth Factor-Induced Human Osteosarcoma MG63 Cell Migration

    Directory of Open Access Journals (Sweden)

    Jinyang Wang

    2018-05-01

    Full Text Available Osteosarcoma, the most common primary bone tumor, occurs most frequently in children and adolescents and has a 5-year survival rate, which is unsatisfactory. As epidermal growth factor receptor (EGFR positively correlates with TNM (tumor-node-metastasis stage in osteosarcoma, EGFR may play an important role in its progression. The purpose of this study was to explore potential mechanisms underlying this correlation. We found that EGF promotes MG63 cell migration and invasion as well as stress fiber formation via Rho A activation and that these effects can be reversed by inhibiting Rho A expression. In addition, molecules downstream of Rho A, including ROCK1, LIMK2, and Cofilin, are activated by EGF in MG63 cells, leading to actin stress fiber formation and cell migration. Moreover, inhibition of ROCK1, LIMK2, or Cofilin in MG63 cells using known inhibitors or short hairpin RNA (shRNA prevents actin stress fiber formation and cell migration. Thus, we conclude that Rho A/ROCK1/LIMK2/Cofilin signaling mediates actin microfilament formation in MG63 cells upon EGFR activation. This novel pathway provides a promising target for preventing osteosarcoma progression and for treating this cancer.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. Stimulation by parathyroid hormone of sup 45 Ca sup 2+ uptake in osteoblast-like cells: Possible involvement of alkaline phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Fukayama, S.; Tashjian, A.H. Jr. (Harvard School of Public Health, Boston, MA (USA))

    1990-04-01

    We have investigated the actions of human PTH (hPTH-(1-34)) on the association of 45Ca2+ with two human (SaOS-2 and MG-63) and two rat (ROS 17/2.8 and UMR-106) osteoblast-like cell types. In SaOS-2 cells, hPTH-(1-34) binds to specific membrane receptors to activate adenylate cyclase. Treatment of SaOS-2 cells with hPTH-(1-34) resulted in an increase in 45Ca2+ uptake, in a dose-dependent fashion, up to 2- to 4-fold above control values. The increase was first evident at 10 min and persisted for at least 30 min. Treatment with nimodipine, a calcium channel antagonist, was without effect on the stimulatory action of PTH. A similar enhancement of cell-associated 45Ca2+ was observed when the cells were incubated with vasoactive intestinal peptide, which acts via different receptors to activate adenylate cyclase in SaOS-2 cells. Treatment with (Bu)2cAMP also induced an increase in cell-associated 45Ca2+. Pretreatment of SaOS-2 cells with hPTH-(1-34) for 4 h, which induced homologous desensitization to a second challenge with the same peptide for stimulation of cAMP production, did not attenuate the further enhancement of cell-associated 45Ca2+ by a second treatment with hPTH-(1-34). We then examined a possible relationship between alkaline phosphatase (ALPase) and 45Ca2+ uptake. SaOS-2 cells contained high levels of alkaline phosphatase activity and continuously released the enzyme into the medium. Release was enhanced by treatment with hPTH-(1-34) for 10 min. Incubation of cells with levamisole (an inhibitor of the liver/bone/kidney type of ALPase) resulted in a rapid decrease in basal and PTH-stimulated 45Ca2+ uptake, while treatment with L-Phe-Gly-Gly was without effect. Treatment of the cells with ALPase (bovine kidney) enhanced 45Ca2+ uptake. In MG-63 cells, a stimulatory effect of hPTH-(1-34) on cell-associated 45Ca2+ was also observed; however, hPTH-(1-34) did not stimulate cAMP production in MG-63 cells.

  3. 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)

  4. Response of the MG-63 human osteosarcoma cell line grown as multicellular spheroids to neutron irradiation

    International Nuclear Information System (INIS)

    Kubota, Nobuo; Kakehi, Masae; Matsubara, Shou; Koike, Sachiko; Ando, Koichi.

    1993-01-01

    Multicellular tumor spheroids are composed of the mixed populations of cells with regard to cell proliferation, nutrition, oxygenation and radiosensitivity. Human osteogenic sarcoma is generally considered clinically radioresistant. However, the in vitro cell survival curves for human osteogenic sarcoma cell lines do not differ from those of other tumor cell lines. In this study, the responses of human osteogenic sarcoma cell line to gamma ray and neutrons were investigated by using spheroid system. The spheroids of the osteogenic sarcoma cell line are considered to be a good in vitro model of radioresistant tumors. The purpose of this study is to measure the response of the spheroids to fast neutron irradiation. MG-63 human osteogenic sarcoma cell line was used for this study. The cell line was cultured in alpha-MEM with supplement. Cell survival was estimated after the trypsinization of spheroids 24 hours after irradiation. The method of measuring spheroid cure is explained. The mean number of surviving cells per spheroid can be obtained from the mean clonogenic number and cell survival curve. The cell survival of MG-63 spheroids exposed to gamma ray and neutrons and the dose effect curves for spheroid cure after irradiation are shown. (K.I.)

  5. Support for the initial attachment, growth and differentiation of MG-63 cells: a comparison between nano-size hydroxyapatite and micro-size hydroxyapatite in composites

    Directory of Open Access Journals (Sweden)

    Filová E

    2014-08-01

    Full Text Available Elena Filová,1 Tomáš Suchý,2,3 Zbynek Sucharda,2 Monika Šupová,2 Margit Žaloudková,2 Karel Balík,2 Vera Lisá,1 Miroslav Šlouf,4 Lucie Bacáková11Department of Biomaterials and Tissue Engineering, Institute of Physiology, 2Department of Composite and Carbon Materials, Institute of Rock Structure and Mechanics, Academy of Sciences of the Czech Republic, 3Laboratory of Biomechanics, Department of Mechanics, Biomechanics and Mechatronics, Faculty of Mechanical Engineering, CTU in Prague, 4Department of Morphology and Rheology of Polymer Materials, Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech RepublicAbstract: Hydroxyapatite (HA is considered to be a bioactive material that favorably influences the adhesion, growth, and osteogenic differentiation of osteoblasts. To optimize the cell response on the hydroxyapatite composite, it is desirable to assess the optimum concentration and also the optimum particle size. The aim of our study was to prepare composite materials made of polydimethylsiloxane, polyamide, and nano-sized (N or micro-sized (M HA, with an HA content of 0%, 2%, 5%, 10%, 15%, 20%, 25% (v/v (referred to as N0–N25 or M0–M25, and to evaluate them in vitro in cultures with human osteoblast-like MG-63 cells. For clinical applications, fast osseointegration of the implant into the bone is essential. We observed the greatest initial cell adhesion on composites M10 and N5. Nano-sized HA supported cell growth, especially during the first 3 days of culture. On composites with micro-size HA (2%–15%, MG-63 cells reached the highest densities on day 7. Samples M20 and M25, however, were toxic for MG-63 cells, although these composites supported the production of osteocalcin in these cells. On N2, a higher concentration of osteopontin was found in MG-63 cells. For biomedical applications, the concentration range of 5%–15% (v/v nano-size or micro-size HA seems to be optimum

  6. 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

  7. Apoptosis and autophagy induced by pyropheophorbide-α methyl ester-mediated photodynamic therapy in human osteosarcoma MG-63 cells.

    Science.gov (United States)

    Huang, Qiu; Ou, Yun-Sheng; Tao, Yong; Yin, Hang; Tu, Ping-Hua

    2016-06-01

    Pyropheophorbide-α methyl ester (MPPa) was a second-generation photosensitizer with many potential applications. Here, we explored the impact of MPPa-mediated photodynamic therapy (MPPa-PDT) on the apoptosis and autophagy of human osteosarcoma (MG-63) cells as well as the relationships between apoptosis and autophagy of the cells, and investigated the related molecular mechanisms. We found that MPPa-PDT demonstrated the ability to inhibit MG-63 cell viability in an MPPa concentration- and light dose-dependent manner, and to induce apoptosis via the mitochondrial apoptosis pathway. Additionally, MPPa-PDT could also induce autophagy of MG-63 cell. Meanwhile, the ROS scavenger N-acetyl-L-cysteine (NAC) and the Jnk inhibitor SP600125 were found to inhibit the MPPa-PDT-induced autophagy, and NAC could also inhibit Jnk phosphorylation. Furthermore, pretreatment with the autophagy inhibitor 3-methyladenine or chloroquine showed the potential in reducing the apoptosis rate induced by MPPa-PDT in MG-63 cells. Our results indicated that the mitochondrial pathway was involved in MPPa-PDT-induced apoptosis of MG-63 cells. Meanwhile the ROS-Jnk signaling pathway was involved in MPPa-PDT-induced autophagy, which further promoted the apoptosis in MG-63 cells.

  8. In vitro study of vancomycin release and osteoblast-like cell growth on structured calcium phosphate-collagen

    International Nuclear Information System (INIS)

    Pon-On, Weeraphat; Charoenphandhu, Narattaphol; Teerapornpuntakit, Jarinthorn; Thongbunchoo, Jirawan; Krishnamra, Nateetip; Tang, I-Ming

    2013-01-01

    A drug delivery vehicle consisting of spherical calcium phosphate-collagen particles covered by flower-like (SFCaPCol) blossoms composed of nanorod building blocks and their cellular response is studied. The spherical structure was achieved by a combination of sonication and freeze-drying. The SFCaPCol blossoms have a high surface area of approximately 280 m 2 g −1 . The blossom-like formation having a high surface area allows a drug loading efficiency of 77.82%. The release profile for one drug, vancomycin (VCM), shows long term sustained release in simulated body fluid (SBF), in a phosphate buffer saline (PBS, pH 7.4) solution and in culture media over 2 weeks with a cumulative release ∼ 53%, 75% and 50%, respectively, over the first 7 days. The biocompatibility of the VCM-loaded SFCaPCol scaffold was determined by in vitro cell adhesion and proliferation tests of rat osteoblast-like UMR-106 cells. MTT tests indicated that UMR-106 cells were viable after exposure to the VCM loaded SFCaPCol, meaning that the scaffold (the flower-like blossoms) did not impair the cell's viability. The density of cells on the substrate was seen to increase with increasing cultured time. - Graphical abstract: A spherical calcium phosphate-collagen with flower-like blossoms consisting of nanorod building blocks (SFCaPCol) particles was achieved by a combination of sonication and freeze-drying. In vitro drug release profile and the biocompatibility of the VCM-loaded SFCaPCol composite cell adhesion and proliferation in rat osteoblast-like UMR-106 cells were determined for biomaterial applications. Highlights: ► SFCaPCol and VCM-loaded SFCaPCol composite were synthesized by a combination of ultra sonication and freeze-drying. ► VCM drug-loaded SFCaPCol composite was used as substrate for the growth of rat osteoblast-like UMR-106 cells. ► Controlled release of VCM from the composite is critically medium dependent. ► The VCM-loaded SFCaPCol composite is also bioactive by in

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

    Science.gov (United States)

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

    2013-04-01

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

  10. 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.

  11. 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.

  12. Key role of the expression of bone morphogenetic proteins in increasing the osteogenic activity of osteoblast-like cells exposed to shock waves and seeded on bioactive glass-ceramic scaffolds for bone tissue engineering.

    Science.gov (United States)

    Muzio, Giuliana; Martinasso, Germana; Baino, Francesco; Frairia, Roberto; Vitale-Brovarone, Chiara; Canuto, Rosa A

    2014-11-01

    In this work, the role of shock wave-induced increase of bone morphogenetic proteins in modulating the osteogenic properties of osteoblast-like cells seeded on a bioactive scaffold was investigated using gremlin as a bone morphogenetic protein antagonist. Bone-like glass-ceramic scaffolds, based on a silicate experimental bioactive glass developed at the Politecnico di Torino, were produced by the sponge replication method and used as porous substrates for cell culture. Human MG-63 cells, exposed to shock waves and seeded on the scaffolds, were treated with gremlin every two days and analysed after 20 days for the expression of osteoblast differentiation markers. Shock waves have been shown to induce osteogenic activity mediated by increased expression of alkaline phosphatase, osteocalcin, type I collagen, BMP-4 and BMP-7. Cells exposed to shock waves plus gremlin showed increased growth in comparison with cells treated with shock waves alone and, conversely, mRNA contents of alkaline phosphatase and osteocalcin were significantly lower. Therefore, the shock wave-mediated increased expression of bone morphogenetic protein in MG-63 cells seeded on the scaffolds is essential in improving osteogenic activity; blocking bone morphogenetic protein via gremlin completely prevents the increase of alkaline phosphatase and osteocalcin. The results confirmed that the combination of glass-ceramic scaffolds and shock waves exposure could be used to significantly improve osteogenesis opening new perspectives for bone regenerative medicine. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  13. Paracrine-mediated osteoclastogenesis by the osteosarcoma MG63 cell line: is RANKL/RANK signalling really important?

    Science.gov (United States)

    Costa-Rodrigues, J; Teixeira, C A; Fernandes, M H

    2011-08-01

    Although in the past little attention has been paid to the influence of osteosarcoma cells in osteoclast function, recent studies suggest a close relationship between osteosarcoma aggressiveness and osteoclastic activity. The present study addresses the paracrine effects of MG63 cells, a human osteosarcoma-derived cell line, on the differentiation of peripheral blood osteoclast precursor cells (PBMC). PBMC were cultured for 21 days in the presence of conditioned media from MG63 cell cultures (CM) collected at 48 h (CM_MG1), 7 days (CM_MG2) and 14 days (CM_MG3). MG63 cell cultures displayed the expression of ALP and BMP-2 and, also, the osteoclastogenic genes M-CSF and RANKL, although with a low expression of RANKL. PBMC cultures supplemented with CM presented an evident osteoclastogenic behavior, which was dependent on the culture period of the MG63 cells. The inductive effect appeared to be more relevant for the differentiation and activation genes, c-myc and c-src, and lower for genes associated with osteoclast function. In addition, PBMC cultures displayed increased functional parameters, including calcium phosphate resorbing activity. Assessment of the PBMC cultures in the presence of U0126, PDTC, and indomethacin suggested that in addition to MEK and NFkB pathways, other signaling mechanisms, probably not involving RANKL/RANK interaction, might be activated in the presence of conditioned medium from MG63. In conclusion, MG63 cell line appears to induce a significant paracrine-mediated osteoclastogenic response. Understanding the mechanisms underlying the interaction of osteosarcoma cells and osteoclasts may contribute to the development of new potential approaches in the treatment of such bone metabolic diseases.

  14. Influence of collagen and chondroitin sulfate (CS) coatings on poly-(lactide-co-glycolide) (PLGA) on MG 63 osteoblast-like cells

    Czech Academy of Sciences Publication Activity Database

    Vandrovcová, Marta; Douglas, T.; Hauk, D.; Grössner-Schreiber, B.; Wiltfang, J.; Bačáková, Lucie; Warnke, P. H.

    2011-01-01

    Roč. 60, č. 5 (2011), s. 797-813 ISSN 0862-8408 R&D Projects: GA AV ČR(CZ) KAN101120701 Institutional research plan: CEZ:AV0Z50110509 Keywords : collagen * biodegradable polymer * osteoblast Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.555, year: 2011

  15. 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

  16. 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

  17. 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

  18. 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.

  19. [Effect of M007 mediated photodynamic therapy on proliferation of human osteosarcoma MG63 cells in vitro].

    Science.gov (United States)

    Zhou, Yu-Kai; Wu, Wen-Zhi; Zhang, Lan; Yang, Chun-Hui; Wang, Yan-Ping

    2012-01-01

    To investigate the effect of a new photosensitizer, M007 mediated photodynamic therapy on proliferation of human osteosarcoma MG63 cells in vitro. Human osteosarcoma MG63 cells were prepared as 1 x 10(6) /mL single-cell suspension, and 1 mL cells were transferred into 60 mL culture dish, then treated with 5 different gradient dosages (0, 2, 4, 8, 16 micromol/L) of M007 followed by photodynamic therapy or dark reaction for 10 min. The survival rate of the cells and the mode of cell death were detected by flow cytometry with the stain of Annexin V-FITC/PI. The effect on proliferation of survival cells was observed by MTT assay and colony-forming assay. M007 mediated photodynamic therapy induced the inactivation of MG63 human osteosarcoma cells in the way of late apoptosis/necrosis or becoming naked nucleus predominately. More than 90% MG63 cells in M007-PDT group were dead under the treatment of 2-16 micromol/L M007. The survival rates of 4-16 micromol/L M007-PDT group were steadily less than 1%. The optical densities did not increase with extension of culture time in 2-8 micromol/L M007-PDT group (P > 0.05). There were 16 survival alive cells found occasionally in 2 micromol/L M007-PDT group, but no colonies found in other groups. M007 mediated photodynamic therapy totally inactivated human osteosarcoma MG63 cells in vitro with the dosage more than 4 micromol/L.

  20. Effect of low-level laser irradiation on osteoblast-like cells cultured on porous hydroxyapatite scaffolds

    Directory of Open Access Journals (Sweden)

    Serena Incerti Parenti

    2013-09-01

    Full Text Available OBJECTIVE: To determine the effect of laser irradiation at a low dose on human osteoblastlike cells. Materials and methods: 32 porous hydroxyapatite scaffolds currently used for bone tissue engineering were seeded with MG63 cells and irradiated or not with a GaAlAs diode laser (wavelength 915 nm, dose 2 J/cm² using different power density and exposure duration. RESULTS: After 72-h incubation, cells showed well spread morphology and good adhesion on both laser-treated and untreated scaffolds. Laser irradiation did not interfere in cell viability and proliferation as compared with the non-irradiated controls. CONCLUSION: This study suggests that there is no effect of 915 nm laser irradiation at a dose of 2 J/cm² on the proliferation rate of MG63 cells. Future investigations are needed to compare different dose and wavelength regimens in order to determine the optimal set of laser parameters for maximum cell yield and safe clinical application.

  1. Bufalin inhibits the differentiation and proliferation of human osteosarcoma cell line hMG63-derived cancer stem cells.

    Science.gov (United States)

    Chang, Yuewen; Zhao, Yongfang; Zhan, Hongsheng; Wei, Xiaoen; Liu, Tianjin; Zheng, Bo

    2014-02-01

    Cancer stem cells (CSCs) play an important role in drug resistance of tumor and are responsible for high recurrence rates. Agents that can suppress the proliferation and differentiation of CSCs would provide new opportunity to fight against tumor recurrence. In this study, we developed a new strategy to enrich CSCs in human osteosarcoma cell line hMG63. Using these CSCs as model, we tested the effect of bufalin, a traditional Chinese medicine, on the proliferation and differentiation of CSCs. hMG63 cells were cultured in poly-HEMA-treated dish and cancer stem cell-specific medium. In this nonadhesive culture system, hMG63 formed spheres, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every 3 days for five times. The enriched xenograft tumors were cultured in cancer stem cell-specific medium again to form tumor spheres. Expression of cancer stem cell markers of these cells was measured by flow cytometry. These cells were then treated with bufalin, and the proliferation and differentiation ability were indicated by the expression level of molecular markers and the formation of sphere again in vitro. We obtained a low CD133+/CD44 cell population with high-level stem cell marker. When treated with bufalin, the sphere could not get attached to the flask and failed to differentiate, which was indicated by the stable expression of stem cell marker CD133 and OCT-4 in the condition permissive to differentiation. Treatment of bufalin also suppressed the single cells isolated from the sphere to form sphere again in the nonadhesive culture system, and a decreased expression of proliferation marker Ki67 was also detected in these cells. Sphere-formed and chemoresistant colon xenograft tumors in immunodeficient mice could enrich cancer stem cell population. Bufalin could inhibit proliferation and differentiation of CSCs.

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

    Science.gov (United States)

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

    2016-10-01

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

  3. 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

  4. 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

  5. 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.

  6. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells

    International Nuclear Information System (INIS)

    Ramp, W.K.; Lenz, L.G.; Galvin, R.J.

    1991-01-01

    Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of [ 3 H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [ 3 H]hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of [ 3 H]thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone

  7. Synergistic effects of arsenic trioxide combined with ascorbic acid in human osteosarcoma MG-63 cells: a systems biology analysis.

    Science.gov (United States)

    Huang, X C; Maimaiti, X Y M; Huang, C W; Zhang, L; Li, Z B; Chen, Z G; Gao, X; Chen, T Y

    2014-01-01

    To further understand the synergistic mechanism of As2O3 and asscorbic acid (AA) in human osteosarcoma MG-63 cells by systems biology analysis. Human osteosarcoma MG-63 cells were treated by As2O3 (1 µmol/L), AA (62.5 µmol/L) and combined drugs (1 µmol/L As2O3 plus 62.5 µmol/L AA). Dynamic morphological characteristics were recorded by Cell-IQ system, and growth rate was calculated. Illumina beadchip assay was used to analyze the differential expression genes in different groups. Synergic effects on differential expression genes (DEGs) were analyzed by mixture linear model and singular value decomposition model. KEGG pathway annotations and GO enrichment analysis were performed to figure out the pathways involved in the synergic effects. We captured 1987 differential expression genes in combined therapy MG-63 cells. FAT1 gene was significantly upregulated in all three groups, which is a promising drug target as an important tumor suppressor analogue; meanwhile, HIST1H2BD gene was markedly downregulated in the As2O3 monotherapy group and the combined therapy group, which was found to be upregulated in prostatic cancer. These two genes might play critical roles in synergetic effects of AA and As2O3, although the exact mechanism needs further investigation. KEGG pathway analysis showed many DEGs were related with tight junction, and GO analysis also indicated that DEGs in the combined therapy cells gathered in occluding junction, apical junction complex, cell junction, and tight junction. AA potentiates the efficacy of As2O3 in MG-63 cells. Systems biology analysis showed the synergic effect on the DEGs.

  8. Biological characteristics of the MG-63 human osteosarcoma cells on composite tantalum carbide/amorphous carbon films.

    Directory of Open Access Journals (Sweden)

    Yin-Yu Chang

    Full Text Available Tantalum (Ta is a promising metal for biomedical implants or implant coating for orthopedic and dental applications because of its excellent corrosion resistance, fracture toughness, and biocompatibility. This study synthesizes biocompatible tantalum carbide (TaC and TaC/amorphous carbon (a-C coatings with different carbon contents by using a twin-gun magnetron sputtering system to improve their biological properties and explore potential surgical implant or device applications. The carbon content in the deposited coatings was regulated by controlling the magnetron power ratio of the pure graphite and Ta cathodes. The deposited TaC and TaC/a-C coatings exhibited better cell viability of human osteosarcoma cell line MG-63 than the uncoated Ti and Ta-coated samples. Inverted optical and confocal imaging was used to demonstrate the cell adhesion, distribution, and proliferation of each sample at different time points during the whole culture period. The results show that the TaC/a-C coating, which contained two metastable phases (TaC and a-C, was more biocompatible with MG-63 cells compared to the pure Ta coating. This suggests that the TaC/a-C coatings exhibit a better biocompatible performance for MG-63 cells, and they may improve implant osseointegration in clinics.

  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. 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.

  11. 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

  12. Videography supported adhesion, and proliferation behavior of MG-63 osteoblastic cells on 2.5D titania nanotube matrices.

    Science.gov (United States)

    Manurung, Robeth Viktoria; Fu, Pei-Wen; Chu, Yeh-Shiu; Lo, Chun-Min; Chattopadhyay, Surojit

    2016-04-01

    Human osteosarcoma cells MG-63 were cultured on anodically etched titania nanotubes (TiO2 NT), with diameters ranging from 40-100 nm, to study the correlations between cell proliferation and adhesion on the 2.5 dimensional (2.5D) extracellular matrix (ECM). Unlike other reports, mostly based on mouse stem cells, and 2D cell culture, our studies indicate that the 2.5D NT promote higher proliferation and activity, but less 2D adhesion. Proliferation of the MG-63 cells was significantly higher in the NTs, the best being the 70 nm diameter sample, compared to planar titania (control). This is consistent with previous studies. However, cellular adhesion was stronger on TiO2 NT with increasing diameter, and highest on the control as obtained from shear stress measurement, paxilin imaging, and western blot measurements probing focal adhesion kinase, p130 CAS, and extracellular-regulated kinase, in addition to cell morphology imaging by fluorescence microscopy. We provide direct videography of cell migration, and cell speed data indicating faster filopodial activity on the TiO2 NT surfaces having lower adhesion. This evidence was not available previously. The NT matrices promote cells with smaller surface area, because of less 2D stretching. In contrast, on comparatively planar 2D-like surfaces uniaxial stretching of the cell body with strong anchoring of the filopodia, resulted in larger cell surface area, and demonstrated stronger adhesion. The difference in the results, with those previously published, may be generally attributed to, among others, the use of mouse stem cells (human osteosarcoma used here), and unannealed as-grown TiO2 NTs used previously (annealed ECMs used here). © 2015 Wiley Periodicals, Inc.

  13. Adhesion and proliferation of OCT-1 osteoblast-like cells on micro- and nano-scale topography structured poly(L-lactide).

    Science.gov (United States)

    Wan, Yuqing; Wang, Yong; Liu, Zhimin; Qu, Xue; Han, Buxing; Bei, Jianzhong; Wang, Shenguo

    2005-07-01

    The impact of the surface topography of polylactone-type polymer on cell adhesion was to be concerned because the micro-scale texture of a surface can provide a significant effect on the adhesion behavior of cells on the surface. Especially for the application of tissue engineering scaffold, the pore size could have an influence on cell in-growth and subsequent proliferation. Micro-fabrication technology was used to generate specific topography to investigate the relationship between the cells and surface. In this study the pits-patterned surfaces of polystyrene (PS) film with diameters 2.2 and 0.45 microm were prepared by phase-separation, and the corresponding scale islands-patterned PLLA surface was prepared by a molding technique using the pits-patterned PS as a template. The adhesion and proliferation behavior of OCT-1 osteoblast-like cells morphology on the pits- and islands-patterned surface were characterized by SEM observation, cell attachment efficiency measurement and MTT assay. The results showed that the cell adhesion could be enhanced on PLLA and PS surface with nano-scale and micro-scale roughness compared to the smooth surfaces of the PLLA and PS. The OCT-1 osteoblast-like cells could grow along the surface with two different size islands of PLLA and grow inside the micro-scale pits of the PS. However, the proliferation of cells on the micro- and nano-scale patterned surface has not been enhanced compared with the controlled smooth surface.

  14. The structurally effect of surface coated rhamnogalacturonan I on response of the osteoblast-like cell line SaOS-2

    DEFF Research Database (Denmark)

    Svava, Rikke; Gurzawska, Katarzyna; Yihau, Yu

    2014-01-01

    Osseointegration is important when implants are inserted into the bone and can be improved by biochemical surface coating of the implant. In this paper enzymatically modified rhamnogalacturonan I (RG-I) from apple and lupin was used for biochemical coating of aminated surfaces and the importance...... remove small fragments of galacturonic acid (GalA) and binding studies showed that the purity of the RG-I molecules was important for the quality of the coating. The structure of RG-I and osteoblast-like cells' viability were positively correlated so that high content of 1,4-linked galactose (Gal...

  15. Biocompatible nanogel derived from functionalized dextrin for targeted delivery of doxorubicin hydrochloride to MG 63 cancer cells.

    Science.gov (United States)

    Das, Dipankar; Rameshbabu, Arun Prabhu; Ghosh, Paulomi; Patra, Priyapratim; Dhara, Santanu; Pal, Sagar

    2017-09-01

    The present article demonstrates the targeted delivery of doxorubicin hydrochloride to human osteosarcoma cancer cell lines (MG 63) using functionalized dextrin based crosslinked, pH responsive and biocompatible nanogel. The nanogel has been prepared through Michael-type addition reaction using dextrin (Dxt), N, N'-methylene bisacrylamide (MBA, as crosslinker), acrylic acid (AA, as monomer) and potassium persulfate (KPS, as initiator). The structure, composition, morphology of the nanogel have been explored using FTIR and 1 H NMR spectroscopy, XRD, TGA, DSC, CHN and AFM analyses. The TEM analysis confirmed that the size of nanogel appeared within 100nm, while DLS study indicates that the diameter of the nanogel remained between 113 and 126nm. The AFM study implied the porous morphology of the synthesized nanogel. The rheological study suggests the gel behaviour of the synthesized nanogel at 37±0.1°C. Difference in% swelling at pH 5.5 and 7.4 indicates pH-responsiveness of the nanogel. The in vitro cytocompatibility results ascertained that the nanogel is non-toxic to human mesenchymal stem cells (hMSCs). In vitro cellular uptake study confirmed that FITC-loaded nanogel can cross the cellular membrane and be well uptake by the cell cytoplasm. The nanogel could efficiently encapsulate doxorubicin hydrochloride (Dox) with the loading efficiency of 27±0.2% after 72h. The Dox-loaded nanogel demonstrates anti-cancer activity towards MG 63 cancer cells and release the encapsulated drug in a controlled way. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. 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.

  17. Fibrous scaffolds for bone tissue engineering: static and dynamic in vitro studies with MG63 cells

    Czech Academy of Sciences Publication Activity Database

    Buczynska, J.; Pamula, E.; Blazewicz, S.; Bačáková, Lucie; Pařízek, Martin; Chlupáč, Jaroslav; Mikolajczyk, T.; Boguň, M.; Dobrzynski, P.

    2007-01-01

    Roč. 10, 65-66 (2007), s. 1-6 ISSN 1429-7248 R&D Projects: GA ČR(CZ) GA106/06/1576 Grant - others:-(PL) 3T08D 023 30 Institutional research plan: CEZ:AV0Z50110509 Keywords : resorbable scaffolds * bioarteficial bone * dynamic cell culture system Subject RIV: EI - Biotechnology ; Bionics

  18. Effect of biomimetic 3D environment of an injectable polymeric scaffold on MG-63 osteoblastic-cell response

    International Nuclear Information System (INIS)

    Verma, Shalini; Kumar, Neeraj

    2010-01-01

    Solid PLGA microspheres were fabricated and characterized in terms of their in vitro degradation behaviour. Microsphere scaffolds were then modified covalently by P-15 (GTPGPQGIAGQRGVV) to obtain a 3D bioactive collagen surrogate matrix for bone filling applications. These scaffolds were characterized for surface topography, hydrophilicity and evaluated for their effect on osteoblastic activity of MG-63 cell line vis-a-vis 2D monolayer culture. AFM and contact angle experiments indicated enhanced nano-level roughness and hydrophilicity on P-15 modification. Modified scaffolds showed enhanced cell attachment, proliferation, extracellular matrix formation, mineralization and collagen type-I expression when compared to unmodified microspheres, prerequisite for bone filling applications. On long term in vitro cell culture, however, decreased cell viability was observed which may be attributed to the acidic microenvironment generated due to polymer degradation and reduction in nutrient diffusion through the copious ECM formed in 3D scaffolds. Though a higher cell count could be obtained in 2D monolayer cell culture, it was overshadowed by weak cell attachment, poor phenotypic characteristics, decreased cell viability and low mineralization levels, over 28 day cell culture studies. Results indicate that P-15 modified microsphere scaffolds may provide a natural, biomimetic 3D environment and may be successfully exploited for non-invasive bone filling applications.

  19. Influence of boron addition to Ti–13Zr–13Nb alloy on MG63 osteoblast cell viability and protein adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Majumdar, P., E-mail: m.pallab@gmail.com [School of Mechanical Science, Indian Institute of Technology, Bhubaneswar (India); Singh, S.B. [Department of Metallurgical and Materials Engineering, Indian Institute of Technology, Kharagpur (India); Dhara, S. [School Medical Science and Technology, Indian Institute of Technology, Kharagpur (India); Chakraborty, M. [School of Mechanical Science, Indian Institute of Technology, Bhubaneswar (India)

    2015-01-01

    Cell proliferation, cell morphology and protein adsorption on near β-type Ti–13Zr–13Nb (TZN) alloy and Ti–13Zr–13Nb–0.5B (TZNB) composite have been investigated and compared to evaluate the effect of boron addition which has been added to the Ti alloy to improve their poor tribological properties by forming in situ TiB precipitates. MG63 cell proliferation on substrates with different chemistry but the same topography was compared. The MTT assay test showed that the cell viability on the TZN alloy was higher than the boron containing TZNB composite after 36 h of incubation and the difference was pronounced after 7 days. However, both the materials showed substantially higher cell attachment than the control (polystyrene). For the same period of incubation in fetal bovine serum (FBS), the amount of protein adsorbed on the surface of boron free TZN samples was higher than that in the case of boron containing TZNB composite. The presence of boron in the TZN alloy influenced protein adsorption and cell response and they are lower in TZNB than in TZN as a result of the associated difference in chemical characteristics. - Highlights: • The influence of boron addition on biocompatibility of Ti–13Zr–13Nb • Boron forms in situ TiB in TZN matrix and decreases cell proliferation on TZN surfaces. • Protein adsorption is lower in TZNB than in TZN. • Compared to TZNB composite, TZN alloy is more suitable for bone grafting applications.

  20. 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.

  1. 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

  2. Characterization of [125I]omega-conotoxin binding to brain N calcium channels and (-)[3H] desmethoxyverapamil binding to novel calcium channels in osteoblast-like osteosarcoma cells

    International Nuclear Information System (INIS)

    Wagner, J.A.

    1987-01-01

    This dissertation provides molecular evidence for a diversity of Ca 2+ channels in neuronal and non-neuronal tissues. First, I demonstrated specific, reversible, saturable binding sites for omega [ 125 I]conotoxin GVIA (omega[ 125 I]CTX) in rat brain and rabbit sympathetic ganglion. Omega [ 125 I]CTX binding has a unique pharmacology, ion selectivity, and anatomical distribution in rat brain. Omega [ 125 I]CTX binding was solubilized, retaining an appropriate pharmacology and ion selectivity. Omega[ 125 I]CTX binding may be associated with a Ca 2+ channel because the K/sub D/ of omega [ 125 I]CTX is similar to the IC 50 of inhibition of depolarization-induced 45 Ca 2+ flux into rat brain synaptosomes. Specific (-)[ 3 H]desmethoxyverapamil ((-)[ 3 H]DMV) binding sites were demonstrated on osteoblast-like osteosarcoma cell membranes

  3. Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy.

    Science.gov (United States)

    Indovina, Paola; Collini, Maddalena; Chirico, Giuseppe; Santini, Maria Teresa

    2007-02-20

    Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stably transfected with a vector constructed with multiple copies of the HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1alpha (hypoxia-inducible factor-1alpha) is stabilized, the binding of HIF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfected monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1alpha expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. Caryophyllene oxide exhibits anti-cancer effects in MG-63 human osteosarcoma cells via the inhibition of cell migration, generation of reactive oxygen species and induction of apoptosis

    Directory of Open Access Journals (Sweden)

    Zheng Pan

    2016-12-01

    Full Text Available The main objective of the present study was to evaluate the antitumor and apoptotic effects of caryophyllene oxide in MG-63 human osteosarcoma cells. Cell viability of these cells was evaluated by MTT assay while as in vitro wound healing assay was used to study the effect of caryophyllene oxide on cell migration. Fluorescence microscopy and transmission electron microscopy were used to study the changes in cell morphology once the cells undergo apoptosis. Caryophyllene oxide significantly led to cytotoxicity in MG-63 cells showing dose-dependent as well as time-dependent effects. Caryophyllene oxide led to an inhibition of wound closure significantly. At caryophyllene oxide doses of 20, 80 and 120 µM, the percentage of cell migration was shown to be 94.2, 67.1 and 14.8% respectively. With an increase in the caryophyllene oxide dose, the extent of apoptosis also increased characterized by cellular shrinkage, membrane blebbing, chromatin condensation and apoptotic body formation.

  10. 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

  11. 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.

  12. 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

  13. Acidic preparations of lysed platelets upregulate proliferative pathways in osteoblast-like cells as demonstrated by genome-wide microarray analysis.

    Science.gov (United States)

    Wahlström, Ola; Linder, Cecilia Halling; Ansell, Anna; Kalén, Anders; Söderström, Mats; Magnusson, Per

    2011-01-01

    Platelets contain numerous growth factors essential for wound and fracture healing. We investigated the gene expression in human osteoblast-like cells stimulated with lysed platelets prepared in acidic, neutral, or alkaline buffers. Lysed platelets prepared in buffers at pH 5.4, 7.4, and 7.9, were added after neutralization to hFOB 1.19 cells. Genome-wide microarray analysis was performed using the Affymetrix GeneChip 7G Scanner. Biometric, cluster, and pathway analyses were performed with GeneSpring GX. Biometric analyses demonstrated that 53 genes were differentially regulated (p ≤ 0.005, ≥2-fold increase). Pathway analysis revealed 10 significant pathways of which eight are common ones regulating bone formation and cancer growth. Eleven genes were selected for quantitative real-time polymerase chain reaction (PCR) based on the microarray analysis of the lysed platelets prepared in the pH 5.4 experiments. In conclusion, acidic preparations of lysed platelet concentrates release factors essential for cell proliferation and particularly cell metabolism under hypoxic conditions. The genetic response from these factors was dominated by genes associated with the same pathways observed in bone formation and cancer growth. Activation of TGF-β in the acidic preparation could be a stimulatory key factor of cell proliferation. These results support the hypothesis that acidification of platelets modifies the stimulatory response of mesenchymal cells in vitro, which is analogous with the observed milieu of a low pH present in wound and fracture sites, as well as in growing tumors.

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

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

  15. The influence of fluid shear stress on the expression of Cbfa1 in MG-63 cells cultured under different gravitational conditions

    Science.gov (United States)

    Zhang, S.; Wang, B.; Cao, X. S.; Yang, Z.; Sun, X. Q.

    2008-12-01

    AuthorPurposeThis study was aimed to explore the effect of flow shear stress on the expression of Cbfa1 in human osteosarcoma cells and to survey its functional alteration in simulated microgravity. After culture for 48 h in two different gravitational environments, i.e. 1 G terrestrial gravitational condition and simulated microgravity condition, human osteosarcoma cells (MG-63) were treated with 0.5 or 1.5 Pa fluid shear stress (FSS) in a flow chamber for 15, 30, and 60 min, respectively. The total RNA in cells was isolated. RT-PCR analysis was made to examine the gene expression of Cbfa1. The total protein of cells was extracted and the expression of Cbfa1 protein was detected by means of Western blotting. ResultsMG-63 cells cultured in 1 G condition reacted to FSS treatment with an enhanced expression of Cbfa1. Compared with no-FSS control group, Cbfa1 mRNA expression increased significantly at 30 and 60 min with the treatment of FSS ( P cells cultured in simulated microgravity by using clinostat, the expression of Cbfa1 was significantly different between 1 G and simulated microgravity conditions at each test time ( P cultured in simulated microgravity, Cbfa1 mRNA expression increased significantly at 30 and 60 min with the treatment of FSS ( P osteosarcoma cells. And this inducible function of FSS was adversely affected by simulated microgravity.

  16. Effect of simulated weightlessness on the expression of Cbfα1 induced by fluid shear stress in MG-63 osteosarcoma cells.

    Science.gov (United States)

    Yang, Z.; Zhang, S.; Wang, B.; Sun, X. Q.

    Objective The role of mechanical load in the functional regulation of osteoblasts becomes an emphasis in osseous biomechanical researches recently This study was aim to explore the effect of flow shear stress on the expression of Cbf alpha 1 in human osteosarcoma cells and to survey its functional alteration in simulated weightlessness Method After cultured for 72 h in two different gravitational environments i e 1G terrestrial gravitational condition and simulated weightlessness condition human osteosarcoma cells MG-63 were treated with 0 5 Pa or 1 5 Pa fluid shear stress FSS in a flow chamber for 15 30 60 min respectively The total RNA in cells was isolated Transcription PCR analysis was made to examine the gene expression of Cbf alpha 1 And the total protein of cells was extracted and the expression of Cbf alpha 1 protein was detected by means of Western Blotting Results MG-63 cultured in 1G condition reacted to FSS treatment with an enhanced expression of Cbf alpha 1 Compared with no FSS control group Cbf alpha 1 mRNA and protein expression increased significantly at 30 and 60 min with the treatment of FSS P 0 01 And there was remarkable difference on the Cbf alpha 1 mRNA and protein expression between the treatments of 0 5 Pa and 1 5 Pa FSS at 30 min or 60 min P 0 01 As to the osteoblasts cultured in simulated weightlessness by using clinostat the expression of Cbf alpha 1 was significantly different between 1G and simulated weightlessness conditions at each test time P 0 05 Compared with no FSS

  17. Evaluation of chemically modified Ti–5Mo–3Fe alloy surface: Electrochemical aspects and in vitro bioactivity on MG63 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, A. Madhan [Center of Research Excellence in Corrosion, King Fahd University of Petroleum and Minerals, Dhahran 31261 (Saudi Arabia); Sudhagar, P. [Energy Materials Laboratory, WCU Program Department of Energy Engineering, Hanyang University, Seoul 133-791 (Korea, Republic of); Ramakrishna, Suresh [Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Sungdong-gu, Seoul (Korea, Republic of); Kang, Yong Soo [Energy Materials Laboratory, WCU Program Department of Energy Engineering, Hanyang University, Seoul 133-791 (Korea, Republic of); Kim, Hyongbum [Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Sungdong-gu, Seoul (Korea, Republic of); Gasem, Zuhair M. [Center of Research Excellence in Corrosion, King Fahd University of Petroleum and Minerals, Dhahran 31261 (Saudi Arabia); Rajendran, N., E-mail: nrajendran@annauniv.edu [Department of Chemistry, Anna University, Chennai (India)

    2014-07-01

    Ti–5Mo–3Fe (TMF) alloy is a newly developed β-titanium alloy with low modulus, and it has been deemed as suitable material for dental or orthopaedic implant. The aim of the present study is to evaluate the effect of alkali and hydrogen peroxide treatment on the corrosion and biological performance of TMF surface. The phases, morphology with chemical composition and topography of the treated surface were examined by X-ray diffraction, scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDAX) analysis and atomic force microscopy (AFM), respectively. Micro hardness of treated substrates was measured using Vicker's micro hardness method. The electrochemical studies were carried out using potentiodynamic polarisation and electrochemical impedance spectroscopy (EIS) measurements. In order to describe the bio-activity, contact angle measurements, in vitro characterisation and cell culture studies were performed for treated TMF surfaces in simulated body fluid (SBF) and MG63 cells. All these observations showed that the NaOH treatment is the most appropriate method for TMF alloy which exhibited superior biocompatibility and enhanced corrosion protection performance due to their hydrophilic, smooth, compact porous surface morphology than that of other substrates.

  18. Selective inhibition of MG-63 osteosarcoma cell proliferation induced by curcumin-loaded self-assembled arginine-rich-RGD nanospheres.

    Science.gov (United States)

    Chang, Run; Sun, Linlin; Webster, Thomas J

    2015-01-01

    Osteosarcoma is the most frequent primary malignant form of bone cancer, comprising 30% of all bone cancer cases. The objective of this in vitro study was to develop a treatment against osteosarcoma with higher selectivity toward osteosarcoma cells and lower cytotoxicity toward normal healthy osteoblast cells. Curcumin (or diferuloylmethane) has been found to have antioxidant and anticancer effects by multiple cellular pathways. However, it has lower water solubility and a higher degradation rate in alkaline conditions. In this study, the amphiphilic peptide C18GR7RGDS was used as a curcumin carrier in aqueous solution. This peptide contains a hydrophobic aliphatic tail group leading to their self-assembly by hydrophobic interactions, as well as a hydrophilic head group composed of an arginine-rich and an arginine-glycine-aspartic acid structure. Through characterization by transmission electron microscopy, self-assembled structures of spherical amphiphilic nanoparticles (APNPs) with diameters of 10-20 nm in water and phosphate-buffered saline were observed, but this structure dissociated when the pH value was reduced to 4. Using a method of codissolution with acetic acid and dialysis tubing, the solubility of curcumin was enhanced and a homogeneous solution was formed in the presence of APNPs. Successful encapsulation of curcumin in APNPs was then confirmed by Fourier transform infrared and X-ray diffraction analyses. The cytotoxicity and cellular uptake of the APNP/curcumin complexes on both osteosarcoma and normal osteoblast cell lines were also evaluated by methyl-thiazolyl-tetrazolium assays and confocal fluorescence microscopy. The results showed that the curcumin-loaded APNPs had significant selective cytotoxicity against MG-63 osteosarcoma cells when compared with normal osteoblasts. We have demonstrated for the first time that APNPs can encapsulate hydrophobic curcumin in their hydrophobic cores, and curcumin-loaded APNPs could be an innovative treatment

  19. Selective inhibition of MG-63 osteosarcoma cell proliferation induced by curcumin-loaded self-assembled arginine-rich-RGD nanospheres

    Directory of Open Access Journals (Sweden)

    Chang R

    2015-05-01

    cytotoxicity against MG-63 osteosarcoma cells when compared with normal osteoblasts. We have demonstrated for the first time that APNPs can encapsulate hydrophobic curcumin in their hydrophobic cores, and curcumin-loaded APNPs could be an innovative treatment for the selective inhibition of osteosarcoma cells. Keywords: osteosarcoma, selective inhibition, curcumin, arginine-rich, arginine-glycine-aspartic acid, self-assembly

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

    Science.gov (United States)

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

    2018-06-01

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

  1. 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

  2. An investigation of the initial attachment and orientation of osteoblast-like cells on laser grooved Ti-6Al-4V surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Chen, J., E-mail: jianboc@Princeton.EDU [Princeton Institute of Science and Technology of Materials, Princeton University, Princeton, NJ 08544 (United States); Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544 (United States); Ulerich, J.P. [Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544 (United States); Abelev, E. [Department of Chemistry, Princeton University, Princeton, NJ 08544 (United States); Fasasi, A. [Princeton Institute of Science and Technology of Materials, Princeton University, Princeton, NJ 08544 (United States); Center for Energy Research, Obafemi Awolowo University, Ile-Ife (Nigeria); Arnold, C.B.; Soboyejo, W.O. [Princeton Institute of Science and Technology of Materials, Princeton University, Princeton, NJ 08544 (United States); Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544 (United States)

    2009-05-05

    This paper presents the results of an experimental study of the initial cell spreading and adhesion on longitudinally- and transversally-oriented micro-grooves produced by the laser irradiation of laser grooved Ti-6Al-4V surfaces. The initial spreading and orientations of human osteosarcoma (HOS) cells were observed and quantified after 15-min, 1-hour, 4-hour and 24-hour cell culture periods. Immuno-fluorescence staining of adhesion proteins (actin and vinculin) was then used to study the spreading and adhesion of HOS cells in 1 hour and 4 hour culture experiments. The initial cell adhesion was also quantified using enzymatic detachment tests. The results showed that cell spreading and adhesion were enhanced by longitudinally- and transversally-oriented micro-grooves. The effects, which increase with time, were not remarkable after 1 hour, but obvious after 4 hours. Contact guidance was found to promote cell adhesion due to the increase in interactions between the focal adhesions and the patterned extra-cellular matrix (ECM) proteins on the laser micro-grooved surfaces.

  3. An investigation of the initial attachment and orientation of osteoblast-like cells on laser grooved Ti-6Al-4V surfaces

    International Nuclear Information System (INIS)

    Chen, J.; Ulerich, J.P.; Abelev, E.; Fasasi, A.; Arnold, C.B.; Soboyejo, W.O.

    2009-01-01

    This paper presents the results of an experimental study of the initial cell spreading and adhesion on longitudinally- and transversally-oriented micro-grooves produced by the laser irradiation of laser grooved Ti-6Al-4V surfaces. The initial spreading and orientations of human osteosarcoma (HOS) cells were observed and quantified after 15-min, 1-hour, 4-hour and 24-hour cell culture periods. Immuno-fluorescence staining of adhesion proteins (actin and vinculin) was then used to study the spreading and adhesion of HOS cells in 1 hour and 4 hour culture experiments. The initial cell adhesion was also quantified using enzymatic detachment tests. The results showed that cell spreading and adhesion were enhanced by longitudinally- and transversally-oriented micro-grooves. The effects, which increase with time, were not remarkable after 1 hour, but obvious after 4 hours. Contact guidance was found to promote cell adhesion due to the increase in interactions between the focal adhesions and the patterned extra-cellular matrix (ECM) proteins on the laser micro-grooved surfaces.

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

    Science.gov (United States)

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

    2016-06-01

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

  5. Immature osteoblastic MG63 cells possess two calcitonin gene-related peptide receptor subtypes that respond differently to [Cys(Acm)(2,7)] calcitonin gene-related peptide and CGRP(8-37).

    Science.gov (United States)

    Kawase, Tomoyuki; Okuda, Kazuhiro; Burns, Douglas M

    2005-10-01

    Calcitonin gene-related peptide (CGRP) is clearly an anabolic factor in skeletal tissue, but the distribution of CGRP receptor (CGRPR) subtypes in osteoblastic cells is poorly understood. We previously demonstrated that the CGRPR expressed in osteoblastic MG63 cells does not match exactly the known characteristics of the classic subtype 1 receptor (CGRPR1). The aim of the present study was to further characterize the MG63 CGRPR using a selective agonist of the putative CGRPR2, [Cys(Acm)(2,7)]CGRP, and a relatively specific antagonist of CGRPR1, CGRP(8-37). [Cys(Acm)(2,7)]CGRP acted as a significant agonist only upon ERK dephosphorylation, whereas this analog effectively antagonized CGRP-induced cAMP production and phosphorylation of cAMP response element-binding protein (CREB) and p38 MAPK. Although it had no agonistic action when used alone, CGRP(8-37) potently blocked CGRP actions on cAMP, CREB, and p38 MAPK but had less of an effect on ERK. Schild plot analysis of the latter data revealed that the apparent pA2 value for ERK is clearly distinguishable from those of the other three plots as judged using the 95% confidence intervals. Additional assays using 3-isobutyl-1-methylxanthine or the PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89) indicated that the cAMP-dependent pathway was predominantly responsible for CREB phosphorylation, partially involved in ERK dephosphorylation, and not involved in p38 MAPK phosphorylation. Considering previous data from Scatchard analysis of [125I]CGRP binding in connection with these results, these findings suggest that MG63 cells possess two functionally distinct CGRPR subtypes that show almost identical affinity for CGRP but different sensitivity to CGRP analogs: one is best characterized as a variation of CGRPR1, and the second may be a novel variant of CGRPR2.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Youn Hee Joung

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

  8. Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3): Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells.

    Science.gov (United States)

    Kopp, Mathis; Rotan, Olga; Papadopoulos, Chrisovalantis; Schulze, Nina; Meyer, Hemmo; Epple, Matthias

    2017-01-01

    Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE). Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1) and lysosomes (shown by the marker Lamp1). There, it was still intact and functional (i.e. properly folded) as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic) effect of a protein inside a cell.

  9. Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3: Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells.

    Directory of Open Access Journals (Sweden)

    Mathis Kopp

    Full Text Available Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE. Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1 and lysosomes (shown by the marker Lamp1. There, it was still intact and functional (i.e. properly folded as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic effect of a protein inside a cell.

  10. Maturation of osteoblast-like SaoS2 induced by carbon nanotubes

    International Nuclear Information System (INIS)

    Li Xiaoming; Uo, Motohiro; Akasaka, Tsukasa; Abe, Shigeaki; Watari, Fumio; Gao Hong; Sato, Yoshinori; Feng Qingling; Cui Fuzhai

    2009-01-01

    Osteogenic maturation of the osteoblast is crucial for bone formation. In this study, multi-walled carbon nanotubes (MWCNTs) and graphite (GP) were pressed as compacts. The greater ability of carbon nanotubes to adsorb proteins, compared with graphite, was shown. Human osteoblast-like SaoS2 cells were cultured and the cell response to the two kinds of compacts was compared in vitro. Meanwhile, we used cell culture on the culture plate as a control. Assays for osteonectin, osteopontin and osteocalcin gene expression, total protein (TP) amount, alkaline phosphatase activity (ALP) and DNA of cells cultured on the samples were done. During the conventional culture, significantly higher osteonectin, osteopontin and osteocalcin gene expression level, ALP/DNA and TP/DNA on carbon nanotubes were found. To confirm the hypothesis that the larger amount of specific proteins adsorbed on the carbon nanotubes was crucial for this, the compacts were pre-soaked in culture medium having additional recombinant human bone morphogenetic protein-2 (rhBMP-2) before cell culture. Compared with GP, osteonectin, osteopontin and osteocalcin gene expression level, ALP/DNA and TP/DNA of the cells tested increased more on the MWCNTs after the compacts were pre-soaked in the culture medium with rhBMP-2. The results indicated that the carbon nanotubes might induce osteogenic maturation of the osteoblast by adsorbing more specific proteins.

  11. 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.

  12. 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.

  13. Aplasia Ras homologue member Ⅰ overexpression inhibits tumor growth and induces apoptosis through inhibition of PI3K/Akt survival pathways in human osteosarcoma MG-63 cells in culture.

    Science.gov (United States)

    Ye, Kaishan; Wang, Shuanke; Yang, Yong; Kang, Xuewen; Wang, Jing; Han, Hua

    2015-09-01

    Aplasia Ras homologue member Ⅰ (ARHI), an imprinted tumor-suppressor gene, is downregulated in various types of cancer. However, the expression, function and specific mechanisms of ARHI in human osteosarcoma (OS) cells remain unclear. The aim of the present study was to assess the effect of ARHI on OS cell proliferation and apoptosis and its associated mechanism. In the study, ARHI mRNA and protein levels were markedly downregulated in OS cells compared with the human osteoblast precursor cell line hFOB1.19. By generating stable transfectants, ARHI was overexpressed in OS cells that had low levels of ARHI. Overexpression of ARHI inhibited cell viability and proliferation and induced apoptosis. However, caspase‑3 activity was not changed by ARHI overexpression. In addition, phosphorylated Akt protein expression decreased in the ARHI overexpression group compared to that in the control vector group. The knockdown of ARHI also resulted in the promotion of cell proliferation and the attenuation of apoptosis in MG‑63 cells. Additionally, ARHI silencing increased the level of p‑Akt. The present results indicate that ARHI inhibits OS cell proliferation and may have a key role in the development of OS.

  14. Pro-apoptotic and pro-autophagic effects of the Aurora kinase A inhibitor alisertib (MLN8237 on human osteosarcoma U-2 OS and MG-63 cells through the activation of mitochondria-mediated pathway and inhibition of p38 MAPK/PI3K/Akt/mTOR signaling pathway

    Directory of Open Access Journals (Sweden)

    Niu NK

    2015-03-01

    mesenchymal transition (EMT and the underlying mechanisms in two human OS cell lines U-2 OS and MG-63. The results showed that ALS had potent growth inhibitory, pro-apoptotic, pro-autophagic, and EMT inhibitory effects on U-2 OS and MG-63 cells. ALS remarkably induced G2/M arrest and down-regulated the expression levels of cyclin-dependent kinases 1 and 2 and cyclin B1 in both U-2 OS and MG-63 cells. ALS markedly induced mitochondria-mediated apoptosis with a significant increase in the expression of key pro-apoptotic proteins and a decrease in main anti-apoptotic proteins. Furthermore, ALS promoted autophagic cell death via the inhibition of phosphatidylinositol 3-kinase (PI3K/protein kinase B (Akt/mammalian target of rapamycin (mTOR and p38 mitogen-activated protein kinase (p38 MAPK signaling pathways, and activation of 5'-AMP-dependent kinase (AMPK signaling pathway. Inducers or inhibitors of apoptosis or autophagy simultaneously altered ALS-induced apoptotic and autophagic death in both U-2 OS and MG-63 cells, suggesting a crosstalk between these two primary modes of programmed cell death. Moreover, ALS suppressed EMT-like phenotypes with a marked increase in the expression of E-cadherin but a decrease in N-cadherin in U-2 OS and MG-63 cells. ALS treatment also induced reactive oxygen species (ROS generation but inhibited the expression levels of sirtuin 1 and nuclear factor-erythroid-2-related factor 2 (Nrf2 in both cell lines. Taken together, these findings show that ALS promotes apoptosis and autophagy but inhibits EMT via PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways with involvement of ROS- and sirtuin 1-associated pathways in U-2 OS and MG-63 cells. ALS is a promising anticancer agent in OS treatment and further studies are needed to confirm its efficacy and safety in OS chemotherapy. Keywords: ALS, autophagy, apoptosis, osteosarcoma, PI3K/Akt/mTOR pathway, EMT

  15. Osteoarticular cells tolerate short-term exposure to nitisinone-implications in alkaptonuria.

    Science.gov (United States)

    Mistry, J B; Jackson, D J; Bukhari, M; Taylor, A M

    2016-02-01

    Alkaptonuria (AKU) is a rare genetic disease resulting in severe, rapidly progressing, early onset multi-joint osteoarthropathy. A potential therapy, nitisinone, is being trialled that reduces the causative agent; homogentisic acid (HGA) and in a murine model has shown to prevent ochronosis. Little is currently known about the effect nitisinone has on osteoarticular cells; these cells suffer most from the presence of HGA and its polymeric derivatives. This led us to investigate nitisinone's effect on chondrocytes and osteoblast-like cells in an in vitro model. Human C20/A4 immortalized chondrocytes, and osteosarcoma cells MG63 cultured in DMEM, as previously described. Confluent cells were then plated into 24-well plates at 4 × 10(4) cells per well in varying concentrations of nitisinone. Cells were cultured for 7 days with medium changes every third day. Trypan blue assay was used to determine viability and the effect of nitisinone concentration on cells. Statistical analysis was performed using analysis of variance, and differences between groups were determined by Newman-Keuls post-test. Analysis of C20/A4 chondrocyte and MG63 osteoblast-like cell viability when cultured in different concentrations of nitisinone demonstrates that there is no statistically significant difference in cell viability compared to control cultures. There is currently no literature surrounding the use of nitisinone in human in vitro models, or its effect on chondrocytes or osteoblast like cells. Our results show that nitisinone does not appear detrimental to cell viability of chondrocytes or osteoblast-like cells, which adds to the evidence that this therapy could be useful in treating AKU.

  16. 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.

  17. Biodegradation and Osteosarcoma Cell Cultivation on Poly(aspartic acid) Based Hydrogels.

    Science.gov (United States)

    Juriga, Dávid; Nagy, Krisztina; Jedlovszky-Hajdú, Angéla; Perczel-Kovách, Katalin; Chen, Yong Mei; Varga, Gábor; Zrínyi, Miklós

    2016-09-14

    Development of novel biodegradable and biocompatible scaffold materials with optimal characteristics is important for both preclinical and clinical applications. The aim of the present study was to analyze the biodegradability of poly(aspartic acid)-based hydrogels, and to test their usability as scaffolds for MG-63 osteoblast-like cells. Poly(aspartic acid) was fabricated from poly(succinimide) and hydrogels were prepared using natural amines as cross-linkers (diaminobutane and cystamine). Disulfide bridges were cleaved to thiol groups and the polymer backbone was further modified with RGD sequence. Biodegradability of the hydrogels was evaluated by experiments on the base of enzymes and cell culture medium. Poly(aspartic acid) hydrogels possessing only disulfide bridges as cross-links proved to be degradable by collagenase I. The MG-63 cells showed healthy, fibroblast-like morphology on the double cross-linked and RGD modified hydrogels. Thiolated poly(aspartic acid) based hydrogels provide ideal conditions for adhesion, survival, proliferation, and migration of osteoblast-like cells. The highest viability was found on the thiolated PASP gels while the RGD motif had influence on compacted cluster formation of the cells. These biodegradable and biocompatible poly(aspartic acid)-based hydrogels are promising scaffolds for cell cultivation.

  18. Direct covalent coupling of proteins to nanostructured plasma polymers: a route to tunable cell adhesion

    International Nuclear Information System (INIS)

    Melnichuk, Iurii; Choukourov, Andrei; Bilek, Marcela; Weiss, Anthony; Vandrovcová, Marta; Bačáková, Lucie; Hanuš, Jan; Kousal, Jaroslav; Shelemin, Artem; Solař, Pavel

    2015-01-01

    Highlights: • Flat and nanostructured interfaces were overcoated by hydrocarbon plasma polymer. • Linker-free covalent attachment of proteins to resultant surfaces was validated. • Ultra-thin hydrocarbon overcoat (<2 nm) secured prolonged effective binding. • Pre-adsorbed tropoelastin promoted proliferation of osteoblast-like MG-63 cells. • Nanostructured films were multi-affine and impeded cell adhesion. - Abstract: Flat and nanostructured thin films were fabricated by deposition of ultra-thin (<2 nm) layer of hydrocarbon plasma polymer over polished silicon and over a pattern of 8 nm-thick poly(ethylene) islands on silicon. Linker-free radical-based covalent binding of bovine serum albumin and tropoelastin was confirmed for both types of films. The binding capability of albumin was found to be stable over many days of ambient air storage time. Tropoelastin-mediated flat plasma polymers favored adhesion and proliferation of osteoblast-like MG-63 cells. Nanostructured plasma polymers were multi-affine and their hierarchical surface represented an additional barrier for cell attachment

  19. An investigation into the stability of commercial versus MG63-derived hepatocyte growth factor under flow cultivation conditions.

    Science.gov (United States)

    Meneghello, Giulia; Storm, Michael P; Chaudhuri, Julian B; De Bank, Paul A; Ellis, Marianne J

    2015-03-01

    The scale-up of tissue engineering cell culture must ensure that conditions are maintained while also being cost effective. Here we analyse the stability of hepatocyte growth factor (HGF) to investigate whether concentrations change under dynamic conditions, and compare commercial recombinant human HGF as an additive in 'standard medium', to HGF secreted by the osteosarcoma cell line MG63 as a 'preconditioned medium'. After 3 h under flow conditions, HGF in the standard medium degraded to 40% of its original concentration but HGF in the preconditioned medium remained at 100%. The concentration of secreted HGF was 10 times greater than the working concentration of commercially-available HGF. Thus HGF within this medium has increased stability; MG63-derived HGF should therefore be investigated as a cost-effective alternative to current lyophilised powders for use in in vitro models. Furthermore, we recommend that those intending to use HGF (or other growth factors) should consider similar stability testing before embarking on experiments with media flow.

  20. 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.

  1. The dependence of MG63 osteoblast responses to (meth)acrylate-based networks on chemical structure and stiffness.

    Science.gov (United States)

    Smith, Kathryn E; Hyzy, Sharon L; Sunwoo, Moonhae; Gall, Ken A; Schwartz, Zvi; Boyan, Barbara D

    2010-08-01

    The cell response to an implant is regulated by the implant's surface properties including topography and chemistry, but less is known about how the mechanical properties affect cell behavior. The objective of this study was to evaluate how the surface stiffness and chemistry of acrylate-based copolymer networks affect the in vitro response of human MG63 pre-osteoblast cells. Networks comprised of poly(ethylene glycol) dimethacrylate (PEGDMA; Mn approximately 750) and diethylene glycol dimethacrylate (DEGDMA) were photopolymerized at different concentrations to produce three compositions with moduli ranging from 850 to 60 MPa. To further decouple chemistry and stiffness, three networks comprised of 2-hydroxyethyl methacrylate (2HEMA) and PEGDMA or DEGDMA were also designed that exhibited a range of moduli similar to the PEGDMA-DEGDMA networks. MG63 cells were cultured on each surface and tissue culture polystyrene (TCPS), and the effect of copolymer composition on cell number, osteogenic markers (alkaline phosphatase specific activity and osteocalcin), and local growth factor production (OPG, TGF-beta1, and VEGF-A) were assessed. Cells exhibited a more differentiated phenotype on the PEGDMA-DEGDMA copolymers compared to the 2HEMA-PEGDMA copolymers. On the PEGDMA-DEGDMA system, cells exhibited a more differentiated phenotype on the stiffest surface indicated by elevated osteocalcin compared with TCPS. Conversely, cells on 2HEMA-PEGDMA copolymers became more differentiated on the less stiff 2HEMA surface. Growth factors were regulated in a differential manner. These results indicate that copolymer chemistry is the primary regulator of osteoblast differentiation, and the effect of stiffness is secondary to the surface chemistry. 2010 Elsevier Ltd. All rights reserved.

  2. Human Periodontal Cells Demonstrate Osteoblast-Like and Fibroblast-Like Characteristics in Tissue Culture

    Science.gov (United States)

    1989-05-05

    osteonectin (Triffitt, 1987). Osteonectin is also found in platelets ( Stenner et al., 1986). Osteonectin is present at mineral formation sites and may bind...cal and histologic observations: one year postsurgery. J. Periodontol., 54: 325-338. 59 Stenner , D. D., Tracy, R. P., Riggs, B. L. and K. G. Mann. 1986

  3. Polylactide nanofibers with hydroxyapatite as growth substrates for osteoblast-like cells

    Czech Academy of Sciences Publication Activity Database

    Novotná, Katarína; Zajdlová, Martina; Suchý, Tomáš; Hadraba, Daniel; Lopot, F.; Žaloudková, Margit; Douglas, T.E.L.; Munzarová, M.; Juklíčková, M.; Stránská, D.; Kubies, Dana; Schaubroeck, D.; Wille, S.; Balcaen, L.; Jarošová, Markéta; Kozak, Halyna; Kromka, Alexander; Švindrych, Zdeněk; Lisá, Věra; Balík, Karel; Bačáková, Lucie

    2014-01-01

    Roč. 102, č. 11 (2014), s. 3918-3930 ISSN 1549-3296 R&D Projects: GA ČR(CZ) GA106/09/1000; GA ČR(CZ) GAP108/10/1106 Institutional support: RVO:67985823 ; RVO:61389013 ; RVO:67985891 ; RVO:68378271 Keywords : electrospinning * nanofibers * polyactide * hydroxyapatite Subject RIV: EI - Biotechnology ; Bionics; CD - Macromolecular Chemistry (UMCH-V) Impact factor: 3.369, year: 2014

  4. The effect of cerium valence states at cerium oxide nanoparticle surfaces on cell proliferation

    KAUST Repository

    Naganuma, Tamaki

    2014-05-01

    Understanding and controlling cell proliferation on biomaterial surfaces is critical for scaffold/artificial-niche design in tissue engineering. The mechanism by which underlying integrin ligates with functionalized biomaterials to induce cell proliferation is still not completely understood. In this study, poly-l-lactide (PL) scaffold surfaces were functionalized using layers of cerium oxide nanoparticles (CNPs), which have recently attracted attention for use in therapeutic application due to their catalytic ability of Ce4+ and Ce3+ sites. To isolate the influence of Ce valance states of CNPs on cell proliferation, human mesenchymal stem cells (hMSCs) and osteoblast-like cells (MG63) were cultured on the PL/CNP surfaces with dominant Ce4+ and Ce3+ regions. Despite cell type (hMSCs and MG63 cells), different surface features of Ce4+ and Ce3+ regions clearly promoted and inhibited cell spreading, migration and adhesion behavior, resulting in rapid and slow cell proliferation, respectively. Cell proliferation results of various modified CNPs with different surface charge and hydrophobicity/hydrophilicity, indicate that Ce valence states closely correlated with the specific cell morphologies and cell-material interactions that trigger cell proliferation. This finding suggests that the cell-material interactions, which influence cell proliferation, may be controlled by introduction of metal elements with different valence states onto the biomaterial surface. © 2014 Elsevier Ltd.

  5. 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

  6. Effects of Surface Structure and Chemical Composition of Binary Ti Alloys on Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Ok-Sung Han

    2016-07-01

    Full Text Available Binary Ti alloys containing Fe, Mo, V and Zr were micro-arc oxidized and hydrothermally treated to obtain micro- and nano-porous layers. This study aimed to investigate cell differentiation on micro and micro/nanoporous oxide layers of Ti alloys. The properties of the porous layer formed on Ti alloys were characterized by X-ray diffraction pattern, microstructural and elemental analyses and inductively coupled plasma mass spectrometry (ICP-MS method. The MTT assay, total protein production and alkaline phosphatase (ALPase activity were evaluated using human osteoblast-like cells (MG-63. Microporous structures of micro-arc oxidized Ti alloys were changed to micro/nanoporous surfaces after hydrothermal treatment. Micro/nanoporous surfaces consisted of acicular TiO2 nanoparticles and micron-sized hydroxyapatite particles. From ICP and MTT tests, the Mo and V ions released from porous oxide layers were positive for cell viability, while the released Fe ions were negative for cell viability. Although the micro/nanoporous surfaces led to a lower total protein content than the polished and microporous Ti surfaces after cell incubation for 7 days, they caused higher ALPase activities after 7 days and 14 days of incubation except for V-containing microporous surfaces. The micro/nanoporous surfaces of Ti alloys were more efficient in inducing MG-63 cell differentiation.

  7. 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

  8. Effect of polygonimitin C on bone formation and resorption in human ...

    African Journals Online (AJOL)

    Purpose: To investigate the effect of polygonimitin C (PC) on bone formation and resorption in human osteoblast-like MG63 cells. Methods: MG63 cells were treated with PC at doses of 0, 20, 40 or 80 μg/mL for 48 h, with an untreated group as control. The effect of PC on alkaline phosphatase (ALP) activity in MG63 cells ...

  9. Nanocomposite Ti/hydrocarbon plasma polymer films from reactive magnetron sputtering as growth support for osteoblast-like and endothelial cells

    Czech Academy of Sciences Publication Activity Database

    Grinevich, A.; Bačáková, Lucie; Choukourov, A.; Boldyryeva, H.; Pihosh, Y.; Slavinska, D.; Nosková, Lenka; Škuciová, Mária; Lisá, Věra; Biederman, H.

    88A, č. 4 (2009), s. 952-966 ISSN 1549-3296 R&D Projects: GA AV ČR(CZ) KAN101120701 Institutional research plan: CEZ:AV0Z50110509 Keywords : titanium * maturation * plasma Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.816, year: 2009

  10. Adhesion and growth of human osteoblast-like cells on aliphatic polyesters with different chemical composition, surface roughness and modification with hydroxyapatite

    Czech Academy of Sciences Publication Activity Database

    Vagaská, Barbora; Bačáková, Lucie; Pamula, E.; Lisá, Věra; Dobrzynski, P.

    2006-01-01

    Roč. 9, č. 58-60 (2006), s. 4-7 ISSN 1429-7248 R&D Projects: GA ČR(CZ) GA106/06/1576 Institutional research plan: CEZ:AV0Z50110509 Keywords : bone tissue engineering * polymers * ceramics Subject RIV: EI - Biotechnology ; Bionics

  11. Adhesion, growth and differentiation markers in human osteoblast-like cells cultured on surface-modified metallic materials designed for bone implants

    Czech Academy of Sciences Publication Activity Database

    Bačáková, Lucie; Kabátová, J.; Lisá, Věra; Starý, V.; Fencl, J.

    2006-01-01

    Roč. 9, č. 58-60 (2006), s. 1-3 ISSN 1429-7248 R&D Projects: GA ČR(CZ) GA101/06/0226 Institutional research plan: CEZ:AV0Z50110509 Keywords : bone tissue engineering * metals * surface modifications Subject RIV: EI - Biotechnology ; Bionics

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

    NARCIS (Netherlands)

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

    2009-01-01

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

  13. Aging effects of plasma polymerized ethylenediamine (PPEDA) thin films on cell-adhesive implant coatings

    International Nuclear Information System (INIS)

    Testrich, H.; Rebl, H.; Finke, B.; Hempel, F.; Nebe, B.; Meichsner, J.

    2013-01-01

    Thin plasma polymer films from ethylenediamine were deposited on planar substrates placed on the powered electrode of a low pressure capacitively coupled 13.56 MHz discharge. The chemical composition of the plasma polymer films was analyzed by Fourier Transform Infrared Reflection Absorption Spectroscopy (FT-IRRAS) as well as by X-ray photoelectron spectroscopy (XPS) after derivatization of the primary amino groups. The PPEDA films undergo an alteration during the storage in ambient air, particularly, due to reactions with oxygen. The molecular changes in PPEDA films were studied over a long-time period of 360 days. Simultaneously, the adhesion of human osteoblast-like cells MG-63 (ATCC) was investigated on PPEDA coated corundum blasted titanium alloy (Ti-6Al-4V), which is applied as implant material in orthopedic surgery. The cell adhesion was determined by flow cytometry and the cell shape was analyzed by scanning electron microscopy. Compared to uncoated reference samples a significantly enhanced cell adhesion and proliferation were measured for PPEDA coated samples, which have been maintained after long-time storage in ambient air and additional sterilization by γ−irradiation. - Highlights: • Development of cell-adhesive nitrogen-rich coatings for biomedical applications. • Plasma polymer films from low pressure 13.56 MHz discharge in argon-ethylenediamine. • Enhanced osteoblast adhesion/proliferation on coated implant material (Ti-6Al-4V). • Despite film aging over 360 days the enhanced cell adhesion of the coating remains. • No influence of additional y-sterilization on the enhanced cell adhesion

  14. Tuning the Density of Poly(ethylene glycol Chains to Control Mammalian Cell and Bacterial Attachment

    Directory of Open Access Journals (Sweden)

    Ahmed Al-Ani

    2017-08-01

    Full Text Available Surface modification of biomaterials with polymer chains has attracted great attention because of their ability to control biointerfacial interactions such as protein adsorption, cell attachment and bacterial biofilm formation. The aim of this study was to control the immobilisation of biomolecules on silicon wafers using poly(ethylene glycol(PEG chains by a “grafting to” technique. In particular, to control the polymer chain graft density in order to capture proteins and preserve their activity in cell culture as well as find the optimal density that would totally prevent bacterial attachment. The PEG graft density was varied by changing the polymer solubility using an increasing salt concentration. The silicon substrates were initially modified with aminopropyl-triethoxysilane (APTES, where the surface density of amine groups was optimised using different concentrations. The results showed under specific conditions, the PEG density was highest with grafting under “cloud point” conditions. The modified surfaces were characterised with X-ray photoelectron spectroscopy (XPS, ellipsometry, atomic force microscopy (AFM and water contact angle measurements. In addition, all modified surfaces were tested with protein solutions and in cell (mesenchymal stem cells and MG63 osteoblast-like cells and bacterial (Pseudomonas aeruginosa attachment assays. Overall, the lowest protein adsorption was observed on the highest polymer graft density, bacterial adhesion was very low on all modified surfaces, and it can be seen that the attachment of mammalian cells gradually increased as the PEG grafting density decreased, reaching the maximum attachment at medium PEG densities. The results demonstrate that, at certain PEG surface coverages, mammalian cell attachment can be tuned with the potential to optimise their behaviour with controlled serum protein adsorption.

  15. Support for the initial attachment, growth and differentiation of MG-63 cells: a comparison between nano-size hydroxyapatite and micro-size hydroxyapatite in composites

    Czech Academy of Sciences Publication Activity Database

    Filová, Elena; Suchý, Tomáš; Sucharda, Zbyněk; Šupová, Monika; Žaloudková, Margit; Balík, Karel; Lisá, Věra; Šlouf, Miroslav; Bačáková, Lucie

    2014-01-01

    Roč. 9, č. 1 (2014), s. 3687-3706 E-ISSN 1178-2013 R&D Projects: GA MZd(CZ) NT13297; GA ČR(CZ) GA106/09/1000; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 ; RVO:67985891 ; RVO:61389013 Keywords : nanoparticles * hydroxyapatie * osteoblasts * composite materials Subject RIV: EI - Biotechnology ; Bionics Impact factor: 4.383, year: 2014

  16. Electrochemical construction of a bio-inspired micro/nano-textured structure with cell-sized microhole arrays on biomedical titanium to enhance bioactivity

    International Nuclear Information System (INIS)

    Liang, Jianhe; Song, Ran; Huang, Qiaoling; Yang, Yun; Lin, Longxiang; Zhang, Yanmei; Jiang, Pinliang; Duan, Hongping; Dong, Xiang; Lin, Changjian

    2015-01-01

    Highlights: • The bio-inspired structure mimicked mulit-level structures of natural bone. • Ordered cell-sized microhole arrays were employed as microscale structure. • High surface roughness and superhydrophilicity were achieved on the titanium surface. • The bio-inspired titanium surface showed superior ability of biomineralization. • Cell responses were enhanced on the bio-inspired micro/nano-texutred surface. - Abstract: Biomimetic surface design of medical implants is vitally crucial to improve cellular responses and the integration of tissue onto materials. In this study, a novel hierarchical cell-sized microhole array combined with a nano-network structure was fabricated on a medical titanium surface to mimic multi-level bone structure. A three-step procedure was developed as follows: 1) electrochemical self-organization of etching on titanium substrate to create highly ordered cell-sized microhole arrays, 2) suitable dual acid etching to increase the roughness of the microholes, and then 3) electrochemical anodization in a NaOH electrolyte to construct a nano-network porous titania layer on the above micro-roughened surface. The bio-inspired micro/nano-textured structure presented the enhanced wettability and superhydrophilicity. The ability of in vitro biomineralization and corrosion resistance of the bio-inspired micro/nano-textured structure were enhanced after annealing treatment. More importantly, the bio-inspired micro/nano-textured structure on the titanium surface possessed a favourable interfacial environment to enhance attachment and proliferation of human osteoblast-like MG63 cells. All of the results demonstrated that such a bio-inspired surface of micro/nano-textured porous TiO 2 is a most promising candidate for the next generation of titanium implants

  17. Bioactivity and cell proliferation in radiopaque gel-derived CaO-P2O5-SiO2-ZrO2 glass and glass-ceramic powders.

    Science.gov (United States)

    Montazerian, Maziar; Yekta, Bijan Eftekhari; Marghussian, Vahak Kaspari; Bellani, Caroline Faria; Siqueira, Renato Luiz; Zanotto, Edgar Dutra

    2015-10-01

    In this study, 10 mol% ZrO2 was added to a 27CaO-5P2O5-68SiO2 (mol%) base composition synthesized via a simple sol-gel method. This composition is similar to that of a frequently investigated bioactive gel-glass. The effects of ZrO2 on the in vitro bioactivity and MG-63 cell proliferation of the glass and its derivative polycrystalline (glass-ceramic) powder were investigated. The samples were characterized using thermo-gravimetric and differential thermal analysis (TG/DTA), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) coupled to energy dispersive X-ray spectroscopy (EDS). Release of Si, Ca, P and Zr into simulated body fluid (SBF) was determined by inductively coupled plasma (ICP). Upon heat treatment at 1000 °C, the glass powder crystallized into an apatite-wollastonite-zirconia glass-ceramic powder. Hydroxycarbonate apatite (HCA) formation on the surface of the glass and glass-ceramic particles containing ZrO2 was confirmed by FTIR and SEM. Addition of ZrO2 to the base glass composition decreased the rate of HCA formation in vitro from one day to three days, and hence, ZrO2 could be employed to control the rate of apatite formation. However, the rate of HCA formation on the glass-ceramic powder containing ZrO2 crystal was equal to that in the base glassy powder. Tests with a cultured human osteoblast-like MG-63 cells revealed that the glass and glass-ceramic materials stimulated cell proliferation, indicating that they are biocompatible and are not cytotoxic in vitro. Moreover, zirconia clearly increased osteoblast proliferation over that of the Zr-free samples. This increase is likely associated with the lower solubility of these samples and, consequently, a smaller variation in the media pH. Despite the low solubility of these materials, bioactivity was maintained, indicating that these glassy and polycrystalline powders are potential candidates for bone graft substitutes and bone cements with

  18. Study of detonation nanodiamond - plasma polymerized hexamethildisiloxan composites for medical application

    International Nuclear Information System (INIS)

    Pramatarova, L D; Krasteva, N A; Radeva, E I; Pecheva, E V; Dimitrova, R P; Hikov, T A; Mitev, D P; Hristova, K T; Altankov, G

    2010-01-01

    The present study reports on how detonation nanodiamond (DND) - plasma poly(hexamethyldisiloxane) composites (PPHMDS) affect osteoblast cell behavior. It has been established that various modified DND nanoparticles (Ag-DND and Si-DND) can be readily integrated into virtually all polymer matrices. In particular, PPHDMS composites have been developed over the past few years because of the variety of their application as medical devices and implants. By incubation of MG-63 osteoblast-like cells on the surface of DND (Ag-DND and Si-DND) - PPHMDS composite, we tested the hypothesis that DND-based polymer composites can influence the adhesion behavior of MG-63 osteoblast-like cells. Morphological and structural characterization of DND, Ag-DND and Si-DND powders was carried out by XRD, HRTEM and EDS. For the study of the composite layers, deposited on cover glass (CG), FTIR spectroscopy has been performed in order to determine if the DND nanofiller can potentially modify the structural and chemical dynamics of the polymer matrix. The kinetic of static water contact angle of composite surfaces as a function of the as-used nanofiller DND's in polymer matrix was measured The results with MG-63 osteoblast-like cells suggest the potential of using DND-based polymer composites for application in engineering implantable scaffolds and devices.

  19. Study of detonation nanodiamond - plasma polymerized hexamethildisiloxan composites for medical application

    Energy Technology Data Exchange (ETDEWEB)

    Pramatarova, L D; Krasteva, N A; Radeva, E I; Pecheva, E V; Dimitrova, R P; Hikov, T A; Mitev, D P; Hristova, K T; Altankov, G, E-mail: lpramat@issp.bas.b

    2010-11-01

    The present study reports on how detonation nanodiamond (DND) - plasma poly(hexamethyldisiloxane) composites (PPHMDS) affect osteoblast cell behavior. It has been established that various modified DND nanoparticles (Ag-DND and Si-DND) can be readily integrated into virtually all polymer matrices. In particular, PPHDMS composites have been developed over the past few years because of the variety of their application as medical devices and implants. By incubation of MG-63 osteoblast-like cells on the surface of DND (Ag-DND and Si-DND) - PPHMDS composite, we tested the hypothesis that DND-based polymer composites can influence the adhesion behavior of MG-63 osteoblast-like cells. Morphological and structural characterization of DND, Ag-DND and Si-DND powders was carried out by XRD, HRTEM and EDS. For the study of the composite layers, deposited on cover glass (CG), FTIR spectroscopy has been performed in order to determine if the DND nanofiller can potentially modify the structural and chemical dynamics of the polymer matrix. The kinetic of static water contact angle of composite surfaces as a function of the as-used nanofiller DND's in polymer matrix was measured The results with MG-63 osteoblast-like cells suggest the potential of using DND-based polymer composites for application in engineering implantable scaffolds and devices.

  20. 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

  1. 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.

  2. Sanguinarine induces apoptosis of human osteosarcoma cells through the extrinsic and intrinsic pathways

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hyunjin; Bergeron, Eric; Senta, Helena; Guillemette, Kim [Cell-Biomaterial Biohybrid Systems, Universite de Sherbrooke, Department of Chemical Engineering and Biotechnological Engineering, Canada J1K 2R1 (Canada); Beauvais, Sabrina [Cell-Biomaterial Biohybrid Systems, Universite de Sherbrooke, Department of Chemical Engineering and Biotechnological Engineering, Canada J1K 2R1 (Canada); Development of Bioprocess, Universite de Sherbrooke, Department of Chemical Engineering and Biotechnological Engineering, Canada J1K 2R1 (Canada); Blouin, Richard [Universite de Sherbrooke, Department of Biology, Canada J1K 2R1 (Canada); Sirois, Joel [Development of Bioprocess, Universite de Sherbrooke, Department of Chemical Engineering and Biotechnological Engineering, Canada J1K 2R1 (Canada); Faucheux, Nathalie, E-mail: Nathalie.Faucheux@Usherbrooke.ca [Cell-Biomaterial Biohybrid Systems, Universite de Sherbrooke, Department of Chemical Engineering and Biotechnological Engineering, Canada J1K 2R1 (Canada)

    2010-08-27

    Research highlights: {yields} We show for the first time the effect of sanguinarine (SA) on MG63 and SaOS-2 cells. {yields} SA altered osteosarcoma cell viability in a concentration and time dependent manner. {yields} SA induced osteosarcoma cell apoptosis and increased caspase-8 and -9 activities. {yields} SA decreased dose dependently the Bcl-2 protein level only in MG63 cells. {yields} SaOS-2 which are osteoblast-derived, seemed more resistant to SA than MG63. -- Abstract: The quaternary benzo[c]phenanthridine alkaloid sanguinarine inhibits the proliferation of cancerous cells from different origins, including lung, breast, pancreatic and colon, but nothing is known of its effects on osteosarcoma, a primary malignant bone tumour. We have found that sanguinarine alters the morphology and reduces the viability of MG-63 and SaOS-2 human osteosarcoma cell lines in concentration- and time-dependent manner. Incubation with 1 {mu}mol/L sanguinarine for 4 and 24 h killed more efficiently MG-63 cells than SaOS-2 cells, while incubation with 5 {mu}mol/L sanguinarine killed almost 100% of both cell populations within 24 h. This treatment also changed the mitochondrial membrane potential in both MG-63 and SaOS-2 cells within 1 h, caused chromatin condensation and the formation of apoptotic bodies. It activated multicaspases, and increased the activities of caspase-8 and caspase-9 in both MG-63 and SaOS-2 cells. These data highlight sanguinarine as a novel potential agent for bone cancer therapy.

  3. The overexpression of MRP4 is related to multidrug resistance in osteosarcoma cells

    Directory of Open Access Journals (Sweden)

    Zhonghui He

    2015-01-01

    Full Text Available Doxorubicin (Adriamycin, ADM is an antimitotic drug used in the treatment of a wide range of malignant tumors, including acute leukemia, lymphoma, osteosarcoma, breast cancer, and lung cancer. Multidrug resistance-associated proteins (MRPs are members of a superfamily of ATP-binding cassette (ABC transporters, which can transport various molecules across extra- and intra-cellular membranes. The aim of this study was to investigate whether there was a correlation between MRP4 and primary ADM resistance in osteosarcoma cells. In this paper, we chose the human osteosarcoma cell line MG63, ADM resistant cell line MG63/DOX, and the patient′s primary cell GSF-0686. We checked the ADM sensitivity and cytotoxicity of all the three cells by cell proliferation assay. The intracellular drug concentrations were measured by using LC-MS/MS. We also examined MRP4 gene expression by RT-PCR and Western Blot. We found that the intracellular ADM concentration of the parent osteosarcoma cell line MG63 was higher than the ADM resistant osteosarcoma MG63/DOX cell line or the GSF-0686 cell after ADM treatment (P < 0.05. In addition, MRP4 mRNA and protein levels in ADM resistant osteosarcoma cells were higher than in MG63 cell (P < 0.05. Taking together, this work suggests that overexpression of MRP4 may confer ADM resistance in osteosarcoma cells.

  4. Phytase-mediated enzymatic mineralization of chitosan-enriched hydrogels

    DEFF Research Database (Denmark)

    Lišková, Jana; Douglas, Timothy E.L.; Wijnants, Robbe

    2018-01-01

    Hydrogels mineralized with calcium phosphate (CaP) are increasingly popular bone regeneration biomaterials. Mineralization can be achieved by phosphatase enzyme incorporation and incubation in calcium glycerophosphate (CaGP). Gellan gum (GG) hydrogels containing the enzyme phytase and chitosan...... oligomer were mineralized in CaGP solution and characterized with human osteoblast-like MG63 cells and adipose tissue-derived stem cells (ADSC). Phytase induced CaP formation. Chitosan concentration determined mineralization extent and hydrogel mechanical reinforcement. Phytase-induced mineralization...... promoted MG63 adhesion and proliferation, especially in the presence of chitosan, and was non-toxic to MG63 cells (with and without chitosan). ADSC adhesion and proliferation were poor without mineralization. Chitosan did not affect ADSC osteogenic differentiation....

  5. Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

    International Nuclear Information System (INIS)

    Nishida, Yoshihiro; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki

    2005-01-01

    Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion

  6. An experimental study on the low-dose radiosensitivity of tumor cell lines

    International Nuclear Information System (INIS)

    Kim, Min Sook; Koh, Kwang Joon

    1994-01-01

    The purpose of this study was to aid in the radiation therapy of head and neck cancer patients. For this study, radiation survival curves were generated for B16, MG-63 and YAC-1 cell lines using semiautomated MTT assay and Dye Exclusion Assay. Irradiation of 2, 4, 6, 8, 10 Gy were delivered at room temperature at a dose rate of 210.2 cGy/min using 60 COγ-ray irradiator ALDORADO 8. The viable cells were determined for each radiation dose and compared to control values. The obtained results were as follows: 1. The was significantly different absorbance at 10 Gy on B16 cell line in MTT assay (P<0.05). 2. There was significantly different absorbance at 4, 6, 8, 10 Gy on MG-63 cell line in MTT assay (P<0.05). 3. YAC-1 cell line was more sensitive than B16 or MG-63 cell line to all doses of radiation (P<0.05). 4. There was significantly different absorbance among all tumor cell lines except between B16 and MG-63 cell line at 2 Gy in MTT assay (P<0.05). 5. Good correlation was obtained between MTT assay and DEA (P<0.05). The efficient of correlation of B16, MG-63 and YAC-1 cell line was 0.845-0.824 and 0.906, respectively.

  7. Influence of surfaces modified with biomimetic extracellular matrices on adhesion and proliferation of mesenchymal stem cells and osteosarcoma cells.

    Science.gov (United States)

    Cai, Rong; Kawazoe, Naoki; Chen, Guoping

    2015-02-01

    Preparation of surfaces modified with biomimetic extracellular matrices (ECMs) is important for investigation of the interaction between ECMs and cells. In the present study, surfaces modified with ECMs from normal somatic cells, stem cells and tumor cells were prepared by cell culture method. The ECMs derived from bone marrow-derived mesenchymal stem cells (MSCs), dermal fibroblasts (FBs), osteoblasts (OBs) and MG63 osteosarcoma cells were deposited on the surfaces of cell-culture polystyrene plates (TCPS). The ECMs from different cell types had different compositions. The effects of the ECM-deposited surfaces on the adhesion, spreading and proliferation of MSCs and MG63 human osteosarcoma cells were dependent on the type of both ECMs and cells. The surfaces deposited with ECMs from MSCs, FBs and OBs promoted cell adhesion more strongly than surfaces deposited with ECMs from MG63 cells and TCPS. Compared to TCPS, the ECM-deposited surfaces promoted proliferation of MSCs while they inhibited the proliferation of MG63 cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Polycrystalline Silicon: a Biocompatibility Assay

    International Nuclear Information System (INIS)

    Pecheva, E.; Fingarova, D.; Pramatarova, L.; Hikov, T.; Laquerriere, P.; Bouthors, Sylvie; Dimova-Malinovska, D.; Montgomery, P.

    2010-01-01

    Polycrystalline silicon (poly-Si) layers were functionalized through the growth of biomimetic hydroxyapatite (HA) on their surface. HA is the mineral component of bones and teeth and thus possesses excellent bioactivity and biocompatibility. MG-63 osteoblast-like cells were cultured on both HA-coated and un-coated poly-Si surfaces for 1, 3, 5 and 7 days and toxicity, proliferation and cell morphology were investigated. The results revealed that the poly-Si layers were bioactive and compatible with the osteoblast-like cells. Nevertheless, the HA coating improved the cell interactions with the poly-Si surfaces based on the cell affinity to the specific chemical composition of the bone-like HA and/or to the higher HA roughness.

  9. Cell uptake survey of pegylated nanographene oxide.

    Science.gov (United States)

    Vila, M; Portolés, M T; Marques, P A A P; Feito, M J; Matesanz, M C; Ramírez-Santillán, C; Gonçalves, G; Cruz, S M A; Nieto, A; Vallet-Regi, M

    2012-11-23

    Graphene and more specifically, nanographene oxide (GO) has been proposed as a highly efficient antitumoral therapy agent. Nevertheless, its cell uptake kinetics, its influence in different types of cells and the possibility of controlling cellular internalization timing, is still a field that remains unexplored. Herein, different cell types have been cultured in vitro for several incubation periods in the presence of 0.075 mg ml(-1) pegylated GO solutions. GO uptake kinetics revealed differences in the agent's uptake amount and speed as a function of the type of cell involved. Osteoblast-like cells GO uptake is higher and faster without resulting in greater cell membrane damage. Moreover, the dependence on the commonly used PEG nature (number of branches) also influences the viability and cell uptake speed. These facts play an important role in the future definition of timing parameters and selective cell uptake control in order to achieve an effective therapy.

  10. Cell uptake survey of pegylated nanographene oxide

    International Nuclear Information System (INIS)

    Vila, M; Nieto, A; Vallet-Regi, M; Portolés, M T; Feito, M J; Matesanz, M C; Ramírez-Santillán, C; Marques, P A A P; Gonçalves, G; Cruz, S M A

    2012-01-01

    Graphene and more specifically, nanographene oxide (GO) has been proposed as a highly efficient antitumoral therapy agent. Nevertheless, its cell uptake kinetics, its influence in different types of cells and the possibility of controlling cellular internalization timing, is still a field that remains unexplored. Herein, different cell types have been cultured in vitro for several incubation periods in the presence of 0.075 mg ml −1 pegylated GO solutions. GO uptake kinetics revealed differences in the agent’s uptake amount and speed as a function of the type of cell involved. Osteoblast-like cells GO uptake is higher and faster without resulting in greater cell membrane damage. Moreover, the dependence on the commonly used PEG nature (number of branches) also influences the viability and cell uptake speed. These facts play an important role in the future definition of timing parameters and selective cell uptake control in order to achieve an effective therapy. (paper)

  11. 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.

  12. Biphasic calcium sulfate dihydrate/iron-modified alpha-tricalcium phosphate bone cement for spinal applications: in vitro study

    International Nuclear Information System (INIS)

    Vlad, M D; Lopez, J; Torres, R; Barraco, M; Fernandez, E; Valle, L J; Poeata, I

    2010-01-01

    In this study, the cytocompatibility of new 'iron-modified/alpha-tricalcium phosphate (IM/α-TCP) and calcium sulfate dihydrate (CSD)' bone cement (IM/α-TCP/CSD-BC) intended for spinal applications has been approached. The objective was to investigate by direct-contact osteoblast-like cell cultures (from 1 to 14 days) the in vitro cell adhesion, proliferation, morphology and cytoskeleton organization of MG-63 cells seeded onto the new cements. The results were as follows: (a) quantitative MTT-assay and scanning electron microscopy (SEM) showed that cell adhesion, proliferation and viability were not affected with time by the presence of iron in the cements; (b) double immunofluorescent labeling of F-actin and α-tubulin showed a dynamic interaction between the cell and its porous substrates sustaining the locomotion phenomenon on the cements' surface, which favored the colonization, and confirming the biocompatibility of the experimental cements; (c) SEM-cell morphology and cytoskeleton observations also evidenced that MG-63 cells were able to adhere, to spread and to attain normal morphology on the new IM/α-TCP/CSD-BC which offered favorable substratum properties for osteoblast-like cells proliferation and differentiation in vitro. The results showed that these new iron-modified cement-like biomaterials have cytocompatible features of interest not only as possible spinal cancellous bone replacement biomaterial but also as bone tissue engineering scaffolds.

  13. Laser Phototherapy Enhances Mesenchymal Stem Cells Survival in Response to the Dental Adhesives

    Directory of Open Access Journals (Sweden)

    Ivana Márcia Alves Diniz

    2015-01-01

    Full Text Available Background. We investigated the influence of laser phototherapy (LPT on the survival of human mesenchymal stem cells (MSCs submitted to substances leached from dental adhesives. Method. MSCs were isolated and characterized. Oral mucosa fibroblasts and osteoblast-like cells were used as comparative controls. Cultured medium conditioned with two adhesive systems was applied to the cultures. Cell monolayers were exposed or not to LPT. Laser irradiations were performed using a red laser (GaAlAs, 780 nm, 0.04 cm2, 40 mW, 1 W/cm2, 0.4 J, 10 seconds, 1 point, 10 J/cm2. After 24 h, cell viability was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide reduction assay. Data were statistically compared by ANOVA followed by Tukey’s test (P<0.05. Results. Different cell types showed different viabilities in response to the same materials. Substances leached from adhesives were less cytotoxic to MSCs than to other cell types. Substances leached from Clearfil SE Bond were highly cytotoxic to all cell types tested, except to the MSCs when applied polymerized and in association with LPT. LPT was unable to significantly increase the cell viability of fibroblasts and osteoblast-like cells submitted to the dental adhesives. Conclusion. LPT enhances mesenchymal stem cells survival in response to substances leached from dental adhesives.

  14. Che-1 gene silencing induces osteosarcoma cell apoptosis by inhibiting mutant p53 expression

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ming; Wang, Dan, E-mail: danwangwdd@163.com; Li, Ning

    2016-04-22

    The transcriptional cofactor Che-1 is an RNA polymerase II (Pol II) which is involved in tumorigenesis, such as breast cancer and multiple myeloma. Che-1 can also regulate mutant p53 expression, which plays roles in many types of cancer. In this study, we aimed to investigate the effects and specific mechanism of Che-1 in the regulation of osteosarcoma (OS) cell growth. We found that Che-1 is highly expressed in several kinds of OS cells compared with osteoblast hFOB1.19 cells. MTT and flow cytometry assays showed that Che-1 depletion by siRNA markedly suppressed MG-63 and U2OS cell proliferation and promoted apoptosis. The chromatin immunoprecipitation (ChIP) assay verified the presence of Che-1 on the p53 promoter in MG-63 and U2OS cells carrying mutant p53. Further studies showed that Che-1 depletion inhibited mutant p53 expression. Notably, our study showed that the loss of Che-1 inhibits proliferation and promotes apoptosis in MG-63 cells by decreasing the level of mutant p53. Therefore, these findings open the possibility that silencing of Che-1 will have therapeutic benefit in OS. - Highlights: • Che-1 is highly expressed in several kinds of OS cells. • Che-1 depletion suppressed MG-63 and U2OS cell growth. • Che-1 is existed in the p53 promoter in MG-63 and U2OS cells. • Che-1 depletion inhibited mutant p53 expression. • Che-1 depletion inhibits cell growth by decreasing the level of mutant p53.

  15. Titanium phosphate glass microcarriers induce enhanced osteogenic cell proliferation and human mesenchymal stem cell protein expression

    Directory of Open Access Journals (Sweden)

    Nilay J Lakhkar

    2015-11-01

    Full Text Available In this study, we have developed 50- to 100-µm-sized titanium phosphate glass microcarriers (denoted as Ti5 that show enhanced proliferation of human mesenchymal stem cells and MG63 osteosarcoma cells, as well as enhanced human mesenchymal stem cell expression of bone differentiation markers, in comparison with commercially available glass microspheres at all time points. We also demonstrate that these microcarriers provide superior human mesenchymal stem cell proliferation with conventional Dulbecco’s Modified Eagle medium than with a specially developed commercial stem cell medium. The microcarrier proliferative capacity is revealed by a 24-fold increase in MG63 cell numbers in spinner flask bioreactor studies performed over a 7-day period, versus only a 6-fold increase in control microspheres under the same conditions; the corresponding values of Ti5 and control microspheres under static culture are 8-fold and 7-fold, respectively. The capability of guided osteogenic differentiation is confirmed by ELISAs for bone morphogenetic protein-2 and osteopontin, which reveal significantly greater expression of these markers, especially osteopontin, by human mesenchymal stem cells on the Ti5 microspheres than on the control. Scanning electron microscopy and confocal laser scanning microscopy images reveal favorable MG63 and human mesenchymal stem cell adhesion on the Ti5 microsphere surfaces. Thus, the results demonstrate the suitability of the developed microspheres for use as microcarriers in bone tissue engineering applications.

  16. MicroRNA-22 inhibits the proliferation and migration, and increases the cisplatin sensitivity, of osteosarcoma cells

    Science.gov (United States)

    Zhou, Xiang; Natino, Dimple; Zhai, Xu; Gao, Zhongyang; He, Xijing

    2018-01-01

    Osteosarcoma (OS) is the major type of primary bone tumor and is associated with a poor prognosis due to chemotherapy resistance. Accumulating evidence indicates that microRNAs (miRNAs/miRs) may influence the tumor progression of OS and cell sensitivity to chemotherapy. In the present study, a total of 7 patients with OS and 7 healthy volunteers were recruited. Reverse transcription-quantitative polymerase chain reaction and ELISA were performed to determine the expression of miRNAs and mRNAs in the serum of participants. Furthermore, the biological function of miR-22 and S100A11 was examined in MG-63 cells using Cell Counting Kit-8 assays, Transwell migration assays and western blot analysis to determine the effects on cell proliferation, migration and protein expression, respectively, while MG-63 cell sensitivity to cisplatin was assessed by measuring cell viability following cisplatin treatment and calculating the half maximal inhibitory concentration (IC50). Additionally, the association between miR-22 and S100 calcium-binding protein A11 (S100A11) was validated using a luciferase reporter assay. The results demonstrated that miR-22 expression was significantly reduced in patients with OS and the MG-63 OS cell line, compared with healthy volunteers and the normal osteoblast hFOB 1.19 cell line, respectively, while the expression of S100A11 was negatively associated with miR-22 levels in the MG-63 cell line. Furthermore, overexpression of miR-22 inhibited the proliferation and migratory ability of MG-63 cells, and increased the sensitivity of MG-63 cells to cisplatin treatment; however, overexpression of S100A11 partially attenuated the alterations in proliferation, migratory ability and chemosensitivity that were induced by miR-22 overexpression. In addition, it was confirmed that S100A11 is a direct target gene of miR-22 in MG-63 cells. In conclusion, to the best of our knowledge, the present study is the first to demonstrate that miR-22 may be a promising

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

    Directory of Open Access Journals (Sweden)

    Huiyun Xu

    2012-01-01

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

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

    Science.gov (United States)

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

    2012-07-01

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

  19. Research on DNA methylation of human osteosarcoma cell MGMT and its relationship with cell resistance to alkylating agents.

    Science.gov (United States)

    Guo, Jun; Cui, Qiu; Jiang, WeiHao; Liu, Cheng; Li, DingFeng; Zeng, Yanjun

    2013-08-01

    The objective of this study was to explore the O(6)-methylguanine-DNA methyltransferase (MGMT) gene methylation status and its protein expression, as well as the effects of demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) on MGMT gene expression and its resistance to alkylating agents, and to elucidate MGMT expression mechanism and significance in osteosarcoma. The human osteosarcoma cell lines Saos-2 and MG-63 were collected and treated with 5-Aza-CdR for 6 days. The cells not treated with 5-Aza-CdR were set as a negative control. The genomic DNA was extracted from the Saos-2 and MG-63 cells using methylation-specific PCR to detect the promoter CpG island methylation status of the MGMT gene. Cell sensitivity to alkylating agents before and after drug administration was detected by the MTT method. The variation in MGMT gene mRNA and protein was detected by reverse transcription PCR (RT-PCR) and Western blotting. The MGMT promoter gene of normal Saos-2 cells was methylated, with reduced MGMT mRNA and protein expression; the MGMT mRNA and protein expression of Saos-2 cells treated with 5-Aza-CdR was obviously enhanced, and its sensitivity to alkylating agents was reversed. Meanwhile, with promoter CpG island unmethylation of the MGMT gene, MGMT protein was expressed in the normal MG-63 cells and the MG-63 cells treated with 5-Aza-CdR, and both showed resistance to alkylating agents. The methylation status of the MGMT gene promoter in human osteosarcoma cells reflected the cells' ability to induce MGMT protein expression and can be used as a molecular marker to project the sensitivity of cancer tissues to alkylating agent drugs.

  20. A Chinese Herbal Decoction, Danggui Buxue Tang, Stimulates Proliferation, Differentiation and Gene Expression of Cultured Osteosarcoma Cells: Genomic Approach to Reveal Specific Gene Activation

    Directory of Open Access Journals (Sweden)

    Roy C. Y. Choi

    2011-01-01

    Full Text Available Danggui Buxue Tang (DBT, a Chinese herbal decoction used to treat ailments in women, contains Radix Astragali (Huangqi; RA and Radix Angelicae Sinensis (Danggui; RAS. When DBT was applied onto cultured MG-63 cells, an increase of cell proliferation and differentiation of MG-63 cell were revealed: both of these effects were significantly higher in DBT than RA or RAS extract. To search for the biological markers that are specifically regulated by DBT, DNA microarray was used to reveal the gene expression profiling of DBT in MG-63 cells as compared to that of RA- or RAS-treated cells. Amongst 883 DBT-regulated genes, 403 of them are specifically regulated by DBT treatment, including CCL-2, CCL-7, CCL-8, and galectin-9. The signaling cascade of this DBT-regulated gene expression was also elucidated in cultured MG-63 cells. The current results reveal the potential usage of this herbal decoction in treating osteoporosis and suggest the uniqueness of Chinese herbal decoction that requires a well-defined formulation. The DBT-regulated genes in the culture could serve as biological responsive markers for quality assurance of the herbal preparation.

  1. Aluminum Templates of Different Sizes with Micro-, Nano- and Micro/Nano-Structures for Cell Culture

    Directory of Open Access Journals (Sweden)

    Ming-Liang Yen

    2017-10-01

    Full Text Available This study investigates the results of cell cultures on aluminum (Al templates with flat-structures, micro-structures, nano-structures and micro/nano-structures. An Al template with flat-structure was obtained by electrolytic polishing; an Al template with micro-structure was obtained by micro-powder blasting; an Al template with nano-structure was obtained by aluminum anodization; and an Al template with micro/nano-structure was obtained by micro-powder blasting and then anodization. Osteoblast-like cells were cultured on aluminum templates with various structures. The microculture tetrazolium test assay was utilized to assess the adhesion, elongation, and proliferation behaviors of cultured osteoblast-like cells on aluminum templates with flat-structures, micro-structures, nano-structures, and micro/nano-structures. The results showed that the surface characterization of micro/nano-structure of aluminum templates had superhydrophilic property, and these also revealed that an aluminum template with micro/nano-structure could provide the most suitable growth situation for cell culture.

  2. Strong adhesion of Saos-2 cells to multi-walled carbon nanotubes

    International Nuclear Information System (INIS)

    Matsuoka, Makoto; Akasaka, Tsukasa; Totsuka, Yasunori; Watari, Fumio

    2010-01-01

    In recent years, carbon nanotubes (CNTs) have been considered potential biomedical materials because of their unique character. The aim of this study was to investigate the response of a human osteoblast-like cell line - Saos-2 - on single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs). The surface of a culture dish was coated with CNTs, and Saos-2 cells were cultured for three days. Cell morphology, viability, alkaline phosphatase (ALP) activity, adhesion, and vinculin expression were evaluated. The result showed high cell viability and strong adhesion to MWCNTs. Saos-2 cultured on MWCNTs exhibited vinculin expression throughout the cell body, while the cells attached to SWCNTs and glass were mostly limited to their periphery. Our results suggest that CNT coatings promote cell activity and adhesiveness. These findings indicate that MWCNTs could be used as surface coating materials to promote cell adhesion.

  3. Ginsenoside Rg3 induces DNA damage in human osteosarcoma cells and reduces MNNG-induced DNA damage and apoptosis in normal human cells.

    Science.gov (United States)

    Zhang, Yue-Hui; Li, Hai-Dong; Li, Bo; Jiang, Sheng-Dan; Jiang, Lei-Sheng

    2014-02-01

    Panax ginseng is a Chinese medicinal herb. Ginsenosides are the main bioactive components of P. ginseng, and ginsenoside Rg3 is the primary ginsenoside. Ginsenosides can potently kill various types of cancer cells. The present study was designed to evaluate the potential genotoxicity of ginsenoside Rg3 in human osteosarcoma cells and the protective effect of ginsenoside Rg3 with respect to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage and apoptosis in a normal human cell line (human fibroblasts). Four human osteosarcoma cell lines (MG-63, OS732, U-2OS and HOS cells) and a normal human cell line (human fibroblasts) were employed to investigate the cytotoxicity of ginsenosides Rg3 by MTT assay. Alkaline comet assay and γH2AX focus staining were used to detect the DNA damage in MG-63 and U-2OS cells. The extent of cell apoptosis was determined by flow cytometry and a DNA ladder assay. Our results demonstrated that the cytotoxicity of ginsenoside Rg3 was dose-dependent in the human osteosarcoma cell lines, and MG-63 and U-2OS cells were the most sensitive to ginsenoside Rg3. As expected, compared to the negative control, ginsenoside Rg3 significantly increased DNA damage in a concentration-dependent manner. In agreement with the comet assay data, the percentage of γH2AX-positive MG-63 and U-2OS cells indicated that ginsenoside Rg3 induced DNA double-strand breaks in a concentration-dependent manner. The results also suggest that ginsenoside Rg3 reduces the extent of MNNG-induced DNA damage and apoptosis in human fibroblasts.

  4. Breast cancer cells obtain an osteomimetic feature via epithelial-mesenchymal transition that have undergone BMP2/RUNX2 signaling pathway induction.

    Science.gov (United States)

    Tan, Cong-Cong; Li, Gui-Xi; Tan, Li-Duan; Du, Xin; Li, Xiao-Qing; He, Rui; Wang, Qing-Shan; Feng, Yu-Mei

    2016-11-29

    Bone is one of the most common organs of breast cancer metastasis. Cancer cells that mimic osteoblasts by expressing bone matrix proteins and factors have a higher likelihood of metastasizing to bone. However, the molecular mechanisms of osteomimicry formation of cancer cells remain undefined. Herein, we identified a set of bone-related genes (BRGs) that are ectopically co-expressed in primary breast cancer tissues and determined that osteomimetic feature is obtained due to the osteoblast-like transformation of epithelial breast cancer cells that have undergone epithelial-mesenchymal transition (EMT) followed by bone morphogenetic protein-2 (BMP2) stimulation. Furthermore, we demonstrated that breast cancer cells that transformed into osteoblast-like cells with high expression of BRGs showed enhanced chemotaxis, adhesion, proliferation and multidrug resistance in an osteoblast-mimic bone microenvironment in vitro. During these processes, runt-related transcription factor 2 (RUNX2) functioned as a master mediator by suppressing or activating the transcription of BRGs that underlie the dynamic antagonism between the TGF-β/SMAD and BMP/SMAD signaling pathways in breast cancer cells. Our findings suggest a novel mechanism of osteomimicry formation that arises in primary breast tumors, which may explain the propensity of breast cancer to metastasize to the skeleton and contribute to potential strategies for predicting and targeting breast cancer bone metastasis and multidrug resistance.

  5. Microfibrous {beta}-TCP/collagen scaffolds mimic woven bone in structure and composition

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Shen; Zhang Xin; Cai Qing; Yang Xiaoping [Key Laboratory of Beijing City on Preparation and Processing of Novel Polymer Materials, College of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing 100029 (China); Wang Bo; Deng Xuliang, E-mail: yangxp@mail.buct.edu.c [Department of VIP Dental Service, School and Hospital of Stomatology, Peking University, Beijing 100081 (China)

    2010-12-15

    Woven bone, as the initial form of bone tissue, is always found in developing and repairing bone. It is thought of as a temporary scaffold for the deposition of osteogenic cells and the laying down of lamellar bone. Thus, we hypothesize that a matrix which resembles the architecture and components of woven bone can provide an osteoblastic microenvironment for bone cell growth and new bone formation. In this study, woven-bone-like beta-tricalcium phosphate ({beta}-TCP)/collagen scaffolds were fabricated by sol-gel electrospinning and impregnating methods. Optimization studies on sol-gel synthesis and electrospinning process were conducted respectively to prepare pure {beta}-TCP fibers with dimensions close to mineralized collagen fibrils in woven bone. The collagen-coating layer prepared by impregnation had an adhesive role that held the {beta}-TCP fibers together, and resulted in rapid degradation and matrix mineralization in in vitro tests. MG63 osteoblast-like cells seeded on the resultant scaffolds showed three-dimensional (3D) morphologies, and merged into multicellular layers after 7 days culture. Cytotoxicity test further revealed that extracts from the resultant scaffolds could promote the proliferation of MG63 cells. Therefore, the woven-bone-like matrix that we constructed favored the attachment and proliferation of MG63 cells in three dimensions. It has great potential ability to shorten the time of formation of new bone.

  6. Microfibrous β-TCP/collagen scaffolds mimic woven bone in structure and composition

    International Nuclear Information System (INIS)

    Zhang Shen; Zhang Xin; Cai Qing; Yang Xiaoping; Wang Bo; Deng Xuliang

    2010-01-01

    Woven bone, as the initial form of bone tissue, is always found in developing and repairing bone. It is thought of as a temporary scaffold for the deposition of osteogenic cells and the laying down of lamellar bone. Thus, we hypothesize that a matrix which resembles the architecture and components of woven bone can provide an osteoblastic microenvironment for bone cell growth and new bone formation. In this study, woven-bone-like beta-tricalcium phosphate (β-TCP)/collagen scaffolds were fabricated by sol-gel electrospinning and impregnating methods. Optimization studies on sol-gel synthesis and electrospinning process were conducted respectively to prepare pure β-TCP fibers with dimensions close to mineralized collagen fibrils in woven bone. The collagen-coating layer prepared by impregnation had an adhesive role that held the β-TCP fibers together, and resulted in rapid degradation and matrix mineralization in in vitro tests. MG63 osteoblast-like cells seeded on the resultant scaffolds showed three-dimensional (3D) morphologies, and merged into multicellular layers after 7 days culture. Cytotoxicity test further revealed that extracts from the resultant scaffolds could promote the proliferation of MG63 cells. Therefore, the woven-bone-like matrix that we constructed favored the attachment and proliferation of MG63 cells in three dimensions. It has great potential ability to shorten the time of formation of new bone.

  7. Positively charged polymers modulate the fate of human mesenchymal stromal cells via ephrinB2/EphB4 signaling

    Directory of Open Access Journals (Sweden)

    Ilenia De Luca

    2016-09-01

    Full Text Available Understanding the mechanisms by which mesenchymal stromal cells (MSCs interact with the physical properties (e.g. topography, charge, ζ-potential, and contact angle of polymeric surfaces is essential to design new biomaterials capable of regulating stem cell behavior. The present study investigated the ability of two polymers (pHM1 and pHM3 with different positive surface charge densities to modulate the differentiation of MSCs into osteoblast-like phenotype via cell-cell ephrinB2/EphB4 signaling. Although pHM1 promoted the phosphorylation of EphB4, leading to cell differentiation, pHM3, characterized by a high positive surface charge density, had no significant effect on EphB4 activation or MSCs differentiation. When the MSCs were cultured on pHM1 in the presence of a forward signaling blocking peptide, the osteoblast differentiation was compromised. Our results demonstrated that the ephrinB2/EphB4 interaction was required for MSCs differentiation into an osteoblast-like phenotype and that the presence of a high positive surface charge density altered this interaction.

  8. Farnesyl diphosphate synthase is involved in the resistance to zoledronic acid of osteosarcoma cells. : resistance of osteosarcoma to nitrogen bisphosphonates

    OpenAIRE

    Ory , Benjamin; Moriceau , Gatien; Trichet , Valérie; Blanchard , Frédéric; Berreur , Martine; Rédini , Françoise; Rogers , Michael; Heymann , Dominique

    2008-01-01

    International audience; We recently demonstrated original anti-tumor effects of zoledronic acid (Zol) on osteosarcoma cell lines independently of their p53 and Rb status. The present study investigated the potential Zol-resistance acquired by osteosarcoma cells after prolonged treatment. After 12 weeks of culture in the presence of 1 microm Zol, the effects of high doses of Zol (10-100 microm) were compared between the untreated rat (OSRGA, ROS) and human (MG63, SAOS2) osteosarcoma cells and ...

  9. Effects of the micro-nano surface topography of titanium alloy on the biological responses of osteoblast.

    Science.gov (United States)

    Yin, Chengcheng; Zhang, Yanjing; Cai, Qing; Li, Baosheng; Yang, Hua; Wang, Heling; Qi, Hua; Zhou, Yanmin; Meng, Weiyan

    2017-03-01

    In clinical applications, osseointegration is essential for the long-term stability of dental implants. Inspired by the hierarchical structure of natural bone, we applied the electrochemical etching (EC) technique to form a micro-nano structure on a titanium alloy (Ti6Al4V) substrate, called EC surface. Sand blasting and acid etching (SLA) and machined (M) methods were employed to generate micro and smooth textures, respectively, as the control groups. The surface topographies of the three substrates were characterized using scanning electron microscopy (SEM). Then, human osteoblast-like cells (MG63) were cultured on substrates, and adhesion, proliferation, morphology, alkaline phosphatase activity (ALP), and gene expression levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN), and type I collagen (COLIA 1) were analyzed. MG63 cells cultured on the EC Ti alloy substrates displayed better cell adhesion, significant proliferation, and a higher production level of ALP, gene expressions of RUNX2, OCN, OPN and COLIA 1 (p < 0.01 or p < 0.05) compared with those of SLA and M substrates. These results indicate that the micro-nano structure fabricated by electrochemical etching method is beneficial for the biological functions of MG63 cells and may be a promising candidate in dental implants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 757-769, 2017. © 2016 Wiley Periodicals, Inc.

  10. Different effects of a novel CaO-MgO-SiO₂-based multiphase glass-ceramic on cell behaviors of normal and cancer cells in vitro.

    Science.gov (United States)

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

    2014-04-01

    The effects in vitro of a novel multiphase glass-ceramic (with nominal composition of 43.19% CaO, 7.68% MgO, and 49.13% SiO2 in weight percent) on cell adhesion, proliferation, differentiation and ultrastructure of human osteosarcoma cell line MG63, mouse fibroblasts L929, and human lung adenocarcinoma epithelial cell line A549 were investigated in this research. Scanning electron microscopy (SEM) micrographs revealed that the surface morphology of this glass-ceramic was beneficial to cell adhesion. The glass-ceramic extracts at certain concentrations could stimulate the proliferation and differentiation of MG63 and L929 cells, whereas inhibit A549 proliferation, which might be resulted from the released Si ions. In addition, when cultured with 0.1mg/mL glass-ceramic powder suspension, the cell ultrastructure of MG63 showed abundant organelles and L929 displayed the phenomena of cellular stress response. While more interestingly, A549 exhibited chromatin condensation, mitochondria swell and RER expansion, which was presumed to be early signs of apoptosis. These results suggest that this novel CaO-MgO-SiO2-based multiphase glass-ceramic has potential for bone regeneration and tissue engineering applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Direct determination of phosphatase activity from physiological substrates in cells.

    Directory of Open Access Journals (Sweden)

    Zhongyuan Ren

    Full Text Available A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1 mg(-1 for PPi, to 56 ± 11 nmol min(-1 mg(-1 for AMP, to 79 ± 23 nmol min(-1 mg(-1 for beta-glycerophosphate and to 73 ± 15 nmol min(-1 mg(-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

  12. 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...

  13. Cytotoxicity and Bioactivity of Calcium Silicate Cements Combined with Niobium Oxide in Different Cell Lines.

    Science.gov (United States)

    Mestieri, Leticia Boldrin; Gomes-Cornélio, Ana Lívia; Rodrigues, Elisandra Márcia; Faria, Gisele; Guerreiro-Tanomaru, Juliane Maria; Tanomaru-Filho, Mário

    2017-01-01

    The aim of this study was to evaluate the cytotoxicity and bioactivity of calcium silicate-based cements combined with niobium oxide (Nb2O5) micro and nanoparticles, comparing the response in different cell lines. This evaluation used four cell lines: two primary cultures (human dental pulp cells - hDPCs and human dental follicle cells - hDFCs) and two immortalized cultures (human osteoblast-like cells - Saos-2 and mouse periodontal ligament cells - mPDL). The tested materials were: White Portland Cement (PC), mineral trioxide aggregate (MTA), white Portland cement combined with microparticles (PC/Nb2O5µ) or nanoparticles (PC/Nb2O5n) of niobium oxide (Nb2O5). Cytotoxicity was evaluated by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) and trypan blue exclusion assays and bioactivity by alkaline phosphatase (ALP) enzyme activity. Results were analyzed by ANOVA and Tukey test (a=0.05). PC/Nb2O5n presented similar or higher cell viability than PC/Nb2O5µ in all cell lines. Moreover, the materials presented similar or higher cell viability than MTA. Saos-2 exhibited high ALP activity, highlighting PC/Nb2O5µ material at 7 days of exposure. In conclusion, calcium silicate cements combined with micro and nanoparticles of Nb2O5 presented cytocompatibility and bioactivity, demonstrating the potential of Nb2O5 as an alternative radiopacifier agent for these cements. The different cell lines had similar response to cytotoxicity evaluation of calcium silicate cements. However, bioactivity was more accurately detected in human osteoblast-like cell line, Saos-2.

  14. Fabrication of chitin-chitosan/nano TiO2-composite scaffolds for tissue engineering applications.

    Science.gov (United States)

    Jayakumar, R; Ramachandran, Roshni; Divyarani, V V; Chennazhi, K P; Tamura, H; Nair, S V

    2011-03-01

    In this study, we prepared chitin-chitosan/nano TiO(2) composite scaffolds using lyophilization technique for bone tissue engineering. The prepared composite scaffold was characterized using SEM, XRD, FTIR and TGA. In addition, swelling, degradation and biomineralization capability of the composite scaffolds were evaluated. The developed composite scaffold showed controlled swelling and degradation when compared to the control scaffold. Cytocompatibility of the scaffold was assessed by MTT assay and cell attachment studies using osteoblast-like cells (MG-63), fibroblast cells (L929) and human mesenchymal stem cells (hMSCs). Results indicated no sign of toxicity and cells were found attached to the pore walls within the scaffolds. These results suggested that the developed composite scaffold possess the prerequisites for tissue engineering scaffolds and it can be used for tissue engineering applications. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. Cobalt-Doped Brushite Cement: Preparation, Characterization, and In Vitro Interaction with Osteosarcoma Cells

    Science.gov (United States)

    Cummings, Haley; Han, Weiguo; Vahabzadeh, Sahar; Elsawa, Sherine F.

    2017-08-01

    Brushite cement (BrC) is being widely used in bone and dental tissue engineering application because of its significant biocompatibility, bioresorbability, and moldability. Here, we have reported the effects of cobalt (Co) and its concentration on physical and biological properties of BrC. Our results show that Co addition stabilizes the tricalcium phosphate structure and decreases the amount of BrC phase in the final product. The in vitro interaction of samples with osteosarcoma MG-63 cells proved the cytocompatibility of all compositions in both normoxic and hypoxic conditions. Although the cell viability increased under hypoxia, the change was insignificant compared with normoxic conditions. Our data show that Co addition reduced hypoxia inducible factor-1α and glioma-associated oncogene family zinc finger 2 expression in MG-63, suggesting Co may provide the benefit of reducing the effects of hypoxia on gene expression in the osteosarcoma cell line.

  16. MicroRNA-224 promotes the sensitivity of osteosarcoma cells to cisplatin by targeting Rac1.

    Science.gov (United States)

    Geng, Shuo; Gu, Lina; Ju, Fang; Zhang, Hepeng; Wang, Yiwen; Tang, Han; Bi, ZhengGang; Yang, Chenglin

    2016-09-01

    Osteosarcoma is the most common primary bone tumour in children and adolescents. Accumulating evidence has shown that microRNAs (miRNAs) participate in the development of almost all types of cancer. Here, we investigated the role of miR-224 in the development and progression of osteosarcoma. We demonstrated that miR-224 was down-regulated in osteosarcoma cell lines and tissues. Lower miR-224 levels were correlated with shorter survivalin osteosarcoma patients. Furthermore, overexpression of miR-224 suppressed osteosarcoma cell proliferation, migration and invasion and contributed to the increased sensitivity of MG-63 cells to cisplatin. We identified Rac1 as a direct target gene of miR-224 in osteosarcoma. Rac1 expression was up-regulated in the osteosarcoma cell lines and tissues, and there was an inverse correlation between Rac1 and miR-224 expression in osteosarcoma tissues. Furthermore, rescuing Rac1 expression decreased the sensitivity of miR-224-overexpressing MG-63 cells to cisplatin. We also demonstrated that ectopic expression of Rac1 promoted the proliferation, migration and invasion of miR-224-overexpressing MG-63 cells. These data suggest that miR-224 plays a tumour suppressor role in the development of osteosarcoma and is related to the sensitivity of osteosarcoma to cisplatin. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  17. Signal transduction and downregulation of C-MET in HGF stimulated low and highly metastatic human osteosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Husmann, Knut, E-mail: khusmann@research.balgrist.ch [Laboratory for Orthopedic Research, Department of Orthopedics, Balgrist University Hospital, University of Zurich, Zurich (Switzerland); Ducommun, Pascal [Laboratory for Orthopedic Research, Department of Orthopedics, Balgrist University Hospital, University of Zurich, Zurich (Switzerland); Division of Plastic Surgery and Hand Surgery, Department of Surgery, University Hospital Zurich, Zurich (Switzerland); Sabile, Adam A.; Pedersen, Else-Marie; Born, Walter; Fuchs, Bruno [Laboratory for Orthopedic Research, Department of Orthopedics, Balgrist University Hospital, University of Zurich, Zurich (Switzerland)

    2015-09-04

    The poor outcome of osteosarcoma (OS), particularly in patients with metastatic disease and a five-year survival rate of only 20%, asks for more effective therapeutic strategies targeting malignancy-promoting mechanisms. Dysregulation of C-MET, its ligand hepatocyte growth factor (HGF) and the fusion oncogene product TPR-MET, first identified in human MNNG-HOS OS cells, have been described as cancer-causing factors in human cancers. Here, the expression of these molecules at the mRNA and the protein level and of HGF-stimulated signaling and downregulation of C-MET was compared in the parental low metastatic HOS and MG63 cell lines and the respective highly metastatic MNNG-HOS and 143B and the MG63-M6 and MG63-M8 sublines. Interestingly, expression of TPR-MET was only observed in MNNG-HOS cells. HGF stimulated the phosphorylation of Akt and Erk1/2 in all cell lines investigated, but phospho-Stat3 remained at basal levels. Downregulation of HGF-stimulated Akt and Erk1/2 phosphorylation was much faster in the HGF expressing MG63-M8 cells than in HOS cells. Degradation of HGF-activated C-MET occurred predominantly through the proteasomal and to a lesser extent the lysosomal pathway in the cell lines investigated. Thus, HGF-stimulated Akt and Erk1/2 signaling as well as proteasomal degradation of HGF activated C-MET are potential therapeutic targets in OS. - Highlights: • Expression of TPR-MET was only observed in MNNG-HOS cells. • HGF stimulated the phosphorylation of Akt and Erk1/2 but not of Stat3 in osteosarcoma cell lines. • Degradation of HGF-activated C-MET occurred predominantly through the proteasomal pathway.

  18. Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis.

    Science.gov (United States)

    Takagi, Satoshi; Takemoto, Ai; Takami, Miho; Oh-Hara, Tomoko; Fujita, Naoya

    2014-08-01

    The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the proliferation of osteosarcomas. Co-culture of platelets with MG63 or HOS osteosarcoma cells, which could induce platelet aggregation, enhanced the proliferation of each cell line in vitro. Analysis of phospho-antibody arrays revealed that co-culture of MG63 cells with platelets induced the phosphorylation of platelet derived growth factor receptor (PDGFR) and Akt. The addition of supernatants of osteosarcoma-platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated-PDGFR and -Akt. Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet-induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt. Our results suggest that inhibitors that specifically target osteosarcoma-platelet interactions may eradicate osteosarcomas. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  19. Cell Phenotype Transitions in Cardiovascular Calcification

    Directory of Open Access Journals (Sweden)

    Luis Hortells

    2018-03-01

    Full Text Available Cardiovascular calcification was originally considered a passive, degenerative process, however with the advance of cellular and molecular biology techniques it is now appreciated that ectopic calcification is an active biological process. Vascular calcification is the most common form of ectopic calcification, and aging as well as specific disease states such as atherosclerosis, diabetes, and genetic mutations, exhibit this pathology. In the vessels and valves, endothelial cells, smooth muscle cells, and fibroblast-like cells contribute to the formation of extracellular calcified nodules. Research suggests that these vascular cells undergo a phenotypic switch whereby they acquire osteoblast-like characteristics, however the mechanisms driving the early aspects of these cell transitions are not fully understood. Osteoblasts are true bone-forming cells and differentiate from their pluripotent precursor, the mesenchymal stem cell (MSC; vascular cells that acquire the ability to calcify share aspects of the transcriptional programs exhibited by MSCs differentiating into osteoblasts. What is unknown is whether a fully-differentiated vascular cell directly acquires the ability to calcify by the upregulation of osteogenic genes or, whether these vascular cells first de-differentiate into an MSC-like state before obtaining a “second hit” that induces them to re-differentiate down an osteogenic lineage. Addressing these questions will enable progress in preventative and regenerative medicine strategies to combat vascular calcification pathologies. In this review, we will summarize what is known about the phenotypic switching of vascular endothelial, smooth muscle, and valvular cells.

  20. 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.

  1. PDLLA honeycomb-like scaffolds with a high loading of superhydrophilic graphene/multi-walled carbon nanotubes promote osteoblast in vitro functions and guided in vivo bone regeneration.

    Science.gov (United States)

    Silva, Edmundo; Vasconcellos, Luana Marotta Reis de; Rodrigues, Bruno V M; Dos Santos, Danilo Martins; Campana-Filho, Sergio P; Marciano, Fernanda Roberta; Webster, Thomas J; Lobo, Anderson Oliveira

    2017-04-01

    Herein, we developed honeycomb-like scaffolds by combining poly (d, l-lactic acid) (PDLLA) with a high amount of graphene/multi-walled carbon nanotube oxides (MWCNTO-GO, 50% w/w). From pristine multi-walled carbon nanotubes (MWCNT) powders, we produced MWCNTO-GO via oxygen plasma etching (OPE), which promoted their exfoliation and oxidation. Initially, we evaluated PDLLA and PDLLA/MWCNTO-GO scaffolds for tensile strength tests, cell adhesion and cell viability (with osteoblast-like MG-63 cells), alkaline phosphatase (ALP, a marker of osteoblast differentiation) activity and mineralized nodule formation. In vivo tests were carried out using PDLLA and PDLLA/MWCNTO-GO scaffolds as fillers for critical defects in the tibia of rats. MWCNTO-GO loading was responsible for decreasing the tensile strength and elongation-at-break of PDLLA scaffolds, although the high mechanical performance observed (~600MPa) assures their application in bone tissue regeneration. In vitro results showed that the scaffolds were not cytotoxic and allowed for osteoblast-like cell interactions and the formation of mineralized matrix nodules. Furthermore, MG-63 cells grown on PDLLA/MWCNTO-GO significantly enhanced osteoblast ALP activity compared to controls (cells alone), while the PDLLA group showed similar ALP activity when compared to controls and PDLLA/MWCNTO-GO. Most impressively, in vivo tests suggested that compared to PDLLA scaffolds, PDLLA/MWCNTO-GO had a superior influence on bone cell activity, promoting greater new bone formation. In summary, the results of this study highlighted that this novel scaffold (MWCNTO-GO, 50% w/w) is a promising alternative for bone tissue regeneration and, thus, should be further studied. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Ai-Rong Qian

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

  3. 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

  4. Treatment of poly(ethylene terephthalate) foils by atmospheric pressure air dielectric barrier discharge and its influence on cell growth

    Science.gov (United States)

    Kuzminova, Anna; Vandrovcová, Marta; Shelemin, Artem; Kylián, Ondřej; Choukourov, Andrei; Hanuš, Jan; Bačáková, Lucie; Slavínská, Danka; Biederman, Hynek

    2015-12-01

    In this contribution an effect of dielectric barrier discharge (DBD) sustained in air at atmospheric pressure on surface properties of poly(ethylene terephthalate) (PET) foils is studied. It is found that exposure of PET to DBD plasma leads to rapid changes of surface chemical composition, wettability, surface morphology as well as mechanical properties of PET surface. In addition, based on biological tests that were performed using two cell types (Saos-2 human osteoblast-like cells and HUVEC human umbilical vein endothelial cells), it may be concluded that DBD plasma treatment positively influences cell growth on PET. This effect was found to be connected predominantly with increased surface energy and oxygen content of the surface of treated PET foils.

  5. Development and characterization of multi-element doped hydroxyapatite bioceramic coatings on metallic implants for orthopedic applications

    International Nuclear Information System (INIS)

    Furko, M.; Havasi, V.; Kónya, Z.; Grünewald, D.; Detsch, R.; Boccaccini, A.R.; Balázsi, C.

    2018-01-01

    Multi-element modified bioactive hydroxyapatite bioceramic (mHAp) coatings were successfully developed onto surgical grade titanium alloy material (Ti6Al4V). The coatings were prepared by pulse current deposition from electrolyte containing adequate amounts of calcium nitrate and ammonium dihydrogen phosphate at 70C. The pure HAp layer was doped and co-deposited with Ag, Zn, Mg, Sr ions. The biocompatible properties of layers were investigated by seeding osteoblast-like MG-63 cells onto the samples’ surface. The biocompatible measurements revealed enhanced bioactivity of modified HAp compared to uncoated implant materials and pure bioceramic coating. The morphology and structure of coatings and cells were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) as well as FT-IR and XRD measurements. The biodegradable properties of samples were investigated by electrochemical potentiodynamic measurements. [es

  6. Silver-loaded chitosan coating as an integrated approach to face titanium implant-associated infections: analytical characterization and biological activity.

    Science.gov (United States)

    Cometa, Stefania; Bonifacio, Maria A; Baruzzi, Federico; de Candia, Silvia; Giangregorio, Maria M; Giannossa, Lorena C; Dicarlo, Manuela; Mattioli-Belmonte, Monica; Sabbatini, Luigia; De Giglio, Elvira

    2017-12-01

    The present work focuses on the idea to prevent and/or inhibit the colonization of implant surfaces by microbial pathogens responsible for post-operative infections, adjusting antimicrobial properties of the implant surface prior to its insertion. An antibacterial coating based on chitosan and silver was developed by electrodeposition techniques on poly(acrylic acid)-coated titanium substrates. When a silver salt was added during the chitosan deposition step, a stable and scalable silver incorporation was achieved. The physico-chemical composition of the coating was studied by X-ray photoelectron spectroscopy (XPS), while atomic force microscopy in intermittent contact mode (ICAFM) was used to explore the coating morphology. The amount of silver released from the coating up to 21 days was evaluated by inductively coupled plasma mass spectrometry (ICP-MS). The capability of the proposed coating to interact in vitro with the biological environment in terms of compatibility and antibacterial properties was assessed using MG-63 osteoblast-like cell line and S. aureus and P. aeruginosa strains, respectively. These studies revealed that a coating showing a silver surface atomic percentage equal to 0.3% can be effectively used as antibacterial system, while providing good viability of osteoblast-like cells after 7 days. The antibacterial effectiveness of the prepared coating is mainly driven by a contact killing mechanism, although the low concentration of silver released (below 0.1 ppm up to 21 days) is enough to inhibit bacterial growth, advantaging MG-63 cells in the race for the surface.

  7. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Further studies on the intermediary metabolism of bone in vitro and a monoclonal cell line (MMB-1) derived from bone: effects of parathyroid hormone and acetazolamide

    International Nuclear Information System (INIS)

    Nichols, F.C.

    1981-01-01

    The mechanism/mechanisms by which PTH affects Ca homeostasis between blood and bone have not been clearly established. Most studies of metabolic acid production in bone took place during the late 1950s and early 1960s. Since that time, assay techniques for metabolic acid production have been improved for greater sensitivity, more has been learned as to how PTH mediates its cellular responses, and techniques for cell isolation and culture have dramatically improved. These improvements have made possible new approaches to the study of short term effects of PTH on metabolic acid production in bone. Chapter 1 explores the potential of bone to utilize substrates other than glucose for metabolic energy transfer. Chapter 2 characterizes glucose metabolism in mouse calvaria in vitro and explores effects of PTH, acetazolamide, and C1 13,850 on calvaria oxidative metabolism. Chapter 3 describes PTH effects on glucose metabolism of MMB-1 cells, a monoclonal cell line reported to possess osteoblast-like characteristics

  10. Carbon foam/hydroxyapatite coating for carbon/carbon composites: Microstructure and biocompatibility

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Leilei, E-mail: zhangleilei1121@aliyun.com; Li, Hejun; Li, Kezhi; Zhang, Shouyang; Lu, Jinhua; Li, Wei; Cao, Sheng; Wang, Bin

    2013-12-01

    To improve the surface biocompatibility of carbon/carbon composites, a carbon foam/hydroxyapatite coating was applied using a combination method of slurry procedure and ultrasound-assisted electrochemical deposition procedure. The morphology, microstructure and chemical composition of the coating were investigated by scanning electron microscopy, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy and X-ray diffraction. The biocompatibility of the carbon foam/hydroxyapatite coating was investigated by osteoblast-like MG63 cell culture tests. The results showed that the carbon foam could provide a large number of pores on the surface of carbon/carbon composites. The hydroxyapatite crystals could infiltrate into the pores and form the carbon foam/hydroxyapatite coating. The coating covered the carbon/carbon composites fully and uniformly with slice morphology. The cell response tests showed that the MG63 cells on carbon foam/hydroxyapatite coating had a better cell adhesion and cell proliferation than those on uncoated carbon/carbon composites. The carbon foam/hydroxyapatite coatings were cytocompatible and were beneficial to improve the biocompatibility. The approach presented here may be exploited for fabrication of carbon/carbon composite implant surfaces.

  11. Carbon foam/hydroxyapatite coating for carbon/carbon composites: Microstructure and biocompatibility

    International Nuclear Information System (INIS)

    Zhang, Leilei; Li, Hejun; Li, Kezhi; Zhang, Shouyang; Lu, Jinhua; Li, Wei; Cao, Sheng; Wang, Bin

    2013-01-01

    To improve the surface biocompatibility of carbon/carbon composites, a carbon foam/hydroxyapatite coating was applied using a combination method of slurry procedure and ultrasound-assisted electrochemical deposition procedure. The morphology, microstructure and chemical composition of the coating were investigated by scanning electron microscopy, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy and X-ray diffraction. The biocompatibility of the carbon foam/hydroxyapatite coating was investigated by osteoblast-like MG63 cell culture tests. The results showed that the carbon foam could provide a large number of pores on the surface of carbon/carbon composites. The hydroxyapatite crystals could infiltrate into the pores and form the carbon foam/hydroxyapatite coating. The coating covered the carbon/carbon composites fully and uniformly with slice morphology. The cell response tests showed that the MG63 cells on carbon foam/hydroxyapatite coating had a better cell adhesion and cell proliferation than those on uncoated carbon/carbon composites. The carbon foam/hydroxyapatite coatings were cytocompatible and were beneficial to improve the biocompatibility. The approach presented here may be exploited for fabrication of carbon/carbon composite implant surfaces.

  12. Evaluation of Osseointegration of Titanium Alloyed Implants Modified by Plasma Polymerization

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    Carolin Gabler

    2014-02-01

    Full Text Available By means of plasma polymerization, positively charged, nanometre-thin coatings can be applied to implant surfaces. The aim of the present study was to quantify the adhesion of human bone cells in vitro and to evaluate the bone ongrowth in vivo, on titanium surfaces modified by plasma polymer coatings. Different implant surface configurations were examined: titanium alloy (Ti6Al4V coated with plasma-polymerized allylamine (PPAAm and plasma-polymerized ethylenediamine (PPEDA versus uncoated. Shear stress on human osteoblast-like MG-63 cells was investigated in vitro using a spinning disc device. Furthermore, bone-to-implant contact (BIC was evaluated in vivo. Custom-made conical titanium implants were inserted at the medial tibia of female Sprague-Dawley rats. After a follow-up of six weeks, the BIC was determined by means of histomorphometry. The quantification of cell adhesion showed a significantly higher shear stress for MG-63 cells on PPAAm and PPEDA compared to uncoated Ti6Al4V. Uncoated titanium alloyed implants showed the lowest BIC (40.4%. Implants with PPAAm coating revealed a clear but not significant increase of the BIC (58.5% and implants with PPEDA a significantly increased BIC (63.7%. In conclusion, plasma polymer coatings demonstrate enhanced cell adhesion and bone ongrowth compared to uncoated titanium surfaces.

  13. Engineered Protein Coatings to Improve the Osseointegration of Dental and Orthopaedic Implants

    Science.gov (United States)

    Raphel, Jordan; Karlsson, Johan; Galli, Silvia; Wennerberg, Ann; Lindsay, Christopher; Haugh, Matthew; Pajarinen, Jukka; Goodman, Stuart B.; Jimbo, Ryo; Andersson, Martin; Heilshorn, Sarah C.

    2016-01-01

    Here we present the design of an engineered, elastin-like protein (ELP) that is chemically modified to enable stable coatings on the surfaces of titanium-based dental and orthopaedic implants by novel photocrosslinking and solution processing steps. The ELP includes an extended RGD sequence to confer bio-signaling and an elastin-like sequence for mechanical stability. ELP thin films were fabricated on cp-Ti and Ti6Al4V surfaces using scalable spin and dip coating processes with photoactive covalent crosslinking through a carbene insertion mechanism. The coatings withstood procedures mimicking dental screw and hip replacement stem implantations, a key metric for clinical translation. They promoted rapid adhesion of MG63 osteoblast-like cells, with over 80% adhesion after 24 hours, compared to 38% adhesion on uncoated Ti6Al4V. MG63 cells produced significantly more mineralization on ELP coatings compared to uncoated Ti6Al4V. Human bone marrow mesenchymal stem cells (hMSCs) had an earlier increase in alkaline phosphatase activity, indicating more rapid osteogenic differentiation and mineral deposition on adhesive ELP coatings. Rat tibia and femur in vivo studies demonstrated that cell-adhesive ELP-coated implants increased bone-implant contact area and interfacial strength after one week. These results suggest that ELP coatings withstand surgical implantation and promote rapid osseointegration, enabling earlier implant loading and potentially preventing micromotion that leads to aseptic loosening and premature implant failure. PMID:26790146

  14. 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

  15. In vitro evaluation of biomimetic chitosan–calcium phosphate scaffolds with potential application in bone tissue engineering

    International Nuclear Information System (INIS)

    Tanase, C E; Popa, M I; Sartoris, A; Unger, R E; Kirkpatrick, C J; Verestiuc, L

    2013-01-01

    This work reports on the physicochemical properties and in vitro cytotoxicity assessment of chitosan–calcium phosphate (Cs–CP) scaffolds for bone tissue engineering, which were synthesized by a novel biomimetic co-precipitation method. X-ray diffraction (XRD) along with scanning electron microscopy (SEM) analysis confirmed the porous morphology of the scaffolds and the amorphous nature of the inorganic phase with different crystallite sizes and the formation of various forms of calcium phosphate. Compressive mechanical testing revealed that the Young's modulus of the biomaterials is in the range of human trabecular bone. In vitro tests were performed on the biomaterials for up to 14 days to study the behavior of the osteoblast-like human cell line (MG63), primary human osteoblasts (HOS) and human dermal microvascular endothelial cells (HDMEC). The cytotoxicity was evaluated by the MTS assay for cell metabolism and the detection of membrane integrity (lactate dehydrogenase-LDH release). An expression of the vascular endothelial growth factor (VEGF) in the cell supernatants was quantified by ELISA. Cell viability gave values close to untreated controls for MG63 and HOS, while in the case of HDMEC the viability after 2 weeks in the cell culture was between 80–90%. The cytotoxicity induced by the Cs–CP scaffolds on MG63, HOS and HDMEC in vitro was evaluated by the amount of LDH released, which is a sensitive and accurate marker for cellular toxicity. The increased levels of VEGF obtained in the osteoblast culture highlights its important role in the regulation of vascularization and bone remodeling. The biological responses of the Cs–CP scaffolds demonstrate a similar proliferation and differentiation characteristics of the cells comparable to the controls. These results reveal that biomimetic Cs–CP composite scaffolds are promising biomaterials for bone tissue engineering; their in vivo response remains to be tested. (paper)

  16. Hydroxyapatite/collagen bone-like nanocomposite.

    Science.gov (United States)

    Kikuchi, Masanori

    2013-01-01

    Our group has succeeded to synthesize material with bone-like nanostructure and bone-like inorganic and organic composition via self-organization mechanism between them using simultaneous titration method under controlled pH and temperature. The hydroxyapatite/collagen (HAp/Col) bone-like nanocomposite completely incorporated into bone remodeling process to be substituted by new bone. Cells cultured on the HAp/Col revealed very interesting reactions. Osteoblast-like MG63 cells showed upregulation of alkaline phosphatase >3 times greater than MG63 cells cultured on tissue culture polystyrene (TCPS). MG63 cells 3-dimensionally cultured in a "HAp/Col sponge," a porous HAp/Col having sponge-like viscoelasticity, accumulated calcium phosphate nodules on extracellular matrices they secreted. Bone marrow cells co-cultured with osteoblasts on HAp/Col differentiated to osteoclasts without differentiation supplements. This phenomenon is not found in cells cultured on hydroxyapatite ceramics and TCPS, and rarely in cells cultured on dentin. These results suggest that HAp/Col is a good candidate for tissue engineering of bone as well as bone filler. In a clinical test as a bone filler, the HAp/Col sponge was significantly better than porous β-tricalcium phosphate. The HAp/Col sponge has been approved by the Japanese government and will be used as greatly needed bone filler in patients. In addition to the above, HAp/Col coating on titanium revealed higher osteo-conductivity than HAp-coated titanium and bare titanium and improved direct bonding between titanium and newly formed bone. The HAp/Col coating may be used for metal devices requiring osseointegration.

  17. Bioactive Sr(II/Chitosan/Poly(ε-caprolactone Scaffolds for Craniofacial Tissue Regeneration. In Vitro and In Vivo Behavior

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    Itzia Rodríguez-Méndez

    2018-03-01

    Full Text Available In craniofacial tissue regeneration, the current gold standard treatment is autologous bone grafting, however, it presents some disadvantages. Although new alternatives have emerged there is still an urgent demand of biodegradable scaffolds to act as extracellular matrix in the regeneration process. A potentially useful element in bone regeneration is strontium. It is known to promote stimulation of osteoblasts while inhibiting osteoclasts resorption, leading to neoformed bone. The present paper reports the preparation and characterization of strontium (Sr containing hybrid scaffolds formed by a matrix of ionically cross-linked chitosan and microparticles of poly(ε-caprolactone (PCL. These scaffolds of relatively facile fabrication were seeded with osteoblast-like cells (MG-63 and human bone marrow mesenchymal stem cells (hBMSCs for application in craniofacial tissue regeneration. Membrane scaffolds were prepared using chitosan:PCL ratios of 1:2 and 1:1 and 5 wt % Sr salts. Characterization was performed addressing physico-chemical properties, swelling behavior, in vitro biological performance and in vivo biocompatibility. Overall, the composition, microstructure and swelling degree (≈245% of scaffolds combine with the adequate dimensional stability, lack of toxicity, osteogenic activity in MG-63 cells and hBMSCs, along with the in vivo biocompatibility in rats allow considering this system as a promising biomaterial for the treatment of craniofacial tissue regeneration.

  18. Structural and biocompatible characterization of TiC/a:C nanocomposite thin films

    International Nuclear Information System (INIS)

    Balázsi, K.; Vandrovcová, M.; Bačáková, L.; Balázsi, Cs.

    2013-01-01

    In this work, sputtered TiC/amorphous C thin films have been developed in order to be applied as potential barrier coating for interfering of Ti ions from pure Ti or Ti alloy implants. Our experiments were based on magnetron sputtering method, because the vacuum deposition provides great flexibility for manipulating material chemistry and structure, leading to films and coatings with special properties. The films have been deposited on silicon (001) substrates with 300 nm thick oxidized silicon sublayer at 200 °C deposition temperature as model substrate. Transmission electron microscopy has been used for structural investigations. Thin films consisted of ∼ 20 nm TiC columnar crystals embedded by 5 nm thin amorphous carbon matrix. MG63 osteoblast cells have been applied for in vitro study of TiC nanocomposites. The cell culture tests give strong evidence of thin films biocompatibility. Highlights: ► The main goal of this work is the relatively easy preparation of nanocomposite TiC thin films by dc magnetron sputtering. ► TEM and HREM were applied for structural characterization of columnar TiC nanocrystals and amorphous carbon matrix. ► The biocompatibility of films was showed by MG63 human osteoblast like cells during 1, 3 and 7 days seeding

  19. Electrochemical construction of micro–nano spongelike structure on titanium substrate for enhancing corrosion resistance and bioactivity

    International Nuclear Information System (INIS)

    Jiang, Pinliang; Lin, Longxiang; Zhang, Fan; Dong, Xiang; Ren, Lei; Lin, Changjian

    2013-01-01

    Highlights: • A hierarchical micro–nano spongelike TiO 2 layer was constructed on Ti substrate. • The micro–nano TiO 2 surface presented good corrosion resistance. • Excellent biomineration ability was observed on such micro–nano TiO 2 layer. • Superior MG63 cell viability was discerned on the micro–nano structured surface. -- Abstract: Surface structures of medical implants generally play a crucial role in tissue growth and healing while implanted into a living body. The surface design and modification of implants can effectively promote its biocompatibility and integration ability. In this study, a hierarchically superhydrophilic structure on titanium surface with a nano-spongelike titania layer on the micro-roughened titanium surface was constructed through dual acid etching and electrochemical treatments. It is shown that the structure of micro/nano-spongelike TiO 2 provides not only better corrosion resistance and less oxygen vacancies, but also much higher ability of biomineralization after immersion in simulated body fluid (SBF) for 14 days. It is evident, by the cell culture for the different samples, that the micro–nano spongelike structured surface on Ti significantly promotes human osteoblast-like MG63 cell attachment and proliferation. All evaluations of electrochemical behavior and biological responses in this study indicate that the micro/nano-spongelike structure on Ti surface is of excellent chemical stability, bioactivity as well as biocompatibility for biomedical implant applications

  20. Bioinformatics prediction of miR-30a targets and its inhibition of cell proliferation of osteosarcoma by up-regulating the expression of PTEN

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    Biao Zhong

    2017-11-01

    Full Text Available Abstract Background MiRNAs are frequently abnormally expressed in the progression of human osteosarcoma. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN is one of the tumor suppressors in various types of human cancer. In the present study, we detected how hsa-miR-30a-3p regulated PTEN and further tested the role of hsa-miR-30a-3p in the cell proliferation of osteosarcoma cells. Methods The levels of miR-30a were determined by real time PCR. The expression of PTEN was tested by western blotting analysis. Cell distribution of PTEN was observed with confocal laser scanning microscope. Cell viability was determined by MTT assay. Results The expression of miR-30a and PTEN was obviously decreased in MG-63, 143B and Saos-2 cells compared with primary osteoblasts. TargetScan analysis data showed miR-30a might bind with position 30-57 of 3’UTR of PTEN. Transfection with miR-30a-3p increased the level of PTEN in MG-63 cells, while transfection with miR-30a-3p inhibitor significantly decreased the expression of PTEN in osteosarcoma cells. Transfection with miR-30a-3p significantly inhibited cell proliferation of osteosarcoma cells, while miR-30a inhibitor obviously promoted cell viability of MG63 cells and Saos-2 cells. Inhibition of PTEN eliminated the proliferation inhibitory effect of miR-30a-3p. Conclusion Thus, all these findings revealed the anti-tumor effects of miR-30a in human osteosarcoma cells, which could be mediated by regulating the level of PTEN.

  1. Glutamic acid ameliorates estrogen deficiency-induced menopausal-like symptoms in ovariectomized mice.

    Science.gov (United States)

    Han, Na-Ra; Kim, Hee-Yun; Yang, Woong Mo; Jeong, Hyun-Ja; Kim, Hyung-Min

    2015-09-01

    Some amino acids are considered alternative therapies for improving menopausal symptoms. Glutamic acid (GA), which is abundant in meats, fish, and protein-rich plant foods, is known to be a neurotransmitter or precursor of γ-aminobutyric acid. Although it is unclear if GA functions in menopausal symptoms, we hypothesized that GA would attenuate estrogen deficiency-induced menopausal symptoms. The objective to test our hypothesis was to examine an estrogenic effect of GA in ovariectomized (OVX) mice, estrogen receptor (ER)-positive human osteoblast-like MG-63 cells, and ER-positive human breast cancer MCF-7 cells. The results demonstrated that administration with GA to mice suppressed body weight gain and vaginal atrophy when compared with the OVX mice. A microcomputed tomographic analysis of the trabecular bone showed increases in bone mineral density, trabecular number, and connectivity density as well as a significant decrease in total porosity of the OVX mice treated with GA. In addition, GA increased serum levels of alkaline phosphatase and estrogen compared with the OVX mice. Furthermore, GA induced proliferation and increased ER-β messenger RNA (mRNA) expression, estrogen response element (ERE) activity, extracellular signal-regulated kinase phosphorylation, and alkaline phosphatase activity in MG-63 cells. In MCF-7 cells, GA also increased proliferation, Ki-67 mRNA expression, ER-β mRNA expression, and ERE activity. Estrogen response element activity increased by GA was inhibited by an estrogen antagonist. Taken together, our data demonstrated that GA has estrogenic and osteogenic activities in OVX mice, MG-63 cells, and MCF-7 cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. The role of human umbilical cord tissue-derived mesenchymal stromal cells (UCX® in the treatment of inflammatory arthritis

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    Santos Jorge M

    2013-01-01

    Full Text Available Abstract Background ECBio has developed proprietary technology to consistently isolate, expand and cryopreserve a well-characterized population of stromal cells from human umbilical cord tissue (UCX® cells. The technology has recently been optimized in order to become compliant with Advanced Medicine Therapeutic Products. In this work we report the immunosuppressive capacity of UCX® cells for treating induced autoimmune inflammatory arthritis. Methods UCX® cells were isolated using a proprietary method (PCT/IB2008/054067 that yields a well-defined number of cells using a precise proportion between tissue digestion enzyme activity units, tissue mass, digestion solution volume and void volume. The procedure includes three recovery steps to avoid non-conformities related to cell recovery. UCX® surface markers were characterized by flow cytometry and UCX® capacity to expand in vitro and to differentiate into adipocyte, chondrocyte and osteoblast-like cells was evaluated. Mixed Lymphocyte Reaction (MLR assays were performed to evaluate the effect of UCX® cells on T-cell activation and Treg conversion assays were also performed in vitro. Furthermore, UCX® cells were administered in vivo in both a rat acute carrageenan-induced arthritis model and rat chronic adjuvant induced arthritis model for arthritic inflammation. UCX® anti-inflammatory activity was then monitored over time. Results UCX® cells stained positive for CD44, CD73, CD90 and CD105; and negative for CD14, CD19 CD31, CD34, CD45 and HLA-DR; and were capable to differentiate into adipocyte, chondrocyte and osteoblast-like cells. UCX® cells were shown to repress T-cell activation and promote the expansion of Tregs better than bone marrow mesenchymal stem cells (BM-MSCs. Accordingly, xenogeneic UCX® administration in an acute carrageenan-induced arthritis model showed that human UCX® cells can reduce paw edema in vivo more efficiently than BM-MSCs. Finally, in a chronic adjuvant

  3. Changes in the gene expression of co-cultured human fibroblast cells and osteosarcoma cells: the role of microenvironment.

    Science.gov (United States)

    Salvatore, Viviana; Focaroli, Stefano; Teti, Gabriella; Mazzotti, Antonio; Falconi, Mirella

    2015-10-06

    The progression of malignant tumors does not depend exclusively on the autonomous properties of cancer cells; it is also influenced by tumor stroma reactivity and is under strict microenvironmental control. By themselves, stromal cells are not malignant, and they maintain normal tissue structure and function. However, through intercellular interactions or by paracrine secretions from cancer cells, normal stromal cells acquire abnormal phenotypes that sustain cancer cell growth and tumor progression. In their dysfunctional state, fibroblast and immune cells produce chemokines and growth factors that stimulate cancer cell growth and invasion. In our previous work, we established an in vitro model based on a monolayer co-culture system of healthy human fibroblasts (HFs) and human osteosarcoma cells (the MG-63 cell line) that simulates the microenvironment of tumor cells and healthy cells. The coexistence between MG-63 cells and HFs allowed us to identify the YKL-40 protein as the main marker for verifying the influence of tumor cells grown in contact with healthy cells. In this study, we evaluated the interactions of HFs and MG-63 cells in a transwell co-culture system over 24 h, 48 h, 72 h, and 96 h. We analyzed the contributions of these populations to the tumor microenvironment during cancer progression, as measured by multiple markers. We examined the effect of siRNA knockdown of YKL-40 by tracking the subsequent changes in gene expression within the co-culture. We validated the expression of several genes, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis: TNF alpha, IL-6, MMP-1, MMP-9, and VEGF. We compared the results to those from a transwell co-culture without the YKL-40 knockdown. In a pro-inflammatory environment promoted by TNF alpha and IL-6, siRNA knockdown of YKL-40 caused a down-regulation of VEGF and MMP-1 expression in HFs. These findings demonstrated that the tumor microenvironment has an influence on the

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

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    Yun-Yun Ma

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

  5. Heterogeneity of osteosarcoma cell lines led to variable responses in reprogramming.

    Science.gov (United States)

    Choong, Pei Feng; Teh, Hui Xin; Teoh, Hoon Koon; Ong, Han Kiat; Choo, Kong Bung; Sugii, Shigeki; Cheong, Soon Keng; Kamarul, Tunku

    2014-01-01

    Four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotent state using Yamanaka factors retroviral transduction method. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. The reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, as in ESC up to Passage 15. All reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension in a low attachment dish for up to 10 days. Further testing on the directed differentiation capacity of the reprogrammed sarcomas showed all four reprogrammed sarcoma lines could differentiate into adipocytes while reprogrammed Saos-2-REP, MG-63-REP and G-292-REP could differentiate into osteocytes. Among the 4 osteosarcoma cell lines, U-2 OS reported the highest transduction efficiency but recorded the lowest reprogramming stability under long term culture. Thus, there may be intrinsic differences governing the variable responses of osteosarcoma cell lines towards reprogramming and long term culture effect of the reprogrammed cells. This is a first report to associate intrinsic factors in different osteosarcoma cell lines with variable reprogramming responses and effects on the reprogrammed cells after prolonged culture.

  6. Heterogeneity of Osteosarcoma Cell Lines Led to Variable Responses in Reprogramming

    Science.gov (United States)

    Choong, Pei Feng; Teh, Hui Xin; Teoh, Hoon Koon; Ong, Han Kiat; Choo, Kong Bung; Sugii, Shigeki; Cheong, Soon Keng; Kamarul, Tunku

    2014-01-01

    Four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotent state using Yamanaka factors retroviral transduction method. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. The reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, as in ESC up to Passage 15. All reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension in a low attachment dish for up to 10 days. Further testing on the directed differentiation capacity of the reprogrammed sarcomas showed all four reprogrammed sarcoma lines could differentiate into adipocytes while reprogrammed Saos-2-REP, MG-63-REP and G-292-REP could differentiate into osteocytes. Among the 4 osteosarcoma cell lines, U-2 OS reported the highest transduction efficiency but recorded the lowest reprogramming stability under long term culture. Thus, there may be intrinsic differences governing the variable responses of osteosarcoma cell lines towards reprogramming and long term culture effect of the reprogrammed cells. This is a first report to associate intrinsic factors in different osteosarcoma cell lines with variable reprogramming responses and effects on the reprogrammed cells after prolonged culture. PMID:25170299

  7. Scaffold preferences of mesenchymal stromal cells and adipose-derived stem cells from green fluorescent protein transgenic mice influence the tissue engineering of bone.

    Science.gov (United States)

    Wittenburg, Gretel; Flade, Viktoria; Garbe, Annette I; Lauer, Günter; Labudde, Dirk

    2014-05-01

    We have analysed the growth and differentiation of mesenchymal stromal cells (MSC) from bone marrow, and of adipose derived stem cells (ASC) from murine abdominal fat tissue, of green fluorescent protein (GFP) transgenic animals grown directly on two types of hydroxyapatite ceramic bone substitutes. BONITmatrix® and NanoBone® have specific mechanical and physiochemical properties such as porosity and an inner surface that influence cellular growth. Both MSC and ASC were separately seeded on 200mg of each biomaterial and cultured for 3 weeks under osteogenic differentiation conditions. The degree of mineralisation was assessed by alizarin red dye and the specific alkaline phosphatase activity of the differentiated cells. The morphology of the cells was examined by scanning electron microscopy and confocal microscopy. The osteoblastic phenotype of the cells was confirmed by analysing the expression of bone-specific genes (Runx2, osteocalcin, osteopontin, and osteonectin) by semiquantitative reverse transcriptase polymerase chain reaction (PCR). Comparison of BONITmatrix® and NanoBone® showed cell type-specific preferences in terms of osteogenic differentiation. MSC-derived osteoblast-like cells spread optimally on the surface of NanoBone® but not BONITmatrix® granules. In contrast BONITmatrix® granules conditioned the growth of osteoblast-like cells derived from ASC. The osteoblastic phenotype of the cultured cells on all matrices was confirmed by specific gene expression. Our results show that the in vitro growth and osteogenic differentiation of murine MSC or ASC of GFP transgenic mice are distinctly influenced by the ceramic substratum. While NanoBone® granules support the proliferation and differentiation of murine MSC isolated from bone marrow, the growth of murine ASC is supported by BONITmatrix® granules. NanoBone® is therefore recommended for use as scaffold in tissue engineering that requires MSC, whereas ASC can be combined with BONITmatrix® for

  8. 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

  9. 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.

  10. Cytotoxicity of TSP in 3D Agarose Gel Cultured Cell.

    Directory of Open Access Journals (Sweden)

    Song-I Chun

    Full Text Available A reference reagent, 3-(trimethylsilyl propionic-2, 2, 3, 3-d4 acid sodium (TSP, has been used frequently in nuclear magnetic resonance (NMR and magnetic resonance spectroscopy (MRS as an internal reference to identify cell and tissue metabolites, and determine chemical and protein structures. This reference material has been exploited for the quantitative and dynamic analyses of metabolite spectra acquired from cells. The aim of this study was to evaluate the cytotoxicity of TSP on three-dimensionally, agarose gel, cultured cells.A human osteosarcoma cell line (MG-63 was selected, and cells were three dimensionally cultured for two weeks in an agarose gel. The culture system contained a mixture of conventional culture medium and various concentrations (0, 1, 3, 5, 7, 10, 20 30 mM of TSP. A DNA quantification assay was conducted to assess cell proliferation using Quant-iT PicoGreen dsDNA reagent and kit, and cell viability was determined using a LIVE/DEAD Viability/Cytotoxicity kit. Both examinations were performed simultaneously at 1, 3, 7 and 14 days from cell seeding.In this study, the cytotoxicity of TSP in the 3D culture of MG-63 cells was evaluated by quantifying DNA (cell proliferation and cell viability. High concentrations of TSP (from 10 to 30 mM reduced both cell proliferation and viability (to 30% of the control after one week of exposure, but no such effects were found using low concentrations of TSP (0-10 mM.This study shows that low concentrations of TSP in 3D cell culture medium can be used for quantitative NMR or MRS examinations for up to two weeks post exposure.

  11. miR-340 alleviates chemoresistance of osteosarcoma cells by targeting ZEB1.

    Science.gov (United States)

    Yan, Haibin; Zhang, Bingyun; Fang, Chongbin; Chen, Liqiu

    2018-06-01

    Chemoresistance during treatment of osteosarcoma (OS) is attracting more and more attention as the main clinical obstacle. The purpose of this study was to elucidate the role of miR-340 in chemoresistance of OS. Plasmid construction and transfection, miRNA arrays, PCR analyses, and western blot analysis, as well as MTT, apoptosis, and luciferase assays were carried out in MG-63 cells and MG-63/cisplatin (DDP)-resistant cells. The results showed that miR-340 was downregulated in OS tissues and drug-resistant OS cells. Moreover, a negative correlation was observed between miR-340 and ZEB1 expression in OS tissues. Forced expression of miR-340 in drug-resistant OS cells significantly reduced multidrug resistance-1 and P-gp expression. Overexpression of miR-340 enhanced sensitivity to DDP by inhibiting viability and promoting apoptosis. The luciferase assay and western blot analysis identified ZEB1 as a direct target of miR-340, and miR-340 negatively regulated ZEB1 expression. Ectopic expression of ZEB1 reversed the effects of miR-340 on P-gp expression, cell viability, and apoptosis. miR-340 alleviated chemoresistance of OS cells by targeting ZEB1. Our results indicate that targeting miR-340 may be a potential therapeutic approach to treat drug-resistant OS.

  12. 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

  13. Uptake of the proteins HTRA1 and HTRA2 by cells mediated by calcium phosphate nanoparticles

    Directory of Open Access Journals (Sweden)

    Olga Rotan

    2017-02-01

    Full Text Available The efficient intracellular delivery of (biomolecules into living cells remains a challenge in biomedicine. Many biomolecules and synthetic drugs are not able to cross the cell membrane, which is a problem if an intracellular mode of action is desired, for example, with a nuclear receptor. Calcium phosphate nanoparticles can serve as carriers for small and large biomolecules as well as for synthetic compounds. The nanoparticles were prepared and colloidally stabilized with either polyethyleneimine (PEI; cationic nanoparticles or carboxymethyl cellulose (CMC; anionic nanoparticles and loaded with defined amounts of the fluorescently labelled proteins HTRA1, HTRA2, and BSA. The nanoparticles were purified by ultracentrifugation and characterized by dynamic light scattering and scanning electron microscopy. Various cell types (HeLa, MG-63, THP-1, and hMSC were incubated with fluorescently labelled proteins alone or with protein-loaded cationic and anionic nanoparticles. The cellular uptake was followed by light and fluorescence microscopy, confocal laser scanning microscopy (CLSM, and flow cytometry. All proteins were readily transported into the cells by cationic calcium phosphate nanoparticles. Notably, only HTRA1 was able to penetrate the cell membrane of MG-63 cells in dissolved form. However, the application of endocytosis inhibitors revealed that the uptake pathway was different for dissolved HTRA1 and HTRA1-loaded nanoparticles.

  14. Nanoscale crystallinity modulates cell proliferation on plasma sprayed surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Alan M. [School of Applied Sciences, University of Huddersfield, Huddersfield HD1 3DH (United Kingdom); Paxton, Jennifer Z.; Hung, Yi-Pei; Hadley, Martin J.; Bowen, James; Williams, Richard L. [School of Chemical Engineering, University of Birmingham, Edgbaston, B15 2TT (United Kingdom); Grover, Liam M., E-mail: l.m.grover@bham.ac.uk [School of Chemical Engineering, University of Birmingham, Edgbaston, B15 2TT (United Kingdom)

    2015-03-01

    Calcium phosphate coatings have been applied to the surface of metallic prostheses to mediate hard and soft tissue attachment for more than 40 years. Most coatings are formed of high purity hydroxyapatite, and coating methods are often designed to produce highly crystalline surfaces. It is likely however, that coatings of lower crystallinity can facilitate more rapid tissue attachment since the surface will exhibit a higher specific surface area and will be considerably more reactive than a comparable highly crystalline surface. Here we test this hypothesis by growing a population of MC3T3 osteoblast-like cells on the surface of two types of hip prosthesis with similar composition, but with differing crystallinity. The surfaces with lower crystallinity facilitated more rapid cell attachment and increased proliferation rate, despite having a less heterogeneous surface topography. This work highlights that the influence of the crystallinity of HA at the nano-scale is dominant over macro-scale topography for cell adhesion and growth. Furthermore, crystallinity could be easily adjusted by without compromising coating purity. These findings could facilitate designing novel coated calcium phosphate surfaces that more rapidly bond tissue following implantation. - Highlights: • Crystallinity of HA at the nano-scale was dominant over macro-scale topography. • Lower crystallinity caused rapid cell attachment and proliferation rate. • Crystallinity could be easily adjusted by without compromising coating purity.

  15. Nanoscale crystallinity modulates cell proliferation on plasma sprayed surfaces

    International Nuclear Information System (INIS)

    Smith, Alan M.; Paxton, Jennifer Z.; Hung, Yi-Pei; Hadley, Martin J.; Bowen, James; Williams, Richard L.; Grover, Liam M.

    2015-01-01

    Calcium phosphate coatings have been applied to the surface of metallic prostheses to mediate hard and soft tissue attachment for more than 40 years. Most coatings are formed of high purity hydroxyapatite, and coating methods are often designed to produce highly crystalline surfaces. It is likely however, that coatings of lower crystallinity can facilitate more rapid tissue attachment since the surface will exhibit a higher specific surface area and will be considerably more reactive than a comparable highly crystalline surface. Here we test this hypothesis by growing a population of MC3T3 osteoblast-like cells on the surface of two types of hip prosthesis with similar composition, but with differing crystallinity. The surfaces with lower crystallinity facilitated more rapid cell attachment and increased proliferation rate, despite having a less heterogeneous surface topography. This work highlights that the influence of the crystallinity of HA at the nano-scale is dominant over macro-scale topography for cell adhesion and growth. Furthermore, crystallinity could be easily adjusted by without compromising coating purity. These findings could facilitate designing novel coated calcium phosphate surfaces that more rapidly bond tissue following implantation. - Highlights: • Crystallinity of HA at the nano-scale was dominant over macro-scale topography. • Lower crystallinity caused rapid cell attachment and proliferation rate. • Crystallinity could be easily adjusted by without compromising coating purity

  16. Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro

    OpenAIRE

    Carsten Geiss; Zoltán Kis; Barbara Leuchs; Monika Frank-Stöhr; Jörg R. Schlehofer; Jean Rommelaere; Christiane Dinsart; Jeannine Lacroix

    2017-01-01

    Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts...

  17. LncRNA GAS5 Represses Osteosarcoma Cells Growth and Metastasis via Sponging MiR-203a

    Directory of Open Access Journals (Sweden)

    Yang Wang

    2018-01-01

    Full Text Available Background/Aims: LncRNA GAS5, a growth suppressor, has been reported to exert anti-tumor actions in various cancers, whereas the exact mechanism underling the anti-tumor action is still unclear. This study was aimed to investigate the effect of lncRNA GAS5 on osteosarcoma and tried to decode the underling mechanisms. Methods: Expressions of lncRNA GAS5 in MG-63 cells were silenced by shRNA transfection, while were overexpressed by vector transfection. Cell viability, migration, invasion and apoptosis were respectively assessed by MTT, Transwell assay and flow cytometry. Regulations between lncRNA GAS5 and miR-203a, as well as between miR-203a and TIMP2 were detected by qPCR, western blot and dual luciferase activity assay. Results: LncRNA GAS5 was down-regulated in MG-63 and OS-732 cells compared to hFOB1.19 cells. Silence of lncRNA GAS5 significantly promoted MG-63 cells viability, migration and invasion, and up-regulated Cyclin D1, Cyclin B1, CDK1 and CDK4 expressions. miR-203a was negatively regulated by lncRNA GAS5. The promoting activities of lncRNA GAS5 silence on MG-63 cells growth and metastasis were reversed by miR-203a suppression. TIMP2 was a target of miR-203a and the anti-growth and anti-metastasis actions of miR-203a suppression were reversed by TIMP2 silence. Further, lncRNA GAS5 silence, miR-203a overexpression, and TIMP2 silence could activate PI3K/AKT/GSK3β signaling while block NF-κB signaling. Conclusion: LncRNA GAS5 might be a tumor suppressor in osteosarcoma via sponging miR-203a, sequestering miR-203a away from TIMP2.

  18. Influence of Cu content on the cell biocompatibility of Ti–Cu sintered alloys

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Erlin, E-mail: zhangel@atm.neu.edu.cn [Key Lab. for Anisotropy and Texture of Materials, Education Ministry of China, Northeastern University, Shenyang 110819 (China); Jiamusi University, Jiamusi 154007 (China); Zheng, Lanlan [Jiamusi University, Jiamusi 154007 (China); Liu, Jie [Key Lab. for Anisotropy and Texture of Materials, Education Ministry of China, Northeastern University, Shenyang 110819 (China); Dept. of Prosthodontics, The Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003 (China); Bai, Bing [Dept. of Prosthodontics, School of Stomatology, China Medical University, Liaoning Institute of Dental Research, Shenyang 110001 (China); Liu, Cong [Jiamusi University, Jiamusi 154007 (China)

    2015-01-01

    The cell toxicity and the cell function of Ti–Cu sintered alloys with different Cu contents (2, 5, 10 and 25 wt.%, respectively) have been investigated in comparison with commercial pure titanium in order to assess the influence of Cu content on the cell biocompatibility of the Ti–Cu alloys. The cytotoxicity was studied by examining the MG63 cell response by CCK8 assessment. The cell morphology was evaluated by acridine orange/ethidium bromide (AO/EB) fluorescence and observed under scanning electronic microscopy (SEM). The cell function was monitored by measuring the AKP activity. It has been shown by the AO/EB morphology results that the cell death on both cp-Ti sample and Ti–Cu samples is due to apoptosis rather than necrosis. Although more apoptotic cells were found on the Ti–2Cu and Ti–5Cu samples, no evidence of Cu content dependent manner of apoptosis has been found. SEM observation indicated very good cell adhesion and spread on the cp-Ti sample and the Ti–Cu samples with different Cu contents. CCK8 results displayed that increase in the Cu content in Ti–Cu alloys does not bring about any difference in the cell viability. In addition, AKP test results indicated that no difference in the differentiation of MG63 was found between the cp-Ti and the Ti–Cu samples and among the Ti–Cu samples. All results indicated that Ti–Cu alloys exhibit very good cell biocompatibility and the Cu content up to 25 wt.% in the Ti–Cu alloys has no influence on the cell proliferation and differentiation. - Highlights: • The effect of Cu content on the cell biocompatibility has been investigated. • Cu content shows no influence on the cell proliferation. • Cu content shows no effect on the cell differentiation.

  19. Influence of Cu content on the cell biocompatibility of Ti–Cu sintered alloys

    International Nuclear Information System (INIS)

    Zhang, Erlin; Zheng, Lanlan; Liu, Jie; Bai, Bing; Liu, Cong

    2015-01-01

    The cell toxicity and the cell function of Ti–Cu sintered alloys with different Cu contents (2, 5, 10 and 25 wt.%, respectively) have been investigated in comparison with commercial pure titanium in order to assess the influence of Cu content on the cell biocompatibility of the Ti–Cu alloys. The cytotoxicity was studied by examining the MG63 cell response by CCK8 assessment. The cell morphology was evaluated by acridine orange/ethidium bromide (AO/EB) fluorescence and observed under scanning electronic microscopy (SEM). The cell function was monitored by measuring the AKP activity. It has been shown by the AO/EB morphology results that the cell death on both cp-Ti sample and Ti–Cu samples is due to apoptosis rather than necrosis. Although more apoptotic cells were found on the Ti–2Cu and Ti–5Cu samples, no evidence of Cu content dependent manner of apoptosis has been found. SEM observation indicated very good cell adhesion and spread on the cp-Ti sample and the Ti–Cu samples with different Cu contents. CCK8 results displayed that increase in the Cu content in Ti–Cu alloys does not bring about any difference in the cell viability. In addition, AKP test results indicated that no difference in the differentiation of MG63 was found between the cp-Ti and the Ti–Cu samples and among the Ti–Cu samples. All results indicated that Ti–Cu alloys exhibit very good cell biocompatibility and the Cu content up to 25 wt.% in the Ti–Cu alloys has no influence on the cell proliferation and differentiation. - Highlights: • The effect of Cu content on the cell biocompatibility has been investigated. • Cu content shows no influence on the cell proliferation. • Cu content shows no effect on the cell differentiation

  20. Perfusion directed 3D mineral formation within cell-laden hydrogels.

    Science.gov (United States)

    Sawyer, Stephen William; Shridhar, Shivkumar Vishnempet; Zhang, Kairui; Albrecht, Lucas; Filip, Alex; Horton, Jason; Soman, Pranav

    2018-06-08

    Despite the promise of stem cell engineering and the new advances in bioprinting technologies, one of the major challenges in the manufacturing of large scale bone tissue scaffolds is the inability to perfuse nutrients throughout thick constructs. Here, we report a scalable method to create thick, perfusable bone constructs using a combination of cell-laden hydrogels and a 3D printed sacrificial polymer. Osteoblast-like Saos-2 cells were encapsulated within a gelatin methacrylate (GelMA) hydrogel and 3D printed polyvinyl alcohol (PVA) pipes were used to create perfusable channels. A custom-built bioreactor was used to perfuse osteogenic media directly through the channels in order to induce mineral deposition which was subsequently quantified via microCT. Histological staining was used to verify mineral deposition around the perfused channels, while COMSOL modeling was used to simulate oxygen diffusion between adjacent channels. This information was used to design a scaled-up construct containing a 3D array of perfusable channels within cell-laden GelMA. Progressive matrix mineralization was observed by cells surrounding perfused channels as opposed to random mineral deposition in static constructs. MicroCT confirmed that there was a direct relationship between channel mineralization within perfused constructs and time within the bioreactor. Furthermore, the scalable method presented in this work serves as a model on how large-scale bone tissue replacement constructs could be made using commonly available 3D printers, sacrificial materials, and hydrogels. © 2018 IOP Publishing Ltd.

  1. Role of Galectin-3 in Bone Cell Differentiation, Bone Pathophysiology and Vascular Osteogenesis

    Directory of Open Access Journals (Sweden)

    Carla Iacobini

    2017-11-01

    Full Text Available Galectin-3 is expressed in various tissues, including the bone, where it is considered a marker of chondrogenic and osteogenic cell lineages. Galectin-3 protein was found to be increased in the differentiated chondrocytes of the metaphyseal plate cartilage, where it favors chondrocyte survival and cartilage matrix mineralization. It was also shown to be highly expressed in differentiating osteoblasts and osteoclasts, in concomitance with expression of osteogenic markers and Runt-related transcription factor 2 and with the appearance of a mature phenotype. Galectin-3 is expressed also by osteocytes, though its function in these cells has not been fully elucidated. The effects of galectin-3 on bone cells were also investigated in galectin-3 null mice, further supporting its role in all stages of bone biology, from development to remodeling. Galectin-3 was also shown to act as a receptor for advanced glycation endproducts, which have been implicated in age-dependent and diabetes-associated bone fragility. Moreover, its regulatory role in inflammatory bone and joint disorders entitles galectin-3 as a possible therapeutic target. Finally, galectin-3 capacity to commit mesenchymal stem cells to the osteoblastic lineage and to favor transdifferentiation of vascular smooth muscle cells into an osteoblast-like phenotype open a new area of interest in bone and vascular pathologies.

  2. Treatment of poly(ethylene terephthalate) foils by atmospheric pressure air dielectric barrier discharge and its influence on cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Kuzminova, Anna [Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holešovickách 2, 180 00 Praha 8 (Czech Republic); Vandrovcová, Marta [Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4 (Czech Republic); Shelemin, Artem [Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holešovickách 2, 180 00 Praha 8 (Czech Republic); Kylián, Ondřej, E-mail: ondrej.kylian@gmail.com [Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holešovickách 2, 180 00 Praha 8 (Czech Republic); Choukourov, Andrei; Hanuš, Jan [Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holešovickách 2, 180 00 Praha 8 (Czech Republic); Bačáková, Lucie [Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4 (Czech Republic); Slavínská, Danka; Biederman, Hynek [Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holešovickách 2, 180 00 Praha 8 (Czech Republic)

    2015-12-01

    Highlights: • Effect of atmospheric pressure DBD plasma on PET foils was investigated. • DBD treatment causes increase in surface density of O-containing functional groups. • DBD plasma causes increase of wettability, roughness and complex modulus of PET. • DBD treatment positively influences cells growth on PET. • Enhancement of cell growth on treated PET depends on the cell type. - Abstract: In this contribution an effect of dielectric barrier discharge (DBD) sustained in air at atmospheric pressure on surface properties of poly(ethylene terephthalate) (PET) foils is studied. It is found that exposure of PET to DBD plasma leads to rapid changes of surface chemical composition, wettability, surface morphology as well as mechanical properties of PET surface. In addition, based on biological tests that were performed using two cell types (Saos-2 human osteoblast-like cells and HUVEC human umbilical vein endothelial cells), it may be concluded that DBD plasma treatment positively influences cell growth on PET. This effect was found to be connected predominantly with increased surface energy and oxygen content of the surface of treated PET foils.

  3. Combination of etoposide and fisetin results in anti-cancer efficiency against osteosarcoma cell models.

    Science.gov (United States)

    Ferreira de Oliveira, José Miguel P; Pacheco, Ana Rita; Coutinho, Laura; Oliveira, Helena; Pinho, Sónia; Almeida, Luis; Fernandes, Eduarda; Santos, Conceição

    2018-03-01

    Osteosarcoma chemotherapy is often limited by chemoresistance, resulting in poor prognosis. Combined chemotherapy could, therefore, be used to prevent resistance to chemotherapeutics. Here, the effects of fisetin on osteosarcoma cells were investigated, as well as cytostatic potential in combination with the anti-cancer drug etoposide. For this, different osteosarcoma cell lines were treated with fisetin, with etoposide and with respective combinations. Fisetin was associated with decrease in colony formation in Saos-2 and in U2OS cells but not in MG-63 cells. Notwithstanding, upon evaluation of cellular growth by crystal violet assay, MG-63 and Saos-2 cells showed decreased cell proliferation at 40 and 20 µM fisetin, respectively. Depending on the relative concentrations, fisetin:etoposide combinations showed negative-to-positive interactions on the inhibition of cell proliferation. In addition, fisetin treatment up to 50 µM for 48 h resulted in G2-phase cell cycle arrest. Regardless of the combination, fisetin:etoposide increased % cells in G2-phase and decreased % cells in G1-phase. In addition, mixtures with more positive combined effects induced increased % cells in S-phase. Compared to etoposide treatment, these combinations resulted in decreased levels of cyclins B1 and E1, pointing to the role of these regulators in fisetin-induced cell cycle arrest. In conclusion, these results show that the combination of fisetin with etoposide has higher anti-proliferative effects in osteosarcoma associated with cell cycle arrest, allowing the use of lower doses of the chemotherapeutic agent, which has important implications for osteosarcoma treatment.

  4. Zr61Ti2Cu25Al12 metallic glass for potential use in dental implants: biocompatibility assessment by in vitro cellular responses.

    Science.gov (United States)

    Li, Jing; Shi, Ling-ling; Zhu, Zhen-dong; He, Qiang; Ai, Hong-jun; Xu, Jian

    2013-05-01

    In comparison with titanium and its alloys, Zr61Ti2Cu25Al12 (ZT1) bulk metallic glass (BMG) manifests a good combination of high strength, high fracture toughness and lower Young's modulus. To examine its biocompatibility required for potential use in dental implants, this BMG was used as a cell growth subtract for three types of cell lines, L929 fibroblasts, human umbilical vein endothelial cells (HUVEC), and osteoblast-like MG63 cells. For a comparison, these cell lines were in parallel cultured and grown also on commercially pure titanium (CP-Ti) and Ti6-Al4-V alloy (Ti64). Cellular responses on the three metals, including adhesion, morphology and viability, were characterized using the SEM visualization and CCK-8 assay. Furthermore, real-time RT-PCR was used to measure the activity of integrin β, alkaline phosphatase (ALP) and type I collagen (COL I) in adherent MG63 cells. As indicated, in all cases of three cell lines, no significant differences in the initial attachment and viability/proliferation were found between ZT1, CP-Ti, and Ti64 until 5d of incubation period. It means that the biocompatibility in cellular response for ZT1 BMG is comparable to Ti and its alloys. For gene expression of integrin β, ALP and COL I, mRNA level from osteoblast cells grown on ZT1 substrates is significantly higher than that on the CP-Ti and Ti64. It suggests that the adhesion and differentiation of osteoblasts grown on ZT1 are even superior to those on the CP-Ti and Ti64 alloy, then promoting bone formation. The good biocompatibility of ZT1 BMG is associated with the formation of zirconium oxide layer on the surface and good corrosion-resistance in physiological environment. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Clonal distribution of osteoprogenitor cells in cultured chick periostea: Functional relationship to bone formation

    International Nuclear Information System (INIS)

    McCulloch, C.A.; Fair, C.A.; Tenenbaum, H.C.; Limeback, H.; Homareau, R.

    1990-01-01

    Folded explants of periosteum from embryonic chick calvaria form bone-like tissue when grown in the presence of ascorbic acid, organic phosphate, and dexamethasone. All osteoblast-like cells in these cultures arise de novo by differentiation of osteoprogenitor cells present in the periosteum. To study the spatial and functional relationships between bone formation and osteoprogenitor cells, cultures were continuously labeled with [3H]thymidine for periods of 1-5 days. Radioautographs of serial 2-microns plastic sections stained for alkaline phosphatase (AP) showed maximal labeling of 30% of fibroblastic (AP-negative) cells by 3 days while osteogenic cells (AP-positive) exhibited over 95% labeling by 5 days. No differential shifts in labeling indices, grain count histograms of fibroblastic and osteogenic cells or numbers of AP-positive cells were observed, indicating no significant recruitment of cells from the fibroblastic to the osteogenic compartment. Despite the continuous presence of [3H]thymidine, less than 35% of both osteoblasts and osteocytes were labeled at 5 days, indicating that only one-third of the osteoprogenitor cells had cycled prior to differentiation. Spatial clustering of [3H]thymidine-labeled cells was measured by computer-assisted morphometry and application of the Poisson distribution to assess contagion. Cluster size and number of labeled cells per cluster did not vary between 1-3 days, but the number of clusters increased 20-fold between Day 1 and Day 3. Three-dimensional reconstruction from serial sections showed that clusters formed long, tubular arrays of osteogenic cells up to eight cells in length and located within 2-3 cell layers from the bone surface. Selective killing of S-phase cells with two pulse labels of high specific activity [3H]thymidine at 1 and 2 days of culture completely blocked bone formation

  6. Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca2+]i elevation

    International Nuclear Information System (INIS)

    Chou, C.-T.; He Shiping; Jan, C.-R.

    2007-01-01

    Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca 2+ ] i increases which involved the mobilization of intracellular Ca 2+ stored in the endoplasmic reticulum and Ca 2+ influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca 2+ chelator, to prevent paroxetine-induced [Ca 2+ ] i increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca 2+ -independent apoptosis via inducing p38 MAPK-associated caspase-3 activation

  7. Combined Effects of Surface Morphology and Mechanical Straining Magnitudes on the Differentiation of Mesenchymal Stem Cells without Using Biochemical Reagents

    Directory of Open Access Journals (Sweden)

    Ji-Yeon Jang

    2011-01-01

    Full Text Available Existing studies examining the control of mesenchymal stem cell (MSC differentiation into desired cell types have used a variety of biochemical reagents such as growth factors despite possible side effects. Recently, the roles of biomimetic microphysical environments have drawn much attention in this field. We studied MSC differentiation and changes in gene expression in relation to osteoblast-like cell and smooth muscle-like cell type resulting from various microphysical environments, including differing magnitudes of tensile strain and substrate geometries for 8 days. In addition, we also investigated the residual effects of those selected microphysical environment factors on the differentiation by ceasing those factors for 3 days. The results of this study showed the effects of the strain magnitudes and surface geometries. However, the genes which are related to the same cell type showed different responses depending on the changes in strain magnitude and surface geometry. Also, different responses were observed three days after the straining was stopped. These data confirm that controlling microenvironments so that they mimic those in vivo contributes to the differentiation of MSCs into specific cell types. And duration of straining engagement was also found to play important roles along with surface geometry.

  8. 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.

  9. Preparation and characterization of carbon/SiC nanowire/Na-doped carbonated hydroxyapatite multilayer coating for carbon/carbon composites

    International Nuclear Information System (INIS)

    Leilei, Zhang; Hejun, Li; Kezhi, Li; Shouyang, Zhang; Qiangang, Fu; Yulei, Zhang; Jinhua, Lu; Wei, Li

    2014-01-01

    Highlights: • CSH coatings were prepared by combination of magnetron sputter ion plating, CVD and UECD. • Na + and CO 3 2− were developed to co-substitute hydroxyapatite. • SiC nanowires were introduced into Na-doped carbonated hydroxyapatite. • CSH coatings showed excellent cell activity and cell proliferation behavior. - Abstract: A carbon/SiC nanowire/Na-doped carbonated hydroxyapatite multilayer coating (CSH coating) was prepared on carbon/carbon composites using a combination method of magnetron sputter ion plating, chemical vapor deposition and ultrasound-assisted electrochemical deposition procedure. The morphology, microstructure and chemical composition of the coating were investigated by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). The results showed that the CSH coating was consisted of three components: carbon layer, SiC nanowires and Na-doped carbonated hydroxyapatite. The carbon layer provided a dense and uniform surface structure for the growth of SiC nanowires. The SiC nanowires exhibited a porous structure, favoring the infiltration of Na-doped carbonated hydroxyapatite crystals. The Na-doped carbonated hydroxyapatite could infiltrate into the pores of SiC nanowires and finally cover the SiC nanowires entirely with a needle shape. The osteoblast-like MG63 cells were employed to assess the in vitro biocompatibility of the CSH coating. The MG63 cells favorably spread and grew well across the CSH coating surface with plenty of filopods and microvilli, exhibiting excellent cell activity. Moreover, the CSH coating elicited higher cell proliferation as compared to bare carbon/carbon composites. In conclusion, the CSH offers great potential as a coating material for future medical application in hard tissue replacement

  10. Preparation and characterization of carbon/SiC nanowire/Na-doped carbonated hydroxyapatite multilayer coating for carbon/carbon composites

    Energy Technology Data Exchange (ETDEWEB)

    Leilei, Zhang, E-mail: zhangleilei1121@aliyun.com; Hejun, Li; Kezhi, Li; Shouyang, Zhang; Qiangang, Fu; Yulei, Zhang; Jinhua, Lu; Wei, Li

    2014-09-15

    Highlights: • CSH coatings were prepared by combination of magnetron sputter ion plating, CVD and UECD. • Na{sup +} and CO{sub 3}{sup 2−} were developed to co-substitute hydroxyapatite. • SiC nanowires were introduced into Na-doped carbonated hydroxyapatite. • CSH coatings showed excellent cell activity and cell proliferation behavior. - Abstract: A carbon/SiC nanowire/Na-doped carbonated hydroxyapatite multilayer coating (CSH coating) was prepared on carbon/carbon composites using a combination method of magnetron sputter ion plating, chemical vapor deposition and ultrasound-assisted electrochemical deposition procedure. The morphology, microstructure and chemical composition of the coating were investigated by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). The results showed that the CSH coating was consisted of three components: carbon layer, SiC nanowires and Na-doped carbonated hydroxyapatite. The carbon layer provided a dense and uniform surface structure for the growth of SiC nanowires. The SiC nanowires exhibited a porous structure, favoring the infiltration of Na-doped carbonated hydroxyapatite crystals. The Na-doped carbonated hydroxyapatite could infiltrate into the pores of SiC nanowires and finally cover the SiC nanowires entirely with a needle shape. The osteoblast-like MG63 cells were employed to assess the in vitro biocompatibility of the CSH coating. The MG63 cells favorably spread and grew well across the CSH coating surface with plenty of filopods and microvilli, exhibiting excellent cell activity. Moreover, the CSH coating elicited higher cell proliferation as compared to bare carbon/carbon composites. In conclusion, the CSH offers great potential as a coating material for future medical application in hard tissue replacement.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  12. 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.

  13. 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.

  14. Methotrexate induces DNA damage and inhibits homologous recombination repair in choriocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Xie L

    2016-11-01

    Full Text Available Lisha Xie,1,* Tiancen Zhao,1,2,* Jing Cai,1 You Su,1 Zehua Wang,1 Weihong Dong1 1Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Department of Obstetrics and Gynecology, Central Hospital of Wuhan, Wuhan, China *These authors contributed equally to this work Objective: The objective of this study was to investigate the mechanism of sensitivity to methotrexate (MTX in human choriocarcinoma cells regarding DNA damage response. Methods: Two choriocarcinoma cancer cell lines, JAR and JEG-3, were utilized in this study. An MTX-sensitive osteosarcoma cell line MG63, an MTX-resistant epithelial ovarian cancer cell line A2780 and an MTX-resistant cervical adenocarcinoma cell line Hela served as controls. Cell viability assay was carried out to assess MTX sensitivity of cell lines. MTX-induced DNA damage was evaluated by comet assay. Quantitative reverse transcription polymerase chain reaction was used to detect the mRNA levels of BRCA1, BRCA2, RAD51 and RAD52. The protein levels of γH2AX, RAD 51 and p53 were analyzed by Western blot. Results: Remarkable DNA strand breaks were observed in MTX-sensitive cell lines (JAR, JEG-3 and MG63 but not in MTX-resistant cancer cells (A2780 and Hela after 48 h of MTX treatment. Only in the choriocarcinoma cells, the expression of homologous recombination (HR repair gene RAD51 was dramatically suppressed by MTX in a dose- and time-dependent manner, accompanied with the increase in p53. Conclusion: The MTX-induced DNA strand breaks accompanied by deficiencies in HR repair may contribute to the hypersensitivity to chemotherapy in choriocarcinoma. Keywords: choriocarcinoma, chemotherapy hypersensitivity, DNA double-strand break, RAD51, p53

  15. 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.

  16. Drug-releasing nano-engineered titanium implants: therapeutic efficacy in 3D cell culture model, controlled release and stability

    Energy Technology Data Exchange (ETDEWEB)

    Gulati, Karan [School of Chemical Engineering, The University of Adelaide, SA 5005 (Australia); Kogawa, Masakazu; Prideaux, Matthew; Findlay, David M. [Discipline of Orthopaedics and Trauma, The University of Adelaide, SA 5005 (Australia); Atkins, Gerald J., E-mail: gerald.atkins@adelaide.edu.au [Discipline of Orthopaedics and Trauma, The University of Adelaide, SA 5005 (Australia); Losic, Dusan, E-mail: dusan.losic@adelaide.edu.au [School of Chemical Engineering, The University of Adelaide, SA 5005 (Australia)

    2016-12-01

    There is an ongoing demand for new approaches for treating localized bone pathologies. Here we propose a new strategy for treatment of such conditions, via local delivery of hormones/drugs to the trauma site using drug releasing nano-engineered implants. The proposed implants were prepared in the form of small Ti wires/needles with a nano-engineered oxide layer composed of array of titania nanotubes (TNTs). TNTs implants were inserted into a 3D collagen gel matrix containing human osteoblast-like, and the results confirmed cell migration onto the implants and their attachment and spread. To investigate therapeutic efficacy, TNTs/Ti wires loaded with parathyroid hormone (PTH), an approved anabolic therapeutic for the treatment of severe bone fractures, were inserted into 3D gels containing osteoblast-like cells. Gene expression studies revealed a suppression of SOST (sclerostin) and an increase in RANKL (receptor activator of nuclear factor kappa-B ligand) mRNA expression, confirming the release of PTH from TNTs at concentrations sufficient to alter cell function. The performance of the TNTs wire implants using an example of a drug needed at relatively higher concentrations, the anti-inflammatory drug indomethacin, is also demonstrated. Finally, the mechanical stability of the prepared implants was tested by their insertion into bovine trabecular bone cores ex vivo followed by retrieval, which confirmed the robustness of the TNT structures. This study provides proof of principle for the suitability of the TNT/Ti wire implants for localized bone therapy, which can be customized to cater for specific therapeutic requirements. - Highlights: • Ti wire with titania nanotubes (TNTs) are proposed as ‘in-bone’ therapeutic implants. • 3D cell culture model is used to confirm therapeutic efficacy of drug releasing implants. Osteoblasts migrated and firmly attached to the TNTs and the micro-scale cracks. • Tailorable drug loading from few nanograms to several hundred

  17. Effects of hematin and Moessbauer gamma-radiation on human osteosarcoma cell lines

    International Nuclear Information System (INIS)

    Ortalli, I.; Pedrazzi, G.; Fano, V.; Ma, W.; Cai, S.; Giuliani, N.; Passeri, M.

    1996-01-01

    In the present work it is reported an investigation on cultures of the human osteosarcoma cell line 'MG-63' and the human osteoblastic cell line 'HOBIT'. It is examined the combined effects of hematin and 14,4keV gamma-radiation from a Moessbauer source. Preliminary results seem to confirm the trend already observed for the bone marrow system. Different degrees of growth inhibition were observed when hematin alone and hematin plus gamma-rays were administered to the cultures. It was previously found that hematin has no toxic effects on normal cells up to a concentration of 10 -4 M, but the same concentration produces various degrees of inhibition on cultures of tumor cells. While no significant effect could be attributed to irradiation alone, hematin plus irradiations show a larger inhibition than that expected for pure additive effects

  18. In vitro bone formation using muscle-derived cells: a new paradigm for bone tissue engineering using polymer-bone morphogenetic protein matrices.

    Science.gov (United States)

    Lu, Helen H; Kofron, Michelle D; El-Amin, Saadiq F; Attawia, Mohammed A; Laurencin, Cato T

    2003-06-13

    Over 800,000 bone grafting procedures are performed in the United States annually, creating a demand for viable alternatives to autogenous bone, the grafting standard in osseous repair. The objective of this study was to examine the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype and in vitro bone formation by muscle-derived cells. Specifically, we evaluated the ability of bone morphogenetic protein-7 (BMP-7), delivered from a poly(lactide-co-glycolide) (PLAGA) matrix, to induce the differentiation of cells derived from rabbit skeletal muscle into osteoblast-like cells and subsequently form mineralized tissue. Results confirmed that muscle-derived cells attached and proliferated on the PLAGA substrates. BMP-7 released from PLAGA induced the muscle-derived cells to increase bone marker expression and form mineralized cultures. These results demonstrate the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype by muscle-derived cells and present a new paradigm for bone tissue engineering.

  19. Vitamin K2 Ameliorates Damage of Blood Vessels by Glucocorticoid: a Potential Mechanism for Its Protective Effects in Glucocorticoid-induced Osteonecrosis of the Femoral Head in a Rat Model.

    Science.gov (United States)

    Zhang, Yuelei; Yin, Junhui; Ding, Hao; Zhang, Changqing; Gao, You-Shui

    2016-01-01

    Glucocorticoid has been reported to decrease blood vessel number and harm the blood supply in the femoral head, which is recognized to be an important mechanism of glucocorticoid-induced osteonecrosis of the femoral head (ONFH). To prevent glucocorticoid-induced ONFH, medication that promotes both bone formation and angiogenesis would be ideal. Vitamin K2 has been revealed to play an important role in bone metabolism; however, few studies have focused on the effect of Vitamin K2 on new vascular formation. Thus, this study aimed to investigate whether Vitamin K2 promoted new blood vessel formation in the presence of glucocorticoids, both in vitro and in vivo. The effect of Vitamin K2 on viability, migration, in vitro tube formation, and VEGF, vWF, CD31, KDR, Flt and PDGFB in EAhy926 incubated with or without dexamethasone were elucidated. VEGF, TGF-β and BMP-2, angiogenesis-related proteins secreted by osteoblasts, were also detected in the osteoblast-like cell line of MG63. In addition, blood vessels of the femoral head in rats administered with or without methylprednisolone and Vitamin K2 were evaluated using angiography and CD31 staining. In vitro studies showed that Vitamin K2 significantly protected endothelial cells from dexamethasone-induced apoptosis, promoted endothelial cell migration and in vitro tube formation. Angiogenesis-related proteins both in EAhy926 and MG63 were also upregulated by Vitamin K2 when cotreated with dexamethasone. In vivo studies showed enhanced blood vessel volume and CD31-positive staining cells in rats cotreated with VK2 and methylprednisolone compared to rats treated with methylprednisolone only. Collectively, Vitamin K2 has the ability to promote angiogenesis in vitro and to ameliorate vessels of the femoral head in glucocorticoid-treated rats in vivo, indicating that Vitamin K2 is a promising drug that may be used to prevent steroid-induced ONFH.

  20. Exosomes Derived from Human Bone Marrow Mesenchymal Stem Cells Promote Tumor Growth Through Hedgehog Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Jin Qi

    2017-08-01

    Full Text Available Background/Aims: Mesenchymal stem/stromal cells (MSCs are known to home to sites of tumor microenvironments where they participate in the formation of the tumor microenvironment and to interplay with tumor cells. However, the potential functional effects of MSCs on tumor cell growth are controversial. Here, we, from the view of bone marrow MSC-derived exosomes, study the molecular mechanism of MSCs on the growth of human osteosarcoma and human gastric cancer cells. Methods: MSCs derived from human bone marrow (hBMSCs were isolated and cultured in complete DMEM/F12 supplemented with 10% exosome-depleted fetal bovine serum and 1% penicillin-streptomycin, cell culture supernatants containing exosomes were harvested and exosome purification was performed by ultracentrifugation. Osteosarcoma (MG63 and gastric cancer (SGC7901 cells, respectively, were treated with hBMSC-derived exosomes in the presence or absence of a small molecule inhibitor of Hedgehog pathway. Cell viability was measured by transwell invasion assay, scratch migration assay and CCK-8 test. The expression of the signaling molecules Smoothened, Patched-1, Gli1 and the ligand Shh were tested by western blot and RT-PCR. Results: In this study, we found that hBMSC-derived exosomes promoted MG63 and SGC7901 cell growth through the activation of Hedgehog signaling pathway. Inhibition of Hedgehog signaling pathway significantly suppressed the process of hBMSC-derived exosomes on tumor growth. Conclusion: Our findings demonstrated the new roles of hedgehog signaling pathway in the hBMSCs-derived exosomes induced tumor progression.

  1. [Inhibitory effect of baicalein on the proliferation and invasion of osteosarcoma cells and mechanism].

    Science.gov (United States)

    Tang, Zhibin; Li, Chun; Chen, Zhiwei

    2015-03-01

    To explore the effect of baicalein on the proliferation and invasion of osteosarcoma cells and its related mechanism. Osteosarcoma MG-63 cells that were cultured in vitro were respectively treated with 20 μL culture medium (control group), dehydrated alcohol (0 μmol/L baicalein group), 100 and 200 μmol/L baicalein solution for 48 hours. Cell proliferation was analyzed by MTT assay. The cell invasion ability was detected using Transwell(TM) invasion assay. The expression of ezrin mRNA was examined by real-time quantitative PCR. The expressions of ezrin protein and p-ezrin protein were measured using Western blotting. Apoptosis index (AI) was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The inhibitory rates of cell proliferation significantly increased in 100 and 200 μmol/L baicalein groups as compared with 0 μmol/L baicalein group. Moreover, that was higher in 200 μmol/L baicalein group than in 100 μmol/L baicalein group. In comparison with control and 0 μmol/L baicalein groups, the mean cell numbers of permeated membrane and levels of ezrin mRNA, ezrin protein and p-ezrin protein gradually decreased, but AI was gradually elevated with the increase of baicalein concentrations, whereas there was no significant difference in these indicators between 0 μmol/L baicalein group and control group. Baicalein can inhibit the proliferation and invasion of osteosarcoma MG-63 cells. The mechanism may be associated with the inhibited expression and activity of ezrin protein and the promoted tumor cell apoptosis.

  2. Bioactivity and cytocompatibility of dicalcium phosphate/poly (amino acid) biocomposite with degradability

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Yunfei [College of Chemistry, Sichuan University, Chengdu 610064 (China); Shan Wenpeng; Li Xiangde [Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Wei Jie, E-mail: biomater2006@yahoo.com.cn [Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Li Hong [College of Physical Science and Technology, Sichuan University, Chengdu 610064 (China); Ma Jian [Hospital of Stomatology, Tongji University, Shanghai 200072 (China); Yan Yonggang, E-mail: yan_yonggang@vip.163.com [College of Physical Science and Technology, Sichuan University, Chengdu 610064 (China)

    2012-01-15

    A bioactive composite of dicalcium phosphate (DCP) and poly (amino acid) (PAA) was fabricated, and the in vitro bioactivity, degradability, and cellular responses to the DCP/PAA composite (DPC) were investigated as compared to PAA. Apatite formation on DPC surfaces occurred after immersion into simulated body fluid (SBF) for 7 days, but not on the surface of PAA. The weight loss ratio of DPC could reach 18.6 {+-} 0.3 wt% after soaking into phosphate buffered saline (PBS) for 2 months, which was higher than PAA (11.0 {+-} 0.2 wt%). Cell attachment and proliferation of MG-63 cells on DPC was obviously higher than on PAA. Moreover, the cells spread and formed confluent layer on the DPC surfaces. The alkaline phosphatase activity (ALP) of the cells on DPC was significantly greater than PAA at day 5 and day 7. The results suggested that introducing DCP into PAA makes the composite bioactive and more degradable, and meanwhile enhances osteoblast-like cells attach, proliferation and osteogenic differentiation.

  3. Raman spectroscopy for grading of live osteosarcoma cells.

    Science.gov (United States)

    Chiang, Yi-Hung; Wu, Stewart H; Kuo, Yi-Chun; Chen, How-Foo; Chiou, Arthur; Lee, Oscar K

    2015-04-18

    Osteosarcoma is the most common primary malignant bone tumor, and the grading of osteosarcoma cells relies on traditional histopathology and molecular biology methods, which require RNA extraction, protein isolation and immunohistological staining. All these methods require cell isolation, lysis or fixation, which is time-consuming and requires certain amount of tumor specimen. In this study, we report the use of Raman spectroscopy for grading of malignant osteosarcoma cells. We demonstrate that, based on the detection of differential production of mineral species, Raman spectroscopy can be used as a live cell analyzer to accurately assess the grades of osteosarcoma cells by evaluating their mineralization levels. Mineralization level was assessed by measuring amount of hydroxyapatite (HA), which is highly expressed in mature osteoblasts, but not in poorly differentiated osteosarcoma cell or mesenchymal stem cells, the putative cell-of-origin of osteosarcoma. We found that under Raman spectroscopy, the level of HA production was high in MG-63 cells, which are low-grade. Moreover, hydroxyapatite production was low in high-grade osteosarcoma cells such as 143B and SaOS2 cells (p Raman spectroscopy for the measurement of HA production by the protocol reported in this study may serve as a useful tool to rapidly and accurately assess the degree of malignancy in osteosarcoma cells in a label-free manner. Such application may shorten the period of pathological diagnosis and may benefit patients who are inflicted with osteosarcoma.

  4. Bone morphogenetic proteins regulate osteoprotegerin and its ligands in human vascular smooth muscle cells

    DEFF Research Database (Denmark)

    Knudsen, Kirsten Quyen Nguyen; Olesen, Ping; Ledet, Thomas

    2007-01-01

    The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved in the transformat......The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved...... in the transformation of human vascular smooth muscle cells (HVSMC) to osteoblast-like cells. In this study, we evaluated the effect of BMP-2, BMP-7 and transforming growth factor beta (TGF-beta1) on the secretion and mRNA expression of OPG and its ligands receptor activator of nuclear factor-kappabeta ligand (RANKL......) and TNF-related apoptosis-inducing ligand (TRAIL) in HVSMC. All three growth factors decreased OPG protein production significantly; these results were paralleled by reduced OPG mRNA expression. TRAIL mRNA levels were also decreased. RANKL mRNA expression declined when treated with TGF-beta1 but were...

  5. Bone tissue engineering with human mesenchymal stem cell sheets constructed using magnetite nanoparticles and magnetic force.

    Science.gov (United States)

    Shimizu, Kazunori; Ito, Akira; Yoshida, Tatsuro; Yamada, Yoichi; Ueda, Minoru; Honda, Hiroyuki

    2007-08-01

    An in vitro reconstruction of three-dimensional (3D) tissues without the use of scaffolds may be an alternative strategy for tissue engineering. We have developed a novel tissue engineering strategy, termed magnetic force-based tissue engineering (Mag-TE), in which magnetite cationic liposomes (MCLs) with a positive charge at the liposomal surface, and magnetic force were used to construct 3D tissue without scaffolds. In this study, human mesenchymal stem cells (MSCs) magnetically labeled with MCLs were seeded onto an ultra-low attachment culture surface, and a magnet (4000 G) was placed on the reverse side. The MSCs formed multilayered sheet-like structures after a 24-h culture period. MSCs in the sheets constructed by Mag-TE maintained an in vitro ability to differentiate into osteoblasts, adipocytes, or chondrocytes after a 21-day culture period using each induction medium. Using an electromagnet, MSC sheets constructed by Mag-TE were harvested and transplanted into the bone defect in the crania of nude rats. Histological observation revealed that new bone surrounded by osteoblast-like cells was formed in the defect area 14 days after transplantation with MSC sheets, whereas no bone formation was observed in control rats without the transplant. These results indicated that Mag-TE could be used for the transplantation of MSC sheets using magnetite nanoparticles and magnetic force, providing novel methodology for bone tissue engineering.

  6. Regulation of mesenchymal stromal cells through fine tuning of canonical Wnt signaling

    Directory of Open Access Journals (Sweden)

    Jin-A Kim

    2015-05-01

    Full Text Available Mesenchymal stromal cells (MSCs have been extensively utilized for various cell therapeutic trials, but the signals regulating their stromal function remain largely unclear. Here, we show that canonical Wnt signals distinctively regulate MSCs in a biphasic manner depending on signal intensity, i.e., MSCs exhibit proliferation and progenitor self-renewal under low Wnt/β-catenin signaling, whereas they exhibit enhanced osteogenic differentiation with priming to osteoblast-like lineages under high Wnt/β-catenin signaling. Moreover, low or high levels of β-catenin in MSCs distinctly regulated the hematopoietic support of MSCs to promote proliferation or the undifferentiated state of hematopoietic progenitors, respectively. A gene expression study demonstrated that different intracellular levels of β-catenin in MSCs induce distinct transcriptomic changes in subsets of genes belonging to different gene function categories. Different β-catenin levels also induced differences in intracellular levels of the β-catenin co-factors, Tcf-1 and Lef-1. Moreover, nano-scale mass spectrometry of proteins that co-precipitated with β-catenin revealed distinctive spectra of proteins selectively interacting with β-catenin at specific expression levels. Together, these results show that Wnt/β-catenin signals can coax distinct transcription milieu to induce different transcription profiles in MSCs depending on the signal intensity and that fine-tuning of the canonical Wnt signaling intensity can regulate the phase-specific functionality of MSCs.

  7. Coaxial electrospun aligned tussah silk fibroin nanostructured fiber scaffolds embedded with hydroxyapatite–tussah silk fibroin nanoparticles for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Weili [Key Laboratory of Advanced Textile Composites, Ministry of Education, Institute of Textile Composites, Tianjin Polytechnic University, Tianjin 300387 (China); He, Jianxin, E-mail: hejianxin771117@163.com [College of Textiles, Zhongyuan University of Technology, Zhengzhou 450007 (China); Collaborative Innovation Center of Textile and Garment Industry, Henan Province, Zhengzhou 450007 (China); Sang, Feng [Department of Acquired Immune Deficiency Syndrome Treatment and Research Center, The First Affiliated Hospital of Henan University of Traditional Chinese Medicine, Zhengzhou 450007 (China); Ding, Bin [College of Textiles, Zhongyuan University of Technology, Zhengzhou 450007 (China); Collaborative Innovation Center of Textile and Garment Industry, Henan Province, Zhengzhou 450007 (China); Chen, Li, E-mail: chenli@tjpu.edu.cn [Key Laboratory of Advanced Textile Composites, Ministry of Education, Institute of Textile Composites, Tianjin Polytechnic University, Tianjin 300387 (China); Cui, Shizhong; Li, Kejing; Han, Qiming; Tan, Weilin [College of Textiles, Zhongyuan University of Technology, Zhengzhou 450007 (China); Collaborative Innovation Center of Textile and Garment Industry, Henan Province, Zhengzhou 450007 (China)

    2016-01-01

    The bone is a composite of inorganic and organic materials and possesses a complex hierarchical architecture consisting of mineralized fibrils formed by collagen molecules and coated with oriented hydroxyapatite. To regenerate bone tissue, it is necessary to provide a scaffold that mimics the architecture of the extracellular matrix in native bone. Here, we describe one such scaffold, a nanostructured composite with a core made of a composite of hydroxyapatite and tussah silk fibroin. The core is encased in a shell of tussah silk fibroin. The composite fibers were fabricated by coaxial electrospinning using green water solvent and were characterized using different techniques. In comparison to nanofibers of pure tussah silk, composite notably improved mechanical properties, with 90-fold and 2-fold higher initial modulus and breaking stress, respectively, obtained. Osteoblast-like MG-63 cells were cultivated on the composite to assess its suitability as a scaffold for bone tissue engineering. We found that the fiber scaffold supported cell adhesion and proliferation and functionally promoted alkaline phosphatase and mineral deposition relevant for biomineralization. In addition, the composite were more biocompatible than pure tussah silk fibroin or cover slip. Thus, the nanostructured composite has excellent biomimetic and mechanical properties and is a potential biocompatible scaffold for bone tissue engineering. - Highlights: • A designing scaffold strategy to imitate the mineralized collagen bundles in natural bone was presented. • Aligned nanostructured composite fibers were fabricated by coaxial electrospinning using green water solvent. • Mechanical properties of aligned TSF nanofiber had been significantly improved by embedding with composite nanoparticles. • Composite scaffolds effectively supported proliferation of MG-63 cells and promoted biomineralization.

  8. Enhanced Hydrophilicity and Biocompatibility of Dental Zirconia Ceramics by Oxygen Plasma Treatment

    Directory of Open Access Journals (Sweden)

    Ching-Chou Wu

    2015-02-01

    Full Text Available Surface properties play a critical role in influencing cell responses to a biomaterial. The objectives of this study were (1 to characterize changes in surface properties of zirconia (ZrO2 ceramic after oxygen plasma treatment; and (2 to determine the effect of such changes on biological responses of human osteoblast-like cells (MG63. The results indicated that the surface morphology was not changed by oxygen plasma treatment. In contrast, oxygen plasma treatment to ZrO2 not only resulted in an increase in hydrophilicity, but also it retained surface hydrophilicity after 5-min treatment time. More importantly, surface properties of ZrO2 modified by oxygen plasma treatment were beneficial for cell growth, whereas the surface roughness of the materials did not have a significant efficacy. It is concluded that oxygen plasma treatment was certified to be effective in modifying the surface state of ZrO2 and has the potential in the creation and maintenance of hydrophilic surfaces and the enhancement of cell proliferation and differentiation.

  9. Novel cerium doped glass-reinforced hydroxyapatite with antibacterial and osteoconductive properties for bone tissue regeneration

    International Nuclear Information System (INIS)

    Morais, D S; Fernandes, S; Santos, J D; Lopes, M A; Gomes, P S; Fernandes, M H; Sampaio, P; Ferraz, M P; Sooraj Hussain, N

    2015-01-01

    The aim of this work was to develop a bioactive bone substitute with an effective antibacterial ability based on a cerium (Ce) doped glass-reinforced hydroxyapatite (GR-HA) composite. Developed composites were physicochemically characterized, using x-ray diffraction (XRD) analysis, SEM, energy dispersive x-ray spectroscopy (EDS), and flexural bending strength (FBS) tests. X-ray photoelectron spectroscopy (XPS) analysis was performed to analyze the oxidation state of Ce in the prepared doped glass. The antimicrobial activity of the composites was evaluated against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa; whether the cytocompatibility profile was assayed with human osteoblastic-like cells (Mg-63 cell line). The results revealed that the Ce inclusion in the GR-HA matrix induced the antimicrobial ability of the composite. In addition, Ce-doped materials reported an adequate biological behavior following seeding of osteoblastic populations, by inducing cell adhesion and proliferation. Developed materials were also found to enhance the expression of osteoblastic-related genes. Overall, the developed GR-HA-Ce composite is a prospective candidate to be used within the clinical scenario with a successful performance due to the effective antibacterial properties and capability of enhancing the osteoblastic cell response. (paper)

  10. Preliminary research on a novel bioactive silicon doped calcium phosphate coating on AZ31 magnesium alloy via electrodeposition

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Xun [Department of Materials Science and Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Institute of Metal Research, Chinese Academy of Science, Shenyang 110016 (China); Wan, Peng, E-mail: pwan@imr.ac.cn [Institute of Metal Research, Chinese Academy of Science, Shenyang 110016 (China); Tan, LiLi [Institute of Metal Research, Chinese Academy of Science, Shenyang 110016 (China); Fan, XinMin [Department of Materials Science and Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Ke [Institute of Metal Research, Chinese Academy of Science, Shenyang 110016 (China)

    2014-03-01

    A silicon doped calcium phosphate coating was obtained successfully on AZ31 alloy substrate via pulse electrodeposition. A novel dual-layer structure was observed with a porous lamellar-like and outer block-like apatite layer. In vitro immersion tests were adopted in simulated body fluid within 28 days of immersion. Slow degradation rate obtained from weight loss was observed for the Si-doped Ca–P coating, which was also consistent with the results of electrochemical experiments showing an enhanced corrosion resistance for the coating. Further formation of an apatite-like layer on the surface after immersion proved better integrity and biomineralization performance of the coating. Biological characterization was carried out for viability, proliferation and differentiation of MG63 osteoblast-like cells. The coating showed a good cell growth and an enhanced cell proliferation. Moreover, an increased activity of osteogenic marker ALP was found. All the results demonstrated that the Si-doped calcium phosphate was perspective to be used as a coating for magnesium alloy implants to control the degradation rate and enhance the bioactivity, which would facilitate the rapidity of bone tissue repair. - Highlights: • A Si-doped calcium phosphate coating was achieved via pulse ED on AZ31 alloy. • The coating was composed of a porous lamellar-like layer and outer block-like apatite. • The coating showed slow degradation rate and better biomineralization property. • The coating improved cell proliferation and activity of osteogenic marker ALP.

  11. Preliminary research on a novel bioactive silicon doped calcium phosphate coating on AZ31 magnesium alloy via electrodeposition

    International Nuclear Information System (INIS)

    Qiu, Xun; Wan, Peng; Tan, LiLi; Fan, XinMin; Yang, Ke

    2014-01-01

    A silicon doped calcium phosphate coating was obtained successfully on AZ31 alloy substrate via pulse electrodeposition. A novel dual-layer structure was observed with a porous lamellar-like and outer block-like apatite layer. In vitro immersion tests were adopted in simulated body fluid within 28 days of immersion. Slow degradation rate obtained from weight loss was observed for the Si-doped Ca–P coating, which was also consistent with the results of electrochemical experiments showing an enhanced corrosion resistance for the coating. Further formation of an apatite-like layer on the surface after immersion proved better integrity and biomineralization performance of the coating. Biological characterization was carried out for viability, proliferation and differentiation of MG63 osteoblast-like cells. The coating showed a good cell growth and an enhanced cell proliferation. Moreover, an increased activity of osteogenic marker ALP was found. All the results demonstrated that the Si-doped calcium phosphate was perspective to be used as a coating for magnesium alloy implants to control the degradation rate and enhance the bioactivity, which would facilitate the rapidity of bone tissue repair. - Highlights: • A Si-doped calcium phosphate coating was achieved via pulse ED on AZ31 alloy. • The coating was composed of a porous lamellar-like layer and outer block-like apatite. • The coating showed slow degradation rate and better biomineralization property. • The coating improved cell proliferation and activity of osteogenic marker ALP

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

    Science.gov (United States)

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

    2017-03-01

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

  13. Zr{sub 61}Ti{sub 2}Cu{sub 25}Al{sub 12} metallic glass for potential use in dental implants: Biocompatibility assessment by in vitro cellular responses

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jing [School of Stomatology, China Medical University, 117 Nanjing North Sreet, Shenyang, 110002 (China); Shi, Ling-ling; Zhu, Zhen-dong; He, Qiang [Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang, 110016 (China); Ai, Hong-jun, E-mail: aih0620@yahoo.com.cn [School of Stomatology, China Medical University, 117 Nanjing North Sreet, Shenyang, 110002 (China); Xu, Jian, E-mail: jianxu@imr.ac.cn [Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang, 110016 (China)

    2013-05-01

    In comparison with titanium and its alloys, Zr{sub 61}Ti{sub 2}Cu{sub 25}Al{sub 12} (ZT1) bulk metallic glass (BMG) manifests a good combination of high strength, high fracture toughness and lower Young's modulus. To examine its biocompatibility required for potential use in dental implants, this BMG was used as a cell growth subtract for three types of cell lines, L929 fibroblasts, human umbilical vein endothelial cells (HUVEC), and osteoblast-like MG63 cells. For a comparison, these cell lines were in parallel cultured and grown also on commercially pure titanium (CP-Ti) and Ti6–Al4–V alloy (Ti64). Cellular responses on the three metals, including adhesion, morphology and viability, were characterized using the SEM visualization and CCK-8 assay. Furthermore, real-time RT-PCR was used to measure the activity of integrin β, alkaline phosphatase (ALP) and type I collagen (COL I) in adherent MG63 cells. As indicated, in all cases of three cell lines, no significant differences in the initial attachment and viability/proliferation were found between ZT1, CP-Ti, and Ti64 until 5 d of incubation period. It means that the biocompatibility in cellular response for ZT1 BMG is comparable to Ti and its alloys. For gene expression of integrin β, ALP and COL I, mRNA level from osteoblast cells grown on ZT1 substrates is significantly higher than that on the CP-Ti and Ti64. It suggests that the adhesion and differentiation of osteoblasts grown on ZT1 are even superior to those on the CP-Ti and Ti64 alloy, then promoting bone formation. The good biocompatibility of ZT1 BMG is associated with the formation of zirconium oxide layer on the surface and good corrosion-resistance in physiological environment. Quantitative analysis of Real-time PCR for MG63 cells cultured on Zr{sub 61}Ti{sub 2}Cu{sub 25}Al{sub 12} BMG, CP-Ti, and Ti64 as well as plastic as a control at several incubation periods. Relative amounts of (a) integrin β, (b) ALP, and (c) COL I (*p < 0

  14. Zr61Ti2Cu25Al12 metallic glass for potential use in dental implants: Biocompatibility assessment by in vitro cellular responses

    International Nuclear Information System (INIS)

    Li, Jing; Shi, Ling-ling; Zhu, Zhen-dong; He, Qiang; Ai, Hong-jun; Xu, Jian

    2013-01-01

    In comparison with titanium and its alloys, Zr 61 Ti 2 Cu 25 Al 12 (ZT1) bulk metallic glass (BMG) manifests a good combination of high strength, high fracture toughness and lower Young's modulus. To examine its biocompatibility required for potential use in dental implants, this BMG was used as a cell growth subtract for three types of cell lines, L929 fibroblasts, human umbilical vein endothelial cells (HUVEC), and osteoblast-like MG63 cells. For a comparison, these cell lines were in parallel cultured and grown also on commercially pure titanium (CP-Ti) and Ti6–Al4–V alloy (Ti64). Cellular responses on the three metals, including adhesion, morphology and viability, were characterized using the SEM visualization and CCK-8 assay. Furthermore, real-time RT-PCR was used to measure the activity of integrin β, alkaline phosphatase (ALP) and type I collagen (COL I) in adherent MG63 cells. As indicated, in all cases of three cell lines, no significant differences in the initial attachment and viability/proliferation were found between ZT1, CP-Ti, and Ti64 until 5 d of incubation period. It means that the biocompatibility in cellular response for ZT1 BMG is comparable to Ti and its alloys. For gene expression of integrin β, ALP and COL I, mRNA level from osteoblast cells grown on ZT1 substrates is significantly higher than that on the CP-Ti and Ti64. It suggests that the adhesion and differentiation of osteoblasts grown on ZT1 are even superior to those on the CP-Ti and Ti64 alloy, then promoting bone formation. The good biocompatibility of ZT1 BMG is associated with the formation of zirconium oxide layer on the surface and good corrosion-resistance in physiological environment. Quantitative analysis of Real-time PCR for MG63 cells cultured on Zr 61 Ti 2 Cu 25 Al 12 BMG, CP-Ti, and Ti64 as well as plastic as a control at several incubation periods. Relative amounts of (a) integrin β, (b) ALP, and (c) COL I (*p < 0.05). Highlights: ► Cellular response to Zr

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-11-01

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

  17. miR-367 regulation of DOC-2/DAB2 interactive protein promotes proliferation, migration and invasion of osteosarcoma cells.

    Science.gov (United States)

    Cai, Wei; Jiang, Haitao; Yu, Yifan; Xu, Yong; Zuo, Wenshan; Wang, Shouguo; Su, Zhen

    2017-11-01

    Recently, miR-367 is reported to exert either oncogenic or tumor suppressive effects in human malignancies. Recent study reports that miR-367 is up-regulated in OS tissues and cell lines, and abrogates adriamycin-induced apoptosis. The clinical significance of miR-367 and its function in OS need further investigation. In our study, miR-367 expression in OS was markedly elevated compared with corresponding non-tumor tissues. High miR-367 expression was associated with malignant clinical features and poor prognosis of OS patients. In accordance, the levels of miR-367 were dramatically up-regulated in OS cells. Loss of miR-367 expression in Saos-2 cells obviously inhibited the proliferation, migration and invasion of cancer cells in vitro. Meanwhile, miR-367 restoration promoted these malignant behaviors of MG-63 cells. Mechanistically, miR-367 negatively regulated DOC-2/DAB2 interactive protein (DAB2IP) abundance in OS cells. Hereby, DAB2IP was recognized as a direct target gene of miR-367 in OS. DAB2IP mRNA level was down-regulated and inversely correlated with miR-367 expression in OS specimens. DAB2IP overexpression prohibited proliferation, migration and invasion in Saos-2 cells, while DAB2IP knockdown showed promoting effects on proliferation, migration and invasion of MG-63 cells. Furthermore, the role of miR-367 might be mediated by DAB2IP-regulated phosphorylation of ERK and AKT in OS cells. To conclude, miR-367 may function as a biomarker for prediction of prognosis and a target for OS therapy. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  18. Microcalcifications in breast cancer: an active phenomenon mediated by epithelial cells with mesenchymal characteristics

    International Nuclear Information System (INIS)

    Scimeca, Manuel; Giannini, Elena; Antonacci, Chiara; Pistolese, Chiara Adriana; Spagnoli, Luigi Giusto; Bonanno, Elena

    2014-01-01

    hypothesize that under specific stimuli, mammary cells, which despite retaining a minimal epithelial phenotype (confirmed by cytokeratin expression), may acquire some mesenchymal characteristics transforming themselves into cells with an osteoblast-like phenotype, and are able to contribute to the production of breast microcalcifications

  19. Evaluation of antibacterial and cytotoxic effects of nano-sized bioactive glass/collagen composites releasing tetracycline hydrochloride.

    Science.gov (United States)

    Rivadeneira, J; Di Virgilio, A L; Audisio, M C; Boccaccini, A R; Gorustovich, A A

    2014-06-01

    To evaluate the antibacterial efficacy of silicate bioactive glass nanoparticles/collagen composites functionalized with tetracycline hydrochloride (TCH). Different concentrations of tetracycline hydrochloride (TCH) were incorporated on silicate bioactive glass nanoparticles/collagen composites by dipping these biomaterials for 48 h at 37°C in a solution of simulated body fluid (SBF) plus 0·05, 0·20 or 0·35 mg ml(-1) of the antibiotic. TCH release was assessed in double-distilled water at 37°C up to 72 h. The antibacterial activity of the samples has been evaluated in two ways: inhibition zone test and plate count method. The experiments were performed in vitro up to 48 h on four staphylococci strains (Staphylococcus aureus ATCC29213, ATCC25923, ATCC6538P and Staphylococcus epidermidis ATCC12228). The new composites were also tested for cytotoxicity on MG-63 human osteosarcoma cells. The results showed that the incorporation and release of TCH was dependent on the initial concentration of TCH in SBF. The biomaterials also inhibited the Staph. aureus cell growth even though the efficacy was similar for all concentration. On the other hand, no cytotoxic effects were found on osteoblast-like cells, even at the highest concentration. Considering all results, it can be concluded that the new composite acts as a suitable bioactive carrier of TCH and could have potential in the prevention of biomaterial related infections. The results suggest a potential application as wound dressing. © 2014 The Society for Applied Microbiology.

  20. A comparative study of thermal calcination and an alkaline hydrolysis method in the isolation of hydroxyapatite from Thunnus obesus bone

    International Nuclear Information System (INIS)

    Venkatesan, Jayachandran; Ryu, BoMi; Thomas, Noel Vinay; Kim, Se Kwon; Qian Zhongji

    2011-01-01

    In the present study, hydroxyapatite (HAp) was isolated from Thunnus obesus bone using alkaline hydrolysis and thermal calcination methods. The obtained ceramic has been characterized by thermal gravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), powder x-ray diffraction analysis (XRD), field-emission scanning electron microscopy, energy-dispersive x-ray analysis, transmission electron microscopy (TEM), selected area diffraction analysis, cytotoxic analysis and cell proliferation analysis. The results indicate that there are significant differences between the ceramics and T. obesus bone. FT-IR and TGA results affirmed that the collagen and organic moieties have been eliminated by both the proposed methods. XRD results were in agreement with JCPDS data. TEM and selective area diffraction images have signified that the thermal calcination method produces good crystallinity with dimensions 0.3-1.0 μm, whereas the alkaline hydrolysis method produces nanostructured HAp crystals with 17-71 nm length and 5-10 nm width. Biocompatibility of HAp crystals was evaluated by cytotoxicity and cell proliferation with human osteoblast-like cell MG-63.

  1. A comparative study of thermal calcination and an alkaline hydrolysis method in the isolation of hydroxyapatite from Thunnus obesus bone

    Energy Technology Data Exchange (ETDEWEB)

    Venkatesan, Jayachandran; Ryu, BoMi; Thomas, Noel Vinay; Kim, Se Kwon [Department of Chemistry, Pukyong National University, Busan 608-737 (Korea, Republic of); Qian Zhongji, E-mail: sknkim@pknu.ac.kr [Marine Bioprocess Research Center, Pukyong National University, Busan 608-737 (Korea, Republic of)

    2011-06-15

    In the present study, hydroxyapatite (HAp) was isolated from Thunnus obesus bone using alkaline hydrolysis and thermal calcination methods. The obtained ceramic has been characterized by thermal gravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), powder x-ray diffraction analysis (XRD), field-emission scanning electron microscopy, energy-dispersive x-ray analysis, transmission electron microscopy (TEM), selected area diffraction analysis, cytotoxic analysis and cell proliferation analysis. The results indicate that there are significant differences between the ceramics and T. obesus bone. FT-IR and TGA results affirmed that the collagen and organic moieties have been eliminated by both the proposed methods. XRD results were in agreement with JCPDS data. TEM and selective area diffraction images have signified that the thermal calcination method produces good crystallinity with dimensions 0.3-1.0 {mu}m, whereas the alkaline hydrolysis method produces nanostructured HAp crystals with 17-71 nm length and 5-10 nm width. Biocompatibility of HAp crystals was evaluated by cytotoxicity and cell proliferation with human osteoblast-like cell MG-63.

  2. Microwave sintering and in vitro study of defect-free stable porous multilayered HAp–ZrO2 artificial bone scaffold

    Directory of Open Access Journals (Sweden)

    Dong-Woo Jang, Thi-Hiep Nguyen, Swapan Kumar Sarkar and Byong-Taek Lee

    2012-01-01

    Full Text Available Continuously porous hydroxyapatite (HAp/t-ZrO2 composites containing concentric laminated frames and microchanneled bodies were fabricated by an extrusion process. To investigate the mechanical properties of HAp/t-ZrO2 composites, the porous composites were sintered at different temperatures using a microwave furnace. The microstructure was designed to imitate that of natural bone, particularly small bone, with both cortical and spongy bone sections. Each microchannel was separated by alternating lamina of HAp, HAp–(t-ZrO2 and t-ZrO2. HAp and ZrO2 phases existed on the surface of the microchannel and the core zone to increase the biocompatibility and mechanical properties of the HAp-ZrO2 artificial bone. The sintering behavior was evaluated and the optimum sintering temperature was found to be 1400 °C, which produced a stable scaffold. The material characteristics, such as the microstructure, crystal structure and compressive strength, were evaluated in detail for different sintering temperatures. A detailed in vitro study was carried out using MTT assay, western blot analysis, gene expression by polymerase chain reaction and laser confocal image analysis of cell proliferation. The results confirmed that HAp-ZrO2 performs as an artificial bone, showing excellent cell growth, attachment and proliferation behavior using osteoblast-like MG63 cells.

  3. 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.

  4. 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

  5. Novel poly(butylene succinate nanocomposites containing strontium hydroxyapatite nanorods with enhanced osteoconductivity for tissue engineering applications

    Directory of Open Access Journals (Sweden)

    M. Nerantzaki

    2015-09-01

    Full Text Available Three series of poly(butylene succinate (PBSu nanocomposites containing 0.5, 1 and 2.5 wt% strontium hydroxyapatite [Sr5(PO43OH] nanorods (SrHAp nrds were prepared by in situ polymerisation. The structural effects of Sr5(PO43OH nanorods, for the different concentrations, inside the polymeric matrix (PBSu, were studied through high angle annular dark field scanning transmission electron microscopy (HAADF-STEM. HAADF-STEM measurements revealed that the SrHAp nanorods at low concentrations are dispersed inside the polymeric PBSu matrix while in 1 wt% some aggregates are formed. These aggregations affect the mechanical properties giving an enhancement for the concentration of 0.5 wt% SrHAp nrds in tensile strength, while a reduction is recorded for higher loadings of the nanofiller. Studies on enzymatic hydrolysis revealed that all nanocomposites present higher hydrolysis rates than neat PBSu, indicating that nanorods accelerate the hydrolysis degradation process. In vitro bioactivity tests prove that SrHAp nrds promote the formation of hydroxyapatite on the PBSu surface. All nanocomposites were tested also in relevant cell culture using osteoblast-like cells (MG-63 to demonstrate their biocompatibility showing SrHAp nanorods support cell attachment.

  6. Biomimetic modification of synthetic hydrogels by incorporation of adhesive peptides and calcium phosphate nanoparticles: in vitro evaluation of cell behavior

    Directory of Open Access Journals (Sweden)

    M Bongio

    2011-12-01

    Full Text Available The ultimate goal of this work was to develop a biocompatible and biomimetic in situ crosslinkable hydrogel scaffold with an instructive capacity for bone regenerative treatment. To this end, synthetic hydrogels were functionalized with two key components of the extracellular matrix of native bone tissue, i.e. the three-amino acid peptide sequence RGD (which is the principal integrin-binding domain responsible for cell adhesion and survival of anchorage-dependent cells and calcium phosphate (CaP nanoparticles in the form of hydroxyapatite (which are similar to the inorganic phase of bone tissue. Rat bone marrow osteoblast-like cells (OBLCs were encapsulated in four different biomaterials (plain oligo(poly(ethylene glycol fumarate (OPF, RGD-modified OPF, OPF enriched with CaP nanoparticles and RGD-modified OPF enriched with CaP nanoparticles and cell survival, cell spreading, proliferation and mineralized matrix formation were determined via cell viability assay, histology and biochemical analysis for alkaline phosphatase activity and calcium. This study showed that RGD peptide sequences promoted cell spreading in OPF hydrogels and hence play a crucial role in cell survival during the early stage of culture, whereas CaP nanoparticles significantly enhanced cell-mediated hydrogel mineralization. Although cell spreading and proliferation activity were inhibited, the combined effect of RGD peptide sequences and CaP nanoparticles within OPF hydrogel systems elicited a better biological response than that of the individual components. Specifically, both a sustained cell viability and mineralized matrix production mediated by encapsulated OBLCs were observed within these novel biomimetic composite systems.

  7. Curcumin Analog DK1 Induces Apoptosis in Human Osteosarcoma Cells In Vitro through Mitochondria-Dependent Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Muhammad Nazirul Mubin Aziz

    2018-01-01

    Full Text Available Osteosarcoma is one of the primary malignant bone tumors that confer low survival rates for patients even with intensive regime treatments. Therefore, discovery of novel anti-osteosarcoma drugs derived from natural products that are not harmful to the normal cells remains crucial. Curcumin is one of the natural substances that have been extensively studied due to its anti-cancer properties and is pharmacologically safe considering its ubiquitous consumption for centuries. However, curcumin suffers from a poor circulating bioavailability, which has led to the development of a chemically synthesized curcuminoid analog, namely (Z-3-hydroxy-1-(2-hydroxyphenyl-3-phenylprop-2-en-1-one (DK1. In this study, the cytotoxic effects of the curcumin analog DK1 was investigated in both U-2OS and MG-63 osteosarcoma cell lines using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and cell death was microscopically examined via acridine orange/propidium iodide (AO/PI double staining. Flow cytometer analysis including Annexin V/Fluorescein isothiocyanate (FITC, cell cycle analysis and JC-1 were adapted to determine the mode of cell death. Subsequently in order to determine the mechanism of cell death, quantitative polymerase chain reaction (qPCR and proteome profiling was carried out to measure the expression of several apoptotic-related genes and proteins. Results indicated that DK1 induced U-2 OS and MG-63 morphological changes and substantially reduced cell numbers through induction of apoptosis. Several apoptotic genes and proteins were steadily expressed after treatment with DK1; including caspase 3, caspase 9, and BAX, which indicated that apoptosis occurred through a mitochondria-dependent signaling pathway. In conclusion, DK1 could be considered as a potential candidate for an anti-osteosarcoma drug in the near future, contingent upon its ability to induce apoptosis in osteosarcoma cell lines.

  8. Micropatterned Silica Films with Nanohydroxyapatite for Y-TZP Implants.

    Science.gov (United States)

    Miranda, R B P; Grenho, L; Carvalho, A; Fernandes, M H; Monteiro, F J; Cesar, P F

    2018-03-01

    This investigation aimed at developing micropatterned silica thin films (MSTFs) containing nanohydroxyapatite (nano-HA) microaggregates that were not completely covered by silica so that they could directly interact with the surrounding cells. The objectives were 1) to evaluate the effect of the presence of 2 films (MSTF with or without nano-HA addition) on the characteristic strength (σ 0 ) and Weibull modulus ( m) of a yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) and 2) to evaluate the effect of these 2 films, as applied onto the Y-TZP surface, on the morphology, orientation, and proliferation of MG63 cells. Sol-gel process and soft lithography were used to apply the MSTF onto the Y-TZP specimens. Three experimental groups were produced: Y-TZP, Y-TZP + MSTF, and Y-TZP + MSTF + sprayed nano-HA. All surfaces were characterized by scanning electron microscopy and energy-dispersive X-ray spectroscopy and tested for 4-point flexural strength ( n = 30) in water at 37 °C. Weibull analysis was used to determine m and σ 0 (maximum likelihood method). In vitro biological behavior was performed with human osteoblast-like cells (MG63). Y-TZP was successfully coated with MSFT and MSFT + nano-HA. Scanning electron microscopy micrographs indicated that the microaggregates of nano-HA were not entirely covered by the silica. There was no statistically significant difference among the experimental groups for σ 0 and m. In the groups containing the films, the cells were elongated and aligned along the lines. The MSFT + nano-HA group showed significantly higher cell metabolic activity than that obtained for the Y-TZP group at day 7. This investigation was successful in producing an MSTF containing nano-HA microaggregates that remained exposed to the environment. The developed films did not jeopardize the structural reliability of a commercial Y-TZP, as confirmed by the Weibull statistics. The MG63 cells seeded over the films became elongated and aligned along the

  9. 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

  10. 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

  11. Effect of ethanol on human osteosarcoma cell proliferatation, differentiation and mineralization

    International Nuclear Information System (INIS)

    Vignesh, R.C.; Sitta Djody, S.; Jayasudha, E.; Gopalakrishnan, V.; Ilangovan, R.; Balaganesh, M.; Veni, S.; Sridhar, M.; Srinivasan, N.

    2006-01-01

    The habitual consumption of even moderate quantities of alcoholic beverages is clearly associated with reduced bone mass, increased prevalence of skeletal fracture and also it is the major risk factor for the development of secondary osteoporosis. The present in vitro study was designed to determine the dose response effects of ethanol on osteoblast-like human osteosarcoma cells (SaOS-2) proliferation, differentiation, mineralization and cyto-toxicity. SaOS-2 cells were plated in 48 and 6 well culture plates and exposed to different concentrations of ethanol (1, 10, 100, 200 and 300 mM) for 24, 48 and 72 h. At the end of incubation, proliferation of cells was studied using crystal violet Bioassay. The cell lysate was utilized to determine ALP activity and conditioned media were used to measure LDH activity. Histochemical localization of ALP and mineralized nodules were studied from cells treated with ethanol (10 and 100 mM) for 21 days. At higher doses, there was a significant reduction in cell number, whereas at lower doses there were variable effects. In 24 h treatment, the higher doses showed a significant increase in ALP activity, whereas 48 and 72 h treatments showed an opposite trend. Ethanol treatment caused a dose- and time-dependent increase in LDH activity. Ethanol treatment altered the quality of mineralization at 10 mM dose whereas completely inhibited mineralization at 100 mM dose, despite the presence of serum. In conclusion, the toxic effect of ethanol is reflected on cell proliferation, differentiation and mineralization even at low doses and at extended treatment duration

  12. 3D Powder Printed Bioglass and β-Tricalcium Phosphate Bone Scaffolds

    Directory of Open Access Journals (Sweden)

    Michael Seidenstuecker

    2017-12-01

    Full Text Available The use of both bioglass (BG and β tricalcium phosphate (β-TCP for bone replacement applications has been studied extensively due to the materials’ high biocompatibility and ability to resorb when implanted in the body. 3D printing has been explored as a fast and versatile technique for the fabrication of porous bone scaffolds. This project investigates the effects of using different combinations of a composite BG and β-TCP powder for 3D printing of porous bone scaffolds. Porous 3D powder printed bone scaffolds of BG, β-TCP, 50/50 BG/β-TCP and 70/30 BG/β-TCP compositions were subject to a variety of characterization and biocompatibility tests. The porosity characteristics, surface roughness, mechanical strength, viability for cell proliferation, material cytotoxicity and in vitro bioactivity were assessed. The results show that the scaffolds can support osteoblast-like MG-63 cells growth both on the surface of and within the scaffold material and do not show alarming cytotoxicity; the porosity and surface characteristics of the scaffolds are appropriate. Of the two tested composite materials, the 70/30 BG/β-TCP scaffold proved to be superior in terms of biocompatibility and mechanical strength. The mechanical strength of the scaffolds makes them unsuitable for load bearing applications. However, they can be useful for other applications such as bone fillers.

  13. Electrochemical and biological characterization of coatings formed on Ti-15Mo alloy by plasma electrolytic oxidation.

    Science.gov (United States)

    Kazek-Kęsik, Alicja; Krok-Borkowicz, Małgorzata; Pamuła, Elżbieta; Simka, Wojciech

    2014-10-01

    β-Type titanium alloys are considered the future materials for bone implants. To improve the bioactivity of Ti-15Mo, the surface was modified using the plasma electrolytic oxidation (PEO) process. Tricalcium phosphate (TCP, Ca3PO4), wollastonite (CaSiO3) and silica (SiO2) were selected as additives in the anodizing bath to enhance the bioactivity of the coatings formed during the PEO process. Electrochemical analysis of the samples was performed in Ringer's solution at 37°C. The open-circuit potential (EOCP) as a function of time, corrosion potential (ECORR), corrosion current density (jCORR) and polarization resistance (Rp) of the samples were determined. Surface modification improved the corrosion resistance of Ti-15Mo in Ringer's solution. In vitro studies with MG-63 osteoblast-like cells were performed for 1, 3 and 7 days. After 24h, the cells were well adhered on the entire surfaces, and their number increased with increasing culture time. The coatings formed in basic solution with wollastonite exhibited better biological performance compared with the as-ground sample. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. 3D Biomimetic Magnetic Structures for Static Magnetic Field Stimulation of Osteogenesis

    Directory of Open Access Journals (Sweden)

    Irina Alexandra Paun

    2018-02-01

    Full Text Available We designed, fabricated and optimized 3D biomimetic magnetic structures that stimulate the osteogenesis in static magnetic fields. The structures were fabricated by direct laser writing via two-photon polymerization of IP-L780 photopolymer and were based on ellipsoidal, hexagonal units organized in a multilayered architecture. The magnetic activity of the structures was assured by coating with a thin layer of collagen-chitosan-hydroxyapatite-magnetic nanoparticles composite. In vitro experiments using MG-63 osteoblast-like cells for 3D structures with gradients of pore size helped us to find an optimum pore size between 20–40 µm. Starting from optimized 3D structures, we evaluated both qualitatively and quantitatively the effects of static magnetic fields of up to 250 mT on cell proliferation and differentiation, by ALP (alkaline phosphatase production, Alizarin Red and osteocalcin secretion measurements. We demonstrated that the synergic effect of 3D structure optimization and static magnetic stimulation enhances the bone regeneration by a factor greater than 2 as compared with the same structure in the absence of a magnetic field.

  15. Influence of processing parameters on pore structure of 3D porous chitosan-alginate polyelectrolyte complex scaffolds.

    Science.gov (United States)

    Florczyk, Stephen J; Kim, Dae-Joon; Wood, David L; Zhang, Miqin

    2011-09-15

    Fabrication of porous polymeric scaffolds with controlled structure can be challenging. In this study, we investigated the influence of key experimental parameters on the structures and mechanical properties of resultant porous chitosan-alginate (CA) polyelectrolyte complex (PEC) scaffolds, and on proliferation of MG-63 osteoblast-like cells, targeted at bone tissue engineering. We demonstrated that the porous structure is largely affected by the solution viscosity, which can be regulated by the acetic acid and alginate concentrations. We found that the CA PEC solutions with viscosity below 300 Pa.s yielded scaffolds of uniform pore structure and that more neutral pH promoted more complete complexation of chitosan and alginate, yielding stiffer scaffolds. CA PEC scaffolds produced from solutions with viscosities below 300 Pa.s also showed enhanced cell proliferation compared with other samples. By controlling the key experimental parameters identified in this study, CA PEC scaffolds of different structures can be made to suit various tissue engineering applications. Copyright © 2011 Wiley Periodicals, Inc.

  16. 3D Biomimetic Magnetic Structures for Static Magnetic Field Stimulation of Osteogenesis.

    Science.gov (United States)

    Paun, Irina Alexandra; Popescu, Roxana Cristina; Calin, Bogdan Stefanita; Mustaciosu, Cosmin Catalin; Dinescu, Maria; Luculescu, Catalin Romeo

    2018-02-07

    We designed, fabricated and optimized 3D biomimetic magnetic structures that stimulate the osteogenesis in static magnetic fields. The structures were fabricated by direct laser writing via two-photon polymerization of IP-L780 photopolymer and were based on ellipsoidal, hexagonal units organized in a multilayered architecture. The magnetic activity of the structures was assured by coating with a thin layer of collagen-chitosan-hydroxyapatite-magnetic nanoparticles composite. In vitro experiments using MG-63 osteoblast-like cells for 3D structures with gradients of pore size helped us to find an optimum pore size between 20-40 µm. Starting from optimized 3D structures, we evaluated both qualitatively and quantitatively the effects of static magnetic fields of up to 250 mT on cell proliferation and differentiation, by ALP (alkaline phosphatase) production, Alizarin Red and osteocalcin secretion measurements. We demonstrated that the synergic effect of 3D structure optimization and static magnetic stimulation enhances the bone regeneration by a factor greater than 2 as compared with the same structure in the absence of a magnetic field.

  17. In-vitro studies with 188Re-HEDP, a clinically used bone pain palliating agent, on bone cancer cells

    International Nuclear Information System (INIS)

    Sharma, Rohit; Kumar, Chandan; Mallia, Madhava B.; Banerjee, Sharmila; Kameswaran, Mythili

    2017-01-01

    Rhenium-188 is an attractive radioisotope for a wide variety of radiotherapy applications. 188 Re-HEDP (HEDPhydroxyethylidene- 1,1-diphosphonic acid) is one such, clinically useful, radiopharmaceutical for palliation of bone pain due to osseous metastasis. Herein, our aim was to study the uptake and retention of 188 Re-HEDP in mineralized bone and to assess its cellular toxicity, along with its underlying mechanism in human osteocarcinoma (MG-63 and Soas-2) cell lines. 188 Re-HEDP uptake was found to be significantly higher in mineralized bone. The 188 Re-HEDP complex also induces G2-M cell cycle arrest and thus contributing to apoptosis and cellular toxicity in bone cancer cells. (author)

  18. Microstructure, corrosion resistance and cytocompatibility of Mg-5Y-4Rare Earth-0.5Zr (WE54) alloy

    Energy Technology Data Exchange (ETDEWEB)

    Smola, Bohumil, E-mail: bohumil.smola@mff.cuni.cz [Charles University Prague, Faculty of Mathematics and Physics, Ke Karlovu 5, 121 16 Praha 2 (Czech Republic); Joska, Ludek [Institute of Chemical Technology Prague, Faculty of Chemical Technology, Technicka 5, 166 28 Praha 6 (Czech Republic); Brezina, Vitezslav [University of South Bohemia, Institute of Physical Biology, Zamek 136, 373 33 Nove Hrady (Czech Republic); Stulikova, Ivana [Charles University Prague, Faculty of Mathematics and Physics, Ke Karlovu 5, 121 16 Praha 2 (Czech Republic); Hnilica, Frantisek [Czech Technical University in Prague, Faculty of Mechanical Engineering, Karlovo namesti 13, 121 35 Praha 2 (Czech Republic)

    2012-05-01

    Conventionally cast Mg-5Y-4Rare Earth-0.5Zr alloy (WE54) was solution treated (525 Degree-Sign C/8 h - T4) and one part subsequently aged (200 Degree-Sign C/16 h - T6). Powder from the cast WE54 alloy prepared by gas atomizing was consolidated by extrusion at 250 Degree-Sign C or 400 Degree-Sign C. Dense triangular arrangement of prismatic plates of transient D0{sub 19} and C-base centered orthorhombic phases precipitated in the {alpha}-Mg matrix during the T6 treatment. Both alloys prepared by powder metallurgy exhibit similar microstructure consisting of {approx} 4-6 {mu}m {alpha}-Mg matrix fibers surrounded by particles of the equilibrium Mg{sub 5}(Y, Nd) phase and of oxides. Open circuit potential and polarization resistance in the isotonic saline (9 g/l NaCl/H{sub 2}O) were monitored for 24 h. The corrosion rate of the T4 and T6 treated alloys was about 80 times lower than that of commercial Mg. Both alloys prepared by powder metallurgy exhibited approximately 8 times higher corrosion resistance than commercial Mg. The human MG-63 osteoblast-like cells spreading and division in the extracts (0.28 g in 28 ml of EMEM) of all 4 alloys were monitored by cinemicrography for 24 h. The MG-63 cells proliferate without cytotoxicity in all extracts. - Highlights: Black-Right-Pointing-Pointer T6 treated WE54 alloy exhibit dense triangular arrangement of {beta} Double-Prime and {beta} Prime phase prismatic plates. Black-Right-Pointing-Pointer Microstructure of PM prepared WE54 alloy consists of {alpha}-Mg phase cells surrounded by {beta} phase particles. Black-Right-Pointing-Pointer PM produced WE54 corroded 10 times faster in physiological solution thanT4 and T6 treated WE54. Black-Right-Pointing-Pointer MG63 cell spreading in EMEM extracts of PM prepared WE54 is comparable to that in control EMEM. Black-Right-Pointing-Pointer Cell mitosis is enhanced in PM WE54 extracts compared to the control and extracts of T4 and T6 WE54.

  19. Gene expression of runx2, Osterix, c-fos, DLX-3, DLX-5, and MSX-2 in dental follicle cells during osteogenic differentiation in vitro.

    Science.gov (United States)

    Morsczeck, C

    2006-02-01

    Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast- like cells under in vitro conditions. However, mechanisms for osteogenic differentiation are not known in detail. Dental follicle cell long-term cultures supplemented with dexamethasone or with insulin resulted in mineralized nodules, whereas no mineralization or alkaline phosphatase activity was detected in the control culture without an osteogenic stimulus. A real-time reverse-transcriptase polymerase chain reaction (PCR) analysis was developed to investigate gene expression during osteogenic differentiation in vitro. Expression of the alkaline phosphatase (ALP) gene was detected during differentiation in the control culture and was similar to that in cultures with dexamethasone and insulin. DLX-3, DLX-5, runx2, and MSX-2 are differentially expressed during osteogenic differentiation in bone marrow mesenchymal stem cells. In dental follicle cells, gene expression of runx2, DLX-5, and MSX-2 was unaffected during osteogenic differentiation in vitro. Osteogenic differentiation appeared to be independent of MSX-2 expression; the same was true of runx2 and DLX-5, which were protagonists of osteogenic differentiation and osteocalcin promoter activity in bone marrow mesenchymal stem cells. Like in bone marrow-derived stem cells, DLX-3 gene expression was increased in dental follicle cells during osteogenic differentiation but similar to control cultures. However, gene expression of osterix was not detected in dental follicle cells during osteogenic differentiation; this gene is expressed during osteogenic differentiation in bone marrow stem cells. These real-time PCR results display molecular mechanisms in dental follicle precursor cells during osteogenic differentiation that are different from those in bone marrow-derived mesenchymal stem cells.

  20. Paris polyphylla Suppresses Proliferation and Vasculogenic Mimicry of Human Osteosarcoma Cells and Inhibits Tumor Growth In Vivo.

    Science.gov (United States)

    Yao, Nan; Ren, Ke; Wang, Yimin; Jin, Qiaomei; Lu, Xiao; Lu, Yan; Jiang, Cuihua; Zhang, Dongjian; Lu, Jun; Wang, Chen; Huo, Jiege; Chen, Yong; Zhang, Jian

    2017-01-01

    Paris polyphylla, a traditional antipyretic-detoxicate chinese medicinal herb, has been applied extensively in cancer treatments for nearly 2000 years. The purpose of the present study is to evaluate the potential anti-osteosarcoma effects of Paris polyphylla ethanol extract (PPEE) and to investigate its underlying mechanisms. The antiproliferation activity of PPEE was tested on 143B, MG-63, U-2 OS and hFOB1.19 cells using MTT assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by Hoechst 33342 staining and flow cytometry. The antimigratory, anti-invasive and antivasculogenic mimicry (VM) effects of PPEE were investigated by wound healing, Transwell and 3D culture assays. Mouse xenograft model was used to examine its anti-osteosarcoma efficacy in vivo. Hematologic profiles and hepatorenal functions were evaluated to assess the toxicity of PPEE. PPEE evidently suppressed cell proliferation of 143B, MG-63 and U-2 OS with IC50 values of 10-60[Formula: see text][Formula: see text]g/mL, but showed little cytotoxicity against normal osteoblastic cell. PPEE promoted apoptosis in 143B cell via caspase activation, increased Bax/Bcl-2 ratio and PARP cleavage. It also induced G2/M phase arrest associated with elevated phosphorylation of CDK1, Cdc25C, Chk2 and down-regulation of cyclin B1, CDK1, Cdc25C expression. Additionally, PPEE inhibited 143B cell migration, invasion and VM formation at noncytotoxic concentrations through decreasing the expression of FAK, Mig-7, MMP2 and MMP9. Finally, daily oral administration of PPEE for four weeks exhibits potent antitumor and anti-VM activity in 143B xenograft model with low toxicity. Taken together, these findings demonstrated PPEE possesses anti-osteosarcoma and anti-VM activity in vitro and in vivo, and therefore is a potential candidate for osteosarcoma treatment.

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

    Directory of Open Access Journals (Sweden)

    Rudkin George H

    2001-07-01

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

  2. Effect of human granulocyte macrophage-colony stimulating factor on differentiation and apoptosis of the human osteosarcoma cell line SaOS-2

    Directory of Open Access Journals (Sweden)

    L Postiglione

    2009-06-01

    Full Text Available We investigated the effects of human granulocyte macrophage- colony stimulating factor (GM-CSF on the relation between differentiation and apoptosis in SaOS-2 cells, an osteoblast-like cell line. To determine the relationship between these cellular processes, SaOS-2 cells were treated in vitro for 1, 7 and 14 days with 200 ng/mL GM-CSF and compared with untreated cells. Five nM insulin-like growth factor (IGF-I and 30 nM okadaic acid were used as negative and positive controls of apoptosis, respectively. Effects on cell differentiation were determined by ECM (extracellular matrix mineralization, morphology of some typical mature osteoblast differentiation markers, such as osteopontin and sialoprotein II (BSP-II, and production of bone ECM components such as collagen I. The results showed that treatment with GM-CSF caused cell differentiation accompanied by increased production of osteopontin and BSP-II, together with increased ECM deposition and mineralization. Flow cytometric analysis of annexin V and propidium iodide incorporation showed that GM-CSF up-regulated apoptotic cell death of SaOS-2 cells after 14 days of culture in contrast to okadaic acid, which stimulated SaOS-2 apoptosis only during the early period of culture. Endonucleolytic cleavage of genomic DNA, detected by “laddering analysis”, confirmed these data. The results suggest that GM-CSF induces osteoblastic differentiation and long-term apoptotic cell death of the SaOS-2 human osteosarcoma cell line, which in turn suggests a possible in vivo physiological role for GM-CSF on human osteoblast cells.

  3. Cell Adhesion on Surface-Functionalized Magnesium.

    Science.gov (United States)

    Wagener, Victoria; Schilling, Achim; Mainka, Astrid; Hennig, Diana; Gerum, Richard; Kelch, Marie-Luise; Keim, Simon; Fabry, Ben; Virtanen, Sannakaisa

    2016-05-18

    The biocompatibility of commercially pure magnesium-based (cp Mg) biodegradable implants is compromised of strong hydrogen evolution and surface alkalization due to high initial corrosion rates of cp Mg in the physiological environment. To mitigate this problem, the addition of corrosion-retarding alloying elements or coating of implant surfaces has been suggested. In the following work, we explored the effect of organic coatings on long-term cell growth. cp Mg was coated with aminopropyltriehtoxysilane + vitamin C (AV), carbonyldiimidazole (CDI), or stearic acid (SA). All three coatings have been previously suggested to reduce initial corrosion and to enhance protein adsorption and hence cell adhesion on magnesium surfaces. Endothelial cells (DH1+/+) and osteosarcoma cells (MG63) were cultured on coated samples for up to 20 days. To quantify Mg corrosion, electrochemical impedance spectroscopy (EIS) was measured after 1, 3, and 5 days of cell culture. We also investigated the speed of initial cell spreading after seeding using fluorescently labeled fibroblasts (NIH/3T3). Hydrogen evolution after contact with cell culture medium was markedly decreased on AV- and SA-coated Mg compared to uncoated Mg. These coatings also showed improved cell adhesion and spreading after 24 h of culture comparable to tissue-treated plastic surfaces. On AV-coated cp Mg, a confluent layer of endothelial cells formed after 5 days and remained intact for up to 20 days. Together, these data demonstrate that surface coating with AV is a viable strategy for improving long-term biocompatibility of cp Mg-based implants. EIS measurements confirmed that the presence of a confluent cell layer increased the corrosion resistance.

  4. Effect of sterilization procedures on properties of plasma polymers relevant to biomedical applications

    International Nuclear Information System (INIS)

    Artemenko, A.; Kylián, O.; Choukourov, A.; Gordeev, I.; Petr, M.; Vandrovcová, M.; Polonskyi, O.; Bačáková, L.; Slavinska, D.; Biederman, H.

    2012-01-01

    This study is focused on the evaluation of resistance of plasma polymers toward common sterilization techniques, i.e. property important for possible use of such materials in biomedical applications. Three kinds of plasma polymers having different bioadhesive natures were studied: plasma polymerized poly(ethylene oxide), fluorocarbon plasma polymers, and nitrogen-rich plasma polymers. These plasma polymers were subjected to dry heat, autoclave and UV radiation treatment. Their physical, chemical and bioresponsive properties were determined by means of different techniques (ellipsometry, atomic force microscopy, wettability measurements, X-ray photoelectron spectroscopy and biological tests with osteoblast-like cells MG63). The results clearly show that properties of thin films of plasma polymers may be significantly altered by a sterilization process. Moreover, observed changes induced by selected sterilization methods were found to depend strongly on the sterilized plasma polymer. - Highlights: ► Effect of common sterilization methods on three kinds of plasma polymers is studied. ► Physical, chemical and bioresponsive properties of plasma polymers are analyzed. ► Changes induced by sterilization depend strongly on type of the plasma polymer.

  5. Effect of sterilization procedures on properties of plasma polymers relevant to biomedical applications

    Energy Technology Data Exchange (ETDEWEB)

    Artemenko, A. [Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, V Holesovickach 2, 180 00 Prague (Czech Republic); Kylian, O., E-mail: ondrej.kylian@gmail.com [Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, V Holesovickach 2, 180 00 Prague (Czech Republic); Choukourov, A.; Gordeev, I.; Petr, M. [Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, V Holesovickach 2, 180 00 Prague (Czech Republic); Vandrovcova, M. [Institute of Physiology, Academy of Sciences of the Czech Republic, Department of Growth and Differentiation of Cell Populations, Videnska 1083, 142 20, Prague 4 (Czech Republic); Polonskyi, O. [Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, V Holesovickach 2, 180 00 Prague (Czech Republic); Bacakova, L. [Institute of Physiology, Academy of Sciences of the Czech Republic, Department of Growth and Differentiation of Cell Populations, Videnska 1083, 142 20, Prague 4 (Czech Republic); Slavinska, D.; Biederman, H. [Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, V Holesovickach 2, 180 00 Prague (Czech Republic)

    2012-10-01

    This study is focused on the evaluation of resistance of plasma polymers toward common sterilization techniques, i.e. property important for possible use of such materials in biomedical applications. Three kinds of plasma polymers having different bioadhesive natures were studied: plasma polymerized poly(ethylene oxide), fluorocarbon plasma polymers, and nitrogen-rich plasma polymers. These plasma polymers were subjected to dry heat, autoclave and UV radiation treatment. Their physical, chemical and bioresponsive properties were determined by means of different techniques (ellipsometry, atomic force microscopy, wettability measurements, X-ray photoelectron spectroscopy and biological tests with osteoblast-like cells MG63). The results clearly show that properties of thin films of plasma polymers may be significantly altered by a sterilization process. Moreover, observed changes induced by selected sterilization methods were found to depend strongly on the sterilized plasma polymer. - Highlights: Black-Right-Pointing-Pointer Effect of common sterilization methods on three kinds of plasma polymers is studied. Black-Right-Pointing-Pointer Physical, chemical and bioresponsive properties of plasma polymers are analyzed. Black-Right-Pointing-Pointer Changes induced by sterilization depend strongly on type of the plasma polymer.

  6. Biomimetic fabrication of antibacterial calcium phosphates mediated by polydopamine.

    Science.gov (United States)

    Forte, Lucia; Torricelli, Paola; Bonvicini, Francesca; Boanini, Elisa; Gentilomi, Giovanna Angela; Lusvardi, Gigliola; Della Bella, Elena; Fini, Milena; Vecchio Nepita, Edoardo; Bigi, Adriana

    2018-01-01

    In this work we developed new antibacterial composite materials using polydopamine (PDA) to trigger the deposition of silver nanoparticles (AgNPs) onto calcium phosphates, namely octacalcium phosphate (OCP) and α-tricalcium phosphate (αTCP). Functionalization of OCP and αTCP with a self-polymerized polydopamine layer was obtained by soaking the calcium phosphates in dopamine solution. The PDA surface of functionalized calcium phosphates (OCPd and αTCPd) promoted the deposition of AgNPs by reducing silver ions when soaked in a silver nitrate solution. The amount of deposited AgNPs can be modulated by varying the concentration of silver nitrate solution and the type of substrate. The results of in vitro tests carried out with osteoblast-like MG63 cells indicate that the combination of AgNPs with OCP provides more biocompatible materials than those obtained using αTCP as substrate. In particular, the study of osteoblast activity and differentiation was focused on the samples OCPdAg5 (silver content=8.2wt%) and αTCPdAg5 (silver content=4.7wt%), which did not show any cytotoxicity, and compared with those obtained on pure OCP and αTCP. The results demonstrate that the AgNPs loaded materials support osteoblast viability and differentiation, whereas they significantly inhibit the growth of relevant antibiotic-resistant pathogenic bacteria. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Nano-hydroxyapatite/chitosan-starch nanocomposite as a novel bone construct: Synthesis and in vitro studies.

    Science.gov (United States)

    Shakir, Mohammad; Jolly, Reshma; Khan, Mohd Shoeb; Iram, Noor e; Khan, Haris M

    2015-09-01

    A novel ternary nanocomposite system incorporating hydroxyapatite, chitosan and starch (n-HA/CS-ST) has been synthesized by co-precipitation method at room temperature, addressing the issues of biocompatibility, mechanical strength and cytotoxicity required for bone tissue engineering. The interactions, crystallite size, surface morphology and thermal stability against n-HA/CS nanocomposite have been obtained by comparing the results of FTIR, SEM, TEM, DLS, XRD and TGA/DTA. A comparative study of bioactivity and thermal stability of n-HA/CS and n-HA/CS-ST nanocomposites revealed that the incorporation of starch as templating agent enhanced these properties in n-HA/CS-ST nanocomposite. A lower swelling rate of n-HA/CS-ST relative to n-HA/CS indicates a higher mechanical strength supportive of bone tissue ingrowths. The MTT assay on murine fibroblast L929 and human osteoblasts-like MG-63 cells and in vitro bioactivity of n-HA/CS-ST matrix referred superior non-toxic nature of n-HA/CS-ST nanocomposite and greater possibility of osteointegration in vivo respectively. Furthermore n-HA/CS-ST exhibited improved antibacterial property against both Gram-positive and Gram-negative bacteria relative to n-HA/CS. Copyright © 2015. Published by Elsevier B.V.

  8. Reinforced nanohydroxyapatite/polyamide66 scaffolds by chitosan coating for bone tissue engineering.

    Science.gov (United States)

    Huang, Di; Zuo, Yi; Zou, Qin; Wang, Yanying; Gao, Shibo; Wang, Xiaoyan; Liu, Haohuai; Li, Yubao

    2012-01-01

    High porosity of scaffold is always accompanied by poor mechanical property; the aim of this study was to enhance the strength and modulus of the highly porous scaffold of nanohydroxyapatite/polyamide66 (n-HA/PA66) by coating chitosan (CS) and to investigate the effect of CS content on the scaffold physical properties and cytological properties. The results show that CS coating can reinforce the scaffold effectively. The compress modulus and strength of the CS coated n-HA/PA66 scaffolds are improved to 32.71 and 2.38 MPa, respectively, being about six times and five times of those of the uncoated scaffolds. Meanwhile, the scaffolds still exhibit a highly interconnected porous structure and the porosity is approximate about 78%, slightly lower than the value (84%) of uncoated scaffold. The cytological properties of scaffolds were also studied in vitro by cocultured with osteoblast-like MG63 cells. The cytological experiments demonstrate that the reinforced scaffolds display favorable cytocompatibility and have no significant difference with the uncoated n-HA/PA66 scaffolds. The CS reinforced n-HA/PA66 scaffolds can meet the basic mechanical requirement of bone tissue engineering scaffold, presenting a potential for biomedical application in bone reconstruction and repair. Copyright © 2011 Wiley Periodicals, Inc.

  9. Bio-composites composed of a solid free-form fabricated polycaprolactone and alginate-releasing bone morphogenic protein and bone formation peptide for bone tissue regeneration.

    Science.gov (United States)

    Kim, MinSung; Jung, Won-Kyo; Kim, GeunHyung

    2013-11-01

    Biomedical scaffolds should be designed with highly porous three-dimensional (3D) structures that have mechanical properties similar to the replaced tissue, biocompatible properties, and biodegradability. Here, we propose a new composite composed of solid free-form fabricated polycaprolactone (PCL), bone morphogenic protein (BMP-2) or bone formation peptide (BFP-1), and alginate for bone tissue regeneration. In this study, PCL was used as a mechanical supporting component to enhance the mechanical properties of the final biocomposite and alginate was used as the deterring material to control the release of BMP-2 and BFP-1. A release test revealed that alginate can act as a good release control material. The in vitro biocompatibilities of the composites were examined using osteoblast-like cells (MG63) and the alkaline phosphatase (ALP) activity and calcium deposition were assessed. The in vitro test results revealed that PCL/BFP-1/Alginate had significantly higher ALP activity and calcium deposition than the PCL/BMP-2/Alginate composite. Based on these findings, release-controlled BFP-1 could be a good growth factor for enhancement of bone tissue growth and the simple-alginate coating method will be a useful tool for fabrication of highly functional biomaterials through release-control supplementation.

  10. Andrographolide Induces Autophagic Cell Death and Inhibits Invasion and Metastasis of Human Osteosarcoma Cells in An Autophagy-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Ying Liu

    2017-11-01

    Full Text Available Background/Aims: Osteosarcoma (OS is the most common primary malignant tumor of bone tissue. Although treatment effectiveness has improved, the OS survival rate has fluctuated in recent years. Andrographolide (AG has been reported to have antitumor activity against a variety of tumors. Our aim was to investigate the effects and potential mechanisms of AG in human osteosarcoma. Methods: Cell viability and morphological changes were assessed by MTT and live/dead assays. Apoptosis was detected using Annexin V-FITC/PI double staining, DAPI, and caspase-3 assays. Autophagy was detected with mRFP-GFP-LC3 adenovirus transfection and western blot. Cell migration and invasion were detected by wound healing assay and Transwell® experiments. Results: AG dose-dependently reduced the viability of osteosarcoma cells. No increase in apoptosis was detected in AG-treated human OS MG-63 and U-2OS cells, and the pan-caspase inhibitor z-VAD did not attenuate AG-induced cell death. However, AG induced autophagy by suppressing PI3K/Akt/mTOR and enhancing JNK signaling pathways. 3-MA and Beclin-1 siRNA could reverse the cytotoxic effects of AG. In addition, AG inhibited the invasion and metastasis of OS, and this effect could be reversed with Beclin-1 siRNA. Conclusion: AG inhibits viability and induces autophagic death in OS cells. AG-induced autophagy inhibits the invasion and metastasis of OS.

  11. 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.

  12. Behavior of bone cells in contact with magnesium implant material.

    Science.gov (United States)

    Burmester, Anna; Willumeit-Römer, Regine; Feyerabend, Frank

    2017-01-01

    Magnesium-based implants exhibit several advantages, such as biodegradability and possible osteoinductive properties. Whether the degradation may induce cell type-specific changes in metabolism still remains unclear. To examine the osteoinductivity mechanisms, the reaction of bone-derived cells (MG63, U2OS, SaoS2, and primary human osteoblasts (OB)) to magnesium (Mg) was determined. Mg-based extracts were used to mimic more realistic Mg degradation conditions. Moreover, the influence of cells having direct contact with the degrading Mg metal was investigated. In exposure to extracts and in direct contact, the cells decreased pH and osmolality due to metabolic activity. Proliferating cells showed no significant reaction to extracts, whereas differentiating cells were negatively influenced. In contrast to extract exposure, where cell size increased, in direct contact to magnesium, cell size was stable or even decreased. The amount of focal adhesions decreased over time on all materials. Genes involved in bone formation were significantly upregulated, especially for primary human osteoblasts. Some osteoinductive indicators were observed for OB: (i) an increased cell count after extract addition indicated a higher proliferation potential; (ii) increased cell sizes after extract supplementation in combination with augmented adhesion behavior of these cells suggest an early switch to differentiation; and (iii) bone-inducing gene expression patterns were determined for all analyzed conditions. The results from the cell lines were inhomogeneous and showed no specific stimulus of Mg. The comparison of the different cell types showed that primary cells of the investigated tissue should be used as an in vitro model if Mg is analyzed. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 165-179, 2017. © 2015 Wiley Periodicals, Inc.

  13. Activation of PERK signaling through fluoride-mediated endoplasmic reticulum stress in OS732 cells

    International Nuclear Information System (INIS)

    Xu, Hui; Zhou, Yu-lai; Zhang, Xiu-yun; Lu, Peng; Li, Guang-sheng

    2010-01-01

    Our proteomical analysis of osteoblasts exposed to fluoride revealed a distinctive upregulation of proteins in osteoblast. These upregulated proteins play key roles in the protein folding. The PRK-like ER kinase (PERK) signaling, one branch of unfolded protein response (UPR) to combat ER stress, is a transcription factor needed for osteoblast proliferation and differentiation. The mechanism of skeletal fluorosis by which fluoride regulates osteoblast is not fully defined. Here we studied the effect of fluoride on PERK signaling genes and x-box binding protein 1 (xbp-1) in OS7232 cells (human osteoblast-like cell line). Meantime, genes associated with bone turnover were examined in this study. We found that early and continuous fluoride exposure increased the binding immunoglobulin protein (BiP) expression and activated the PERK signaling pathway, resulting in activation of transcription factor 4 (ATF4) and nuclear factor erythroid 2-related factor 2 (Nrf2). The altered expression of cbfa1, osteoprotegerin (OPG)/nuclear factor kappa B ligand (RANKL) were viewed in this study. These results showed fluoride impelled a distinctive ER stress response in OS732 cells, primarily by activating PERK and PERK-dependent signaling. Little effects were viewed for activating xbp-1, a common target of the other two canonical sensors of ER stress, ATF6 and IRE1. In this study the altered expression of bone turnover genes were consistent with activation of ER stress and PERK signaling. This study proved that PERK signaling play major roles in action of fluoride on osteoblast, and suggested that bone response in skeletal fluorosis may be due in part to PERK signaling pathway.

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

    Science.gov (United States)

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

    2017-11-01

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

  15. Automated measurement of cell motility and proliferation

    Directory of Open Access Journals (Sweden)

    Goff Julie

    2005-04-01

    Full Text Available Abstract Background Time-lapse microscopic imaging provides a powerful approach for following changes in cell phenotype over time. Visible responses of whole cells can yield insight into functional changes that underlie physiological processes in health and disease. For example, features of cell motility accompany molecular changes that are central to the immune response, to carcinogenesis and metastasis, to wound healing and tissue regeneration, and to the myriad developmental processes that generate an organism. Previously reported image processing methods for motility analysis required custom viewing devices and manual interactions that may introduce bias, that slow throughput, and that constrain the scope of experiments in terms of the number of treatment variables, time period of observation, replication and statistical options. Here we describe a fully automated system in which images are acquired 24/7 from 384 well plates and are automatically processed to yield high-content motility and morphological data. Results We have applied this technology to study the effects of different extracellular matrix compounds on human osteoblast-like cell lines to explore functional changes that may underlie processes involved in bone formation and maintenance. We show dose-response and kinetic data for induction of increased motility by laminin and collagen type I without significant effects on growth rate. Differential motility response was evident within 4 hours of plating cells; long-term responses differed depending upon cell type and surface coating. Average velocities were increased approximately 0.1 um/min by ten-fold increases in laminin coating concentration in some cases. Comparison with manual tracking demonstrated the accuracy of the automated method and highlighted the comparative imprecision of human tracking for analysis of cell motility data. Quality statistics are reported that associate with stage noise, interference by non-cell

  16. Extracellular KCl effect on organic bound tritium in human cells

    International Nuclear Information System (INIS)

    Gonen, Rafi; Uzi, German; Priel, Esther; Alfassi, Zeev B.

    2008-01-01

    Tritium atoms can replace hydrogen atoms in organic compounds, forming Organic Bound Tritium. Therefore, exposure of the body to tritium may lead to binding of tritium in tissue molecules, retaining it in the body longer than HTO, and causing higher doses. Ignoring this effect when evaluating inner exposures, may lead to under-estimation of tritium exposures. It was published, that tritium bound to some organic molecules has the potential to accumulate in organisms at higher levels as in the surrounding media. In order to investigate this effect and to identify physiological factors, OBT production in human malignant MG-63 osteoblast cells was studied. The purpose of the present work was to investigate the influence of the ionic extracellular potassium concentration on the amount of tritium in cells. Potassium is known as an ionic compound present in the body, which has the potential to cause cells swelling. Therefore, cells were exposed to isotonic and hypotonic media, supplemented with different concentrations of KCl, and the tritium accumulations were determined after incubation with HTO. An increase in the total Organic Bound Tritium production was observed, as well as an increase of the intracellular HTO content when increasing the KCl concentration. (author)

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

    Science.gov (United States)

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

    2010-02-25

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

  18. Covalent attachment of cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) to poly(etheretherketone) surface by tailored silanization layers technique

    International Nuclear Information System (INIS)

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

    2014-01-01

    Highlights: • The carbonyl groups on PEEK surface were effectively reduced to hydroxyl groups using sodium borohydride. • Silanization layers technique was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on hydroxylation-pretreated PEEK sheet surface by covalent chemical attachment. • XPS, surface profiler and water contact angle measurements proved the presence of GRGD on PEEK surface. • Osteoblast-like cells (MC3T3-E1) attachment and proliferation were improved effectively on GRGD-modified PEEK surface. - Abstract: Poly(etheretherketone) (PEEK) is a rigid semicrystalline polymer that combines excellent mechanical properties, broad chemical resistance and bone-like stiffness and is widely used in biomedical fields. However, PEEK is naturally bioinert, leading to limited biomedical applications, especially when a direct bone-implant osteointegration is desired. In this study, a three-step reaction procedure was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on the surface of PEEK sheet by covalent chemical attachment to favor cell adhesion and proliferation. First, hydroxylation-pretreated PEEK surfaces were silanized with 7-Oct-1-enyltrichlorosilane (OETS) in dry cyclohexane, resulting in a silanization layer with terminal ethenyl. Second, the terminal ethylenic double bonds of the silanization layer on PEEK surface were converted to carboxyl groups through acidic potassium manganate oxidation. Finally, GRGD was covalently attached by carbodiimide mediated condensation between the carboxyl on PEEK surface and amine presents in GRGD. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, surface profiler and water contact angle measurements were applied to characterize the modified surfaces. The effect of cells attachment and proliferation on each specimen was investigated. Pre-osteoblast cells (MC3T3-E1) attachment, spreading and proliferation

  19. Covalent attachment of cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) to poly(etheretherketone) surface by tailored silanization layers technique

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-11-30

    Highlights: • The carbonyl groups on PEEK surface were effectively reduced to hydroxyl groups using sodium borohydride. • Silanization layers technique was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on hydroxylation-pretreated PEEK sheet surface by covalent chemical attachment. • XPS, surface profiler and water contact angle measurements proved the presence of GRGD on PEEK surface. • Osteoblast-like cells (MC3T3-E1) attachment and proliferation were improved effectively on GRGD-modified PEEK surface. - Abstract: Poly(etheretherketone) (PEEK) is a rigid semicrystalline polymer that combines excellent mechanical properties, broad chemical resistance and bone-like stiffness and is widely used in biomedical fields. However, PEEK is naturally bioinert, leading to limited biomedical applications, especially when a direct bone-implant osteointegration is desired. In this study, a three-step reaction procedure was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on the surface of PEEK sheet by covalent chemical attachment to favor cell adhesion and proliferation. First, hydroxylation-pretreated PEEK surfaces were silanized with 7-Oct-1-enyltrichlorosilane (OETS) in dry cyclohexane, resulting in a silanization layer with terminal ethenyl. Second, the terminal ethylenic double bonds of the silanization layer on PEEK surface were converted to carboxyl groups through acidic potassium manganate oxidation. Finally, GRGD was covalently attached by carbodiimide mediated condensation between the carboxyl on PEEK surface and amine presents in GRGD. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, surface profiler and water contact angle measurements were applied to characterize the modified surfaces. The effect of cells attachment and proliferation on each specimen was investigated. Pre-osteoblast cells (MC3T3-E1) attachment, spreading and proliferation

  20. Laser-direct writing by two-photon polymerization of 3D honeycomb-like structures for bone regeneration.

    Science.gov (United States)

    Paun, Irina Alexandra; Popescu, Roxana Cristina; Mustaciosu, Cosmin Catalin; Zamfirescu, Marian; Calin, Bogdan Stefanita; Mihailescu, Mona; Dinescu, Maria; Popescu, Andrei; Chioibasu, Diana; Soproniy, Mihai; Luculescu, Catalin Romeo

    2018-02-05

    A major limitation of existing 3D implantable structures for bone tissue engineering is that most of the cells rapidly attach on the outer edges of the structure, restricting the cells penetration into the inner parts and causing the formation of a necrotic core. Furthermore, these structures generally possess a random spatial arrangement and do not preserve the isotropy on the whole volume. Here, we report on the fabrication and testing of an innovative 3D hierarchical, honeycomb-like structure (HS), with reproducible and isotropic arhitecture, that allows in 'volume' migration of osteoblasts. In particular, we demonstrate the possibility to control the 3D spatial cells growth inside these complex architectures by adjusting the free spaces inside the structures. The structures were made of vertical microtubes arranged in a mulitlayered configuration, fabricated via laser direct writing by two photons polymerization of the IP-L780 photopolymer. In vitro tests performed in MG-63 osteoblast-like cells demonstrated that the cells migration inside the 3D structures is conducted by the separation space between the microtubes layers. Specifically, for layers separation between 2 and 10 μm, the cells gradually penetrated between the microtubes. Furthermore, these structures induced the strongest cells osteogenic differentiation and mineralization, with ALP activity 1.5 times stronger, amount of calcified minerals 1.3 times higher and osteocalcin secretion increased by 2.3 times compared to the other structures. On the opposite, for layers separation less than 2 μm and above 10 μm, the cells were not able to make interconnections and exhibited poor mineralization ability.

  1. Nanosurface design of dental implants for improved cell growth and function

    Science.gov (United States)

    Pan, Hsu-An; Hung, Yao-Ching; Chiou, Jin-Chern; Tai, Shih-Ming; Chen, Hsin-Hung; Huang, G. Steven

    2012-08-01

    A strategy was proposed for the topological design of dental implants based on an in vitro survey of optimized nanodot structures. An in vitro survey was performed using nanodot arrays with dot diameters ranging from 10 to 200 nm. MG63 osteoblasts were seeded on nanodot arrays and cultured for 3 days. Cell number, percentage undergoing apoptotic-like cell death, cell adhesion and cytoskeletal organization were evaluated. Nanodots with a diameter of approximately 50 nm enhanced cell number by 44%, minimized apoptotic-like cell death to 2.7%, promoted a 30% increase in microfilament bundles and maximized cell adhesion with a 73% increase in focal adhesions. An enhancement of about 50% in mineralization was observed, determined by von Kossa staining and by Alizarin Red S staining. Therefore, we provide a complete range of nanosurfaces for growing osteoblasts to discriminate their nanoscale environment. Nanodot arrays present an opportunity to positively and negatively modulate cell behavior and maturation. Our results suggest a topological approach which is beneficial for the design of dental implants.

  2. Spontaneous gene transfection of human bone cells using 3D mineralized alginate-chitosan macrocapsules.

    Science.gov (United States)

    Green, David W; Kim, Eun-Jung; Jung, Han-Sung

    2015-09-01

    The effectiveness of nonviral gene therapy remains uncertain because of low transfection efficiencies and high toxicities compared with viral-based strategies. We describe a simple system for transient transfection of continuous human cell lines, with low toxicity, using mineral-coated chitosan and alginate capsules. As proof-of-concept, we demonstrate transfection of Saos-2 and MG63 human osteosarcoma continuous cell lines with gfp, LacZ reporter genes, and a Sox-9 carrying plasmid, to illustrate expression of a functional gene with therapeutic relevance. We show that continuous cell lines transfect with significant efficiency of up to 65% possibly through the interplay between chitosan and DNA complexation and calcium/phosphate-induced translocation into cells entrapped within the 3D polysaccharide based environment, as evidenced by an absence of transfection in unmineralized and chitosan-free capsules. We demonstrated that our transfection system was equally effective at transfection of primary human bone marrow stromal cells. To illustrate, the Sox-9, DNA plasmid was spontaneously expressed in primary human bone marrow stromal cells at 7 days with up to 90% efficiency in two repeats. Mineralized polysaccharide macrocapsules are gene delivery vehicles with a number of biological and practical advantages. They are highly efficient at self-transfecting primary bone cells, with programmable spatial and temporal delivery prospects, premineralized bone-like environments, and have no cytotoxic effects, as compared with many other nonviral systems. © 2015 Wiley Periodicals, Inc.

  3. 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)

  4. 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.

  5. 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.

  6. Improving osteoblasts cells proliferation via femtosecond laser surface modification of 3D-printed poly-ɛ-caprolactone scaffolds for bone tissue engineering applications

    Science.gov (United States)

    Daskalova, A.; Ostrowska, B.; Zhelyazkova, A.; Święszkowski, W.; Trifonov, A.; Declercq, H.; Nathala, C.; Szlazak, K.; Lojkowski, M.; Husinsky, W.; Buchvarov, I.

    2018-06-01

    Synthetic polymer biomaterials incorporating cells are a promising technique for treatment of orthopedic injuries. To enhance the integration of biomaterials into the human body, additional functionalization of the scaffold surface should be carried out that would assist one in mimicking the natural cellular environment. In this study, we examined poly-ɛ-caprolactone (PCL) fiber matrices in view of optimizing the porous properties of the constructs. Altering the porosity of a PCL scaffold is expected to improve the material's biocompatibility, thus influencing its osteoconductivity and osteointegration. We produced 3D poly-ɛ-caprolactone (PCL) matrices by a fused deposition modeling method for bone and cartilage tissue engineering and performed femtosecond (fs) laser modification experiments to improve the surface properties of the PCL construct. Femtosecond laser processing is one of the useful tools for creating a vast diversity of surface patterns with reproducibility and precision. The processed surface of the PCL matrix was examined to follow the effect of the laser parameters, namely the laser pulse energy and repetition rate and the number ( N) of applied pulses. The modified zones were characterized by scanning electron microscopy (SEM), confocal microscopy, X-ray computed tomography and contact angle measurements. The results obtained demonstrated changes in the morphology of the processed surface. A decrease in the water contact angle was also seen after fs laser processing of fiber meshes. Our work demonstrated that a precise control of material surface properties could be achieved by applying a different number of laser pulses at various laser fluence values. We concluded that the structural features of the matrix remain unaffected and can be successfully modified through laser postmodification. The cells tests indicated that the micro-modifications created induced MG63 and MC3T3 osteoblast cellular orientation. The analysis of the MG63 and MC3T3

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

    DEFF Research Database (Denmark)

    Skindersø, Mette Elena; Krogfelt, Karen Angeliki; Blom, Ashley

    2015-01-01

    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 (osseo)integration of TJRs is therefore highly desirable and continues to represent a research priority in current biomaterial...

  8. Immobilization of chitosan film containing semaphorin 3A onto a microarc oxidized titanium implant surface via silane reaction to improve MG63 osteogenic differentiation

    Directory of Open Access Journals (Sweden)

    Fang K

    2014-10-01

    Full Text Available Kaixiu Fang,1,* Wen Song,2,* Lifeng Wang,1 Sen Jia,3 Hongbo Wei,1 Shuai Ren,1 Xiaoru Xu,1 Yingliang Song1 1State Key Laboratory of Military Stomatology, Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi’an, People’s Republic of China; 2State Key Laboratory of Military Stomatology, Department of Prosthetic Dentistry, School of Stomatology, Fourth Military Medical University, Xi’an, People’s Republic of China; 3State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an, People’s Republic of China *These authors contributed equally to this work Abstract: Improving osseointegration of extensively used titanium (Ti implants still remains a main theme in implantology. Recently, grafting biomolecules onto a Ti surface has attracted more attention due to their direct participation in the osseointegration process around the implant. Semaphorin 3A (Sema3A is a new proven osteoprotection molecule and is considered to be a promising therapeutic agent in bone diseases, but how to immobilize the protein onto a Ti surface to acquire a long-term effect is poorly defined. In our study, we tried to use chitosan to wrap Sema3A (CS/Sema and connect to the microarc oxidized Ti surface via silane glutaraldehyde coupling. The microarc oxidization could formulate porous topography on a Ti surface, and the covalently bonded coating was homogeneously covered on the ridges between the pores without significant influence on the original topography. A burst release of Sema3A was observed in the first few days in phosphate-buffered saline and could be maintained for >2 weeks. Coating in phosphate-buffered saline containing lysozyme was similar, but the release rate was much more rapid. The coating did not significantly affect cellular adhesion, viability, or cytoskeleton arrangement, but the osteogenic-related gene expression was dramatically increased and calcium deposition was also abundantly detected. In conclusion, covalent bonding of CS/Sema could strongly improve osteogenic differentiation of osteoblasts and might be applied for Ti implant surface biofunctionalization. Keywords: titanium, semaphorin 3A, silane reaction, microarc oxidation, osteogenic differentiation

  9. Nano Petri dishes: a new polystyrene platform for studying cell-nanoengineered surface interactions

    Science.gov (United States)

    Cha, Kyoung Je; Na, Moon-Hee; Kim, Hyung Woo; Kim, Dong Sung

    2014-05-01

    In this study, we fabricated and fully characterized a new type of polystyrene (PS) cell-culture platform containing nanoengineered surfaces (NES), referred to as a nano Petri dish, which can be used at the transition stage of basic cell-NES interaction studies for clinical applications. Nano-injection molding in this study was used for the mass production of the nano Petri dish having nanopore arrays. The effects of processing parameters of the injection molding on the replication quality of the nanopore arrays were quantitatively evaluated by means of design of experiments based on the Taguchi method. This allowed efficient and reliable cell culture studies by providing large numbers of the same dishes, in addition to removing the fixation step of the NES plates inside the cell-culture container. Physical, chemical and mechanical properties of the NES, as well as cell behavior including attachment and proliferation of human osteosarcoma MG-63 cells on the NES, were then characterized, with and without the oxygen plasma surface treatment.

  10. 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.

  11. Comparison of the reaction of bone-derived cells to enhanced MgCl2-salt concentrations.

    Science.gov (United States)

    Burmester, Anna; Luthringer, Bérengère; Willumeit, Regine; Feyerabend, Frank

    2014-01-01

    Magnesium-based implants exhibit various advantages such as biodegradability and potential for enhanced in vivo bone formation. However, the cellular mechanisms behind this possible osteoconductivity remain unclear. To determine whether high local magnesium concentrations can be osteoconductive and exclude other environmental factors that occur during the degradation of magnesium implants, magnesium salt (MgCl2) was used as a model system. Because cell lines are preferred targets in studies of non-degradable implant materials, we performed a comparative study of 3 osteosarcoma-derived cell lines (MG63, SaoS2 and U2OS) with primary human osteoblasts. The correlation among cell count, viability, cell size and several MgCl2 concentrations was used to examine the influence of magnesium on proliferation in vitro. Moreover, bone metabolism alterations during proliferation were investigated by analyzing the expression of genes involved in osteogenesis. It was observed that for all cell types, the cell count decreases at concentrations above 10 mM MgCl2. However, detailed analysis showed that MgCl2 has a relevant but very diverse influence on proliferation and bone metabolism, depending on the cell type. Only for primary cells was a clear stimulating effect observed. Therefore, reliable results demonstrating the osteoconductivity of magnesium implants can only be achieved with primary osteoblasts.

  12. Collagen-embedded hydroxylapatite-beta-tricalcium phosphate-silicon dioxide bone substitute granules assist rapid vascularization and promote cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Ghanaati, Shahram M; Thimm, Benjamin W; Unger, Ronald E; Orth, Carina; Barbeck, Mike; Kirkpatrick, C James [Institute of Pathology, Johannes Gutenberg-University Mainz, Langenbeckstr.1, 55101 Mainz (Germany); Kohler, Thomas; Mueller, Ralph, E-mail: ghanaati@uni-mainz.d [Institute for Biomechanics, ETH Zuerich, Wolfgang-Pauli-Str.10, 8093 Zuerich (Switzerland)

    2010-04-15

    In the present study we assessed the biocompatibility in vitro and in vivo of a low-temperature sol-gel-manufactured SiO{sub 2}-based bone graft substitute. Human primary osteoblasts and the osteoblastic cell line, MG63, cultured on the SiO{sub 2} biomatrix in monoculture retained their osteoblastic morphology and cellular functionality in vitro. The effect of the biomaterial in vivo and its vascularization potential was tested subcutaneously in Wistar rats and demonstrated both rapid vascularization and good integration within the peri-implant tissue. Scaffold degradation was progressive during the first month after implantation, with tartrate-resistant acid phosphatase-positive macrophages being present and promoting scaffold degradation from an early stage. This manuscript describes successful osteoblastic growth promotion in vitro and a promising biomaterial integration and vasculogenesis in vivo for a possible therapeutic application of this biomatrix in future clinical studies.

  13. Design and development of novel MRI compatible zirconium- ruthenium alloys with ultralow magnetic susceptibility.

    Science.gov (United States)

    Li, H F; Zhou, F Y; Li, L; Zheng, Y F

    2016-04-19

    In the present study, novel MRI compatible zirconium-ruthenium alloys with ultralow magnetic susceptibility were developed for biomedical and therapeutic devices under MRI diagnostics environments. The results demonstrated that alloying with ruthenium into pure zirconium would significantly increase the strength and hardness properties. The corrosion resistance of zirconium-ruthenium alloys increased significantly. High cell viability could be found and healthy cell morphology observed when culturing MG 63 osteoblast-like cells and L-929 fibroblast cells with zirconium-ruthenium alloys, whereas the hemolysis rates of zirconium-ruthenium alloys are zirconium-ruthenium alloys (1.25 × 10(-6) cm(3)·g(-1)-1.29 × 10(-6) cm(3)·g(-1) for zirconium-ruthenium alloys) are ultralow, about one-third that of Ti-based alloys (Ti-6Al-4V, ~3.5 × 10(-6) cm(3)·g(-1), CP Ti and Ti-6Al-7Nb, ~3.0 × 10(-6) cm(3)·g(-1)), and one-sixth that of Co-Cr alloys (Co-Cr-Mo, ~7.7 × 10(-6) cm(3)·g(-1)). Among the Zr-Ru alloy series, Zr-1Ru demonstrates enhanced mechanical properties, excellent corrosion resistance and cell viability with lowest magnetic susceptibility, and thus is the optimal Zr-Ru alloy system as therapeutic devices under MRI diagnostics environments.

  14. Design and development of novel MRI compatible zirconium- ruthenium alloys with ultralow magnetic susceptibility

    Science.gov (United States)

    Li, H.F.; Zhou, F.Y.; Li, L.; Zheng, Y.F.

    2016-01-01

    In the present study, novel MRI compatible zirconium-ruthenium alloys with ultralow magnetic susceptibility were developed for biomedical and therapeutic devices under MRI diagnostics environments. The results demonstrated that alloying with ruthenium into pure zirconium would significantly increase the strength and hardness properties. The corrosion resistance of zirconium-ruthenium alloys increased significantly. High cell viability could be found and healthy cell morphology observed when culturing MG 63 osteoblast-like cells and L-929 fibroblast cells with zirconium-ruthenium alloys, whereas the hemolysis rates of zirconium-ruthenium alloys are alloys and Ti-based alloys, the magnetic susceptibilities of the zirconium-ruthenium alloys (1.25 × 10−6 cm3·g−1–1.29 × 10−6 cm3·g−1 for zirconium-ruthenium alloys) are ultralow, about one-third that of Ti-based alloys (Ti–6Al–4V, ~3.5 × 10−6 cm3·g−1, CP Ti and Ti–6Al–7Nb, ~3.0 × 10−6 cm3·g−1), and one-sixth that of Co–Cr alloys (Co–Cr–Mo, ~7.7 × 10−6 cm3·g−1). Among the Zr–Ru alloy series, Zr–1Ru demonstrates enhanced mechanical properties, excellent corrosion resistance and cell viability with lowest magnetic susceptibility, and thus is the optimal Zr–Ru alloy system as therapeutic devices under MRI diagnostics environments. PMID:27090955

  15. 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.

  16. Proliferation and mineralization ability of dental pulp cells derived from primary and permanent teeth

    Directory of Open Access Journals (Sweden)

    Suttatip Kamolmatyakul

    2011-04-01

    Full Text Available The aims of this study were to compare the proliferation and mineralization ability of CFU-F selected dental pulp cellsderived from primary and permanent teeth. Those cells were isolated by enzyme digestion and analyzed for their colonyformingcapacity. The cell proliferation was measured by the MTT assay on day 1, day 7, and day14. Alizarin Red S stainingwas used to detect mineralized nodule formation of the cells on day 7, 14, 21, and 28. Proliferation of CFU-F selected pulpcells from primary teeth was significantly higher than that of CFU-F selected pulp cells from permanent teeth in all periods ofthe experiment. Upon cultured cells in mineralization inducing media, the mineralized nodules appeared as early as day 14 inCFU-F selected pulp cells from primary teeth and MG-63, whereas those of CFU-F selected pulp cells from permanent teethcan be found at day 21. On day 21 and day 28, the mineralized nodules of the CFU-F selected pulp cells from the primaryteeth group were more than those in the CFU-F selected pulp cells from the permanent teeth group. Mineralized noduleformation in the CFU-F selected pulp cells from the permanent teeth group appeared later and were less than those ofCFU-F selected pulp cells from primary teeth. However, mineralized nodules in CFU-F selected pulp cells from the permanentteeth group increased very fast after their appearance. Those results suggest that CFU-F selected pulp cells from primaryteeth had a higher proliferation rate and mineralization rate when compared to CFU-F selected pulp cells from permanentteeth.

  17. Nano Petri dishes: a new polystyrene platform for studying cell-nanoengineered surface interactions

    International Nuclear Information System (INIS)

    Cha, Kyoung Je; Na, Moon-Hee; Kim, Hyung Woo; Kim, Dong Sung

    2014-01-01

    In this study, we fabricated and fully characterized a new type of polystyrene (PS) cell-culture platform containing nanoengineered surfaces (NES), referred to as a nano Petri dish, which can be used at the transition stage of basic cell–NES interaction studies for clinical applications. Nano-injection molding in this study was used for the mass production of the nano Petri dish having nanopore arrays. The effects of processing parameters of the injection molding on the replication quality of the nanopore arrays were quantitatively evaluated by means of design of experiments based on the Taguchi method. This allowed efficient and reliable cell culture studies by providing large numbers of the same dishes, in addition to removing the fixation step of the NES plates inside the cell-culture container. Physical, chemical and mechanical properties of the NES, as well as cell behavior including attachment and proliferation of human osteosarcoma MG-63 cells on the NES, were then characterized, with and without the oxygen plasma surface treatment. (paper)

  18. MAPLE deposition of polypyrrole-based composite layers for bone regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Paun, Irina Alexandra, E-mail: irina.paun@physics.pub.ro [Faculty of Applied Sciences, University Politehnica of Bucharest, RO-060042 (Romania); National Institute for Laser, Plasma and Radiation Physics, Magurele, Bucharest RO-077125 (Romania); Acasandrei, Adriana Maria [Horia Hulubei National Institute for Physics and Nuclear Engineering IFIN-HH, Magurele, Bucharest RO-077125 (Romania); Luculescu, Catalin Romeo, E-mail: catalin.luculescu@inflpr.ro [National Institute for Laser, Plasma and Radiation Physics, Magurele, Bucharest RO-077125 (Romania); Mustaciosu, Cosmin Catalin [Horia Hulubei National Institute for Physics and Nuclear Engineering IFIN-HH, Magurele, Bucharest RO-077125 (Romania); Ion, Valentin [National Institute for Laser, Plasma and Radiation Physics, Magurele, Bucharest RO-077125 (Romania); Mihailescu, Mona; Vasile, Eugenia [Faculty of Applied Sciences, University Politehnica of Bucharest, RO-060042 (Romania); Dinescu, Maria, E-mail: dinescum@nipne.ro [National Institute for Laser, Plasma and Radiation Physics, Magurele, Bucharest RO-077125 (Romania)

    2015-12-01

    Highlights: • PPy-based composite layers for bone regeneration were produced by MAPLE. • Conductive PPy nanograins were embedded in insulating PLGA and PU matrices. • PLGA was chosen for providing biodegradability and PU for toughness and elasticity. • The layers conductivities reached 10{sup −2} S/cm for PPy loadings of 1:10 weight ratios. • The layers promoted osteoblast viability, proliferation and mineralization. - Abstract: We report on biocompatible, electrically conductive layers of polypyrrole (PPy)-based composites obtained by Matrix Assisted Pulsed Laser Evaporation (MAPLE) for envisioned bone regeneration. In order to preserve the conductivity of the PPy while overcoming its lack of biodegradability and low mechanical resilience, conductive PPy nanograins were embedded in two biocompatible, insulating polymeric matrices, i.e. poly(lactic-co-glycolic)acid (PLGA) and polyurethane (PU). PLGA offers the advantage of full biodegradability into non-toxic products, while PU provides toughness and elasticity. The PPy nanograins formed micro-domains and networks within the PLGA and PU matrices, in a compact spatial arrangement favorable for electrical percolation. The proposed approach allowed us to obtain PPy-based composite layers with biologically meaningful conductivities up to 10{sup −2} S/cm for PPy loadings as low as 1:10 weight ratios. Fluorescent staining and viability assays showed that the MG63 osteoblast-like cells cultured on the PPy-based layers deposited by MAPLE were viable and retained their capacity to proliferate. The performance of the proposed method was demonstrated by quantitative evaluation of the calcium phosphate deposits from the cultured cells, as indicative for cell mineralization. Electrical stimulation using 200 μA currents passing through the PPy-based layers, during a time interval of 4 h, enhanced the osteogenesis in the cultured cells. Despite their lowest conductivity, the PPy/PU layers showed the best

  19. Combining 3D human in vitro methods for a 3Rs evaluation of novel titanium surfaces in orthopaedic applications.

    Science.gov (United States)

    Stevenson, G; Rehman, S; Draper, E; Hernández-Nava, E; Hunt, J; Haycock, J W

    2016-07-01

    In this study, we report on a group of complementary human osteoblast in vitro test methods for the preclinical evaluation of 3D porous titanium surfaces. The surfaces were prepared by additive manufacturing (electron beam melting [EBM]) and plasma spraying, allowing the creation of complex lattice surface geometries. Physical properties of the surfaces were characterized by SEM and profilometry and 3D in vitro cell culture using human osteoblasts. Primary human osteoblast cells were found to elicit greater differences between titanium sample surfaces than an MG63 osteoblast-like cell line, particularly in terms of cell survival. Surface morphology was associated with higher osteoblast metabolic activity and mineralization on rougher titanium plasma spray coated surfaces than smoother surfaces. Differences in osteoblast survival and metabolic activity on titanium lattice structures were also found, despite analogous surface morphology at the cellular level. 3D confocal microscopy identified osteoblast organization within complex titanium surface geometries, adhesion, spreading, and alignment to the biomaterial strut geometries. Mineralized nodule formation throughout the lattice structures was also observed, and indicative of early markers of bone in-growth on such materials. Testing methods such as those presented are not traditionally considered by medical device manufacturers, but we suggest have value as an increasingly vital tool in efficiently translating pre-clinical studies, especially in balance with current regulatory practice, commercial demands, the 3Rs, and the relative merits of in vitro and in vivo studies. Biotechnol. Bioeng. 2016;113: 1586-1599. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

  20. Electrically responsive microreservoires for controllable delivery of dexamethasone in bone tissue engineering

    Science.gov (United States)

    Paun, Irina Alexandra; Zamfirescu, Marian; Luculescu, Catalin Romeo; Acasandrei, Adriana Maria; Mustaciosu, Cosmin Catalin; Mihailescu, Mona; Dinescu, Maria

    2017-01-01

    A major concern in orthopedic implants is to decrease the chronic inflammation using specific drug therapies. The newest strategies rely on the controlled delivery of antiinflammatory drugs from carrier biointerfaces designed in the shape of 3D architectures. We report on electrically responsive microreservoires (ERRs) acting as microcontainers for antiinflammatory drugs, as potential biointerfaces in orthopedic implants. The ERRs consist in arrays of vertical microtubes produced by laser direct writing using two photon polymerization effects (2PP_LDW) of a commercially available photoresist, IP-L780. A polypyrrole (conductive)/dexamethasone (drug model) (PPy/Dex) mixture was loaded into the ERRs via a simple immersion process. Then, the ERRs were sealed with a poly(lactic-co-glycolic acid)(PLGA) layer by Matrix Assisted Pulsed Laser Evaporation. ERRs stimulation using voltage cycles between -1 V and +1 V, applied at specific time intervals, at a scan rate of 0.1 V s-1, enabled to control the Dex release. The release time scales were between 150 and 275 h, while the concentrations of Dex released were between 450-460 nM after three applied voltage cycles, for different microreservoires dimensions. The proposed approach was validated in osteoblast-like MG-63 cell cultures. Cell viability and adhesion assays showed that the Dex-loaded ERRs sustained the cells growth and preserved their characteristic polygonal shape. Importantly, for the electrically-stimulated Dex release, the level of the alkaline phosphatase activity increased twice, the osteogenic differentiation surpassed by 1.6 times and the relative level of osteocalcin gene expression was 2.2 times higher as compared with the unstimulated drug release. Overall, the ERRs were able to accelerate the cells osteogenic differentiation via electrically controlled release of Dex.

  1. Pressure-activated microsyringe (PAM) fabrication of bioactive glass-poly(lactic-co-glycolic acid) composite scaffolds for bone tissue regeneration.

    Science.gov (United States)

    Mattioli-Belmonte, M; De Maria, C; Vitale-Brovarone, C; Baino, F; Dicarlo, M; Vozzi, G

    2017-07-01

    The aim of this work was the fabrication and characterization of bioactive glass-poly(lactic-co-glycolic acid) (PLGA) composite scaffolds mimicking the topological features of cancellous bone. Porous multilayer PLGA-CEL2 composite scaffolds were innovatively produced by a pressure-activated microsyringe (PAM) method, a CAD/CAM processing technique originally developed at the University of Pisa. In order to select the optimal formulations to be extruded by PAM, CEL2-PLGA composite films (CEL2 is an experimental bioactive SiO 2 -P 2 O 5 -CaO-MgO-Na 2 O-K 2 O glass developed at Politecnico di Torino) were produced and mechanically tested. The elastic modulus of the films increased from 30 to > 400 MPa, increasing the CEL2 amount (10-50 wt%) in the composite. The mixture containing 20 wt% CEL2 was used to fabricate 2D and 3D bone-like scaffolds composed by layers with different topologies (square, hexagonal and octagonal pores). It was observed that the increase of complexity of 2D topological structures led to an increment of the elastic modulus from 3 to 9 MPa in the composite porous monolayer. The elastic modulus of 3D multilayer scaffolds was intermediate (about 6.5 MPa) between the values of the monolayers with square and octagonal pores (corresponding to the lowest and highest complexity, respectively). MG63 osteoblast-like cells and periosteal-derived precursor cells (PDPCs) were used to assess the biocompatibility of the 3D bone-like scaffolds. A significant increase in cell proliferation between 48 h and 7 days of culture was observed for both cell phenotypes. Moreover, qRT-PCR analysis evidenced an induction of early genes of osteogenesis in PDPCs. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  2. Evaluation of short-term effects of rare earth and other elements used in magnesium alloys on primary cells and cell lines.

    Science.gov (United States)

    Feyerabend, Frank; Fischer, Janine; Holtz, Jakob; Witte, Frank; Willumeit, Regine; Drücker, Heiko; Vogt, Carla; Hort, Norbert

    2010-05-01

    Degradable magnesium alloys for biomedical application are on the verge of being used clinically. Rare earth elements (REEs) are used to improve the mechanical properties of the alloys, but in more or less undefined mixtures. For some elements of this group, data on toxicity and influence on cells are sparse. Therefore in this study the in vitro cytotoxicity of the elements yttrium (Y), neodymium (Nd), dysprosium (Dy), praseodymium (Pr), gadolinium (Gd), lanthanum (La), cerium (Ce), europium (Eu), lithium (Li) and zirconium (Zr) was evaluated by incubation with the chlorides (10-2000 microM); magnesium (Mg) and calcium (Ca) were tested at higher concentrations (200 and 50mM, respectively). The influence on viability of human osteosarcoma cell line MG63, human umbilical cord perivascular (HUCPV) cells and mouse macrophages (RAW 264.7) was determined, as well as the induction of apoptosis and the expression of inflammatory factors (TNF-alpha, IL-1alpha). Significant differences between the applied cells could be observed. RAW exhibited the highest and HUCPV the lowest sensitivity. La and Ce showed the highest cytotoxicity of the analysed elements. Of the elements with high solubility in magnesium alloys, Gd and Dy seem to be more suitable than Y. The focus of magnesium alloy development for biomedical applications should include most defined alloy compositions with well-known tissue-specific and systemic effects. Copyright (c) 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  3. Three-dimensional printing of stem cell-laden hydrogels submerged in a hydrophobic high-density fluid

    International Nuclear Information System (INIS)

    Duarte Campos, Daniela F; Blaeser, Andreas; Weber, Michael; Fischer, Horst; Jäkel, Jörg; Neuss, Sabine; Jahnen-Dechent, Wilhelm

    2013-01-01

    Over the last decade, bioprinting technologies have begun providing important tissue engineering strategies for regenerative medicine and organ transplantation. The major drawback of past approaches has been poor or inadequate material-printing device and substrate combinations, as well as the relatively small size of the printed construct. Here, we hypothesise that cell-laden hydrogels can be printed when submerged in perfluorotributylamine (C 12 F 27 N), a hydrophobic high-density fluid, and that these cells placed within three-dimensional constructs remain viable allowing for cell proliferation and production of extracellular matrix. Human mesenchymal stem cells and MG-63 cells were encapsulated into agarose hydrogels, and subsequently printed in high aspect ratio in three dimensional structures that were supported in high density fluorocarbon. Three-dimensional structures with various shapes and sizes were manufactured and remained stable for more than six months. Live/dead and DAPI stainings showed viable cells 24 h after the printing process, as well as after 21 days in culture. Histological and immunohistochemical analyses after 14 and 21 days revealed viable cells with marked matrix production and signs of proliferation. The compressive strength values of the printed gels consequently increased during the two weeks in culture, revealing encouraging results for future applications in regenerative medicine. (paper)

  4. Development of rheological characterization and twin-screw extrusion/spiral winding processing methods for functionally-graded tissue engineering scaffolds and characterization of cell/biomaterial interactions

    Science.gov (United States)

    Ozkan, Seher

    solvents). Finally, the surface topographies of melt processed poly(L-lactic acid) (ranging from nanoindentations to spherulitic protrusions) were determined to affect the orientation directions of fibroblast and osteoblast-like cells and the spherulitic surfaces giving rise to reduced proliferation rates of fibroblasts.

  5. Menaquinone-4 enhances osteogenic potential of human amniotic fluid mesenchymal stem cells cultured in 2D and 3D dynamic culture systems.

    Science.gov (United States)

    Mandatori, Domitilla; Penolazzi, Letizia; Pipino, Caterina; Di Tomo, Pamela; Di Silvestre, Sara; Di Pietro, Natalia; Trevisani, Sara; Angelozzi, Marco; Ucci, Mariangela; Piva, Roberta; Pandolfi, Assunta

    2018-02-01

    Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment. Copyright © 2017 John Wiley & Sons, Ltd.

  6. Antibacterial activity and biocompatibility of three-dimensional nanostructured porous granules of hydroxyapatite and zinc oxide nanoparticles—an in vitro and in vivo study

    International Nuclear Information System (INIS)

    Grenho, L; Salgado, C L; Monteiro, F J; Fernandes, M H; Ferraz, M P

    2015-01-01

    Ceramic scaffolds are widely studied in the bone tissue engineering field due to their potential in regenerative medicine. However, adhesion of microorganisms on biomaterials with subsequent formation of antibiotic-resistant biofilms is a critical factor in implant-related infections. Therefore, new strategies are needed to address this problem. In the present study, three-dimensional and interconnected porous granules of nanostructured hydroxyapatite (nanoHA) incorporated with different amounts of zinc oxide (ZnO) nanoparticles were produced using a simple polymer sponge replication method. As in vitro experiments, granules were exposed to Staphylococcus aureus and Staphylococcus epidermidis and, after 24 h, the planktonic and sessile populations were assessed. Cytocompatibility towards osteoblast-like cells (MG63 cell line) was also evaluated for a period of 1 and 3 days, through resazurin assay and imaging flow cytometry analysis. As in vivo experiments, nanoHA porous granules with and without ZnO nanoparticles were implanted into the subcutaneous tissue in rats and their inflammatory response after 3, 7 and 30 days was examined, as well as their antibacterial activity after 1 and 3 days of S. aureus inoculation. The developed composites proved to be especially effective at reducing bacterial activity in vitro and in vivo for a weight percentage of 2% ZnO, with a low cell growth inhibition in vitro and no differences in the connective tissue growth and inflammatory response in vivo. Altogether, these results suggest that nanoHA–ZnO porous granules have a great potential to be used in orthopaedic and dental applications as a template for bone regeneration and, simultaneously, to restrain biomaterial-associated infections. (paper)

  7. Preparation of Nanofibrous Structure of Mesoporous Bioactive Glass Microbeads for Biomedical Applications

    Directory of Open Access Journals (Sweden)

    Shiao-Wen Tsai

    2016-06-01

    Full Text Available A highly ordered, mesoporous (pore size 2~50 nm bioactive glass (MBG structure has a greater surface area and pore volume and excellent bone-forming bioactivity compared with traditional bioactive glasses (BGs. Hence, MBGs have been used in drug delivery and bone tissue engineering. MBGs can be developed as either a dense or porous block. Compared with a block, microbeads provide greater flexibility for filling different-shaped cavities and are suitable for culturing cells in vitro. In contrast, the fibrous structure of a scaffold has been shown to increase cell attachment and differentiation due to its ability to mimic the three-dimensional structure of natural extracellular matrices. Hence, the aim of this study is to fabricate MBG microbeads with a fibrous structure. First, a sol-gel/electrospinning technique was utilized to fabricate the MBG nanofiber (MBGNF structure. Subsequently, the MBGNF microbeads (MFBs were produced by an electrospraying technology. The results show that the diameter of the MFBs decreases when the applied voltage increases. The drug loading and release profiles and mechanisms of the MFBs were also evaluated. MFBs had a better drug entrapment efficiency, could reduce the burst release of tetracycline, and sustain the release over 10 days. Hence, the MFBs may be suitable drug carriers. In addition, the cellular attachment of MG63 osteoblast-like cells is significantly higher for MFBs than for glass microbeads after culturing for 4 h. The nanofibrous structure of MFBs could provide an appropriate environment for cellular spreading. Therefore, MFBs have great potential for use as a bone graft material in bone tissue engineering applications.

  8. Antibacterial activity and biocompatibility of three-dimensional nanostructured porous granules of hydroxyapatite and zinc oxide nanoparticles—an in vitro and in vivo study

    Science.gov (United States)

    Grenho, L.; Salgado, C. L.; Fernandes, M. H.; Monteiro, F. J.; Ferraz, M. P.

    2015-08-01

    Ceramic scaffolds are widely studied in the bone tissue engineering field due to their potential in regenerative medicine. However, adhesion of microorganisms on biomaterials with subsequent formation of antibiotic-resistant biofilms is a critical factor in implant-related infections. Therefore, new strategies are needed to address this problem. In the present study, three-dimensional and interconnected porous granules of nanostructured hydroxyapatite (nanoHA) incorporated with different amounts of zinc oxide (ZnO) nanoparticles were produced using a simple polymer sponge replication method. As in vitro experiments, granules were exposed to Staphylococcus aureus and Staphylococcus epidermidis and, after 24 h, the planktonic and sessile populations were assessed. Cytocompatibility towards osteoblast-like cells (MG63 cell line) was also evaluated for a period of 1 and 3 days, through resazurin assay and imaging flow cytometry analysis. As in vivo experiments, nanoHA porous granules with and without ZnO nanoparticles were implanted into the subcutaneous tissue in rats and their inflammatory response after 3, 7 and 30 days was examined, as well as their antibacterial activity after 1 and 3 days of S. aureus inoculation. The developed composites proved to be especially effective at reducing bacterial activity in vitro and in vivo for a weight percentage of 2% ZnO, with a low cell growth inhibition in vitro and no differences in the connective tissue growth and inflammatory response in vivo. Altogether, these results suggest that nanoHA-ZnO porous granules have a great potential to be used in orthopaedic and dental applications as a template for bone regeneration and, simultaneously, to restrain biomaterial-associated infections.

  9. Magnetic biodegradable Fe3O4/CS/PVA nanofibrous membranes for bone regeneration

    International Nuclear Information System (INIS)

    Wei Yan; Zhang Xuehui; Hu Xiaoyang; Deng Xuliang; Song Yu; Lin Yuanhua; Han Bing; Wang Xinzhi

    2011-01-01

    In recent years, interest in magnetic biomimetic scaffolds for tissue engineering has increased considerably. The aim of this study is to develop magnetic biodegradable fibrous materials with potential use in bone regeneration. Magnetic biodegradable Fe 3 O 4 /chitosan (CS)/poly vinyl alcohol (PVA) nanofibrous membranes were achieved by electrospinning with average fiber diameters ranging from 230 to 380 nm and porosity of 83.9-85.1%. The influences of polymer concentration, applied voltage and Fe 3 O 4 nanoparticles loading on the fabrication of nanofibers were investigated. The polymer concentration of 4.5 wt%, applied voltage of 20 kV and Fe 3 O 4 nanoparticles loading of lower than 5 wt% could produce homogeneous, smooth and continuous Fe 3 O 4 /CS/PVA nanofibrous membranes. X-ray diffraction (XRD) data confirmed that the crystalline structure of the Fe 3 O 4 , CS and PVA were maintained during electrospinning process. Fourier transform infrared spectroscopy (FT-IR) demonstrated that the Fe 3 O 4 loading up to 5 wt% did not change the functional groups of CS/PVA greatly. Transmission electron microscopy (TEM) showed islets of Fe 3 O 4 nanoparticles evenly distributed in the fibers. Weak ferrimagnetic behaviors of membranes were revealed by vibrating sample magnetometer (VSM) test. Tensile test exhibited Young's modulus of membranes that were gradually enhanced with the increase of Fe 3 O 4 nanoparticles loading, while ultimate tensile stress and ultimate strain were slightly reduced by Fe 3 O 4 nanoparticles loading of 5%. Additionally, MG63 human osteoblast-like cells were seeded on the magnetic nanofibrous membranes to evaluate their bone biocompatibility. Cell growth dynamics according to MTT assay and scanning electron microscopy (SEM) observation exhibited good cell adhesion and proliferation, suggesting that this magnetic biodegradable Fe 3 O 4 /CS/PVA nanofibrous membranes can be one of promising biomaterials for facilitation of osteogenesis.

  10. 2-methoxyestradiol-mediated anti-tumor effect increases osteoprotegerin expression in osteosarcoma cells.

    Science.gov (United States)

    Benedikt, Michaela B; Mahlum, Eric W; Shogren, Kristen L; Subramaniam, Malayannan; Spelsberg, Thomas C; Yaszemski, Michael J; Maran, Avudaiappan

    2010-04-01

    Osteosarcoma is a bone tumor that frequently develops during adolescence. 2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17beta-estradiol, induces cell cycle arrest and cell death in human osteosarcoma cells. To investigate whether the osteoprotegrin (OPG) protein plays a role in 2-ME actions, we studied the effect of 2-ME treatment on OPG gene expression in human osteosarcoma cells. 2-ME treatment induced OPG gene promoter activity and mRNA levels. Also, Western blot analysis showed that 2-ME treatment increased OPG protein levels in MG63, KHOS, 143B and LM7 osteosarcoma cells by 3-, 1.9-, 2.8-, and 2.5-fold, respectively, but did not affect OPG expression in normal bone cells. In addition, increases in OPG protein levels were observed in osteosarcoma cell culture media after 3 days of 2-ME treatment. The effect of 2-ME on osteosarcoma cells was ligand-specific as parent estrogen, 17beta-estradiol and a tumorigenic estrogen metabolite, 16alpha-hydroxyestradiol, which do not affect osteosarcoma cell cycle and cell death, had no effect on OPG protein expression. Furthermore, co-treating osteosarcoma cells with OPG protein did not further enhance 2-ME-mediated anti-tumor effects. OPG-released in 2-ME-treated cultures led to an increase in osteoblastic activity and a decrease in osteoclast number, respectively. These findings suggest that OPG is not directly involved in 2-ME-mediated anti-proliferative effects in osteosarcoma cells, but rather participates in anti-resorptive functions of 2-ME in bone tumor environment. Copyright 2010 Wiley-Liss, Inc.

  11. 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

  12. Acceleration of segmental bone regeneration in a rabbit model by strontium-doped calcium polyphosphate scaffold through stimulating VEGF and bFGF secretion from osteoblasts

    International Nuclear Information System (INIS)

    Gu, Zhipeng; Zhang, Xu; Li, Li; Wang, Qiguang; Yu, Xixun; Feng, Ting

    2013-01-01

    The development of suitable bioactive three-dimensional scaffold for the promotion of bone regeneration is critical in bone tissue engineering. The purpose of this study was to investigate in vivo osteogenesis of the porous strontium-doped calcium polyphosphate (SCPP) scaffolds for bone repair, as well as the relationship between osteogenic properties of SCPP scaffolds and the secretion of bFGF and VEGF from osteoblasts stimulated by SCPP. Besides, the advantages of scaffolds seeded with mesenchymal stem cells (MSCs) for bone repair were also studied. Firstly, the bone repair evaluation of scaffolds was performed on a rabbit segmental bony defects model over a period of 16 weeks by histology combined with X-ray microradiography. And then, in order to avoid the influence from the other factors such as hypoxia which emerge in vivo study and affect the secretion of VEGF and bFGF from host cells, human osteoblast-like cells (MG63) were seeded to SCPP, CPP and HA scaffolds in vitro to determine the ability of these scaffolds to stimulate the secretion of angiogenic growth factors (VEGF and bFGF) from MG63 and further explore the reason for the better osteogenic properties of SCPP scaffolds. The histological and X-ray microradiographic results showed that the SCPP scaffolds presented better osteogenic potential than CPP and HA scaffolds, when combined with MSCs, the SCPP scaffolds could further accelerate the bone repair. And the amounts of VEGF measured by ELISA assay in SCPP, CPP and HA groups after cultured for 7 days were about 364.989 pg/mL, 244.035 pg/mL and 232.785 pg/mL, respectively. Accordingly, the amounts of bFGF were about 27.085 pg/mL, 15.727 pg/mL and 8.326 pg/mL. The results revealed that the SCPP scaffolds significantly enhanced the bFGF and VEGF secretion compared with other scaffolds. The results presented in vivo and in vitro study demonstrated that the SCPP could accelerate bone formation through stimulating the secretion of VEGF and bFGF from

  13. Synthesis and Characterization of Novel Polycarbonate Based Polyurethane/Polymer Wrapped Hydroxyapatite Nanocomposites: Mechanical Properties, Osteoconductivity and Biocompatibility.

    Science.gov (United States)

    Selvakumar, M; Jaganathan, Saravana Kumar; Nando, Golok B; Chattopadhyay, Santanu

    2015-02-01

    The present investigation reports the preparation of two types of 2D rod-like nano-hydroxyapatite (nHA) (unmodified and Polypropylene glycol (PPG) wrapped) of varying high-aspect ratios, by modified co-precipitation methods, without any templates. These nHA were successfully introduced into novel synthesized Thermoplastic Polyurethane (TPU) matrices based on polycarbonate soft segments, by both in-situ and ex-situ techniques. Physico-mechanical properties of the in-situ prepared TPU/nHA nanocomposites were found to be superior compared to the ex-situ counterparts, and pristine nHA reinforced TPU. Improved biocompatibility of the prepared nanocomposites was confirmed by MTT assays using osteoblast-like MG63 cells. Cell proliferation was evident over an extended period. Osteoconductivity of the nanocomposites was observed by successful formation of an apatite layer on the surface of the samples, after immersion into simulated body fluid (SBF). Prothrombin time (PT) and activated partial thromboplastin time (APTT), as calculated from coagulation assays, displayed an increase in the clotting time, particularly for the PPG-wrapped nHA nanocomposites, prepared through the in-situ technique. Only 0.3% of hemolysis was observed for the in-situ prepared nanocomposites, which establishes the antithrombotic property of the material. The key parameters for enhancing the technical properties and biocompatibility of the nanocomposites are: the interfacial adhesion parameter (B(σy)), the polymer-filler affinity, the aspect ratio of filler and non-covalent modifications, and the state of dispersion. Thus, the novel TPU/polymer wrapped nHA nanocomposites have great potential for biomedical applications, in particular for vascular prostheses, cardiovascular implants, scaffolds, and soft and hard tissues implants.

  14. Characterization of mechanical and biological properties of 3-D scaffolds reinforced with zinc oxide for bone tissue engineering.

    Directory of Open Access Journals (Sweden)

    Pei Feng

    Full Text Available A scaffold for bone tissue engineering should have highly interconnected porous structure, appropriate mechanical and biological properties. In this work, we fabricated well-interconnected porous β-tricalcium phosphate (β-TCP scaffolds via selective laser sintering (SLS. We found that the mechanical and biological properties of the scaffolds were improved by doping of zinc oxide (ZnO. Our data showed that the fracture toughness increased from 1.09 to 1.40 MPam(1/2, and the compressive strength increased from 3.01 to 17.89 MPa when the content of ZnO increased from 0 to 2.5 wt%. It is hypothesized that the increase of ZnO would lead to a reduction in grain size and an increase in density of the strut. However, the fracture toughness and compressive strength decreased with further increasing of ZnO content, which may be due to the sharp increase in grain size. The biocompatibility of the scaffolds was investigated by analyzing the adhesion and the morphology of human osteoblast-like MG-63 cells cultured on the surfaces of the scaffolds. The scaffolds exhibited better and better ability to support cell attachment and proliferation when the content of ZnO increased from 0 to 2.5 wt%. Moreover, a bone like apatite layer formed on the surfaces of the scaffolds after incubation in simulated body fluid (SBF, indicating an ability of osteoinduction and osteoconduction. In summary, interconnected porous β-TCP scaffolds doped with ZnO were successfully fabricated and revealed good mechanical and biological properties, which may be used for bone repair and replacement potentially.

  15. A Simple Method to Functionalize the Surface of Plasma Electrolytic Oxidation Produced TiO2 Coatings for Growing Hydroxyapatite

    International Nuclear Information System (INIS)

    Teng, Huan-Ping; Yang, Chia-Jung; Lin, Jia-Fu; Huang, Yu-Hsin; Lu, Fu-Hsing

    2016-01-01

    Highlights: • TiO 2 coatings with porous surfaces were produced by plasma electrolytic oxidation. • Simple pre-immersion in K 2 HPO 4 could functionalize the surfaces of the TiO 2 . • Such pre-immersion enhanced substantially the growth of hydroxyapatite in SBF. • Growth mechanisms of hydroxyapatite via the pre-immersion have been proposed. • MTT assay shows great osteoblast-like cell activity on the obtained hydroxyapatite. - Abstract: Conventionally, hydrothermal treatment was often used to modify the TiO 2 surface prior to the growth of hydroxyapatite (HA) that is one of the most important implant biomaterials. In this work, a simple pre-immersion of the obtained TiO 2 in a weak base, instead of the conventionally high pressure-temperature hydrothermal pre-treatment, was conducted prior to the growth of HA. Firstly, anatase TiO 2 coatings with porous surfaces were produced by plasma electrolytic oxidation with optimized processing parameters. X-ray diffraction patterns and field-emission microscopy reveal that the anatase TiO 2 films with porous surfaces were produced by plasma electrolytic oxidation. Subsequently, the films were pre-immersed in 0.1–2 M K 2 HPO 4 solutions for only 10 min. Fourier transform infrared spectroscopy shows that the −OH functional groups were generated after such pre-immersion, which could enhance significantly the growth of a single phase of HA in simulated body fluid (SBF). Growth mechanisms of HA via the pre-immersion treatment and soaking in SBF have been proposed. Moreover, the proliferation rate and attachment of the MG-63 osteoblast cells were greatly enhanced on the obtained HA compared to that without the immersion pre-treatment from the MTT assay and morphology analyses. This simple immersion pre-treatment evidently provides an easy route for the growth of HA and has great potential for biomedical applications.

  16. 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.

  17. Antiproliferative and apoptosis-inducing activity of an oxidovanadium(IV) complex with the flavonoid silibinin against osteosarcoma cells.

    Science.gov (United States)

    Leon, I E; Porro, V; Di Virgilio, A L; Naso, L G; Williams, P A M; Bollati-Fogolín, M; Etcheverry, S B

    2014-01-01

    Flavonoids are a large family of polyphenolic compounds synthesized by plants. They display interesting biological effects mainly related to their antioxidant properties. On the other hand, vanadium compounds also exhibit different biological and pharmacological effects in cell culture and in animal models. Since coordination of ligands to metals can improve or change the pharmacological properties, we report herein, for the first time, a detailed study of the mechanisms of action of an oxidovanadium(IV) complex with the flavonoid silibinin, Na2[VO(silibinin)2]·6H2O (VOsil), in a model of the human osteosarcoma derived cell line MG-63. The complex inhibited the viability of osteosarcoma cells in a dose-dependent manner with a greater potency than that of silibinin and oxidovanadium(IV) (p cell cycle arrest and activated caspase 3, triggering apoptosis as determined by flow cytometry. As a whole, these results show the main mechanisms of the deleterious effects of VOsil in the osteosarcoma cell line, demonstrating that this complex is a promising compound for cancer treatments.

  18. Growth and Potential Damage of Human Bone-Derived Cells on Fresh and Aged Fullerene C60 Films

    Directory of Open Access Journals (Sweden)

    Jiri Vacik

    2013-04-01

    Full Text Available Fullerenes are nanoparticles composed of carbon atoms arranged in a spherical hollow cage-like structure. Numerous studies have evaluated the therapeutic potential of fullerene derivates against oxidative stress-associated conditions, including the prevention or treatment of arthritis. On the other hand, fullerenes are not only able to quench, but also to generate harmful reactive oxygen species. The reactivity of fullerenes may change in time due to the oxidation and polymerization of fullerenes in an air atmosphere. In this study, we therefore tested the dependence between the age of fullerene films (from one week to one year and the proliferation, viability and metabolic activity of human osteosarcoma cells (lines MG-63 and U-2 OS. We also monitored potential membrane and DNA damage and morphological changes of the cells. After seven days of cultivation, we did not observe any cytotoxic morphological changes, such as enlarged cells or cytosolic vacuole formation. Furthermore, there was no increased level of DNA damage. The increasing age of the fullerene films did not cause enhancement of cytotoxicity. On the contrary, it resulted in an improvement in the properties of these materials, which are more suitable for cell cultivation. Therefore, fullerene films could be considered as a promising material with potential use as a bioactive coating of cell carriers for bone tissue engineering.

  19. Growth and potential damage of human bone-derived cells on fresh and aged fullerene c60 films.

    Science.gov (United States)

    Kopova, Ivana; Bacakova, Lucie; Lavrentiev, Vasily; Vacik, Jiri

    2013-04-26

    Fullerenes are nanoparticles composed of carbon atoms arranged in a spherical hollow cage-like structure. Numerous studies have evaluated the therapeutic potential of fullerene derivates against oxidative stress-associated conditions, including the prevention or treatment of arthritis. On the other hand, fullerenes are not only able to quench, but also to generate harmful reactive oxygen species. The reactivity of fullerenes may change in time due to the oxidation and polymerization of fullerenes in an air atmosphere. In this study, we therefore tested the dependence between the age of fullerene films (from one week to one year) and the proliferation, viability and metabolic activity of human osteosarcoma cells (lines MG-63 and U-2 OS). We also monitored potential membrane and DNA damage and morphological changes of the cells. After seven days of cultivation, we did not observe any cytotoxic morphological changes, such as enlarged cells or cytosolic vacuole formation. Furthermore, there was no increased level of DNA damage. The increasing age of the fullerene films did not cause enhancement of cytotoxicity. On the contrary, it resulted in an improvement in the properties of these materials, which are more suitable for cell cultivation. Therefore, fullerene films could be considered as a promising material with potential use as a bioactive coating of cell carriers for bone tissue engineering.

  20. Therapeutic potential of the metabolic modulator Metformin on osteosarcoma cancer stem-like cells.

    Science.gov (United States)

    Paiva-Oliveira, Daniela I; Martins-Neves, Sara R; Abrunhosa, Antero J; Fontes-Ribeiro, Carlos; Gomes, Célia M F

    2018-01-01

    Osteosarcoma is the most common primary bone tumour appearing in children and adolescents. Recent studies demonstrate that osteosarcoma possesses a stem-like cell subset, so-called cancer stem-like cells, refractory to conventional chemotherapeutics and pointed out as responsible for relapses frequently observed in osteosarcoma patients. Here, we explored the therapeutic potential of Metformin on osteosarcoma stem-like cells, alone and as a chemosensitizer of doxorubicin. Stem-like cells were isolated from human osteosarcoma cell lines, MNNG/HOS and MG-63, using the sphere-forming assay. Metformin cytotoxicity alone and combined with doxorubicin were evaluated using MTT/BrdU assays. Protein levels of AMPK and AKT were evaluated by Western Blot. Cellular metabolic status was assessed based on [ 18 F]-FDG uptake and lactate production measurements. Sphere-forming efficiency and expression of pluripotency transcription factors analysed by qRT-PCR were tested as readout of Metformin effects on stemness features. Metformin induced a concentration-dependent decrease in the metabolic activity and proliferation of sphere-forming cells and improved doxorubicin-induced cytotoxicity. This drug also down-regulated the expression of master regulators of pluripotency (OCT4, SOX2, NANOG), and decreased spheres' self-renewal ability. Metformin effects on mitochondria led to the activation and phosphorylation of the energetic sensor AMPK along with an upregulation of the pro-survival AKT pathway in both cell populations. Furthermore, Metformin-induced mitochondrial stress increased [ 18 F]-FDG uptake and lactate production in parental cells but not in the quiescent stem-like cells, suggesting the inability of the latter to cope with the energy crisis induced by metformin. This preclinical study suggests that Metformin may be a potentially useful therapeutic agent and chemosensitizer of osteosarcoma stem-like cells to doxorubicin.

  1. Melatonin releasing PLGA micro/nanoparticles and their effect on osteosarcoma cells.

    Science.gov (United States)

    Altındal, Damla Çetin; Gümüşderelioğlu, Menemşe

    2016-02-01

    Melatonin loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles and microparticles in the diameter of ∼200 nm and 3.5 μm, respectively, were prepared by emulsion-diffusion-evaporation method. Melatonin entrapment into the particles was significantly improved with the addition of 0.2% (w/v) melatonin into the aqueous phase and encapsulation efficiencies were found as 14 and 27% for nanoparticles and microparticles, respectively. At the end of 40 days, ∼70% of melatonin was released from both of particles, with high burst release. Both blank and melatonin loaded PLGA nanoparticles caused toxic effect on the MG-63 cells due to their uptake by the cells. However, when 0.05 mg microparticle that is carrying ∼1.7 μg melatonin was added to the cm(2) of culture, inhibitory effect of melatonin on the cells were obviously observed. The results would provide an expectation about the usage of melatonin as an adjunct to the routine chemotherapy of osteosarcoma by encapsulating it into a polymeric carrier system.

  2. In vitro testing of curcumin based composites coatings as antitumoral systems against osteosarcoma cells

    Science.gov (United States)

    Tirca, I.; Mitran, V.; Marascu, V.; Brajnicov, S.; Ion, V.; Stokker-Cheregi, F.; Popovici, I. A.; Cimpean, A.; Dinca, V.; Dinescu, M.

    2017-12-01

    In this work, we propose a new design for biodegradable composite coatings obtained by laser methods, which are aimed at evaluating the effects of active antitumoral elements on osteosarcoma cells. Our approach relies on embedding curcumin, which is a natural polyphenol having antitumoral properties, within biodegradable copolymer coatings (i.e. polyvinyl alcohol-polyethylene glycol - PVA-PEG) by using matrix assisted pulsed laser evaporation (MAPLE). The structural and morphological characteristics of the coatings were tailored by using different solvents (water, ethanol, benzene, dimethylsufoxide) as deposition matrix. The morphological characteristics of the resulting films were investigated by atomic force microscopy (AFM), whereas their chemical composition was characterized by Fourier transform infrared spectroscopy (FTIR). These characteristics were correlated with the degradation behavior by using ellipsometry (SE) and AFM measurements data. The in vitro study of the MG-63 osteosarcoma cell behavior indicates that the developed hybrid coatings significantly decreased osteosarcoma cell viability and proliferation potential. The physico-chemical characteristics of the thin films, along with the preliminary in vitro analyses, suggest that our developed polymeric hybrid coatings represent an efficient way to tackle the design of antitumoral surfaces, with applications in biomedicine.

  3. Tubular Scaffold with Shape Recovery Effect for Cell Guide Applications

    Directory of Open Access Journals (Sweden)

    Kazi M. Zakir Hossain

    2015-07-01

    Full Text Available Tubular scaffolds with aligned polylactic acid (PLA fibres were fabricated for cell guide applications by immersing rolled PLA fibre mats into a polyvinyl acetate (PVAc solution to bind the mats. The PVAc solution was also mixed with up to 30 wt % β-tricalcium phosphate (β-TCP content. Cross-sectional images of the scaffold materials obtained via scanning electron microscopy (SEM revealed the aligned fibre morphology along with a significant number of voids in between the bundles of fibres. The addition of β-TCP into the scaffolds played an important role in increasing the void content from 17.1% to 25.3% for the 30 wt % β-TCP loading, which was measured via micro-CT (µCT analysis. Furthermore, µCT analyses revealed the distribution of aggregated β-TCP particles in between the various PLA fibre layers of the scaffold. The compressive modulus properties of the scaffolds increased from 66 MPa to 83 MPa and the compressive strength properties decreased from 67 MPa to 41 MPa for the 30 wt % β-TCP content scaffold. The scaffolds produced were observed to change into a soft and flexible form which demonstrated shape recovery properties after immersion in phosphate buffered saline (PBS media at 37 °C for 24 h. The cytocompatibility studies (using MG-63 human osteosarcoma cell line revealed preferential cell proliferation along the longitudinal direction of the fibres as compared to the control tissue culture plastic. The manufacturing process highlighted above reveals a simple process for inducing controlled cell alignment and varying porosity features within tubular scaffolds for potential tissue engineering applications.

  4. Opposite Effects of Soluble Factors Secreted by Adipose Tissue on Proliferating and Quiescent Osteosarcoma Cells.

    Science.gov (United States)

    Avril, Pierre; Duteille, Franck; Ridel, Perrine; Heymann, Marie-Françoise; De Pinieux, Gonzague; Rédini, Françoise; Blanchard, Frédéric; Heymann, Dominique; Trichet, Valérie; Perrot, Pierre

    2016-03-01

    Autologous adipose tissue transfer may be performed for aesthetic needs following resection of osteosarcoma, the most frequent primary malignant tumor of bone, excluding myeloma. The safety of autologous adipose tissue transfer regarding the potential risk of cancer recurrence must be addressed. Adipose tissue injection was tested in a human osteosarcoma preclinical model induced by MNNG-HOS cells. Culture media without growth factors from fetal bovine serum were conditioned with adipose tissue samples and added to two osteosarcoma cell lines (MNNG-HOS and MG-63) that were cultured in monolayer or maintained in nonadherent spheres, favoring a proliferation or quiescent stage, respectively. Proliferation and cell cycle were analyzed. Adipose tissue injection increased local growth of osteosarcoma in mice but was not associated with aggravation of lung metastasis or osteolysis. Adipose tissue-derived soluble factors increased the in vitro proliferation of osteosarcoma cells up to 180 percent. Interleukin-6 and leptin were measured in higher concentrations in adipose tissue-conditioned medium than in osteosarcoma cell-conditioned medium, but the authors' results indicated that they were not implicated alone. Furthermore, adipose tissue-derived soluble factors did not favor a G0-to-G1 phase transition of MNNG-HOS cells in nonadherent oncospheres. This study indicates that adipose tissue-soluble factors activate osteosarcoma cell cycle from G1 to mitosis phases, but do not promote the transition from quiescent G0 to G1 phases. Autologous adipose tissue transfer may not be involved in the activation of dormant tumor cells or cancer stem cells.

  5. Combined Use of Zoledronic Acid Augments Ursolic Acid-Induced Apoptosis in Human Osteosarcoma Cells through Enhanced Oxidative Stress and Autophagy

    Directory of Open Access Journals (Sweden)

    Chia-Chieh Wu

    2016-11-01

    Full Text Available Ursolic acid (UA, a naturally occurring pentacyclic triterpene acid found in many medicinal herbs and edible plants, triggers apoptosis in several tumor cell lines but not in human bone cancer cells. Most recently, we have demonstrated that UA exposure reduces the viability of human osteosarcoma MG-63 cells through enhanced oxidative stress and apoptosis. Interestingly, an inhibitor of osteoclast-mediated bone resorption, zoledronic acid (ZOL, also a third-generation nitrogen-containing bisphosphonate, is effective in the treatment of bone metastases in patients with various solid tumors. In this present study, we found that UA combined with ZOL to significantly suppress cell viability, colony formation, and induce apoptosis in two lines of human osteosarcoma cells. The pre-treatment of the antioxidant had reversed the oxidative stress and cell viability inhibition in the combined treatment, indicating that oxidative stress is important in the combined anti-tumor effects. Moreover, we demonstrated that ZOL combined with UA significantly induced autophagy and co-administration of autophagy inhibitor reduces the growth inhibitory effect of combined treatment. Collectively, these data shed light on the pathways involved in the combined effects of ZOL and UA that might serve as a potential therapy against osteosarcoma.

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

    Science.gov (United States)

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

    2015-01-01

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

  7. Calcium silicate ceramic scaffolds toughened with hydroxyapatite whiskers for bone tissue engineering

    International Nuclear Information System (INIS)

    Feng, Pei; Wei, Pingpin; Li, Pengjian; Gao, Chengde; Shuai, Cijun; Peng, Shuping

    2014-01-01

    Calcium silicate possessed excellent biocompatibility, bioactivity and degradability, while the high brittleness limited its application in load-bearing sites. Hydroxyapatite whiskers ranging from 0 to 30 wt.% were incorporated into the calcium silicate matrix to improve the strength and fracture resistance. Porous scaffolds were fabricated by selective laser sintering. The effects of hydroxyapatite whiskers on the mechanical properties and toughening mechanisms were investigated. The results showed that the scaffolds had a uniform and continuous inner network with the pore size ranging between 0.5 mm and 0.8 mm. The mechanical properties were enhanced with increasing hydroxyapatite whiskers, reached a maximum at 20 wt.% (compressive strength: 27.28 MPa, compressive Young's modulus: 156.2 MPa, flexural strength: 15.64 MPa and fracture toughness: 1.43 MPa·m 1/2 ) and then decreased by addition of more hydroxyapatite whiskers. The improvement of mechanical properties was due to whisker pull-out, crack deflection and crack bridging. Moreover, the degradation rate decreased with the increase of hydroxyapatite whisker content. A layer of bone-like apatite was formed on the scaffold surfaces after being soaked in simulated body fluid. Human osteoblast-like MG-63 cells spread well on the scaffolds and proliferated with increasing culture time. These findings suggested that the calcium silicate scaffolds reinforced with hydroxyapatite whiskers showed great potential for bone regeneration and tissue engineering applications. - Highlights: • HA whiskers were incorporated into CS to improve the properties. • The scaffolds were successfully fabricated by SLS. • Toughening mechanisms was whisker pull-out, crack deflection and bridging. • The scaffolds showed excellent apatite forming ability

  8. Development of macroporous calcium phosphate scaffold processed via microwave rapid drying

    Energy Technology Data Exchange (ETDEWEB)

    Jamuna-Thevi, K., E-mail: jamuna@sirim.my [Advanced Materials Research Centre (AMREC), SIRIM Berhad, Lot 34, Jalan Hi-Tech 2/3, Kulim Hi-Tech Park, 09000 Kulim, Kedah (Malaysia); Zakaria, F.A. [Advanced Materials Research Centre (AMREC), SIRIM Berhad, Lot 34, Jalan Hi-Tech 2/3, Kulim Hi-Tech Park, 09000 Kulim, Kedah (Malaysia); Othman, R. [Materials and Mineral Resources Engineering, Engineering Campus, Universiti Sains Malaysia, Seri Ampangan, 14300 Nibong Tebal, Penang (Malaysia); Muhamad, S. [Bioassay Unit, Herbal Medicine Research Centre (HMRC), Institute for Medical Research (IMR), Jalan Pahang, 50588 Kuala Lumpur (Malaysia)

    2009-06-01

    Porous hydroxyapatite (HA) scaffold has great potential in bone tissue engineering applications. A new method to fabricate macroporous calcium phosphate (CP) scaffold via microwave irradiation, followed by conventional sintering to form HA scaffold was developed. Incorporation of trisodium citrate dihydrate and citric acid in the CP mixture gave macroporous scaffolds upon microwave rapid drying. In this work, a mixture of {beta}-tricalcium phosphate ({beta}-TCP), calcium carbonate (CaCO{sub 3}), trisodium citrate dihydrate, citric acid and double distilled de-ionised water (DDI) was exposed to microwave radiation to form a macroporous structure. Based on gross eye examinations, addition of trisodium citrate at 30 and 40 wt.% in the CP mixture ({beta}-TCP and CaCO{sub 3}) without citric acid indicates increasing order of pore volume where the highest porosity yield was observed at 40 wt.% of trisodium citrate addition and the pore size was detected at several millimeters. Therefore, optimization of pore size was performed by adding 3-7 wt.% of citric acid in the CP mixture which was separately mixed with 30 and 40 wt.% of trisodium citrate for comparison purposes. Fabricated scaffolds were calcined at 600 deg. C and washed with DDI water to remove the sodium hydroxycarbonate and sintered at 1250 deg. C to form HA phase as confirmed in the X-ray diffraction (XRD) results. Based on Archimedes method, HA scaffolds prepared from 40 wt.% of trisodium citrate with 3-7 wt.% of citric acid added CP mixture have an open and interconnected porous structure ranging from 51 to 53 vol.% and observation using Scanning electron microscope (SEM) showed the pore size distribution between 100 and 500 {mu}m. The cytotoxicity tests revealed that the porous HA scaffolds have no cytotoxic potential on MG63 osteoblast-like cells which might allow for their use as biomaterials.

  9. Calcium silicate ceramic scaffolds toughened with hydroxyapatite whiskers for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Pei [State Key Laboratory of High Performance Complex Manufacturing, Central South University, Changsha 410083, PR China, (China); Wei, Pingpin [Cancer Research Institute, Central South University, Changsha 410078 (China); Li, Pengjian; Gao, Chengde [State Key Laboratory of High Performance Complex Manufacturing, Central South University, Changsha 410083, PR China, (China); Shuai, Cijun, E-mail: shuai@csu.edu.cn [State Key Laboratory of High Performance Complex Manufacturing, Central South University, Changsha 410083, PR China, (China); Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425 (United States); Peng, Shuping, E-mail: shuping@csu.edu.cn [Cancer Research Institute, Central South University, Changsha 410078 (China)

    2014-11-15

    Calcium silicate possessed excellent biocompatibility, bioactivity and degradability, while the high brittleness limited its application in load-bearing sites. Hydroxyapatite whiskers ranging from 0 to 30 wt.% were incorporated into the calcium silicate matrix to improve the strength and fracture resistance. Porous scaffolds were fabricated by selective laser sintering. The effects of hydroxyapatite whiskers on the mechanical properties and toughening mechanisms were investigated. The results showed that the scaffolds had a uniform and continuous inner network with the pore size ranging between 0.5 mm and 0.8 mm. The mechanical properties were enhanced with increasing hydroxyapatite whiskers, reached a maximum at 20 wt.% (compressive strength: 27.28 MPa, compressive Young's modulus: 156.2 MPa, flexural strength: 15.64 MPa and fracture toughness: 1.43 MPa·m{sup 1/2}) and then decreased by addition of more hydroxyapatite whiskers. The improvement of mechanical properties was due to whisker pull-out, crack deflection and crack bridging. Moreover, the degradation rate decreased with the increase of hydroxyapatite whisker content. A layer of bone-like apatite was formed on the scaffold surfaces after being soaked in simulated body fluid. Human osteoblast-like MG-63 cells spread well on the scaffolds and proliferated with increasing culture time. These findings suggested that the calcium silicate scaffolds reinforced with hydroxyapatite whiskers showed great potential for bone regeneration and tissue engineering applications. - Highlights: • HA whiskers were incorporated into CS to improve the properties. • The scaffolds were successfully fabricated by SLS. • Toughening mechanisms was whisker pull-out, crack deflection and bridging. • The scaffolds showed excellent apatite forming ability.

  10. Novel approach to the fabrication of an artificial small bone using a combination of sponge replica and electrospinning methods

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yang-Hee; Lee, Byong-Taek, E-mail: lbt@sch.ac.kr [Department of Biomedical Engineering and Materials, School of Medicine, Soonchunhyang University 366-1, Ssangyong-dong, Cheonan, Chungnam 330-090 (Korea, Republic of)

    2011-06-15

    In this study, a novel artificial small bone consisting of ZrO{sub 2}-biphasic calcium phosphate/polymethylmethacrylate-polycaprolactone-hydroxyapatite (ZrO{sub 2}-BCP/PMMA-PCL-HAp) was fabricated using a combination of sponge replica and electrospinning methods. To mimic the cancellous bone, the ZrO{sub 2}/BCP scaffold was composed of three layers, ZrO{sub 2}, ZrO{sub 2}/BCP and BCP, fabricated by the sponge replica method. The PMMA-PCL fibers loaded with HAp powder were wrapped around the ZrO{sub 2}/BCP scaffold using the electrospinning process. To imitate the Haversian canal region of the bone, HAp-loaded PMMA-PCL fibers were wrapped around a steel wire of 0.3 mm diameter. As a result, the bundles of fiber wrapped around the wires imitated the osteon structure of the cortical bone. Finally, the ZrO{sub 2}/BCP scaffold was surrounded by HAp-loaded PMMA-PCL composite bundles. After removal of the steel wires, the ZrO{sub 2}/BCP scaffold and bundles of HAp-loaded PMMA-PCL formed an interconnected structure resembling the human bone. Its diameter, compressive strength and porosity were approximately 12 mm, 5 MPa and 70%, respectively, and the viability of MG-63 osteoblast-like cells was determined to be over 90% by the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. This artificial bone shows excellent cytocompatibility and is a promising bone regeneration material.

  11. Generating 3D tissue constructs with mesenchymal stem cells and a cancellous bone graft for orthopaedic applications

    International Nuclear Information System (INIS)

    Arca, Turkan; Genever, Paul; Proffitt, Joanne

    2011-01-01

    Bone matrix (BM) is an acellular crosslinked porcine-derived cancellous bone graft, and therefore may provide advantages over other synthetic and naturally derived materials for use in orthopaedic surgery. Here, we analysed the potential of BM to support the growth and differentiation of primary human multipotent stromal cells/mesenchymal stem cells (MSCs) in order to predict in vivo bone regeneration events. Imaging with laser scanning confocal microscopy and scanning electron microscopy showed that 1 day after static seeding, a dense population of viable MSCs could be achieved on scaffolds suggesting they could be used for in vivo delivery of cells to the implant site. Long-term growth analysis by confocal imaging and histology demonstrated that BM was permissive to the growth and the 3D population of primary MSCs and an enhanced green fluorescent protein expressing osteosarcoma cell line, eGFP.MG63s, over several days in culture. Measurement of alkaline phosphatase (ALP) activities and mRNA expression levels of osteogenic markers (Runx-2, ALP, collagen type I, osteonectin, osteocalcin and osteopontin) indicated that BM supported osteogenesis of MSCs when supplemented with osteogenic stimulants. Upregulation of some of these osteogenic markers on BM, but not on tissue culture plastic, under non-osteogenic conditions suggested that BM also had osteoinductive capacities.

  12. Growth and Potential Damage of Human Bone-Derived Cells Cultured on Fresh and Aged C60/Ti Films

    Science.gov (United States)

    Kopova, Ivana; Lavrentiev, Vasily; Vacik, Jiri; Bacakova, Lucie

    2015-01-01

    Thin films of binary C60/Ti composites, with various concentrations of Ti ranging from ~ 25% to ~ 70%, were deposited on microscopic glass coverslips and were tested for their potential use in bone tissue engineering as substrates for the adhesion and growth of bone cells. The novelty of this approach lies in the combination of Ti atoms (i.e., widely used biocompatible material for the construction of stomatological and orthopedic implants) with atoms of fullerene C60, which can act as very efficient radical scavengers. However, fullerenes and their derivatives are able to generate harmful reactive oxygen species and to have cytotoxic effects. In order to stabilize C60 molecules and to prevent their possible cytotoxic effects, deposition in the compact form of Ti/C60 composites (with various Ti concentrations) was chosen. The reactivity of C60/Ti composites may change in time due to the physicochemical changes of molecules in an air atmosphere. In this study, we therefore tested the dependence between the age of C60/Ti films (from one week to one year) and the adhesion, morphology, proliferation, viability, metabolic activity and potential DNA damage to human osteosarcoma cells (lines MG-63 and U-2 OS). After 7 days of cultivation, we did not observe any negative influence of fresh or aged C60/Ti layers on cell behavior, including the DNA damage response. The presence of Ti atoms resulted in improved properties of the C60 layers, which became more suitable for cell cultivation. PMID:25875338

  13. Generating 3D tissue constructs with mesenchymal stem cells and a cancellous bone graft for orthopaedic applications

    Energy Technology Data Exchange (ETDEWEB)

    Arca, Turkan; Genever, Paul [Department of Biology, University of York, York, YO10 5DD (United Kingdom); Proffitt, Joanne, E-mail: paul.genever@york.ac.uk [TSL Centre of Biologics, Covidien, Allerton Bywater, Castleford, WF10 2DB (United Kingdom)

    2011-04-15

    Bone matrix (BM) is an acellular crosslinked porcine-derived cancellous bone graft, and therefore may provide advantages over other synthetic and naturally derived materials for use in orthopaedic surgery. Here, we analysed the potential of BM to support the growth and differentiation of primary human multipotent stromal cells/mesenchymal stem cells (MSCs) in order to predict in vivo bone regeneration events. Imaging with laser scanning confocal microscopy and scanning electron microscopy showed that 1 day after static seeding, a dense population of viable MSCs could be achieved on scaffolds suggesting they could be used for in vivo delivery of cells to the implant site. Long-term growth analysis by confocal imaging and histology demonstrated that BM was permissive to the growth and the 3D population of primary MSCs and an enhanced green fluorescent protein expressing osteosarcoma cell line, eGFP.MG63s, over several days in culture. Measurement of alkaline phosphatase (ALP) activities and mRNA expression levels of osteogenic markers (Runx-2, ALP, collagen type I, osteonectin, osteocalcin and osteopontin) indicated that BM supported osteogenesis of MSCs when supplemented with osteogenic stimulants. Upregulation of some of these osteogenic markers on BM, but not on tissue culture plastic, under non-osteogenic conditions suggested that BM also had osteoinductive capacities.

  14. Plasma-deposited fluorocarbon polymer films on titanium for preventing cell adhesion: a surface finishing for temporarily used orthopaedic implants

    Science.gov (United States)

    Finke, B.; Testrich, H.; Rebl, H.; Walschus, U.; Schlosser, M.; Zietz, C.; Staehlke, S.; Nebe, J. B.; Weltmann, K. D.; Meichsner, J.; Polak, M.

    2016-06-01

    The design of a titanium implant surface should ideally support its later application in clinical use. Temporarily used implants have to fulfil requirements different from permanent implants: they should ensure the mechanical stabilization of the bone stock but in trauma surgery they should not be integrated into the bone because they will be removed after fracture healing. Finishing of the implant surface by a plasma-fluorocarbon-polymer (PFP) coating is a possible approach for preventing cell adhesion of osteoblasts. Two different low pressure gas-discharge plasma processes, microwave (MW 2.45 GHz) and capacitively coupled radio frequency (RF 13.56 MHz) plasma, were applied for the deposition of the PFP film using a mixture of the precursor octafluoropropane (C3F8) and hydrogen (H2). The thin films were characterized by x-ray photoelectron spectroscopy, Fourier transform infrared reflection absorption spectroscopy, and water contact angle measurements. Cell culture experiments show that cell adhesion and spreading of MG-63 osteoblasts were clearly reduced or nonexistent on these surfaces, also after 24 h of storage in the cell culture medium. In vivo data demonstrated that the local inflammatory tissue response for the PFP films deposited in MW and RF plasma were comparable to uncoated controls.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-11-01

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

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  17. miR-335 negatively regulates osteosarcoma stem cell-like properties by targeting POU5F1.

    Science.gov (United States)

    Guo, Xiaodong; Yu, Ling; Zhang, Zhengpei; Dai, Guo; Gao, Tian; Guo, Weichun

    2017-01-01

    Evidence is accumulating to link cancer stem cells to the pathogenesis and progression of osteosarcoma. The aim of this study is to investigate the role of miR-335 in osteosarcoma stem cells. Tumor spheroid culture and flow cytometry were applied to screen out osteosarcoma stem cells. Real-time quantitative PCR was used to detect the expression level of miR-335 in MG63, U2OS and 143B osteosarcoma stem cells. The relationship of miR-335 expression with osteosarcoma stem cells was then analyzed. Transwell assay and transplantation assay were performed to elucidate biological effects of miR-335 on cell invasion and vivo tumor formation. Western Blot and luciferase assays were executed to investigate the regulation of POU5F1 by miR-335. The expression of miR-335 in osteosarcoma stem cells was lower than their differentiated counterparts. Cells expressing miR-335 possessed decreased stem cell-like properties. Gain or loss of function assays were applied to find that miR-335 antagonist promoted stem cell-like properties as well as invasion. Luciferase report and transfection assay showed that POU5F1 was downregulated by miR-335. Pre-miR-335 resulted in tumor enhanced sensitivity to traditional chemotherapy, whereas anti-miR-335 promoted chemoresistance. Finally, the inhibitory effect of miR-335 on in vivo tumor formation showed that combination of pre-miR-335 with cisplatin further reduced the tumor size, and miR-335 brought down the sphere formation capacity induced by cisplatin. The current study demonstrates that miR-335 negatively regulates osteosarcoma stem cell-like properties by targeting POU5F1, and miR-335 could target CSCs to synergize with traditional chemotherapeutic agents to overcome osteosarcoma.

  18. Influence of low contents of superhydrophilic MWCNT on the properties and cell viability of electrospun poly (butylene adipate-co-terephthalate) fibers

    International Nuclear Information System (INIS)

    Rodrigues, Bruno V.M.; Silva, Aline S.; Melo, Gabriela F.S.; Vasconscellos, Luana M.R.; Marciano, Fernanda R.; Lobo, Anderson O.

    2016-01-01

    The use of poly (butylene adipate-co-terephthalate) (PBAT) in tissue engineering, more specifically in bone regeneration, has been underexplored to date due to its poor mechanical resistance. In order to overcome this drawback, this investigation presents an approach into the preparation of electrospun nanocomposite fibers from PBAT and low contents of superhydrophilic multi-walled carbon nanotubes (sMWCNT) (0.1–0.5 wt.%) as reinforcing agent. We employed a wide range of characterization techniques to evaluate the properties of the resulting electrospun nanocomposites, including Field Emission Scanning Electronic Microscopy (FE-SEM), Transmission Electronic Microscopy (TEM), tensile tests, contact angle measurements (CA) and biological assays. FE-SEM micrographs showed that while the addition of sMWCNT increased the presence of beads on the electrospun fibers' surfaces, the increase of the neat charge density due to their presence reduced the fibers' average diameter. The tensile test results pointed that sMWCNT acted as reinforcement in the PBAT electrospun matrix, enhancing its tensile strength (from 1.3 to 3.6 MPa with addition of 0.5 wt.% of sMWCNT) and leading to stiffer materials (lower elongation at break). An evaluation using MG63 cells revealed cell attachment into the biomaterials and that all samples were viable for biomedical applications, once no cytotoxic effect was observed. MG-63 cells osteogenic differentiation, measured by ALP activity, showed that mineralized nodules formation was increased in PBAT/0.5%CNTs when compared to control group (cells). This investigation demonstrated a feasible novel approach for producing electrospun nanocomposites from PBAT and sMWCNT with enhanced mechanical properties and adequate cell viability levels, which allows for a wide range of biomedical applications for these materials. - Highlights: • Nanocomposites from PBAT and superhydrophilic MWCNT (sMWCNT) were successfully prepared by electrospinning

  19. SaOS-2 cell response to macro-porous boron-incorporated TiO{sub 2} coating prepared by micro-arc oxidation on titanium

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Qianli [State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Elkhooly, Tarek A. [State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Department of Ceramics, Inorganic Chemical Industries Division, National Research Centre, Dokki, 12622 Cairo (Egypt); Liu, Xujie [State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055 (China); Zhang, Ranran; Yang, Xing; Shen, Zhijian [State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Feng, Qingling, E-mail: biomater@mail.tsinghua.edu.cn [State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Key Laboratory of Advanced Materials of Ministry of Education of China, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China)

    2016-10-01

    The aims of the present study were to develop boron-incorporated TiO{sub 2} coating (B-TiO{sub 2} coating) through micro-arc oxidation (MAO) and subsequently evaluate the effect of boron incorporation on the in vitro biological performance of the coatings. The physicochemical properties of B-TiO{sub 2} coating and its response to osteoblast like cells (SaOS-2) were investigated compared to the control group without boron (TiO{sub 2} coating). The morphological and X-ray diffraction results showed that both coatings exhibited similar surface topography and phase composition, respectively. However, the incorporation of B led to an enhancement in the surface hydrophilicity of B-TiO{sub 2} coating. The spreading of SaOS-2 cells on B-TiO{sub 2} coating was faster than that on TiO{sub 2} coating. The proliferation rate of SaOS-2 cells cultured on B-TiO{sub 2} decreased after 5 days of culture compared to that on TiO{sub 2} coating. SaOS-2 cells cultured on B-TiO{sub 2} coating exhibited an enhanced alkaline phosphatase (ALP) activity, Collagen I synthesis and in vitro mineralization compared to those on TiO{sub 2} coating. The present findings suggest that B-TiO{sub 2} coating is a promising candidate surface for orthopedic implants. - Highlights: • SaOS-2 cell response to pure TiO{sub 2} and B-TiO{sub 2} coatings was investigated. • Initial cell spreading on B-TiO{sub 2} coating was accelerated compared to that on TiO{sub 2} coating. • Cell proliferation on B-TiO{sub 2} coating was inhibited compared to that on TiO{sub 2} coating. • Cell differentiation on B-TiO{sub 2} coating was enhanced compared to that on TiO{sub 2} coating.

  20. Additive influence of extracellular pH, oxygen tension, and pressure on invasiveness and survival of human osteosarcoma cells

    Directory of Open Access Journals (Sweden)

    Takao eMatsubara

    2013-07-01

    Full Text Available BACKGROUND/PURPOSE:The effects of chemical and physical interactions in the microenvironment of solid tumors have not been fully elucidated. We hypothesized that acidosis, hypoxia, and elevated interstitial fluid pressure (eIFP have additive effects on tumor cell biology and lead to more aggressive behavior during tumor progression. We investigated this phenomenon using 3 human osteosarcoma cell lines and a novel in vitro cell culture apparatus. MATERIALS AND METHODS:U2OS, SaOS, and MG63 cell lines were cultured in media adjusted to various pH levels, oxygen tension (hypoxia 2% O2, normoxia 20% O2, and hydrostatic gauge pressure (0 or 50 mm Hg. Growth rate, apoptosis, cell cycle parameters, and expression of mRNA for proteins associated with invasiveness and tumor microenvironment (CA IX, VEGF-A, HIF-1A, MMP-9, and TIMP-2 were analyzed. Levels of CA IX, HIF-1α, and MMP-9 were measured using immunofluorescence. The effect of pH on invasiveness was evaluated in a Matrigel chamber assay.RESULTS: Within the acidic–hypoxic–pressurized conditions that simulate the microenvironment at a tumor’s center, invasive genes were upregulated, but the cell cycle was downregulated. The combined influence of acidosis, hypoxia, and IFP promoted invasiveness and angiogenesis to a greater extent than did pH, pO2, or eIFP individually. Significant cell death after brief exposure to acidic conditions occurred in each cell line during acclimation to acidic media, while prolonged exposure to acidic media resulted in reduced cell death. Furthermore, 48-hour exposure to acidic conditions promoted tumor invasiveness in the Matrigel assay. CONCLUSION: Our findings demonstrate that tumor microenvironmental parameters—particularly pH, pO2, and eIFP—additively influence tumor proliferation, invasion, metabolism, and viability to enhance cell survival.

  1. Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro.

    Science.gov (United States)

    Geiss, Carsten; Kis, Zoltán; Leuchs, Barbara; Frank-Stöhr, Monika; Schlehofer, Jörg R; Rommelaere, Jean; Dinsart, Christiane; Lacroix, Jeannine

    2017-10-17

    Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.

  2. Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro

    Directory of Open Access Journals (Sweden)

    Carsten Geiss

    2017-10-01

    Full Text Available Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.

  3. Modified n-HA/PA66 scaffolds with chitosan coating for bone tissue engineering: cell stimulation and drug release.

    Science.gov (United States)

    Zou, Qin; Li, Junfeng; Niu, Lulu; Zuo, Yi; Li, Jidong; Li, Yubao

    2017-09-01

    The dipping-drying procedure and cross-linking method were used to make drug-loaded chitosan (CS) coating on nano-hydroxyapatite/polyamide66 (nHA/PA66) composite porous scaffold, endowing the scaffold controlled drug release functionality. The prefabricated scaffold was immersed into an aqueous drug/CS solution in a vacuum condition and then crosslinked by vanillin. The structure, porosity, composition, compressive strength, swelling ratio, drug release and cytocompatibility of the pristine and coating scaffolds were investigated. After coating, the scaffold porosity and pore interconnection were slightly decreased. Cytocompatibility performance was observed through an in vitro experiment based on cell attachment and the MTT assay by MG63 cells which revealed positive cell viability and increasing proliferation over the 11-day period in vitro. The drug could effectively release from the coated scaffold in a controlled fashion and the release rate was sustained for a long period and highly dependent on coating swelling, suggesting the possibility of a controlled drug release. Our results demonstrate that the scaffold with drug-loaded crosslinked CS coating can be used as a simple technique to render the surfaces of synthetic scaffolds active, thus enabling them to be a promising high performance biomaterial in bone tissue engineering.

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

    International Nuclear Information System (INIS)

    Wihlidal, P.

    2010-01-01

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

  5. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Qiang [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhao, Zhi-Ning [Clinical Laboratory, 451 Hospital of Chinese PLA, Xi' an 710054 (China); Cheng, Jing-Tao [Department of Special Dentistry, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhang, Bin [Department of Orthodontics, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Xu, Jie [Department of Periodontology, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Huang, Fei; Zhao, Rui-Ni [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Chen, Yong-Jin, E-mail: cyj1229@fmmu.edu.cn [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China)

    2011-01-07

    Research highlights: {yields} Ibandronate significantly promote the proliferation of PDLSC cells. {yields} Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. {yields} The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. {yields} Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. {yields} Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation

  6. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    International Nuclear Information System (INIS)

    Zhou, Qiang; Zhao, Zhi-Ning; Cheng, Jing-Tao; Zhang, Bin; Xu, Jie; Huang, Fei; Zhao, Rui-Ni; Chen, Yong-Jin

    2011-01-01

    Research highlights: → Ibandronate significantly promote the proliferation of PDLSC cells. → Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. → The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. → Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. → Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation-related genes via miRNAs. The exact

  7. Osteogenic cell differentiation on H-terminated and O-terminated nanocrystalline diamond films

    Directory of Open Access Journals (Sweden)

    Liskova J

    2015-01-01

    Full Text Available Jana Liskova,1 Oleg Babchenko,2 Marian Varga,2 Alexander Kromka,2 Daniel Hadraba,1 Zdenek Svindrych,1 Zuzana Burdikova,1 Lucie Bacakova1 1Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 2Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Abstract: Nanocrystalline diamond (NCD films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination or oxygen atoms (O-termination. Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers – type I collagen, alkaline phosphatase, and osteocalcin – was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix

  8. Imatinib mesylate exerts anti-proliferative effects on osteosarcoma cells and inhibits the tumour growth in immunocompetent murine models.

    Directory of Open Access Journals (Sweden)

    Bérengère Gobin

    Full Text Available Osteosarcoma is the most common primary malignant bone tumour characterized by osteoid production and/or osteolytic lesions of bone. A lack of response to chemotherapeutic treatments shows the importance of exploring new therapeutic methods. Imatinib mesylate (Gleevec, Novartis Pharma, a tyrosine kinase inhibitor, was originally developed for the treatment of chronic myeloid leukemia. Several studies revealed that imatinib mesylate inhibits osteoclast differentiation through the M-CSFR pathway and activates osteoblast differentiation through PDGFR pathway, two key cells involved in the vicious cycle controlling the tumour development. The present study investigated the in vitro effects of imatinib mesylate on the proliferation, apoptosis, cell cycle, and migration ability of five osteosarcoma cell lines (human: MG-63, HOS; rat: OSRGA; mice: MOS-J, POS-1. Imatinib mesylate was also assessed as a curative and preventive treatment in two syngenic osteosarcoma models: MOS-J (mixed osteoblastic/osteolytic osteosarcoma and POS-1 (undifferentiated osteosarcoma. Imatinib mesylate exhibited a dose-dependent anti-proliferative effect in all cell lines studied. The drug induced a G0/G1 cell cycle arrest in most cell lines, except for POS-1 and HOS cells that were blocked in the S phase. In addition, imatinib mesylate induced cell death and strongly inhibited osteosarcoma cell migration. In the MOS-J osteosarcoma model, oral administration of imatinib mesylate significantly inhibited the tumour development in both preventive and curative approaches. A phospho-receptor tyrosine kinase array kit revealed that PDGFRα, among 7 other receptors (PDFGFRβ, Axl, RYK, EGFR, EphA2 and 10, IGF1R, appears as one of the main molecular targets for imatinib mesylate. In the light of the present study and the literature, it would be particularly interesting to revisit therapeutic evaluation of imatinib mesylate in osteosarcoma according to the tyrosine-kinase receptor

  9. 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.

  10. 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.

  11. The effect of taurine chloramine on human osteosarcoma cell-lines

    International Nuclear Information System (INIS)

    Pilz, M.

    2010-01-01

    Osteosarcoma is the most frequent nonhaematogenic primary malignant tumor of the bone. The rather rare tumour occurs mainly in children and adolescents. This osteogenic tumour is a high-aggressive mesenchymal neoplasm typically producing osteoid. The surgical resection of the complete tumour must be carried out with wide or radical margins to prevent recurrence. Even though the combination of surgery with chemotherapy has noticeably improved the survival rate of osteosarcoma patients, the application of anticancer drugs is still associated with significant adverse reactions, for example the common acquisition of drug-resistant phenotypes, indicating the need of new chemotherapeutical substances. Taurine is a sulfur-containing β-amino acid, which is present in high concentrations in mammalian cells and plasma, in granulocytes and lymphocytes. Large quantities of taurine are released by stimulated neutrophiles and rapidly chlorinated by the reaction with hypochlorous acid (HOCl) generating taurine monochloramine (NCT). NCT is noted to play quite a few roles in the modulation of the immune response and sizeably decreases the production of plenty proinflammatory mediators from adherent as well as non-adherent leucocytes. NCT inhibits the intracellular transcription of nitric-oxide synthase and the production of nitric oxide and switches cell death from the less advantageous necrosis to the more physiologically beneficial apoptosis. Aim of this study was to research, if different concentrations of NCT are capable to induce apoptosis in the human osteosarcoma cell lines HOS, MG-63 and SAOS-2. Therefore the cell proliferation assay EZ4U, a fluorescence staining with acridine-orange, an ELISA for the detection of DNA-fragments and a JC-1 mitochondrial FACS-analysis were performed. The results of these four independent experiments show, that NCT has a pro-apoptotic effect on these osteosarcoma cell lines. (author) [de

  12. ZnO nanoparticle incorporated nanostructured metallic titanium for increased mesenchymal stem cell response and antibacterial activity

    Science.gov (United States)

    Elizabeth, Elmy; Baranwal, Gaurav; Krishnan, Amit G.; Menon, Deepthy; Nair, Manitha

    2014-03-01

    Recent trends in titanium implants are towards the development of nanoscale topographies that mimic the nanoscale properties of bone tissue. Although the nanosurface promotes the integration of osteoblast cells, infection related problems can also occur, leading to implant failure. Therefore it is imperative to reduce bacterial adhesion on an implant surface, either with or without the use of drugs/antibacterial agents. Herein, we have investigated two different aspects of Ti surfaces in inhibiting bacterial adhesion and concurrently promoting mammalian cell adhesion. These include (i) the type of nanoscale topography (Titania nanotube (TNT) and Titania nanoleaf (TNL)) and (ii) the presence of an antibacterial agent like zinc oxide nanoparticles (ZnOnp) on Ti nanosurfaces. To address this, periodically arranged TNT (80-120 nm) and non-periodically arranged TNL surfaces were generated by the anodization and hydrothermal techniques respectively, and incorporated with ZnOnp of different concentrations (375 μM, 750 μM, 1.125 mM and 1.5 mM). Interestingly, TNL surfaces decreased the adherence of staphylococcus aureus while increasing the adhesion and viability of human osteosarcoma MG63 cell line and human mesenchymal stem cells, even in the absence of ZnOnp. In contrast, TNT surfaces exhibited an increased bacterial and mammalian cell adhesion. The influence of ZnOnp on these surfaces in altering the bacterial and cell adhesion was found to be concentration dependent, with an optimal range of 375-750 μM. Above 750 μM, although bacterial adhesion was reduced, cellular viability was considerably affected. Thus our study helps us to infer that nanoscale topography by itself or its combination with an optimal concentration of antibacterial ZnOnp would provide a differential cell behavior and thereby a desirable biological response, facilitating the long term success of an implant.

  13. ZnO nanoparticle incorporated nanostructured metallic titanium for increased mesenchymal stem cell response and antibacterial activity

    International Nuclear Information System (INIS)

    Elizabeth, Elmy; Baranwal, Gaurav; Krishnan, Amit G; Menon, Deepthy; Nair, Manitha

    2014-01-01

    Recent trends in titanium implants are towards the development of nanoscale topographies that mimic the nanoscale properties of bone tissue. Although the nanosurface promotes the integration of osteoblast cells, infection related problems can also occur, leading to implant failure. Therefore it is imperative to reduce bacterial adhesion on an implant surface, either with or without the use of drugs/antibacterial agents. Herein, we have investigated two different aspects of Ti surfaces in inhibiting bacterial adhesion and concurrently promoting mammalian cell adhesion. These include (i) the type of nanoscale topography (Titania nanotube (TNT) and Titania nanoleaf (TNL)) and (ii) the presence of an antibacterial agent like zinc oxide nanoparticles (ZnOnp) on Ti nanosurfaces. To address this, periodically arranged TNT (80–120 nm) and non-periodically arranged TNL surfaces were generated by the anodization and hydrothermal techniques respectively, and incorporated with ZnOnp of different concentrations (375 μM, 750 μM, 1.125 mM and 1.5 mM). Interestingly, TNL surfaces decreased the adherence of staphylococcus aureus while increasing the adhesion and viability of human osteosarcoma MG63 cell line and human mesenchymal stem cells, even in the absence of ZnOnp. In contrast, TNT surfaces exhibited an increased bacterial and mammalian cell adhesion. The influence of ZnOnp on these surfaces in altering the bacterial and cell adhesion was found to be concentration dependent, with an optimal range of 375−750 μM. Above 750 μM, although bacterial adhesion was reduced, cellular viability was considerably affected. Thus our study helps us to infer that nanoscale topography by itself or its combination with an optimal concentration of antibacterial ZnOnp would provide a differential cell behavior and thereby a desirable biological response, facilitating the long term success of an implant. (paper)

  14. Reaming debris as a novel source of autologous bone to enhance healing of bone defects

    NARCIS (Netherlands)

    Bakker, A.D.; Kroeze, R.J.; Korstjens, C.; de Kleine, R.H.; Frolke, J.P.M.; Klein-Nulend, J.

    2011-01-01

    Reaming debris is formed when bone defects are stabilized with an intramedullary nail, and contains viable osteoblast-like cells and growth factors, and might thus act as a natural osteoinductive scaffold. The advantage of using reaming debris over stem cells or autologous bone for healing bone

  15. Reaming debris as a novel source of autologous bone to enhance healing of bone defects

    NARCIS (Netherlands)

    Bakker, Astrid D.; Kroeze, Robert Jan; Korstjens, Clara; de Kleine, Ruben H.; Frolke, Jan Paul M.; Klein-Nulend, Jenneke

    Reaming debris is formed when bone defects are stabilized with an intramedullary nail, and contains viable osteoblast-like cells and growth factors, and might thus act as a natural osteoinductive scaffold. The advantage of using reaming debris over stem cells or autologous bone for healing bone

  16. 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

  17. MicroRNA-127-3p inhibits proliferation and invasion by targeting SETD8 in human osteosarcoma cells

    International Nuclear Information System (INIS)

    Zhang, Jun; Hou, Wengen; Chai, Mingxiang; Zhao, Hongxing; Jia, Jinling; Sun, Xiaohui; Zhao, Bin; Wang, Ran

    2016-01-01

    MicroRNAs (miRNAs) play an essential role in cancer development. Several studies have indicated that miRNAs mediate tumorigenesis processes, such as, inflammation, proliferation, apoptosis and invasion. In the present study, we focused on the influence of the miR-127-3p on the proliferation, migration and invasion of osteosarcoma (OS). MiR-127-3p was found at reduced levels in OS tissues and cell lines. Overexpression of miR-127-3p in the OS cell lines significantly inhibited the cell proliferation, migration and invasion; however, inhibition of miR-127-3p increased the proliferation, migration and invasion of OS in vitro. SETD8 was identified as a direct target of miR-127-3p, and SETD8 expression decreased post miR-127-3p overexpression, while SETD8 overexpression could reverse the potential influence of miR-127-3p on the migration and invasion of OS cells. MiR-127-3p is suggested to act mainly via the suppression of SETD8 expression. Overall, the results revealed that miR-127-3p acts as a tumor suppressor and that its down-regulation in cancer may contribute to OS progression and metastasis, suggesting that miR-127-3p could be a potential therapeutic target in the treatment of OS. - Highlights: • MiR-127-3p is decreased in osteosarcoma tissues and cell lines. • MiR-127-3p overexpression suppresses cell migration and invasion in MG63 and U2OS. • SETD8 overexpression abolishes the roles of miR-127-3p in osteosarcoma.

  18. Bone repair by cell-seeded 3D-bioplotted composite scaffolds made of collagen treated tricalciumphosphate or tricalciumphosphate-chitosan-collagen hydrogel or PLGA in ovine critical-sized calvarial defects.

    Science.gov (United States)

    Haberstroh, Kathrin; Ritter, Kathrin; Kuschnierz, Jens; Bormann, Kai-Hendrik; Kaps, Christian; Carvalho, Carlos; Mülhaupt, Rolf; Sittinger, Michael; Gellrich, Nils-Claudius

    2010-05-01

    The aim of this study was to investigate the osteogenic effect of three different cell-seeded 3D-bioplotted scaffolds in a ovine calvarial critical-size defect model. The choice of scaffold-materials was based on their applicability for 3D-bioplotting and respective possibility to produce tailor-made scaffolds for the use in cranio-facial surgery for the replacement of complex shaped boneparts. Scaffold raw-materials are known to be osteoinductive when being cell-seeded [poly(L-lactide-co-glycolide) (PLGA)] or having components with osteoinductive properties as tricalciumphosphate (TCP) or collagen (Col) or chitosan. The scaffold-materials PLGA, TCP/Col, and HYDR (TCP/Col/chitosan) were cell-seeded with osteoblast-like cells whether gained from bone (OLB) or from periost (OLP). In a prospective and randomized design nine sheep underwent osteotomy to create four critical-sized calvarial defects. Three animals each were assigned to the HYDR-, the TCP/Col-, or the PLGA-group. In each animal, one defect was treated with a cell-free, an OLB- or OLP-seeded group-specific scaffold, respectively. The fourth defect remained untreated as control (UD). Fourteen weeks later, animals were euthanized for histo-morphometrical analysis of the defect healing. OLB- and OLP-seeded HYDR and OLB-seeded TCP/Col scaffolds significantly increased the amount of newly formed bone (NFB) at the defect bottom and OLP-seeded HYDR also within the scaffold area, whereas PLGA-scaffolds showed lower rates. The relative density of NFB was markedly higher in the HYDR/OLB group compared to the corresponding PLGA group. TCP/Col had good stiffness to prepare complex structures by bioplotting but HYDR and PLGA were very soft. HYDR showed appropriate biodegradation, TCP/Col and PLGA seemed to be nearly undegraded after 14 weeks. 3D-bioplotted, cell-seeded HYDR and TCP/Col scaffolds increased the amount of NFB within ovine critical-size calvarial defects, but stiffness, respectively, biodegradation of

  19. Effect and Mechanism of EGFL7 Downregulation in Human Osteosarcoma Cells on the Biological Function of Co-cultured HUVEC

    Directory of Open Access Journals (Sweden)

    Xia Li

    2018-03-01

    Full Text Available Background: Even though epidermal growth factor-like domain 7 is known to be overexpressed in osteosarcoma and is associated with poor clinical outcome, few reports are available regarding its mechanism. Aims: The objective of this study was to explore the effect and mechanism of downregulating epidermal growth factor-like domain 7 expression in a human osteosarcoma cell line on the biological function of co-cultured human umbilical vein endothelial cells. Study Design: Cell study. Methods: In the present study, human osteosarcoma cell lines U2OS, Saos-2, HOS, and MG63, and normal human osteoblasts were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum and 1x antibiotics at 37 °C and 5% CO2 in an incubator. Of the four osteosarcoma cell lines, U2OS expresses the highest level of epidermal growth factor-like domain 7 mRNA as determined using quantitative reverse transcription polymerase chain reaction. With the knockdown of epidermal growth factor-like domain 7 in U2OS and human umbilical vein endothelial cells by lentivirus, the proliferation and apoptosis of U2OS and human umbilical vein endothelial cells were investigated using MTT and flow cytometry assays. After the co-culture of human umbilical vein endothelial cells and epidermal growth factor-like domain 7-knockdown U2OS, the in vitro effects on cell proliferation, apoptosis, adhesion, migration, and the angiogenic ability of human umbilical vein endothelial cells were detected using MTT, flow cytometry, Transwell, and tube formation assays, respectively. The expressions of phosphoinositide 3-kinase, phospho-Akt, total Akt, and vascular endothelial growth factor in human umbilical vein endothelial cells were detected using western blot assay. Results: Lentivirus with epidermal growth factor-like domain 7 shRNA could not significantly affect the proliferation and apoptosis of both U2OS and human umbilical vein endothelial cells, whereas the knockdown of

  20. Cell adhesion and growth on ultrananocrystalline diamond and diamond-like carbon films after different surface modifications

    Energy Technology Data Exchange (ETDEWEB)

    Miksovsky, J. [Institute of Nanostructure Technologies and Analytics, Center for Interdisciplinary Nanostructure Science and Technology, University of Kassel (Germany); Institute of Physics ASCR, Prague (Czech Republic); Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno (Czech Republic); Voss, A. [Institute of Nanostructure Technologies and Analytics, Center for Interdisciplinary Nanostructure Science and Technology, University of Kassel (Germany); Kozarova, R. [Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia (Bulgaria); Kocourek, T.; Pisarik, P. [Institute of Physics ASCR, Prague (Czech Republic); Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno (Czech Republic); Ceccone, G. [Unit Nanobiosciences, European Commission Joint Research Centre, Ispra (Italy); Kulisch, W. [Institute of Nanostructure Technologies and Analytics, Center for Interdisciplinary Nanostructure Science and Technology, University of Kassel (Germany); Jelinek, M. [Institute of Physics ASCR, Prague (Czech Republic); Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno (Czech Republic); Apostolova, M.D. [Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia (Bulgaria); Reithmaier, J.P. [Institute of Nanostructure Technologies and Analytics, Center for Interdisciplinary Nanostructure Science and Technology, University of Kassel (Germany); Popov, C., E-mail: popov@ina.uni-kassel.de [Institute of Nanostructure Technologies and Analytics, Center for Interdisciplinary Nanostructure Science and Technology, University of Kassel (Germany)

    2014-04-01

    Graphical abstract: - Highlights: • UNCD and DLC films were modified by UV/O{sub 3} treatments, O{sub 2} or NH{sub 3}-containing plasmas. • Surface composition, wettability and surface energy change upon modifications. • Higher efficiency of UNCD modifications was observed. • Cell attachment and growth were influenced by the surface termination and roughness. - Abstract: Diamond and diamond-like carbon (DLC) films possess a set of excellent physical and chemical properties which together with a high biocompatibility make them attractive candidates for a number of medical and biotechnological applications. In the current work thin ultrananocrystalline diamond (UNCD) and DLC films were comparatively investigated with respect to cell attachment and proliferation after different surface modifications. The UNCD films were prepared by microwave plasma enhanced chemical vapor deposition, the DLC films by pulsed laser deposition (PLD). The films were comprehensively characterized with respect to their basic properties, e.g. crystallinity, morphology, chemical bonding nature, etc. Afterwards the UNCD and DLC films were modified applying O{sub 2} or NH{sub 3}/N{sub 2} plasmas and UV/O{sub 3} treatments to alter their surface termination. The surface composition of as-grown and modified samples was studied by X-ray photoelectron spectroscopy (XPS). Furthermore the films were characterized by contact angle measurements with water, formamide, 1-decanol and diiodomethane; from the results obtained the surface energy with its dispersive and polar components was calculated. The adhesion and proliferation of MG63 osteosarcoma cells on the different UNCD and DLC samples were assessed by measurement of the cell attachment efficiency and MTT assays. The determined cell densities were compared and correlated with the surface properties of as-deposited and modified UNCD and DLC films.

  1. Low-level light therapy potentiates NPe6-mediated photodynamic therapy in a human osteosarcoma cell line via increased ATP.

    Science.gov (United States)

    Tsai, Shang-Ru; Yin, Rui; Huang, Ying-Ying; Sheu, Bor-Ching; Lee, Si-Chen; Hamblin, Michael R

    2015-03-01

    Low-level light therapy (LLLT) is used to stimulate healing, reduce pain and inflammation, and preserve tissue from dying. LLLT has been shown to protect cells in culture from dying after various cytotoxic insults, and LLLT is known to increase the cellular ATP content. Previous studies have demonstrated that maintaining a sufficiently high ATP level is necessary for the efficient induction and execution of apoptosis steps after photodynamic therapy (PDT). We asked whether LLLT would protect cells from cytotoxicity due to PDT, or conversely whether LLLT would enhance the efficacy of PDT mediated by mono-l-aspartyl chlorin(e6) (NPe6). Increased ATP could lead to enhanced cell uptake of NPe6 by the energy dependent process of endocytosis, and also to more efficient apoptosis. In this study, human osteosarcoma cell line MG-63 was subjected to 1.5J/cm(2) of 810nm near infrared radiation (NIR) followed by addition of 10μM NPe6 and after 2h incubation by 1.5J/cm(2) of 652nm red light for PDT. PDT combined with LLLT led to higher cell death and increased intracellular reactive oxygen species compared to PDT alone. The uptake of NPe6 was moderately increased by LLLT, and cellular ATP was increased. The mitochondrial respiratory chain inhibitor antimycin A abrogated the LLLT-induced increase in cytotoxicity. Taken together, these results demonstrate that LLLT potentiates NPe6-mediated PDT via increased ATP synthesis and is a potentially promising strategy that could be applied in clinical PDT. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Integration of porosity and bio-functionalization to form a 3D scaffold: cell culture studies and in vitro degradation

    Energy Technology Data Exchange (ETDEWEB)

    Mittal, Anupama; Negi, Poonam; Garkhal, Kalpna; Verma, Shalini; Kumar, Neeraj, E-mail: neeraj@niper.ac.i [Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar-160 062, Punjab (India)

    2010-08-01

    In this study, porous poly(lactide-co-glycolide) (PLGA) (50/50) microspheres have been fabricated by the gas-foaming technique using ammonium bicarbonate as a gas-foaming agent. Microspheres of different porosities have been formulated by varying the concentration of the gas-foaming agent (0%, 5%, 10% and 15% w/v). These microspheres were characterized for particle size, porosity and average pore size, morphology, water uptake ratio and surface area and it was found that the porosity, pore size and surface area increased on increasing the concentration of the gas-foaming agent. Further, the effect of porosity on degradation behavior was evaluated over a 12 week period by measuring changes in mass, pH, molecular weight and morphology. Porosity was found to have an inverse relationship with degradation rate. To render the surface of the microspheres biomimetic, peptide P-15 was coupled to the surface of these microspheres. In vitro cell viability, proliferation and morphological evaluation were carried out on these microsphere scaffolds using MG-63 cell line to study the effect of the porosity and pore size of scaffolds and to evaluate the effect of P-15 on cell growth on porous scaffolds. MTT assay, actin, alizarin staining and SEM revealed the potential of biomimetic porous PLGA (50/50) microspheres as scaffolds for tissue engineering. As shown in graphical representation, an attempt has been made to correlate the cell behavior on the scaffolds (growth, proliferation and cell death) with the concurrent degradation of the porous microsphere scaffold as a function of time.

  3. Integration of porosity and bio-functionalization to form a 3D scaffold: cell culture studies and in vitro degradation

    International Nuclear Information System (INIS)

    Mittal, Anupama; Negi, Poonam; Garkhal, Kalpna; Verma, Shalini; Kumar, Neeraj

    2010-01-01

    In this study, porous poly(lactide-co-glycolide) (PLGA) (50/50) microspheres have been fabricated by the gas-foaming technique using ammonium bicarbonate as a gas-foaming agent. Microspheres of different porosities have been formulated by varying the concentration of the gas-foaming agent (0%, 5%, 10% and 15% w/v). These microspheres were characterized for particle size, porosity and average pore size, morphology, water uptake ratio and surface area and it was found that the porosity, pore size and surface area increased on increasing the concentration of the gas-foaming agent. Further, the effect of porosity on degradation behavior was evaluated over a 12 week period by measuring changes in mass, pH, molecular weight and morphology. Porosity was found to have an inverse relationship with degradation rate. To render the surface of the microspheres biomimetic, peptide P-15 was coupled to the surface of these microspheres. In vitro cell viability, proliferation and morphological evaluation were carried out on these microsphere scaffolds using MG-63 cell line to study the effect of the porosity and pore size of scaffolds and to evaluate the effect of P-15 on cell growth on porous scaffolds. MTT assay, actin, alizarin staining and SEM revealed the potential of biomimetic po